Introduction: Canine monocytic ehrlichiosis is an emerging tick-borne disease among dogs around the world, including Iran. It is caused by infection of monocytes/macrophages with a bacterium called Ehrlichia chaffeensis. This obligate intracellular Gram-negative bacterium can enter the white blood cells where it reproduces and infect the lymph nodes, spleen, liver and bone marrow. The chronic phase of the illness can lead to pancytopenia (very low blood cell counts), bleeding, lameness, neurological and ophthalmic disorders, and kidney disease and even death. So far, Treatment options are limited to the broad-spectrum antibiotic doxycycline and no vaccine is currently available. The best strategy for managing the disease is to interrupt the cycle of infection by directing the immune responses toward key virulence factors as potential targets by vaccination. Genomic and proteomic data relevant to the living organism immune systems and diseases and wide variety of bioinformatics tools have given rise to the field known as immunoinformatics, and in particular reverse vaccinology approach. This method is useful in the development of multi-epitope vaccine which is generally considered to be safe in comparison to conventional vaccine design approaches such as attenuated or subunit vaccines that have high risk of toxicity and autoimmunity. Our study aimed to design a vaccine construct against E. chaffeensis based on reverse vaccinology for the first time.
Methods: In this study, OMP-1B which is a major outer membrane protein in E. chaffeensis was selected as an antigen to obtain epitopes. Antigenicity of the selected antigen was predicted by the VaxiJen v2.0 server and the default threshold of 0.5 for bacteria was maintained. Prediction of B-cell and T-cell epitopes from selected target proteins is done through ABCpred and IEDB MHC I binding tool online servers. For quality prediction of formation of MHC-epitope complex structures GalaxyPepDock server was employed to perform docking of DLA-8803401, DLA-8850101, and DLA-8850801 with CTLs. Furthermore, the potent epitopes were screened on the basis of antigenicity, and toxicity, as evaluated by VaxiJen v2.0 and ToxinPred servers. The predicted antigenic epitopes were linked together and then fused to Beta-defensin as adjuvant to enhance immunogenicity of the vaccine. It linked by the “EAAAK” linker followed by CTL epitopes which were linked together by the “AYY” linkers. Further, the B cell epitopes were linked by KK linkers. The ProtParam server was used to determine the physical and chemical characteristics of the final vaccine. I-TASSER server was used to generate the 3D model of linear vaccine construct. Then, the refinement of 3D structure model was done using GalaxyRefine Server. The tertiary structure was validated using ERRAT score followed by ProSA-web analysis. Further, the overall quality of the generated model of vaccine was determined by Ramachandran plot analysis using Procheck server. Also, interactions of the vaccine with TLR5 is studied as it induce immune response when activated by the vaccine. FTsite server was utilized for predicting the active residues for the interactions. The docking of was performed by HADDOCK 2.4. The best cluster was chosen from the docked clusters based on lowest HADDOCK score. HADDOCK Refinement Interface was used to refine the chosen cluster. The best structure after refinement was chosen and their binding affinity was calculated using PRODIGY web server. Then, the interacting residues between the vaccine and the TLR5 was mapped using PDBsum. Finally, the recombinant plasmid was designed by inserting the adapted codon sequence into pET-28a (+) vector using SnapGene software.
Results: Through epitope mapping 8 T-cell and 5 B-cell were predicted and fused together. Further analysis showed that the constructed vaccine was highly antigenic, and non-toxic with a molecular mass of 35.3kDa. The 3D model depicted that 82.8% of residues are in favorable region, 13.5% of residues in allowed region and 3.7% in outer region according to Ramachandran plot analysis. Further analyses by ProSA revealed Z- score is - 1. Moreover, the refined model score was 51.4 in quality check analysis through ERRAT. All these results showed that the refined model have good quality. Also, the results of docking demonstrated that the vaccine is potentially able to interact with TLR5 and is energetically feasible.
Conclusion: Overall, the designed vaccine holds promise as a potential preventive approach for Canine monocytic ehrlichiosis.