Characterize HIV-Infected T-Cell Clones on ART and Impact of Experimental Interventions

Division Of Basic Sciences - Nci

Investigators

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Abstract

We and others showed that HIV-infected cells can clonally expand and persist despite ART, and that the proviral integration site may influence this phenomenon. A major focus of this project is to determine how commonly such clonally expanded, infected cells that persist during ART carry intact HIV proviruses and in which tissues and cell types they persist. To answer this question, we improved the throughput of our method called multiple-displacement amplification single-genome sequencing (MDA-SGS) (Patro et al., PNAS 116:25891-25899, 2019) and we are developing a new method called PIT (provirus, integration, TCR)-seq which will allow us to analyze HIV proviruses that persist on ART and to characterize the cells that harbor them. To date, we have used the MDA-SGS assay to determine if HIV proviruses that persist during ART in blood and tissues have intact sequences or contain lethal mutations or deletions. Analysis of the data generated using PIT-seq will provide a better understanding of T cell proliferation in maintaining the HIV reservoir and may reveal new avenues for development of personalized approaches to achieving HIV remission without ART. In PIT-seq, RNA and DNA from individual infected cells are compartmentalized in nanoliter scale droplets or capsules, and these collections of genetic material are segregated both from collections of uninfected cell RNA and DNA and from each other. The droplets or capsules are dispensed into separate wells of a microtiter plate, enabling discrete downstream single-cell analyses such as full-length HIV sequencing, integration sites analysis (ISA), and T-cell receptor (TCR) sequencing of the host cells. Future iterations of the assay will also include single-cell RNA sequencing to profile transcriptomes of single infected cells with intact and defective proviruses, including measuring the expression of KRAB-ZNF genes with integrated HIV compared to their expression levels in uninfected cells. PIT-seq will be used to characterize hundreds of single infected cells in PWH on ART to answer questions such as, "What cell types are infected with HIV", "Do proviral structures within clones change over time (e.g., become solo LTRs)? What are some of the specific cognate antigens of clonotypes carrying intact HIV proviruses?" and many more. To date, we have successfully generated both droplets and capsules housing single cells and have optimized the approach to detect and segregate them into separate wells of a 96-well plate. In Project 1, we are also continuing our studies in children born with HIV. Recent reports have claimed that naïve CD4+ T cells are an important reservoir of HIV. However, skeptics claim that the lack of, or only transient, expression of the HIV co-receptor CCR5 prevents this cell type from being infected in vivo and that previous studies may have detected HIV proviruses in memory T cells that contaminated their cell sorts. As an intramurally funded lab, we have the opportunity to dedicate resources to perform investigations on controversial areas of research and to perform the many controls needed to address possible contamination. Because approximately 90% of CD4+ T cells in infants and children are naïve, addressing this question in children born with the virus provides the best opportunity to collect naïve cells of high purity, allowing us to address questions in children that have not previously been investigated including, "Are naïve T cells in children infected with HIV? Do they persist on ART? Can they proliferate to form infected cell clones? Do they contain intact proviruses? Are their levels, integration sites, or proviral structures different than in memory T cells?" To answer these questions, we collaborated with Drs. Maud Mavigner and Ann Chahroudi (both at Emory) to collect naïve and memory CD4+ T cells from children born with HIV and on long-term ART from the CFAR specimen repository study (IRB00009146, Emory University). We performed our MDA-SGS assay to quantify the number of infected naïve and memory cells, obtain their sites of integration in the host genomes, and sequence their proviral structures. With 95% statistical confidence, we consistently found naïve CD4+ T cells containing HIV proviruses at levels that cannot be explained by memory cell contamination, though naïve T cell infection frequencies were 20-fold lower than in the memory subsets from the same children. A median of 4.7% of infected naïve CD4+ T cells also contained HIV genetic elements consistent with intactness (Psi+RRE), and we confirmed intactness of one of these proviruses by full-length proviral sequencing. ISA revealed several large, infected T-cell clones in both sorted naïve and memory cell collections, although none of these were found in both cell subsets. Also notable is that the divergent integration site profiles significantly reflected the differential gene expression patterns of naïve and memory T-cells from RNA-seq data. These results demonstrate that HIV persists in children in both naïve and memory CD4+ T cells and that they can undergo clonal expansion and harbor intact proviruses, consistent with reports in adults. Our findings highlight the need to address and target this important reservoir for HIV on ART (Katusiime, Neer, et al. JCI 2025). Although we previously published our finding that the largest infected T cell clones in children contain solo-LTR proviruses and that intact proviruses that persist on ART are present in both naïve and memory CD4+ T cells, other proviral structures in children on long-term ART had not been characterized as fully as they have in adults. In Project 1a, we applied near full-length (NFL)-SGS to children who were born with HIV and had their viremia suppressed on long-term ART. The proviruses in these children, compared to children before ART or on short-term ART who received either immediate or delayed treatment, contained more large internal deletions, especially of the env gene. Only 2.5% of the proviruses were intact, lower than in comparable datasets from adults. Of the proviruses that retained the env gene, >80% had two or more defects, the most common being stop codons and/or gag start mutations. Significantly fewer defects in the major spice donor site (MSD) and packaging signal (Psi) were found in the children on long-term ART compared to adults, and tat was more frequently defective in children. These findings suggest that different selection pressures may shape the proviral landscape in children compared to adults, consistent with a recent study showing a profound reduction of intact proviruses in children born with HIV and on ART for 28 years, and reveal potentially different genetic regions to target in HIV reservoir intactness proxy assays, such as the gag start codon rather than Psi, and provide new information that may be informative in the design of strategies to control HIV infection in children (Hasson, et al. Viruses 2025).