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

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Authors

Nkone, Paballo
Loubser, Shayne
Quinn, Thomas C.
Redd, Andrew D.
Ismail, Arshad
Tiemessen, Caroline T.
Mayaphi, Simnikiwe Horatious

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BMC

Abstract

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.

Description

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 infection
Additional 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.

Keywords

HIV-1, Vaccine, Infection, Minority variant epitopes, Cytotoxic T-lymphocyte (CTL)

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Citation

Nkone, P., Loubser, S., Quinn, T.C. et al. 2022, 'Deep sequencing of the HIV‑1 polymerase gene for characterisation of cytotoxic T‑lymphocyte epitopes during early and chronic disease stages', Virology Journal, vol. 19, no. 1, pp. 1-16, doi : 10.1186/s12985-022-01772-8.