ANTIGEN PRESENTATION DURING NUCLEIC ACID VACCINATION

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The overall goal of an immunization strategy is to induce specific immune responses which confer lifetime protection from the pathogen of interest. However, it is well known that the specific immune response required to provide such protection varies from one disease to the next. Nucleic acid immunization is a novel vaccination technique which induces antigen- specific immune responses. We have been interested in taking advantage of DNA vaccine technology to tailor immune responses according to the known correlates of protection for specific pathogens. As part of this effort, we developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules which play an important role in the induction of T cell-mediated immune responses. We co- immunized these expression plasmids along with plasmids DNA encoding for HIV-1 antigens and analyzed the magnitude of antigen-specific humoral and cellular immune responses. Although we did not see any significant change in the humoral response, we observed a dramatic increase in cytotoxic T lymphocyte (CTL) induction as well as T helper cell proliferation after the co-administration of CD86 genes. In contrast, co-immunization with a CD80 expression cassette resulted in a minor, but positive increase T helper cell or CTL responses. We have decided to ask a related question:can muscle be converted to a functional APC? For this experiment, we developed bone marrow chimeras, transferring bone marrow from beta-2 microglobulin (beta2m-/-) knockout mice into MHC identical C57BL/6J mice. These beta2m-/-mice lack expression of functional class I MHC molecules. The converse transfer was also performed. The chimerism was confirmed by flow cytometry, which showed more then 90% of peripheral blood cells class I positive in the beta2m+/+ -- more than beta2m-/- mice, with the beta2m -/- --more then beta2m+/+ mice showing less then 10% class I MHC expression. The functional absence of class I MHC molecules on the surface of APC in the beta2m -/- --more then beta2m+/+ mice let us ask whether we could functionally convert their muscle cells (which are class I MHC positive) into antigen presenting cells. Finally, using CD86/CD80 hybrid molecules we will identify in vivo functional region/s in the V- domains of human CD86 in enhancement of cellular immune responses to HIV envelop in mice. Different hybrids between CD80 and CD86 glycoproteins will be constructed using overlap PCR extension technique and they will be analyzed in vivo. The results will allow us to understand in general mechanism of antigen-presentation during DNA immunization, and specifically for construction of more powerful vaccine against AIDS.