Project Summary / Abstract This proposal describes the framework of an R01 grant for Alejandro Balazs, PhD. Dr. Balazs is currently an assistant professor at Harvard Medical School working as a principal investigator at the Ragon Institute of MGH, MIT & Harvard. Dr. Balazs’ research is focused on engineering the immune system via gene transfer as a novel means of creating protection against HIV. Broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus (HIV) show great promise in HIV prevention and therapy as they potently neutralize a significant breadth of globally circulating HIV strains. Two recent Phase I clinical studies have demonstrated the safety and potential for adeno associated virus (AAV) vectors to deliver bNAbs to human participants following intramuscular injection. These have resulted in markedly different outcomes: A lack of expression and significant immunogenicity from AAV1-PG9, or long-lived expression and less immunogenicity from AAV8-VRC07. The mechanisms underlying these differences are poorly defined. This proposal will uncover the elements of vectored immunoprophylaxis which are responsible for the immunogenicity seen in human trials. It will dissect the immunological response during vector administration in patients at single-cell resolution using multi-modal approaches. It will engineer novel AAV capsids designed to avoid stimulating the immune system through directed evolution to avoid antigen presenting cells. Furthermore, it will test the functional capacity of next-generation antibodies delivered by novel AAV capsids in animal models of sustained virologic remission and evaluate the immune response to novel capsid designs. Together this work will greatly enhance our understanding of the human immune response to AAV vectors and yield capsids that eliminate the development of transgene immunogenicity to enable the use of vectored antibody delivery for HIV cure strategies.