A live attenuated strain of Shigella flexneri has been constructed and preliminarily tested as a novel vaccine vector for generating humoral and cell-mediated immunity against HIV. The parent bacterial strain is a strain of Shigella flexneri, a variant of which has been thoroughly tested in trials with human volunteers. We recognized that this strain, in which the gene, icsA or virG, for intracellular spread, has been inactivated, icsA-, could be exploited for surface display of antigens. We have developed a novel technique to express foreign antigens on the outer membrane by modifying the icsA- strain. In tissue culture and animal studies, this genetically engineered bacterium is fully competent to invade the host cell cytoplasm, but is unable to spread from cell to cell or cause serious illness in animals. Of most interest is that attenuated intracellular bacteria may be uniquely suited to deliver immunodominant B and T cell epitopes. Equally important is the realization that in the normal infective process of these bacteria, mucosal surfaces and underlying mucosal-associated lymphoid tissue (MALT) are the initial and primary sites of colonization. It is in this region that the bacteria contact directly B cells and are phagocytosed and processed for peptide presentation by antigen presenting cells (APCs) and/or macrophages. T cell activation may occur at this time through both the MHC I and MHC II pathways of antigen presentation leading to epitope-specific memory T cell populations. Ultimately the immune system when challenged by this type of antigen presentation will develop a strong protective immunity at all three HIV entry sites: mucosal tissue, regional lymph nodes and blood. We have constructed our vaccine vector to surface display either HIV p24 or gp120. We have demonstrated by Western blots that the HIV antigens are anchored in the outer membrane for display on the surface of the bacteria. We have tested these as potential vaccine vectors in an intranasally challenged mouse model and have preliminary data indicating significant levels of antigen-specific serum IgG and low levels of IgA in vaginal mucosal secretions. We now propose to: 1) Further refine our vaccine vector for increased level and stability of antigen surface display in order to maximize IgG and IgA antibody responses. 2) Measure specific T cell responses and the extent and specificity of the mucosal immune response to our vaccine constructs.
