HIV-1 Genomes Are Enriched in Memory CD4+ T-Cells with Short Half-Lives.

Future HIV-1 curative therapies require a thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets during antiretroviral therapy (ART) and the cellular mechanisms that maintain this reservoir. Therefore, we sequenced near-full-length HIV-1 genomes and identified genetically-intact and genetically-defective genomes from resting naive, stem-cell memory, central memory, transitional memory, effector memory, and terminally-differentiated CD4+ T-cells with known cellular half-lives from 11 participants on ART. We find that a higher infection frequency with any HIV-1 genome was significantly associated with a shorter cellular half-life, such as transitional and effector memory cells. A similar enrichment of genetically-intact provirus was observed in these cells with relatively shorter half-lives. We found that effector memory and terminally-differentiated cells also had significantly higher levels of expansions of genetically-identical sequences, while only transitional and effector memory cells contained genetically-intact proviruses that were part of a cluster of identical sequences. Expansions of identical sequences were used to infer cellular proliferation from clonal expansion. Altogether, this indicates that specific cellular mechanisms such as short half-life and proliferative potential contribute to the persistence of genetically-intact HIV-1. IMPORTANCE The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically-intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir. Our investigations into the possible cellular mechanisms maintaining the HIV-1 reservoir in different T-cell subsets have revealed a link between the half-lives of T-cells and the level of proviruses they contain. Taken together, we believe our study shows that more differentiated and proliferative cells, such as transitional and effector memory T-cells, contain the highest levels of genetically-intact proviruses, and the rapid turnover rate of these cells contributes to the expansion of genetically-intact proviruses within them. Therefore, our study delivers an in-depth assessment of the cellular mechanisms, such as cellular proliferation and half-life, that contribute to and maintain the latent HIV-1 reservoir

High levels of genetically intact HIV in HLA-DR+ memory T cells indicates their value for reservoir studies.

ObjectiveThe contribution of HLA-DR+ memory CD4 T cells to the HIV reservoir during prolonged antiretroviral therapy is unclear as these cells are commonly excluded when assessing for replication-competent HIV. To address this issue, we examined the distribution of genetically intact HIV DNA within HLA-DR- and HLA-DR+ memory CD4 T cells and the RNA transcriptional profile of these cells during antiretroviral therapy.Design/methodsFull-length DNA sequencing was used to examine the HIV DNA landscape within HLA-DR+ and HLA-DR- memory CD4 T cells. RNA quantification and sequencing was used to interrogate the relationship between HLA-DR status and HIV RNA transcription.ResultsHLA-DR+ CD4 T cells contained a high frequency of genetically intact HIV genomes, contributing over half of the genetically intact viral sequences to the reservoir. Expansions of genetically identical sequences were identified in all T-cell subsets, indicating that cellular proliferation maintains genetically intact and defective viral DNA during therapy. Intracellular HIV RNA levels in HLA-DR+ and HLA-DR- T cells were not statistically different by either long terminal repeat quantitative PCR quantification or single-genome RNA sequencing of the p6-RT region.ConclusionThe high proportion of intact viral DNA sequences in the proliferative HLA-DR+ subset suggests they are critical in maintaining HIV infection during effective therapy. As such, these cells should be included in any immune intervention targeting HIV during effective therapy

Identification of Genetically Intact HIV-1 Proviruses in Specific CD4+ T Cells from Effectively Treated Participants

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir

Impact of Antiretroviral Therapy Duration on HIV-1 Infection of T Cells within Anatomic Sites.

Understanding the impact of antiretroviral therapy (ART) duration on HIV-infected cells is critical for developing successful curative strategies. To address this issue, we conducted a cross-sectional/inter-participant genetic characterization of HIV-1 RNA from pre- and on-therapy plasmas and HIV-1 DNA from CD4+ T cell subsets derived from peripheral blood (PB), lymph node (LN), and gut tissues of 26 participants after 3 to 17.8 years of ART. Our studies revealed in four acute/early participants who had paired PB and LN samples a substantial reduction in the proportion of HIV-infected cells per year on therapy within the LN. Extrapolation to all 12 acute/early participants estimated a much smaller reduction in the proportion of HIV-1-infected cells within LNs per year on therapy that was similar to that in the participants treated during chronic infection. LN-derived effector memory T (TEM) cells contained HIV-1 DNA that was genetically identical to viral sequences derived from pre- and on-therapy plasma samples. The proportion of identical HIV-1 DNA sequences increased within PB-derived TEM cells. However, the infection frequency of TEM cells in PB was stable, indicating that cellular proliferation that compensates for T cell loss over time contributes to HIV-1 persistence. This study suggests that ART reduces HIV-infected T cells and that clonal expansion of HIV-infected cells maintains viral persistence. Importantly, LN-derived TEM cells are a probable source of HIV-1 genomes capable of producing infectious HIV-1 and should be targeted by future curative strategies.IMPORTANCE HIV-1 persists as an integrated genome in CD4+ memory T cells during effective therapy, and cessation of current treatments results in resumption of viral replication. To date, the impact of antiretroviral therapy duration on HIV-infected CD4+ T cells and the mechanisms of viral persistence in different anatomic sites is not clearly elucidated. In the current study, we found that treatment duration was associated with a reduction in HIV-infected T cells. Our genetic analyses revealed that CD4+ effector memory T (TEM) cells derived from the lymph node appeared to contain provirus that was genetically identical to plasma-derived virions. Moreover, we found that cellular proliferation counterbalanced the decay of HIV-infected cells throughout therapy. The contribution of cellular proliferation to viral persistence is particularly significant in TEM cells. Our study emphasizes the importance of HIV-1 intervention and provides new insights into the location of memory T cells infected with HIV-1 DNA, which is capable of contributing to viremia

Impact of Antiretroviral Therapy Duration on HIV-1 Infection of T Cells within Anatomic Sites.

Understanding the impact of antiretroviral therapy (ART) duration on HIV-infected cells is critical for developing successful curative strategies. To address this issue, we conducted a cross-sectional/inter-participant genetic characterization of HIV-1 RNA from pre- and on-therapy plasmas and HIV-1 DNA from CD4+ T cell subsets derived from peripheral blood (PB), lymph node (LN), and gut tissues of 26 participants after 3 to 17.8 years of ART. Our studies revealed in four acute/early participants who had paired PB and LN samples a substantial reduction in the proportion of HIV-infected cells per year on therapy within the LN. Extrapolation to all 12 acute/early participants estimated a much smaller reduction in the proportion of HIV-1-infected cells within LNs per year on therapy that was similar to that in the participants treated during chronic infection. LN-derived effector memory T (TEM) cells contained HIV-1 DNA that was genetically identical to viral sequences derived from pre- and on-therapy plasma samples. The proportion of identical HIV-1 DNA sequences increased within PB-derived TEM cells. However, the infection frequency of TEM cells in PB was stable, indicating that cellular proliferation that compensates for T cell loss over time contributes to HIV-1 persistence. This study suggests that ART reduces HIV-infected T cells and that clonal expansion of HIV-infected cells maintains viral persistence. Importantly, LN-derived TEM cells are a probable source of HIV-1 genomes capable of producing infectious HIV-1 and should be targeted by future curative strategies.IMPORTANCE HIV-1 persists as an integrated genome in CD4+ memory T cells during effective therapy, and cessation of current treatments results in resumption of viral replication. To date, the impact of antiretroviral therapy duration on HIV-infected CD4+ T cells and the mechanisms of viral persistence in different anatomic sites is not clearly elucidated. In the current study, we found that treatment duration was associated with a reduction in HIV-infected T cells. Our genetic analyses revealed that CD4+ effector memory T (TEM) cells derived from the lymph node appeared to contain provirus that was genetically identical to plasma-derived virions. Moreover, we found that cellular proliferation counterbalanced the decay of HIV-infected cells throughout therapy. The contribution of cellular proliferation to viral persistence is particularly significant in TEM cells. Our study emphasizes the importance of HIV-1 intervention and provides new insights into the location of memory T cells infected with HIV-1 DNA, which is capable of contributing to viremia

Impact of Allogeneic Hematopoietic Stem Cell Transplantation on the HIV Reservoir and Immune Response in 3 HIV-Infected Individuals

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) can lead to significant changes to the HIV reservoir and HIV immune responses, indicating that further characterization of HIV-infected patients undergoing HSCT is warranted.MethodsWe studied 3 patients who underwent HSCT after either reduced intensity conditioning or myeloablative conditioning regimen. We measured HIV antigens and antibodies (Ag/Ab), HIV-specific CD4 T-cell responses, HIV RNA, and DNA in plasma, peripheral blood mononuclear cells, isolated CD4 T cells from peripheral blood, and lymph node cells. The patients remained on antiretroviral therapy throughout the follow-up period.ResultsAll patients have been in continued remission for 4-6 years post-HSCT. Analyses of HIV RNA and DNA levels showed substantial reductions in HIV reservoir-related measurements in all 3 patients, changes in immune response varied with pronounced reductions in 2 patients and a less dramatic reduction in 1 patient. One patient experienced unexpected viral rebound 4 years after HSCT.ConclusionsThese 3 cases highlight the substantial changes to the HIV reservoir and the HIV immune response in patients undergoing allogeneic HSCT. The viral rebound observed in 1 patient indicates that replication competent HIV can re-emerge several years after HSCT despite these marked changes