HIV-1 DNA Sequences Research Articles

Proviral HIV DNA sequencing to inform suitability for long-acting injectable cabotegravir/rilpivirine.

Increasingly, people attending HIV services are requesting long-acting injectable (LAI) antiretroviral treatment (ART). However, without HIV RNA resistance-associated mutation results, individuals are considered unsuitable for LAI ART. We present our experience of sequencing proviral HIV DNA in 30 individuals to inform suitability for LAI ART, of whom 23 were considered suitable. In conclusion, optimization of diagnostic tools such as proviral HIV DNA sequencing to confirm suitability for LAI ART would be a welcome addition.

Comparison of short-read and long-read next-generation sequencing technologies for determining HIV-1 drug resistance.

Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.

Use of genotypic HIV DNA testing: a DELPHI-type consensus.

As many disparities in the clinical use of HIV DNA sequencing are observed, a DELPHI-type consensus was initiated in France to homogenize use, techniques and interpretation of results. Based on a literature review and clinical experience, a steering committee (SC) of eight virologists and one infectious disease specialist formulated statements. Statements were submitted to an independent and anonymous electronic vote of virologists and HIV clinicians in France, between October 2022 and December 2022. The SC developed 20 statements grouped into six categories: clinical situations for the use of HIV DNA genotyping; techniques for performing HIV DNA genotyping; consideration of apolipoprotein B mRNA editing enzyme (APOBEC) mutations; genotyping results reporting; recycling of antiretrovirals; and availability of HIV DNA genotyping tests and delays. Twenty-one virologists and 47 clinicians participated in two voting rounds and 18/20 (90%) assertions reached a 'strong' consensus. For example, that prior genotyping on HIV DNA is useful for clinical decision-making when considering switching to some long-acting regimens or to reduce the number of antiretroviral agents in virologically suppressed patients for whom RNA data are unavailable/not exploitable/not sufficiently informative. Two statements achieved no consensus: reporting any detected viral minority population for discussion in multidisciplinary meetings (virologists), and possible risk of virological failure when using a second-generation InSTI plus lamivudine or emtricitabine regimen in patients with undetectable viral load within ≥1 year and in the presence of a documented M184V mutation within the last 5 years (clinicians). This DELPHI-type consensus will facilitate the strengthening and harmonization of good practice when performing HIV DNA sequencing.

Comparison of HIV-1 DNA load measurements in blood and in relation to successful proviral sequencing

ObjectiveHIV DNA sequencing is now routinely used for HIV-infected individuals on antiretroviral therapy (ART) with or without partial genotypic history. Successful amplification of HIV pol gene has yet to be correlated with HIV DNA levels. Here, we assessed the relationship between HIV DNA load and sequencing results. MethodsWe analyzed three different qPCR measurements of total (LTR and LTR-gag) and integrated (Alu-LTR) HIV DNA in blood samples collected from viremic as well as virally suppressed HIV-infected individuals on ART. HIV DNA levels were compared to HIV DNA Sanger sequencing and clinical and therapeutic parameters. ResultsAmong the 135 individuals analyzed for HIV DNA measurements and sequencing, all three HIV DNA measurements were associated with HIV DNA Sanger sequencing results. A threshold of around 2 and 1.5 log copies/million leukocytes of total HIV DNA was identified for LTR and LTR-gag qPCRs, respectively. Integrated HIV DNA positivity was also associated with successful sequencing. We further compared HIV DNA measurement techniques in an extended cohort of 312 individuals and showed that all measurements correlated between the different techniques, regardless of the HIV-1 subtypes analyzed. However, higher detection rates were observed with LTR (96%) compared to LTR-gag (86%) and Alu-LTR (59%) qPCRs. Duration of virological control on ART and CD4 nadir were the main determinants of HIV reservoir size. ConclusionsHIV DNA measurement is associated with Sanger sequencing success, regardless of the technique used. In a clinical setting, Application of HIV DNA quantification before sequencing should be further evaluated.

HIV-1 reservoir evolution in infants infected with clade C from Mozambique

BackgroundThe persistence of HIV-1-infected cells during antiretroviral therapy is well documented but may be modulated by early initiation of antiretroviral therapy in infants. MethodsHere, we longitudinally analyzed the proviral landscape in nine infants with vertical HIV-1 infection from Mozambique over a median period of 24 months, using single-genome, near full-length, next-generation proviral sequencing. ResultsWe observed a rapid decline in the frequency of intact proviruses, leading to a disproportional under-representation of intact HIV-1 sequences within the total number of HIV-1 DNA sequences after 12-24 months of therapy. In addition, proviral integration site profiling in one infant demonstrated clonal expansion of infected cells harboring intact proviruses and indicated that viral rebound was associated with an integration site profile dominated by intact proviruses integrated into genic and accessible chromatin locations. ConclusionTogether, these results permit rare insight into the evolution of the HIV-1 reservoir in infants infected with HIV-1 and suggest that the rapid decline of intact proviruses, relative to defective proviruses, may be attributed to a higher vulnerability of genome-intact proviruses to antiviral immunity. Technologies to analyze combinations of intact proviral sequences and corresponding integration sites permit a high-resolution analysis of HIV-1 reservoir cells after early antiretroviral treatment initiation in infants.

HIV DNA Sequencing to Detect Archived Antiretroviral Drug Resistance.

Proviral HIV DNA integrated within CD4 T-cells maintains an archive of viral variants that replicate during the course of the infection, including variants with reduced drug susceptibility. We considered studies that investigated archived drug resistance, with a focus on virologically suppressed patients and highlighted interpretative caveats and gaps in knowledge. Either Sanger or deep sequencing can be used to investigate resistance-associated mutations (RAMs) in HIV DNA recovered from peripheral blood. Neither technique is free of limitations. Furthermore, evidence regarding the establishment, maintenance, expression and clinical significance of archived drug-resistant variants is conflicting. This in part reflects the complexity of the HIV proviral landscape and its dynamics during therapy. Clinically, detection of RAMs in cellular HIV DNA has a variable impact on treatment outcomes, modulated by the drugs affected, treatment duration and additional determinants of virological failure, including those leading to suboptimal drug exposure. Sequencing cellular HIV DNA can provide helpful complementary information in treatment-experienced patients with suppressed plasma HIV RNA who require a change of regimen. However, care should be taken when interpreting the results. Presence of RAMs is not necessarily a barrier to treatment success. Conversely, even the most sensitive sequencing techniques will fail to provide a comprehensive view of the HIV DNA archive. To inform treatment decisions appropriately, the overall clinical and treatment history of a patient must always be considered alongside the results of resistance testing. Prospective controlled studies are needed to validate the utility of drug resistance testing using cellular HIV DNA.

Near-Full-Length Single-Genome HIV-1 DNA Sequencing.

HIV-1 integrates into human chromosomes to establish a lifelong reservoir of virally infected cells. However, the majority of integrated viral DNA shows lethal defects, likely due to errors introduced during reverse transcription of viral RNA. Identifying and quantifying HIV-1 DNA sequences that are genome-intact and can give rise to rebound viremia during antiretroviral treatment interruption are critical steps for understanding the complexity and evolutionary dynamics of HIV-1 reservoir cells. Here, we describe FLIP-Seq, (Full-Length Individual Proviral Sequencing) a near full-length, single-genome next-generation sequencing approach for analyzing HIV-1 DNA in human cells. Briefly, this technique involves sequential dilution of proviral DNA to single genomes, amplification of near full-length viral DNA, deep sequencing of amplification products, and a biocomputational analysis designed to distinguish genome-intact HIV-1 DNA from defective viral DNA species. This procedure can be performed with small numbers of cells from highly purified CD4 T cell subsets, allows to generate an absolute quantification of viral sequences present in a given cell population, provides insight into phylogenetic associations of intact proviruses, and can identify proportions of sequence-identical proviruses likely derived from clonally expanded reservoir cells.

Single center, open label dose escalating trial evaluating once weekly oral ixazomib in ART-suppressed, HIV positive adults and effects on HIV reservoir size in vivo.

BackgroundAchieving a functional or sterilizing cure for HIV will require identification of therapeutic interventions that reduce HIV reservoir size in infected individuals. Proteasome inhibitors, such as ixazomib, impact multiple aspects of HIV biology including latency, transcription initiation, viral replication, and infected cell killing through the HIV protease – Casp8p41 pathway, resulting in latency reversal and reduced measures of HIV reservoir size ex vivo.MethodsWe conducted a phase 1b/2a dose escalating, open label trial of weekly oral ixazomib for 24 weeks in antiretroviral (ART)-suppressed, HIV positive adults (NCT02946047). The study was conducted from March 2017 to August 2019 at two tertiary referral centers in the United States. The primary outcomes were safety and tolerability of oral ixazomib. Secondary outcomes included changes in immunologic markers and estimates of HIV reservoir size after ixazomib treatment.FindingsSixteen participants completed the study. Ixazomib up to 4mg weekly was safe and well-tolerated, yielding no treatment-emergent events above grade 1. In exploratory analyses, ixazomib treatment was associated with detectable viremia that was below the lower limit of quantification (LLQ) in 9 participants, and viremia that was above LLQ in 4 of 16 participants. While treatment was associated with reduced CD4 counts [baseline 783 cells/ mm3 vs. week-24 724 cells/ mm3 p=0.003], there were no changes in markers of cellular activation, exhaustion or inflammation. Total HIV DNA and proviral sequencing were not altered by ixazomib treatment. Intact proviral DNA assay (IPDA) identified intact proviruses in 14 patients pre-treatment, and in 10/14 of those subjects post treatment values were reduced (P=0.068), allowing a calculated intact proviral half life of 0.6 years (95% CI 0.3, 2.5), compared to 7.1 years (95% CI 3.9, 18, p=0.004) in historical controls. Differentiation Quantitative Viral Outgrowth Assays (dQVOA) identified measurable proviruses in 15 subjects pre-treatment; post-treatment values were numerically reduced in 9, but overall differences were not significantly different.InterpretationOur study successfully met its primary endpoint of demonstrating the safety of ixazomib for 24 weeks in HIV infected persons. Exploratory analyses suggest that the effects observed ex vivo of latency reversal and reductions in HIV reservoir size, also occur in vivo. Future controlled studies of ixazomib are warranted.FundingThis study was funded by Millennium Pharmaceuticals Inc..; the Mayo Clinic Foundation; the National Institutes of Health, including the National Institute of Allergy and Infectious Diseases, Division of AIDS, the National Heart, Lung and Blood Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Neurological Disorders and Stroke, and the National Institute on Drug Abuse. Mayo Clinic also acknowledges generous funding support from Mr. Joseph T. and Mrs. Michele P. Betten.

Genital Shedding of Human Immunodeficiency Virus Type-1 (HIV) When Antiretroviral Therapy Suppresses HIV Replication in the Plasma.

During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding (DS). We hypothesized that proliferating cells produce virions without HIV replication. ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points. Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation. HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.

Polymorphisms and drug resistance analysis of HIV-1 isolates from patients on first line antiretroviral therapy (ART) in South-eastern Nigeria.

Acquisition of resistance mutations by HIV-1 isolates causes treatment failure among infected patients receiving antiretroviral therapy (ART). This study determined patterns of drug-resistance mutations (DRMs) among HIV-1 isolates from patients receiving first-line ART in South-eastern Nigeria. Blood samples were collected from HIV-1 infected patients accessing antiretroviral treatment centers at General Hospital Awo-Omamma, Imo state, State Hospital Asaba, Delta state and St Joseph's Catholic Hospital Adazi, Anambra state and used for HIV-1 DNA sequencing and phylogenetic analysis. DRMs were scored using combination of Stanford algorithm and the 2015 International Antiviral Society-USA list while drug susceptibility was predicted using Stanford algorithm. Twenty eight of the HIV-1 isolates were sequenced and identified as subtypes G (35.7%), CRF02_AG (57.1%) and unclassifiable, UG (7.1%). Major PI resistance-associated mutations were identified at two sites including M46L (16.7% of subtype G/UG) and V82L (6.3% of CRF02_AG). Minor PI resistance-associated mutations identified among subtype G/UG are L10V/I (8.3%) and K20I (100%) while L10V/I (50%), K20I (100%), L33F (6.3%) and N88D (6.3%) were identified among CRF02_AG. Other polymorphisms found include; I13V/A, E35Q, M36I/L, N37D/S/E/H, R57K/G, L63T/P/S/Q, C67E/S, H69K/R, K70R, V82I and L89M in the range of 28.6% to 100% among the different subtypes. Interpretation based on Stanford algorithm showed that Darunavir/ritonavir is the only regimen whose potency was not compromised by the circulating mutations. Identification of major and minor PI resistance mutations in this study underscores the need for drug resistance testing prior to initiation of second line antiretroviral therapy in Nigeria.

Differential detection of M184V/I between plasma historical HIV genotypes and HIV proviral DNA from PBMCs.

The M184V/I reverse transcriptase mutation, which confers major resistance to lamivudine and emtricitabine, is still quite frequent in people living with HIV. The underlying presence of the M184V/I mutation may undermine virological outcomes of ART, particularly in the context of proposed treatment with two-drug combinations that include drugs affected by M184V, such as lamivudine. In suppressed patients for whom historical data are seldom available, resistance assays evaluating integrated viral DNA can help select a fully active switch regimen. To compare detectability of M184V/I in historical HIV-1 RNA analyses versus HIV-1 DNA sequencing. We analysed the detection of the M184V/I mutation in a prospective study and compared HIV historical genotypes (plasma) versus integrated HIV DNA (PBMCs) obtained via a validated commercial proviral HIV DNA assay. Eligible participants had HIV-1 RNA <50 copies/mL for ≥6 consecutive months prior to screening. A plasma historical genotypic report (HGR) showing the presence of M184V/I was required for all participants and proviral HIV DNA analysis was conducted prior to enrolment. All 84 participants had evidence of M184V or M184I in their HGR (100%), whereas the mutation was detected in only 40/84 participants by proviral HIV DNA sequencing analysis (48%). Differential detection of M184V/I was not associated with timing differences between the HGR and proviral HIV DNA sampling, the overall duration of ART, or CD4 cell counts and HIV-1 viral load at baseline. Our results suggest that undetected M184V/I should be considered when switching virologically suppressed patients to new regimens, particularly two-drug lamivudine- or emtricitabine-containing combinations.

New Frontiers in Measuring and Characterizing the HIV Reservoir

A cure for HIV infection remains elusive due to the persistence of replication-competent HIV proviral DNA during suppressive antiretroviral therapy (ART). With the exception of rare elite or post-treatment controllers of viremia, withdrawal of ART invariably results in the rebound of viremia and progression of HIV disease. A thorough understanding of the reservoir is necessary to develop new strategies in order to reduce or eliminate the reservoir. However, there is significant heterogeneity in the sequence composition, genomic location, stability, and expression of the HIV reservoir both within and across individuals, and a majority of proviral sequences are replication-defective. These factors, and the low frequency of persistently infected cells in individuals on suppressive ART, make understanding the reservoir and its response to experimental reservoir reduction interventions challenging. Here, we review the characteristics of the HIV reservoir, state-of-the-art assays to measure and characterize the reservoir, and how these assays can be applied to accurately detect reductions in reservoir during efforts to develop a cure for HIV infection. In particular, we highlight recent advances in the development of direct measures of provirus, including intact proviral DNA assays and full-length HIV DNA sequencing with integration site analysis. We also focus on novel techniques to quantitate persistent and inducible HIV, including RNA sequencing and RNA/gag protein staining techniques, as well as modified viral outgrowth methods that seek to improve upon throughput, sensitivity and dynamic range.

Memory CD4 + T-Cells Expressing HLA-DR Contribute to HIV Persistence During Prolonged Antiretroviral Therapy.

To date, most assays for measuring the human immunodeficiency virus (HIV-1) reservoir do not include memory CD4+ T-cells expressing the activation marker, human leukocyte antigen-antigen D related (HLA-DR). However, little is known concerning the role these cells play in maintaining persistent HIV-1 during effective antiretroviral therapy (ART). To address this issue, we examined, cellular activation/exhaustion markers (Ki67, CCR5, PD-1, Lag-3 and Tim-3) and viral gag-pol DNA sequences within HLA-DR− and HLA-DR+ memory CD4+ T-cell subsets longitudinally from the peripheral blood of six participants over 3 to ≥15 years of effective therapy. HLA-DR expression was readily detected during the study period in all participants. The average expression levels of CCR5, PD-1 and Tim-3 were higher on the HLA-DR+ T-cell subset whereas the average of LAG-3 expression was higher on their HLA-DR− counterpart. The proportion of HIV-infected cells increased within the HLA-DR+ subset by an average of 18% per year of ART whereas the frequency of infected HLA-DR− T-cells slightly decreased over time (5% per year). We observed that 20–33% of HIV-DNA sequences from the early time points were genetically identical to viral sequences from the last time point within the same cell subset during ART. This indicates that a fraction of proviruses persists within HLA-DR+ and HLA-DR− T-cell subsets during prolonged ART. Our HIV-DNA sequence analyses also revealed that cells transitioned between the HLA-DR+ and HLA-DR− phenotypes. The Ki67 expression, a marker for cellular proliferation, and the combined markers of Ki67/PD-1 averaged 19-fold and 22-fold higher on the HLA-DR+ T-cell subset compared to their HLA-DR− counterpart. Moreover, cellular proliferation, as reflected by the proportion of genetically identical HIV-DNA sequences, increased within both T-cell subsets over the study period; however, this increase was greater within the HLA-DR+ T-cells. Our research revealed that cellular transition and proliferation contribute to the persistence of HIV in HLA-DR+ and HLA-DR− T-cell subsets during prolonged therapy. As such, the HIV reservoir expands during effective ART when both the HLA-DR+ and HLA-DR− cell subsets are included, and therapeutic interventions aimed at reducing the HIV-1 reservoir should target HLA-DR+ and HLA-DR− T-cells.

An apparent paradox: resistance mutations in HIV-1 DNA predict improved virological responses to antiretroviral therapy.

In sub-Saharan Africa, detecting resistance-associated mutations (RAMs) at failure of first-line ART with two NRTIs plus an NNRTI predicts improved virological responses to second-line therapy with two NRTIs plus a ritonavir-boosted PI (PI/r). This indicates residual NRTI activity in the presence of RAMs, although additional factors may contribute to the effect. The aim of this study was to investigate the influence of pre-existing RAMs on the outcomes of maintenance monotherapy with ritonavir-boosted darunavir within a randomized trial in Cameroon. RAMs were detected in HIV-1 DNA using PBMCs collected at initiation of darunavir/ritonavir monotherapy. Adherence was assessed by pill count and visual analogue scale (VAS). Predictors of virological failure (confirmed or last available viral load >400 copies/mL) were explored by logistic regression analysis. Trial name = MANET (NCT02155101). After NNRTI-based therapy, participants (n = 81) had received PI/r-based therapy for a median of 3.2 years and had a confirmed viral load <60 copies/mL and a median CD4 count of 466 cells/mm3. NRTI and NNRTI RAMs were detected in 39/60 (65.0%) and 41/60 (68.3%) HIV-1 DNA sequences, respectively. Over 48 weeks of monotherapy, 16/81 (19.8%) patients experienced virological failure. After adjusting for age, HIV-1 DNA load, adherence by VAS and RAM status, virological failure was less likely with higher VAS-measured adherence (adjusted OR 0.04, 95% CI 0.01-0.37; P = 0.004) and detectable HIV-1 DNA RAMs (adjusted OR 0.15, 95% CI 0.03-0.82; P = 0.028). Pre-existing NRTI and NNRTI RAMs are associated with improved virological responses to NRTI-sparing ART in sub-Saharan Africa, indicating a predictive effect that is independent of residual NRTI activity.

Routine drug resistance testing in proviral HIV-1 DNA: Prevalence of stop codons and hypermutation, and associated factors.

We investigated the presence of stop codons (SC) and/or hypermutation (HM) in HIV-1 DNA sequences generated for routine drug resistance testing in proviral HIV-1 DNA, and sought for associated factors. At least one SC was identified in 6.2% of HIV-1 DNA sequences, among which 54.8% were hypermutated. The defective virus group (SC w/o HM) was similar to the non-SC group regarding the characteristics of HIV-1 infection, and before drug exposure. In addition, the HIV-1 DNA levels were not different between both groups. Sequences with SC/HM displayed a higher proportion of RAMs. The impact of the SC/HM associated RAMs on clinical responses requires further investigation.

Detection of pretreatment minority HIV-1 reverse transcriptase inhibitor-resistant variants by ultra-deep sequencing has a limited impact on virological outcomes.

Ultra-deep sequencing (UDS) is a powerful tool for exploring the impact on virological outcome of minority variants with low frequencies, some even <1% of the virus population. Here, we compared HIV-1 minority variants at baseline, through plasma RNA and PBMC DNA analyses, and the dominant variants at the virological failure (VF) point, to evaluate the impact of minority drug-resistant variants (MDRVs) on virological outcomes. Single-molecule real-time sequencing (SMRTS) was performed on baseline RNA and DNA. The Stanford HIV-1 drug resistance database was used for the identification and evaluation of drug resistance-associated mutations (DRAMs). We classified 50 patients into virological success (VS) and VF groups. We found that the rates of reverse transcriptase inhibitor (RTI) DRAMs determined by SMRTS did not differ significantly within or between groups, whether based on RNA or DNA analyses. There was no significant difference in the level of resistance to specific drugs between groups, in either DNA or RNA analyses, except for the DNA-based analysis of lamivudine, for which there was a trend towards a higher prevalence of intermediate/high-level resistance in the VF group. The RNA MDRVs corresponded to DNA MDRVs, except for M100I and Y188H. Sequencing from DNA appeared to be more sensitive than from RNA to detect MDRVs. Detection of pretreatment minority HIV-1 RTI-resistant variants by UDS showed that MDRVs at baseline were not significantly associated with virological outcome. However, HIV-1 DNA sequencing by UDS was useful for detecting pretreatment drug resistance mutations in patients, potentially affecting virological responses, suggesting a potential clinical relevance for ultra-deep DNA sequencing.

HIV-1 DNA Is Maintained in Antigen-Specific CD4+ T Cell Subsets in Patients on Long-Term Antiretroviral Therapy Regardless of Recurrent Antigen Exposure

Memory CD4+ T cells (mCD4s) containing integrated HIV DNA are considered the main barrier to a cure for HIV infection. Here, we analyzed HIV DNA reservoirs in antigen-specific subsets of mCDs to delineate the mechanisms by which HIV reservoirs persist during antiretroviral therapy (ART). HIV Gag, cytomegalovirus (CMV), and tetanus toxoid (TT)-specific mCD4s were isolated from peripheral blood samples obtained from 11 individual subjects, 2-11 years after commencing ART. Antigen-specific mCD4s were identified by the sensitive OX40 assay and purified by cell sorting. Total HIV DNA levels were quantified by real-time PCR, and clonal viral sequences generated from mCD4 subsets and pre-ART plasma samples. Quantitative results and sequence analysis were restricted to five and three study participants, respectively, which was likely due to the low frequency of the antigen-specific mCD4s and relatively low HIV DNA proviral loads. Median HIV Gag-, CMV-, and TT-specific mCD4s were 0.61%, 2.46%, and 0.78% of total mCD4s, and they contained a median of 2.50, 2.38, and 2.55 log10 copies of HIV DNA per 106 cells, respectively. HIV DNA sequences were derived from antigen-specific mCD4s clustered with sequences derived from pre-ART plasma samples. There was a trend toward increased viral diversity in clonal viral sequences derived from CMV-specific mCD4s relative to TT-specific mCD4s. Despite limitations, this study provides direct evidence that HIV reservoirs persist in memory CD4+ T cell subsets maintained by homeostatic proliferation (TT) and adds to growing evidence against viral evolution during ART. Similar future studies require techniques that sample diverse HIV reservoirs and with improved sensitivity.

Electrochemical detection of sequence-specific DNA based on formation of G-quadruplex-hemin through continuous hybridization chain reaction

A high-sensitive detection of sequence-specific DNA was established based on the formation of G-quadruplex-hemin complex through continuous hybridization chain reaction (HCR). Taking HIV DNA sequence as an example, a capture probe complementary to part of HIV DNA was firstly self-assembled onto the surface of Au electrode. Then a specially designed assistant probe with both terminals complementary to the target DNA and a G-quadruplex-forming sequence in the center was introduced into the detection solution. In the presence of both the target DNA and the assistant probe, the target DNA can be captured on the electrode surface and then a continuous HCR can be conducted due to the mutual recognition of the target DNA and the assistant probe, leading to the formation of a large number of G-quadruplex on the electrode surface. With the help of hemin, a pronounced electrochemical signal can be observed in differential pulse voltammetry (DPV), due to the formation of G-quadruplex-hemin complex. The peak current is linearly related with the logarithm of the concentration of the target DNA in the range from 10 fM to 10 pM. The electrochemical sensor has high selectivity to clearly discriminate single-base mismatched and three-base mismatched sequences from the original HIV DNA sequence. Moreover, the established DNA sensor was challenged by detection of HIV DNA in human serum samples, which showed the low detection limit of 6.3 fM. Thus it has great application prospect in the field of clinical diagnosis and environmental monitoring.

Phylogenetic and temporal dynamics of human immunodeficiency virus type 1 CRF01_AE and CRF07_BC among recently infected antiretroviral therapy-naïve men who have sex with men in Jiangsu province, China, 2012 to 2015: A molecular epidemiology-based study.

The prevalence and incidence of human immunodeficiency virus type 1 (HIV-1) among men who have sex with men (MSM) are on the rise throughout China. With a large population of MSM, Jiangsu Province is facing an escalating HIV-1 epidemic.The aim of this study was to explore the phylogenetic and temporal dynamics of HIV-1 CRF01_AE and CRF07_BC among antiretroviral therapy (ART)-naïve MSM recently infected with HIV-1 in Jiangsu Province.We recruited MSM in Jiangsu Province (Suzhou, Wuxi, Nantong, Taizhou and Yancheng) 2012 to 2015. We collected information on demographics and sexual behaviors and a blood sample for HIV genome RNA extraction, RT-PCR amplification, and DNA sequencing. Multiple alignments were made using Gene Cutter, with the selected reference sequences of various subtypes/recombinants from the Los Alamos HIV-1 database. Phylogenetic and Bayesian evolutionary analysis was performed by MEGA version 6.0, Fasttree v2.1.7. and BEAST v1.6.2. Categorical variables were analyzed using χ2 test (or Fisher exact test where necessary). χ2 test with trend was used to assess the evolution of HIV-1 subtype distribution over time. All data were analyzed using SPSS20.0 software package (IBM Company, New York, NY).HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (60.06%), CRF07_BC (22.29%), subtype B (5.88%), CRF67_01B (5.26%), CRF68_01B (2.79%), CRF55_01B (1.55%), CRF59_01B (0.93%), and CRF08_BC (0.62%). Two unique recombination forms (URFs) (0.62%) were also detected. Four epidemic clusters and 1 major cluster in CRF01_AE and CRF07_BC were identified. The introduction of CRF01_AE strain (2001) was earlier than CRF07_BC strain (2004) into MSM resided in Jiangsu based on the time of the most recent common ancestor.Our study demonstrated HIV-1 subtype diversity among ART-naïve MSM recently infected with HIV-1 in Jiangsu. We first depicted the spatiotemporal dynamics, traced the dates of origin for the HIV-1 CRF01_AE/07_BC strains and made inference for the effective population size among newly infected ART-naïve MSM in Jiangsu from 2012 to 2015. A real-time surveillance of HIV-1 viral diversity and phylodynamics of epidemic cluster would be of great value to the monitoring of the epidemic and control of transmission, improvement of antiretroviral therapy strategies, and design of vaccines.

Clinical Outcomes Following the Use of Archived HIV-1 DNA Sequencing to Guide Antiretroviral Therapy Regimen Adjustment and Simplification

Abstract Background While favorable new antiretroviral therapy (ART) options are available for HIV disease, the Department of Health and Human Services guidelines recommend against switching suppressive regimens unless there is evidence that the new regimen will be fully active. A new assay analyzes archived HIV pro-viral DNA and can detect resistance mutations when HIV RNA is below the limit of detection, when standard genotyping (GT) is not possible. Small studies have correlated archived DNA GT to historical plasma RNA GT, but there is minimal available data on treatment outcomes when using this assay to determine antiretroviral therapy switch strategies. We evaluated clinical outcomes following ART adjustment based upon results of archived DNA GT testing. Methods A retrospective review of electronic medical records was performed at our medical center from October 2014 to October 2016. Inclusion criteria included age ≥ 18 years, archived DNA GT result available, ART changed after archived GT result, and follow up HIV RNA available after ART switch. Data was collected prior to and after ART switching. McNemar’s test was used for categorical variables and paired t-test for continuous variables. Results A total of 38 patients were included. Most patients were male (89%), Caucasian (66%), had a history of AIDS diagnosis (45%), had HIV for &amp;gt;10 years (74%), and had baseline ART resistance (24% resistant to 1 class, 37% resistant to ≥ 2 classes). Median baseline CD4 was 532 cells/mm3. At baseline, 31 (82%) patients had HIV RNA &amp;lt; 50 copies/mL. Compared with baseline, 35 (92%) patients were undetectable at furthest follow up (P = 0.22). Median time to furthest follow up was 146.5 days (range 12–485). Overall, 36 (95%) patients had at least one undetectable HIV RNA after switching. None of the patients with an initial undetectable HIV RNA became detectable after switching ART. Average number of pills per day and administrations per day decreased from 3.84 to 1.97 (P &amp;lt; 0.001) and 1.47 to 1.05 (P &amp;lt; 0.001) respectively. The number of patients on protease inhibitors (PIs) decreased from 66% to 21% (P &amp;lt; 0.001). Conclusion The use of archived DNA GT to guide ART adjustment may result in maintained viral suppression while allowing for regimens with optimized long-term safety and decreased pill burden. Disclosures All authors: No reported disclosures.