Background:  CD8⁺ cytotoxic T-lymphocyte (CTL) responses are associated with control of HIV-1 infection. CTL epitopes at which escape mutants were detected, as well as epitopes that persist at high frequencies in HIV-1 infection have been reported. It is not clear why at some epitopes escape mutants are detected while at others they are not. However, viral polymorphisms of high representation in the HIV Sequence Database have been correlated with higher viral fitness.

Methods:  We sequenced whole or multiple HIV-1 viral genome segments from 1 person at 17 time points over the first 4 years of infection, and characterized the recognition of epitopic or mutant peptides spanning the entire proteome by interferon-gamma (IFN-g)-secreting T cells at 16 time points. For each amino acid site associated with cytotoxic T lymphocyte (CTL) escape, frequencies of the amino acid at the respective positions in its epitope and escape mutant forms were calculated and compared using the Wilcoxon signed rank test. Furthermore, for each amino acid site in a recognized CTL epitope, its Shannon entropy from the HIV Sequence Database was calculated:  lower entropy indicates lower variability, suggesting a stronger fitness constraint. Entropies of amino acid sites associated with CTL escape were compared with those of sites not associated with escape using the Mann-Whitney test.

Results:  We detected 25 CTL epitopes. Mutations at 25 sites in 16 epitopes were associated with CTL escape, 20 of which showed lower database frequencies. Database frequencies of the mutant forms were significantly lower than that of the epitope forms (p = 0.0015). Compared with amino acids in CTL epitopes not exhibiting escape mutations, amino acids related to escape mutations were associated with higher Shannon entropy (p = 0.002) and we infer, weak functional constraints. This association was further supported when all 42 reported escape mutants of defined CTL epitopes in the HIV Immunology Database were studied. Finally, we found that cross-reactivity of CTL responses to a mutant form of an epitope was associated with a failure to rise in frequency in the subject.

Conclusions:  Although CTL responses selected for escape mutants, many potentially have fitness costs. The balance between the advantage of escaping from immune responses and the disadvantage of losing replicative fitness plays an important role in determining whether a CTL epitope will persist or by replaced by escape mutants during infection.