CXCR4-Using HIV Strains Predominate in Naive and Central Memory CD4+ T Cells in People Living with HIV on Antiretroviral Therapy: Implications for How Latency Is Established and Maintained.

HIV can persist in people living with HIV (PLWH) on antiretroviral therapy (ART) in multiple CD4+ T cell subsets, including naive cells, central memory (CM) cells, transitional (TM) cells, and effector memory (EM) cells. Since these cells express different levels of the viral coreceptors CXCR4 and CCR5 on their surface, we sought to determine whether the HIV envelope protein (Env) was genotypically and phenotypically different between CD4+ T cell subsets isolated from PLWH on suppressive ART (n = 8). Single genome amplification for the HIV env gene was performed on genomic DNA extracts from different CD4+ T cell subsets. We detected CXCR4-using (X4) strains in five of the eight participants studied, and in these participants, the prevalence of X4 strains was higher in naive CD4+ T cells than in the memory subsets. Conversely, R5 strains were mostly found in the TM and EM populations. Identical sets of env sequences, consistent with clonal expansion of some infected cells, were more frequent in EM cells. These expanded identical sequences could also be detected in multiple CD4+ T cell subsets, suggesting that infected cells can undergo T cell differentiation. These identical sequences largely encoded intact and functional Env proteins. Our results are consistent with a model in which X4 HIV strains infect and potentially establish latency in naive and CM CD4+ T cells through direct infection, in addition to maintenance of the reservoir through differentiation and proliferation of infected cells.IMPORTANCE In people living with HIV (PLWH) on suppressive ART, latent HIV can be found in a diverse range of CD4+ T cells, including quiescent naive and central memory cells that are typically difficult to infect in vitro It is currently unclear how latency is established in these cells in vivo We show that in CD4+ T cells from PLWH on suppressive ART, the use of the coreceptor CXCR4 was prevalent among viruses amplified from naive and central memory CD4+ T cells. Furthermore, we found that expanded numbers of identical viral sequences were most common in the effector memory population, and these identical sequences were also found in multiple different CD4+ T cell subsets. Our results help to shed light on how a range of CD4+ T cell subsets come to harbor HIV DNA, which is one of the major barriers to eradicating the virus from PLWH.

Human Immunodeficiency Virus (HIV)-Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy.

BACKGROUND: Identifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy. METHODS: Cell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry. RESULTS: Integrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines. CONCLUSIONS: HIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.

Human Immunodeficiency Virus Persistence and T-Cell Activation in Blood, Rectal, and Lymph Node Tissue in Human Immunodeficiency Virus-Infected Individuals Receiving Suppressive Antiretroviral Therapy.

Background: Immune activation and inflammation remain elevated in human immunodeficiency virus (HIV)-infected individuals receiving antiretroviral therapy (ART) and may contribute to HIV persistence. Methods: Using flow cytometry expression of CD38, HLA-DR and PD-1 were measured in blood (n = 48), lymph node (LN; n = 9), and rectal tissue (n = 17) from virally suppressed individuals. Total and integrated HIV DNA, 2-LTR circles, and cell-associated unspliced HIV RNA were quantified. Results: CD4+ T cells from rectal tissue had a higher frequency of integrated HIV DNA compared with blood (4.26 fold-change in DNA; 95% confidence interval [CI] = 2.61-7.00; P < .001) and LN (2.32 fold-change in DNA; 95% CI = 1.22-4.41; P = .01). In rectal tissue, there were positive associations between integrated HIV DNA with PD-1+ CD4+ T-cells (1.44 fold-change in integrated HIV DNA per 10-unit increase in PD-1+ CD4+ T cells; 95% CI = 1.01-2.05; P = .045) and CD38+HLA-DR+ CD8+ T cells (1.40 fold-change in integrated HIV DNA per 1-unit increase in CD38+HLA-DR+ CD8+ T cells; 95% CI = 1.05-1.86; P = .02). Both associations were independent of current and nadir CD4+ T-cell counts. Conclusions: During ART, rectal tissue is an important reservoir for HIV persistence with a high frequency of activated CD4+ and CD8+ T cells. PD-1 may represent a marker of HIV persistence in rectal tissue.

CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART.

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

The antiviral compound BIT225 inhibits HIV-1 replication in myeloid dendritic cells.

BACKGROUND: Previous studies with BIT225 (N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide) have demonstrated a unique antiviral activity that blocks the release of HIV-1 from monocyte-derived macrophages (MDM). Antagonising the ion channel formed by HIV-1 Vpu, BIT225 preferentially targets de novo intracellular virus produced in 'virus-containing compartments' of MDM. In primary infections, dendritic cells (DC) are one of the first cells infected by HIV-1 and can transfer virus to more permissive CD4(+) T cells, making these cells an important target for novel antiviral therapies. To extend previous findings with BIT225, we aimed to further characterise the antiviral activity of BIT225 on HIV-1 replication in monocyte-derived DC (MDDC). RESULTS: The anti-HIV-1 activity of BIT225 was evaluated in vitro within MDDC alone and in co-cultures with activated CD4(+) T cells to examine the effect of the drug on HIV-1 transfer. Antiviral activity was determined by measuring HIV-1 reverse transcriptase activity in the culture supernatant of BIT225 treated and DMSO control cultures. A single dose of BIT225 resulted in a mean (SE) peak inhibition of HIV-1 release from MDDC by 74.5 % (±0.6) following 14 days of culture and a 6-fold reduction of HIV-1 transfer to activated uninfected CD4(+) T cells in co-culture. CONCLUSIONS: HIV-1 release from MDDC was inhibited by BIT225. This data broadens the drug's antiviral activity profile within cells of the myeloid lineage. These findings suggest a potential role for BIT225 in reducing HIV-1 production and preventing viral dissemination in early and chronic infection and may assist in limiting virus spread with any ongoing viral replication during antiretroviral therapy.

Persistence of integrated HIV DNA in CXCR3 + CCR6 + memory CD4+ T cells in HIV-infected individuals on antiretroviral therapy.

BACKGROUND: HIV latent infection can be established in vitro by treating resting CD4 T cells with chemokines that bind to chemokine receptors (CKR), CCR7, CXCR3, and CCR6, highly expressed on T cells. OBJECTIVE: To determine if CKR identify CD4 T cells enriched for HIV in HIV-infected individuals receiving suppressive antiretroviral therapy (ART). DESIGN: A cross-sectional study of CKR expression and HIV persistence in blood from HIV-infected individuals on suppressive ART for more than 3 years (n = 48). A subset of 20 individuals underwent leukapheresis and sorting of specific CD4 T-cell subsets. METHODS: We used flow cytometry to quantify CCR5, CCR6, CXCR3, and CXCR5 expression on CD4 T cells. HIV persistence was quantified using real-time Polymerase Chain Reaction to detect total, integrated HIV DNA, 2-long terminal repeat circles and cell-associated unspliced (CA-US) HIV RNA in total CD4 T cells from blood or sorted T-cell subsets. Associations between CKR and HIV persistence in CD4 T cells in blood were determined using regression models and adjusted for current and nadir CD4 T-cell counts. RESULTS: The frequency of cells harbouring integrated HIV DNA was inversely associated with current CD4 T-cell count and positively associated with CCR5+ CD4 T cells, CXCR3+CCR6+ and CXCR3+CCR6- expression on total memory CD4 T cells (P < 0.001, 0.048, 0.015, and 0.016, respectively). CXCR3+CCR6+ CM CD4 T cells contained the highest amount of integrated HIV DNA and lowest ratio of CA-US HIV RNA to DNA compared to all T-cell subsets examined. CONCLUSION: CXCR3 and CCR6 coexpression defines a subset of CD4 T cells that are preferentially enriched for HIV DNA in HIV-infected individuals on ART.

Ex vivo response to histone deacetylase (HDAC) inhibitors of the HIV long terminal repeat (LTR) derived from HIV-infected patients on antiretroviral therapy.

Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.

Targeting antigen to bone marrow stromal cell-2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation.

Bone marrow stromal cell-2 (BST-2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST-2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST-2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST-2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8(+) and CD8(-) DCs was determined. T-cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST-2 was compared with that via receptors DEC205 or Siglec-H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST-2, BST-2-targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST-2 was inferior in its capacity to deliver antigen to the MHCI cross-presentation pathway. In contrast, BST-2 was superior to Siglec-H at initiating either MHCI or MHCII antigen presentation. In summary, BST-2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.

HIV persistence: chemokines and their signalling pathways.

Latently infected resting CD4+ T cells are the major barrier to curing HIV. We have recently demonstrated that chemokines, which bind to the chemokine receptors CCR7, CXCR3 and CCR6, facilitate efficient HIV nuclear localisation and integration in resting CD4+ T cells, leading to latency. As latently infected cells are enriched in lymphoid tissues, where chemokines are highly concentrated, this may provide a mechanism for the generation of latently infected cells in vivo. Here we review the role of chemokines in HIV persistence; the main signalling pathways that are involved; and how these pathways may be exploited to develop novel strategies to reduce or eliminate latently infected cells.

Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells.

BACKGROUND: We recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency. RESULTS: High levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the nef open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat. CONCLUSIONS: In this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with ex vivo findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent in vitro model of HIV latency.

The role of naïve T-cells in HIV-1 pathogenesis: an emerging key player.

Functional naïve T-cells are critical for an effective immune response to multiple pathogens. HIV leads to a significant reduction in CD4+ naïve T-cell number and impaired function and there is incomplete recovery following combination antiretroviral therapy (cART). Here we review the basic homeostatic mechanisms that maintain naïve CD4+ T-cells and discuss recent developments in understanding the impact of HIV infection on naïve CD4+ T-cells. Finally we review therapeutic interventions in HIV-infected individuals aimed at specifically enhancing recovery of naïve CD4+ T-cells.

Both CD31(+) and CD31⁻ naive CD4(+) T cells are persistent HIV type 1-infected reservoirs in individuals receiving antiretroviral therapy.

BACKGROUND: Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31⁻ naive T cells to immune reconstitution and viral persistence is unknown. METHODS: In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31⁻ naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS: Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31⁻ naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS: After ART, both CD31(+) and CD31⁻ naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.

Antiviral efficacy of the novel compound BIT225 against HIV-1 release from human macrophages.

Building on previous findings that amiloride analogues inhibit HIV-1 replication in monocyte-derived macrophages (MDM), Biotron Limited has generated a library of over 300 small-molecule compounds with significant improvements in anti-HIV-1 activity. Our lead compound, BIT225, blocks Vpu ion channel activity and also shows anti-HIV-1 activity, with a 50% effective concentration of 2.25+/-0.23 microM (mean+/-the standard error) and minimal in vitro toxicity (50% toxic concentration, 284 microM) in infected MDM, resulting in a selectivity index of 126. In this study, we define the antiretroviral efficacy of BIT225 activity in macrophages, which are important drug targets because cells of the monocyte lineage are key reservoirs of HIV-1, disseminating virus to the peripheral tissues as they differentiate into macrophages. In assays with acutely and chronically HIV-1Ba-L-infected MDM, BIT225 resulted in significant reductions in viral integration and virus release as measured by real-time PCR and a reverse transcriptase (RT) activity assay at various stages of monocyte-to-macrophage differentiation. Further, the TZM-bl assay showed that the de novo virus produced at low levels in the presence of BIT225 was less infectious than virus produced in the absence of the compound. No antiviral activity was observed in MDM chronically infected with HIV-2, which lacks Vpu, confirming our initial targeting of and screening against this viral protein. The activity of BIT225 is post-virus integration, with no direct effects on the HIV-1 enzymes RT and protease. The findings of this study suggest that BIT225 is a late-phase inhibitor of the viral life cycle, targeting Vpu, and is a drug capable of significantly inhibiting HIV-1 release from both acute and chronically infected macrophages.