Study design/RVA design.

<p>Viral RNA was extracted from patient plasma RT-PCR amplified. Env amplicons spanning the Env ectodomain were further amplified through an inner PCR. Five independent PCRs were pooled to minimize PCR-selection. Recombinant viruses were produced by co-transfecting HEK293T cells with Afe I-linearized, luciferase-tagged, Env-deleted, viral backbone and patient-derived PCR amplicon. Normalized amounts of recombinant viruses were used to infect U87.CD4.CCR5 or U87.CD4.CXCR4 indicator cells. Infection was monitored by quantifying luminescence in the cell lysates. Depending on the outcome of the infection, viruses were classified as either CCR5 tropic, CXCR4 tropic or dual/mixed. The same patient-derived PCR amplicon used for viral production was sequenced and tropism inferred by Geno2Pheno_[coreceptor] and webPSSM algorithms. The phenotypic and genotypic results were compared. Abbreviations: Env EC: Env ectodomain; gp41-TM-CT: gp41 Transmembrane+cytoplasmic tail.</p

Characteristics of phenotypic assays developed for determination of HIV-1 coreceptor usage.

<p>Abbreviations: ESTA: Enhanced Sensitivity Trofile Assay; Env: Envelope; eGFP: enhanced Green Fluorescent protein; X4: CXCX4-using strains; R5:CCR5-using strains.</p

Detection of minority CXCR4 and CCR5 using variants within mixed viral populations.

<p>Mixtures containing known proportions of pNLAD8 and pNL4-3 (100∶0, i.e. pure NLAD8, 99∶1, 97.5∶2.5, 95∶5, 90∶10, 80∶20, 50∶50, 20∶80, 10∶90, 5∶95, 2.5∶97.5, 1∶99, 0∶100, i.e. pure NL4-3) were PCR-amplified and used to generate recombinant viruses. U87.CD4.CCR5 and U87.CD4.CXCR4 indicator cells were infected with serial 2-fold dilutions (x-axis) of mixtures (z-axis) to determine the threshold for detection of minority variants. Infection was quantified 48 hours after infection by measuring luciferase activity in cell lysates (y-axis). Black bars report infection of U87.CD4.CXCR4 cells and grey bars report infection of U87.CD4.CCR5 cells. Panels A and B report the same data, oriented to focus on NL4-3 minority variants (A) or on NLAD8 minority variants (B).</p

Distribution of PCR amplification success stratified by viral load.

<p>The Env ectodomain was amplified from plasma viral RNA by a one-step RT-PCR followed by an inner PCR. Five independent PCR amplifications were pooled to minimize primer-related selection. 292 samples from patients infected with HIV subtypes A1, B, C, D, F, G, CRF01_AE and CRF02_AG were included. Viral load ranged from 466 to 1,350,000 RNA copies/mL.</p

Concordance between tropism measured phenotypically and inferred genotypically using the Geno2pheno_(coreceptor) and webPSSM algorithms.

<p>(<b>A</b>) Concordance for subtype B (black bars) and non-B subtype (grey bars) strains with Geno2Pheno (G2P) at different FPR cutoffs and webPSSM. (<b>B</b>) Concordance with Geno2pheno_(coreceptor) with a FPR set at 10% (black bars) and webPSSM (grey bars) for different HIV-1 subtypes. The webPSSM X4/R5 matrix was used for all subtypes, except for subtype C, for which the subtype C SI/NSI matrix was used.</p

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy, Viral Evolution and Compartmentalization

<div><p>Background</p><p>Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.</p><p>Methods & Results</p><p>We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No <i>de novo</i> CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.</p><p>Conclusions</p><p>The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.</p></div

Kaplan-Meier curves of overall survival according to HER2 (A), p95HER2 (B), HER3 (C), and HER2/HER3 (D) level.

<p><i>P</i>-values by log-rank test.</p

Selection of a CXCR4-using variant above the 454 sequencing error threshold during persistent viremia suppression in Subject 26.

<p><b><i>Panel A, antiretroviral treatment history, virological and immunological evolution</i></b><b>.</b> Continuous line, HIV-1 RNA levels; dashed line, CD4+ counts; horizontal bars, time period during which a given antiretroviral drug was prescribed. Vertical lines indicate the timepoints when 454 sequencing was performed. LPVr, lopinavir/ritonavir; AZT, zidovudine; ddI, didanosine; RAL, raltegravir. <b><i>Panel B, maximum likelihood nucleotide-based phylogenetic tree</i></b> including V3-loop haplotypes present at a frequency ≥0.6% in the virus population in plasma (triangles), PBMCs before therapy initiation (circles) and PBMCs after persistent viremia suppression (squares). The tree is rooted at the most frequent plasma sequence before antiretroviral treatment initiation. Filled symbols show predicted CXCR4-using viruses; open symbols show predicted CCR5-using viruses. Symbol size increases proportionally to the V3-loop haplotype frequency in the virus population in 10% intervals. Node reliability was tested using 1000 bootstraps; bootstrap values ≥50% are shown. The V3-loop aminoacid sequence translation is shown next to each taxon. Aminoacid changes relative to the predominant sequence in plasma are highlighted in bold and underlined. Gaps correspond to aminoacid indeterminations. A Geno2Pheno _[coreceptor] false positive rate (FPR) equal or lower than 10% was used to define CXCR4 use. The actual false positive rate of each sequence is shown. *Sequence #2 was identical to one detected in 0.04% of PBMC-associated viruses, below the error threshold, before treatment initiation.</p

Accuracy of Tropism Assays Relative to the Enhanced-Sensitivity Trofile™ Assay.^{^a}

a<p>Values are mean percentages (95% confidence interval of the mean), calculated assuming a binomial distribution of the data.</p>b<p><i>G2P FPR</i>, Geno2Pheno_[coreceptor] false positive rate used for population and 454 sequencing to assign CXCR4 use . The Geno2Pheno_[coreceptor] clonal model was always used.</p>c<p><i>MT-2</i>, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells.</p>d<p><i>PPV</i>, Positive Predictive Value.</p>e<p><i>NPV</i>, Negative Predictive Value.</p>f<p>“Accuracy” is defined as: (True positives + True negatives)/Total.</p

Best overall response and receptor level.

<p><i>P</i>-values are by Kruskal-Wallis test. Bars indicate median values.</p