Friday, April 19, 2024
4/19/2024
PROJECT SUMMARY/ABSTRACT HIV-1 cannot be eradicated by antiretroviral therapies alone. The major obstacle to eradicating HIV-1 is the ability of integrated, replication competent viral DNA to persist latently in cellular reservoirs. Despite there being a significant and extensive knowledge base regarding HIV-1 infection of the CNS and the development of neurological disorders in HIV-1 infected viremic individuals, there is only a very limited understanding of the mechanisms of persistence in the CNS following viral suppression. Despite some data supporting the CNS as a potential reservoir of HIV-1 in virally suppressed individuals, there remains critical gaps in our knowledge regarding the location, frequency and nature of viral persistence in the CNS. This data is critical to both developing strategies aimed at the development of both a functional and/or sterilising cure. The goal of our proposal is to characterise the persistent reservoir of HIV in the CNS, specifically the myeloid reservoir, and the replication competence of the persistent virus. The goals of this proposal, in response to the “Role of Myeloid Cells in Persistence and Eradication of HIV-1 Reservoirs from the Brain” RFA-MH-20-702 are to (i) use highly sensitive PCR based to quantify and characterise the intact and defective HIV composing the myeloid reservoir in the CNS of virally suppressed patients, and (ii) characterise the replication competence of the CNS myeloid reservoir. In Aim 1 we will use highly sensitive and well established PCR assays, including the intact proviral DNA assay (IPDA) in conjunction with sensitive immunohistochemistry and a modified laser microdissection technology to determine the quantity and phenotypical location of the potentially intact HIV viral genomes CNS and non-CNS tissue from a cohort virally suppressed individuals. We will determine the compartmentalisation of CNS derived genomes by comparison to non-CNS tissues. In Aim 2, we will comprehensively characterise the replication competent nature of proviruses from the myeloid CNS reservoir. Briefly, a subset of intact or defective proviruses isolated from myeloid cells of the CNS of virally suppressed individuals (as determined by IPDA) will be confirmed by full-length individual proviral sequencing (FLIPS) and cloned into expression vectors to characterise the replication competence in both macrophage and T cells. Importantly, the ability of defective proviruses to continue to produce RNA and/or neurotoxic proteins including tat and nef will be measured using retroviral vector systems or transfection. We hypothesise based on our preliminary data that whilst the majority of proviruses in the myeloid CNS reservoir will not generate infectious virions, a subset of proviruses will be replication competent. Furthermore, a subset of defective proviruses will produce viral proteins. These data are essential to the development of HIV cure strategies.
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