Rotaviruses are the most important cause of severe acute diarrhea in children worldwide, causing up to 1 million deaths per year. Current vaccines have not been effective in controlling the disease, and the mechanisms involved in generation of protective immunity are poorly understood. We propose to characterize immune responses and mechanisms involved in immunity by use of plasmid DNAs (DNA vaccines) encoding for specific rotavirus proteins. Immune responses will be examined in an adult mouse model with DNA vaccines encoding for murine rotavirus proteins, and DNA vaccines encoding for human rotavirus proteins will be examined in gnotobiotic pigs, the only animal model which can be clinically infected with human rotaviruses. Unlike other subunit vaccines, DNA vaccines allow for proteins to be endogenously expressed in cells in association with class I MHC antigens, which is necessary for eliciting CD8+ cytotoxic T cell responses. In preliminary experiments we have found that DNA vaccines encoding for murine rotaviral proteins VP4 and VP7 induced rotavirus specific neutralizing and ELISA antibodies, and protected mice against rotavirus challenge. A DNA vaccine encoding for VP6, an antigenically conserved rotavirus protein, elicited rotavirus specific ELISA antibodies but not neutralizing antibodies. However, protection was also provided against rotavirus challenge. The mechanism of this protection will be investigated in mice that are deficient in B cells, in T cells, and in T-cells depleted mice, and by testing for intracellular IgA-mediated neutralization. The antibodies and specific isotypes induced will be characterized to determine virus neutralizing activity and epitope specificity. Antibody secreting cells (ASC) will be assayed in spleen and intestinal mononuclear cells (mouse) and in spleen, peripheral blood, lymph nodes, and small intestine (gnotobiotic pigs). Cell mediated immune responses to be examined in the mouse model will include cytotoxic T cell responses and specific T helper cell subsets induced. Studies in gnotobiotic pigs will include testing lymphoproliferative responses in cells from spleen, peripheral blood, lymph nodes, and small intestine. Plasmid DNAs expressing specific, endogenously expressed rotavirus proteins in a porcine model for human disease provide a method to study mechanisms of immunity to rotavirus infection and candidate rotavirus vaccines. The ultimate goal of the work is to demonstrate and define protective immune responses against human rotaviruses which will provide the basis for human immunization with rotavirus DNA vaccines.
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