ROTAVIRUS VACCINES AND IMPACT OF MATERNAL ANTIBODIES AND CYTOKINES ON NEONATAL IMMUNE RESPONSES IN SWINE DISSERTATION

Transcription

1 ROTAVIRUS VACCINES AND IMPACT OF MATERNAL ANTIBODIES AND CYTOKINES ON NEONATAL IMMUNE RESPONSES IN SWINE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Trang Van Nguyen, B.Sc. ***** The Ohio State University 2005 Dissertation Committee: Distinguished University Professor Dr. Linda J. Saif, Adviser Professor John H. Hughes Approved by Assistant Professor Dr. Jeffrey T. Lejeune Adjunct Assistant Professor Dr. Lijuan Yuan Adviser Graduate Program in Veterinary Preventive Medicine

2

3 ABSTRACT Group A rotavirus (RV) is the most important cause of viral gastroenteritis in young children and animals worldwide and the need for effective vaccines against RV is urgent. Because infections occur in infants and nursing pigs, the vaccine should be delivered as early as the first few days or weeks of life, which poses problems because of the impact of maternal antibodies (MatAb) on vaccine responses. The transfer of cytokines from colostrum/milk to neonates which influences the development of neonatal immunity has not been studied in humans or animals. The main goals of this thesis were: (1) to study the efficacy, immunogenicity and correlates of protective immunity after oral vaccination with virus-like particles (VLP) with or without attenuated human rotavirus (AttHRV) in a prime/boost strategy; (2) and (3) to understand the impact of high and low titer circulating MatAb to RV on protective immunity and B cell responses induced by RV vaccines (replicating vs. non-replicating); and (4) to examine the transfer of cytokines from mothers to neonates and the implications of such transfer for immunomodulation of neonatal immunity. ii

4 We evaluated antibody responses and protection induced by a replicating vaccine consisting of an oral dose of AttHRV for priming and two intranasal doses of a 2/6VLP(100 or 250ug)/ISCOM vaccine for boosting (AttHRV/VLP) or an intranasal VLP/ISCOM prime/boost vaccine (VLP/ISCOM3x) in gnotobiotic (Gn) pigs and compared them with the 3 doses AttHRV (AttHRV3x) oral vaccine. The AttHRV- VLP250µg/ISCOM and AttHRV3x groups had significantly higher serum IgA and IgG and intestinal IgA antibody titers to HRV pre-challenge than the 3-dose- VLP100µg/ISCOM group (VLP/ISCOM3x) and controls (diluent/iscommatrix). The pre-challenge serum virus neutralizing and IgA antibodies correlated moderately with protection against challenge with virulent HRV. Protection rates against viral shedding and diarrhea were highest in the AttHRV-VLP250µg/ISCOM and AttHRV3x groups, lower in the AttHRV-VLP100µg/ISCOM group, and with no protection in the VLP/ISCOM3x group and controls. Thus the VLP/ISCOM vaccine boosted antibody titers and protection after priming with the AttHRV vaccine, but not after priming with VLP/ISCOM. We next investigated effects of high titer MatAb on protection and immune responses induced by the above AttHRV/VLP and VLP/ISCOM3x vaccines. Passive circulating MatAb (hyperimmune sow serum) injected into Gn pigs contributed to partial protection against virulent HRV challenge; however, MatAb interference led to no or low intestinal IgM, IgA and IgG antibody titers and significantly reduced intestinal IgA and IgG antibody secreting cell (ASC) and memory B cell responses in the AttHRV/VLP pigs pre- and post-challenge. The MatAb suppression was not alleviated by extending the vaccination/challenge interval from 28 to 42 days. The suppression by high titer MatAb iii

5 was also observed in the non-replicating 3xVLP/ISCOM vaccinated pigs, as indicated by a reduction in intestinal IgG ASC responses, serum and intestinal IgA antibody titers and the pre-challenge memory B cell responses. Consequently, alternative strategies are needed to overcome the high titer MatAb suppression of immune responses to vaccines in neonates. We then investigated the effect of low titer circulating MatAb (LoMatAb) on protection and immune responses induced by these two vaccine regimens. Protection rates in the AttHRV/VLP groups with and without LoMatAb were similar against viral shedding and diarrhea when challenged with virulent HRV. The LoMatAb had both enhancing and suppressive effects on B cell responses, depending on antibody isotype, tissue and vaccine. For the AttHRV/VLP (replicating) vaccine, LoMatAb enhanced intestinal IgM and IgA-ASC numbers pre- or post-challenge, but it suppressed systemic (spleen and blood) IgA-ASC numbers and intestinal IgA antibody responses pre- and post- challenge. For the VLP (non-replicating) vaccine, LoMatAb also enhanced intestinal and splenic IgM-ASC numbers post-challenge, but it suppressed IgG-ASC numbers pre-challenge in all tissues. Pre-challenge IgA memory B cells in ileum and spleen were unaffected, but they were suppressed in blood of both vaccine groups. The differential effects of LoMatAb on the IgA responses suggests that LoMatAb did not suppress the induction of IgA-ASC and memory B cells at the induction site (ileum) but it impaired the homing of activated B cells to secondary lymphoid or effector tissues, reducing IgA-ASC and antibodies at these sites. Thus, even LoMatAb exerts different effects on B cell responses to replicating versus non-replicating vaccines. iv

6 In addition to MatAb, other important immune regulators such as cytokines may also play significant roles in the development of the neonatal immune system and the characteristics of the reduced responsiveness and Th2 biased immune responses in neonates. The effects of maternally-acquired cytokines on development of neonatal immunity are undefined. We investigated IL-6 and TNF-α (pro-inflammatory), IFN-γ and IL-12, (Th1), IL-10 and IL-4 (Th2) and TGF-β1 (Th3) cytokine concentrations in sow serum and colostrum/milk and in serum and intestinal contents of their suckling piglets at 0-13 post-partum days (PPD) or post-weaning. All cytokines except for TNF-α were detected in sow colostrum/milk. No IL-6, TNF-α, IFN-γ, IL-4 and IL-10 were detected in piglet sera at PPD0 documenting absence of transplacental transfer, whereas IL-12 and TGF-β1, present at birth, may be constitutively produced or maternallyderived. The peak mean cytokine concentrations in piglet sera were detected at PPD1-2 (IL-4>TGF-β1>IL-6>IL-12>IFN-γ>IL-10) with high concentrations of IL-4 and TGF-β1 likely contributing to the neonatal Th2 bias. Increased concentrations of intestinal IL-6, IL-12 and TNF-α in suckling piglets and of serum IL-6 and IL-12 in weaned piglets were potentially induced by intestinal colonization with commensal bacteria. Low concentrations of IFN-γ in suckling and weaned piglets may be due to its downregulation by TGF-β. In summary, we comprehensively documented the transfer of maternal cytokines from colostrum/milk to neonates and have provided new evidence for their potential role in the Th2 bias of neonatal immune responses. These findings have improved our understanding of the differential effects of circulating MatAb on different RV vaccines and protection and should facilitate the v

7 design of new rotavirus vaccines to overcome such interference. Our research also provides new information about the cytokines in maternal milk, their transfer and persistence in neonates and their potential role in the development of neonatal immune responses. vi

8 DEDICATION I dedicate this dissertation to my parents, Nguyen Ngoc Thien and Tran Thi Binh An, whose love and devotion always motivated me To my husband, Hoang, whose love supported and encouraged me from thousands miles away vii

9 ACKNOWLEDGMENTS I am in debt to my adviser, Dr Linda J. Saif for giving me the opportunity to work in her lab. I am grateful for her guidance, support and patience during my study, the preparation of the manuscripts and this dissertation and during my unexpected delay in Vietnam. I learned a lot from her critical thinking and original ideas. Her knowledge and dedication to research was an inspiration during my study. My deep gratitude goes to my committee member Dr. Lijuan Yuan for innovative ideas and extensive revision of my manuscripts and dissertation. I thank her for the constant encouragement from the first day I started this PhD program. I sincerely thank Dr Jeoffrey LeJeune for helpful comments on the manuscripts and my dissertation, in special his help with the statistical analysis. I thank Dr. John Hughes for valuable comments on my dissertation. Many thanks to Dr. Mo Saif, Dr. Kenneth Theil, Dr. Joy Pate and Dr. Srinand Sreevatsan for reviewing my manuscripts. To my colleagues, graduate students and post-doctors: Dr. Marli Azevedo, Dr. Kwang-il Jeong, Dr. Cristiana Iosef and Ana Gonzalez with whom I shared the hard work in rotavirus vaccine research in the pig model. Thank you for the friendship in special to viii

10 Dr. Quihong Wang, Menira Souza, Veronica Costantini, Sonia Cheetham, Peggy Lewis, Marcela Azevedo, Severin Pouly, Stacie Shafer and Ke Wei. Many thanks go to Hong Liu for his valuable help and suggestions with statistical analyses. To Dr. Juliette Hanson and Greg Myers for their technical assistance and efforts to make my work with animals possible. To Terry Meek, Todd Root and Richard McCormick for their technical assistance. To Hannah Gehman and Robin Weimer, the secretaries of the Department for their indispensable support. Thanks to the Food Animal Health Research Program-OARDC for giving me the fellowship for my PhD (from September 2002 to August 2005). Thanks to the OARDC for granting me the Charles Thorne fellowship for the year My thanks to the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam in special to Dr Vu Tan Trao for her support and encouragement. My sincere appreciation to Samar Al Maalouf for her precious friendship and support during stressful times. I also thank her for her invaluable comments on my dissertation. My appreciation once again to my husband, Hoang, for his patience, love and understanding that helped me to pass through this great challenge. ix

11 VITA July 20, Born- Hanoi, Vietnam Bachelor of Science The University of Western Australia, Perth, Australia Research Scientist National Institute of Hygiene and Epidemiology, Hanoi, Vietnam Graduate Student Food Animal Health Research Department Department of Veterinary Preventive Medicine, OARDC, The Ohio State University PUBLICATIONS 1. Azevedo, M. S., Yuan, L., Jeong, K. I., Gonzalez, A., Nguyen, T. V., Pouly, S., Gochnauer, M., Zhang, W., Azevedo, A., and Saif, L. J. (2005). Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus. J Virol 79(9), x

12 2. Yuan, L., Azevedo, M. S., Gonzalez, A. M., Jeong, K. I., Van Nguyen, T., Lewis, P., Iosef, C., Herrmann, J. E., and Saif, L. J. (2005). Mucosal and systemic antibody responses and protection induced by a prime/boost rotavirus-dna vaccine in a gnotobiotic pig model. Vaccine 23(30), Azevedo, M. S., Yuan, L., Iosef, C., Chang, K. O., Kim, Y., Nguyen, T. V., and Saif, L. J. (2004). Magnitude of serum and intestinal antibody responses induced by sequential replicating and nonreplicating rotavirus vaccines in gnotobiotic pigs and correlation with protection. Clin Diagn Lab Immunol 11(1), Gonzalez, A. M., Nguyen, T. V., Azevedo, M. S., Jeong, K., Agarib, F., Iosef, C., Chang, K., Lovgren-Bengtsson, K., Morein, B., and Saif, L. J. (2004). Antibody responses to human rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM. Clin Exp Immunol 135(3), Nguyen, T. V., Iosef, C., Jeong, K., Kim, Y., Chang, K. O., Lovgren- Bengtsson, K., Morein, B., Azevedo, M. S., Lewis, P., Nielsen, P., Yuan, L., and Saif, L. J. (2003). Protection and antibody responses to oral priming by attenuated human rotavirus followed by oral boosting with 2/6-rotavirus-like particles with immunostimulating complexes in gnotobiotic pigs. Vaccine 21(25-26), Iosef, C., Van Nguyen, T., Jeong, K., Bengtsson, K., Morein, B., Kim, Y., Chang, K. O., Azevedo, M. S., Yuan, L., Nielsen, P., and Saif, L. J. (2002). Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-likeparticles associated with immunostimulating complexes. Vaccine 20(13-14), Nguyen, V. M., Hoang, T. N., Huynh, T. P., Nguyen, T. V., Nguyen, K. G., Nguyen, M. L., Nguyen, T. T., Dunia, I., Cohen, J., and Benedetti, E. L. (2001a). Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines. C R Acad Sci III 324(9), Nguyen, V. M., Nguyen, V. T., Huynh, P. L., Dang, D. T., Nguyen, T. H., Phan, V. T., Nguyen, T. L., Le, T. L., Ivanoff, B., Gentsch, J. R., and Glass, R. I. (2001b). The epidemiology and disease burden of rotavirus in Vietnam: sentinel surveillance at 6 hospitals. J Infect Dis 183(12), Yuan, L., Iosef, C., Azevedo, M. S., Kim, Y., Qian, Y., Geyer, A., Nguyen, T. V., Chang, K. O., and Saif, L. J. (2001). Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant Escherichia coli heat-labile toxin in gnotobiotic pigs. J Virol 75(19), xi

13 PUBLISHED ABSTRACTS 1. Yuan, L.J., Nguyen, T.V., Azevedo, M.S.P., Gonzalez, A.M., Jeong, K-I and Saif, L.J. (2005). Maternal cytokines in serum and intestinal contents of suckling pigs. The 12 th International Congress of Mucosal Immunology, Boston, Massachusetts, June 26-30, Gonzalez, A.M., Yuan, L., Azevedo, M.S.P., Nguyen, T.V., Jeong, K-I, Lovgren-Bengtsson, K., Morein, B. and Saif, L.J. (2005). B cell responses elicited by oral/intranasal (IN) immunization of gnotobiotic pigs with a rotavirus-like particle prime/boost vaccine. The 12 th International Congress of Mucosal Immunology, Boston, Massachusetts, June 26-30, Nguyen, T., Yuan, L., Azevedo, M.S.P., Gonzalez, A.M., K.-I Jeong and Saif, L.J. (2005). Transfer of cytokines from sows to newborn piglets via colostrum and milk. Swine in Biomedical Research Conference, Chicago, IL, January, Jeong, K-I, Azevedo, M., Gonzalez, A.M., Nguyen, T., Yuan, L. and Saif, L. Cell-mediated immune responses in Gnotobiotic pigs immunized with sequential attenuated human rotavirus (AttHRV) and 2/6-rotavirus-like-particles vaccines and challenge with virulent HRV, 24 th American Society for Virology, Pensylvania, June 18-22, Nguyen, T.V., Yuan, L., Azevedo, M.S.P., Jeong, K., Gonzalez, A.M., Lovgren-Bengtsson, K., Morein, B and Saif, L.J. Effects of maternal antibodies on effector and memory B cell responses to rotavirus vaccines. 23 rd American Society for Virology, Monstreal, Canada, July 10-14, Yuan, L., Jeong, K., Nguyen, T. V., Gonzalez, A.M., Azevedo, M.S.P., Zhang, W. and Saif, L.J. Effects of maternal antibodies on T cell responses to rotavirus vaccine. 23 rd American Society for Virology, Montreal, Canada, July 10-14, Saif, L.J., Yuan, L., Azevedo, M.S.P., Jeong, K., Gonzalez, A.M., Nguyen, T. V. and Herrmann, J.E. Protective immunity induced by live attenuated (Att) human rotavirus (HRV) priming and bovine rotavirus VP6 DNA boosting in a gnotobiotic (Gn) pig model. 22 nd American Society for Immunology, Denver, CO, May 6-10, Yuan, L., Azevedo, M.S.P., Nguyen, T. V., Gonzalez, A.M., Jeong, K., Chang, K. and Saif, L. J. Booster effects of rotavirus 2/6 virus-like particle (VLP) vaccine on antibody responses to rotavirus outer-capsid protein VP4 and VP7 primed by oral attenuated human rotavirus (Att HRV) vaccine in gnotobiotic pigs. 22 nd American Society for Virology, Davis, CA, July 12-16, Jeong, K-I, Azevedo, M., Nguyen, T., Gonzalez, A., Iosef, C., Chang, K., Yuan, L., Herrmann, J.E. and Saif, L. Cellular immune responses and protection in gnotobiotic pigs vaccinated with a VP6 DNA plasmid vaccine regimen with or without attenuated human rotavirus (AttHRV). 22 nd American Society for Virology, Davis, CA, July 12-16, xii

14 10. Nguyen, T.V., Yuan, L., Azevedo, M.S.P., Jeong, K., Gonzalez, A.M., Lovgren-Bengtsson, K., Morein, B. and Saif, L.J. Effects of maternal antibodies on immune responses and protection induced by immunostimulating complexes (ISCOM)- VP2/6 rotavirus-like-particles (VLP) vaccine. 22 nd American Society for Virology, Davis, CA, July 12-16, Azevedo, M.S.P., Jeong, K., Nguyen, T.V., Gonzalez, A.M., Nielsen, P., Lewis, P., Lovgren-Bengtsson, K., Morein, B., Yuan, L. and Saif, L. J. Protective immunity in gnotobiotic (Gn) pigs after intranasal (IN) or oral priming with attenuated human rotavirus (AttHRV) and boosting with 2/6-rotavirus-like-particles (VLPs) and immunostimulating complexes (ISCOM), and detection of nasal shedding of AttHRV. 22 nd American Society for Virology, Davis, CA, July 12-16, Saif, L.J., Azevedo, M.S.P., Yuan, L., Jeong, K. I., Gonzalez, A., Nguyen, T. V., Pouly, S. and Gochnauer, M Nasal and rectal shedding and viraemia in gnotobiotic pigs after oral or intranasal inoculation with Wa human rotavirus. Abstract W th International Symposium on ds-rna viruses, II Ciocco, Lucca, Italy. September 13-18, Yuan, L., Azevedo, M. S., Gonzalez, A.M., Jeong, K. I., Van Nguyen, T., Lewis, P., Iosef, C., Herrmann, J.E., and Saif, L.J. (2005). Evaluation of a live attenuated human rotavirus priming and bovine VP6 DNA boosting vaccination strategy in a gnotobiotic pig model. Abstract PR.4. 8 th International Symposium on ds-rna viruses, II Ciocco, Lucca, Italy. September 13-18, Azevedo, M., Iosef, C., K. Jeong, Nguyen, T.V., Gonzalez, A.M., Kim, Y., Agarib, F. and Saif, L.J. Rotavirus and rotavirus-like particle (VLP) vaccines with immunostimulating complexes (ISCOM) as a prime/boost strategy induce intestinal IgA antibody secreting cells and protective immunity to human rotavirus in a gnotobiotic pig disease model. 11 th International Congress Mucosal Immunology, Orlando, FL, June 16-20, Azevedo, M., Iosef, C., K. Jeong, Nguyen, T.V., Gonzalez, A.M., Agarib, F., Chang, K., Lovgren-Bengtsson, K., Morein, B., Nielsen, P., Nguyen, T.V. and Saif, L. J. Antibody secreting cell responses and protection in gnotobiotic pigs vaccinated orally with attenuated human rotavirus and intranasally (IN) with 2/6-rotavirus-like particles (VLPs) and immunostimulating complexes (ISCOM). 21 st American Society for Virology, Lexington, KY, July 20-24, Gonzalez, A. M., Nguyen, T. V., Azevedo, M. S., Jeong, K., Agarib, F., Iosef, C., Chang, K., Lewis, P., Lovgren-Bengtsson, K., Morein, B. and Saif, L.J. Vaccination of gnotobiotic pigs orally with attenuated human rotavirus (AttHRV) and intranasal (IN) VP2/6 rotavirus-like particles (2/6VLP) with ISCOM induces similar protection rates but higher antibody titers than AttHRV alone. 21 st American Society for Virology, Lexington, KY, July 20-24, Jeong, K-I, Gonzalez, A.M., Iosef, C., Azevedo, M., Nguyen, T., Chang, K., Agarib, F., Lovgren-Bengtsson, K., Morein, B. and Saif, L. Lymphoproliferative responses in gnotobiotic pigs inoculated orally with attenuated human rotavirus xiii

15 (AttHRV) and intranasally (IN) with 2/6 rotavirus-like particles (VLP) with immunostimulating complexes (ISCOM). 21 st American Society for Virology, Lexington, KY, July 20-24, Iosef, C., Nguyen, T. V., Jeong, K., Kim, Y., Lovgren-Bengtsson, K., Morein, B., Nielsen, P., Azevedo, M.S.P. and Saif, L.J. Analysis of antibody secreting cell responses and protection to Wa human rotavirus (HRV) in gnotobiotic pigs immunized with attenuated Wa HRV (AttHRV) and 2/6 rotavirus-like particles (VLP) administered with immunostimulating complexes (ISCOM). 20 th American Society for Virology, Madison, WI, July 22-25, Nguyen, T. V., Iosef, C., Jeong, K., Kim, Y., Lovgren-Bengtsson, K., Morein, B., Nielsen, P., Azevedo, M. S.P. and Saif, L.J. Antibody responses to oral rotaviruslike particles (VLP) administered with immunostimulating complexes (ISCOM) and attenuated rotavirus vaccines in gnotobiotic pigs 20 th American Society for Virology, Madison, WI, July 22-25, Saif, L. J., Yuan, L., Iosef, C., Azevedo, M.S.P., Nguyen, T.V., Jeong, K., Kim, Y. Vaccine strategies to induce intestinal antibody secreting cells (ASC) and memory B-cells to rotavirus and protection in gnotobiotic pigs. Proceedings to Sixth International Veterinary Immunology Symposium, Uppsala, Sweden, July 15-20, Saif, L. J., Yuan, L., Iosef, C., Azevedo, M.S.P., Nguyen, T.V., Jeong, K., Kim, Y. Vaccination stratergies to induce protective immunity to human rotavirus in a neonatal gnotobiotic pigs disease model. Proceedings to Vaccines for Enteric Diseases, Tampere, Finland, September 12-14, Saif, L. J., Yuan, L., Iosef, C., Azevedo, M.S.P., Nguyen, T.V., Jeong, K., Kim, Y. Evaluation of vaccines to induce intestinal antibody secreting cell (ASC) and memory B-cells to rotavirus and protection in a gnotobiotic pigs model of human rotavirus diarrhea. Brazillian Society for Virology, Caldas Novas, Goias, Brazil, Nov , FIELDS OF STUDY Major Field: Veterinary Preventive Medicine Studies in Virology and Immunology xiv

16 TABLE OF CONTENT Page Abstract.....ii Dedications...vii Acknowledgment.....viii VITA...x List of tables.. xx List of figures....xxi List of abbreviation. xxiii CHAPTER I: LITERATURE REVIEW ROTAVIRUS VACCINES AND INFLUENCE OF MATERNAL ANTIBODIES AND CYTOKINES ON NEONATAL IMMUNE RESPONSES Rotavirus and rotavirus vaccine Impact of rotavirus diseases- diarrhea morbidity and mortality in children worldwide Rotavirus structure and proteins Rotavirus classification Rotavirus disease Rotavirus replication Rotavirus pathogenesis and mechanisms of diarrhea Cross-species infections The viral enterotoxin NSP Structure and functions of NSP Genotypes of NSP Mechanisms of diarrhea caused by NSP Diarrhea induction by NSP xv

17 Immune responses to NSP Homotypic and heterotypic NSP4 specific antibodies and protection Passive immunity conferred by NSP4 antibodies Immunity to RV Role of innate immunity Natural killer cells Adaptive immunity to RV: Studies of different animal models and humans Mouse model Rabbit model Pig model Non-human primate models Immunity to RV-human studies: The immune determinants of protection against RV Serum and fecal IgA antibodies Markers on B and T cells and the association with RV infection and protection Role of cytokines in RV infections Rotavirus specific antibody levels in children worldwide Interference by MatAb with RV infections RV antibodies in milk and passive protection Circulating antibody and protection against RV Maternal immunization Protection against RV by other milk components Interference with active immune responses to RV in infants and in animal models Rotavirus vaccines Jennerian and modified Jennerian approaches to RV vaccines Non Jennerian approach to RV vaccines Rotavirus-like particles as vaccines in different animal models Individual RV proteins as potential vaccines The mucosal immune system General structure Innate immunity Mucosal DC Toll-like receptors Roles of TLR in the recognition of microbial components Distribution of TLRs in different tissues of humans and animals Role of TLRs in the regulation of adaptive immunity Adaptive immunity CD4 + T cells CD8 + T cells B cells Biological roles of IgA antibody Inhibition of adherence...74 xvi

18 Neutralization of viruses, enzymes and toxins Inhibition of antigen presentation Leukocyte trafficking in the mucosal immune system Neonatal immune responses Overview Innate immunity Dendritic cells Monocytes Natural killer (NK) cells Adaptive immunity T effector and memory cell responses Neonatal B cell repertoire Activation of neonatal B cells- in vitro system Mucosal immune system in neonatal pigs Gnotobiotic pig model for human enteric viruses and mucosal immunity Maternal interference with neonatal active immune responses: sources and mechanisms of interference Sources of maternal antibodies (MatAb) Maternal antibodies from milk Transfer of siga antibodies into mucosal secretion Structure of the pigr Functions of pigr Transfer of IgG across the placenta and from milk to neonates Structure of the FcRn Functions of FcRn Mechanisms of FcRn mediated IgG homeostasis Cells in passive immunity Origin of plasma cells in the mammary gland Transfer of functional immune cells in milk to neonates Lactoferrin Cytokines Transfer of cytokines via the placenta CD Levels of MatAb transferred and factors influencing the transfer Interference of neonatal responses by MatAb Inhibition by Mat Ab Enhancement of the immune responses by MatAb Mechanisms of MatAb interference Neutralization of live viral vaccines Interference of passive antibodies via the Fc receptor Interference via idiotypic interaction xvii

19 1.5 Vaccine strategies to overcome the immaturity of neonatal immunity and interference by MatAb Type of vaccines Virus-like particles (VLPs) DNA vaccines Antigen delivery system Aluminum salts Liposomes, virosomes and Archaeosomes Microspheres Vectored vaccines Monophosphoryl lipid A (MPL) Saponins and derivatives Muramyl dipeptide derivatives (MDP) Hormones Cytokines CpG Oligodeoxynucleotides REFERENCES CHAPTER PROTECTION AND ANTIBODY RESPONSES TO ORAL PRIMING BY ATTENUATED HUMAN ROTAVIRUS FOLLOWED BY ORAL BOOSTING WITH 2/6-ROTARUS-LIKE-PARTICLES WITH IMMUNOSTIMULATING COMPLEXES IN GNOTOBIOTIC PIGS Summary Introduction Materials and methods Results Discussion Acknowledgments References CHAPTER HIGH TITER SERUM MATERNAL ANTIBODIES IMPACT PROTECTIVE IMMUNITY AND B CELL RESPONSES INDUCED BY ATTENUATED ROTAVIRUS PRIMING AND A ROTAVIRUS-LIKE PARTICLE ISCOM BOOSTING VACCINE REGIMEN Summary Introduction Materials and Methods Results Discussion Acknowledgments References xviii

20 CHAPTER LOW TITER MATERNAL ANTIBODIES CAN BOTH ENHANCE AND SUPPRESS B CELL RESPONSES TO A COMBINED LIVE ATTENUATED HUMAN ROTAVIRUS AND IMMUNOSTIMULATING COMPLEX-BASED ROTAVIRUS- LIKE PARTICLE VACCINE Summary Introduction Materials and Methods Results Discussion Acknowledgments References CHAPTER CYTOKINES TRANSFERRED FROM MOTHER TO NEONATES IN SWINE: IMPLICATIONS FOR IMMUNOMODULATION OF NEONATAL IMMUNITY BY MATERNAL CYTOKINES Summary Introduction Materials and Methods Results Discussion Acknowledgements References BIBLIOGRAPHY 376 xix

21 Table LIST OF TABLES Page Table 1.1: Level of circulating rotavirus specific antibody of infants in some developing countries Table 1.2: Level of circulating rotavirus specific antibody of mothers and infants in some developed countries Table 1.3: Routes of transmission of MatAb across taxa Table 1.4: Expected loss (days) of MatAb from the newborn in different species Table 1.5: Levels of cytokines in human colostrum and milk (pg/ml) Table 1.6: Virus-like particles for various virus families Table 2.1: Summary of protection rates against virus shedding and diarrhea in gnotobiotic pigs receiving different vaccine treatments Table 2.2: Isotype antibody responses to Wa HRV in serum of pigs at PID 28/PCD0 and the correlation between antibody titers and protection against virus shedding and diarrhea at this time point 239 Table 3.1: Rotavirus shedding and diarrhea in gnotobiotic pigs after challenge with Vir HRV Table 3.2: Comparison of memory B cell responses in pigs given AttHRV/VLP vaccines in the presence or absence of MatAb and between vaccine groups in Exp.I and II..286 Table 4.1: Rotavirus shedding and diarrhea in gnotobiotic pigs after challenge with VirHRV Table 5.1: Concentrations (pg/ml) of IL-12 and TGF-β1 in serum and small intestinal contents (SIC) of neonatal piglets derived by hysterectomy or natural birth..365 xx

22 LIST OF FIGURES Figure Page Figure 1.1: Structure of rotavirus.209 Figure 1.2: Structure of FcRn and pigr Figure 1.3: Expected influence of maternal antibodies to neonatal immune responses.211 Figure 1.4: Association of RV 2/6-VLP with immunostimulating complexes Figure 2.1: Immunoblot of protein components in 2/6VLP and double-layered inactivated HRV (dl-inact-hrv) Figure 2.2: Geometric mean virus neutralizing (VN) antibody titers in sera of pigs that received different vaccine regimens and their correlation with the protection rates against viral shedding and diarrhea..241 Figure 2.3: GMT of isotype-specific antibody to Wa HRV in serum of gnotobiotic pigs at various time-points from each vaccine treatment group..242 Figure 2.4: The IgG subclass responses to Wa HRV in serum samples at PID28/PCD0 from different vaccine groups..244 Figure 2.5: GMT of isotype-specific antibodies to Wa HRV in small (SIC) and large (LIC) intestinal contents of gnotobiotic pigs euthanised at various time-points from each vaccine group (1-7)..245 Figure 3.1: Virus neutralizing (VN) and isotype specific geometric mean antibody titer of pigs vaccinated and challenged in the presence or absence of MatAb Figure 3.2: Isotype specific geometric mean antibody titers in small intestinal contents of pigs following vaccination and challenged in the presence or absence of MatAb..290 Figure 3.3: IgM ASC responses to Wa HRV in pigs vaccinated in the presence or absence of MatAb 292 xxi

23 Figure 3.4: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of MatAb Figure 3.5: IgG ASC responses to Wa HRV in pigs vaccinated in the presence or absence of MatAb.296 Figure 4.1: Virus neutralizing (VN) and isotype specific geometric mean antibody titers in serum of pigs vaccinated and challenged in the presence or absence of LoMatAb 329 Figure 4.2: Isotype specific geometric mean antibody titers in small intestinal contents of pigs following vaccination and challenged in the presence or absence of LoMatAb 330 Figure 4.3: IgM ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb Figure 4.4: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb Figure 4.5: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb Figure 4.6: Memory B cell responses in pigs vaccinated with AttHRV/VLP, VLP vaccine regimens or ISCOM control in the presence and absence of LoMatAb at PID28/PCD Figure 5.1: Cytokine concentrations in colostrum/milk and the relationship with the cytokine concentrations in sows serum samples.366 Figure 5.2: Different forms of TGF-β1 in sows colostrum/milk 368 Figure 5.3: Cytokines in sera of suckling piglets and correlations with the concentrations in sows colostrum/milk..369 Figure 5.4: Comparison of cytokine concentrations in sera of weaned and suckling piglets Figure 5.5: The concentrations of IL-6 and TNF-α (pro-inflammatory cytokines) and TGF-β (Th3 cytokine) in the intestinal contents and serum of suckling piglets..372 Figure 5.6: The concentrations of Th1 cytokines (IFN-γ and IL-12) in the intestinal contents and serum of suckling piglets.374 Figure 5.7: The concentrations of Th2 cytokines (IL-10 and IL-4) in the intestinal contents and serum of suckling piglets.375 xxii

24 LIST OF ABBREVIATIONS Aluminium hydroxide (AlOH) Antibody secreting cells (ASC) Bacille Calmette-Guerin (BCG) Bovine rotavirus (BRV) Bovine serum albumin (BSA) Cell culture immunofluorescence (CCIF) Chemokine (C-C motif) ligand 25 CCL25 Chemokine (C-C motif) receptor (CCR) Chemokine (CXC motif) receptor 5 (CXCR5) Cholera toxin (CT) Cluster differentiation (CD) (human) colonic carcinomas cell line (Caco-2) Cytotoxic T cells (Tc) Dendritic cells (DC) 2,4 dinitrophenyl (DNP) Endoplasmic reticulum (ER) Enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunospot assay (ELISPOT) Fluorescence forming unit (FFU) Gnotobiotic (Gn) pig Granulocyte-macrophage colony-stimulating factor (GM-CSF) Gut associated lymphoid tissue (GALT) Hen egg white lyzozyme (HEL ) Hepatitis B surface antigen (HBsAg) High endothelial venules (HEV) Human immunodeficiency virus (HIV) Human leukocyte antigen (HLA) Human rotavirus (HRV) Human synsytial virus (HSV) Idiotope (Id) Immunoglobulin (Ig) Immunostimulatory complexes (ISCOM TM ) Interferon (IFN) Interferon regulatory factor (IRF) Interleukin (IL) Intraepithelial lymphocytes (IEL) Intramammary route (IMm) xxiii

25 Intramuscular route (IM) Intranasal route (IN) Keyhole limpet hemocyanin (KLH) Lamina propia (LP) LCMV (lymphocytic choriomeningitis virus) Lymphocyte proliferation assays (LPA) Major histocompatibility complex (MHC) Maternal antibodies (MatAb) Mesenteric lymph node (MLN) Microfold (M) cell Monoclonal antibodies (Mab) Mononuclear cells (MNC) Mucosal adhesion cell adhesion molecule (MadCAM-1) Mucosal associate lymphoid tissues (MALT) Mutant Escherichia coli heat labile enterotoxin (mlt) Nasal associate lymphoid tissues (NALT) Natural Killer (NK) Neonatal Fc Receptor (FcRn) Non-structural protein (NSP) Pathogen associated molecular pattern (PAMP) Pattern recognition receptor (PRR) Peyer s Patches (PP) Phosphate buffered saline (PBS) PLG (Poly DL-lactide-co-glycolide) Polymeric Ig receptor (pigr) Porcine respiratory and reproductive virus (PRRV) Post challenge day (PCD) Post inoculation day (PID) Protection receptor of serum IgG (FcRp) Quillaja saponina 21 (QS21) Recombinase-activating gene (RAG) Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES) Rhesus monkey kidney cells (MA104) Rhesus rotavirus strain (RRV) RRV rhesus rotavirus RRV-TV rhesus rotavirus based tetravalent vaccine Secretory IgA (siga) Severe combined immune deficient mice (SCID) Spodoptera frugiperda (Sf-9 cells) T helper cells (Th) Toll-like receptor (TLR) Transfroming growth factor (TGF) Tulane University and Cincinnati Children s hospital (TICH) Tumor necrosy factor (TNF) Tetravalent rhesus rotavirus vaccine (TV-RRV) xxiv

26 Vascular cell adhesion molecule (VCAM-1) Viral protein (VP) Virus neutralization (VN) Virus-like-particle (VLP) xxv

27 CHAPTER 1: LITERATURE REVIEW ROTAVIRUS VACCINES AND INFLUENCE OF MATERNAL ANTIBODIES AND CYTOKINES ON NEONATAL IMMUNE RESPONSES 1.1 Rotavirus and rotavirus vaccine Impact of rotavirus diseases- diarrhea morbidity and mortality in children worldwide Rotaviruses (RV) are the most common cause of gastroenteritis in children worldwide. They are responsible for about 608,000 deaths annually (74). In a ten year study, from , in 20 countries, the estimated incidence of diarrhea was 3.8 episodes per child per year for children less than 11 months old, and 2.1 episodes per child per year for children between the age of 1-4 years (374). Based on these numbers, it is estimated that 1.4 billion diarrhea episodes per year occur in children less than 5 years of age (520). More importantly, the incidence rate of RV disease is similar in children from developing and developed countries, suggesting that the virus infection does not discriminate between economic status. Nevertheless, children from developing countries showed a higher rate of diarrhea related deaths due to poor medical care systems and malnutrition. In Mexico, RV causes 2148 deaths a year, whereas in the US 1

28 the numbers were 102 (23). It is estimated that 1205 children die from rotavirus disease each day in developing countries with 82% of deaths occurring in the poorest countries. Rotavirus infection occurs year-round in developing countries with tropical climates, whereas the infection peaks in the winter season and decreases in the summer in developed countries with temperate climates (157). This difference in seasonality of RV infection explains the younger median age of illness in children from developing compared to developed countries (6-8 months vs months, respectively) (86). As a consequence, it has been suggested that RV vaccine be delivered to children in developing countries at an earlier age, even as early as when they receive the Bacillus Calmette-Guerin (BCG) vaccine (from birth to 3 months of age) (494). Because of an increasing awareness of the disease, the estimated annual mortality rate from RV has decreased over the past 2 decades from 873,000 deaths (1985) to 608,000 (2004) (74). Yet a study in Mexico showed that although the overall mortality rates decreased, the decline in the winter season was not evident (709). The economic burden caused by RV is high in industrialized countries with the estimated hospitalization rate among children of 445 per 100,000 children (520). In the United States, it is estimated that RV cause 24, ,000 hospitalizations and deaths in young children annually. The costs associated with RV disease in the US were estimated at $ million annually. Thus the incidence of RV can not be eliminated just by improvement of living standards, i.e. improving hygiene and sanitation and water supplies: a vaccine is 2

29 necessary to prevent and reduce the disease mortality. The use of a RV vaccine could reduce hospitalizations by 40-60% and could decrease RV associated deaths by 10-20% (1, 173) Rotavirus structure and proteins Rotavirus is a non-enveloped double stranded (ds) RNA virus, in the Reoviridae family (197). The RV genome is composed of 11 segments of dsrna ranging from bp. The genome encodes 12 proteins, six structural proteins (VP) 1, 2, 3, 4, 6 and 7 and six non-structural proteins (NSP) 1, 2, 3, 4, 5 and 6. The gene segment 11 encodes both NSP 5 and 6 (196). The inner shell of the virion is composed of 60 dimers of VP2, which surrounds the viral genome and 12 copies each of VP1 (RNA polymerase) and VP3 (guanylyl-transferase and methylase). Addition of 780 molecules of VP6 on the VP2 produces double-layered particles. The outer layer of the virion is composed of 780 molecules of VP7 forming a smooth surface, organized as trimers plus sixty dimers of VP4 forming spikes extending outward [Figure 1.1] (324). The VP4 plays an important role in virus replication including receptor binding and cell penetration (25). The VP7 interacts with the cell surface molecules after the attachment of the virus via the VP4 (256). Detailed steps of the virus replication will be discussed subsequently. The non-structural proteins. The NSP1, encoded by gene segment 5, has been implicated as a virulence factor in mice but not in pigs or rabbits (87, 91, 142). A recent study suggested that NSP1 subverts the innate immune response by inducing degradation of interferon regulatory factor (IRF)3 (46). The NSP2, encoded by gene segment 8, is an oligomeric NTPase, possessing helix destabilizing activity and may be involved in RNA 3

30 encapsidation and virulence (119, 349, 669, 704). The NSP3, encoded by gene segment 7, binds to eif4g and inhibits cellular protein synthesis as well as enhances translation of viral mrna (536, 703). The exact functions of NSP5 and NSP6 are not yet known; due to their RNA binding capacity, it is postulated that they may function as a transporter of other viral proteins from the sites of synthesis into the viroplasms or they may facilitate RNA encapsidation or they may be involved in the movement of viral particles from the viroplasms into the ER membrane (523). The NSP4, encoded by gene segment 10, plays a role in viral morphogenesis, acting as the receptor for double layer particles, mediating the budding of these particles into the ER lumen (42). It is also an important enterotoxin, involved in the pathogenesis of RV. The detailed functions of the NSP4 will be discussed later Rotavirus classification Rotavirus classification is based on VP4 and VP7 proteins corresponding to P and G types, respectively. There are currently 15 G serotypes/genotypes identified. Of 22 different P genotypes reported, only 13 P serotypes have been identified (301, 303, 412, 433, 506). Common serotypes of RV infected humans include P[8]G1, P[4]G2, P[8]G3 and P[8]G4 and smaller percentages of G5, G8 and G9. Rotavirus G9 strain, initially detected in the US, is becoming more common in Asian countries such as Australia, China (up to 18.1%) and Japan (71.4%) (114, 216, 413, 436, 594, 778). Animal RV has been suggested to cross the species barrier and cause infection in humans. The RV G5 strain which is common in swine has also been found in Brazilian children with an incidence ranging from 5.6% to 57% (406, 435). Rotavirus G8 strains, 4

31 which are found in cows, are also present in children with increasing frequency, from few cases in India (320) to as high frequency as 27.7% and 34.1% of RV strains in Nigeria and Malawi, respectively (4, 166). Rotavirus G6 strains, commonly found in cows, sheep and goats were isolated from children with acute diarrhea in Italy, US, Australia, Belgium and Hungary in combination with P[9] or P[14] strains or in India with unknown P specificity (594). Sporadic cases of RV-G12 strains were found in Thailand, Korea, India, Philippines and the US (594). A single case with P[14]G3 lapine-like RV was found in a 6-year old Belgium child with severe diarrhea (171). Rotavirus strain P[11]G10 commonly found in cattle was reported to cause both asymptomatic and symptomatic infections in human neonates and infants in India (317). These potential cross species infections can be traced to the ability of RV to undergo genomic rearrangement and reassortment between homologous and heterologous strains, due to the distinctive segmented RV genome. Molecular characterization of a human RV (HRV) strain RMC321 from an outbreak of RV diarrhea in India revealed porcine characteristics in most of the genes including VP4, VP6 and NSP1-5 (95-99% amino acid identity) (699). This study provided strong evidence that porcine RV can cross the species barrier and cause severe gastroenteritis in humans. Apart from the extensive genome rearrangements and genetic reassortments, evidence of point mutations and accumulations have been reported (594). Monotypes within serotype G1, G2, G3, G4 and G5 RV strains have been proposed due to the existence of distinct VP7 gene phylogenetic lineages. The antigenic differences found between the vaccine G1 strain in the reassortant rhesus vaccine and the circulating G1 strain might be the reason for vaccine failure (331). Antisera to one lineage of the G9 5

32 strains failed to neutralize another lineage of the G9 strains efficiently (302). Likewise genetic diversity between VP4 genes of the same P type was identified (594). Thus the pool of RV infecting humans has the potential to increase, with different combinations of P and G serotypes and different monotypes within serotypes, complicating the development of a globally effective vaccine for RV. However, heterotypic protection has been induced by a single RV serotype against other serotypes, thus it might not be necessary to include all the RV serotypes in vaccine. A monovalent G3 Rhesus RV (RRV) vaccine induced a 58.5% heterotypic protection rate against the circulating G1 serotype, compared to the 72.8% homotypic protection rate induced by the G1 vaccine (427). Interestingly, a study in Peru showed that the G3 RRV monovalent vaccine had a 29% heterotypic protection rate against the circulating G1 or G2 RV strains, whereas neither the G1 nor G2 RRV reassortant vaccines induced homotypic protection against G1 or G2 RV (393). The P6[1]G6 vaccine (RIT 4237) also showed high heterotypic protection against RV G1, G2 and G3 strains in several preliminary studies (531, 707). A RV vaccine of the G1 strain (RIX4414 vaccine) induced protection against severe diarrhea caused by both G1 and non-g1 RV strains (172). Thus it is expected that inclusion of at least the most common RV strains may increase the vaccine efficacy by induction of both homotypic and heterotypic protection Rotavirus disease Rotaviruses are transmitted via the oral-fecal route. The symptoms of RV disease include diarrhea, vomiting, fever, nausea, anorexia, cramping and malaise. The symptoms can be either mild or severe; in the latter case resulting in severe dehydration and death. 6

33 Rotavirus infections occur in children as early as 3 months and up to 5 years of age; however 90% of children in both developing and developed countries encounter RV infection before the age of 3. Severe symptoms often occur in young children from 6-24 months of age. Rotavirus infections also occur in neonates, but are usually asymptomatic (263). For this reason, the neonatal strains have been evaluated as candidate RV vaccines in some settings. Rotavirus infection also occurs in adults; the symptoms however are usually mild (128, 201, 268) Rotavirus was first observed in the intestine of mice (EDIM) and monkeys (SA11) by electron microscopy in 1963 (5, 430). After this discovery, RVs were associated with diarrhea in calves (461), humans (62) and pigs (71, 583). Human and animal rotaviruses belong to 7 groups (A-G) based on the common group antigen (VP6). Rotavirus groups A-C are associated with human infections of which Group A rotaviruses account for the majority of cases. However, group B-RV which causes adult diarrhea and group C-RV caused large outbreaks in China and Japan (296, 308, 330, 438, 577, 585). Porcine RVs are recognized as an important pathogen associated with diarrhea in both nursing and weaned pigs (71, 323, 746). Rotaviruses in swine are also classified into several antigenically distinct groups: A, B, C and E (585). Group A RV causes diarrhea in pigs and belongs to G types 1-6 and 8-11 and P types [5]-[8], [13], [19] and [23] (432). Two G serotypes of Group C porcine RV have been identified (684). Porcine RVs cause both clinical infections, manifested by diarrhea, anorexia, and depressed growth rates, and subclinical infections (50, 69, 224). 7

34 1.1.5 Rotavirus replication Rotavirus replication consists of the following steps: attachment, penetration, uncoating, replication of the genome and translation of viral proteins, assembly and release. Attachment. Many attempts were made to identify the cellular receptors for RV. Some animal rotaviruses require sialic acid (SA) on the cell surface for efficient binding and infection whereas the human viruses do not (140). Thus the SA binding is not an essential step for effective infection. Neuraminidase (NA) resistant strains, which do not require SA to infect cells, have been isolated (419). Ciarlet et al.(143) further classified the need for SA according to P genotypes of the RV strains: RV of P genotypes [1], [2], [3] and [7] require SA residues for infection whereas RV strains with other P genotypes are SA independent. Gangliosides GM1 and GM3 were also suggested as receptors for RV (262, 564). The integrin α2β1 binds to the VP4 via the DGE tripeptide motif, whereas α4β1 and αxβ2 integrins bind to the VP7 via distinct motifs. The heat shock protein (HSP) 70 and the β3 integrin have also been identified as post-attachment receptors (260, 261). The removal of these proteins by the non-ionic detergent octyl-β-glucoside under non-lytic conditions renders the cell resistant to the virus binding. Antibodies against αvβ3 reduce the infectivity of RV RRV, but do not block attachment to the cells. The HSP70 binds specifically to triple, but not to double layered particles and this interaction can be blocked by antibodies against the VP4 and VP7. Antibodies against α2β1 and αvβ3 combined also suppressed the virus infection, suggesting that the two integrins may be involved in different steps of virus entry. All of the above mentioned receptors must be organized in a lipid microdomain of the cell membrane, called lipid rafts to maintain 8

35 the ability to bind to RV. The search for RV cellular receptors is ongoing. However, the available information suggests that there are at least 3 receptors involved in the attachment and post-attachment stages of viral entry. Penetration. Upon infection, RV VP4 acts as a receptor, binding and allowing the virus to penetrate the cell (26). The cleavage of VP4 by trypsin into VP8 and VP5 is associated with the virus entry. The mechanism of activation by trypsin is not yet known; however, it is postulated that VP4 cleavage leads to either a conformational change or a new terminal region in VP4 molecules which favors penetration. Evidence supporting both endocytosis and direct cell penetration as the routes of viral entry are available but still do not give a definite conclusion of the mode of entry of RV. Uncoating. Once inside the cell, the viral transcriptase is activated to transcribe the viral genome. This process is triggered by the uncoating and removal of VP4 and VP7 proteins from the triple-layered virus particles. The removal of VP4 and VP7 proteins can be achieved by removing Ca 2+ ions from the medium. The low intracellular Ca 2+ concentration is not essential for initiating viral replication because increasing the calcium levels in the cell did not affect viral protein synthesis (165). Replication of the viral genome and translation of the viral proteins. Once the uncoating process is completed, the RNA polymerase activity in the VP1-VP3 enzyme complex (which is latent in the triple-layered particles) is activated in the double-layered virus particles due to the removal of the outer capsid, resulting in genome transcription and extrusion of the eleven viral mrnas from such particles. The mrnas direct both 9

36 protein synthesis and minus-strand synthesis to yield dsrnas. It was shown that the positive and negative stranded RNAs can be detected 3h post infection (PI) (648). The level of transcription peaks between 9-12h. Viral assembly. The synthesis of dsrnas, which are still associated with subviral particles, is an event that occurs following packaging of viral mrnas into corelike assembly intermediates with the viral RNA-dependent RNA polymerases and the capping enzyme (524). Most of RV structural and non-structural proteins are produced on the free ribosomes. The VP7 and NSP4 are synthesized on ribosomes associated with the ER membranes and then cotranslationally inserted into the ER membranes. The subviral particles are assembled in the cytoplasm, and then bud through the ER membranes which is modified with VP7 and NSP4 to form transient enveloped particles. The transient envelope is lost as the particles move into the ER and VP4 and VP7 assemble to form the outer capsid while NSP4 is excluded from the particles (415). The membrane destabilizing activities of NSP4 and/or VP4 and the high Ca 2+ concentration in the ER lumen are implicated in the removal of the transient lipid envelope (176, 679) Rotavirus pathogenesis and mechanisms of diarrhea The common recognized mechanism for RV-induced diarrhea in humans and other animals is the malabsorption caused by loss of the absorptive villous epithelial cells in the intestine (346). Diarrhea is also accompanied by loss of sugar digestive enzymes, loss of sodium, potassium, bicarbonate and water and osmotic loss of fluid due to undigested lactose (37, 170, 211, 311, 468, 745) Other mechanisms of diarrhea induced by RV infection have been demonstrated only in the mouse model. In neonatal mice, 10

37 NSP4 functions as an enterotoxin that mediates a secretory diarrhea (44). Shaw et al (615) and Ball et al showed (44) that RV associated diarrhea also occurs independently of viral replication in mice. Diarrhea can be induced upon inoculation of mice with a large number of virus particles genetically inactivated using psoralen or long-wave ultraviolet light which cross-links the viral RNA. It was hypothesized that morphological changes in murine intestinal cells and diarrhea are associated with attachment of virus to the cells. Thus the mechanism of RV-induced diarrhea is similar to a toxin effect, which is associated with the function of the NSP4 (44), as discussed subsequently. Another mechanism is the intestinal inflammatory response which causes disruption of the intestinal mucosa and leaky membranes that allows toxins or bacteria to penetrate though the intestinal epithelial cell barrier (66). In mice, RV infection also stimulates the enteric nervous system (ENS), leading to intestinal secretion (422). Drugs that inhibit the ENS could attenuate the intestinal secretory response induced by RRV in mice. Therefore the involvement of ENS was proposed to be another mechanism of RV diarrhea. In pigs, RV infection is initiated in the upper small intestine after oral inoculation, causing cytolytic infections of the villous epithelium resulting in shortening of the villi (460, 715). The virus replicates in the nondividing mature enterocytes near the tips of the villi leading to the extensive destruction of these cells (100, 674). The villi become covered with immature, non-differentiated cuboidal epithelium. Extensive destruction of enterocytes causes an impaired digestive-absorptive process, leading to an acute and malabsorptive diarrhea. Rotavirus also infects a few cecal and colonic epithelial cells without colonic lesions (675). The villous tips are denuded and mononuclear cells 11

38 migrate into the underlying lamina propria (LP). The infection starts at the proximal end of the small intestine and advances distally (88). After infection of gnotobiotic (Gn) pigs with HRV Wa strain, moderate to severe villous atrophy occurred in the small intestine by 48-96h PI (715). The lesion was most severe in the caudal region of the intestine, whereas lesion in the proximal part was less extensive and persisted for shorter time (24-48h PI) (584). The low pathogenic strains tend to infect the proximal part of the small intestine, whereas the highly virulent strains infect the mid and distal small intestine (88). In neonatal Gn and colostrum-deprived calves infected with HRV-D strain, diarrhea occurred within half an hour of inoculation with denuding of the villi in the upper small intestine, whereas the lower small intestine remained intact (462). Morphological changes in the lower small intestine occurred later (7h PI), yet RV antigens were not detectable by immunofluorescence. In pigs, orally inoculated with focus-forming units (FFU) of virulent (Vir) Wa HRV, porcine OSU or SB1A, diarrhea was induced within 12-18h PI, in parallel with the presence of RV antigen within villous epithelial cells (125, 305, 306, 715). Villous atrophy was observed at 24-72h PI, coincident with the peak of virus replication and was prominent in the caudal small intestine. Destruction of the cells on the villous tips causes a malabsorptive and osmotic diarrhea in pigs. Hyperplasia occurs in lymphoid tissues such as Peyer s patches (PP) and mesenteric lymph nodes (MLN) (715). The duration of diarrhea and viral shedding for VirHRV in pigs is between 4-7 days PI. Pigs inoculated with the attenuated (Att) Wa HRV strain showed no signs of diarrhea and no villous atrophy; instead, the villous epithelial cells remained attached to the basement membrane with only minor morphological alterations. The pathophysiological changes contributing 12

39 to the maladsorptive diarrhea include impaired glucose-coupled sodium transport (170), decreased disaccharidase activity (255, 781) and increased thymidine kinase activity (170). The degree of villous atrophy and the distribution of these damaged villi vary according to RV strains (152), serogroups (580) and the age of the pigs (577, 614). Rotavirus groups A-C generally replicate and cause villous atrophy in the lower intestine, but not the colon. The groups A and C RV cause infection of cells forming scattered foci throughout the intestine, but limited to the tip or sides of the villi (72, 460, 676). Group B RV produced scattered foci of infection in the distal small intestine and generated mild but highly acute diarrhea (585). Younger piglets exhibit more severe villous atrophy than older pigs. Group B RV caused an acute, transient but non-fatal diarrhea in pigs less than 6 days of age whereas Groups A and C RV infections led to dehydration and death in most Gn pigs less than 6 day old (577). Infections with non-group A RV have been reported in chickens (Group D), Gn piglets (Cowden porcine Group C and several Group B strains (577)) and calves (bovine Ohio Group B) resulting in villous atrophy and diarrhea similar to that induced by Group A RV [review in Saif et al (577)]. Infections of Gn pigs and Calves with group B or C RV lead to a more rapid onset of diarrhea, with fewer shedding virus for shorter period of time, compared to Group A RV (577). In children, similarly to pigs, RV replicates in the nondividing mature enterocytes near the tips of the villi (169). Histopathologic changes vary from broadening of the villi to complete villous atrophy and from mild to heavy mononuclear cell filtration (373, 13

40 584).This variation in histopathologic changes may represent the spectrum of illness or may indicate the limited information obtained from the small size of biopsy duodenal or jejunal specimens. Diarrhea is due to sugar maladsorption including lactose and D-xylose (211, 442) and enterocyte loss (345). Human intestinal biopsies showed mononuclear cell infiltration, mitochodria swelling and denudation of the microvilli (169, 294). Diarrhea induced by RV in children has an incubation period of 24-48h, and lasts up to 7 days. Symptomatic infections with RV are restricted to the young infants, between 6-24 months of age (258, 346). Asymptomatic infections also occur in premature and newborn babies, which may be due to the attenuated nature of the virus or the presence of maternal antibodies (14, 258). In mice, RV associated diarrhea occurs only during the first 2 weeks of life (514). In neonatal mice, murine RV causes diarrhea with villous ischemia (514) but with little or no villous atrophy and inflammation (5). In adult mice, RV infection occurs without causing disease or lesions. Acute inflammatory cell recruitment and mucosal ulceration observed in calves and piglets are not seen in the mucosa of mice upon heterologous RV infection (503, 649). Homologous RV infection in mice (EDIM) is associated with shortening of the villi, disordered microvilli and epithelial vacuolation, whereas heterologous RV infection results in little change in villous structure (346, 503). In addition, malabsorption is not the major diarrhea mechanism on the murine RV disease model (151). Two phases of villous morphological change were observed after infection of neonatal mice with murine RV (514). In the first phase (18-48 h PI), ischemia and transient shortening of the villi occurred throughout the small intestine. The villi recovered to their normal height at 72h PI due to a slow rate of cell division, yet initial 14

41 signs of hyperemic microcirculation were observed in the villi. The decreased rate of cell division failed to replace the lost cells leading to hypersecretion. The second phase occurred around h PI with few villous shortening and the changes were restricted to the upper and middle regions of the intestine with hyperemic microcirculation in the villi. The increased number of red blood cells in the villi led to perturbance in the countercurrent system and decreases in the osmolarity in the villous tips thus leading to impaired water absorption and more prolonged diarrhea. Recovery from diarrhea occurred at 168h PI, coincident with the recovery of the villous microcirculation. Thus the villous atrophy upon RV infection in neonatal mice is not as pronounced as observed of humans, pigs or calves. Rotaviruses infect mainly enterocytes of the tip of the small intestinal villi although not exclusively. Blutt et al. (67) recently demonstrated antigenemia and viremia associated with of different RV strains infecting adult humans, mice and rabbits and neonatal humans, rats and calves. Furthermore, these viruses from serum were shown to be infectious in neonatal and adult mice. A study of neonatal Gn pigs (3-5 days of age) demonstrated viral antigen in serum between post infection day (PID) 1 to PID 7 after inoculation with VirHRV (34). This study also showed that serum from pigs infected with VirHRV Wa strain was infectious for pigs, providing further proof for viremia associated with HRV infection of pigs. Rotavirus antigens and RNA have been observed in the central nervous system, liver and kidney of immunodeficient children (244, 409, 496), and in the blood, spleen, liver, kidneys, lungs and mesenteric lymph nodes (MLN) of RV inoculated mice (93, 15

42 177). Viral RNA has also been detected in the blood of 64% of RV infected immunocompetent children, suggesting that viremia is a common phenomenon (126). The RV also spreads to mucosal tissues other than the intestine. Oral or gavage inoculation of pigs with Wa VirHRV also caused nasal virus shedding from PID 1 (oral) or PID2 (gavage) to PID 7, longer than the duration of rectal virus shedding (34). Similarly, RV antigen has also been found in nasopharyngeal secretions of children with respiratory symptoms (218). Virus induced hepatitis has been described in a neonatal mouse model (1-3 dayold) inoculated with RRV (687). Liver spread of RRV and hepatitis were demonstrated in both BALB/c mice and the severe combined immunodeficiency (SCID) mice. Not all strains of RV can spread to the liver. The RRV, but not the SA11-Cl4 strain can infect the liver following oral inoculation of CD-1 neonatal mice. The gene segment 7 encoding NSP3 has been correlated with the spread of RV to the liver (477). Rotavirus infection occurs via the oral-fecal route, yet bypassing of the intestinal barrier by an intraperitoneal (IP) injection allows the infection and replication of RV in the liver regardless of whether the strain is liver prone (RRV) or not (SA11-Cl4) (477). One of the mechanisms used by RV to escape the intestine and enter the circulation is the transepithelial transport of the virus through M cells of the Peyer s patches (PP), which act as an intermediate among the immune, lymphatic and circulatory systems (100). Rotavirus has been demonstrated to enter the lymphatic system (476). Infection of neonatal mice with RRV or the spread-competent clone SA11-CI4 led to the progression of virus particles from the intestine to the MLN and then to the peripheral tissues. Gene segment 7 (encodes NSP3) was identified as the primary determinant of the 16

43 spread of virus to the MLN and this spread via a lymphatic pathway could be modified by gene segment 6 (encodes VP6) (476). Rotavirus can also be taken up by macrophages which travel in the blood circulation and cause infections in other tissues (93). Evidence for the spread of RV via the circulation to cause infection in other tissues can be found in a study by Azevedo et al (34). Pigs inoculated intravenously with serum or intestinal contents from the viremic VirHRV-inoculated pigs developed diarrhea, rectal and nasal virus shedding, and viremia, similar to orally inoculated pigs Cross-species infections Although many RV strains are species specific, cross species infections are not uncommon as mentioned earlier for animal viruses infecting humans. In addition, infection of adult mice with the non-human primate strains RRV and SA11, has been widely studied for pathogenesis and immunological responses in the mouse model. Infection of mice with these heterologous viruses does not induce the typical RV virus related symptom, i.e. diarrhea. On the other hand, experimental infection with the PP-1 strain of bovine RV, Wa or M strain of HRV caused diarrhea in pigs (266, 589). The mechanism of cross species infection is still under investigation. It has been hypothesized that the VP4 phenotype is the determinant of cross species specificity and pathogenicity (186). This hypothesis was supported by the finding that the PP-1 strain, which was isolated from calves in which it was asymptomatic, caused diarrhea in pigs. This strain has 96-97% homology in VP4 amino acid sequence with the porcine P[7] strain but only 62-79% homology in VP4 amino acid sequence with other bovine strains (186). Similarly, Gentsch et al (235) showed that the 116E strain HRV which causes 17

44 asymptomatic infections in human infants, has high homology in VP4 nucleotide and amino acid sequences with the bovine B223 strain. In addition, RV may exist as a population of reassortants from which a new RV species appears under special conditions, i.e. a new species emerges during infection of pigs with the bovine PP-1 strain (254). From an original fecal sample containing P[5] and P[7] gene profiles, the bovine pathogenic P[5] and porcine pathogenic P[7] strains were isolated after serial passage in Gn calves and pigs, respectively. Naturally occurring assumingly reassortants have been reported between HRV with VP4 from porcine or bovine strains; all of which induce asymptomatic infections in human infants (168, 251). The NSP4, although it plays a crucial role in RV diarrhea induction in mice, hasn t been clearly associated with RV species specific virulence due to contradictory reports (186). The VP7 gene might also contribute to inter-species transmission of RV. Homologies in amino acid sequences (90%) were identified between the human G6 strain (PA151 and PA169) and the bovine G6 strain (UK and NCDV) and monoclonal antibodies against the bovine G6 strain cross reacted with the human G6 strains (239, 240, 312). Human RV G10 isolated in Thailand may have originated from a bovine strain (688). A bovine RV G1 strain (T449) isolated in Argentina had 90% homology in the VP7 amino acid sequence with a human G1 serotype (Wa strain) (65). An outbreak of gastroenteritis in infants in Manipur, India identified a new HRV strain, RMC321 in which the genes encoding VP4, VP6, VP7, NSP 1-5 showed identity to porcine RV but not to HRV (699). As previously mentioned, reassortment is a common event due to the 18

45 segmented genome of rotavirus, which allows gene segment(s) of one origin to reassort with rotavirus segments of other origins including between animal and human strains The viral enterotoxin NSP4 The NSP4, encoded by gene segment 10, serves as an intracellular receptor during subviral particle morphogenesis (30). It was also identified as a viral enterotoxin and was recognized as the diarrhea inducing mechanism before morphological damages of intestinal cells occur in neonatal mice (44). The NSP4 induces mobilization of intracellular Ca 2+ in the intestinal epithelial cells, leading to an efflux of Cl - across the plasma membrane, thus initiating secretory diarrhea independently of camp. Other ion channels are also activated by the increase in Ca 2+ concentrations which increase fluid secretion and reduce absorption (474) Structure and functions of NSP4 Rotavirus NSP4 was the first viral enterotoxin identified (42, 44). There are only two other enterotoxins which were identified following the discovery of RV NSP4: the surface unit glycoproteins of simian immunodeficiency virus (SIV) and those of equine infectious anemia virus (43, 657). They are called enterotoxins because they stimulate intestinal secretion without altering the tissue morphology (42). Other enterotoxins are found in bacteria such as Vibrio cholerae (cholera toxin), E.coli (heat-labile and stable toxins) and other cholera-like enterotoxins in A.hydrophila, Campylobacter jejuni, Salmonella spp, Bacillus sp, Clostridioum difficile and Yersinia enterocolitica. The NSP4, a 28kD glycoprotein acts as an intracellular receptor for VP6. Because NSP4 resides in the ER, the binding of NSP4 with VP6 is crucial to the budding of 19

46 double-layered particles through the ER membrane, which facilitates the addition of VP7 and VP4 and a transient ER membrane to form the mature particles. The NSP4 contains a transmembrane domain (residues 24-44) with the C terminus spanning the ER bilayer, the N terminus remaining in the lumen of the ER and residues extending in the cytoplasmic domain. The NSP4 doesn t contain the classical ER retention signal; thus it was unclear how NSP4 retains at this site. The double-layered RV receptor activity of NSP4 is localized at the C terminus of the protein in which the methionine residue is essential for ligand binding (671). The NSP4 also binds to VP4 and VP7 and facilitates the addition of the outer coat proteins to the double layer to form a triple layer. The binding site of NSP4 to VP4 is localized to residues (672). A functional study of NSP4 was achieved using RNA interference (RNAi), which is the small interference RNA (sirna) triggered event in which any RNA with 100% sequence match with the sirna is degraded (245). Silencing of NSP4 by RNAi led to 75% reduction in RV replication, whereas the silencing was removed when there were 4 nucleotide mismatches in the sequence of sirna compared to NSP4 (415). The NSP4 silencing led to a reduction in the number of double-layered and triple-layered particles and redistribution of other RV proteins. In the presence of NSP4 silencing, the VP6 forms filaments at the periphery of the cell whereas VP2 scatters throughout the cytoplasm instead of in the viroplasm. The VP4 is found in the cytoplasm instead of the nucleus and the VP7 diffuses in the perinuclear area instead of into the ER (250, 490). Thus NSP4 is the crucial protein for the later stages of RV morphogenesis. 20

47 Genotypes of NSP4 The NSP4 genes of mammalian RV are classified into 4 groups (genotypes A, B, C and D) based on the nucleotide and amino acid sequences. Genotypes A, B and C are detected in human viruses and show 87-99% identity between genotypes (142, 297, 298, 368). Murine RV-NSP4 is classified into genotype D due to only 60-65% sequence identity to other human genotypes. Avian RV NSP4 is classified into 2 genotypes (E and F) that are distinct from the mammalian RV NSP4s because of low identities (31-37%) between them (473). Turkey and pigeon RV NSP4 belong to the same genotypes, whereas the chicken RV NSP4 is of a separate genotype Mechanisms of diarrhea caused by NSP4 Diarrhea occurs early in RV infection before the initiation of any inflammatory response with little infiltration of mononuclear cells and limited vacuolization of some enterocytes at the tips of the villi (44). Thus the mucosal destruction is not the cause of diarrhea at this early stage. In mice, diarrhea occurs 4-8h post IP inoculation of the purified NSP4 and persists for 24h. Diarrhea induced by NSP4 is age dependent in that 6-7 days old mice developed diarrhea after IP injection of the protein whereas mice of up to 9 days old developed diarrhea when infected intraileally with NSP4. The region responsible for Ca 2+ mobilization encompasses the peptide segment, which is localized in the cytoplasmic domain of the molecule, folded as an amphipathic helix. The crystal structure of the NSP peptide segment showed a homotetrameric pore which could span the ER membrane and act as a Ca 2+ channel. 21

48 Multiple pathways may be involved in diarrhea induction by NSP4 (42). The NSP induces chloride secretory currents across the mouse intestinal mucosa that potentiate the camp induced effects (44). The NSP4 also induces an iodide influx into mouse crypt cells. This influx only occurs in neonatal mice and requires Ca 2+ (475). The NSP4 of RV group A-C mobilizes Ca 2+ in all regions of crypt cells. Thus NSP4- induced diarrhea may occur via activation of the anionic halide permeability pathway, which is dependent on the age of the mice and Ca 2+ (475). The NSP4 is also responsible for inhibition of sodium-dependent glucose transporter (SGLT1) and sodium leucine symporter activity in the intestinal border of young rabbits which leads to fluid accumulation in the intestinal lumen, because the SGLT functions in water reabsorption (265). The NSP4 is hypothesized to function as an enteric nerve secretagogue, i.e. NSP4 may activate the secretory reflexes of the ENS to induce fluid loss and this process is also Ca 2+ dependent (422). Because of the association of NSP4 with diarrhea induction, it was hypothesized that NSP4 may play a role in virulence. This hypothesis was supported by sequence comparisons and site-directed mutational study of NSP4 sequences of the virulent and attenuated strains of both OSU and Gottfried RVs (774). However the comparison of the NSP4 nucleotide sequence between the virulent unpassaged HRV strain and the attenuated strains resulting after 33 passages of this virus failed to document any difference (720). It is possible that a mutation in the NSP4 sequence is not the only mechanism for loss of virulence (774). 22

49 Diarrhea induction by NSP4 Residues in the NSP4 protein are responsible for the enterotoxigenic activity of this protein. Synthetic peptides encompassing these residues induced diarrhea in mice in a dose-dependent and age-dependent manner (44). Administration of purified NSP4 IP ( 1µmol) or intraileally (IL) (0.5nmol) caused diarrhea in infant mice within 1-4 hours PI; the diarrhea could last up to 8h PI. The rate of diarrhea was the highest in 6-7 days old mice (IP route) or 8-9 day-old (IL route), but lower in older animals and no diarrhea occurred in day-old mice. The route of administration is important in the diarrhea induction by NSP4. Intramuscular inoculation of NSP4 of the same dose as for the IP route did not induce diarrhea in CD1 mice. The administration of the synthetic NSP4 corresponding to residues led to a lower rate of diarrhea (60 and 71% by IP and IL, respectively) in 6-7-day-old mice, whereas IP or IL inoculation of older mice (11 days or more) led to low or no diarrhea even when the dose was increased 2-4 fold. The sensitivity to NSP4 induced diarrhea increased when the protein was delivered directly into the intestinal lumen. The effective dose of synthetic NSP4 was higher than the purified full length NSP4. Diarrhea in neonatal mice can be induced by NSP4 from different virus strains: the full-length SA11-NSP4 and the peptide, the RV EW-NSP4 in CD1 mice, or by the group C-NSP4 (44, 298, 597). The ability of NSP4 to induce diarrhea was also demonstrated in vitro. The addition of rnsp4 to the intestinal cell culture induced enterotoxigenic signaling activity, i.e. an increase in Ca 2+ concentrations (775). It is 23

50 speculated that the concentrations of the receptor for NSP4 in the intestine were higher in infant mice than in adult mice, resulting in reduction of the disease caused by NSP4 in older animals. Avian RV NSP4 also induced diarrhea in suckling mice despite the low homology (only 31-37% identity) between avian and mammalian RV NSP4. The enterotoxin domain in avian RV NSP4 lies in a similar region (residues ) as in SA11 NSP4 (472) Immune responses to NSP4 The NSP4, although a non-structural protein, is able to stimulate humoral and cellular immune responses in humans (both children and adults) and other animals, e.g. rabbits, pigs, etc (154, 313, 315, 332, 766, 768). In human infants, the serum IgG antibody responses to recombinant NSP4 (rnsp4) and native NSP segments were observed in all children naturally infected or in 90% of vaccinated infants (332). In that study, following vaccination with reassortant tetravalent vaccine, none of the children had detectable serum IgA antibody responses to rnsp4 or NSP In naturally infected infants, NSP4 specific IgG and IgA antibodies could be measured in serum samples and no differences in these responses between different RV strains of different NSP4 genotypes were observed (544). However, Yuan et al (768) showed that broad heterotypic responses to NSP4 occurred after natural RV infections, with 50%-70% seroconversion to IgA and IgG serotype specific antibodies to NSP4 from RV groups A, 24

51 B or C. Both homotypic and heterotypic NSP4 antibody responses also were detected in patients given various live oral RV vaccines, including the reassortant tetravalent vaccine. In humans, the cellular immune responses to NSP4 (indicated by the production of IL-2 and IFN-γ or by the increase of T cell proliferation) can be detected after injection with NSP4, which suggests that Th1-like responses and cytotoxic T cell responses occur (332). However the secretion of IL-2 and IFN-γ were demonstrated in RV infected adults only, whereas T cell proliferation responses were observed in less than 50% of RV infected infants. Thus NSP4 is a poor inducer of T cell responses in infants. Similarly, in animal models, such as Gn pigs, infection with a Vir or Att HRV led to 100% seroconversion to the homotypic NSP4 by 28 days post-inoculation (PID) (313). However, VirHRV induced 5-6-fold higher NSP4-specific antibody titers than the AttHRV. Virulent HRV also induced NSP4-IgA antibody secreting cells (ASC) in the intestine of the pigs and vaccination and challenge with the VirHRV induced a high number of NSP4-IgG ASC in the MLN. The presence of NSP4-ASC may be related to the extracellular release of NSP4 from infected cells (120). In mice, however, no measurable amounts of antibody against NSP4 were found after homologous and heterologous infections (315), which was due to the low homology between genotypes D and C of NSP4s used in the study Homotypic and heterotypic NSP4 specific antibodies and protection. Evidence for the role of NSP4 in diarrhea induction in mice and immunogenicity of NSP4 in mice, humans, calves and pigs suggest the possible role of NSP4 in protection 25

52 against RV diarrhea by the integration of NSP4 into a RV vaccine containing VP4, VP6 and VP7. Potatoes expressing the fusion protein of NSP4 with cholera toxin-subunit B (CTB) were fed to mouse pups, which showed a significant decrease in diarrhea when challenged with RV (24, 761). Another NSP4 fusion protein was expressed in potatoes using the HIV-Tat transduction domain, which allowed direct entry of the protein into the cytosol of mammalian cells (205). The CTB-NSP induced higher serum IgG antibody titers than CTB-NSP , Tat-NSP or NSP in orally immunized mice. Mice immunized with a combination of CTB-NSP and Tat- NSP showed lower IgG antibody titers but higher IgG2 antibody titers than CTB-NSP alone, indicating an increase of Th1 responses in the presence of Tat fusion protein (364). The Tat fusion protein was shown to induce Th1 responses leading to cytotoxic T lymphocyte (CTL) responses, but not mucosal IgA antibodies, whereas a CTB fusion protein generated high secretory IgA (siga) antibodies. Unlike the fecal and serum IgA antibody responses to the whole virus which correlates with protection against RV infection and disease, the serum NSP4-IgA may not play any role in protection against RV disease and it is not yet clear whether mucosal NSP4-IgA serves any role. Antibodies against NSP4 might just represent the number of exposures to RV. It was observed that Nicaraguan adults showed higher, but not statistically significant, NSP4- IgG antibody titers than those of Swedish adults (550). The NSP4 antibody responses and protection against RV disease conferred by NSP4 antibodies are species specific but not genotype specific. Following primary infection and challenge with Vir RV in Gn calves and pigs, higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous 26

53 host heterotypic NSP4s were induced (766). In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine RV bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine RV in primary infection Passive immunity conferred by NSP4 antibodies All of the passive immunity studies using NSP4 specific antibodies were conducted in the mouse model. The NSP4 antibodies can block induction of disease in mice. The administration of antiserum to NSP4 peptide , 5 min before IP delivery of nmol of NSP4 peptide caused a 90% reduction in diarrhea in mice (44). In the absence of the NSP4 antibody, 67% of mice showed diarrhea upon IP injection with NSP4. In addition, pups born to dams immunized with NSP4 peptide showed significant reduction in severity and duration of diarrhea when challenged with SA11 virulent RV. Pups infected with SA11 RV and fed with NSP4 serum every 4-6h for 60h also showed a significantly reduced diarrhea compared to infected pups fed with control serum. Thus in the mouse model, the presence of NSP4 antibodies in the circulation and in the intestinal tract provides protection against NSP4 and RV induced diarrhea, respectively Immunity to RV Role of innate immunity The role of innate immunity has been explored for RV infection only recently, although RV, being a dsrna virus, can induce the production of interferon type I from macrophages and fibroblasts (440). The involvement of dendritic cell (DC) in the 27

54 recognition of RV antigen via the toll-like receptor (TLR) might play a very important role in antigen presentation to cells of the adaptive immune response (440). Unknown innate immune factors have been suggested as the mechanisms for RV clearance in SCID mice (C57BL/6 background) (219). The involvement of natural killer (NK) cells in immunity against RV has also been investigated (471, 486). Rotavirus antigens are found in monocytes/macrophages during infection and the presence of RV in these cells was suggested to cause spread of RV to tissues other than the intestine (93). Microcapsules containing RV were observed to bind specifically to antigen presenting cells (APCs), i.e. B cells, macrophages and DC in vitro which might explain the enhanced immune response when microencapsulated RV antigens are used for immunization (96). The role of DC is further supported by a study in which mice lacking chemokine receptor (CCR) 6 showed a deficiency in DCs expressing the DC and monocyte/macrophage markers CD11b and CD11c from the subepithelial dome of the PP and impaired humoral responses to orally inoculated RV antigens (156). The CCR6 mediates the migration of DCs and lymphocytes during immune responses. In addition, human immature DCs produced IL-12p70, IFN-γ and IFN-β after stimulation with dsrna from RV (440). Of note, only professional APCs produce the p40 component of the biologically active IL-12p70, whereas other cells only synthesize the p35 subunits of IL-12 (2). Further understanding of the role of DCs in RV infection requires knowledge of the interaction between the RV and the TLRs. However RV also exhibits mechanism(s) to escape the recognition by innate immune surveillance. In knock out mice lacking signal transducers and activators of transcription-1 [Stat1(-/-)] leading to lack of IFN type I and II responses, oral infection of 28

55 suckling Stat1(-/-) and immunocompetent mice with RV induced diarrhea and virus shedding of similar intensity (697). A later study identified the RV-NSP1 (encoded by gene segment 5) which causes rapid degradation of the interferon regulatory factor (IRF)3 during the replication cycle, thus antagonizing the IFN-signaling pathway of the innate immune response (46). Of note, IRF3, a constitutively expressed protein in the cytoplasm, is an important effector of the innate immune responses Natural killer cells Apart from the cytotoxic effect conferred by CD8 T cells, the cytotoxic activity of NK cells also contributes to innate immunity and clearance of RV. Prolonged shedding of RV has been observed in elderly patients and impaired NK cell cytotoxic activity has been suggested to be the possible cause (471). Cytotoxicity by NK cells was identified in intestinal intraepithelial cells of chickens infected with RV. This activity was not MHC restricted (486) and it was not restricted to cells from RV infected chickens. Upregulation of NK cytotoxicity via IL-15 was observed in an in vitro reovirus infection (204). Further studies are needed to expand an understanding of the role of NK cells in RV immunity Adaptive immunity to RV: Studies of different animal models and humans In response to RV infection, both arms of the adaptive immune system are involved, i.e. B and T cells (CD4 and CD8 T cells). However, to what extent each 29

56 component contributes to protective immunity against RV varies considerably in studies of humans and different animal models. The roles of all components are summarized in regard to humans and each model in the following sections Mouse model In this model, both adult and neonatal mice have been used which yield different interpretations of the determinants of protection against RV. In the adult mouse model, protection against live virus doesn t correlate with the serum or intestinal neutralizing antibodies, but does correlate with the serum and stool RV IgA antibodies and IgA ASC responses (207, 511). Long-term protection also depends on antibody production as B cell deficient mice shed virus for a prolonged time (220). Rotavirus IgG antibodies also play an important role in protection in the adult mice model, especially in gene knockout mice that do not produce IgA antibodies (511). The defense mechanism of intestinal IgA antibodies in this model operates via surface exclusion and intracellular neutralization of the viruses. In the adult mouse model, the VP6 which does not induce virus neutralizing (VN) antibodies induces a protective response (208). Non-neutralizing IgA monoclonal antibodies against the VP6 protein can confer both homologous and heterologous protection (572). It is postulated that the transcytosis of the dimeric IgA antibody with the pigr blocks crucial steps in the viral replication cycle (102, 572, 607). Protection induced by VP6 proteins in the adult mouse model also depends on the route of vaccination and 30

57 adjuvants (134, 136). Intranasal (IN) immunization of adult mice with EDIM VP6 with E.coli mutant heat labile toxin mlt (LT-R192G) adjuvant induced more than 99% protection against viral shedding after homologous virus challenge. Less protection was induced by IN administration of VP6 with CTA1-DD, Adjumer and CpGoligodeoxynucleotide (CpG ODN) adjuvants (95, 80 and 74%, respectively). Of note, CTA1-DD is a gene fusion protein which combine the active subunit A1 of cholera toxin (CT) with a B cell targeting peptide, D, derived from protein A of S.aureus (424). The use of VP6 with QS21 adjuvant (purified form of Quil A) via the IN route induced only 43% protection compared to a 16% protection rate when the VP6 was administered alone. The oral route was less effective in inducing protection with all of the above adjuvants. In the adult mouse model, B cells, CD8 and CD4 T cells were all identified as effectors that play different roles in protection and resolution of the viral infection. The resolution of RV shedding and protection against subsequent infection was associated with RV-specific CD8 T cells. In B cell deficient mice, the depletion of CD8 cells prevented the resolution of infection (221). In immunocompetent mice, CD8 T cell depletion only delayed the resolution of shedding. The CD8 T cells therefore appear to play an effector role in short term protection, whereas long term protection is mediated by RV antibodies. The CD4 T cells are essential for the complete resolution of viral shedding as they provide help for B cell development and antibody production. A study by McNeal et al (459) indicates that CD4 T cells are the only lymphocytes required for protection, as B cell deficient mice, depleted of CD8 T cells, were completely protected against reinfection whereas the removal of CD4 T cells led to a loss of protection. 31

58 In the neonatal mouse model, unlike the adult mouse model, the role of VN antibodies was emphasized in protection against RV. The neutralizing IgA antibodies against VP4, but not against VP6, in a backpack implanted hybridoma protected against RV induced diarrhea in neonatal mice (572). Infant mice receiving milk from dams immunized IN with VP2/VP6 protein expressed by recombinant S.typhi (virus-like particles (VLP) were not formed in this system) were not protected against diarrhea (158). Similarly, pups born to mice immunized with recombinant vaccinia viruses expressing RV VP7 but not VP6 were protected against diarrhea (21). The protection against diarrhea in the newborn mice was mediated by VN antibodies present in milk, but not by serum antibodies because mice born to unimmunized dams but raised by dams immunized with VP8 were protected against diarrhea upon challenge with homologous virus (243). Thus in this model, local but not serum IgA antibodies against neutralizing epitopes is suggested to play a role in protection against diarrhea. However, in this model, the protection against viral shedding was not assessed. On the contrary, in the adult mouse model, the protection against virus shedding, but not diarrhea has been used throughout as adult mice do not develop diarrhea after RV challenge. Despite the ease of studies using mice, this model is far from being a true representative of the human system. The human newborn immune system at birth is more developed than that of the mouse: only 7-day old mice are similar to human newborns (11). In addition, neonatal mice can quickly loose the bias for Th2 response by 5-6 days after birth whereas human infants remain still under the influence of Th2 cytokines for a longer time (10). 32

59 Rabbit model In the rabbit model, Ciarlet et al reported that VP4 and VP7 were the perquisites for protection against infection after challenge with a lapine RV (G3 ALA) (139). Parenteral injection of SA11 derived 2/6/7, 2/4/6 or 2/4/6/7 VLP into rabbits induced fecal IgG and not IgA antibodies which were also associated with protection or partial protection against ALA virus (155). Similar to mice, induction of diarrhea in rabbits by RV is age dependent (141). Only rabbits less than 2 weeks old develop diarrhea and shed virus when infected with lapine RV. According to this study, the rabbits up to 11 months of age, although not having diarrhea, still exhibit histological changes in intestinal villi, typical of diarrhea induced symptoms. The rabbit model has only been used in very limited studies of rotavirus immunity Pig model Compared to the mouse (both neonates and adults) and rabbit models, the neonatal pig model is a more accurate representation of what happens in human neonates. Pigs are the only animals in which diarrhea can be induced by HRV strains, which make them more relevant for the study of RV vaccines for use in human infants. The window of susceptibility of pigs is longer (up to 8 weeks or more after birth), which allows studies of the immune responses and the evaluation of vaccine efficacy with booster doses (589, 770). In mice, on the other hand, diarrhea can be induced only within the first two week, in comparison to the week lifetime of mice. Within the pig model, there are currently two approaches for the study of RV induced protection and immune responses: conventional pigs (raised under normal 33

60 management conditions) and the Gn pigs (raised in germ-free conditions). The former has been used by many investigators, but pigs varied in age, the presence or absence of MatAb (colostrum fed or deprived pigs) and the confounding effects of extraneous RV infectious. Some conventional pigs raised by dams with previous exposure to RV still shed RV (25% of pigs shed during nursing and 70% during the post-nursing period). Naturally occurring RV-associated diarrhea is reported in 1- to 41-day-old suckling pigs (29, 71, 174, 558, 656, 752) or within 7 days following weaning (71, 403, 686, 746). Uncomplicated RV diarrhea in suckling pigs usually resolves in 2 3 days. Morbidity is usually less than 20% and mortality due to dehydration is typically less than 15% in diarrheic pigs. Mortality is highest in young pigs. Neonatal pigs remain susceptible to RV infection and exhibit pathological signs until 12 weeks of age (233). The MatAb usually provides protection against disease and infection only until 2 weeks of ages (225). Natural infection of pigs with RV occurs when MatAb levels in these pigs drops to antibody titers of 1600 (ELISA) (225). High persisting levels of passive IgG RV antibodies transudated from serum back to the gut were also protective (714). Post-weaning and neonatal pigs have currently been used to study diarrhea pathophysiology and the immune responses and the determinants of protection against RV. Enterotoxigenic E.coli (ETEC) or RV were detected in the feces of weaned pigs with diarrhea (487). Colostrum-deprived pigs and 3-week-old newly weaned pigs were exposed to RV before oral treatment with natural human IFN-α (402). The IFN-α treatment was successful in reducing virus secretion and mortality rates in colostrumdeprived pigs, but not in weaned pigs. However the use of conventional pigs only allows 34

61 limited interpretation of the development of active neonatal immune responses to RV due to the presence of other pathogens, extraneous RV infection and interference by MatAb. Rotavirus has been detected in stools of weaned pigs without diarrhea (487). One of the approaches to avoid such interference was to infect fetal pigs in utero, days before birth and orally challenge the newborn pigs at 2-4h after derivation in germ-free conditions to assess protection against diarrhea and shedding (683). In this model, diarrhea was prevented in 12 of 14 pigs and reduced in the other two pigs, but fecal viral shedding was still detected in 12 out of 14 pigs. Neonatal Gn pigs have been used extensively to study immune responses to RV, RV vaccines and the correlates of protective immunity. In this model, intestinal villous atrophy was induced by infection with the VirHRV Wa strain, but not by the attenuated strain (589, 715). Studies were conducted of the magnitude and kinetics of IgM, IgA and IgG ASC in pigs inoculated with porcine RV SB1A and Gottfrield strains or HRV Wa strain using an ELISPOT assay (125, 771). The IgM ASC appears as early as 3 days after infection and peak at 7 PID in MLN and spleen and later in lamina propria (LP) of the intestine. The IgA and IgG ASC numbers peak later at PID and the IgA ASC responses are found mostly in intestinal lymphoid tissues. The magnitude of the antibody responses are highest in the tissues close to the site of viral replication, i.e. the intestine. In the Gn pig model, a correlation of protection against infection and disease has been associated with intestinal IgA ASC, whereas the IgA ASC and antibodies in blood can serve as indicator of the IgA intestinal response, and hence protection (493, 681, 771). Unlike the mouse model, in Gn pigs, protection against diarrhea requires the presence of antibodies to the neutralizing antigens VP4 and/or VP7. The 2/6-VLP with 35

62 mlt, ISCOM or a DNA plasmid vaccine encoding VP6 did not induce protection against viral shedding and diarrhea in Gn pigs (249, 314, 493, 763, 765). Repeated inoculations with an inactivated vaccine via either the oral or the intramuscular (IM) route (with incomplete Freund s adjuvant for the IM vaccine) induced low levels of protection, and were less effective in inducing intestinal IgA ASC (769). In contrast with the mouse model, when RV was inoculated via the IM route, the high IgG ASC and antibody responses induced in systemic tissues did not confer protection (769). However, a combination of priming with Wa AttHRV followed by two booster doses of VP6-DNA vaccine IM induced moderate to high levels of protection (30-70%) against diarrhea and virus shedding, respectively (763). Of interest, the subsequent boosting with 2/6VLP (contains no neutralizing epitopes) following the initial priming with Wa AttHRV enhanced the responses to other neutralizing epitopes on VP4 and VP7, as indicated by the enhanced levels of neutralizing antibodies (763). This cross induction and enhancement between epitopes of the same virus or different closely related viruses has also been observed for vaccinia virus, LCMV etc. (610). The route of vaccine delivery has also been investigated intensively in the Gn model. The administration via mucosal routes has shown clear advantages over the systemic route in inducing mucosal antibody responses as well as in the ease of delivery of the vaccine. The oral administration of 3 doses of Wa AttHRV induces higher protection against RV than the intranasal (IN) route (Azevedo and Saif, unpublished). Boosting of the Wa HRV orally primed pigs with 250µg VLP IN resulted in higher protection against shedding and diarrhea than boosting with 2/6VLP/ISCOM orally (249, 314). The order of the routes used for priming and boosting also determines the level of 36

63 protection in pigs. Oral priming with attenuated HRV followed by IN boosting with 2/6VLP or IM boosting with VP6-DNA vaccine, but not the reverse, induced high protection against viral infection and disease (763, 767) Non-human primate models Recently pigtailed macaques have been used as host for simian RV infections (731). Infection of seronegative macaques aged months with YK-1 simian RV led to high shedding of RV antigen in stools for 2-10 days after challenge, but without signs of diarrhea. Infection of the seropositive animals also led to shedding of RV antigen in stools but with shorter duration. The infection induced RV specific antibody responses and conferred protection of the macaques against challenge virus after 28 days. This model also demonstrated the role of existing RV IgG antibodies in reduction of virus shedding when macaques were infected with YK-1 virus. However the macaques, similar to the adult mice and rabbit models, do not exhibit RV-related diarrhea. Even the very young macaques (4-6 months old) did not develop symptoms upon infection. In another non-human primate model for RV, a new strain of simian RV namely TUCH (Tulane University and Cincinnati Children s hospital), characterized as a new P[23]G3 strain, was used to infect juvenile rhesus macaques (458, 611). The T cell immune responses were induced by this infection, as demonstrated by increased IL-6 and IL-12 in secretions from the peripheral T lymphocyte cultures. In this model, the CD4 + and CD8 + T cells exhibiting intracellular IL-6 and IFN-γ and the HLA-DR + differentiated monocyte-derived DCs were detected after virus infection. The baboons and the velvet 37

64 monkeys were also investigated as animal infection models for HRV. Shedding of RV in the feces of 5 of 5 velvet monkeys and 1 of 2 baboons was observed with increased IgG and VN antibody titers (122). These non-human primates could be useful to study candidate vaccines for human use. Yet like other infection models, the lack of diarrhea related symptoms remains a disadvantage for these models Immunity to RV-human studies: The immune determinants of protection against RV Serum and fecal IgA antibodies The association of fecal IgA antibodies with protection against RV infection is indicated by human studies. Fecal IgA antibody responses are considered a good marker for protection (161, 437). A study of RV outbreaks in children in day care centers in the US showed that the geometric mean RV fecal IgA antibody titers before exposure were highest in children who remained free of infection, lower in children with asymptomatic infections and the lowest in children with diarrhea (437). Thus a RV-fecal IgA antibody titer 80 correlates with protection against infection, whereas titers 20 correlate with protection against diarrhea. Intestinal IgA antibodies are an even better correlate of protection (92, 288). A study of Danish children with acute gastroenteritis showed an increase in IgA antibodies in duodenal fluids (intestinal IgA antibody) 10 days after infection and increased fecal IgA antibody titers at 5-7 months after infection (288). Furthermore, the presence of IgA ASC in the small intestine of children was correlated with serum IgA antibody titers (92). Furthermore, salivary IgA antibody titers reflected 38

65 mucosal IgA RV antibodies and could be a substitute measure for intestinal rotavirus antibody, as suggested by a RV (CJN strain) challenge study of adult volunteers (722). The correlation between serum antibody and protection is still controversial as contradictory conclusions were drawn from studies of adults and infants with natural infection or following RV vaccination. Studies of adult volunteers inoculated with HRV-D strain showed a correlation between serum antibody titers and protection against diarrhea. Pre-challenge VP7 antibody titers 20 correlated with protection against diarrhea or shedding of the virus upon challenge at 19 months after inoculation (257, 347). The presence of the strainspecific or cross-reactive VP4 antibodies also correlated with protection in this study. Ward et al (717) also found that serum IgG and VN antibodies in jejunal fluid (obtained endoscopically using a jejunal catheter) correlated with protection of adult volunteers administered 2 doses of CJN (P[8]G1)-HRV. However this correlation was not consistently found in another study by the same investigators (718). It is postulated that the existing antibodies in adults due to previous exposure to RV were the reason for the inconsistency of these studies (328). Studies of young children with natural infection also showed contradictory results regarding the role of serum antibody in protection against RV. A study of Danish children suggested that serum IgA but not IgG antibodies correlated with a decreased severity of symptoms (287). But a study in Bangladesh found that serum IgG antibodies correlated with protection against severe illnesses (147). Serum RV specific IgA, IgA1, IgG, IgG1 and IgG3 antibodies are recognized as markers for RV infection in Bangladeshi children with RV (36). A study of US children, who previously developed 39

66 gastroenteritis, showed that both an IgA antibody titer of >200 and an IgG antibody titer of >800 obtained after the first RV season correlated with protection in these children in the second RV season (512). Similarly a study in Mexico demonstrated that both IgA and IgG RV antibodies were protective, yet the antibody titers required to achieve protection were higher than those in the US study (702). A serum VN antibody of 128 was protective in Japanese children against subsequent illness due to G1-RV (127). Homotypic antibodies acquired via the placenta were associated with protection against dehydration in infants 1-6 months of age during RV induced diarrhea (561). Some vaccine trials of young children also showed a correlation of serum VN and IgA RV antibody responses with protection (708, 719). In contrast, Ward et al (721) found that protection against RV diarrhea after natural infection did not depend on homologous serum VN antibodies. Similarly, a lack of correlation between serum antibodies and protection were reported in vaccine trials with RRV-TV, RIT 4237, WC3, Wa, RRV and DxRRV (328). Jiang et al (328) postulated that these discrepancies may result from differences in sample size, vaccine type and dose, demographics and laboratory diagnostics used Markers on B and T cells and the association with RV infection and protection The use of a flow cytometry assay allows the study of circulating B and T cells and their subsets using surface markers and the association with RV infection and protection. The presence of large IgD- B cells expressing RV specific surface immunoglobulin (sig) correlates with RV-specific ASC during acute but not during 40

67 convalescent RV infection (248). Lymphocytes detected during acute infection were RV ASC whereas those detected during convalescence were memory B cells. The human cells expressing RV-sIg also express the integrin α 4 β 7, an intestinal homing receptor, which indicates that RV-sIg B cells are primed to traffic back to the intestine. Therefore the flow cytometric assay of blood lymphocytes bearing α4β7 is considered as an indirect measure of intestinal RV ASC which are responsible for protection against RV infection (321). Chemokines TECK/CCL25 (thymus-expressed chemokine/chemokine (C-C motif) ligand 25), MEC/CCL28 (mucosa associated epithelial cytokines) and the receptors CCR9 and CCR10 respectively, play an important role in the process of B cell homing to the gut (388, 397, 772). The CCL25 is exclusively expressed in the small intestine and attracts B cells that express CCR9 and IgA. The CCL25 and CCR9 may serve to compartmentalize the small intestine immune response because RV-IgA ASC migrated preferentially to tissues expressing CCL25. In addition, B cells expressing CCR9 are mainly found in the small intestine whereas they are rarely found in the colon and absent in other epithelial tissue (79, 385). The roles of CCR9 and CC10 were confirmed in that RV specific IgM and IgA ASC, predominantly large lymphocytes, also express CCR9 and CCR10 in response to acute RV infection, which likely targets these cells to the gut (321). During convalescence, the B cell population consists of both small and large cells expressing low or no CCR9 and CCR10, most likely representing RV specific memory B cells with both gut and systemic trafficking profiles. The CCR6 which is expressed by most B cells, subsets of CD4 and CD8 memory T cells and subsets of DC helps positioning leukocytes at mucosal locations (739). The RV memory cells express CCR6, which allows their recruitment upon inflammation. 41

68 Role of cytokines in RV infections A number of cytokines have been implied in disease manifestations and protection against RV infection in humans. In a study of Bangladeshi children with RV diarrhea, IFN-γ was higher in children with RV diarrhea than those with diarrhea unassociated with any enteric pathogen (36). In children with persistent diarrhea, plasma IFN-γ levels were higher than in those with acute diarrhea, indicating that plasma IFN-γ levels may be associated with subsequent development of persisting RV infections (35). In this study, TNF-α correlates with the acute phase of the diarrhea, related to the proinflammatory effect of this cytokine. In another study of US children with RV diarrhea, it was shown that serum IL-6 was correlated with fever, whereas serum IL-6, IL-10 and IFN-γ correlated with acute RV infection. In regard to the other symptoms of RV infection, the presence of TNF-α was associated with fever and multiple diarrhea episodes whereas IFN-γ levels were an indicator of vomiting (329). Cytokines in RV infections in Gn pigs have been studied (32). The study was conducted using Gn pigs inoculated with either VirHRV or AttHRV. Generally, higher cytokine levels occurred early after infection with VirHRV compared to AttHRV. The pro-inflammatory cytokine TNF-α levels peaked and remained elevated in serum of the VirHRV inoculated pigs early in the infection (PID 3) and later (PID 21) in the AttHRV inoculated pigs. In serum, IL-6 was significantly elevated at PID 1 in the VirHRV group and at PID 3 in both HRV groups. For Th1 cytokines, only low and transient IFN-γ responses (PID 3-5) occurred in serum and intestinal contents of the AttHRV-infected 42

69 pigs, compared to significantly higher and prolonged IFN-γ responses (PID 3-28) in the VirHRV-infected pigs which correlated with viremia and diarrhea induced by VirHRV but not by AttHRV. The role for IL-12 in the induction of immune responses to rotavirus infection was confirmed in both groups. Early in the infection, there were higher levels of the TH2 cytokine IL-10 in the serum of the VirHRV group compared to the AttHRV group. A delayed initiation of Th2 responses occurred after AttHRV infection of pigs as indicated by higher IL-10 cytokine secreting cell numbers in ileum and spleen of the AttHRV group compared to the VirHRV group at later times, PID 14 and 28. Similarly to pigs, in mice, a mixed pattern of Th1 and Th2 cytokines were induced upon infection of mice either with heterologous (SA11) or homologous (EHPw) RV (223). In mice IL-6 does not play a significant role in protection against RV nor in Th1 and IgA development (696). Unlike in pigs, type I and II IFN did not play a role in protection of neonatal mice against RV shedding (22, 697). In knock out mice lacking signal transducers and activators of transcription-1 [Stat1(-/-)], oral infection of suckling Stat1(-/-) and immunocompetent mice with RV induced diarrhea and virus shedding of similar intensity. Clearance of RV from stools of adult Stat1(-/-) mice occurred at the same time as in wild-type mice. However, adult Stat1(-/-) mice shed up to 100-fold more rotavirus antigen in stools than did immunocompetent mice after infection. Type I IFN receptor -/- suckling mice and IFN-γ -/- suckling mice developed diarrhea with similar duration and had comparable quantities of viral antigen in their intestines as did immunocompetent mice (22). Intestinal epithelial cells also produce chemokines, IFNs and GM-CSF in RV infections, yet the roles in these cytokines and chemokines in protection of mice against RV are unknown (563). 43

70 Rotavirus specific antibody levels in children worldwide A number of studies in the 1980s and 1990s have focused on the antibody levels specific to RV in infants, children and their mothers. The results of these studies are summarized in Tables 1.1 and 1.2. Our studies of the influence of MatAb were based on these antibodies levels (Chapters 3 and 4). Due to the variety of techniques used to measure the antibody titers, it is not possible to unify all of the studies to get the full spectrum of antibody levels in children and mothers in many countries. In addition, the obvious challenge is to identify which are the RV antibodies transferred from the mother to the infants and which antibodies are due to previous encounter with RV and active immune responses in infants Interference by MatAb with RV infections RV antibodies in milk and passive protection The roles of milk antibodies in passive protection against RV have been suggested in humans and demonstrated in animals. In humans, it is suggested that breast milk IgA antibodies confer partial protection against RV infection. A study of Nicaraguan infants from birth until 2 years of age showed a positive correlation between RV IgA antibodies in colostrum and the time of onset of viral shedding (195). In particular, colostral IgA antibody titers were significantly lower in children first demonstrated RV excretion before 6 months of age than in children first demonstrated excretion of the virus after 6 months of age. This study also suggested a relationship between the duration of breast 44

71 feeding and asymptomatic infections (195). Breast-fed infants who did not excrete RV over the 5-day period received milk with significantly higher RV siga antibody titers than breast-fed infants who were infected with RV (456). Infants fed daily with colostrum from cows hyperimmunized with RV were protected from RV associated diarrhea whereas 70% of infants not receiving the bovine colostrum developed diarrhea (184). In pigs, MatAb were detected in the feces of suckling pigs up to 18 days of age. Natural RV infection of these pigs occurred when the geometric mean ELISA titers of MatAb in their sera declined to 1600, suggesting that MatAb is protective against RV infection in pigs but only for the first one or two weeks (225). The passive antibody doses which conferred protection against RV were quantitated in Gn pigs fed with colostrum/milk antibody from cows immunized with different strains of HRV. Dose levels of 15.8 x 10 6 and 19.5 x 10 6 VN antibody units were required to achieve a 50% reduction of diarrhea and viral shedding, respectively (599). However, in nursing piglets, Saif et al (578) showed that maternal vaccination IMm with live attenuated porcine RV serotypes I and II did not prevent natural infection with OSU and Gottfried RV of suckling pigs, but it did decrease the duration and delay the onset of RV shedding and diarrhea. In mice, the milk antibodies specific to VP4 but not VP2 or VP6 were associated with protection against RV induced diarrhea. After nasal administration live recombinant Salmonella expressing RV VP2 and VP6 to female mice, high antibody responses specific to both VP2 and VP6 were induced in serum and milk, but these antibodies failed to protect pups against diarrhea after challenge with bovine RV (BRV) (RF strain) (158, 45

72 159). However VP4 antibodies in milk provided homotypic protection in the mouse model. Pups born to dams immunized with SA11 VP8* were protected from diarrhea when orally challenge with the SA11 strain. In addition, pups born to naive dams but nursed by VP8*-immunized dams were also protected against diarrhea after challenged with the SA11 strain, whereas pups born to VP8*-immunized dams but nursed by naïve dams were not. Thus neutralizing antibodies in the milk rather than serum antibodies transferred through the placenta provided protection against RV induced diarrhea (243). The RV-specific IgY antibodies from egg yolk provided complete homotypic protection against RV MO strain-challenge in BALB/c mice. Rotavirus specific colostrum from cows also protected mice against infection with four different RV G types (184). Colostrum from cows immunized with the adjuvanted modified live Ohio Agricultural Research and Development Center (OARDC) RV vaccine provided complete protection to calves against virulent bovine RV when given as a 1% dietary supplement and partial protection when given as 0.1% supplement (586). Thus passive protection by RV milk antibodies is dose dependent. Shortly after cessation of colostrum feeding, two of three calves shed virus at 14 days post exposure, highlighting the importance of continued presence of passive antibody in colostrum/milk (579) Circulating antibody and protection against RV Circulating antibodies specific to RV were also implicated in protection. A study of children from New Delhi, India showed that neonates infected with the neonatal RV strain 116E-like had significantly lower levels of cord blood neutralizing antibodies to 46

73 116E than the neonates who did not become infected, suggesting that MatAb acquired transplacentally provided passive protection against neonatal RV (543). The effect of circulating RV specific IgG antibodies was investigated in the pigtailed macaque model (730). Upon oral challenge with YK-1 virus, the animals which did not receive the antibody containing serum, shed virus starting at 1-3 days after challenge and the shedding lasted for 6-8 days. On the contrary, in animals passively given IV the immune or control serum (RV specific antibody titers of 10,000 or 300, respectively) did not shed virus or showed delayed shedding at low titers for a limited time. The Gn pigs injected IP with high antibody titer serum from RV hyperimmunized sows exhibited 76 and 36% protection against diarrhea and shedding, respectively when orally challenged with virulent HRV 5 days after IP injection (289). In contrast, the low antibody titer serum derived from naturally infected sows did not provide any protection. Oral feeding with colostrum/milk from RV hyperimmune sows in addition to IP injection of the hyperimmune serum did not improve the protection against virus shedding and diarrhea but significantly delayed the onset of virus shedding (289) Maternal immunization Maternal immunization may provide protection against RV in newborns. The stability of RV antibody in milk was assessed to evaluate the duration of milk antibody to provide protection against RV. Significantly higher concentrations of antibody to RV in milk persisted for 4 months in postpartum women who received a RRV monovalent reassortant vaccine or the tetravalent vaccine (535). When hyperimmune bovine colostrum containing different levels of RV antibody was administered to children, 3 47

74 times a day, for a period of 6 days, the antibody activity was detected as early as 8 hours after ingestion of hyperimmune colostrum and up to 72 hours after consumption had ceased. These results showed that anti-rv activity survived passage through the gut; therefore, passive immunotherapy may be used to prevent or treat infectious diseases that affect the gastrointestinal tract (517). Vaccination of pregnant baboons intramuscularly with a RRV vaccine with ISCOM repeatedly at 1-2 and 14 weeks after delivery caused significant increases in RV-specific maternal serum IgG and VN antibodies and in milk IgA, IgG and VN antibodies (639). The combined IND/2292B VLP vaccine specific for two different serotypes of BRV induced comparable VN responses to each BRV serotype in serum, colostrum and milk compared to the responses induced by the individual IND or 2292B VLP vaccines (366). Maternal immunization can also be enhanced by feeding probiotic bacteria to mothers, which translated to better protection against RV diarrhea in the mouse model. Mouse pups born to and nursed by dams fed with Bifidobacterium breve YIT4064 and immunized orally with the simian RV SA11 were more highly protected against SA11- induced diarrhea than those born to and nursed by dams immunized with RV only (757). In addition, the titers of RV IgA antibody in milk and feces of dams fed B. breve YIT4064 and immunized orally with RV were higher or significantly higher than those of dams immunized with RV only. Passive protection to heterologous RV depends on the route and the type of antigen. Parenteral immunization of dams with RV, homotypic or heterotypic to the challenge virus protected suckling mice against diarrhea, whereas oral immunization with homotypic, but not heterotypic RV strains to the challenge virus conferred protection 48

75 (502). Colostrum from cows immunized with the simian RV SA11-VLP but not inactivated SA11 afforded a high rate of protection to colostrum-fed unsuckled calves against challenge with heterologous BRV (209, 210). The highest titers of RV antibodies in mammary secretions of pregnant cows were induced by IM immunization at ~9 week pre-partum and IMm inoculation at 2 weeks pre-partum with the OARDC modified live NCDV bovine RV, but not after IM immunization with a commercial inactivated Rota- Coronavirus vaccine (587). Feeding colostrum from IM plus IMm immunized cows to newborn calves challenged by RV prevented diarrhea and shedding of RV (587). The antibody titer induced by this vaccine via these routes remained significantly elevated in milk for at least 30 days post-partum. The rationale for this vaccination scheme was that parenteral stimulation near involution resulted in seeding of sensitized plasmablasts to the mammary gland with boosting by IMm RV injection. The antigen dose in the vaccine plays a significant role in RV maternal vaccination. Saif and Smith (587) also showed that the antigen dose of the live OARDC RV vaccine was 1x10 8 plaque forming unit (pfu) /ml, whereas that of the commercial live RV vaccine was 1x10 4 pfu/ml, which likely influenced the RV antibody titers in milk. The authors also indicated that the efficacy of inactivated vaccine could be influenced by the inactivating reagent. Binary ethylenimine (BEI) inactivated RV induced 10-fold greater antibody titers in cow s milk compared to β-propiolactone as inactivating agent. The use of adjuvants is important in enhancing antibody titers in milk. Enhanced colostral RV antibody titers were achieved by incorporating Freund s incomplete adjuvant with the commercial RV-coronavirus vaccine, compared to the same vaccine without adjuvant or with AlOH adjuvant (587). 49

76 Protection against RV by other milk components Human but not bovine lactadherin in milk has been shown to inhibit Wa HRV infection in vitro (in RV-infected MA104 and Caco-2 cell lines) (390). Milk-fat globule membrane protein MUC1 in bovine milk inhibits the neuraminidase-sensitive RV RRV strain efficiently but not the Wa strain which is NA resistant. Therefore it is possible that milk feeding can interfere with a live RV vaccine based on the RRV data (e.g. RRV-TV). A study also suggested an association of trypsin inhibitors in human milk with protection of neonates against RV infection in the first 5 days of life (457). Breast-fed infants were significantly less likely to become infected with RV and showed significantly lower stool tryptic activity or higher trypsin-inhibitory capacity than did bottle-fed infants. Human milk mucin can bind to RV and inhibit viral replication in vitro and in vivo, also providing protection (759) Interference with active immune responses to RV in infants and in animal models Interference by circulating MatAb with the development of active immune responses after RV infection or vaccination has only been studied in animal models, whereas interference by milk antibodies has been widely studied in both humans and animal models. In a comparison of milk from New York and Venezuelan mothers, both milk and infants' serum pre-immunization RRV VN antibody titers had a negative effect on seroconversion after RRV vaccination (P =.008 and.02, respectively). Infants fed milk containing RRV VN antibody titers 160 showed a lower RRV seroconversion rate compared to infants fed milk containing RRV VN antibody titers 160 after vaccination 50

77 with RRV. In addition, VP4-specific milk antibodies may interfere with RRV seroconversion (553). A study of milk from women in Caracas, Venezuela and in Rochester, New York, USA, also indicated the role of VP4 antibodies, but not VP7 antibodies in the reduction of vaccine responses to RRV in children (416). Seventy-five percent of RRV vaccinated infants who were breast-fed with milk containing antibodies against VP4 did not exhibit a seroresponse whereas 45% of infants who consumed breast milk with VP7 antibodies did not respond to the vaccine. On the contrary, among those children fed with breast milk negative for VP4 antibodies and positive for VP7 antibodies, 71% experienced a seroresponse. Interference by RV MatAb, although not proven, was suggested as the reason for the failure of vaccine trials such as RIT4237, WC3, or RRV-TV (54, 110, 371, 416). In the Gn pig model, various titers of RV specific MatAb at vaccination have been shown to influence the outcome of active immunity. Hodgins et al (289) demonstrated that in Gn pigs, induction of ASC responses was suppressed after Wa VirHRV primary infection and challenge in the presence of circulating high titer MatAb. The RV antibodies from milk enhanced suppression of the intestinal IgA ASC responses before and after challenge with VirHRV. In addition, the serum and intestinal IgA antibody responses were also suppressed in pigs receiving high titer MatAb in circulation and/or in milk diet (522). At low titer MatAb, only IgG ASC, but not IgA ASC numbers in duodenum and MLN were significantly reduced after HRV challenge (289). A passive antibody study of calves showed that calves fed with colostrum from normal cows actively produced serum IgG1 antibodies at 14 days post exposure with the virus, compared to 7 days in calves without colostrum feeding, whereas calves fed with 51

78 colostrum from immunize cows did not show any increase in serum IgG responses (579). Similarly, calves fed with colostrum before inoculation with BRV showed an inverse relationship between the IgG1 titers in colostrum and the ASC responses (521). In particular, fewer IgG ASC numbers in response to BRV inoculation were observed in calves fed with colostrum from naturally infected cows (low titers of RV antibody, or control colostrum, CC), whereas significantly lower ASC responses of all isotypes occurred in calves receiving BRV hyperimmune colostrum (IC). In addition, calves fed with IC only developed fecal IgM antibody upon inoculation with BRV. Calves fed with CC showed significantly lower serum and fecal IgA and IgG1 antibody titers compared to colostrum-deprived calves after BRV inoculation. Thus passive RV antibodies interfere with neonatal active immune responses in a dose-dependent manner, similar to observations for measles virus in neonatal mice (624) Rotavirus vaccines Jennerian and modified Jennerian approaches to RV vaccines Initially, several RV vaccines using animal viruses were based on the Jennerian approach to vaccination. Of the four vaccines developed in this way, 3 were discontinued before licensure (RIT4237, bovine P[1]G6; WC3, bovine P[5]G6 and RRV, rhesus P[3]G3) due to variable efficacy between the different countries where the vaccines were tested. Only the RV lamb strain LLR vaccine (P[12]G10) is still used in China (85). Vaccination of infants with WC and RIT 4237 bovine RV gave good protection in Finland, but not in other developing countries (706). Similarly the bovine WC3 RV was effective in Philadelphia, but not in Cincinnati where only 9% of children produced 52

79 antibodies and no difference in the numbers with illness and the severity of diarrhea were observed between vaccinated and placebo groups (54, 144). The RRV was also tested in Venezuelan and Swedish children (217, 252) but gave inconsistent results. Because of the failure of animal RV vaccines, the subsequent vaccine approaches attempted to include HRV components (VP7 or VP4) to the animal viruses (Modified Jennerian approach). Reassortants were created, either monovalent (Wa x UK, P[8]G6; Wa x DS-1xUK, P[8]G2) or tetravalent (Rotashield and UK-based reassortants) or pentavalent (RotaTeq, P[5]G1-4 and P[8]G6), which cover the four most common human G1-G4 serotypes in the background of rhesus(p[3]) and bovine P[5]RV. The first vaccine for RV approved by the US Food and Drug Administration (FDA) in 1998 was RotaShield, a tetravalent vaccine of simian/human reassortants, created by Dr. Kapikian s group at the National Institutes of Health and licensed for production to Wyeth-Ayerst Laboratories for use in infants at 2, 4 and 6 months of age. However due to its association with a high rate of intussusception among vaccinees, it was withdrawn one year after appearing in the market. During a phase III trial of this vaccine in Caracas, Venezuela, the vaccine induced 88% protection against severe diarrhea (531, 635). The tetravalent vaccine, licensed as Rotashield in 1998, is now licensed to a biotech company BioVirX for global marketing. Merck Incorporation produces RotaTeq, composed of five bovine x human reassortant rotaviruses of the most prevalent human serotypes (G1, G2, G3, G4 and P1A[8]), which is in Phase III trials. In a clinical study of a G1 reassortant strain WI79-9 (G1 serotype VP7 on a BRV WC3 strain background), a component of the pentavalent vaccine RotaTeq, the vaccine induced a 95% response rate in vaccinees against the WC3 VP4 (P7) and significant antibody responses to VP7 53

80 were achieved in more than 70% of infants after three oral doses (146). A combination G1 and G2 human-bovine reassortant RV vaccine has been tried without any adverse effects (145). A lower rate of seroconversion to WC3 (45-59%) was observed after 3 doses. However, the IgA coproconversion rate was intermediate (~80%) among vaccinees. It is of great importance to know whether vaccination with 5 strains of recombinant viruses can improve the immunogenicity of the vaccine. Other RV vaccines are also in consideration. Because these rare strains of RV are becoming more common each year, as mentioned previously, the need for a vaccine that carries the full spectrum of HRV serotypes becomes more apparent. A subunit vaccine based on RV VP4 recombinant protein is being considered because the need for the coldchain is eliminated making it ideal for use in developing countries (304). Hoshino et al (300) generated pentavalent or hexavalent RV vaccines containing the existing reassortants of the RRV tetravalent vaccine with the addition of VP4 gene substitution reassortants of human P1A[8] or P1B[4] in the RRV background Non Jennerian approach to RV vaccines The non-jennerian approach to RV vaccination includes the use of monovalent human neonatal strains based on the observation that these strains are asymptomatic in neonates but immunogenic in both neonates and older children. These vaccines include M37 (P[6]G1), 116E (P[11]G9 and I321 (P[11]G10) which are in phase I vaccine trials. A naturally attenuated human neonatal strain RV3 (G3P2A[6]) was tested as a RV vaccine in a limited phase II trial (45). The virus was propagated on African green monkey kidney (AGMK) cells and given at 6.5x10 5 FFU to children at 3, 5 and 7 months 54

81 of age. With this strategy, only 46% of vaccinated infants developed any immune response (serum neutralizing antibodies, serum IgG, IgA, IgM or copro IgA antibodies) and only 2 of 39 infants developed both seroconversion and coproconversion. Of those that developed immune responses, 76% were protected against RV infection and diarrhea following the first epidemic season, compared to the 55% in those with no immune response or those who received placebo. However the severity of the disease was not reduced compared to the non-vaccinated group. This low rate of immune response and protection rates can be attributed to the presence of MatAb, the lack of heterotypic protective immunity induced by the P[2]G3 strain against the more common P[8]G1, P[8]G2 or P[6]G2 strains, or the low vaccine dose used (594) Another non-jennerian monovalent live oral vaccine is based on the human strain (P[8]G1) called Rotarix. GlaxoSmithKline PLC (GSK) is responsible for the development of the Rotarix vaccine which was licensed in Mexico in Phase II and III trials of Rotarix were conducted in Finland and 11 Latin American countries with a total of 65,000 subjects enrolled (557). This vaccine induced a high rate of seroresponses and intestinal IgA responses, yet only 7 out of 20 subjects experienced more than a 4-fold increase in VN antibody titers (55). The protection against severe diarrhea due to G1 or G9 strains was both 77% for each (531), indicating cross protection of the vaccine G1 strain against the other heterotypic strains Rotavirus-like particles as vaccines in different animal models Virus-like particles (VLP) offer attractive approaches for neonatal RV vaccines due to their potential safety (non-replicating so lack of infectivity) compared to live 55

82 vaccines. The viral VP2 and VP6 proteins spontaneously form a double-shelled structure and the addition of VP7 or both VP7 and VP4 results in triple layered particles. The VLPs were constructed from simian, bovine and human strains or a hybrid structure between RV proteins from different species e.g. bovine and human, bovine and simian or proteins from different RV groups, e.g. hybrid VLP from group A VP2 with group C Shintoku VP6 (153, 365, 765). The synthesis of VLPs can be achieved in an insect cell expression system using Spodoptera frugiperda 9 (Sf9) or High Five insect cells (326). In the insect cell system, recombinant baculoviruses expressing different RV proteins were coinfected into insect cells. The majority of VLPs are found in the culture medium. A higher yield of VLPs per cell (5-fold) was achieved using the High five cultures compared to that of the Sf9 cultures (326). The immunogenicity and efficacy of VLPs as a vaccine were assessed in different animal models including mice, rabbits, cows and Gn pigs. In adult mice, 2/6VLPs containing bovine RF VP2 and simian SA11 VP6 administered IN with cholera toxin (CT), E.coli heat labile toxin (LT) or mutant LT with an amino acid substitution (LT- R192G) induced a high level of protection (91-100%) against challenge with live murine RV strains including ECwt (509, 510). Even in the absence of an adjuvant 100ug of 2/6VLP administered IN, also resulted in a 38% reduction in virus shedding (510). Similarly, adjuvant was not necessary for 2/6VLP (0-100µg) to induce systemic and mucosal immune responses when administered orally or IP, suggesting that in mice the VLPs themselves can act as adjuvant (619). The inclusion of VP7 in the particles as 2/6/7 VLP did not improve the immunogenicity or protective efficacy against challenge 56

83 with wild type murine RV. A high level of protection was found using 2/6VLP produced from a dual recombinant baculovirus vector expressing both VP2 and VP6 of simian RV origin (SA11), especially when administered by IM or IN routes (57). In addition, the 2/6-VLP primed for heterotypic (non-g3) immune responses, but the induction of heterotypic neutralizing antibodies required replication of the challenge virus. Thus VP6- T helper cells can provide cognate help to B cells specific for neutralizing epitopes on the VP7 and VP4 of the challenge virus. Similarly, in the Gn pig model, a vaccination scheme consisting of an oral dose of Wa AttHRV followed by 2 IN doses of 2/6VLP with either mlt or ISCOM induced significantly higher VN antibody responses and serum IgA and IgG antibody titers to VP4 and VP7 compared to one oral dose of Wa AttHRV alone at PID28 (764). Similar results were found in studies using the rabbit model of RV infection (without disease). In this model, 2/6-VLP from bovine RF-VP2 and simian SA11 VP6 in Freund s adjuvant administered parenterally resulted in a 41% reduction rate in virus antigen shedding (139). A lower protection rate (5-26%) was achieved by using 2/6-VLP with QS-21 adjuvant. A high protection rate against viral shedding ( % reduction in virus shedding) was induced when the 2/6VLP particles were combined with CT-E29H, the detoxified version of CT adjuvant, and oral or IN administration (620). In both mouse and rabbit models, the presence of VP7 in the VLP did not improve protection against virus shedding. Nevertheless, in the rabbit model, the role of VP4 in protection was realized when 2/4/6/7 VLP conferred higher mean rates of protection than 2/6/7VLP. Contradicting results have been observed in neonatal mouse, Gn pig and calf models. Only suckling newborn mice, born to mothers immunized with 2/6/7-VLP but 57

84 not 2/6-VLP were protected against RV diarrhea when challenged with RF RV 4-days after parturition (159). In Gn pigs, 2/6 VLP, composed of RF BRV-VP2 and Wa HRV VP6 alone did not induce any protection against virus shedding and diarrhea when pigs were challenged with Wa VirHRV. No protection was induced when 2/6-VLP were administered alone or with mlt or ISCOM adjuvants via IN or oral routes (33, 249, 314, 493, 765). In the calf model, only conventional colostrum-deprived calves fed with colostrum and milk from cows immunized with VLP 2/4/6/7 were protected against diarrhea, and 60% of these calves were protected against viral shedding (209). All the calves fed with 2/6VLP immunize colostrum shed virus and one calf out of 5 (20%) had diarrhea when challenged with virulent IND BRV. Differences in the ability of 2/6VLP to induce protective immunity in different animal models may be attributed to use of the adult vs. neonatal of animals and the species origin of the VLPs. Bovine VLPs of different serotypes P[5]G6 and P[11]G10 have been used to coimmunize and they induced lactogenic antibody responses in cow colostrum and milk against both serotypes, confirming the efficacy of these VLPs for passive immunization of cows (366). The VLPs also have potential for the induction of heterotypic protection. The G1-2/6/7 VLPs in which VP7 was derived from the human G1 Houston strain 8697 strain and the VP2 and VP6 from the bovine RF and C486 strains, respectively, induced partial protection (88%) in adult mice against oral challenge with the murine EC RV (G3) when given IM in 2 doses with QS21 adjuvant (327). However, this heterotypic protection was highly dependent on type of adjuvant because the G1-VLP in AlOH adjuvant did not protect mice from G3 RV challenge. Similarly in rabbits, two IM doses G1-2/6/7 VLP or G1-2/4/6/7 VLP or alternative G1 then G3-VLP doses in QS-21 adjuvant induced 58

85 protection against G3 ALA virus similar to the homotypic protection induced by G3-VLP (162). Furthermore, the inclusion of VP4 in the VLPs was not critical for the induction of heterotypic neutralizing antibody. In calves, colostrum from cows immunized with SA11 2/4/6/7VLP (P[2]G[3]) provided complete protection to calves against diarrhea when challenged with virulent IND BRV (P[5]G[6]) and partial protection against viral shedding (209). This cross protection of different G and P serotypes was the basis for monovalent G1 vaccine used for a human infants (594) Individual RV proteins as potential vaccines Individual RV proteins have been studied as candidate vaccines. The VP6, which is highly antigenic and the major component of the RV capsid is conserved among all group A RV was the first choice. Oral and IN administration of RV chimeric VP6- mannose binding protein (MBP) in combination with LT(R192G) adjuvants to mice induced between % reduction in fecal viral shedding (130, ). Other adjuvants were also tested with this chimeric protein for the induction of immune responses and protection in the mouse model (136). The VP6-MBP vaccines delivered with Adjumer, CpG-ODN and chimeric A1 subunit of CT induced a high titer of serum IgG antibody when delivered IN, but not orally. Intranasal delivery with Adjumer increased Th2 responses, whereas CpG-ODN shifted the response more toward Th1. In contrast, adjuvants CTA1-DD, LT (R192G) and QS-21, did not alter the Th1/Th2 pattern. All adjuvants, except QS-21, induced a high level of protection against virus shedding (74-99%) when using the IN route. Intranasal delivery with QS-21 induced only 43% protection only, which was not significantly different from the administration of chimeric 59

86 VP6 alone. Oral immunization with QS-21 improved the protection rate (71%), whereas oral immunization with other adjuvants resulted in decreased protection rates. Thus in the mouse model, the IN route of immunization with the VP6 protein is more effective than the oral route. Because mlt is not likely to be approved for human use via mucosal routes, skin immunization with this adjuvant was explored as an alternative to mucosal routes (137). Needle-free transcutaneous (tci) delivery was achieved by gentle abrasion of the shaved skin of mice and the gauze pads containing the vaccine were applied to the shaved skin for 24h. Subcutaneous injection of the antigen was also tested using a biojet liquid jet injection apparatus. However, the tci delivery of VP6/mLT or VP6 alone did not induced significant protection against virus shedding whereas biojet injection induced low levels of protection in mice compared to IN administration, although VP6 specific serum IgG antibody was induced by using all methods. Delivery of the VP6 plasmid from the EDIM strain of RV using a Powerjet particle device induced serum IgG antibodies but failed to protect mice from homologous virus challenge (132). Similarly three doses of VP6 plasmid administered IM to Gn pigs did not induce protection against virus shedding and diarrhea (763). However, significant protection rates were induced when attenuated Wa HRV was used as a priming dose followed by 2 IM booster doses of VP6 plasmid. The use of other RV proteins has also been explored. Epidermal immunization with VP4 and VP7 genes alone did not induce protection in mice (131). The VP4 fused with maltose binding protein (MBP) expressed in the cytoplasm of Shigella flexneri 60

87 induced immune responses in mice, which suggested that the use of Shigella as a delivery system can induce immune responses to both Shigella and RV (417). A single dose of VP4 or VP7 DNA vaccines encapsulated in PLG microparticles delivered orally also induced protection and intestinal and serum IgA antibody in mice when challenged with the virulent virus 6 weeks later. The VP7 DNA vaccine was less effective than the VP4 vaccine (283). Using a tobacco mosaic virus (TMV) vector, the VP8 fragment of the VP4 protein of BRV was expressed in plants. Immunization of dam with this VP8 fragment induced immune responses and provided protection to suckling pups against challenge with BRV (529, 737). Maternal immunization of pregnant cows with the VP8 protein of BRV resulted in high VN antibody titers in colostrum and milk, which neutralize in vitro BVR P5 serotype (B641) at significant level and P11 serotype (B223) moderately (760). Thus passive protection to calves against BRV by VP8* immune colostrum and milk was suggested. Similarly the VP7 protein expressed in potato tubers induced serum IgG and mucosal IgA antibodies specific for VP7 when fed to mice, suggesting the possibility of employing the transgenic plant as an edible RV vaccine (747). Yet in another study, particle bombardment by VP4 or VP7 plasmids did not confer any protection, or induce serum and fecal IgA antibodies or neutralizing antibodies in mice, indicating the importance of the immunization route in the induction of protective responses against rotavirus in infants (131). In summary, the expression of individual RV proteins as vaccines has been studied extensively in the mouse model. In this model, the RV proteins VP4, VP6 or VP7 can be used as effective vaccines that induce protection when administered with adjuvants or in expression vectors but not alone. There are few reports in other animal 61

88 models about the use of these individual proteins for RV vaccines. Current research results indicate the need for VP4 and/or VP7 for the induction of protection against RV challenge in animal models other than the mouse. 1.2 The mucosal immune system General structure The mucosal-associated lymphoid tissue (MALT) is the largest mammalian lymphoid organ system that provides protection of the respiratory, digestive and urogenital tracts, the eye conjunctiva, the inner ear and the exocrine glands (465). The highly compartmentalized MALT consists of defined lymphoid compartments such as the PP, MLN, the appendix and follicles in the intestine and the tonsils and adenoids in the respiratory tract (369, 478). These compartments act as the site where immune responses are initiated. The MALT also contains the effector sites which are the diffuse accumulation of lymphoid cells in the parenchyma of the mucosal organs and in the glands (295). The antigen is taken up by the absorptive epithelial cells and specialized epithelial cells, called M cells, that reside in the inductive sites and shuttle the antigen to the antigen presenting cells (APC) (295). The antigen can also be captured directly by the professional APC such as DCs, B cells and macrophages. The APCs present the antigen to the CD4 and CD8 α/β T cells in the inductive sites. Intraepithelial cells (IEL) can also present and process the antigen to T cells directly. Different types of responses can be followed: either Th1 or Th2 responses, or suppression responses leading to either oral tolerance or an inflammatory response which involves both humoral and cellular immune 62

89 responses. The sensitized B and T cells then leave the inductive sites, via the lymph, enter the circulation and seed the mucosal effector sites where they differentiate into memory or effector cells (581). One of the properties of MALT is the ability of an immunocyte to be activated at one site but travel to other remote mucosal tissues; this phenomenon is called the common mucosal immune system. On the other hand, the MALT is also highly compartmentalized, i.e. it links specific inductive sites with specific effector sites, such as the gut with the mammary gland, the nose with the respiratory and genital tracts. Thus consideration should be taken in regard of the route of mucosal immunization to induce immune responses in distant sites. Oral immunization induced strong antibody responses in the small intestine, colon, mammary and salivary glands, but induced only limited responses in the genital tract, rectum or tonsils (194, 376, 588). Similarly, IN immunization resulted in strong antibody responses in saliva, nasal secretions and the genital tract but limited responses in the gut (334) Innate immunity Innate immune recognition is mediated by the molecules, pattern-recognition receptors (PRR) which recognize pathogen associated molecular patterns (PAMPs)(12). These PRR include LPS-binding protein, CD14, β2 integrins and TLRs. Natural killer cells, phagocytic neutrophils and macrophages present in the epithelium lining of mucosal tissues play important roles in the first line of defense 63

90 against pathogens. The NK cells are present in low numbers and carry CD16 and CD56 markers (humans, mice). These cells of the innate immune system express TLRs that mediate the recognition of pathogens in the mucosal system (12) Mucosal DC. The DCs are present in both the PP and LP, lining the villi, or in MLN and peripheral lymphoid organs (479). Some DCs also enter the epithelium to sample antigen in the lumen, similar to the M cell function (548). The prominent population of DCs in PP express a high level of MHC class II, indicating their role as APC (491). There is also a subset of DCs in PP with CD11c - phenotype associated with the induction of T cell tolerance. Tolerance of T cells can be achieved by the production of IL-10 by DCs (318). These special subsets of DC in the LP exhibit CD11c lo and MHC class II lo. In addition to APC function, DCs also influence T cell homing. Furthermore mucosal DCs in MLN and PP express the mucosal homing receptor α4β7 and CCR9, which guide the homing of the effector/memory CD8 T cells to the intestine as mentioned previously Toll-like receptors Members of the TLR family recognize PAMP leading to activation of NF-kB and other signaling pathways, important for initiation of the immune response. The common structure of the TLR family includes an extracellular domain with leucine rich repeats, a cytoplamic domain called Toll-IL1 receptor homology domain with a signaling function (439). In humans, ten members of the TLR family have been identified (7, 13, 559). They are further classified into subfamilies TLR2 (consisting of TLRs 1, 2, 6 and 10), TLR 3, TLR 4, TLR 5 and TLR 9 (consisting of TLRs 7, 8, and 9). The TLR 1, 2, 4, 5 64

91 and 6 are involved in the recognition of antigens of bacterial origin such as LPS, peptidoglycan, lipoprotein and flagellin. The TLRs 7 and 8 recognize single-stranded RNA and CpG whereas TLRs 3 and 9 recognize dsrna. The TLR 9 also recognizes CpG. Previous studies of immunity to viral or bacterial infection have focused heavily on the adaptive immune response, i.e. T and B cell responses. Only recently has emphasis been put on the innate immune response which appears much sooner in the course of an infection. The TLRs can be found on NK cells, monocytes/macrophages and DCs. Binding of TLR4 to its ligand leads to the activation of DCs which in turn activate B cells in the mouse mammary tumor virus (MMTV) infection (105). Direct binding of MMTV to TLR4 induced maturation of bone marrow derived DCs (upregulated expression of CD40 and CD80 markers) and induced them to secrete cytokines or chemokines such as TNF-α, IL-6 and IL-12p40. These chemoattractants cause massive recruitment of naïve B and T cells to PP on day 2 of infection (105) The activation of NK cells via TLR3 in response to viral dsrna or polyinosini-polycytidylic (poly-ic) increases the cytotoxic effect of NK cells, independently of APC activation (602) Roles of TLR in the recognition of microbial components Different TLRs can bind to various ligands derived from microbial components or can be activated by endogenous ligands causing autoimmune disease (552). The TLR4 responses to LPS via CD14 are expressed in monocytes/macrophages and neutrophils which recognize LPS-LPS-binding protein complexes. The response of TLR4 to LPS can be enhanced by a protein, MD-2, expressed in macrophages, DCs and B cells. The MD-2 65

92 acts as a chaperone to direct TLR4 from ER/cis Golgi to the cell surface (488). A protein named RP105 on the B cell surface also associates with TLR4 to recognize LPS (504). The TLR4 also recognizes various groups of ligands, e.g. HSP60 and 70, Taxol (354, 505, 689) and more importantly some viruses e.g. RSV, MMTV and MMLV (105, 281). For RSV, the F glycoprotein of the virus induces TLR4 activation via the CD14 dependent pathway (389). In the TLR2 family, TLR2 recognizes many types of microbial agents, e.g. lipoprotein from Gram negative bacteria, mycoplasma and spirochetes, peptidoglycan and lipoteichoic acid (LTA) from Gram positive bacteria (663). The TLR2 can also associate with TLR1 and 6 for the recognition of distinct microorganisms (666, 749). The TLR5 which contains immunostimulatory activity at the highly conserved amino and carboxyl termini responds to flagella of Gram negative bacteria(638). The TLR3 recognizes dsrna either as the viral genome, as intermediates during viral RNA synthesis or as byproduct of transcription of DNA virus genomes or even as synthetic dsrna e.g. poly IC (439). Stimulation by dsrna leads to the production of type I IFN for antiviral and immunostimulatory activities. The dsrna also promotes maturation of DCs. The TLR3 expresses several characteristics which are not found in other TLRs, i.e. its preferential expression in mature DCs and its different genomic organization and structure from other TLRs (439). The TLR9 family consists of TLR7, 8 and 9. The TLR9 directly recognizes unmethylated CpG DNA which is the motif found only in bacterial DNA and very immunopotent (48). The CpG DNA activates DCs to produce IL-12 which favors Th1 66

93 like immune responses. The recognition of CpG DNA by TLR9 occurs in endosomes, whereas that of TLRs 1, 2 and 4 occurs at the cell surface (421). The presence of TLR9 in the endosome, as well as of that of TLR2 in the phagosomes suggests the possible use of these receptors as vaccine adjuvants. The TLR7 identifies synthetic chemicals, nucleic acid like structures, imidazoquinolines, R848 and others (340) Distribution of TLRs in different tissues of humans and animals The classification of DCs is rather complex; they can be classified according to species, phenotype, tissue origin, function and location. In the previous lineage system, DCs were classified into cells developed from myeloid restricted and plasmacytoid restricted precursors. However, cells unable to form T cells still produce thymic CD8 + DCs, which were originally thought to be lymphoid derived (112). Rather each lymphoid restricted or myeloid-restricted precursor can produce mature splenic and thymic DC subtypes with some bias in the subset balance (618). Distinct DC subtypes can be observed in mice differentiated by a number of markers. Currently, murine DCs are classified into 5 subtypes based on the T cell markers CD4 and CD8, integrin CD11b and interdigitating DC marker CD205. Human DCs are also classified into plasmacytoid and myeloid lineages. In addition, another classification of human DCs is based on pathways of development, since most of results came from studies of DC development in culture from immature DCs or mature DCs (618). Human monocytes/macrophages express most TLRs except for TLR3 (485). Human DCs express different TLRs depending on the DC subsets. Myeloid DCs express TLRs 1, 2, 4 and 5 while plasmacytoid DCs express TLRs 7 and 9 (343). Expression of 67

94 TLR also depends on the maturation status of human DCs. Immature DCs express TLRs 1,2,4 and 5 whereas the expression of these TLRs decreases in the mature DCs; the TLR3 is expressed only in mature DCs (485, 710). In mice, phagocytes express all known TLRs, whereas B cells have several types of TLRs and mast cells express TLRs 2, 4, 6 and 8 (655). The TLRs are expressed on a wide range of tissues. Each tissue expresses at least one TLR in humans (773). However TLRs are also expressed by non-immune cells, e.g. epithelial cells lining the respiratory and intestinal tracts at the basolateral surfaces, possibly to provide the initial proinflammatory signals to attract professional immune cells to the site of infection (481, 601). Thus TLRs can be expressed on either apical or basolateral surfaces of the cells. Furthermore, TLR4 was localized in the Golgi apparatus of the epithelial cells, which is also the final location of LPS for innate immune recognition (299). The invasion of bacteria from apical to lateral sides of the intestine elicits an inflammatory response. Human intestinal epithelial cells also express TLR4 in the Golgi where LPS is delivered and induce an LPS response (113). Renal epithelial cells express TLRs 2 and 4 to prevent invasion of bacteria to the lumen of the renal tubules (744). Corneal epithelial cells express TLR4 leading to inflammatory responses to parasitic infections of the eyes (590). The TLR 2, 4 and TLR9 homologous to those of humans have been found in pigs and guinea pigs (352, 617, 682). The TLR2 and TLR9 were preferentially expressed in MLN and PP of adult swine, more than in the spleen. The TLR2 and TLR-9 expressing 68

95 cells were found both immune cells, such as T cells and B cells and in M cells in pigs. The TLR2 was strongly expressed, not only in the cytoplasm, but also in the apical membrane of the pocket-like M cells. Thus the presences of TLR2 and TLR9 on the MLN and PP enables the host defense to respond to a variety of bacterial cell wall components (682). In neonatal pigs, however, elevated expression of TLR9 was detected only in MLN (617) Role of TLRs in the regulation of adaptive immunity Because monocytes/dcs express at least one kind of TLR and these APCs participate in the signaling of adaptive immunity, TLRs no doubt play crucial roles in both innate and adaptive immunity. Immature DCs in the periphery are activated by microbial components to undergo maturation and increase the expression of TLRs, costimulatory molecules CD80/CD86 and inflammatory cytokines. The mature DCs migrate into the draining LN, and present the antigen to naïve T cells and thus initiate antigen specific responses. The type of T cell responses depends on the type of TLRs activated (540, 712). Activation of TLRs 4 and 9 in DCs induces IL-12 production and Th1 type responses whereas TLR2 engagement leads to Th2 type responses. The activation of TLR7 induces the production of IL-12 in myeloid DCs and IFN-α in plasmacytoid DCs. Different lineages of DCs also determine the lineage of Th responses. Myeloid DCs produce IL-12 in response to LPS whereas plasmacytoid DCs produce IFNα in response to viral antigen and CpG DNA (116, 621). The activation of TLRs induces an antimicrobial activity via macrophages or by the production of antimicrobial peptides. The activation of TLR2 by Mycobacteria 69

96 tuberculosis (MTB) leads to nitric oxide (NO) dependent or independent killing by macrophages (677). Intracellular infection of macrophages lead to apoptosis, triggered by LPS and lipoprotein-tlr2 interaction via FADD and caspase-8 pathways (15). In addition, antimicrobial peptides can be produced by the epithelial surface of the small intestine and in the lungs upon engagement of TLRs 4 and 2, respectively (61) Adaptive immunity CD4 + T cells In the LP, CD4 + T cells represent 60-70% of the T lymphocytes, mainly expressing αβ-tcr (84). Most of the CD4 T cells in the LP express mature or memory phenotypes, CD45RO (humans) and the homing receptor α4β7 integrin (355). The LP CD4 T cells have lower proliferative activity than the CD4 T cells in PBL, but are capable of producing cytokines [IL-2, IL-4, IL-5 (Th2) and IFN-γ (Th1)], that provide help to support B cell antibody production (322). The CD4 T cells in PP contain different populations of T cells that play regulatory roles in IgA antibody production, oral tolerance induction and inflammatory responses. Both Th cell subsets regulate IgA production by the secretion of IL-6 and IL-10 (Th2) and IFN-γ (Th1) (149, 275). Mucosal lymphoid tissues also contain an abundance of CD4 T reg cells which down regulate CD4 Th1 or Th2 responses. These regulatory cells express CD25 +, bind to CTLA-4 and produce regulatory cytokines such as IL-10 and TGF-β1. These T reg cells produce TGF-β1, which is considered as a putative switch for T cells for µ α antibody isotype switching. The T cell clones from the murine intestine mixed 70

97 with noncommitted sigm B cells induced isotype switching to B cells expressing surface IgA (353). Evidence also indicates that CD3 +, CD4 + CD8 - T clones are responsible for this switch of B cells as well as the terminal differentiation of siga+ B cells into IgA producing plasma cells (51). The TGF-β1 induces Cα germ line transcripts and induces mouse spleen B cells to switch to IgA upon LPS stimulation (149). However, TGF-β1 alone only induced 2-5% of B cells to switch to IgA antibody production (149, 400). The TGF-β1 with IL-4 and IL-5 induced up to 15-20% of B cell switching to IgA antibody production (454). The CD4 + T reg but not CD8 cells play a role in tolerance induction through the secretion of TGF-β1 to down regulate the host responses to low doses of oral antigen (353). These suppressor T cells also carry the CD25 + marker and do not proliferate in vitro (565). They downregulate the expression of the costimulatory signals CD80 and CD86 on DCs, thus suppressing the APC activity of DCs (705). It appears that these cells are derived from anergic T cells which have reduced IL-2R expression and the ability to proliferate. Through production of IL-10, these cells develop into T reg cells that suppress the immune responses of other cells (99). It is not known where these cells are located but many cells with suppressor activity are found in the liver (723). In pig LP, large proportions of cells are CD45 + of which 40% are T cells and 40% are monocytes/granulocytes (700, 701). In 6-month-old pigs, there are 4-fold more T cells in the villi than in the crypts, whereas B cell numbers are ten-fold more in the crypts. In newborn pigs, in contrast, the T cells are more numerous in crypts than in the villi, most of which are double negative (DN). During the first week of life, the number 71

98 of CD4 T cells increase rapidly whereas CD8 T cell numbers increase at much slower rates. A significant increase in CD8 T cells occurs by 5-7 weeks of age. In germ free pigs not exposed to any extraneous antigen, the differentiation of CD4 and CD8 subsets is not clear even at 7 weeks of age (566). The CD25 (IL-2R-α) T cells (activated T cells) are scattered throughout the LP, and the numbers are not influenced by age or environment. The pig LP contains a significant population of activated cells (651) CD8 + T cells The CD8 + T cells account for 30-40% of LP T cells with CTL activity (501, 613). Mucosal CTL are crucial for the clearance of enteric and respiratory viruses as well as intracellular parasites. The CD8 T cells in the mouse PP are rare and are the source of CTL in the LP. Studies showed that upon infection of mice with RV or of rats with vaccinia virus, CD8-CTL were found in PP and MLN (414). The IEL CD8 γ/δ T cells in the respiratory and intestinal mucosa play roles in innate immunity, IgA B cell differentiation and tolerance. In humans this γ/δ T cell subset comprises 10-15% of IEL in the small intestine and 20-40% in the colon. The majority of human CD3 IEL are CD45RO +, indicating that a high proportion of cells are memory or recently activated cells (267). In pigs, among IEL, 90% express the T cell marker CD2, 77% are expressed CD8 and only 5% express CD4 (39). The IEL T cells in pigs are recently activated cells, expressing the CD25 marker (39). 72

99 B cells The B cells in PP are organized into follicles and carry IgM + /IgD + surface markers surrounding germinal centers (GC) to form B cell centrocytes and centroblasts. The GC B cells are associated with a network of follicular DC (FDC) and CD4 T cells. Thus both direct B cell signaling and indirect B cell activation through T cell help are present in the follicles for B cell isotype switching to IgA (642). In pig LP, there are only a few IgM + cells at birth and the number of these IgM + cells exceeds IgA + cell number during the first 3 weeks of life. The IgA + B cells become dominate afterwards (95). Pig Ig delta genes have been identified and are transcriptionally active (776). The pig IgD H chains are structurally similar to those of human IgD. The B cells in the LP are highly differentiated into plasma cells, of which 80% are IgA ASC (455). Mucosal B cells produce mainly siga, which is the major mediator of humoral immune responses at the mucosal surface. The siga antibodies neutralize the pathogens or act by molecular exclusion to prevent the pathogens from invading the mucosal surface. The transcytosis of IgA antibody across the intestine removes RV and prevents entry of the virus (572). The siga antibody is resistant to proteases due to the presence of the secretory component, making it most suitable for the effector functions in the mucosal surface. The presence of CD40L/CD40 interaction and TGF-β leads to the large production of IgA antibodies (175). The T cell subset γ/δ IEL also has an impact on IgA B cell differentiation (226). This subset has been shown to produce Th1 and Th2 cytokines as well as TGF-β for IgA class switching (660). 73

100 Biological roles of IgA antibody The roles of siga antibody in the mucosal secretions have been demonstrated for different pathogens Inhibition of adherence From the structure of siga, it is postulated that siga antibodies surround the microbes with a hydrophilic shell of the Fc portion secretory components (Fc-SC) part of the molecule, thus preventing the attachment of pathogens to the mucosal surface. The siga can also inhibit colonization via agglutination as observed for H.infuenzae (350). The IgA and siga antibodies can bind to bacteria and antigen via their carbohydrate chains. The IgA2 can agglutinate E.coli through mannose rich glycan chains (742). Adhesion of S-fimbriated E.coli to human epithelial cells is inhibited by sialyloligosaccharides on s-iga (605) Neutralization of viruses, enzymes and toxins The siga antibodies neutralize virus possibly by inhibiting viral binding to cellular receptors. High concentrations of siga to influenza virus HA inhibit the cellular attachment (28). The polymeric IgA (piga), formed during the transepithelial transport of IgA mediated by the polymeric immunoglobulin receptor (pigr) can also provide virus neutralization activities. The piga antibodies to influenza HA inhibit the internalization or intracellular replication of the virus (28). Transcytosis of IgA RV antibodies inhibits RV replication in mice (572). Even IgA antibodies against the non-neutralizing epitope VP6 inhibits RNA transcription (208). Intraepithelial virus neutralization by IgA 74

101 antibodies was also observed for many other viruses e.g. HIV, measles, Sendai, etc (446, 741, 753). Enzymes such as neuraminidase glycosyltranserase of S.mutants, cholera toxin, heat labile enterotoxin or C.difficile toxin can be effectively neutralized by IgA (335, 425, 637) Inhibition of antigen presentation. It was suggested that the pigr mediates the transport of piga by enterocytes and helps to remove antigens if they form complexes with piga in the LP (527). In addition, siga inhibits the uptake of reovirus through M cells (629). The siga can interact synergically with other antimicrobial factors such as lactoferrin, causing antibody interference with channels of iron uptake, used by many mucosal pathogens (228). The siga can act in concert with lyzozymes and complement to lyse E.coli (8). Other isotypes such as IgM and IgG antibodies, produced at the mucosal surface at lower levels, can contribute to the immune defense. The IgG antibodies can be transferred across the intestine via FcRn receptors, which will be discussed in detail later. The numbers of IgG plasma cells in the LP increase during inflammation Leukocyte trafficking in the mucosal immune system Both mucosal T and B cells, activated in PP, express α4β7 integrin and use this integrin for their entry into the mucosal tissues (20, 358). The counter receptor for this integrin is MadCAM-1, which is expressed on mucosal high endothelial venules (HEVs) (52). In addition, L-selectin contribute to the mucosal homing process (81). Naïve cells in PP express CD45RA + and IgG + B cells express high levels of L-selectin but only 75

102 intermediate levels of α4β7. In contrast, mature T and B cells (CD45RO + memory T cells and sigd - B cells) express both L-selectin high and α4β7 high (203). The expression of adhesion molecules also depend on the route of immunization. Most circulating IgA and IgG ASCs induced by oral or rectal route express α4β7, whereas the ASCs induced by IN route coexpress L-selectin and α4β7 (202, 541). Trafficking patterns of IgG and IgA ASCs are different. The IgA B cells activated in the PP or MLN seed other mucosal sites including intestine, urogenital tract, mammary glands, salivary gland and respiratory tract, whereas cells activated in the lymphoid tissues of the upper aerodigetive tract e.g. oral cavity, oesophagus, bronchi and lungs migrate preferentially to the salivary gland and respiratory tract with less traffic to the intestine (385). The activated IgG B cells, on the contrary, preferentially migrate to nonmucosal lymphoid tissues regardless of whether they are originated from mucosal or peripheral lymph nodes The CC chemokine receptors contribute to the trafficking of leukocytes in the mucosal system. The entry of B cells into PP involves contact with HEV via CXCR5, CCR7 and CXCR4 (507). The CCR6 is expressed on most B cells, CD4 and CD8 T cells and CD11b + DCs (383, 410) and CCR6 is involved in the localization of DCs to PP (386). On T cells, this receptor is mostly found on CD45RO + memory cells (humans) or on CD44 hi and CD62L lo (mice), suggesting the role of this chemokine receptor in trafficking of T memory cells. Mice deficient in CCR6 showed an abnormal expansion of LP T cells, IEL T cells, smaller PP and defects in IgA antibody responses (739). The CCR9 is present on T cells in IEL including the γ/δ subset and intestinal homing T cells and is 76

103 preferentially expressed on small intestinal T cells (387). The CCR9 T cells found in blood express the α4β7 integrins which are the mucosal homing receptors (772). Mice deficient in CCR9 showed an increase in γ/δ TCR T cells in peripheral LNs, but not in the IEL (748). The CCR10 is the receptor for chemokine CCL28, which is expressed by epithelial cells at various mucosal sites. The CCR10 directs IgA producing plasma cells into mucosal sites where the CCL28 expression takes place (385, 397). 1.3 Neonatal immune responses Overview The first challenge to neonatal vaccination is the immature immune system of the newborn. This immaturity is attributed to both the limited source of immune cells and the immature cell function. The newborn has a limited resource of immune cells. A germfree animal has about 10% of ASC and very few T cells in the gut compared to a conventional animal. Because the gut immune system makes up 2/3 of total immune cells, this dictates the limited size of the neonatal immune system (270). The immaturity of immune cell function, exhibited by inadequate phagocyte and lymphocyte functions, leads to poor and delayed immune responses. There is insufficient help for B cell to produce antibody as well as for CTL and memory cells to develop. The other challenge to neonatal vaccination is the Th2 bias in the immune response. It is postulated that the Th2 environment of the mother plays a role in this Th2 bias in infant immune response (270). This Th2 condition is produced to protect the fetus from rejection by the mother s immune system during pregnancy. The activities of NK cells and macrophages which are enhanced by Th1 IFN- γ and CTL are down-regulated 77

104 to avoid destruction of a foreign body the fetus, by the mother (726). This down regulation is achieved mostly via IL-10 which at the same time upregulates Th2 responses (121). The presence of FAS-ligand in the placenta induces CTL to undergo apoptosis (309). The T cell suppressors in the placenta also inhibit the cytotoxic activity of CTL (659). The human leukocyte antigen (HLA) expression in trophoblasts at the placenta is inhibited. In addition, expression of HLA-G exclusively in placenta helps it to escape the attack by NK cells which is HLA-I and II independent (525). The function of T regulatory cells in maternal tolerance to the fetus is also important. The T regulatory cells (CD4 + CD25 + ), found in the human deciduas which are in direct contact with the fetus, play an important role in the suppression of maternal immune responses against the fetus (282). Aluvihare et al (17) showed that depletion of CD25 T cells in mice led to gestation failure. The following sections focus on the properties and functions of some immune cells of the innate and adaptive immunity in neonates in comparison to those of adults in humans and animals Innate immunity Dendritic cells Dendritic cells are central APC, essential to induction of non-specific or specific immune responses. The decrease in antigen responses in neonates may reside in this APC population and others (B cells and monocytes) (336). The function of neonatal DCs as APC is reduced compared to adults and shows a bias against Th1 responses (396). Human DCs from cord blood failed to produce IL-12 upon stimulation with 78

105 lipopolysaccharide (LPS). The kinetics of the upregulation of the DC surface markers such as HLA-DR and CD86, CD25, CD83 are much reduced in cord DCs compared to adult DCs. Cord blood DCs also fail to down-regulate the expression of CCR5 and induce low levels of IFN- γ production. However cord blood DCs exhibit similar rates of TNF-α and IL-10 production upon LPS stimulation compared to adult DCs. Thus human neonatal DCs are programmed to favor the induction of Th2 immune responses (via reduced IFN-γ and IL-12 production). This bias contributes to the overall Th2 bias of the neonatal immune system in humans, which will be discussed in later sections. A number of studies of mice have led to different conclusions from those of humans about the characterization and function of neonatal DCs compared to adult DCs. The murine neonatal DC subsets differ from adult DC subsets (653). In mice, neonatal DCs exhibit higher a frequency of CD11 c low marker compared to adult DCs which is mainly CD11 chigh (653). In regard to CD4 and CD8 markers, at birth, almost all DCs are CD4 - CD8 - (DN) and remain so until the 2 nd week of life. Neonatal DN DCs represent a transitional state, which first gives rise to CD8α + and later generate CD4 + DCs. The CD4 + DC population continues to increase and becomes the major subset in the adult spleen. Fifty per cent of adult murine DCs express CD4 +, 25-30% express CD8α + and the rest is DN. The CD4 + DC population in the adult spleen also express the myeloid markers e.g. CD11b, F4/80 and 33D1, only low levels of CD1d and CD205, which are associated with CD8α + CD4 - DCs. In comparison, neonatal DCs, mainly DN, express CD205 and CD1d of the CD8α + but express poorly CD11b and 33D1. The CD205 marker which is rich in carbohydrate recognition domains allows the binding and recognition of bacteria by DCs. The CD1d marker facilitates the recognition of glycolipid antigens and represents the link 79

106 between DCs and NK T cells. However in spite of these subset differences, the murine neonatal DCs are fully competent in their innate immune function. The neonatal DCs produce IL-12 more efficiently than adult cells. The major producers of IL-12 are DN DCs in neonatal mice compared to CD8α + in adult mice. When stimulated through TLR9 and TLR3, neonatal DCs are more efficient in the production of IFN-α and IFN-γ for antiviral activity and NO-mediated anti-bacterial activity. The murine neonatal DCs also exhibit up-regulation of co-stimulatory signals including MHC class II, CD40, CD86 (a costimulator for T lymphocyte activation, expressed in B cells, monocytes, DCs and some T cells) and CD25 (subunits of IL-2 receptor), which will be discussed subsequently (396) Monocytes Functional immaturity of neonatal immune responses could also reside in the reduced co-stimulation signals provided by monocytes to T cells during antigen uptake and presentation (336). These costimulation signals in monocytes are MHC class II molecules, CD40/CD40L, CD80/CD86 and CD28/CTLA-4. Fewer cord blood monocytes express MHC class II than adult cells. However, there is an increase in the percentage of MHC class II monocytes during gestation, although still lower than the levels in adults. The numbers of CD40 + and CD86 + monocytes in the fetus and neonate were comparable to adults, yet the expression of CD80 is absent on fetal/neonatal cells (188, 189, 336). Monocytes are also involved in the first line of defense against microorganisms. Receptors on the monocyte surface involved in monocyte responses to microbial products are CD11b, CD35 (type 1 complement receptor), CD14 and TLR4. The expression of 80

107 CD14 increases with gestational age: the CD11b and CD35 are also coexpressed with CD14, although the intensity is not stable (336, 338). The TLR-4 expression in murine lung tissues also increases extensively after birth (271) Natural killer (NK) cells There are contradictory reports about the maturity of neonatal NK cells. In a study of cells from umbilical cord, the NK cytotoxicity in neonatal umbilical cord lymphocytes was found to be similar to that of maternal peripheral blood cells and correlated with a similar proportion of CD56 NK and CD8 CD56 LAK cells (185). In contrast, a study of preterm and term infants showed significant impairment of NK cytotoxicity activity compared to that of adult NK cells (232). It is not clear why there is such discrepancy in the neonatal NK activity between different studies Adaptive immunity T effector and memory cell responses There are significant differences between neonatal and adult T cell phenotypes, suggesting a reduction in the T cell responses of neonates (232). In humans and pigs, newborns exhibit a higher percentage of CD4 but a lower percentage of CD8 T cells resulting in higher CD4/CD8 ratios in blood. Neonates also exhibit lower numbers of CD4 and CD8 IFN-γ producing cells. In human neonates, the CD4/CD8 ratio decreases with age, suggesting a gradual maturation of cytotoxic responses. In humans, all neonatal T cells express high levels of CD38 (activation marker for B and T cells), similar to most thymocytes, indicating immature transition from thymocytes to mature adult T cells 81

108 (545). The CD45RA marker is expressed in more than 90% of neonatal T cells indicating the unprimed state of the cells, whereas only 40-60% of adult T cells express this marker (148). A higher percentage of CD4 + /CD45RA + (naïve T cell marker) but a lower percentage of CD4 + /CD45RO + (memory T cell marker) were found in newborns than in children and adult cells, indicating the naïve phenotype of T cells (232). However, almost all newborn CD4 T cells express the dim isoforms of CD45 markers (~97%), whereas 28.4% and 53% of adult T cells express CD45RA + bright and CD45RO + bright markers, respectively (276). Because cells with different CD45 isoforms reflect differences in T cell receptor signaling, these findings suggest differences between neonatal and adult T cell function (500). The CD40 ligand, whose expression is restricted to the activation status of CD4 T cells, is absent or reduced significantly on cord blood T cells compared to adult T cells (182). This reduction correlates with the reduced antibody production in newborns and infants. Stimulation of human T cells with mitogens, alloantigens, mitogenic antibodies and superantigens also demonstrated the differences between human adult and neonatal T cell functions (545). The immune activities of neonatal umbilical cord are lower than of maternal peripheral blood cells with a lower degree of polyclonality and repertoire diversity, and lower T cell responses to phytohaemagglutinin (PHA) (185). Yet other studies showed that the response of T cells to PHA is similar between newborn and adult cells, whereas significantly lower responses to anti-cd3 or anti-cd2 antibodies occurs (418, 762). The difference in the T cell stimulation by mitogens between the two populations, cord blood vs. newborn cells, was not clear, but could be due to the difference in proportion of T cells in each population. The IL-2 receptor expression, IL-2 82

109 synthesis and proliferation are also reduced in cord blood CD4 T cells (238). Neonatal T cells also respond strongly to IL-4 proliferation signals, whereas adult T cells remain unresponsive which is another example of Th2 bias in neonatal immune responses (183). Thus many studies demonstrated a deficiency in neonatal T cell functions, whereas others documented that neonatal T cells, although naïve, can induce responses under appropriate stimuli. When stimulated with anti-cd3 antibody, neonatal T cells express similar levels of CD40 ligand as adult cells (545). Although the level of CD40 ligand expression depends on the concentration of the antigens, adult T cells required much lower phorbol myristate acetate (PMA) concentrations for CD40 expression compared to neonatal T cells. In response to TCR, neonatal cord blood T cells can be stimulated to produce cytokines of both Th1 and Th2 types (129). The neonatal T cell population, however, is composed of high numbers of self-reactive cells, which indicates the incomplete T cell tolerance process in neonates (3). For alloantigens presented on APCs, cord blood T cells appear unresponsive, even upon IL-2 stimulation, suggesting the anergy state of the cells and the activity of CD8 + suppressor cells (556). In murine neonates, it has been shown that the Th1 function is impaired (9). Many studies demonstrated the Th2 bias of neonatal immune responses, with different antigens, live or attenuated viruses and aluminum salt adjuvants, (75, 76, 375). The Th2 biased responses in neonates are not related to the antigen dose, the carrier and do not require the continual presence of the thymus or spleen to replenish the Th2 cell pool. Splenectomized neonates showed no difference in Th2 bias memory responses in LN, which indicates that the spleen is not required for Th2 dominant memory responses in neonates, and thus these biased responses are more likely controlled by events in the 83

110 effector development (9). The major differences between neonates and adults reside in IL-4 production and this difference persists until 5 weeks after immunization with keyhole limpet hemocyanin (KLH). At the same time, the level of INF-γ does not differ considerably between neonatal and adult LNs and spleens 1, 2 or 5 week after KLH immunization. This Th2 memory response induced by KLH immunization of neonates continues to develop when the same neonates were immunized with KLH as adults. Thus the increase in IL-4 production during primary immunization skews the development of memory effectors to a Th2 lineage and impaired Th1 function. It has been postulated that failure to develop Th1 memory effector function results in failure to develop Th1 effector cells, leading to the Th2 bias (9). In young pigs, T cells exhibit reduced responses from birth up to 4 weeks, as indicated by low proliferative responses of blood mononuclear cells when exposed to a low antigen dose of a T-cell dependent antigen. The responses were not enhanced by the addition of IL-2 (606).The T lymphocytes from newborn pigs are immature in providing cognate help for B cells in antibody production. The IgG and IgM antibody production by the newborn B cells was enhanced when cocultured with adult T cells, but not with newborn T cells (652). The newborn porcine T cells also show suppressive activity during the suckling period, this activity; however, decreases with in adult pigs Neonatal B cell repertoire The poor and restricted antibody responses of neonates can be partially explained by the immaturity of B cells, which possess restricted repertoires. The repertoire restriction was demonstrated in the biased expression of proximal V gene 84

111 families coding for the B cell receptor, the absence of N region diversity and the low rate of mutations in V genes in humans and mice (539). The diversification of neonatal B cell repertoires after immunization is mediated by activation of recombinase-activating genes (RAG), which induce V(D)J rearrangement and light chain replacement (73). Upon influenza infection, the human neonatal B cells expressed mainly one VH gene family in the first week of life, and diversification of the VH gene only started from the second week with preferred usage of only a few VH genes (73). However, a study by Weitkamp et al (728) suggested that adult B cells specific to RV also exhibit bias in the use of VH gene segments, similar to cells from infants aged 2-11 months. In particular, infant and adult RV specific B cells from blood showed frequent usage of V H 4 and V H 1-46, a uniform bias toward the D3 and J H 4 gene segments and similar distributions of Vκ, Jκ and Jλ families. Thus the poor antibody responses to viruses in infants can not be explained only by the residual fetal bias of the B cell repertoires. In addition, neonatal swine B cells are fully functional at birth. The repertoires of oligoclonal IgA and IgM antibodies in a non-inductive site of the mucosal immune system (parotid gland) become polyclonal in Gn pigs, suggesting that the expansion of the fetal and neonatal B cells that undergo isotype switching is not driven by environmental antigens (106). Neonatal isotype switch B cells can differentiate into ASC in the presence of intestinal commensal bacteria in a T-cell dependent manner or via direct stimulation by bacterial products in a T-cell independent manner (108). Upon infection with PRRSV, Gn pigs showed dramatic increases in serum IgM, IgG and IgA 85

112 antibody responses which were not dependent on bacterial colonization (407). The serum antibody levels of PRRSV infected Gn pigs were 42-94% of adult levels depending on isotypes, similar to those of bacteria colonized pigs. The pigs have B cells that produce antibodies even 2 days before birth (60). The generation of Ig-producing cells in PBL from suckling pigs increased with age, and reached about half the adult mean numbers at six weeks of age (652) Activation of neonatal B cells- in vitro system The activation of B cells involves the interaction of many types of cells, cytokines, growth factors, etc. Replication of this process in vitro allows assessment of individual components in this interaction. The lack of adequate numbers of effector cells and accessory cells and the antigen naivety of B cells make it difficult to activate neonatal B cells. Stimulation of neonatal B cells with antigen is even more difficult due to the low number of antigen specific B cells in the population (a few cells per thousand). Fayette et al (206) successfully established an in vitro system for the differentiation of human naïve B cells into plasma cells. In this system, the role of DCs was strongly emphasized. The B cell primary responses take place in the T cell-rich area of the GC of secondary lymphoid tissues in which DCs also reside. Upon activation, 12% of naïve B cells differentiated into plasma cells and this proportion increased in the presence of IL-2 and IL-10 without CD40 signaling and DCs. Addition of DCs to this system enhanced the plasma cell differentiation to 57% B cells, secreting IgM, and IgA and IgG antibodies (206). It was shown that DCs act in synergy with IL-2 in the early stage of differentiation which is CD40 dependent, whereas in the later stage, IL-2 and IL-10 act together to 86

113 promote plasma cell differentiation in a CD40 independent manner. Therefore a two-step culture was developed in which B cells were cultured with DCs, CD40-L transfected cells and IL-2 in the first step for 7 days. After that the B cells were harvested and seeded with DCs in the presence of IL-2 and IL-10 for 4 days (206). The IL-12 was also found to contribute to DC-induced differentiation of naïve B cells, demonstrated in in vitro systems with cells from different animal origins. The IL- 12, produced by CD40-activated DCs was essential for promoting human plasma cell differentiation and IgM antibody secretion (181). Similarly, B, T cells and DCs of porcine origin cocultured in the presence of IL-12 induced plasma cell differentiation, whereas the formation of memory B cells was IL-12 independent (163). In addition, interaction of T cells with the CD11c + CD4 + CD3 - DC subset was crucial in providing signals for B cell differentiation into memory cells. The bovine B cells and DCs can be cocultured in the presence of a CD40L transfected cell line to induce IgG1 antibody responses (40) Although antibody secretion can be measured in these systems, it is not clear in any of these studies whether antigen-specific antibody production can readily be detected due to the low percentage of the specific antigen responsive cells in the antibody producing pool. Measurement of antigen-specific antibodies from naïve B cells was accomplished by Colino et al. in which the mouse DCs pulsed with S.pneumonia antigen in vitro were introduced into recipient mice to induce antigen specific IgM and IgG antibodies 14 days after cell transfer (150). The reason for the difficulty in stimulating naïve B cell differentiation into antigen specific plasma cells was proposed by Tangye et al (667). In regard to naïve and 87

114 memory B cells, the human memory B cells entered the first cell division 20-30h earlier than naïve cells in response to stimulation with CD40 ligand alone or with IL-10, whereas subsequent cell division time was similar in both groups. In addition, increased proliferation of memory B cells can be mediated by IL-2, whereas this cytokine did not improve proliferation of naive cells. Naïve B cells remained undivided for 72h, and only a small portion (up to 20%) entered cell division after this period Mucosal immune system in neonatal pigs In pigs the mucosal immune system doesn t develop before birth, whereas the systemic immunity has already developed. However, two days before birth, most organs in pigs contain some ASC, suggesting the immunocompetence of the pig immune system (60). The T cells represent 70% of lymphocyte population in MLN and IEL of neonatal pigs (733). The IEL in neonatal pigs do not proliferate well in response to Con A, yet they exhibit a high proliferative response to exogenous IL-2, in relation to the change in T cell phenotype from CD2 - CD4 - CD8 - cells to CD2 + CD4 - CD8 - cells as the age of pigs increases. In contrast to IEL T cells, T cells in MLN proliferate in response to Con A, but become unresponsive to IL-2, indicating the different stages of activation and responsiveness in neonatal pigs. Mucosal T cells in neonatal pigs are more susceptible to apoptosis, which is one of the regulatory mechanism to eliminate the excess mucosal effector cells produced by the active thymus in the young (38). However unlike adults, the involvement of T suppressors as a regulatory mechanisms doesn t exist in neonates because by 5 weeks the T suppressive activity declines (652). 88

115 In conventionally raised neonatal pigs, PP follicles and T cell areas expand rapidly within 12 days after birth to reflect the structures in the mature animal (38). The T cells that enter the intestinal villi mainly express the CD2 marker but not CD4 and CD8 markers. Expression of MHC class II antigen in the intestinal villi increases with age. The CD4 T cells enter the intestine by 2-4 weeks, at the same position as in the intestine of adult pigs. The movement of CD8 T cells takes place following the CD4 movement to the epithelium and LP of the intestinal villi. The normal distribution of T cells as in adult animals is as follows: T cells are confined in the villi, not around the crypts, CD8 T cells reside immediately bellow the epithelium of the villi, whereas CD4 cells are located deep in the villi (38) Gnotobiotic pig model for human enteric viruses and mucosal immunity The Gn neonatal pig model represents a good model to study the mucosal immune responses to enteric viruses, in particular RV. The pig gastrointestinal physiology is comparable with that of humans (38). The Gn pigs are susceptible to infection and diseases induced by several HRV strains for up to 8 weeks of age (589). The histopathologic lesions in the small intestine of pigs after HRV infection are similar to those in humans (715). Because of the impervious nature of the sow placenta, the Gn pigs, derived by hysterectomy, are devoid of MatAb, but are immunocompetent, allowing the study of the true neonatal immune responses. In addition MatAb, cytokines and colonizing bacteria can be added to study the effects of these components on neonatal immune responses (32, 522, 589). Similarly, Gn pigs infected with PRRSV developed bronchial and submandibular lymph nodes that were 5-10 times larger than those in 89

116 E.coli colonized animals (407). Typical lesions, infiltration of inflammatory cells, deposition of antibody to the basement membrane and vascular endothelium of kidney were observed in Gn pigs similar to conventional pigs infected with PRRSV. Thus Gn pigs were demonstrated to serve as a useful model to study virus-induced immunopathology. Despite these advantages, there are disadvantages in using the Gn pig model. Under the Gn conditions, little development of the mucosal immune system is observed, which indicates a role for the normal intestinal flora or exposure to common enteric viruses (enteroviruses, RV, etc) to mature the immune system (516). In fetuses and newborn pigs as in human infants, limited Vh and Dh segments are used, leading to a limited B-cell repertoire (654). This limitation persists longer for the Gn pigs. In comparison, there is an increase in V segment usage resulting in increasing B cell repertoires in conventional pigs and in germfree pigs colonized with intestinal flora (107). In the Gn pigs, the T cell population is also limited. The T cell population of 49- day old the Gn pigs is similar to that of 5-day old conventional pigs (566). However, all the above mentioned studies of Gn pigs are limited to pigs that were not exposed to any extraneous antigen. When stimulated with RV antigen (live virus infection or vaccine regimens), the immunocompetent neonatal Gn pigs developed strong intestinal and systemic T and B cell effector and memory responses (589, 716, 770). Similarly, Gn pigs infected with PRRSV showed elevated serum antibody responses, independent of bacterial colonization and hyperplastic lymph nodes of larger size than 90

117 those of bacterial colonized pigs (407). Thus viral antigen stimulation can also substituted for the effects of microflora colonization in promoting maturation/development of the mucosal immune system in neonatal Gn pigs. 1.4 Maternal interference with neonatal active immune responses: sources and mechanisms of interference Sources of maternal antibodies (MatAb) Depending on the species, MatAb transferred to neonates can be derived from different sources, as summarized in Table 1.3 (259). In mammals, MatAb are transferred across the placenta prenatally or supplied postnatally as colostrum and milk or by both routes. In particular, the hemendothelial and hemochorial structures of the placenta of humans, primates and rodents allow passive transfer of immunoglobulins mainly IgG across the placenta to the fetus (Table 1.3). In guinea pigs and rabbits, MatAb are transferred to fetuses via yolk sacs whereas postnatal transfer of MatAb is not available in these species. In ruminants and other animals such as pigs, horses and donkeys, on the contrary, no transfer of MatAb occurs across the placenta and therefore the newborns acquire passive immunity entirely from colostrum and milk. In fish, reptiles and birds, MatAb are deposited in eggs to be transferred to the offspring via yolk sac. The persistence of MatAb in neonates depends on the species; from 10 days in fish to 9 months in humans, as a function of body size and catabolic rates (Tables 1.3 and 1.4). In addition, the half-life (t 1/2 ) of MatAb specific to a particular pathogen might vary 91

118 according to the pathogen, the specificity and the amount of antibodies. For some viruses, specific antibodies from immunized cows transferred to neonatal calves have longer halflives than those from unimmunized animals (227) Maternal antibodies from milk In humans and most animals except cattle, IgA antibodies are the major immunoglobulin in colostrum and milk of these species. However in pigs and horses, IgG antibody is the major isotype in colostrum, but later changes to the predominance of IgA antibodies in milk after the transition from colostrum to milk. In cattle, IgG1 antibodies persist as the major isotype in both colostrum and milk and they are selectively transported from serum into the colostrums and milk. These antibodies play important roles in the early immune defense of neonates. The secretory IgA and IgM antibodies present on the mucosal surface of the intestine mask the surface receptors on the enterocytes and prevent adherence and entry of enteric pathogens. These antibodies can also neutralize infectious virus and bacterial enterotoxins. They activate the complement pathway and exhibit bactericidal action. The presence of siga antibodies was shown to prevent the entry of viruses such as RV to the cytosol (445). Antibodies can be either transferred from blood to mammary secretions or synthesized by local ASC in the mammary glands. The IgG is transudated to the mammary secretions via the Fcγ receptors on the surface of epithelial cells, similar to the 92

119 neonatal receptor (FcRn) for epithelial cell transport of IgG through the placenta. The structure and the role of FcRn in the transfer of maternal IgG antibodies to neonates will be discussed subsequently. The siga antibodies secreted in milk play an important role in passive immunity of infants. The IgA dimer-j chain-producing lymphocytes migrate to the mammary gland from the intestinal lymphoid tissues. Secretion of IgA antibodies into milk occurs via the pigr. These siga antibodies are specific to many pathogens that the mothers have been exposed to in their lives, thus they provide protection to infants. Milk also has prolonged effects even after breast feeding is terminated, as confirmed by higher numbers of hypersensitivity reactions and respiratory infections in non breast-fed infants compared to breast-fed infants beyond the period of breast-feeding (103, 627). Antibodies, e.g. antiidiotypic antibodies from milk can also enhance infant responses to vaccines; the detailed mechanisms will be discussed in a later section. The following sections explain the mechanisms of transfer of antibodies (IgA) into mammary secretions Transfer of siga antibodies into mucosal secretion Structure of the pigr The polymeric Ig receptor (pigr) is an important receptor for the transfer of IgA and IgM antibodies into mucosal secretions. The pigr is a glycosylated membrane protein, a homolog of the Ig superfamily. The extracellular portion of the molecule consists of five homologous domains, resembling the V domain of the Ig superfamily (542) (Figure 1.2). Interaction between the pigr and IgM is high with Ka=6x10-8 to 93

120 2x10-9 M, whereas the IgA binds to the pigr via both covalent and noncovalent bonds (253, 464). Interaction between this receptor and IgA occurs via the extracellular Ig domains 1 and 5 of the pigr and Cα2 and Cα3 domains of both IgA molecules (234, 542) Functions of pigr The pigr performs several important functions including protection of IgA from proteolysis by adding the secretory components (SC) to mediate immune exclusion by siga at the mucosal surface. Secretory component can be released as the free form into the lumen and serves as a scavenger to prevent the interaction between the pathogen and intestinal epithelial cells. Yet some pathogens can utilize pigr to invade the mucosal defense, such as Streptococcus pneumoniae (89). However the inefficiency of the apical to basolateral movement of pigr and the cleavage of pigr at the luminal surface prevent the recycling of this receptor, and thus reduces the chance for the pathogens to use it for internalization (533). In mice, milk siga antibodies are produced locally in the mammary gland. During the initial stage of lactation, milk IgA antibody may originate from blood, although the derivation and contribution of milk IgA antibody derived from blood remains controversial (89). In sows, 70% of colostrum/milk IgG and >90% of IgM and IgA antibodies were synthesized in the mammary gland (78). In addition, IgA ASCs that arise in the intestinal lymphoid tissue after oral immunization with transmissible gastroenteritis virus (TGEV) were the first description of trafficking of IgA ASC to populate the mammary gland (70, 582, 695). 94

121 The levels of pigr in the mammary gland vary according to the stages of pregnancy and parturition. In the mammary gland, the pigr is expressed on the secretory epithelial cells surrounding the ducts and alveoli. In mice, the pigr levels increase gradually during mid-pregnancy, peak at parturition then drop to minimal levels around post parturition day 7 (691). Then the pigr level increases again and maintains a high level even at the end of lactation period, corresponding to the increased level of siga in milk. Similarly the number of ASC in the mammary gland also differs according to the pregnancy and lactation stage (691). In mice, the IgA ASC number is low in the mammary gland during late pregnancy, increases around parturition and increases substantially during mid and late lactation. When labeling lymphocytes from the MLN and inguinal (ILN), it was observed that lymphocytes in the mammary gland were derived from both the MLN and ILN (593). In particular, T cells and large B cells (plasma cells) migrated from the MLN whereas small and medium B cells (memory cells) migrated from the ILN. The ASC from the MLN also traveled to the mammary gland via interaction with MadCAM-1, which together with the pigr was expressed in early-mid pregnancy. The levels of MadCAM-1 declined during lactation whereas the levels of pigr and the IgA-ASC numbers continued to rise until the end of lactation (691). However, MadCAM-1 was not considered as the rate limiting step for the B cell traffic from blood to the mammary gland since the rise in the MadCAM-1 level was out of phase with numbers of IgA ASC. In contrast, the rise of the pigr level was sufficient to explain the increase siga levels in milk. Of interest, expression of the pigr is regulated by lactogenic hormones and IFN-γ (555). 95

122 Transfer of IgG across the placenta and from milk to neonates The only antibody which is able to across the placenta of mammals is IgG, which mediated by the neonatal Fc receptor, called FcRn. The following section describes the structure of this receptor and the mode of transfer of the IgG antibody across the placenta using this receptor. This receptor is also postulated to play role in the transfer of milk IgG antibodies to neonatal ruminants and in the maintenance of the passive IgG antibody levels in the gut and in serum for passive protection. In ruminants, pigs and horses, this is the only method of acquiring passive immunity in these animals due to the impermeability of their placenta. Of note, transport of IgG differs from that of siga antibodies as the FcRn is recycled whereas the pigr is degraded after transcytosis Structure of the FcRn The FcRn was first isolated from rats in 1989 (632) but its function in maintaining the homeostatic status of serum IgG antibody was postulated in 1970 by Brambell (83, 179). The FcRn is encoded for by a gene lying outside of the MHC region, splitted into two exons for the cytoplasmic and transmembrane domains, respectively (631). The FcRn, an MHC class I homolog, is a heterodimer of β2 microglobulin and a larger subunit α (45-53 kda) with 3 extracellular domains (631) (Figure 1.2). The β2m subunit, similar to one found in MHC class I molecules, mediates contact with IgG. The larger α subunit is glycosylated at 4 different sites and alteration of these glycosylation sites lowers the binding of FcRn to IgG. The FcRn binds to IgG at a ratio of 2:1; that is FcRn is required to form a dimer to bind to IgG with high affinity. The FcRn 96

123 extracellular and transmembrane domains share homology with MHC class I respective regions, whereas the cytoplasmic domain differs, corresponding to differences between functions of MHC class I and FcRn molecules. Consistent with the conserved role of FcRn across different animal species, the amino acid residues in the binding region of FcRn with IgG antibodies are also highly conserved between different species. There is between 50-60% homology between humans and rats in the α and β chain domains of the FcRn (631). Thus most functions of FcRn derived from studies in rodents are implicated for humans. The FcRn interacts with IgG antibody via the Fc residues at the CH2-CH3 interface. This interaction is ph dependent: a ph of is the maximum condition for binding. This is due to the presence of a single highly conserved histidine residue at position 433 (or 435, depending on the studies). Other residues at the CH2-CH3 interface are also involved in the interaction with the Fc region of IgG antibody, which explains the variation in affinity of FcRn with different IgG isotypes Functions of FcRn (i). The FcRn has been suggested to play a major role in transfer of IgG antibody to the fetus via the placenta (633). The FcRn is also found on the epithelial layer of the placenta in humans. The FcRn found in the syncytiotrophoblast cells tends to be expressed only 2-3 weeks before parturition and drops considerably after birth (634). In humans, the concentration of maternal IgG concentrations in fetal blood increase from early in the second trimester and peak at term; most antibodies are transferred during the third trimester. These findings coincided with the observation that pre-term infants have lower 97

124 concentrations of anti-rubella IgG antibodies compared to those of full-term infants (180). An in vitro placental model was used to analyze the materno-fetal transfer using recombinant IgG1 antibody with a mutation at His 435 site (H435A). The mutated form of antibody showed reduction in the amount transferred across the placenta compared to the wild-type antibody indicating that binding of an IgG molecule to FcRn is a prerequisite for transport across the placenta (214). However the mechanism of IgG transport across the endothelium of fetal blood vessels is not understood (630). (ii).role of FcRn in general homeostasis of serum levels of IgG in different species. Brambell suggested the existence of a protection receptor for serum IgG (FcRp), which explained why serum IgG can survive longer than other isotypes in blood (82, 339). Brambel proposed that serum IgG is bound to FcRp in pinocytic vacuoles for its protection from degradation and redirection of the antibody into the circulation. When the FcRp is saturated, any excess IgG is directed to lysosomes for degradation. Later FcRn was identified in the intestinal epithelium of neonate mice for transfer of IgG from ingested milk to serum, which was later also found in other species including humans, pigs, cows and sheep (242, 337). The proposed FcRp has a similar role and structure to FcRn. The roles of FcRn receptor in IgG homeostasis and transfer of passive immunity to neonates were demonstrated in various species, including humans, primates, rodents, marsupials, ruminants etc (560). The expression of FcRn can be temporal or life long, depending on the species. In rodents, the FcRn is expressed at high levels in intestinal epithelial cells during suckling to mediate the absorption of IgG antibodies from milk. Expression decreases with time and is completely lost at weaning (56, 241). Thus in 98

125 mice, FcRn is not expressed throughout life long. In humans, on the contrary, the intestinal FcRn is expressed in both neonates and adults (612). In human fetal intestinal cells (18-22 weeks of age), similar to the adult intestine, FcRn is found in the apical region of both villous and crypt enterocytes and in stomach and colon. The expression of FcRn was enhanced in the ileum of children and adolescents (612). In fetal tissues, it was also suggested, but not proven that enhanced expression of the FcRn occurred in the fetal ileum with reduced expression in the fetal stomach. Using human malignant intestinal epithelial cell lines, FcRn was shown to mediate IgG transport in both directions. However, uni-directional transfer (from basolateral to apical surface) of IgG antibody, which was demonstrated in neonatal rodents, was suggested to human FcRn. The receptor can also be found on the bronchial epithelial cells of adult humans, non-human primates and mice in which FcRn gives rise to a steady and dynamic distribution of IgG across the respiratory epithelium (643). The FcRn has been also found in human skin, muscle and liver (178). The receptor was also found in the intestine of the brushtail possum (732), nonhuman primates (342, 643) and cows (342). The FcRn transcripts were also found in adult possums, suggesting a role in regulation of serum IgG in this species. In possums, FcRn α and β2m expression is biphasic; high level occur shortly after the birth and after 110 th days in the mother s pouch, whereas low or undetectable levels were observed from birth until the 110th day (732). The FcRn also is expressed in the bovine intestine (342). Newborn lamb FcRn is expressed by intestinal crypt cells mainly at the apical surface (444). However, FcRn was not detectable in lamb duodenal enterocytes. Thus the initial uptake of IgG antibody from 99

126 colostrum in lamb during the first 18-24h is not receptor mediated and may be due to a non-specific mechanism(444). However in calves, the bovine FcRn was still hypothesized to play an important role in colostral IgG transport because different haplotypes of the bovine FcRn genes (FCGRT) are associated with differences in serum IgG levels in newborn calves (392). The haplotype 3 of FCGRT in dams was associated with an increased likelihood of failure of passive transfer of antibody to calves; the haplotype 2 was less likely to have high level of passive transfer. The expression of FcRn has not been demonstrated in the porcine intestine. (iii). The FcRn plays a role in transport of IgG antibody during colostrum formation (443). In cattle and sheep, the FcRn expressed by epithelial cells of the mammary gland selectively binds and transports IgG1 into the mammary gland s lumen, contributing to mucosal immunoprotection (341, 342, 444). Bovine FcRn expression was found in different tissues including the intestine and the mammary gland, suggesting its involvement in IgG transcytosis (342). In sheep, FcRn was localized in the epithelial cells of the acini and ducts in the mammary gland around parturition (443, 444). The presence of FcRn in the mammary gland was demonstrated in pigs (108, 603), mice (138), brushtail possums (6), lambs (443) and cows (342). Expression of the receptor in the sheep s mammary gland is time dependent. Before parturition, the receptor is expressed homogeneously in the cytoplasm of epithelial cells. After lambing, more FcRn molecules move to the apical side of the cells (443). The regulation of FcRn expression by hormones has not been extensively studied. Using the rat alveolar epithelial cell lines, Kim et al showed that dexamethasone caused a decreased mrna level of rat lung FcRn α -subunit and the apical to basal (but not basal 100

127 to apical) flux of IgG antibody (362). A study of rat intestine showed that spermidine and cortisone decreased the neonatal Fc receptor expression in the intestine (111). Dihydrotestosterone decreased FcγRIIB2, but not FcRn in patients with autoimmune Grave s disease (198). (iv). Function of FcRn in neonatal immunity. The binding of IgG to the FcRn receptor could have both positive and negative consequences (316). The receptor allows the passage of IgG antibody from colostrum/milk into the circulation of the neonate, providing systemic passive immunity to animals especially ruminants, horses and pigs. The reverse transfer of IgG from serum to the intestines of calves accounts for clearance of up to 70% of passively acquired antibodies (59). This transport is mediated by FcRn on crypt cells of the intestine. If the level of serum IgG antibody is sufficiently high, at least short term protection can be achieved at the mucosal surface (59, 492). Because the absorption of colostrum/milk in rodents can last up to 19 days, the FcRn is expected to a play role in absorption of milk to the neonatal circulation of mice (680). However the presence of passive antibodies in the intestine could interfere with vaccine responses; more detailed discussion about this topic is in the following section. (v). Function of FcRn in active immunity. A new function for FcRn has been proposed recently, that FcRn mediates transport of IgG antibody across the intestine in active immunity (562). In this model, IgG antibody is endocytosed at the basolateral side of the cells, transported by FcRn to the apical surface and released into the intestinal lumen. In the presence of antigen in the 101

128 lumen, antigen-antibody complexes are formed. Via their interaction with FcRn or via internalization by fluid-phase endocytosis, the complexes are transcytosed back to the basolateral surface and delivered to the LP for immune activation or tolerance. The model is supported by a study in which mouse Fc-eythropoetin chimeric molecules can be shuttled by FcRn across the neonatal mouse intestinal epithelium or the adult mouse respiratory lining and stimulate production of red blood cell progenitors (643) Mechanisms of FcRn mediated IgG homeostasis The endothelial cells, which line the blood vessels may be responsible for the regulation and maintenance of a steady state of serum IgG in mice and humans (242). In humans, expression of FcRn was found in endothelial cells of various tissues such as in intestine (612), liver (673), kidney (280) and renal proximal epithelial cells(372). Thus cells throughout the body are involved in IgG catabolism. There are two proposed models for the mechanism of maintaining serum IgG homeostasis but neither is fully proven (242). In both models, IgG antibodies are internalized nonspecifically into endosomes. The low ph of the endosome (ph=6) allows the tight binding of IgG antibody and FcRn. Once the receptor is saturated, any excess unbound IgG antibodies would be directed to the lysosome. The release of IgG antibodies is mediated by the fusion of the endosome with the plasma membrane. The two models differ in the movement of the endosome inside the cell. In one model, the IgG antibodies in the endosome do not traverse the cell, instead they cycle back to the membrane from which they were pinocytosed (called the circular recycling model). In the second model (trancellular recycling model), IgG molecules traverse the cells so that serum IgG 102

129 antibodies can be transported to the interstitial space and vice versa. The exact events that might occur are not yet known, but the second model is more likely to be the case as endothelial cells exhibit bi-directional transport of vesicles and endosomes can be found in these cells (242). These two models of IgG transversing the cells were derived from studies using a wild type human IgG and a mutant type (at one of the Histidine residues) that can t bind to FcRn in the polarized cell lines (713). These models also suggest a mechanism of regulation of IgG antibody levels. The IgG antibody levels might depend considerably on FcRn receptor levels which could be up or down regulated by cytokines (suggested but not proven). It was also shown that phosphorylation of FcRn at the serine residue 313 regulates the transcytosis of the receptor across the rat inner medullary collecting duct cells. Mutation at this site reverse transcytosis from apical to basolateral surface of the cells (447). The stability of the cell cytoskeleton and actin network also regulates the transcytosis of FcRn as drugs that destabilize the network result in blocking of FcRn trancytosis in one or both directions (187, 650). The mechanism of IgG transport across mucosal surfaces is unknown. Transudation of IgG from the serum could be one possible mechanism which would result in the same proportion of IgG isotypes in the secretion and in the serum. As mentioned previously, FcRn is found in the apical region of both villous and crypt enterocytes and in stomach and colon in humans. The FcRn has not been identified in porcine intestine. However evidence has shown that IgG is locally produced in the intestine and transported to the lumen mediated by a receptor, rather than simply by transudation. It was shown that the pattern of IgG subclasses in serum and mucosal 103

130 secretions differed; the IgG1 is more prevalent in mucosal secretions than in serum (53). In peridontitis individuals, the concentration of IgG4 antibodies in gingival crevicular fluid was higher than that in the serum (53) Cells in passive immunity Origin of plasma cells in the mammary gland There are two major inductor/effector axes that are responsible for the presence of antigen specific antibodies in breast milk. In the first axis (the gut mammary axis), primed cells formed following antigenic stimulation in the gut can home to the mammary gland (70, 582). These cells then mature into plasma cells in the mammary gland and secrete dimeric IgA (567). The IgG-ASC, although present in small numbers in the mammary gland, also may originate from the gut (451). The second inductor/effector axis, the broncho -mammary axis, can also transfer the maternal encounter of mucosal pathogens or maternal immunization into breast-milk antibodies (269). The gutmammary immunologic axis was first suggested by Bohl et al (70) and Saif et al (582) in studies of lactogenic immunity to TGEV in swine. In these studies, antigenic stimulation at one mucosal site (intestine) led to siga antibody responses at distant site(s) such as the mammary gland. Maturation of these B cells into IgA ASC occurs at the mucosal effector sites in response to antigens, T cells and cytokines (580). In pigs, the contribution of immunocytes sensitized in the intestine to the IgA ASC pool in the mammary gland appears to be more important than local production of IgA antibodies because IMm injections of live attenuated TGEV failed to produce any appreciable level of TGEV IgA antibodies in milk (70). These important findings, confirmed by subsequent studies in 104

131 mice (727), pigs (273) and humans (453) were an important tenet of the common mucosal immune system. However, there is little evidence for the existence of a gutmammary gland immunologic axis in cattle and sheep. Antibody responses in milk were not detected in cattle orally vaccinated with E.coli K99 (469). Harp et al (274) found that most lymphocytes from the intestinal lymph nodes of cattle and sheep returned to the intestine. At the same time, in another study by the same authors, trafficking of cells to the mammary gland of sows was observed (273). It was suggested that the plasma cells in the mammary gland of ruminants may be derived from cells stimulated in the draining lymph nodes for the mammary gland and not derived from gut (109) Transfer of functional immune cells in milk to neonates Milk also contains many functional immune cells, billions of which are provided to neonates during the first few days. Sow milk contains 2x10 5 to 2x10 7 cells/ml (592). The proportion of different cells are in sow milk as follows: epithelial cells: 31%, neutrophils: 47%, lymphocytes: 12% and macrophages: 9% (604). The T cells account for 70-90% of the sow colostral lymphocytes. The presence of epithelial cells, NK cells, neutrophils and macrophages in milk are responsible for innate immune responses, providing early protection to the neonate. Activities of sow NK cells have been shown to provide resistance to TGEV when transferred to newborn pigs (117). The CD8 and CD4 T cells are both found in the mammary gland with CD8 being more dominant, however, these cells exhibit properties of memory cells: weak reactivities to mitogens but strong proliferative responses to enteric antigens. 105

132 The lymphocyte population in mammary secretions is dynamic during the pregnancy and lactation period. In dairy cows, 80-90% of lymphocytes in the involuting gland are CD2 +, and proportions of CD4 and CD8 are 55% and 40%, respectively. Numbers of CD4 + decrease strongly at birth and always remain lower than 20%, whereas the numbers of CD8 cells remain constant during lactation (755). The absorption of colostral cells has been demonstrated in neonatal pigs (399, 685, 740). The neonatal pigs were estimated to ingest on average million colostral cells/days (398). Twenty-four hours post feeding, the maternal lymphocytes can be found in liver, lung, spleen, lymph nodes (LN) and gastrointestinal tract of pigs (740). These cells carrying immunological information such as antigen specificity or memory from previous exposure of the mothers can be passed on to neonates Lactoferrin Lactoferrin, the major protein in milk, about 1-4g/L, together with siga make up 30% of milk proteins (only 5% in cow s milk). Lactoferrin possesses microbicidal, immunostimulatory and anti-inflammatory properties. Human and bovine lactoferrins inhibit IL-6 production of a monocytic cell line as early as minutes after stimulation with LPS (441). Lactoferrin exhibits antiviral activity against both DNA- and RNA-viruses, including RV, respiratory syncytial virus, adenovirus, herpes viruses and HIV(693). Lactoferrin from bovine milk was shown to prevent RV attachment to intestinal cells by binding to viral particles and inhibiting a post adsorption step of the virus (609). Lactoferrin interferes with an early infection step of poliovirus and together 106

133 with Zn +2 ions inhibits viral replication after the viral adsorption phase. Lactoferrin also competes for common glycosaminoglycan receptors to prevent the attachment of adenovirus to cell membranes Cytokines Human milk contains high levels of TGF-β, TNF-α, IL-1, IL-6, IL-8 and IFN-γ (247). In human milk, the cells expressed transcripts for TGF-β (1 and 2) and IL-6 and production of these cytokines by mammary epithelial cells were detected (518, 646). The TGF-β synergistically enhances the secretion of IL-6 by epithelial cells (452). Interferon-γ was shown to be secreted by T cells from human milk in vitro (58). Table 1.5 summarizes the levels of different cytokines in human colostrum/milk. Limited data are available on the levels of cytokines as well as the cytokine producing cells in porcine colostrum and milk (711). The level of the latent form of TGFβ is high only in the sow colostrum period and the concentration of this form rapidly declines and is partially replaced by the free form. These milk cytokines have been suggested to play roles in immunomodulation of neonatal immune development. The TGF-β, present at high concentrations in milk, helps to control inflammation in neonates due to its immuno-down regulatory function (526). Uncontrolled inflammatory responses were shown to be fatal in TGF-β knock-out mice after weaning. When provided as supplement, TGF-β also helps to restore the level of pro IL-18 in the intestine. Of note, IL-18 has been shown to play a role in homeostasis by influencing the development of Th1 or Th2 responses depending on the cytokine environment at the time of antigen priming (489, 751). In another study, the important 107

134 immunoregulatory role of TGF-β was demonstrated in a study where TGF-β1 supplied to the fetus by injection into the mother s circulation during gestation or to the neonate via milk during suckling was shown to rescue TGF-β1-/- newborn pups from severe cardiac abnormalities (408). The presence of TGF-β1 in the fetus could play a role in limiting the development of fetal primary immune responses and favoring the neonatal Th2 responses, which explains the Th2 bias responses in human infants and murine neonates as mentioned in previous sections (420, 538). Milk cytokines such as TGF-β, IL-4 and IL-10, which can also influence antigen priming (526). The immunomodulatory antiinflammatory activity of IL-10 in milk has also been suggested (231). However the effects of these cytokines have not been clearly validated in human milk in vivo (690). Certain cytokines such as IL-4, IL-5 and IL-13 were elevated in colostrum of allergic mothers compared to non-allergic mothers, which might contribute to the Th2 bias of neonatal immunity development in breast-fed children (49). However, it is also suggested that the presences of IFN-γ and IFN-γ producing cells also enhance Th1-IgG2 production in neonates (628). Thus milk cytokines and cytokine secreting cells can modulate both types of Th responses. The IL-18 was present in milk of women with complication in their pregnancy (661). The acute phase cytokines (TNF-α and IL-6) were minimal in human whey in one study using ELISA (646), but other studies (569, 570) reported much higher levels of TNF-α (620pg/ml) and IL-6 (151pg/ml) using radioactive immunoassay. Cytokines in milk also function in trafficking of immune cells. Higher levels of chemoattractant factors IL-8 and RANTES (regulated on activation, normal T cell expressed and secreted) were found in colostrum of allergic mothers which may enhance 108

135 the traffic to breast milk of specific cell populations involved in allergic reactions, e.g. IgE secreting cells and their transfer to neonates (77). A high level of TGF-β in milk has also been associated with high levels of serum IgA and allergen specific IgA ASC responses in infants (344, 574). Other cytokines that are present in colostrum and milk are IL-6, which is known to enhance IgA antibody production. Milk TNF-α enhances the production of secretory component associated with siga (246) Transfer of cytokines via the placenta In humans, TGF-β1 and IL-10 can be detected in human umbilical cord fluid (538). Elevation of TGF-β levels in maternal and fetal circulations from pregnancy to birth have been reported in humans (538). In humans, TGF-β1 was present at the maternal-fetal interface including the placenta. However there is also no correlation between maternal and fetal TGF-β1 levels in humans suggesting that either restriction of its passage across the placenta or its polarized secretion by the placenta at the fetal side (538). It has also been shown that IL-4, but not IFN-γ and IL-12 are constitutively produced by cord blood mononuclear cells upon stimulation with PHA (94). In mice, there was no transfer of maternal interferon to the fetus even when the level of interferon injected into the mothers was high (199). Transfer of cytokines via the sow placenta has not been reported. It is well established that the sow placenta which consists of 6 tissue layers doesn t support transfer of immunoglobulins (382). Uteroferrin, the iron-containing glycoprotein composed of a single oligosaccharide chain has been shown to be transported across the pig placenta via a receptor (598). Monomers such as glucose and amino acids are actively transported to 109

136 the fetus as nutrients whereas the transfer of fatty acids is limited (678). In chapter 5, we showed that cytokines such as IFN-γ, IL-4, IL-6 and IL-10 were not detectable in serum of germ-free pigs or in pre-suckling pigs. However, IL-12 and TGF-β were present in serum of these pigs. The presence of these cytokines in piglet serum can be constitutive or via ingestion of placental fluids or possible transfer via the placenta or colostrum/milk CD14 CD14, the co-receptor for bacterial LPS on mature monocytes, macrophages and neutrophils, can be found in colostrum and milk in the soluble form. These CD14 molecules in milk were shown to stimulate B cell growth and differentiation in neonates (212). The soluble form of CD14 (scd14) in mammary secretions was enriched fold compared to the serum levels, and this level persists up to 400 days post partum in humans (300 days in cows). The LPS-induced soluble CD14 signaling and B cell activation are mediated by TLR-4. Intramammary administration of soluble CD14 with E.coli was shown to induce an early increase of scd14 levels in milk, resulting in rapid clearance of bacteria and reduction of the severity of E.coli infection in the mammary gland (404). It is postulated that the scd14 might induce early recruitment of neutrophils to milk, which help with the clearance of bacteria. Similarly, the presence of the soluble form of TLR2 produced by blood monocytes in breast milk and plasma can modulate the activation of monocytes (401) Levels of MatAb transferred and factors influencing the transfer The level of MatAb transferred and its variation between individuals are partially hereditary. It was reported that the level of IgG antibody in the colostrum of female cattle 110

137 is constant overtime (167). Chicks born to lines selected for an antigen-specific antibody response leads to similar consequences in the offspring as in the parent (68, 101). The environment experienced by the mother also plays a role in the variation of antibody transmission from mothers to offsprings. If the mother is exposed to a particular pathogen and develops antibody responses to the pathogen with the specific antibody transmitted to her offspring, these offspring are more likely to and more efficiently mount similar specific antibody responses (259). The MatAb level is also dietary dependent. A study of rats showed that restriction of the dietary proteins resulted in 2-fold lower IgG antibody levels in colostrum (466). Similarly, birds raised under restriction of vitamin E showed decreased antibody transmission to chicken eggs (319). The age of the mother is also an important factor for MatAb level transference to neonates. Offspring from 3 year-old cows had significantly higher serum IgG levels than those from younger (2 year-old) or older (more than 3 year-old) cows. The numbers of IgG ASC in the ovary of hens at 50 days of age or more than 450 days of age were significantly lower than those of mature but young hens (180 days). The influence of MatAb on neonatal and infant immune responses is long-term, persisting even after being catabolized completely in the offspring. This influence is inducible early during the development of infants (259). The effects of MatAb on both the humoral and cellular arms of the immune system will be discussed in next section. The MatAb influence is not only applied to that particular individual but also to the next generation (423). In the first generation, there was no difference in antibody responses to 111

138 E.coli in normal pups vs. pups inoculated with mouse antibodies against E.coli antigen. In the next generation, however, the responses to E.coli were significantly higher in pups from the mouse dams with antibodies than those from dams which were not inoculated with antibodies. This is partially due to the expansion of B and T cell repertoires, as was similarly observed during vaccination of an individual with multiple antigen doses. The MatAb also improves the intensity of the offspring immune responses (259). Offspring from B cell deficient mice expressed 2-3-fold lower pre-b and B cell numbers in bone marrow and spleen (429). The frequency of MHC II expressing cells also decreased in these mice (756). The antibody level after primary immunization of the F 2 offspring of immunized mothers was equal to that obtained after secondary responses in offspring from unimmunized mothers. Infants may have different expression levels of the IgG receptor (FcRn) and the receptor affinity to IgG, different metabolic rates of antibodies, as well as incubation/gestation periods (411, 480). Thus the level of MatAb transmitted is also a function of interaction between maternal genome (antibody transferred) and offspring genome (antibody absorbed). However the ability to absorb MatAb by the offspring tends to co-adapt with the level of MatAb transmitted (743) Interference of neonatal responses by MatAb Inhibition by Mat Ab Inhibition of B cell responses by MatAb has been documented for many vaccines. The humoral responses in infants were reduced or inhibited following vaccination with measles, hepatitis A, the combined diphtheria, pertusis and tetanus (DPT) and 112

139 Hemophilus influenza vaccines (63, 190, 213, 499, 595, 758). Again, the inhibitory effects of MatAb can be long lasting: after 3 doses of Hepatitis A vaccine in children before 6 months of age, only 24% of children born to Hepatitis A antibody positive mothers showed protective antibody levels at 6 years of age, compared to the 68% in children born to Hepatitis A antibody negative mothers. Feeding or IP injection of rats as neonates with monoclonal IgG2a or IgG1 antibodies led to suppression of humoral responses in these rats as adults; thus, the suppression by MatAb lasted for up to 5 months (528). This suppression was only observed when the rat received the monoclonal antibody before weaning or gut closure. Feeding at a subsequent time did not cause suppression. Thus the maternal IgG antibodies determine the immune repertoire of the offspring. The T cells responses to vaccines appear to be more resistant to the suppression by MatAb. The T cell proliferation, IFN-γ and IL-12 production specific to measles vaccine were not affected by the presence of MatAb (230). In the murine model, the in vitro T cell responses to measles and tetanus vaccine in pups were unaffected by high titer MatAb (623). However, there are many other studies which have described a suppressive impact of MatAb on T cell responses. Both humoral and CTL responses were decreased in newborn rhesus macaques immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin and fusion proteins in the presence of measles immunoglobulin (780). Similarly, pups from rabies virus-immune dams showed a decrease in the specific B- and T-cell responses to rabies virus immunization, measured by serum antibody level and cytokine release from in vitro lymphocyte cultures, 113

140 respectively, leading to the failure of the vaccine. The degree and duration of the vaccine failures was inversely correlated with the amount of maternally transferred antibodies (750) Enhancement of the immune responses by MatAb Maternal antibodies can also enhance vaccine responses. It has been demonstrated that IgG antibodies in the immune complex form can stimulate the formation of GC, activate somatic mutation of B cells and suppress differentiation of ASC simultaneously (641). Antibody dependent immune enhancement has been reported previously for both vaccines and infectious agents. Maternal tetanus IgG antibodies enhanced the T cell responses by increasing IL-4, IL-5 and IL-13 responses to tetanus vaccine in infants (568). Low or moderate levels of pneumococcal polysaccharide-specific MatAb not only provided protection against pneumococcal infections but also enhanced the immune responses elicited by pneumococcal vaccine in neonatal and infant mice (551). The immune enhancement by passive antibodies is dose dependent. In vivo and in vitro studies showed that mice receiving Japanese Encephalitis virus-immune mouse serum at dilutions of 1:10 and 1:100 but not undiluted serum, had a significant reduction in average time to death when challenged with the related Murray Valley Encephalitis virus (90). Low titer MatAb present in conventional pigs was shown to enhance PRRSV virus infection, as indicated by an increase in viremia as well as the tissue distribution of virus likely mediated by an antibody dependent enhancement mechanism of PRRSV infection of macrophages (616). 114

141 The immune enhancement is not only applicable for antigen specific antibodies but also to natural antibodies. Chicken given pooled plasma from non-klh immunized hens (containing natural antibodies), then immunized with KLH antigen showed increased IgM and IgY antibody titers to KLH compared to those that received phosphate buffered saline (PBS) (734) Mechanisms of MatAb interference Neutralization of live viral vaccines It is hypothesized that MatAb neutralize and hence reduce the viral load, preventing the induction of effective B and T cell responses. It has been demonstrated that MatAb reduced the replication of human RSV in the lower respiratory tract of mice (164). Oral vaccination of young foxes with a modified live vaccine against rabies in the presence of MatAb did not protect the animals from infection with virulent virus (482). The lack of protection against rabies in these animals was correlated with the failure of this vaccine to induce active antibody responses. Homologous but not heterologous MatAb to Marek s Disease virus delayed the development of viremia induced by the live vaccine virus and also inhibited immune responses to the vaccine in chicks (367). The level of inhibition of MatAb to infant vaccine responses is the function of MatAb levels at the time of vaccination and the antigen load, as proposed by Siegrist et al (622), Figure1.3. Interference by MatAb is dose dependent; some studies suggest micrograms of MatAb may lead to suppression whereas nanogram amounts lead to enhancement of an infant s immune response (665, 738). 115

142 Interference of passive antibodies via the Fc receptor At least 3 mechanisms have been proposed for IgG mediated suppression: (1) IgG masks antigenic epitopes and prevents B cells from recognizing and responding to the antigen; (2) the immune complexes are removed by phagocytoxic cells by FcγR before they can activate B cells and (3) IgG inhibits B cell activation by cross-linking the B cell receptors (BCR) and FcγRIIB (284). The FcγRIIB, expressed on the macrophages and B cells, acts as an inhibitory receptor that terminates the activation signals initiated by the BCR (2). At the same time, enhancement by IgG antibody has also implied the involvement of complement and FcγR. The first mechanism, epitope masking by IgG, helps to explain the inhibition of B cell responses, but not T cell responses in the majority of studies as mentioned in the previous sections. Because epitope masking is not expected to prevent the uptake of the IgG/antigen complexes by the APC, the presentation of antigen to T cells would be unaffected which was demonstrated by Karlsson et al (348). By this mechanism, the induction of memory and secondary responses may not be effected by passive antibody (284). The epitope masking but not the suppression via FcR is likely the mechanism of immune response suppression because non- antigen specific antibody also causes similar suppression (348). Thus antibody bound to one epitope on an antigen can sterically block the recognition of the neighboring epitopes (284). There has been no significant support for the second mechanisms of suppression. The role of FcγRII in feedback inhibition in the third mechanism is still controversial. First, IgG mediated suppression is often observed for particulate antigens whereas 116

143 stimulation is observed for protein antigens (284). Secondly, co-ligation of the BCR and of FcγRIIB results in an inhibitory signal (534). The FcγRIIB has an immunoreceptor tyrosine-based inhibitory motif (ITIM), which is the predominant form in both cord blood and adult B cells (325). However this suppression was always observed in adult cells, but was not seen or was less pronounced in cord blood cells, suggesting a lower level of CD32 (human FcγRII) on cord blood cells, making cord blood B cell less susceptible to feedback inhibition by antibody (325). In addition, studies showed that mice lacking FcγRII still showed IgG mediated suppression and suppression was still observed with Fab fragments. Furthermore, FcγRIIB deficient mice given the IgG1/antigen complexes (anti-tnp/tnp-bsa) still produced BSA antibody (662). It is postulated that FcγRIIB only prevents the IgG/antigen complexes from reaching abnormal levels in the body. Thus most data suggested that epitope masking is the most important mechanism of suppression of antibody responses by passive antibodies. The IgG mediated enhancement of antibody responses involves complement or FcγR or both. The role of complement was demonstrated in complement depleted mice in which the ability of IgG to prime DNP (2,4 Dinitrophenyl) specific B cells is abolished (192). Monoclonal antibody to DNP complexes with DNP-KLH can be found in splenic follicles and the presence of these complexes in these sites correlated with enhancement of the memory cell responses (160, 735). However other studies suggest that IgG antibody can enhance antibody responses without involvement of the complement system. Mutant IgG, which fails to activate complement, still enhanced the efficiency of antibody responses to KLH (736). 117

144 On the contrary, the FcγR mediated antibody enhancement is considered to be the major mechanism. Mice deficient in this receptor showed impaired responses to antigen/igg complexes (729). In FcγR deficient mice, where FcγRIIB was normally present, the responses to antigen/igg complexes were impaired Interference via idiotypic interaction Maternal antibodies result in an altered primary antigen-specific antibody repertoire of the offsprings via altering the idiotypic composition of the offspring s immune responses (394). Somatic mutations that produce a pool of antibodies specific to different antigens can be regarded as a step in the learning process of the mother. Maternal immunological experience in turn educates the nascent immune system of the newborn. The transfer of antibodies from mother or grandmother influences the repertoires in the early primary response of the offspring. When a panel of monoclonal antibodies (Mab) to hen egg-white lyzozyme (HEL) was administered to the mother, these antibodies did not have equal effects on infant s immune responses (640). Only Mab that are idiotypically connected in the anti-hel Mab panel induced suppression of the offspring s responses. The findings suggest that idiotypic suppression occurs through the reaction of anti-idiotopes with idiotopes on the surface of newly generated B cells. There are two types of suppression: either direct blocking of B cell maturation which is short-lived, or induction of regulatory cells via the idiotope-anti-idiotype complexes, which is long-lived (665). 118

145 Transfer of protective idiotopes from mother to the child could both prime the neonate for a booster effect of antigen and provide passive protection against infection (19). Of note, antibodies against antigen are labeled Ab1; antibodies raised against Ab1 antibodies are called Ab2. The Ab2 that recognizes an idiotope outside the antigen binding site of Ab1 is called Ab2α. The Ab2 that recognizes the paratope inside the antigen binding site is called Ab2β and is an internal image antibody since it mimics the shape of the original antigen. Antibodies against Ab2 are called Ab3 and so on and so forth. Some of these Ab3 can cross react with the original antigen and can be labeled Ab1. This chain of reaction is called a network response (19). This network has been found in young animals. Any given antibody species will bind to more than 20% of other antibodies of the same repertoire (291, 292). Immunization of mice with monoclonal Ab1 to Schistosoma mansoni induces high levels of Ab2 and Ab3 whereas Ab2 can inhibit % of Ab1 binding (277). This immune network can be formed when MatAb are transferred to neonates. This network could be responsible for translating the maternal signal Ab1 into Ab2 and antibodies Ab3 that cross react with the original antigen or Ab1. Okamoto et al (508) measured both Ab1 and Ab2 in neonatal rat serum, indicating that neonates do mount antibody response against the incoming Ab1, but these levels decline shortly after birth. By the fifth month, the level of Ab2 becomes immeasurable whereas the suppression still persists in rats (528). It is hypothesized that idiotypic specific T suppressor cells are responsible for the suppression (47, 664, 665). This immune network is found largely in neonates, whereas the antibodies don t bind to each other at a similar frequency in adult animals (293). It is possible that the 119

146 adult T cells undergo extensive selection to avoid the anti-self responses, thus there is no help available to develop this network of B cell responses in adults. It is also hypothesized that the expansion of non-self reactive clones will overtake other clones with age. Anderson et al. built a model for maternal-neonatal idiotypic interaction, namely the molecular attention hypothesis (19). The neonatal immune network is said to amplify and translate the maternal signal Ab1 into a dynamic activity for all clonal species. There will be thousands of copies of id and anti-id. The id and anti-id complex can be taken up by APC and the critical region of Ab1 and Ab2β can be presented in association with MHC II. The Ab1-Ab2β complex can also bind to Fcγ receptor on the surface of FDCs. These FDCs can retain the complexes over long periods of time, then travel to spleen and LN and deposit in GC. Thus these cells help to maintain the dynamic state of the anti-id B cell clones. Alternatively, Th cells for internal image antibodies can be formed. First, there is an overlap between B cell epitopes and T cell determinants. Fragments of Ab1 can be processed and associated with MHC molecules. If the fragments contain the VH-VL parts, they would trigger binding of T cells (115). 1.5 Vaccine strategies to overcome the immaturity of neonatal immunity and interference by MatAb The following sections focus on the types of vaccines, modes of vaccine delivery and adjuvants, which have demonstrated efficacy or have shown the potential for effective vaccination of neonates in the presence of MatAb 120

147 1.5.1 Type of vaccines If neutralization by MatAb remains a limitation of live vaccines, the use of nonreplicating vaccines, such as virus-like particles and DNA plasmids may be advantageous Virus-like particles (VLPs) The VLPs have been produced for more than 30 different viruses (Table 1.6), including viruses with single capsid proteins (Caliciviridae and Retroviridae family), viruses with multiple capsid proteins (poliovirus, infectious bursa disease virus etc), enveloped viruses (Hepatitis B virus, influenza virus etc) and non-enveloped viruses (RV, parvovirus etc) (497). The VLP possess the overall structure of the virus, but without the infectious genetic material. The use of non-replicating vaccines is much safer than live vaccines even attenuated vaccines because recombination, reversion to virulent strains or re-assortant can not occur with the VLPs. The VLPs can be used alone without adjuvants to induce both humoral and cellular responses in some cases. The hepatitis E VLP or foreign epitopes expressed as chimeric proteins on hepatitis E VLP induces both systemic and mucosal IgA antibody responses (495). Use of the VLP generated from lymphocytic choriomeningitis virus (LCMV) with CpG in a prime/boost strategy demonstrated effectiveness in induction of CTL responses (571, 608). However, the suppressive effects of MatAb were also observed using nonreplicating vaccines including VLPs. In Chapters 3 and 4, the pre-challenge IgG ASC numbers and the serum IgA and IgG antibody responses induced by 3 doses of RV 2/6 VLP with ISCOM were suppressed in the presence of high and low titer passively 121

148 transferred RV specific antibodies. Thus interference by MatAb is not restricted to neutralization of live viral vaccines. An inactivated pertussis toxin vaccine is a clear example in which infant immune responses can only be observed in infants without MatAb (104). In addition, larger amounts or higher concentrations of the non-replicating vaccines will be required for induction of effective immune responses. Therefore an antigen delivery system or adjuvant is normally required to reduce the antigen dose, to prevent the degradation of the vaccine as well as to avoid tolerance when the vaccine is delivered via oral route. Thus the use of non-replicating vaccines alone might not be sufficient to overcome the interference by MatAb. A combination of different strategies will be necessary to improve vaccine efficacy in the presence of MatAb DNA vaccines The DNA vaccines can be administered with adjuvants or supplied with cytokines that might deviate from the Th2 bias of the neonate. Nucleic acid vaccines have been shown to be strong inducers of Th1 responses, which enhance Th1 CD4 T cells and induce CD8 CTL. The DNA vaccination might also reduce the number of injections required. Substantial efforts have been made in developing an effective way to deliver DNA vaccines. The DNA vaccines are normally administered via systemic or subcutaneous routes. Mucosal DNA vaccines are less common. Most such studies used mucosal DNA vaccines targeted to human immunodeficiency virus (HIV) infection; only a limited number of vaccines for other mucosal infections. In BALB/c mice, protective immune responses were obtained after oral immunization with one oral dose of RV VP6 122

149 DNA vaccines encapsulated in poly(lactide-coglycolide) (PLG) microparticles, indicated by RV-specific serum and intestinal IgA antibody responses and significantly reduced fecal RV antigen after challenge with homologous RV(124). Similarly, one dose of VP4 DNA and VP7 DNA vaccines, given to BALB/c mice by oral gavage induced RV serum antibodies and intestinal IgA antibodies 6 weeks after immunization and protection against viral shedding at 12 weeks post-immunization. Unencapsulated VP7 DNA vaccines delivered orally also induced protection against viral shedding in these mice but to a lesser degree (283). Priming by a mucosal route followed by boosting via a systemic route can induce both mucosal and systemic immunity which are important for vaccines against pathogens such as human herpes simplex virus (HSV) and RV (193, 763). Yuan et al (763) demonstrated protection against RV induced diarrhea (30%) and shedding (70%) in Gn pigs using a prime/boost vaccine strategy consisting of an oral dose of AttHRV and two intramuscular doses of VP6-DNA vaccine. High levels of IgA ASC responses in ileum and intestinal antibody responses were induced by this vaccine regimen. The DNA vaccines overcame the Th2 bias of neonatal immune responses as mentioned previously. Intramuscular injection of murine VP6 DNA vaccines raised a Th1-like antibody response, high titers of RV-specific serum IgG and serum but not fecal IgA antibodies in BALB/c mice (754). In addition, both partial homologous and heterologous protection against murine RV challenge as measured by reduction of RV antigen shedding in feces were achieved by murine and bovine VP6 DNA vaccines, respectively. Immunization of VP6 DNA vaccines via the abdominal epidermis or anorectal epidermis using a gene-gun (PowderJet, Inc) induced protection against 123

150 challenge virus, yet stimulated Th-2 type responses even in adult BALB/c mice (123). Thus the routes of DNA vaccination are important in the induction of the Th1/Th2 balance in neonatal immune responses. Mucosal DNA vaccines have been delivered by other routes. The most interesting approach is via the oral route in utero to the fetus, which has been tried in lambs (237) against HSV and in baboons against hepatitis B (724). In these studies, 75-80% of the animals produced both systemic and mucosal immune responses. Strong memory responses were still detected 3 months after birth. The reason for the success of the in utero approach is that the fetal T cells might have a longer life span than the neonatal T cells. In addition, mucosal epithelial cells in the fetus might have much lower turnover rates, which allows sufficient time for transfection and gene expression after oral DNA immunization of the fetus (236). Thus the rate of survival of memory T cells can be improved in fetuses or for at least 3 months in this study. Several drawbacks of this method of DNA vaccine delivery were noted. The presence of high levels of cortisol in serum of the mammalian fetus might have significant effects on the peripheral T cell function, T cell trafficking or T cell commitment to Th1 or Th2 responses, as well as a reduction in T cell survival rates. Also because DNA vaccines often require long time to develop immune responses, in utero immunization should be performed weeks before the delivery date, for examples 4 weeks in the lamb study (237), which can pose a danger to the fetus. Other modes of administration have been tried for mucosal DNA vaccines. The DNA vaccines administered via eye-drops preferably induced IgA antibodies in tear and bile of young chickens (573). The DNA vaccines can also encode antigens together with 124

151 cytokines e.g. IL-12 and GM-CSF, which exert immunomodulating effects or enhance antigen presentation, useful for the induction of immune responses in early life. The DNA vaccines containing genes encoding both IL-12 and IL-18 have been tested to enhance the immune responses and protection in vaccines against HSV and Leshmania (668, 779). A DNA vaccine codelivered with IL-2 or GM-CSF increased the antibody and lymphoproliferative responses to bovine viral diarrhea virus (498). The common concern with DNA vaccines is that if the vaccine is expressed in a bacterial genome or plasmid, the presence of the CpG motif can have significant effects on both innate and adaptive immune responses. The CpG can promote the development of autoimmune disease, or cause strong local inflammatory reactions. Although the halflife of the plasmid is short (2-4h in amniotic fluid, degraded completely within 8h), there is still a brief time for bacterial DNA to interact with the innate immune system (237). The DNA plasmid vaccines also pose a potential risk of integration into the chromosomal DNA (375). In spite of these concerns, DNA vaccines are the most promising vaccines for use in infants to overcome MatAb suppression. This type of vaccine has been tried in different animal models and against different pathogens. The DNA vaccines have been demonstrated to induce adult-like Th1 and CTL responses in newborn and young mice against measles, Sendai, influenza, LCMV, retrovirus and rabies viruses (75, 278, 434, 596). Some formulations of DNA vaccines such as those used for HSV do not require adjuvants. Naked DNA encoding for a HSV protein could be administered intramuscularly and induced effective humoral and T cell responses in the presence of MatAb (426). The DNA immunization targets APCs to increase their uptake of antigen. 125

152 However, there are contradictory studies concerning whether DNA vaccines can escape interference by MatAb. The gd glycoprotein gene of the pseudorabies virus (PRV) inoculated in pigs born and suckled by immune mothers did not induce immune response or protection against PRV. On the contrary, Fischer et al. described a DNA vaccine that induces both humoral immune responses and protection against PRV in pigs suckled by PRV seropositive sows (215). The difference between these studies in the time of challenge of the pigs (16 weeks in the former vs. 20 weeks in the latter) and the duration between priming and boosting (6 weeks in the former vs. 11 weeks in the latter), which led to further declining MatAb titers in the pigs might explain the conflicting results. In turkeys, vaccination with DNA plasmids expressing the major outer membrane protein of avian Chlamydophila psittaci induced protective immunity against homologous challenge in the presence of MatAb. Although the antibody responses induced by the vaccine were affected, the cell-mediated-immunity was unchanged (694). The effects of MatAb are not the same for different proteins of the same virus. The MatAb inhibited the antibody responses induced by DNA vaccines encoding HA but did not have impact on the antibody responses to the nucleoprotein (NP) nor the generation of cellular immune responses to HA or NP (532). A DNA vaccine against Plasmodium yoelii induced tolerance rather than immunity in neonatal suckling mice at 2-5 days of age (470). Thus not all DNA vaccines are effective in preventing the disease in the presence of MatAb. Further studies are needed to analyze the mechanisms by which DNA vaccines and the various modes of delivery/adjuvant which can escape MatAb interference. 126

153 1.5.2 Antigen delivery system Aluminum salts Aluminum salts form antigen depots at the site of injection from which the antigen is slowly released. This is an adjuvant that favors Th2 responses in mice and humans; therefore it is only advantageous to any infection that needs strong antibody responses e.g. tetanus, diphtheria toxoid or to viral agents such as poliovirus vaccine. Therefore its role in neonatal vaccination to overcome MatAb effects is limited although this adjuvant is the only one licensed for human use. However alum can be combined with other adjuvants to enhance the immune responses. Immunization of newborn (1-7- day-old) BALB/c mice against HBsAg with alum and CpG ODN induced HBsAgspecific CTL responses in the presence of high levels of MatAb (anti-hbs) (725). However, the B cell responses to HBsAg/alum/CpG ODN remained weak Liposomes, virosomes and Archaeosomes Vaccines encapsulated within liposome microspheres have the tendency to move to the cytosol and be processed by the MHC class I pathway and presented to CD8 T cells. Virosomes are a special form of liposomes whereby the virus fusion protein is inserted into the liposome bilayer. These methods of antigen delivery enhance cell binding, endocytic uptake of the antigen and delivery into the cytosol. A liposome-based vaccine against Hepatitis A virus (Epaxal, Swiss Serum and Vaccine Institute), induced 100% seroconversion after 2 doses and being well tolerated by vaccinees, has been licensed for human use (18). In addition, liposomes have been combined with cholera toxin, lipid A or cytokines to enhance adjuvancity (272, 391, 549). 127

154 Another lipid based adjuvant recently being investigated is Archaeosomes. Archaeosomes are a polar lipid structure or triphospholipid (TPLs) derived from Achaebacteria which is able to form stable liposomal vesicles (645). Unlike the conventional liposomes which are made from ester phospholipids, often with unsaturated lipid chains of variable length, Archaesomes are composed of fully saturated, branched chains attached via ether bonds to a glycerol backbone. This unique ether structure may facilitate the formation of unique head group structures that interact with the specific receptor on APCs. Archaeosomes induced antibody responses, CD4 + and CD8 + CTL responses, recruited and activated DCs and macrophages and attracted NK cells to tumor sites ( ). Protection of mice against Listeria monocytogenes was induced after immunization with the protective antigen entrapped in Archaeosomes. Several studies indicated that Archaeosome-based vaccines are safe (379, 380). An Archaeosome species such as Methanobrevibacter smithii is a nonpathogenic inhabitant of the human colon, which suggests a high tolerance when used as vaccine (467). However the safety of this adjuvant group is still in question as other Archaeosomes of species such as Planococcus still evoked unnecessarily non-specific inflammatory cytokine secretion in the absence of antigen, whereas the liposomes of H.volcanii species did not (644). In addition, it is unknown whether this lipid can enter the circulation of the host, which helps to accelerate immune responses. These adjuvant systems have not been tested in the presence of MatAb. 128

155 Microspheres Several studies have documented the use of microspheres to deliver vaccines. In this system, antigens are delivered by microspheres of biodegradable polymers. The polymers can be manipulated to release antigens gradually or as pulses. Microspheres have been shown to retain antigens for up to 9 days. Presentation of antigen-loaded microspheres to macrophages can extend over 7 days whereas the presentation of soluble antigen has a half-life of 18.6h (31). Rotavirus VP6 encapsulated in alginate microspheres delivered orally to BALB/c mice induced high level of fecal IgA antibodies, similar to those induced by live virus (359). In contrast, RV VP6 in incomplete Freund s adjuvant induced only IgG, but not IgA antibodies. Microspheres were observed in the follicles-associated-epithelium of PP (359, 360). Microspheres of more than 5um which are taken up and remain in the dome area of PP are likely to induce strong mucosal immune responses. Branched aliphatic oligoester microspheres with incorporated RV administered to mice IP or orally resulted in the production of systemic IgG and IgA antibodies Repeated oral vaccination with an increased dose was required to obtain mucosal antibody responses (537). Neither the liposome (previous section) or microsphere adjuvants have been evaluated in the presence of MatAb Vectored vaccines There are numbers of microorganisms including bacteria and viruses which can be used as vaccine vectors. The BCG strains can be used as the vector for vaccine delivery in the newborn due to its safety record. They have been shown in newborn mice to induce preferentially Th1 responses (384). They also induce mucosal responses in 129

156 macaques and guinea pigs (286, 351, 395, 463). The normal flora such as Lactobacillus, Streptococcus and Staphylococcus can also be used as vaccine vectors (357, 405, 600). Non-replicating live viral vectors such as genetically engineered poxvirus NY- VAC or naturally attenuated vectors such as AL-VAC, TRO-VAC or vectors from avipoxviruses e.g. canarypox, which are unable of replicating in mammalian cells, have been under investigation as a possible safe vaccine vectors (519). The deletion of virulence and host range genes in the NYVAC vector prevents reversion to the virulent phenotype. Canarypox-carrying rabies vaccines induced CD4 and CTL responses and conferred protection against rabies in both young and adult dogs and in humans (222, 670). However it has been shown that this vector is not able to overcome the Th2 bias of neonatal immune responses (626). When used with the HA of measles virus in young mice, high levels of IL-5, but lower IFN-γ and poor CTL responses were obtained, which differed quantitively from the Th1/CTL-prone responses in adult (623). This vaccine strategy was also unable to induce measles specific responses in pups born to immune mothers (623). However, high titers of MatAb did not affect the induction of vaccinespecific Th1/Th2 responses, as assessed by proliferation and levels of IFN-γ and IL-5 productions nor the CTL responses in infant mice Monophosphoryl lipid A (MPL) Monophosphoryl lipid A (MPL) is another promising adjuvant for use in infant vaccines. The MPL is the product derived from bacterial cell walls, a detoxified form of lipid A from the LPS of Salmonella Minnesota R595 (41). Its immunostimulating effects include activating APCs to produce IFN-γ, TNF-α, IL-1β (Th1 response) and inducing 130

157 IgG antibody production (an implication for long term immunity). A study of mice using an inactivated RV vaccine administered IM with MPL reported high levels of serum IgG and VN antibody titers, but low fecal IgG and IgA antibody titers even after the third dose (333). The MPL can also be a good inducer of mucosal immunity or of IgA antibody responses in particular. Intranasal immunization of mice with HIV-1 oligomeric gp160 (o-gp160), formulated with liposomes containing MPL induced strong antigen specific IgG and IgA responses in serum, vaginal, lung, and intestinal washes and in feces (698) Saponins and derivatives Saponins have been used in veterinary vaccines for a long time. Examples of vaccines using saponins are the vaccines against foot and mouth disease, Rhipicephalus sanguineus ticks in dogs, Fusobacterium necrophorum in steers, feline leukemia virus in cats and rabies vaccine in mice (290, 310, 428, 576, 658). The more frequently used saponin is the one derived from the bark of the Quillajia saponaria trees. The mode of action of saponins includes intercalating with cholesterol in the cell membranes, forming pores to allow the antigen transport across the membranes and facilitating antigen uptake and processing (636). Several derivatives/purified forms of saponins have been tested for use in mucosal vaccines. The Quil A, the fragments from saponins with immunostimulating activity can either be mixed with cholesterol and phospholipids to create immunostimulating complexes (ISCOM), or further purified as QS-21 both of which have been tested as adjuvants for mucosal vaccines for humans. These forms of saponins have been known to 131

158 induce CTL, both Th1 and Th2 types of responses and increase the magnitude of antibody responses. The ISCOM associated with RV 2/6-VLP (Figure 1.4) induced more balanced Th1/Th2 responses in Gn pigs (489).This immunity enhancing ability is the result of the ability of ISCOM to recruit accessory cells e.g. DCs, macrophages to the PP, MLN, evident at 3.5 and 7h and peaked at 48h after feeding of the vaccine (229). The ISCOM increased the uptake of protein significantly and induced an earlier peak of antigen uptake into the circulation after feeding, compared to vaccination with the antigen alone (30min vs. 60min) (229). Especially, ISCOM have been suggested to overcome MatAb suppression (515). Calves immunized with the bovine respiratory syncytial virus (BRSV) vaccine with ISCOM, but not with the inactivated vaccine were protected against challenge with virulent virus even in the presence of BRSV-specific MatAb (264). In addition, significantly higher BRSV-specific nasal IgG, serum IgG1 and IgG2 antibody titers were detected before and after challenge in animals immunized with ISCOM compared to those immunized with inactivated vaccine. The ISCOM has a higher potential for human use compared to QS-21 due to its safety record. The ISCOM has been pronounced safe in field trials in several animal models e.g. pigs, cattle, horses, macaques, etc (307, 314, 483) and in human adult volunteers (554). The use of QS-21, on the other hand, has reported several side effects such as moderate to severe pain in many of the volunteers with vasovagal episodes and hypertension (200). A combination of QS-21 and other adjuvants such as MPL, CpG, CRL-1005 (a non-ionic block copolymer), Titermax, Titermax plus CpG have been tried to improve the adjuvancity (363). The GPI-0100, a new semi-synthetic saponin adjuvant 132

159 containing the docecylamide derivative of the hydrolyzed saponins, was found to be more potent than QS-21 for antibody responses, delayed-type hypersensitivity reactions and IFN-γ production in the mouse model (363). Further testing to confirm a similar adjuvancity of GPI-0100 as ISCOM in humans is needed Muramyl dipeptide derivatives (MDP) In the search for a safe but effective adjuvant for clinical use, the N- acetylmuramyl-l-alanyl-d-isoglutamine, the minimal active component of the Freund s complete adjuvant (FCA) was found. The antigens with MDP in emulsion (Syntex adjuvant formulation SAF) has shown efficacy against several viruses such as influenza virus, hepatitis B virus, HSV and lentivirus (16) Hormones It is possible to use hormones such as Vitamin D3 to enhance both mucosal and systemic immune responses. Vitamin D3 has been observed to induce DC migration from the skin to PP (191). There is still controversy about the beneficiary effects of Vitamin D3. Positive effects to the systemic and mucosal immune responses of Vitamin D3 have been reported in pigs and cattle but not in humans receiving influenza vaccines (378, 546, 692). In the study by Kriesel et al (378), an unusual mode of vaccination was employed. Instead of mixing the vaccine with the adjuvant, Calcitriol was injected at 1cm from the site of influenza antigen injection. Administering antigens with Vitamin D3 on the skin can induce both systemic and mucosal immune responses due to migration of DCs from skin to PP (191). 133

160 Cytokines Cytokines which have immunomodulating activities such as IL-12 or proinflammatory cytokines such as IL-1 and IL-6 have been tested as adjuvants. Administration of Leshmania antigen with ril-12 at 200ng/dose or more induced 2-fold or more increases in antigen specific IgG antibody titers, whereas no increase in antibody titer was observed in the absence of ril-12 (356). Partial protection against the parasite was observed in the presence of either ril-12 or alum as adjuvants, whereas complete protection was found when both alum and 2ug ril-12 were used in combination. Interleukin-1, IL-6 and IL-12 promote either Th2 or Th1 while IL-1 and IL-12, not IL-6 can be used as adjuvants to induce mucosal immune responses (80). Interleukin-1 promotes IgG1 and IgG2b, and little IgG2a, an indication of Th2-type response. Interleukin-18, similar to IL-1β, which plays an important role in Th1 responses and often acts synergistically with IL-12, has also been demonstrated as a potential adjuvant (80). Cytokine adjuvants are especially effective when given IN with little or low side effects as compared with systemic injection (80). The nasal route also requires much less cytokine than the systemic route to achieve similar responses, yet the kinetics of these responses achieved via the IN route are also faster (431). Interleukin-12 is also able to induce IFN-γ, more efficiently by the IN than the parental route. Mice immunized IN with IL-1α or IL-1β produced specific IgG and siga antibody responses, similar to those induced by cholera toxin (647). A combination of different cytokines can also 134

161 enhance the vaccine responses. A DNA vaccine against HIV induced increased antibody responses and T cell proliferative responses when coinjected with IL-18 and INF-γ (361). However, cytokine adjuvants have not yet been documented in vaccine strategies to overcome MatAb effects CpG Oligodeoxynucleotides The unmethylated cytosine-guanine dinucleotides (CpG motifs) in the synthetic oligodeoxynucleotide (ODN) or in bacterial DNA possess immunostimulatory properties (377). The TLR-9 in DCs and B cells recognize CpG motifs which trigger a series of signaling cascades that cause B cell proliferation and antibody production. The CpG also activates cytokine secretion by macrophages, monocytes and DCs, which in turn stimulate T cells to secrete other cytokines and NK cells to increase cytotoxic activity (370, 450). More importantly, CpG induces predominantly Th1 responses (IFN-γ and IL- 12) and very little Th2 responses; thus, this adjuvant has the potential to overcome the Th2 bias in neonatal immune responses. The CpG was demonstrated to have adjuvant effects when delivered IN, orally or intrarectally with Hepatitis B surface antigen (HBsAg), tetanus toxoid and killed influenza virus (448, 449). The HBsAg-CpG vaccine induced antibodies with higher antigen-binding affinity but with minor side effects in humans (625). The HBsAg/CpG ODN vaccine induced CTL responses in young mice, but weak B cell responses in the presence of high levels of MatAb against HBsAg (725). Thus more optimization is necessary to improve CpG for use in humans in the presence of MatAb. 135

162 1.6. REFERENCES Research priorities for diarrhoeal disease vaccines: memorandum from a WHO meeting. Bull World Health Organ 69: Abbas, A. K., Lichtman, A.H. and Pober, J.S Cellular and Molecular Immunology, Fourth ed, vol. 1. W.B Saunders Company, Philadelphia. 3. Achtnich, M., and M. Zoller Autoreactive antibodies in thymus and spleen of neonatal and young adult BALB/c mice: influence of prenatal tolerization. Scand J Immunol 33: Adah, M. I., A. Wade, and K. Taniguchi Molecular epidemiology of rotaviruses in Nigeria: detection of unusual strains with G2P[6] and G8P[1] specificities. J Clin Microbiol 39: Adams, W. R., and L. M. Kraft Epizootic diarrhea of infant mice: indentification of the etiologic agent. Science 141: Adamski, F. M., A. T. King, and J. Demmer Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum). Mol Immunol 37: Aderem, A., and R. J. Ulevitch Toll-like receptors in the induction of the innate immune response. Nature 406: Adinolfi, M., and A. Glynn The interaction of antibacterial factors in breast milk. Dev Med Child Neurol 21: Adkins, B., Y. Bu, and P. Guevara The generation of Th memory in neonates versus adults: prolonged primary Th2 effector function and impaired development of Th1 memory effector function in murine neonates. J Immunol 166: Adkins, B., A. Ghanei, and K. Hamilton Developmental regulation of IL-4, IL-2, and IFN-gamma production by murine peripheral T lymphocytes. J Immunol 151: Adkins, B., C. Leclerc, and S. Marshall-Clarke Neonatal adaptive immunity comes of age. Nat Rev Immunol 4: Akira, S Toll-like receptors and innate immunity. Adv Immunol 78: Akira, S., K. Takeda, and T. Kaisho Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2:

163 14. Albert, M. J., L. E. Unicomb, G. L. Barnes, and R. F. Bishop Cultivation and characterization of rotavirus strains infecting newborn babies in Melbourne, Australia, from 1975 to J Clin Microbiol 25: Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, and A. Zychlinsky Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285: Alison, M. R., C. E. Sarraf, V. E. Emons, S. A. Hill, M. Maghsoudloo, and G. M. Murphy Effect of alpha-difluoromethylornithine on the polyamine levels and proliferation in two transplantable tumours. Virchows Arch A Pathol Anat Histopathol 419: Aluvihare, V. R., M. Kallikourdis, and A. G. Betz Regulatory T cells mediate maternal tolerance to the fetus. Nat Immunol 5: Ambrosch, F., G. Wiedermann, S. Jonas, B. Althaus, B. Finkel, R. Gluck, and C. Herzog Immunogenicity and protectivity of a new liposomal hepatitis A vaccine. Vaccine 15: Anderson, R. W On the maternal transmission of immunity: a 'molecular attention' hypothesis. Biosystems 34: Andrew, D. P., L. S. Rott, P. J. Kilshaw, and E. C. Butcher Distribution of alpha 4 beta 7 and alpha E beta 7 integrins on thymocytes, intestinal epithelial lymphocytes and peripheral lymphocytes. Eur J Immunol 26: Andrew, M. E., D. B. Boyle, B. E. Coupar, D. Reddy, A. R. Bellamy, and G. W. Both Vaccinia-rotavirus VP7 recombinants protect mice against rotavirusinduced diarrhoea. Vaccine 10: Angel, J., M. A. Franco, H. B. Greenberg, and D. Bass Lack of a role for type I and type II interferons in the resolution of rotavirus-induced diarrhea and infection in mice. J Interferon Cytokine Res 19: Anonymous Hopes and fears for rotavirus vaccines. Lancet 365: Arakawa, T., J. Yu, D. K. Chong, J. Hough, P. C. Engen, and W. H. Langridge A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat Biotechnol 16: Arias, C. F., M. A. Dector, L. Segovia, T. Lopez, M. Camacho, P. Isa, R. Espinosa, and S. Lopez RNA silencing of rotavirus gene expression. Virus Res 102:

164 26. Arias, C. F., C. A. Guerrero, E. Mendez, S. Zarate, P. Isa, R. Espinosa, P. Romero, and S. Lopez Early events of rotavirus infection: the search for the receptor(s). Novartis Found Symp 238:47-60; discussion Arias, C. F., S. Lopez, J. D. Mascarenhas, P. Romero, P. Cano, Y. B. Gabbay, R. B. de Freitas, and A. C. Linhares Neutralizing antibody immune response in children with primary and secondary rotavirus infections. Clin Diagn Lab Immunol 1: Armstrong, S. J., and N. J. Dimmock Neutralization of influenza virus by low concentrations of hemagglutinin-specific polymeric immunoglobulin A inhibits viral fusion activity, but activation of the ribonucleoprotein is also inhibited. J Virol 66: Askaa, J., B. Bloch, G. Bertelsen, and K. O. Rasmussen Rotavirus associated diarrhoea in nursing piglets and detection of antibody against rotavirus in colostrum, milk and serum. Nord Vet Med 35: Au, K. S., W. K. Chan, J. W. Burns, and M. K. Estes Receptor activity of rotavirus nonstructural glycoprotein NS28. J Virol 63: Audran, R., K. Peter, J. Dannull, Y. Men, E. Scandella, M. Groettrup, B. Gander, and G. Corradin Encapsulation of peptides in biodegradable microspheres prolongs their MHC class-i presentation by dendritic cells and macrophages in vitro. Vaccine 21: Azevedo, M. S Efficacy of Rotavirus-like particle vaccines and pathogenesis of human rotavirus evaluated in a gnotobiotic pig model. PhD. The Ohio State University, Columbus. 33. Azevedo, M. S., L. Yuan, C. Iosef, K. O. Chang, Y. Kim, T. V. Nguyen, and L. J. Saif Magnitude of serum and intestinal antibody responses induced by sequential replicating and nonreplicating rotavirus vaccines in gnotobiotic pigs and correlation with protection. Clin Diagn Lab Immunol 11: Azevedo, M. S., L. Yuan, K. I. Jeong, A. Gonzalez, T. V. Nguyen, S. Pouly, M. Gochnauer, W. Zhang, A. Azevedo, and L. J. Saif Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus. J Virol 79: Azim, T., S. M. Ahmad, E. K. Sefat, M. S. Sarker, L. E. Unicomb, S. De, J. D. Hamadani, M. A. Salam, M. A. Wahed, and M. J. Albert Immune response of children who develop persistent diarrhea following rotavirus infection. Clin Diagn Lab Immunol 6:

165 36. Azim, T., M. H. Zaki, G. Podder, N. Sultana, M. A. Salam, S. M. Rahman, K. Sefat e, and D. A. Sack Rotavirus-specific subclass antibody and cytokine responses in Bangladeshi children with rotavirus diarrhoea. J Med Virol 69: Backman, P. A. a. H., R.G Comparative aspects of pathogenesis of immunity in animals, p In D. A. a. K. Tyrrell, A.Z (ed.), Viral infections of the gastrointestinal tract. Marcel Dekker, New York. 38. Bailey, M., F. J. Plunkett, H. J. Rothkotter, M. A. Vega-Lopez, K. Haverson, and C. R. Stokes Regulation of mucosal immune responses in effector sites. Proc Nutr Soc 60: Bailey, M., K. Stevens, P. W. Bland, and C. R. Stokes A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors. J Immunol Methods 153: Bajer, A. A., D. Garcia-Tapia, K. R. Jordan, K. M. Haas, D. Werling, C. J. Howard, and D. M. Estes Peripheral blood-derived bovine dendritic cells promote IgG1-restricted B cell responses in vitro. J Leukoc Biol 73: Baldrick, P., D. Richardson, G. Elliott, and A. W. Wheeler Safety evaluation of monophosphoryl lipid A (MPL): an immunostimulatory adjuvant. Regul Toxicol Pharmacol 35: Ball, J. M., D. M. Mitchell, T. F. Gibbons, and R. D. Parr Rotavirus NSP4: a multifunctional viral enterotoxin. Viral Immunol 18: Ball, J. M., C. L. Swaggerty, X. Pei, W. S. Lim, X. Xu, V. C. Cox, and S. L. Payne SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties. Virology 333: Ball, J. M., P. Tian, C. Q. Zeng, A. P. Morris, and M. K. Estes Agedependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272: Barnes, G. L., J. S. Lund, S. V. Mitchell, L. De Bruyn, L. Piggford, A. L. Smith, J. Furmedge, P. J. Masendycz, H. C. Bugg, N. Bogdanovic-Sakran, J. B. Carlin, and R. F. Bishop Early phase II trial of human rotavirus vaccine candidate RV3. Vaccine 20: Barro, M., and J. T. Patton Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci U S A 102: Bartnes, K., and K. Hannestad Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules. Eur J Immunol 27:

166 48. Bauer, S., and H. Wagner Bacterial CpG-DNA licenses TLR9. Curr Top Microbiol Immunol 270: Bednar-Tantscher, E., G. C. Mudde, and A. Rot Maternal antigen stimulation downregulates via mother's milk the specific immune responses in young mice. Int Arch Allergy Immunol 126: Benfield, D. A., I. Stotz, R. Moore, and J. P. McAdaragh Shedding of rotavirus in feces of sows before and after farrowing. J Clin Microbiol 16: Benson, E. B., and W. Strober Regulation of IgA secretion by T cell clones derived from the human gastrointestinal tract. J Immunol 140: Berlin, C., E. L. Berg, M. J. Briskin, D. P. Andrew, P. J. Kilshaw, B. Holzmann, I. L. Weissman, A. Hamann, and E. C. Butcher Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74: Berneman, A., L. Belec, V. A. Fischetti, and J. P. Bouvet The specificity patterns of human immunoglobulin G antibodies in serum differ from those in autologous secretions. Infect Immun 66: Bernstein, D. I., V. E. Smith, D. S. Sander, K. A. Pax, G. M. Schiff, and R. L. Ward Evaluation of WC3 rotavirus vaccine and correlates of protection in healthy infants. J Infect Dis 162: Bernstein, D. I., V. E. Smith, J. R. Sherwood, G. M. Schiff, D. S. Sander, D. DeFeudis, D. R. Spriggs, and R. L. Ward Safety and immunogenicity of live, attenuated human rotavirus vaccine Vaccine 16: Berryman, M., and R. Rodewald Beta 2-microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc receptor throughout the transepithelial transport pathway of the neonatal rat. J Cell Sci 108 ( Pt 6): Bertolotti-Ciarlet, A., M. Ciarlet, S. E. Crawford, M. E. Conner, and M. K. Estes Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice. Vaccine 21: Bertotto, A., R. Gerli, G. Fabietti, S. Crupi, C. Arcangeli, F. Scalise, and R. Vaccaro Human breast milk T lymphocytes display the phenotype and functional characteristics of memory T cells. Eur J Immunol 20: Besser, T. E., C. C. Gay, T. C. McGuire, and J. F. Evermann Passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen. J Virol 62:

167 60. Bianchi, A. T., R. J. Zwart, S. H. Jeurissen, and H. W. Moonen-Leusen Development of the B- and T-cell compartments in porcine lymphoid organs from birth to adult life: an immunohistological approach. Vet Immunol Immunopathol 33: Birchler, T., R. Seibl, K. Buchner, S. Loeliger, R. Seger, J. P. Hossle, A. Aguzzi, and R. P. Lauener Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein. Eur J Immunol 31: Bishop, R. F., G. P. Davidson, I. H. Holmes, and B. J. Ruck Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 2: Bjorkholm, B., M. Granstrom, J. Taranger, M. Wahl, and L. Hagberg Influence of high titers of maternal antibody on the serologic response of infants to diphtheria vaccination at three, five and twelve months of age. Pediatr Infect Dis J 14: Black, R. E., H. B. Greenberg, A. Z. Kapikian, K. H. Brown, and S. Becker Acquisition of serum antibody to Norwalk Virus and rotavirus and relation to diarrhea in a longitudinal study of young children in rural Bangladesh. J Infect Dis 145: Blackhall, J., R. Bellinzoni, N. Mattion, M. K. Estes, J. L. La Torre, and G. Magnusson A bovine rotavirus serotype 1: serologic characterization of the virus and nucleotide sequence determination of the structural glycoprotein VP7 gene. Virology 189: Blikslager, A. T., and M. C. Roberts Mechanisms of intestinal mucosal repair. J Am Vet Med Assoc 211: Blutt, S. E., C. D. Kirkwood, V. Parreno, K. L. Warfield, M. Ciarlet, M. K. Estes, K. Bok, R. F. Bishop, and M. E. Conner Rotavirus antigenaemia and viraemia: a common event? Lancet 362: Boa-Amponsem, K., E. A. Dunnington, and P. B. Siegel Antibody transmitting ability of hens from lines of chickens differing in response to SRBC antigen. Br Poult Sci 38: Bohl, E. H Comments on passive immunity in rotaviral infections. J Am Vet Med Assoc 173: Bohl, E. H., R. K. Gupta, M. V. Olquin, and L. J. Saif Antibody responses in serum, colostrum, and milk of swine after infection or vaccination with transmissible gastroenteritis virus. Infect Immun 6:

168 71. Bohl, E. H., E. M. Kohler, L. J. Saif, R. F. Cross, A. G. Agnes, and K. W. Theil Rotavirus as a cause of diarrhea in pigs. J Am Vet Med Assoc 172: Bohl, E. H., L. J. Saif, K. W. Theil, A. G. Agnes, and R. F. Cross Porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs. J Clin Microbiol 15: Bona, C., D. Radu, and T. Kodera Molecular studies on the diversification of hemagglutinin-specific human neonatal repertoire subsequent to immunization with naked DNA. Vaccine 22: Bonn, D New vaccines to fight killer rotavirus. Lancet Infect Dis 4: Bot, A., S. Antohi, S. Bot, A. Garcia-Sastre, and C. Bona Induction of humoral and cellular immunity against influenza virus by immunization of newborn mice with a plasmid bearing a hemagglutinin gene. Int Immunol 9: Bot, A., M. Shearer, S. Bot, C. Woods, J. Limmer, R. Kennedy, S. Casares, and C. Bona Induction of antibody response by DNA immunization of newborn baboons against influenza virus. Viral Immunol 12: Bottcher, M. F., M. C. Jenmalm, B. Bjorksten, and R. P. Garofalo Chemoattractant factors in breast milk from allergic and nonallergic mothers. Pediatr Res 47: Bourne, F. J., and J. Curtis The transfer of immunoglobins IgG, IgA and IgM from serum to colostrum and milk in the sow. Immunology 24: Bowman, E. P., N. A. Kuklin, K. R. Youngman, N. H. Lazarus, E. J. Kunkel, J. Pan, H. B. Greenberg, and E. C. Butcher The intestinal chemokine thymusexpressed chemokine (CCL25) attracts IgA antibody-secreting cells. J Exp Med 195: Boyaka, P. N., and J. R. McGhee Cytokines as adjuvants for the induction of mucosal immunity. Adv Drug Deliv Rev 51: Bradley, L. M., M. E. Malo, S. L. Tonkonogy, and S. R. Watson L- selectin is not essential for naive CD4 cell trafficking or development of primary responses in Peyer's patches. Eur J Immunol 27: Brambell, F. W The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet 2: Brambell, F. W. R The transmission of passive immunity from mother to young, Amsterdam, North Holland. 142

169 84. Brandtzaeg, P Research in gastrointestinal immunology. State of the art. Scand J Gastroenterol Suppl 114: Breese, J Presented at the 6th Canadian Immunization Conference, Montreal. 86. Bresee, J. S., R. I. Glass, B. Ivanoff, and J. R. Gentsch Current status and future priorities for rotavirus vaccine development, evaluation and implementation in developing countries. Vaccine 17: Bridger, J. C., W. Dhaliwal, M. J. Adamson, and C. R. Howard Determinants of rotavirus host range restriction--a heterologous bovine NSP1 gene does not affect replication kinetics in the pig. Virology 245: Bridger, J. C., G. A. Hall, and K. R. Parsons A study of the basis of virulence variation of bovine rotaviruses. Vet Microbiol 33: Brock, S. C., P. A. McGraw, P. F. Wright, and J. E. Crowe, Jr The human polymeric immunoglobulin receptor facilitates invasion of epithelial cells by Streptococcus pneumoniae in a strain-specific and cell type-specific manner. Infect Immun 70: Broom, A. K., M. J. Wallace, J. S. Mackenzie, D. W. Smith, and R. A. Hall Immunisation with gamma globulin to murray valley encephalitis virus and with an inactivated Japanese encephalitis virus vaccine as prophylaxis against australian encephalitis: evaluation in a mouse model. J Med Virol 61: Broome, R. L., P. T. Vo, R. L. Ward, H. F. Clark, and H. B. Greenberg Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence. J Virol 67: Brown, K. A., J. A. Kriss, C. A. Moser, W. J. Wenner, and P. A. Offit Circulating rotavirus-specific antibody-secreting cells (ASCs) predict the presence of rotavirus-specific ASCs in the human small intestinal lamina propria. J Infect Dis 182: Brown, K. A., and P. A. Offit Rotavirus-specific proteins are detected in murine macrophages in both intestinal and extraintestinal lymphoid tissues. Microb Pathog 24: Brown, M. A., P. Y. Rad, and M. J. Halonen Method of birth alters interferon-gamma and interleukin-12 production by cord blood mononuclear cells. Pediatr Allergy Immunol 14: Brown, P. J., and F. J. Bourne Development of immunoglobulincontaining cell populations in intestine, spleen, and mesenteric lymph node of the young pig, as demonstrated by peroxidase-conjugated antiserums. Am J Vet Res 37:

170 96. Brubaker, J. O., K. K. Macartney, T. J. Speaker, and P. A. Offit Specific attachment of aqueous-based microcapsules to macrophages, B cells, and dendritic cells in vitro. J Microencapsul 19: Brussow, H., H. Werchau, W. Liedtke, L. Lerner, C. Mietens, J. Sidoti, and J. Sotek Prevalence of antibodies to rotavirus in different age-groups of infants in Bochum, West Germany. J Infect Dis 157: Bryan, D. L., J. S. Hawkes, and R. A. Gibson Interleukin-12 in human milk. Pediatr Res 45: Buer, J., A. Lanoue, A. Franzke, C. Garcia, H. von Boehmer, and A. Sarukhan Interleukin 10 secretion and impaired effector function of major histocompatibility complex class II-restricted T cells anergized in vivo. J Exp Med 187: Buller, C. R., and R. A. Moxley Natural infection of porcine ileal dome M cells with rotavirus and enteric adenovirus. Vet Pathol 25: Bumstead, N., R. L. Reece, and J. K. Cook Genetic differences in susceptibility of chicken lines to infection with infectious bursal disease virus. Poult Sci 72: Burns, J. W., M. Siadat-Pajouh, A. A. Krishnaney, and H. B. Greenberg Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272: Burr, M. L., E. S. Limb, M. J. Maguire, L. Amarah, B. A. Eldridge, J. C. Layzell, and T. G. Merrett Infant feeding, wheezing, and allergy: a prospective study. Arch Dis Child 68: Burstyn, D. G., L. J. Baraff, M. S. Peppler, R. D. Leake, J. St Geme, Jr., and C. R. Manclark Serological response to filamentous hemagglutinin and lymphocytosis-promoting toxin of Bordetella pertussis. Infect Immun 41: Burzyn, D., J. C. Rassa, D. Kim, I. Nepomnaschy, S. R. Ross, and I. Piazzon Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus. J Virol 78: Butler, J. E., J. Sun, P. Weber, S. P. Ford, Z. Rehakova, J. Sinkora, and K. Lager Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification. J Immunol 167: Butler, J. E., J. Sun, P. Weber, P. Navarro, and D. Francis Antibody repertoire development in fetal and newborn piglets, III. Colonization of the 144

171 gastrointestinal tract selectively diversifies the preimmune repertoire in mucosal lymphoid tissues. Immunology 100: Butler, J. E., P. Weber, M. Sinkora, D. Baker, A. Schoenherr, B. Mayer, and D. Francis Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens. J Immunol 169: Butler, J. E. a. K., M.E Immunoglobulins and Immunocytes in mammary gland and its secretion, p In J. Mestecky, Lamm, M.E., Strober, W., Bienenstock, J., McGhee, J.R. and Mayer, L. (ed.), Mucosal Immunology, vol. 2. Elsevier Academic Press, San Diego Cadranel, S., S. Zeglache, T. Jonckheer, G. Zissis, F. E. Andre, H. Bogaerts, and A. Delem Factors affecting antibody response of newborns to repeated administrations of the rotavirus vaccine RIT J Pediatr Gastroenterol Nutr 6: Capano, G., K. J. Bloch, E. J. Schiffrin, J. A. Dascoli, E. J. Israel, and P. R. Harmatz Influence of the polyamine, spermidine, on intestinal maturation and dietary antigen uptake in the neonatal rat. J Pediatr Gastroenterol Nutr 19: Carbone, F. R., and W. R. Heath The role of dendritic cell subsets in immunity to viruses. Curr Opin Immunol 15: Cario, E., and D. K. Podolsky Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 68: Castello, A. A., M. L. Arvay, R. I. Glass, and J. Gentsch Rotavirus strain surveillance in Latin America: a review of the last nine years. Pediatr Infect Dis J 23:S Celada, F., and E. E. Sercarz Preferential pairing of T-B specificities in the same antigen: the concept of directional help. Vaccine 6: Cella, M., F. Facchetti, A. Lanzavecchia, and M. Colonna Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nat Immunol 1: Cepica, A., and J. B. Derbyshire The effect of adoptive transfer of mononuclear leukocytes from an adult donor on spontaneous cell-mediated cytotoxicity and resistance to transmissible gastroenteritis in neonatal piglets. Can J Comp Med 48: Ceyhan, M., G. Kanra, G. Secmeer, K. Midthun, B. L. Davidson, E. T. Zito, and T. Vesikari Take of rhesus-human reassortant tetravalent rotavirus vaccine in breast-fed infants. Acta Paediatr 82:

172 119. Chang, K. O., A. V. Parwani, and L. J. Saif Comparative nucleotide and deduced amino acid sequence analysis of VP7 gene of the NCDV Cody (I-801) strain of group A bovine rotavirus. Arch Virol 140: Chang, K. O., O. H. Vandal, L. Yuan, D. C. Hodgins, and L. J. Saif Antibody-secreting cell responses to rotavirus proteins in gnotobiotic pigs inoculated with attenuated or virulent human rotavirus. J Clin Microbiol 39: Chaouat, G., A. Assal Meliani, J. Martal, R. Raghupathy, J. Elliot, T. Mosmann, and T. G. Wegmann IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-tau. J Immunol 154: Chege, G. K., A. D. Steele, C. A. Hart, D. R. Snodgrass, E. O. Omolo, and J. M. Mwenda Experimental infection of non-human primates with a human rotavirus isolate. Vaccine 23: Chen, S. C., E. F. Fynan, H. B. Greenberg, and J. E. Herrmann Immunity obtained by gene-gun inoculation of a rotavirus DNA vaccine to the abdominal epidermis or anorectal epithelium. Vaccine 17: Chen, S. C., D. H. Jones, E. F. Fynan, G. H. Farrar, J. C. Clegg, H. B. Greenberg, and J. E. Herrmann Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles. J Virol 72: Chen, W. K., T. Campbell, J. VanCott, and L. J. Saif Enumeration of isotype-specific antibody-secreting cells derived from gnotobiotic piglets inoculated with porcine rotaviruses. Vet Immunol Immunopathol 45: Chiappini, E., C. Azzari, M. Moriondo, L. Galli, and M. de Martino Viraemia is a common finding in immunocompetent children with rotavirus infection. J Med Virol 76: Chiba, S., T. Yokoyama, S. Nakata, Y. Morita, T. Urasawa, K. Taniguchi, S. Urasawa, and T. Nakao Protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies. Lancet 2: Chimura, Y., M. Annaka, S. Shibazaki, K. Adachi, T. Shinkai, K. Sadamasu, Y. Noguchi, Y. Masuda, and T. Inamatsu [An epidemic of rotavirus infection in a nursing home for the elderly in Japan]. Kansenshogaku Zasshi 76: Chipeta, J., Y. Komada, X. L. Zhang, E. Azuma, H. Yamamoto, and M. Sakurai Neonatal (cord blood) T cells can competently raise type 1 and 2 immune responses upon polyclonal activation. Cell Immunol 205:

173 130. Choi, A. H., M. Basu, M. M. McNeal, J. D. Clements, and R. L. Ward Antibody-independent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein. J Virol 73: Choi, A. H., M. Basu, M. N. Rae, M. M. McNeal, and R. L. Ward Particle-bombardment-mediated DNA vaccination with rotavirus VP4 or VP7 induces high levels of serum rotavirus IgG but fails to protect mice against challenge. Virology 250: Choi, A. H., D. R. Knowlton, M. M. McNeal, and R. L. Ward Particle bombardment-mediated DNA vaccination with rotavirus VP6 induces high levels of serum rotavirus IgG but fails to protect mice against challenge. Virology 232: Choi, A. H., M. M. McNeal, M. Basu, J. A. Bean, J. L. VanCott, J. D. Clements, and R. L. Ward Functional mapping of protective epitopes within the rotavirus VP6 protein in mice belonging to different haplotypes. Vaccine 21: Choi, A. H., M. M. McNeal, M. Basu, J. A. Flint, S. C. Stone, J. D. Clements, J. A. Bean, S. A. Poe, J. L. VanCott, and R. L. Ward Intranasal or oral immunization of inbred and outbred mice with murine or human rotavirus VP6 proteins protects against viral shedding after challenge with murine rotaviruses. Vaccine 20: Choi, A. H., M. M. McNeal, M. Basu, and R. L. Ward Immunity to homologous rotavirus infection in adult mice: response. Trends Microbiol 8: Choi, A. H., M. M. McNeal, J. A. Flint, M. Basu, N. Y. Lycke, J. D. Clements, J. A. Bean, H. L. Davis, M. J. McCluskie, J. L. VanCott, and R. L. Ward The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant. Vaccine 20: Choi, A. H., K. Smiley, and M. Basu Induction of immune responses and partial protection in mice after skin immunization with rotavirus VP6 protein and the adjuvant LT(R192G). Vaccine 23: Cianga, P., C. Medesan, J. A. Richardson, V. Ghetie, and E. S. Ward Identification and function of neonatal Fc receptor in mammary gland of lactating mice. Eur J Immunol 29: Ciarlet, M., S. E. Crawford, C. Barone, A. Bertolotti-Ciarlet, R. F. Ramig, M. K. Estes, and M. E. Conner Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity. J Virol 72: Ciarlet, M., and M. K. Estes Human and most animal rotavirus strains do not require the presence of sialic acid on the cell surface for efficient infectivity. J Gen Virol 80 ( Pt 4):

174 141. Ciarlet, M., M. A. Gilger, C. Barone, M. McArthur, M. K. Estes, and M. E. Conner Rotavirus disease, but not infection and development of intestinal histopathological lesions, is age restricted in rabbits. Virology 251: Ciarlet, M., F. Liprandi, M. E. Conner, and M. K. Estes Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses. Arch Virol 145: Ciarlet, M., J. E. Ludert, M. Iturriza-Gomara, F. Liprandi, J. J. Gray, U. Desselberger, and M. K. Estes Initial interaction of rotavirus strains with N- acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype, not species of origin. J Virol 76: Clark, H. F., F. E. Borian, L. M. Bell, K. Modesto, V. Gouvea, and S. A. Plotkin Protective effect of WC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus season. J Infect Dis 158: Clark, H. F., C. J. Burke, D. B. Volkin, P. Offit, R. L. Ward, J. S. Bresee, P. Dennehy, W. M. Gooch, E. Malacaman, D. Matson, E. Walter, B. Watson, D. L. Krah, M. J. Dallas, F. Schodel, M. Kaplan, and P. Heaton Safety, immunogenicity and efficacy in healthy infants of G1 and G2 human reassortant rotavirus vaccine in a new stabilizer/buffer liquid formulation. Pediatr Infect Dis J 22: Clark, H. F., D. Lawley, D. Shrager, D. Jean-Guillaume, P. A. Offit, S. Y. Whang, J. J. Eiden, P. S. Bennett, K. M. Kaplan, and A. R. Shaw Infant immune response to human rotavirus serotype G1 vaccine candidate reassortant WI79-9: different dose response patterns to virus surface proteins VP7 and VP4. Pediatr Infect Dis J 23: Clemens, J. D., R. L. Ward, M. R. Rao, D. A. Sack, D. R. Knowlton, F. P. van Loon, S. Huda, M. McNeal, F. Ahmed, and G. Schiff Seroepidemiologic evaluation of antibodies to rotavirus as correlates of the risk of clinically significant rotavirus diarrhea in rural Bangladesh. J Infect Dis 165: Clement, L. T Isoforms of the CD45 common leukocyte antigen family: markers for human T-cell differentiation. J Clin Immunol 12: Coffman, R. L., H. F. Savelkoul, and D. A. Lebman Cytokine regulation of immunoglobulin isotype switching and expression. Semin Immunol 1: Colino, J., Y. Shen, and C. M. Snapper Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms. J Exp Med 195: Collins, J., W. G. Starkey, T. S. Wallis, G. J. Clarke, K. J. Worton, A. J. Spencer, S. J. Haddon, M. P. Osborne, D. C. Candy, and J. Stephen Intestinal 148

175 enzyme profiles in normal and rotavirus-infected mice. J Pediatr Gastroenterol Nutr 7: Collins, J. E., D. A. Benfield, and J. R. Duimstra Comparative virulence of two porcine group-a rotavirus isolates in gnotobiotic pigs. Am J Vet Res 50: Conner, M. E., S. E. Crawford, C. Barone, C. O'Neal, Y. J. Zhou, F. Fernandez, A. Parwani, L. J. Saif, J. Cohen, and M. K. Estes Rotavirus subunit vaccines. Arch Virol Suppl 12: Conner, M. E., M. K. Estes, and D. Y. Graham Rabbit model of rotavirus infection. J Virol 62: Conner, M. E., C. D. Zarley, B. Hu, S. Parsons, D. Drabinski, S. Greiner, R. Smith, B. Jiang, B. Corsaro, V. Barniak, H. P. Madore, S. Crawford, and M. K. Estes Virus-like particles as a rotavirus subunit vaccine. J Infect Dis 174 Suppl 1:S Cook, D. N., D. M. Prosser, R. Forster, J. Zhang, N. A. Kuklin, S. J. Abbondanzo, X. D. Niu, S. C. Chen, D. J. Manfra, M. T. Wiekowski, L. M. Sullivan, S. R. Smith, H. B. Greenberg, S. K. Narula, M. Lipp, and S. A. Lira CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue. Immunity 12: Cook, S. M., R. I. Glass, C. W. LeBaron, and M. S. Ho Global seasonality of rotavirus infections. Bull World Health Organ 68: Coste, A., J. Cohen, M. Reinhardt, J. P. Kraehenbuhl, and J. C. Sirard Nasal immunisation with Salmonella typhimurium producing rotavirus VP2 and VP6 antigens stimulates specific antibody response in serum and milk but fails to protect offspring. Vaccine 19: Coste, A., J. C. Sirard, K. Johansen, J. Cohen, and J. P. Kraehenbuhl Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea. J Virol 74: Coulie, P. G., and J. Van Snick Enhancement of IgG anti-carrier responses by IgG2 anti-hapten antibodies in mice. Eur J Immunol 15: Coulson, B. S., K. Grimwood, P. J. Masendycz, J. S. Lund, N. Mermelstein, R. F. Bishop, and G. L. Barnes Comparison of rotavirus immunoglobulin A coproconversion with other indices of rotavirus infection in a longitudinal study in childhood. J Clin Microbiol 28: Crawford, S. E., M. K. Estes, M. Ciarlet, C. Barone, C. M. O'Neal, J. Cohen, and M. E. Conner Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles. J Virol 73:

176 163. Crawley, A., C. Raymond, and B. N. Wilkie Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines. Vet Immunol Immunopathol 91: Crowe, J. E., Jr Influence of maternal antibodies on neonatal immunization against respiratory viruses. Clin Infect Dis 33: Cuadras, M. A., C. F. Arias, and S. Lopez Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle. J Virol 71: Cunliffe, N. A., W. Dove, J. E. Bunn, M. Ben Ramadam, J. W. Nyangao, R. L. Riveron, L. E. Cuevas, and C. A. Hart Expanding global distribution of rotavirus serotype G9: detection in Libya, Kenya, and Cuba. Emerg Infect Dis 7: Dardillat, J., G. Trillat, and P. Larvor Colostrum immunoglobulin concentration in cows: relationship with their calf mortality and with the colostrum quality of their female offspring. Ann Rech Vet 9: Das, M., S. J. Dunn, G. N. Woode, H. B. Greenberg, and C. D. Rao Both surface proteins (VP4 and VP7) of an asymptomatic neonatal rotavirus strain (I321) have high levels of sequence identity with the homologous proteins of a serotype 10 bovine rotavirus. Virology 194: Davidson, G. P., and G. L. Barnes Structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis. Acta Paediatr Scand 68: Davidson, G. P., D. G. Gall, M. Petric, D. G. Butler, and J. R. Hamilton Human rotavirus enteritis induced in conventional piglets. Intestinal structure and transport. J Clin Invest 60: De Leener, K., M. Rahman, J. Matthijnssens, L. Van Hoovels, T. Goegebuer, I. van der Donck, and M. Van Ranst Human infection with a P[14], G3 lapine rotavirus. Virology 325: De Vos, B., T. Vesikari, A. C. Linhares, B. Salinas, I. Perez-Schael, G. M. Ruiz-Palacios, L. Guerrero Mde, K. B. Phua, A. Delem, and K. Hardt A rotavirus vaccine for prophylaxis of infants against rotavirus gastroenteritis. Pediatr Infect Dis J 23:S de Zoysa, I., and R. G. Feachem Interventions for the control of diarrhoeal diseases among young children: rotavirus and cholera immunization. Bull World Health Organ 63: Debouck, P., and M. Pensaert Rotavirus excretion in suckling pigs followed under field circumstances. Ann Rech Vet 14:

177 175. Defrance, T., B. Vanbervliet, F. Briere, I. Durand, F. Rousset, and J. Banchereau Interleukin 10 and transforming growth factor beta cooperate to induce anti-cd40-activated naive human B cells to secrete immunoglobulin A. J Exp Med 175: Denisova, E., W. Dowling, R. LaMonica, R. Shaw, S. Scarlata, F. Ruggeri, and E. R. Mackow Rotavirus capsid protein VP5* permeabilizes membranes. J Virol 73: Dharakul, T., M. Riepenhoff-Talty, B. Albini, and P. L. Ogra Distribution of rotavirus antigen in intestinal lymphoid tissues: potential role in development of the mucosal immune response to rotavirus. Clin Exp Immunol 74: Dickinson, B. L., K. Badizadegan, Z. Wu, J. C. Ahouse, X. Zhu, N. E. Simister, R. S. Blumberg, and W. I. Lencer Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line. J Clin Invest 104: Dima, S., C. Medesan, G. Mota, I. Moraru, J. Sjoquist, and V. Ghetie Effect of protein A and its fragment B on the catabolic and Fc receptor sites of IgG. Eur J Immunol 13: Doroudchi, M., A. Samsami Dehaghani, K. Emad, and A. Ghaderi Placental transfer of rubella-specific IgG in fullterm and preterm newborns. Int J Gynaecol Obstet 81: Dubois, B., C. Massacrier, B. Vanbervliet, J. Fayette, F. Briere, J. Banchereau, and C. Caux Critical role of IL-12 in dendritic cell-induced differentiation of naive B lymphocytes. J Immunol 161: Durandy, A., G. De Saint Basile, B. Lisowska-Grospierre, J. F. Gauchat, M. Forveille, R. A. Kroczek, J. Y. Bonnefoy, and A. Fischer Undetectable CD40 ligand expression on T cells and low B cell responses to CD40 binding agonists in human newborns. J Immunol 154: Early, E. M., and D. J. Reen Antigen-independent responsiveness to interleukin-4 demonstrates differential regulation of newborn human T cells. Eur J Immunol 26: Ebina, T Prophylaxis of rotavirus gastroenteritis using immunoglobulin. Arch Virol Suppl 12: Eisenthal, A., A. Hassner, M. Shenav, S. Baron, and B. Lifschitz-Mercer Phenotype and function of lymphocytes from the neonatal umbilical cord compared to paired maternal peripheral blood cells isolated during delivery. Exp Mol Pathol 75:

178 186. El-Attar, L., W. Dhaliwal, C. R. Howard, and J. C. Bridger Rotavirus cross-species pathogenicity: molecular characterization of a bovine rotavirus pathogenic for pigs. Virology 291: Ellinger, I., A. Rothe, M. Grill, and R. Fuchs Apical to basolateral transcytosis and apical recycling of immunoglobulin G in trophoblast-derived BeWo cells: effects of low temperature, nocodazole, and cytochalasin D. Exp Cell Res 269: Elliott, S. R., P. J. Macardle, D. M. Roberton, and H. Zola Expression of the costimulator molecules, CD80, CD86, CD28, and CD152 on lymphocytes from neonates and young children. Hum Immunol 60: Elliott, S. R., D. M. Roberton, H. Zola, and P. J. Macardle Expression of the costimulator molecules, CD40 and CD154, on lymphocytes from neonates and young children. Hum Immunol 61: Englund, J. A., E. L. Anderson, G. F. Reed, M. D. Decker, K. M. Edwards, M. E. Pichichero, M. C. Steinhoff, M. B. Rennels, A. Deforest, and B. D. Meade The effect of maternal antibody on the serologic response and the incidence of adverse reactions after primary immunization with acellular and whole-cell pertussis vaccines combined with diphtheria and tetanus toxoids. Pediatrics 96: Enioutina, E. Y., D. Visic, and R. A. Daynes The induction of systemic and mucosal immune responses to antigen-adjuvant compositions administered into the skin: alterations in the migratory properties of dendritic cells appears to be important for stimulating mucosal immunity. Vaccine 18: Enriquez-Rincon, F., and G. G. Klaus Follicular trapping of haptenerythrocyte-antibody complexes in mouse spleen. Immunology 52: Eo, S. K., M. Gierynska, A. A. Kamar, and B. T. Rouse Prime-boost immunization with DNA vaccine: mucosal route of administration changes the rules. J Immunol 166: Eriksson, K., M. Quiding-Jarbrink, J. Osek, A. Moller, S. Bjork, J. Holmgren, and C. Czerkinsky Specific-antibody-secreting cells in the rectums and genital tracts of nonhuman primates following vaccination. Infect Immun 66: Espinoza, F., M. Paniagua, H. Hallander, L. Svensson, and O. Strannegard Rotavirus infections in young Nicaraguan children. Pediatr Infect Dis J 16: Estes, M. K Rotaviruses and Their Replication, p In D. M. Knipe, P.M. Howley (ed.), Fields Virology, Fourth ed, vol. 2. Lippincott Williams and Wilkins, Philadelphia. 152

179 197. Estes, M. K., and J. Cohen Rotavirus gene structure and function. Microbiol Rev 53: Estienne, V., C. Duthoit, M. Reichert, A. Praetor, P. Carayon, W. Hunziker, and J. Ruf Androgen-dependent expression of FcgammaRIIB2 by thyrocytes from patients with autoimmune Graves' disease: a possible molecular clue for sex dependence of autoimmune disease. Faseb J 16: Evans, A. T., G. Carandang, E. J. Quilligan, and T. C. Cesario Interferon responses in maternal and fetal mice. Am J Obstet Gynecol 152: Evans, T. G., M. J. McElrath, T. Matthews, D. Montefiori, K. Weinhold, M. Wolff, M. C. Keefer, E. G. Kallas, L. Corey, G. J. Gorse, R. Belshe, B. S. Graham, P. W. Spearman, D. Schwartz, M. J. Mulligan, P. Goepfert, P. Fast, P. Berman, M. Powell, and D. Francis QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immunization in humans. Vaccine 19: Fang, Z. Y., Q. Ye, M. S. Ho, H. Dong, S. Qing, M. E. Penaranda, T. Hung, L. Wen, and R. I. Glass Investigation of an outbreak of adult diarrhea rotavirus in China. J Infect Dis 160: Farstad, I. N., T. S. Halstensen, A. I. Lazarovits, J. Norstein, O. Fausa, and P. Brandtzaeg Human intestinal B-cell blasts and plasma cells express the mucosal homing receptor integrin alpha 4 beta 7. Scand J Immunol 42: Farstad, I. N., J. Norstein, and P. Brandtzaeg Phenotypes of B and T cells in human intestinal and mesenteric lymph. Gastroenterology 112: Fawaz, L. M., E. Sharif-Askari, and J. Menezes Up-regulation of NK cytotoxic activity via IL-15 induction by different viruses: a comparative study. J Immunol 163: Fawell, S., J. Seery, Y. Daikh, C. Moore, L. L. Chen, B. Pepinsky, and J. Barsoum Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci U S A 91: Fayette, J., I. Durand, J. M. Bridon, C. Arpin, B. Dubois, C. Caux, Y. J. Liu, J. Banchereau, and F. Briere Dendritic cells enhance the differentiation of naive B cells into plasma cells in vitro. Scand J Immunol 48: Feng, N., J. W. Burns, L. Bracy, and H. B. Greenberg Comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus. J Virol 68:

180 208. Feng, N., J. A. Lawton, J. Gilbert, N. Kuklin, P. Vo, B. V. Prasad, and H. B. Greenberg Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mab. J Clin Invest 109: Fernandez, F. M., M. E. Conner, D. C. Hodgins, A. V. Parwani, P. R. Nielsen, S. E. Crawford, M. K. Estes, and L. J. Saif Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines. Vaccine 16: Fernandez, F. M., M. E. Conner, A. V. Parwani, D. Todhunter, K. L. Smith, S. E. Crawford, M. K. Estes, and L. J. Saif Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP). Vaccine 14: Fiehring, C., H. J. Korting, and G. Jung [Rotavirus and malabsorption. Immunofluorescence microscopy studies of small intestine specimens]. Dtsch Z Verdau Stoffwechselkr 44: Filipp, D., K. Alizadeh-Khiavi, C. Richardson, A. Palma, N. Paredes, O. Takeuchi, S. Akira, and M. Julius Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation. Proc Natl Acad Sci U S A 98: Fiore, A. E., C. N. Shapiro, K. Sabin, K. Labonte, K. Darling, D. Culver, B. P. Bell, and H. S. Margolis Hepatitis A vaccination of infants: effect of maternal antibody status on antibody persistence and response to a booster dose. Pediatr Infect Dis J 22: Firan, M., R. Bawdon, C. Radu, R. J. Ober, D. Eaken, F. Antohe, V. Ghetie, and E. S. Ward The MHC class I-related receptor, FcRn, plays an essential role in the maternofetal transfer of gamma-globulin in humans. Int Immunol 13: Fischer, L., S. Barzu, C. Andreoni, N. Buisson, A. Brun, and J. C. Audonnet DNA vaccination of neonate piglets in the face of maternal immunity induces humoral memory and protection against a virulent pseudorabies virus challenge. Vaccine 21: Fischer, T. K., N. A. Page, D. D. Griffin, J. Eugen-Olsen, A. G. Pedersen, P. Valentiner-Branth, K. Molbak, H. Sommerfelt, and N. M. Nielsen Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections. Virology 311: Flores, J., I. Perez-Schael, M. Gonzalez, D. Garcia, M. Perez, N. Daoud, W. Cunto, R. M. Chanock, and A. Z. Kapikian Protection against severe rotavirus diarrhoea by rhesus rotavirus vaccine in Venezuelan infants. Lancet 1:

181 218. Fragoso, M., A. Kumar, and D. L. Murray Rotavirus in nasopharyngeal secretions of children with upper respiratory tract infections. Diagn Microbiol Infect Dis 4: Franco, M. A., and H. B. Greenberg Immunity to rotavirus infection in mice. J Infect Dis 179 Suppl 3:S Franco, M. A., and H. B. Greenberg Role of B cells and cytotoxic T lymphocytes in clearance of and immunity to rotavirus infection in mice. J Virol 69: Franco, M. A., C. Tin, and H. B. Greenberg CD8+ T cells can mediate almost complete short-term and partial long-term immunity to rotavirus in mice. J Virol 71: Fries, L. F., J. Tartaglia, J. Taylor, E. K. Kauffman, B. Meignier, E. Paoletti, and S. Plotkin Human safety and immunogenicity of a canarypox-rabies glycoprotein recombinant vaccine: an alternative poxvirus vector system. Vaccine 14: Fromantin, C., L. Piroth, I. Petitpas, P. Pothier, and E. Kohli Oral delivery of homologous and heterologous strains of rotavirus to BALB/c mice induces the same profile of cytokine production by spleen cells. Virology 244: Fu, Z. F., and D. J. Hampson Group A rotavirus excretion patterns in naturally infected pigs. Res Vet Sci 43: Fu, Z. F., D. J. Hampson, and C. R. Wilks Transfer of maternal antibody against group A rotavirus from sows to piglets and serological responses following natural infection. Res Vet Sci 48: Fujihashi, K., T. Taguchi, W. K. Aicher, J. R. McGhee, J. A. Bluestone, J. H. Eldridge, and H. Kiyono Immunoregulatory functions for murine intraepithelial lymphocytes: gamma/delta T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while alpha/beta TCR+ T cells provide B cell help. J Exp Med 175: Fulton, R. W., R. E. Briggs, M. E. Payton, A. W. Confer, J. T. Saliki, J. F. Ridpath, L. J. Burge, and G. C. Duff Maternally derived humoral immunity to bovine viral diarrhea virus (BVDV) 1a, BVDV1b, BVDV2, bovine herpesvirus-1, parainfluenza-3 virus bovine respiratory syncytial virus, Mannheimia haemolytica and Pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination. Vaccine 22: Funakoshi, S., T. Doi, T. Nakajima, T. Suyama, and M. Tokuda Antimicrobial effect of human serum IgA. Microbiol Immunol 26:

182 229. Furrie, E., R. E. Smith, M. W. Turner, S. Strobel, and A. M. Mowat Induction of local innate immune responses and modulation of antigen uptake as mechanisms underlying the mucosal adjuvant properties of immune stimulating complexes (ISCOMS). Vaccine 20: Gans, H., R. DeHovitz, B. Forghani, J. Beeler, Y. Maldonado, and A. M. Arvin Measles and mumps vaccination as a model to investigate the developing immune system: passive and active immunity during the first year of life. Vaccine 21: Garofalo, R., S. Chheda, F. Mei, K. H. Palkowetz, H. E. Rudloff, F. C. Schmalstieg, D. K. Rassin, and A. S. Goldman Interleukin-10 in human milk. Pediatr Res 37: Gasparoni, A., L. Ciardelli, A. Avanzini, A. M. Castellazzi, R. Carini, G. Rondini, and G. Chirico Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults. Biol Neonate 84: Gelberg, H. B Studies on the age resistance of swine to group A rotavirus infection. Vet Pathol 29: Geneste, C., S. Iscaki, R. Mangalo, and J. Pillot Both Fc alpha domains of human IgA are involved in in vitro interaction between secretory component and dimeric IgA. Immunol Lett 13: Gentsch, J. R., B. K. Das, B. Jiang, M. K. Bhan, and R. I. Glass Similarity of the VP4 protein of human rotavirus strain 116E to that of the bovine B223 strain. Virology 194: Gerdts, V., L. A. Babiuk, H. van Drunen Littel-van den, and P. J. Griebel Fetal immunization by a DNA vaccine delivered into the oral cavity. Nat Med 6: Gerdts, V., M. Snider, R. Brownlie, L. A. Babiuk, and P. J. Griebel Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate. J Immunol 168: Gerli, R., A. Bertotto, S. Crupi, C. Arcangeli, I. Marinelli, F. Spinozzi, C. Cernetti, P. Angelella, and P. Rambotti Activation of cord T lymphocytes. I. Evidence for a defective T cell mitogenesis induced through the CD2 molecule. J Immunol 142: Gerna, G., A. Sarasini, M. Parea, S. Arista, P. Miranda, H. Brussow, Y. Hoshino, and J. Flores Isolation and characterization of two distinct human rotavirus strains with G6 specificity. J Clin Microbiol 30:

183 240. Gerna, G., A. D. Steele, Y. Hoshino, M. Sereno, D. Garcia, A. Sarasini, and J. Flores A comparison of the VP7 gene sequences of human and bovine rotaviruses. J Gen Virol 75 ( Pt 7): Ghetie, V., J. G. Hubbard, J. K. Kim, M. F. Tsen, Y. Lee, and E. S. Ward Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur J Immunol 26: Ghetie, V., and E. S. Ward FcRn: the MHC class I-related receptor that is more than an IgG transporter. Immunol Today 18: Gil, M. T., C. O. de Souza, M. Asensi, and J. Buesa Homotypic protection against rotavirus-induced diarrhea in infant mice breast-fed by dams immunized with the recombinant VP8* subunit of the VP4 capsid protein. Viral Immunol 13: Gilger, M. A., D. O. Matson, M. E. Conner, H. M. Rosenblatt, M. J. Finegold, and M. K. Estes Extraintestinal rotavirus infections in children with immunodeficiency. J Pediatr 120: Gitlin, L., and R. Andino Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing. J Virol 77: Goldman, A. S The immune system of human milk: antimicrobial, antiinflammatory and immunomodulating properties. Pediatr Infect Dis J 12: Goldman, A. S., S. Chheda, R. Garofalo, and F. C. Schmalstieg Cytokines in human milk: properties and potential effects upon the mammary gland and the neonate. J Mammary Gland Biol Neoplasia 1: Gonzalez, A. M., M. C. Jaimes, I. Cajiao, O. L. Rojas, J. Cohen, P. Pothier, E. Kohli, E. C. Butcher, H. B. Greenberg, J. Angel, and M. A. Franco Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha4beta7. Virology 305: Gonzalez, A. M., T. V. Nguyen, M. S. Azevedo, K. Jeong, F. Agarib, C. Iosef, K. Chang, K. Lovgren-Bengtsson, B. Morein, and L. J. Saif Antibody responses to human rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM. Clin Exp Immunol 135: Gonzalez, R. A., R. Espinosa, P. Romero, S. Lopez, and C. F. Arias Relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells. Arch Virol 145:

184 251. Gorziglia, M., K. Nishikawa, Y. Hoshino, and K. Taniguchi Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains. J Virol 64: Gothefors, L., G. Wadell, P. Juto, K. Taniguchi, A. Z. Kapikian, and R. I. Glass Prolonged efficacy of rhesus rotavirus vaccine in Swedish children. J Infect Dis 159: Goto, Y., and K. Aki Interaction of the fluorescence-labeled secretory component with human polymeric immunoglobulins. Biochemistry 23: Gouvea, V., and M. Brantly Is rotavirus a population of reassortants? Trends Microbiol 3: Graham, D. Y., and M. K. Estes Pathogenesis and treatment of rotavirus diarrhea. Gastroenterology 101: Graham, K. L., P. Halasz, Y. Tan, M. J. Hewish, Y. Takada, E. R. Mackow, M. K. Robinson, and B. S. Coulson Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaxbeta2 and alphavbeta3 by using VP7 during cell entry. J Virol 77: Green, K. Y., and A. Z. Kapikian Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers. J Virol 66: Greenberg, H. B., H. F. Clark, and P. A. Offit Rotavirus pathology and pathophysiology. Curr Top Microbiol Immunol 185: Grindstaff, J. L., E. D. Brodie, 3rd, and E. D. Ketterson Immune function across generations: integrating mechanism and evolutionary process in maternal antibody transmission. Proc R Soc Lond B Biol Sci 270: Guerrero, C. A., D. Bouyssounade, S. Zarate, P. Isa, T. Lopez, R. Espinosa, P. Romero, E. Mendez, S. Lopez, and C. F. Arias Heat shock cognate protein 70 is involved in rotavirus cell entry. J Virol 76: Guerrero, C. A., E. Mendez, S. Zarate, P. Isa, S. Lopez, and C. F. Arias Integrin alpha(v)beta(3) mediates rotavirus cell entry. Proc Natl Acad Sci U S A 97: Guo, C. T., O. Nakagomi, M. Mochizuki, H. Ishida, M. Kiso, Y. Ohta, T. Suzuki, D. Miyamoto, K. I. Hidari, and Y. Suzuki Ganglioside GM(1a) on the cell surface is involved in the infection by human rotavirus KUN and MO strains. J Biochem (Tokyo) 126: Haffejee, I. E Neonatal rotavirus infections. Rev Infect Dis 13:

185 264. Hagglund, S., K. F. Hu, L. E. Larsen, M. Hakhverdyan, J. F. Valarcher, G. Taylor, B. Morein, S. Belak, and S. Alenius Bovine respiratory syncytial virus ISCOMs--protection in the presence of maternal antibodies. Vaccine 23: Halaihel, N., V. Lievin, J. M. Ball, M. K. Estes, F. Alvarado, and M. Vasseur Direct inhibitory effect of rotavirus NSP4( ) peptide on the Na(+)-Dglucose symporter of rabbit intestinal brush border membrane. J Virol 74: Hall, G. A., J. C. Bridger, R. L. Chandler, and G. N. Woode Gnotobiotic piglets experimentally infected with neonatal calf diarrhoea reovirus-like agent (Rotavirus). Vet Pathol 13: Halstensen, T. S., I. N. Farstad, H. Scott, O. Fausa, and P. Brandtzaeg Intraepithelial TcR alpha/beta+ lymphocytes express CD45RO more often than the TcR gamma/delta+ counterparts in coeliac disease. Immunology 71: Halvorsrud, J., and I. Orstavik An epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly. Scand J Infect Dis 12: Hanson, L. A., S. Ahlstedt, B. Andersson, J. R. Cruz, U. Dahlgren, S. P. Fallstrom, O. Porras, C. Svanborg Eden, T. Soderstrom, and B. Wettergren The immune response of the mammary gland and its significance for the neonate. Ann Allergy 53: Hanson, L. A., L. Ceafalau, I. Mattsby-Baltzer, M. Lagerberg, A. Hjalmarsson, R. Ashraf, S. Zaman, and F. Jalil The mammary gland-infant intestine immunologic dyad. Adv Exp Med Biol 478: Harju, K., V. Glumoff, and M. Hallman Ontogeny of Toll-like receptors Tlr2 and Tlr4 in mice. Pediatr Res 49: Harokopakis, E., G. Hajishengallis, and S. M. Michalek Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system. Infect Immun 66: Harp, J. A., and H. W. Moon Lymphocyte localization in lymph nodes of swine: changes induced by lactation. Vet Immunol Immunopathol 18: Harp, J. A., P. L. Runnels, and B. A. Pesch Lymphocyte recirculation in cattle: patterns of localization by mammary and mesenteric lymph node lymphocytes. Vet Immunol Immunopathol 20: Harriman, G. R., A. Bradley, S. Das, P. Rogers-Fani, and A. C. Davis IgA class switch in I alpha exon-deficient mice. Role of germline transcription in class switch recombination. J Clin Invest 97:

186 276. Hassan, J., and D. J. Reen Cord blood CD4+ CD45RA+ T cells achieve a lower magnitude of activation when compared with their adult counterparts. Immunology 90: Hassan, M. M., N. Mostafa, M. Sultan, and M. Gaafar Idiotypic regulation in Schistosomiasis mansoni. J Egypt Soc Parasitol 29: Hassett, D. E., J. Zhang, and J. L. Whitton Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge. J Virol 71: Hawkes, J. S., D. L. Bryan, M. J. James, and R. A. Gibson Cytokines (IL-1beta, IL-6, TNF-alpha, TGF-beta1, and TGF-beta2) and prostaglandin E2 in human milk during the first three months postpartum. Pediatr Res 46: Haymann, J. P., J. P. Levraud, S. Bouet, V. Kappes, J. Hagege, G. Nguyen, Y. Xu, E. Rondeau, and J. D. Sraer Characterization and localization of the neonatal Fc receptor in adult human kidney. J Am Soc Nephrol 11: Haynes, L. M., D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 75: Heikkinen, J., M. Mottonen, A. Alanen, and O. Lassila Phenotypic characterization of regulatory T cells in the human decidua. Clin Exp Immunol 136: Herrmann, J. E., S. C. Chen, D. H. Jones, A. Tinsley-Bown, E. F. Fynan, H. B. Greenberg, and G. H. Farrar Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles. Virology 259: Heyman, B Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu Rev Immunol 18: Hines, H. C Immunology and Immunogenetics. The Ohio State University Hiroi, T., H. Goto, K. Someya, M. Yanagita, M. Honda, N. Yamanaka, and H. Kiyono HIV mucosal vaccine: nasal immunization with rbcg-v3j1 induces a long term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient conditions. J Immunol 167: Hjelt, K., P. C. Grauballe, A. Paerregaard, O. H. Nielsen, and P. A. Krasilnikoff Protective effect of preexisting rotavirus-specific immunoglobulin A against naturally acquired rotavirus infection in children. J Med Virol 21:

187 288. Hjelt, K., P. C. Grauballe, P. O. Schiotz, L. Andersen, and P. A. Krasilnikoff Intestinal and serum immune response to a naturally acquired rotavirus gastroenteritis in children. J Pediatr Gastroenterol Nutr 4: Hodgins, D. C., S. Y. Kang, L. dearriba, V. Parreno, L. A. Ward, L. Yuan, T. To, and L. J. Saif Effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs. J Virol 73: Hoffman, S. L., R. Edelman, J. P. Bryan, I. Schneider, J. Davis, M. Sedegah, D. Gordon, P. Church, M. Gross, C. Silverman, and et al Safety, immunogenicity, and efficacy of a malaria sporozoite vaccine administered with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane as adjuvant. Am J Trop Med Hyg 51: Holmberg, D., A. Andersson, L. Carlsson, and S. Forsgren Establishment and functional implications of B-cell connectivity. Immunol Rev 110: Holmberg, D., S. Forsgren, F. Ivars, and A. Coutinho Reactions among IgM antibodies derived from normal, neonatal mice. Eur J Immunol 14: Holmberg, D., G. Wennerstrom, L. Andrade, and A. Coutinho The high idiotypic connectivity of "natural" newborn antibodies is not found in adult mitogenreactive B cell repertoires. Eur J Immunol 16: Holmes, I. H., and R. F. Bishop Letter: Naming viruses. Lancet 1: Holmgren, J., and C. Czerkinsky Mucosal immunity and vaccines. Nat Med 11:S Hong, T Human group B rotavirus: adult diarrhea rotavirus. Chin Med J (Engl) 109: Horie, Y., O. Masamune, and O. Nakagomi Three major alleles of rotavirus NSP4 proteins identified by sequence analysis. J Gen Virol 78 ( Pt 9): Horie, Y., O. Nakagomi, Y. Koshimura, T. Nakagomi, Y. Suzuki, T. Oka, S. Sasaki, Y. Matsuda, and S. Watanabe Diarrhea induction by rotavirus NSP4 in the homologous mouse model system. Virology 262: Hornef, M. W., T. Frisan, A. Vandewalle, S. Normark, and A. Richter- Dahlfors Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells. J Exp Med 195: Hoshino, Y., R. W. Jones, R. M. Chanock, and A. Z. Kapikian Generation and characterization of six single VP4 gene substitution reassortant rotavirus 161

188 vaccine candidates: each bears a single human rotavirus VP4 gene encoding P serotype 1A[8] or 1B[4] and the remaining 10 genes of rhesus monkey rotavirus MMU18006 or bovine rotavirus UK. Vaccine 20: Hoshino, Y., R. W. Jones, and A. Z. Kapikian Characterization of neutralization specificities of outer capsid spike protein VP4 of selected murine, lapine, and human rotavirus strains. Virology 299: Hoshino, Y., R. W. Jones, J. Ross, S. Honma, N. Santos, J. R. Gentsch, and A. Z. Kapikian Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3. J Virol 78: Hoshino, Y., and A. Z. Kapikian Classification of rotavirus VP4 and VP7 serotypes. Arch Virol Suppl 12: Hoshino, Y., A. Z. Kapikian, and R. M. Chanock Selection of coldadapted mutants of human rotaviruses that exhibit various degrees of growth restriction in vitro. J Virol 68: Hoshino, Y., L. J. Saif, S. Y. Kang, M. M. Sereno, W. K. Chen, and A. Z. Kapikian Identification of group A rotavirus genes associated with virulence of a porcine rotavirus and host range restriction of a human rotavirus in the gnotobiotic piglet model. Virology 209: Hoshino, Y., L. J. Saif, M. M. Sereno, R. M. Chanock, and A. Z. Kapikian Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J Virol 62: Hubschle, O. J., G. Tjipura-Zaire, I. Abusugra, G. di Francesca, F. Mettler, A. Pini, and B. Morein Experimental field trial with an immunostimulating complex (ISCOM) vaccine against contagious bovine pleuropneumonia. J Vet Med B Infect Dis Vet Public Health 50: Hung, T., G. M. Chen, C. G. Wang, H. L. Yao, Z. Y. Fang, T. X. Chao, Z. Y. Chou, W. Ye, X. J. Chang, S. S. Den, and et al Waterborne outbreak of rotavirus diarrhoea in adults in China caused by a novel rotavirus. Lancet 1: Hunt, J. S., D. Vassmer, T. A. Ferguson, and L. Miller Fas ligand is positioned in mouse uterus and placenta to prevent trafficking of activated leukocytes between the mother and the conceptus. J Immunol 158: Hunter, P The performance of southern African territories serotypes of foot and mouth disease antigen in oil-adjuvanted vaccines. Rev Sci Tech 15:

189 311. Hyams, J. S., P. J. Krause, and P. A. Gleason Lactose malabsorption following rotavirus infection in young children. J Pediatr 99: Iizuka, M., E. Kaga, M. Chiba, O. Masamune, G. Gerna, and O. Nakagomi Serotype G6 human rotavirus sharing a conserved genetic constellation with natural reassortants between members of the bovine and AU-1 genogroups. Arch Virol 135: Iosef, C., K. O. Chang, M. S. Azevedo, and L. J. Saif Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pig model of human rotavirus disease. J Med Virol 68: Iosef, C., T. Van Nguyen, K. Jeong, K. Bengtsson, B. Morein, Y. Kim, K. O. Chang, M. S. Azevedo, L. Yuan, P. Nielsen, and L. J. Saif Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-like-particles associated with immunostimulating complexes. Vaccine 20: Ishida, S. I., N. Feng, J. M. Gilbert, B. Tang, and H. B. Greenberg Immune responses to individual rotavirus proteins following heterologous and homologous rotavirus infection in mice. J Infect Dis 175: Israel, E. J., S. Taylor, Z. Wu, E. Mizoguchi, R. S. Blumberg, A. Bhan, and N. E. Simister Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells. Immunology 92: Iturriza Gomara, M., G. Kang, A. Mammen, A. K. Jana, M. Abraham, U. Desselberger, D. Brown, and J. Gray Characterization of G10P[11] rotaviruses causing acute gastroenteritis in neonates and infants in Vellore, India. J Clin Microbiol 42: Iwasaki, A., and B. L. Kelsall Mucosal immunity and inflammation. I. Mucosal dendritic cells: their specialized role in initiating T cell responses. Am J Physiol 276:G Jackson, D. W., G. R. Law, and C. F. Nockels Maternal vitamin E alters passively acquired immunity of chicks. Poult Sci 57: Jagannath, M. R., R. R. Vethanayagam, B. S. Reddy, S. Raman, and C. D. Rao Characterization of human symptomatic rotavirus isolates MP409 and MP480 having 'long' RNA electropherotype and subgroup I specificity, highly related to the P6[1],G8 type bovine rotavirus A5, from Mysore, India. Arch Virol 145: Jaimes, M. C., O. L. Rojas, E. J. Kunkel, N. H. Lazarus, D. Soler, E. C. Butcher, D. Bass, J. Angel, M. A. Franco, and H. B. Greenberg Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children. J Virol 78:

190 322. James, S. P., and A. S. Graeff Effect of IL-2 on immunoregulatory function of intestinal lamina propria T cells in normal non-human primates. Clin Exp Immunol 70: Janke, B. H., J. K. Nelson, D. A. Benfield, and E. A. Nelson Relative prevalence of typical and atypical strains among rotaviruses from diarrheic pigs in conventional swine herds. J Vet Diagn Invest 2: Jayaram, H., M. K. Estes, and B. V. Prasad Emerging themes in rotavirus cell entry, genome organization, transcription and replication. Virus Res 101: Jessup, C. F., J. Ridings, A. Ho, S. Nobbs, D. M. Roberton, P. Macardle, and H. Zola The Fc receptor for IgG (Fc gamma RII; CD32) on human neonatal B lymphocytes. Hum Immunol 62: Jiang, B., V. Barniak, R. P. Smith, R. Sharma, B. Corsaro, B. Hu, and H. P. Madore Synthesis of rotavirus-like particles in insect cells: comparative and quantitative analysis. Biotechnol Bioeng 60: Jiang, B., M. K. Estes, C. Barone, V. Barniak, C. M. O'Neal, A. Ottaiano, H. P. Madore, and M. E. Conner Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles. Vaccine 17: Jiang, B., J. R. Gentsch, and R. I. Glass The role of serum antibodies in the protection against rotavirus disease: an overview. Clin Infect Dis 34: Jiang, B., L. Snipes-Magaldi, P. Dennehy, H. Keyserling, R. C. Holman, J. Bresee, J. Gentsch, and R. I. Glass Cytokines as mediators for or effectors against rotavirus disease in children. Clin Diagn Lab Immunol 10: Jiang, B. M., L. J. Saif, S. Y. Kang, and J. H. Kim Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus. J Virol 64: Jin, Q., R. L. Ward, D. R. Knowlton, Y. B. Gabbay, A. C. Linhares, R. Rappaport, P. A. Woods, R. I. Glass, and J. R. Gentsch Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses. Arch Virol 141: Johansen, K., J. Hinkula, F. Espinoza, M. Levi, C. Zeng, U. Ruden, T. Vesikari, M. Estes, and L. Svensson Humoral and cell-mediated immune responses in humans to the NSP4 enterotoxin of rotavirus. J Med Virol 59: Johansen, K., U. Schroder, and L. Svensson Immunogenicity and protective efficacy of a formalin-inactivated rotavirus vaccine combined with lipid adjuvants. Vaccine 21:

191 334. Johansson, E. L., C. Bergquist, A. Edebo, C. Johansson, and A. M. Svennerholm Comparison of different routes of vaccination for eliciting antibody responses in the human stomach. Vaccine 22: Johnson, S., W. D. Sypura, D. N. Gerding, S. L. Ewing, and E. N. Janoff Selective neutralization of a bacterial enterotoxin by serum immunoglobulin A in response to mucosal disease. Infect Immun 63: Jones, C. A., J. A. Holloway, and J. O. Warner Phenotype of fetal monocytes and B lymphocytes during the third trimester of pregnancy. J Reprod Immunol 56: Jones, E. A., and T. A. Waldmann The mechanism of intestinal uptake and transcellular transport of IgG in the neonatal rat. J Clin Invest 51: Jones, S. L., and A. T. Blikslager Role of the enteric nervous system in the pathophysiology of secretory diarrhea. J Vet Intern Med 16: Junghans, R. P., and C. L. Anderson The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor. Proc Natl Acad Sci U S A 93: Jurk, M., F. Heil, J. Vollmer, C. Schetter, A. M. Krieg, H. Wagner, G. Lipford, and S. Bauer Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 3: Kacskovics, I Fc receptors in livestock species. Vet Immunol Immunopathol 102: Kacskovics, I., Z. Wu, N. E. Simister, L. V. Frenyo, and L. Hammarstrom Cloning and characterization of the bovine MHC class I-like Fc receptor. J Immunol 164: Kadowaki, N., S. Ho, S. Antonenko, R. W. Malefyt, R. A. Kastelein, F. Bazan, and Y. J. Liu Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 194: Kalliomaki, M., A. Ouwehand, H. Arvilommi, P. Kero, and E. Isolauri Transforming growth factor-beta in breast milk: a potential regulator of atopic disease at an early age. J Allergy Clin Immunol 104: Kapikian, A. Z A rotavirus vaccine for prevention of severe diarrhoea of infants and young children: development, utilization and withdrawal. Novartis Found Symp 238:153-71; discussion Kapikian, A. Z Rotaviruses. In D. M. Knipe, Howley, P.M. (ed.), Fields Virology, 4th ed, vol. 1. Lippincott Williams and Wilkins, Philadelphia. 165

192 347. Kapikian, A. Z., R. G. Wyatt, M. M. Levine, R. H. Yolken, D. H. VanKirk, R. Dolin, H. B. Greenberg, and R. M. Chanock Oral administration of human rotavirus to volunteers: induction of illness and correlates of resistance. J Infect Dis 147: Karlsson, M. C., S. Wernersson, T. Diaz de Stahl, S. Gustavsson, and B. Heyman Efficient IgG-mediated suppression of primary antibody responses in Fcgamma receptor-deficient mice. Proc Natl Acad Sci U S A 96: Kattoura, M. D., X. Chen, and J. T. Patton The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase. Virology 202: Kauppi-Korkeila, M., L. van Alphen, D. Madore, L. Saarinen, and H. Kayhty Mechanism of antibody-mediated reduction of nasopharyngeal colonization by Haemophilus influenzae type b studied in an infant rat model. J Infect Dis 174: Kawahara, M., A. Hashimoto, I. Toida, and M. Honda Oral recombinant Mycobacterium bovis bacillus Calmette-Guerin expressing HIV-1 antigens as a freeze-dried vaccine induces long-term, HIV-specific mucosal and systemic immunity. Clin Immunol 105: Kawahara, T., Y. Kuwano, S. Teshima-Kondo, T. Kawai, T. Nikawa, K. Kishi, and K. Rokutan Toll-like receptor 4 regulates gastric pit cell responses to Helicobacter pylori infection. J Med Invest 48: Kawanishi, H., and W. Strober Regulatory T-cells in murine Peyer's patches directing IgA-specific isotype switching. Ann N Y Acad Sci 409: Kawasaki, K., S. Akashi, R. Shimazu, T. Yoshida, K. Miyake, and M. Nishijima Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharidemimetic signal transduction by Taxol. J Biol Chem 275: Kelsall, B. L., W.Strober Gut-associated lymphoid tissue: Antigen handling and T-lymphocyte responses, p In P. L. Ogra, Mestecky, J., Lamm, M.E., Strober, W., Bienenstock, J. and McGhee, J.R. (ed.), Mucosal Immunology, Second ed, vol. 1. Academic Press, San Diego-London Kenney, R. T., D. L. Sacks, J. P. Sypek, L. Vilela, A. A. Gam, and K. Evans- Davis Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis. J Immunol 163: Kernodle, D. S., R. K. Voladri, B. E. Menzies, C. C. Hager, and K. M. Edwards Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model. Infect Immun 65:

193 358. Kilshaw, P. J., and S. J. Murant Expression and regulation of beta 7(beta p) integrins on mouse lymphocytes: relevance to the mucosal immune system. Eur J Immunol 21: Kim, B., T. Bowersock, P. Griebel, A. Kidane, L. A. Babiuk, M. Sanchez, S. Attah-Poku, R. S. Kaushik, and G. K. Mutwiri Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres. J Control Release 85: Kim, C. K., and S. J. Lim Recent progress in drug delivery systems for anticancer agents. Arch Pharm Res 25: Kim, J. J., L. K. Nottingham, A. Tsai, D. J. Lee, H. C. Maguire, J. Oh, T. Dentchev, K. H. Manson, M. S. Wyand, M. G. Agadjanyan, K. E. Ugen, and D. B. Weiner Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-gamma, IL-12, or IL-18 gene adjuvants. J Med Primatol 28: Kim, K. J., T. E. Fandy, V. H. Lee, D. K. Ann, Z. Borok, and E. D. Crandall Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers. Am J Physiol Lung Cell Mol Physiol 287:L Kim, S. K., G. Ragupathi, S. Cappello, E. Kagan, and P. O. Livingston Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates. Vaccine 19: Kim, T. G., and W. H. Langridge Synthesis of an HIV-1 Tat transduction domain-rotavirus enterotoxin fusion protein in transgenic potato. Plant Cell Rep 22: Kim, Y., K. O. Chang, W. Y. Kim, and L. J. Saif Production of hybrid double- or triple-layered virus-like particles of group A and C rotaviruses using a baculovirus expression system. Virology 302: Kim, Y., P. R. Nielsen, D. Hodgins, K. O. Chang, and L. J. Saif Lactogenic antibody responses in cows vaccinated with recombinant bovine rotaviruslike particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines. Vaccine 20: King, D., D. Page, K. A. Schat, and B. W. Calnek Difference between influences of homologous and heterologous maternal antibodies on response to serotype- 2 and serotype-3 Marek's disease vaccines. Avian Dis 25: Kirkwood, C. D., J. R. Gentsch, Y. Hoshino, H. F. Clark, and R. I. Glass Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the United States. Virology 256:

194 369. Kiyono, H., and S. Fukuyama NALT- versus Peyer's-patch-mediated mucosal immunity. Nat Rev Immunol 4: Klinman, D. M., F. Takeshita, I. Gursel, C. Leifer, K. J. Ishii, D. Verthelyi, and M. Gursel CpG DNA: recognition by and activation of monocytes. Microbes Infect 4: Kobayashi, M., J. Thompson, S. J. Tollefson, G. W. Reed, and P. F. Wright Tetravalent rhesus rotavirus vaccine in young infants. J Infect Dis 170: Kobayashi, N., Y. Suzuki, T. Tsuge, K. Okumura, C. Ra, and Y. Tomino FcRn-mediated transcytosis of immunoglobulin G in human renal proximal tubular epithelial cells. Am J Physiol Renal Physiol 282:F Kohler, T., U. Erben, H. Wiedersberg, and N. Bannert [Histological findings of the small intestinal mucosa in rotavirus infections in infants and young children]. Kinderarztl Prax 58: Kosek, M., C. Bern, and R. L. Guerrant The global burden of diarrhoeal disease, as estimated from studies published between 1992 and Bull World Health Organ 81: Kovarik, J., and C. A. Siegrist Optimization of vaccine responses in early life: the role of delivery systems and immunomodulators. Immunol Cell Biol 76: Kozlowski, P. A., S. Cu-Uvin, M. R. Neutra, and T. P. Flanigan Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect Immun 65: Krieg, A. M CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20: Kriesel, J. D., and J. Spruance Calcitriol (1,25-dihydroxy-vitamin D3) coadministered with influenza vaccine does not enhance humoral immunity in human volunteers. Vaccine 17: Krishnan, L., C. J. Dicaire, G. B. Patel, and G. D. Sprott Archaeosome vaccine adjuvants induce strong humoral, cell-mediated, and memory responses: comparison to conventional liposomes and alum. Infect Immun 68: Krishnan, L., S. Sad, G. B. Patel, and G. D. Sprott Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help. J Immunol 165:

195 381. Krishnan, L., S. Sad, G. B. Patel, and G. D. Sprott The potent adjuvant activity of archaeosomes correlates to the recruitment and activation of macrophages and dendritic cells in vivo. J Immunol 166: Kruse, P. E The importance of colostral immunoglobulins and their absorption from the intestine of the newborn animals. Ann Rech Vet 14: Kucharzik, T., J. T. Hudson, 3rd, R. L. Waikel, W. D. Martin, and I. R. Williams CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock-in mouse. Eur J Immunol 32: Kumar, M., A. K. Behera, H. Matsuse, R. F. Lockey, and S. S. Mohapatra A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice. Immunology 97: Kunkel, E. J., and E. C. Butcher Plasma-cell homing. Nat Rev Immunol 3: Kunkel, E. J., D. J. Campbell, and E. C. Butcher Chemokines in lymphocyte trafficking and intestinal immunity. Microcirculation 10: Kunkel, E. J., J. J. Campbell, G. Haraldsen, J. Pan, J. Boisvert, A. I. Roberts, E. C. Ebert, M. A. Vierra, S. B. Goodman, M. C. Genovese, A. J. Wardlaw, H. B. Greenberg, C. M. Parker, E. C. Butcher, D. P. Andrew, and W. W. Agace Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J Exp Med 192: Kunkel, E. J., C. H. Kim, N. H. Lazarus, M. A. Vierra, D. Soler, E. P. Bowman, and E. C. Butcher CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J Clin Invest 111: Kurt-Jones, E. A., L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 1: Kvistgaard, A. S., L. T. Pallesen, C. F. Arias, S. Lopez, T. E. Petersen, C. W. Heegaard, and J. T. Rasmussen Inhibitory effects of human and bovine milk constituents on rotavirus infections. J Dairy Sci 87: Lachman, L. B., B. Ozpolat, and X. M. Rao Cytokine-containing liposomes as vaccine adjuvants. Eur Cytokine Netw 7:

196 392. Laegreid, W. W., M. P. Heaton, J. E. Keen, W. M. Grosse, C. G. Chitko- McKown, T. P. Smith, J. W. Keele, G. L. Bennett, and T. E. Besser Association of bovine neonatal Fc receptor alpha-chain gene (FCGRT) haplotypes with serum IgG concentration in newborn calves. Mamm Genome 13: Lanata, C. F., R. E. Black, J. Flores, F. Lazo, B. Butron, A. Linares, A. Huapaya, G. Ventura, A. Gil, and A. Z. Kapikian Immunogenicity, safety and protective efficacy of one dose of the rhesus rotavirus vaccine and serotype 1 and 2 human-rhesus rotavirus reassortants in children from Lima, Peru. Vaccine 14: Lange, H., J. Kobarg, S. Yazynin, M. Solterbeck, M. Henningsen, H. Hansen, and H. Lemke Genetic analysis of the maternally induced affinity enhancement in the non-ox1 idiotypic antibody repertoire of the primary immune response to 2- phenyloxazolone. Scand J Immunol 49: Langermann, S., S. Palaszynski, A. Sadziene, C. K. Stover, and S. Koenig Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi. Nature 372: Langrish, C. L., J. C. Buddle, A. J. Thrasher, and D. Goldblatt Neonatal dendritic cells are intrinsically biased against Th-1 immune responses. Clin Exp Immunol 128: Lazarus, N. H., E. J. Kunkel, B. Johnston, E. Wilson, K. R. Youngman, and E. C. Butcher A common mucosal chemokine (mucosae-associated epithelial chemokine/ccl28) selectively attracts IgA plasmablasts. J Immunol 170: Le Jan, C Cellular components of mammary secretions and neonatal immunity: a review. Vet Res 27: Le Jan, C Secretory component and IgA expression by epithelial cells in sow mammary gland and mammary secretions. Res Vet Sci 55: Lebman, D. A., F. D. Lee, and R. L. Coffman Mechanism for transforming growth factor beta and IL-2 enhancement of IgA expression in lipopolysaccharide-stimulated B cell cultures. J Immunol 144: LeBouder, E., J. E. Rey-Nores, N. K. Rushmere, M. Grigorov, S. D. Lawn, M. Affolter, G. E. Griffin, P. Ferrara, E. J. Schiffrin, B. P. Morgan, and M. O. Labeta Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk. J Immunol 171: Lecce, J. G., J. M. Cummins, and A. B. Richards Treatment of rotavirus infection in neonate and weanling pigs using natural human interferon alpha. Mol Biother 2:

197 403. Lecce, J. G., and M. W. King Role of rotavirus (reo-like) in weanling diarrhea of pigs. J Clin Microbiol 8: Lee, J. W., M. J. Paape, T. H. Elsasser, and X. Zhao Recombinant soluble CD14 reduces severity of intramammary infection by Escherichia coli. Infect Immun 71: Lee, S. F Oral colonization and immune responses to Streptococcus gordonii: Potential use as a vector to induce antibodies against respiratory pathogens. Curr Opin Infect Dis 16: Leite, J. P., A. A. Alfieri, P. A. Woods, R. I. Glass, and J. R. Gentsch Rotavirus G and P types circulating in Brazil: characterization by RT-PCR, probe hybridization, and sequence analysis. Arch Virol 141: Lemke, C. D., J. S. Haynes, R. Spaete, D. Adolphson, A. Vorwald, K. Lager, and J. E. Butler Lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs. J Immunol 172: Letterio, J. J., A. G. Geiser, A. B. Kulkarni, N. S. Roche, M. B. Sporn, and A. B. Roberts Maternal rescue of transforming growth factor-beta 1 null mice. Science 264: Li, N., and Z. Y. Wang Viremia and extraintestinal infections in infants with rotavirus diarrhea. Di Yi Jun Yi Da Xue Xue Bao 23: Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, and J. M. Farber CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3 alpha. J Immunol 162: Linder, N., I. Waintraub, Z. Smetana, A. Barzilai, D. Lubin, E. Mendelson, and L. Sirota Placental transfer and decay of varicella-zoster virus antibodies in preterm infants. J Pediatr 137: Liprandi, F., M. Gerder, Z. Bastidas, J. A. Lopez, F. H. Pujol, J. E. Ludert, D. B. Joelsson, and M. Ciarlet A novel type of VP4 carried by a porcine rotavirus strain. Virology 315: Lo, J. Y., K. C. Szeto, D. N. Tsang, K. H. Leung, and W. W. Lim Changing epidemiology of rotavirus G-types circulating in Hong Kong, China. J Med Virol 75: London, S. D., J. A. Cebra-Thomas, D. H. Rubin, and J. J. Cebra CD8 lymphocyte subpopulations in Peyer's patches induced by reovirus serotype 1 infection. J Immunol 144:

198 415. Lopez, T., M. Camacho, M. Zayas, R. Najera, R. Sanchez, C. F. Arias, and S. Lopez Silencing the morphogenesis of rotavirus. J Virol 79: Losonsky, G. A., and H. D'Alessandra de Rimer Rotavirus specific breast milk antibody in two populations and possible correlates of protection. Adv Exp Med Biol 310: Loy, A. L., G. Allison, C. F. Arias, and N. K. Verma Immune response to rotavirus VP4 expressed in an attenuated strain of Shigella flexneri. FEMS Immunol Med Microbiol 25: Lucivero, G., L. Dalla Mora, E. Bresciano, M. P. Loria, L. Pezone, and D. Mancino Functional characteristics of cord blood T lymphocytes after lectin and anti-cd3 stimulation. Differences in the way T cells express activation molecules and proliferate. Int J Clin Lab Res 26: Ludert, J. E., B. B. Mason, J. Angel, B. Tang, Y. Hoshino, N. Feng, P. T. Vo, E. M. Mackow, F. M. Ruggeri, and H. B. Greenberg Identification of mutations in the rotavirus protein VP4 that alter sialic-acid-dependent infection. J Gen Virol 79 ( Pt 4): Ludviksson, B. R., D. Seegers, A. S. Resnick, and W. Strober The effect of TGF-beta1 on immune responses of naive versus memory CD4+ Th1/Th2 T cells. Eur J Immunol 30: Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 198: Lundgren, O., A. T. Peregrin, K. Persson, S. Kordasti, I. Uhnoo, and L. Svensson Role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea. Science 287: Lundin, B. S., A. Dahlman-Hoglund, I. Pettersson, U. I. Dahlgren, L. A. Hanson, and E. Telemo Antibodies given orally in the neonatal period can affect the immune response for two generations: evidence for active maternal influence on the newborn's immune system. Scand J Immunol 50: Lycke, N From toxin to adjuvant: the rational design of a vaccine adjuvant vector, CTA1-DD/ISCOM. Cell Microbiol 6: Lycke, N., L. Eriksen, and J. Holmgren Protection against cholera toxin after oral immunization is thymus-dependent and associated with intestinal production of neutralizing IgA antitoxin. Scand J Immunol 25:

199 426. Macphail, S., and O. Stutman Specific neonatal induction of functional tolerance to allogeneic Mls determinants occurs intrathymically and the tolerant state is Mls haplotype-specific. J Immunol 143: Madore, H. P., C. Christy, M. Pichichero, C. Long, P. Pincus, D. Vosefsky, A. Z. Kapikian, and R. Dolin Field trial of rhesus rotavirus or human-rhesus rotavirus reassortant vaccine of VP7 serotype 3 or 1 specificity in infants. The Elmwood, Panorama, and Westfall Pediatric Groups. J Infect Dis 166: Maharaj, I., K. J. Froh, and J. B. Campbell Immune responses of mice to inactivated rabies vaccine administered orally: potentiation by Quillaja saponin. Can J Microbiol 32: Malanchere, E., F. Huetz, and A. Coutinho Maternal IgG stimulates B lineage cell development in the progeny. Eur J Immunol 27: Malherbe, H., and R. Harwin The cytopathic effects of vervet monkey viruses. S Afr Med J 37: Marinaro, M., P. N. Boyaka, R. J. Jackson, F. D. Finkelman, H. Kiyono, E. Jirillo, and J. R. McGhee Use of intranasal IL-12 to target predominantly Th1 responses to nasal and Th2 responses to oral vaccines given with cholera toxin. J Immunol 162: Martella, V., A. Pratelli, G. Greco, M. Tempesta, M. Ferrari, M. N. Losio, and C. Buonavoglia Genomic characterization of porcine rotaviruses in Italy. Clin Diagn Lab Immunol 8: Martella, V., V. Terio, G. Del Gaudio, M. Gentile, P. Fiorente, S. Barbuti, and C. Buonavoglia Detection of the emerging rotavirus G9 serotype at high frequency in Italy. J Clin Microbiol 41: Martinez, X., C. Brandt, F. Saddallah, C. Tougne, C. Barrios, F. Wild, G. Dougan, P. H. Lambert, and C. A. Siegrist DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life. Proc Natl Acad Sci U S A 94: Mascarenhas, J. D., R. H. Gusmao, C. R. Barardi, F. L. Paiva, C. O. Simoes, Y. B. Gabbay, T. A. Monteiro, and A. C. Linhares Characterization of rotavirus P genotypes circulating among paediatric inpatients in northern brazil. Rev Inst Med Trop Sao Paulo 41: Masendycz, P., N. Bogdanovic-Sakran, E. Palombo, R. Bishop, and G. Barnes Annual report of the Rotavirus Surveillance Programme, 1999/2000. Commun Dis Intell 24:

200 437. Matson, D. O., M. L. O'Ryan, I. Herrera, L. K. Pickering, and M. K. Estes Fecal antibody responses to symptomatic and asymptomatic rotavirus infections. J Infect Dis 167: Matsumoto, K., M. Hatano, K. Kobayashi, A. Hasegawa, S. Yamazaki, S. Nakata, S. Chiba, and Y. Kimura An outbreak of gastroenteritis associated with acute rotaviral infection in schoolchildren. J Infect Dis 160: Matsumoto, M., K. Funami, H. Oshiumi, and T. Seya Toll-Like Receptor 3: A Link between Toll-Like Receptor, Interferon and Viruses. Microbiol Immunol 48: Matsumoto, M., K. Funami, M. Tanabe, H. Oshiumi, M. Shingai, Y. Seto, A. Yamamoto, and T. Seya Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 171: Mattsby-Baltzer, I., A. Roseanu, C. Motas, J. Elverfors, I. Engberg, and L. A. Hanson Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells. Pediatr Res 40: Mavromichalis, J., N. Evans, A. S. McNeish, A. S. Bryden, H. A. Davies, and T. H. Flewett Intestinal damage in rotavirus and adenovirus gastroenteritis assessed by d-xylose malabsorption. Arch Dis Child 52: Mayer, B., A. Zolnai, L. V. Frenyo, V. Jancsik, Z. Szentirmay, L. Hammarstrom, and I. Kacskovics Localization of the sheep FcRn in the mammary gland. Vet Immunol Immunopathol 87: Mayer, B., A. Zolnai, L. V. Frenyo, V. Jancsik, Z. Szentirmay, L. Hammarstrom, and I. Kacskovics Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs. Immunology 107: Mazanec, M. B., C. L. Coudret, and D. R. Fletcher Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J Virol 69: Mazanec, M. B., C. S. Kaetzel, M. E. Lamm, D. Fletcher, and J. G. Nedrud Intracellular neutralization of virus by immunoglobulin A antibodies. Proc Natl Acad Sci U S A 89: McCarthy, K. M., M. Lam, L. Subramanian, R. Shakya, Z. Wu, E. E. Newton, and N. E. Simister Effects of mutations in potential phosphorylation sites on transcytosis of FcRn. J Cell Sci 114:

201 448. McCluskie, M. J., and H. L. Davis CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice. J Immunol 161: McCluskie, M. J., and H. L. Davis Oral, intrarectal and intranasal immunizations using CpG and non-cpg oligodeoxynucleotides as adjuvants. Vaccine 19: McCluskie, M. J., and R. D. Weeratna Novel adjuvant systems. Curr Drug Targets Infect Disord 1: McDermott, M. R., and J. Bienenstock Evidence for a common mucosal immunologic system. I. Migration of B immunoblasts into intestinal, respiratory, and genital tissues. J Immunol 122: McGee, D. W., K. W. Beagley, W. K. Aicher, and J. R. McGhee Transforming growth factor-beta and IL-1 beta act in synergy to enhance IL-6 secretion by the intestinal epithelial cell line, IEC-6. J Immunol 151: McGhee, J. R., J. Mestecky, M. T. Dertzbaugh, J. H. Eldridge, M. Hirasawa, and H. Kiyono The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10: McIntyre, T. M., M. R. Kehry, and C. M. Snapper Novel in vitro model for high-rate IgA class switching. J Immunol 154: McIntyre, T. M. a. S., W Gut-Associated lymphoid tissue Regulation of IgA B-Cell Development, p In P. L. Ogra, Mestecky, J., Lamm, M.E., Strober, W., Bienenstock, J. and McGhee, J.R. (ed.), Mucosal Immunology, second ed, vol. 1. Academic Press, San Diego McLean, B., and I. H. Holmes Transfer of antirotaviral antibodies from mothers to their infants. J Clin Microbiol 12: McLean, B. S., and I. H. Holmes Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection. J Clin Microbiol 13: McNeal, M. M., K. Sestak, A. H. Choi, M. Basu, M. J. Cole, P. P. Aye, R. P. Bohm, and R. L. Ward Development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new P genotype. J Virol 79: McNeal, M. M., J. L. VanCott, A. H. Choi, M. Basu, J. A. Flint, S. C. Stone, J. D. Clements, and R. L. Ward CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G). J Virol 76:

202 460. Mebus, C. A., M. B. Rhodes, and N. R. Underdahl Neonatal calf diarrhea caused by a virus that induces villous epithelial cell syncytia. Am J Vet Res 39: Mebus, C. A., N. R. Underdahl, M. B. Rhodes, and M. J. Twiehaus Further studies on neonatal calf diarrhea virus. Proc Annu Meet U S Anim Health Assoc 73: Mebus, C. A., R. G. Wyatt, and A. Z. Kapikian Intestinal lesions induced in gnotobiotic calves by the virus of human infantile gastroenteritis. Vet Pathol 14: Mederle, I., R. Le Grand, B. Vaslin, E. Badell, B. Vingert, D. Dormont, B. Gicquel, and N. Winter Mucosal administration of three recombinant Mycobacterium bovis BCG-SIVmac251 strains to cynomolgus macaques induces rectal IgAs and boosts systemic cellular immune responses that are primed by intradermal vaccination. Vaccine 21: Mestecky, J., and J. R. McGhee Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv Immunol 40: Mestecky, J., Z. Moldoveanu, and C. O. Elson Immune response versus mucosal tolerance to mucosally administered antigens. Vaccine 23: Michalek, S. M., A. F. Rahman, and J. R. McGhee Rat immunoglobulins in serum and secretions: comparison of IgM, IgA and IgG in serum, colostrum, milk and saliva of protein malnourished and normal rats. Proc Soc Exp Biol Med 148: Miller, T. L., and M. J. Wolin Enumeration of Methanobrevibacter smithii in human feces. Arch Microbiol 131: Moon, H. W Pathophysiology of viral diarrhea, p In A. Z. Kapikian (ed.), viral infections of the gastrointestinal tract. Marcel Dekker, New York Moon, H. W., and J. S. McDonald Antibody response of cows to Escherichia coli pilus antigen K99 after oral vaccination with live or dead bacteria. Am J Vet Res 44: Mor, G., G. Yamshchikov, M. Sedegah, M. Takeno, R. Wang, R. A. Houghten, S. Hoffman, and D. M. Klinman Induction of neonatal tolerance by plasmid DNA vaccination of mice. J Clin Invest 98: Mori, I., K. Matsumoto, K. Sugimoto, M. Kimura, N. Daimon, T. Yokochi, and Y. Kimura Prolonged shedding of rotavirus in a geriatric inpatient. J Med Virol 67:

203 472. Mori, Y., M. A. Borgan, N. Ito, M. Sugiyama, and N. Minamoto Diarrhea-inducing activity of avian rotavirus NSP4 glycoproteins, which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice. J Virol 76: Mori, Y., M. A. Borgan, N. Ito, M. Sugiyama, and N. Minamoto Sequential analysis of nonstructural protein NSP4s derived from Group A avian rotaviruses. Virus Res 89: Morris, A. P., and M. K. Estes Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin. Am J Physiol Gastrointest Liver Physiol 281:G Morris, A. P., J. K. Scott, J. M. Ball, C. Q. Zeng, W. K. O'Neal, and M. K. Estes NSP4 elicits age-dependent diarrhea and Ca(2+)mediated I(-) influx into intestinal crypts of CF mice. Am J Physiol 277:G Mossel, E. C., and R. F. Ramig A lymphatic mechanism of rotavirus extraintestinal spread in the neonatal mouse. J Virol 77: Mossel, E. C., and R. F. Ramig Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse. J Virol 76: Mowat, A. M Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 3: Mowat, A. M Dendritic cells and immune responses to orally administered antigens. Vaccine 23: Muggli, N. E., W. D. Hohenboken, L. V. Cundiff, and K. W. Kelley Inheritance of maternal immunoglobulin G1 concentration by the bovine neonate. J Anim Sci 59: Muir, A., G. Soong, S. Sokol, B. Reddy, M. I. Gomez, A. Van Heeckeren, and A. Prince Toll-like receptors in normal and cystic fibrosis airway epithelial cells. Am J Respir Cell Mol Biol 30: Muller, T. F., P. Schuster, A. C. Vos, T. Selhorst, U. D. Wenzel, and A. M. Neubert Effect of maternal immunity on the immune response to oral vaccination against rabies in young foxes. Am J Vet Res 62: Mumford, J. A., D. M. Jessett, E. A. Rollinson, D. Hannant, and M. E. Draper Duration of protective efficacy of equine influenza immunostimulating complex/tetanus vaccines. Vet Rec 134: Munoz, C., S. Endres, J. van der Meer, L. Schlesinger, M. Arevalo, and C. Dinarello Interleukin-1 beta in human colostrum. Res Immunol 141:

204 485. Muzio, M., D. Bosisio, N. Polentarutti, G. D'Amico, A. Stoppacciaro, R. Mancinelli, C. van't Veer, G. Penton-Rol, L. P. Ruco, P. Allavena, and A. Mantovani Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164: Myers, T. J., and K. A. Schat Natural killer cell activity of chicken intraepithelial leukocytes against rotavirus-infected target cells. Vet Immunol Immunopathol 26: Nabuurs, M. J Weaning piglets as a model for studying pathophysiology of diarrhea. Vet Q 20 Suppl 3:S Nagai, Y., S. Akashi, M. Nagafuku, M. Ogata, Y. Iwakura, S. Akira, T. Kitamura, A. Kosugi, M. Kimoto, and K. Miyake Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 3: Nakanishi, K., T. Yoshimoto, H. Tsutsui, and H. Okamura Interleukin- 18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev 12: Nejmeddine, M., G. Trugnan, C. Sapin, E. Kohli, L. Svensson, S. Lopez, and J. Cohen Rotavirus spike protein VP4 is present at the plasma membrane and is associated with microtubules in infected cells. J Virol 74: Nelson, D. J., and P. G. Holt Defective regional immunity in the respiratory tract of neonates is attributable to hyporesponsiveness of local dendritic cells to activation signals. J Immunol 155: Newby, T. J., and F. J. Bourne Relative resistance of bovine and porcine immunoglobulins to proteolysis. Immunol Commun 5: Nguyen, T. V., C. Iosef, K. Jeong, Y. Kim, K. O. Chang, K. Lovgren- Bengtsson, B. Morein, M. S. Azevedo, P. Lewis, P. Nielsen, L. Yuan, and L. J. Saif Protection and antibody responses to oral priming by attenuated human rotavirus followed by oral boosting with 2/6-rotavirus-like particles with immunostimulating complexes in gnotobiotic pigs. Vaccine 21: Nguyen, V. M., V. T. Nguyen, P. L. Huynh, D. T. Dang, T. H. Nguyen, V. T. Phan, T. L. Nguyen, T. L. Le, B. Ivanoff, J. R. Gentsch, and R. I. Glass The epidemiology and disease burden of rotavirus in Vietnam: sentinel surveillance at 6 hospitals. J Infect Dis 183: Niikura, M., S. Takamura, G. Kim, S. Kawai, M. Saijo, S. Morikawa, I. Kurane, T. C. Li, N. Takeda, and Y. Yasutomi Chimeric recombinant hepatitis E virus-like particles as an oral vaccine vehicle presenting foreign epitopes. Virology 293:

205 496. Nishimura, S., H. Ushijima, H. Shiraishi, C. Kanazawa, T. Abe, K. Kaneko, and Y. Fukuyama Detection of rotavirus in cerebrospinal fluid and blood of patients with convulsions and gastroenteritis by means of the reverse transcription polymerase chain reaction. Brain Dev 15: Noad, R., and P. Roy Virus-like particles as immunogens. Trends Microbiol 11: Nobiron, I., I. Thompson, J. Brownlie, and M. E. Collins Cytokine adjuvancy of BVDV DNA vaccine enhances both humoral and cellular immune responses in mice. Vaccine 19: Nohynek, H., L. Gustafsson, M. R. Capeding, H. Kayhty, R. M. Olander, L. Pascualk, and P. Ruutu Effect of transplacentally acquired tetanus antibodies on the antibody responses to Haemophilus influenzae type b-tetanus toxoid conjugate and tetanus toxoid vaccines in Filipino infants. Pediatr Infect Dis J 18: Novak, T. J., D. Farber, D. Leitenberg, S. C. Hong, P. Johnson, and K. Bottomly Isoforms of the transmembrane tyrosine phosphatase CD45 differentially affect T cell recognition. Immunity 1: Offit, P. A., D. B. Boyle, G. W. Both, N. L. Hill, Y. M. Svoboda, S. L. Cunningham, R. J. Jenkins, and M. A. McCrae Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes. Virology 184: Offit, P. A., and H. F. Clark Maternal antibody-mediated protection against gastroenteritis due to rotavirus in newborn mice is dependent on both serotype and titer of antibody. J Infect Dis 152: Offit, P. A., H. F. Clark, M. J. Kornstein, and S. A. Plotkin A murine model for oral infection with a primate rotavirus (simian SA11). J Virol 51: Ogata, H., I. Su, K. Miyake, Y. Nagai, S. Akashi, I. Mecklenbrauker, K. Rajewsky, M. Kimoto, and A. Tarakhovsky The toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells. J Exp Med 192: Ohashi, K., V. Burkart, S. Flohe, and H. Kolb Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 164: Okada, J., T. Urasawa, N. Kobayashi, K. Taniguchi, A. Hasegawa, K. Mise, and S. Urasawa New P serotype of group A human rotavirus closely related to that of a porcine rotavirus. J Med Virol 60:

206 507. Okada, T., V. N. Ngo, E. H. Ekland, R. Forster, M. Lipp, D. R. Littman, and J. G. Cyster Chemokine requirements for B cell entry to lymph nodes and Peyer's patches. J Exp Med 196: Okamoto, Y., J. Freihorst, and P. L. Ogra Maternal determinants of neonatal immune response to ovalbumin: effect of breast feeding on development of antiovalbumin antibody in the neonate. Int Arch Allergy Appl Immunol 89: O'Neal, C. M., J. D. Clements, M. K. Estes, and M. E. Conner Rotavirus 2/6 viruslike particles administered intranasally with cholera toxin, Escherichia coli heat-labile toxin (LT), and LT-R192G induce protection from rotavirus challenge. J Virol 72: O'Neal, C. M., S. E. Crawford, M. K. Estes, and M. E. Conner Rotavirus virus-like particles administered mucosally induce protective immunity. J Virol 71: O'Neal, C. M., G. R. Harriman, and M. E. Conner Protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin A. J Virol 74: O'Ryan, M. L., D. O. Matson, M. K. Estes, and L. K. Pickering Acquisition of serum isotype-specific and G type-specific antirotavirus antibodies among children in day care centers. Pediatr Infect Dis J 13: O'Ryan, M. L., D. O. Matson, M. K. Estes, and L. K. Pickering Antirotavirus G type-specific and isotype-specific antibodies in children with natural rotavirus infections. J Infect Dis 169: Osborne, M. P., S. J. Haddon, K. J. Worton, A. J. Spencer, W. G. Starkey, D. Thornber, and J. Stephen Rotavirus-induced changes in the microcirculation of intestinal villi of neonatal mice in relation to the induction and persistence of diarrhea. J Pediatr Gastroenterol Nutr 12: Osterhaus, A., G. van Amerongen, and R. van Binnendijk Vaccine strategies to overcome maternal antibody mediated inhibition of measles vaccine. Vaccine 16: Pabst, R., M. Geist, H. J. Rothkotter, and F. J. Fritz Postnatal development and lymphocyte production of jejunal and ileal Peyer's patches in normal and gnotobiotic pigs. Immunology 64: Pacyna, J., K. Siwek, S. J. Terry, E. S. Roberton, R. B. Johnson, and G. P. Davidson Survival of rotavirus antibody activity derived from bovine colostrum after passage through the human gastrointestinal tract. J Pediatr Gastroenterol Nutr 32:

207 518. Palkowetz, K. H., C. L. Royer, R. Garofalo, H. E. Rudloff, F. C. Schmalstieg, Jr., and A. S. Goldman Production of interleukin-6 and interleukin-8 by human mammary gland epithelial cells. J Reprod Immunol 26: Paoletti, E., J. Taylor, B. Meignier, C. Meric, and J. Tartaglia Highly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC. Dev Biol Stand 84: Parashar, U. D., E. G. Hummelman, J. S. Bresee, M. A. Miller, and R. I. Glass Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 9: Parreno, V., C. Bejar, A. Vagnozzi, M. Barrandeguy, V. Costantini, M. I. Craig, L. Yuan, D. Hodgins, L. Saif, and F. Fernandez Modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus. Vet Immunol Immunopathol 100: Parreno, V., D. C. Hodgins, L. de Arriba, S. Y. Kang, L. Yuan, L. A. Ward, T. L. To, and L. J. Saif Serum and intestinal isotype antibody responses to Wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies. J Gen Virol 80 ( Pt 6): Patton, J. T Structure and function of the rotavirus RNA-binding proteins. J Gen Virol 76 ( Pt 11): Patton, J. T., R. Vasquez-Del Carpio, and E. Spencer Replication and transcription of the rotavirus genome. Curr Pharm Des 10: Pazmany, L., O. Mandelboim, M. Vales-Gomez, D. M. Davis, H. T. Reyburn, and J. L. Strominger Protection from natural killer cell-mediated lysis by HLA-G expression on target cells. Science 274: Penttila, I. A., I. E. Flesch, A. L. McCue, B. C. Powell, F. H. Zhou, L. C. Read, and H. Zola Maternal milk regulation of cell infiltration and interleukin 18 in the intestine of suckling rat pups. Gut 52: Peppard, J., E. Orlans, A. W. Payne, and E. Andrew The elimination of circulating complexes containing polymeric IgA by excretion in the bile. Immunology 42: Peppard, J. V Feeding neonatal rats with IgG antibodies leads to humoral hyporesponsiveness in the adult. Immunology 77: Perez Filgueira, D. M., M. Mozgovoj, A. Wigdorovitz, M. J. Dus Santos, V. Parreno, K. Trono, F. M. Fernandez, C. Carrillo, L. A. Babiuk, T. J. Morris, and M. 181

208 V. Borca Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector. Arch Virol 149: Perez-Schael, I., R. Gonzalez, R. Fernandez, E. Alfonzo, D. Inaty, Y. Boher, and L. Sarmiento Epidemiological features of rotavirus infection in Caracas, Venezuela: implications for rotavirus immunization programs. J Med Virol 59: Perez-Schael, I., M. J. Guntinas, M. Perez, V. Pagone, A. M. Rojas, R. Gonzalez, W. Cunto, Y. Hoshino, and A. Z. Kapikian Efficacy of the rhesus rotavirus-based quadrivalent vaccine in infants and young children in Venezuela. N Engl J Med 337: Pertmer, T. M., A. E. Oran, J. M. Moser, C. A. Madorin, and H. L. Robinson DNA vaccines for influenza virus: differential effects of maternal antibody on immune responses to hemagglutinin and nucleoprotein. J Virol 74: Phalipon, A., and B. Corthesy Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins. Trends Immunol 24: Phillips, N. E., and D. C. Parker Fc-dependent inhibition of mouse B cell activation by whole anti-mu antibodies. J Immunol 130: Pickering, L. K., A. L. Morrow, I. Herrera, M. O'Ryan, M. K. Estes, S. E. Guilliams, L. Jackson, S. Carter-Campbell, and D. O. Matson Effect of maternal rotavirus immunization on milk and serum antibody titers. J Infect Dis 172: Piron, M., T. Delaunay, J. Grosclaude, and D. Poncet Identification of the RNA-binding, dimerization, and eif4gi-binding domains of rotavirus nonstructural protein NSP3. J Virol 73: Pokorova, D., S. Reschova, J. Franz, J. Hampl, and M. Dittrich Antigenic and adjuvant activities of branched aliphatic oligoester (M-DL-LA) microspheres with incorporated bovine rotavirus. Drug Deliv 9: Power, L. L., E. J. Popplewell, J. A. Holloway, N. D. Diaper, J. O. Warner, and C. A. Jones Immunoregulatory molecules during pregnancy and at birth. J Reprod Immunol 56: Press, J. L Neonatal immunity and somatic mutation. Int Rev Immunol 19: Pulendran, B., P. Kumar, C. W. Cutler, M. Mohamadzadeh, T. Van Dyke, and J. Banchereau Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo. J Immunol 167:

209 541. Quiding-Jarbrink, M., I. Nordstrom, G. Granstrom, A. Kilander, M. Jertborn, E. C. Butcher, A. I. Lazarovits, J. Holmgren, and C. Czerkinsky Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunizations. A molecular basis for the compartmentalization of effector B cell responses. J Clin Invest 99: Raghavan, M., and P. J. Bjorkman Fc receptors and their interactions with immunoglobulins. Annu Rev Cell Dev Biol 12: Ramachandran, M., A. Vij, R. Kumar, B. K. Das, J. R. Gentsch, M. K. Bhan, and R. I. Glass Lack of maternal antibodies to P serotypes may predispose neonates to infections with unusual rotavirus strains. Clin Diagn Lab Immunol 5: Ray, P., J. Malik, R. K. Singh, S. Bhatnagar, R. Bahl, R. Kumar, and M. K. Bhan Rotavirus nonstructural protein NSP4 induces heterotypic antibody responses during natural infection in children. J Infect Dis 187: Reen, D. J Activation and functional capacity of human neonatal CD4 T- cells. Vaccine 16: Reinhardt, T. A., J. R. Stabel, and J. P. Goff ,25-dihydroxyvitamin D3 enhances milk antibody titers to Escherichia coli J5 vaccine. J Dairy Sci 82: Rennels, M. B., S. S. Wasserman, R. I. Glass, and V. A. Keane Comparison of immunogenicity and efficacy of rhesus rotavirus reassortant vaccines in breastfed and nonbreastfed children. US Rotavirus Vaccine Efficacy Group. Pediatrics 96: Rescigno, M., B. Valzasina, R. Bonasio, M. Urbano, and P. Ricciardi- Castagnoli Dendritic cells, loaded with recombinant bacteria expressing tumor antigens, induce a protective tumor-specific response. Clin Cancer Res 7:865s-870s Richards, R. L., M. Rao, N. M. Wassef, G. M. Glenn, S. W. Rothwell, and C. R. Alving Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen. Infect Immun 66: Richardson, S. C., K. Grimwood, and R. F. Bishop Analysis of homotypic and heterotypic serum immune responses to rotavirus proteins following primary rotavirus infection by using the radioimmunoprecipitation technique. J Clin Microbiol 31: Richter, M. Y., H. Jakobsen, J. F. Haeuw, U. F. Power, and I. Jonsdottir Protective levels of polysaccharide-specific maternal antibodies may enhance the immune response elicited by pneumococcal conjugates in neonatal and infant mice. Infect Immun 73:

210 552. Rifkin, I. R., E. A. Leadbetter, L. Busconi, G. Viglianti, and A. Marshak- Rothstein Toll-like receptors, endogenous ligands, and systemic autoimmune disease. Immunol Rev 204: Rimer, H. C., S. S. Wasserman, J. Flores, M. E. Pichichero, and G. A. Losonsky Rotavirus-specific breast milk antibody in two populations and possible correlates of interference with rhesus rotavirus vaccine seroconversion. J Infect Dis 165: Rimmelzwaan, G. F., N. Nieuwkoop, A. Brandenburg, G. Sutter, W. E. Beyer, D. Maher, J. Bates, and A. D. Osterhaus A randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza ISCOM vaccines and conventional vaccines. Vaccine 19: Rincheval-Arnold, A., L. Belair, and J. Djiane Developmental expression of pigr gene in sheep mammary gland and hormonal regulation. J Dairy Res 69: Risdon, G., J. Gaddy, M. Horie, and H. E. Broxmeyer Alloantigen priming induces a state of unresponsiveness in human umbilical cord blood T cells. Proc Natl Acad Sci U S A 92: Roberts, L Vaccines. Rotavirus vaccines' second chance. Science 305: Roberts, L., E. J. Walker, D. R. Snodgrass, and K. W. Angus Diarrhoea in unweaned piglets associated with rotavirus and coccidial infections. Vet Rec 107: Rock, F. L., G. Hardiman, J. C. Timans, R. A. Kastelein, and J. F. Bazan A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A 95: Rodewald, R., and D. R. Abrahamson Receptor-mediated transport of IgG across the intestinal epithelium of the neonatal rat. Ciba Found Symp: Rojas, A. M., Y. Boher, M. J. Guntinas, and I. Perez-Schael Homotypic immune response to primary infection with rotavirus serotype G1. J Med Virol 47: Rojas, R., and G. Apodaca Immunoglobulin transport across polarized epithelial cells. Nat Rev Mol Cell Biol 3: Rollo, E. E., K. P. Kumar, N. C. Reich, J. Cohen, J. Angel, H. B. Greenberg, R. Sheth, J. Anderson, B. Oh, S. J. Hempson, E. R. Mackow, and R. D. Shaw The epithelial cell response to rotavirus infection. J Immunol 163:

211 564. Rolsma, M. D., T. B. Kuhlenschmidt, H. B. Gelberg, and M. S. Kuhlenschmidt Structure and function of a ganglioside receptor for porcine rotavirus. J Virol 72: Roncarolo, M. G., and M. K. Levings The role of different subsets of T regulatory cells in controlling autoimmunity. Curr Opin Immunol 12: Rothkotter, H. J., H. Ulbrich, and R. Pabst The postnatal development of gut lamina propria lymphocytes: number, proliferation, and T and B cell subsets in conventional and germ-free pigs. Pediatr Res 29: Roux, M. E., M. McWilliams, J. M. Phillips-Quagliata, P. Weisz-Carrington, and M. E. Lamm Origin of IgA-secreting plasma cells in the mammary gland. J Exp Med 146: Rowe, J., J. T. Poolman, C. Macaubas, P. D. Sly, R. Loh, and P. G. Holt Enhancement of vaccine-specific cellular immunity in infants by passively acquired maternal antibody. Vaccine 22: Rudloff, H. E., F. C. Schmalstieg, Jr., A. A. Mushtaha, K. H. Palkowetz, S. K. Liu, and A. S. Goldman Tumor necrosis factor-alpha in human milk. Pediatr Res 31: Rudloff, H. E., F. C. Schmalstieg, Jr., K. H. Palkowetz, E. J. Paszkiewicz, and A. S. Goldman Interleukin-6 in human milk. J Reprod Immunol 23: Ruedl, C., K. Schwarz, A. Jegerlehner, T. Storni, V. Manolova, and M. F. Bachmann Virus-like particles as carriers for T-cell epitopes: limited inhibition of T-cell priming by carrier-specific antibodies. J Virol 79: Ruggeri, F. M., K. Johansen, G. Basile, J. P. Kraehenbuhl, and L. Svensson Antirotavirus immunoglobulin A neutralizes virus in vitro after transcytosis through epithelial cells and protects infant mice from diarrhea. J Virol 72: Russell, P. H., and A. Mackie Eye-drop DNA can induce IgA in the tears and bile of chickens. Vet Immunol Immunopathol 80: Saarinen, K. M., O. Vaarala, P. Klemetti, and E. Savilahti Transforming growth factor-beta1 in mothers' colostrum and immune responses to cows' milk proteins in infants with cows' milk allergy. J Allergy Clin Immunol 104: Sack, D. A., R. H. Gilman, A. Z. Kapikian, and K. M. Aziz Seroepidemiology of rotavirus infection in rural Bangladesh. J Clin Microbiol 11: Saginala, S., T. G. Nagaraja, Z. L. Tan, K. F. Lechtenberg, M. M. Chengappa, K. E. Kemp, and P. M. Hine Serum neutralizing antibody response 185

212 and protection against experimentally induced liver abscesses in steers vaccinated with Fusobacterium necrophorum. Am J Vet Res 57: Saif, L Nongroup A Rotaviruses, p In L. J. Saif, Theil, K.W. (ed.), Viral Diarrhea of Man and Animals, vol. 1. CRC Press, Inc, Boca Raton Saif, L Passive immunity to coronavirus and rotavirus infections in swine and cattle: enhancement by maternal vaccination, p In S. Tzipori (ed.), Infectious diarrhoea in the young. Elsevier Science Saif, L., Weilnau, P., Miller, K and Stitzlein, L Isotypes of intestinal and systemic antibodies in colostrum-fed and colostrum-deprived calves challenged with rotavirus. In J. Mestecky, McGhee, J.R., Bienenstock, J. and Ogra, P.L. (ed.), Recent Advances in Mucosal Immunology. Plenum Publishing Corporation Saif, L. J Enteric viral infections of pigs and strategies for induction of mucosal immunity. Adv Vet Med 41: Saif, L. J Mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease. Vet Immunol Immunopathol 54: Saif, L. J., E. H. Bohl, and R. K. Gupta Isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies. Infect Immun 6: Saif, L. J., E. H. Bohl, K. W. Theil, R. F. Cross, and J. A. House Rotavirus-like, calicivirus-like, and 23-nm virus-like particles associated with diarrhea in young pigs. J Clin Microbiol 12: Saif, L. J., H.G. Greenberg Rotaviral Gastroenteritits, p In D. H. Connor, Chandler, F.W. (ed.), Pathology of Infectious Diseases. Appleton and Lange Publisher Saif, L. J., and B. Jiang Nongroup A rotaviruses of humans and animals. Curr Top Microbiol Immunol 185: Saif, L. J., D. R. Redman, K. L. Smith, and K. W. Theil Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from immunized or nonimmunized cows. Infect Immun 41: Saif, L. J., and K. L. Smith Enteric viral infections of calves and passive immunity. J Dairy Sci 68: Saif, L. J., J. L. van Cott, and T. A. Brim Immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine. Vet Immunol Immunopathol 43:

213 589. Saif, L. J., L. A. Ward, L. Yuan, B. I. Rosen, and T. L. To The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses. Arch Virol Suppl 12: Saint Andre, A., N. M. Blackwell, L. R. Hall, A. Hoerauf, N. W. Brattig, L. Volkmann, M. J. Taylor, L. Ford, A. G. Hise, J. H. Lass, E. Diaconu, and E. Pearlman The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295: Saito, S., M. Yoshida, M. Ichijo, S. Ishizaka, and T. Tsujii Transforming growth factor-beta (TGF-beta) in human milk. Clin Exp Immunol 94: Salmon, H The mammary gland and neonate mucosal immunity. Vet Immunol Immunopathol 72: Salmon, H Surface markers of swine lymphoctes: application to the study of local immune system or mammary gland and transplanted gut, p In E. M. Tumleson (ed.), Swine in Biomedical Research. Plenum Press, New York Santos, N., and Y. Hoshino Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol 15: Sarvas, H., S. Kurikka, I. J. Seppala, P. H. Makela, and O. Makela Maternal antibodies partly inhibit an active antibody response to routine tetanus toxoid immunization in infants. J Infect Dis 165: Sarzotti, M., T. A. Dean, M. P. Remington, C. D. Ly, P. A. Furth, and D. S. Robbins Induction of cytotoxic T cell responses in newborn mice by DNA immunization. Vaccine 15: Sasaki, S., Y. Horie, T. Nakagomi, M. Oseto, and O. Nakagomi Group C rotavirus NSP4 induces diarrhea in neonatal mice. Arch Virol 146: Saunders, P. T., R. H. Renegar, T. J. Raub, G. A. Baumbach, P. H. Atkinson, F. W. Bazer, and R. M. Roberts The carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver. J Biol Chem 260: Schaller, J. P., L. J. Saif, C. T. Cordle, E. Candler, Jr., T. R. Winship, and K. L. Smith Prevention of human rotavirus-induced diarrhea in gnotobiotic piglets using bovine antibody. J Infect Dis 165: Scheppler, L., M. Vogel, A. W. Zuercher, M. Zuercher, J. E. Germond, S. M. Miescher, and B. M. Stadler Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle. Vaccine 20:

214 601. Schmausser, B., M. Andrulis, S. Endrich, S. K. Lee, C. Josenhans, H. K. Muller-Hermelink, and M. Eck Expression and subcellular distribution of tolllike receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection. Clin Exp Immunol 136: Schmidt, K. N., B. Leung, M. Kwong, K. A. Zarember, S. Satyal, T. A. Navas, F. Wang, and P. J. Godowski APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA. J Immunol 172: Schnulle, P. M., and W. L. Hurley Sequence and expression of the FcRn in the porcine mammary gland. Vet Immunol Immunopathol 91: Schollenberger, A., T. Frymus, and A. Degorski Cells of sow mammary secretions. II. Characterization of lymphocyte populations. Zentralbl Veterinarmed A 33: Schroten, H., C. Stapper, R. Plogmann, H. Kohler, J. Hacker, and F. G. Hanisch Fab-independent antiadhesion effects of secretory immunoglobulin A on S-fimbriated Escherichia coli are mediated by sialyloligosaccharides. Infect Immun 66: Schwager, J., and J. Schulze Maturation of the mitogen responsiveness, and IL2 and IL6 production by neonatal swine leukocytes. Vet Immunol Immunopathol 57: Schwartz-Cornil, I., Y. Benureau, H. Greenberg, B. A. Hendrickson, and J. Cohen Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins. J Virol 76: Schwarz, K., E. Meijerink, D. E. Speiser, A. C. Tissot, I. Cielens, R. Renhof, A. Dishlers, P. Pumpens, and M. F. Bachmann Efficient homologous primeboost strategies for T cell vaccination based on virus-like particles. Eur J Immunol 35: Seganti, L., A. M. Di Biase, M. Marchetti, A. Pietrantoni, A. Tinari, and F. Superti Antiviral activity of lactoferrin towards naked viruses. Biometals 17: Selin, L. K., S. M. Varga, I. C. Wong, and R. M. Welsh Protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory T cell populations. J Exp Med 188: Sestak, K., M. M. McNeal, A. Choi, M. J. Cole, G. Ramesh, X. Alvarez, P. P. Aye, R. P. Bohm, M. Mohamadzadeh, and R. L. Ward Defining T-cellmediated immune responses in rotavirus-infected juvenile rhesus macaques. J Virol 78:

215 612. Shah, U., B. L. Dickinson, R. S. Blumberg, N. E. Simister, W. I. Lencer, and W. A. Walker Distribution of the IgG Fc receptor, FcRn, in the human fetal intestine. Pediatr Res 53: Shanahan, F., R. Deem, R. Nayersina, B. Leman, and S. Targan Human mucosal T-cell cytotoxicity. Gastroenterology 94: Shaw, D. P., L. G. Morehouse, and R. F. Solorzano Experimental rotavirus infection in three-week-old pigs. Am J Vet Res 50: Shaw, R. D., S. J. Hempson, and E. R. Mackow Rotavirus diarrhea is caused by nonreplicating viral particles. J Virol 69: Shibata, I., M. Mori, and K. Uruno Experimental infection of maternally immune pigs with porcine reproductive and respiratory syndrome (PRRS) virus. J Vet Med Sci 60: Shimosato, T., M. Tohno, H. Kitazawa, S. Katoh, K. Watanabe, Y. Kawai, H. Aso, T. Yamaguchi, and T. Saito Toll-like receptor 9 is expressed on follicleassociated epithelia containing M cells in swine Peyer's patches. Immunol Lett 98: Shortman, K., and Y. J. Liu Mouse and human dendritic cell subtypes. Nat Rev Immunol 2: Shuttleworth, G., D. C. Eckery, and P. Awram Oral and intraperitoneal immunization with rotavirus 2/6 virus-like particles stimulates a systemic and mucosal immune response in mice. Arch Virol 150: Siadat-Pajouh, M., and L. Cai Protective efficacy of rotavirus 2/6-viruslike particles combined with CT-E29H, a detoxified cholera toxin adjuvant. Viral Immunol 14: Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, S. Ho, S. Antonenko, and Y. J. Liu The nature of the principal type 1 interferonproducing cells in human blood. Science 284: Siegrist, C. A Mechanisms by which maternal antibodies influence infant vaccine responses: review of hypotheses and definition of main determinants. Vaccine 21: Siegrist, C. A., C. Barrios, X. Martinez, C. Brandt, M. Berney, M. Cordova, J. Kovarik, and P. H. Lambert Influence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early primeboost strategies in mice. Eur J Immunol 28:

216 624. Siegrist, C. A., M. Cordova, C. Brandt, C. Barrios, M. Berney, C. Tougne, J. Kovarik, and P. H. Lambert Determinants of infant responses to vaccines in presence of maternal antibodies. Vaccine 16: Siegrist, C. A., M. Pihlgren, C. Tougne, S. M. Efler, M. L. Morris, M. J. AlAdhami, D. W. Cameron, C. L. Cooper, J. Heathcote, H. L. Davis, and P. H. Lambert Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response. Vaccine 23: Siegrist, C. A., F. Saddallah, C. Tougne, X. Martinez, J. Kovarik, and P. H. Lambert Induction of neonatal TH1 and CTL responses by live viral vaccines: a role for replication patterns within antigen presenting cells? Vaccine 16: Silfverdal, S. A., L. Bodin, S. Hugosson, O. Garpenholt, B. Werner, E. Esbjorner, B. Lindquist, and P. Olcen Protective effect of breastfeeding on invasive Haemophilus influenzae infection: a case-control study in Swedish preschool children. Int J Epidemiol 26: Silverman, E., M. Mamula, J. A. Hardin, and R. Laxer Importance of the immune response to the Ro/La particle in the development of congenital heart block and neonatal lupus erythematosus. J Rheumatol 18: Silvey, K. J., A. B. Hutchings, M. Vajdy, M. M. Petzke, and M. R. Neutra Role of immunoglobulin A in protection against reovirus entry into Murine Peyer's patches. J Virol 75: Simister, N. E Placental transport of immunoglobulin G. Vaccine 21: Simister, N. E., and J. C. Ahouse The structure and evolution of FcRn. Res Immunol 147:333-7; discussion Simister, N. E., and K. E. Mostov An Fc receptor structurally related to MHC class I antigens. Nature 337: Simister, N. E., and C. M. Story Human placental Fc receptors and the transmission of antibodies from mother to fetus. J Reprod Immunol 37: Simister, N. E., C. M. Story, H. L. Chen, and J. S. Hunt An IgGtransporting Fc receptor expressed in the syncytiotrophoblast of human placenta. Eur J Immunol 26: Sinkora, M., J. Sinkorova, and J. E. Butler B cell development and VDJ rearrangement in the fetal pig. Vet Immunol Immunopathol 87:

217 636. Sjolander, A., D. Drane, E. Maraskovsky, J. P. Scheerlinck, A. Suhrbier, J. Tennent, and M. Pearse Immune responses to ISCOM formulations in animal and primate models. Vaccine 19: Smith, D. J., M. A. Taubman, and J. L. Ebersole Salivary IgA antibody to glucosyltransferase in man. Clin Exp Immunol 61: Smith, K. D., E. Andersen-Nissen, F. Hayashi, K. Strobe, M. A. Bergman, S. L. Barrett, B. T. Cookson, and A. Aderem Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 4: Snodgrass, D. R., I. Campbell, J. M. Mwenda, G. Chege, M. A. Suleman, B. Morein, and C. A. Hart Stimulation of rotavirus IgA, IgG and neutralising antibodies in baboon milk by parenteral vaccination. Vaccine 13: Song, C. H., G. B. Calandra, C. J. Palmer, A. Miller, E. E. Sercarz, and M. A. Keller Inhibition of offspring response to HEL-CFA by administration of anti- HEL MAB to the mother is not related to the predominant idiotype, IdXE, or specificity of the MAB. Cell Immunol 131: Song, H., X. Nie, S. Basu, M. Singh, and J. Cerny Regulation of VH gene repertoire and somatic mutation in germinal centre B cells by passively administered antibody. Immunology 98: Spalding, D. M., and J. A. Griffin Different pathways of differentiation of pre-b cell lines are induced by dendritic cells and T cells from different lymphoid tissues. Cell 44: Spiekermann, G. M., P. W. Finn, E. S. Ward, J. Dumont, B. L. Dickinson, R. S. Blumberg, and W. I. Lencer Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in the mammalian lung. J Exp Med 196: Sprott, G. D., C. J. Dicaire, K. Gurnani, L. A. Deschatelets, and L. Krishnan Liposome adjuvants prepared from the total polar lipids of Haloferax volcanii, Planococcus spp. and Bacillus firmus differ in ability to elicit and sustain immune responses. Vaccine 22: Sprott, G. D., D. L. Tolson, and G. B. Patel Archaeosomes as novel antigen delivery systems. FEMS Microbiol Lett 154: Srivastava, M. D., A. Srivastava, B. Brouhard, R. Saneto, S. Groh-Wargo, and J. Kubit Cytokines in human milk. Res Commun Mol Pathol Pharmacol 93:

218 647. Staats, H. F., and F. A. Ennis, Jr IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens. J Immunol 162: Stacy-Phipps, S., and J. T. Patton Synthesis of plus- and minus-strand RNA in rotavirus-infected cells. J Virol 61: Starkey, W. G., J. Collins, T. S. Wallis, G. J. Clarke, A. J. Spencer, S. J. Haddon, M. P. Osborne, D. C. Candy, and J. Stephen Kinetics, tissue specificity and pathological changes in murine rotavirus infection of mice. J Gen Virol 67 ( Pt 12): Stefaner, I., A. Praetor, and W. Hunziker Nonvectorial surface transport, endocytosis via a Di-leucine-based motif, and bidirectional transcytosis of chimera encoding the cytosolic tail of rat FcRn expressed in Madin-Darby canine kidney cells. J Biol Chem 274: Stokes, C. R., M. Bailey, and A. D. Wilson Immunology of the porcine gastrointestinal tract. Vet Immunol Immunopathol 43: Suganuma, A., A. Ishizuka, Y. Sakiyama, Y. Maede, and S. Namioka B lymphocyte differentiation and suppressor activity by T lymphocytes derived from neonatal and sucking piglets. Res Vet Sci 40: Sun, C. M., L. Fiette, M. Tanguy, C. Leclerc, and R. Lo-Man Ontogeny and innate properties of neonatal dendritic cells. Blood 102: Sun, J., C. Hayward, R. Shinde, R. Christenson, S. P. Ford, and J. E. Butler Antibody repertoire development in fetal and neonatal piglets. I. Four VH genes account for 80 percent of VH usage during 84 days of fetal life. J Immunol 161: Supajatura, V., H. Ushio, A. Nakao, S. Akira, K. Okumura, C. Ra, and H. Ogawa Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 109: Svensmark, B., K. Nielsen, K. Dalsgaard, and P. Willeberg Epidemiological studies of piglet diarrhoea in intensively managed Danish sow herds. III. Rotavirus infection. Acta Vet Scand 30: Swaggerty, C. L., A. A. Frolov, M. J. McArthur, V. W. Cox, S. Tong, R. W. Compans, and J. M. Ball The envelope glycoprotein of simian immunodeficiency virus contains an enterotoxin domain. Virology 277: Szabo, M. P., and G. H. Bechara Immunisation of dogs and guinea pigs against Rhipicephalus sanguineus ticks using gut extract. Vet Parasitol 68:

219 659. Tafuri, A., J. Alferink, P. Moller, G. J. Hammerling, and B. Arnold T cell awareness of paternal alloantigens during pregnancy. Science 270: Taguchi, T., W. K. Aicher, K. Fujihashi, M. Yamamoto, J. R. McGhee, J. A. Bluestone, and H. Kiyono Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, gamma/delta TCR+ T cells produce IFN-gamma and IL-5. J Immunol 147: Takahata, Y., H. Takada, A. Nomura, K. Ohshima, H. Nakayama, T. Tsuda, H. Nakano, and T. Hara Interleukin-18 in human milk. Pediatr Res 50: Takai, T., M. Ono, M. Hikida, H. Ohmori, and J. V. Ravetch Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice. Nature 379: Takeda, K., T. Kaisho, and S. Akira Toll-like receptors. Annu Rev Immunol 21: Takemori, T., and K. Rajewsky Mechanism of neonatally induced idiotype suppression and its relevance for the acquisition of self-tolerance. Immunol Rev 79: Takemori, T., and K. Rajewsky Specificity, duration and mechanism of idiotype suppression induced by neonatal injection of monoclonal anti-idiotope antibodies into mice. Eur J Immunol 14: Takeuchi, O., T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky, K. Takeda, and S. Akira Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 13: Tangye, S. G., D. T. Avery, E. K. Deenick, and P. D. Hodgkin Intrinsic differences in the proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune responses. J Immunol 170: Tapia, E., E. Perez-Jimenez, L. Lopez-Fuertes, R. Gonzalo, M. M. Gherardi, and M. Esteban The combination of DNA vectors expressing IL-12 + IL-18 elicits high protective immune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines, followed by a booster with a vaccinia virus recombinant expressing p36/lack. Microbes Infect 5: Taraporewala, Z., D. Chen, and J. T. Patton Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity. J Virol 73: Taylor, C. E Cytokines as adjuvants for vaccines: antigen-specific responses differ from polyclonal responses. Infect Immun 63:

220 671. Taylor, J. A., J. C. Meyer, M. A. Legge, J. A. O'Brien, J. E. Street, V. J. Lord, C. C. Bergmann, and A. R. Bellamy Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding. J Virol 66: Taylor, J. A., J. A. O'Brien, V. J. Lord, J. C. Meyer, and A. R. Bellamy The RER-localized rotavirus intracellular receptor: a truncated purified soluble form is multivalent and binds virus particles. Virology 194: Telleman, P., and R. P. Junghans The role of the Brambell receptor (FcRB) in liver: protection of endocytosed immunoglobulin G (IgG) from catabolism in hepatocytes rather than transport of IgG to bile. Immunology 100: Theil, K. W Group A Rotavirus, p In L. Saif, Theil, K.W. (ed.), Viral Diarrheas of man and animals, vol. 1. CRC Press, Inc, Boca Raton Theil, K. W., E. H. Bohl, R. F. Cross, E. M. Kohler, and A. G. Agnes Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs. Am J Vet Res 39: Theil, K. W., L. J. Saif, P. D. Moorhead, and R. E. Whitmoyer Porcine rotavirus-like virus (group B rotavirus): characterization and pathogenicity for gnotobiotic pigs. J Clin Microbiol 21: Thoma-Uszynski, S., S. Stenger, O. Takeuchi, M. T. Ochoa, M. Engele, P. A. Sieling, P. F. Barnes, M. Rollinghoff, P. L. Bolcskei, M. Wagner, S. Akira, M. V. Norgard, J. T. Belisle, P. J. Godowski, B. R. Bloom, and R. L. Modlin Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 291: Thulin, A. J., G. L. Allee, D. L. Harmon, and D. L. Davis Uteroplacental transfer of octanoic, palmitic and linoleic acids during late gestation in gilts. J Anim Sci 67: Tian, P., J. M. Ball, C. Q. Zeng, and M. K. Estes The rotavirus nonstructural glycoprotein NSP4 possesses membrane destabilization activity. J Virol 70: Tizard, I. R Veterinary Immunology: an introduction, 7th ed. Saunders, Philadelphia To, T. L., L. A. Ward, L. Yuan, and L. J. Saif Serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Gen Virol 79 ( Pt 11): Tohno, M., T. Shimosato, H. Kitazawa, S. Katoh, I. D. Iliev, T. Kimura, Y. Kawai, K. Watanabe, H. Aso, T. Yamaguchi, and T. Saito Toll-like receptor 2 194

221 is expressed on the intestinal M cells in swine. Biochem Biophys Res Commun 330: Torres, A., and L. Ji-Huang Diarrheal response of gnotobiotic pigs after fetal infection and neonatal challenge with homologous and heterologous human rotavirus strains. J Virol 60: Tsunemitsu, H., B. Jiang, Y. Yamashita, M. Oseto, H. Ushijima, and L. J. Saif Evidence of serologic diversity within group C rotaviruses. J Clin Microbiol 30: Tuboly, S., and S. Bernath Intestinal absorption of colostral lymphoid cells in newborn animals. Adv Exp Med Biol 503: Tzipori, S., D. Chandler, T. Makin, and M. Smith Escherichia coli and rotavirus infections in four-week-old gnotobiotic piglets fed milk or dry food. Aust Vet J 56: Uhnoo, I., M. Riepenhoff-Talty, T. Dharakul, P. Chegas, J. E. Fisher, H. B. Greenberg, and P. L. Ogra Extramucosal spread and development of hepatitis in immunodeficient and normal mice infected with rhesus rotavirus. J Virol 64: Urasawa, S., A. Hasegawa, T. Urasawa, K. Taniguchi, F. Wakasugi, H. Suzuki, S. Inouye, B. Pongprot, J. Supawadee, S. Suprasert, and et al Antigenic and genetic analyses of human rotaviruses in Chiang Mai, Thailand: evidence for a close relationship between human and animal rotaviruses. J Infect Dis 166: Vabulas, R. M., P. Ahmad-Nejad, S. Ghose, C. J. Kirschning, R. D. Issels, and H. Wagner HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J Biol Chem 277: Van de Perre, P Transfer of antibody via mother's milk. Vaccine 21: van der Feltz, M. J., N. de Groot, J. P. Bayley, S. H. Lee, M. P. Verbeet, and H. A. de Boer Lymphocyte homing and Ig secretion in the murine mammary gland. Scand J Immunol 54: Van der Stede, Y., E. Cox, F. Verdonck, S. Vancaeneghem, and B. M. Goddeeris Reduced faecal excretion of F4+-E coli by the intramuscular immunisation of suckling piglets by the addition of 1alpha,25-dihydroxyvitamin D3 or CpG-oligodeoxynucleotides. Vaccine 21: van der Strate, B. W., L. Beljaars, G. Molema, M. C. Harmsen, and D. K. Meijer Antiviral activities of lactoferrin. Antiviral Res 52:

222 694. Van Loock, M., S. Lambin, G. Volckaert, B. M. Goddeeris, and D. Vanrompay Influence of maternal antibodies on Chlamydophila psittaci-specific immune responses in turkeys elicited by naked DNA. Vaccine 22: VanCott, J. L., T. A. Brim, J. K. Lunney, and L. J. Saif Contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge. J Immunol 152: VanCott, J. L., M. A. Franco, H. B. Greenberg, S. Sabbaj, B. Tang, R. Murray, and J. R. McGhee Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally. J Virol 74: Vancott, J. L., M. M. McNeal, A. H. Choi, and R. L. Ward The role of interferons in rotavirus infections and protection. J Interferon Cytokine Res 23: VanCott, T. C., R. W. Kaminski, J. R. Mascola, V. S. Kalyanaraman, N. M. Wassef, C. R. Alving, J. T. Ulrich, G. H. Lowell, and D. L. Birx HIV-1 neutralizing antibodies in the genital and respiratory tracts of mice intranasally immunized with oligomeric gp160. J Immunol 160: Varghese, V., S. Das, N. B. Singh, K. Kojima, S. K. Bhattacharya, T. Krishnan, N. Kobayashi, and T. N. Naik Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4. Arch Virol 149: Vega-Lopez, M. A., G. Arenas-Contreras, M. Bailey, S. Gonzalez-Pozos, C. R. Stokes, M. G. Ortega, and R. Mondragon-Flores Development of intraepithelial cells in the porcine small intestine. Dev Immunol 8: Vega-Lopez, M. A., E. Telemo, M. Bailey, K. Stevens, and C. R. Stokes Immune cell distribution in the small intestine of the pig: immunohistological evidence for an organized compartmentalization in the lamina propria. Vet Immunol Immunopathol 37: Velazquez, F. R., D. O. Matson, M. L. Guerrero, J. Shults, J. J. Calva, A. L. Morrow, R. I. Glass, L. K. Pickering, and G. M. Ruiz-Palacios Serum antibody as a marker of protection against natural rotavirus infection and disease. J Infect Dis 182: Vende, P., M. Piron, N. Castagne, and D. Poncet Efficient translation of rotavirus mrna requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eif4g and the mrna 3' end. J Virol 74:

223 704. Vende, P., M. A. Tortorici, Z. F. Taraporewala, and J. T. Patton Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis. Virology 313: Vendetti, S., J. G. Chai, J. Dyson, E. Simpson, G. Lombardi, and R. Lechler Anergic T cells inhibit the antigen-presenting function of dendritic cells. J Immunol 165: Vesikari, T Clinical trials of live oral rotavirus vaccines: the Finnish experience. Vaccine 11: Vesikari, T., E. Isolauri, E. D'Hondt, A. Delem, F. E. Andre, and G. Zissis Protection of infants against rotavirus diarrhoea by RIT 4237 attenuated bovine rotavirus strain vaccine. Lancet 1: Vesikari, T., T. Ruuska, K. Y. Green, J. Flores, and A. Z. Kapikian Protective efficacy against serotype 1 rotavirus diarrhea by live oral rhesus-human reassortant rotavirus vaccines with human rotavirus VP7 serotype 1 or 2 specificity. Pediatr Infect Dis J 11: Villa, S., H. Guiscafre, H. Martinez, O. Munoz, and G. Gutierrez Seasonal diarrhoeal mortality among Mexican children. Bull World Health Organ 77: Visintin, A., A. Mazzoni, J. H. Spitzer, D. H. Wyllie, S. K. Dower, and D. M. Segal Regulation of Toll-like receptors in human monocytes and dendritic cells. J Immunol 166: Wagstrom, E. A., K. J. Yoon, and J. J. Zimmerman Immune components in porcine mammary secretions. Viral Immunol 13: Wang, P. L., Y. Azuma, M. Shinohara, and K. Ohura Toll-like receptor 4-mediated signal pathway induced by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts. Biochem Biophys Res Commun 273: Ward, E. S., J. Zhou, V. Ghetie, and R. J. Ober Evidence to support the cellular mechanism involved in serum IgG homeostasis in humans. Int Immunol 15: Ward, L. A., E. D. Rich, and T. E. Besser Role of maternally derived circulating antibodies in protection of neonatal swine against porcine group A rotavirus. J Infect Dis 174: Ward, L. A., B. I. Rosen, L. Yuan, and L. J. Saif Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs. J Gen Virol 77 ( Pt 7):

224 716. Ward, L. A., L. Yuan, B. I. Rosen, T. L. To, and L. J. Saif Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model. Clin Diagn Lab Immunol 3: Ward, R. L., D. I. Bernstein, R. Shukla, M. M. McNeal, J. R. Sherwood, E. C. Young, and G. M. Schiff Protection of adults rechallenged with a human rotavirus. J Infect Dis 161: Ward, R. L., D. I. Bernstein, R. Shukla, E. C. Young, J. R. Sherwood, M. M. McNeal, M. C. Walker, and G. M. Schiff Effects of antibody to rotavirus on protection of adults challenged with a human rotavirus. J Infect Dis 159: Ward, R. L., D. R. Knowlton, E. T. Zito, B. L. Davidson, R. Rappaport, and M. E. Mack Serologic correlates of immunity in a tetravalent reassortant rotavirus vaccine trial. US Rotavirus Vaccine Efficacy Group. J Infect Dis 176: Ward, R. L., B. B. Mason, D. I. Bernstein, D. S. Sander, V. E. Smith, G. A. Zandle, and R. S. Rappaport Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene. J Virol 71: Ward, R. L., M. M. McNeal, and J. F. Sheridan Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody. Virology 188: Ward, R. L., K. A. Pax, J. R. Sherwood, E. C. Young, G. M. Schiff, and D. I. Bernstein Salivary antibody titers in adults challenged with a human rotavirus. J Med Virol 36: Watanabe, T., M. Yoshida, Y. Shirai, M. Yamori, H. Yagita, T. Itoh, T. Chiba, T. Kita, and Y. Wakatsuki Administration of an antigen at a high dose generates regulatory CD4+ T cells expressing CD95 ligand and secreting IL-4 in the liver. J Immunol 168: Watts, A. M., J. R. Stanley, M. H. Shearer, P. S. Hefty, and R. C. Kennedy Fetal immunization of baboons induces a fetal-specific antibody response. Nat Med 5: Weeratna, R. D., C. L. Brazolot Millan, M. J. McCluskie, C. A. Siegrist, and H. L. Davis Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies. FEMS Immunol Med Microbiol 30: Wegmann, T. G., H. Lin, L. Guilbert, and T. R. Mosmann Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 14:

225 727. Weisz-Carrington, P., M. E. Roux, M. McWilliams, J. M. Phillips-Quagliata, and M. E. Lamm Hormonal induction of the secretory immune system in the mammary gland. Proc Natl Acad Sci U S A 75: Weitkamp, J. H., N. Kallewaard, K. Kusuhara, E. Bures, J. V. Williams, B. LaFleur, H. B. Greenberg, and J. E. Crowe, Jr Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires. J Immunol 171: Wernersson, S., M. C. Karlsson, J. Dahlstrom, R. Mattsson, J. S. Verbeek, and B. Heyman IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice. J Immunol 163: Westerman, L. E., H. M. McClure, B. Jiang, J. W. Almond, and R. I. Glass Serum IgG mediates mucosal immunity against rotavirus infection. Proc Natl Acad Sci U S A Westerman, L. E., J. Xu, B. Jiang, H. M. McClure, and R. I. Glass Experimental infection of pigtailed macaques with a simian rotavirus, YK-1. J Med Virol 75: Western, A. H., D. C. Eckery, J. Demmer, J. L. Juengel, K. P. McNatty, and A. E. Fidler Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young. Mol Immunol 39: Whary, M. T., A. Zarkower, F. L. Confer, and F. G. Ferguson Agerelated differences in subset composition and activation responses of intestinal intraepithelial and mesenteric lymph node lymphocytes from neonatal swine. Cell Immunol 163: Wieland, W. H., D. Orzaez, A. Lammers, H. K. Parmentier, M. W. Verstegen, and A. Schots A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity. Biochem J Pt Wiersma, E. J., P. G. Coulie, and B. Heyman Dual immunoregulatory effects of monoclonal IgG-antibodies: suppression and enhancement of the antibody response. Scand J Immunol 29: Wiersma, E. J., M. Nose, and B. Heyman Evidence of IgG-mediated enhancement of the antibody response in vivo without complement activation via the classical pathway. Eur J Immunol 20: Wigdorovitz, A., M. Mozgovoj, M. J. Santos, V. Parreno, C. Gomez, D. M. Perez-Filgueira, K. G. Trono, R. D. Rios, P. M. Franzone, F. Fernandez, C. Carrillo, 199

226 L. A. Babiuk, J. M. Escribano, and M. V. Borca Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants. J Gen Virol 85: Wilke, J., G. Lehle, and E. Weiler Isogeneic monoclonal antibodies against anti-alpha(1----3)dextran idiotypes. II. Neonatally induced idiotope-specific suppression: a comparative analysis. Eur J Immunol 17: Williams, I. R Chemokine receptors and leukocyte trafficking in the mucosal immune system. Immunol Res 29: Williams, P. P Immunomodulating effects of intestinal absorbed maternal colostral leukocytes by neonatal pigs. Can J Vet Res 57: Wolbank, S., R. Kunert, G. Stiegler, and H. Katinger Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12. J Virol 77: Wold, A. E., J. Mestecky, M. Tomana, A. Kobata, H. Ohbayashi, T. Endo, and C. S. Eden Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin. Infect Immun 58: Wolf, L. B., and E. D. Brodie, 3rd The coadaptation of parental and offspring characters. Evolution 52: Wolfs, T. G., W. A. Buurman, A. van Schadewijk, B. de Vries, M. A. Daemen, P. S. Hiemstra, and C. van 't Veer In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated upregulation during inflammation. J Immunol 168: Woodard, J. P., W. Chen, E. O. Keku, S. C. Liu, J. G. Lecce, and J. M. Rhoads Altered jejunal potassium (Rb+) transport in piglet rotavirus enteritis. Am J Physiol 265:G Woode, G. N., J. Bridger, G. A. Hall, J. M. Jones, and G. Jackson The isolation of reovirus-like agents (rota-viruses) from acute gastroenteritis of piglets. J Med Microbiol 9: Wu, Y. Z., J. T. Li, Z. R. Mou, L. Fei, B. Ni, M. Geng, Z. C. Jia, W. Zhou, L. Y. Zou, and Y. Tang Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA. Virology 313: Wurbel, M. A., M. Malissen, D. Guy-Grand, E. Meffre, M. C. Nussenzweig, M. Richelme, A. Carrier, and B. Malissen Mice lacking the CCR9 CCchemokine receptor show a mild impairment of early T- and B-cell development and a 200

227 reduction in T-cell receptor gammadelta(+) gut intraepithelial lymphocytes. Blood 98: Wyllie, D. H., E. Kiss-Toth, A. Visintin, S. C. Smith, S. Boussouf, D. M. Segal, G. W. Duff, and S. K. Dower Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J Immunol 165: Xiang, Z. Q., and H. C. Ertl Transfer of maternal antibodies results in inhibition of specific immune responses in the offspring. Virus Res 24: Xu, D., V. Trajkovic, D. Hunter, B. P. Leung, K. Schulz, J. A. Gracie, I. B. McInnes, and F. Y. Liew IL-18 induces the differentiation of Th1 or Th2 cells depending upon cytokine milieu and genetic background. Eur J Immunol 30: Yaeger, M., N. Funk, and L. Hoffman A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus. J Vet Diagn Invest 14: Yan, H., M. E. Lamm, E. Bjorling, and Y. T. Huang Multiple functions of immunoglobulin A in mucosal defense against viruses: an in vitro measles virus model. J Virol 76: Yang, K., S. Wang, K. O. Chang, S. Lu, L. J. Saif, H. B. Greenberg, J. P. Brinker, and J. E. Herrmann Immune responses and protection obtained with rotavirus VP6 DNA vaccines given by intramuscular injection. Vaccine 19: Yang, T. J., I. A. Ayoub, and M. J. Rewinski Lactation stage-dependent changes of lymphocyte subpopulations in mammary secretions: inversion of CD4+/CD8+ T cell ratios at parturition. Am J Reprod Immunol 37: Yasuda, M., S. Furusawa, H. Matsuda, Y. Taura, T. Urano, Y. Yokomizo, and S. Ekino Development of maternal IgG-free chick obtained from surgically bursectomized hen. Comp Immunol Microbiol Infect Dis 21: Yasui, H., J. Kiyoshima, and H. Ushijima Passive protection against rotavirus-induced diarrhea of mouse pups born to and nursed by dams fed Bifidobacterium breve YIT4064. J Infect Dis 172: Yeager, A. S., J. H. Davis, L. A. Ross, and B. Harvey Measles immunization. Successes and failures. Jama 237: Yolken, R. H., C. Ojeh, I. A. Khatri, U. Sajjan, and J. F. Forstner Intestinal mucins inhibit rotavirus replication in an oligosaccharide-dependent manner. J Infect Dis 169: Yoo, D., J. Lee, R. Harland, E. Gibbons, Y. Elazhary, and L. A. Babiuk Maternal immunization of pregnant cattle with recombinant VP8* protein of 201

228 bovine rotavirus elicits neutralizing antibodies to multiple serotypes. Colostral neutralizing antibody by rotavirus VP8*. Adv Exp Med Biol 412: Yu, J., and W. H. Langridge A plant-based multicomponent vaccine protects mice from enteric diseases. Nat Biotechnol 19: Yu, V. Y., C. A. Waller, I. C. Maclennan, and J. D. Baum Lymphocyte reactivity in pregnant women and newborn infants. Br Med J 1: Yuan, L., M. S. Azevedo, A. M. Gonzalez, K. I. Jeong, T. Van Nguyen, P. Lewis, C. Iosef, J. E. Herrmann, and L. J. Saif Mucosal and systemic antibody responses and protection induced by a prime/boost rotavirus-dna vaccine in a gnotobiotic pig model. Vaccine 23: Yuan, L., Azevedo,M., Nguyen, T.V, Gonzalez,A.M.,Jeong, K-I.,Chang,K.O. and Saif, L.J Presented at the American Society for Virology, Davis, CA, July 12-16, Yuan, L., A. Geyer, D. C. Hodgins, Z. Fan, Y. Qian, K. O. Chang, S. E. Crawford, V. Parreno, L. A. Ward, M. K. Estes, M. E. Conner, and L. J. Saif Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heatlabile toxin (LT-R192G) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs. J Virol 74: Yuan, L., S. Honma, S. Ishida, X. Y. Yan, A. Z. Kapikian, and Y. Hoshino Species-specific but not genotype-specific primary and secondary isotype-specific NSP4 antibody responses in gnotobiotic calves and piglets infected with homologous host bovine (NSP4[A]) or porcine (NSP4[B]) rotavirus. Virology 330: Yuan, L., C. Iosef, M. S. Azevedo, Y. Kim, Y. Qian, A. Geyer, T. V. Nguyen, K. O. Chang, and L. J. Saif Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant Escherichia coli heat-labile toxin in gnotobiotic pigs. J Virol 75: Yuan, L., S. Ishida, S. Honma, J. T. Patton, D. C. Hodgins, A. Z. Kapikian, and Y. Hoshino Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus nonstructural protein 4 and viral protein (VP) 4, VP6, and VP7 in infants who received selected live oral rotavirus vaccines. J Infect Dis 189: Yuan, L., S. Y. Kang, L. A. Ward, T. L. To, and L. J. Saif Antibodysecreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus. J Virol 72: Yuan, L., and L. J. Saif Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model. Vet Immunol Immunopathol 87:

229 771. Yuan, L., L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Virol 70: Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, E. C. Ebert, N. Kassam, S. Qin, M. Zovko, G. J. LaRosa, L. L. Yang, D. Soler, E. C. Butcher, P. D. Ponath, C. M. Parker, and D. P. Andrew Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis. J Exp Med 190: Zarember, K. A., and P. J. Godowski Tissue expression of human Tolllike receptors and differential regulation of Toll-like receptor mrnas in leukocytes in response to microbes, their products, and cytokines. J Immunol 168: Zhang, M., C. Q. Zeng, Y. Dong, J. M. Ball, L. J. Saif, A. P. Morris, and M. K. Estes Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence. J Virol 72: Zhang, M., C. Q. Zeng, A. P. Morris, and M. K. Estes A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells. J Virol 74: Zhao, Y., I. Kacskovics, Q. Pan, D. A. Liberles, J. Geli, S. K. Davis, H. Rabbani, and L. Hammarstrom Artiodactyl IgD: the missing link. J Immunol 169: Zheng, B. J., R. X. Chang, G. Z. Ma, J. M. Xie, Q. Liu, X. R. Liang, and M. H. Ng Rotavirus infection of the oropharynx and respiratory tract in young children. J Med Virol 34: Zhou, Y., L. Li, B. Kim, K. Kaneshi, S. Nishimura, T. Kuroiwa, T. Nishimura, K. Sugita, Y. Ueda, S. Nakaya, and H. Ushijima Rotavirus infection in children in Japan. Pediatr Int 42: Zhu, M., X. Xu, H. Liu, X. Liu, S. Wang, F. Dong, B. Yang, and G. Song Enhancement of DNA vaccine potency against herpes simplex virus 1 by coadministration of an interleukin-18 expression plasmid as a genetic adjuvant. J Med Microbiol 52: Zhu, Y., P. Rota, L. Wyatt, A. Tamin, S. Rozenblatt, N. Lerche, B. Moss, W. Bellini, and M. McChesney Evaluation of recombinant vaccinia virus--measles vaccines in infant rhesus macaques with preexisting measles antibody. Virology 276: Zijlstra, R. T., S. M. Donovan, J. Odle, H. B. Gelberg, B. W. Petschow, and H. R. Gaskins Protein-energy malnutrition delays small-intestinal recovery in neonatal pigs infected with rotavirus. J Nutr 127:

230 Sites of studies Age of Subject Serum IgA antibody titers Bangladesh 0-4 months 4-7 months 0-24 months Serum IgG antibody titers a a Virus Neutralizing Antibody titers (RV strain specificity) Methods References ELISA (147) Bangladesh 0-6 months >200 NA 8 Complement fixing antibody Brazil - Belem VN test (777) 1-7 months 50 (anti-wa) VN b test (27) China- x = 8 months NA b NA 67c Guangzhou 3426 d Venezuela- NA 90 VN, ELISA (530) Caracas Venezuela- Caracas 0-1 month 2 months 3 months 4 months 5 months 6-11 months Newborn 4-10 months (anti-rrv), 49 (anti-p) 30 (anti-rrv), 27 (anti-p) (64) (575) (553) 1-6 months 13 (anti-rrv), 14 (anti-p) Table 1.1: Level of circulating rotavirus specific antibody of infants in some developing countries a b c d titers range from children with no infection to infection with non-severe or severe diarrhea abbreviation: NA; Not available, VN: virus neutralizing antibody titer measured in children with rotavirus in stool antibody titer measured in children without rotavirus in stool

231 Sites of studies Subject (age) Serum IgA antibody titers Serum IgG antibody titers Australia Mother (serum) 313 (cord blood) Virus Neutralizing Antibody titers Germany-Bochum Mother NA NA 490 (anti-wa) 330 (anti-sa11) Methods References (RV strain specificity) NA a ELISA (456) VN a (97) Germany-Bochum Infants (1-4 months) NA NA (anti-wa) 40 (anti-sa11) VN (97) Texas -United States Infants (< 18 months) b NA ELISA (513) United States Infants (2-6 months) United States Infants (1-6.5 months) NA NA VN, ELISA (719) NA NA VN (547) United States Infants NA NA ELISA (118) Table 1.2: Level of circulating rotavirus specific antibody of mothers and infants in some developed countries a abbreviation: NA; Not available, VN; virus neutralizing b preexisting antibody titers range from children infected to children not infected with rotavirus during the following season

232 Species Type of placenta Route of transfer of immunoglobulin Duration of passive immunity prenatal route Postnatal route Fish NA Yolk sac Yolk sac ~10 days Reptile NA Yolk sac Yolk sac <1 year Chicken/duck NA Yolk sac Yolk sac (<5 days for chicken 2 weeks Ruminant Syndesmochorial None gut (24-36h) Pig Epitheliochorial None gut (24-36h) 3-4 weeks Horse Epitheliochorial None gut (24h) days Dog, cat Endotheliochorial placenta (+) gut Rat/mouse Hemendothelial placenta (+++) gut (20 days) 4-10 weeks Primates Hemochorial placenta gut Human Hemochorial placenta (+++) gut 9 months Table 1.3: Routes of transmission of MatAb across taxa [modified from Grindstaff et al (259)] Species IgM IgA IgG t (½) a 97% b loss t (½) 97% loss t (½) 97% loss Chicken?? Bovine Sheep Pig Horse Dog Table 1.4: Expected loss (days) of MatAb in serum of the newborn in different species a t(½): half-life (days) of MatAb in newborn serum b time for 97% MatAb loss=5x half-lives [modified from Hines, H.C. (285)] 206

233 Preterm mother At-term mother Colostrum/milk TGF-β1 TGF-β2 IL-10 IL-6 TNF-α IL-8 IL-12 IL-1β Colostrum (0-5days) Transitional milk (6-30days) Mature milk (>30days) Colostrum (0-5days) Mean ± SD Mean ± SD Mean Mean ± SD Latent Free Latent Free Range (mean) Range (mean) 240 a ND b 2300 a ND 0-10 (8.5) a 0-24 (10) a 440 a ND 3560 a ND 0 a (6.8) a 520 a ND 5400 a ND 0-1.5(0.4)a 0-32 (14.3) a 3280 a 1366 ± 243 c 728 ± 249 c a 130 ± 108 d 0-6 (3.5) a (3304) d 8-34 (21) a 151 ± 89 f Mean±SD Mean±SD Mean±SD range 620±183 g 3684 ± 99 ± 10 h 2910 i 1408 ± k 2256 j Transitional milk (6-30days) Mature milk (>30days) 435 a 3560 a 0 a 0-14 (6.6) a 328 a 953 ± 213 c 179 ± 157 c 5310 a (13.5) a 0-25 (3.5) a Ref. a (646) c (591) a (646) c (591) a (646) d (518) a (646) e (231) a (646) f (570) g (569) h (484) i (518) j (98) k (279) Table 1.5: Concentrations of cytokines in human colostrum and milk (pg/ml) b: ND: not detectable (646)

234 Virus family Representative Genome Envelope Major Minor virus proteins proteins Caliciviridae Norwalk Ss RNA No Picornaviridae Poliovirus No 4 0 Flaviviridae Hepatitis C Yes 1 0 Retrovirus Human immunodeficiency virus Yes 1 0 Paramyxoviridae Newcastle disease Yes 4 0 Bunyaviridae Hantaan Yes 1 0 Orthomyxoviridae Influenza A Yes 2 3 Birnaviridae Infectious bursal disease No 3 0 Reoviridae Rotavirus No Parvoviridae Parvovirus No 1 2 Papillomarividae Papillomavirus Ds DNA No 1 1 Polyomaviridae SV40 No 1 2 Hepadnaviridae Hepatitis B Discontinuous Yes 1 0 Ds DNA Table 1.6: Virus-like particles for various virus families Modified from Noad and Roy (497). 208

235 VP2 VP3 VP4 VP4 VP7 Figure 1.1: Rotavirus structures Modified from Jayaram et.al., 2004 (324). 209

236 pigr FcRn Figure 1.2: Structure of FcRn and pigr Modified from Simister and Ahouse, 1996 (631) The pigr is a glycosylated membrane protein, homolog of Ig superfamily. The extracellular portion of the molecule consists of five homologous domains, resembling to the V domain of Ig superfamily. The FcRn, a MHC class I homolog, is a heterodimer of β2 microglobulin and a larger subunit α (45-53 kda) with 3 extracellular domains. 210

237 100 MatAb concentration Inhibition (%) 50 Antigen load 0 Excess MatAb Insufficient Ag Equivalent Insufficient MatAb Excess Ag Inhibition of T and B cell responses Preservation of T +/- B cell Preservation of T and B cell responses Enhancements? Figure 1.3: Expected influence of maternal antibody (MatAb) on neonatal immune responses to vaccines [Modified from Siegrist et al. 2003, (622) ] 211

238 ISCOM 2/6-VLP Figure 1.4: Association of RV 2/6VLP with immunostimulatory complexes (ISCOM) 212