Immunoinformatics design of multi-epitope vaccine using OmpA, OmpD and enterotoxin against non-typhoidal salmonellosis

Abstract

Background: Non-typhoidal Salmonella (NTS) is one of the important bacteria that cause foodborne diseases and invasive infections in children and elderly people. Since NTS infection is difficult to control due to the emergence of antibiotic-resistant species and its adverse effect on immune response, the development of a vaccine against NTS would be necessary. This study aimed to develop a multi-epitope vaccine against the most prevalent serovars of NTS (Salmonella Typhimurium, Salmonella Enteritidis) using an immunoinformatics approach and targeting OmpA, OmpD, and enterotoxin (Stn).

Results: Initially, the B cell and T cell epitopes were predicted. Then, epitopes and suitable adjuvant were assembled by molecular linkers to construct a multi-epitope vaccine. The computational tools predicted the tertiary structure, refined the tertiary structure and validated the final vaccine construct. The effectiveness of the vaccine was evaluated via molecular docking, molecular dynamics simulation, and in silico immune simulation. The vaccine model had good binding affinity and stability with MHC-I, MHC-II, and toll-like receptors (TLR-1, 2, 4) as well as activation of T cells, IgM, IgG, IFN-γ and IL-2 responses. Furthermore, after codon optimization of the vaccine sequence, this sequence was cloned in E. coli plasmid vector pET-30a (+) within restriction sites of HindIII and BamHI.

Conclusions: This study, for the first time, introduced a multi-epitope vaccine based on OmpA, OmpD and enterotoxin (Stn) of NTS that could stimulate T and B cell immune responses and produced in the prokaryotic system. This vaccine was validated in-silico phase which is an essential study to reduce challenges before in vitro and in vivo studies.

Keywords: Enterotoxin; Immunoinformatics; Multi-epitope vaccine; Non-typhoidal Salmonella; OmpA; OmpD.

Fig. 1

A. The final schematic multi-epitope vaccine with 237 amino acids. The adjuvant (Griselimycin) sequence was linked by EAAAK linker at both N and the C terminal of the vaccine the B cell, HTL and CTL epitopes were connected by GPGPG and AYY linkers respectively. B. The prediction of secondary structure and solubility of vaccine constructs. C. The analysis of secondary structure in SOPMA tool shows that the vaccine contains 36.29% alpha-helix, 13.08% extended strands, 6.75% beta turns and 43.88% random coils

Fig. 2

The 3D structure, refinement and validation of multi-epitope vaccine. A. The 3D structure of multi-epitope vaccine was predicted by I-TASSER server. B. The 3D structure of multi-epitope vaccine after refinement using GalaxyRefine server. C. Ramachardan plot indicating 89.3% of the residues were in the most favored region, 8.5% were in the allowed region, and 1.7% were in the disallowed region. D. The ERRAT quality factor was 84.4%. E. ProSA-web, with a Z score of -6.88

Fig. 3

1–5: Discontinuous B cell epitopes of multi-epitope vaccine predicted by ElliPro server. 1–4. The Yellow surface shows discontinuous B cell epitopes. 5. The residues and score discontinuous B cell epitopes

Fig. 4

The peptide-protein docking of vaccine model with HLA receptors. A. OmpA (ALDQLYSQLSNLDPK: red color) docked with HLA-DRB1 (green color). B. Stn (EAIFTPYFTE: red color) docked with HLA-C (green color)

Fig. 5

The protein–protein docking of vaccine model with TLR-1, TLR-2, TLR-4 and molecular dynamics simulation. A. Protein–protein docking of vaccine model with TLR-1, TLR-2 and TLR-4 by ClusPro 2.0 server B. Molecular dynamics simulation: deformability. C. B-factor. D. Eigenvalues (lower value means easier deformation). E. Variance (red color: individual variances and green color: cumulative variances). F. Covariance map (red: correlated, white: uncorrelated, blue: anti-correlated). G. Elastic network (darker regions mean stiffer regions)

Fig. 6

In silico immune system simulations produced by C-ImmSim server. A. Increase IgM and IgG responses (cream peak) and decrease antigen (black peak) after the second and third injection B. Activation of B cell population (purple peak) C. Increase memory B cells (green peak) D. Activation of TH cells (purple peak) E. Increase memory TH cells (green peak) (F). polarization of T cells response to Th1 (purple peak) G. Increase IFN-γ (purple peak) and IL-2 (cream peak) in response to the vaccine

Fig. 7

In-silico cloning of the multi-epitope vaccine sequence into the pET30a (+). The magenta area is place the vaccine sequence was inserted