Suppressive Antiretroviral Therapy Research Articles

Elevation of cell-associated HIV-1 transcripts in CSF CD4+ T cells, despite effective antiretroviral therapy, is linked to brain injury

Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in sixteen PLHIV. 14/16 CSF cell samples had quantifiable CA-HIV-1 RNA, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T-cells; p<0.0001). In individual PLHIV, higher levels of HIV-1 transcripts in CSF cells were associated with greater brain injury in the frontal white matter (Std β=-0.73; p=0.007) and posterior cingulate (Std β=-0.61; p=0.03). 18-colour flow cytometry revealed that the CSF cells were 91% memory T-cells, equally CD4+ and CD8+ T-cells, but fewer B cells (0.4 %), and monocytes (3.1%). CXCR3+CD49d+integrin β7-, CCR5+CD4+ T-cells were highly enriched in CSF, compared with PBMC (p <0.001). However, CA-HIV-1 RNA could not be detected in 10/16 preparations of highly purified monocytes from PBMC, and was extremely low in the other six. Our data show that elevated HIV-1 transcripts in CSF cells were associated with brain injury, despite suppressive ART. The cellular source is most likely memory CD4+ T cells from blood, rather than trafficking monocytes. Future research should focus on inhibitors of this transcription to reduce local production of potentially neurotoxic and inflammatory viral products.

Stable HIV Reservoir Despite Prolonged Low-Dose Mycophenolate to Limit CD4+ T-cell Proliferation.

The HIV reservoir of latently infected CD4+ T cells represents the barrier to cure. CD4+ T-cell proliferation is a mechanism that sustains the reservoir even during prolonged antiretroviral therapy (ART). Blocking proliferation may therefore deplete the reservoir. We conducted an unblinded, uncontrolled clinical trial of mycophenolate, a T-cell antiproliferative compound, in people with HIV on chronic suppressive ART. Study drug dose selection was based on calibration to an observed ex vivo antiproliferative effect. The primary outcome was clinically significant reduction (>0.25 log10) in the HIV reservoir, measured by total and intact HIV DNA per million T cells in blood over 48 weeks. Five participants enrolled in the trial. Four participants took mycophenolate mofetil (MMF). One had a per-protocol switch to enteric-coated mycophenolate sodium (Myfortic) due to nausea but left the study for personal reasons. One participant developed finger cellulitis, but there were no opportunistic infections. In the 4 participants who completed the protocol, there was no clinically significant reduction in total or intact HIV DNA. There was no change in blood CD4+ T-cell subset composition within the HIV reservoir or the entire CD4+ T-cell population, although total CD4+ T cells decreased slightly in all 4 participants. An ex vivo antiproliferative effect was observed using participant serum obtained 1 hour after dosing, but this effect was severely diminished at drug trough. Mycophenolate given over 48 weeks did not reduce the volume or composition of the HIV reservoir. NCT03262441.

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

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). 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). 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. 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.

Association Between the Development of Subclinical Cardiovascular Disease and Human Immunodeficiency Virus (HIV) Reservoir Markers in People With HIV on Suppressive Antiretroviral Therapy.

We report that people with human immunodeficiency virus (HIV) diagnosed with coronary artery atherosclerotic plaques display higher levels of HIV DNA compared with those without atherosclerotic plaques. In a multivariable prediction model that included 27 traditional and HIV-related risk factors, measures of HIV DNA were among the most important predictors of atherosclerotic plaque formation.

HIV proviral genetic diversity, compartmentalization and inferred dynamics in lung and blood during long-term suppressive antiretroviral therapy.

The lung is an understudied site of HIV persistence. We isolated 898 subgenomic proviral sequences (nef) by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 8% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harboured at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. The extent of blood proviral diversity on ART was also a strong indicator of diversity in lung (Spearman's ρ = 0.98, p<0.0001). For three participants, insufficient lung sequences were recovered to reliably investigate genetic compartmentalization. Of the remainder, only two participants showed statistically significant support for compartmentalization when analysis was restricted to distinct proviruses per site, and the extent of compartmentalization was modest in both cases. When all within-host sequences (including duplicates) were considered, the number of compartmentalized datasets increased to four. Thus, while a subset of individuals harbour somewhat distinctive proviral populations in blood and lung, this can simply be due to unequal distributions of clonally-expanded sequences. For two participants, on-ART proviruses were also phylogenetically analyzed in context of plasma HIV RNA populations sampled up to 18 years prior, including pre-ART and during previous treatment interruptions. In both participants, on-ART proviruses represented the most ancestral sequences sampled within-host, confirming that HIV sequences can persist in the body for decades. This analysis also revealed evidence of re-seeding of the reservoir during treatment interruptions. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individual's proviral composition. Personalized HIV remission and cure strategies may be needed to overcome these challenges.

Non-AIDS Events in Individuals With Spontaneous Control of HIV-1: A Systematic Review.

Despite antiretroviral therapy (ART), people living with HIV (PLHIV) are at increased risk for non-AIDS-defining events (nADEs), including cardiovascular events, non-AIDS malignances, hepatic disease, and bacterial pneumonia. This systematic review seeks to answer the question: are PLHIV who spontaneously control HIV-1 subject to an increased risk of various nADEs relative to noncontrolling PLHIV on ART and people without HIV? Databases were searched on June 9, 2021 with a search syntax focused on the elements "HIV," "spontaneous control," and "clinical outcomes": Embase.com (includes Embase and Medline), Medline Ovid (includes PubMed), Cochrane library, Web of Science, and Google Scholar. Included were studies reporting non-AIDS events in spontaneous controllers. Excluded were case reports, conference papers, editorials, and reviews. Of 1134 identified records, 34 were assessed for full-text and 12 studies were included in the qualitative synthesis: 5 cohorts, 2 cross-sectional prevalence studies, 4 cross-sectional imaging studies, and one case series. Four of 5 cohort studies showed that spontaneous controllers have a similar risk to develop nADEs compared with PLHIV on suppressive ART, specifically cardiovascular events, non-AIDS malignancies, hepatic disease, and bacterial pneumonia. Cross-sectional imaging studies showed a higher presence of subclinical cardiovascular disease in spontaneous controllers, than in people without HIV. Individuals with spontaneous control of HIV-1 do not seem to be at a greater risk to develop different nADEs compared with PLHIV on suppressive ART. More data are needed, because the present conclusions are based on a limited number of studies that show large heterogeneity among them.

Forging a Functional Cure for HIV: Transcription Regulators and Inhibitors.

Current antiretroviral therapy (ART) increases the survival of HIV-infected individuals, yet it is not curative. The major barrier to finding a definitive cure for HIV is our inability to identify and eliminate long-lived cells containing the dormant provirus, termed viral reservoir. When ART is interrupted, the viral reservoir ensures heterogenous and stochastic HIV viral gene expression, which can reseed infection back to pre-ART levels. While strategies to permanently eradicate the virus have not yet provided significant success, recent work has focused on the management of this residual viral reservoir to effectively limit comorbidities associated with the ongoing viral transcription still observed during suppressive ART, as well as limit the need for daily ART. Our group has been at the forefront of exploring the viability of the block-and-lock remission approach, focused on the long-lasting epigenetic block of viral transcription such that without daily ART, there is no risk of viral rebound, transmission, or progression to AIDS. Numerous studies have reported inhibitors of both viral and host factors required for HIV transcriptional activation. Here, we highlight and review some of the latest HIV transcriptional inhibitor discoveries that may be leveraged for the clinical exploration of block-and-lock and revolutionize the way we treat HIV infections.

HIV but Not CMV Replication Alters the Blood Cytokine Network during Early HIV Infection in Men.

Objective: CMV coinfection contributes to sustained immune activation in people with chronic HIV. In particular, asymptomatic CMV shedding in semen has been associated with increased local and systemic immune activation, even during suppressive antiretroviral therapy (ART). However, the effect of seminal CMV shedding in people with HIV in the earliest phase of HIV infection is not known. Methods: Using Luminex, we measured the concentration of 34 cytokines in the blood plasma of sixty-nine men who had sex with men with or without HIV and in subgroups of CMV shedders vs. non-shedders. Differences in blood plasma cytokines between groups were investigated using the multivariate supervised partial least squares discriminant analysis method. Results: Independently of CMV, we found that concentrations of IP-10, MIG, MCP-1, I-TAC 10, IL-16, and MIP-1β were modulated in the earliest phase of HIV infection compared with control individuals without HIV. In people with HIV, there was no difference in blood cytokines among CMV shedders vs. non-shedders. Conclusion: In early/acute HIV infection, asymptomatic CMV shedding in semen does not drive additional cytokine changes in blood. Early ART initiation should remain the priority, while the added benefit of CMV suppression during the various stages of HIV infection needs to be further investigated.

Mucosal immune cell populations and the bacteriome of adenoids and tonsils from people living with HIV on suppressive antiretroviral therapy.

Ear, nose, and throat (ENT) conditions are prevalent in people living with HIV (PLWH) and occur at all strata of CD4 counts and despite antiretroviral therapy (ART). ENT conditions are underreported in PLWH. Also, little is known about the adenotonsillar microbiota and its relation to resident adaptive and innate immune cells. To bridge this gap, we characterized immune cell populations and the bacterial microbiota of two anatomical sites (adenoids, tonsils) and the oral cavity. Adenoids and tonsils were obtained from PLWH (n = 23) and HIV-seronegative individuals (SN, n = 16) after nasal surgery and tonsillectomy and processed for flow cytometry. Nasopharyngeal, oropharyngeal swabs, and oral rinses were collected prior to surgery for 16S sequencing. Wilcoxon rank sum test, principal coordinate analysis, permutational multivariate analysis of variance, and linear discriminant analysis (LEfSe) were used to assess differences between PLWH and SN. Spearman’s correlations were performed to explore interactions between the bacteriome and mucosal immune cells. Of the 39 individuals included, 30 (77%) were men; the median age was 32 years. All PLWH were on ART, with a median CD4 of 723 cells. ENT conditions were classified as inflammatory or obstructive, with no differences observed between PLWH and SN. PLWH had higher frequencies of activated CD4+ and CD8+ T cells, increased T helper (Th)1 and decreased Th2 cells; no differences were observed for B cells and innate immune cells. Alpha diversity was comparable between PLWH and SN at all 3 anatomical sites (adenoids, tonsils, and oral cavity). The impact of HIV infection on the bacterial community structure at each site, as determined by Permutational multivariate analysis of variance, was minor and not significant. Two discriminant genera were identified in adenoids using LEfSe: Staphylococcus for PLWH and Corynebacterium for SN. No discriminant genera were identified in the oropharynx and oral cavity. Niche-specific differences in microbial diversity and communities were observed. PLWH shared less of a core microbiota than SN. In the oropharynx, correlation analysis revealed that Th17 cells were inversely correlated with bacterial richness and diversity, Filifactor, Actinomyces and Treponema; and positively correlated with Streptococcus. Our study contributes toward understanding the role of the adenotonsillar microbiota in the pathophysiology of ENT conditions.

Plasma Cell-Free Mitochondrial DNA as a Marker of Geriatric Syndromes in Older Adults With HIV.

Older people with HIV experience more comorbidities and geriatric syndromes than their HIV-negative peers, perhaps due to residual inflammation despite suppressive antiretroviral therapy. Cell-free mitochondrial DNA (cfmtDNA) released during necrosis-mediated cell death potentially acts as both mediator and marker of inflammatory dysregulation. Thus, we evaluated plasma cfmtDNA as a potential biomarker of geriatric syndromes. Participants underwent the Montreal Cognitive Assessment (MoCA), frailty testing, and measurement of plasma cfmtDNA by qPCR and inflammatory markers including C-reactive protein, interleukin-6 (IL-6), interferon gamma, and tumor necrosis factor alpha in this cross-sectional study. Across 155 participants, the median age was 60 years (Q1, Q3: 56, 64), one-third were female, and 92% had HIV-1 viral load <200 copies/mL. The median MoCA score was 24 (21, 27). The plasma cfmtDNA level was higher in those with cognitive impairment (MoCA <23) ( P = 0.02 by the t test) and remained significantly associated with cognitive impairment in a multivariable logistic regression model controlling for age, sex, race, CD4 T-cell nadir, HIV-1 viremia, and depression. Two-thirds of participants met the criteria for a prefrail or frail state; higher plasma cfmtDNA was associated with slow walk and exhaustion but not overall frailty state. Cognitive dysfunction was not associated with C-reactive protein, IL-6, interferon gamma, or tumor necrosis factor alpha, and frailty state was only associated with IL-6. Plasma cfmtDNA may have a role as a novel biomarker of cognitive dysfunction and key components of frailty. Longitudinal investigation of cfmtDNA is warranted to assess its utility as a biomarker of geriatric syndromes in older people with HIV.

Adenovirus 36 Infection in People Living with HIV-An Epidemiological Study of Seroprevalence and Associations with Cardiovascular Risk Factors.

Background. With the life expectancy of people living with HIV (PLHIV) rapidly approaching that of the general population, cardiovascular health in this group is as relevant as ever. Adenovirus 36 (Adv36) is one of the few viruses suspected to be a causative factor in promoting obesity in humans, yet there is a lack of data on this infection in PLHIV. Methods. PLHIV on stable suppressive antiretroviral therapy were included in the study, with assessment of anthropometric measures, blood pressure, serum lipid levels, fasting serum glucose and insulin, non-classical serum cardiovascular risk markers related to inflammation (hsCRP, resistin, calprotectin), and anti-Adv36 antibodies during a routine check-up. Results. 91 participants were recruited, of which 26.4% were Adv36-seropositive (Adv36(+)). Compared to Adv36-seronegative (Adv36(−)) controls, Adv36(+) individuals had a lower waist circumference (Adv36(+) 89.6 ± 7.7 cm, Adv36(−) 95.5 ± 11.7 cm, p = 0.024) and a lower waist-to-hip ratio (Adv36(+) 0.88 ± 0.06, Adv36(−) 0.92 ± 0.09, p = 0.014), but this did not reach statistical significance in the multivariate analysis (p > 0.05). Adv36(+) participants were less likely to be on lipid-lowering treatment (Adv36(+) 12.5%, Adv36(−) 34.3%, p = 0.042), even after adjustment for relevant baseline characteristics (OR = 0.23, 95%CI = 0.04–0.91), but no differences in cholesterol or triglyceride levels were found. No other statistically significant associations were observed. Conclusions. We found no evidence to support the claim that past Adv36-infection is associated with an increased prevalence of cardiovascular risk factors or with elevated inflammatory markers in PLHIV. More research is needed to replicate these findings in other samples of PLHIV and to compare them with the HIV-negative population.

Neurodegenerative phagocytes mediate synaptic stripping in Neuro-HIV.

Glial cell activation is a hallmark of several neurodegenerative and neuroinflammatory diseases. During HIV infection, neuroinflammation is associated with cognitive impairment, even during sustained long-term suppressive antiretroviral therapy. However, the cellular subsets contributing to neuronal damage in the CNS during HIV infection remain unclear.Using post-mortem brain samples from eight HIV patients and eight non-neurological disease controls, we identify a subset of CNS phagocytes highly enriched in LGALS3, CTSB, GPNMB and HLA-DR, a signature identified in the context of ageing and neurodegeneration.In HIV patients, the presence of this phagocyte phenotype was associated with synaptic stripping, suggesting an involvement in the pathogenesis of HIV-associated neurocognitive disorder.Taken together, our findings elucidate some of the molecular signatures adopted by CNS phagocytes in HIV-positive patients and contribute to the understanding of how HIV might pave the way to other forms of cognitive decline in ageing HIV patient populations.

Evaluation of markers of fibrinolysis and coagulation in pregnant women with human immunodeficiency virus

IntroductionHuman immunodeficiency virus (HIV) in pregnant women is characterized by immune activation and inflammation despite suppressive antiretroviral therapy (ART). The extent to which ongoing inflammation contributes to activation of coagulation and fibrinolysis is unknown. Materials and methodsThis cross-sectional study included pregnant women in the following three groups: HIV negative (n = 109), HIV infected virologically suppressed (n = 109) and HIV infected with HIV viral load (VL) of >50 copies/mL (n = 80). Fibrinolytic activity was evaluated by measuring d-dimer and plasminogen activator inhibitor-1 (PAI-1) as well as thrombin-antithrombin (TAT) complex concentrations, as an index of coagulation, in the first, second and third trimesters. ResultsIn this population, with a mean age of 33 ± 6 years, pregnancy outcomes were recorded for 277 (93.0 %) participants with live births. HIV infected participants with virological suppression and VL of >50 copies/mL showed significantly increasing levels of d-dimer and PAI-1 in the first, second and third trimesters, as compared to HIV negative participants. No significant differences were observed between HIV infected participants with virological suppression and HIV infected participants with VL > 50 copies/mL for levels of first and third trimester d-dimer and PAI-1 in each trimester. In addition, TAT complex levels in the first trimester were significantly increased in HIV infected virologically suppressed participants as compared to HIV negative participants. ConclusionHIV infected virologically suppressed pregnant women show evidence of persistently impaired markers of fibrinolysis. Future research should explore the risk of adverse pregnancy complications among HIV infected pregnant women in the modern era of ART.

No evidence that circulating HIV-specific immune responses contribute to persistent inflammation and immune activation in persons on long-term ART.

People with HIV (PWH) have persistently elevated levels of inflammation and immune activation despite suppressive antiretroviral therapy (ART), with specific biomarkers showing associations with non-AIDS-defining morbidities and mortality. We investigated the potential role of the HIV-specific adaptive immune response, which also persists under ART, in driving levels of these clinically relevant biomarkers. Cohort-based study. HIV-specific IFN-γ-producing T-cell responses and antibody concentrations were measured in blood at study entry in the ACTG A5321 cohort, following a median of 7 years of suppressive ART. HIV persistence measures including cell-associated (CA)-DNA, CA-RNA, and plasma HIV RNA (single-copy assay) were also assessed at study entry. Plasma inflammatory biomarkers and T-cell activation and cycling were measured at a pre-ART time point and at study entry. Neither the magnitudes of HIV-specific T-cell responses nor HIV antibody levels were correlated with levels of the inflammatory or immune activation biomarkers, including hs-CRP, IL-6, neopterin, sCD14, sCD163, TNF-α, %CD38 + HLA-DR + CD8 + and CD4 + cells, and %Ki67 + CD8 + and CD4 + cells - including after adjustment for pre-ART biomarker level. Plasma HIV RNA levels were modestly correlated with CD8 + T-cell activation ( r = 0.25, P = 0.027), but other HIV persistence parameters were not associated with these biomarkers. In mediation analysis, relationships between HIV persistence parameters and inflammatory biomarkers were not influenced by either HIV-specific T-cell responses or antibody levels. Adaptive HIV-specific immune responses do not appear to contribute to the elevated inflammatory and immune activation profile in persons on long-term ART.

Immunometabolic Reprogramming in Response to HIV Infection Is Not Fully Normalized by Suppressive Antiretroviral Therapy.

Background: HIV infection results in immunometabolic reprogramming. While we are beginning to understand how this metabolic reprogramming regulates the immune response to HIV infection, we do not currently understand the impact of ART on immunometabolism in people with HIV (PWH). Methods: Serum obtained from HIV-infected (n = 278) and geographically matched HIV seronegative control subjects (n = 300) from Rakai Uganda were used in this study. Serum was obtained before and ~2 years following the initiation of ART from HIV-infected individuals. We conducted metabolomics profiling of the serum and focused our analysis on metabolic substrates and pathways assocaited with immunometabolism. Results: HIV infection was associated with metabolic adaptations that implicated hyperactive glycolysis, enhanced formation of lactate, increased activity of the pentose phosphate pathway (PPP), decreased β-oxidation of long-chain fatty acids, increased utilization of medium-chain fatty acids, and enhanced amino acid catabolism. Following ART, serum levels of ketone bodies, carnitine, and amino acid metabolism were normalized, however glycolysis, PPP, lactate production, and β-oxidation of long-chain fatty acids remained abnormal. Conclusion: Our findings suggest that HIV infection is associated with an increased immunometabolic demand that is satisfied through the utilization of alternative energetic substrates, including fatty acids and amino acids. ART alone was insufficient to completely restore this metabolic reprogramming to HIV infection, suggesting that a sustained impairment of immunometabolism may contribute to chronic immune activation and comorbid conditions in virally suppressed PWH.

Stochastic HIV model coupled with pharmacokinetics and drug adherence may explain intermittent viral blips

We develop a stochastic HIV model by integrating drug adherence into a pharmacokinetic model and by coupling a pharmacodynamic model with a viral dynamic model. Numerical simulations show that the proposed model can generate viral blips, which have been observed in HIV patients receiving suppressive antiretroviral therapy. We calculate the probability density function of infected CD4+ T cells by developing the generalized density evolution equation. We fit the model to the clinical data of four HIV patients exhibiting viral blips. The results demonstrate that poor drug adherence can be a reason explaining the occurrence of viral blips in treated HIV patients. We also find that viral dynamics are sensitive to drug-adherence parameters, which have a significant impact on the frequency and amplitude of viral blips. The modeling and methods can be applied to the study of long-term therapy of other chronic diseases in which drug adherence might also be an issue.

Deep Sequencing Analysis of Individual HIV-1 Proviruses Reveals Frequent Asymmetric Long Terminal Repeats.

ABSTRACTEffective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5′-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5′ and 3′ long terminal repeats (LTRs), commonly in the form of major 3′ deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1.IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3′ long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.

People With Human Immunodeficiency Virus Receiving Suppressive Antiretroviral Therapy Show Typical Antibody Durability After Dual Coronavirus Disease 2019 Vaccination and Strong Third Dose Responses.

BackgroundLonger-term humoral responses to two-dose COVID-19 vaccines remain incompletely characterized in people living with HIV (PLWH), as do initial responses to a third dose.MethodsWe measured antibodies against the SARS-CoV-2 spike protein receptor-binding domain, ACE2 displacement and viral neutralization against wild-type and Omicron strains up to six months following two-dose vaccination, and one month following the third dose, in 99 PLWH receiving suppressive antiretroviral therapy, and 152 controls.ResultsThough humoral responses naturally decline following two-dose vaccination, we found no evidence of lower antibody concentrations nor faster rates of antibody decline in PLWH compared to controls after accounting for sociodemographic, health and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after two doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though Omicron-specific responses were consistently weaker than against wild-type. Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses.ConclusionPLWH receiving suppressive antiretroviral therapy mount strong antibody responses after two- and three-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

Neuropathic pain correlates with worsening cognition in people with human immunodeficiency virus.

Neuropathic pain and cognitive impairment are among the HIV-related conditions that have most stubbornly resisted amelioration by virally suppressive antiretroviral therapy. Overlaps between the regional brain substrates and mechanisms of neuropathic pain and cognitive disorders are increasingly recognized, yet no studies have examined the longitudinal relationship between these two disorders. Participants in the prospective, observational CNS HIV AntiRetroviral Therapy Effects Research (CHARTER) cohort underwent standardized clinical evaluations for clinical examination findings of distal sensory polyneuropathy, reporting distal neuropathic pain and neurocognitive performance at study entry (baseline) and an average of 12 years later. Change in neuropathic pain and neuropathy status from baseline to follow-up was by self-report and repeat examination, and change in neurocognitive performance was assessed using a previously published summary regression-based change score. Relationships between incident or worsened neuropathic pain and neurocognitive change were evaluated using uni- and multivariable regressions, including age at baseline and other relevant covariates. Participants were 385 people with HIV, 91 (23.6%) females, mean ± standard deviation (SD) age at baseline 43.5 (7.81) years, ethnicity 44.9% African American, 10.6% Hispanic, 42.6% non-Hispanic white and 1.82% other. Baseline median (interquartile range) nadir CD4 was 175 (34 309) cells/µl and current CD4 was 454 (279 639). Incident or worsened distal neuropathic pain occurred in 98 (25.5%) over the follow-up period. People with HIV with incident or worsened distal neuropathic pain had significantly worsened neurocognitive performance at follow-up compared to those without incident or worsened distal neuropathic pain (summary regression-based change score mean ± SD -0.408 ± 0.700 versus -0.228 ± 0.613; P = 0.0158). This effect remained significant when considering viral suppression on antiretroviral therapy, incident diabetes and other covariates as predictors. Overall neurocognitive change related to neuropathic pain was driven primarily by changes in the domains of executive function and speed of information processing. Those with incident distal neuropathy signs did not have neurocognitive worsening, nor did individuals who used opioid analgesics or other pain-modulating drugs such as amitriptyline. Worsened neurocognitive performance in people with HIV was associated with worsened neuropathic pain but not with changes in physical signs of neuropathy, and this was not attributable to therapies for pain or depression or to differences in viral suppression. This finding implies that incident or worsened pain may signal increased risk for neurocognitive impairment, and deserves more investigation, particularly if better pain management might stabilize or improve neurocognitive performance.

Sex differences in cytokine profiles during suppressive antiretroviral therapy.

Despite lower plasma HIV RNA levels, women progress faster to AIDS than men. The reasons for these differences are not clear but might be a consequence of an elevated inflammatory response in women. We investigated sex differences in cytokine profiles by measuring the concentrations of 36 cytokines/chemokines by Luminex in blood of women and men (sex at birth) with chronic HIV infection under suppressive therapy. We initially performed a principal component analysis to see if participants clustered by sex, and then fit a partial least squares discriminant analysis (PLS-DA) model where we used cytokines to predict sex at birth. The significance of the difference in nine cytokines with VIP greater than 1 was tested using Wilcoxon test-rank. Further, potential confounding factors were tested by multivariate linear regression models. Overall, we predicted sex at birth in the PLS-DA model with an error rate of approximately 13%. We identified five cytokines, which were significantly higher in women compared with men, namely the pro-inflammatory chemokines CXCL1 (Gro-α), CCL5 (RANTES), CCL3 (MIP-1α), CCL4 (MIP-1β), as well as the T-cell homeostatic factor IL-7. The effect of sex remained significant after adjusting for CD4 + , age, ethnicity, and race for all cytokines, except for CCL3 and race. The observed sex-based differences in cytokines might contribute to higher immune activation in women compared with men despite suppressive therapy. Increased levels of IL-7 in women suggest that homeostatic proliferation may have a differential contribution to HIV reservoir maintenance in female and male individuals. Our study emphasizes the importance of sex-specific studies of viral pathogenesis.