Positional Bias of MHC Class I Restricted T-Cell Epitopes in Viral Antigens Is Likely due to a Bias in Conservation

<div><p>The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from <b>D</b>efective <b>Ri</b>bosomal <b>P</b>roducts (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.</p> </div

Positional bias of conservation for viral proteins.

<p>For each viral protein which also contained at least one tested peptide from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi-1002884-t001" target="_blank">Table 1</a>, conservation scores at the residue-level were calculated using Rate4Site <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi.1002884-Mayrose1" target="_blank">[26]</a>. Residue positions were converted into normalized positions and corresponding conservation scores were binned (5 bins of equal size). Higher conservation score indicates higher degree of conservation. Conservation scores were normalized for each protein (i.e. mean = 0; sd = 1). One thousand bootstrapping over proteins was used to estimate confidence intervals for the means of conservation scores. Each box covers 25^th and 75^th percentiles. Whiskers extend out from each box 1.0 times the interquartile range.</p

Distributions of conservation scores of peptides with positive and negative T-cell assay results for viruses in the context of immunization with peptides.

<p>The difference between the two distributions is not statistically significant (Welch's t-test; one-sided; p-value = 0.62).</p

MHC class I restricted T-cell epitopes retrieved from the Immune Epitope Database for viral species.

<p>For each organism, total number of tested peptides as well as numbers of those with positive and negative assay outcomes are shown. A total of 93 viruses were studied. For brevity, 20 viruses with the highest number of tested peptides are shown in the table.</p

Positional biases of predicted binders for 12 HLA supertypes.

<p>For each supertype, 9-mer peptide binding predictions were carried out and ratios of probability masses of predicted ‘binders’ and ‘non-binders’ were calculated. Peptide binding predictions were made for alleles belonging to each supertype, using SMM^PMBEC method. All possible 9-mer peptides were generated from a set of viral proteins that contain at least one tested peptide from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi-1002884-t001" target="_blank">Table 1</a>. Relationships between HLA molecules and supertypes are provided in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi.1002884-Sidney1" target="_blank">[11]</a>.</p

Estimating positional bias of epitopes from conservation data.

<p>(A) Bootstrap sampling of conservation scores for positive and negative peptides are shown as two boxplots placed next to each other. The bins used are of variable lengths to ensure a sufficient count in each bin. Each bin contains ∼20% of data points. Middle positions of bins are indicated on the x-axis. The difference between the means of the two conservation score distributions is statistically significant (Welch's t-test; one-sided; p-value = 5.9×10⁻⁶). (B) Estimating probability ratios from the conservation score distributions. This is simply taking a ratio of positive and negative peptide probabilities as a function of a conservation score. Confidence intervals are derived from bootstrap sampling. (C) Estimated probability ratios as a function of normalized position, using the mapping shown in the second panel. As input, distributions of means of conservation scores shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002884#pcbi-1002884-g003" target="_blank">Fig. 3</a> were used. (D) Positional bias curves derived from observed normalized positions (black) and conservation scores (gray).</p

Epitope distribution among host species.

<p>Data represent the number of epitopes identified (in parentheses) for each host species reported to date.</p

Detailed reactivity maps for HCV structural proteins.

<p>Data represent individual RFscores for antibody, CD4⁺ and CD8⁺ T cell responses plotted for each antigen translated from the HCV H77 reference polyprotein: A–C = core; D–F = E1; G–I = E2. The red line denotes average frequency for all antibody, CD4 and CD8 T cell responses. Note: Data displayed for antibody reactivity only include linear epitopes. Note: decimal values for RF scores on the Y-axis are converted to percent in the text.</p

Detailed reactivity maps for HCV non-structural proteins.

<p>Data represent individual RFscores for antibody, CD4⁺ and CD8⁺ T cell responses plotted for each antigen translated from the HCV H77 reference polyprotein: A–C = NS2; D–F = NS3; G–I = NS4a; J–L = NS4b; M–O = NS5a; P–R = NS5b. The red line denotes average frequency for all antibody, CD4 and CD8 T cell responses. Note: decimal values for RF scores on the Y-axis are converted to percent in the text.</p

Summary of immune epitope data for HCV.

<p>The data represent all antibody and T cell epitopes reported for all HCV genotypes in all hosts. MHC binding data has been excluded. Non-peptidic epitopes and MHC ligand elution structures have not been described for HCV to date. ‘References’ denotes the number of individual peer-reviewed papers reporting HCV epitope data. ‘Total structures’ include positive and negative peptides. ‘Epitopes’ represents all positive structures. Clustering was performed using default 80% sequence homology of positive epitopes using IEDB Search interface. Note: total numbers for individual T cell and antibody responses may be more than the number of total reported epitopes because many structures are reported as both T and antibody.</p