The mechanisms underlying HIV-1 control by protective HLA alleles are not fully understood and could involve targeting of functionally important epitopes in Gag, resulting in selection of escape mutations with a fitness cost. Therefore, this study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its impact on HIV-1 pathogenesis.
Our results showed an association between protective HLA alleles (HLA-B*57, HLA-B*5801, and HLA-B*81) and lower Gag-protease replication capacities. Since (i) protective HLA alleles were associated with lower viral loads, (ii) Gag-protease replication capacity correlated with viral loads even on removal of protective HLA alleles from the analysis and within individuals with protective alleles, and (iii) replication capacity ranked according to HLA-A, -B, and -C alleles correlated significantly with the ranks according to viral load, the possibility that HLA alleles and replication capacity are indirectly related to each other through association with viral load cannot be excluded. However, since mutations in Gag selected by the protective HLA alleles B*5703 and B*5801 were shown to significantly decrease the overall replication capacity of isolates and to confer benefits on infant and adult recipients (7
), except in the presence of compensatory mutations (39
), it seems very likely that a direct relationship exists between HLA alleles and Gag-protease replication capacity. Gag-protease replication capacity varied significantly between the different HLA-B but not HLA-A or HLA-C alleles, consistent with the idea that HLA alleles influence Gag-protease replication capacity through selecting mutations, as HLA-B alleles have the greatest selection pressure (20
). Moreover, increasing numbers of HLA-B-associated mutations in or flanking epitopes (likely HLA-selected escape mutations) correlated with decreased HIV replication capacities. In further support of a direct relationship between protective HLA alleles and replication capacity, HLA-B*81 was by far the allele most strongly associated with lower replication capacity, even though HLA-B*5703-positive individuals had a lower average viral load than HLA-B*81-positive individuals, and 186S present in the HLA-B*81-restricted epitope TL9 (positions 180 to 188) was the mutation most strongly associated with lowered replication capacity, thereby providing a possible mechanism for the influence of HLA-B*81 on replication capacity. TL9 was previously described as one of the key Gag epitopes under strong selection pressure by a beneficial HLA allele, with variance mainly at residues 182 and 186 (both with changes predominantly to serine) (14
). Interestingly, in a recent study, the number of public T-cell clonotypes specific for simian immunodeficiency virus (SIV) Gag CM9 (residues 181 to 189), which occurs in nearly the exact same position as TL9 in HIV, correlated strongly and negatively (r2
= −0.71) with the viral set point in rhesus macaques (34
). Residue 186 in HIV Gag has also been classified as a site where mutations revert upon transmission to a host lacking the HLA allele that selected them, presumably due to a fitness cost (26
). It should be noted, though, that differences in fitness associated with variability at position 186 did not translate into viral load differences in this chronic infection cohort (data not shown), which could suggest that the fitness cost of the 186S mutation may be compensated in some cases, and therefore not of lasting benefit, and that the balance between the fitness cost of 186S and an effective CTL response to TL9 may be important in determining the outcome. However, taking the results together, it seems likely that protective HLA alleles, in particular HLA-B*81, influence Gag-protease replication capacity through CTL selection pressure and that this may partly contribute to their protective effect. From the present data, this seems likely to be a more prominent mechanism of protection for HLA-B*81 than for HLA-B*57 and HLA-B*5801 in subtype C infection.
Given our observation that lower Gag-protease replication capacities were related to protective HLA types, lower baseline viral loads, and higher baseline CD4 counts, we wished to investigate whether viral replication capacity may also correlate with the subsequent rate of CD4 decline during chronic infection. However, such a correlation was not observed in the present study. This may be explained partly by the balance that exists between Gag CTL responses and replication capacity in influencing clinical outcomes. Accumulation of escape mutations in HIV carries a fitness cost to the virus, but the disadvantage to the virus is offset by the advantage of escaping effective CTL responses that were holding replication in check, resulting in increased viral loads and accelerated disease progression despite a replication-deficient virus (8
). Another consideration is that replication capacity is not static and compensatory mutations may have developed at a time point later than that measured, influencing the subsequent rate of CD4 decline. Data from the present study and previous studies suggest that mutations with a fitness cost are readily compensated. The T186S mutation was most strongly associated with decreased replication capacity, yet in the presence of covarying mutations at positions 182 and 190, the mean replication capacity was not significantly different from the mean for the entire cohort, suggesting that the possible fitness cost of this mutation was compensated in these cases. Therefore, although there may be a benefit to decreased replication capacity (as supported by cross-sectional correlations with viral loads and CD4 counts), the data do not support an enduring benefit or a lasting significant impact of Gag-protease replication capacity on the rate of disease progression, at least once the chronic infection stage has been reached. The results of Brockman et al. (submitted) are consistent with this notion. However, acute infection studies and/or longitudinal analysis of replication capacity and sequence changes, together with CTL responses, may be necessary to better assess the relative impact of each on disease progression. Site-directed mutagenesis experiments would also be necessary to confirm the suspected fitness costs and compensatory roles of some of the mutations described above.
The data support the hypothesis that mutations at conserved residues/regions, in particular in conserved Gag p24 as opposed to the less-conserved Gag p17, are more likely to result in a fitness cost: HLA-associated escape mutations at conserved sites were associated with lower replication capacities, there were significantly more variant p24 epitopes in the least-fit viruses than in the fittest viruses, and most of the mutations significantly associated with altered replication capacities in p24 decreased replication capacity, while most in p17 increased replication capacity. In agreement with these data, beneficial HLA alleles in an African cohort were associated with strong selection at key epitopes which occurred mostly in Gag p24 (14
), and there is recent evidence that HLA-B*57 mediates its protective effect mainly through attenuating mutations in Gag p24 (39
). Furthermore, the breadth of Gag p24, but not p17 or p15, CD8 T-cell responses in HLA-B*13-positive individuals was significantly associated with decreasing viral loads (17
). Taken together, the data generally support the inclusion of conserved regions such as Gag p24 in a vaccine that is aimed at driving HIV toward a less-fit state.
Interestingly, a larger number of amino acid differences from the consensus subtype C Gag sequence were weakly but significantly associated with increasing viral fitness. The percent amino acid similarity to the consensus subtype C Gag sequence also correlated negatively with viral load and positively with CD4 count (data not shown), suggesting that more changes from consensus and increased fitness of viruses may occur with disease progression. In fact, the fitness of HIV isolates was previously shown to increase with disease progression (44
). Consensus amino acids could, in some instances, be escape mutations in response to common HLA alleles, but we speculate that they represent the nonescape form in the majority of cases and that nonconsensus residues represent escape and compensatory mutations in response to CTL and non-CTL immune pressure, although they could also represent random mutations. Based on this conjecture, we suggest that more changes away from consensus likely indicate more compensation, and therefore fitter viruses. Another explanation is that the majority of mutations introduced into HIV are likely to have no or little fitness cost or to actually increase fitness. Consistent with this idea, p17 and p7 were significantly more divergent from the consensus than p24 was, i.e., significantly more mutations occurred in p17 and p7 than in p24, and the percent similarity to consensus for both p17 and p7 was negatively correlated overall with fitness, while there was no correlation for p24. The direct relationship between replication capacity and the entropy of mutated sites in the present study, as well as the recent finding that escape mutations in conserved Gag p24 carry significant fitness costs while most of the escape mutations in the highly variable env
gene are fitness neutral or increase fitness (45
), lends further support to this argument.
Another interesting finding was that most of the mutations in Gag associated with altered replication capacity were not HLA associated (71%). It should be noted, however, that a limitation of this study was the insertion of subtype C Gag-protease into a subtype B backbone, and therefore some Gag-protease mutations associated with altered replication capacity might represent those that interact with other components of the backbone. A significantly lower replication capacity of subtype C/B recombinants than that of subtype B recombinants was observed, which could suggest that mixing of subtypes results in suboptimal replication. Alternatively, this finding could mean that Gag-protease function is inferior in subtype C versus subtype B viruses, which may partly explain previously described fitness differences between subtypes (1
). Further experiments are required to discriminate between these possibilities. Supporting the latter rather than the former possibility, convergence of subtype C Gag sequences to the consensus subtype B sequence was not associated with fitter recombinant viruses. Furthermore, the findings of the present study are in agreement with those of Brockman et al. (submitted), which show that subtype B Gag-protease NL4-3 recombinant viruses correlate with cross-sectional viral load and CD4 count data as well as with specific HLA types, strongly supporting the hypothesis that the current assay system is clinically and biologically relevant.
In summary, there is evidence that protective HLA alleles, especially HLA-B*81, influence subtype C HIV replication capacity through selection of mutations in Gag that incur a fitness cost. Moreover, mutations in conserved rather than more-variable regions of Gag are more likely to carry a fitness cost, suggesting that conserved regions such as Gag p24 should be included in a vaccine aiming to drive HIV toward a less-fit state. However, the long-term clinical impact of immune-driven fitness costs requires further investigation, given the evidence for compensation and the observation that replication capacity does not correlate with the subsequent rate of CD4 decline in chronic infection.