Non-synonymous Substitutions in HIV-1 GAG Are Frequent in Epitopes Outside the Functionally Conserved Regions and Associated With Subtype Differences

Abstract

In 2019, 38 million people lived with HIV-1 infection resulting in 690,000 deaths. Over 50% of this infection and its associated deaths occurred in Sub-Saharan Africa. The West African region is a known hotspot of the HIV-1 epidemic. There is a need to develop an HIV-1 vaccine if the HIV epidemic would be effectively controlled. Few protective cytotoxic T Lymphocytes (CTL) epitopes within the HIV-1 GAG (HIV_gagconsv) have been previously identified to be functionally conserved among the HIV-1 M group. These epitopes are currently the focus of universal HIV-1 T cell-based vaccine studies. However, these epitopes' phenotypic and genetic properties have not been observed in natural settings for HIV-1 strains circulating in the West African region. This information is critical as the usefulness of universal HIV-1 vaccines in the West African region depends on these epitopes' occurrence in strains circulating in the area. This study describes non-synonymous substitutions within and without HIV_gagconsv genes isolated from 10 infected Nigerians at the early stages of HIV-1 infection. Furthermore, we analyzed these substitutions longitudinally in five infected individuals from the early stages of infection till after seroconversion. We identified three non-synonymous substitutions within HIV_gagconsv genes isolated from early HIV infected individuals. Fourteen and nineteen mutations outside the HIV_gagconsv were observed before and after seroconversion, respectively, while we found four mutations within the HIV_gagconsv. These substitutions include previously mapped CTL epitope immune escape mutants. CTL immune pressure likely leaves different footprints on HIV-1 GAG epitopes within and outside the HIV_gagconsv. This information is crucial for universal HIV-1 vaccine designs for use in the West African region.

Keywords: HIV cure; HIV-1 GAG; HIV-1 subtypes; MHR; non-synonymous substitutions.

FIGURE 1

Phylogenetic tree of the P17/P24 regions of the GAG gene of HIV-1. Reference subtypes are indicated with Ref. before their accession numbers. Other sequences are shown with their accession numbers and country of isolation. Subtypes obtained from samples in this study are shown with their accession numbers only. Subtypes A, G, and CRF02_AG were identified with green diamond, blue circle, and pink square symbols respectively. Multiple sequence alignment and phylogenetic tree were constructed using MAFFTS and Maximum Parsimony algorithm in MEGA 6 software. Statistical significance of the tree topology was tested by 1000 bootstrap replication.

FIGURE 2

Distribution of mutations associated with escape in EHIV012 and EHIV013 before seroconversion. Three individuals had mutations previously associated with escape, diminished responses, non-susceptible forms, etc. These mutations were compiled from the Los Alamos National Laboratory HIV Immunology Database for CTL/CD8 + Epitope Variants and Escape Mutations⁸. The list of all the identified mutations is presented in Supplementary Table 1. Two individuals (EHIV012 and EHIV013) had mutations outside the GAG HIV_GAGCONSV, while EHIV022 had a mutation corresponding to escape (Murakoshi) – E203D. Figure 3 shows the distribution of mutations associated with escape in EHIV 012(Red Bars) and EHIV013 (Green Bars). The number of occurrences of the mutations in GAG sequences is shown in the Y-axis while the X-axis shows aa mutations.

FIGURE 3

Distribution of mutations associated with escape in EHIV012 and EHIV013 after seroconversion. Three individuals had mutations previously associated with escape, diminished responses, non-susceptible forms, etc. These mutations were compiled from the Los Alamos National Laboratory HIV Immunology Database for CTL/CD8 + Epitope Variants and Escape Mutations⁸. The list of all the identified mutations is presented in Supplementary Table 1. Two individuals (EHIV012 and EHIV013) had mutations outside the GAG HIV_GAGCONSV, while EHIV022 had a mutation corresponding to escape (Murakoshi) – E203D. Figure 4 shows the distribution of mutations associated with escape in EHIV 012(Red Bars) and EHIV013 (Green Bars). The number of occurrences of the mutations in GAG sequences is shown in the Y-axis while the X-axis shows aa mutations.

FIGURE 4

Serum creatinine concentration during longitudinal follow up of three individuals from early infection till after seroconversion. The three individuals followed up are represented in blue, yellow, and green. Serum creatinine levels for these individuals were measured in three-time, Baseline, second-time, and after seroconversion. Significant differences in the levels of serum creatinine (measured in mg/dl) observed across the three-time points (P < 0.44) were calculated using 1-way ANOVA.