Predominance of the heterozygous CCR5 delta-24 deletion in African individuals resistant to HIV infection might be related to a defect in CCR5 addressing at the cell surface.

INTRODUCTION: The chemokine receptor CCR5 is the main co-receptor for R5-tropic HIV-1 variants. We have previously described a novel 24-base pair deletion in the coding region of CCR5 among individuals from Rwanda. Here, we investigated the prevalence of hCCR5Δ24 in different cohorts and its impact on CCR5 expression and HIV-1 infection in vitro. METHODS: We screened hCCR5Δ24 in a total of 3232 individuals which were either HIV-1 uninfected, high-risk HIV-1 seronegative and seropositive partners from serodiscordant couples, Long-Term Survivors, or HIV-1 infected volunteers from Africa (Rwanda, Kenya, Guinea-Conakry) and Luxembourg, using a real-time PCR assay. The role of the 24-base pair deletion on CCR5 expression and HIV infection was assessed in cell lines and PBMC using mRNA quantification, confocal analysis, flow and imaging cytometry. RESULTS AND DISCUSSION: Among the 1661 patients from Rwanda, 12 individuals were heterozygous for hCCR5Δ24 but none were homozygous. Although heterozygosity for this allele may not confer complete resistance to HIV-1 infection, the prevalence of the mutation was 2.41% (95%CI: 0.43; 8.37) in 83 Long-Term Survivors (LTS) and 0.99% (95%CI: 0.45; 2.14) in 613 HIV-1 exposed seronegative members as compared with 0.35% (95% Cl: 0.06; 1.25) in 579 HIV-1 seropositive members. The prevalence of hCCR5Δ24 was 0.55% (95%CI: 0.15; 1.69) in 547 infants from Kenya but the mutation was not detected in 224 infants from Guinea-Conakry nor in 800 Caucasian individuals from Luxembourg. Expression of hCCR5Δ24 in cell lines and PBMC showed that the hCCR5Δ24 protein is stably expressed but is not transported to the plasma membrane due to a conformational change. Instead, the mutant receptor was retained intracellularly, colocalized with an endoplasmic reticulum marker and did not mediate HIV-1 infection. Co-transfection of hCCR5Δ24 and wtCCR5 did not indicate a transdominant negative effect of CCR5Δ24 on wtCCR5. CONCLUSIONS: Our findings indicate that hCCR5Δ24 is not expressed at the cell surface. This could explain the higher prevalence of the heterozygous hCCR5Δ24 in LTS and HIV-1 exposed seronegative members from serodiscordant couples. Our data suggest an East-African localization of this deletion, which needs to be confirmed in larger cohorts from African and non-African countries.

Characteristics and spread to the native population of HIV-1 non-B subtypes in two European countries with high migration rate.

BACKGROUND: Non-B subtypes account for at least 50 % of HIV-1 infections diagnosed in Belgium and Luxembourg. They are considered to be acquired through heterosexual contacts and infect primarily individuals of foreign origin. Information on the extent to which non-B subtypes spread to the local population is incomplete. METHODS: Pol and env gene sequences were collected from 410 non-subtype B infections. Profound subtyping was performed using 5 subtyping tools and sequences of both pol and env. Demographic information, disease markers (viral load, CD4 count) and viral characteristics (co-receptor tropism) were compared between subtypes. Maximum likelihood phylogenetic trees were constructed and examined for clustering. RESULTS: The majority of non-B infections were diagnosed in patients originating from Africa (55.8 %), individuals born in Western Europe represented 30.5 %. Heterosexual transmission was the most frequently reported transmission route (79.9 %), MSM transmission accounted for 12.2 % and was significantly more frequently reported for Western Europeans (25.7 % versus 4.3 % for individuals originating from other regions; p < 0.001). Subtypes A and C and the circulating recombinant forms CRF01_AE and CRF02_AG were the most represented and were included in the comparative analysis. Native Western Europeans were underrepresented for subtype A (14.5 %) and overrepresented for CRF01_AE (38.6 %). The frequency of MSM transmission was the highest for CRF01_AE (18.2 %) and the lowest for subtype A (0 %). No differences in age, gender, viral load or CD4 count were observed. Prevalence of CXCR4-use differed between subtypes but largely depended on the tropism prediction algorithm applied. Indications for novel intersubtype recombinants were found in 20 patients (6.3 %). Phylogenetic analysis revealed only few and small clusters of local transmission but could document one cluster of CRF02_AG transmission among Belgian MSM. CONCLUSIONS: The extent to which non-B subtypes spread in the native Belgian-Luxembourg population is higher than expected, with 30.5 % of the non-B infections diagnosed in native Western Europeans. These infections resulted from hetero- as well as homosexual transmission. Introduction of non-B variants in the local high at risk population of MSM may lead to new sub-epidemics and/or increased genetic variability and is an evolution that needs to be closely monitored.

COMET: adaptive context-based modeling for ultrafast HIV-1 subtype identification.

Viral sequence classification has wide applications in clinical, epidemiological, structural and functional categorization studies. Most existing approaches rely on an initial alignment step followed by classification based on phylogenetic or statistical algorithms. Here we present an ultrafast alignment-free subtyping tool for human immunodeficiency virus type one (HIV-1) adapted from Prediction by Partial Matching compression. This tool, named COMET, was compared to the widely used phylogeny-based REGA and SCUEAL tools using synthetic and clinical HIV data sets (1,090,698 and 10,625 sequences, respectively). COMET's sensitivity and specificity were comparable to or higher than the two other subtyping tools on both data sets for known subtypes. COMET also excelled in detecting and identifying new recombinant forms, a frequent feature of the HIV epidemic. Runtime comparisons showed that COMET was almost as fast as USEARCH. This study demonstrates the advantages of alignment-free classification of viral sequences, which feature high rates of variation, recombination and insertions/deletions. COMET is free to use via an online interface.

HIV-1 tropism determination using a phenotypic Env recombinant viral assay highlights overestimation of CXCR4-usage by genotypic prediction algorithms for CRF01_AE and CRF02_AG [corrected].

BACKGROUND: Human Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno[coreceptor] and webPSSM. METHODS: Co-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno[coreceptor] (5-20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile® Enhanced-Sensitivity-Tropism-Assay. RESULTS: The lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno[coreceptor] was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno[coreceptor] was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno[coreceptor] and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A. CONCLUSIONS: Phenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors.

Polymorphisms of HIV-2 integrase and selection of resistance to raltegravir.

BACKGROUND: Human Immunodeficiency Virus type 2 is naturally resistant to some antiretroviral drugs, restricting therapeutic options for patients infected with HIV-2. Regimens including integrase inhibitors (INI) seem to be effective, but little data on HIV-2 integrase (IN) polymorphisms and resistance pathways are available. MATERIALS AND METHODS: The integrase coding sequence from 45 HIV-2-infected, INI-naïve, patients was sequenced and aligned against the ROD (group A) or EHO (group B) reference strains and polymorphic or conserved positions were analyzed.To select for raltegravir (RAL)-resistant variants in vitro, the ROD strain was cultured under increasing sub-optimal RAL concentrations for successive rounds. The phenotype of the selected variants was assessed using an MTT assay. RESULTS: We describe integrase gene polymorphisms in HIV-2 clinical isolates from 45 patients. Sixty-seven percent of the integrase residues were conserved. The HHCC Zinc coordination motif, the catalytic triad DDE motif, and AA involved in IN-DNA binding and correct positioning were highly conserved and unchanged with respect to HIV-1 whereas the connecting residues of the N-terminal domain, the dimer interface and C-terminal LEDGF binding domain were highly conserved but differed from HIV-1. The N155 H INI resistance-associated mutation (RAM) was detected in the virus population from one ARV-treated, INI-naïve patient, and the 72I and 201I polymorphisms were detected in samples from 36 and 38 patients respectively. No other known INI RAM was detected.Under RAL selective pressure in vitro, a ROD variant carrying the Q91R+I175M mutations was selected. The Q91R and I175M mutations emerged simultaneously and conferred phenotypic resistance (13-fold increase in IC50). The Q91R+I175M combination was absent from all clinical isolates. Three-dimensional modeling indicated that residue 91 lies on the enzyme surface, at the entry of a pocket containing the DDE catalytic triad and that adding a positive charge (Gln to Arg) might compromise IN-RAL affinity. CONCLUSIONS: HIV-2 polymorphisms from 45 INI-naïve patients are described. Conserved regions as well as frequencies of HIV-2 IN polymorphisms were comparable to HIV-1. Two new mutations (Q91R and I175M) that conferred high resistance to RAL were selected in vitro, which might affect therapeutic outcome.