An Oral versus Intranasal Prime/Boost Regimen Using Attenuated Human Rotavirus or VP2 and VP6 Virus-Like Particles with Immunostimulating Complexes Influences Protection and Antibody-Secreting Cell Responses to Rotavirus in a Neonatal Gnotobiotic Pig Model

We determined the impact of mucosal prime/boost regimens and vaccine type (attenuated Wa human rotavirus [AttHRV] or nonreplicating Wa 2/6 rotavirus-like particles [VLP]) on protection and antibody-secreting cell (ASC) responses to HRV in a neonatal gnotobiotic pig disease model. Comparisons of delivery routes for AttHRV and evaluation of nonreplicating VLP vaccines are important as alternative vaccine approaches to overcome risks associated with live oral vaccines. Groups of neonatal gnotobiotic pigs were vaccinated using combinations of oral (PO) and intranasal (IN) inoculation routes as follows: (i) 3 oral doses of AttHRV (AttHRV3×PO); (ii) AttHRV3×IN; (iii) AttHRVPO, then 2/6VLP2×IN; (iv) AttHRVIN, then 2/6VLP2×IN; and (v) mock-inoculated controls. Subsets of pigs from each group were challenged with virulent Wa HRV [P1A(8) G1] (4 weeks post-primary inoculation) to assess protection. The AttHRVPO+2/6VLP2×IN pigs had the highest protection rates against virus shedding and diarrhea (71% each); however, these rates did not differ statistically among the vaccine groups, except for the AttHRVIN+2/6VLPIN group, which had a significantly lower protection rate (17%) against diarrhea. The isotype, magnitude, and tissue distribution of ASCs were analyzed by enzyme-linked immunospot assay. The highest mean numbers of virus-specific IgG and IgA ASCs were observed pre- and postchallenge in both intestinal and systemic lymphoid tissues of the AttHRVPO+2/6VLPIN group. Thus, the AttHRVPO+2/6VLPIN vaccine regimen using immunostimulating complexes (ISCOM) and multiple mucosal inductive sites, followed by AttHRV3×PO or IN regimens, were the most effective vaccine regimens, suggesting that either AttHRVPO+2/6VLPIN or AttHRV3×IN may be an alternative approach to AttHRV3×PO for inducing protective immunity against rotavirus diarrhea.Safety is a major concern in the use of live vaccines due to potential adverse effects. However, immune responses at mucosal sites are usually most effectively induced in naïve hosts by the administration of living microorganisms. In contrast, nonreplicating vaccines administered orally (PO) require an effective mucosal adjuvant and delivery system to enhance the immunogenicity of the vaccines and to avoid oral tolerance.Rotavirus is the most common cause of infantile gastroenteritis worldwide. Each year, rotavirus causes approximately 25 million clinic visits, 2 million hospitalizations, and 500,000 deaths in children <5 years of age (29). Although two new live attenuated rotavirus (RV) vaccines are licensed, there is an urgent need to develop rotavirus vaccines that are more efficacious in developing countries than the existing vaccines (17, 37). The association of an increased risk of intussusception with the rhesus × human rotavirus reassortant vaccine RotaShield prompted concerns related to possible side effects of live rotavirus vaccines, including excessive virus shedding, fever, diarrhea, vomiting, and irritability, prompting the development of alternative vaccine strategies. Virus-like particles (VLP) composed of rotavirus inner capsid proteins VP2 and VP6 (2/6VLPs) constructed by coexpressing rotavirus gene 2 and gene 6 in a baculovirus expression system using Spodoptera frugiperda (Sf9) insect cells are one strategy (11, 15, 39). However, these 2/6VLP vaccines do not induce rotavirus-neutralizing antibodies. In our previous studies, a vaccine using intranasal (IN) 2/6VLP (2/6VLPIN) (RF VP2, Wa VP6) with mutant Escherichia coli heat-labile toxin (mLT) adjuvant was immunogenic but not protective in gnotobiotic pigs (39). Also, three oral doses of a vaccine of 2/6VLP (2/6VLP3×PO) with immunostimulating complexes (ISCOM) were less immunogenic than three doses of 2/6VLPIN vaccine, but both failed to induce protection against rotavirus diarrhea or shedding (18). However, partial protection against diarrhea and virus shedding was induced when 2/6VLPIN+mLT or 2/6VLPPO+ISCOM was used as a booster vaccine in pigs that were previously primed orally with live attenuated Wa HRV (AttHRVPO+2/6VLPIN+mLT or AttHRVPO+2/6VLPPO+ISCOM) (18, 40). The efficacies of these two vaccine regimens against rotavirus diarrhea (44% and 50%, respectively) were similar or slightly lower than that induced by three oral doses of live attenuated Wa HRV vaccine in pigs (50 to 67%) (18, 40, 41, 43). These results suggest that the prime/boost vaccine regimens may be a more effective approach than multiple doses of either live or VLP vaccines alone.Respiratory symptoms and rotavirus shedding in nasopharyngeal secretions of children have been reported (26, 31), and we previously demonstrated shedding of AttHRV in the respiratory tract (2) of gnotobiotic pigs. Because lymphocytes sensitized in the nasal lymphoid tissue (NALT) can relocate to distant effector sites through the common mucosal immune system (6), the respiratory tract should be explored further as a possible route to improve rotavirus vaccine efficacy. In this study, we first evaluated the dose response to 2/6VLPIN boosting after priming with AttHRVPO. We then assessed the effect of PO versus IN priming with AttHRV using the optimized 2/6VLPIN booster dose. We further compared the impact of IN versus PO priming and boosting using AttHRV alone. Such vaccine regimens, although requiring priming with live AttHRV, may at least reduce the risk associated with live virus boosters or, by using IN delivery, avoid side effects or interference (i.e., intestinal parasites, maternal antibodies, etc.) more prominent in infants in developing countries when live oral rotavirus vaccines are used. Accordingly, the use of 2/6VLP boosters given IN might overcome some of the suppressive effects of maternal antibodies on live oral RV vaccines (19, 25). Bertolotti-Ciarlet et al. (4) showed that oral administration of homologous or heterologous 2/6VLPs to CD-1 mice with or without adjuvant induced low protection rates against rotavirus challenge (10 and 39%), whereas intranasal administration induced higher protection rates (85 and 84%). Their findings together with the findings of others (5) suggest that the harsh environment of the gastrointestinal (GI) tract (3, 34), including the low pH and presence of digestive enzymes which influence the degradation of protein antigens, may account for the differences observed between immunizations using these two routes. Thus, less degradation of VLPs occurs when they are given IN compared to when they are given PO, which may permit lowering of the VLP dosage. The use of an effective and age-appropriate mucosal adjuvant such as ISCOM may also increase the efficacy of the nonreplicating 2/6VLP rotavirus vaccine. Intrarectal immunization was also used to avoid GI degradation. In adult mice, intrarectal immunization using 2/6VLP induced protection against rotavirus shedding and higher intestinal immune responses (1). Similarly, intrarectal inoculation of 8-2/6/7VLP induced complete protection against rotavirus shedding in the same model when administered with LT or cholera toxin (CT) adjuvants (30).The gnotobiotic pig is the only animal model susceptible to HRV diarrhea for at least the first 8 weeks of age, the time necessary to assess protective immunity against disease upon challenge (44), whereas other animal models are susceptible to rotavirus diarrhea only up to 14 to 21 days of age (9, 10). Studies of our selected candidate rotavirus vaccines and new vaccine strategies in neonatal gnotobiotic pigs that mimic immune responses of infants (33) should generate data potentially applicable for the development of similar rotavirus vaccines for infants. Our findings should further improve our understanding of effective prime/boost strategies comparing live versus nonreplicating vaccines and PO versus IN routes to induce intestinal immunity in neonates.(Some data used in Table 16].)

TABLE 2.

Virus shedding and diarrhea in gnotobiotic pigs after challenge with virulent Wa HRV

Vaccine regimen	ne	Virus shedding			Diarrhea			Protection rateb (%) against:
		% shedf	Mean days to onset of sheddinga,g	Mean duration in daysa,g	% with diarrheaf	Mean duration in daysc,g	Mean cumulative scored,g	Shedding	Diarrhea
AttHRV3×PO	9	33 B	2.3 B	0.7 B	56 B	1.4 B	7.4 BC	67	44
AttHRV3×IN	6	33 B	2.0 B	0.3 C	50 B	1.0 B	6.0 C	67	50
AttHRVPO-2/6VLPIN	7	29 B	2.0 B	0.4 C	29 B	0.7 C	6.4 C	71	71
AttHRVIN-2/6VLPIN	6	50 B	2.3 B	0.7 B	83 A	1.2 B	7.5 BC	50	17
Controls	12	100 A	1.6 A	2.5 A	100 A	2.8 A	9.4 AB	0	0

Open in a separate window^aDetermined by ELISA and cell culture immunofluorescence infectivity assay.^bProtection rate = [1 − (percentage of vaccinated pigs in each group with diarrhea/percentage of control pigs with diarrhea)] × 100. Protection data were partially presented previously by Gonzalez et al. (16). Gonzalez et al. reported the protection rates against diarrhea and virus shedding in pigs primed with AttHRVPO and boosted with 25 μg or 250 μg of 2/6VLP-ISCOM IN and their correlation with virus neutralizing antibody titers in serum and serum and fecal antibody isotypes and titers.^cDuration of diarrhea determined by number of days with fecal scores greater than or equal to 2. Feces were scored as follows: 0, normal; 1, pasty; 2, semiliquid; 3, liquid.^dMean cumulative score = (sum of fecal consistency scores for 6 days postchallenge)/n.^en, number of pigs/group.^fProportions in the same column followed by different letters differ significantly (Fisher''s exact test). A, statistically different from B at P < 0.05.^gMeans in the same column followed by different letters differ significantly (One way ANOVA). A, statistically different from B and C; B, statistically different from C at P < 0.05.