Analysis of CadF sequences
The complete sequences of CadF protein contained 319 amino acids, and multiple sequence alignment confirmed that this protein was a highly conserved protein among Campylobacter species. It was shown to belong to the outer member proteins superfamily (ompA), and an ompA-like domain was identified in the 193-287 position of the protein. The result of the phylogenic tree also confirmed CadF classification in the outer membrane proteins superfamily (data not shown).
Characterization of CadF
Using the ProtParam server, the MV (molecular weight) and pI (isoelectric point) parameters were determined as 35979.04 Da and 5.89, respectively. The aliphatic index was 69.12, and the GRAVY (grand average of hydropathicity) of the protein was -0.679. As a result, the amino acids of CadF protein had hydrophobicity and acidity properties (pI≤7.35). The aliphatic index included alanine, valine, isoleucine, and leucine amino acids, indicating the thermostability of the protein. Moreover, the TMHMM server data analysis results also confirmed that CadF was an outer membrane protein. According to the obtained result from the Phyre2 server, it was predicted that CadF was a stable target.
The PSIPRED server showed the graphical results of secondary structures of the protein, indicating a sheet, helix, and extracellular transmembrane structure. In addition, the Phyre2 server showed the three-dimensional structure of the modeled CadF with a 97% confidence score and 192 known-domain alignments. The structural content included 16% alpha-helix, 41% beta strands, and 16% disordered regions. Also, the prediction of CadF protein showed a binding site at glutamate-histidine-lysine residues and a large amount of metallic heterogenic sections in its structure.
Evaluation of antigenicity and allergenicity
The score of antigenic prediction of CadF protein was calculated as ~0.79 by the VaxiJen server. The results showed that the protein was probably an antigen and could be used for further analysis. The AllergenFP server data indicated the highest Tanimoto similarity index of 0.82 for the protein; therefore, it could not be an allergen. The AllerTOP server data analysis results also confirmed the finding.
Prediction of T and B cell epitopes
The best score for predicting T cell epitopes was selected from the SYFPEITH, IEDB, NetCTL, NHLAPred, and NetMHC servers. The epitopes of MHC I (A 0101, A 0201, and B 2705) and MHC II (DRB1 0101 and DRB1 0401) were the most frequent epitopes among Iranian alleles that were considered in this study. Using the Kolaskar & Tongaonkar Antigenicity method on the IEDB server, a graph was plotted, suggesting the yellow areas as B cell epitopes (Supplementary File A). According to the obtained results, some epitopes such as VLFGADNNV, GLASVLFGA, LSDSLALRL, etc. were the most common epitopes among T and B cells. Further detailed information about the predicted MHC I, II, and B cell epitopes are presented in Tables 1a, 1b, and 2, respectively. Overall, the results showed that the best epitope was LSDSLALRL located in 136-144 regions of CadF with antigenic properties and no allergenic specifications. The three-dimensional structure of the final epitope painted by the PyMOL is showed in Supplementary File B. Therefore, it was suggested as a candidate vaccine for further analysis.
Analysis of docking
The bindings of the best epitope to the desired HLA molecules were observed by Molecular Virtual Docker software, and five models were estimated. The proposed models showed the interaction of the epitope side chains with the cavities in the groove of MHC I and II (Supplementary File C). The energies of the bonding models resulted from the binding of LSDSLALRL peptide to HLA-A 0101 consisted of -26.18, -18.62, -12.77, and -12.56 kcal/mol. The best scores of the peptide docking to HLA-DRB1 0101 were computed as -109.86, -99.52, -98.40, and -85.79 kcal/mol. According to the principles of docking energy evaluation, the model with the most negative docking results was selected as the best model with energies of -26.18 and -109.86 kcal/mol, which were related to HLA-A 0101 of MHC I and HLA-DRB1 0101 of MHC II, respectively.