Deep sequencing of the HIV‑1 polymerase gene for characterisation of cytotoxic T‑lymphocyte epitopes during early and chronic disease stages

BACKGROUND : Despite multiple attempts, there is still no effective HIV-1 vaccine available. The HIV-1 polymerase (pol) gene is highly conserved and encodes cytotoxic T-lymphocyte (CTL) epitopes. The aim of the study was to characterise HIV-1 Pol CTL epitopes in mostly sample pairs obtained during early and chronic stages of infection. METHODS : Illumina deep sequencing was performed for all samples while Sanger sequencing was only performed on baseline samples. Codons under immune selection pressure were assessed by computing nonsynonymous to synonymous mutation ratios using MEGA. Minority CTL epitope variants occurring at 5% were detected using lowfrequency variant tool in CLC Genomics. Los Alamos HIV database was used for mapping mutations to known HIV-1 CTL epitopes. RESULTS : Fifty-two participants were enrolled in the study. Their median age was 28 years (interquartile range: 24–32 years) and majority of participants (92.3%) were female. Illumina minority variant analysis identified a significantly higher number of CTL epitopes (n = 65) compared to epitopes (n = 8) identified through Sanger sequencing. Most of the identified epitopes mapped to reverse transcriptase (RT) and integrase (IN) regardless of sequencing method. There was a significantly higher proportion of minority variant epitopes in RT (n = 39, 60.0%) compared to IN (n = 17, 26.2%) and PR (n = 9, 13.8%), p = 0.002 and < 0.0001, respectively. However, no significant difference was observed between the proportion of minority variant epitopes in IN versus PR, p = 0.06. Some epitopes were detected in either early or chronic HIV-1 infection whereas others were detected in both stages. Different distribution patterns of minority variant epitopes were observed in sample pairs; with some increasing or decreasing over time, while others remained constant. Some of the identified epitopes have not been previously reported for HIV-1 subtype C. There were also variants that could not be mapped to reported CTL epitopes in the Los Alamos HIV database. CONCLUSION : Deep sequencing revealed many Pol CTL epitopes, including some not previously reported for HIV-1 subtype C. The findings of this study support the inclusion of RT and IN epitopes in HIV-1 vaccine candidates as these proteins harbour many CTL epitopes.Additional file 1. Supplementary Table 1: The in-house complete HIV pol nested PCR conditions and Sanger sequencing primers.Additional file 2. Supplementary Table 2: Pol CTL epitopes identified through Sanger sequencing and comparison by stage of infectionAdditional file 3. Supplementary Table 3: Minority variant proportions within Pol CTL epitopes by stage of infection.Additional file 4. Supplementary Figure 1: Neighbour-joining phylogenetic analysis of Illumina consensus and Sanger consensus sequences for baseline samples. Sanger and Illumina consensus of the same sample significantly clustered together. Majority of the sequences clustered with HIV-1 subtype C references. HIV group O was used for rooting the tree and a bootstrap value of 1000 was used for analysis. S = Sanger sequencing; D = Deep sequencing.The National Research Foundation of South Africa; Poliomyelitis Research Foundation (PRF); Discovery Foundation; National Health Laboratory Service Research Trust (NHLS-RT); South African Medical Research Council Self-Initiated Research (MRC-SIR); University of Pretoria Faculty of Health Sciences Research Committee; the South African Research Chairs Initiative of the Department of Science and Innovation and ADR—The Division of Intramural Research, NIAID, NIH.http://www.virologyj.comam2023Medical Virolog

Evaluation of the HIV-1 polymerase gene sequence diversity for prediction of recent HIV-1 infections using Shannon entropy analysis

SUPPLEMENTARY MATERIALS : TABLE S1: List of articles from which HIV-1 subtype C reference sequences were obtained; Supplementary TABLE S2: Nucleotide differences within amino acid mutations found to have a different distribution between recent and chronic infection sequences; FIGURE S1: Amino acid sequence alignment to show the difference in sequences between recent and chronic HIV-1 infection; FIGURE S2: Comparison of differences in amino acids E628 and R629 in study sequences and non-subtype C reference sequences, for recent and chronic sequences.DATA AVAILABILITY STATEMENT : All data generated or analysed during this study are included in this manuscript and its supplementary information files.HIV-1 incidence is an important parameter for assessing the impact of HIV-1 interventions. The aim of this study was to evaluate HIV-1 polymerase (pol) gene sequence diversity for the prediction of recent HIV-1 infections. Complete pol Sanger sequences obtained from 45 participants confirmed to have recent or chronic HIV-1 infection were used. Shannon entropy was calculated for amino acid (aa) sequences for the entire pol and for sliding windows consisting of 50 aa each. Entropy scores for the complete HIV-1 pol were significantly higher in chronic compared to recent HIV-1 infections (p < 0.0001) and the same pattern was observed for some sliding windows (p-values ranging from 0.011 to <0.001), leading to the identification of some aa mutations that could discriminate between recent and chronic infection. Different aa mutation groups were assessed for predicting recent infection and their performance ranged from 64.3% to 100% but had a high false recency rate (FRR), which was decreased to 19.4% when another amino acid mutation (M456) was included in the analysis. The pol-based molecular method identified in this study would not be ideal for use on its own due to high FRR; however, this method could be considered for complementing existing serological assays to further reduce FRR.The National Research Foundation (NRF), Poliomyelitis Research Foundation, Discovery Foundation, National Health Laboratory Service Research Trust (NHLS-RT), South African Medical Research Council Self-Initiated Research (MRC-SIR), University of Pretoria Faculty of Health Sciences Research Committee, the South African Research Chairs Initiative of the Department of Science and Innovation and National Research Foundation of South Africa and the Division of Intramural Research.https://www.mdpi.com/journal/virusesam2023Medical Virolog