Group A Rotaviruses are highly important human pathogens. Estimates of their impact on man include worldwide mortality rates of 800,000 to 1 million infant deaths annually. Despite substantial progress over the past two decades in determining rotavirus gene structure and function, we still do not have a clear understanding of the basis of rotavirus immunity, virulence or host tropism. It is the purpose of this proposal to continue our investigation of several aspects of rotavirus pathogenesis, including host range restriction, virulence and immunity.
The specific aims of this application are: 1) To identify the viral and immunologic determinants of protective immunity in vivo. We will carry out a series of experiments in a mouse model to correlate the quantity and specificity of the local and systemic humoral immune response with protection. In order to determine why homologous infection induces local IgA responses more efficiently than heterologous infection, we will characterize the T helper cell response in the gut and spleen after murine and non-murine rotavirus infection of suckling mice. We further propose to identify and characterize the viral targets of protective immunity. Protection studies will be carried out in the adult mouse challenge model with homologous, heterologous and ressortant viruses that vary serologically. Immunization studies will also be carried out using specific recombinant viral gene products or killed virion delivered to various mucosal or systemic sites. We plan to determine which specific arm of the immune system mediates protective immunity. Passive transfer studies will be carried out employing isolated populations of immune effectors cells (CD4+, and/or CD8+T cells and/or B cells derived from various tissues) from immune MHC-matched donors. We will also study the cellular determinants of protective immunity using mice deficient in specific components of the immune system. 2) To identify the viral and immunologic determinants for the resolution of rotavirus infection. We will determine if CD4+ as well as CD8+ T cells or local IgA antibody are capable of mediating resolution of infection by themselves when passively transferred into chronically infected SCID mice. Mice deficient in specific immune functions will be also be examined to determine if they ar fully or partially deficient in their ability to resolve infection and/or if immune cells taken from such animals can eliminate rotavirus infection in chronically infected SCID mice. 3) To determine the target of and mechanism by which IgA and other monoclonal antirotavirus antibodies prevent, and possibly resolve, rotarvirus infection in vitro and in vivo. In order to study the role of IgA directly we will carry out challenge studies in mice bearing transplanted rotavirus-specific IgA secreting hybridomas. Finally, we will investigate the mechanism by which IgA and IgG neutralizing monoclonals directed at VP7 or VP4 inhibit viral replication. 4) To determine the genetic and molecular basis of host range restriction and virulence. It is our hypothesis that the surface proteins of rotavirus are important but not exclusive determinants of virulence; that virulence and host range determinants vary depending on the genetic and physiological context of the analysis and that genes encoding non- structural as well as structural proteins may be important determinants of host range restriction. In order to determine the genetic basic of host range restrictions and virulence, we will carry out genotypic and phenotypic analyses of rotavirus reassortants in the infant mouse model. In order to better understand the molecular and virologic basis of virulence and/or host tropism, we will identify relevant steps in the viral replication cycle in vivo that are restricted on the basis of host range.
