ABSTRACT We aim to develop a one-time treatment for durable remission of human immunodeficiency virus (HIV) for patients who are not suppressed by antiretroviral therapy (ART), including patients who are ART naïve or ART non-compliant. Our treatment is an autologous HIV-specific chimeric antigen receptor (CAR)-T cell therapy that employs the CXCR5 chemokine receptor as a homing device to direct anti-HIV killer T cells into immune- protected “hidden” viral reservoirs in lymphoid B cell follicles, where most virus-producing cells are located during chronic infections. Virus-specific CD8 T cells exert potent antiviral activity against HIV-1 and Simian immunodeficiency virus (SIV), an animal model of HIV. Nevertheless, despite abundant CD8 T cell responses in HIV-1-infected humans and SIV-infected macaques, these cells do not fully suppress virus replication, likely because the majority of HIV-1 and SIV replication occurs in CD4+ T cells concentrated within B cell follicles in secondary lymphoid tissues where few virus-specific CD8 T cells reside. In fact, we showed that the ratio of in vivo effector virus-specific CD8 T cells to target SIV RNA+ cells is >40-fold lower inside compared to outside of B cell follicles in lymphoid tissues in SIV-infected macaques. Furthermore, the majority of virus-specific CD8 T cells fail to express the follicular homing molecule CXCR5, likely explaining low levels of virus-spcific CD8 T cells localizing to and surveilling B cell follicles. These data suggest that the inability of HIV- and SIV-specific CD8 T cells to fully suppress virus replication may be due to a deficiency of virus-specific CD8 T cells in B cell follicles. As the vast majority of virus-producing cells are CD4 T cells located in secondary lymphoid tissue during chronic HIV and SIV infections, we targeted lymphoid cells by autologously infusing anti-viral CAR (specifically CAR/CXCR5) T cells into chronically SIV-infected rhesus macaques. The treated animals showed CAR/CXCR5- T cell localization to B cell follicles and decreased virus replication in the follicles compared to control animals. In addition, this product showed safety and efficacy in a pilot preclinical study of ART-suppressed SIV-infected macaques. Based on these findings and the success of our preclinical studies in ART-suppressed macaques, we propose to: (Phase 1, Aim 1) modify our current CAR/CXCR5 construct by producing and characterizing an optimized (opt) CAR/CXCR5 construct that contains a tri-specific HIV binding domain, an alternate co-stimulatory domain, and an HIV resistance domain; (Phase 2, Aim 2) develop a GMP-scalable method to produce CAR-T cells using the optCAR/CXCR5 construct and PBMCs from a) SIV-infected non-ART-treated rhesus macaques for use in an IND-enabling preclinical study; and b) HIV-infected non-ART treated individuals to prepare for the first-in-human Phase 1 clinical trial; and (Phase 2, Aim 3) assess the safety and efficacy of CAR-T cells produced with the...