The mechanisms underlying HIV-1 control by protective HLA class I alleles are not fully understood and could involve selection of escape mutations in functionally important Gag epitopes resulting in fitness costs. This study was undertaken to investigate, at the population level, the impact of HLA-mediated immune pressure in Gag on viral fitness and its influence on HIV-1 pathogenesis. Replication capacities of 406 recombinant viruses encoding plasma-derived Gag-protease from patients chronically infected with HIV-1 subtype C were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. Viral replication capacities varied significantly with respect to the specific HLA-B alleles expressed by the patient, and protective HLA-B alleles, most notably HLA-B*81, were associated with lower replication capacities. HLA-associated mutations at low-entropy sites, especially the HLA-B*81-associated 186S mutation in the TL9 epitope, were associated with lower replication capacities. Most mutations linked to alterations in replication capacity in the conserved p24 region decreased replication capacity, while most in the highly variable p17 region increased replication capacity. Replication capacity also correlated positively with baseline viral load and negatively with baseline CD4 count but did not correlate with the subsequent rate of CD4 decline. In conclusion, there is evidence that protective HLA alleles, in particular HLA-B*81, significantly influence Gag-protease function by driving sequence changes in Gag and that conserved regions of Gag should be included in a vaccine aiming to drive HIV-1 toward a less fit state. However, the long-term clinical benefit of immune-driven fitness costs is uncertain given the lack of correlation with longitudinal markers of disease progression.

f HIV-1 attenuation resulting from immune escape mutations selected in Gag may contribute to slower disease progression in HIV-1-infected individuals expressing certain HLA class I alleles. We previously showed that the protective allele HLA-B*81 and the HLA-B*81-selected Gag T186S mutation are strongly associated with a lower viral replication capacity of recombinant viruses encoding Gag-protease derived from individuals chronically infected with HIV-1 subtype C. In the present study, we directly tested the effect of this mutation on viral replication capacity. In addition, we investigated potential compensatory effects of various polymorphisms, including other HLA-B*81-associated mutations that significantly covary with the T186S mutation. Mutations were introduced into a reference subtype B backbone and into patient-derived subtype C sequences in subtype B and C backbones by site-directed mutagenesis. The exponential-phase growth of mutant and wild-type viruses was assayed by flow cytometry of a green fluorescent protein reporter T cell line or by measurement of HIV-1 reverse transcriptase activity in culture supernatants. Engineering of the T186S mutation alone into all patient-derived subtype C sequences failed to yield replicationcompetent viruses, while in the subtype B sequence, the T186S mutation resulted in impaired replication capacity. Only the T186S mutation in combination with the T190I mutation yielded replication-competent viruses for all virus backbones tested; however, these constructs replicated slower than the wild type, suggesting that only partial compensation is mediated by the T190I mutation. Constructs encoding the T186S mutation in combination with other putative compensatory mutations were attenuated or defective. These results suggest that the T186S mutation is deleterious to HIV-1 subtype C replication and likely requires complex compensatory pathways, which may contribute to the clinical benefit associated with HLA-B*81.

HLA class I-mediated selection of immune escape mutations in functionally important Gag epitopes may partly explain slower disease progression in HIV-1-infected individuals with protective HLA alleles. To investigate the impact of Gag function on disease progression, the replication capacities of viruses encoding Gag-protease from 60 individuals in early HIV-1 subtype C infection were assayed in an HIV-1-inducible green fluorescent protein reporter cell line and were correlated with subsequent disease progression. Replication capacities did not correlate with viral load set points (P ‫؍‬ 0.37) but were significantly lower in individuals with below-median viral load set points (P ‫؍‬ 0.03), and there was a trend of correlation between lower replication capacities and lower rates of CD4 decline (P ‫؍‬ 0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (P ‫؍‬ 0.04), but host HLA-B-specific polymorphisms were associated with higher viral load set points (P ‫؍‬ 0.01). Further, polymorphisms associated with host-specific protective HLA alleles were linked with higher viral load set points (P ‫؍‬ 0.03). These data suggest that transmission or early HLA-driven selection of Gag polymorphisms results in reduced early cytotoxic T-lymphocyte (CTL) responses and higher viral load set points. In support of the former, 46% of individuals with nonprotective alleles harbored a Gag polymorphism exclusively associated with a protective HLA allele, indicating a high rate of their transmission in sub-Saharan Africa. Overall, HIV disease progression is likely to be affected by the ability to mount effective Gag CTL responses as well as the replication capacity of the transmitted virus.

These data demonstrate that the majority of CD8(+) responses are not elicited during acute HIV infection despite the presence of the cognate epitope in the infecting strain. There is a need to define factors that influence lack of induction of effective immune responses and the parameters that dictate immunodominance in acute infection.

Immune escape mutations selected by human leukocyte antigen class I-restricted CD8+ cytotoxic T lymphocytes (CTLs) can result in biologically and clinically relevant costs to HIV-1 replicative fitness. This phenomenon may be exploited to design an HIV-1 vaccine capable of stimulating effective CTL responses against highly conserved, mutationally constrained viral regions, where immune escape could occur only at substantial functional costs. Such a vaccine might ‘channel’ HIV-1 evolution towards a less-fit state, thus lowering viral load set points, attenuating the infection course and potentially reducing the risk of transmission. A major barrier to this approach, however, is the accumulation of immune escape variants at the population level, possibly leading to the loss of immunogenic CTL epitopes and diminished vaccine-induced cellular immune responses as the epidemic progresses. Here, we review the evidence supporting CTL-driven replicative defects in HIV-1 and consider the implications of this work for CTL-based vaccines designed to attenuate the infection course.