Deep Sequencing Detects V3 loop Forms Present in Functional X4 Viruses Growing in MT 2 assays Christian Pou 1, Rocío Bellido 1, Francisco M. Codoñer 1, Alexander Thielen 3, Cecilia Cabrera 1, Judith Dalmau 1, Martin Däumer 4, Bonaventura Clotet 1,2, Roger Paredes 1,2 and the Barcelona Tropism Study Group. 1 Institut de Recerca de la SIDA irsicaixa HIVACAT & 2 HIV Unit Fundació Lluita contra la SIDA, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Catalonia, Spain; 3 Max Planck Institut für Informatik, Saarbücken, Germany; 4 Institut für Immunologie und Genetik, Kaiserslautern, Germany.
BACKGROUND HIV Tropism testing is mandatory before treatment with CCR5 antagonists. Deep V3 loop sequencing (454 Life Sciences/Roche) Achieves similar diagnostic accuracy to ESTA in plasma Can be performed in proviral DNA in subjects with undetectable VL Proviral DNA could act as a repository of non functional viruses stored along infection. Does 454 detect functional viruses? We investigated if V3 loop forms of X4 viruses growing in MT2 assays were also detected by deep sequencing in plasma and/or proviral DNA.
METHODS Study design and participants Retrospective proof of concept study Inclusion criteria: Chronically HIV 1 infected adults with HIV 1 RNA <50 copies/ml during at least 2 years after starting first line ART without CCR5 antagonists. Subjects had to have cryopreserved samples available for testing: Within 6 months before ART initiation (T1). After at least 2 years of undetectable viremia (T2). ART without CCR5 antagonists Viral Load <50 copies/ml 96 weeks QDS: Plasma ARN Proviral DNA MT2 assay QDS: Proviral DNA MT2 assay
METHODS Assays and Internal Controls MT2 assay Patient derived PBMCs + MT2 cell line Syncytium formation and p24 production (ELISA) Virus extraction from supernatant and sequencing by Sanger Parallel control to asses virus growth ability Patient derived PBMCs + PBMC from healthy seronegative donors Quantitative Deep Sequencing HXB2 genome 1st round PCR 2nd round PCR X4: Geno2Pheno FPR=10% Parallel control to assess error induced during polymerization events Sequencing of commercial pnl4.3 DNA clone
RESULTS Per subject and timepoint determination of viral load, CD4+ cells count and tropism prediction Phylogenetic analysis Filtering of sequences achieved by QDS (Error established at 0.6) Population sequences from MT2 V3 Alignment with HAMMER Test of the best nucleotide evolutionary model using FindModel Construction of Maximum likelihood trees using PhyML Edition of trees using MEGA
RESULTS Comparison between V3 loop sequences from MT2 stocks and QDS before ART administration. Subject 1 Subject 3 Subject 4 Subject 5 64 65 60 66 81 69 51 63 80 85 61 57 Subject 2 55 87 71 99 98 99 51 67 55 69 68 Empty R5 / Filled X4 QDS Plasma ARN QDS Proviral DNA MT2 59 76 54
RESULTS Comparison between V3 loop sequences from MT2 stocks and QDS during viremia suppression Subject 1 Subject 3 Subject 4 100 80 55 54 72 92 80 100 96 96 63 88 96 99 100 77 Empty R5 / Filled X4 QDS Proviral DNA T2 MT2
CONCLUSIONS At least one of the predominant V3 forms with predicted CXCR4 use detected by deep sequencing in plasma or PBMCs is shared by functional X4 viruses growing in MT 2 assays. This suggests that deep sequencing detects functional X4 viruses with the potential to escape from CCR5 antagonist selective pressure, adding clinical value to the technology.
CAVEATS This does not mean that all X4 viruses detected by 454 are functional. V3 is the main determinant of co receptor specificity, but tropism also depends on the env background where this V3 form exists.
ACKNOWLEDGMENTS Institut de Recerca de la SIDA IrsiCaixa (Badalona) Rocío Bellido Francisco Codoñer Cecília Cabrera Judith Dalmau Mattia Schiaulini Marc Noguera Maria Casadellà Cristina Rodríguez Roger Paredes Bonaventura Clotet Max-Planck-Institut für Informatik (Saarbücken) Alexander Thielen Institut für Immunologie und Genetik (Kaiserslautern) Martin Däumer Pfizer, CHAIN