infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal ...
JOURNAL OF VIROLOGY, Nov. 1998, p. 9233–9246 0022-538X/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 11
Subunit Rotavirus Vaccine Administered Parenterally to Rabbits Induces Active Protective Immunity MAX CIARLET,1 SUE E. CRAWFORD,1 CHRISTOPHER BARONE,1 ANDREA BERTOLOTTI-CIARLET,1 ROBERT F. RAMIG,1 MARY K. ESTES,1 AND MARGARET E. CONNER1,2* Division of Molecular Virology, Baylor College of Medicine,1 and Veterans Affairs Medical Center,2 Houston, Texas 77030 Received 23 February 1998/Accepted 24 July 1998
Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund’s adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 103 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r 5 0.55; Pearson’s correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund’s adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund’s adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund’s adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine. alone (47). Coexpression of VP2 and VP6 alone or with VP4 results in the production of double-layered 2/6- or 2/4/6-VLPs, respectively (26, 47). Coexpression of VP2, VP6, and VP7, with or without VP4, results in triple-layered 2/6/7- or 2/4/6/7-VLPs (26). All VLPs maintained the structural and functional characteristics of native particles (26, 63, 65, 69), including binding to and internalization of 2/4/6/7-VLPs into MA104 cells (26, 28). Models of rotavirus infection, without disease, were developed in rabbits (14, 17–19, 38, 73) and in adult mice (58, 79, 80) to monitor the development of active serum and mucosal immunity as well as protection from infection following a live rotavirus challenge. We demonstrated that parenteral vaccination with live or inactivated rotavirus induces active immunity and protection in the rabbit model (19). Preliminary results for rabbits showed that VLPs administered parenterally in Freund’s adjuvants and aluminum phosphate (AlP) were immunogenic and induced active protection from homologous serotype G3 oral rotavirus challenge (21, 23). Here, we report the immunogenicity and protective efficacy of parenterally administered VLPs of different compositions (2/6, 2/6/7, and 2/4/ 6/7) in rabbits, using different adjuvants: Freund’s, AlP, and QS-21. QS-21 has the advantage that it may be licensed for use in humans (46) and has been tested with VLPs in mice (23, 24, 42, 50).
Rotaviruses are the most common etiologic agents of acute viral gastroenteritis in young children throughout the world, and a worldwide effort is under way to design an effective vaccination strategy. In the United States, rotavirus infection is primarily a problem of morbidity and associated health care costs (48, 52), whereas in developing countries, mortality is high, with .870,000 deaths per year attributed to rotavirus (39). Live attenuated rotavirus vaccine candidates consisting of human-animal (simian or bovine) rotavirus reassortants were tested in children but showed variable effectiveness in different settings (20, 74). In recent trials, these vaccines provided approximately 70% effectiveness against severe diarrhea (16, 43, 44, 61, 69, 75, 76). Rotaviruses belong to the Reoviridae family and are composed of three protein layers surrounding 11 segments of double-stranded RNA (32). The innermost layer is composed of VP1, VP2, VP3, and the genome, the middle layer is composed of VP6; and the outer layer is composed of the glycoprotein VP7 and spikes of VP4 dimers (32, 64, 70). VP4 and VP7 possess distinct antigenic activities, defining P serotypes and G serotypes, respectively. VP4 and VP7 independently elicit antibodies capable of neutralizing rotavirus infectivity and inducing protective immunity (32). Rotavirus genes encoding the rotavirus structural proteins VP2, VP6, VP4, and VP7 have been cloned in baculovirus, and the recombinant rotavirus proteins have been coexpressed in the baculovirus expression system (26, 31, 47). Stable virus-like particles (VLPs) self-assemble following expression of VP2
MATERIALS AND METHODS Cells and viruses. The lapine rotavirus ALA (P[14], G3) strain (11), used for rabbit rotavirus challenge inoculations or enzyme-linked immunosorbent assays (ELISAs) and fluorescent-focus neutralization assays (FFNAs), was passaged in the presence of trypsin 10 times in fetal rhesus monkey kidney MA104 cells or was passaged in MA104 cells and plaque purified three times, respectively, as described previously (17). The simian rotavirus SA11 clone 3 (SA11 Cl3; P[2], G3) (5, 30, 51), bovine rotavirus B223 (P8[11], G10), and SA11 Cl3 3 B233
* Corresponding author. Mailing address: Division of Molecular Virology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-3590. Fax: (713) 798-3586. E-mail: mconner @bcm.tmc.edu. 9233
9234
CIARLET ET AL.
rotavirus reassortant viruses R-N33 (P8[11], G3) and R-A32 (P[2], G10) used in FFNAs were propagated in MA104 cells with trypsin and plaque purified two or three times as described elsewhere (29). Original derivation and characterization of the SA11 Cl3 3 B223 rotavirus reassortant viruses R-N33 and R-A32 were described previously (66). R-N33 (P8[11], G3) contains genome segments 1 and 4 from B223 and the remainder of its genes from SA11 Cl3, and R-A32 (P[2], G10) contains genome segments 2 and 4 from SA11 Cl3 and all other genome segments from B223 (66, 67). Baculovirus recombinants used in this study encoded full-length genes of bovine RF VP2 (BacRF2A [47]), simian SA11 Cl3 VP4 (pVL941/SA11-4; genotype P[2] [26]), simian SA11 Cl3 VP6 (pAc461/SA11-6 [30]), and simian SA11 Cl3 VP7 (pVL941/SA11-9; serotype G3 [27]). Spodoptera frugiperda 9 (Sf9) cells were grown in a 1.5-liter bioreactor unit (Celligen Cell Culture system; New Brunswick Scientific, Edison, N.J.) and maintained on TNM-FH medium containing 10% fetal bovine serum (26). Preparation of rotavirus SA11 Cl3 virus. Live or formalin-inactivated SA11 Cl3-infected cell lysates were prepared as described elsewhere (19). Following formalin inactivation, SA11 Cl3 preparations were characterized by ELISA as described previously (26) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies (MAbs) to confirm that virus inactivation did not result in loss of epitope reactivity. SA11 Cl3 triple-layered particles (TLPs) were purified by CsCl density gradient centrifugation (26). Purified SA11 Cl3 TLPs were inactivated with 49-aminomethyltrioxalin-hydrochloride (psoralen; Lee Biomolecular Research Laboratories, Inc., San Diego, Calif.) (37). Before and after psoralen inactivation, SA11 Cl3 TLPs were (i) examined by electron microscopy (EM) to confirm their integrity; (ii) analyzed by staining of polyacrylamide gels with silver nitrate following sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) to determine purity; and (iii) following SDS-PAGE, analyzed by Western blotting (immunoblotting) using a mouse hyperimmune serum prepared against triple-layered SA11 Cl3 virus to determine protein composition as described previously (5, 26). Psoralen-inactivated CsCl-purified SA11 Cl3 TLPs were not analyzed by ELISA with the panel of MAbs for epitope reactivity since psoralen inactivation of rotavirus results in antigenically intact particles (37). To determine whether virus was completely inactivated, both formalin- and psoralen-treated viruses were passaged three times in cell culture; no cytopathic effect or plaques were seen, indicating that inactivation was complete. Complete virus inactivation was more readily obtained with psoralen than with formalin; therefore, in the later studies with CsCl-purified virus, psoralen inactivation was used. Production and characterization of VLPs. Sf9 cells were coinfected with the baculovirus recombinants as described previously (26) to produce 2/6, (G3) 2/6/7, and (P[2], G3) 2/4/6/7-VLPs. Therefore, 2/6/7- or 2/4/6/7-VLPs contained a VP7 of serotype G3 specificity, while 2/4/6/7-VLPs contained a VP4 of a P[2] type. VLPs were purified by isopycnic CsCl gradient centrifugation and characterized essentially as described previously (26). Briefly, VLP composition, purity, and integrity were confirmed by Western blotting (using a mouse hyperimmune serum prepared against triple-layered SA11 Cl3 virus as described elsewhere [5, 26]), staining of polyacrylamide gels with silver nitrate, and EM, respectively. The G3 serotype of the 2/6/7- and 2/4/6/7-VLPs was confirmed by immunoelectron microscopy and a MAb-based ELISA as described previously (26). The total protein concentration was determined by the Bio-Rad (Hercules, Calif.) protein assay, using bovine immunoglobulin G (IgG) as the standard, and endotoxin levels were quantitated by the Limulus amebocyte lysate test (,0.05 endotoxin unit [EU]/dose); Associates of Cape Cod, Inc., Woods Hole, Mass.). Preparation of rotavirus or VLP vaccines. Vaccine preparations were either (i) adsorbed to AlP (kindly supplied by Wyeth Lederle Vaccines and Pedriatics, West Henrietta, N.Y.) as described previously (19); (ii) mixed 1:1 with Freund’s adjuvants (complete for the first dose and incomplete for the second dose); or (iii) mixed with 20 mg (unless otherwise specified) of QS-21 adjuvant (kindly supplied by Wyeth Lederle Vaccines and Pediatrics), a purified triterpene glycoside (saponin) of aquillaic acid derivative from the bark of the South American Molina tree Quillaja saponaria (45, 46). Animals. The 3- to 6-month-old rotavirus antibody- and specific-pathogen-free New Zealand White rabbits of either sex used in this study were either from our own rabbit breeding colony (14, 17–19) or from a commercial source (Charles River Laboratories, Inc., Wilmington, Mass.). During the immunization phase of the study, rabbits were housed in open cages in two rooms in a BL2 containment facility. During the rotavirus challenge, rabbits were housed individually in negative-pressure isolator units. Animal inoculations and sample collection. Approximately 1 to 2 months prior to rotavirus or VLP inoculations, all rabbits, except those parenterally immunized with 10 or 20 mg of 2/4/6/7-VLPs in Freund’s adjuvants or absorbed to AlP, were vaccinated twice intramuscularly with a Clostridium spiroforme toxoid (developed and kindly supplied by R. Carman, TechLabs, Inc., Blacksburg, Va.), to prevent disease due to endemic C. spiroforme and its associated toxin (3, 19, 60). Rabbits were vaccinated twice intramuscularly (quadriceps muscle of the leg) at 0 and 21 or 28 days postvaccination (dpv) with a volume of 0.5 ml of (i) live or formalin-inactivated SA11 Cl3 rotavirus cell lysate (1.4 3 107 PFU), (ii) 50 mg of live or psoralen-inactivated purified SA11 Cl3 rotavirus, (iii) 10, 20, or 50 mg of 2/4/6/7-VLPs, (iv) 50 mg of 2/6/7-VLPs, (v) 50 or 200 mg of 2/6-VLPs, (vi) MA104 cell lysate, or (vii) TNC buffer (10 mM Tris [pH 7.4], 140 mM NaCl, 10
J. VIROL. mM Ca2Cl). All rabbits were challenged at 56 or 69 dpv with 3.5 3 105 PFU (approximately 103 50% infectious doses [ID50]) of lapine ALA rotavirus. Individual serum and lavage or fecal samples were collected from all rabbits at 0, 28, 56, or 69, and 84 or 97 dpv to measure antirotavirus antibodies. In the first experiment, where we compared the immunogenicity of 10 or 20 mg of VLPs with Freund’s adjuvants and AlP, fluid from the entire intestine was collected by intestinal lavages as described previously (6, 13, 18, 19). Due to time required for the lavage procedure, lavage and corresponding serum samples were collected over 2 days (consecutive whenever possible) for each time point. For data analysis, no distinction was made between the two sampling days. For all subsequent experiments, only fecal samples were collected to measure intestinal antirotavirus antibodies (13, 14, 25). Comparison of intestinal antibody titers in lavage or fecal samples in rabbits revealed that rotavirus-specific total (IgM, IgG, and IgA) or IgG antibody titers measured from fecal samples were generally fourfold lower than those measured in lavage fluids (25). The reason for this fourfold decrease in antibody titers in fecal samples is not known, but it may be a consequence of degradation or dilution. Collection of fecal samples to detect antirotavirus antibodies was much less time-intensive and allowed experimental designs with increased numbers of rabbits. To assess efficacy of protection from rotavirus infection, fecal samples from individual rabbits were collected 0 to 14 days postchallenge (dpc). Detection of rotavirus in fecal samples by ELISA. The presence of rotavirus antigen in fecal samples was determined 0 to 14 dpc by ELISA as described previously (13, 14, 17, 19, 25). A sample was considered positive if the optical density (OD) value of the virus well minus the OD value of the control well was $0.1 and this OD value was at least 2 standard deviations greater than the OD values of the negative control samples (18). Virus antigen shedding curves (OD versus dpc) for each animal were plotted, and the area under the shedding curve for each animal was calculated. Percent reduction in virus shedding compared to control animals was calculated for each animal by comparing the area under the curve for each individual animal to the mean area under the curves of virus shedding in the control group within each experiment. Percent reduction in virus antigen shedding was then calculated for each vaccine group by determining the mean percent reduction of virus antigen shedding for each vaccine group. Detection of infectious virus from fecal samples by FFA. The titers of infectious virus from selected fecal samples collected from 17 rabbits following ALA rotavirus challenge were measured by fluorescent-focus assay (FFA) (10, 12). The 10% fecal suspensions were serially diluted (10-fold), and each dilution was assayed in duplicate on MA104 cell monolayers. Infectivity titers were expressed as fluorescent focus units (FFU) per milliliter. Due to toxicity, the lowest dilution tested for infectivity titers was 1:10. When fluorescent foci in 1/10 dilutions could not be visualized by fluorescence microscopy, the samples were considered negative, and a value of 5 FFU/ml was arbitrarily assigned. Detection of antibody responses by Western blotting, ELISA, and FFNA. Western blots to examine rotavirus-specific antibodies were performed by a procedure modified from those used by Burns et al. (4) and Crawford et al. (26). Briefly, SDS-PAGE was performed as described above, using SA11 Cl3-infected cell lysate (3.5 3 108 PFU/ml) as the antigen. Following immunoblotting, the nitrocellulose membranes were blocked for 30 min with 5% BLOTTO and were then cut into strips prior to overnight incubation at room temperature with the different primary antibodies diluted in 0.5% BLOTTO. Monoclonal or polyclonal antibodies used as controls were (i) purified anti-VP4 MAbs 3D8, 9E3, and 5E4 (4) at a final concentration of 2 mg/ml (used conjointly), (ii) polyclonal sera specific to VP6 (guinea pig) (33) and to VP7 (rabbit) (62) at dilutions of 1:600 and 1:1,000, respectively, and (iii) anti-SA11 polyclonal mouse serum at a dilution of 1:7,500. Pre- and postchallenge (0 and 28 dpc, respectively) serum samples from all rabbits were diluted 1:100 for Western blot analyses. The strips were then washed three times with 0.5% BLOTTO and incubated for 2 h with a 1:3,000 dilution of either horseradish peroxidase-conjugated goat anti-rabbit total immunoglobulin (IgM, IgG, and IgA) antibody (Cappel Research Products, Durham, N.C.), anti-mouse total immunoglobulin (IgM, IgG, and IgA) antibody (Southern Biotechnology Associates, Inc., Birmingham, Ala.), or anti-guinea pig IgG antibody (Cappel Research Products) as appropriate. The strips were washed and developed with 3,39,5,59-tetramethylbenzidine–peroxidase membrane enhancer (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) according to the manufacturer’s directions. The ELISAs to measure total (IgA, IgM, and IgG) and IgG- or IgA-specific antirotavirus antibodies present in serum, intestinal contents, or fecal samples were described previously (13, 17–19, 25). A positive reaction was defined as an OD value of $0.1 after subtraction of the OD values of the antigen-negative well (mock) from those of the antigen-positive well (13, 18). Additionally, the endpoint titer of the positive control serum (run on each plate) had to be within one dilution of an established standard for the assay to be acceptable. Neutralization tests (FFNA) were performed essentially as described previously (10, 12), with the endpoint determined as the serum dilution producing a $66% reduction in the number of fluorescent foci. Statistical analysis. Statistical analyses were performed with SPSS version 7.5 for Windows (SPSS, Inc., Chicago, Ill.). Percent reductions in shedding were compared by the Mann-Whitney U test. Antibody titers before and after virus challenge within a group were compared by the Wilcoxon signed ranks test, and titers between groups were compared by using the Kruskal-Wallis test followed
VOL. 72, 1998
ROTAVIRUS RECOMBINANT SUBUNIT VACCINE
9235
by the Mann-Whitney U test. Trend analysis was performed by linear regression, and correlation coefficients were calculated by Pearson’s correlation coefficients.
RESULTS Preparation and characterization of VLPs used for inoculation of rabbits. Coinfection of Sf9 cells with the respective baculovirus recombinants resulted in the formation of 2/6-, 2/6/7-, and 2/4/6/7-VLPs. 2/6-VLPs resembled double-layered particles and 2/6/7- and 2/4/6/7-VLPs resembled rotavirus TLPs (data not shown). EM analysis revealed that the structural integrity of all VLP types was high ($90% [data not shown]). The total protein concentration of each VLP type was determined by the Bio-Rad protein assay. The endotoxin levels in the VLP preparations used for rabbit inoculations were quantitated at 0.037 EU/10 mg, 0.016 EU/20 mg, and 0.015 to 0.05 EU/50 mg. The purity and protein composition of all VLPs used in this study were confirmed by electrophoretic analysis and staining of gels with silver nitrate and by Western blot analysis (data not shown). Immune response and protection from challenge of rabbits parenterally immunized with 10 or 20 mg of P[2], G3 2/4/6/7VLPs in Freund’s adjuvants or absorbed to AlP. Two doses of live or formalin-inactivated simian rotavirus SA11 was previously shown to induce protection in rabbits (19). We next examined whether VLPs would afford the same protection and whether the adjuvant used would influence the immunogenicity or protective efficacy of the VLPs. In an initial experiment, rabbits were immunized twice parenterally (0 and 21 dpv) with either 10 or 20 mg of 2/4/6/7-VLPs either in Freund’s adjuvants (n 5 5) or absorbed to AlP (n 5 5). Control animals were immunized with either live or formalin-inactivated SA11-infected cell lysates (1.4 3 107 PFU) either in Freund’s adjuvants (n 5 3) or absorbed to AlP (n 5 2). Negative control rabbits were immunized with MA104 cell lysates in either adjuvant. Doses of 10 or 20 mg of 2/4/6/7-VLPs in Freund’s adjuvants or AlP were immunogenic (Fig. 1). VLPs in Freund’s adjuvants induced high levels of rotavirus-specific total Ig (IgM, IgG, and IgA) in serum (Fig. 1A). Compared to responses with Freund’s adjuvants, SA11 rotavirus or VLPs in AlP induced significantly lower titers of serum antirotavirus antibody (P # 0.049) (Fig. 1A). A dosage of 10 or 20 mg VLPs in both Freund’s adjuvants and AlP induced equivalent high levels of serum neutralizing antibodies (N-Abs) against homologous SA11 serotype G3 (geometric mean titers [GMTs] of 6,400 and 5,572, respectively [Table 1]). Only one rabbit, vaccinated with SA11 with Freund’s adjuvants, developed low levels of rotavirus-specific intestinal IgA following vaccination (Fig. 1B). IgG antirotavirus antibody was detected in the intestines of all rabbits after parenteral vaccination with VLPs or SA11 (Fig. 1C). The intestinal IgG titers were at least two- to eightfold lower in rabbits immunized with SA11 or VLPs in AlP than in rabbits immunized with VLPs in Freund’s adjuvants, although the titers were not statistically different (P 5 0.063 and P 5 0.059, respectively). Since there was no significant difference (P 5 0.623) in mean protective efficacy between rabbits vaccinated with either live (n 5 2; 81%) or formalin-inactivated (n 5 1; 100%) SA11-
FIG. 1. Serum and intestinal antibody responses following two parenteral immunizations (0 and 21 dpv) of rabbits with formalin-inactivated SA11 rotavirus, 10 or 20 mg of G3 2/4/6/7-VLPs, or MA104 cell lysate in either Freund’s adjuvants or AlP. Total (IgM, IgG, and IgA) serum (A), intestinal IgA (B), and intestinal IgG (C) antirotavirus antibodies were measured by ELISA before (56 dpv) and after (84 dpv) oral challenge with lapine ALA rotavirus. Formulation
of vaccine and adjuvant are indicated along the x axis. Antibody titers were measured for individual rabbits, and results are plotted as the GMTs of the groups. Error bars represent 1 standard error of the mean. For titers of ,50 and ,5, 25 and 2.5, respectively, were used to calculate the GMT. A GMT of 25 or 2.5, for serum or intestinal antibodies, respectively, was considered negative. A significant (P , 0.05, Wilcoxon signed ranks test) increase in rotavirus antibody GMTs following challenge is indicated by an asterisk.
9236
CIARLET ET AL.
J. VIROL.
TABLE 1. Summary of rotavirus antibody responses and percent protection of rabbits challenged with 103 ID50 of homologous lapine ALA rotavirus following two intramuscular vaccinations with VLPs or SA11 rotavirus GMTa Vaccineb
Vaccine dose
Adjuvant
No. of rabbits
Total serum (IgM, IgG, IgA) and isotype-specific intestinal antibody Serum
SA11d
Serum N-Abs against:
Intestinal IgG
IgA
% Reduction of virus antigen sheddingc
SA11 Cl3
ALA
None (lysate) None (lysate) 50 mg
AlP Freund’s f QS-21g
2 3 7
72,408ADEF 2,755,449B 1,483,938BCFI
28AC 2,560B 119C
2.5A 4A 2.5A
2,400A 6,400A 6,400A
ND ND 1,767A
10–20 mg 10–20 mg 50 mg 50 mg 50 mg 50 mg 50 mg 50 mg 200 mg 50 mg
AlP Freund’s Freund’s Freund’s Freund’s QS-21 QS-21 QS-21 QS-21 QS-21*
5 5 5 9 5 10 10 5 5 5
25,600AD 77,605AE 713,155CFGI 378,929FGH 1,080,994I 145,096AE AE 102,400 155,209AEH 178,289AEH 117,627AE
6A 17A 211CD 118C 368D 35A 25A 53AC 61AC 53AC
2.5A 2.5A 2.5A 2.5A 2.5A 2.5A 2.5A 2.5A 2.5A 2.5A
5,572A 6,400A 77,605B 75,250B 50C 4,222A 4,222A 50C 50C 50C
ND ND 16,890B 10,973B 50C 1,213A 1,493A 5C 50C 50C
MA104 cells
0.5 ml 0.5 ml
AlP Freund’s
1 2
25J 25J
2.5E 2.5E
2.5A 2.5A
5C 50C
50C 50C
0L 0L
TNC buffer
0.5 ml
QS-21
11
25J
2.5E
2.5A
50C
50C
0KL
SA11e VLPs 2/4/6/7 2/6/7 2/6 2/4/6/7 2/6/7 2/6
76ABCFM 87AD 83AE 36CFGHM 71AFHP 89AO 58BC 41BCDEFHIJMNOP 56BH 26GIMN 26FMN 5JK 18CFJKMN
a Serum total and intestinal IgG and IgA antibodies were measured by ELISA at 56 dpv (0 dpc). For titers of ,50 and ,5, 25 and 2.5, respectively, were used to calculate the GMT. A GMT of 25 or 2.5, for serum or intestinal antibodies, respectively, was considered negative. Serum N-Abs were measured by FFNA at 56 dpv (0 dpc). For titers of ,100, 50 was used to calculate the GMT. A GMT of 50 was considered negative. Within columns, statistical similarities (P . 0.05, Mann-Whitney U test) in rotavirus antibody responses or in reduction of virus antigen shedding are indicated by the same capital letter; those followed by capital different letters are significantly different (P , 0.05 Mann-Whitney U test). Bold face titers indicate that a significant (P , 0.05, Mann-Whitney U test) increase in rotavirus antibody GMTs was induced following challenge. ND, not done. b The P genotype of all 2/4/6/7-VLPs is P[2]; the G serotype of all 2/6/7- or 2/4/6/7-VLPs is G3. c Virus antigen shedding curves were plotted for individual rabbits, the mean area under the curve was calculated for each group, and reduction of virus antigen shedding was determined by comparing it to the mean area under the curve for the control group. d Live or formalin-inactivated SA11 Cl3 (P[2], G3) rotavirus cell lysate (1.4 3 107 PFU). e Live or psoralen-inactivated CsCl-purified triple-layered SA11 Cl3 (P[2], G3) rotavirus. f Complete adjuvant for the first dose and incomplete adjuvant for the second dose. g Dose of QS-21 was 20 mg except for the group vaccinated with 2/6-VLPs indicated by an asterisk, which was 50 mg.
infected cell lysates in Freund’s adjuvants and the number of animals was small, the two groups were combined (mean protective efficacy, 87%) to perform statistical analyses (Fig. 2). The mean protective efficacy of the rabbits (n 5 2) vaccinated with formalin-inactivated SA11-infected cell lysates absorbed to AlP was 76% (Fig. 2) and was not significantly different from that afforded by SA11 in Freund’s adjuvants (p 5 0.564). Four of five rabbits immunized with 10 or 20 mg of VLPs in Freund’s adjuvants showed complete or partial protection from challenge (59 to 100%), while one rabbit had very low level protection (16%) affording a mean protective efficacy of 71% (Fig. 2). The mean protective efficacy of the VLPs in AlP was low, 36% (range, 24 to 52%), with protection from infection of $50% observed in only one rabbit immunized with 10 mg of VLPs. Although the mean protection achieved with VLPs in Freund’s adjuvants (71%) was higher than that achieved with VLPs in AlP (36%) (Fig. 2), the difference was not significant (P 5 0.115). Following challenge, all mock-immunized control rabbits developed serum and intestinal antibodies (Fig. 1). Rotavirusspecific IgA was induced after challenge in all but the two rabbits that were totally protected from challenge (Fig. 1B). Following challenge, there was no significant increase in the GMTs of total serum, total intestinal, intestinal IgG, or intestinal IgA of rabbits vaccinated with SA11 rotavirus or 10 or 20
mg of VLPs in Freund’s adjuvants (0.109 # P # 0.593). In contrast, following challenge of rabbits immunized with VLPs in AlP, GMTs of total serum, total intestinal, intestinal IgG, or intestinal IgA were significantly higher (0.038 # P # 0.042). The levels of serum N-Abs against SA11 serotype G3 in rabbits vaccinated with 10 or 20 mg of VLPs in either Freund’s adjuvants or AlP did not increase significantly following challenge (P # 0.180). Immune response and protection from challenge of rabbits parenterally immunized twice with 50 mg of P[2], G3 2/4/6/7or G3 2/6/7-VLPs in Freund’s adjuvants or QS-21. We next examined whether increasing the dose of 2/4/6/7-VLPs would increase protective efficacy from ALA rotavirus challenge (103 ID50). Since the response with AlP was weaker than with Freund’s adjuvants, we also examined whether QS-21, a saponin adjuvant, would be able to induce higher antibody responses and protection rates than AlP. In addition, to investigate the contribution of the outer capsid protein VP4, to induction of protective immunity, we compared the immunogenicity and protective efficacies of 50 mg of 2/4/6/7- or 2/6/7VLPs in Freund’s adjuvants (n 5 5 and n 5 9, respectively) and QS-21 (n 5 10 and n 5 10, respectively). Control rabbits were either mock vaccinated with TNC buffer or vaccinated with CsCl-purified live or psoralen-inactivated SA11 Cl3 virus in
VOL. 72, 1998
FIG. 2. Protection from lapine ALA rotavirus challenge in rabbits immunized twice parenterally with formalin-inactivated SA11 rotavirus, 10 or 20 mg of G3 2/4/6/7-VLPs, or MA104 cell lysate in either Freund’s adjuvant or AlP. Virus antigen shedding curves were plotted for individual rabbits, and the mean area under the curve was calculated for each group. Results are plotted as percent reduction in shedding for each individual animal (F), as well as the mean for each vaccine group (O), calculated by comparing the mean area under the curve for each vaccine group to the mean area under the curve for the control group. The number of rabbits with overlapping levels of protective efficacy is indicated next to the dots (n 5 2). Formulation of vaccine, adjuvant, and number of rabbits per group are indicated along the x axis.
QS-21 only, in the same way as controls in Freund’s adjuvants were performed previously. Serologic and intestinal antibody responses were evaluated at 56 dpv (28 days after the second vaccination) prior to virus challenge (Fig. 3). As expected, negative control animals inoculated with TNC buffer with QS-21 or Freund’s adjuvants remained rotavirus antibody negative until after challenge (Fig. 1 and 3). Rabbits vaccinated with CsCl-purified live (n 5 4) or psoralen-inactivated (n 5 3) SA11 in QS-21 developed equivalent high serum and IgG intestinal antibody GMTs (P $ 0.390). Therefore, the groups were combined (total n 5 7) for statistical comparison of immunogenicity and protective efficacy of virus and VLP immunization (Fig. 3D to F). Rabbits immunized with 2/4/6/7- and 2/6/7-VLPs in Freund’s adjuvants developed higher serum (P # 0.007) and intestinal (P # 0.014) GMTs than those immunized with the corresponding formulation in QS-21 (Fig. 3). No significant difference in serum or intestinal IgG antibody GMTs was induced by 2/4/ 6/7- or 2/6/7-VLPs in each adjuvant group (P . 0.05), indicating that the presence of VP4 did not affect the magnitude of the immune response. Levels of anti-G3 SA11 rotavirus serum N-Abs induced by 2/4/6/7- or 2/6/7-VLPs in Freund’s adjuvants (GMTs 5 77,605 and 72,250, respectively) were significantly higher than levels induced by the corresponding formulations in QS-21 (GMTs 5 4,222) (P 5 0.002) (Table 1). Anti-G3 ALA rotavirus serum N-Abs induced by 2/4/6/7- or 2/6/7-VLPs in Freund’s adjuvants or QS-21 (Table 1) were significantly lower (P # 0.042) than the serum N-Abs to SA11 rotavirus of the corresponding formulations in the same adjuvant (Table 1). Although both SA11 and ALA share the same VP7 serotype, these results suggest that serum N-Abs are preferentially made to the specific immunizing VP7 strain (12, 13, 72). However, the presence or absence of VP4 in the VLPs did not affect the levels of serum N-Abs to serotype G3 compared within the same adjuvant group (P $ 0.541) (Table 1).
ROTAVIRUS RECOMBINANT SUBUNIT VACCINE
9237
Both 2/4/6/7- and 2/6/7-VLPs in Freund’s adjuvants provided greater mean protection (89 and 58%, respectively) than the equivalent VLP formulation in QS-21 (56 and 26%, respectively) (P # 0.007) (Fig. 4). In rabbits, VP4 appears to play a role in protection, as 2/4/6/7-VLPs in either adjuvant afforded significantly more protection than 2/6/7-VLPs (P # 0.034) (Fig. 4). Although 50 mg of 2/4/6/7-VLPs in Freund’s adjuvants induced significantly higher (P # 0.012) GMTs of total serum, serum neutralizing, and intestinal IgG antibody than 10 or 20 mg of 2/4/6/7-VLPs, the increased VLP dose did not significantly increase the protective efficacy (P $ 0.435) against ALA challenge. These data suggest that immunogenicity (P 5 0.027, linear regression), but not protection (P 5 0.294, linear regression), is VLP dose dependent. However, our ability to discern significant differences in protective efficacy by using Freund’s adjuvants may require the use of lower doses of VLPs and larger sample sizes. Since there was no significant difference (P 5 0.724) in mean protective efficacy between rabbits immunized with live purified SA11 (n 5 4, 81%) and psoralen-inactivated purified SA11 (n 5 3; 86%), the two groups were combined (mean protective efficacy, 83% to perform the statistical analyses (Fig. 4). When protection afforded by SA11 was compared between all groups, mean protection values among the groups that received either Freund’s adjuvants, QS-21, or AlP were not statistically different (P 5 0.462 to 1.000) (Fig. 2 and 4). Therefore, choice of adjuvant was not critical when rabbits were vaccinated with SA11 virus, although significant differences might have been observed with larger group sizes. All rabbits except those (n 5 5) that were 100% protected from ALA challenge developed an intestinal IgA antirotavirus response after challenge (Fig. 3C and F). Following challenge, GMTs of total serum, serum N-Abs, total intestinal, intestinal IgG, and intestinal IgA antibodies of animals vaccinated with SA11, TNC buffer, 2/4/6/7- or 2/6/7-VLPs in QS-21 and 2/6/7VLPs in Freund’s adjuvants (except serum N-Abs) increased significantly (P # 0.042) (Table 1). The titers of rabbits immunized with SA11 in Freund’s adjuvants or AlP or with 10, 20, or 50 mg of 2/4/6/7-VLPs in Freund’s adjuvant did not increase significantly (P . 0.109). These results correspond with the level of protection from infection (Fig. 4), since the level of protection was higher in the latter groups. Immune response and protection from challenge of rabbits parenterally immunized twice with 50 mg of 2/6-VLPs in Freund’s adjuvants or QS-21. We recently showed that 2/6VLPs administered parenterally, orally, or intranasally to mice provide protection (42 to 100%) from homologous ECwt murine rotavirus challenge (2, 33, 42, 58, 59). To determine if either of the outer capsid proteins, VP7 or VP4, is required to achieve protection from infection in the rabbit model, 50 mg of 2/6-VLPs in Freund’s adjuvants (n 5 5) and QS-21 (n 5 5) was administered parenterally to rabbits. In Freund’s adjuvants, the total serum antibody responses induced by 2/6-VLPs were not significantly different (P 5 0.278) from those induced by 2/4/6/7-VLPs; however, they were significantly higher (P 5 0.012) and lower (P 5 0.042) than those induced by 2/6/7-VLPs and SA11, respectively (Fig. 3A). Similarly, rotavirus-specific IgG intestinal antibodies induced by 2/6-VLPs were not significantly different from those induced by 2/4/6/7-VLPs (P 5 0.118) but were significantly higher (P 5 0.02) and lower (P 5 0.024) than those induced by 2/6/7-VLPs and SA11, respectively (Fig. 3B). Rabbits immunized with 2/6-VLPs in QS-21 developed lower serum (P 5 0.008) and intestinal (P 5 0.011) GMTs than the corresponding formulation in Freund’s adjuvants. The total serum antibody responses induced by 2/6- VLPs in QS-21
9238
CIARLET ET AL.
J. VIROL.
FIG. 3. Serum and intestinal antibody responses following two parenteral immunizations (0 and 21 or 28 dpv) of rabbits with formalin-inactivated SA11 rotavirus, MA104 cell lysate, or 50 mg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in Freund’s adjuvants (A to C) and with 50 mg of live/psoralen-inactivated SA11 rotavirus, TNC buffer, or 50 mg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in QS-21 (D to F). Total (IgM, IgG, and IgA) serum (A and D), intestinal IgG (B and E), and intestinal IgA (C and F) antirotavirus antibodies were measured by ELISA before (56 or 69 dpv) and after (84 or 97 dpv) oral challenge with lapine ALA rotavirus. Antibody titers were measured for individual rabbits, and results are plotted as GMTs of the groups. The GMT of intestinal IgG antibody titers for both 0 and 28 dpc of the rabbits vaccinated with 2/6-VLPs in Freund’s adjuvants is 368. Therefore, for this group, only one box appears on the graph (B). Error bars represent 1 standard error of the mean. For titers of ,50 and ,5, 25 and 2.5, respectively, were used to calculate the GMT. A GMT of 25 or 2.5, for serum or intestinal antibodies, respectively, was considered negative. A significant (P , 0.05, Wilcoxon signed ranks test) increase in rotavirus antibody GMTs following challenge is indicated by an asterisk.
VOL. 72, 1998
ROTAVIRUS RECOMBINANT SUBUNIT VACCINE
9239
FIG. 4. Protection from lapine ALA rotavirus challenge in rabbits immunized twice parenterally with formalin-inactivated SA11 rotavirus, MA104 cell lysate, or 50 mg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in Freund’s adjuvant (A) or with 50 mg of live/psoralen-inactivated SA11 rotavirus, TNC buffer, or 50 mg of G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in QS-21 (B). Virus antigen shedding curves were plotted for individual rabbits, and the mean area under the curve calculated for each group. Results are plotted as percent reduction in shedding for each individual animal (F), as well as the mean for each vaccine group (O), calculated by comparing the mean area under the curve for each vaccine group to the mean area under the curve for the control group. The number of rabbits with overlapping levels of protective efficacy is indicated next to the dots (n 5 2 or 3); formulation of vaccine and number of rabbits per group are indicated along the x axis.
were not significantly different (P $ 0.204) from those of any other VLP formulations in QS-21 but were lower (P # 0.004) than SA11-vaccinated rabbits (Fig. 3D). Titers of rotavirusspecific IgG intestinal antibodies induced by 2/6-VLPs were not significantly different from those induced by any VLP formulation in QS-21 (P $ 0.125) or from those induced by SA11 in QS-21 (P 5 0.093) (Fig. 3E). As expected, none of the rabbits vaccinated with 2/6-VLPs in either Freund’s adjuvants or QS-21 developed serum or intestinal N-Abs against SA11 and ALA rotavirus (Table 1). The protective efficacies of 2/6-VLPs in the two adjuvants were equivalent (P 5 0.917). In Freund’s adjuvants, the mean protective efficacy afforded by 2/6-VLPs (41%) was not significantly different (P $ 0.071) from that afforded by SA11 (87%), 2/4/6/7-VLPs (89%), or 2/6/7-VLPs (58%) (Fig. 4A). The inability to detect significant differences in protective efficacy between 2/6-VLPs and any other VLP formulation or SA11 may have been due to the number of animals in the 2/6-VLP group (n 5 5). 2/6/7-VLPs (n 5 9), which afforded a similar level of mean protection as 2/6-VLPs, provided significantly lower protection (P 5 0.034) than 2/4/6/7-VLPs (n 5 5). Trend analysis of protective efficacy of 2/6-, 2/6/7-, and 2/4/6/7-VLPs in Freund’s adjuvants revealed that the protective efficacy increased as the number of proteins in the VLPs increased (P 5 0.009, linear regression). Therefore, the level of protection of VLPs in Freund’s adjuvants was dependent on VLP formulation. In QS-21, protection afforded by 2/6-VLPs (26%) was significantly lower than the protection afforded by 2/4/6/7VLPs (56%, P 5 0.014) or SA11 (83%, P 5 0.004) (Fig. 4B). However, 2/6-VLPs provided the same levels (p 5 1.000) of protection (26%) against challenge as 2/6/7-VLPs in QS-21 (Fig. 4B). Since 2/6-VLPs did not induce significantly lower total serum or intestinal IgG titers than 2/4/6/7-VLPs (P 5 0.445) or SA11 virus (P 5 0.093) in QS-21 (Fig. 3D and E), these results
suggest that the presence of antirotavirus IgG in the intestine alone is not sufficient to protect rabbits from challenge. Intestinal VP4- but not VP7-specific antibodies may be required to induce higher levels of protection. Nevertheless, although our results indicate a need for VP4 in the VLP formulation, the correlation is not absolute, as individual rabbits (4 of 14) immunized with VLPs that lacked VP4 in Freund’s adjuvants showed high levels (.80%) of protection (Fig. 4A). Following challenge, total serum and intestinal IgG and IgA antibody titers increased significantly (P # 0.043) in 2/6-VLPvaccinated rabbits in QS-21 (Fig. 3D to F). However, in rabbits vaccinated with 2/6-VLPs in Freund’s adjuvants, only total serum and intestinal IgA antibody titers increased significantly after challenge (P # 0.043) (Fig. 3A to C). Following challenge, all 2/6-VLP-vaccinated rabbits developed serum N-Abs to the serotype G3 simian SA11 and lapine ALA viruses. The serum N-Ab GMT to SA11 (800) observed after challenge was significantly lower (p 5 0.008) than that to ALA (11,143), suggesting that N-Abs were preferentially made to the challenge ALA virus, although SA11 and ALA rotaviruses have the same VP7 type (G3) (12, 13, 72). Immune response and protection from challenge of rabbits parenterally immunized twice with different concentrations of 2/6-VLPs and QS-21. Experiments in mice showed that parenteral administration of 2/6-VLPs in QS-21 induced high levels of protection from challenge (2, 33, 42). However, 2/6-VLPs in QS-21 administered parenterally to rabbits failed to induce high levels of protection, and 2/6-VLPs in Freund’s adjuvants afforded high levels of protection in only two of five rabbits (Fig. 4). To determine if either an increase of VLP or QS-21 dose would afford higher levels of protection in rabbits, 200 mg of 2/6-VLPs in 20 mg of QS-21 (n 5 5) or 50 mg of 2/6-VLPs in 50 mg of QS-21 (n 5 5) was administered parenterally to rabbits. Increases in the dose of either 2/6-VLPs (200 mg) or QS-21 (50 mg) failed to increase the levels of total serum
9240
CIARLET ET AL.
J. VIROL.
FIG. 5. Serum immune responses to individual rotavirus proteins before (56 or 69 dpv) or after (84 or 97 dpv) oral challenge with lapine ALA rotavirus. Representative results are shown for rabbits vaccinated parenterally either with 2/4/6/7-VLPs in Freund’s adjuvants (F) or with 2/4/6/7-, 2/6/7-, or 2/6-VLPs, psoralen-inactivated SA11, or TNC buffer in QS-21 (Q). Proteins in a SA11-infected cell lysate were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with MAbs directed to VP4, VP6, or VP7, with a hyperimmune serum to SA11, or with paired sera from rabbits immunized with the indicated vaccine formulations. Percent protection from infection for individual rabbits is indicated below each lane. Locations of the viral proteins are indicated on the left.
antibodies (P $ 0.072), intestinal IgG antibodies (P $ 0.228), or mean protective efficacy (5 and 18%, respectively) (P $ 0.141) compared to 50 mg of 2/6-VLPs in 20 mg of QS-21 (data not shown). As observed with lower doses of VLPs or QS-21, the total serum and intestinal IgG antibody levels or mean protective efficacy with either the increased dose of VLPs or adjuvant were significantly lower (P # 0.048) than those induced by 2/6-VLPs in Freund’s adjuvants (data not shown). Analyses of the immune response to individual rotavirus proteins and correlation of protective efficacy and immune responses detected by Western blotting and FFNA. Table 1 summarizes the rabbit immune responses (as measured by ELISA and FFNA), percent protective efficacy, and the corresponding statistical analyses among all vaccine groups. Immunization with virus or with the inclusion of VP4 in 2/4/6/7-VLP vaccines resulted in significantly higher (P , 0.05, Mann-Whitney U test) mean protective efficacy than in the 2/6/7-VLP vaccines. Therefore, we investigated whether rabbits vaccinated with 50 mg of live or psoralen-inactivated SA11 rotavirus or 2/4/6/7-, 2/6/7-, or 2/6-VLPs in Freund’s adjuvants or QS-21 developed VP4-specific immune responses. To detect rotavirus protein-specific antibody responses, Western blot analyses with denatured SA11 Cl3 proteins were performed on all pre- and postchallenge serum samples from individual rabbits. Prior to challenge, all rabbits developed serum antibodies to all, or a subset of, the proteins found in SA11 or VLP vaccine. Figure 5 shows the pre- and postchallenge serum antibody responses of a representative group of rabbits as measured by Western blotting and the correspond-
ing percent protective efficacy against virus antigen shedding following challenge. Prior to challenge, none of the mockvaccinated rabbits possessed antibodies to any rotavirus proteins. Rabbits vaccinated with live or inactivated SA11 rotavirus developed antibodies only to structural rotavirus proteins, indicating that no rotavirus replication occurred. The predominant antibody responses was directed to VP6 irrespective of virus or VLP vaccine administered to rabbits. Antibodies to VP2 were observed seldom with Freund’s adjuvants and not at all with QS-21. Antibodies to VP7 were detected in about 50% of the samples of rabbits vaccinated with VP7-containing formulations in either Freund’s adjuvants or QS-21. VP4-specific antibodies were not detected in any prechallenge serum samples from rabbits immunized with SA11 or 2/4/6/7-VLPs in either adjuvant. Following ALA challenge, rabbits that were 98 to 100% protected from virus antigen shedding did not develop antibodies to additional rotavirus structural or nonstructural proteins. Conversely, rabbits that were not protected from challenge developed antibodies to most rotavirus proteins, including nonstructural proteins. The failure to detect antibody to VP4 by Western blotting does not indicate that rabbits did not develop serum antibodies to VP4 but rather suggests that this method may not be sensitive enough to detect antibodies to individual proteins or the induced antibodies do not react with denatured proteins because the immunodominant epitopes are conformational. To determine if antibodies to VP4 play a role in protection, the prechallenge serum samples of all rabbits vaccinated with 2/4/ 6/7-, 2/6/7-, or 2/6-VLPs in Freund’s adjuvants or QS-21 or with
VOL. 72, 1998
ROTAVIRUS RECOMBINANT SUBUNIT VACCINE
9241
FIG. 6. Correlation of higher protection rates with presence of serum N-Abs to VP4. (A) Serum N-Ab responses following two parenteral immunizations (0 and 28 dpv) of rabbits with 50 mg of P[2], G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in Freund’s adjuvants or with 50 mg of live or psoralen-inactivated SA11, TNC buffer, or 50 mg of P[2], G3 2/4/6/7-, G3 2/6/7-, or 2/6-VLPs in QS-21 were measured by FFNA against rotavirus strains SA11 (P[2], G3) and B223 (P8[11], G10) and against reassortant virus strains R-N33 (P8[11], G3) and R-A32 (P[2], G10) prior to (56 or 69 dpv) oral challenge with lapine ALA rotavirus. Serum N-Ab titers were measured for individual rabbits, and results are plotted as the GMTs of the groups. Error bars represent 1 standard error of the mean. For titers of ,100, 50 was used to calculate the GMT. A GMT of 50 and its log were considered negative. A significant difference (P # 0.021, Mann-Whitney U test) in VP4-specific N-Abs induced by 2/4/6/7-VLPs vaccines measured by comparing serum N-Abs measured against B223 and R-A32 is indicated by an asterisk. (B) Scatterplot of the log of the SA11 VP4-specific-NAb titers and the percent reduction of virus antigen shedding for individual rabbits (n 5 58). Results were graphed to indicate whether rabbits were immunized with both VP4 and VP7 (■; SA11 or 2/4/6/7-VLPs in either adjuvant), absence of VP4 but presence of VP7 (F; 2/6/7-VLPs in either adjuvant), or absence of both VP4 and VP7 (Œ; 2/6-VLPs in either adjuvant or TNC buffer in QS-21). Correlation coefficient was calculated by Pearson’s correlation coefficient.
live or psoralen-inactivated SA11 rotavirus in QS-21 were tested by FFNA. SA11 C13 3 B223 reassortant rotavirus strains R-N33 (P8[11], G3) and R-A32 (P[2], G10), and their parental rotavirus strains, were used in FFNAs to measure directly SA11 VP4-specific serum N-Abs. The N-Ab GMTs induced to SA11 and R-N33 (both G3) by
SA11 rotavirus in QS-21 or 2/4/6/7- or 2/6/7-VLPs in Freund’s adjuvants or QS-21 were equivalent (P $ 0.643), although the N-Ab titers to the reassortant R-N33 were slightly lower than those obtained to the parental strain SA11 (Fig. 6A). Rabbits vaccinated with 2/4/6/7- and 2/6/7-VLPs in Freund’s adjuvants developed low-level N-Abs to the heterologous B223 rotavirus
9242
CIARLET ET AL.
J. VIROL.
FIG. 7. Correlation between reduction of virus antigen shedding as measured by ELISA and reduction of infectious virus titers as measured by FFA independently of vaccine formulation administered to rabbits. (A) Scatterplot of the areas under the curve for virus antigen shedding (as measured by ELISA) versus the log of infectious virus titers (as measured by FFA). Analysis was performed on a subset of rabbits (n 5 17) vaccinated parenterally with buffer, SA11 virus, or different VLP formulations. Correlation coefficient was calculated by Pearson’s correlation coefficient. Line depicts linear regression analysis [f(x) 5 0.247X 2 0.969]. Boxes indicate clusters of rabbits according to percent protection ranges. (B) Daily mean infectious (0 to 10 dpc) ALA rotavirus titers measured by FFA were determined for rabbits grouped based on protection, independent of vaccine formulation administered to rabbits. Based on the scatterplot, rabbits were grouped based on 0% (■), 20 to 65% (F), 68 to 98% (Œ), and 100% (}) protection as measured by ELISA. For infectious titers of ,10, 5 was used to calculate the mean infectious virus titer. An infectious titer of 5 was considered negative.
strain (GMTs 5 528 and 147, respectively) that were significantly lower (P # 0.023) than those obtained with the homologous G3 VP7 type (Fig. 6A). Rabbits vaccinated with live or psoralen-inactivated SA11 virus or with 2/4/6/7- or 2/6/7-VLPs in QS-21 developed low N-Abs to B223 (Fig. 6A). The ability of 2/4/6/7- and 2/6/7-VLPs to induce heterotypic N-Abs to other additional strains of different serotypes is described elsewhere (27). VP4-specific N-Abs induced by SA11 rotavirus or 2/4/6/7-VLP vaccines were directly measured by comparing the N-Ab responses obtained against strain B223 and the reassortant rotavirus strain R-A32, since the latter possesses an SA11 VP4 type (P[2]) and a B223 VP7 type (G10). The serum N-Ab GMTs of rabbits vaccinated with 2/4/6/7-VLPs in Freund’s adjuvants measured with R-A32 were significantly higher (P 5 0.021) than those measured by B223 alone (Fig. 6A). N-Ab GMTs of serum samples of rabbits vaccinated with 2/6/7-VLPs measured with B223 and R-A32 were equivalent (P 5 0.864), indicating that the difference in N-Ab GMT observed with R-A32 and the serum of rabbits vaccinated with 2/4/6/7-VLPs in Freund’s adjuvants was due to N-Abs to VP4 present in the serum samples (Fig. 6A). Similar significant results (P , 0.001) were observed when N-Abs were measured in the serum samples of rabbits vaccinated with 2/4/6/7-VLPs in QS-21 against B223 and R-A32. The N-Ab GMTs of serum samples of rabbits vaccinated with 2/6/7-VLPs in QS-21 were similar (P 5 0.909) against both viruses tested, as were those of rabbits vaccinated with SA11 rotavirus in QS-21 (P 5 0.197) (Fig. 6A). As expected, no N-Abs to the reassortant or parental rotavirus strains were detected in any of the rabbits vaccinated with 2/6-VLPs in either adjuvant (Fig. 6A). Although significant differences were observed in SA11 VP4-specific N-Ab GMTs, not all rabbits that were immunized with, and protected by, a vaccine that included VP4 in the formulation developed a detectable SA11 VP4-specific neutralizing immune response. To determine whether higher protection from challenge correlated with an specific immune
response to VP4, the Pearson’s correlation coefficient between the protective efficacy and the log of SA11 VP4-specific serum N-Ab titers for all rabbits was calculated. Our results showed that higher protection levels correlated with the presence of N-Abs to VP4 (P , 0.001, r 5 0.55, Pearson’s correlation coefficient) (Fig. 6B). Therefore, generally the inclusion of VP4 significantly increases the protective efficacy against infection in rabbits parenterally immunized with VLPs, but antibodies to VP4 alone may not explain the enhanced protection levels in some animals. Unfortunately, we were unable to isolate the reassortants necessary to measure VP4-specific N-Abs to ALA, the challenge rotavirus strain (68). Detection of infectious virus in fecal samples collected 0 to 10 dpc by FFA. To determine if protective efficacy measured by percent reduction in virus antigen shedding by ELISA predicts the amount of infectious virus shed, the infectivity titers of the ALA virus shed following challenge were measured by FFA (Fig. 7). For comparison, a subset of rabbits (n 5 17) were chosen to cover the whole range of protection, independent of vaccine formulation. To establish a correlation, Pearson’s correlation coefficient was calculated by plotting the areas under the curve for the virus antigen shedding curves and the virus titer curves. A significant (P , 0.001, r 5 0.979) correlation coefficient was found (Fig. 7A). Based on the scatterplot (Fig. 7A), two distinct groups were readily evident, controls (n 5 3) that were not protected (0%) and rabbits (n 5 3) with that were completely protected (100%). Rabbits (n 5 11) with intermediate protection rates (20 to 98%) cluster in the middle of the plot and were subdivided into two groups, one with rabbits exhibiting relatively high protection levels (66 to 98%) and another with those exhibiting lower protection levels (20 to 65%). These results show a strong association between percent protection from infection to both virus antigen shedding and infectious virus titers.
VOL. 72, 1998
ROTAVIRUS RECOMBINANT SUBUNIT VACCINE
The amount of infectious virus shed by rabbits with 20 to 65% protection was approximately 1 log lower than that for control rabbits (0%) (Fig. 7B). In contrast, there was a 2- to 3-log difference in the titer of virus shed in the group that was 68 to 98% protected with respect to the control group. Also, the duration of shedding of infectious virus was 2 to 3 days shorter for the protected group than for the control group. Rabbits that were 100% protected from challenge did not shed detectable infectious ALA virus and showed at least a 4-log decrease in the amount of infectious virus shed compared to the 0% control group. Therefore, protection as measured by reduction in antigen virus shedding (68 to 100%) corresponded with 2- to 4-log reductions of infectious virus shedding. DISCUSSION We examined the immunogenicity and protective efficacy of different formulations of parenterally administered candidate rotavirus VLP subunit vaccines in the rabbit model of rotavirus infection. Previously, we showed that parenteral vaccination with live or inactivated rotavirus induces complete active protective immunity in rabbits against virus challenge. This protection correlated with the presence of rotavirus-specific IgG, but not IgA, in the intestine (19). In this study, we showed that VLPs administered parenterally are immunogenic and can induce active protective immunity in rabbits. Vaccinated rabbits developed high levels of total (IgM, IgG, and IgA) rotavirusspecific antibody in the serum. Rabbits vaccinated with VLPs or with live or inactivated SA11 virus developed antirotavirus intestinal antibody of the IgG, but not IgA, isotype. Protective efficacy varied with both VLP formulation and adjuvant. With all adjuvants tested, 2/4/6/7-VLPs consistently induced the highest levels of protection from homotypic (G3) ALA rotavirus challenge. There was no significant difference in the mean protective efficacy afforded by live or formalin-inactivated SA11 in either Freund’s adjuvants (87%) or AlP (76%) or purified live or psoralen-inactivated SA11 in QS-21 (83%) (P $ 0.462). Although the results are similar to our previous work comparing live or inactivated SA11 virus administered in Freund’s adjuvants and AlP (19), in the present study, unlike the previous study, total protection from rotavirus challenge was not achieved in rabbits vaccinated with SA11 in either adjuvant. The live and formalin-inactivated SA11 vaccine preparations were the same vaccine preparations used previously (19), but antigen ELISA results indicated that limited degradation of the immunogen occurred in this study (data not shown). Therefore, the lower protective efficacy in this study is likely due to immunization with a lower dose of antigen. The difference in virus inactivation procedures probably did not influence the immune responses because (i) we did not observe any significant difference in either immunogenicity or protective efficacy between live, formalin-inactivated, and psoralen-inactivated SA11 virus, (ii) we had previously shown (19) that following formalin treatment of virus, the reactivity levels of MAbs for several viral epitopes remain unchanged on formalin-inactivated virus compared to live virus, and (iii) likewise, others have shown that psoralen-inactivated virus retains epitope reactivity to the same panel of MAbs (37). Therefore, the effect of the differences in inactivation procedures on the immune responses elicited was not a major concern. Live or inactivated SA11 rotavirus elicited the highest antibody responses and levels of protection in all three adjuvants tested. The high levels of antibody were not due to replication of the SA11 rotavirus administered intramuscularly to rabbits because prior to challenge none of the SA11-vaccinated rabbits
9243
developed serum antibodies to nonstructural proteins (Fig. 5 and data not shown), and fecal samples collected 2 to 14 dpv from rabbits vaccinated intramuscularly with live or formalininactivated SA11 rotavirus did not contain rotavirus antigen, indicating that virus replication did not occur (19). It is probable that the higher responses induced by SA11 compared to VLPs were due to differences in the immunizing dose. The doses of SA11 and VLPs administered to rabbits were based on total protein concentration determined by the Bio-Rad protein assay. Other studies have determined that if vaccine dose is based on the amount of VP7 in VLP and SA11 preparations, then SA11 has to be administered at a lower total protein concentration to yield equivalent doses of VP7 (50). Therefore, SA11 may have provided better responses in all adjuvants in this study compared to VLPs because of slightly higher doses, not differences in inherent properties between SA11 and VLPs. Since the rabbit is an infection, rather than a disease, model, we determined protective efficacy of the VLPs by calculating percent reduction in antigen virus shedding measured between vaccinated and mock-vaccinated rabbits. Prevention of virus antigen shedding may be a more stringent measure of protection than protection from clinical disease, since detectable levels of virus antigen shedding can occur in the absence of clinical signs (21). Based on the strong correlation (r 5 0.979, P , 0.001, Pearson’s correlation coefficient) between the amount of virus antigen and infectious virus shedding, calculation of percent protection from infection reflects both the amplitude and the duration of both infectious virus and virus antigen shedding. The significant reduction of infectious virus shedding in vaccinated rabbits that had $68% protection from virus antigen shedding indicates that substantially less virus replication occurred in the intestines of vaccinated rabbits. A decrease in the amount of virus shedding could lead to a considerable decrease in the amount of infectious virus disseminated among the population. Although VLPs administered parenterally can effect a significant reduction in rotavirus shedding, it is difficult to predict how the reduced shedding relates to protection from any rotavirus diarrhea or severe rotavirus diarrhea because comparable measurements of these parameters have not been reported for naturally infected children or for any disease animal model. To assess the immunogenicity and protective efficacy of different VLP formulations in the rabbit model, Freund’s adjuvants, AlP, and QS-21 were tested as adjuvants. Only AlP is licensed for use in humans. Although our results indicate that VLPs can induce homotypic protection from challenge by the parenteral route, our data combined with data obtained for mice (42, 50) indicate that AlP and aluminum hydroxide are not potent adjuvants for use in conjunction with VLPs. Nevertheless, in previous rabbit experiments (14), the antibody titers induced by SA11 virus in AlP was sufficient to provide protection. It is probable that higher doses of VLPs in AlP will be needed to induce complete protective immunity. QS-21 has been shown to be a powerful immunopotentiator with VLPs in mice (42, 50) and has been tested with soluble protein antigens in preclinical studies in humans (45, 46, 56). Although Freund’s adjuvant proved to be superior to QS-21 in our study, QS-21 may prove to be a better adjuvant than AlP. Studies with children and animals have both supported and refuted protection based on the development of N-Abs (1, 15, 16, 40, 49, 53, 57, 77, 78, 81, 82). Recently, we showed that mucosally administered 2/6-VLPs, parenterally administered 2/6-VLPs, or inactivated double-layered murine EDIM rotavirus with QS-21 induced protection from rotavirus challenge in mice (2, 33, 55, 58, 59), indicating that neither VP7 nor VP4 is
9244
CIARLET ET AL.
absolutely required for protection. However, parenteral immunization of rabbits with 2/6/7- or 2/6-VLPs with QS-21 resulted in levels of protection lower than those achieved with the 2/4/6/7-VLPs. Increases in the dose of either 2/6-VLPs or QS-21 failed to improve the protective efficacy. The presence of neutralizing VP4 antibodies in VLP- or SA11-vaccinated rabbits correlated (P , 0.001, r 5 0.55, Pearson’s correlation coefficient) with enhanced protection rates. Therefore, it appears that the presence of antibodies to VP4 plays an important role in protection of rabbits from infection. Although an absolute requirement for VP7 is not supported by our results with rabbits, VP7 may be needed to stabilize VP4 or to achieve proper expression of the epitopes on VP4 required for the induction of protective antibodies (26). It remains unclear whether N-Abs are required to induce high levels of protection; however, if N-Abs are needed for protection, then a limited number of VLP formulations may be sufficient to produce a broadly protective VLP subunit vaccine (27). A parenteral 2/4/6/7-VLP vaccine as opposed to an inactivated rotavirus vaccine will have the advantages of safety (i.e., it will be nonreplicating), no residual infectious virus or chemicals following inactivation, antigen stability (.6 years at 4°C), purity, and ability to alter protein content as needed (26, 27). Taken together, our results using VLPs and QS-21 in rabbits suggest that both VP4 and VP7 neutralization antigens may be required in a parenteral immunogen to induce protection from rotavirus challenge. Nevertheless, the correlation is not absolute because individual rabbits immunized with 2/6/7- or 2/6VLPs in Freund’s adjuvants achieved high levels of protection (87 to 100%). The widely variable protective efficacies obtained with the 2/6-VLPs in Freund’s adjuvants may be due to variation in the immunodominance of epitopes or immunological regulatory mechanisms of the outbred rabbit, which may be important factors influencing the outcome of the immune response. The dichotomy in results obtained in mice and rabbits may be influenced (i) by differences in the P types of the mouse (P[16]) and rabbit (P[14]) virus strains used for challenge relative to the immunizing P type (P[2]) or (ii) by inherent differences in antigen uptake, processing, and epitope recognition between rabbits and mice. Following oral infection of mice with a heterologous virus, the majority of the intestinal antibody-secreting cells were directed toward VP2 and VP6, and only about 1% of the total virus-specific response was directed to VP4 (71). It would be interesting to perform similar experiments in rabbits. Ultimately, clinical testing of VLPs in the gnotobiotic piglet rotavirus disease model and humans will be required to determine whether results from the rabbit or mouse model will be more predictive of protection against disease in children. Results from studies of children naturally infected with human rotaviruses or bovine-human virus reassortants have suggested that VP4 may be the immunodominant neutralization antigen in a homotypic response (9, 81), while data from another study of children suggested that VP7 is the immunodominant neutralization antigen (78). Currently, virus reassortants with different VP4-VP7 combinations are being applied to study immune responses to each neutralization antigen after primary rotavirus infections and reinfections in humans (16, 36). In our study, we were unable to measure the VP4-specific immune response to ALA, the challenge strain, but it will be of interest to determine if N-Abs to ALA VP4 (P[14]) were also induced in 2/4/6/7-VLP- or SA11-vaccinated rabbits. Alternatively, the use of VLPs with ALA VP4 and VP7 proteins may increase the protection of 2/4/6/7-VLPs in the rabbit model because of the effects of protein interactions on immunogenicity and epitope presentations (7, 8).
J. VIROL.
The difference in protective efficacy afforded by the different VLP formulations in QS-21 did not result from variable immunogenicity of the different VLPs since significant differences in total serum or intestinal IgG antibody titers were not observed. However, protection in VLP-vaccinated rabbits correlated with the presence of rotavirus-specific intestinal IgG (P 5 0.001, r 5 0.495; Pearson’s correlation coefficient). Our studies in mice also indicated that VLPs administered by parenteral or mucosal routes preferentially induced intestinal IgG with limited induction of intestinal IgA and that protection in mice immunized intranasally correlated with serum and intestinal IgG (51, 52). In contrast, our data for rabbits suggest that the presence of VP4-specific intestinal IgG may be required to induce high levels of protection since protective efficacy of 2/6/7- or 2/6-VLPs in QS-21 was poor. Studies of the immune responses to individual proteins in the intestine prior to virus challenge may be useful to determine if the differences in protective efficacy correlate with the presence of protein-specific intestinal antibodies in the rabbit (41). However, new sensitive assays that can detect native epitopes need to be developed for such studies. Although intestinal antibody responses are expected to play a major role in protection, recent data in gene-knockout mice indicate that CD81 T cells can mediate total short-term and partial long-term protection from rotavirus challenge (34, 35, 59). We do not yet know if VLPs can induce CD81 T cells; therefore, we cannot exclude the possibility that CD81 T cells also play a role in protection following immunization of rabbits with VLPs. Protection studies in rabbits and mice suggest that VLPs may provide a safe and efficacious alternative to live oral rotavirus vaccines. A parenteral vaccine might overcome two of the problems observed with oral live rotavirus vaccines: (i) interference by maternal antibodies and (ii) poor replication of the vaccine virus. Because a parenteral vaccine would not require replication of the rotavirus vaccine strains, formulation of the vaccine might be easier since differences in immunogenicity based on growth properties of individual viruses would not come into play. However, the need for multivalent parenteral vaccines to induce broad immunity against the various human rotavirus serotypes will need to be examined. The results of our studies support the further development of rotavirus subunit vaccines as well as evaluation of combined parenteral-oral vaccination regimens. ACKNOWLEDGMENTS We thank Reginald Semiens for excellent work in the maintenance of rabbits, Robert L. Atmar for helpful discussions, WLVP for provision of QS-21 and AlP, Carl Q.-Y. Zeng and Sharon Krater for insect and mammalian cell culture work, respectively, and Robert Carman for supplying the C. spiroforme toxoid. The technical assistance and help in the maintenance of rabbits by Silvia Krasuk, Ramo ´n Simo ´n, Suzie Groner, Rhonda Carter, and Sheryl Henderson is greatly appreciated. This work was supported by Public Health Service grants AI 24998 and AI 16687 from the National Institute of Allergy and Infectious Disease. REFERENCES 1. Andrew, M. E., D. B. Boyle, B. E. H. Coupar, D. Reddy, A. R. Bellamy, and G. W. Both. 1992. Vaccinia-rotavirus VP7 recombinants protect mice against rotavirus-induced diarrhea. Vaccine 10:185–191. 2. Bertolotti-Ciarlet, A., S. E. Crawford, M. E. Conner, and M. K. Estes. Unpublished data. 3. Borriello, S. P., and R. J. Carman. 1983. Association of iota-like toxin and Clostridium spiroforme with both spontaneous and antibiotic-associated diarrhea and colitis in rabbits. J. Clin. Microbiol. 17:414–418. 4. Burns, J. W., H. B. Greenberg, R. D. Shaw, and M. K. Estes. 1988. Func-
VOL. 72, 1998
5. 6. 7. 8. 9. 10. 11.
12. 13.
14. 15.
16. 17. 18. 19. 20. 21.
22.
23.
24. 25. 26. 27. 28. 29. 30.
tional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11. J. Virol. 62:2164–2172. Burns, J. W., D. Chen, M. K. Estes, and R. F. Ramig. 1989. Biological and immunological characterization of a simian rotavirus SA11 variant with an altered genome segment 4. Virology 169:427–435. Burr, D. H., D. T. Kerner, C. S. Blanco, A. L. Bourgeois, and R. Wistar, Jr. 1987. Gastric lavage: a simple method to obtain IgA-rich intestinal secretions from the rabbit. J. Immunol. Methods 99:277–281. Chen, D., J. W. Burns, M. K. Estes, and R. F. Ramig. 1989. Phenotypes of rotavirus reassortants depend upon the recipient genetic background. Proc. Natl. Acad. Sci. USA 86:3743–3747. Chen, D., M. K. Estes, and R. F. Ramig. 1992. Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope. J. Virol. 66:432–439. Christy, C., P. Offit, F. H. Clark, and J. Treanor. 1993. Evaluation of a bovine-human rotavirus reassortant vaccine in infants. J. Infect. Dis. 168: 1598–1599. Ciarlet, M., M. Hidalgo, M. Gorziglia, and F. Liprandi. 1994. Characterization of neutralization epitopes on the VP7 surface protein of serotype G11 porcine rotaviruses. J. Gen. Virol. 75:1867–1873. Ciarlet, M., M. K. Estes, and M. E. Conner. 1997. Comparative amino acid sequence analysis of the outer capsid protein VP4 from four lapine rotavirus strains reveals identity with genotype P[14] human rotavirus. Arch. Virol. 142:1059–1069. Ciarlet, M., Y. Hoshino, and F. Liprandi. 1997. Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum? J. Virol. 71:8213–8220. Ciarlet, M., M. K. Estes, C. Barone, R. F. Ramig, and M. E. Conner. 1998. Analysis of host range restriction determinants in the rabbit model: comparison of homologous and heterologous rotavirus infections. J. Virol. 72:2341– 2351. 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 histophatological lesions, is age-restricted in rabbits. Virology, in press. Clark, H. F., F. E. Borian, L. M. Bell, K. Modesto, V. Gouvea, and S. A. Plotkin. 1988. Protective effect of WC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus season. J. Infect. Dis. 158:570–587. Clark, H. F., P. A. Offit, R. W. Ellis, D. Krah, A. R. Shaw, J. J. Eiden, M. Pichichero, and J. J. Treanor. 1996. WC3 reassortant vaccines in children. Arch. Virol. Suppl. 12:187–198. Conner, M. E., M. K. Estes, and D. Y. Graham. 1988. Rabbit model of rotavirus infection. J. Virol. 62:1625–1633. Conner, M. E., M. A. Gilger, M. K. Estes, and D. Y. Graham. 1991. Serologic and mucosal immune response to rotavirus infection in the rabbit model. J. Virol. 65:2562–2571. Conner, M. E., S. E. Crawford, C. Barone, and M. K. Estes. 1993. Rotavirus vaccine administered parenterally induces protective immunity. J. Virol. 67: 6633–6641. Conner, M. E., D. O. Matson, and M. K. Estes. 1994. Rotavirus vaccines and vaccination potential. Curr. Top. Microbiol. Immunol. 185:286–337. Conner, M. E., S. E. Crawford, C. Barone, and M. K. Estes. 1994. Rotavirus or virus-like particles administered parenterally induce active immunity, p. 351–355. In F. Brown, R. Chanock, H. Ginsberg, and E. Norrby (eds.), Vaccines 94. Proceedings of Modern Approaches to New Vaccines including Prevention of AIDS, Cold Spring Harbor 1993. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Conner, M. E., and R. F. Ramig. 1996. Enteric diseases, p. 713–743. In N. Nathanson, R. Ahmed, F. Gonza´lez-Scarano, D. E. Griffin, K. V. Homes, F. A. Murphy, and H. L. Robinson, (ed.), Viral pathogenesis. LippincottRaven Publishers, Philadelphia, Pa. 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. E. Crawford, and M. K. Estes. 1996. Virus-like particles as a rotavirus subunit vaccine. J. Infect. Dis. 174 Suppl. 1:S88–S92. Conner, M. E., S. E. Crawford, C. Barone, C. M. O’Neal, Y.-J. Zhou, F. Ferna ´ndez, A. Parwani, L. J. Saif, J. Cohen, and M. K. Estes. 1996. Rotavirus subunit vaccines. Arch. Virol. Suppl. 12:199–206. Conner, M. E., D. Y. Graham, and M. K. Estes. 1997. Determination of the duration of a primary immune response and the ID50 of ALA rabbit rotavirus in rabbits. Arch. Virol. 142:2281–2294. Crawford, S. E., M. Labbe´, J. Cohen, M. H. Burroghs, Y.-J. Zhou, and M. K. Estes. 1994. Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J. Virol. 68:5945–5952. Crawford, S. E., M. K. Estes, M. Ciarlet, C. Barone, C. M. O’Neal, J. Cohen, and M. E. Conner. Induction of heterotypic neutralizing antibodies by rotavirus-like particles. Submitted for publication. Crawford, S. E., and M. K. Estes. Unpublished results. Estes, M. K., D. Y. Graham, E. M. Smith, and C. P. Gerba. 1979. Rotavirus stability and inactivation. J. Gen. Virol. 43:403–409. Estes, M. K., D. Y. Graham, R. F. Ramig, and B. L. Ericson. 1982. Heter-
ROTAVIRUS RECOMBINANT SUBUNIT VACCINE
31.
32. 33.
34. 35. 36. 37. 38. 39. 40.
41. 42. 43. 44.
45. 46.
47. 48.
49. 50.
51. 52. 53.
54. 55.
9245
ogeneity in the structural glycoprotein (VP7) of simian rotavirus SA11. Virology 122:8–14. Estes, M. K., S. E. Crawford, M. E. Pen ˜ aranda, B. L. Petrie, J. W. Burns, W.-K. Chan, B. L. Ericson, G. E. Smith, and M. D. Summers. 1987. Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system. J. Virol. 61:1488–1494. Estes, M. K. 1996. Rotaviruses and their replication, p. 1625–1655. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Lippincott-Raven Publishers, Philadelphia, Pa. Feng, N., M. E. Conner, P. T. Vo, S. E. Crawford, M. K. Estes, and H. B. Greenberg. 1997. Both CD81 T cells and antibody are involved in protection against rotavirus infection after systemic immunization with rotavirus-likeparticles or inactivated rotavirus, abstr. W20-4, p. 129. In Abstracts of the 16th Annual Meeting of the American Society for Virology. Franco, M. A., and H. B. Greenberg. 1995. Role of B cells and cytotoxic T lymphocytes in clearance of and immunity to rotavirus infection in mice. J. Virol. 69:7800–7806. Franco, M. A., C. Tin, and H. B. Greenberg. 1997. CD81 T cells can mediate complete short-term and partial long-term protection against reinfection by rotavirus. J. Virol. 71:4165–4170. Gorrell, R. J., and R. F. Bishop. 1997. Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection. Clin. Diagn. Lab. Immunol. 4:509–514. Groene, W. S., and R. D. Shaw. 1992. Psoralen preparation of antigenically intact noninfectious rotavirus particles. J. Virol. Methods 38:93–102. Hambraeus, B. A. M., L. E. J. Hambraeus, and G. Wadell. 1989. Animal model of rotavirus infection in rabbits—protection obtained without shedding of viral antigen. Arch. Virol. 107:237–251. Ho, M.-S., R. I. Glass, P. F. Pinsky, N. Young-Okoh, W. M. Sappenfield, J. W. Buehler, N. Gunter, and L. J. Anderson. 1988. Diarrheal deaths in American children. Are they preventable? JAMA 260:3281–3285. Hoshino, Y., L. J. Saif, M. M. Sereno, R. M. Chanock, and A. Z. Kapikian. 1988. 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. 62:744–748. Ishida, S. I., N. Feng, J. M. Gilbert, B. Tang, and H. B. Greenberg. 1997. Immune responses to individual rotavirus proteins following heterologous rotavirus infection in mice. J. Infect. Dis. 175:1317–1323. Jiang, B., V. Barniak, C. Barone, C. M. O’Neal, R. Smith, H. P. Madore, M. K. Estes, and M. E. Conner. Rotavirus G1 VLPs induce heterotypic protection in a murine model. Vaccine, in press. Joensuu, J., E. Koskenniemi, X.-L. Pang, and T. Vesikari. 1997. Randomised placebo-controlled trial of rhesus-human reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis. Lancet 350:1205–1209. Kapikian, A. Z., Y. Hoshino, R. M. Chanock, and I. Pe´rez-Schael. 1996. Jennerian and modified Jennerian approach to vaccination against rotavirus diarrhea using a quadrivalent rhesus rotavirus (RRV) and human-RRV reassortant vaccine. Arch. Virol. Suppl. 12:163–175. Kensil, C. R., U. Patel, M. Lennick, and D. Marciani. 1991. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria molina cortex. J. Immunol. 146:431–437. Kensil, C. R., J.-Y. Wan, and S. Soltysik. 1995. Structural and immunological characterization of the vaccine adjuvant QS-21, p. 525–541. In M. F. Powell and M. J. Newman (ed.), Vaccine design: the subunit and adjuvant approach. Plenum Press, New York, N.Y. Labbe´, M., A. Charpilienne, S. E. Crawford, M. K. Estes, and J. Cohen. 1991. Expression of rotavirus VP2 produces empty corelike particles. J. Virol. 65: 2946–2952. LeBaron, C. W., J. Lew, R. I. Glass, J. M. Weber, and G. M. Ruiz-Palacios. 1990. Annual rotavirus epidemic patterns in North America: results of a five-year retrospective survey of 88 centers in Canada, Mexico, and the United States. JAMA 264:983–988. Mackow, E. R., P. T. Vo, R. Broome, D. Bass, and H. B. Greenberg. 1990. Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge. J. Virol. 64:1698–1703. Madore, H. P., M. K. Estes, C. D. Zarley, B. Hu, S. Parsons, D. Drabinski, S. Greiner, R. Smith, B. Jiang, B. Corsaro, V. Barniak, S. Crawford, and M. E. Conner. Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine. Vaccine, in press. Malherbe, H. H., and M. Strickland-Cholmley. 1967. Simian virus SA11 and the related O agent. Arch. Gesamte Virusforsch. 22:235–245. Matson, D. O., and M. K. Estes. 1990. Impact of rotavirus infection at a large pediatric hospital. J. Infect. Dis. 162:598–604. Matsui, S. M., P. A. Offit, P. T. Vo, E. R. Mackow, D. A. Benfield, R. D. Shaw, L. Padilla-Noriega, and H. B. Greenberg. 1989. Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to the heterotypic neutralization domain of VP7 and the VP8 fragment of VP4. J. Clin. Microbiol. 27:780–782. McNeal, M. M., K. S. Barone, M. N. Rae, and R. L. Ward. 1995. Effector functions of antibody and CD81 cells in resolution of rotavirus infection and protection against reinfection in mice. Virology 214:387–397. McNeal, M. M., M. N. Rae, M. E. Conner, and R. L. Ward. 1998. Stimulation
9246
56.
57. 58. 59.
60. 61.
62.
63. 64. 65. 66. 67. 68. 69.
CIARLET ET AL.
of local immunity and protection in mice by intramuscular immunization with triple- or double-shelled rotavirus particles and QS-21. Virology 243: 158–166. Newman, M. J., J.-Y. Wu, B. H. Gardner, C. A. Anderson, C. R. Kensil, J. Recchia, R. T. Coughlin, and M. F. Powell. 1997. Induction of cross-reactive cytotoxic T-lymphocyte responses specific for HIV-1 gp120 using saponin adjuvant (QS-21) supplemented subunit vaccine formulations. Vaccine 15: 1001–1007. Offit, P. A., R. D. Shaw, and H. B. Greenberg. 1986. Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins VP3 and VP7. J. Virol. 58:700–703. O’Neal, C. M., S. E. Crawford, M. K. Estes, and M. E. Conner. 1997. Rotavirus virus-like particles administered mucosally induce protective immunity. J. Virol. 71:8707–8717. O’Neal, C. M., J. D. Clemments, M. K. Estes, and M. E. Conner. 1997. 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. 72:3390–3393. O’Neal, C. M., R. Carman, and M. E. Conner. Unpublished data. Pe´rez-Schael, I., M. J. Guntin ˜ as, M. Pe´rez, V. Pagone, A. M. Rojas, R. Gonza ´lez, W. Cunto, Y. Hoshino, and A. Z. Kapikian. 1997. Efficacy of the rhesus rotavirus-based quadrivalent vaccine in infants and young children in Venezuela. N. Engl. J. Med. 337:1181–1187. Poruchynsky, M. S., C. Tyndall, G. W. Both, F. Sato, A. R. Bellamy, and P. H. Atkinson. 1985. Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein. J. Cell Biol. 101:2199–2209. Prasad, B. V. V., G. J. Wang, J. P. M. Clerx, and W. Chiu. 1988. Threedimensional structure of rotavirus. Mol. Biol. 199:269–275. Prasad, B. V. V., J. W. Burns, E. Marietta, M. K. Estes, and W. Chiu. 1990. Localization of VP4 neutralization sites in rotavirus by 3D cryo-electron microscopy. Nature 343:476–479. Prasad, B. V. V., R. Rothangel, C. Q.-Y. Zeng, J. Jakana, J. A. Lawton, and M. K. Estes. 1996. Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus. Nature 382:471–473. Ramig, R. F., and K. L. Galle. 1990. Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2). J. Virol. 64:1044– 1049. Ramig, R. F. Unpublished data. Ramig, R. F., and M. E. Conner. Unpublished data. Santosham, M., L. H. Moulton, R. Reid, J. Croll, R. Weatherholt, R. Ward, J. Forro, M. Mack, G. Brenneman, and B. L. Davidson. 1997. Efficacy and
J. VIROL.
70. 71. 72. 73. 74. 75. 76. 77.
78.
79. 80. 81.
82.
safety of high-dose rhesus-human reassortant rotavirus vaccine in Native American populations. J. Pediatr. 131:632–638. Shaw, A. L., R. Rothangel, D. Chen, R. F. Ramig, W. Chiu, and B. V. V. Prasad. 1993. Three-dimensional visualization of the rotavirus hemagglutinin structure. Cell 74:693–701. Shaw, R. D., W. S. Groene, E. R. Mackow, A. A. Merchant, and E. H. Cheng. 1991. VP4-specific intestinal response to rotavirus in a murine model of heterotypic infection. J. Virol. 65:3052–3059. Tanaka, T. N., M. E. Conner, D. Y. Graham, and M. K. Estes. 1988. Molecular characterization of three rabbit rotavirus strains. Arch. Virol. 98:253– 265. Thouless, M. E., R. DiGiacomo, B. J. Deeb, and H. Howard. 1988. Pathogenicity of rotavirus in rabbits. J. Clin. Microbiol. 26:943–947. Vesikari, T. 1993. Clinical trials of live oral rotavirus vaccines: the Finnish experience. Vaccine 11:255–261. Vesikari, T. 1996. Trials of oral bovine and rhesus rotavirus vaccines in Finland: a historical account and present status. Arch. Virol. Suppl. 12:177– 186. Vesikari, T. 1997. Rotavirus vaccines against diarrhoeal disease. Lancet 350: 1538–1541. Ward, R. L., D. I. Bernstein, R. Shukla, E. C. Young, J. R. Sherwood, M. M. McNeal, M. C. Walker, and G. M. Schiff. 1989. Effects of antibody to rotavirus on protection of adults challenged with a human rotavirus. J. Infect. Dis. 159:79–88. Ward, R. L., D. R. Knowlton, H. B. Greenberg, G. M. Schiff, and D. I. Bernstein. 1990. Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus. J. Virol. 64:2687– 2691. Ward, R. L., M. M. McNeal, and J. F. Sheridan. 1990. Development of an adult mouse model for studies on protection against rotavirus. J. Virol. 64: 5070–5075. Ward, R. L., M. M. McNeal, and J. F. Sheridan. 1992. Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody. Virology 188:57–66. Ward, R. L., M. M. McNeal, D. S. Sander, H. B. Greenberg, and D. I. Bernstein. 1993. Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children. J. Virol. 67:464– 468. Yuan, L., L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif. 1996. Systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J. Virol. 70: 3075–3083.
