We studied mechanisms for T cell recognition of antigens in association with major histocompatibility complex (MHC)-encoded molecules, and applications to the design of synthetic vaccines for AIDS and cancer. We have been characterizing the helper and cytotoxic T lymphocyte (CTL) responses to HIV envelope and reverse transcriptase, mapping the key epitopes, and defining the role of individual residues in these epitopes to be able to modify the structures to make more potent immunogens as vaccines. We have made vaccine constructs in which clusters of helper epitopes are synthesized coupled to a peptide that is a CTL epitope presented promiscuously by multiple class I MHC molecules in the human and mouse as well as a neutralizing antibody epitope. These constructs can induce all three arms of the immune response, neutralizing antibodies, CTL, and Th1 helper cells. Results of the first arm of a phase I clinical trial with one of these peptides show ability to induce CTL, helper T cell responses, and neutralizing antibodies to HIV in at least a subset of human recipients. Meanwhile, we are developing new approaches in mouse models to improve on the peptide vaccine constructs. We have now shown proof of principle that we can modify the sequence of a helper epitope of HIV to make it more immunogenic and also much more potent, when coupled to a CTL epitope, in eliciting CTL. We are applying this ?epitope enhancement? approach to conserved helper epitopes presented by human class II HLA molecules, as well as to hepatitis C virus (HCV) epitopes presented by human HLA-A2.1 (see below). We have discovered ways of increasing CTL, helper, and antibody responses and steering them toward desired phenotypes, such as Th1 or Th2 or particular antibody isotypes, by incorporating cytokines into the emulsion adjuvant with the antigen. We compared a panel of 8 cytokines for their effects on 8 types of immune response, and discovered a novel synergy between GM-CSF and IL-12 and between TNF and IL-12 in induction of CTL. We found that all 3 cytokines provide triple synergy for induction of CTL with a peptide vaccine, for induction of interferon-gamma, and for protection against viral challenge in vivo. The mechanism of this synergy appears to relate to the upregulation of antigen presenting function and cytokine receptors. We have shown that high avidity CTL specific for HIV-1 envelope peptide are much more effective at clearing a recombinant vaccinia virus expressing HIV gp160 from SCID mice than are low avidity CTL specific for the same peptide-MHC complex, and have worked out one mechanism involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced. However, we found that high avidity CTL are exquisitely sensitive to high dose antigen and will undergo programmed cell death, mediated by TNF and the TNF receptor II, but also requiring a permissive state involving a decrease in Bcl-2, IAP1, and TRAF2, and correlating with downmodulation of the T cell receptor. This effect may explain clonal exhaustion in viral infections. Finally, we have shown for the first time that protection against mucosal transmission of virus can be mediated by CD8 CTL without antibodies, but requires that the CTL be present at the mucosal site of transmission, whereas systemic CTL are not sufficient. The protection can be accomplished by intrarectal immunization with a peptide vaccine and increased by inclusion of IL-12 and GM-CSF with the vaccine. We observed an asymmetry between mucosal and systemic immune responses in that systemic immunization induced only systemic CTL whereas mucosal immunization induced both mucosal and systemic CTL. This observation led us to develop an approach to overcome the problem of preexisting poxvirus immunity from smallpox vaccination in order to use recombinant vaccinia vector vaccines by taking advantage of the naivete of the mucosal immune system after systemic immunization to still be able to immunize with recombinant vaccinia vaccines through the mucosal route in vaccinia-immune animals. With regard to cancer, we identified several CTL epitopes in proteins of hepatitis C virus (HCV), that causes liver cancer, using a novel approach, and have analyzed the role of each amino acid residue in order to modify one of the peptides to make a more potent vaccine. Using this ?epitope enhancement approach, we could increase the immunogenicity of an epitope of the HCV core protein, presented by the most common human class I HLA molecule, HLA-A2.1, both for HLA-A2.1-transgenic mice in vivo and for human T cells in vitro. This ?enhanced? epitope is being incorporated into a vaccine. We also demonstrated striking protection of HLA-A2.1-transgenic mice from challenge with a recombinant vaccinia virus expressng HCV core protein by immunization with a DNA vaccine expressing HCV core, and showed that the protection was CD8-T cell dependent and correlated with HLA-A2.1-restricted CTL. Further, we found that T cell help against the hypervariable region 1 of HCV envelope was critical for induction of human antibodies to this region, believed to be a neutralizing epitope. We also developed a model of immunosurveillance of cancer in which tumors are rejected by CD8 T cells, but the rejection is incomplete in the presence of normal CD4 regulatory cells, and an escape variant of the tumor recurs. However, depletion of CD4 cells allows complete eradication of the tumor by CD8 cells, and we are exploring the cytokine mechanisms involved using receptor knock-out mice. We developed peptide cancer vaccines inducing CTL immunity to mutant p53 expressed in cancer cells. We found that mutant p53 peptides, coated on dendritic cells, elicit CTL that kill tumor cells expressing the mutation and suppress established tumors in animals. Common mutations in ras peptides were found to enhance binding to HLA-A2.1, but also to influence antigen processing. We also induced murine CTL against fusion proteins from chromosomal translocations in pediatric tumors, alveolar rhabdomyosarcoma and Ewings sarcoma. We also identified novel epitopes spanning these fusion protein junctions in these sarcomas that could bind to several human HLA molecules, HLA-A1, A3, B7 and B27. 29 patients have been treated in a phase I/II clinical trial of the mutant p53/ras peptide vaccine approach to treating cancer, and a large fraction have made CTL or cytokine responses, and no adverse effects have been seen. A trial of translocation fusion peptide immunization of patients with alveolar rhabdomyosarcoma and Ewings sarcoma is underway. We have also started a trial of immunization of cervical cancer patients with peptides from the E6 and E7 oncoproteins of human papillomavirus type 16 that bind to HLA-A2.1 in patients who express this HLA molecule. A phase II trial of autologous dendritic cells pulsed with mutant ras peptides corresponding to the patients tumor in colon cancer patients with HLA-A2.1 that can present these ras peptides has opened and we have treated 3 patients to date. (50% AIDS related)
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