HLA-associated polymorphisms in the HIV-2 capsid highlight key differences between HIV-1 and HIV-2 immune adaptation

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Introduction
HIV-specific cytotoxic T-lymphocyte (CTL) responses are thought to play an important role in HIV-1 control [1][2][3][4]. A hallmark of HIV-1 evolution, however, is the rapid appearance of mutations within CTL epitopes, leading to loss of CTL recognition and immune control [5]. HIV-2 differs from HIV-1 in that a substantial proportion of infected people maintain undetectable plasma viral loads for decades with no signs of immunodeficiency. Many others have viral loads 30-fold lower than HIV-1 at equivalent disease stages [6][7][8][9]. We have previously demonstrated a strong correlation between the presence of high frequency HIV-2 Gagspecific CTLs and viral control [10][11][12]. As HIV-2 is able to generate resistance mutations akin to HIV-1 under antiretroviral pressure [13], HIV-2 should also have the capacity to adapt to immune responses similar to HIV-1.
Establishing similarities and differences between HIV-1 and HIV-2 immune evasion strategies has the potential to enhance our understanding of HIV pathogenesis. HIV-1 p24 and HIV-2 p26 represent the two major CTL-targeted proteins in these viruses. Here, we provide the first comparison of selective pressure in HIV-1 p24 and HIV-2 p26 capsid sequences from a community cohort in Guinea-Bissau, along with HLA-associated viral polymorphisms in HIV-2,whichmayrepresentCTL-drivenadaptivechanges.

Sequence analysis and tests for codon selection
Sites under positive and negative selection in HIV-1 (231 codons) and HIV-2 (230 codons) were identified by comparison of synonymous (dS, no amino acid change) and nonsynonymous (dN, amino acid change) substitution rates using three different methods in the Datamonkey web-server [20]: single-likelihood ancestor counting (SLAC), fixed-effects likelihood (FEL) and fast unbiased Bayesian approximation (FUBAR) [21,22] (Supplementary methods, http://links.lww.com/QAD/ B217). A P-value cut-off of 0.05 (SLAC and FEL) and posterior probability of 0.95 (FUBAR) was used to define significant positive or negative selection at a codon.
Identification of HLA-associated HIV-2 viral polymorphisms HLA-associated HIV-2 viral polymorphisms were identified using a phylogenetically corrected logistic regression, used extensively for identifying HLA-associated HIV-1 viral polymorphisms [23]. This method corrects for phylogenic relatedness, HLA-linkage disequilibrium and codon covariation. Class I HLA types from 73 HIV-2infected adults were available for this analysis. Separate statistical tests were constructed for each HLA-amino acid pair, limited to HLA alleles and amino acids that were observed in at least 5 and at most 68 individuals. To correct for multiple comparisons, we used a 20% false discovery rate (threshold P < 0.0008). Epitope predictions were made by scanning the candidate sequence for peptides of 8-11 amino acids, with each prediction made using the supplied HLA (2% prior probability distribution).
Identification of a potential HLA-associated HIV-2 p26 polymorphism within a known HLA-B M 5801-restricted cytotoxic T-lymphocyte epitope We then identified five associations between HLA alleles and polymorphisms in HIV-2 p26 using a previously described statistical model [23] (Table 1)

Discussion
We report the first analysis of HLA-associated viral polymorphisms in HIV-2 p26, including a codon substitution within a known immunodominant HLA-B Ã 5801-restricted epitope. This may represent CTLdriven adaptation by HIV-2 and allows direct comparison with what is known about the equivalent HLA-B57/ B Ã 5801-restricted epitope in HIV-1. In contrast to HIV-1, wherever a mutation at position 3 of the epitope (T242N) occurs in 63-93% of HLA-B Ã 5801-positive individuals [25], a mutation at position 5 (E245D) is found in 65% of HLA-B58 ST-positive patients. The HIV-1 TW10 epitope lies within a region essential for capsid formation [27] and residue 242 is thought to be critical to stabilizing the electrostatic charge along helix 6 [26]. A T242N mutation reduces this stabilizing effect [26], consistent with viable virus with significantly reduced fitness. It is possible that for HIV-2, with much lower in-vivo viral titres than HIV-1, the fitness costs of such a mutation are too severe, leading to an alternative pathway of immune adaptation. Further functional studies are required to explore this hypothesis.
The E!D mutation found in HIV-2 replaces one hydrophilic, negatively charged, amino acid with another. In contrast to HIV-1 where T242N escape usually results in loss of immune control, robust CTL responses are generated against this HIV-2 mutant. The absence of E245D mutant responses in one participant (B58_8, Supplementary Table 4, http://links.lww.com/QAD/ B217) suggests that these E245D variant responses do not simply represent cross-reactive CTLs. The presence of CTLs specific to both variants in most individuals may reflect low-level persistence of epitope variants in the viral population not detected by bulk PCR sequencing used in this study. These data also imply that this potential HIV-2 immune adaptation is at the level of T-cell receptor recognition of the peptide-MHC complex, as peptide processing and MHC-epitope binding of E245D variants are presumably still maintained.
We also describe the first comparison of selective pressure on HIV-1 and HIV-2 capsids, finding greater purifying selective pressure in HIV-2. We reported similar findings in HIV-2 env, where relative conservation of this highly 712 AIDS 2018, Vol 32 No 6 The 10-mer TSTVEEQIQW has been previously identified as a B58_ST-restricted epitope via functional assays. The prediction algorithm used identifies the 9-mer STVDEQIQW as an optimal B58_ST-restricted epitope. f NPVPVGNIY is a known BM35-restricted epitope.
variable gene is seen despite high magnitude autologous neutralizing antibody titres [28]. Although one explanation for the lower adaptive changes in HIV-2 is lower viral replication compared with HIV-1, previous studies have demonstrated that evolutionary rates are equivalent if not faster in advanced HIV-2 than in HIV-1 infection [29]. Furthermore, the emergence of ART-driven resistance mutations in HIV-2 shows that at least in the reverse transcriptase and protease genes, viral escape can readily occur [13].
A key limitation of our study is the lack of longitudinal data following individual patients from acute HIV-2 infection to demonstrate CTL-driven escape, shown extensively in HIV-1 [1][2][3][4][5]. Although such a study would add considerable insight, the reducing incidence of HIV-2 infection in West Africa, on a background of vastly lower transmission rates than HIV-1, makes this challenging [14,30]. Acute HIV-2 infection is difficult to identify, and therefore, rarely described. As an indirect way of investigating the issue of HIV-2 immune adaptation, we have, therefore, utilized a statistical model of evolution, well validated in HIV-1 cohorts [23,31]. The significant proportion of HIV-2-infected individuals with low viral loads make generating sequence data challenging (approximately 50% success if viral load <100 copies/ml [19]) and could lead to a biased dataset whenever compared with HIV-1. Nevertheless, our dataset represents the largest collection of sequence data generated from HIV-2-infected individuals with viral load less than 100 copies/ml to date [19].
In conclusion, we provide the first evidence of adaptive changes in the HIV-2 capsid. Our data highlight fundamental differences in immune adaptation between HIV-1 and HIV-2, suggesting that HIV-2 evolution may be limited in this region. Further functional studies are required to characterize the polymorphisms identified in HIV-2, validate our findings and explore whether this characteristic explains why robust immune responses can persist in HIV-2-infected individuals for many years. This in turn may underpin the diverse outcomes seen in HIV-1 and HIV-2 infections, providing a crucial clue to the yet unsolved conundrum of the relatively attenuated nature of most HIV-2 infections.