Development of a broad-spectrum epitope-based vaccine against Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (SPN) is a significant pathogen causing pneumonia and meningitis, particularly in vulnerable populations like children and the elderly. Available pneumonia vaccines have limitations since they only cover particular serotypes and have high production costs. The emergence of antibiotic-resistant SPN strains further underscores the need for a new, cost-effective, broad-spectrum vaccine. Two potential vaccine candidates, CbpA and PspA, were identified, and their B-cell, CTL, and HTL epitopes were predicted and connected with suitable linkers, adjivant and PADRE sequence. The vaccine construct was found to be antigenic, non-toxic, non-allergenic, and soluble. The three-dimensional structure of the vaccine candidate was built and validated. Docking analysis of the vaccine candidate by ClusPro demonstrated robust and stable binding interactions between the MEV and toll-like receptor 4 in both humans and animals. The iMOD server and Amber v.22 tool has verified the stability of the docking complexes. GenScript server confirmed the high efficiency of cloning for the construct and in-silico cloning into the pET28a (+) vector using SnapGene, demonstrating successful translation of the epitope region. Immunological responses were shown to be enhanced by the C-IMMSIM server. This study introduced a strong peptide vaccine candidate that has the potential to contribute to the development of a rapid and cost-effective solution for combating SPN. However, experimental verification is necessary to evaluate the vaccine's effectiveness.

Fig 1. Multi-epitope vaccine development process against streptococcus pneumoniae.

This flowchart outlines the key steps in developing a multi-epitope vaccine targeting S. pneumoniae, highlighting the tools and servers used at each stage.

Fig 2. Population coverage analysis of predicted epitopes.

Assessment of the population coverage achieved by the predicted epitopes for MHC-I (A), MHC-II (B), and combined MHC-I and MHC-II (C) alleles. The horizontal axis in the graph depicts the frequency of epitope hits per recognized HLA combination, while the vertical axis represents the proportion of persons and the total proportion of population coverage.

Fig 3. Multi-epitope vaccine construct design.

(A) Schematic representation of the multi-epitope vaccine construct, illustrating the arrangement of B-cell, HTL, and CTL epitopes. (B) Sequence of the final vaccine construct, which is 327 amino acids in length. The construct incorporates various components linked together, including an adjuvant, PADRE sequence, EAAAK, GGGGS, GPGPG, and KK linkers.

Fig 4. Quality assessment of the refined vaccine construct.

Various assessments confirmed the high quality of the refined structure. (A) Refined 3D structure of the vaccine construct, (B) Ramachandran plot analysis, (C) Z-score distribution, (D) ERRAT quality factor, (E) VERIFY-3D score. These results indicate that the refined structure is accurate and reliable, providing a solid foundation for further analysis and simulations.

Fig 5. Vaccine constructs with mutated structure.

The left panel shows the original vaccine construct, while the right panel displays the mutant version. The white arrows in the mutant structure highlight the locations where disulfide bonds (yellow) were introduced, leading to structural alterations.

Fig 6. Predicted B-cell epitopes in the vaccine construct.

The epitopes are visualized as blue and pink spheres on the 3D structure of the protein. The number of residues in each epitope and their corresponding scores are indicated.

Fig 7. Molecular interactions of the vaccine construct with TLR4.

(A) Visual representation of the interactions between the vaccine construct and mouse TLR4 (B) Visual representation of the interactions between the vaccine construct and human TLR4.

Fig 8. Normal mode analysis of human and mouse TLR4-vaccine complexes.

Molecular dynamics simulations of the vaccine constructs with human TLR4 (left column) and mouse TLR4 (right column). (A) Deformability, (B) B-factor, (C) eigenvalues, (D) variance map, (E) covariance map, and (F) elastic network analysis. These results provide insights into the flexibility, stability, and interactions within the vaccine constructs and their complexes with TLR4.

Fig 9. Molecular dynamics simulation of MEVC-human TLR4 complex.

Molecular dynamics simulation of the MEVC-Human TLR4 complex. (A) Root-mean-square deviation (RMSD) of the protein backbone over time. (B) RMSF of individual residues. (C) Hydrogen bond formation and breakage. (D) Radius of gyration (Rg) over time.

Fig 10. Molecular dynamics simulation of MEVC-mice TLR4 complex.

Molecular dynamics simulation of the MEVC-Mice TLR4 complex. (A) Root-mean-square deviation (RMSD) of the protein backbone over time. (B) RMSF of individual residues. (C) Hydrogen bond formation and breakage. (D) Radius of gyration (Rg) over time.

Fig 11. Immune response simulation to vaccine antigen.

The immunological response to the vaccine antigen after three doses given at intervals of 1, 84, and 168 hours was simulated using the C-ImmSim server. The results depict the dynamics of various immune cell populations and parameters, including: (A) antigen and immunoglobulin levels, (B) B cell population, (C) B cell population distribution by state, (D) helper T cell population, (E) cytotoxic T cell population distribution by state, (F) cytokine production, (G) total cytotoxic T cell population, (H) macrophage population distribution by state, and (I) dendritic cell population distribution by state. The conducted simulations offer significant insights into the prospective effectiveness and safety of the vaccination candidate.

Fig 12. Computational cloning of vaccine construct into pET28a (+) vector.

(A) A diagram illustrating the cloning process, showing the pET28a (+) vector, the vaccine construct fragment, and the final cloned product. Between the NdeI and XhoI restriction sites, the vaccine fragment is introduced. (B) Detailed map of the final construct, indicating the various restriction enzyme sites, promoters, terminators, and other elements. The vaccine construct is highlighted in red. The total length of the final construct is 6265 base pairs.