Vijay Prajapati 2019

Vijay Prajapati

Asst Prof Vijay Kumar Prajapati

Central University of Rajasthan, India

Evaluation of immunogenicity of the engineered multi-epitope subunit vaccine against visceral leishmaniasis infection


Poster Abstract

Background: The parasitic infection, visceral leishmaniasis (VL) is tropical neglected disease, whose severity ranges from adverse localized to chronic systemic infection. The predicaments arising due to the Leishmania infection is increasing day-by-day and affecting the world population. Hence, to overcome this severe complication, an instantaneous necessity is required for the development of safe and immunogenic vaccine against VL. 
Methodology: With the aid of immunoinformatic approaches, we screened the Leishmania membrane and secretory proteins and salivary proteins of sandfly (Phlebotomine argentipes) to obtain HTL, CTL and B cell epitopes. All these epitopes were sorted and used for vaccine designing along with suitable adjuvant namely RS09 (a TLR-4 agonist) and linkers. Further, multi-epitope subunit vaccine was evaluated on the different parameters like immunogenicity, physiological properties, and allergenicity for the safety and efficacy concern. Further, the 3D structure was predicted via ITASSER and the validation of 3D structure was assured by Ramachandran plot. Additionally, molecular docking and molecular dynamics simulation were conducted to evaluate the binding affinity of vaccine protein with TLR4 receptor. Besides the entire process, in silico cloning was also accomplished to check the effectiveness of designed vaccine construct in a suitable expression vector pET28a. To validate the immunogenicity of the designed vaccine candidate we have synthesized the gene and cloned it into the pET28a expression vector. Further, the cloned gene product was transformed into the BL21 bacterial strain for the protein expression purpose. 
Results: Following screening and sorting of 6 parasitic membranous and secretory proteins and 4 salivary proteins, total 24 CTL, 10 HTL and 10 B-Cell epitopes were obtained which were used to design the vaccine candidate of 483 amino acid residues. Physiological characterization reveals that the vaccine construct was stable, non-allergen and immunogenic in nature. The developed 3D model was found to be good with ≥90% residues in favoured region. The binding affinity between vaccine protein and TLR-4 receptor was -1009.1 and they form a stable complex as stated by dynamics simulation results. The CAI value obtained for the optimized codons were 1.0. Further, in silico cloning was performed in pET28a bacterial expression vector. For the validation of the work, we have cloned the synthesized vaccine candidate gene into pET28a expression vector. Further, the expression of desired protein was acheieved 
Conclusion: Novel immunoinformatic approaches were applied to design potential and antigenic multi- epitope subunit vaccine which was further subjected to the experimental examination. 



My lab is using system vaccinology to devise an immunogenic vaccine against parasitic infectious diseases such as visceral leishmaniasis and malaria infection. In the visceral leishmaniasis, we are using system vaccinology approaches which include different immunoinformatics tools to devise a potential and immunogenic vaccine molecule. Furthermore, we are doing the expression of this vaccine molecule in E.coli microbial system to perform the investigation of IFN-γ response to confirm the immunogenic response of engineered vaccine. Visceral leishmaniasis is caused by Leishmania donovani, so to devise the vaccine molecule we have collected the proteomics data of L.donovani parasites from UniPort server. We collected all the Leishmania secretory (Sci Rep 2017) and sandfly salivary (J Cell Biochem 2018) proteins and by using different immunoinformatics data predicted helper and cytotoxic T lymphocytes (HTL & CTL) epitopes and B cell epitopes and all these epitopes were assembled to engineer a possible vaccine molecule. Further, we performed molecular docking and molecular dynamics simulation against the receptor expressed on immune cells during Leishmania infection such as TLR-2 and TLR-4 to verify microscopic interaction. These studies confirm that engineered vaccine molecules are having good binding affinity with immune receptor and it will be able to develop the immunogenic response in human body. We have completed the vaccine protein expression study and in the process to investigate the immunogenic response such as IFN-γ response in the experimental model.