Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein

Transcription

1 JOURNAL OF VIROLOGY, Sept. 1999, p Vol. 73, No X/99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein ANTHONY H.-C. CHOI, 1 * MITALI BASU, 1 MONICA M. MCNEAL, 1 JOHN D. CLEMENTS, 2 AND RICHARD L. WARD 1 Division of Infectious Diseases, Children s Hospital Medical Center, Cincinnati, Ohio 45229, 1 and Department of Microbiology and Immunology, Tulane University Medical Center, New Orleans, Louisiana Received 23 March 1999/Accepted 10 June 1999 This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9- g doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9- g doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient Mt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody. Rotaviruses are the primary cause of severe infantile gastroenteritis and are estimated to cause nearly a million deaths worldwide annually. Although a live, orally deliverable rotavirus vaccine has recently been licensed in the United States, it and other experimental live, oral vaccine candidates have provided only partial protection of limited duration against subsequent rotavirus diseases (3, 4, 11, 21, 22, 34, 36 38). To supplement or replace these vaccines, second-generation candidates are being developed by a variety of approaches. Based on studies with animal models, excellent protection against rotavirus infection can be stimulated by parenteral as well as mucosal immunization. For example, with the adult mouse model developed specifically for studies of active immunity (39), inactivated rotavirus particles delivered parenterally stimulated either partial or complete protection against subsequent murine rotavirus challenge, depending on the type and quantity of particles, route of immunization, and use of adjuvant (12 14, 26, 27, 29, 33). More recently, it was established that either intranasal (i.n.) or oral immunization with triple- or double-layered (TL or DL, respectively) inactivated rotavirus particles or virus-like particles could stimulate protection in this model (28, 32). As with parenteral immunization (26, 27), * Corresponding author. Mailing address: Division of Infectious Diseases, Children s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH Phone: (513) Fax: (513) achoi@chmcc.org. inclusion of adjuvants during mucosal immunization significantly enhanced immune responses and protection (28, 32, 33). One goal in the development of alternative vaccines is the identification of the rotavirus proteins that stimulate protection. It has been reported that antibodies to the VP4 and VP7 neutralization proteins passively protect neonatal mice from rotavirus illness in a serotypically specific manner following oral administration (1, 5, 23, 24, 31). Likewise, it has been found that monoclonal immunoglobulin A (IgA) to either VP4 (35) or VP6 (7) protein can protect mice in the hybridoma backpack model. Finally, Herrmann and coworkers reported that immunization of mice with DNA plasmids containing the gene for VP4, VP6, or VP7 stimulated protection against murine rotavirus infection (8, 16, 17). Based on these findings and the excellent protection against rotavirus shedding stimulated by i.n. immunization of mice with rotavirus particles, either with or without VP4 and VP7, we determined whether this route of immunization with Escherichia coli-expressed VP4, VP6, or VP7 could protect mice against subsequent murine rotavirus infection. Since intramuscular (i.m.) immunization of mice with either TL or DL rotavirus particles also stimulated protection in this model (26), we determined whether individual E. coli-expressed rotavirus proteins could induce protection by this route. Finally, because of the significant enhancement of immune responses and protection stimulated by adjuvants by either route of immunization with rotavirus particles (26 28, 32, 33), the effect of adjuvants on the protection induced by individual proteins was also determined. 7574

2 VOL. 73, 1999 VP4 OR VP6 PROTECTS AGAINST ROTAVIRUS INFECTION 7575 MATERIALS AND METHODS Virus. The murine EDIM strain of rotavirus used throughout this study was originally isolated from a fecal specimen of an infected mouse (obtained from M. Collins, Microbiological Associates, Bethesda, Md.) and adapted to grow in cell culture by serial passage in MA-104 cells. After the ninth passage, the virus was triply plaque purified, and this preparation was used for the construction of recombinant plasmids. To challenge mice after immunization, both wild-type rotavirus from mouse stool and cell culture-adapted passage 9 EDIM preparations were used. As has been done in our laboratory since the inception of the adult mouse model (39), passage 9 EDIM was used to challenge BALB/c mice in this study. However, because of their potential resistance to rotavirus infection based on their origin and for reasons described in detail elsewhere (26, 28), the Mt mice were challenged with unpassaged EDIM. The unpassaged EDIM was purified from mouse stools as previously described (25) and had a titer of 10 7 focus-forming units (FFU)/ml. Passage 9 EDIM had a titer of FFU/ml. Construction of recombinant pmal-c2 plasmids. The bacterial expression plasmid pmal-c2 (New England Biolabs, Beverly, Mass.) was used to construct recombinant pmal-c2/edim4, pmal-c2/edim6, and pmal-c2/edimtr7. For cloning, cdnas were synthesized by PCR with plasmids pcdna1/edim4, pcdna1/edim6, and pcdna1/edimtr7 as templates and gene-specific primers. Construction of the pcdna1 plasmids and the rationale for cloning EDIMTr7, the EDIM VP7 gene sequence lacking the sequence encoding the N-terminal leader peptide, have been previously described (9, 10). The forward and reverse primers for VP4 were 5 -ATGGCTTCACTCATTTATAGACAA-3 and 5 -TCACAGTCTACACTGCATAATTAA-3, respectively. The forward primer for VP6 was 5 -ATGGATGTGCTGTACTCTATC-3, and the reverse primer was 5 -TCACTTTACCAGCATGCTTCT-3. Finally, the forward and reverse primers for the truncated VP7 (TrVP7) were 5 -ATTAATCTTCCAAT TACTGGTTCAATGGAC-3 and 5 -TAACTTCAGTTATACCTACACTCT- 3, respectively. cdnas generated by PCR were inserted into the XmnI restriction site of pmal-c2. The inserted sequences were downstream from the E. coli male gene, which encodes the maltose-binding protein (MBP), and immediately after the factor Xa proteolytic cleavage site, which consists of the amino acid sequence Ile-Glu-Gly-Arg. pmal-c2 utilizes the strong tac promoter and the male translation initiation signals for expression of fusion proteins. The plasmid contains the gene for ampicillin resistance to recombinant bacteria and a lacz- gene sequence. Insertional inactivation of lacz- allows blue-to-white selection of recombinants with inserts. Following ligation of cdna and XmnI-digested pmal-c2, recombinant pmal-c2 plasmids were transformed into E. coli DH5- and were then grown on agar plates. Numbers of white colonies of bacteria grown in the presence of IPTG (isopropyl- -D-thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside) on replicate plates were noted, and the corresponding clones were selected from replicate plates for further screening by PCR for gene identity and orientation. Recombinant plasmids were sequenced to ultimately confirm the authenticity of the rotavirus gene sequences. Induction of recombinant proteins. Single colonies of recombinant bacteria expressing MBP::VP4, MBP::VP6, or MBP::TrVP7 were grown as overnight cultures (37 C) in 50 ml of rich broth (10 g of tryptone, 5gofyeast extract, 5 g of NaCl, 2 g of glucose, 100 mg of ampicillin per liter). On the following day, 10 ml of the overnight cell culture was inoculated into 1 liter of rich broth. When the optical density (A 600 ) reached approximately 0.6, IPTG was added to give a final concentration of 0.3 mm to induce expression of fusion proteins. At 3 h postinduction, a 1.5-ml aliquot was taken and a mixture of inhibitors of bacterial proteases (Sigma Chemical Co., St. Louis, Mo.) consisting of 18 mm 4-[2- aminoethyl]benzenesulfonyl fluoride (AEBSF), 1.7 mm bestatin, 0.22 mm transepoxysuccinyl-l-leucyl-amido[4-guanidino]butane (E-64), 2.5 mm pepstatin A, and 86 mm EDTA was immediately added. The aliquot was spun in a microcentrifuge (2 min, 4 C) to obtain a cell pellet, which was resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The sample was kept frozen until it was subjected to SDS-PAGE. The remaining cell suspension was centrifuged (4,000 g, 20 min, 4 C) to harvest the cells, which were washed in phosphate-buffered saline and centrifuged again. The pellet was frozen at 20 C. Preparation of soluble chimeric proteins. Frozen bacteria containing expressed chimeric MBP::VP4, MBP::VP6, or MBP::TrVP7 were processed according to the method of Jarrett and Foster (18). In short, the bacterial pellets were thawed and resuspended in 50 ml of buffer L (5 mm NaH 2 PO 4,10mM Na 2 HPO 4, 30 mm NaCl, 10 mm -mercaptoethanol, 0.2% Tween 20, 1 mm phenylmethylsulfonyl fluoride, 25 mm benzamidine, 200 mg of lysozyme per liter). After digestion (15 min, room temperature), the suspensions were sonicated (Bronwill BioSonic IV, 50% power setting, three 30-s bursts; VWR Scientific, Piscataway, N.J.) in an ice-cold water bath. NaCl and RNase A (final concentrations of 26.5 mg/ml and 5 g/ml, respectively) were then added. The lysates were centrifuged (54,000 g, 30 min) to separate insoluble cell debris from supernatants (soluble fraction) which contained chimeric rotavirus proteins. Affinity chromatography. Fusion proteins in the soluble fractions were purified by affinity chromatography. Amylose resin (New England Biolabs) was used to purify chimeric proteins containing MBP. The resin was prepared by placing 25 ml of the packed resin in a 250-ml centrifuge tube and washing it twice with 8 volumes of buffer C (buffer L containing 0.5 M NaCl). For each wash, the mixture was rocked for 30 min at 4 C and the resin was recovered by centrifugation (2,100 g, 5 min). The supernatants, which contained the fusion proteins, were mixed with amylose resin for 2 h in a 500-ml flask on a magnetic stirrer. After centrifugation (2,100 g, 5 min), the resin was recovered and then resuspended in 50 ml of buffer C, rocked for 30 min, and centrifuged to recover the resin. The resin was washed in this manner three times and finally washed overnight with 500 ml of buffer C. On the following day, the resin was recovered by centrifugation (2,100 g, 5 min), resuspended in 50 ml of buffer D (50 mm Tris-HCl [ph 7.5], 50 mm NaCl, 1 mm EDTA, 10 mm -mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride), and rocked for 30 min. The resin was pelleted by centrifugation (2,100 g, 5 min), and the bound fusion protein was eluted from the resin with 250 ml of 15 mm maltose in buffer D for 2 h. The resin was removed by centrifugation (2,100 g, 5 min), and the supernatant containing the fusion proteins was subjected to buffer exchange to phosphate-buffered saline while simultaneously being concentrated by ultrafiltration with a stirredcell concentrator (model 8400; Amicon Inc., Beverly, Mass.). Concentrations of purified proteins were measured by the method described by Bradford (6). Western blot analyses of chimeric rotavirus proteins. Soluble fractions containing chimeric proteins or preparations of affinity-chromatography-purified chimeric proteins were subjected to SDS-PAGE. Samples were suspended in gel loading buffer (50 mm Tris [ph 6.8], 10% glycerol, 5% SDS, 5% -mercaptoethanol, 0.005% bromophenol blue), heated (95 C, 5 min), and subjected to electrophoresis in SDS 8% polyacrylamide gels. Following SDS-PAGE, separated proteins were blotted to nitrocellulose sheets, which were then blocked with 5% skim milk in TBS (25 mm Tris-HCl [ph 7.5], 0.9% NaCl). The sheets were then incubated with a rabbit anti-mbp serum (1:10,000 dilution; New England Biolabs). After being washed with 0.1% Tween 20 in TBS (TTBS), the sheet was incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase (1:3,000; Life Technologies, Gaithersburg, Md.). The sheet was washed with TTBS and then incubated with nitroblue tetrazolium (NBT; 0.25 mg/ml) and 5-bromo-4-chloro-3-indolylphosphate (BCIP; 0.25 mg/ml; Life Technologies) to visualize bound antibodies. Western blot analyses of immune sera. To determine whether the immune sera obtained from mice vaccinated with MBP::VP4, MBP::VP6, or MBP::TrVP7 generated antibodies against the specific rotavirus proteins, TL rotavirus particles were subjected to SDS-PAGE as described above. Following SDS-PAGE, separated rotavirus proteins were electrophoretically transferred to nitrocellulose sheets, which were cut into strips. The strips were blocked with 5% skim milk in TBS. The strips were then incubated either with rabbit anti-mbp or with antisera obtained from mice immunized with MBP::VP4, MBP::VP6, or MBP::TrVP7, which were used at a 1:100 dilution. After being washed with 0.1% TTBS, the strips were incubated with goat anti-mouse IgG conjugated to alkaline phosphatase. The strips were washed with TTBS and then incubated with NBT and BCIP to visualize bound antibodies as described above. Mice. Rotavirus antibody-free female BALB/c mice were purchased at 6 weeks of age from Harlan-Sprague-Dawley (Indianapolis, Ind.). The B-cell-deficient Mt mice were produced by Kitamura et al. (19), and a breeder pair with the C57BL/6 genetic background was obtained from Jackson Laboratories (Bar Harbor, Maine). They were included in this study with the permission of K. Rajewsky. Although very low levels of IgG were detected in the sera of these mice (i.e., titers of 0.1% compared to those found in immunologically normal C57BL/6 mice), neither rotavirus IgM, IgG, nor IgA was detectable in these mice at any time following immunization or challenge. Experiments were conducted with adult Mt mice between 6 and 20 weeks of age. All mice were housed in groups of three or four in sterile microisolation cages. All procedures were carried out in accordance with protocols reviewed and approved by the Children s Hospital Research Foundation Institutional Animal Care and Use Committee. Immunization of mice with chimeric rotavirus proteins. i.n. immunization was carried out with the mice being under light sedation by administration of 25 l of immunogen per nostril. The inoculum consisted of 9 g of one of the chimeric rotavirus proteins or MBP (New England Biolabs). When the proteins were coadministered with an adjuvant, 10 g of cholera toxin (Sigma Chemical Co.) or 10 g of the attenuated E. coli heat-labile toxin LT(R192G) was used. LT(R192G) carries a mutation in the proteolytic site of its A subunit at amino acid 192, where an arginine is replaced by a glycine residue. The mutation abrogates cleavage of LT(R192G) and attenuates the toxicity of the protein (15). When more than one dose was given, each subsequent dose was administered at a 2-week interval. When administered i.m., groups of mice were immunized with 9 g of one of the test vaccines in the muscle of the hind leg with or without 20 g of the adjuvant QS-21. This saponin adjuvant was manufactured by Aquila Biopharmaceuticals (Worcester, Mass.) and provided by Wyeth-Lederle Vaccines and Pediatrics (Pearl River, N.Y.). Challenge of mice with EDIM rotavirus. Four weeks after the last i.n. or i.m. immunization, mice were orally (gavage) challenged with either FFU of passage 9 EDIM (BALB/c mice) or FFU of unpassaged EDIM ( Mt mice). To study the longevity of protective immunity induced by MBP::VP6 and LT(R192G), BALB/c mice were challenged 3 months after the second (last) immunization with the test vaccine and adjuvant. Detection of rotavirus antigen in stools. Two fecal pellets were collected from each mouse into 0.5 ml of Earle s balanced salt solution on the day of EDIM

3 7576 CHOI ET AL. J. VIROL. rotavirus challenge and for 7 days following challenge. Samples were stored frozen and then homogenized and centrifuged (1,500 g, 5 min, 4 C) to remove debris before being analyzed. Quantities of rotavirus antigen in the fecal samples were determined by enzyme-linked immunosorbent assay (ELISA) as nanograms per stool specimen by methods previously described (28). Determination of rotavirus antibody titers. Blood samples were collected by retroorbital capillary plexus puncture before the last immunization, before challenge, and 21 days after challenge. Stool specimens were collected at the same periods. Titers of rotavirus IgG and IgA in sera, as well as of rotavirus IgA in feces, were determined by ELISA as previously described (10, 28) and were reported in units per milliliter. Subtype-specific rotavirus IgG concentrations were also determined as described previously (10, 28) and reported in nanograms per milliliter. Neutralizing antibody to EDIM was measured by an antigen reduction assay described previously (20). Statistical methods. Statistical analyses of titers of rotavirus-specific antibodies and amounts of shed rotavirus antigen between groups of mice immunized with different chimeric proteins and mock-vaccinated groups were performed by Student s t test (unpaired, two-tailed). Differences between groups were considered significant when the probability level (P) was RESULTS Purification of chimeric VP4, VP6, and TrVP7 by affinity chromatography. In a previous attempt to protect mice from rotavirus infection by immunization with individual rotavirus proteins, we prepared DNA vaccines expressing recombinant VP4, VP6, or TrVP7, a truncated form of VP7 that lacks the N-terminal signal peptide, of murine rotavirus strain EDIM. All three plasmids elicited serum rotavirus IgG responses following gene gun immunization, but protection against rotavirus infection was not observed in immunized mice (9, 10). To determine if vaccination with recombinant VP4, VP6, or TrVP7 protein could induce protective immunity, the coding sequences of these proteins were subcloned from the DNA vaccines into the bacterial plasmid pmal-c2. These recombinant plasmids expressed chimeric rotavirus VP4, VP6, and TrVP7 in E. coli that were genetically fused to the C terminus of a 42.7-kDa MBP whose gene was borne by the plasmid. The chimeric proteins were purified by affinity chromatography with amylose resin and analyzed by Western blot analyses (Fig. 1). Peptides migrating with mobilities expected of MBP::VP4 (129.1 kda), MBP::VP6 (87.7 kda), and MBP::TrVP7 (74.5 kda) were detected in the Western blots. In addition, numerous MBP-containing proteins with smaller molecular sizes were detected even though protease inhibitors were added to all solutions used in the purification procedure. In an attempt to prevent truncation of expressed rotavirus proteins, protease inhibitors were added to IPTG-induced E. coli cells, which were pelleted in a microcentrifuge (4 C, 1 min). These pellets were immediately subjected to SDS-PAGE and then Western blot analyses. In spite of the presence of bacterial protease inhibitors and the short time taken to harvest and analyze the proteins, truncated MBP-containing peptides could still be detected. Thus, truncation may have occurred prior to the processing of the expressed proteins. Observations of a similar profile of truncated chimeric VP6 expressed in E. coli have also been noted in a study by others (2). Rotavirus antibody stimulated by immunization of mice with chimeric rotavirus proteins. Preparations of MBP::VP4, MBP::VP6, and MBP::TrVP7 obtained by amylose resin affinity chromatography were used to vaccinate mice to determine whether they could induce rotavirus antibody. Since VP6 is typically the most immunogenic of these three rotavirus proteins, it was examined first. BALB/c mice (8 or 10 per group) were immunized by i.n. or i.m. administration of three doses (9 g/dose) of MBP::VP6 at 2-week intervals. To examine the effects of adjuvants, MBP::VP6 was delivered by i.n. inoculation with 10 g of the genetically attenuated E. coli heat-labile toxin LT(R192G) or by i.m. injection (9 g/dose) with the saponin adjuvant QS-21 (20 g). Blood and stool specimens FIG. 1. Amylose resin affinity chromatography of chimeric MBP::VP4, MBP::VP6, and MBP::TrVP7. E. coli cells transformed with recombinant plasmids expressing MBP::VP4, MBP::VP6, and MBP::TrVP7 were induced with 0.3 mm IPTG for 3 h. Cell lysates and affinity chromatography-purified chimeric proteins were obtained as described in Materials and Methods. Pure MBP, cell lysates, and purified chimeric proteins were subjected to SDS-PAGE and then electrotransferred to nitrocellulose for Western blot analyses. The blots were incubated with a polyclonal rabbit anti-mbp antiserum. Goat anti-rabbit IgG conjugated to alkaline phosphatase was used as the secondary antibody. BCIP and NBT were used as enzyme substrates to visualize the antibodies bound to chimeric proteins. The arrows indicate putative full-length chimeric MBP::VP4, MBP::VP6, and MBP::TrVP7. Molecular mass markers (in kilodaltons) are noted at the left. collected 1 month after the last immunization (i.e., just before EDIM challenge) were examined for rotavirus antibody by ELISA. i.n. vaccination with MBP::VP6 alone resulted in moderate titers of serum rotavirus IgG (Table 1). However, only one of eight mice developed a serum rotavirus IgA response and no detectable stool rotavirus IgA was generated. When LT(R192G) was included with MBP::VP6, serum rotavirus IgG and IgA were stimulated in all 10 mice and these titers were significantly greater than those obtained without the adjuvant (P 0.01). A low, but detectable, titer of rotavirus IgA was also detected in the stools of 8 of 10 mice. i.m. inoculation with MBP::VP6 also stimulated high titers of serum rotavirus IgG, but only four of eight animals developed serum rotavirus IgA and no animals exhibited stool rotavirus IgA (Table 1). Mice coadministered QS-21 with MBP::VP6 during i.m. immunization generated significant (P 0.002) greater rotavirus IgG titers in serum than those immunized without QS-21. All eight mice developed serum rotavirus IgA, and the titers were significantly (P 0.02) greater than those from mice immunized with MBP::VP6 alone. However, no stool rotavirus IgA was detected. Groups of mice immunized by i.n. inoculation of MBP and LT(R192G) or i.m. with MBP and QS-21 generated no rotavirus antibodies (Table 1). Since MBP::VP6 was so highly immunogenic following both i.n. and i.m. administration, mice were inoculated with MBP::VP4 or MBP::TrVP7 (three doses of 9 g) by the i.n. or i.m. route with the adjuvant LT(R192G) or QS-21, respectively. No serum rotavirus IgG or IgA or stool rotavirus IgA was detected in mice immunized by i.n. inoculation with MBP::VP4 and LT(R192G) (Table 2). In contrast, i.m. inoculation of MBP::VP4 with QS-21 stimulated high titers of rotavirus IgG. None of the mice generated a serum or stool rotavirus IgA response. Mice immunized by either i.n. or i.m. inoculation with MBP::TrVP7 and adjuvant generated similarly low titers of serum rotavirus IgG but no detectable serum or stool rotavirus IgA.

4 VOL. 73, 1999 VP4 OR VP6 PROTECTS AGAINST ROTAVIRUS INFECTION 7577 TABLE 1. Geometric mean titers of rotavirus antibodies following immunization of BALB/c mice by i.n. or i.m. inoculation with MBP::VP6 alone or in combination with LT(R192G) or QS-21 a Route of inoculation Inoculum No. of mice GMT U/ml) b Serum IgG Serum IgA Stool IgA None i.n. MBP LT(R192G) MBP::VP6 8 5,276 4, MBP::VP6 LT(R192G) , ,131 c d i.m. MBP QS MBP::VP6 8 29,665 12, MBP::VP6 QS , ,284 e a Groups of 8 or 10 BALB/c mice were immunized with three i.n. or i.m. doses (9 g/dose) of MBP::VP6 with or without LT(R192G) or QS-21, respectively. Blood specimens were drawn at 4 weeks after the third dose and just before EDIM challenge. b ELISA antibody titers are expressed as geometric mean titers (GMT) standard errors. The symbol indicates the limit of detection, and this titer was used to calculate geometric mean titers. c Significantly (P 0.01) higher than serum rotavirus antibody titers in mice immunized i.n. with MBP::VP6 alone. d Significantly (P 0.002) higher than serum rotavirus antibody titers in mice immunized i.n. with MBP::VP6 alone. e Significant (P 0.04) higher than serum rotavirus antibody titers in mice immunized i.m. with MBP::VP6 alone. To verify that the IgG titers obtained by ELISA were indeed induced by the specific rotavirus protein used for vaccination. Western blot analysis was performed with antisera collected from vaccinated mice. Rotavirus proteins (TL EDIM particles) were subjected to SDS-PAGE and then blotted onto a nitrocellulose sheet, which was then cut into strips. The strips were incubated with immune sera to ascertain the presence of anti- VP4, -VP6, or -VP7 IgG. With the exception of mice immunized i.n. with MBP::VP4 and LT(R192G), all mice immunized with subunit vaccines and adjuvants generated rotavirus protein-specific IgG (Fig. 2), thus verifying results obtained by ELISA. It should be noted that even though VP4 and VP7 in rotavirus particles can independently elicit neutralizing antibodies, none of the sera collected from mice immunized with the chimeric proteins contained detectable neutralizing antibody to EDIM (i.e., titers were all 10 [results not shown]). Vaccination with chimeric VP4 or VP6 stimulates protection against rotavirus shedding. Mice inoculated i.n. or i.m. with chimeric proteins were orally challenged with live EDIM 4 weeks after the last vaccination to measure protection against infection. Shedding of rotavirus was determined between 1 and 7 days after challenge. Immunization with MBP::VP6 alone stimulated only a small (16%), insignificant reduction in EDIM shedding (Fig. 3). In contrast, inclusion of LT(R192G) during immunization with MBP::VP6 resulted in a 93% reduction in rotavirus shedding (P 0.001). i.m. inoculation of MBP::VP6 with QS-21 also resulted in a small (38%) but significant (P 0.02) reduction in shedding. The only other significant reduction in EDIM shedding was stimulated by i.n. inoculation of MBP::VP4 (56%, P 0.001). Interestingly, this was also the only group of mice in which inoculation (i.n.) with a rotavirus protein and adjuvant did not result in a detectable rotavirus IgG response (Tables 1 and 2). Another method that we have routinely used to measure rotavirus infection in this model has been based on significant (i.e., 4-fold) increases in rotavirus antibody titers following EDIM challenge. For example, mice orally immunized with EDIM have been consistently found to develop no significant increase in any rotavirus antibody following a subsequent EDIM challenge and were, therefore, considered to be completely protected against infection (30, 39). In the present study, it was of interest to determine whether mice protected from EDIM shedding following either i.n. or i.m. immunization were also protected against the development of significant increases in rotavirus antibody titers after EDIM challenge. Interestingly, every immunized mouse experienced 4-fold increases in serum rotavirus IgG and IgA as well as stool rotavirus IgA by 21 days after EDIM challenge (results not shown). This result demonstrates that even though both i.n. and i.m. immunization with EDIM proteins (particularly MBP::VP6 with an adjuvant) caused large reductions in shedding after TABLE 2. Geometric mean titers of rotavirus antibodies in mice immunized by i.n. or i.m. inoculation with MBP::VP4 or MBP::TrVP7 and an adjuvant a Route of inoculation Inoculum No. of mice GMT (U/ml) b Serum IgG Serum IgA Stool IgA None i.n. MBP::VP4 LT(R192G) MBP::TrVP7 LT(R192G) i.m. MBP::VP4 QS ,617 14, MBP::TrVP7 QS a Groups of seven or eight mice were immunized with three i.n. or i.m. doses (9 g/dose) of MBP::VP4 or MBP::TrVP7 with LT(R192G) or QS-21. Blood and stool specimens were collected 4 weeks after the third dose. b ELISA antibody titers are expressed as geometric mean titers (GMT) standard errors. The symbol indicates the limit of detection, and this titer was used to calculate geometric mean titers.

5 7578 CHOI ET AL. J. VIROL. FIG. 2. Western blot analyses of sera from mice immunized with chimeric rotavirus proteins. CsCl-purified TL rotavirus particles were subjected to SDS- PAGE and then electrotransferred onto a nitrocellulose sheet, which was then cut into strips for Western blot analyses. The strips were incubated with sera from mice which had been immunized i.n. with MBP::VP4, MBP::VP6, or MBP::TrVP7, each in combination with LT(R192G) (A), or i.m. with the same proteins in combination with QS-21 (B). Goat anti-mouse IgG conjugated to alkaline-phosphatase was used as the secondary antibody. BCIP and NBT were used to visualize antibodies bound to VP4, VP6, or VP7. EDIM challenge, complete protection from infection did not occur in any mouse. Protection following i.n. immunization is not reduced by substitution of cholera toxin for LT(R192G), by a decrease in the number of immunizations from 3 to 1, or by an increase in the time of challenge from 1 to 3 months after immunization. Once it was determined that i.n. immunization with three doses of MBP::VP6 stimulated almost complete protection against rotavirus shedding following EDIM challenge 1 month after the third immunization, it was of interest to determine whether these parameters could be modified and still stimulate the same level of protection. We therefore asked the following questions (i) could cholera toxin substitute for LT(R192G), (ii) could the number of immunizations be reduced, and (iii) would protection diminish between 1 and 3 months after immunization? When i.n. immunization with MBP::VP6 (three doses) was performed with cholera toxin (10 g/dose) in place of LT(R192G), rotavirus antibody titers at 4 weeks after the last immunization were about twofold less than they were with LT(R192G) (results not shown). However, immunization in the presence of either adjuvant reduced rotavirus shedding by 98 to 99% following EDIM challenge 1 month after the third dose. It was next found that i.n. immunization with an increasing number of doses of MBP::VP6 (9 g/dose) with LT(R192G) from 1 to 3 progressively resulted in significantly (P 0.05) higher titers of serum rotavirus IgG and IgA and stool rotavirus IgA. However, levels of protection against rotavirus shedding were comparable in all groups following EDIM challenge 1 month after the last immunization (i.e., 99.5, 97.6, and 97.8% reductions in shedding for one, two, and three doses, respectively). Finally, it was found that levels of rotavirus shedding following EDIM challenge 1 and 3 months after a second immunization with MBP::VP6 and LT(R192G) were equivalent (i.e., 99.1 and 98.6%, respectively). These results indicate that adjuvants other than LT(R192G) can be used to augment protection stimulated by i.n. immunization with MBP::VP6, that a single i.n. immunization is as protective as three, and that protection does not decline between 1 and 3 months following immunization. Inclusion of LT(R192G) during i.n. immunization with MBP::VP6 does not alter rotavirus IgG1/IgG2a ratios. Because inclusion of LT(R192G) as an adjuvant during i.n. immunization with MBP::VP6 had such dramatic effects on rotavirus antibody responses and protection, it was of interest to determine whether inclusion of this adjuvant also modified the relative T-helper-cell responses (i.e., T H 1 versus T H 2 responses). If so, it would suggest that this adjuvant might also modify the mechanism of protection. Although several methods can be used to measure T H 1 and T H 2 responses, we and numerous other investigators have used the concentrations of IgG1 and IgG2a and ratios of IgG1 to IgG2a as surrogate markers of these responses. T H 1 responses are associated with IgG2a production, while T H 1 responses are accompanied by IgG1-dominated antibody production. As already noted (Table 1), inclusion of LT(R192G) during i.n. immunization with MBP::VP6 caused large increases in rotavirus antibody titers, including those of serum IgG. To determine whether the dramatic increase in protection stimulated by inclusion of LT(R192G) was associated with a change in T-helper-cell subset, the ratios of rotavirus IgG1 to IgG2a in postvaccination sera of mice inoculated with MBP::VP6 alone or with LT(R192G) were determined. Inclusion of LT(R192G) during immunization resulted in large increases in the concentrations of both serum rotavirus IgG1 and IgG2a but had little effect on the IgG1/IgG2a ratio (Table 3). Therefore, it appeared that the mechanism by which LT(R192G) increased protection had little effect on the relative T H 1-versus-T H 2 response. i.n. immunization with MBP::VP6 and LT(R192G) stimulates equivalent levels of protection in BALB/c and B-celldeficient Mt mice. We previously reported that protection against EDIM shedding stimulated by i.n. immunization with inactivated TL EDIM particles was partially dependent on rotavirus antibody but that DL EDIM particles stimulated protection by an antibody-independent mechanism (28). This FIG. 3. Protection from shedding of rotavirus antigen in mice inoculated with chimeric subunit vaccines. BALB/c mice were inoculated either i.n. or i.m. with three 9- g doses of either MBP::VP4, MBP::VP6, or MBP::TrVP7 with or without an adjuvant. The adjuvants used for i.n. and i.m. inoculations were LT(R192G) (10 g) and QS-21 (20 g), respectively. One month after the last immunization, mice were challenged by oral gavage with FFU of passage 9 EDIM. Stool samples were collected on the day of challenge and for the subsequent 7 days. Quantities of rotavirus antigen shed were determined by ELISA. The total quantity shed by each group was compared with the total quantity shed by the control group (MBP and adjuvant) and expressed as percent reduction in shedding relative to the level of shedding of the control group. The * symbols indicate that levels of shedding by immunized groups were significantly lower (P 0.02) than the level of shedding of uninoculated mice.

6 VOL. 73, 1999 VP4 OR VP6 PROTECTS AGAINST ROTAVIRUS INFECTION 7579 TABLE 3. Effect of LT(R192G) on the geometric mean titer of postvaccination rotavirus IgG1 and IgG2a titers in sera following i.n. immunization of mice with MBP::VP6 Inoculation IgG1 GMT (ng/ml) a conclusion was based primarily on the relative levels of protection induced by these two particles in BALB/c versus B-celldeficient Mt mice. The same experiment was conducted following i.n. immunization with MBP::VP6 and LT(R192G). Following two doses (9 g/dose) separated by 2 weeks, both BALB/c and Mt mice were challenged with EDIM. Relative to that of unimmunized control mice, rotavirus shedding in immunized mice was reduced 97% in both strains of mice (Fig. 4). As expected, prechallenge titers of rotavirus IgG in sera were high in BALB/c mice but titers of rotavirus IgG, IgA, and IgM in sera were undetectable in immunized Mt mice. Therefore, protection stimulated by MBP::VP6 with LT(R192G) appeared to be independent of antibody. DISCUSSION IgG2a IgG1/IgG2a ratio MBP::VP6 1,890 5, MBP::VP6 85,067 33,647 25,283 15, LT(R192G) a ELISA antibody titers are expressed as geometric mean titers (GMT) standard errors. Because of the severity of rotavirus disease, efforts to develop a rotavirus vaccine that provides complete protection of extended duration have been the focus of intense international investigations. The first and only licensed rotavirus vaccine, which is composed of four live virus strains that are delivered orally, has provided only partial protection, especially against severe rotavirus disease (3, 21, 22, 34, 37). An alternative approach that has yielded promising results in the adult mouse model has been i.n. immunization with noninfectious viral particles. By this model, either TL or DL inactivated rotavirus particles or virus-like particles generated from baculovirusexpressed VP6 and VP2 inner capsid proteins have stimulated excellent protection against murine rotavirus infection (28, 32, 33). In every case, the immune responses and levels of protection were greatly enhanced by inclusion of a mucosal adjuvant (i.e., either cholera toxin or E. coli heat-labile toxin). The ability of individual rotavirus proteins to stimulate active immunity following their administration by any route, either with or without adjuvant, had not been determined prior to this study. The rotavirus proteins chosen to make this determination (i.e., VP4, VP6, and VP7) have all been associated in some way with protection. For example, high concentrations of IgA to VP4 or VP6 generated in mice following injection of hybridoma cells had been reported to confer protection in the backpack model (7, 35). Likewise, Herrmann and coworkers had reported that DNA immunization of mice with plasmids containing the gene for either VP4, VP6, or VP7 of murine rotavirus protected against shedding following murine rotavirus challenge (8, 16, 17). However, in spite of stimulating very high titers of rotavirus IgG using essentially identical procedures, we were unable to show any protection following DNA immunization in this model (9, 10). Therefore, it was unclear whether immunization with individual rotavirus proteins could provide protection against rotavirus infection. The rotavirus proteins used in this study were expressed in E. coli as chimeras with the 42.7-kDa MBP. When purified by affinity chromatography, most of the expressed chimeras of the three rotavirus proteins were found to be smaller than expected. Even so, i.n. inoculation of 10 g of the expressed VP4 or VP6 product, in association with the attenuated E. coli heat-labile toxin LT(R192G), stimulated a 56 or 93% reduction in rotavirus shedding, respectively, following a murine rotavirus challenge 1 month after a third i.n. immunization. In contrast, i.n. administration of the VP7 chimera stimulated nonneutralizing rotavirus IgG titers but provided no protection. i.m. immunization with rotavirus particles in the presence of the saponin adjuvant QS-21 has also been found to induce excellent protection in this model (26). However, only E. coliexpressed chimeric VP6, administered together with QS-21, was found to provide significant protection, providing a reduction in rotavirus shedding of 38% (P 0.02) after three i.m. immunizations compared to the level of shedding in control mice. Therefore, the most effective protection in this study was stimulated by expressed VP6 administered i.n. with LT(R192G). Because baculovirus-expressed VP6 in virus-like particles also containing VP2 elicited excellent protection following i.n. immunization in mice (32, 33), it was of interest to determine whether the E. coli-expressed VP6 in this study had also formed virus-like particles. This seemed highly unlikely, since VP6 was expressed as a fusion protein with MBP (42.7 kda), which has a molecular size similar to that of VP6 (45.0 kda). Analysis of the sedimentation properties of the expressed VP6 fusion product in a sucrose gradient under conditions where DL rotavirus particles were pelleted revealed that VP6 was detected only in the upper 10% of the gradient (results not shown). Therefore, no VP6 particles were found, indicating FIG. 4. Protection from shedding of rotavirus antigen in mice immunized with chimeric VP6. BALB/c and Mt mice were inoculated i.n. with two 9- g doses of MBP::VP6 and LT(R192G). One month after the last immunization, mice were challenged by oral gavage with FFU of passage 9 (BALB/c) or FFU of unpassaged EDIM ( Mt). Stool samples were collected on the day of challenge and for 7 days thereafter. Quantities of rotavirus antigen shed each day were determined by ELISA. The numbers in the graphs are quantities of rotavirus antigen shed by immunized mice.

7 7580 CHOI ET AL. J. VIROL. that protection was induced in the absence of particle formation. Further analysis of protection induced by i.n. immunization with the VP6 fusion product revealed that no loss of protection occurred when the number of doses was reduced from 3 to 1. In that experiment, protection against shedding was consistently 97%. Although the quantity of intact MBP::VP6 was not determinable due to the large amount of truncated fusion product, this result suggested that the quantity of antigen used for immunization (9 g/dose) was in significant excess over that needed to stimulate the observed levels of protection. Had this not been the case, it is likely that one dose would have provided less protection than three. However, since the time of rotavirus challenge up to this point in the study had always been 1 month after the last i.n. immunization, it was possible that comparable levels of protection were found after one, two, and three doses because of the consistent length of time between the last immunization and challenge. That is, the level of protection may have decreased with time, and the amount of protection observed may have been dictated by the time between the last immunization and challenge. This, however, was not the case since protection following two doses did not decrease between 1 and 3 months after the second immunization (i.e., it remained above 98%). It was of interest to note that full protection in this adult mouse model has been equated with the complete absence of shedding and no significant (i.e., 4-fold) increases in rotavirus antibody titers (39). This type of protection had been observed for at least 14 months following oral immunization with live murine rotavirus (30). Based on these criteria, i.n. immunization with expressed VP6 did not induce complete protection in any mouse (i.e., although most mice did not shed detectable amounts of rotavirus antigen following challenge, all had large increases in rotavirus antibody titers). This result suggested that i.n. immunization did not prevent infection but merely blocked most intestinal virus production. It has been reported that monoclonal IgA against the VP6 protein is able to protect mice against murine rotavirus infection by the backpack model (7). To determine whether protection stimulated by i.n. immunization with chimeric VP6 was also due to antibody against this rotavirus protein, shedding was measured in i.n. immunized Mt mice. This B-cell-deficient mouse strain developed no detectable rotavirus antibody, yet immunized mice were protected against shedding to the same degree (i.e., 97%) as immunologically normal BALB/c mice following two i.n. immunizations. Therefore, antibody appeared to play no role in VP6-mediated protection. It should be noted that BALB/c mice immunized i.n. with chimeric VP4 were partially protected (56% reduction in shedding) in the absence of detectable antibody responses and, therefore, also appeared to be protected by an antibody-independent mechanism. Finally, MBP::TrVP7 administered i.n. stimulated serum antibody responses but no protection. Taken together, these data indicate that rotavirus antibody is not the effector of protection following i.n. immunization of mice with rotavirus proteins. Lack of association between antibody titers and protection in this model was also found following immunization by other routes with either rotavirus proteins or DNA that expressed these proteins. In the present study, we observed that i.m. immunization with MBP::VP4 and QS-21 stimulated very large rotavirus titers (Table 2) but that shedding following EDIM challenge was reduced by only 15% (Fig. 3). Furthermore, DNA (gene gun) immunization with plasmids containing genes encoding EDIM VP6, VP4, or TrVP7 all stimulated moderate to large rotavirus IgG responses in serum but these responses did not lead to reduction in shedding following EDIM challenge (9, 10). Therefore, it is unclear in situations where rotavirus antibody has been associated with protection whether, in many instances, this antibody is merely a marker for the true immunological effector and not the actual effector. It is of interest that even though i.m. immunization with EDIM VP4 stimulated large rotavirus IgG responses in sera, we were unable to detect neutralizing antibody to EDIM. This result is presumably due to stimulation of VP4 antibody to nonneutralizing VP4 epitopes. This same observation has been made following both oral immunization with live EDIM and DNA immunization with a plasmid containing the EDIM VP4 gene (10). Because no significant protection was stimulated by i.n. immunization with chimeric VP6 in the absence of an adjuvant, it was of interest to determine whether the types of immune responses stimulated with and without the adjuvant differed. Although inclusion of the adjuvant significantly increased rotavirus antibody responses following immunization, it did not alter the relative amounts of IgG1 and IgG2a. This finding indicated that the relative quantities of rotavirus-specific T- helper cells (i.e., T H 1 versus T H 2 cells) induced following immunization under nonprotective (i.e., absence of adjuvant) and protective (i.e., presence of adjuvant) conditions also did not differ. The utility of these findings will depend primarily on their applicability to larger animals and humans. It is possible that i.n. immunization of VP6 alone may provide long-lasting protection against multiple rotavirus serotypes in vaccines. However, it is also possible that immunization with VP6 alone may provide only partial protection. If this is the case, it may be more practical to use this subunit vaccine to boost immune responses following oral immunization with live rotaviruses. VP6 should be more effective than live virus vaccines for boosting immune responses because live rotavirus vaccines are dependent on virus replication to stimulate immunity, which can be blocked by prior immunization. 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