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.