HIV-1 tropism testing in subjects achieving undetectable HIV-1 RNA: diagnostic accuracy, viral evolution and compartmentalization

Abstract

Background: 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.

Methods & results: 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 de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.

Conclusions: 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.

Figure 1. Prevalence of CXCR4-using viruses using different tropism assays and settings.

Bar plot showing the mean and 95% confidence intervals of the prevalence of subjects with CXCR4-using viruses using different tropism assays and settings. The Geno2Pheno_[coreceptor] clinical model was only used in pre-treatment bulk sequences derived from plasma RNA; otherwise, the clonal model was used. ESTA, Enhanced-Sensitivity Trofile™ Assay; FPR, Geno2Pheno_[coreceptor] false positive rate used to assign tropism; MT-2, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. * p-value<0.05, two-sided exact binomial tst.

Figure 2. Selection of a CXCR4-using variant above the 454 sequencing error threshold during persistent viremia suppression in Subject 26.

Panel A, antiretroviral treatment history, virological and immunological evolution . 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. Panel B, maximum likelihood nucleotide-based phylogenetic tree 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.

Figure 3. V3-loop haplotypes detected by quantitative deep sequencing are also found in CXCR4-using viruses growing in MT2 assays.

Maximum likelihood phylogenetic trees showing that the V3-loop sequence of syncytium-inducing viruses grown in MT-2 assays (diamond) is identical to one of the predominant V3-loop haplotypes detected with quantitative deep sequencing in proviral DNA and/or plasma RNA before antiretroviral therapy initiation (Trees A to E) or after at least 2 years of persistent viremia suppression (trees F to H). Trees include 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); trees are rooted at the predominant plasma (trees A to E) or PBMC (trees G to H) V3-loop haplotype. Filled symbols represent CXCR4-using viruses; open symbols show R5 viruses. Symbol size increases proportionally to the V3-loop haplotype frequency in the virus population in 10% intervals. Node reliability was tested using 1,000 bootstraps; bootstrap values ≥50% are shown. CXCR4 use was defined by a Geno2Pheno _[coreceptor] false positive rate ≤10%.