Persistence of envelopes in different CD4+ T-cell subsets in antiretroviral therapy-suppressed people with HIV.

OBJECTIVES: Despite suppressive antiretroviral therapy (ART), HIV can persist in a diverse range of CD4+ T-cell subsets. Through longitudinal env sampling from people with HIV (PWH) on ART, we characterized the persistence and phenotypic properties of HIV envs over two time-points (T1 and T2). METHODS: Longitudinal blood and lymphoid tissue samples were obtained from eight PWH on suppressive ART. Single genome amplification (SGA) was performed on env to understand the genetic diversity and degree of clonal expansions over time. A subset of envs were used to generate pseudovirus particles to assess sensitivity to autologous plasma IgG and broadly neutralizing antibodies (bNAbs). RESULTS: Identical env sequences indicating clonal expansion persisted between T1 and T2 and within multiple T-cell subsets. At both time-points, CXCR4-tropic (X4) Envs were more prevalent in naive and central memory cells; the proportion of X4 Envs did not significantly change in each subset between T1 and T2. Autologous purified plasma IgG showed variable neutralization of Envs, with no significant difference in neutralization between R5 and X4 Envs. X4 Envs were more sensitive to neutralization with clinical bNAbs, with CD4-binding site bNAbs demonstrating high breadth and potency against Envs. CONCLUSION: Our data suggest the viral reservoir in PWH on ART was predominantly maintained over time through proliferation and potentially differentiation of infected cells. We found the humoral immune response to Envs within the latent reservoir was variable between PWH. Finally, we identified coreceptor usage can influence bNAb sensitivity and may need to be considered for future bNAb immunotherapy approaches.

HIV DNA persists in hepatocytes in people with HIV-hepatitis B co-infection on antiretroviral therapy.

BACKGROUND: HIV can infect multiple cells in the liver including hepatocytes, Kupffer cells and infiltrating T cells, but whether HIV can persist in the liver in people with HIV (PWH) on suppressive antiretroviral therapy (ART) remains unknown. METHODS: In a prospective longitudinal cohort of PWH and hepatitis B virus (HBV) co-infection living in Bangkok, Thailand, we collected blood and liver biopsies from 18 participants prior to and following ART and quantified HIV and HBV persistence using quantitative (q)PCR and RNA/DNAscope. Antiretroviral (ARV) drug levels were quantified using mass spectroscopy. FINDINGS: In liver biopsies taken prior to ART, HIV DNA and HIV RNA were detected by qPCR in 53% (9/17) and 47% (8/17) of participants respectively. Following a median ART duration of 3.4 years, HIV DNA was detected in liver in 61% (11/18) of participants by either qPCR, DNAscope or both, but only at very low and non-quantifiable levels. Using immunohistochemistry, HIV DNA was observed in both hepatocytes and liver infiltrating CD4+ T cells on ART. HIV RNA was not detected in liver biopsies collected on ART, by either qPCR or RNAscope. All ARVs were clearly detected in liver tissue. INTERPRETATION: Persistence of HIV DNA in liver in PWH on ART represents an additional reservoir that warrants further investigation. FUNDING: National Health and Medical Research Council of Australia (Project Grant APP1101836, 1149990, and 1135851); This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024.

Memory CD4+ T cells that co-express PD1 and CTLA4 have reduced response to activating stimuli facilitating HIV latency.

Programmed cell death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) suppress CD4+ T cell activation and may promote latent HIV infection. By performing leukapheresis (n = 21) and lymph node biopsies (n = 8) in people with HIV on antiretroviral therapy (ART) and sorting memory CD4+ T cells into subsets based on PD1/CTLA4 expression, we investigate the role of PD1 and CTLA 4 in HIV persistence. We show that double-positive (PD1+CTLA4+) cells in blood contain more HIV DNA compared with double-negative (PD1-CTLA4-) cells but still have a lower proportion of cells producing multiply spliced HIV RNA after stimulation as well as reduced upregulation of T cell activation and proliferation markers. Transcriptomics analyses identify differential expression of key genes regulating T cell activation and proliferation with MAF, KLRB1, and TIGIT being upregulated in double-positive compared with double-negative cells, whereas FOS is downregulated. We conclude that, in addition to being enriched for HIV DNA, double-positive cells are characterized by negative signaling and a reduced capacity to respond to stimulation, favoring HIV latency.

Neurotoxicity with high-dose disulfiram and vorinostat used for HIV latency reversal.

OBJECTIVE: The aim of this study was to examine whether administering both vorinostat and disulfiram to people with HIV (PWH) on antiretroviral therapy (ART) is well tolerated and can enhance HIV latency reversal. DESIGN: Vorinostat and disulfiram can increase HIV transcription in PWH on ART. Together, these agents may lead to significant HIV latency reversal. METHODS: Virologically suppressed PWH on ART received disulfiram 2000 mg daily for 28 days and vorinostat 400 mg daily on days 8-10 and 22-24. The primary endpoint was plasma HIV RNA on day 11 relative to baseline using a single copy assay. Assessments included cell-associated unspliced RNA as a marker of latency reversal, HIV DNA in CD4+ T-cells, plasma HIV RNA, and plasma concentrations of ART, vorinostat, and disulfiram. RESULTS: The first two participants (P1 and P2) experienced grade 3 neurotoxicity leading to trial suspension. After 24 days, P1 presented with confusion, lethargy, and ataxia having stopped disulfiram and ART. Symptoms resolved by day 29. After 11 days, P2 presented with paranoia, emotional lability, lethargy, ataxia, and study drugs were ceased. Symptoms resolved by day 23. CA-US RNA increased by 1.4-fold and 1.3-fold for P1 and P2 respectively. Plasma HIV RNA was detectable from day 8 to 37 (peak 81 copies ml-1) for P2 but was not increased in P1 Antiretroviral levels were therapeutic and neuronal injury markers were elevated in P1. CONCLUSION: The combination of prolonged high-dose disulfiram and vorinostat was not safe in PWH on ART and should not be pursued despite evidence of latency reversal.

Cell-Associated Human Immunodeficiency Virus (HIV) Ribonucleic Acid Has a Circadian Cycle in Males With HIV on Antiretroviral Therapy.

BACKGROUND: Circadian transcription factors that regulate cell-autonomous circadian clocks can also increase human immunodeficiency virus (HIV) transcription in vitro. We aimed to determine whether circadian variation in HIV transcription exists in people with HIV (PWH) on antiretroviral therapy (ART). METHODS: We performed a prospective observational study of male PWH on ART, sampling blood every 4 hours for 24 hours. Using quantitative polymerase chain reaction, we quantified expression of circadian-associated genes, HIV deoxyribonucleic acid (DNA), and cell-associated unspliced (CA-US) ribonucleic acid (RNA) in peripheral blood CD4+ T cells. Plasma sex hormones were quantified alongside plasma and salivary cortisol. The primary outcome was to identify temporal variations in CA-US HIV RNA using a linear mixed-effect regression framework and maximum likelihood estimation. RESULTS: Salivary and plasma cortisol, and circadian genes including Clock, Bmal1, and Per3, varied with a circadian rhythm. Cell-associated unspliced HIV RNA and the ratio of CA-US HIV RNA/DNA in CD4+ T cells also demonstrated circadian variations, with no variation in HIV DNA. Circulating estradiol was highly predictive of CA-US HIV RNA variation in vivo. CONCLUSIONS: Cell-associated unspliced HIV RNA in PWH on ART varies temporally with a circadian rhythm. These findings have implications for the design of clinical trials and biomarkers to assess HIV cure interventions.

Combination Immune Checkpoint Blockade Enhances IL-2 and CD107a Production from HIV-Specific T Cells Ex Vivo in People Living with HIV on Antiretroviral Therapy.

In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4+ and CD8+ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8+ and CD107a and IL-2 production in HIV-specific CD4+ and CD8+ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4+ and CD8+ T cells in PWH on ART. These combinations should be further explored for an HIV cure.

The impact of immune checkpoint therapy on the latent reservoir in HIV-infected individuals with cancer on antiretroviral therapy.

OBJECTIVE: The aim of this study was to quantify HIV-specific immunological and virological changes in people with HIV (PWH) on antiretroviral therapy (ART) with malignancy who received immune checkpoint blockade (ICB). DESIGN: An observational cohort study. METHODS: Blood samples were collected before and after four cycles of ICB in HIV-positive adults on ART. Virological assessments performed on CD4+ T cells included cell-associated unspliced HIV RNA, cell-associated HIV DNA, Tat/rev-induced limiting dilution assay (TILDA) and plasma HIV RNA using a single copy assay (SCA). Flow cytometry was used to assess the frequency of precursor exhausted T cells (Tpex) and exhausted T cells (Tex), and Gag-specific CD4+ and CD8+ T cells positive for IFN-γ, TNF-α or CD107a by intracellular cytokine staining (ICS). RESULTS: Participant (P)1 received avelumab (anti-PD-L1) for Merkel cell carcinoma. P2 and P3 received ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) for metastatic melanoma. An increase in CA-US RNA following each infusion was noted in all three participants. There were no consistent changes in HIV DNA or the proportion of cells with inducible MS HIV RNA. P2 demonstrated a striking increase in the frequency of gag-specific central and effector memory CD8+ T cells producing IFN-γ, TNF-α and CD107a following anti-PD1 and anti-CTLA-4. The frequency of CD8+ Tpex cells pre-ICB was also highest in this participant. CONCLUSION: In three PWH with cancer on ART, we found that ICB activated latent HIV and enhanced HIV-specific T cell function but with considerable variation.

Multiply spliced HIV RNA is a predictive measure of virus production ex vivo and in vivo following reversal of HIV latency.

BACKGROUND: One strategy being pursued to clear latently infected cells that persist in people living with HIV (PLWH) on antiretroviral therapy (ART) is to activate latent HIV infection with a latency reversing agent (LRA). Surrogate markers that accurately measure virus production following an LRA are needed. METHODS: We quantified cell-associated unspliced (US), multiply spliced (MS) and supernatant (SN) HIV RNA by qPCR from total and resting CD4+ T cells isolated from seven PLWH on ART before and after treatment ex vivo with different LRAs, including histone deacetylase inhibitors (HDACi). MS and plasma HIV RNA were also quantified from PLWH on ART (n-11) who received the HDACi panobinostat. FINDINGS: In total and resting CD4+ T cells from PLWH on ART, detection of US RNA was common while detection of MS RNA was infrequent. Primers used to detect MS RNA, in contrast to US RNA, bound sites of the viral genome that are commonly mutated or deleted in PLWH on ART. Following ex vivo stimulation with LRAs, we identified a strong correlation between the fold change increase in SN and MS RNA, but not the fold change increase in SN and US RNA. In PLWH on ART who received panobinostat, MS RNA was significantly higher in samples with detectable compared to non0detectable plasma HIV RNA. INTERPRETATION: Following administration of an LRA, quantification of MS RNA is more likely to reflect an increase in virion production and is therefore a better indicator of meaningful latency reversal. FUNDING: NHMRC, NIH DARE collaboratory.

Impact of Anti-PD-1 and Anti-CTLA-4 on the Human Immunodeficiency Virus (HIV) Reservoir in People Living With HIV With Cancer on Antiretroviral Therapy: The AIDS Malignancy Consortium 095 Study.

BACKGROUND: Antibodies to programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) may perturb human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) by reversing HIV latency and/or boosting HIV-specific immunity, leading to clearance of infected cells. We tested this hypothesis in a clinical trial of anti-PD-1 alone or in combination with anti-CTLA-4 in people living with HIV (PLWH) and cancer. METHODS: This was a substudy of the AIDS Malignancy Consortium 095 Study. ART-suppressed PLWH with advanced malignancies were assigned to nivolumab (anti-PD-1) with or without ipilimumab (anti-CTLA-4). In samples obtained preinfusion and 1 and 7 days after the first and fourth doses of immune checkpoint blockade (ICB), we quantified cell-associated unspliced (CA-US) HIV RNA and HIV DNA. Plasma HIV RNA was quantified during the first treatment cycle. Quantitative viral outgrowth assay (QVOA) to estimate the frequency of replication-competent HIV was performed before and after ICB for participants with samples available. RESULTS: Of 40 participants, 33 received nivolumab and 7 nivolumab plus ipilimumab. Whereas CA-US HIV RNA did not change with nivolumab monotherapy, we detected a median 1.44-fold increase (interquartile range, 1.16-1.89) after the first dose of nivolumab and ipilimumab combination therapy (P = .031). There was no decrease in the frequency of cells containing replication-competent HIV, but in the 2 individuals on combination ICB for whom we had longitudinal QVOA, we detected decreases of 97% and 64% compared to baseline. CONCLUSIONS: Anti-PD-1 alone showed no effect on HIV latency or the latent HIV reservoir, but the combination of anti-PD-1 and anti-CTL-4 induced a modest increase in CA-US HIV RNA and may potentially eliminate cells containing replication-competent HIV. CLINICAL TRIALS REGISTRATION: NCT02408861.

A clinical trial of non-invasive imaging with an anti-HIV antibody labelled with copper-64 in people living with HIV and uninfected controls.

BACKGROUND: A research priority in finding a cure for HIV is to establish methods to accurately locate and quantify where and how HIV persists in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). Infusing copper-64 (64Cu) radiolabelled broadly neutralising antibodies targeting HIV envelope (Env) with CT scan and positron emission tomography (PET) identified HIV Env in tissues in SIV infected non-human primates . We aimed to determine if a similar approach was effective in people living with HIV (PLWH). METHODS: Unmodified 3BNC117 was compared with 3BNC117 bound to the chelator MeCOSar and 64Cu (64Cu-3BNC117) in vitro to assess binding and neutralization. In a clinical trial 64Cu-3BNC117 was infused into HIV uninfected (Group 1), HIV infected and viremic (viral load, VL >1000 c/mL; Group 2) and HIV infected aviremic (VL <20 c/mL; Group 3) participants using two dosing strategies: high protein (3mg/kg unlabeled 3BNC117 combined with <5mg 64Cu-3BNC117) and trace (<5mg 64Cu-3BNC117 only). All participants were screened for 3BNC117 sensitivity from virus obtained from viral outgrowth. Magnetic resonance imaging (MRI)/PET and pharmacokinetic assessments (ELISA for serum 3BNC117 concentrations and gamma counting for 64Cu) were performed 1, 24- and 48-hours post dosing. The trial (clincialtrials.gov NCT03063788) primary endpoint was comparison of PET standard uptake values (SUVs) in regions of interest (e.g lymph node groups and gastrointestinal tract). FINDINGS: Comparison of unmodified and modified 3BNC117 in vitro demonstrated no difference in HIV binding or neutralisation. 17 individuals were enrolled of which 12 were dosed including Group 1 (n=4, 2 high protein, 2 trace dose), Group 2 (n=6, 2 high protein, 4 trace) and Group 3 (n=2, trace only). HIV+ participants had a mean CD4 of 574 cells/microL and mean age 43 years. There were no drug related adverse effects and no differences in tissue uptake in regions of interest (e.g lymph node gut, pharynx) between the 3 groups. In the high protein dosing group, serum concentrations of 3BNC117 and gamma counts were highly correlated demonstrating that 64Cu-3BNC117 remained intact in vivo. INTERPRETATION: In PLWH on or off ART, the intervention of infusing 64Cu-3BNC117 and MRI/PET imaging over 48 hours, was unable to detect HIV-1 env expression in vivo. Future studies should investigate alternative radiolabels such as zirconium which have a longer half-life in vivo. FUNDING: Funded by the Alfred Foundation, The Australian Centre for HIV and Hepatitis Virology Research with additional support from the Division of AIDS, National Institute of Allergy and Infectious Disease, US National Institutes of Health (USAI126611). JHM and SRL are supported by the Australian National Health and Medical Research Council.

A randomized trial of vorinostat with treatment interruption after initiating antiretroviral therapy during acute HIV-1 infection.

OBJECTIVE AND DESIGN: A randomized, open-label pilot study in individuals treated with antiretroviral therapy (ART) since acute HIV infection (AHI) with a regimen including a histone deacetylase inhibitor to induce HIV from latency and control HIV replication during subsequent treatment interruption (TI). METHODS: Fifteen participants who initiated ART at AHI were randomized to vorinostat/hydroxychloroquine/maraviroc (VHM) plus ART (n â€‹= â€‹10) or ART alone (n â€‹= â€‹5). The VHM arm received three 14-day vorinostat cycles within 10 weeks before TI. ART was resumed for plasma viral load (VL) â€‹> â€‹1,000 HIV RNA copies/mL. Primary outcome was proportion of participants on VHM â€‹+ â€‹ART versus ART only with VL â€‹< â€‹50 copies/mL for 24 weeks after TI. RESULTS: Fifteen participants on ART (median: 178 weeks: range 79-295) enrolled. Two on VHM â€‹+ â€‹ART experienced serious adverse events. Fourteen participants underwent TI; all experienced VL rebound with no difference in time between arms: VHM â€‹+ â€‹ART (n â€‹= â€‹9) median: 4 weeks and ART only (n â€‹= â€‹5) median: 5 weeks. VHM induced a 2.2-fold increase in VL (p â€‹= â€‹0.008) by single-copy HIV RNA assay after the first cycle. Neopterin levels increased significantly following the first two cycles. After VHM treatment, the frequencies of peripheral blood mononuclear cells harboring total HIV DNA and cell-associated RNA were unchanged. All participants achieved VL suppression following ART re-initiation. CONCLUSIONS: Administration of VHM increased HIV VL in plasma, but this was not sustained. VHM did not impact time to viral rebound following TI and had no impact on the size of the HIV reservoir, suggesting that HIV reservoir elimination will require alternative treatment strategies.

Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection.

In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection.

Longitudinal analysis of subtype C envelope tropism for memory CD4+ T cell subsets over the first 3 years of untreated HIV-1 infection.

BACKGROUND: HIV-1 infects a wide range of CD4+ T cells with different phenotypic properties and differing expression levels of entry coreceptors. We sought to determine the viral tropism of subtype C (C-HIV) Envelope (Env) clones for different CD4+ T cell subsets and whether tropism changes during acute to chronic disease progression. HIV-1 envs were amplified from the plasma of five C-HIV infected women from three untreated time points; less than 2 months, 1-year and 3-years post-infection. Pseudoviruses were generated from Env clones, phenotyped for coreceptor usage and CD4+ T cell subset tropism was measured by flow cytometry. RESULTS: A total of 50 C-HIV envs were cloned and screened for functionality in pseudovirus infection assays. Phylogenetic and variable region characteristic analysis demonstrated evolution in envs between time points. We found 45 pseudoviruses were functional and all used CCR5 to mediate entry into NP2/CD4/CCR5 cells. In vitro infection assays showed transitional memory (TM) and effector memory (EM) CD4+ T cells were more frequently infected (median: 46% and 25% of total infected CD4+ T cells respectively) than naïve, stem cell memory, central memory and terminally differentiated cells. This was not due to these subsets contributing a higher proportion of the CD4+ T cell pool, rather these subsets were more susceptible to infection (median: 5.38% EM and 2.15% TM cells infected), consistent with heightened CCR5 expression on EM and TM cells. No inter- or intra-participant changes in CD4+ T cell subset tropism were observed across the three-time points. CONCLUSIONS: CD4+ T cell subsets that express more CCR5 were more susceptible to infection with C-HIV Envs, suggesting that these may be the major cellular targets during the first 3 years of infection. Moreover, we found that viral tropism for different CD4+ T cell subsets in vitro did not change between Envs cloned from acute to chronic disease stages. Finally, central memory, naïve and stem cell memory CD4+ T cell subsets were susceptible to infection, albeit inefficiently by Envs from all time-points, suggesting that direct infection of these cells may help establish the latent reservoir early in infection.

Diverse effects of interferon alpha on the establishment and reversal of HIV latency.

HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4+ T cells. Latently infected CD4+ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4+ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNß and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4+ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4+ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established.

Combination Immune Checkpoint Blockade to Reverse HIV Latency.

In people living with HIV on antiretroviral therapy, HIV latency is the major barrier to a cure. HIV persists preferentially in CD4+ T cells expressing multiple immune checkpoint (IC) molecules, including programmed death (PD)-1, T cell Ig and mucin domain-containing protein 3 (TIM-3), lymphocyte associated gene 3 (LAG-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). We aimed to determine whether these and other IC molecules have a functional role in maintaining HIV latency and whether blocking IC molecules with Abs reverses HIV latency. Using an in vitro model that establishes latency in both nonproliferating and proliferating human CD4+ T cells, we show that proliferating cells express multiple IC molecules at high levels. Latent infection was enriched in proliferating cells expressing PD-1. In contrast, nonproliferating cells expressed IC molecules at significantly lower levels, but latent infection was enriched in cells expressing PD-1, TIM-3, CTL-associated protein 4 (CTLA-4), or B and T lymphocyte attenuator (BTLA). In the presence of an additional T cell-activating stimulus, staphylococcal enterotoxin B, Abs to CTLA-4 and PD-1 reversed HIV latency in proliferating and nonproliferating CD4+ T cells, respectively. In the absence of staphylococcal enterotoxin B, only the combination of Abs to PD-1, CTLA-4, TIM-3, and TIGIT reversed latency. The potency of latency reversal was significantly higher following combination IC blockade compared with other latency-reversing agents, including vorinostat and bryostatin. Combination IC blockade should be further explored as a strategy to reverse HIV latency.

Variation in cell-associated unspliced HIV RNA on antiretroviral therapy is associated with the circadian regulator brain-and-muscle-ARNT-like-1.

OBJECTIVE(S): To determine whether variation in cell-associated unspliced (CA-US) HIV RNA in HIV-infected individuals on antiretroviral therapy (ART) has a circadian basis. METHODS: Prospective observational study of HIV-infected individuals on ART. Blood was collected on three occasions and CA-US HIV RNA and mRNA of the circadian-locomotor-output-cycles-kaput (CLOCK)-associated genes quantified by real time PCR. CLOCK-associated proteins were over-expressed in a cell line stably transfected with an HIV long-terminal repeat (LTR) luciferase reporter. RESULTS: Using a mixed effects model, there was a significant increase in log-CA-US RNA at the third visit compared with the first visit (effect size of 0.619 with standard error (SE) of 0.098, P < 0.001) and an independent effect of time of blood draw (effect size 0.051 (SE 0.025), P = 0.040). The CLOCK-associated gene, brain-and-muscle-ARNT-like-1 (BMAL-1) had a significant relationship with log CA-US HIV RNA (effect size 8.508 (SE 3.777), P = 0.028) and also with time (P = 0.045). Over expression of BMAL-1 and CLOCK in a cell line stably transfected with an HIV-LTR luciferase reporter resulted in an increase in luciferase expression and this was reduced following mutation of the second E-box in the HIV-LTR. CONCLUSION: The basal level of HIV transcription on ART can vary significantly and is modulated by the circadian regulator BMAL-1, amongst other factors.

Programmed cell death-1 contributes to the establishment and maintenance of HIV-1 latency.

OBJECTIVE: In HIV-infected individuals on antiretroviral therapy (ART), latent HIV is enriched in CD4 T cells expressing immune checkpoint molecules, in particular programmed cell death-1 (PD-1). We therefore assessed the effect of blocking PD-1 on latency, both in vitro and in vivo. METHODS: HIV latency was established in vitro following coculture of resting CD4+ T cells with myeloid dendritic cells. Expression of PD-1 was quantified by flow cytometry, and latency assessed in sorted PD-1high and PD-1low/-nonproliferating CD4+ memory T cells. The role of PD-1 in the establishment of latency was determined by adding anti-PD-1 (pembrolizumab) to cocultures before and after infection. In addition, a single infusion of anti-PD-1 (nivolumab) was administered to an HIV-infected individual on ART with metastatic melanoma, and cell-associated HIV DNA and RNA, and plasma HIV RNA were quantified. RESULTS: HIV latency was significantly enriched in PD-1high compared with PD-1low/- nonproliferating, CD4 memory T cells. Sorting for an additional immune checkpoint molecule, T-cell immunoglobulin domain and mucin domain-3, in combination with PD-1, further enriched for latency. Blocking PD-1 prior to HIV infection, in vitro, resulted in a modest but significant decrease in latently infected cells in all donors (n = 6). The administration of anti-PD-1 to an HIV-infected individual on ART resulted in a significant increase in cell-associated HIV RNA in CD4 T cells, without significant changes in HIV DNA or plasma HIV RNA, consistent with reversal of HIV latency. CONCLUSION: PD-1 contributes to the establishment and maintenance of HIV latency and should be explored as a target, in combination with other immune checkpoint molecules, to reverse latency.

The effect of antiretroviral intensification with dolutegravir on residual virus replication in HIV-infected individuals: a randomised, placebo-controlled, double-blind trial.

BACKGROUND: Whether ongoing virus replication occurs in HIV-infected individuals on antiretroviral therapy (ART) is unclear; therefore, whether residual virus replication is a barrier to achieving a cure for HIV is also unknown. We aimed to establish whether ART intensification with dolutegravir would reveal or affect residual virus replication in HIV-infected individuals on suppressive treatment. METHODS: In this randomised, placebo-controlled, double-blind trial, we enrolled HIV-infected adults (aged 18 years and older) receiving combination ART (at least three agents) for at least 3 years from the Alfred Hospital and Melbourne Sexual Health Centre, Melbourne, VIC, Australia. Eligible participants had fewer than 50 copies per mL HIV-1 plasma RNA for more than 3 years and fewer than 20 copies per mL at screening and two CD4 counts higher than 350 cells per µL in the previous 24 months including screening. Participants were randomly assigned (1:1) to receive 50 mg oral dolutegravir or placebo once a day for 56 days in addition to background ART. Follow-up was done at days 1, 3, 7, 14, 28, 56, and 84. The primary outcome was the change from baseline in frequency of 2-long terminal repeat (2-LTR) circles in peripheral blood CD4 cells at day 7. This trial is registered with ClinicalTrials.gov, number NCT02500446. FINDINGS: Between Sept 21, 2015, and Sept 19, 2016, 46 individuals were screened for inclusion. 40 were eligible for inclusion and were randomly assigned to the dolutegravir (n=21) or placebo group (n=19). All enrolled participants completed the study procedures and no individuals were lost to follow up. All participants were on suppressive ART with 12% receiving protease inhibitors and the others non-nucleoside reverse transcriptase inhibitors. Median 2-LTR circles fold-change from baseline to day 7 was -0·17 (IQR -0·90 to 0·90) in the dolutegravir group and -0·26 (-1·00 to 1·17) in the placebo group (p=0·17). The addition of dolutegravir to pre-existing ART regimens was safe and there were no treatment discontinuations or treatment-related serious adverse events. INTERPRETATION: Our findings show that in HIV-infected individuals on modern suppressive ART regimens, residual replication is rare, if at all present, and was not recorded in blood after dolutegravir intensification. Because tissue biopsies were not done we cannot exclude the possibility of residual virus replication in tissue. Strategies other than ART alone are needed to eliminate HIV persistence on treatment. FUNDING: ViiV Healthcare.

Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

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.