Rotavirus (RV) is the major cause of severe gastroenteritis in infants and young children worldwide. The failure of rotavirus vaccines to consistently induce protection against disease in infants and animals underscores a need for more effective vaccines. Our goal is an improved understanding of disease pathogenesis and mucosal immune responses to human rotaviruses (HRV) and identification of correlates of protective immunity in the gnotobiotic (Gn) pig model. The Gn pig is an ideal animal model because of its susceptibility to infection and disease (including intestinal lesions) with HRV, a lack of previous exposure or maternal antibodies to RV and its similarity to human infants in size, milk diet, gastrointestinal physiology and ontogeny of mucosal immune responses. To study disease pathogenesis, the putative role of the RV nonstructural protein, NSP4 as a viral enterotoxin and virulence determinant or target for induction of protective immune responses will be investigated in Gn pigs inoculated with recombinant NSP4 and NSP 4 peptides from virulent and attenuated pairs of RV. Expression of NSP4 and induction of immune responses to NSP4 will be further analyzed in Gn pigs infected with the respective virulent and attenuated pairs of HRV. In previous studies, the magnitude of the intestinal igA antibody secreting cell and lymphoproliferative immune responses coincided with the degree of RV replication and diarrhea induction, and were positively correlated with the level of protection induced. Hence we will further explore the role that viral replication and virulence (lesions and/or diarrhea) play in induction of mucosal immunity and protection against HRV. To assess which viral proteins are targets of protective immunity, B cell responses to RV and to RV structural and nonstructural proteins will be measured by virus neutralization, ELISA and ELISPOT assays and T cell responses will be examined by lymphoproliferative assays and analysis of cytokine profiles (RT- PCR,ELISA,ELISPOT). The type, magnitude, kinetics and correlation with protection of these mucosal immune responses will be compared in Gn pigs inoculated orally with virulent HRV (induces diarrhea and lesions and mimics natural infection) or candidate HRV vaccines including: attenuated HRV (no diarrhea or lesions); or the viral subunits, NSP4 (from virulent RV, induces diarrhea; attenuated RV, no diarrhea) and rotavirus-like particles (VLP) (no diarrhea or lesions), and challenged with virulent homotypic HRV. Finally we will examine if selected immunoenhancers (microencapsulation, mucosal adjuvants) can augment the mucosal immune responses to the attenuated and subunit HRV vaccines and increase vaccine efficacy in the Gn pig model.
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