A New Immunogenic Structure of Polyepitopic Fusion against Leishmania major: In Silico Study

Abstract

Background: The lack of complete protection against leishmaniasis and the challenges of anti-leishmaniasis drug treatment have made the treatment process more difficult. This study aimed to develop a new strategy for preparing a vaccine against cutaneous leishmaniasis using some of the antigenic proteins of the Leishmania parasite.

Methods: This study was carried out in 2022 at Shahid Chamran University of Ahvaz, Ahvaz, Iran. After preparing suitable epitopes of the Leishmania parasite and examining their antiparasitic properties, the process of making a fusion vaccine was performed and with the help of various bioinformatics tools, physicochemical and structural properties as well as immunological and simulation properties were studied and finally optimized. Construction and cloning were performed in the E.coli K12 system and finally, the docking process was performed with Toll-like receptors (TLRs), major histocompatibility complex I (MHC-I), and MHC-II receptors. With the help of selected epitopes of the Leishmania parasite, which had a high percentage of population coverage, a stable, antigenic, and non-allergenic chimeric vaccine was predicted.

Results: The results of the structural analysis of the TLR5\vaccine complex and simulation of its molecular dynamics showed a sufficiently stable binding. It also showed good potential for stimulation and production of active B cells and memory, as well as the potential for CD8+ T, CD4+ T cell production and development of Th2 and Th1-induced immune responses.

Conclusion: Computational results showed that the designed immunogenic structure has the potential to adequately stimulate cellular and humoral immune responses against Leishmania parasitic disease. As a result of evaluating the effectiveness of the candidate vaccine through in vivo and in vitro immunological tests, it can be suggested as a vaccine against Leishmania major.

Keywords: Bioinformatics; Chimeric vaccine; Immunogenicity; Leishmania.

Fig. 1:

Prediction of linear vaccine epitopes by IEDB server

Fig. 2:

Population coverage results of vaccine epitopes

Fig. 3:

Schematic diagram of the designed vaccine structure

Fig. 4:

Access level of the vaccine designed by the IEDB server

Fig. 5:

Graphical representation of secondary structure prediction of the multi-epitope vaccine. Here, the β-strands, α-helix, and random coils are indicated, and evaluation of the composition of the vaccine produced in terms of two-dimensional and three-dimensional structure with the help of software, A) psiPred and B) Prabi

Fig. 6:

Structural validation of the tertiary structure of the vaccine construct. A) Represents the Ramachandran plot of the refined model where the most favored, allowed regions are 97.1% using the Prochek server. B) Indicates ProSA-web validation of 3D structure showing Z-score (− 4.54)

Fig. 7:

Comparative image through PAYMOL alignment software between the two original and modified models vaccine after modifying and calculating the amount of RMSD between them

Fig. 8:

Residual interactions between vaccine structures and and molecules of A) TLR5, B) HLA.DRB1.0101, and C) HLA.A.0201, which shows hydrogen bonds and other bonds between the residues of the molecule and the residues of the vaccine

Fig. 9:

Molecular dynamics simulation, normal mode analysis, and receptor-ligand interactions using the iMODS server. A) This graph represents the deformability potential of each residue. The higher the peak, the higher will be the deformability. B) B-factor. C) Eigenvalue of vaccine-TLR5 complex. This graph shows the eigenvalue of the vaccine-TLR5, which interprets the ease of deforming the structure. The lower the eigenvalue, the higher the chance of deformation. D) Variance. E) Covariance matrix. Most of the region is red so it shows the correlated motion of residues; whereas, the blue region shows uncorrelated motion between residues. F) Elastic network model. In this graph, most of the atoms form stronger springs

Fig. 10:

Image of the results provided by the C-ImmSim server. The in-silico immune response induced by vaccine-TLR5. A) Production of several immunoglobulin subclasses (colored lines) in response to vaccine injection. B) B-cell richness after three injections. C) Evolution of T helper cells (Th cells) after the injections. D) Evolution of cytotoxic T cells (TC cells) after the injections. E) Production of cytokines and interleukins

Fig. 11:

A) In-silico cloning of fusion protein sequence into pET28a (+) vector using EcoRI and BamHI restriction enzymes. Blue-colored semicircle showing fusion protein sequence and black-colored semi-circles indicating pET28a (+) vector backbone. B) Image of vaccine cloning and electrophoresis process using SnapGene software