Next-Generation Sequencing of HIV-1 Single Genome Amplicons

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005-2010; plasma viral load: 5,884-194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage \u3e50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies

AZD1222/ChAdOx1 nCoV-19 vaccination induces a polyfunctional spike protein-specific Th1 response with a diverse TCR repertoire

AZD1222 (ChAdOx1 nCoV-19), a replication-deficient simian adenovirus–vectored vaccine, has demonstrated safety, efficacy, and immunogenicity against coronavirus disease 2019 in clinical trials and real-world studies. We characterized CD4+ and CD8+ T cell responses induced by AZD1222 vaccination in peripheral blood mononuclear cells from 296 unique vaccine recipients aged 18 to 85 years who enrolled in the phase 2/3 COV002 trial. Total spike protein–specific CD4+ T cell helper type 1 (TH1) and CD8+ T cell responses were increased in AZD1222-vaccinated adults of all ages after two doses of AZD1222. CD4+ TH2 responses after AZD1222 vaccination were not detected. Furthermore, AZD1222-specific TH1 and CD8+ T cells both displayed a high degree of polyfunctionality in all adult age groups. T cell receptor β (TCRβ) sequences from vaccinated participants mapped against TCR sequences known to react to SARS-CoV-2 revealed substantial breadth and depth across the SARS-CoV-2 spike protein for both AZD1222-induced CD4+ and CD8+ T cell responses. Overall, AZD1222 vaccination induced a polyfunctional TH1-dominated T cell response, with broad CD4+ and CD8+ T cell coverage across the SARS-CoV-2 spike protein

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

[This corrects the article DOI: 10.1371/journal.ppat.1006510.]

Summary of viral dynamics during AHI.

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

Clinical characteristics of 6 participants from the acute HIV-1 infection cohort RV217.

<p>Clinical characteristics of 6 participants from the acute HIV-1 infection cohort RV217.</p

Estimated escape rates from early CTL responses during acute HIV infection.

<p>Estimated escape rates from early CTL responses during acute HIV infection.</p