Immunization with live, replication-competent Ad-HIV or Ad-SIV envelope recombinant vaccines primes strong antibody responses that develop following administration of booster immunizations with envelope protein. These antibodies display a variety of functional activities. The most desirable for an HIV/AIDS vaccine is neutralizing activity that is able to prevent infection following exposure to the virus. We have elicited such antibodies in the rhesus macaque model that conferred apparent sterilizing immunity following challenge with an HIV/SIV chimeric SHIV virus. Our vaccine regimen also elicits antibodies with other functional activities mediated by Fc-receptor bearing cells such as NK cells. HIV/SIV infection is initially manifested as small foci of infected cells. Within 2 to 6 days, virus spreads from these cell foci to draining lymph nodes, subsequently leading to systemic infection. These additional non-neutralizing functional activities can help control the initial viral burden by limiting the spread of virus from the foci of infection. Such activities include antibody dependent cellular cytotoxicity (ADCC), antibody dependent cell-mediated viral inhibition (ADCVI), and antibody-dependent cellular phagocytosis (ADCP). With regard to the latter, an in-depth investigation of V2 monoclonal antibodies was recently completed. V2 antibodies were identified as a protective immune correlate in the RV144 clinical vaccine trial in Thailand, the only trial to have reached any degree of protective efficacy. Although V2 antibodies were associated with protection, the mechanism(s) by which they conferred protection remained unidentified. Our recent study found that V2 monoclonal antibodies mediate ADCP activity, suggesting a mechanism which may contribute to protection. Further, studies of antibody-dependent complement mediated lysis of virus and virus infected cells have recently been initiated, and represent another functional activity that can contribute to overall protection, as is ADCP activity mediated by neutrophils. Since HIV is transmitted mainly at rectal/genital mucosal sites, a key goal of HIV vaccine development is to elicit mucosal immunity. The Ad-recombinant prime/protein boost strategy induces antibodies in mucosal secretions which can inhibit transcytosis of SIV across an epithelial cell barrier, suggesting another mechanism which may contribute to protection. These mechanisms are all currently under study. Recently we completed a pre-clinical vaccine study using the SIV-rhesus macaque model in which we uncovered a sex bias in vaccine-induced protection. Vaccinated female macaques, but not males, exhibited delayed SIV acquisition. We showed that vaccine-induced SIV envelope-specific mucosal IgA, Envelope-specific memory B cells, and total rectal plasma cells were correlated with this protective outcome. Subsequently we showed that female macaques developed better quality antibodies of the IgG3 subtype which is more effective in mediating protective antibody responses. Moreover, male macaques exhibited a greater proportion of B regulatory cells associated with suppression, suggesting that these cells might have facilitated infection in the males. On-going studies are investigating cytokines secreted by B cells, as these molecules have the ability to regulate immune B cell responses. We have also examined plasma cell niche factors in bone marrow and the rectal mucosa in order to better understand the accumulation of plasma cells in the mucosa. Investigation of the sex bias in vaccine-induced protection is important as approximately 50% of HIV infected individuals are women. A recent pre-clinical study in the macaque model has achieved partial protection, illustrated by reduced viral burdens in animals that became infected. Notably, vaccinated female macaques developed lower viremia levels compared to vaccinated males, indicating that further investigation of mechanisms contributing to the vaccine-induced sex bias is warranted. In this regard, we have previously demonstrated that our vaccination strategy induces viral envelope-specific T follicular helper (Tfh) cells in lymph nodes of immunized non-human primates. These cells are critical for providing the help necessary for B cell development and antibody production, leading to memory B cells and the long-lived plasma cells necessary for long-term vaccine-induced protection. Current studies are pursuing this observation, focusing specifically on the interaction between Tfh and B cells in lymph node germinal centers. Other cells also contribute to B cell development. We have recently found that neutrophils in non-human primates can provide help to B cells, providing another mechanism which might be exploited for elicitation of greater protective efficacy. Finally, a newly initiated pre-clinical study in the SIV rhesus macaque model in collaboration with Dr. Ettore Appella (NCI) and Dr. Daniel Appella (NIDDK) is investigating the hypothesis that a novel microbicide will provide additive or synergistic protection against subsequent viral infection. The microbicide (SAMT-247), a zinc finger inhibitor that prevents proper processing of the viral gag protein, leads to production of non-infectious viral particles which nevertheless possess native envelope on the virion surface. SAMT-247 possesses a novel intracellular cycling mechanism, which results in continual recycling of the active molecule within cells providing longer lasting protection. We have shown that our vaccine regimen elicits strong mucosal immunity. We postulate that in previously vaccinated macaques, topical application of the microbicide prior to intravaginal viral exposure would not only prevent infection, but also boost the anti-envelope antibody of the macaques, resulting in synergistic protection. Initial studies have demonstrated the safety and efficacy of the microbicide in the macaque model. The vaccine phase of the experiment has now been initiated.