Identifying vulnerabilities in the long-lived HIV reservoir to accelerate its decay

点击翻译按钮获取中文摘要

PROJECT SUMMARY Latently infected CD4+ T cells harboring integrated and replication-competent HIV genomes persist during ART and are the main obstacle to HIV eradication. In most people with HIV (PWH), the reservoir is extremely stable with a half-life of over 3 years. All prior attempts to significantly reduce its size or accelerate its decay have failed. Using samples from participants in the MERLIN clade B primary HIV infection cohort (Lima, Peru), we observed 5 to 10-times faster decay of the HIV reservoir in individuals initiating ART during the first 3 months of infection compared to those randomized to start ART later, suggesting that HIV-infected cells in people treated early are more susceptible to elimination. Differences in the half-life of the reservoir, which are maintained during at least the first 4 years of ART, offer a unique opportunity to identify mechanisms that could be harnessed to reduce the reservoir in all PWH on ART. In this project, we propose to unravel the cellular and viral features responsible for the rapid clearance or long-term persistence of individual HIV reservoir cells. We will test the hypothesis that the capacity of reservoir cells to persist for prolonged periods is driven by a combination of cellular and viral features, which differ between early and late treated individuals and result in differential reservoir decay. We will take advantage of the unique MERLIN cohort to study HIV reservoir cells in 12 participants who initiated ART less than 3 months after HIV acquisition (early ART: rapid decay) and 12 participants who deferred treatment for 6 months (late ART: slow decay). We will study the early, intermediate and late reservoirs, using cryopreserved leukaphereses (collected at 1 & 2-3 years of ART) and newly collected leukapheresis from the same continually- suppressed participants at >7 years of ART. In Aim 1, we will test the hypothesis that intrinsic cellular features of HIV reservoir cells underlie differences in reservoir decay. We will employ a single cell approach to identify pro-survival factors associated with reservoir stability (Bcl-2, TCF-1, FOXO3A etc.) or that may protect infected cells from immune clearance (ligands of immune checkpoint molecules, Serpin B9, TGF-β). We will also evaluate the clonality of the reservoir with the hypothesis that clonal expansions of intact genomes will be more common in late treated participants. In Aim 2, we will test the hypothesis that specific viral characteristics also contribute to the persistence of HIV-infected cells and will be gradually enriched over time on ART. To determine if specific viruses are selected against during therapy, we will use an assay that couples integration site sequencing with HIV transcription assessment, to determine the proportion of reservoir cells that are transcriptionally active. We will use a novel approach to reconstruct molecular viral clones from the latent reservoir, and will measure the fitness of these viruses and functionally assess their HIV proteomes. In this way, we will determine if the reservoir is gradually enriched in proviruses encoding functional Nef and Vpu, which cause escape of antigen presentation and impede CTL-mediated killing as well as neutralization and ADCC by autologous antibodies. Results from this study will identify cellular and viral mechanisms that can be targeted to accelerate decay of the HIV reservoir.