Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response

Abstract

Human lymphocyte antigen (HLA)-restricted CD8(+) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.

Figure 1. Experimental Strategy.

In part 1, the days post onset of symptoms (DPS) viral genetic data was obtained from plasma in our previous study. In addition, sequences were obtained from PBMC DNA from days 34 and 298 for the present study (part 2). The remaining parts of the Figure outline the methods used to generate isogenic viruses and perform ex vivo competition assays in PBMC. Additional details are provided in Figure S1 and S2.

Figure 2. Mapping of the CTL escape and secondary mutations of PIC1362 onto the primary amino acid sequence and X-ray crystal structures of the N-terminus of p24 Gag.

CTL epitopes are mapped to the consensus amino acid sequences of p24 in panel (A) (shaded regions). Amino acid positions are indicated using HXB2 amino acid numbering. Sites of mutations are depicted as space filled amino acids (those within the epitopic form are shown) in X-ray crystallography-derived ribbon structure models of the N terminus p24 (B). The “yellow” residues signify sites at which no impact on replication fitness was detected and “pink” – decreased fitness (see Figure 4).

Figure 3. Mapping of the CTL escape and secondary mutations of PIC1362 onto the primary amino acid sequence and X-ray crystal structures of the N-terminus of gp120 Env core.

CTL epitopes are mapped to the consensus amino acid sequences of gp120 in panels (A) (shaded regions). Amino acid positions are indicated using HXB2 amino acid numbering. Sites of mutations are depicted as space filled amino acids (those within the epitopic form are shown) in X-ray crystallography-derived ribbon structure models of the gp120 core complexed to CD4 and an anti-gp120 antibody (D). The “yellow” residues signify sites at which no impact on replication fitness was detected, “pink” – decreased fitness, and “blue” - increased fitness (see Figure 6).

Figure 4. Ex vivo fitness, CTL escape phenotype and sequence evolution of gag p24 CTL escape mutants.

The predicted amino acid sequence derived from 11–12 clones at different DPS is presented for epitopes EW10 (A) and QW11 (C). The effect of each mutation, or combination of mutations, on the CTL responses to the epitopic peptide in PIC1362 was assessed by ELISpot (grey box in panels (B) and (D)). The fold escape conferred by each mutation is displayed here as the ratio of effective peptide concentration (EC₅₀) required to elicit 50% of maximal ELISpot signal relative to the pre-escape peptide . EC₅₀ values>10 were considered to indicate escape. The standard IFN-γ Elispot assay was performed with the defined optimal epitopic peptide at the following concentrations: 0.64, 3.2, 16, 80, 400, 2,000, and 10,000 ng/ml. The effective peptide concentration that elicited 50% of the maximum T-cell response, defined as the EC50, was determined with the Sigmoidal Fit tool in the software. ELISpot assays not done (ND) with some peptides. NR (no response) refers to a lack of response to a mutant peptide by ELISpot. A positive response was defined as twice the negative control and >50 Spot Forming Cells (SFC)/10⁶ cells. In the case where the wild type peptide was recognized and mutant peptide did not induce a response, full escape (“Full”) was given as the value for the EC₅₀ ratio. Mutations that conferred significant CTL escape are displayed in red columns in (B) and (D). Mutations that did not confer significant escape are shown in green type in (A) and (C) and in green bars in (B) and (D). Combinations of these mutations are shown in purple. CTL escape and associated mutations in the p24 coding sequence were introduced individually and in combination into chimeric NL4-3 viruses bearing the consensus initial/infecting strain from 8DPS (Figure 1 and Figure S1). The effect of these mutations was determined by direct competition with isogenic virus bearing the wild-type amino acid. The control involved competitions between 8DPS-p24 virus competed against a virus of identical amino acid sequence (except for synonymous mutations in vif to differentiate the viruses). These viruses were equally represented following dual infection and thus had equal fitness (relative fitness ∼1) (gray bar in (B) and (D)). Significance of any increase or decrease in fitness conferred by a mutation(s) was evaluated using Student's t-test to compare the relative amounts of each virus. Only p values<0.05 are displayed on the graphs and the 95% confidence interval of the mean is shown for each bar. Fitness data presented on the EW10 epitope (B) have been previously published but for this figure, all competitions were repeated in quadruplicate resulting in very similar results.

Figure 5. Sequence of p24 epitopes QW11 and EW10 in HLA-A25 patients with CD8+ T cell responses to these epitopes.

The consensus sequence at each timepoint was determined by bulk population nucleotide sequencing of the QW11 (A) and EW10 (C) epitopes of four HLA A25-restricted patients, PIC1362, PIC1349, PIC1052, and PIC1483. The sequences of the QW11 and EW10 epitopes of PIC1362 are shown at the top of panels A and C. The EW10 sequences from the “HLA-A25” patient subject at the bottom of panel C is from a previously published study . The shaded amino acids refer to CTL escape mutations. Non-shaded epitopic mutations were associated with escape but confirmatory CTL recognition assays were not performed. ELISpot assays were performed with the QW10 and EW10 peptides and PBMC from multiple time points from PIC1362, PIC1349, PIC1052, and PIC1483 (B and D). NR refers to no detectable IFN-γ ELISpot response.

Figure 6. Predicted and ex vivo processing of peptides harboring the EW10 epitope.

Peptides of 27 amino acids in length and representing positions 200 to 226 in the Env gp120 sequence were used in silico in the NetChop 3.0 C-term peptide processing algorithm (http://www.cbs.dtu.dk/services/NetChop/). C-term 3.0 network is trained with a database consisting of 1260 publicly available MHC class I ligands (using only C-terminal cleavage site of the ligands). The probability of cleavage at each amino acid (panel i) is presented for wt 27 mer peptide (A) and peptides containing the E207D/V215L (B) or V215L/I223V (C). The cleavage peptide products were predicted based on a program recommended probability of cleavage (above a 0.5 threshold, black bars). Low probabilities of cleavage (below 0.5) were presented in grey bars. The same three 27 mer peptides were also synthesized and subject to protease processing using cytosol preparation from HIV-negative PBMCs. Panel ii displays the peptides derived from the wt (A), E207D/V215L (B) or V215L/I223V (C) and identified by mass spectrometry.

Figure 7. Ex vivo fitness, CTL escape phenotype and sequence evolution of env gp120 CTL escape mutants.

The predicted amino acid sequences for 10–16 clones from different DPS from subject 1362 are presented for epitopes YW9 (A), NW9 (C), YL10 (E), EY10 (G), and TL9 (E). The effect of each mutation or combination of mutations on the CTL response to the mutant peptide was assessed by ELISpot (grey box in panels B, D, F, H, and J, respectively). ELISpot and EC₅₀ concentrations were determined as described in the legend to Figure 4. Mutations that confer significant CTL escape are displayed in red text and bars in the respective panels. Mutations that did not confer significant escape are shown in green. Combinations of these mutations are shown in purple. Control experiments (gray bars) and significance testing were as described in the legend to Figure 4.