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Human immunodeficiency virus (HIV) persists in cells despite antiretroviral therapy; however, the influence of cellular mechanisms such as activation, differentiation, and proliferation upon the distribution of proviruses over time is unclear. To address this, we used full-length sequencing to examine proviruses within memory CD4+ T-cell subsets longitudinally in 8 participants. Over time, the odds of identifying a provirus increased in effector and decreased in transitional memory cells. In all subsets, more activated (HLA-DR–expressing) cells contained a higher frequency of intact provirus, as did more differentiated cells such as transitional and effector memory subsets. The proportion of genetically identical proviruses increased over time, indicating that cellular proliferation was maintaining the persistent reservoir; however, the number of genetically identical proviral clusters in each subset was stable. As such, key biological processes of activation, differentiation, and proliferation influence the dynamics of the HIV reservoir and must be considered during the development of any immune intervention.

Objective The 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/methods Full-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. Results HLA-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. Conclusion The 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.

Background Subject C135 is one of the members of the Sydney Blood Bank Cohort, infected in 1981 through transfusion with attenuated nef/3′ long terminal repeat (LTR)-deleted HIV-1, and has maintained undetectable plasma viral load and steady CD4 cell count, in the absence of therapy. Uniquely, C135 combines five factors separately associated with control of viraemia: nef/LTR-deleted HIV-1, HLA-B57, HLA-DR13, heterozygous CCR5 Δ32 genotype and vigorous p24-stimulated peripheral blood mononuclear cell (PBMC) proliferation. Therefore, we studied in detail viral burden and immunological responses in this individual. Methods PBMC and gut and lymph node biopsy samples were analysed for proviral HIV-1 DNA by real-time and nested PCRs, and nef/LTR alleles by nested PCR. HIV-specific antibodies were studied by Western blotting, and CD4+ and CD8+ T lymphocyte responses were measured by proliferation and cytokine production in vitro. Results PBMC samples from 1996, but not since, showed amplification of nef alleles with gross deletions. Infectious HIV-1 was never recovered. Proviral HIV-1 DNA was not detected in recent PBMC or gut or lymph node biopsy samples. C135 has a consistently weak antibody response and a substantial CD4+ T cell proliferative response to a previously described HLA-DR13-restricted epitope of HIV-1 p24 in vitro, which augmented a CD8+ T cell response to an immunodominant HLA-B57-restricted epitope of p24, while his T cells show reduced levels of CCR5. Conclusions Subject C135's early PCR and weak antibody results are consistent with limited infection with a poorly replicating nef/LTR-deleted strain of HIV-1. With his HLA-B57-restricted gag-specific CD8 and helper HLA-DR13-restricted CD4 T cell proliferative responses, C135 appears to have cleared his HIV-1 infection 37 years after transfusion.

OBJECTIVE: To investigate the origin of the HIV-1 viremia induced by the latency-reversing agent romidepsin.DESIGN: Six individuals on suppressive antiretroviral therapy received romidepsin administered intravenously once weekly for 3 consecutive weeks. CD4 T cells were obtained at baseline, following the second and third romidepsin infusion, and 10 weeks after the final romidepsin treatment. Plasma samples were collected 24 and 72 h after romidepsin infusions.METHODS: Single-genome sequencing of the env and p24-RT region was used to genetically characterize the virus from proviral DNA, the transcribed cell-associated RNA and the plasma RNA pool.RESULTS: In three of six participants with available plasma samples we identified plasma HIV-1 RNA sequences that were identical to DNA and/or cell-associated RNA sequences from peripheral blood CD4 T cells. In two participants, plasma RNA sequences contained expansions of identical sequences, corresponding to 62 and 100% of the total sequences, respectively. Plasma HIV-1 RNA had very low amounts of defective viruses compared to cell-associated RNA (odds ratio 20.85, P CONCLUSIONS: Romidepsin induced transcription from proviruses in peripheral blood cells, which contributed to viremia in patients on suppressive therapy. The intermingling of these cell-associated HIV-1 RNA with DNA sequences indicates transcription from a diverse range of proviruses, but the expansions of identical viral plasma sequences with few defects indicate that the romidepsin-induced viremia arises from intact proviruses with highly similar or identical genetic backgrounds.

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., 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.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5' and 3' LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Dendritic cells (DCs) and macrophages are present in the tissues of the anogenital tract, where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and represent the first opportunity for the virus to interfere with innate recognition. Previously we have shown that both cell types fail to produce type I interferons (IFNs) in response to HIV-1 but that, unlike T cells, the virus does not block IFN induction by targeting IFN regulatory factor 3 (IRF3) for cellular degradation. Thus, either HIV-1 inhibits IFN induction by an alternate mechanism or, less likely, these cells fail to sense HIV-1. Here we show that HIV-1 (but not herpes simplex virus 2 [HSV-2] or Sendai virus)-exposed DCs and macrophages fail to induce the expression of all known type I and III IFN genes. These cells do sense the virus, and pattern recognition receptor (PRR)-induced signaling pathways are triggered. The precise stage in the IFN-inducing signaling pathway that HIV-1 targets to block IFN induction was identified; phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1) was completely inhibited. Two HIV-1 accessory proteins, Vpr and Vif, were shown to bind to TBK1, and their individual deletion partly restored IFN-β expression. Thus, the inhibition of TBK1 autophosphorylation by binding of these proteins appears to be the principal mechanism by which HIV-1 blocks type I and III IFN induction in myeloid cells. IMPORTANCE Dendritic cells (DCs) and macrophages are key HIV target cells. Therefore, definition of how HIV impairs innate immune responses to initially establish infection is essential to design preventative interventions, especially by restoring initial interferon production. Here we demonstrate how HIV-1 blocks interferon induction by inhibiting the function of a key kinase in the interferon signaling pathway, TBK1, via two different viral accessory proteins. Other viral proteins have been shown to target the general effects of TBK1, but this precise targeting between ubiquitination and phosphorylation of TBK1 is novel.

Summary 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.

OBJECTIVE: To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells.DESIGN: Case report.METHODS: Blood was collected 30 and 18 months prior to presentation with Sezary syndrome, at the time of presentation and during alemtuzumab. T-cell subsets in malignant (CD7-CD26-TCR-VBeta2+) and nonmalignant cells were quantified by flow cytometry. HIV-DNA in total CD4 T cells, in sorted malignant and nonmalignant CD4 T cells, was quantified by PCR and clonal expansion of HIV-DNA assessed by full-length next-generation sequencing.RESULTS: HIV-hepatitis B virus coinfection was diagnosed and antiretroviral therapy initiated 4 years prior to presentation with Sezary syndrome and primary cutaneous anaplastic large cell lymphoma. The patient received alemtuzumab 10 mg three times per week for 4 weeks but died 6 weeks post alemtuzumab. HIV-DNA was detected in nonmalignant but not in malignant CD4 T cells, consistent with expansion of a noninfected CD4 T-cell clone. Full-length HIV-DNA sequencing demonstrated multiple defective viruses but no identical or expanded sequences. Alemtuzumab extensively depleted T cells, including more than 1 log reduction in total T cells and more than 3 log reduction in CD4 T cells. Finally, alemtuzumab decreased HIV-DNA in CD4 T cells by 57% but HIV-DNA remained detectable at low levels even after depletion of nearly all CD4 T cells.CONCLUSION: Alemtuzumab extensively depleted multiple T-cell subsets and decreased the frequency of but did not eliminate HIV-infected CD4 T cells. Studying the effects on HIV persistence following immune recovery in HIV-infected individuals who require alemtuzumab for malignancy or in animal studies may provide further insights into novel cure strategies.

Background: Allogeneic 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. Methods: We 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. Results: All 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. Conclusions: These 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.

Long-lived latently infected resting CD4+ T cells are the main reason why current antiretroviral therapy (ART) is unable to cure HIV infection [1]. Recent work has suggested that the expression of immune checkpoint markers, such as programmed death-1 (PD1), may play a role in viral persistence on ART via either suppression of virus transcription and/or reduced HIV-specific T cell activity [2,3], but the role of cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) in HIV persistence on ART is not clear. Ipilimumab (Yervoy, Bristol-Myers Squibb, New York, New York) is a human immunoglobulin G1 antibody to CTLA-4 that inhibits binding of CTLA-4, expressed on activated T cells and regulatory T cells (Tregs), to its ligands CD80 and CD86. The drug is used to treat metastatic melanoma and has been associated with multiple changes in immune function thought to enhance antitumor T cell function [4]. In HIV-infected individuals, CTLA-4 expression on CD4+ T cells correlates with HIV disease progression [5], and loss of HIV-specific CD4+ T cell function can be reversed in vitro by CTLA-4 blockade [5–7]. In a simian immunodeficiency virus (SIV) macaque model, CTLA-4 blockade led to an increase in T-cell activation and viral replication [8]. Here, we describe changes in the HIV reservoir in an HIV-infected patient on ART who received ipilimumab for the treatment of metastatic melanoma. At initiation of ipilimumab treatment in October 2013 for disseminated melanoma, the patient was a 51-year-old man diagnosed with HIV in 1986 and with a CD4+ nadir of 159 cells/μl in 1995. He was on ART since 1996 and plasma HIV RNA was less than 400 copies/ml from 2004 and less than 20 copies/ml from July 2012 (Fig. 1a). He received four doses of ipilimumab 3 mg/kg given at three-weekly intervals. Fig. 1 Clinical details and changes and impact of ipilimumab on virological and immunological parameters Whilst receiving ipilimumab, there was no overall change in plasma HIV RNA as measured by the Roche viral load assay [lower limit of detection (LLOD) = 20 copies/ml; Fig. 1c]. Using a sensitive single-copy HIV RNA assay (SCA) (LLOD = 0.3 copies/ml) [9], there was a cyclical decrease in plasma HIV RNA following each infusion and an overall decline from 60 to 5 copies/ml (Fig. 1c). Given more frequent sampling was performed with the SCA, we believe that longitudinal changes over time were best assessed with this assay. There was an increase in CD4+ T cells after each infusion (overall change from 610 to 900 cells/μl) (Fig. 1b). This increase was predominantly in total memory (Fig. 1d) and effector memory CD4+ T cells (Fig. 1e). Postinfusion increases in CD4+ T-cell activation were seen as measured by human leukocyte antigen-DR and CD38 and CCR5 expression (Fig. 1f). There were transient increases in CD8+ T cells following the second and third infusions, but no overall change in CD8+ T cell activation (Fig. 1g). Cell-associated unspliced HIV RNA in sorted CD4+ T cells was quantified with increases observed following the first and second infusions, with a maximum change from baseline of 19.6-fold (Fig. 1h). The changes in cell-associated unspliced HIV RNA was greater than those recently reported, following the administration of the histone deacetylase inhibitors vorinostat [10,11] or panobinostat [12], or following disulfiram [13]. There was no change in cell-associated HIV DNA (Fig. 1i), but any change in the small proportion of cells with HIV DNA containing inducible proviruses [14] may not have been detectable with the assays used here. Acknowledging the limitations deriving from this being a single case, we speculate the increase in cell-associated unspliced RNA could have been due to mechanisms, including an increase in HIV RNA transcription secondary to blocking the inhibitory effects of CTLA-4 on T cell transcription, similar to that described following ex-vivo anti-PD1 treatment of CD4+ T cells from HIV-infected patients on ART [15]; redistribution or expansion of effector memory CD4+ T cells that may have a higher ratio of cell-associated HIV RNA to HIV DNA [16] (Satish Pillai, San Francisco, UCSF, San Francisco, California, personal communication); or redistribution or expansion of activated T cells including Tregs. The increase in cell-associated unspliced HIV RNA and decline in SCA was intriguing, perhaps mediated by elimination of latently infected CD4+ T cells that were induced to express viral antigens. But the rapidity of the decline in SCA makes this somewhat unlikely. Blockade of CTLA-4 with ipilimumab in an HIV-infected patient on ART had significant effects on the total number and phenotype of CD4+ T cells and induced a profound increase in cell-associated unspliced HIV RNA with onset after the first dose and was associated with subsequent decline in plasma HIV RNA. Further studies are warranted to determine if ipilimumab could play a role in eliminating latently infected cells in HIV-infected patients on ART.

Author(s): Fromentin, R; Massanella, M; Vandergeeten, C; Barton, K; Hiener, B; Chiu, WW; Looney, D; Ramgopal, M; Richman, DD; Trautmann, L; Palmer, S; Chomont, N

Aims Two HIV-1 infected patients were studied post-haematopoietic stem cell transplant (HSCT) to determine effects on the viral reservoir. Methods HIV-1 Ag/Ab was measured by chemiluminescence microparticle immunoassay (CMIA) and western blot (WB). Specific CD4+T-cell responses were measured by CD25/CD134 up-regulation. HIV-1 RNA was measured in serum and HIV-1 DNA was measured in peripheral blood mononuclear cells (PBMCs) and CD4+T-cells by PCR. CCR5 phenotype in PBMC was determined by PCR. Results Patient A and B underwent HSCT for non-Hodgkin's lymphoma and acute myeloid leukemia, respectively. Both remained on antiretroviral therapy. Patient A experienced systemic graft-versus-host disease (GVHD), whereas Patient B developed skin GVHD. Patient A was heterozygous for the CCR5Δ32 mutation and Patient B was wild-type. Following HSCT, both patients had undetectable HIV-1 RNA in plasma, and undetectable HIV-1 DNA in PBMCs and CD4+T-cells. Antigen-specific CD4+T-cell responses were absent. Pre-transplant, Ag/Abs to HIV-1 was detectable. Post-transplant, patients had low-level Ag/Abs on CMIA, but trace (Patient A) and absent (Patient B) antibody by WB. Conclusion HSCT reduced the HIV-1 reservoir size in these two patients with undetectable HIV-1 RNA in serum and DNA in PBMC/CD4+T-cells and absent antigen-specific CD4+T-cell responses. A profound reduction in Ab to HIV was demonstrated in both patients by WB.