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

The HIV/AIDS pandemic has been a global crisis for four decades [1, 2]. At the end of 2020, there were 37.6 million people living with HIV globally, 1.5 million new HIV-1 infections, and 690,000 AIDS-related deaths [3]. South Africa has 7.9 million people living with HIV-1 (PLWH), making it the country with the highest number of infections in the world [4, 5]. This highlights the need to better understand HIV-1 natural immune responses in order to bolster the efforts of developing effective HIV-1 vaccines.

During early HIV-1 infection, effective cytotoxic T-lymphocyte (CTL) immune responses play an important role in the control of viraemia, by contributing to suppression of the viral load (VL) to a set-point [6]. CTL responses target and kill virus-infected cells, via recognition of viral peptide epitopes and releasing cytokines and cytotoxic granules that facilitate cell killing [7‐9]. Viral peptide epitopes are presented on the surface of infected cells by the human leukocyte antigen type I (HLA I) trans-membrane proteins encoded by HLA-A, B and C alleles [10‐12]. The targeted epitopes undergo immune selection pressure, which leads to the emergence of viral escape mutations [13, 14]. This results in evasion of the immune system, loss of immune control and ultimately progression to AIDS [7, 10]. The majority of epitopes that play a role in the control of viraemia have been extensively studied and reported for HIV-1 Gag [15, 16]. However, some studies show that HIV-1 Pol also harbours important CTL epitopes that contribute to the control of HIV-1 viraemia [17‐19], hence pol is included in some HIV-1 vaccine candidates [20‐25]. There are limited data on the characterisation of HIV-1 Pol CTL epitopes in early and chronic HIV-1 disease stages. The aim of this study was to characterise and assess the evolution of CTL epitopes encoded by HIV-1 pol during early and chronic stages of HIV-1 infection, using deep sequencing methods.

To our knowledge, this is the first study to assess the evolution of HIV-1 Pol CTL epitopes in samples obtained during early and chronic disease stages in a setting where HIV-1 subtype C is predominant. We identified epitopes within all three regions of Pol, unlike previous studies that have only characterised epitopes within RT and PR [16, 29, 33]. This highlights the advantage of using a PCR method that amplifies the complete pol gene. Baseline and follow-up sequences for each participant correctly clustered together, indicating accurate amplification and sequencing of viral genes belonging to the same participant. It was expected that the majority of sequences would cluster with HIV-1 subtype C as this is the most common subtype in the southern African region [34, 35]. A small number of non-subtype C strains was detected, which could have been introduced through migration, and this has also been reported in previous South African studies [36, 37].

Many epitopes were identified through the analysis of minority variants obtained by deep sequencing compared to majority variants obtained by Sanger sequencing. This shows the advantage of using deep sequencing for characterisation of Pol CTL epitopes as this method can detect variants that exist below 20% of the total virus population, which are not readily detected by Sanger sequencing [38, 39]. Some identified minority variant epitopes have never been reported for HIV-1 subtype C and were possibly missed by previous studies that employed Sanger sequencing. The use of Sanger sequencing in most studies has most likely underestimated the presence of CTL epitopes in Pol. Other studies have also shown increased detection of CTL epitopes when using NGS methods [40, 41]. This shows an advantage in the use of NGS for characterising HIV-1 CTL epitopes in future research studies.

In this study, CTL epitopes were identified in all regions of Pol, but mostly in RT and IN. Some of these epitopes such as GKKAIGTVL (PR 68–76) and IAMESIVIW (RT 375–383) (Table 3) have been previously reported to induce CTL responses [42, 43]. These data indicate that all three regions of Pol are immunogenic and may play a role in the control of viraemia and the establishment of a viral set-point during early HIV-1 infection, in agreement with previous studies that assessed CTL responses against the HIV-1 proteome [42‐44]. In a study that assessed CTL responses during acute HIV-1 infection, Kim et al.showed that the larger proportion of targeted epitopes were within Pol as compared to other regions of the HIV-1 proteome, and some of these epitopes stimulated strong CTL responses [42]. Ojwach et al.reported that CTL responses towards epitopes in RT and IN during acute and chronic HIV-1 infection induced mutations that decreased viral replicative fitness, indicating that RT and IN harbour crucial CTL epitopes [45]. Fewer epitopes were identified in PR especially during early HIV-1 infection and this could indicate that this protein harbours more subdominant CTL epitopes that are mostly recognised later during the course of infection [46]. Thus, PR might have a limited role in control of viraemia during early HIV-1 infection. CTL epitopes encoded by pol have also been reported to play a role in the control of viraemia in long-term non-progressors (LTNPs) and elite controllers (ECs) [47, 48].

HLA class I allotypes play an important role in the control of HIV-1 viraemia [29]. Several HLA class I alleles that encode allotypes predicted to present epitopes identified in this study are known to be present in the South African population, including HLA-B*58:01 and HLA-A*02 that present SAAVKAACW (IN 123–131) and MASDFNLPPIV (IN 22–31), respectively [28, 30, 49]. Past gene association studies showed that alleles within the HLA-B group correlate with greater viral control than alleles in the HLA-A and HLA-C groups [28, 29, 50]. Payne et al.found that HLA-B*58:01 was associated with slower HIV-1 disease progression and was protective against HIV-1 infection [51], which could explain why some participants such as 8047 and 6743 recognised more epitopes, as they also recognised an epitope reported to be presented by HLA-B*58:01. A hierarchy in epitope presentation may be an explanation for recognition of more epitopes during chronic HIV-1 infection. Immunodominant epitopes could be preferentially presented earlier during the course of infection, followed by presentation of subdominant epitopes later [52]. Immunodominant epitopes are often located in variable domains whereas subdominant epitopes usually map to more conserved regions of the HIV-1 proteome [18, 53]. The HIV-1 pol gene is more conserved than gag and env [54] and therefore harbours more subdominant epitopes [18]. Examples of subdominant responses being more effective in controlling viraemia have been shown in past studies [55, 56].

The evolution of Pol CTL epitopes was monitored by assessing minority variants in sample pairs. Minority variant epitopes that increased in proportion from baseline to follow-up may have replaced the wild-type epitopes over time and facilitated immune escape [57]. Fitness cost is the likely explanation for minority variant epitopes that decreased in proportion over time, and these may have been out-competed by new variants [57]. The proportion of some minority variant epitopes remained constant between the two time-points, and these could also represent variants that do not replicate efficiently and are hence maintained at lower levels within the HIV-1 quasispecies [40, 57]. Some variant sites were located outside of reported epitopes (Additional file 3: Table S3), and these may represent new unreported epitopes or could highlight important adjacent sites that may affect epitope processing and presentation [13, 58]. Viral evolution was also assessed by comparing sequences from early and chronic HIV-1 stages. Data from this study showed that evolution within CTL epitopes began quite early following infection, as epitopes with escape mutations were detected in participants who had early HIV-1 infection [59]. Many epitopes with escape mutations were detected in participants in the chronic HIV-1 stage, indicating that viral evolution of HIV-1 results from immune selection pressure that occurs throughout the course of infection and diverse viral populations evolve that constantly evade immune responses [57, 60].

All three expressed proteins (PR, RT and IN) of Pol harbour CTL epitopes and this highlights the importance of including pol in the design of a vaccine candidate [2, 20]. Some researchers have included RT and IN in the design of HIV-1 vaccines [22, 23, 53]; this is supported by the findings of our study as the majority of epitopes mapped to these two proteins. Conserved epitopes have been shown to provide cross-clade protection against HIV-1 infection [2, 18]. Some of the conserved Pol epitopes such as IETVEPVKL (RT 5–12) and SVPLDEGFRK (RT 117–126) (Table 3), that were previously observed in a study that looked at using a vaccine candidate with conserved immunogens were also identified in our study [18]. The immune responses directed towards epitopes in conserved regions are usually subdominant but may provide protection against multiple HIV-1 subtypes [17, 53]. Many studies that included Pol epitopes in vaccine candidates showed stimulation of effective CTL responses [18, 56, 61, 62]. Ahmed et al., showed control of HIV-1 replication in vitro through stimulation of Pol CTL responses by a vaccine that contained subdominant epitopes [56].

Limitations of this study include the small sample size and fewer participants with early HIV-1 infection. The duration of infection in participants with chronic HIV-1 infection was not known. HIV-1 VL was not measured in the participants at follow-up, therefore no correlation between VL and the number of minority variant epitopes detected could be made. Functional CTL responses against identified epitopes were not determined and genomic DNA samples were unavailable to conduct HLA class I genotyping. Longitudinal samples were only obtained at two time-points for most of the participants at a median interval of 4 weeks and this might not be sufficient for assessing viral evolution.

Deep sequencing revealed many HIV-1 Pol CTL epitopes, including some which have never been reported for HIV-1 subtype C. Immune selection pressure on Pol CTL epitopes was observed during early HIV-1 infection highlighting the possibility that these epitopes might contribute to the control of viraemia and the establishment of a VL set-point. Variable patterns of distribution of epitopes in sample pairs reflect the ongoing generation of escape mutants capable of evading the immune responses throughout the course of infection. The findings of this study support the inclusion of RT and IN in potential HIV-1 vaccine candidates, as these regions harbour the majority of CTL epitopes. Variants that could not be mapped to known epitopes in the Los Alamos HIV database might represent new unreported epitopes. Future research, using deep sequencing, is needed for better characterisation of Pol epitopes as this protein contains highly conserved epitopes that have the potential to provide HIV-1 cross-clade protection.