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. 2022 Mar 28;19(1):56.
doi: 10.1186/s12985-022-01772-8.

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

Paballo Nkone  1 , Shayne Loubser  2 , Thomas C Quinn  3   4 , Andrew D Redd  3   4 , Arshad Ismail  2 , Caroline T Tiemessen  2 , Simnikiwe H Mayaphi  5   6
Affiliations

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

Paballo Nkone et al. Virol J. .

Erratum in

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 [Formula: see text] 5% were detected using low-frequency 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.

Keywords: Chronic HIV-1 infection; Early HIV-1 infection; Illumina deep sequencing; Minority variants; Pol CTL epitopes.

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Conflict of interest statement

The authors declared no competing interest.

Figures

Fig. 1
Fig. 1
Neighbour-joining phylogenetic tree of all samples including sample pairs. Group O strain of HIV-1 was used for rooting. Only bootstrap values above 70% are shown. Baseline and follow-up (fu) sequences of the same participant correctly clustered
Fig. 2
Fig. 2
Frequently recognized Pol CTL epitopes in early and chronic HIV infection. These are epitopes that were recognized by more than one participant. A Epitopes within the protease (PR) region. B Epitopes within the reverse transcriptase (RT) region. C Epitopes within the integrase (IN) region. Many epitopes were located within RT and IN as compared to PR, and most were found in chronic HIV infection. CTL = cytotoxic T-lymphocytes
Fig. 3
Fig. 3
Dynamics of minority variants in sample pairs during A early HIV infection and B chronic HIV infection. (i) Some variants either increased in proportion between baseline and follow-up or remained at / above the detection limit of 5% (black solid line). (ii) Other variants decreased in proportion over time and were detected below 5% at follow-up. (iii) There were variants that increased in proportion over time to become majority variants and were thus maintained above 20% (black dotted line). (iv) Some minority variants that were detected at follow-up existed as majority variants at baseline. The median interval between baseline and follow-up sampling was 4 weeks (IQR: 3–8 weeks)
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