The vast majority of HIV-1 infections occur at mucosal surfaces in the body. There is therefore an immediate need for potent HIV vaccines that can provide barrier protection at mucosal surfaces. While there is this need, most HIV vaccines have been developed and tested for their ability to drive systemic immune responses and not for mucosal responses. Given that systemic immunization generally does not provoke potent mucosal protection, this project will develop safer replication-defective helper-dependent adenoviral gene delivery vectors for mucosal vaccination. This project will pursue this goal in the following Aims: Specific Aim 1. Test Helper-dependent Adenoviral (HD-Ad) Vectors Expressing Codon-optimized SHIV Antigens for Their Ability to Drive Mucosal and Systemic Immune Responses. Specific Aim 2. Test Vector Immune Evasion Strategies to Enable Multiple Rounds of Priming and Boosting with HD-Ad Vectors. Specific Aim 3. Compare Routes of Administration and Enteric-coated Capsules to Maximize Mucosal T Cell and Neutralizing Antibody Responses. This project will generate HD-Ad vectors expressing HIV gag-pol, SIV gag-pol, and HIV-1 env. These HD-Ad vectors should prove safer than first generation vectors and will be packaged by four serotypes of adenovirus to test serotype switching as a means to evade neutralizing vector-specific immune responses and allow more effective vaccine boosting. Serotype-switching will be compared to polyethylene glycol (PEG) modification of the HD-Ad vectors for their abilities to evade vector neutralizing antibodies. These approaches should allow HD-Ad to be utilized for multiple rounds of immunization in the same host to take advantage of to robust supraphysiology transgene expression of Ad vectors. This project will package these adenoviral vaccines in enteric-coated capsules to develop a simple oral vaccine that delivers the vaccines more effectively to mucosal immune cells. These vectors will be tested in humanized HLA-A*0201 transgenic mice for T cell responses and will be tested in rabbits for neutralizing env antibody production. Successful pursuit of this project will enable more specific and less dangerous adenoviral vaccines for future testing in rhesus macaque SHIV challenge models and for future use in humans.