non-B Subtypes Research Articles

Attenuated HIV-1 Nef But Not Vpu Function in a Cohort of Rwandan Long-Term Survivors.

HIV-1 accessory proteins Nef and Vpu enhance viral pathogenesis through partially overlapping immune evasion activities. Attenuated Nef or Vpu functions have been reported in individuals who display slower disease progression, but few studies have assessed the relative impact of these proteins in non-B HIV-1 subtypes or examined paired proteins from the same individuals. Here, we examined the sequence and function of matched Nef and Vpu clones isolated from 29 long-term survivors (LTS) from Rwanda living with HIV-1 subtype A and compared our results to those of 104 Nef and 62 Vpu clones isolated from individuals living with chronic untreated HIV-1 subtype A from the same geographic area. Nef and vpu coding regions were amplified from plasma HIV RNA and cloned. The function of one intact, phylogenetically-validated Nef and Vpu clone per individual was then quantified by flow cytometry following transient expression in an immortalized CD4+ T-cell line. We measured the ability of each Nef clone to downregulate CD4 and HLA class I, and of each Vpu clone to downregulate CD4 and Tetherin, from the cell surface. Results were normalized to reference clones (Nef-SF2 and Vpu-NL4.3). We observed that Nef-mediated CD4 and HLA downregulation functions were lower in LTS compared to the control cohort (Mann-Whitney p=0.03 and p<0.0001, respectively). Moreover, we found a positive correlation between Nef-mediated CD4 downregulation function and plasma viral load in LTS and controls (Spearman ρ= 0.59, p=0.03 and ρ=0.30, p=0.005, respectively). In contrast, Vpu-mediated functions were similar between groups and did not correlate with clinical markers. Further analyses identified polymorphisms at Nef codon 184 and Vpu codons 60-62 that were associated with function, which were confirmed through mutagenesis. Overall, our results support attenuated function of Nef, but not Vpu, as a contributor to slower disease progression in this cohort of long-term survivors with HIV-1 subtype A.

Soluble CD14 is subtype-dependent in serum but not in cerebrospinal fluid in people with HIV

Monocytes and macrophages activation are crucial in human immunodeficiency virus (HIV) central nervous system (CNS) infection and HIV associated neurocognitive disorders (HAND) pathogenesis. The soluble form of CD14 (sCD14) is a marker of monocyte activation. We hypothesized that sCD14 levels would be lower in people with HIV-1 subtype C (HIV-1C) than in HIV-1B owing to a variant Tat cysteine dimotif (C30S31) with reduced chemotactic activity. A total of 68 paired cerebrospinal fluid (CSF) and blood samples from people with HIV (PWH); 27 samples of the HIV-1B subtype and 40 of the non-B HIV-1 subtypes (including 26,HIV-1C), and 18 HIV-negative controls were included. sCD14 levels were quantified using a high-sensitivity enzyme-linked immunosorbent assay. sCD14 increase in serum, but not in CSF, was higher in samples from HIV-1B than HIV-1C (p = 0.002; Cohen's d, 0.7). CSF or serum sCD14 values were not correlated with global deficit score or specific cognitive domains. The impact of HIV-1 on monocyte stimulation biomarkers evaluated by sCD14 in serum was subtype-dependent, higher in HIV-1B than HIV-1C, consistent with reduced chemotactic activity as hypothesized.

Performance assessment of the new Xpert® HIV-1 viral load XC assay for quantification of HIV-1 viral loads

BackgroundHIV-1 RNA quantification is a key component of treatment monitoring. ObjectivesTo assess the performance of a redesigned HIV-1 RNA quantitative assay that uses a dual-target approach: Xpert® HIV-1 Viral Load (VL) XC. Study designFresh and frozen samples (N = 533) from HIV-1 positive patients tested with Abbott HIV-1 assays (Alinity m and RealTime [m2000]) were retested using the new Xpert XC assay. Three samples with known underquantification using the previous single-target Xpert assay were retested. ResultsThe Xpert XC assay yielded valid results in 98.5% (N = 528/536) of cases and showed high sensitivity in 80 fresh samples that had undetectable VLs or ≤1.7 log copies/mL with Alinity m. Linear regression and Bland-Altman analyses showed high concordance with the Abbott tests for quantified samples over a wide VL range (1.6–6.9 copies/mL), including non-B subtypes (mean difference=-0.1±0.23 log copies/mL). Mutations associated with integrase resistance did not impact the results. Very good linearity and reproducibility was shown for the tested subtypes B, CRF06_cpx, and CRF02_AG. Xpert XC VLs in samples that were previously underquantified using the original single-target Xpert assay were similar to those detected by the Abbott assays (±0.11 log copies/mL). ConclusionsThe Xpert XC assay showed excellent correlation with the Abbott assays for all tested HIV-1 subtypes. Sensitivity, linearity and accuracy were high in the therapeutically relevant VL range. With a time to result of only 90 min, this on-demand decentralized assay is a safe, reliable and fast option for VL monitoring in HIV-1-infected patients.

Impact of HIV-1 subtypes on gross deletion in the nef gene after Korean Red Ginseng treatment

BackgroundThe number of primary human immunodeficiency virus (HIV)-1 non-B subtype infections (non-B) and that of reports regarding the differences in the pathogenesis of subtype B and non-B infections are increasing. However, to the best of our knowledge, there have been no reports on gross deletion in the nef gene (gΔnef) in non-B infections. MethodsTo determine whether there is a difference in the change in CD4+ T cells after treatment with Korean Red Ginseng (KRG) between patients with subtype B and non-B infections, we retrospectively analyzed and compared the annual decrease in CD4+ T cells (AD) and the proportion of gΔnef in 77 patients who were followed for more than 10 years in the absence of combination antiretroviral therapy. ResultsOverall, AD was significantly faster in patients with non-B infections than in those with subtype B infections. Survival analysis showed that the survival probability was significantly higher in subtype B than in non B-infected patients. These differences mainly resulted from significant differences in the amount of KRG and age. In the patients treated with KRG, there was a significant correlation between the amount of KRG and the AD in both subtypes. Interestingly, there was a significant correlation between the amount of KRG and the proportion of gΔnef in patients infected with subtype B, but not in those infected with non-B. The same phenomenon was observed when the KRG dose was adjusted. ConclusionOur results suggest that non-B may be biologically more stable than subtype B.

Detection of Gag C-terminal mutations among HIV-1 non-B subtypes in a subset of Cameroonian patients

Response to ritonavir-boosted-protease inhibitors (PI/r)-based regimen is associated with some Gag mutations among HIV-1 B-clade. There is limited data on Gag mutations and their covariation with mutations in protease among HIV-1 non-B-clades at PI/r-based treatment failure. Thus, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02_AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drug-naïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1–5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This analysis suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 non-B clades.

Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda

BackgroundOur understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia.ResultsIn this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4+ T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4+ counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001).ConclusionsThe smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs.

Revealing the drug resistance mechanism of saquinavir due to G48V and V82F mutations in subtype CRF01_AE HIV-1 protease: molecular dynamics simulation and binding free energy calculations

Human immunodeficiency virus-1 (HIV-1) protease is one of the important targets in AIDS therapy. The majority of HIV infections are caused due to non-B subtypes in developing countries. The co-occurrence of mutations along with naturally occurring polymorphisms in HIV-1 protease cause resistance to the FDA approved drugs, thereby posing a major challenge in the treatment of antiretroviral therapy. In this work, the resistance mechanism against SQV due to active site mutations G48V and V82F in CRF01_AE (AE) protease was explored. The binding free energy calculations showed that the direct substitution of valine at position 48 introduces a bulkier side chain, directly impairing the interaction with SQV in the binding pocket. Also, the intramolecular hydrogen bonding network of the neighboring residues is altered, indirectly affecting the binding of SQV. Interestingly, the substitution of phenylalanine at position 82 induces conformational changes in the 80’s loop and the flap region, thereby favoring the binding of SQV. The V82F mutant structure also maintains similar intramolecular hydrogen bond interactions as observed in AE-WT. Communicated by Ramaswamy H. Sarma

Identification of risk factors associated with national transmission and late presentation of HIV-1, Denmark, 2009 to 2017.

BackgroundDespite availability of pre-exposure prophylaxis (PrEP), the incidence of HIV-1 in Europe remained stable the past decade. Reduction of new HIV-1 infections requires more knowledge about the profiles of high-risk transmitters and late presenters (LP).AimWe aimed to investigate risk factors associated with HIV-1 transmission clusters and late presentation with HIV-1 in Denmark.MethodsBlood samples and epidemiological information were collected from newly diagnosed HIV-1 patients between 2009 and 2017. We genotyped pol genes and performed phylogenetic analyses to identify clusters. Risk factors for clustering and LP were investigated with partial proportional odds and logistic regression. Covariates included transmission mode, HIV-1 subtype, age, origin and cluster activity.ResultsWe included 1,040 individuals in the analysis, 59.6% identified with subtype B and 48.4% in a cluster. Risk factors for clustering included Danish origin (odds ratio (OR): 2.95; 95% confidence interval (CI): 2.21–3.96), non-LP (OR: 1.44; 95% CI: 1.12–1.86), and men who have sex with men (MSM). Increasing age and non-B subtype infection decreased risk (OR: 0.69; 95% CI: 0.50–0.94). Risk for late presentation was lower for active clusters (OR: 0.60; 95% CI: 0.44–0.82) and Danish origin (OR: 0.43; 95% CI: 0.27–0.67). Non-Danish MSM had a lower risk than non-Danish heterosexuals (OR: 0.34; 95% CI: 0.21–0.55).ConclusionHIV-1 transmission in Denmark is driven by early diagnosed, young, subtype B infected MSM. These may benefit most from PrEP. Non-Danish heterosexual HIV-1 patients could benefit from improved communication to achieve earlier diagnosis and treatment.

Five Years With Dolutegravir Plus Lamivudine as a Switch Strategy: Much More Than a Positive Finding.

Results from clinical trials and observational studies suggest that dolutegravir plus lamivudine could be an effective and well-tolerated option for simplification in HIV-1-positive patients. We aimed to assess long-time efficacy and safety in our multicenter cohort. This was a retrospective study enrolling HIV-1-infected, virologically suppressed patients switching to dolutegravir + lamivudine. We performed survival analysis to evaluate time to virological failure (VF, defined by a single HIV-RNA ≥1000 copies/mL or by 2 consecutive HIV-RNA ≥ 50 copies/mL) and treatment discontinuation (defined as the interruption of either 3TC or dolutegravir), assessing predictors via Cox regression analyses. Seven-hundred eighty-five patients were considered for the analysis: 554 were men (70.6%), with a median age of 52 years (interquartile range 45-58 years). Estimated probabilities of maintaining virological suppression at weeks 96, 144, and 240 were 97.7% (SD ±0.6), 96.9% (SD ±0.8), and 96.4% (SD ±0.9), respectively. A non-B HIV subtype (P = 0.014) and a previous VF (P = 0.037) resulted predictors of VF. We did not observe differences in probability of VF in people living with HIV with an M184V resistance mutation (P = 0.689); however, in a deeper analysis, M184V mutation was a predictor of VF (P = 0.038) in patients with time of virological suppression <88 months. Estimated probabilities of remaining on study regimen at 96, 144, and 240 weeks were 82.9% (SD ±1.4), 79.7% (SD ±1.6) and 74.3% (SD ±2.2), respectively. Our findings show the long-term efficacy and tolerability of dolutegravir plus lamivudine in virologically suppressed patients.

Many but small HIV-1 non-B transmission chains in the Netherlands.

Objective:The aim of this study was to investigate introductions and spread of different HIV-1 subtypes in the Netherlands.Design:We identified distinct HIV-1 transmission chains in the Netherlands within the global epidemic context through viral phylogenetic analysis of partial HIV-1 polymerase sequences from individuals enrolled in the ATHENA national HIV cohort of all persons in care since 1996, and publicly available international background sequences.Methods:Viral lineages circulating in the Netherlands were identified through maximum parsimony phylogeographic analysis. The proportion of HIV-1 infections acquired in-country among heterosexuals and MSM was estimated from phylogenetically observed, national transmission chains using a branching process model that accounts for incomplete sampling.Results:As of 1 January 2019, 2589 (24%) of 10 971 (41%) HIV-1 sequenced individuals in ATHENA had non-B subtypes (A1, C, D, F, G) or circulating recombinant forms (CRF01AE, CRF02AG, CRF06-cpx). The 1588 heterosexuals were in 1224, and 536 MSM in 270 phylogenetically observed transmission chains. After adjustments for incomplete sampling, most heterosexual (75%) and MSM (76%) transmission chains were estimated to include only the individual introducing the virus (size = 1). Onward transmission occurred mostly in chains size 2–5 amongst heterosexuals (62%) and in chains size at least 10 amongst MSM (64%). Considering some chains originated in-country from other risk-groups, 40% (95% confidence interval: 36–44) of non-B-infected heterosexuals and 62% (95% confidence interval: 49–73) of MSM-acquired infection in-country.Conclusion:Although most HIV-1 non-B introductions showed no or very little onward transmission, a considerable proportion of non-B infections amongst both heterosexuals and MSM in the Netherlands have been acquired in-country.

Molecular Epidemiology of HIV-1 Virus in Egypt: A Major Change in the Circulating Subtypes.

Human immunodeficiency virus type 1 (HIV-1) is characterized by high genetic diversity due to its high mutation and recombination rates. Although, there is an increasing prevalence of Circulating Recombinant Forms (CRFs) worldwide, subtype B is still recognized as the predominant subtype in the Middle East and North Africa (MENA) region. There is a limited sampling of HIV in this region due to its low prevalence. The main purpose of this study is to provide a summary of the current status of the resident HIV subtypes and their distribution among Egyptian patients. Forty-five HIV-1 patients were included in this study. Partial pol gene covering the protease (PR) and Reverse Transcriptase (RT) was successfully amplified in 21 HIV patients using nested PCR of cDNA of the viral genomic RNA, then sequenced. The sequence data were used for viral HIV-1 subtyping by 5 online subtyping tools: NCBI viral genotyping tool, Stanford University HIV database (HIVDB) subtyping program, REGA tool, Context-Based Modeling for Expeditious Typing (COMET) tool, and Recombinant Identification Program (RIP) tool. The final subtype assignment was based on molecular phylogenetic analysis. Unexpectedly, non-B subtypes are dominating, with the most common circulating one is CRF02_AG (57.1%) followed by subtype B (14.3%), subtype BG recombinant (9.5%), CRF35_ AD (9.5%), subtype A1 and CRF06_cpx (4.8% each). To the best of our knowledge, this is the first study to tackle HIV-1 subtyping among the group of HIV-1 patients in Egypt. CRF02_AG is the most prevalent subtype in Egypt.

The Role of Phylogenetics in Unravelling Patterns of HIV Transmission towards Epidemic Control: The Quebec Experience (2002-2020).

Phylogenetics has been advanced as a structural framework to infer evolving trends in the regional spread of HIV-1 and guide public health interventions. In Quebec, molecular network analyses tracked HIV transmission dynamics from 2002–2020 using MEGA10-Neighbour-joining, HIV-TRACE, and MicrobeTrace methodologies. Phylogenetics revealed three patterns of viral spread among Men having Sex with Men (MSM, n = 5024) and heterosexuals (HET, n = 1345) harbouring subtype B epidemics as well as B and non-B subtype epidemics (n = 1848) introduced through migration. Notably, half of new subtype B infections amongst MSM and HET segregating as solitary transmissions or small cluster networks (2–5 members) declined by 70% from 2006–2020, concomitant to advances in treatment-as-prevention. Nonetheless, subtype B epidemic control amongst MSM was thwarted by the ongoing genesis and expansion of super-spreader large cluster variants leading to micro-epidemics, averaging 49 members/cluster at the end of 2020. The growth of large clusters was related to forward transmission cascades of untreated early-stage infections, younger at-risk populations, more transmissible/replicative-competent strains, and changing demographics. Subtype B and non-B subtype infections introduced through recent migration now surpass the domestic epidemic amongst MSM. Phylodynamics can assist in predicting and responding to active, recurrent, and newly emergent large cluster networks, as well as the cryptic spread of HIV introduced through migration.

Epidemiological and phylogenetic analysis for non-B subtypes of human immunodeficiency virus type 1 in Busan, Korea

Recent data on non-B subtypes’ epidemiology among patients infected with human immunodeficiency virus-1 (HIV-1) in Korea are lacking. We aimed to assess the changing trends in the epidemiology of non-B subtypes of HIV-1 in Korea using phyloepidemiological analyses. We analyzed the demographic records and sequencing data obtained from genotypic drug resistance tests between 2005 and 2019 from 517 patients infected with HIV attending a tertiary care hospital in Busan, Korea. Subtyping and phylogenetic analyses with reference sequences were performed. Additionally, transmission clusters were identified via maximum-likelihood trees. Non-B subtypes accounted for 21.3% of the 517 sequences. CRF01_AE (52.7%) was the most common non-B subtype, followed by CRF02_AG (16.4%), A1 (11.8%), and C (5.5%). The prevalence of non-B subtypes decreased from 36.4 to 13.4% by 2009, while it increased to 27.4% between 2015 and 2019. Among patients with non-B subtypes, the proportion of overseas sailors decreased from 66.7 to 7.5%; contrarily, the proportion of men-who-have-sex-with-men (MSM) increased from 0 to 46.9% over the study period. We identified 8 transmission clusters involving non-B subtypes, with sizes ranging from 2 to 4 patients, including 3 clusters containing MSM. Our results highlight the changes in the epidemiological trends of non-B subtypes of HIV-1 in Korea.

HIV-1 molecular diversity in Brazil unveiled by 10\xa0years of sampling by the national genotyping network

HIV-1 has diversified into several subtypes and recombinant forms that are heterogeneously spread around the world. Understanding the distribution of viral variants and their temporal dynamics can help to design vaccines and monitor changes in viral transmission patterns. Brazil has one of the largest HIV-1 epidemics in the western-world and the molecular features of the virus circulating in the country are still not completely known. Over 50,000 partial HIV-1 genomes sampled between 2008 and 2017 by the Brazilian genotyping network (RENAGENO) were analyzed. Sequences were filtered by quality, duplicate sequences per patient were removed and subtyping was performed with online tools and molecular phylogeny. Association between patients’ demographic data and subtypes were performed by calculating the relative risk in a multinomial analysis and trends in subtype prevalence were tested by Pearson correlation. HIV-1B was found to be the most prevalent subtype throughout the country except in the south, where HIV-1C prevails. An increasing trend in the proportion of HIV-1C and F1 was observed in several regions of the country, while HIV-1B tended to decrease. Men and highly educated individuals were more frequently infected by HIV-1B and non-B variants were more prevalent among women with lower education. Our results suggest that socio-demographic factors partially segregate HIV-1 diversity in Brazil while shaping viral transmission networks. Historical events could explain a preferential circulation of HIV-1B among men who have sex with men (MSM) and non-B variants among heterosexual individuals. In view of an increasing male/female ratio of AIDS cases in Brazil in the last 10–15 years, the decrease of HIV-1B prevalence is surprising and suggests a greater penetrance of non-B subtypes in MSM transmission chains.

Increasing Frequency and Transmission of HIV-1 Non-B Subtypes Among Men Who Have Sex With Men in the Swiss HIV Cohort Study.

In Switzerland, HIV-1 transmission among men who have sex with men (MSM) has been dominated by subtype B, whilst non-B subtypes are commonly attributed to infections acquired abroad among heterosexuals. Here, we evaluated the temporal trends of non-B subtypes and the characteristics of molecular transmission clusters (MTCs) among MSM. Sociodemographic and clinical data and partial pol sequences were obtained from participants enrolled in the Swiss HIV Cohort Study. For non-B subtypes, maximum likelihood trees were constructed, from which Swiss MTCs were identified and analyzed by transmission group. Non-B subtypes were identified in 8.1% (416/5116) of MSM participants. CRF01_AE was the most prevalent strain (3.5%), followed by subtype A (1.2%), F (1.1%), CRF02_AG (1.1%), C (0.9%), and G (0.3%). Between 1990 and 2019, an increase in the proportion of newly diagnosed individuals (0/123 [0%] to 11/32 [34%]) with non-B subtypes in MSM was found. Across all non-B subtypes, the majority of MSM MTCs were European. Larger MTCs were observed for MSM than heterosexuals. We found a substantial increase in HIV-1 non-B subtypes among MSM in Switzerland and the occurrence of large MTCs, highlighting the importance of molecular surveillance in guiding public health strategies targeting the HIV-1 epidemic.

Phenotypic and Genotypic Co-receptor Tropism Testing in HIV-1 Epidemic Region of Tanzania Where Multiple Non-B Subtypes Co-circulate.

HIV human immunodeficiency virus type I (HIV-1) entry inhibitor potency is dependent on viral co-receptor tropisms and thereby tropism determination is clinically important. However, phenotypic tropisms of HIV-1 non-B subtypes have been poorly investigated and the genotypic prediction algorithms remain insufficiently validated. To clarify this issue, we recruited 52 treatment-naïve, HIV-1-infected patients in Tanzania, where multiple HIV-1 non-B subtypes co-circulate. Sequence analysis of 93 infectious envelope clones isolated from their plasma viral RNA revealed the co-circulation of subtypes A1, C, D, and inter-subtype recombinant forms (isRFs). Phenotypic tropism assays revealed that lentivirus reporters pseudotyped with 75 (80.6%) and 5 (5.4%) envelope clones could establish infection toward U87.CD4 cells expressing CCR5 (R5) and CXCR4 (X4), respectively; whereas the remaining 13 (14%) clones could infect both cells. Genotypic analyses by widely used algorithms including V3 net charge, Geno2pheno, WebPSSM, and PhenoSeq showed that almost all phenotypic X4-tropic clones and only 15 of 75 phenotypic R5-tropic clones were concordantly predicted. However, the remaining 60 phenotypic R5-tropic clones were discordantly predicted by at least one algorithm. In particular, 2 phenotypic R5-tropic clones were discordantly predicted by all algorithms tested. Taken together, the results demonstrate the limitation of currently available genotypic algorithms for predicting co-receptor inference among co-circulating multiple non-B subtypes and emerging isRFs. Also, the phenotypic tropism dataset presented here could be valuable for retraining of the widely used genotypic prediction algorithms to enhance their performance.

Factors associated with the emergence of integrase resistance mutations in patients failing dual or triple integrase inhibitor-based regimens in a French national survey.

Successful 2-drug regimens (2DRs) for HIV were made possible by the availability of drugs combining potency and tolerability with a high genetic barrier to resistance. How these deal with resistance development/re-emergence, compared with 3DRs, is thus of paramount importance. A national survey including patients who were either naive or experienced with any 2DR or 3DR but failing integrase strand transfer inhibitor (INSTI)-containing regimens [two consecutive plasma viral load (VL) values >50 copies/mL] was conducted between 2014 and 2019. Genotypic resistance tests were interpreted with the v28 ANRS algorithm. Overall, 1104 patients failing any INSTI-containing regimen (2DRs, n = 207; 3DRs, n = 897) were analysed. Five hundred and seventy-seven (52.3%) patients were infected with a B subtype and 527 (47.3%) with non-B subtypes. Overall, 644 (58%) patients showed no known integrase resistance mutations at failure. In multivariate analysis, factors associated with the emergence of at least one integrase mutation were: high VL at failure (OR = 1.24 per 1 log10 copies/mL increase); non-B versus B subtype (OR = 1.75); low genotypic sensitivity score (GSS) (OR = 0.10 for GSS = 2 versus GSS = 0-0.5); and dolutegravir versus raltegravir (OR = 0.46). Although 3DRs versus 2DRs reached statistical significance in univariate analysis (OR = 0.59, P = 0.007), the variable is not retained in the final model. This study is one of the largest studies characterizing integrase resistance in patients failing any INSTI-containing 2DR or 3DR in routine clinical care and reveals factors associated with emergence of integrase resistance that should be taken into consideration in clinical management. No difference was evidenced between patients receiving a 2DR or a 3DR.

Presence of HIV-1 G-to-A mutations linked to APOBEC editing is more prevalent in non-B HIV-1 subtypes and is associated with lower HIV-1 reservoir.

APOBEC3 editing activity contributes to sequences variation and viral diversification. We aimed to characterize virological and clinical factors associated with G-to-A mutations and stop codons in the HIV-1 reservoir, markers of APOBEC3 footprints, in order to better understand HIV-1 diversity among virologically suppressed HIV-1-infected patients. Immuno-virological and clinical factors were compared between 92 patients harbouring G-to-A mutations and stop codons (APOBEC+) in the reverse transcriptase gene and 92 patients without G-to-A mutations (APOBEC-) and stop codons in their DNA genotypes. Patients were predominantly men (74.5%) and were mostly infected by B-subtype (69.0%), with 44.1% and 55.9% in APOBEC+ and APOBEC- groups, respectively. At time of HIV DNA genotypes, the total cell-associated HIV-1 DNA load was 2.34 log10 copies/106 cells (IQR 1.85-2.67) and 33.2% of them had a detectable ultrasensitive plasma viral load. Hypermutated sequences were identified in 28.2% of the APOBEC+ group. The median total cell-associated HIV-1 DNA level was significantly lower in APOBEC+ than APOBEC- group: 2.13 log10 copies/106 cells (IQR 1.60-2.60) versus 2.52 log10 copies/106 cells (IQR 2.19-2.71) (P < 0.001), respectively. Presence of G-to-A mutations and stop codon was independently associated with HIV-1 subtype non-B (P = 0.017). These results show an independent association between the presence of G-to-A mutations and stop codons with HIV-1 subtype non-B and low proviral DNA that could be explained by the APOBEC3 footprints and restriction of DNA synthesis and integration. However, further investigations are needed to study the contribution of Vif amino acid variability among HIV-1 subtypes.

Cell culture selections reveal favourable drug resistance profiles for doravirine and islatravir.

The newer generation NNRTIs, including doravirine and rilpivirine, were designed to show high potency and overcome K103N, Y181C and G190A resistance. To assess emergent resistance to doravirine and rilpivirine, alone and paired with lamivudine or islatravir through in vitro drug selections. Subtype B (n = 3), non-B subtype (n = 3), and pNL4.3 viral isolates were passaged in cord blood mononuclear cells with progressively increasing concentrations of drug(s). Genotypic analysis compared the acquisition and accumulation of drug resistance mutations at weeks 8 and 24 following drug pressure. Cell-based phenotypic assays assessed cross-resistance patterns to NNRTIs by acquired resistance mutations. Doravirine pressure resulted in the acquisition of V108I (6/7) and V106A/I/M (5/7) mutations at weeks 8, followed by F227L (4/7), Y318F (4/7), M230L (2/7) or L234I (2/7) by weeks 24. In contrast, rilpivirine resulted in E138K (5/7) followed by L100I (3/7), K101E (1/7), or M230L (1/7). Doravirine resistance pathways retained susceptibility to rilpivirine, whereas rilpivirine resistance conferred intermediate resistance (12-152-fold) to doravirine. Dual selections with islatravir or lamivudine delayed and diminished emergent resistance to doravirine, resulting in V108I (9/15) with fewer or no other changes at weeks 24. There was a lesser delay in emergent resistance to rilpivirine when combined with islatravir or lamivudine. The M184V mutation did not arise in dual selections with islatravir or lamivudine. Doravirine showed a more robust resistance profile compared with other NNRTIs. The long intracellular half-life of islatravir and delayed acquisition of resistance in dual selections provide an opportunity for long-acting treatment options.

HIV-1 subtypes and drug resistance in children during antiretroviral therapy in Brazil.

We evaluate the genetic characterization of 132 HIV-1 pol sequences from children and adolescents undergoing antiretroviral therapy in Northeast Brazil. Phylogenetic and recombination analyses were performed using the maximum likelihood method using SeaView version 4 and SIMPLOT software. Most individuals harbored HIV-1 B (84.8%) and BF recombinants (9.8%), although other non-B subtypes were detected: HIV-1 C (1.5%), HIV-1 F (2.4%), and BC recombinants (1.5%). Antiretroviral resistance was 47% (95% confidence interval [CI]: 38.7%-55.4%). Non-nucleoside reverse transcriptase inhibitors (NNRTIs) showed higher frequencies of primary mutations, with 40.9% (95% CI: 32.9%-49.4%), followed by nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PIs) with 34.8% (95% CI: 27.3-43.3) and 6.1% (95% CI: 3.1%-11.5%), respectively. Among NRTIs, higher resistance levels were observed for abacavir, emtricitabine, and lamivudine; for NNRTI, nevirapine and efavirenz. The most common primary mutations found were M184V (29.5%), K103N (25%), M41L (9.8%), T215Y (8.3%), and G190A (8.3%). Our findings highlight the importance of surveillance of resistance mutations, which contributes to the continuous updating and implementation of preventive measures to decrease mother-to-child-transmission and transmitted drug resistance.