CD8 T Cell Epitope Distribution in Viruses Reveals Patterns of Protein Biosynthesis

<div><p>Distinguishing T cell epitope distribution patterns is relevant for epitope-vaccine design. To that end, we invest0069gated the distribution of known CD8 T cell epitopes from Hepatitis C Virus, Human Immunodeficiency Virus-1 and Influenza A Virus using χ² statistics. We found that epitopes are not distributed in the viral proteomes proportionally to the size of the source proteins. Specifically, capsid and matrix proteins pack significantly more epitopes than those expected by their size. Such non-homogeneous distribution cannot be accounted by underlying MHC I-peptide binding preferences nor it is related to sequence variability. Instead, we propose that it might be related to preferential protein translation/biosynthesis. Overall, these results support the prioritization of structural antigens for epitope identification and vaccine design.</p> </div

Correlation between epitope distribution and sequence conservation.

<p>For the proteomes of HCV (panel <b>A</b>), HIV (panel <b>B</b>) and IAV (panel <b>C</b>), we plot the ratio between observed and expected epitopes (Y-axis) against the corresponding conservation factors (<i>CF</i>)(X-axis).</p

Epitope map.

<p>The figure shows the localization of known CD8 T cell epitopes specific of HCV (Panel <b>A</b>), HIV (Panel <b>B</b>) and IAV (Panel <b>C</b>). Epitopes are shown as blue segments underneath of the relevant proteins. IAV proteins that are encoded by the same RNA segment are shown in near proximity. CD8 T cell epitopes used in this work range from 9 to 10 residues and they all differ in at least one amino acid residue (See Material and Methods for details). Therefore, those epitopes that match in the same or near the same location are either epitope variants or overlapping epitopes.</p

Distribution of predicted A*0201-binding peptides in HIV.

<p>Only A*0201-bindig peptides from HIV were not distributed homogeneously by protein size at the α value (0.001) used in the epitope analysis. In panel <b>A</b>, we show the contribution (in percentage) to the χ² statistics of each HIV protein and in panel <b>B</b>, the ratio between observed and expected A*0201-binding peptides.</p

Protein-size distribution of CD8 T cell epitopes in HCV, HIV and IAV.

<p>The expected epitopes in a given protein are those resulting after distributing all of the virus-specific epitopes proportionally to the length of that protein with regard to the total size of the relevant viral proteome.</p>*<p>Conservation Factor of each protein.</p

Protein-size distribution of virus-specific CD8 T cell epitopes.

<p>We depict for each of the viral proteins of HCV, HIV and IAV the contribution (in percentage) to the χ² statistics (Panel <b>A</b>) and the ratio between observed and expected epitopes (Panel <b>B</b>). A value greater than 1 indicates more observed epitopes than expected, while a value lower than 1 reflects fewer epitopes than expected.</p

χ²–statistics resulting of analyzing the distribution of MHC I-binding peptides in HCV, HIV and IAV.

*<p>Statistics obtained with sum of the peptides that are predicted to bind each MHC I molecule.</p

Protein-size distribution of Gag-specific CD8 T cell epitopes.

<p>In panel <b>A</b>, we show the contribution of p17, p24, p7 and p6 to the Gag χ² statistics and in panel <b>B</b>, the ratio between observed and expected epitopes.</p

pCMV-sHAPQ does not protect against lethal ASFV challenge.

<p>(A) Surviving kinetics of pCMV, pCMV-pCMV-PQ and pCMV-sHAPQ immunized pigs after lethal challenge with ASFV (10⁴ UHA of the E75 isolate). (B) Viremia kinetics of these same individuals after ASFV challenge (days 3, 5 and 7 post infection). Results are represented as the logarithm of HAU₅₀/ml serum (mean and standard deviation from each group are shown).</p

pCMV-sHAPQ induces specific antibody responses in pigs.

<p>14 pigs were divided into three groups receiving either: the pCMV-sHAPQ plasmid (6 pigs), the pCMV-PQ plasmid (4 pigs) or the pCMV empty plasmid as controls for the assay (4 pigs). Four pigs from each group were immunized three times and two extra pigs from the pCMV-sHAPQ immunized-group received a fourth dose of this same plasmid. (A) Sera collected 15 days after each DNA vaccine administration were used to follow by ELISA the kinetics of the specific anti-p30 antibodies induced after DNA immunization. (B) Sera collected 15 days after the last DNA vaccination was used to ELISA-titrate the anti-p30 antibodies induced. Data shown correspond to average O.D values and standard deviations obtained per each immunization group. (C) Confirmatory Western-blot using ASFV infected cell extracts as antigen. Immunoreactive bands correspond to the specific recognition of the immunodominant p30 protein (arrow) and to the diverse p54 isoforms (arrow head) found in these extracts, ranging the last ones between 22 and 27 KD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040942#pone.0040942-Rodriguez4" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040942#pone.0040942-Alcaraz2" target="_blank">[66]</a>. Figure shown corresponds to the results obtained with a representative serum (1∶100 dilution) obtained 15 days after the third immunization with pCMV (a), pCMV-PQ (b) or pCMV-sHAPQ (c). The anti-p30 monoclonal antibody (d) and the specific polyclonal anti-p54 antibody (e) confirm the specificity of the reactions.</p