USE OF ACCESSORY GENE MUTANTS FOR THE DEVELOPMENT OF ATTENUATED HIV VACCINES

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The long-term goals of this project are: 1) to evaluate the feasibility of generating live attenuated virus vaccines of HIV-1 and HIV-2 that are rendered non-pathogenic by mutation of accessory genes, either individually or in combination; and 2) to explore the possibility that the accessory gene proteins can be targets for anti-viral therapy. As prerequisites to both the development of candidate live attenuated virus vaccines and the development of anti-HIV drugs directed against the accessory gene products, we have been engaged on studies to determine the role of these proteins in the life cycle of HIV-1 and HIV-2 in vitro, since a knowledge of how they function is critical to both goals. Our earlier work had demonstrated the critical role of HIV-1 Vif to virus replication in primary T cells (peripheral blood mononuclear cells, PBMC), and we have gone on to show that Vif also plays a critical role for HIV-1 replication in primary monocyte-derived macrophages (MDM). In the case of Nef, we have shown that whether or not Nef has a measurable effect on virus replication depends on the particular virus-host system used. While Nef mutants of several HIV-1 strains all replicate slightly less well than wild type in PBMC and in MDM, there can be either no effect or dramatic reductions in virus replication when Nef mutants of HIV-1 and HIV-2 are assayed in CD4-positive cell lines. As part of our goal to develop attenuated HIV vaccine candidates, we have modified an HIV-1 genome to allow the insertion of different genes into the nef open reading frame. Using this virus, we plan to investigate the consequences of ectopic expression of the vpr and vif genes on virus replication and whether different HIV-1 and HIV-2 alleles of these genes and nef can complement the appropriate Vif, Vpr or Nef mutants of HIV-1. As part of our program to ensure the safety of vaccines and other biological products (see project BK 03017), we have been characterizing a sensitive PCR-based RT (PBRT) assay to detect the presence of low levels of retrovirus contamination. This PBRT assay has the capacity to detect between 1 and 10 virions. We have modified the assay to be able to remove background signals and have adapted it for use with three different RNA templates. Because the PBRT assay can detect low amounts of retroviruses, we are applying it to address certain questions in HIV biology. For example, can virus be produced from non-stimulated PBMC, from non-stimulated purifed CD4-positive T cells, and fom elutriated monocytes before differentiation to macrophages? Can virus be detected early after infection?