Immunoinformatic approach to the design of a novel multi-epitope vaccine against Leishmania major fused to human IgG-Fc

Abstract

Background and purpose: Cutaneous leishmaniasis poses significant health and socioeconomic challenges, making vaccine development a top priority, especially in endemic regions. Cysteine proteases, KMP-11, and HASPB proteins are promising candidates for leishmaniasis vaccine development owing to their immunogenic properties and capacity to provoke robust immune responses, as evidenced by different investigations. This study aimed to design a recombinant chimeric protein (MEV-Fc) vaccine using multi-epitopes from these Leishmania major proteins.

Experimental approach: The antigens were subjected to immunoinformatic prediction and screening of HTL, CTL, and B-cell epitopes. The multi-epitope protein was designed with significantly high-scoring epitopes and suitable linkers. Natural adjuvants were then added to enhance immunogenicity. Vaccine potency was innovatively improved by covalently fusing human IgG1 Fc with multi-epitope protein. To investigate how the MEV-Fc vaccine interacts with Toll-like receptors, molecular docking, multi-scale normal mode analysis simulation, and computational immune simulation were employed to study humoral and cellular immune responses.

Findings/results: The results demonstrated the vaccine's antigenicity, stability, and nontoxicity. The structural validation confirmed the accuracy of the 3D models, indicating robust interactions with TLR2 and TLR4, with binding free energies of -1269.9 and -1128.7 (kcal/mol), respectively. Immune simulation results showed significant increases in IgM and IgG antibody levels following three vaccinations, along with enhanced activation of B cells, helper T cells, and cytotoxic T lymphocytes.

Conclusion and implications: These findings provide novel insights for developing effective candidates for cutaneous leishmaniasis vaccines. However, laboratory experiments are necessary to evaluate its protective effects.

Keywords: Adjuvant; IgG-Fc; Immunodominant epitopes; Leishmania major; Subunit vaccine.

Fig. 1

(A) Schematic diagram of the vaccine (MEV-Fc). (B) MEV-Fc amino acid sequence. MEV-FC, Multi-epitope Vaccine-Fc fragment; HTL, Helper T-lymphocyte; CTL, cytotoxic T-lymphocyte.

Fig. 2

3D structure model prediction and validation. (A) Visualization of a 3D model for the multi-epitope vaccine-Fc fragment using PyMOL software; (B) the Ramachandran plot depicts the distribution of amino acid residues in areas that are favored (red color), allowed (yellow/faint yellow color), and disallowed (white color); (C) Z-score plot for the predicted 3D model using ProSA-web; (D) energy plot for each residue in the predicted model.

Fig. 3

Molecular docking interaction analysis of the vaccine and TLR-2. (A) The outcome visualization of molecular docking of the vaccine structures (cyan) and TLR-2 receptor (red) derived from the ClusPro; (B) more investigation into the multi-epitope vaccine-Fc fragment-TLR2 complex interactions and the generation of two-dimensional pictures of these complexes using the Ligplot+ visualization program. TLR2, Toll-like receptor 2.

Fig. 4

Molecular docking interaction analysis of the vaccine and TLR-4. (A) The outcome visualization of molecular docking of the vaccine structures (cyan) and TLR-4 receptor (red) derived from the ClusPro; (B) more investigation into the multi-epitope vaccine-Fc fragment-TLR2 complex interactions and the generation of two-dimensional pictures of these complexes using the Ligplot+ visualization program. TLR4, Toll-like receptor 4.

Fig. 5

The findings of the molecular dynamics simulation of MEV-Fc-TLR2. (A) Deformability analysis; (B) the simulation’s eigenvalues; (C) variance plots display individual variances in red and cumulative variances in green; (D) comparison of B-factors; (E) covariance map, illustrating movements that are correlated (red), uncorrelated (white), and anticorrelated (blue); (F) the depiction of an elastic network, with darker gray regions suggesting more stiffness.

Fig. 6

C-ImmSim server-based immune simulation. (A) Immunoglobulin levels in various states following antigen stimulation; (B) various B-cell subtype distributions in response to antigen stimulation; (C) CD4 HTL generation in response to antigen stimulation; (D) distribution of CD4 HTL according to their activation status following antigen stimulation; (E) the total (active and resting) cytotoxic T lymphocyte count; (F) cytokine production profile following antigen stimulation. CT, cytotoxic T cells; HTL, Helper T-lymphocyte.

Fig. 7

In silico cloning. The MEV-Fc sequence (highlighted in red) was successfully inserted into the pET28-a(+) expression vector at the NdeI and XhoI restriction endonuclease cleavage sites. MEV-Fc, Multi-epitope vaccine-Fc fragment.