The significant amount of data generated by SIV vaccine studies has led to the suggestion that an HIV vaccine is achievable. The capacity of humoral and cellular immune responses in mucosal tissues to block or contain replication at the initial stage of virus transmission may have a profound impact on the ability of a vaccinated host to resist infection. The development of an effective vaccine that restricts viral replication at the mucosal portal of entry may be our best hope for controlling HIV infection. We believe there are two necessary features for a successful vaccine: 1) life-long stimulation of the immune system with viral antigens; and 2) a targeted immune response at the site of primary replication of HIV. A vaccine approach that simultaneously addresses these two issues would have the potential to achieve solid, long-term protection. To fulfill these requirements, we propose an alternative approach to successfully deliver a vaccine to mucosal sites and elicit protective mucosal immune responses. We propose to use the epithelial stem cell as a permanent source of viral antigen and their differentiated offspring as antigen producing presenting cells. Using a single cycle SIV (SIVsc) approach, which has been shown to be a very safe strategy compared to traditional attenuated vaccines, we propose to clone the SIVsc genome under the control of the involucrin promoter, a terminally differentiated keratinocyte specific promoter. This virus will be then administered to target epithelial stem cells from different tissues (epidermal, vaginal, rectal). Basal layer cells will divide and differentiate thus triggering SIV antigen expression and both direct and cross priming. Herein, we propose: 1) To elicit and optimize the SIV antigen expression from a terminally differentiated keratinocyte-specific promoter in rhesus macaques vaccinated by dermal, vaginal and rectal routes; 2) To investigate the nature of immune responses induced by the different inoculation protocols; and, 3) if these animals are able to mount a satisfactory immune response compared to attenuated SIV infected animals, we will attempt to demonstrate protection against multiple low-dose vaginal and rectal challenges with a homologous SIV strain. The nature of the HIV virus has created several barriers to effective immune control by the humoral and cellular arms of adaptive immunity leading to chronic viral replication. Of the vaccine approaches tested in the SIV/macaque model, vaccination with live attenuated lentiviruses has consistently yielded the most effective and durable protection against pathogenic heterologous SIV strains. However, safety issues preclude the use of live attenuated lentiviruses in humans. As highlighted by many Authors, these results lead to the conclusion that only a life-long stimulation of the immune system by the vaccine will be sufficient to achieve long-term protection. Another key issue is transmission which occurs predominantly across genital or rectal mucosal surfaces. The capacity of humoral and cellular immune responses in mucosal tissues to either block or contain HIV replication at the initial stages of virus infection may have a profound impact on the ability of a vaccinated host to resist infection. The development of an effective vaccine that restricts viral replication at the mucosal portal of entry may be our best hope for controlling the HIV pandemic. One of the obstacles to the development of an effective AIDS vaccine has been our inability to deliver antigen for a prolonged period at the mucosal portal of entry. To address these issues a vaccine will have to demonstrate long-term expression of antigens using a persistent vaccine vector and be delivered to the epithelial barrier to generate mucosal homing cellular responses. To that end, we propose to use epidermal stem cell as a permanent source of antigen and their differentiated offspring as antigen producing cells. All stratified squamous epithelia are composed of epithelial layers. Cells at the proliferative basal cell layer upregulate transcription of proteins (such as involucrin) as they differentiate and move upward. Among these promoters, the involucrin promoter has extensively been studied and is restricted to terminally differentiated epithelial cells in epidermis and mucosa. Our concept is to target epithelial stem cells with a lentivirus vector to introduce an antigen construct under the control of the involucrin promoter. These cells will carry the construct without being attacked by the immune system because they do not express the antigens. As the progeny cells differentiate, the promoter will become activated and the daughter cells will become, in a sense, antigen presenting cells leading to induction of humoral and cellular immune response via direct priming and cross-priming.