Evolution of human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitopes: fitness-balanced escape

Abstract

CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.

FIG. 1.

Schematic representation of the protocol used for fitness determination. Viruses were added to PHA- and IL-2-stimulated PBMC alone (mono-infection) or in pairs in equal amounts (dual infection), and viral production was monitored by HTA. X viruses are chimeric viruses with an original p24 (p24_298-3) or p24 mutants with mutations of E75D, V83L, or E75D/V83L in plasmid pNL4-3_VifA. Y viruses are chimeric viruses with p24_298-3 in plasmid pNL4-3_VifB or standard SI/X4 reference HIV-1 isolates (E6, A8, or C08). To control for minor differences in probe binding efficiency, the virus production levels of dual infections and mono-infections were compared. In the mono-infections, the production of virus X was a and the production of virus Y was d. In dual infections, the production of X was b/a and the production of Y was c/d. Relative fitness (W) was defined as the production of an individual virus strain (b/a or c/d) in a dual infection divided by total viral production (b/a + c/d) and its initial proportion (0.5 here) in the inoculum. The equations for the relative fitnesses of viruses X and Y are shown. MOI, multiplicity of infection.

FIG. 2.

Characterization of the ETINEEAAEW p24 epitope (EW10). (A) Frequencies of occurrence of the epitope, its escape mutant, and mutants with a mutation C terminal to the epitope. Amino acids C terminal to the epitope are enclosed within parentheses. (B) Database frequencies of the initial and selected mutations of interest. (C) Relative fitnesses of chimeric viruses, all within the pNL4-3_VifA backbone, containing either the original p24 coding sequence (p24_298-3, the epitopic form from day 8 after the onset of symptoms of primary HIV-1 infection) or a p24 mutant: the E75D mutant (escape mutant), the V83L mutant (with a change 3 amino acids C terminal to the epitope), or the E75D V83L mutant (double mutant). (D) Relative fitnesses of p24_298-3, E75D, V83L, and E75D V83L chimeric viruses, competed against laboratory strains A8, E6, and C08.

FIG. 3.

Comparison of peak CTL responses and functional avidities between epitopes whose frequencies persisted or decreased during the infection of PIC1362. Peak CTL responses correspond to the peak levels of IFN-γ in ELISPOT assays detected over the 4-year time period examined (43). Functional avidity is represented by EC₅₀s. Dotted horizontal lines representmean values. SFC, spot-forming cells.

FIG. 4.

Database frequencies of amino acids at sites associated with CTL escape mutations in epitopic or mutant forms. When multiple mutations were observed for a site, the database frequency of the mutant forms is shown, corresponding to the sum of the frequency of each mutation. Wild-type epitopes are presented above the columns. Underlined amino acids indicate sites at which mutations associated with CTL escape were demonstrated.

FIG. 5.

Comparison of entropies of epitopes with decreasing frequencies and epitopes that persisted in PIC1362 (A), sites associated and not associated with CTL escape in PIC1362 (B), and sites associated and not associated with CTL escape mutations in the HIV immunology database (C). Dotted horizontal lines represent mean values.

FIG. 6.

CTL responses to persistent epitopes containing sites of high entropy and their most frequent mutants found in the HIVDB. (A) LPPVVAKEI IN epitope and the LPPIVAKEI mutant. (B) VIPMFSAL p24 epitope and the VIPMFTAL mutant. (C) YPLTFGWCF Nef epitope and the FPLTFGWCF mutant.