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1 JVI Accepts, published online ahead of print on 11 June 2014 J. Virol. doi: /jvi Copyright 2014, American Society for Microbiology. All Rights Reserved. Newcastle Disease Virus Vector Producing Human Norovirus-like Particles Induces Serum, Cellular, and Mucosal Immune Responses in Mice Shin-Hee Kim, a Shun Chen, a Xi Jiang, b Kim Y. Green, c and Siba K. Samal a* a Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA b Division of Infectious Disease, Cincinnati Children s Hospital Medical Center, University of Cincinnati, College of Medicine, Cincinnati, Ohio, USA c Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, DHHS, Bethesda, Maryland, USA Downloaded from on June 29, 2018 by guest *Corresponding author: Siba K. Samal Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, USA Telephone: (301) ; Fax: (301) ; ssamal@umd.edu 1

2 ABSTRACT Human norovirus infection is the most common cause of viral gastroenteritis worldwide. Development of an effective vaccine is required for reducing norovirus outbreaks. Inability to grow human norovirus in cell culture has hindered development of live attenuated vaccines. To overcome this obstacle, we generated a recombinant Newcastle disease virus (rndv) vectored experimental norovirus vaccine by expressing the capsid protein (VP1) of norovirus strain VA387. We compared two different NDV vectors, a conventional rndv vector and a modified rndv vector for their efficiency to express VP1 protein. Our results showed that the modified vector replicated to higher titers and expressed higher levels of VP1 protein in DF1 cells and in allantoic fluid of embryonated chicken eggs than the conventional vector. We further demonstrated that the VP1 protein produced by rndvs was able to self-assemble into virus-like particles (VLPs), which are morphologically similar to baculovirs-expressed VLPs. Evaluation of their immunogenicity in mice showed that the modified rndv vector induced higher level of IgG response than that induced by the conventional vector and by the baculovirus-expressed VLPs. The rndv vectors predominantly induced subclass IgG2a antibody for Th1 response, and specifically, high levels of IFN-γ, TNF-α, and IL-2 were detected in the splenocytes. In addition, the modified rndv vector induced higher level of fecal IgA response in mice than baculovirusexpressed VLPs. Our findings suggest that rndv vector is an efficient system to produce costeffective VLPs in embryonated chicken eggs as well as has the potential to be used as a live attenuated vaccine in humans. 2

3 IMPORTANCE Noroviruses are the major cause of viral gastroenteritis worldwide. Currently, effective vaccines against norovirus infection are not available. In this study, we have evaluated Newcastle disease virus (NDV) as a vaccine vector for norovirus. Our results suggest that NDV can not only be used as a cost-effective method for large-scale production of norovirus-like particle vaccines but also can be used as a live-attenuated vectored vaccine. Downloaded from on June 29, 2018 by guest 3

4 Norovirus (NoV) is the most frequent cause of viral gastroenteritis in people of all ages, causing an average of deaths, 56,000-71,000 hospitalizations, 400,000 emergency department visits, million outpatient visits, and million total illnesses per year in the U.S (1). NoV infections usually result in an acute and self-limiting gastroenteritis. NoV is also a common cause of severe diarrhea in children (2). Frequent NoV outbreaks cause a significant economic burden due to medical and social expenses (3). For these reasons, there is a great need to develop an effective NoV vaccine. Noroviruses are members of the family Caliciviridae. The genome of norovirus consists of a linear, positive-sense, single-stranded RNA molecule of 7.6 kb with three open reading frames (4). ORF1 encodes a large nonstructural polyprotein that is autocatalytically processed by the viral protease to yield six nonstructural proteins (p48 N-terminal protein, p41 NTPase, p22, VPg, protease, and RNA-dependent RNA polymerase). ORF2 and ORF3 encode structural proteins, the major capsid protein (VP1) and a minor capsid protein (VP2), respectively. Expression of VP1 alone has been shown to produce self-assembled norovirus-like particles (VLPs) that are morphologically and antigenically similar to native virions (5). VP1 has been shown to be the major immunogenic protein of norovirus. The genus Norovirus is composed of more than 40 diverse virus strains divided into 6 genogroups (GI GVI), with the GI and GII genogroups being the most important for human infection (6). The genogroup II, genotype 4 NoV (GII.4) are currently responsible for 70 80% of norovirus outbreaks worldwide (7). The inability of NoV to grow in cell culture has been the major impediment to developing vaccines against norovirus infection. To overcome this obstacle, various in vitro expression systems have been evaluated for production of NoV VLPs as vaccine. Preparation of VLPs in vitro is time-consuming and expensive. Immunization usually requires a high dose of 4

5 VLPs (usually more than 100 µg), multiple booster immunizations, and efficient delivery system and adjuvants (8). The duration of the antigen stimulation may be limited because VLP is a nonreplicating immunogen (9). Alternatively, Venezuelan equine encephalitis (VEE) replicon system has been applied for expression of VLPs in mammalian cells (10). Preliminary clinical trials suggest that immunization efficacy of VLP vaccine needs to be improved and effective delivery approaches of vaccines needs to be developed to achieve efficient immunization (8). Recently, other expression systems, such as vesicular stomatitis virus (VSV), have been used as a vaccine vector to produce VLPs (9). There is a need to evaluate additional expression systems, which are cost-effective for large scale production of VLPs with efficient immunogenicity for use in humans. Newcastle disease virus (NDV) belongs to the genus Avulavirus in the family Paramyxoviridae. The genome of NDV is a single-stranded, negative-sense RNA that contains six genes in the order 3 -N-P-M-F-HN-L-5 and encodes at least seven proteins (11). NDV isolates vary greatly in their pathogenicity for chickens, and are categorized into three main pathotypes: lentogenic (avirulent), mesogenic (moderately virulent), and velogenic (highly virulent) (12). In NDV, the F protein mediates fusion of the viral envelope with the cell membrane. The F protein is synthesized as an inactive processor (F 0 ) that is cleaved by host cell protease into two biologically active F 1 and F 2 subunits. Cleavage of the F protein is a requisite for virus entry and cell-to-cell fusion. The amino acid sequence at the F protein cleavage site has been identified as the primary determinant of virulence (11). Virulent NDV strains have multibasic residues that conform to the preferred cleavage site of the intracellular protease furin (Arg-X-Arg/Lys-Arg ) present in most cell types. In contrast, avirulent NDV strains typically contain a single or two basic residues at the F protein cleavage site and are delivered to the 5

6 plasma membrane in an uncleaved form for cleavage by extracellular proteases, thus restricting viral replication to the respiratory and enteric tracts where secretary proteases for cleavage are available. Lentogenic NDV strains are widely used as live attenuated vaccines, whereas mesogenic strains are used as live vaccines only in some countries. Recombinant lentogenic and mesogenic NDV strains have also been evaluated as vaccine vectors for animal and human pathogens (13-16). NDV accommodates foreign genes (at least 4.2 kb in length) with a good degree of stability. In contrast to the other viral vectors that encode a large number of proteins, such as adeno, herpes, and pox viruses, NDV encodes only seven proteins and so there is less competition for immune responses between vector proteins and the expressed foreign antigens. NDV replicates in the cytoplasm and does not integrate into the host cell DNA. Genetic recombination is either rare or does not occur in NDV, thus making it a stable vaccine vector. Several characteristics of NDV suggest that it is an ideal vaccine vector for human use (17). NDV is safe in humans, due to a natural host range restriction. NDV shares only a low level of amino acid sequence identity with known human paramyxoviruses and are antigenically distinct from common human and animal pathogens, and thus would not be affected by preexisting immunity in humans. NDV infects via the intranasal route and has been shown to induce humoral and cellular immune responses both at the mucosal and systemic levels in murine and nonhuman primate models (13-15, 18, 19). NDV has been used to express protective antigens of simian immunodeficiency virus (20), Ebola virus (15), respiratory syncytial virus (21), human parainfluenza virus type 3 (13), H5N1avian influenza virus (19), and severe acute respiratory syndrome coronavirus (18). NDV vectored vaccines have shown promising results in preventing infection of human pathogens. However, NDV has not been evaluated as a vaccine vector for NoV. 6

7 In this study, we have successfully generated recombinant NDVs (rndvs) expressing the VP1 protein of norovirus (rndv-vp1). Infection of cell culture and embryonated eggs resulted in high levels of VLP production. Furthermore, we have shown that rndvs-vp1 elicited NoVspecific humoral, mucosal, and cellular immune responses in a mouse model. These results suggest that rndv-vp1 could provide a new approach to produce cost-effective VLPs as well as use as a safe live-viral vaccine in humans. MATERIALS AND METHODS Viruses and cells. The chicken embryo fibroblast cell line (DF1) and human epidermoid carcinoma cell line (HEp-2) were grown in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FBS). The modified vaccinia virus strain Ankara (MVA) expressing T7 RNA polymerase was kindly provided by Dr. Bernard Moss (NIAID, NIH). In experiments that required supplementations of exogenous protease for the cleavage of the F protein, normal SPF chicken egg allantoic fluid was added to a concentration of 10%. NDV strain rlasota (22) and the vaccine viruses generated in this study were grown in the allantoic cavities of 9-day-old specific pathogen free (SPF) embryonated chicken eggs. Virus stocks were quantified by hemagglutination (HA) assay with chicken erythrocytes. All experiments involving experimental animals were approved by the committee of IACUC, University of Maryland and conducted following the guidelines. Generation of rndvs expressing norovirus ORF2. The ORF2 (1644 nt) of norovirus strain VA387 (genogroup II.4) was flanked with NDV-specific gene start and gene end transcriptional signals at the upstream and downstream ends, respectively. The construct was designed to conform to the rule of six, which is a requirement for efficient RNA replication (23). 7

8 The expression cassette was inserted using a unique PmeI site between the P and M genes, in a cdna encoding the complete antigenomic RNA of NDV strain rlasota (22). To enhance the levels of ORF2 gene expression, the gene was similarly cloned into a modified NDV vector. (23). A previously described full length cdna of the antigenome of strain BC (24) was modified by changing its naturally-occurring F protein cleavage site motif (RRQKR F) to that of strain LaSota (GRQGR L) to create modified rndv. Furthermore, the HN protein was replaced with that of strain LaSota. The modified vector was found to replicate to high titer and express foreign proteins at high level, but was avirulent in chickens. The recombinant viruses were recovered from NDV antigenomic cdnas by transfection into HEp-2 cells with support plasmids expressing the N, P, and L proteins as previously described (24). To evaluate genetic stability, the recovered viruses were passaged five times in SPF 9-day-old embryonated chicken eggs, and the sequences of the ORF2 and F gene were analyzed. For analysis of the expression of the NoV capsid protein, DF1 cells were infected with rndvs at an MOI of 1. At 24 h postinfection (hpi), cell lysates were collected and analyzed by Western blot using a monoclonal antibody to VP1. We further analyzed the presence of the VP1 protein in the culture medium and allantoic fluid in SPF chicken eggs by Western blot. At various times of postinfection, the culture medium of infected DF1 cells was harvested, clarified at 3,000 rpm for 15 min and further concentrated at 30,000 rpm for 1.5 h. Similarly, allantoic fluids were collected from infected SPF chicken eggs, clarified, and concentrated by ultracentrifugation. All the samples were subjected to Western blot analysis. To determine whether NoV VP1 protein was incorporated into NDV particles, purified virus from the allantoic fluid (25) was analyzed by Western blot. 8

9 Production of VLPs. The ability of rndv-vp1 to produce VLPs in DF1 cells and in embryonated eggs was evaluated using a procedure described previously (26). For purification of VLPs, the culture medium of DF1 cells at 2 days postinfection (dpi) was collected by centrifugation at 1000 rpm for 5 min. Similarly, allantoic fluid of infected embryonated chicken eggs was collected at 3 dpi and centrifuged at 3,000 rpm for 10 min. The VLPs were purified by ultracentrifugation through a 40% sucrose cushion, followed by CsCl isopycnic gradient (1.36 g/cm 3 ) ultracentrifugation. The band containing VLPs was collected for analysis after pelleting by centrifugation. Negative-staining electron microscopy (EM) of purified VLPs was performed as described previously (5). Briefly, 10 μl of VLP suspension was fixed on copper grids and negatively stained with 1% ammonium molybdate. The negatively stained grids were examined for presence of VLPs using an electron microscope. For comparison of VLPs produced using the rndv system with the VLPs produced using the baculovirus system, the ORF2 of NoV strain VA387 was cloned into bacmids and transfected into Spodoptera frugiperda (Sf9) cells (Invitrogen). For purification of VLPs, the Sf9 cells were infected with the recombinant baculovirus at an MOI of 1, and the infected Sf9 cells and cell culture supernatants were harvested at 8 dpi, purified by CsCl isopycnic gradient, and examined by EM as described above. Growth characteristics of rndvs expressing VP1 protein in DF1 cells. The multicycle growth kinetics of the rndv-vp1 was evaluated in DF1 cells in the presence of 10% chicken egg allantoic fluid. Duplicate wells of six-well plates were infected with each rndv- VP1 at an MOI of Supernatants were collected and replaced with an equal volume of fresh medium at 12-h intervals until 56 hpi. Virus titers in the collected supernatants were quantified in 9

10 DF1 cells by limiting dilution in the presence of added allantoic fluid and expressed as 50% tissue culture infectious dose (TCID 50 /ml) by the end-point method of Reed and Muench (27). Pathogenicity of rndvs in embryonated chicken eggs and in 1-day-old chicks. The pathogenicity of rndvs-vp1 was determined by the mean death time (MDT) test in 9-day-old SPF embryonated chicken eggs and by the intracerebral pathogenicity index (ICPI) test in 1-dayold SPF chicks (12). The MDT was determined as mean time (h) for the minimum lethal dose of virus to kill all the inoculated embryos. For the ICPI test, fresh infective allantoic fluid for each virus was inoculated into groups of 10 1-day-old SPF chicks via the intracerebral route. The ICPI is the mean of the score per bird per observation for clinical symptoms and mortality over the 8- day period. At each observation, the birds were scored as follows: 0 if normal; 1 if sick; and 2 if dead. Highly virulent velogenic viruses give values approaching 2, and avirulent or lentogenic strains give values at or close to 0. Immunization of mice. Groups of four-week-old female BALB/c mice (5 mice per group) were immunized individually with conventional vector (rlasota-vp1) or modified rndv-vp1 (30 µl of each, 10 6 EID 50 ) by the intranasal route after inhalational anesthesia. A third group of mice was intranasally inoculated with 30 μg of baculovirus-expressed VLPs (ORF2 of NoV strain VA387) (28). All the viruses and VLPs were prepared in PBS for immunization. Mice received three doses of immunization with a two-week interval (28). The last group of mice was inoculated with PBS as unvaccinated controls. After inoculation, the mice were observed daily for any clinical signs. Serum and fecal samples were collected from each mouse prior to the first immunization and one and two weeks after final immunization for IgG and IgA antibody detection, respectively. At 6 weeks postinoculation (wpi), all mice were sacrificed, and the spleens were collected for detection of cellular immune response. 10

11 Serum IgG and subclass ELISA. Antibody titers in sera were measured by an enzymelinked immunosorbent assay (ELISA). NoV-specific IgG titers were determined by testing individual serum samples against baculovirus VLP antigen-coated plates using a protocol described previously (28). We further evaluated the presence of NoV-specific IgG1 and IgG2a antibodies to evaluate the type of response elicited (Th1 versus Th2) by rndv immunogen (29, 30). Ninety-six-well plates were coated with 50 μl of purified baculovirus-expressed VLPs at 4 C overnight. Sera were serially diluted (starting at 1:100) in a series of two- fold dilutions and the specific antibody titers were defined as the reciprocal of the endpoint dilution with an OD 405 equal to or greater than 0.2. ELISPOT assay. A cytokine specific enzyme-linked immunospot (ELISPOT) assay for detection of IFN-γ, TNF-α, and IL-2 was evaluated. Two weeks after the final immunization, spleens were collected from mice. Suspension of splenocytes was prepared by lysing red blood cells with RBC lysis buffer and subjected to the assay following the manufacturer s instruction (BD Bioscience). For detection of NoV-specific cytokine producing cells, splenocytes were stimulated with synthetic 14-mer peptide to CD4+ T cell epitope (FYQEAAPAQSDVAL) targeting NoV GII.4 strain VA387 (28). Cells without the peptide and cells stimulated with by concanavalin A (10 µg/ml) were used as controls. The results are expressed as mean spotforming cells (SFC) per 10 6 splenocytes of duplicate wells. Fecal IgA ELISA. Stool samples were analyzed for NoV-specific and total IgA responses by ELISA (29). Fecal pellets were diluted in PBS (1:2) containing 0.1% Tween and a Complete EDTA-free proteinase inhibitor cocktail tablet, vortexed, and clarified by centrifugation at 10,000 g for 10 min (9). Ninety-six-well plates were coated with 50 μl of NoV VLPs (1 μg/ml) at 4 C overnight for detection of NoV-specific IgA. The level of NoV- 11

12 specific fecal IgA was calculated from a standard curve that was determined by the absorbance values of the IgA standard. Total fecal IgA was determined by capturing all IgA molecules contained in the fecal extracts with goat anti-mouse IgA. The level of IgA was calculated from a standard curve that was determined by the absorbance values of the mouse IgA standard. Fecal IgA responses were expressed as a ratio of the NoV-specific IgA (ng/ml) to total IgA (μg/ml). Statistical analysis. Statistically significant differences in serum, cellular, and mucosal immune responses between immunized mouse groups (p<0.05) were evaluated by one-way analysis of variance (ANOVA) (SPSS 22.0, IBM SPSS Inc.). RESULTS Generation of rndvs expressing VP1 protein of norovirus. The VP1 protein of NoV is the major viral neutralization and protective antigen (29). The transcription cassette containing the ORF2 of NoV was inserted as an additional gene into the antigenomic cdna of lentogenic NDV strain LaSota between the P and M genes (Fig. 1A). For comparison purpose, the ORF2 cassette was also inserted into the antigenomic cdna of a modified NDV vector. The recombinant NDVs were readily recovered by reverse genetics. The recovered viruses were passaged five times in SPF 9-day-old embryonated chicken eggs. The correct sequence of the NoV insert in each recovered virus was confirmed by RT-PCR and sequence analysis. This demonstrated genetic stability of the NoV insert in the rndv genome without any adventitious mutations. In vitro characterization of rndv-vp1. To detect expression of the VP1 protein, DF1 cell monolayers were infected with rlasota-vp1, modified rndv-vp1, and their parental viruses for 24 h. Cell lysates were analyzed by Western blotting using a VP1 specific antiserum. 12

13 Both the rndv vectors expressed NoV VP1 (58 kda) in DF1 cells (Fig. 1B). However, there was approximately 2.6-fold more VP1 protein synthesized by the modified rndv vector compared to that expressed by the rlasota vector. In order to investigate whether the insertion of the foreign gene in the NDV genome might have differentially affected the growth of one vector versus the other, we evaluated the growth kinetics of parental and rndv-vp1 in DF1 cells after infection at an MO1 of 0.01 in the presence of exogenous protease (Fig. 2). All the viruses were able to replicate efficiently in the DF1 cells. Parental modified rndv replicated more efficiently than the parental rlasota. The maximum titers were TCID 50 /ml at 40 hpi for modified rndv and TCID 50 /ml at 48 hpi for rlasota. In general, the two rndv-vp1 viruses replicated less efficiently than their parental viruses. Similarly to the parental viruses, the modified rndv-vp1 grew to high titer than rlasota-vp1 up to 48 h of infection, reaching a maximum titer of TCID 50 /ml at 40 hpi. The growth kinetics of parental and modified rndv-vp1 also indicated that the modified rndv vector had enhanced replication in vitro compared to rlasota vector. rndvs expressing VP1 are highly attenuated in chickens. The pathogenicity of rndvs was evaluated by the MDT assay in embryonated chicken eggs and by the ICPI in 1-dayold chicks (Table 1). Of the two parental viruses, rlasota (117 h) was more attenuated than the modified rndv (108 h). The pathogenicity tests further confirmed that the two rndvs expressing VP1 protein were more attenuated than their parental viruses (144 h for rlasota-vp1 and 135 h for modified rndv-vp1). Thus, introduction of VP1 protein to rndv genome conferred further attenuation. In addition, the ICPI values of all rndvs were 0.00, and chicks infected with rndvs had no apparent clinical signs during the 8-day period of the ICPI test. This suggests that rndvs are avirulent in chickens. 13

14 Characterization of NoV VLPs produced by rndv vectors. To determine whether the VP1 protein produced by rndv vectors self-assembled to VLPs, we infected DF1 cells and embryonated chicken eggs with rndv vectors expressing VP1 protein. Kinetic analysis of VP1 protein expression showed initial detection of VP1 in cell lysates at 24 hpi and then subsequent detection of VP1 protein in culture medium (24 and 48 hpi), indicating gradual secretion of the VP1 protein into cell culture medium (Fig. 3A). We further evaluated the expression of VP1 protein by rndvs in allantoic fluid of SPF embryonated chicken eggs by Western blotting analysis. Since NDV grows to high titer in eggs, there is a possibility that the VP1 protein is also expressed at high level in allantoic fluid, leading to efficient production of VLPs. Nine-day-old embryonated SPF chicken eggs were inoculated with the rndv vectors expressing VP1 protein. The allantoic fluid samples were collected every 24 h for virus titration and VP1 protein expression. The two rndv vectors expressing VP1 protein grew to high titers (>10 8 pfu/ml) in embryonated chicken eggs at 3 dpi (data not shown) and produced high levels of VP1 protein in allantoic fluids (Fig. 3B). Further, Western blot analysis of rndv purified from allantoic fluid showed that the VP1 protein was not incorporated into NDV particles (not shown), indicating efficient secretion of VP1 protein into allantoic fluid. We next evaluated assembly and morphology of VLPs expressed by rndvs in DF1 cells and in allantoic fluid of embryonated chicken eggs using EM analysis (Fig. 4). For comparison purpose, baculovirus-expressed VLPs were included. All the samples were prepared by CsCl isopycnic gradient purification. VLPs of approximately 35 to 40 nm in diameter, similar to the size of baculovirus-expressed VLPs were observed in DF1 cell and in embryonated egg preparations, indicating self-assembly of VP1 protein produced by rndv vectors into VLPs. 14

15 Induction of NoV-specific immune responses in mice. We first confirmed absence of NoV-specific antibodies in serum and fecal samples collected from all pre-immunized mice. To determine the immunogenicity of VP1 protein produced by rndv vectors, groups of mice (5 mice per group) were immunized with each of the rndv vectors via intranasal route. Baculovirus-expressed VLPs were included as a control. One group of mice was inoculated with PBS as an unvaccinated control. All mice infected with rndvs or VLPs had no apparent clinical signs during the immunization. We first evaluated the immunogenicity of the rndv vectors by collecting sera at 5 and 6 wpi and determining antibody responses using HI assay (log 2 ) against NDV (Fig. 5). NDV-specific antibody response in the sera was analyzed using HI test. rndv vectors induced similar levels of NDV-specific serum antibody in mice (p>0.05), indicating replication of both rndv vectors in mice. The sera collected from groups of PBS and VLP immunized mice showed negative HI titer to NDV. We first evaluated NoV-specific antibody response in mice by determining the serum IgG antibody by ELISA. Similar levels of serum antibody titers were detected in immunized mice at 5 and 6 wpi (p>0.05). In the immunized mouse groups, modified rndv and baculovirusexpressed VLPs induced high titers of NoV specific IgG in mice both at 5 and 6 wpi (Fig. 6). In contrast, rlasota was less efficient for the induction of IgG than modified rndv and baculovirus-expressed VLPs (p<0.05). A group of mice immunized with PBS did not show an NoV specific IgG response. Our results suggest that the level of NoV-specific antibody response induced by intranasal inoculation of modified rndv-vp1 is similar to that induced by baculovirus-expressed VLPs. 15

16 The systemic NoV-specific IgG response was further characterized into Th1 and Th2 responses by measuring IgG antibody subtypes IgG2a and IgG1, respectively. The NoV-specific IgG1 and IgG2a antibody levels in immunized mice were determined by ELISA (Fig. 6B and C). The baculovirus-expressed VLPs induced significantly higher level of IgG1 subtype (titer 8,320) than the two rndvs (p<0.05), resulting in a Th2/Th1 ratio of 4.2. In contrast, immunization of mice with modified rndv-vp1 induced significantly high levels of IgG2a (titer 8,960) (p<0.05) but low levels of IgG1 (titer 380), resulting in a Th1/Th2 ratio of rlasota-vp1 induced lower levels of subtype immune response compared to modified rndv-vp1 (p<0.05). However, the rlasota/ VP1 virus also induced higher levels of IgG2a than IgG1 (a Th1/Th2 ratio of 5.8). These data suggests that variation can occur in helper T cell populations induced by live NDV vaccines and nonreplicating baculovirus VLPs presenting norovirus antigens. Induction of cellular immune response in mice. The difference in the induction of Th1 responses by rndvs and baculovirus-expressed VLPs were determined by measuring cellular immune response from mouse splenocytes using ELISPOT assay (Fig. 7). Spleen samples were collected from infected mice at 6 wpi. Among all the mice groups, cells of mice collected from the group immunized by modified rndv vector produced the highest levels of IFN-γ (371 SFC/10 6 ), IL-2 (239 SFC/10 6 ), and TNF-α (610 SFC/10 6 ) after stimulation with the synthetic peptides representing a T-cell epitope among the immunized mouse groups (p<0.05). In contrast, immunization of mice with rlasota vector and baculovirus-expressed VLPs did not result in efficient induction of a cellular immune response compared to that induced by modified rndv vector (p<0.05). Cell viability was similar in all groups controlled by concanavalin A stimulation. No response to the negative control was detected in any of the groups. 16

17 Induction of intestinal IgA immune response in mice. Since mucosal antibodies play an important role in protection from NoV infection, we next characterized induction of mucosal immune response in mice by determining fecal IgA titers (Fig. 8). Fecal samples were assayed for NoV-specific and total IgA by ELISA. The two rndv vectors induced an intestinal IgA response in all the mice. Modified rndv-vp1 induced significantly higher levels of IgA response than rlasota-vp1 in mice at 5 and 6 wpi (p<0.05). In contrast, baculovirus-expressed VLPs induced the lowest level of IgA response in mice (p<0.05). Our result suggests that liveattenuated rndv vaccines may be more efficient in the stimulation of an IgA immune response than baculovirus-expressed VLPs. DISCUSSION Historically live viral vaccines have provided the most effective protection against viral infection and disease (31). Unfortunately, development of live viral vaccine is not realistic for an uncultivable virus like NoV. Alternatively, replicating viral vector vaccines offer a live vaccine approach that neither requires involvement of the complete pathogen nor cultivation of the pathogen. Therefore, in this study, we have evaluated NDV, a common avian virus, as a vaccine vector for NoV. NDV offers a number of advantages as a vaccine for human use because of its high tolerability, absence of preexisting immunity, robust replication, and ability to induce a mucosal immune response. Although both lentogenic and mesogenic NDV strains can be used as vaccine vectors in humans, our results showed that mesogenic strains replicate to higher titer in nonhuman primates (13). Therefore, in this study, we have evaluated two different NDV backbones, lentogenic strain rlasota (conventional NDV-vectored backbone) and modified rndv from 17

18 mesogenic strain BC. For safety concerns, we altered the cleavage site of the F protein to that of rlasota in the modified rndv backbone. Both vaccine vectors were found to be avirulent in chickens and the modified cleavage site sequence was stable throughout the passage of the virus in vivo. In this study, we have used both rndv vectors to express the VP1 protein of NoV. We demonstrated that the VP1 protein was expressed at high levels by both vectors. However, the level of VP1 produced by the modified rndv vector was higher than that produced by the rlasota vector. Currently, NoV VLPs produced in insect cells by recombinant baculovirus are being evaluated as a vaccine for NoV infection (8). However, preparation of VLPs can hinder vaccine design and manufacture due to its high cost and time-consuming process. Moreover, immunization usually requires a high dose of VLPs with multiple booster immunizations (32, 33). The degree of structural integrity in the VLP preparation can also affect the immunogenicity and protection efficacy of VLP vaccine (34). In this study, we further evaluated whether rndv can be an efficient system to produce VLPs for use as a norovirus vaccine. Detection of VP1 protein both in culture medium of DF1 and in allantoic fluid of chicken eggs suggested that VLPs can be self-assembled and produced by using both cell culture and embryonated egg systems. Subsequently, electron-microscopic analysis confirmed that the VP1 protein expressed by rndv vector self-assembled to NoV VLPs which are morphologically similar to NoV VLPs produced by baculovirus system (Fig. 4A). Baculovirus-expressed NoV VLPs have been studied and extensively and are considered morphologically and antigenically similar to native NoV virions (5). Since preparation of baculovirus-expressed VLPs is expensive and time-consuming, we propose that production of NoV VLPs by rndv vectors in embryonated chicken eggs can improve manufacturing cost of NoV VLPs. rndv vector can replicate to high titer and produce 18

19 large quantities of VLPs in embryonated chicken eggs. For VLP production, allantoic fluid from modified rndv-infected chicken eggs can be collected at 3 dpi, whereas baculovirus infected cell lysates can be collected at 7 dpi. Therefore, this approach can be cost-effective, efficient, time-saving, and feasible for large-scale manufacturing of VLP vaccines. Similarly, rndv vectors can also be applied for VLP production of other non-cultivable viruses. We then evaluated rndvs expressing NoV VP1 as live virus vaccine candidates in mice. The general similarity and difference in the immune system between human and mouse are known, but mice are frequently used in evaluating vaccine candidates before moving to human trials (28). NDV replicates well in mice without causing significant mortality (19). In this study, the mice immunized with rlasota and modified rndv vectors were all healthy without showing any clinical signs during immunization period. Furthermore, NDV is a potent inducer of IFN-α and IFN-β production in mice (21). This can be used as an adjuvant for the induction of humoral and cellular immune responses and is also important for resistance to norovirus infection. We compared the serum, mucosal and cellular immune responses among mouse groups inoculated with live rndvs expressing VP1 and baculovirus-expressed VLPs. A previously established protocol for immunizing with baculovirus VLP-based vaccine was used for the immunization (28). An intranasal route was chosen for immunization of rndv vector vaccines, since this is one of the natural routes of NDV infection in chickens. In agreement with the results of our in vitro study, modified NDV vectored vaccine induced higher levels of immune responses than rlasota vector vaccine in mice, suggesting more efficient replication of modified rndv in mice. In addition, the modified rndv was able to induce higher titers of NoV-specific IgG than baculovirus-expressed VLPs, suggesting the effectiveness of rndv vector as a live NoV vaccine. 19

20 For noroviruses, CD4+ and CD8+ T cells are known to be required for efficient clearance of infection (35). Norovirus infection can induce activation of Th1 cells and the production of IFN-γ (36). In this NoV challenge study conducted in humans, activation of Th1 responses was associated with protection against NoV infection in some volunteers (36). Recent study with murine norovirus strains suggests that noroviruses display virus strain-specific differences in their induction of protective immunity (37). However, in general, antibody and CD4+T cells are essential for protection from a secondary norovirus infection. Therefore, the development of an appropriate CD4+ Th-cell subset may be important for disease resolution and for the effectiveness of the vaccine. To determine the type of Th cellular immune responses in vaccinated mice, serum IgG subtype analysis was conducted and compared to the induction of cellular Th1/Th2 immune responses between rndv-vectored vaccine and baculovirus-expressed VLPs. Specifically, modified rndv predominantly induced serum IgG2a for a Th1 response, whereas inoculation of VLPs led to IgG1 for a Th2 response. In fact, antibodies of the IgG2a isotype are most often induced by viral infections or viral antigens, and IgG2a is known to be effective in complement activation and antibody-dependent, cell mediated cytotoxicity (38). Induction of Th1 response by modified rndv was further confirmed by detection of all three markers of Th1 (IFN-α, IFN-γ and IL-2). In contrast, Th2 is responsible for enhancing humoral immune response. Thus, baculovirus-expressed VLPs predominantly induced Th2 response and lower levels of the three cytokines compared to that induced with modified rndv. Our results demonstrated that a live NDV vectored vaccine can effectively induce cellular immunity for the regulation of antiviral response. Since NoV causes acute gastroenteritis, mucosal immunity plays an important role in protecting humans from disease. However, the induction of IgA responses after immunization 20

21 with VLP-based vaccines requires higher concentrations of antigen than those to induce serum IgG responses in mice (39). For example, oral immunization of mice with VLPs for Norwalk virus showed that use of high doses (200 and 500 μg of VLPs) in the absence of adjuvant only resulted in intestinal IgA responses in the mice (29). Similarly, intranasal immunization of Norwalk virus-like particles in mice showed that both serum IgG and IgA virus-specific titers were higher with increasing doses of the immunogen (39). In addition, the efficacy of VLP-based vaccines has been shown to rely on the addition of biological adjuvants, such as cholera toxin and alphavirus adjuvant particles (40). Our results also indicated that intranasal immunization of VLP vaccine was unable to induce a strong mucosal immunity without use of adjuvant. In contrast, induction of strong mucosal immunity by modified rndv vector further confirmed a potential use of NDV as a live attenuated NoV vaccine. In summary, our findings suggest that rndv can be used as a safe and effective vaccine vector for NoV infection. rndv vector expressed high levels of NoV VP1 protein and formed VLPs in DF1 cells and in allantoic fluid of embryonated chicken eggs. In particular, efficient production of VLPs by rndvs in allantoic fluid can be a novel approach in facilitating generation of cost-effective VLP based vaccines. The rndv vectored vaccine induced higher levels of serum, cellular, and mucosal immune responses than baculovirus-expressed VLPs in mice, suggesting that rndv is an efficient system in delivering the VP1 protein in vivo and can be a good candidate as a live attenuated vaccine against NoV infection. As an initial step, in this study, we have used a mouse model to demonstrate immunogenicity of rndv vectored vaccine. However, the immunogenicity and protective efficacy of rndv vectored vaccine will need to be evaluated further in animal models, such as gnotobiotic pigs, which can support symptomatic infection and shedding of human NoV (41). 21

22 ACKNOWLEDGEMENTS We thank Daniel Rockemann, Girmay Gebreluul, Yonas Araya, and our laboratory members for excellent technical assistance; and Dr. Bernard Moss (NIAID, NIH) for providing the vaccinia T7 recombinant virus and the ptm1 plasmid. This research was supported by NIAID (1R21AI100195). The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services, nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government. REFERENCES 1. Hall AJ, Lopman BA, Payne DC, Patel MM, Gastañaduy PA, Vinjé J, Parashar UD Norovirus disease in the United States. Emerg. Infect. Dis. 19(8): Payne DC, Vinjé J, Szilagyi PG, Edwards KM, Staat MA, Weinberg GA Norovirus and medically attended gastroenteritis in U.S. children. N. Engl. J. Med. 368(12): Yee EL, Palacio H, Atmar RL, Shah U, Kilborn C, Faul M, Gavagan TE, Feigin RD, Versalovic J, Neill FH, Panlilio AL, Miller M, Spahr J, Glass RI Widespread outbreak of norovirus gastroenteritis among evacuees of hurricane Katrina residing in a large megashelter in Houston, Texas: lessons learned for prevention. Clin. Infect. Dis. 44: Green KY Caliciviridae: the noroviruses, p In D. M. Knipe and P. M. Howley (ed.), Fields virology, 6th ed. Wolters Kluwer-Lippincott Williams & Wilkins, Philadelphia, PA. 22

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28 Table 1. Pathogenicity of parental and chimeric viruses in embryonated eggs and in chicks Virus MDT a ICPI b rlasota 117 h 0.00 rlasota/ VP1 144 h 0.00 Modified rndv 108 h 0.00 Modified rndv/ VP1 135 h 0.00 a Mean embryo death time (MDT): the mean time (h) for the minimum lethal dose of virus to kill all of the inoculated embryos. Pathotype definition: virulent strains, <60 h; intermediate virulent strains, 60 to 90 h; avirulent strains, >90 h. b Pathogenicty of NDV in 1-day-old SPF chicks was evaluated by the ICPI assay. Pathotype definition: virulent strains, ; intermediate virulent strains, ; and avirulent strains, Downloaded from on June 29, 2018 by guest 28

29 FIGURE LEGENDS Figure 1. Generation of rndvs containing norovirus ORF2 and production of VP1 protein by rndvs. (A) The ORF2 of capsid gene was flanked by the gene-start and gene-end signals of NDVs and inserted into the intergenic region between the P and M genes in a full-length antigenomic cdna of NDVs. (B) DF1 cells were infected with each virus at MOI 1, and cell lysates were collected at 24 h post-infection for Western blot analysis. Capsid (58 kda) and NDV HN (74 kda) proteins were detected using VP1 and HN specific monoclonal antibodies, respectively. Lanes: 1. rlasota, 2. Modified rndv, 3. rlasota/vp1, and 4. Modified rndv/vp1. Figure 2. In vitro multicycle growth of parental and vaccine viruses in chicken embryo fibroblast DF1 cells following infection with an MOI of Exogenous protease was provided in the infected cells. The viral titers were determined by limiting dilution on DF1 cells. Figure 3. Characterization of VP1 protein expression by rndvs in DF1 cells and embryonated eggs. (A) Kinetic analysis of VP1 expression by rndvs in DF1 cells. Cell lysates and culture medium were collected every 12 h and subjected to Western blot analysis. (B) Detection of norovirus VP1 in allantoic fluid of chicken embryonated eggs. Allantoic fluids were harvested at 72 h post-infection and clarified by centrifugation at 3,000 rpm for 10 min. Figure 4. Production of VLPs by rndv. (A) Electron microscope analysis of norovirus VLPs. VLP suspension (10 µ each) was fixed in copper grids, negatively stained with 1% ammonium molybdate, and visualized by using an electron microscope. (B) Quantification analysis of VP1 29

30 produced by modified rndv and baculovirus. The levels of VP1 expression in Sf9 cells and culture medium infected with baculovirus and in allantoic fluid in chicken eggs infected with modified rndv were analyzed by Western blotting Figure 5. Induction of serum antibodies in 4-week-old mice in response to infection with rndvs expressing VP1 protein. Mice were inoculated with each virus by the intranasal route. Sera were collected at 5 and 6 weeks post-infection. NDV-specific antibodies were measured by a hemagglutination inhibition assay using chicken erythrocytes. Figure 6. Antibody titers in mice after immunization with rndvs and baculovirus-expressed VLPs. Mice were inoculated with each virus and VLPs by the intranasal route for three times in a two-week interval. The titers of NoV-specific total IgG (A) and subtypes IgG1 (B) and IgG2a (C) were determined by ELISA against purified baculovirus-expressed VLPs. The antibody titers were defined as the endpoint dilution with a cut off signal intensity of 0.2. Figure 7. Norovirus-specific cellular response after immunization with rndvs and baculovirusexpressed VLPs. Splenocytes from the immunized mice were stimulated with the synthetic NoVspecific peptides and analyzed for the production of IFN-γ, TNF-α, and IL-2 by the ELISPOT assay. The mean spot-forming cells (SFC)/10 6 cells with the error bars are shown Figure 8. Fecal IgA responses in mice immunization with rndvs and baculovirus-expressed VLPs. Fecal samples were diluted in PBS, vortexed, and clarified by centrifugation. NoV- 30

31 specific and total IgA antibodies were determined by ELISA. The ratio between NoV-specific IgA and total IgA was determined. 31

32 FIGURE 1 A. Downloaded from B. on June 29, 2018 by guest 32

33 Figure 2 33

34 Figure 3 A. Downloaded from B. on June 29, 2018 by guest 34