VACCINE STUDIES: A crucial element in the development of effective prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model since it induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Recent studies suggest that SIV-infection is likely to be more representative of human AIDS pathogenesis than SHIV viruses that utilize CXCR4 as their coreceptor and target naive rather than memory CD4+ T cells. During primary viremia in SHIV-infected macaques, massive elimination of CXCR4 naive CD4+ T cells occurred. In contrast, CCR5+ memory CD4+ T cells were selectively depleted in rapidly progressing SIV-infected macaques. Thus SHIV and SIV target different subsets of CD4+ T cells. These differences explain the different pathogenesis of SIV and SHIV. Importantly, in the context of developing an effective vaccine, regimens that suppress SHIV might not protect monkeys against SIV or humans against HIV. An earlier vaccine effort within my lab has been the evaluation of the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector. MVA-SIV recombinants expressing Env, Gag-pol or both antigens were evaluated. Immunization with MVA-SIV recombinants resulted in a reduction in setpoint plasma viral load that was associated with prolonged survival. All but two vaccinees have progressed to AIDS by nine years after challenge. Recent studies evaluated the effect of vaccination of memory CD4+ T cells in the blood; vaccination was associated with better preservation of memory CD4+ T cells and inversely correlated with virus load. Importantly, preservation of CD4+ T cells early post challenge correlated with improved survival and with titers of neutralizing antibodies to the challenge virus prior to challenge. All of our previous vaccine studies have used SIVsmH4 as an immunogen, which possesses a lab-adapted neutralization-sensitive envelope. Therefore, these previous studies have focused mostly on generating cellular immune responses. The purpose of future experiments will be to use a prime-boost strategy to generate antibody that will neutralize the pathogenic SIVsmE543 and study the role of this antibody in the SIV model. Prior studies have only demonstrated a role for neutralizing antibody in vivo using the less pathogenic SIVmne model or in SHIV. Similar studies using SIVmac239 have been hampered due to the neutralization resistance associated with its envelope glycoprotein. We therefore generated a MVA recombinant that expresses the less neutralization-resistant SIVsmE543-3 envelope and a plasmid of the codon-optimized gp140 that has been used to generate recombinant trimeric SIVsmE543-3 envelope. We are also evaluating neutralizing activity of a large IgG pool that was purified from a group of six SIVsmE660-infected macaques;this IgG effectively neutralizes SIVsmE660 and SIVsmH4 but does not cross-neutralize SIVsmE543-3. We plan to use this IgG in passive prophylactic experiments to determine the predictive power of in vitro neutralization assays. DEVELOPMENT OF ALTERNATIVE CHALLENGE STRAINS The development and characterization of alternative to the major SIVmac239/2251 and SIVsmE660 challenge strains are required for studies evaluating the role of neutralizing antibody in protection. Therefore we wish to develop and characterize additional strains of SIV that are more sensitive to neutralization. One of these is the minimally pathogenic, neutralization sensitive SIVsmH4i strain. This clone has a defective nef gene due to frameshift mutation in the C-terminus of this gene. We generated a corrected version of this clone, SIVsmH4i Nef+. The two clones produced similar levels of virus following transfection of 293T cells, but the correction restored expression of the Nef protein. Intravenous inoculation of macaques with SIVsmH4i resulted in delayed and low levels of peak viremia. This contrasted with robust peak viremia in macaques inoculated with the corrected version and viral kinetics similar to that observed in pathogenic SIV strains. Despite the restoration of in vivo replication ability, neither clone resulted in significant acute memory CD4+ T cell loss or disease in a period of two years of follow-up. We are also presently evaluating two full length infectious clones of SIVsmE660. These both replicate efficiently in macaque PBMC in vivo, are sensitive to neutralization and are being evaluated for replication and pathogenesis in vivo.