Identification of bovine MHC class II reactive peptides that are specific/unique to paratuberculosis and conserved across pathogenic variations of the paratuberculosis proteome will be of high value for development of new vaccines and immune based diagnostics. Here, we present an in silico screening procedure that can identify such peptides. In short, the procedure identifies 20mer peptides that fulfill the following criteria a) Conserved in a set of positive (i.e. pathogenic) genomesb) Absent in a set of negative genomes (i.e. non-pathogenic or related pathogen). c) High density of Bovine MHC-II epitopes As positive genomes were used full genome sequences of two Mycobacterium avium subsp. paratuberculosis strains, strain K-10 and the newly sequenced strain Ejlskov2007. As negative genomes were used five complete genomes of Mycobacterium tuberculosis, bovis and avium. All reading frames were virtually translated and converted into overlapping sub-peptide of length 20. Peptides fully conserved in the positive strains were selected as potential positive hits. All positive hits were compared to the set of 20mers peptides from the negative strains and discarded if an overlap of 8 consecutive amino acids was found. This resulted in approximately 80000 20mers that were 100% conserved between the two positive strains, sharing no 8mer overlap to any negative genome. Residual 20mers were next in-silico checked for binding to each of five prevalent bovine class-II MHC molecules using the NetMHCII-pan method . Studies covering Bovine ligands from public data-resources revealed that this method, even though trained solely on human HLA-DR binding data, could to a large degree predict the binding specificity of Bovine MHC-II molecules. The 51 peptides with the highest predicted binding affinity averaged over all five MHC alleles were selected for experimental validation in EliSpot assays with blood from paratuberculosis infected cattle. The experimental validation is currently awaiting termination. 1. Larsen, T.S. and A. Krogh, EasyGene-a prokaryotic gene finder that ranks ORFs by statistical significance. BMC Bioinformatics, 2003. 4: p. 21. 2. Nielsen, M., et al., Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan. PLoS Comput Biol, 2008. 4(7): p. e1000107.
|Publication status||Published - 2009|
|Event||10th International Colloquium on Paratuberculosis - Minneapolis, MN, United States|
Duration: 9 Aug 2009 → 13 Aug 2009
Conference number: 10
|Conference||10th International Colloquium on Paratuberculosis|
|Period||09/08/2009 → 13/08/2009|