Investigation of Novel Prophylactics Against Human Rotavirus Using Gnotobiotic Pig Models

Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE), which causes severe dehydrating diarrhea in children under the age of five and results in up to 215,000 deaths worldwide each year. There are two live oral attenuated vaccines licensed for use in the United States that are highly effective in high-income countries but much less so in low-and middle-income countries (LMICs). Several factors contributing to decreased efficacy in these areas include chronic malnutrition, gut dysbiosis, and concurrent viral infection. Along with this, currently used vaccines require constant cold-chain storage to maintain vaccine stability, and those resources can be scarce in LMICs. These areas continue to maintain a high burden of HRV morbidity and mortality, and more efficacious vaccines are needed. The gnotobiotic (Gn) pig model of HRV infection and diarrhea has long been used in the evaluation of novel HRV vaccines due to Gn pigs' susceptibility to HRV infection, development of clinical signs, histopathological changes in the intestine, and the infection kinetics that mimic those seen in human infants. The first project in this dissertation used the Gn pig model to evaluate a thermostable live oral attenuated vaccine administered as a dissolvable film. Two doses of the tetravalent dissolvable film vaccine conferred significant protection from virus shedding by delaying its onset and reducing peak titers in feces. It also significantly delayed the onset of diarrhea and reduced the duration and area under the curve (AUC) of diarrhea. The dissolvable film was highly immunogenic, inducing high titers of serum virus neutralizing (VN) antibodies specific to each of the four G-types included in the vaccine formulation, HRV-specific serum IgA and IgG, and intestinal IgA. These data confirm the thermostable platform as a useful alternative to liquid vaccines that require cold-chain. The second project evaluated three mRNA-based nonreplicating vaccine candidates in the Gn pig model. All three mRNA candidates encoded a universal CD4+ T cell epitope, P2, derived from tetanus toxoid, fused with the encoded VP8* from P[4], P[6], and P[8] HRVs. Two candidates also encoded for a lumazine synthase (LS) domain fused with the P2-VP8*. A dose response study of the LS-P2-VP8* candidates was conducted simultaneously. Significant protection against virus shedding was induced by all three candidates, with LS-P2-VP8* candidates inducing significantly higher VP8*-specific serum IgG. All three candidates induced significantly higher numbers of P[8]-VP8*-specific IgG antibody-secreting cells (ASCs) and IFN-γ-producing T cells in the ileum, spleen and blood. These data provide guidance for further development of the relatively new mRNA-based technology for use in HRV vaccine development. In the final study of this dissertation, we used the Gn pig model of both P[8] and P[6] HRV infection to evaluate a cocktail nanoparticle-based HRV vaccine. This vaccine was made up of an S60 nanoparticle, self-assembled from the S domain of the human norovirus capsid protein. The exposed C-termini on the S60 nanoparticle were utilized as an antigen display platform, where VP8* from P[4], P[6] and P[8] HRVs was fused. This vaccine was tested as both a two-dose intramuscular (IM) regimen, or as an IM booster preceded by an oral priming immunization with commercial monovalent Rotarix®. Pigs were challenged with either P[6] or P[8] HRV to evaluate cross-protection of the nanoparticle vaccine. Both regimens were highly immunogenic, inducing high titers of serum VN, IgG and IgA antibodies. Furthermore, the prime-boost regimen conferred significant protection against virus shedding in P[8] HRV-challenged pigs as evidenced by the shortened duration of fecal virus shedding. There was also significant protection in P[6] HRV-challenged pigs vaccinated with the prime-boost regimen, as evidenced by the shortened duration, reduced mean peak titer and AUC of virus shedding. Prime-boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG ASCs in the spleen post-challenge. Prime-boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6] and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. Oral priming followed by parenteral boosting appears to be a promising vaccination strategy for HRV and these data warrant further investigation into this regimen. Through these studies, we improved our understanding of the effect of different vaccination routes and formulations in the effectiveness of conferring protection against an enteric virus. The knowledge will facilitate the development of more effective vaccination strategies against HRV, the leading cause of infantile diarrhea in LMICs, as well as other enteric viruses.