Additional file 3: of NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets

Allele-specific length preference for 24 MHC molecules characterized by 20 or more ligand data points for the allmer and 9mer prediction methods compared to the length preference in the SYFPEITHI data. Length profiles for the allmer and 9mer methods were estimated as described in the text. (XLSX 50 kb

Example of output from the <i>NNAlign</i> server trained on MHC class II binding data for allele HLA-DRB1*0101.

<p>Links on the results page (in pink) redirect to additional files and figures relevant for the analysis. Run ID is a sequential identifier for the current job, and Run Name a user-defined prefix that is added to all files of the run. The “view data distribution” link shows the transformation applied to the data in pre-processing, which can be either a linear or logarithmic transformation. In this case the method was trained with a motif length of 9, including a PFR of size 3 to both ends of the peptide, and encoding in the network input layer peptide length and PFR length. The hidden layer was made of a fixed number of 20 neurons. Peptides were presented to the networks using a Blosum encoding to account for amino acid similarity, for 500 hundred iterations per peptide without stopping on the best test set performance. At each cross-validation step, 10 networks were trained starting from 10 different initial configurations. The subsets for cross-validation were constructed using a Hobohm1 method that groups in the same subset sequences that align with more than 80% identity (thr = 0.8). The model can be downloaded to disk using the dedicated link, and can be resubmitted to <i>NNAlign</i> to find occurrences of the learned pattern in new data. The estimated performance of the trained method is expressed in terms of Root Mean Square Error, Pearson and Spearman correlation. A visual representation of the correlation can be obtained from the scatterplot of predicted versus observed values. The “complete alignment core” link allows downloading the prediction values in cross-validation for each peptide, and where the core was placed within the peptides. Next follows a section on the sequence logo, showing a logo representation of the binding motif learned by the network ensemble. If the relative option is selected, links to logos for the individual networks in the final ensemble are also listed here. Finally, if an evaluation set is uploaded, an additional section shows performance measures and core alignment for these data.</p

Identification of optimal motif length using the <i>NNAlign</i> method.

<p><b>Left panel:</b> Histogram of the optimal motifs lengths for the 14 HLA-DR molecules in the Wang dataset as identified by the <i>NNAlign</i> method. Right panel: Predictive performance measured in terms of the root mean square error (RMSE) between observed and predicted values as a function of the motif length for the two molecules DRB1*0101 and DRB1*1501. <i>NNAlign</i> was trained using the same parameters settings described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026781#pone-0026781-g004" target="_blank">Figure 4</a>. At each motif length are shown the mean and standard error of the mean RMSE as estimated by bootstrap sampling. For DRB1*0101 a single consistent optimal motif length of 9 amino acids is found. For DRB1*1501 all motif length 8–11 had statistically indistinguishable performance (paired t-test).</p

Analysing high-density peptide array data with <i>NNAlign</i>.

<p>a) Fluorescence microscopy picture of a peptide microarray. The image is a magnified segment of the peptide chip used in the trypsin cleavage analysis. b) Trypsin peptide-chip data. The normalized observed (target) likelihood of cleavage as a function of the prediction score for the trypsin data set. Localizations of peptides containing the pairs of amino acids RP, RA or RR are highlighted in the plot. Proline (P) is known to prevent cleavage after arginine (R), whereas cleavage is observed with other amino acids such as R and A. c) Chymotrypsin peptide-chip data. Correlation plot between predicted and measured (target) data from the chymotrypsin data set. Values are binned by their x,y proximity, so that the scatterplot represents the density of data in each bin. <i>NNAlign</i> was trained with linear rescaling of the quantitative data, a motif length of 4 amino acids without inclusion of PFR encoding, Blosum encoding of peptide sequences, a combination of 3,7,15 hidden neurons, 10 initial seeds, 5-fold exhaustive cross-validation, training was stopped on the best test set performance.</p

table_2_Predicting HLA CD4 Immunogenicity in Human Populations.xlsx

Background<p>Prediction of T cell immunogenicity is a topic of considerable interest, both in terms of basic understanding of the mechanisms of T cells responses and in terms of practical applications. HLA binding affinity is often used to predict T cell epitopes, since HLA binding affinity is a key requisite for human T cell immunogenicity. However, immunogenicity at the population it is complicated by the high level of variability of HLA molecules, potential other factors beyond HLA as well as the frequent lack of HLA typing data. To overcome those issues, we explored an alternative approach to identify the common characteristics able to distinguish immunogenic peptides from non-recognized peptides.</p>Methods<p>Sets of dominant epitopes derived from peer-reviewed published papers were used in conjunction with negative peptides from the same experiments/donors to train neural networks and generate an “immunogenicity score.” We also compared the performance of the immunogenicity score with previously described method for immunogenicity prediction based on HLA class II binding at the population level.</p>Results<p>The immunogenicity score was validated on a series of independent datasets derived from the published literature, representing 57 independent studies where immunogenicity in human populations was assessed by testing overlapping peptides spanning different antigens. Overall, these testing datasets corresponded to over 2,000 peptides and tested in over 1,600 different human donors. The 7-allele method prediction and the immunogenicity score were associated with similar performance [average area under the ROC curve (AUC) values of 0.703 and 0.702, respectively] while the combined methods reached an average AUC of 0.725. This increase in average AUC value is significant compared with the immunogenicity score (p = 0.0135) and a strong trend toward significance is observed when compared to the 7-allele method (p = 0.0938). The new immunogenicity score method is now freely available using CD4 T cell immunogenicity prediction tool on the Immune Epitope Database website (http://tools.iedb.org/CD4episcore).</p>Conclusion<p>The new immunogenicity score predicts CD4 T cell immunogenicity at the population level starting from protein sequences and with no need for HLA typing. Its efficacy has been validated in the context of different antigen sources, ethnicities, and disparate techniques for epitope identification.</p

table_4_Predicting HLA CD4 Immunogenicity in Human Populations.xlsx

Background<p>Prediction of T cell immunogenicity is a topic of considerable interest, both in terms of basic understanding of the mechanisms of T cells responses and in terms of practical applications. HLA binding affinity is often used to predict T cell epitopes, since HLA binding affinity is a key requisite for human T cell immunogenicity. However, immunogenicity at the population it is complicated by the high level of variability of HLA molecules, potential other factors beyond HLA as well as the frequent lack of HLA typing data. To overcome those issues, we explored an alternative approach to identify the common characteristics able to distinguish immunogenic peptides from non-recognized peptides.</p>Methods<p>Sets of dominant epitopes derived from peer-reviewed published papers were used in conjunction with negative peptides from the same experiments/donors to train neural networks and generate an “immunogenicity score.” We also compared the performance of the immunogenicity score with previously described method for immunogenicity prediction based on HLA class II binding at the population level.</p>Results<p>The immunogenicity score was validated on a series of independent datasets derived from the published literature, representing 57 independent studies where immunogenicity in human populations was assessed by testing overlapping peptides spanning different antigens. Overall, these testing datasets corresponded to over 2,000 peptides and tested in over 1,600 different human donors. The 7-allele method prediction and the immunogenicity score were associated with similar performance [average area under the ROC curve (AUC) values of 0.703 and 0.702, respectively] while the combined methods reached an average AUC of 0.725. This increase in average AUC value is significant compared with the immunogenicity score (p = 0.0135) and a strong trend toward significance is observed when compared to the 7-allele method (p = 0.0938). The new immunogenicity score method is now freely available using CD4 T cell immunogenicity prediction tool on the Immune Epitope Database website (http://tools.iedb.org/CD4episcore).</p>Conclusion<p>The new immunogenicity score predicts CD4 T cell immunogenicity at the population level starting from protein sequences and with no need for HLA typing. Its efficacy has been validated in the context of different antigen sources, ethnicities, and disparate techniques for epitope identification.</p

table_3_Predicting HLA CD4 Immunogenicity in Human Populations.xlsx

Background<p>Prediction of T cell immunogenicity is a topic of considerable interest, both in terms of basic understanding of the mechanisms of T cells responses and in terms of practical applications. HLA binding affinity is often used to predict T cell epitopes, since HLA binding affinity is a key requisite for human T cell immunogenicity. However, immunogenicity at the population it is complicated by the high level of variability of HLA molecules, potential other factors beyond HLA as well as the frequent lack of HLA typing data. To overcome those issues, we explored an alternative approach to identify the common characteristics able to distinguish immunogenic peptides from non-recognized peptides.</p>Methods<p>Sets of dominant epitopes derived from peer-reviewed published papers were used in conjunction with negative peptides from the same experiments/donors to train neural networks and generate an “immunogenicity score.” We also compared the performance of the immunogenicity score with previously described method for immunogenicity prediction based on HLA class II binding at the population level.</p>Results<p>The immunogenicity score was validated on a series of independent datasets derived from the published literature, representing 57 independent studies where immunogenicity in human populations was assessed by testing overlapping peptides spanning different antigens. Overall, these testing datasets corresponded to over 2,000 peptides and tested in over 1,600 different human donors. The 7-allele method prediction and the immunogenicity score were associated with similar performance [average area under the ROC curve (AUC) values of 0.703 and 0.702, respectively] while the combined methods reached an average AUC of 0.725. This increase in average AUC value is significant compared with the immunogenicity score (p = 0.0135) and a strong trend toward significance is observed when compared to the 7-allele method (p = 0.0938). The new immunogenicity score method is now freely available using CD4 T cell immunogenicity prediction tool on the Immune Epitope Database website (http://tools.iedb.org/CD4episcore).</p>Conclusion<p>The new immunogenicity score predicts CD4 T cell immunogenicity at the population level starting from protein sequences and with no need for HLA typing. Its efficacy has been validated in the context of different antigen sources, ethnicities, and disparate techniques for epitope identification.</p

image_1_Predicting HLA CD4 Immunogenicity in Human Populations.PDF

Background<p>Prediction of T cell immunogenicity is a topic of considerable interest, both in terms of basic understanding of the mechanisms of T cells responses and in terms of practical applications. HLA binding affinity is often used to predict T cell epitopes, since HLA binding affinity is a key requisite for human T cell immunogenicity. However, immunogenicity at the population it is complicated by the high level of variability of HLA molecules, potential other factors beyond HLA as well as the frequent lack of HLA typing data. To overcome those issues, we explored an alternative approach to identify the common characteristics able to distinguish immunogenic peptides from non-recognized peptides.</p>Methods<p>Sets of dominant epitopes derived from peer-reviewed published papers were used in conjunction with negative peptides from the same experiments/donors to train neural networks and generate an “immunogenicity score.” We also compared the performance of the immunogenicity score with previously described method for immunogenicity prediction based on HLA class II binding at the population level.</p>Results<p>The immunogenicity score was validated on a series of independent datasets derived from the published literature, representing 57 independent studies where immunogenicity in human populations was assessed by testing overlapping peptides spanning different antigens. Overall, these testing datasets corresponded to over 2,000 peptides and tested in over 1,600 different human donors. The 7-allele method prediction and the immunogenicity score were associated with similar performance [average area under the ROC curve (AUC) values of 0.703 and 0.702, respectively] while the combined methods reached an average AUC of 0.725. This increase in average AUC value is significant compared with the immunogenicity score (p = 0.0135) and a strong trend toward significance is observed when compared to the 7-allele method (p = 0.0938). The new immunogenicity score method is now freely available using CD4 T cell immunogenicity prediction tool on the Immune Epitope Database website (http://tools.iedb.org/CD4episcore).</p>Conclusion<p>The new immunogenicity score predicts CD4 T cell immunogenicity at the population level starting from protein sequences and with no need for HLA typing. Its efficacy has been validated in the context of different antigen sources, ethnicities, and disparate techniques for epitope identification.</p