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. 2014 Apr 24;7(2):448-463.
doi: 10.1016/j.celrep.2014.03.031. Epub 2014 Apr 13.

HIV-1 Adaptation to Antigen Processing Results in Population-Level Immune Evasion and Affects Subtype Diversification

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Free PMC article

HIV-1 Adaptation to Antigen Processing Results in Population-Level Immune Evasion and Affects Subtype Diversification

Stefan Tenzer et al. Cell Rep. .
Free PMC article

Abstract

The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8(+) T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ~30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ~60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins.

Figures

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Figure 1
Figure 1
Overview of the HIV p24 Gag Regions Analyzed (A) Outline of p24 Gag fragment 1 (F1, amino acids 5–46, HIVHXB2 numbering) and fragment 2 (F2, amino acids 100–143) that highlights the overlapping CTL epitope clusters and subtype-specific positions (27, 41, 116, 120, and 128: orange boxes). The four epitopes presented by protective HLA class I molecules are underlined; all epitopes are listed in Table S1. (B) Outline of the overlapping 25-mer peptides used as proteasomal substrates using the HIV B and HIV C consensus sequences as examples. F1 was divided into two parts (NF1 [N-terminal F1] and CF1 [C-terminal F1]), and F2 was divided into three parts (NF2, MF2 [Middle F2], and CF2). (C) Scatterplots of the relationship between the allelic frequency of a restricting HLA variant (or the sum of HLA variants if the CD8 epitope and/or epitope precursor is presented by more than one HLA molecule or if it contains more than one epitope presented by different HLA variants) in six white and African populations (infected primarily with HIV B or HIV C, respectively) and “epitope yield” (i.e., the abundance of individual epitopes and/or epitope precursors represented as percentages of all peptide fragments) following 4 hr of immunoproteasomal digestion of either the consensus HIV B or HIV C sequence. KI8 and KF11 are grouped together as they are two forms of the same epitope. HLA restriction details are shown in Table S1. Red epitopes and their restricting HLA variants signify epitopes that are not produced but are made following the processing of either the other subtype or one of the sequence combinations (Table 1). Asterisk, the HLA allelic frequency for the PY epitope is set at zero in HIV-B and HIV-C infection because of intraepitope CTL-escape mutations (Matthews et al., 2012, Sabbaj et al., 2003). The figure represents one of three independent experiments. (D) Multilevel analysis with the three-way interaction of fragment (NF1, CF1, or MF2&CF2), the presenting HLA allele frequency and ethnic group on epitope yield. NF2 could not be analyzed due to differential epitope processing and restriction by only two HLA class I variants.
Figure 2
Figure 2
TAP Binding, ERAP Trimming, and CD8+ T Cell Analyses (A) Overview of the abundance of all IW9, KF11, KI8, and TW10 epitope forms produced after 4 hr constitutive (c-p) or immunoproteasomal (i-p) digestion. (B) TAP binding affinity of the IW9, KF11, KI8, and TW10 epitope forms generated by proteasomal digestion. GW12 could not be tested because it was insoluble and WF20, VI14, and VI12 were not tested. The epitope forms are colored as in (A). The results represent one of at least two independent experiments. (C) ERAP1,2 digestion of the TW10 epitope precursor RW18 from HIV B and HIV C. The result represents one of three independent experiments. (D) PBMCs from four untreated HIV-1-infected patients with HLA-B5701 (Table S3) were stimulated with a subset of IW9, KF11, KI8, and TW10 HIV B epitope-precursor peptides because of limited sample availability. IL-2, IFN-γ, TNF-α, and CD107a responses were analyzed using flow cytometry. The percentages of responding CD8+ T cells are shown after background subtraction, and each peptide was tested two to three times. The epitope-peptide form is indicated along the x axis, and the optimal epitope is shown in the top-left corner of the graph. The epitope forms made by the proteasome are colored as in (A) and (B).
Figure 3
Figure 3
Epitope Production after Proteasomal Digestion of F1 and F2 Variants (A) Overview of the NF1 and CF1 subtype-specific motifs (orange boxes), the HIV B and HIV C consensus sequences, and all combinations of the subtype-specific amino acids with a color-code key. The abundances of individual epitopes were represented as percentages of all peptide fragments following constitutive and immunoproteasomal digestion, and the epitopes are shown with the restricting HLA variants. RV9 and EV9 were produced in amounts too small to be easily visible in this figure. The results represent one of three independent experiments. F1 contains the protective epitopes IW9 (NF1) and KF11 (CF1) and overlapping epitopes (Figure 1A, Table S1). Asterisk, QR10; arrow, QW11 (IW9); pin, KF11. (B) As in (A) except that all F2 peptide variants are shown in Table 1. SM10, TT10, EN12, and WV10 were produced in amounts too small to be easily visible in this figure. F2 contains the protective TW10 epitope (NF2) and several KK10 epitope forms (MF and CF2; in brackets) and overlapping epitopes (Table S1). Arrow, TW10; pins, KK10 epitope forms.
Figure 4
Figure 4
Intrahost Selection for the Subtype-Specific 120N→S Substitution Analyses of intrahost selection for the subtype-specific HIV 120N (HIV B) → 120S (HIV C) substitution in HIV-B- and HIV-C-infected subjects with HLA variants restricting CD8 epitopes in F2 (all subtype-specific amino acid substitution analyses are presented in Figure S4 and Table S4). The circles represent the percentages of 120S in HIV from infected individuals who carry a specific HLA variant (y axis: 120S +, HLA X+) and the percentage of 120S in infected individuals without this HLA variant (x axis: 120S +, HLA X−). Therefore, the data illustrate the extent to which a specific HLA variant is associated with HIV evolution at position 120; specifically, circles above the line suggest positive selection, circles on the line suggest no selection, and circles below the line suggest a negative selection of 120S in patents with HLA X; Fisher’s exact test is used to estimate significance and asterisks indicate that the two-tailed p value is <0.05. Fewer than three HIV-B-infected patients carried HLA-B63, and an insufficient number of sequences were available from patients with HLA-B5801 and from HIV-C-infected patients with HLA-A11, -B53, -B63, and -B27 to test for significance. The frequencies of 120S in HIV B and HIV C from patients without HLA variants that could present epitopes from F2 were 6% and 72%, respectively.
Figure 5
Figure 5
Selective Production of the Internal PY9 Epitope and KK10 Epitope Variants (A) Outline of the overlapping epitope region comprising the B27-KK10 epitope; prolines 122, 123, and 125 are underlined, the internal B35/53-PY epitope is shaded red, and the cytoplasmic nardilysin (NRDc) recognition motif 131KR132 is gray (Kessler et al., 2011). Epitopes are boxed and HLA restriction elements are displayed in the same color as the box. Arrow, NRDc cut site. (B) ERAP1,2 digestion of the HLA-A02, -A03, -A11, -A33, and -B2705-restricted GI15 precursor epitope. The result represents one of three independent experiments. (C) ERAP1,2 digestion of the HLA-B08-restricted GI8 precursor epitope. The result represents one of three independent experiments. (D) Outline of all proteasomal digestion fragments with different KK10 epitope forms produced following 4 hr of immunoproteasomal digestion of HIV B F2. The position of prolines 122, 123, and 125 and the NRDc cut site are indicated; production of the internal PY9 epitope and the KK10 epitope forms are delineated. HLA restriction elements are displayed next to the epitope precursors using the same color as in Figure 5A.
Figure 6
Figure 6
HIV B Adaptation to the HLA Profiles of Host Populations (A) One sequence from each HIV-B-infected patient was selected from the HIV database. The frequencies of HIV-C-like p24 Gag 27I, 41T, 116A, 120S, and 128D amino acids in HIV B were calculated across the data set at the indicated time periods in five geographic regions (region, n from time periods 1, 2, and 3), all minus Caribbean, 160, 575, and 556; Europe, 69, 254, and 375; Caribbean, 2, 68, and 31; Asia, 27, 96, and 97; North America, 47, 111, and 40; and South America, 17, 114, and 44. Fisher’s exact test was used to compare mutation frequencies at time period 3 between sequences from the Caribbean and all other cohorts combined (p < 0.05, ∗∗∗p < 0.0001). (B) The frequency of an HLA variant in black Trinidadians (BT)/US whites (USW) versus the HIV B 120N→S substitution frequencies in subjects with or without that HLA variant is illustrated using log scales (all analyses, Figure S5 and Table S4). (C) The summed allelic frequencies of HLA variants selecting for HIV B wild-type (“WT”) and HIV-C-like amino acids (“Mutant” or “M”), respectively, at each of the five subtype-specific positions for BT and USW; p values for the ethnic differences were calculated by fitting a binomial generalized linear model (GLM) (∗∗∗p < 0.0001) (Table S5). (D) The ratios of the summed weighted HLA allelic frequencies in BT and USW that select for M/WT amino acids at each subtype-specific position (∗∗∗p < 0.0001) (Table S4; Table S5; Figure S5). (E) A binomial GLM produced the graph of the frequency of p24 Gag 27I, 41T, 120S, and 128D in HIV B sequences from the Caribbean or North America versus the ratios of the summed HLA allelic frequencies in BT or USW that select for M/WT amino acids at each subtype-specific position. Position 116 was excluded because of competing selective pressures. (F) HLA-B2705 frequencies versus the frequencies of HIV B p24 Gag 120N and 120S in sequences from each country during 2000–2009. The superposed curve was obtained by fitting a binomial GLM to the points. n = number of sequences, AR, Argentina (n = 78); AU, Australia (n = 21); BB, Barbados (n = 64); BR, Brazil (n = 68); CN, China (n = 75); CO, Columbia (n = 7); DE, Germany (n = 14); DK, Denmark (n = 23); ES, Spain (n = 8); FR, France (n = 7); GB, United Kingdom (n = 526); HT, Haiti (n = 16); JM, Jamaica (n = 14); JP, Japan (n = 27); KR, Korea (n = 18); TH, Thailand (n = 17); and US, USA (n = 122).

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