Designation Of The Novel Multi-epitope Vaccine Against Clostridium Difficile By Immunoinformatics Approach

Objective:A multi-epitope vaccine against Clostridium difficile infection(CDI)has been designed using the immunoinformatics approach to prevent CDI by blocking the adhesion of Clostridium dfficile spore and propagule to the host intestine.Methodology:(1)The amino acid sequences of CdeC,FliD and the adjuvant LT-Ⅱb were obtained from the National Center of Biotechnology Information(NCBI)database,and the PDB structures of TLR2,TLR4,TLR5,MHC Ⅰ and MHC Ⅱ molecules were downloaded from the Protein Data Bank(PDB).(2)NetCTL 1.2,NetMHCⅡ pan-3.2 and ABCpred servers were used to predict cytotoxic T lymphocyte(CTL)epitopes,Helper T lymphocytes(HTL)epitopes and linear B-cell epitopes,respectively.And the antigenicity,immunogenicity,allergenicity,toxicity and the similarity with human proteins of the epitopes were predicted by different servers.(3)The selected epitopes and adjuvant were connected with appropriate liners to design the multi-epitope vaccine.The physicochemical properties of the vaccine were further predicted by the ProtParam tool.Meanwhile,the different servers were used to predict the antigenicity,allergenicity,toxicity,the transmembrane helix region of the vaccine protein,and the probability of dissolubility of the vaccine protein upon overexpression in Escherichia coli(E.coli).(4)The Prabi and RaptorX servers were employed to predict the secondary and tertiary structures of the vaccine,respectively.The Galaxy WEB server was used to refine the vaccine’s tertiary structure,and the PROCHECK,ERRAT and ProSA-web servers were used to check the quality of the refined vaccine’s tertiary structure.(5)The HADDOCK 2.4 server and Rosetta v3.2 software were used to perform the molecular docking of the vaccine with TLRs and MHC molecules,and the Amberl8 software was used to perform the molecular dynamics(MD)simulation of the protein docking complex system.(6)The population coverage predicted server among the Immune Epitope Database(IEDB)was used to predict the population coverage of the multi-epitope vaccine in different areas.(7)To predict the immunogenicity of the multi-epitope vaccine,the computer immune stimulation of the multi-epitope vaccine was carried out using the C-IMMSIM server.(8)Using Escherichia coli(E.coli)K12 as the host,the JCAT online server was used to perform the reverse translation and Condon optimization of the vaccine,and the vaccination sequence was inserted into the pET28a(+)plasmid with the help of SnapGene software.Result:The final vaccine construct was composed of four parts,CTL epitopes,HTL epitopes,linear B cell epitopes and the LT-γb(adjuvant).The predicted results of the ProtParam tool showed that the multi-epitope vaccine contained 389 amino acids with a molecular weight of 41632.89Da,an isoelectric point(pI)of 9.11,an instability index of 31.98,an aliphatic index of 59.43,and a grand average of hydropathicity(GRAVY)of-0.585,indicating that the vaccine was stable,thermotolerant and hydrophilic in theory.Meanwhile,the predicted half-life of the multi-epitope vaccine was 30 hours(h)in mammalian reticulocytes,greater than 20 h in yeast,and greater than 10 h in E.coli.The antigenicity of the vaccine predicted by Vaxijenv2.0 and ANTIGENpro server was 0.97 and 0.94,respectively,showing that the vaccine had the strong binding ability to host immune system products in theory.Moreover,the designed vaccine was theoretically non-allergenic and non-toxic.When the vaccine sequence overexpressed in E.coli,the probability of dissolubility of the multi-epitope vaccine in E.coli was 0.61,and the vaccine was an extracellular protein,indicating that the multi-epitope vaccine protein could be solubly expressed and efficiently secreted in E.coli in theory.The secondary structure of the vaccine sequence consisted of 25.19%alpha-helices,55.27%random colis and 19.54%extended strands.The Ramachandran plot of the refined tertiary structure of the vaccine showed that 83.0%of residues situated in the most favored region,and only 0.9%lie in the disallowed region.The ERRAT score was 92.42 and the Z score predicted by ProSA-web was-5.01,which indicated that the side-chain and overall quality of the vaccine were good.The docked results and molecular dynamics simulation results between the vaccine and TLRs and MHC molecules showed that the multi-epitope vaccine could stably and flexibly bind to TLRs and MHC molecules in theory,thus inducing effective host immune responses.Theoretically,the population coverage rates of the multi-epitope vaccine in the world,Europe,the United States and China were 95.54%,96.84%,96.86%and 89.01%,respectively.Finally,the results of computer immune simulation suggested that the vaccine was strongly immunogenic in theory,and the codon optimization results showed that the vaccine theoretically could be well cloned and expressed with pET28a(+)as vector and E.coli as host.Conclusion:In this study,the multi-epitope vaccine designed by the immunoinformatics approach has good efficacy to prevent CDI in theory,but it still needs to be synthesized and further tested in vivo and in vitro to verify this conclusion.