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Selectively Eliminating HIV Latently Infected Cells Without Viral Reactivation
Grant R. Campbell1, Rachel S. Bruckman1, Yen-Lin Chu1, Stephen A. Spector1
1 Pediatrics, University of California San Diego, La Jolla, CA, United States.
Background: Efforts to disrupt the establishment and maintenance of the latent reservoir have focused on the “shock-and-kill” therapeutic approach to reverse HIV latency from CD4+ T cells with subsequent killing of the infected cells. The X-linked inhibitor of apoptosis protein (XIAP) is up regulated in latently infected cell lines. In this study, we investigated whether this molecular signature existed in primary latently-infected resting central memory CD4+ T cells and whether this could be used to selectively target and kill latent HIV harboring cells.
Methods: CCL19-treated naïve CD4+ T cells isolated from HIV-uninfected donors were infected with HIV then expanded in the presence of IL2 for 12 d. Memory CD4+ T cells were then isolated and cultured in the presence of IL7 for a further 20 d then analyzed by flow cytometry. HIV integration was analyzed by Alu-LTR qPCR. Expression of XIAP was assessed using Western blotting. HIV p24 antigen was quantified by ELISA. Long-lived, resting memory CD4+ T cells were then treated with the XIAP antagonist GDC-0152, the SMAC mimetic antagonist birinapant or the inhibitor of XIAP embelin. Apoptosis was assessed using annexin-V combined with propidium iodide staining as well as by Western blotting for PARP and caspase 3 cleavage. Data were analyzed using the Student's t test.
Results: After the 32 d infection, CD4+ T cells displayed a resting central memory CD4+ T cell phenotype (CD45R0+ CD62L+ CCR7+ CD25- Ki-67-). Alu-LTR qPCR and p24 ELISA demonstrated that these cells contained the equivalent of 1 copy of integrated HIV DNA in the absence of viral release into the culture supernatant. Moreover, XIAP expression was significantly increased compared with uninfected cells (P < 0.05). Targeting XIAP with birinapant, GDC-0152, and embelin resulted in a significant dose-dependent increase in the number of latently infected resting central memory CD4+ T cells undergoing apoptosis which was not observed in mock-infected cells. Moreover, we did not observe an increase in p24 antigen expression.
Conclusions: We have identified XIAP as a molecular signature of latently infected primary long-lived, resting central memory CD4+ T cells. Moreover, these cells are more sensitive to XIAP-agonist-induced apoptosis than uninfected cells. Therefore, by targeting XIAP with selective inhibitors and antagonists we have developed a novel approach that selectively eliminates latently-infected cells that does not require reactivation of HIV gene expression.