The failure of rotavirus vaccines in infants and animals underscores a need for more effective vaccination strategies for human rotavirus. An improved understanding of mucosal immune responses to rotaviral antigens and identification of correlates or protective immunity to human rotavirus should aid in accomplishing this goal. The gnotobiotic (Gn) pig is an ideal animal model for this research due to its extended susceptibility (up to 6 weeks) to infection and disease with human rotaviruses, a lack of exposure or antibodies to undefined rotaviruses and its similarity to humans in gastrointestinal physiology and immunity. Accordingly, we will define specific antibody immune responses to the two outer capsid proteins of rotavirus (VP4 and VP7) that induce neutralizing antibodies and assess their correlation with recovery from disease and resistance to viral challenge in Gn pigs. In the first phase of the project, Gn pigs will be orally inoculated with virulent rotaviruses to mimic natural infection, then challenged with homotypic or heterotypic strains to evaluate protection. Mucosal antibody responses to specific rotavirus proteins, antigens and epitopes will be determined to identify potential correlates of immunity. Emphasis will be on neutralization, isotype, ELISA and ELISPOT, competition ELISA and Western blotting assays to detect and quantitate antibodies to rotavirus. We will then evaluate oral rotavirus candidate vaccines (reassortants, attenuated and killed viruses, recombinant proteins) for their ability to stimulate protective immune responses similar to those induced by natural infection. The data from these experiments will provide a basis upon which to evaluate novel approaches to mucosal immunity, including oral delivery systems and adjuvants to enhance the efficacy of rotavirus candidate vaccines. The recombinant (r) rotavirus proteins to be evaluated in this proposal require new approaches to realize their potential as subunit vaccines including preserving their antigenic integrity and enhancing their immunogenicity and delivery to mucosal sites. Intact virus and r proteins will be enteric-coated or microencapsulated and administered with/without adjuvant (avridine) to determine the effectiveness of these approaches to enhance immune responses by controlled release of antigen in the gut and by targeting of antigen to Peyer's patches. An increased understanding of intestinal immune responses is critical for the development of oral vaccines, including r vaccines designed to invoke the common mucosal immune system to induce protection against enteric and other mucosal pathogens.
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