Analysis of co‐receptor usage of circulating viral and proviral HIV genome quasispecies by ultra‐deep pyrosequencing in patients who are candidates for CCR5 antagonist treatment

Abstract

UDPS combined with genotypic algorithms for prediction of HIV-1 co-receptor usage may provide quantitative data about the tropism of each variant present in the viral quasispecies. The aim of the present study was to assess co-receptor usage by ultra-deep pyrosequencing (UDPS), in comparison with the reference phenotypic test (Trofile), in patients who are candidates for CCR5 antagonist treatment, in both circulating and proviral HIV-1. Seventeen patients who were tested by Trofile were enrolled. UDPS of the V3 loop region was carried out on both plasma RNA and proviral DNA. Genotypic prediction of co-receptor usage was established by position-specific score matrices (PSSM) and confirmed, in discordant cases, with geno2pheno. Genetic heterogeneity of the RNA and DNA quasispecies was assessed as well. A total of 196,729 V3 sequences were considered (mean coverage per site, 6346). Concordance between phenotypic test and UDPS with PSSM was 0.82. Geno2pheno results were in line with those obtained with PSSM. Proviral quasispecies were more heterogeneous than those found in circulating HIV. In most patients eligible for CCR5 antagonist treatment, X4 variants were detected in proviral DNA, ranging from 1.0% to 52.7%. UDPS combined with genotypic algorithms for co-receptor usage prediction highlighted the presence of minority variants, with a discordant tropism with respect to the predominant population, in both circulating viral and proviral HIV. In most patients treated with Maraviroc the virological response was independent of the presence of X4 in proviral DNA. The clinical impact of minority X4 variants present in patients who are candidates for anti-CCR5 antagonists remains a crucial point to be addressed.

Detection of quasispecies variants predicted to use CXCR4 by ultra-deep pyrosequencing during early HIV infection

“…However, standard genotypic test performed on bulk sequences may provide information only on the predominant circulating variants, whereas HIV-1 is present in each infected individual as a swarm of highly related variants referred to as quasispecies. We and others have recently reported that the classification obtained with the combined use of ultra-deep pyrosequencing (UDPS) with genotypic algorithms for prediction of HIV-1 coreceptor usage, such as position specific score matrix (PSSM) analysis or geno2pheno, is in good agreement with the standard phenotypic classification, and, in addition, may provide a detailed description of the viral HIV quasispecies, being able to quantify each variant with different coreceptor usage [19][20][21][22].…”

“…To maximize the genetic heterogeneity to be amplified and sequenced, for each sample, amplicons from at least four replicate PCR reactions were pooled, to analyze , for viral RNA, the viral heterogeneity present in 1 ml of plasma, and, for proviral DNA, that of associated with 2-6 Â 10 6 PBMC. To minimize the noise due to the high-throughput pyrosequencing platform, a correction pipeline, based on the translation of nucleotide sequences and conservation of only the coding ones, was adopted as previously described [20,21]. The application of this pipeline reduced the mean pyrosequencing error rate to 0.058%.…”

“…Taking into account the estimated error rate for the high-fidelity polymerase used to obtain the amplicons ($1 Â 10 À6 mutations/bp per duplication), frequencies of variants exceeding by at least five times the corrected error rate were considered to reflect true variability and not procedural/experimental errors. Considering the number of viral templates actually undergoing UDPS and the corrected error rate, the threshold of sensitivity was set to 0.3% [21,22], which also corresponds to the sensitivity of X4 variants detection of the latest version 'enhanced' Trofile test.…”

“…Sequencing UDPS was carried out with the 454 Life Sciences platform (GS-FLX; Roche Applied Science, Monza, Italy) as described in [20,21] on plasma virus from all the 20 enrolled patients at enrollment and, in eight patients from group B, after 6 months (T 6 ). In a subgroup of five patients (two from group A and three from group B), UDPS was also performed on proviral DNA present in peripheral blood mononuclear cell (PBMC) at enrollment.…”

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

“…Maraviroc (MVC) became the first, and so far only, FDA-approved smallmolecule HIV inhibitor/CCR5 antagonist for use in HIV-infected patients. Other CCR5 antagonists, agonists, and binding antibodies reached various stages of preclinical and clinical development but were eventually abandoned due to off-target complications (10), poor pharmacodynamics and pharmacokinetics (11), and difficulties in screening appropriate patients for treatment due to FDA requirements to counterscreen for CXCR4-using HIV-1 (12,13).…”