HIV-1 DNA Research Articles

Estimating the contribution of CD4 T cell subset proliferation and differentiation to HIV persistence

Persistence of HIV in people living with HIV (PWH) on suppressive antiretroviral therapy (ART) has been linked to physiological mechanisms of CD4+ T cells. Here, in the same 37 male PWH on ART we measure longitudinal kinetics of HIV DNA and cell turnover rates in five CD4 cell subsets: naïve (TN), stem-cell- (TSCM), central- (TCM), transitional- (TTM), and effector-memory (TEM). HIV decreases in TTM and TEM but not in less-differentiated subsets. Cell turnover is ~10 times faster than HIV clearance in memory subsets, implying that cellular proliferation consistently creates HIV DNA. The optimal mathematical model for these integrated data sets posits HIV DNA also passages between CD4 cell subsets via cellular differentiation. Estimates are heterogeneous, but in an average participant’s year ~10 (in TN and TSCM) and ~104 (in TCM, TTM, TEM) proviruses are generated by proliferation while ~103 proviruses passage via cell differentiation (per million CD4). In simulations, therapies blocking proliferation and/or enhancing differentiation could reduce HIV DNA by 1-2 logs over 3 years. In summary, HIV exploits cellular proliferation and differentiation to persist during ART but clears faster in more proliferative/differentiated CD4 cell subsets and the same physiological mechanisms sustaining HIV might be temporarily modified to reduce it.

Characterisation of HIV-1 reservoirs in paediatric populations: protocol for a systematic review and meta-analysis

IntroductionThe success of antiretroviral therapy (ART) has changed HIV from a deadly to a chronic infection, thus increasing the transitioning from infancy toward adulthood. However, the virostatic nature of antiretrovirals...

Identification of system-level features in HIV migration within a host.

Identify system-level features in HIV migration within a host across body tissues. Evaluate heterogeneity in the presence and magnitude of these features across hosts. Using HIV DNA deep sequencing data generated across multiple tissues from 8 people with HIV, we represent the complex dependencies of HIV migration among tissues as a network and model these networks using the family of exponential random graph models (ERGMs). ERGMs allow for the statistical assessment of whether network features occur more (or less) frequently in viral migration than might be expected by chance. The analysis investigates five potential features of the viral migration network: (1) bi-directional flow between tissues; (2) preferential migration among tissues in the same biological system; (3) heterogeneity in the level of viral migration related to HIV reservoir size; (4) hierarchical structure of migration; and (5) cyclical migration among several tissues. We calculate the Cohran's Q statistic to assess heterogeneity in the magnitude of the presence of these features across hosts. The analysis adjusts for missing data on body tissues. We observe strong evidence for bi-directional flow between tissues; migration among tissues in the same biological system; and hierarchical structure of the viral migration network. This analysis shows no evidence for differential level of viral migration with respect to the HIV reservoir size of a tissue. There is evidence that cyclical migration among three tissues occurs less frequent than expected given the amount of viral migration. The analysis also provides evidence for heterogeneity in the magnitude that these features are present across hosts. Adjusting for missing tissue data identifies system-level features within a host as well as heterogeneity in the presence of these features across hosts that are not detected when the analysis only considers the observed data. Identification of common features in viral migration may increase the efficiency of HIV cure efforts as it enables targeting specific processes.

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo

Persistent HIV-1 reservoirs of infected CD4 Tcells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained invivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 Tcells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

Kinetics of TTV Loads in Peripheral Blood Mononuclear Cells of Early Treated Acute HIV Infections.

Torquetenovirus (TTV) is the most abundant component of the human blood virome and its replication is controlled by a functioning immune system. In this study, TTV replication was evaluated in 21 people with acute HIV infection (AHI) and immune reconstitution following antiretroviral therapy (ART). PBMC-associated TTV and HIV-1 DNA, as well as plasma HIV-1 RNA, were measured by real-time PCR. CD4 and CD8 differentiation, activation, exhaustion, and senescence phenotypes were analyzed by flow cytometry. Thirteen healthy donors (HD) and twenty-eight chronically infected HIV individuals (CHI), late presenters at diagnosis, were included as control groups. TTV replication in AHI seems to be controlled by the immune system being higher than in HD and lower than in CHI. During ART, a transient increase in TTV DNA levels was associated with a significant perturbation of activation and senescence markers on CD8 T cells. TTV loads were positively correlated with the expansion of CD8 effector memory and CD57+ cells. Our results shed light on the kinetics of TTV replication in the context of HIV acute infection and confirm that the virus replication is strongly regulated by the modulation of the immune system.

Virological safety of the UK blood supply in the era of individual risk assessments and HIV PrEP.

A more individualised donor selection policy was implemented in the UK in 2021, which replaced the previous 3-month deferral for men who have sex with men (MSM). Other blood services have a variety of policies in place to ensure the virological safety of blood components, ranging from an indefinite ban on MSM, to a defined period of exclusion, or to an individualised risk assessment that is not based on gender or sexual orientation. Justification of these policies should be based on scientific evidence including assessment of lengths of virological window periods, infectious disease epidemiology within donor populations and donation screening assay sensitivities. Developments in molecular technology and assays which can detect both antibodies and antigens in the very early stages of infection have significantly reduced the risk in most developed countries. However, the increasing usage of pre-exposure prophylaxis (PrEP) to prevent acquisition of HIV infection after possible high-risk sexual contact within the UK blood donor population has been recently noted. It has brought with it new diagnostic challenges within blood screening, notably possible non-detection of HIV RNA and serological markers following PrEP use despite potential infectivity. The use of other testing strategies such as detection of HIV DNA and screening for non-declared PrEP usage should be investigated further.

Near full-length HIV sequencing in multiple tissues collected postmortem reveals shared clonal expansions across distinct reservoirs during ART

HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

An active HIV reservoir during ART is associated with maintenance of HIV-specific CD8+ T cell magnitude and short-lived differentiation status

Before initiation of antiretroviral therapy (ART), HIV-specific CD8+ Tcells are dysfunctional and short lived. To better understand the relationship between the HIV reservoir in CD4+ Tcells and the magnitude and differentiation status of HIV-specific CD8+ Tcells, we investigated these cells from acute and chronic HIV-infected individuals after 2 years of ART. Although both the HIV reservoir and the CD8+ Tcell responses declined significantly after 2 years of ART, sustained HIV-specific CD8+ Tcell responses correlated with a greater reduction of integrated HIV provirus. However, the magnitude of CD8+ Tcells specific for HIV Gag, Pol, Nef, and Vif proteins positively associated with the active reservoir size during ART, measured as cell-associated RNA. Importantly, high HIV DNA levels strongly associate with maintenance of short-lived HIV-specific CD8+ Tcells, regardless of ART initiation time. Our data suggest that the active reservoir maintains HIV-specific CD8+ Tcell magnitude but prevents their differentiation into functional cells.

Venetoclax, alone and in combination with the BH3 mimetic S63845, depletes HIV-1 latently infected cells and delays rebound in humanized mice

HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4+ Tcells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4+ Tcells from PLWH exvivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound.

Effect of high dose vitamin D3 on the HIV-1 reservoir: A pilot randomised controlled trial

IntroductionAntiretroviral therapy for people living with HIV-1 must be taken lifelong due to the persistence of latent virus in long-lived and proliferating CD4+ T cells. Vitamin D3 is a steroidal gene transcription regulator which exerts diverse effects on immune and epithelial cells including reductions in CD4+ T cell proliferation and improvement in gut barrier integrity. We hypothesised that a high dose of vitamin D3 would reduce the size of the HIV-1 reservoir by reducing CD4+ T cell proliferation. MethodsWe performed a randomised placebo-controlled trial evaluating the effect of 24 weeks of vitamin D3 (10,000 international units per day) on the HIV-1 reservoir and immunologic parameters in 30 adults on antiretroviral therapy; participants were followed for 12 weeks post-treatment. The primary endpoint was the effect on total HIV-1 DNA at week 24. Parameters were assessed using mixed-effects models. ResultsWe found no effect of vitamin D3 on the change in total HIV-1 DNA from week 0 to week 24 relative to placebo. There were also no changes in integrated HIV-1 DNA, 2-long-terminal repeat (2-LTR) circles or cell-associated HIV-1 RNA. Vitamin D3 induced a significant increase in the proportion of central memory CD4+ and CD8+ T cells, a reduction in the proportion of senescent CD8+ T cells and a reduction in the natural killer cell frequency at all time points including week 36, 12 weeks after the study drug cessation. At week 36, there was a significant reduction in total HIV-1 DNA relative to placebo and persistently elevated 25-hydroxyvitamin D levels. No significant safety issues were identified. ConclusionsVitamin D3 administration had a significant impact on the T cell differentiation but overall effects on the HIV-1 reservoir were limited and a reduction in HIV-1 DNA was only seen following cessation of the study drug. Additional studies are required to determine whether the dose and duration of vitamin D3 can be optimised to promote a continued depletion of the HIV-1 reservoir over time. Trial registrationClinicalTrials.gov NCT03426592.

Sensitive HIV-1 DNA Pol Next-Generation Sequencing for the Characterisation of Archived Antiretroviral Drug Resistance.

Modern HIV-1 treatment effectively suppresses viral amplification in people living with HIV. However, the persistence of HIV-1 DNA as proviruses integrated into the human genome remains the main barrier to achieving a cure. Next-generation sequencing (NGS) offers increased sensitivity for characterising archived drug resistance mutations (DRMs) in HIV-1 DNA for improved treatment options. In this study, we present an ultra-sensitive targeted PCR assay coupled with NGS and a robust pipeline to characterise HIV-1 DNA DRMs from buffy coat samples. Our evaluation supports the use of this assay for Pan-HIV-1 analyses with reliable detection of DRMs across the HIV-1 Pol region. We propose this assay as a new valuable tool for monitoring archived HIV-1 drug resistance in virologically suppressed individuals, especially in clinical trials investigating novel therapeutic approaches.

Impact of Cannabis Use on Immune Cell Populations and the Viral Reservoir in People With HIV on Suppressive Antiretroviral Therapy.

Human immunodeficiency virus (HIV) infection remains incurable due to the persistence of a viral reservoir despite antiretroviral therapy (ART). Cannabis (CB) use is prevalent amongst people with HIV (PWH), but the impact of CB on the latent HIV reservoir has not been investigated. Peripheral blood cells from a cohort of PWH who use CB and a matched cohort of PWH who do not use CB on ART were evaluated for expression of maturation/activation markers, HIV-specific T-cell responses, and intact proviral DNA. CB use was associated with increased abundance of naive T cells, reduced effector T cells, and reduced expression of activation markers. CB use was also associated with reduced levels of exhausted and senescent T cells compared to nonusing controls. HIV-specific T-cell responses were unaffected by CB use. CB use was not associated with intact or total HIV DNA frequency in CD4 T cells. This analysis is consistent with the hypothesis that CB use reduces activation, exhaustion, and senescence in the T cells of PWH, and does not impair HIV-specific CD8 T-cell responses. Longitudinal and interventional studies with evaluation of CB exposure are needed to fully evaluate the impact of CB use on the HIV reservoir.

Regional Analysis of Intact and Defective HIV Proviruses in the Brain of Viremic and Virally Suppressed People with HIV.

Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798-802.

Prevalence of HIV-1 drug resistance mutations in proviral DNA in the Swiss HIV Cohort Study, a retrospective study from 1995 to 2018.

Genotypic resistance testing (GRT) is routinely performed upon diagnosis of HIV-1 infection or during virological failure using plasma viral RNA. An alternative source for GRT could be cellular HIV-1 DNA. A substantial number of participants in the Swiss HIV Cohort Study (SHCS) never received GRT. We applied a method that enables access to the near full-length proviral HIV-1 genome without requiring detectable viraemia. Nine hundred and sixty-two PBMC specimens were received. Our two-step nested PCR protocol was applied to generate two overlapping long-range amplicons of the HIV-1 genome, sequenced by next-generation sequencing (NGS) and analysed by MinVar, a pipeline to detect drug resistance mutations (DRMs). Six hundred and eighty-one (70.8%) of the samples were successfully amplified, sequenced and analysed by MinVar. Only partial information of the pol gene was contained in 82/681 (12%), probably due to naturally occurring deletions in the proviral sequence. All common HIV-1 subtypes were successfully sequenced. We detected at least one major DRM at high frequency (≥15%) in 331/599 (55.3%) individuals. Excluding APOBEC-signature (G-to-A mutation) DRMs, 145/599 (24.2%) individuals carried at least one major DRM. RT-inhibitor DRMs were most prevalent. The experienced time on ART was significantly longer in DRM carriers (P = 0.001) independent of inclusion or exclusion of APOBEC-signature DRMs. We successfully applied a reliable and efficient method to analyse near full-length HIV-1 proviral DNA and investigated DRMs in individuals with undetectable or low viraemia. Additionally, our data underscore the need for new computational tools to exclude APOBEC-related hypermutated NGS sequence reads for reporting DRMs.

Gut dysbiosis associates with cytokine production capacity in viral-suppressed people living with HIV.

People living with human immunodeficiency virus (PLHIV) are exposed to chronic immune dysregulation, even when virus replication is suppressed by antiretroviral therapy (ART). Given the emerging role of the gut microbiome in immunity, we hypothesized that the gut microbiome may be related to the cytokine production capacity of PLHIV. To test this hypothesis, we collected metagenomic data from 143 ART-treated PLHIV and assessed the ex vivo production capacity of eight different cytokines [interleukin-1β (IL-1β), IL-6, IL-1Ra, IL-10, IL-17, IL-22, tumor necrosis factor, and interferon-γ] in response to different stimuli. We also characterized CD4+ T-cell counts, HIV reservoir, and other clinical parameters. Compared with 190 age- and sex-matched controls and a second independent control cohort, PLHIV showed microbial dysbiosis that was correlated with viral reservoir levels (CD4+ T-cell-associated HIV-1 DNA), cytokine production capacity, and sexual behavior. Notably, we identified two genetically different P. copri strains that were enriched in either PLHIV or healthy controls. The control-related strain showed a stronger negative association with cytokine production capacity than the PLHIV-related strain, particularly for Pam3Cys-incuded IL-6 and IL-10 production. The control-related strain is also positively associated with CD4+ T-cell level. Our findings suggest that modulating the gut microbiome may be a strategy to modulate immune response in PLHIV.

HIV-1-Host Interaction in Gut-Associated Lymphoid Tissue (GALT): Effects on Local Environment and Comorbidities.

HIV-1 replication in the gastrointestinal (GI) tract causes severe CD4+ T-cell depletion and disruption of the protective epithelial barrier in the intestinal mucosa, causing microbial translocation, the main driver of inflammation and immune activation, even in people living with HIV (PLWH) taking antiretroviral drug therapy. The higher levels of HIV DNA in the gut compared to the blood highlight the importance of the gut as a viral reservoir. CD4+ T-cell subsets in the gut differ in phenotypic characteristics and differentiation status from the ones in other tissues or in peripheral blood, and little is still known about the mechanisms by which the persistence of HIV is maintained at this anatomical site. This review aims to describe the interaction with key subsets of CD4+ T cells in the intestinal mucosa targeted by HIV-1 and the role of gut microbiome and its metabolites in HIV-associated systemic inflammation and immune activation that are crucial in the pathogenesis of HIV infection and related comorbidities.

Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA.

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.

Impact of misclassified defective proviruses on HIV reservoir measurements

Most proviruses persisting in people living with HIV (PWH) on antiretroviral therapy (ART) are defective. However, rarer intact proviruses almost always reinitiate viral rebound if ART stops. Therefore, assessing therapies to prevent viral rebound hinges on specifically quantifying intact proviruses. We evaluated the same samples from 10 male PWH on ART using the two-probe intact proviral DNA assay (IPDA) and near full length (nfl) Q4PCR. Both assays admitted similar ratios of intact to total HIV DNA, but IPDA found ~40-fold more intact proviruses. Neither assay suggested defective proviruses decay over 10 years. However, the mean intact half-lives were different: 108 months for IPDA and 65 months for Q4PCR. To reconcile this difference, we modeled additional longitudinal IPDA data and showed that decelerating intact decay could arise from very long-lived intact proviruses and/or misclassified defective proviruses: slowly decaying defective proviruses that are intact in IPDA probe locations (estimated up to 5%, in agreement with sequence library based predictions). The model also demonstrates how misclassification can lead to underestimated efficacy of therapies that exclusively reduce intact proviruses. We conclude that sensitive multi-probe assays combined with specific nfl-verified assays would be optimal to document absolute and changing levels of intact HIV proviruses.

Impact of obefazimod on viral persistence, inflammation, and immune activation in people with HIV on suppressive antiretroviral therapy.

Persistence of viral reservoirs has been observed in people with HIV (PWH), despite long-term ART, and likely contributes to chronic immune activation and inflammation. Obefazimod is a novel drug that inhibits HIV-1 replication and reduces inflammation. Here we assess whether obefazimod is safe and might impact HIV-1 persistence, chronic immune activation, and inflammation in ART-suppressed PWH. We evaluated obefazimod-related adverse events, changes in cell-associated HIV-1 DNA and RNA, residual viraemia, immunophenotype, and inflammation biomarkers in blood and rectal tissue. We compared 24 ART-suppressed PWH who received daily doses of 50 mg of obefazimod for 12wks (n = 13), or 150 mg for 4wks (n = 11) and 12 HIV-negative individuals who received 50 mg for 4wks. 50 or 150 mg doses of obefazimod were safe, although 150 mg dose showed inferior tolerability. 150 mg dose reduced HIV-1 DNA (p = 0.008, median fold-change = 0.6), and residual viraemia in all individuals with detectable viraemia at baseline. Furthermore, obefazimod upregulated miR-124 in all participants, reduced the activation markers CD38, HLA-DR, and PD-1, and several inflammation biomarkers. The effect of obefazimod by reducing chronic immune activation and inflammation suggests a potential role for the drug in virus remission strategies involving other compounds that can activate immune cells, such as latency-reversing agents.

Brief Report: Comparative Analysis of Pre-existing HIV Drug Resistance Mutations in Proviral DNA Using Next-Generation Sequencing and Routine HIV RNA Genotyping.

We investigated whether deep sequencing of archived HIV DNA of antiretroviral-naive persons with acute/early HIV infection could identify transmitted drug resistance mutations (DRM), per the IAS drug resistance algorithm, which are not detected by routine bulk (consensus) sequencing. Deep sequencing of HIV DNA from peripheral blood mononuclear cells and consensus sequencing from concurrent blood plasma (BP) was performed from antiretroviral (ART)-naive adults with recent infection. We compared the prevalence of low-frequency (2%-20%) and high-frequency (>20%) nonnucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor (NRTI), and protease inhibitor (PI) DRM. Overall, 190 individuals were included, 72 (37.9%) with acute, 20 (10.5%) with very early, and 98 (51.6%) with recent HIV infection. Although all DRM detected in plasma appeared in archived proviral DNA, 9 high-frequency mutations were only detected in HIV DNA. These included 3 NRTI mutations, 4 NNRTI mutations, 1 PI mutation, and 1 H221Y (associated rilpivirine resistance) mutation. When considering DRM <20%, 11 NNRTI, 7 NRTI, 6 PI, and 3 F227L (associated doravirine resistance) mutations were found exclusively in HIV DNA. Interestingly, although 2 high-frequency M184V appeared in both DNA and RNA, low-frequency M184I were exclusive to HIV DNA (n = 6). No participants experienced virologic failure after initiating ART during the median 25.39 ± 3.13 months of follow-up on treatment. Although most high-frequency DRMs were consistently detected in HIV RNA and HIV DNA, the presence of low-frequency DRM in proviral DNA may be relevant for clinicians because these mutations could become dominant under drug selection pressure.