Rare HIV-1 transmitted/founder lineages identified by deep viral sequencing contribute to rapid shifts in dominant quasispecies during acute and early infection

Abstract

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection.

Fig 1. Viral load dynamics and pre-peak viremia HIV-1 genetic diversity in 6 participants from the RV217 cohort.

a) Plasma viral load (red) and CD4+ T-cell counts (blue) are shown for six volunteers with documented NAT-conversion. Day 0 represents the first date of NAT-positivity. Black-bordered circles depict the time points where HIV-1 sequencing was performed, and asterisks indicate samples obtained during Fiebig stage I/II. The dotted line depicts the lower limit of detection of the plasma viral load assay. b) Highlighter plots depicting the SGS-based analysis. For each SGS sequence, differences from the consensus of the major T/F virus are indicated by colored tic marks: green = A, blue = C, orange = G, red = T, and gray = deletion. c) Using TDS, the low-level presence of minor T/F viruses was detected in 5 participants; the time of first detection and their frequencies are indicated in pie charts (ranges depict measurements in different HIV-1 sub-genomic regions). Sequences of the minor T/F viruses were obtained during AHI either by SGS or by sequence-specific PCR (SSP); highlighter plots show that minor T/F viruses were highly related but distinct from cognate major T/F viruses.

Fig 2. T/F virus dynamics during AHI in participants with infections established by multiple T/F viruses.

a) The frequency of major and minor T/F lineages, and b) their contribution to the total viral load (gray area) are shown. For clarity, different variants within a T/F lineage were combined.

Fig 3

Escape from CTL responses to epitopes a) Pol SP10 (participant 20225), b) Rev VL9 (participant 20225), c) Env LV9 (participant 40100), d) Gag SM9 (participant 40061), e) putative epitope Nef DQ11 (participant 10463), f) putative epitope in Rev (codons 49–66) (participant 40265), and g) putative epitope in Env (codons 765–782) (participant 40265). In all of these epitopes, evolution of CTL escape happened through epitope shattering. The frequency of each variant and the contribution of main variants to the plasma viral load (gray area) are depicted.

Fig 4. 40100 major and minor T/F viruses present distinct phenotypes in in vitro competition assays.

a) The replication capacity of FLIMCs from 40100 major and minor T/F viruses was compared. Lines represent the proportion of infected cells that carried each T/F virus. Colors code for each experiment, which were conducted on PBMCs or the A3R5 cell line, as indicated. b) Ratio of cells infected with minor vs. major T/F virus after 6-day culture in PBMCs in control conditions (black), and in the presence of IFN alpha (blue) or IFN beta (red). c) Ratio of cells infected with minor vs. major T/F virus after 6-day culture in RA-treated PBMCs in the absence (blue, green) or presence (red, orange) of α4β7-blocking mAb Act-1. Data for two different PBMC donor pools, 50+94 and 78+150, are shown. In all the experiments, the initial inoculum was a 1:1 mixture of the two viruses (dotted lines). Whiskers represent interquartile intervals.

Fig 5. Summary of viral dynamics during AHI.

For the 6 participants considered in the current study, we compare the dynamics of pVL, of the frequency of the major T/F lineage, and of viral escape from CTL responses. For the sake of clarity, the curves were aligned based on day of peak viremia. For epitope Gag SM9 in participant 40061, the initial replacement of the major T/F by the minor T/F sequence is indicated separately from the later escape that proceeded through epitope shattering (1 and 2, respectively). For participant 40265, the CTL epitope in Rev has not been mapped.