Recognition of CTL epitopes by donor SC2 bulk cultured lymphocytes. (a) HLA B8 Nef (FLKEKGGL) response (Nov. 3, 1995). (b and c) HLA B8 p17-3 Gag (GGKKKYKL) response (Oct. 19, 1995). Screening peptides used were: GGKKKYKL (p17-3 Gag; ref. 13), GEIYKRWII (p24-13 Gag; ref. 13), DCKTILKAL (p24-20 Gag; ref. 13), GPKVKQWPL (Pol; ref. 14), and FLKEKGGL (Nef; P.J.R.G., unpublished work).

Nucleotide and amino acid sequence variation in the region of Nef containing the HLA B8 epitope. For donor SC2, a number of clones from each time point (19 from each of the first three time points, 18 from the fourth time point) were sequenced further to incorporate regions flanking the epitope (nucleotides 114–395, amino acids 38–132, numbered according to the nef reference sequence HIV-LAI). (a) The Kabat–Wu index (KWI), calculated at each amino acid position as the number of different residues/frequency of the commonest residue, provides a measure of variability; an index of 1 indicates that no variation was detected at the relevant position (15). The HLA B8 epitope (amino acids 90–97) is shaded. (b) The rates of nonsynonymous (dN) and synonymous (dS) nucleotide substitutions were calculated in a sliding window of 60 nt over all pairwise comparisons between clones at a given time point, according to the method of Nei and Gojobori (16). The window is advanced in steps of three nucleotides, and the values of dN and dS are plotted against the central nucleotide in the window. dN rises sharply at the border of the region encoding the HLA B8 epitope, and exceeds dS over this region. The apparent extension of positive selection beyond the limits of the epitope reflects window width, not flanking mutations. Dotted line, synonymous substitutions; solid line, nonsynonymous substitutions.

Truncations and frameshifts within donor SC2 nef clones (deduced amino acid sequence). Identical single nucleotide deletions (clone 2/17, clone 4/19) resulting in premature termination of the open reading frame 6 aa before the B8 epitope are present at two separate time points, suggesting proviral replicative capacity. Index refers to proviral sequence from 19/10/95. ∗, Stop codon. Shading denotes the epitope. These sequences were not included in either the Kabat–Wu or dN/dS analyses.

Proviral sequence variation in HIV-1 epitopes from SC2. (a) HLA B8 Nef. (b) HLA B8 p17-3 Gag. Variant residues are highlighted.

(a) Altered recognition of variant Nef epitopes by donor SC2 CTLs. ⁵¹Cr-labeled HLA B8-matched targets were added directly to 96-well plates containing peptide, and therefore peptide was present at the concentrations drawn for the duration of the assay. Targets and peptide were incubated at room temperature for 15 min before the addition of effector cells. (b) Functional HLA binding assay. Target cells pulsed with variant peptides containing mutations at position 5 (a HLA B8 anchor position) were not recognized by donor SC2 CTLs after 6 h (N5 and E5) and 10 h (Q5), suggesting that these peptides bind poorly and have high off-rates. (c) The p17-3 Gag Q5 variant is poorly recognized by donor SC2-derived GGKKKYKL-specific CTLs.