Structural topology defines protective CD8+ T cell epitopes in the HIV proteome

Abstract

Mutationally constrained epitopes of variable pathogens represent promising targets for vaccine design but are not reliably identified by sequence conservation. In this study, we employed structure-based network analysis, which applies network theory to HIV protein structure data to quantitate the topological importance of individual amino acid residues. Mutation of residues at important network positions disproportionately impaired viral replication and occurred with high frequency in epitopes presented by protective human leukocyte antigen (HLA) class I alleles. Moreover, CD8⁺ T cell targeting of highly networked epitopes distinguished individuals who naturally control HIV, even in the absence of protective HLA alleles. This approach thereby provides a mechanistic basis for immune control and a means to identify CD8⁺ T cell epitopes of topological importance for rational immunogen design, including a T cell-based HIV vaccine.

Fig. 1.. Structure-based network analysis identifies amino acid residues with low mutational tolerance.

(A) Scatter plot of average mutational tolerance and network score for TEM-1 b-lactamase residues (red, active site residues Ser⁷⁰, Lys⁷³, Glu¹⁶⁶, and Asn¹⁷⁰). (B) Comparative receiver operator curves (ROC) and area under the curve (AUC) characteristics for network score, RSA, and sequence entropy to identify the bottom 10% of residues of low mutational tolerance in TEM-1 b-lactamase. (C) Structure-based network schematic for Gag p24 monomer (PDB ID: 3J34, chain C), including amino acid residues (nodes) and noncovalent interactions (edges). Edge width indicates interaction strength and node size indicates relative network score. (D and E) Comparison of Gag p24 and HIV proteome network scores (binned by quintile: low, 2nd, 3rd, 4th, and high; top 5% in gray) with viral sequence entropy. (F) Comparison of viral infectivity of TZM-bl cells and (G and H) viral spreading within GXR cells after mutation of conserved, highly networked residues (blue); conserved, poorly networked residues (red); and nonconserved, poorly networked residues (green). Statistical analyses by one-way analysis of variance and Wilcoxon matched pairs test. (I and J) Scatter plot of sequence conservation and network scores with mutant virus infectivity. Correlations were calculated by Spearman’s rank correlation coefficient. Statistical comparisons were made using Mann-Whitney U test. For comparisons of more than two groups, Kruskal-Wallis test with Dunn’s post hoc analyses was used. Calculated P values are as follows: NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.. Network score distinguishes HIV proteins and CTL epitopes associated with protective, neutral, and risk-associated HLA alleles.

(A) Ranked median second-order degree centrality values (red dots) for HIV proteins (median, interquartile range). (B and C) Network scores for risk and protective allele epitopes B*35Px-DL9 and B*57-KF11 (HLA anchor residues, red; TCR contact residues, blue). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (D) Network-based depiction of B*35Px-DL9 in open gp120 (left, red) (PDB ID: 3J70, chain D) and B*57-KF11 in monomeric Gag p24 (right, blue) (PDB ID: 3J34, chain C). (E) Ranked median epitope network scores for individual HLA alleles (median, interquartile range). GWAS-defined protective and risk HLA alleles indicated in blue and red, respectively. (F) Comparison of epitope network scores presented by protective, neutral, and risk HLA alleles. (G) Comparison of epitope network scores of immunodominant epitopes presented by HLA alleles associated with protection (blue; B*5701-TW10, B*5201-RI8, B*2705-KK10, B*1402-DA9) and risk (red; B*0801-FL8, B*3501-DL9, B*0702-RV9, Cw*07-RY11). (H) Scatter plot of GWAS-defined protective HLA allele odds ratios (OR) to median epitope network score. (I) Ranked network scores of each amino acid type across the HIV proteome (median, interquartile range). HLA-B*57 anchor residues denoted in blue. Statistical comparisons were made using Mann-Whitney U test. For comparisons of more than two groups, Kruskal-Wallis test with Dunn’s post hoc analyses was used. Correlations were calculated by Spearman’s rank correlation coefficient. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.. Targeting of topologically important epitopes distinguishes HIV controllers from progressors irrespective of HLA allele.

(A and B) Proliferative CTL responses in a single representative controller (top) and progressor (bottom) after 6-day incubation of carboxyfluorescein succinimidyl ester (CFSE)–loaded peripheral blood mononuclear cells with optimal epitopes matched to the person’s HLA haplotype. VL, viral load. (C) Residue network scores of epitopes targeted in (B) (red, HLA anchor; blue, TCR contact). (D) Network-based depiction of A*02-KL9 and B*07-RI10 in open (top; PDB ID: 3J70, chain D) and closed (bottom; PDB ID: 5T3X) gp120 trimeric conformations. A single subunit of trimeric, open gp120 is presented for ease of epitope visualization. (E) Proliferative responses of controllers (blue), intermediates (green), and progressors (red). The x axis depicts all CTL epitopes ranked by epitope network score from lowest to highest. (F to H) Comparison of controllers (C; n = 46 individuals), intermediates (I; n = 25), and progressors (P; n = 43) by summed epitope network scores, summed proliferative responses, and summed epitope network scores scaled by proliferative CTL response. (I and J) Comparison of controllers with nonprotective alleles (NPC; n = 18) and protective alleles (PC; n = 28) by summed epitope network scores (I) and summed epitope network scores scaled by magnitude of proliferative CTL response (J). (K) Comparison of B*57⁺ controllers (C; n = 17) and B*57⁺ progressors (P; n = 7) by summed epitope network scores scaled by magnitude of CTL proliferation. (L) CFSE dilution of immunodominant CTL responses from a single representative B*57 ⁺ controller and B*57⁺ progressor. (M) Residue network scores of CTL epitopes targeted in (L). (N and O) Comparison of controllers (C; n = 14) and progressors (P; n = 14) with similar magnitude of summed CTL proliferation (upper) by CTL proliferation (N) and epitope network scores (O). Statistical comparisons were made using Mann-Whitney U test. For comparisons of more than two groups, Kruskal-Wallis test with Dunn’s post hoc analyses was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.. Topologically important CTL epitopes targeted by HIV controllers are infrequently mutated in vivo.

(A and D) CFSE dilution of immunodominant CTL responses from a single representative controller and progressor. (B and E) Network scores of the B*53-YF9 and B*08-FL8 epitopes (red, HLA anchor; blue, TCR contact). (C and F) WebLogo of B*53-YF9 and B*08-FL8 sequence data (red, HLA anchor; blue, TCR contact; green, flanking). (G) Network representation of the B*08-FL8 and B*53-YF9 epitopes within the Nef dimer (PDB ID: 2XI1). (H) Comparison of average number of mutations within epitopes targeted by controllers (n = 9) and progressors (n = 15) for each individual patient. (I) Comparison of the percent frequency of mutations at HLA anchor (blue), TCR contact (red), and flanking residues (black) between controller-targeted (open bars) and progressor-targeted epitopes (filled bars). Statistical comparisons were made using Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.