Investigating the latent HIV-1 reservoir in lymphoid tissue using multiplexed imaging and spatial transcriptomics

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PROJECT SUMMARY/ABSTRACT The introduction of combination antiretroviral therapy (ART) has led to a dramatic improvement in lifespan for individuals infected with HIV-1, but any interruption of treatment leads to rapid resumption of viral replication, requiring patients to follow a strict drug regimen. HIV-1 establishes a stable reservoir within days of transmission before the virus can be detected in the peripheral blood and can persist as replication-competent proviruses inside cells, known as the latent reservoir. ART is incapable of targeting these latent reservoirs; therefore, a cure for HIV-1 is needed. Treatments that target the latent reservoir have thus far been unsuccessful, largely because the latent reservoir is poorly understood. Due to ease of sample access, the vast majority of studies of the latent viral reservoir have been performed with peripheral blood. However, lymphoid tissue are the principal sites of viral replication and HIV-1 has been shown to persist in lymphoid tissue of patients on ART who have undetectable viral loads in the blood. The few prior studies of the latent reservoir in lymphoid tissue were limited by approaches that could only target a few markers or required the preparation of cell suspensions, which can lead to changes in cell phenotype or loss of cells, as well as loss of spatial information. Our group has previously developed Multiplexed Ion Beam Imaging, which uses secondary ion mass spectrometry to visualize up to 40 proteins at subcellular resolution in a single tissue. By combining this novel imaging technology with spatial transcriptomics to assess immune features in lymphoid tissue, we will be able to identify the cells and mechanisms important for viral persistence. We recently applied this method in rhesus macaque tissue, demonstrating an immunosuppressive microenvironment in SIV+ macaque lymph nodes. Building on this work, I hypothesize that applying novel imaging and spatial transcriptomic methods will reveal an immunosuppressive phenotype in lymphoid tissue of HIV+ patients. This work will yield a comprehensive picture of the infected cells, cell function, and tissue structure within the vicinity of the HIV-1 reservoir. In Aim 1, I will extend MIBI to the imaging of HIV RNA and DNA using in situ hybridization, enabling the simultaneous quantification of viral RNA and DNA and cellular protein to fully characterize infected cells. In Aim 2, I will assess the spatial structure and follicle heterogeneity within the lymphoid tissue of viremic patients to identify features of the microenvironment in which cells are actively responding against the virus. In Aim 3, I will compare the immune features between viremic patients, aviremic patients, and healthy individuals to uncover features of the immune microenvironment that are important to viral persistence after ART. This work will improve our understanding of the latent viral reservoir in lymphoid tissue, which is a critical barrier to HIV-1 eradication. This fellowship will give me the scientific training to prepare me for a postdoctoral fellowship and development into an independent scientist.