Harnessing Heterogeneity to Isolate and Eradicate the Clonal CD4 HIV Reservoir

PROJECT SUMMARY/ABSTRACT HIV remains a critical global health issue, particularly in Sub-Saharan Africa, where infection rates remain high, and a significant pediatric burden persists. Whilst antiretroviral therapies exist, adherence remains an issue, and the need for a cure remains urgent. A major barrier to a cure is the HIV reservoir, which persists under suppressive treatment. The HIV reservoir consists of clonally expanded populations of infected CD4 cells, which can remain latent for extended periods of time, thereby allowing the virus to evade immune responses and treatment. Clonal expansion can occur over time under suppressive treatment due to various mechanisms including homeostatic proliferation, antigenic stimulation, and CTL resistance. Furthermore, the CD4 reservoir, under suppressive antiretroviral treatment, is a rare population of cells that are difficult to isolate and culture. Early in my postdoctoral career, I developed a methodology to isolate pure CD4 HIV reservoir clones from people living with HIV on suppressive antiretroviral treatment. From this, I was able to isolate pure CD4 HIV reservoir clonal populations and culture them ex vivo. I was able to begin phenotypically, genotypically, and functionally characterizing these clones using flow cytometry based functional assays and CITE-Seq. This method development has laid the foundation for this proposal in optimizing CD4 HIV clonal reservoir isolation and establishing a system to interrogate clone characteristics and to describe the clonal landscape, whilst pursuing functional assays that will inform mechanisms of clone persistence. CD4 HIV reservoir clones can now be analyzed for intrinsic mechanisms of resistance to CTL-mediated killing via flow cytometry functional assays including proliferation assays, CTL-mediated killing assays, and latency reversal agent assays. These experimental efforts will inform clone elimination efforts through enhancement of autologous cytotoxic T cells (CTLs). In Aim 1 I hypothesize that clonal CD4 HIV reservoir isolation methodology will need to be adapted based on the phenotypic characteristics of each unique HIV reservoir to enable successful isolation of pure clonal populations. In Aim 2 I propose priming of autologous CTLs will improve CD4 HIV reservoir elimination through CTL-mediated killing by a slow clearing of active or partially active reservoir clones. And in Aim 3 I propose extending this research into Subtype C HIV populations and pediatric populations, who I hypothesize will be more difficult to isolate CD4 HIV reservoir clones from given the reduced reservoir size in pediatric populations infected with HIV. I propose novel approaches to obtain pure clonal populations of CD4 T cells carrying HIV proviruses and phenotypic, genotypic, and functional characterization of clones. This study will enhance our understanding of the HIV reservoir by providing a unique opportunity to study the HIV reservoir landscape in pure infected clonal populations.