A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

Abstract

dKey clinical studies for HIV coreceptor antagonists have used the phenotyping-based Trofile test. Meanwhile various simplerto-do genotypic tests have become available that are compatible with standard laboratory equipment and Web-based interpretation tools. However, these systems typically analyze only the most prominent virus sequence in a specimen. We present a new diagnostic HIV tropism test not needing DNA sequencing. The system, XTrack, uses physical properties of DNA duplexes after hybridization of single-stranded HIV-1 env V3 loop probes to the clinical specimen. Resulting "heteroduplexes" possess unique properties driven by sequence relatedness to the reference and resulting in a discrete electrophoretic mobility. A detailed optimization process identified diagnostic probe candidates relating best to a large number of HIV-1 sequences with known tropism. From over 500 V3 sequences representing all main HIV-1 subtypes (Los Alamos database), we obtained a small set of probes to determine the tropism in clinical samples. We found a high concordance with the commercial TrofileES test (84.9%) and the Web-based tool Geno2Pheno (83.0%). Moreover, the new system reveals mixed virus populations, and it was successful on specimens with low virus loads or on provirus from leukocytes. A replicative phenotyping system was used for validation. Our data show that the XTrack test is favorably suitable for routine diagnostics. It detects and dissects mixed virus populations and viral minorities; samples with viral loads (VL) of <200 copies/ml are successfully analyzed. We further expect that the principles of the platform can be adapted also to other sequence-divergent pathogens, such as hepatitis B and C viruses. The predominant virus variant in early stages of the clinical manifestation of disease, CCR5-tropic HIV, is found in approximately 80% of treatment-naive patients (1, 2). Although this number can vary for the different virus subtypes, the percentage of CXCR4-tropic HIV isolates is generally low and tends to rise with disease progression (3-6). Nevertheless, the fraction of CCR5-tropic viruses in clinical specimens continues to stay at Ͼ50% throughout the course of infection (7,8). As such, the molecular interactions between the viral envelope and the cellular chemokine receptor CCR5 were recognized as potentially attractive targets for drug development and have yielded compounds and drugs able to specifically block CCR5-tropic HIV (9-11). It is this selectivity of the inhibition of one (CCR5) and not the other (CXCR4) viral coreceptor that necessitates tropism testing prior to prescribing drugs of this particular class. Although the chemokine receptor binding site in the HIV envelope is constituted mainly by the V3 loop, the V1/V2 regions, and the C4 conserved region in the HIV protein gp120, coreceptor tropism is dictated predominantly by amino acid sequences of the V3 region (12, 13). But also sequences of other variable env regions can contribute as secondary sites to the viral tropism (14-17).Initially, all...

Phenotypic co-receptor tropism and Maraviroc sensitivity in HIV-1 subtype C from East Africa

“…Plasmids pNL4-3 and pMJ4 were also amplified with 6435F and 8329R primers. The resultant fragments were gel purified using QIAquickGel Extraction Kit (Qiagen, USA) and cloned into a pMN-K7-Luc-IRESs-NefΔgp120 plasmid 25 following digestion with NgoMIV and MluI-HF (New England Biolabs, US) and ligation with T4 DNA ligase (New England Biolabs, US). The recombinant viruses were produced transfecting the plasmids using FuGENE® HD Transfection Reagent (Promega, US) in 293 T cell line.…”

“…Phenotypic tropism testing was performed using two methods: (1) Tropism testing in GHOST cell-lines 27 of the viruses generated from individual clones (cPTT) and (2) replicative phenotypic tropism test rPhenotyping (pPTT) whereas described previously 25 . In cPTT co-receptor tropism was determined by measuring Renilla luciferase activity (relative light units [RLU]) using Bright - Glo ™ Luciferase Assay System (Promega, US).…”

Correlating HIV tropism with immunological response under combination antiretroviral therapy

“…Although there are currently no clinically available entry inhibitors that target X4, a better understanding of the mechanisms that drive viral infection via X4 is of central relevance. Reliable genotypic tropism tests have been established for identifying the viral tropism and to dissect mixed virus populations .…”

Performance Evaluation of a Genotypic Tropism Test Using HIV-1 CRF01_AE Isolates in Japan

“…Subsequently, 2 algorithms, namely, Geno-2Pheno (coreceptor) and Geno2pheno-C_NGS-Sanger (Geno2Pheno [Sanger]) (cutoff: X4-risk < 36%), were used to evaluate chemokine receptor preference. In parallel to env sequencing for tropism evaluation, we performed phenotypic testing by using the replicative format of PhenXR (InPheno AG, Basel, Switzerland), according to a previously described protocol (14). In brief, the env-amplicons were cloned into a plasmid cassette where they were reconstituted into fully functional HIV genomes.…”