Varied Patterns of Decay of Intact Human Immunodeficiency Virus Type 1 Proviruses Over 2 Decades of Antiretroviral Therapy.

Fourteen people with human immunodeficiency virus type 1 had longitudinal measurements of intact, defective, and total proviral DNA over the course of two decades of antiretroviral therapy. Three patterns of intact proviral DNA decay were revealed: (1) biphasic decline with markedly slower second-phase decline, (2) initial decline that transitions to a zero-slope plateau, and (3) initial decline followed by later increases in intact proviral DNA. Defective proviral DNA levels were essentially stable. Mechanisms of slowing or reversal of second-phase decay of intact proviral DNA may include the inability to clear cells with intact but transcriptionally silent proviruses and clonal expansion of cells with intact proviruses.

Associations Between Multiple Measures of HIV-1 Persistence in Persons on Suppressive Antiretroviral Therapy.

Clinical research to achieve antiretroviral therapy-free remission requires quantitative assays of the HIV-1 reservoir. Intact proviral DNA (IPD) measurement has greater throughput than the quantitative viral outgrowth assay (QVOA). In 25 individuals with well-documented long-term viral suppression, IPD levels and infectious units per million CD4+ T cells by QVOA strongly correlated (r = 0.59, P = .002), and IPD correlated with total cell-associated HIV-1 DNA and cell-associated HIV-1 RNA (r = 0.62 and r = 0.59, P ≤ .002). IPD may provide an accessible marker of inducible replication-competent virus, total numbers of infected cells, and cellular expression of HIV-1 RNA.

Association of Male Sex and Obesity With Residual Plasma Human Immunodeficiency Virus 1 Viremia in Persons on Long-Term Antiretroviral Therapy.

BACKGROUND: Although adipose tissue has been proposed to harbor part of the human immunodeficiency virus 1 (HIV-1) reservoir, the influence of host characteristics, including sex and body mass index (BMI), on measures of HIV-1 persistence during antiretroviral therapy (ART) are incompletely understood. METHODS: We evaluated age, sex, BMI, waist circumference, years on ART, pre-ART HIV-1 RNA, pre-ART CD4+ T-cell count, and initial ART regimen with measures of HIV-1 persistence in blood (residual viremia, cellular HIV-1 DNA and RNA) in a cohort of 295 individuals with well-documented long-term virologic suppression (HIV-1 RNA <50 copies/mL) on ART (AIDS Clinical Trials Group study A5321). RESULTS: Men were more likely than women to have detectable plasma HIV-1 RNA by single-copy assay (52% vs 29%; P = .003), and the proportion of participants with detectable residual viremia increased in a stepwise fashion by BMI category (normal weight or underweight, 38%; overweight, 50%; and obese, 55%). ART regimen type was not associated with measures of HIV-1 persistence after controlling for ART duration. CONCLUSIONS: Sex and obesity are independently associated with residual viremia in people on long-term ART. Additional studies to confirm these relationships and to define the mechanisms by which sex and obesity affect HIV-1 persistence are needed to inform HIV-1 cure strategies.

Selective Decay of Intact HIV-1 Proviral DNA on Antiretroviral Therapy.

BACKGROUND: HIV-1 proviruses persist in people on antiretroviral therapy (ART) but most are defective and do not constitute a replication-competent reservoir. The decay of infected cells carrying intact compared with defective HIV-1 proviruses has not been well defined in people on ART. METHODS: We separately quantified intact and defective proviruses, residual plasma viremia, and markers of inflammation and activation in people on long-term ART. RESULTS: Among 40 participants tested longitudinally from a median of 7.1 years to 12 years after ART initiation, intact provirus levels declined significantly over time (median half-life, 7.1 years; 95% confidence interval [CI], 3.9-18), whereas defective provirus levels did not decrease. The median half-life of total HIV-1 DNA was 41.6 years (95% CI, 13.6-75). The proportion of all proviruses that were intact diminished over time on ART, from about 10% at the first on-ART time point to about 5% at the last. Intact provirus levels on ART correlated with total HIV-1 DNA and residual plasma viremia, but there was no evidence for associations between intact provirus levels and inflammation or immune activation. CONCLUSIONS: Cells containing intact, replication-competent proviruses are selectively lost during suppressive ART. Defining the mechanisms involved should inform strategies to accelerate HIV-1 reservoir depletion.

A Phase 1/2 Randomized, Placebo-Controlled Trial of Romidespin in Persons With HIV-1 on Suppressive Antiretroviral Therapy.

BACKGROUND: Romidepsin (RMD) is a histone deacetylase inhibitor reported to reverse HIV-1 latency. We sought to identify doses of RMD that were safe and induced HIV-1 expression. METHODS: Enrollees had HIV-1 RNA <40 copies/mL on antiretroviral therapy. Measurements included RMD levels, plasma viremia by single-copy HIV-1 RNA assay, HIV-1 DNA, cell-associated unspliced HIV-1 RNA (CA-RNA), acetylation of histone H3-lysine-9 (H3K9ac+), and phosphorylation of transcription factor P-TEFb. Wilcoxon tests were used for comparison. RESULTS: In the single-dose cohorts 1-3, 43 participants enrolled (36 participants 0.5, 2, 5 mg/m 2 RMD; 7 placebo) and 16 enrolled in the multidose cohort 4 (13 participants 5 mg/m 2 RMD; 3 placebo). One grade 3 event (neutropenia) was possibly treatment related. No significant changes in viremia were observed in cohorts 1-4 compared to placebo. In cohort 4, pharmacodynamic effects of RMD were reduced proportions of CD4+ T cells 24 hours after infusions 2-4 (median, -3.5% to -4.5%) vs placebo (median, 0.5% to 1%; P ≤ .022), and increased H3K9ac+ and phosphorylated P-TEFb in CD4 + T cells vs placebo (P ≤ .02). CONCLUSIONS: RMD infusions were safe but did not increase plasma viremia or unspliced CA-RNA despite pharmacodynamic effects on CD4 + T cells. CLINICAL TRIALS REGISTRATION: NCT01933594.

Vesatolimod, a Toll-like Receptor 7 Agonist, Induces Immune Activation in Virally Suppressed Adults Living With Human Immunodeficiency Virus-1.

BACKGROUND: Treatment with vesatolimod, an investigational, oral, toll-like receptor 7 (TLR7) agonist, leads to sustained viral remission in some non-human primates when combined with anti-envelope antibodies or therapeutic vaccines. We report results of a Phase Ib study evaluating safety, pharmacokinetics, and pharmacodynamics of vesatolimod in adults living with human immunodeficiency virus (HIV)-1. METHODS: In this double-blind, multicenter, placebo-controlled trial, participants on antiretroviral therapy with screening plasma HIV-1 RNA levels <50 copies/mL were randomized (6:2) to receive 6-10 doses of vesatolimod (1-12 mg) or matching placebo orally every other week in sequential dose-escalation cohorts. The primary study objectives included establishing the safety and virologic effects of vesatolimod (change from baseline in plasma HIV-1 RNA). Pharmacokinetics and pharmacodynamic/immunologic activity were assessed as secondary objectives. RESULTS: A total of 48 individuals were randomly assigned to vesatolimod (n = 36) or placebo (n = 12). Vesatolimod was generally well tolerated, with no study drug-related serious adverse events or adverse events leading to study drug discontinuation. There were no statistically significant changes from baseline in plasma HIV-1 RNA in the vesatolimod groups, compared to placebo.Vesatolimod plasma exposures increased dose proportionally; consistent responses in cytokines, interferon-stimulated gene expression, and lymphocyte activation were observed with increasing dose levels above 4 mg. Peak elevations 24 hours after receipt of a 6 mg dose were >3.9-fold higher for interferon gamma-induced protein 10 (IP-10), interleukin-1 receptor antagonist (IL-1RA), interferon-inducible T-cell alpha chemoattractant (ITAC) when compared to baseline values. CONCLUSIONS: Vesatolimod was well tolerated at doses ranging from 1 to 12 mg. Immune stimulation was observed at doses above 4 mg, providing rationale for future combination trials in people living with HIV. CLINICAL TRIALS REGISTRATION: NCT02858401.

A Randomized, Placebo-Controlled, Pilot Clinical Trial of Dipyridamole to Decrease Human Immunodeficiency Virus-Associated Chronic Inflammation.

BACKGROUND: Adenosine is a potent immunoregulatory nucleoside produced during inflammatory states to limit tissue damage. We hypothesized that dipyridamole, which inhibits cellular adenosine uptake, could raise the extracellular adenosine concentration and dampen chronic inflammation associated with human immunodeficiency virus (HIV) type 1. METHODS: Virally suppressed participants receiving antiretroviral therapy were randomized 1:1 for 12 weeks of dipyridamole (100 mg 4 times a day) versus placebo capsules. All participants took open-label dipyridamole during weeks 12-24. Study end points included changes in markers of systemic inflammation (soluble CD163 and CD14, and interleukin 6) and levels of T-cell immune activation (HLA-DR+CD38+). RESULTS: Of 40 participants who were randomized, 17 dipyridamole and 18 placebo recipients had baseline and week 12 data available for analyses. There were no significant changes in soluble markers, apart from a trend toward decreased levels of soluble CD163 levels (P = .09). There was a modest decrease in CD8+ T-cell activation (-17.53% change for dipyridamole vs +13.31% for placebo; P = .03), but the significance was lost in the pooled analyses (P = .058). Dipyridamole also reduced CD4+ T-cell activation (-11.11% change; P = .006) in the pooled analyses. In post hoc analysis, detectable plasma dipyridamole levels were associated with higher levels of inosine, an adenosine surrogate, and of cyclic adenosine monophosphate. CONCLUSION: Dipyridamole increased extracellular adenosine levels and decreased T-cell activation significantly among persons with HIV-1 infection receiving virally suppressive therapy.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Allogeneic Hematopoietic Cell Transplant for HIV Patients with Hematologic Malignancies: The BMT CTN-0903/AMC-080 Trial.

We set out to assess feasibility and safety of allogeneic hematopoietic cell transplant in 17 persons with HIV in a phase II prospective multicenter trial. The primary endpoint was 100-day nonrelapse mortality (NRM). Patients had an 8/8 HLA-matched related or at least a 7/8 HLA-matched unrelated donor. Indications for transplant were acute leukemia, myelodysplasia, and lymphoma. Conditioning was myeloablative or reduced intensity. There was no NRM at 100 days. The cumulative incidence of grades II to IV acute graft-versus-host disease (GVHD) was 41%. At 1 year, overall survival was 59%; deaths were from relapsed/progressive disease (n = 5), acute GVHD (n = 1), adult respiratory distress syndrome (n = 1), and liver failure (n = 1). In patients who achieved complete chimerism, cell-associated HIV DNA and inducible infectious virus in the blood were not detectable. Blood and Marrow Transplant Clinical Trials Network 0903/AIDS Malignancy Consortium 080 was registered at www.clinicaltrials.gov (no. NCT01410344).

A Simpler and More Sensitive Single-Copy HIV-1 RNA Assay for Quantification of Persistent HIV-1 Viremia in Individuals on Suppressive Antiretroviral Therapy.

A real-time quantitative reverse transcriptase PCR assay with single-copy sensitivity targeting the integrase region of HIV-1 (integrase single-copy assay [iSCA] v1.0) has been widely used to quantify plasma viremia in individuals on antiretroviral therapy (ART). iSCA v1.0 requires the use of an ultracentrifuge, and only about half of the nucleic acid extracted from plasma is assayed for HIV-1 RNA. We sought to simplify sample processing using microcentrifugation and improve assay sensitivity by testing more than 75% of the total extracted nucleic acid for HIV-1 RNA (iSCA v2.0). We evaluated the limit of detection (LoD) of iSCA v2.0 by testing replicates of low-copy plasma HIV-1 RNA standards. By probit analysis, the 95% LoD was 1 copy of HIV-1 RNA per milliliter for a 5-ml plasma sample. To compare the sensitivity of iSCA v1.0 and v2.0, we tested plasma samples with both assays from 60 participants on ART with HIV-1 RNA below 20 cps/ml. Of the 31 samples that had no detectable HIV-1 RNA by iSCA v1.0, 17 (55%) were detectable by v2.0 with an HIV-1 RNA mean value of 3.5 cps/ml. Twenty-nine samples were detectable with both assay versions, but average values of HIV-1 RNA cps/ml were 2.7-fold higher for v2.0 than v1.0. These results support the adoption of a new, more sensitive and simpler single-copy HIV-1 RNA assay (iSCA v2.0) to quantify residual viremia on ART and to assess the impact of experimental interventions designed to decrease HIV-1 reservoirs.

Levels of HIV-1 persistence on antiretroviral therapy are not associated with markers of inflammation or activation.

Antiretroviral therapy (ART) reduces levels of HIV-1 and immune activation but both can persist despite clinically effective ART. The relationships among pre-ART and on-ART levels of HIV-1 and activation are incompletely understood, in part because prior studies have been small or cross-sectional. To address these limitations, we evaluated measures of HIV-1 persistence, inflammation, T cell activation and T cell cycling in a longitudinal cohort of 101 participants who initiated ART and had well-documented sustained suppression of plasma viremia for a median of 7 years. During the first 4 years following ART initiation, HIV-1 DNA declined by 15-fold (93%) whereas cell-associated HIV-1 RNA (CA-RNA) fell 525-fold (>99%). Thereafter, HIV-1 DNA levels continued to decline slowly (5% per year) with a half-life of 13 years. Participants who had higher HIV-1 DNA and CA-RNA before starting treatment had higher levels while on ART, despite suppression of plasma viremia for many years. Markers of inflammation and T cell activation were associated with plasma HIV-1 RNA levels before ART was initiated but there were no consistent associations between these markers and HIV-1 DNA or CA-RNA during long-term ART, suggesting that HIV-1 persistence is not driving or driven by inflammation or activation. Higher levels of inflammation, T cell activation and cycling before ART were associated with higher levels during ART, indicating that immunologic events that occurred well before ART initiation had long-lasting effects despite sustained virologic suppression. These findings should stimulate studies of viral and host factors that affect virologic, inflammatory and immunologic set points prior to ART initiation and should inform the design of strategies to reduce HIV-1 reservoirs and dampen immune activation that persists despite ART.

T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy.

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal.

Cumulative Antiretroviral Exposure Measured in Hair Is Not Associated With Measures of HIV Persistence or Inflammation Among Individuals on Suppressive ART.

Data on the relationship of antiretroviral exposure to measures of human immunodeficiency virus (HIV) persistence are limited. To address this gap, multiple viral, immunologic, and pharmacologic measures were analyzed from individuals with sustained virologic suppression on therapy (median 7 years) in the AIDS Clinical Trials Group A5321 cohort. Among 110 participants on tenofovir-(TFV)-disoproxil-fumarate (TDF)/emtricitabine (FTC)-containing regimens, we found no significant correlation between hair concentrations of individual antiretrovirals (ARVs) in the regimen and measures of HIV persistence (plasma HIV-1 RNA by single copy assay, cell-associated-DNA, cell-associated RNA) or soluble markers of inflammation. These findings suggest that higher systemic ARV exposure may not impact HIV persistence or inflammation.

IL-10 inhibits mature fibrotic granuloma formation during Mycobacterium tuberculosis infection.

Protective immunity and latent Mycobacterium tuberculosis infection in humans are associated with the formation of mature protective granulomas within the lung. Unfortunately, understanding the importance of such structures has been hindered by the lack of small-animal models that can develop mature granulomas. In this article, we describe for the first time, to our knowledge, the formation of mature, fibrotic M. tuberculosis-containing pulmonary granulomas in a mouse model of IL-10 deficiency (CBA/J IL-10(-/-)). Long-term control of M. tuberculosis infection in the absence of IL-10 was also associated with an early and enhanced capacity for Ag presentation and a significant increase in the generation of multifunctional T cells. Although IL-10 deficiency is known to enhance Th1 immune responses in general, we demonstrate in this study using transient anti-IL-10R treatment that it is the presence of IL-10 in vivo during the first month of M. tuberculosis infection that plays a definitive role in the inhibition of optimum protective immunity that can establish the environment for mature granuloma formation. Although the importance of IL-10 during M. tuberculosis infection has been debated, our data demonstrate that in CBA/J mice, IL-10 plays a significant early inhibitory role in preventing the development of protective immunity associated with containment of M. tuberculosis infection.

HIV-1 envelope diversity and sensitivity to broadly neutralizing antibodies across stages of acute HIV-1 infection.

We studied the relationship between viral diversity and susceptibility to broadly neutralizing antibodies (bNAbs) in longitudinal plasma and peripheral blood mononuclear cells from 89 people with HIV who initiated antiretroviral therapy (ART) during acute and early HIV-1 infection (AEHI). HIV-1 diversity and predicted bNAb susceptibility were comparable across AEHI. Diversity evolution was not observed during ART, suggesting (pro)viruses at initiation or during treatment may identify individuals with susceptible virus for bNAb interventional trials.

HIV-1-infected T cell clones are shared across cerebrospinal fluid and blood during ART.

The central nervous system HIV reservoir is incompletely understood and is a major barrier to HIV cure. We profiled people with HIV (PWH) and uninfected controls through single-cell transcriptomic and T cell receptor (TCR) sequencing to understand the dynamics of HIV persistence in the CNS. In PWH on ART, we found that most participants had single cells containing HIV-1 RNA, which was found predominantly in CD4 central memory T cells, in both cerebrospinal fluid (CSF) and blood. HIV-1 RNA-containing cells were found more frequently in CSF than blood, indicating a higher burden of reservoir cells in the CNS than blood for some PWH. Most CD4 T cell clones containing infected cells were compartment specific, while some (22%) - including rare clones with members of the clone containing detectable HIV RNA in both blood and CSF - were found in both CSF and blood. These results suggest that infected T cells trafficked between tissue compartments and that maintenance and expansion of infected T cell clones contributed to the CNS reservoir in PWH on ART.

Impact of antiretroviral therapy during acute or early HIV infection on virologic and immunologic outcomes: results from a multinational clinical trial.

OBJECTIVE: To assess how antiretroviral therapy (ART) initiation during acute or early HIV infection (AEHI) affects the viral reservoir and host immune responses. DESIGN: Single-arm trial of ART initiation during AEHI at 30 sites in the Americas, Africa, and Asia. METHODS: HIV DNA was measured at week 48 of ART in 5 million CD4 + T cells by sensitive qPCR assays targeting HIV gag and pol . Peripheral blood mononuclear cells were stimulated with potential HIV T cell epitope peptide pools consisting of env , gag , nef, and pol peptides and stained for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines. RESULTS: From 2017 to 2019, 188 participants initiated ART during Fiebig stages I ( n  = 6), II ( n  = 43), III ( n  = 56), IV ( n  = 23), and V ( n  = 60). Median age was 27 years (interquartile range 23-38), 27 (14%) participants were female, and 180 (97%) cisgender. Among 154 virally suppressed participants at week 48, 100% had detectable HIV gag or pol DNA. Participants treated during Fiebig I had the lowest HIV DNA levels ( P  < 0.001). Week 48 HIV DNA mostly did not correlate with concurrent CD4 + or CD8 + T cell HIV-specific immune responses (rho range -0.11 to +0.19, all P  > 0.025). At week 48, the magnitude, but not polyfunctionality, of HIV-specific T cell responses was moderately reduced among participants who initiated ART earliest. CONCLUSION: Earlier ART initiation during AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood. These findings explain the rapid viral rebound observed after ART cessation in early-treated individuals with undetectable HIV DNA by less sensitive methods.

Killer cell lectin-like receptor G1 deficiency significantly enhances survival after Mycobacterium tuberculosis infection.

The expression of T cell differentiation markers is known to increase during Mycobacterium tuberculosis infection, and yet the biological role of such markers remains unclear. We examined the requirement of the T cell differentiation marker killer cell lectin-like receptor G1 (KLRG1) during M. tuberculosis infection using mice deficient in KLRG1. KLRG1(-/-) mice had a significant survival extension after M. tuberculosis infection compared to wild-type controls, and maintained a significantly lower level of pulmonary M. tuberculosis throughout chronic infection. Improved control of M. tuberculosis infection was associated with an increased number of activated pulmonary CD4(+) T cells capable of secreting gamma interferon (IFN-γ). Our report is the first to show an in vivo impact of KLRG1 on disease control.

CBA/J mice generate protective immunity to soluble Ag85 but fail to respond efficiently to Ag85 during natural Mycobacterium tuberculosis infection.

In CBA/J mice, susceptibility to Mycobacterium tuberculosis (M.tb) is associated with low interferon-gamma (IFN-γ) responses to antigens (Antigen 85 (Ag85) and early secreted antigenic target-6 (ESAT-6)) that have been defined as immunodominant. Here, we asked whether the failure of CBA/J mice to recognize Ag85 is a consequence of M.tb infection or whether CBA/J mice have a general defect in generating specific T-cell responses to this protein antigen. We compared CBA/J mice during primary M.tb infection, Ag85 vaccination followed by M.tb challenge, or M.tb memory immune mice for their capacity to generate Ag85-specific IFN-γ responses and to control M.tb infection. CBA/J mice did not respond efficiently to Ag85 in the context of natural infection or re-infection. In contrast, CBA/J mice could generate Ag85-specific IFN-γ responses and protective immunity when this antigen was delivered as a soluble protein. Our data indicate that although M.tb infection of CBA/J mice does not drive an Ag85 response, these mice can fully and protectively respond to Ag85 if it is delivered as a vaccine. The data from this experimental model suggest that the Ag85-containing vaccines in clinical trials should protect M.tb susceptible humans.