Evaluation of the consistence between the results of immunoinformatics predictions and real-world animal experiments of a new tuberculosis vaccine MP3RT

Abstract

Background: Our previous study developed a novel peptide-based vaccine, MP3RT, to fight against tuberculosis (TB) infection in a mouse model. However, the consistency between the immunoinformatics predictions and the results of real-world animal experiments on the MP3RT vaccine remains unclear.

Method: In this study, we predicted the antigenicity, immunogenicity, physicochemical parameters, secondary structure, and tertiary structure of MP3RT using bioinformatics technologies. The immune response properties of the MP3RT vaccine were then predicted using the C-ImmSim server. Finally, humanized mice were used to verify the characteristics of the humoral and cellular immune responses induced by the MP3RT vaccine.

Results: MP3RT is a non-toxic and non-allergenic vaccine with an antigenicity index of 0.88 and an immunogenicity index of 0.61, respectively. Our results showed that the MP3RT vaccine contained 53.36% α-helix in the secondary structure, and the favored region accounted for 98.22% in the optimized tertiary structure. The binding affinities of the MP3RT vaccine to the human leukocyte antigen (HLA)-DRB1*01:01 allele, toll-like receptor-2 (TLR-2), and TLR-4 receptors were -1234.1 kcal/mol, -1066.4 kcal/mol, and -1250.4 kcal/mol, respectively. The results of the C-ImmSim server showed that the MP3RT vaccine could stimulate T and B cells to produce immune responses, such as high levels of IgM and IgG antibodies, IFN-γ, TNF-α, and IL-2 cytokines. Results from real-world animal experiments showed that the MP3RT vaccine could stimulate the humanized mice to produce high levels of IgG and IgG2a antibodies and IFN-γ⁺ T lymphocytes. Furthermore, the levels of IFN-γ, IL-2, and IL-6 cytokines in mice immunized with the MP3RT vaccine were significantly higher than those in the control group.

Conclusion: MP3RT is a highly antigenic and immunogenic potential vaccine that can effectively induce Th1-type immune responses in silico analysis and animal experiments. This study lays the foundation for evaluating the value of computational tools and immunoinformatic techniques in reverse vaccinology research.

Keywords: MP3RT; computational tools; immune responses; immunoinformatics; tuberculosis (TB); vaccine.

Figure 1

The solubility, secondary structure, tertiary structure, and optimized tertiary structure of the MP3RT vaccine. (A) Solubility of the MP3RT vaccine was predicted by the Protein–Sol server. (B) Secondary structure of the MP3RT vaccine was predicted by the PSIPRED server. (C) The 3Dpro serve was used to predict the tertiary structure of the MP3RT vaccine before optimization. (D) The optimized tertiary structure of the MP3RT vaccine was predicted by the Galaxy WEB server. The colored parts in the picture are improvements to all sidechain structures that initialize the model, improving the structure accuracy and physical correctness of the global and partial parts through repeated relaxation.

Figure 2

Ramachandran diagrams for the MP3RT vaccine. Ramachandran map is a method of visualizing energy preference regions, which can be used to see if protein structure is reasonable. The dark green area is the favored region. The other two green areas are the outlier region. The white area is the rotamer region. The rotamer region can be interpreted as the high-energy region, and the protein needs to spend some energy to drive the residues into this region. (A) Before optimization, the Ramachandran diagram showed that the favored region, outlier region, and rotamer region of the MP3RT vaccine were 91.34%, 3.15%, and 5.32%. (B) After optimization, the Ramachandran diagram showed that the favored region, outlier region, and rotamer region of the MP3RT vaccine were 98.22%, 0.36%, and 0.99%, respectively.

Figure 3

Molecular docking between the MP3RT vaccine and the HLA-DRB1*01:01 molecule, TLR-2, and TLR-4 receptors. Molecular docking of the MP3RT vaccine (blue) with HLA-DRB1*01:01 (A, green), TLR2 (C, green), and TLR4 (E, green) was performed using ClusPro2.0 server (https://cluspro.bu.edu/home.php). PDB files for TLR2 (6NIG) and TLR4 (4G8A) receptors were obtained from the Molecular Modeling Database (MMDB, https://www.ncbi.nlm.nih.gov/structure/). PyMOL2.5.3 was used to visualize and analyze the amino acid sites where the MP3RT vaccine (blue) docked with HLA molecule (B, red), TLR2 (D, red), and TLR4 (F, red). The detailed information of ligand amino acid, distance, and acceptor amino acid for molecular docking of the MP3RT vaccine with HLA-DRB1*01:01, TLR-2, and TLR-4 can be found in Table S1 , Table S2 , and Table S3 , respectively.

Figure 4

Conformational B cell epitopes predicted by the ElliPro server. The positions of the yellow balls in (A–D) indicate the positions of conformational B cell epitopes, and the rest were shown in gray.

Figure 5

Immune stimulation of innate immune cells predicted by the C-ImmSim Server. (A) The macrophage population per state. (B) The DCs population per state.

Figure 6

Immune stimulation of adaptive immune cells predicted by the C-ImmSim Server. (A) The TH cell population. (B) The TH cell population per state. (C) The Tc cell population. (D) The Tc cell population per state. (E) The B cell population. (F) The B cell population per state.

Figure 7

The levels of MP3RT-specific IgG, IgM, IgG1, and IgG2 antibodies predicted by the C-ImmSim server.

Figure 8

Schematic diagram of mouse immunization and detection of MP3RT-specific antibodies in mice. (A) Time of injection of the MP3RT vaccine and PBS in humanized mice. (B–E) The levels of the MP3RT-specific IgG, IgG₁, IgG_2a antibodies, and IgG_2a/IgG₁ ratio in mice vaccinated with MP3RT and PBS. Six serum samples in PBS group or 7 serum samples in MP3RT group were pooled together and tested in quadruplicate for antibody detection. The results were analyzed with the Unpaired t-test or Mann-Whitney test according to the normality. All data were shown as mean + SEM (n = 4). P < 0.05 was considered significantly different. ****, P < 0.0001.

Figure 9

The levels of cytokines predicted by the C-ImmSim server.

Figure 10

IFN-γ⁺ T lymphocytes detection with ELISPOT in mice. (A) The splenocytes collected from mice immunized with PBS or MP3RT vaccine were stimulated with MP3RT in vitro, and the SFCs were detected using a mouse ELISPOT kit. (B) The number of IFN-γ⁺ T lymphocytes (showed as SI) stimulated by the MP3RT vaccine in PBS and MP3RT groups were compared. In order to reduce individual differences, the spleens of mice in each group were mixed to prepare spleen cell suspensions, and then ELISPOT experiments were performed in quadruplicate. The results were analyzed with the Unpaired t-test or Mann-Whitney test according to the normality. All data were shown as mean + SEM (n = 4). P < 0.05 was considered significantly different.

Figure 11

The levels of cytokines induced by the MP3RT vaccine in mice. The levels of IFN-γ (A), TNF-α (B), IL-2 (C), IL-4 (D), IL-6 (E), IL-10 (F), and IL-17A (G) cytokines induced by the MP3RT vaccine in the PBS group and MP3RT group were detected by a Mouse Th1/Th2/Th17 Cytokine Kit. In order to reduce individual differences, the spleens of mice in each group were mixed to prepare spleen cell suspensions, and then cytokine detection were performed in quadruplicate. The results were analyzed with the Unpaired t-test or Mann-Whitney test according to the normality. All data were shown as mean + SEM (n = 4). P < 0.05 was considered significantly different.