Cellular and viral determinants of the persistent HIV reservoir

Eradication of the latent HIV reservoir remains the major stumbling block to achieving cure. To eliminate this
reservoir, accurate definition (a biomarker) of latently infected cells of different types and within different
tissues is highly needed. Several biomarkers proposed previously were able to very modestly enrich (~10-fold)
for latently infected cells, but failed to capture the substantial portion of the latent reservoir. Without the
detailed characterization of latently infected cells, identification of a suitable biomarker to capture the majority
of the reservoir cells will remain challenging. Our long-term goal is to identify a biomarker of HIV latency that
can be translated into strategies to target latently infected cells for elimination. The overall objectives of this
application are to identify cellular and viral determinants of the persistent HIV reservoir and to test selected
biomarkers for their ability to capture latently infected cells in vitro and ex vivo. Our central hypothesis is that a
successful biomarker will be represented by reservoir determinants identified individually for cells of different
phenotypes and states. The term “phenotype” refers to the canonical phenotypic subsets defined with widely
used surface protein markers (for example, maturation phenotype – central memory; functional phenotype – T
helper 17). The term “state” refers to a cell state more broadly defined by the cell's total transcriptomic
signature: gene sets and pathways that are actively expressed. The rationale of the proposed research is the
expected improvement in the efficiency of the reservoir capture when heterogeneity of the reservoir cells is
taken into account. We will test our central hypothesis by pursuing the following specific aims: (1) Identify
cellular determinants of different reservoir subsets and test selected biomarkers for cell enrichment in vitro; (2)
Identify viral determinants of different reservoir subsets in vitro; (3) Validate the reservoir determinants and
selected biomarkers using samples from people with HIV. To identify cellular and viral determinants of the
persistent reservoir, latest innovations in RNA sequencing (RNA-Seq) technologies will be used. Single cell
RNA-Seq coupled with immunophenotyping will be used to characterize the phenotypes and states of cells that
can be infected with either CXCR4- or CCR5-tropic virus. Genes that can discriminate between latently
infected and uninfected cells will be identified individually within each cell type. To inform on how the identified
cellular determinants of the persistent reservoir relate to the type of provirus that they define, proviral activity in
different cell types will be characterized using single cell RNA-Seq data, full length sequencing of HIV
transcripts, and the PrimeFlow assay to quantify responsiveness of provirus to reactivation stimuli. Biomarkers
will be selected from sets of cellular determinants of the HIV reservoir in different cell subsets. Antibodies
against these proteins will be tested for the ability to efficiently capture the latently infected cells. Our ultimate
goal is to ensure that identified biomarkers can accurately define latently infected cells in clinical samples, and
specifically in different tissue compartments, where as much as 98% of the persistent reservoir resides in vivo.
In collaboration with the Last Gift cohort, we will have a unique opportunity to conduct studies to validate the
identified determinants and biomarkers using blood and lymphoid tissue samples from persons with HIV. When
these studies are complete, we will have identified biomarkers that can be used to capture latently infected
cells in vitro and ex vivo, with the efficiency of at least 500-fold greater than is currently achievable. These
results will be significant because identified biomarkers can be used to isolate reservoir cells from different
tissues to provide better characterization of the latent reservoir across the human body. In the future, these
biomarkers can serve as a platform for development of strategies to target latently infected cells for elimination.
Such research is important to address the needs of people living with HIV, including the large cohort of HIV-
infected patients within the national VA Healthcare System.

Grant Number: 5I01BX005285-04
NIH Institute/Center: VA
Principal Investigator: Nadejda Beliakova-Bethell