TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage.

Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1+ and TIGIT+ CD4 T Cells.

Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

Determinants in HIV-2 Env and tetherin required for functional interaction.

BACKGROUND: The interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity. RESULTS: By examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin. CONCLUSIONS: These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.

Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells.

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.

Impact of baseline HIV-1 tropism on viral response and CD4 cell count gains in HIV-infected patients receiving first-line antiretroviral therapy.

BACKGROUND: Viral tropism influences the natural history of human immunodeficiency type 1 (HIV-1) disease: X4 viruses are associated with faster decreases in CD4 cell count. There is scarce information about the influence of viral tropism on treatment outcomes. METHODS: Baseline plasma samples from patients recruited to the ArTEN (Atazanavir/ritnoavir vs. Nevirapine on a background of Tenofovir and Emtricitabine) trial were retrospectively tested for HIV-1 tropism using the genotypic tool geno2pheno(FPR=5.75%). ArTEN compared nevirapine with atazanavir-ritonavir, both along with tenofovir-emtricitabine, in drug-naïve patients. RESULTS: Of 569 ArTEN patients, 428 completed 48 weeks of therapy; 282 of these received nevirapine and 146 of these received atazanavir-ritonavir. Overall, non-B subtypes of HIV-1 were recognized in 96 patients (22%) and X4 viruses were detected in 55 patients (14%). At baseline, patients with X4 viruses had higher plasma HIV RNA levels (5.4 vs 5.2 log copies/mL, respectively; P = .044) and lower CD4 cell counts (145 vs 188 cells/µL, respectively; P < .001) than those with R5 strains. At week 48, virologic responses were lower in patients with X4 viruses than in patients with R5 viruses (77% vs 92%, respectively; P = .009). Multivariate analysis confirmed HIV-1 tropism as an independent predictor of virologic response at week 24 (P = .012). This association was extended to week 48 (P = .007) in clade B viruses. Conversely, CD4 cell count recovery was not influenced by baseline HIV-1 tropism. CONCLUSIONS: HIV-1 tropism is an independent predictor of virologic response to first-line antiretroviral therapy. In contrast, it does not seem to influence CD4 cell count recovery. CLINICAL TRIALS REGISTRATION: NCT00389207.

High concordance between the position-specific scoring matrix and geno2pheno algorithms for genotypic interpretation of HIV-1 tropism: V3 length as the major cause of disagreement.

The agreement between the position-specific scoring matrix (PSSM) and geno2pheno as tools for genotypic interpretation of HIV-1 tropism using 800 clinical specimens was assessed. There was an overall concordance of 88%. Disagreement was found mostly in specimens with short V3 lengths (<35 amino acids). Thus, consideration of V3 lengths should improve the predictability of HIV-1 tropism using genotypic algorithms.

[Variability in HIV viral tropism determination using different genotypic algorithms in patients infected with B versus non-B HIV-1 subtypes]. / Variabilidad en la determinación del tropismo viral del VIH utilizando diferentes algoritmos genotípicos de interpretación en pacientes VIH-1 infectados con subtipos B versus no-B.

BACKGROUND: Genotypic tools based on the analysis of the V3 region are seen as an alternative to phenotypic assays for viral tropism determination before prescribing maraviroc. The concordance between different genotypic algorithms has been evaluated in HIV+ patients infected with B versus non-B subtypes. METHODS: HIV-infected patients on regular follow up at Hospital Universitario de Santiago de Compostela (Spain) were selected. The env-V3 region was sequenced from plasma samples and viral tropism was estimated using 8 different genotypic algorithms. Concordance among predictors was statistically evaluated by the calculation of the kappa index. Phylogenetic analyses were performed to determine the genetic subtype. RESULTS: A total of 92 HIV-infected patients were selected, 72 B and 20 non-B subtypes. Regarding the B subtype group, significant kappa values were obtained among all 28 possible combinations between the genotypic predictors evaluated. The best concordance among non-related predictors was observed for webPSSM(SINSI)/Wetcat(PART) (k: 0.771) and webPSSM(SINSI)/geno2pheno (k: 0.574). Conversely, among non-B subtypes, a significative kappa index was only obtained for 13 combinations. Among non-B subtypes, the best concordance values were obtained for webPSSM(X4R5)/Wetcat(PART) (k: 0.600) and webPSSM(SINSI)/Charge rule (k: 0.590). CONCLUSION: A high concordance was observed between different genotypic algorithms to determine viral tropism among HIV-1 B subtypes infected patients, especially between webPSSM(SINSI) and geno2pheno or Wetcat. Conversely, the overall concordance among non-B subtypes was lower. This heterogeneity could be justified by the low prevalence of non B subtypes in the datasets in which the genotypic tropism predictors were trained.

Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes.

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.

Dynamics of HIV tropism under suppressive antiretroviral therapy: implications for tropism testing in subjects with undetectable viraemia.

OBJECTIVES: The use of maraviroc as part of a simplification of antiretroviral therapy (ART) is hampered by the difficulty of assessing viral tropism in patients with undetectable viraemia. In this context, information on tropism might be obtained from testing either older stored viraemic sera collected before initiation of ART or current proviral DNA in peripheral blood cells. METHODS: HIV-1-infected individuals who had initiated ART and had undetectable viraemia for >2 years were identified. V3 genotyping was performed in parallel from plasma HIV-RNA and proviral DNA before starting ART and from proviral DNA while on suppressive ART. Viral tropism was interpreted using geno2pheno (false positive rate = 10%) and an optimized version of position specific scoring matrices (PSSM) with a greater sensitivity to detect X4 variants (PSSM(X4/R5-8)). RESULTS: A total of 78 HIV-1 infected individuals were examined. Mean time under suppressive ART was 3.5 years (interquartile range: 2.3-4.4). The rate of X4 variants in plasma and proviral DNA samples at baseline was 32.8% and 34.0%, respectively. It was 33.9% after >2 years of suppressive ART in DNA samples. Paired RNA/DNA tropism results at baseline could be obtained for 38 patients, with an overall 82% concordance. After >2 years of suppressed plasma viraemia, HIV tropism was re-assessed in proviral DNA; tropism switches were uncommon, especially comparing baseline and most recent DNA longitudinal specimens (12%). CONCLUSIONS: HIV tropism switches over time under suppressive ART are rare. There is a relatively good correlation between RNA and DNA tropism estimations using genotypic tests. Thus, HIV-1 tropism might confidently be examined either in older stored viraemic plasma specimens or in current proviral DNA samples.

High sensitivity of specific genotypic tools for detection of X4 variants in antiretroviral-experienced patients suitable to be treated with CCR5 antagonists.

OBJECTIVES: Evaluation of the reliability of several V3-based genotypic predictors to infer viral tropism in patients infected with B and non-B strains of HIV-1. METHODS: Several genotypic tropism predictors were evaluated in plasma (RNA) samples from 198 HIV-1-infected patients, taking as gold standard the results of the phenotypic recombinant virus assay Phenoscript((R)). In addition, for 37 B subtype HIV-1 patients the phenotypic results from plasma samples were also compared with tropism predictions based on V3 amplification from paired peripheral blood mononuclear cells (PBMCs). RESULTS: A total of 150 paired genotypic/phenotypic results were obtained from plasma specimens. Concordance values ranged from 63% to 85%, depending on the genotypic algorithm used. The best predictors in terms of sensitivity/specificity to detect X4 variants were WebPSSM(X4/R5) (77%/87%), Geno2pheno(FPR) (=) (5%) (80%/77%) and an algorithm combining the '11/25' and 'Net charge' rules, termed Garrido's rule (80%/79%). The performance of genotypic predictors was better testing B than non-B clades. The overall sensitivity ranged from 28% to 94%, reaching 100% in subtype B antiretroviral-experienced patients using WebPSSM(SI/NSI), Geno2pheno(FPR) (> or =) (5%) and Garrido's rule. Conversely, the sensitivity when testing non-B subtypes was poorer, ranging from 17% to 67%. Interestingly, the correlation between genotypic and phenotypic results was better when testing PBMCs than plasma using all genotypic predictors. CONCLUSIONS: Genotypic tools based on V3 sequences may provide reliable information on HIV-1 tropism when testing clade B viruses, especially in antiretroviral-experienced patients. The sensitivity to detect X4 variants using genotypic tools may improve by testing proviral DNA instead of plasma RNA.

Primary resistance to maraviroc in a large set of R5-V3 viral sequences from HIV-1-infected patients.

OBJECTIVES: Evaluation of the prevalence of V3 mutational patterns associated with maraviroc resistance in R5-using variants. METHODS: V3 sequences were obtained from 809 plasma specimens collected from maraviroc-naive HIV-1-infected individuals on regular follow-up at Hospital Carlos III. Sequences considered to harbour R5-tropic viruses were examined for the presence of primary maraviroc resistance mutational patterns, as found in both in vitro and in vivo studies. RESULTS: A total of 498 R5-V3 sequences were identified. They belonged to recent HIV-1 seroconverters (55.6%), chronically antiretroviral-naive subjects (20.1%) and antiretroviral-experienced patients (24.3%). Most individuals (93.8%) were infected with HIV-1 subtype B. The overall prevalence of maraviroc resistance mutational patterns was low (≤5%). Likewise, specific polymorphisms 4L, 11R or 19S, recently found to be associated with lower clinical response to maraviroc, were found in <2% of tested samples. The rate of maraviroc resistance patterns did not differ significantly according to length of HIV-1 infection, antiretroviral exposure or HIV-1 subtype. CONCLUSIONS: The prevalence of maraviroc resistance mutations is low in maraviroc-naive HIV-1-infected individuals.

Design and validation of new genotypic tools for easy and reliable estimation of HIV tropism before using CCR5 antagonists.

BACKGROUND: Genotypic tools may allow easier and less expensive estimation of HIV tropism before prescription of CCR5 antagonists compared with the Trofile assay (Monogram Biosciences, South San Francisco, CA, USA). METHODS: Paired genotypic and Trofile results were compared in plasma samples derived from the maraviroc expanded access programme (EAP) in Europe. A new genotypic approach was built to improve the sensitivity to detect X4 variants based on an optimization of the webPSSM algorithm. Then, the new tool was validated in specimens from patients included in the ALLEGRO trial, a multicentre study conducted in Spain to assess the prevalence of R5 variants in treatment-experienced HIV patients. RESULTS: A total of 266 specimens from the maraviroc EAP were tested. Overall geno/pheno concordance was above 72%. A high specificity was generally seen for the detection of X4 variants using genotypic tools (ranging from 58% to 95%), while sensitivity was low (ranging from 31% to 76%). The PSSM score was then optimized to enhance the sensitivity to detect X4 variants changing the original threshold for R5 categorization. The new PSSM algorithms, PSSM(X4R5-8) and PSSM(SINSI-6.4), considered as X4 all V3 scoring values above -8 or -6.4, respectively, increasing the sensitivity to detect X4 variants up to 80%. The new algorithms were then validated in 148 specimens derived from patients included in the ALLEGRO trial. The sensitivity/specificity to detect X4 variants was 93%/69% for PSSM(X4R5-8) and 93%/70% for PSSM(SINSI-6.4). CONCLUSIONS: PSSM(X4R5-8) and PSSM(SINSI-6.4) may confidently assist therapeutic decisions for using CCR5 antagonists in HIV patients, providing an easier and rapid estimation of tropism in clinical samples.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

Prevalence of natural polymorphisms at the HCV NS5A gene associated with resistance to daclatasvir, an NS5A inhibitor.

BACKGROUND: Daclatasvir (BMS-790052) is an investigational molecule that inhibits the HCV NS5A protein and shows potent antiviral activity apparently across all HCV genotypes. Selection of drug resistance mutations has been reported only for HCV genotype 1, and no information exists for other HCV variants and/or in HIV-HCV-coinfected individuals. METHODS: All interferon-α-naive, HIV-HCV-coinfected patients newly attended at Hospital Carlos III (Madrid, Spain) in 2011 were identified. Changes reported to be associated with daclatasvir resistance in the in vitro replication system for HCV genotype/subtypes 1a/1b (M28T, Q30H/R, L31F/M/V, P32L and Y93C/H/N) were examined. RESULTS: A total of 78 HIV-HCV-coinfected individuals as well as 635 NS5A sequences deposited at Los Alamos HCV database were analysed. None of the NS5A sequences from HCV-1a or HCV-3 showed changes associated with daclatasvir resistance. By contrast, all NS5A sequences from HCV-4 harboured L31M. The double mutant L31M+Y93H was found in 7% of HCV-1b and 13% of HCV-4. Finally, all NS5A sequences from HCV-1b and HCV-4 harboured changes at codon 28 (M28L) and 30 (L30R), which are of unknown significance. The rate of all these NS5A polymorphisms did not differ significantly when comparing HIV-HCV-coinfected patients and sequences from HCV-monoinfected subjects deposited at Los Alamos HCV database. CONCLUSIONS: Primary resistance mutations to daclatasvir, an investigational HCV NS5A inhibitor, are not seen in HCV-1a or in HCV-3 as natural polymorphisms. By contrast, they can be recognized in most HCV-1b and HCV-4 strains, regardless HIV coinfection.

Short communication: severe immune suppression in patients infected with R5-tropic HIV-1 strains is associated with increased gp120 net charge at variable regions.

CXCR4-tropic viruses have been associated with advanced immune suppression. However, 50% of patients with AIDS exclusively harbor CCR5-tropic viruses. The net charge at HIV-1 envelope gp120 variable regions was examined in 66 HIV-1-infected individuals with CCR5-tropic viruses, of whom 30 had less than 200 cells/mm(3). A positive net charge at gp120 variable regions was significantly associated with lower CD4 counts. Thus, the net charge at gp120 variable regions could influence HIV-1 disease progression in subjects with CCR5-tropic viruses.

High prevalence of natural polymorphisms in Gag (CA-SP1) associated with reduced response to Bevirimat, an HIV-1 maturation inhibitor.

Mutations H358Y, L363F/M, A364V and A366T/V confer in-vitro resistance to bevirimat. Moreover, polymorphisms at the Glutamine-Valine-Threonine (QVT) motif (369-371) have been associated with reduced bevirimat activity in vivo. The rate of these changes was assessed in 389 HIV+ patients naïve for bevirimat. QVT polymorphisms were frequent (47%), especially in non-B subtypes (93%). Conversely, only four patients (1%) harbored major bevirimat resistance mutations. Finally, specific gag changes were associated with protease inhibitor resistance mutations in subtype B viruses.

Safety and efficacy of tenofovir/emtricitabine plus nevirapine in HIV-infected patients.

All 178 HIV-infected individuals who had initiated tenofovir-emtricitabine-nevirapine (TDF/FTC/NVP) at our institution and were adherent to their medication were retrospectively examined. Only 22% were antiretroviral naive. After a median follow-up of 16 months, only five (2.8%) individuals (all with prior exposure to other antiretroviral regimens) experienced virological failure. In all instances, viral rebound occurred after 12 weeks of therapy. These results do not support an increased risk of early virological failure using TDF/FTC/NVP.

Variabilidad en la determinación del tropismo viral del VIH utilizando diferentes algoritmos genotípicos de interpretación en pacientes VIH-1 infectados con subtipos B versus no-B / Variability in HIV viral tropism determination using different genotypic algorithms in patients infected with B versus non-B HIV-1 subtypes

Antecedentes Las herramientas genotípicas basadas en el análisis de la región V3 de la envuelta viral se perfilan como la alternativa a los ensayos fenotípicos para la determinación del tropismo del VIH por los receptores de quimiocinas CCR5 y CXCR4 en la práctica clínica. Este trabajo evalúa la concordancia entre los distintos algoritmos de interpretación genotípica actualmente disponibles en pacientes VIH infectados con subtipo B versus subtipos no-B .Métodos Se seleccionaron pacientes VIH positivos, procedentes del Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), España. A partir de muestras de plasma, se amplificó y secuenció la región V3 de la envuelta viral. El tropismo viral se determinó usando 8 algoritmos genotípicos distintos. La concordancia entre los distintos predictores se evaluó calculando el índice de concordancia kappa. El subtipo genético fue determinado por análisis filogenético. Resultados Se incluyeron un total de 92 pacientes, 72 infectados por subtipo B y 20 por no-B. En pacientes con subtipo B, se obtuvieron valores significativos de kappa para todas combinaciones posibles (n=28) entre los algoritmos genotípicos analizados. La mejores valores entre predictores no relacionados se obtuvieron para webPSSMSINSI/WetcatPART (k: 0,771) y webPSSMSINSI/geno2pheno (k: 0,574). En subtipos no-B solo se obtuvieron valores significativos para 13 combinaciones, correspondiendo los mejores a PSSMX4R5/WetcatPART (k: 0,600) y PSSMSINSI/Charge rule (k: 0,590) (..) (AU)

Background Genotypic tools based on the analysis of the V3 region are seen as an alternative to phenotypic assays for viral tropism determination before prescribing maraviroc. The concordance between different genotypic algorithms has been evaluated in HIV+ patients infected with B versus non-B subtypes. Methods HIV-infected patients on regular follow up at Hospital Universitario de Santiago de Compostela (Spain) were selected. The env-V3 region was sequenced from plasma samples and viral tropism was estimated using 8 different genotypic algorithms. Concordance among predictors was statistically evaluated by the calculation of the kappa index. Phylogenetic analyses were performed to determine the genetic subtype. Results A total of 92 HIV-infected patients were selected, 72 B and 20 non-B subtypes. Regarding the B subtype group, significant kappa values were obtained among all 28 possible combinations between the genotypic predictors evaluated. The best concordance among non-related predictors was observed for webPSSMSINSI/WetcatPART (k: 0.771) and webPSSMSINSI/geno2pheno (k: 0.574). Conversely, among non-B subtypes, a significative kappa index was only obtained for 13 combinations. Among non-B subtypes, the best concordance values were obtained for webPSSMX4R5/WetcatPART (k: 0.600) and webPSSMSINSI/Charge rule (k: 0.590).Conclusion A high concordance was observed between different genotypic algorithms to determine viral tropism among HIV-1 B subtypes infected patients, especially between webPSSMSINSI and geno2pheno or Wetcat. Conversely, the overall concordance among non-B subtypes was lower. This heterogeneity could be justified by the low prevalence of non B subtypes in the datasets in which the genotypic tropism predictors were trained (AU)