Michelle Cazabat

Correlation between genotypic predictions based on V3 sequences and phenotypic determination of HIV-1 tropism.

Objective:Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. Design:Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. Methods:HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. Results:The 103 patients had a median CD4+ T lymphocyte count of 268 × 106 cells/l and nadirs of 98 × 106 cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. Conclusion:Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.

Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals

Depletion of CD4+ T cells from the gut occurs rapidly during acute HIV-1 infection. This has been linked to systemic inflammation and disease progression as a result of translocation of microbial products from the gut lumen into the bloodstream. Combined antiretroviral therapy (cART) substantially restores CD4+ T cell numbers in peripheral blood, but the gut compartment remains largely depleted of such cells for poorly understood reasons. Here, we show that a lack of recruitment of CD4+ T cells to the gut could be involved in the incomplete mucosal immune reconstitution of cART-treated HIV-infected individuals. We investigated the trafficking of CD4+ T cells expressing the gut-homing receptors CCR9 and integrin α4β7 and found that many of these T cells remained in the circulation rather than repopulating the mucosa of the small intestine. This is likely because expression of the CCR9 ligand CCL25 was lower in the small intestine of HIV-infected individuals. The defective gut homing of CCR9+β7+ CD4+ T cells - a population that we found included most gut-homing Th17 cells, which have a critical role in mucosal immune defense - correlated with high plasma concentrations of markers of mucosal damage, microbial translocation, and systemic T cell activation. Our results thus describe alterations in CD4+ T cell homing to the gut that could prevent efficient mucosal immune reconstitution in HIV-infected individuals despite effective cART.

R5 to X4 switch of the predominant HIV-1 population in cellular reservoirs during effective highly active antiretroviral therapy.

Summary:HIV-1 coreceptor usage plays a critical role for virus tropism and pathogenesis. A switch from CCR5 to CXCR4-using viruses can occur in the natural course of infection and correlates with subsequent disease progression. To investigate whether HIV-1 genetic evolution might lead to changes in virus coreceptor usage during highly active antiretroviral therapy (HAART), a longitudinal genotypic analysis of the virus found in cellular reservoirs was conducted in 32 patients with undetectable viral loads on HAART for 5 years. The genotype of the 3rd variable region of the env gene predicting coreceptor usage was retrospectively determined in the plasma or in peripheral blood mononuclear cells (PBMC) at baseline and then in PBMCs at months 30 and 60 of HAART. There was a switch from R5 to X4 variants in 11 of the 23 patients who harbored a majority virus population of R5 variants at baseline. X4 variants remained predominant in the 9 patients who harbored mainly X4 variants at baseline. The patients harboring predominantly X4 variants during HAART, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously a majority population of R5 variants. These results suggest that potent antiretroviral therapy produces the conditions necessary for the gradual emergence of X4 variants in cellular reservoirs.

Population-Based Sequencing of the V3 Region of env for Predicting the Coreceptor Usage of Human Immunodeficiency Virus Type 1 Quasispecies

ABSTRACT Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.

Persistence of distinct HIV-1 populations in blood monocytes and naive and memory CD4 T cells during prolonged suppressive HAART.

Objective:Reservoirs of HIV-1 are a major obstacle to virus eradication. There is therefore a need to clearly understand the molecular nature of the virus populations that persist in patients with sustained suppression of plasma viraemia on highly active antiretroviral therapy (HAART). Design:We performed a detailed analysis of the genotypes of HIV-1 quasispecies isolated from highly purified blood cell types taken from three selected patients with sustained undetectable viral loads on HAART for 7 years. Methods:We used polychromatic flow cytometry to sort naive and memory CD4 T cells, CD14 monocytes, and CD56+CD3− natural killer (NK) cells from the total peripheral blood mononuclear cells after 7 years of HAART. Clonal analysis was used to determine coreceptor use and drug-resistance genotypes of HIV-1 quasispecies in the sorted blood cell types. Results:We detected HIV-1 DNA in memory and naive CD4 T cells and in CD14 monocytes, but not in the CD56+CD3− NK cells. Phylogenetic analysis demonstrated that the various blood cells types of two of the three patients harboured genetically distinct HIV-1 quasispecies. Drug-resistance mutations were also distributed differently from one cell type to another. This compartmentalization suggests a minimal virus trafficking between blood cell types during suppressive HAART. Conclusions:We observed a cell-specific compartmentalization of the residual virus populations during prolonged suppressive HAART. The coexistence of numerous HIV-1 quasispecies with different resistance genotypes and coreceptor use in cellular reservoirs may be relevant for future antiretroviral treatment strategies.

Prediction of HIV Type 1 Subtype C Tropism by Genotypic Algorithms Built From Subtype B Viruses

Background:Genotypic predictions of HIV-1 tropism could simplify CCR5 antagonist usage. However, the genotypic algorithms built from subtype B viruses could be inadequate for non-B subtypes. We therefore performed paired genotypic and phenotypic determination of subtype C tropism. Methods:We studied 52 patients recruited in Malawi and 21 patients recruited in France. We directly sequenced the V3 env region and performed a recombinant virus phenotypic entry assay in parallel. Results:The Malawi patients had 29% of CXCR4-using subtype C viruses compared with only 5% in the patients from France. For detecting CXCR4-using subtype C viruses, the genotypic rule combining the amino acids at positions 11/25 and the net charge of V3 was 93.3% sensitive and 96.4% specific. The Geno2pheno tool was 86.7% sensitive and 89.1% specific. The WebPSSM tool with the SI/NSI matrix was 80% sensitive and 98.2% specific in its subtype B version and 93.3% sensitive and 81.8% specific in its subtype C version. Conclusions:The genotypic determinants of coreceptor usage for HIV-1 subtype C were mainly in V3 and were globally similar to those previously reported for subtype B viruses. The main genotypic algorithms built from subtype B viruses perform well when applied to subtype C viruses.

Genotypic Prediction of Human Immunodeficiency Virus Type 1 CRF02-AG Tropism

ABSTRACT We assessed the performance of genotypic algorithms for predicting the tropism of human immunodeficiency virus type 1 coreceptor usage in 52 patients infected with the CRF02-AG subtype. The combined criteria of the 11/25 and net charge rules accurately detected CXCR4-using CRF02-AG viruses, whereas the Geno2pheno tool lacked sensitivity and the position-specific scoring matrix (PSSM) tool WebPSSM lacked specificity.

Naïve T-Cell Depletion Related to Infection by X4 Human Immunodeficiency Virus Type 1 in Poor Immunological Responders to Highly Active Antiretroviral Therapy

ABSTRACT The reasons for poor CD4+ T-cell recovery in some human immunodeficiency virus (HIV)-infected subjects despite effective highly active antiretroviral therapy (HAART) remain unclear. We recently reported that CXCR4-using (X4) HIV-1 could be gradually selected in cellular reservoirs during sustained HAART. Because of the differential expression of HIV-1 coreceptors CCR5 and CXCR4 on distinct T-cell subsets, the residual replication of R5 and X4 viruses could have different impacts on T-cell homeostasis during immune reconstitution on HAART. We examined this hypothesis and the mechanisms of CD4+ T-cell restoration by comparing the virological and immunological features of 15 poor and 15 good immunological responders to HAART. We found a high frequency of X4 viruses in the poor immunological responders. But the levels of intrathymic proliferation of the two groups were similar regardless of whether they were infected by R5 or X4 virus. The frequency of recent thymic emigrants in the poor immunological responders was also similar to that found in the good immunological responders, despite their reduced numbers of naïve CD4+ T cells. Our data, rather, suggest that the naïve T-cell compartment is drained by a high rate of mature naïve cell loss in the periphery due to bystander apoptosis or activation-induced differentiation. X4 viruses could play a role in the depletion of naïve T cells in poor immunological responders to HAART by triggering persistent T-cell activation and bystander apoptosis via gp120-CXCR4 interactions.

Frequency of CXCR4-using viruses in primary HIV-1 infections using ultra-deep pyrosequencing.

We used ultra-deep pyrosequencing and the Toulouse Tropism Test phenotypic assay to determine the prevalence of CXCR4-using viruses in 21 patients with primary HIV-1 infections. We found X4-containing virus populations in 9% of patients by ultra-deep pyrosequencing using position-specific scoring matrices (PSSMX4/R5) or geno2pheno5.75 and in 14% using the combined 11/25 and net charge rule. The phenotypic assay identified 9% of CXCR4-using viruses. This confirms that R5 viruses are predominant in primary HIV-1 infections.

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission.

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%-90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed. Electron microscopy showed the accumulation of viral particles either into large intracellular viral-containing compartments or outside the cells, indicating that endosomal accumulation of BMP could block intracellular biogenesis of viral particles while inhibition of macropinocytosis would prevent viral particle uptake. This is the first report linking BMP metabolism with a natural steroid such as progesterone or with involvement of a phospholipase A1 activity. BMP cellular content could be used as a biomarker for efficient anti-viral drugs.