PROJECT SUMMARY HIV-1 broadly neutralizing antibodies (bNAbs) are currently under clinical evaluation for their ability to prevent transmission. Until a conventional vaccine is realized, repetitive dosing of a bNAb would be necessary to maintain protective antibody concentrations. An alternative to passive infusion of bNAbs is to use adeno- associated virus (AAV) vectors that can turn muscles into antibody production factories. This one-time treatment would have clear cost advantages over the continuous production, purification, and administration of recombinant monoclonal antibodies. However, host immune responses limit the efficacy of AAV vectors. CD8+ T cell clearance of AAV transduced muscle cells limits the total number of cells producing the antibody, and pre-existing immunity to AAV capsids limit the number of possible individuals that can receive AAV vectors. We and others have previously shown that host immune responses are detrimental to AAV-delivered HIV-1 antibodies resulting in low to no detectable serum concentrations. Thus, overcoming the host immune response to the AAV vector and expressed transgene is critical for future evaluation of AAV-delivered antibody studies in non-human primates. One area of investigation for limiting a host immune response would be to utilize immune checkpoints that regulate immune system pathways. To this end, in a pilot study, we have observed about a 21-fold increase in concentrations of an HIV-1 antibody in rhesus macaques when macaques were co-inoculated with an AAV vector encoding rhesus macaque PD-L1. PD-L1 functions in binding T cell expressed PD-1 to inhibit the cytolytic and degranulation functions. It also helps in the development of regulatory T cells. Thus, we hypothesize that expression of PD-L1 on muscle cells transduced by AAV vectors to express antibodies will avoid T cell clearance and maintain expression of the antibody. Here we seek to demonstrate that co-inoculation of vectors encoding PD-L1 will result in serum concentrations of a bNAb that will protect rhesus macaques from repetitive, low-dose SHIV challenges. Additionally, we will develop this system by evaluating strategies to reduce the dose of AAV vector. Furthermore, we will engineer AAV transgene cassettes and assess novel a novel AAV capsid for increasing expression from intramuscular inoculation. Together, the results from these studies will provide a foundation for AAV gene therapy studies in non-human primates as well as lead to the development of novel AAV vectors for expressing HIV-1 bNAbs.