The contribution of innate immunity to the early containment of HIV-1 replication remains unknown. We have initiated studies to evaluate the role of NK cells in primate lentivirus control in a 10 monkey study in which NK cells have been eliminated from the circulation of animals by infusion of an anti-CD16 monoclonal antibody. In this ongoing study we have demonstrated that elimination of NK cells (CD3-, CD20-, NKG2+ cells) from the peripheral blood of monkeys for 7 days does not effect the kinetics of plasma viral RNA levels or loss of memory CD4+ T lymphocytes in the first weeks following an infection with SIVmac251. This observation suggests that NK cells are playing a negligible role in the early control of SIVmac251 replication. The observation that HIV-1 envelope sequences evolve over time in infected humans to escape from antibody neutralization suggests that humoral immune responses may be contributing to viral control and, in the process, exerting selection pressure on the virus. We have recently optimized the administration regimen of an anti- CD20 monoclonal antibody to eliminate B lymphocytes for up to a year from the peripheral blood and lymph nodes of rhesus monkeys. We are using this antibody as a tool in an ongoing 20 monkey study to evaluate the contribution of antibody to early containment of SIVmac251 infection. One hundred days following infection, the anti-CD20 infused monkeys still do not have detectable peripheral blood B lymphocytes. Plasma viral RNA levels from these animals are still pending. However, we have not observed a significant difference in peripheral blood CD4+ T cell numbers and SIV-gag ELISPOT responses between the monkeys that received the experimental and the control monoclonal antibody infusions, suggesting that viral replication during this period of time is not significantly different in the experimental and control monkeys. We will initiate a complementing study in May, 2007 to deplete B cells in the setting of SIVmac239 delta nef vaccination and SIVmac251 challenge to assess the contribution of antibody generation to the protection conferred by this live attenuated vaccine. Although the frequency of HIV-1 superinfection is not known, there is increasing evidence that individuals already infected with HIV-1 can be infected with a heterologous strain of the virus. This clinical setting may provide an important window through which to view the immune and virologic correlates of protection against HIV-1 infection. The SIV-infected rhesus monkey model provides a powerful system for studying events associated with lentivirus superinfection. This model provides a means of controlling potential variables that might impact on transmission, such as the genetic relatedness of the two viruses, the route and timing of virus exposure, and the immune status of the previously infected individual. We have created a model for superinfection in monkeys using two SIV isolates that are as disparate in sequence as any two circulating HIV-1 isolates. We will also include in these studies cohorts of monkeys that have received received nef-deleted SIVmac239 virus so that the degree of protection afforded by a prior infection with replication-competent virus can be compared with that provided by a live attenuated virus vaccine. We have initiated studies in this nonhuman primate model of lentivirus superinfection. To differentiate and quantify SIVmac251 and SIVsmE660 in the blood, we have developed a quantitative PCR method for determining the RNA levels of the two virus strains in plasma samples. We have established two cohorts of rhesus monkeys, one infected with SIVmac251 and the other with SIVsmE660. We have defined the baseline immune function of these monkeys by characterizing their CD4+ and CD8+ T lymphocyte memory subsets and their SIV-specific T lymphocyte function by Elispot assay and intracellular cytokine staining. These monkeys were then exposed to the heterologous virus via 6 weekly intrarectal challenges. Their CD4+ T lymphocyte memory subsets, total CD4+ T lymphocyte counts, and plasma viral loads were assessed prospectively. At the completion of 6 intrarectal challenges, these animals had no evidence of superinfection as determined by quantitative PCR. We will monitor these animals for an additional four weeks, and if there is no evidence of the second virus in their blood, we will expose them to another round of 6 intrarectal challenges. If these monkeys remain protected against superinfection, we will begin studies to determine the mechanism mediating this protection. The first of these studies will be to assess the role of CD8+ T lymphocytes in this protection by depleting these cells through monoclonal antibody infusion and assessing the protection afforded by the prior infection. It will be essential to define the nature of the transmitted virus to create an effective HIV-1 vaccine. A report by Hunter and colleagues suggested that the transmitted virus may have shortened variable loops. To elucidate whether deletions in the variable loops of Env reflect a transmission bottleneck, we are examining viral sequence evolution in a cohort of 5 rhesus monkeys that were infected with a genetically-defined stock of SIVmac251 via repeated intrarectal inoculation. At the time of peak viral replication and up to 6 months after infection, we did not observe variable loop deletions in the env gene. Interestingly, as these monkeys progressed from acute to chronic infection at 16 months after infection, selection occurred for viruses with deletions in the variable loops VI and V4 in all five animals. The data suggest that these variants evolved as a result of phenotypic selection. To evaluate the biological properties of these variants which have been selected for in the host, we will evaluate whether these viral variants emerged as a result of viral escape from neutralization antibodies, change in receptor usage, or variable viral fitness. We also have a study ongoing in rhesus monkeys to evaluate the quasi-speciation of SIVmac251 following the initiation of infection to determine whether there are particular selection pressures in mucosal compartments that shape the transmitted virus. In a pilot study of 4 rhesus monkeys, we are evaluating virus evolution in mucosal and systemic anatomic compartments to determine whether a local immunologic milieu has an impact on the nature of the mutational changes that the virus accumulates over time. Preliminary data indicate that CTL pressure may be greater on seminal virus than on circulating virus in the first weeks following infection. Although chimeric viruses that express an HIV-1 envelope on an SIV backbone hold promise for facilitating the elucidation of important envelope-associated events in HIV-1 infection and early pathogenesis, the R5-tropic SHIVs that have been generated to date do not cause sufficiently consistent pathogenic consequences to be useful for such studies. To address the need for a consistently pathogenic R5-tropic SHIV, we have constructed SHIV-KB9 viruses that express the V3 loop of two R5-tropic HIV-1 isolates, ADA and YU2. These viruses demonstrate an in vitro tropism for CD4+ T cells that express the CCR5 co-receptor, suggesting that altered tropism of SHFV-KB9 can be achieved by substitution of the gp!20 V3 loop. In a pilot study of the in vivo pathogenic properties of these newly constructed R5-tropic SHIVs, both chimeric constructs replicated to high levels during the period of primary infection and caused a dramatic loss of memory CD4+ T lymphocytes. The most consistently pathogenic of these viruses will be chosen for further development. A cell-free stock will be prepared and titered in vivo for rectal challenge studies to evaluate novel vaccine candidates in monkeys. This chimeric virus will also serve as a backbone for creating a construct to evaluate the pathogenic ramifications of the newly defined signature sequences associated with transmitted HIV-1. Building on previous descriptions of human, mouse, and cynomologous monkey B cell subsets, antibody panels were developed to detect activation, maturation, and function-associated molecules on peripheral blood B cells of rhesus monkeys. To establish baseline data on B cell subsets, peripheral blood from 20 naive rhesus monkeys was evaluated. We then applied these B cell panels to the study of peripheral blood of SlV-infected animals 56 days and 2 years after infection. While the overall number of B cells did not differ significantly different between naive and infected animals, we found clear evidence of pathogenic subset changes, including a loss of naive B cells and an increase in number of activated, memory-type B cells. We are currently extending these studies to include an evaluation lymphatic tissue, including lymph nodes and gastrointestinal tissue, as well as an application of antibodies that detect plasma cell subsets.