PRODUCTION OF ANTI-HIV-1 THERAPEUTIC VACCINE

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(1) Goals of Project: To identify a carrier which will increase the immunogenicity of the HIV-1 subunits, and will be able to recall anti-HIV B memory cells in patients with pre-existing immune deficiency. To identify a carrier which would augment the TH1/TH2 ratio of infected individuals and will favor generation of cellular responses including cytotxic cells (CTL). (2) Experimental approaches: The gram negative Brucella abortus (Ba), and LPS derived from its cell wall (Ba-LPS), were tested as carriers for either inactivated HIV-1 virions, gp120 (SF2) glycoprotein, or peptide derived from the V3-loop of HIV-1 (MN) env. The different conjugates were used to immunize mice with different degrees of T cell deficiency. Four Rhesus macaques were also vaccinated with a Ba-V3 conjugate. Both humoral and cytotxic immune responses were measured including systemic and mucosal antibody responses. Biologically relevant antibodies were measured in syncytia inhibition assays. In vitro studies with human T cells and elutriated monocytes from normal as well as HIV-1 infected patients were assessed for their lymphokine production in response to Ba and Ba-LPS by PCR and biological assays. (3) Major findings: Ba conjugated to a peptide containing B-cell epitope and CTL epitope (N3v3), generated both neutralizing antibodies and cytotoxic T cells capable of killing HIV-infected targets. CD4-depleted mice retained their ability to generate anti-HIV neutralizing Ab and CTL after immunization with the Ba-N3V3 conjugate. From a safety point of view, Ba or its LPS are much less toxic to animals than E. coli derived LPS. Ba and Ba-LPS were found to directly activate purified human CD4-positive TH1 cells, and to a lesser degree, CD8-positive cells, as judged by induction of the lymphokines IL2 and IFN-gamma. PBL from HIV-1 infected individuals were also responsive. Ba and Ba-LPS can also activate IL12 secretion by human monocytes. Rhesus macacques produced high titer HIV-1-neutralizing antibodies in the serum and mucosal surfaces.