Evaluating the combination effect of autophagy induction and CAR T cell therapy on "Kick-and-Kill" HIV reservoir depletion

PROJECT SUMMARY The Human Immunodeficiency Virus (HIV), when left untreated, can result in the destruction of the host’s adaptive and innate immune responses, leading to negative pathologies by opportunistic pathogens (AIDS). Some of the greatest barriers in finding an HIV cure is the ability of the virus to establish latency within CD4+ cells, evade human immune clearance mechanisms, as well as induce immune cell exhaustion as a result of chronic infection. An investigatory approach for eliminating latently infected cells, is to use latency reversing agents (LRAs) to force the virus to enter a transcriptionally active state, allowing for host immune mechanisms to identify and kill infected cells. The “Kick-and-Kill” approach has its inherent problems, as it does not address the aforementioned barriers to finding an HIV cure. Additionally, some of the best LRAs, Protein Kinase C (PKC) modulators, are highly toxic in vivo by mechanisms that are not well understood but have been speculated to be toxic due to generalized immune cell activation, or excessive platelet activation/aggregation. The overall objective of this proposal is to augment the “Kick-and-Kill” approach so that the barriers to finding an HIV cure are surmounted, and effective viral clearance in an infection model can be achieved. Current literature has demonstrated that the limitations of the “Kick-and-Kill” approach/HIV cure may be addressed by pairing latency reversal, HIV-specific chimeric antigen receptor (CAR) expressing T cells, and autophagy induction. My preliminary data thus far has shown that PKC modulators are not toxic via an immune cell activation mediated mechanism, and that the immunosuppressive functionality of rapamycin does not prevent HIV latency reversal in our “Kick-and-Kill” approach. The combined effect of the current literature, as well as my preliminary data has allowed us to hypothesize that tolerable PKC modulator-mediated latency reversal, paired with HIV-specific CAR T cells and autophagy induction via rapamycin will effectively deplete the viral reservoir within an HIV-infected humanized mouse model. Aim 1 of this proposal will investigate the role of platelet activation/aggregation in bryostatin-1-mediated toxicity in vivo, by measuring mouse survival and platelet activation/aggregation in byrostatin-1 and platelet aggregation inhibitor treated C57BL/6 mice. Aim 2 will evaluate the effects of bryostatin- 1 and the autophagy inducer rapamycin on T cell exhaustion markers, HIV latency reversal, viral load and time to viral rebound in HIV-infected, ART-suppressed, humanized mice after ART cessation. Finally, Aim 3 will utilize a CAR T cell-expressing (D1D2CAR 4-1BB CAR T cell), ART-suppressed, humanized mouse HIV infection model to test for T cell exhaustion markers, viral load, and HIV latency reversal in tissues and blood upon bryostatin-1 and rapamycin treatment, as well as time to viral rebound after ART cessation. Together, these data will elucidate the curative potential of autophagy induction and HIV-specific CAR T cells in a “Kick-and-Kill” oriented HIV cure approach. At the same time, this work will also shed light on the role of platelet activation/aggregation in PKC modulator-induced toxicity, providing insight into future drug development.