Search

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., Competing Interests: The authors have declared that no competing interests exist.

Background: Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position., Results: We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells., Conclusion: Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

BackgroundAntibodies 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.MethodsThis 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.ResultsOf 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.ConclusionsAnti-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 registrationNCT02408861.

BackgroundOne 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.MethodsWe 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.FindingsIn 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.InterpretationFollowing 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.FundingNHMRC, NIH DARE collaboratory.

BackgroundIn HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels.MethodsThirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition.ResultsThe addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling.ConclusionsTreatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes. [ABSTRACT FROM AUTHOR]

In people living with HIV (PLWH) on antiretroviral therapy (ART), virus persists in a latent form where there is minimal transcription or protein expression. Latently infected cells are a major barrier to curing HIV. Increasing HIV transcription and viral production in latently infected cells could facilitate immune recognition and reduce the pool of infected cells that persist on ART. Given that programmed cell death protein 1 (PD-1) expressing CD4+ T cells are preferentially infected with HIV in PLWH on ART, we aimed to determine whether administration of antibodies targeting PD-1 would reverse HIV latency in vivo. We therefore evaluated the impact of intravenous administration of pembrolizumab every 3 weeks on HIV latency in 32 PLWH and cancer on ART. After the first infusion of anti–PD-1, we observed a median 1.32-fold increase in unspliced HIV RNA and 1.61-fold increase in unspliced RNA:DNA ratio in sorted blood CD4+ T cells compared to baseline. We also observed a 1.65-fold increase in plasma HIV RNA. The frequency of CD4+ T cells with inducible virus evaluated using the tat/rev limiting dilution assay was higher after 6 cycles compared to baseline. Phylogenetic analyses of HIV env sequences in a participant who developed low concentrations of HIV viremia after 6 cycles of pembrolizumab did not demonstrate clonal expansion of HIV-infected cells. These data are consistent with anti–PD-1 being able to reverse HIV latency in vivo and support the rationale for combining anti–PD-1 with other interventions to reduce the HIV reservoir. PD-1 blockade for HIV: Strategies to target and reverse the latent HIV reservoir are required to cure HIV. Because CD4+ T cells expressing programmed cell death protein 1 (PD-1) are preferentially infected with HIV in an individual on antiretroviral therapy, PD-1 is an attractive target to manipulate the latent reservoir. To this end, Uldrick et al. measured the impact of anti–PD-1 treatment with pembrolizumab on the HIV reservoir in 32 individuals living with HIV on suppressive antiretroviral therapy and diagnosed with cancer. They observed evidence of increased unspliced HIV RNA and of the unspliced RNA:DNA ratio, consistent with anti–PD-1 treatment having the potential to reverse HIV latency. Further studies are needed to evaluate the dose and frequency of anti–PD-1 treatment required for latency reversal. [ABSTRACT FROM AUTHOR]

Background: A sterilizing cure of HIV-1 infection has been reported in 2 persons living with HIV-1 who underwent allogeneic hematopoietic stem cell transplantations from donors who were homozygous for the CCR5Δ32 gene polymorphism. However, this has been considered elusive during natural infection.Objective: To evaluate persistent HIV-1 reservoir cells in an elite controller with undetectable HIV-1 viremia for more than 8 years in the absence of antiretroviral therapy.Design: Detailed investigation of virologic and immunologic characteristics.Setting: Tertiary care centers in Buenos Aires, Argentina, and Boston, Massachusetts.Patient: A patient with HIV-1 infection and durable drug-free suppression of HIV-1 replication.Measurements: Analysis of genome-intact and replication-competent HIV-1 using near-full-length individual proviral sequencing and viral outgrowth assays, respectively; analysis of HIV-1 plasma RNA by ultrasensitive HIV-1 viral load testing.Results: No genome-intact HIV-1 proviruses were detected in analysis of a total of 1.188 billion peripheral blood mononuclear cells and 503 million mononuclear cells from placental tissues. Seven defective proviruses, some of them derived from clonally expanded cells, were detected. A viral outgrowth assay failed to retrieve replication-competent HIV-1 from 150 million resting CD4+ T cells. No HIV-1 RNA was detected in 4.5 mL of plasma.Limitations: Absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses. A sterilizing cure of HIV-1 can never be empirically proved.Conclusion: Genome-intact and replication-competent HIV-1 were not detected in an elite controller despite analysis of massive numbers of cells from blood and tissues, suggesting that this patient may have naturally achieved a sterilizing cure of HIV-1 infection. These observations raise the possibility that a sterilizing cure may be an extremely rare but possible outcome of HIV-1 infection.Primary Funding Source: National Institutes of Health and Bill & Melinda Gates Foundation. [ABSTRACT FROM AUTHOR]

Background Hepatitis C virus (HCV) coinfection among people with human immunodeficiency virus (HIV) might perturb immune function and HIV persistence. We aimed to evaluate the impact of HCV clearance with direct-acting antivirals (DAAs) on immune activation and HIV persistence in HIV/HCV-coinfected individuals on antiretroviral therapy (ART). Methods In a prospective observational study, ART-treated participants with HIV/HCV coinfection received sofosbuvir/daclatasvir ± ribavirin (n = 19). Blood samples were collected before DAA therapy, at the end of treatment, and 12 months after DAA termination (12MPT). T- and natural killer (NK)-cell phenotype, soluble plasma factors, cell-associated (CA)-HIV deoxyribonucleic acid (DNA) forms (total, integrated, 2LTR), CA-unspliced (US) and multiple-spliced ribonucleic acid (RNA), and plasma HIV RNA were evaluated. Results Hepatitis C virus clearance was associated with (1) a downmodulation of activation and exhaustion markers in CD4+, CD8+ T, and NK cells together with (2) decreased plasma levels of Interferon gamma-induced protein 10 (IP10), interleukin-8 (IL-8), soluble (s)CD163 and soluble intercellular adhesion molecule (sICAM). Cell-associated US HIV RNA was significantly higher at 12MPT compared to baseline, with no change in HIV DNA or plasma RNA. Conclusions Elimination of HCV in HIV/HCV-coinfected individuals alters immune function and the transcriptional activity of latently infected cells. This report provides insights into the effects of HCV coinfection in HIV persistence and regards coinfected subjects as a population in which HIV remission might prove to be more challenging. [ABSTRACT FROM AUTHOR]

Co-infection with hepatitis B (HBV) and human immunodeficiency virus (HIV) increases overall and liver-related mortality. In order to identify interactions between these two viruses in vivo, full-length HIV proviruses were sequenced from a cohort of HIV-HBV coinfected participants and from a cohort of HIV mono-infected participants recruited from Bangkok, Thailand, both before the initiation of antiretroviral therapy (ART) and after at least 2 years of ART. The co-infected individuals were found to have higher levels of geneticallyintact HIV proviruses than did mono-infected individuals pre-therapy. In these co-infected individuals, higher levels of genetically-intact HIV proviruses or proviral genetic-diversity were also associated with higher levels of sCD14 and CXCL10, suggesting that immune activation is linked to more genetically-intact HIV proviruses. Three years of ART decreased the overall level of HIV proviruses, with fewer genetically-intact proviruses being identified in co-infected versus mono-infected individuals. However, ART increased the frequency of certain genetic defects within proviruses and the expansion of identical HIV sequences. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Background: The persistence of latently infected T cells remains the principal barrier to HIV cure. Understanding how the early immune responses shape persistence of HIV on antiretroviral therapy (ART) will be fundamental for potential eradication. Here, we aimed to determine the relationship between CD8+ T-cell function and phenotype before therapy and HIV persistence on ART. Methods: Blood samples from 29 individuals enrolled during primary HIV infection (at baseline and every 3 months up to 2 years post-ART initiation) were obtained. HIV-specific T-cell function and expression of the activation markers were evaluated before ART by flow cytometry. Cell-associated HIV DNA and unspliced (US)-RNA were quantified in purified CD4+ T cells by real-time polymerase chain reaction. Data were analyzed using nonparametric statistics. Results: Elevated immune activation, dominance of monofunctional CD8+ T cells, and skewed distribution of memory profile were observed before ART. After ART initiation, HIV DNA and US-RNA levels rapidly diminished, reaching a plateau by 30 weeks after ART. The proportion of baseline HIV-specific effector memory and terminal effector CD8+ T cells directly correlated with HIV DNA levels at 1 year after ART. A strong positive correlation was observed between the proportion of bulk and HIV-specific PD-1High CD8+ T cells measured before ART and HIV DNA at 1 year after ART. Conclusions: A higher proportion of terminally differentiated CD8+ T cells and increased PD1 expression were associated with HIV persistence on ART after treatment of primary infection. Thus, the quality of the early CD8+ T-cell immune response may serve as a predictor of HIV persistence on ART. [ABSTRACT FROM AUTHOR]

HIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4+ T cells. The induction of virus expression from latently infected cells occurs following T cell receptor (TCR) activation, but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19-treated resting CD4+ (rCD4+) T cells (preactivation latency) or activated CD4+ T cells that returned to a resting state (postactivation latency). We isolated latently infected cells by sorting for EGFP-negative (EGFP-) cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti- CD28, mitogens, and latency-reversing agents (LRAs) and cocultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in postactivation than in preactivation latency. Stimulation of latently infected cells with monocytes/ anti-CD3 resulted in an increase in EGFP expression compared to that for unstimulated controls using the preactivation latency model but led to a reduction in EGFP expression in the postactivation latency model. The reduced EGFP expression was not associated with reductions in the levels of viral DNA or T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the postactivation latency model during the establishment of latency reduced spontaneous virus expression, suggesting that monocyte-T cell interactions at an early time point postinfection can maintain HIV latency. This direct comparison of preand postactivation latency suggests that effective strategies needed to reverse latency will depend on how latency is established. IMPORTANCE One strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus-mediated cytolysis or immune-mediated clearance of infected cells. The gold standard for the activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus activates only a small proportion of latently infected cells. We show clear differences in the responses of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of noninduced intact proviruses in HIV-infected individuals on ART. [ABSTRACT FROM AUTHOR]