Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Nussenzweig Laboratory

Abstract

More than 40 years after its onset, the Human Immunodeficiency Virus Type 1 (HIV-1) epidemic remains a major global health burden. HIV-1 is a retrovirus that infects and destroys predominantly CD4+ T cells. Since CD4+ T cells are crucial for the innate and adaptive immune system, their progressive loss in the course of HIV-1 infection leads to the Acquired Immune Deficiency Syndrome (AIDS). Antiretroviral therapy can suppress the virus in people living with HIV and prevent progression to AIDS, but does not provide a cure. Therefore, lifelong antiretroviral therapy is necessary. The barrier to HIV-1 cure is the HIV-1 reservoir, a scarce pool of HIV-1 infected CD4+ T cells that has a very long lifetime, can multiply, and is not affected by antiretroviral therapy. These persisting infected cells have been the focus of HIV research, with the aim to identify potential biomarkers and characterize their gene expression profile in order to tailor a therapeutic approach to HIV-1. Since these cells are exceedingly rare and cannot be distinguished from uninfected CD4+ T cells, ex vivo analysis on a single cell level has not been feasible. In the first part of this thesis, a method for the enrichment of clonally expanded cells harboring an intact HIV-1 provirus that does not require latency reversal is presented. The enrichment is based on CD45RA, a marker that distinguishes naïve from memory CD4+ T cells, as well as on the variable and constant domain of the T cell receptor (TCR) b chain, that are shared by all members of a given infected clone. The resulting enrichment enables the identification of the unique TCRab sequence of infected clones, which is presented in the second part. The TCRab sequence is a unique molecular identifier of a given infected clone and can be read out by commercially available high throughput single cell gene expression sequencing platforms. In the third part, the gene expression profile of six enriched infected clones harboring an intact provirus from six individuals living with HIV-1 is analyzed by single cell gene expression and TCR sequencing on the 10x Genomics platform. Cells pertaining to expanded infected clones harboring an intact provirus are predominantly found in the CD4+ T effector memory compartment, but also belong to other CD4+ memory cell populations. Thus, expanded infected clones display a diverse gene expression profile and are not a homogenous cell population. Furthermore, the infected clones analyzed in this study did not display a unique gene expression profile that would set them apart from uninfected CD4+ T cells. Thus, proviral integration does not appear to shape the host cell neither in a uniform, nor in a unique way.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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