2572 Background and Methods The development of vaccination strategies targeted to tumor antigens holds great promise for cancer therapy. Prostate-specific membrane antigen (PSMA) is an attractive candidate self-antigen for vaccination based on its expression signature. PSMA is abundantly expressed on the surface of prostate cancer cells and on the neovasculature of a wide array of other tumors, but PSMA has limited expression in normal extraprostatic tissues. In prostate cancer, PSMA expression increases with disease progression, becoming highest in high-grade, metastatic, hormone-refractory disease. Our main goals are to overcome tolerance to PSMA by active immunization and to induce recognition and rejection of tumor cells expressing PSMA. We are investigating non-replicating vaccine replicon particles (VRP) based on the alphavirus, Venezuelan equine encephalitis virus. VRP have demonstrated potency in animal models, and infect cells in the lymph nodes where the replicon drives production of large quantities of the heterologous antigen. Robust immune responses can be further amplified by VRP-induced cell apoptosis leading to cross-priming. We are also evaluating a complementary purified protein vaccine based on a novel recombinant soluble PSMA (rsPMSA) immunogen both alone and with VRP in prime-boost regimens. Results When used as single immunogens, VRP encoding full-length human PSMA elicited vigorous, durable Th1-biased cellular and humoral responses in normal BALB/c mice, whereas rsPSMA vaccines induced high levels of Th2-biased antibodies. We have now extended this analysis to human HLA-A2 transgenic mice, a more pertinent model for quantification of human Class I-restricted cellular responses, and to prime-boost regimens. Mice were immunized with VRP alone or in combination with rsPSMA boosts. PSMA-specific immune responses were measured with a panel of cellular and humoral immunoassays, including ELISPOT and intracellular cytokine staining for IFN-γ and IL-4 in T cell subsets. Conclusions Based on these translational findings, the rsPSMA protein vaccine has been advanced into a dose-escalating Phase I clinical trial in advanced prostate cancer, while the VRP-PSMA vaccine is in late-stage preclinical testing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AlphaVax, PSMA Development Company, LLC

A functional Nef protein is crucial in vivo for viral replication leading to pathogenesis in SIV-infected macaques. Moreover, a full-length Nef protein is required for optimal virus replication in primary cells, and both HIV and SIV Nef proteins enhance virion infectivity. Enhanced infectivity may result in part from the ability of Nef to incorporate cellular kinases into virions. In two previous reports, we compared in vitro kinase profiles of SIV recombinant clones that express nef genes derived either from the prototypic lymphocyte-tropic SIVmac239, clone SIV/Fr-2, or from our neurovirulent clone SIV/17E-Fr. While the SIV/Fr-2 Nef protein associated with the previously described PAK-related kinase and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC), SIV/17E-Fr Nef was found to associate with a novel serine kinase activity that was biochemically distinct from both PAK and NAKC. Interestingly, while both Nef proteins were incorporated into virus particles, Nef-associated kinase activity was detected only in virions containing the SIV/17E-Fr Nef protein. Because sequence analysis identified only five amino acids that differed between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr, we were able to evaluate the contribution of each amino acid to Nef-associated kinase activity as well as virus infectivity by constructing a panel of SIV clones containing individual reversions of each differing amino acid in SIV/17E-Fr Nef to the corresponding amino acid in SIV/Fr-2 Nef. In this report, we identify previously uncharacterized amino acids in the N terminus and the conserved core domain of Nef that are essential for the detection of Nef/kinase interactions as well as Nef phosphorylation during SIV infection. Further, via a novel infectivity assay recently developed in our laboratory that utilizes CEMX174 reporter cells stably expressing an SIV/LTR-luciferase construct, we find no direct correlation between specific Nef kinase associations and enhanced virion infectivity.