Bioinformatics analysis and consistency verification of a novel tuberculosis vaccine candidate HP13138PB

Abstract

Background: With the increasing incidence of tuberculosis (TB) and the shortcomings of existing TB vaccines to prevent TB in adults, new TB vaccines need to be developed to address the complex TB epidemic.

Method: The dominant epitopes were screened from antigens to construct a novel epitope vaccine termed HP13138PB. The immune properties, structure, and function of HP13138PB were predicted and analyzed with bioinformatics and immunoinformatics. Then, the immune responses induced by the HP13138PB were confirmed by enzyme-linked immunospot assay (ELISPOT) and Th1/Th2/Th17 multi-cytokine detection kit.

Result: The HP13138PB vaccine consisted of 13 helper T lymphocytes (HTL) epitopes, 13 cytotoxic T lymphocytes (CTL) epitopes, and 8 B-cell epitopes. It was found that the antigenicity, immunogenicity, and solubility index of the HP13138PB vaccine were 0.87, 2.79, and 0.55, respectively. The secondary structure prediction indicated that the HP13138PB vaccine had 31% of α-helix, 11% of β-strand, and 56% of coil. The tertiary structure analysis suggested that the Z-score and the Favored region of the HP13138PB vaccine were -4.47 88.22%, respectively. Furthermore, the binding energies of the HP13138PB to toll-like receptor 2 (TLR2) was -1224.7 kcal/mol. The immunoinformatics and real-world experiments showed that the HP13138PB vaccine could induce an innate and adaptive immune response characterized by significantly higher levels of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and IL-10.

Conclusion: The HP13138PB is a potential vaccine candidate to prevent TB, and this study preliminarily evaluated the ability of the HP13138PB to generate an immune response, providing a precursor target for developing TB vaccines.

Keywords: bioinformatics; epitope vaccines; immune responses; immunoinformatics; tuberculosis.

Figure 1

Schematic representation of the HP13138PB vaccine. The amino acid sequence of the epitopes, adjuvants, Pan DR reactive epitope (PADRE), and linkers have been shown in different colors.

Figure 2

(A) The molecular solubility of the HP13138PB vaccine was predicted by the Protein-Sol server. (B) The secondary structure of the HP13138PB vaccine.

Figure 3

The three-dimensional (3-D) model, Z-score, and Ramachandran diagram of the HP13138PB vaccine. (A) The model of the HP13138PB vaccine was predicted by the I-TASSER server. (B) The Galaxy Refine web server indicated a model of the HP13138PB vaccine. The colored part repackaged the amino acid side chain and the overall structural relaxation that minimizes energy. (C) The Z-score of the HP13138PB vaccine was -4.47 determined by the ProSA-web server. The light blue and dark blue represent the Z-score of all proteins in the PDB experimentally solved by X-Ray and NMR (nuclear magnetic resonance) spectroscopy, respectively. (D) The amino acid sequence position of the HP13138PB vaccine showed local model quality by the ProSA-web server. (E) The Ramachandran diagram before optimization. The two green areas represent the favored region and the outlier region. The white area is the rotamer region. The favored, outlier, and rotamer regions of the HP13138PB vaccine were 56.97%, 14.09%, and 14.25%. (F) The Ramachandran diagram after optimization. The favored, outlier, and rotamer regions of the HP13138PB vaccine were 88.22%, 4.13%, and 0.88%.

Figure 4

The conformational B-cell epitopes of the HP13138PB vaccine predicted by the ElliPro server. Three conformational B-cell epitopes (A–C) were predicted and identified in the amino acid sequence of HP13138PB vaccine, which are indicated by yellow balls and other amino acid residues in their amino acid sequence are indicated by gray lines.

Figure 5

The interaction of the HP13138PB vaccine with toll-like receptor 2 (TLR2). (A) Diagram of HP13138PB docking with TLR2 molecule. Blue represents the HP13138PB vaccine, and green means the TLR2 receptor. (B) The interaction of the HP13138PB vaccine and the TLR2 was described in a 2D plot. The B chain represents the residence of the HP13138PB vaccine with hydrogen bonds. The A chain represents the residue of the TLR2 receptor with hydrogen bonds. The temperature plot and the pressure graph of HP13138PB-TLR2. (C, D) The temperature plot and the pressure graph of HP13138PB-TLR2 at 300K and 1 bar under the AMBER99 force field using Gromacs v5.1.515.

Figure 6

Prediction results of the C-ImmSim Server on macrophages (MA) and dendritic cells (DCs). (A) MA population per state (cells per mm³), (B) DC population per state (cells per mm³).

Figure 7

Prediction results of the C-ImmSim Server on helper T (TH) cell, cytotoxic T (TC) cell, B cell, and antibody. (A) TH cell population (cells per mm³), (B) TH cell population per state (cells per mm³), (C) TC cell population (cells per mm³), (D) TC cell population per state (cells per mm³), (E) B cell population per state (cells per mm³), (F) antibody titers of IgM, IgG1, and IgG2.

Figure 8

Construction of pET28a-HP13138PB recombinant plasmid in silico and its expression in vitro. (A) The nucleotide sequence of the HP13138PB vaccine was inserted into the arrangement of the pET28a plasmid through the restriction sites BamHI and XhoI, and the recombinant plasmid pET28a-HP13138PB had a gene length of 7453 base pairs. (B) The expression result of the HP13138PB vaccine in E coli..

Figure 9

The levels of cytokines induced by the HP13138PB vaccine in the C-ImmSim Server. Three times injection of the HP13138PB vaccine was simulated in the C-ImmSim Server, and the levels of IFN-γ, IL-4, IL-12, TGF-β, TNF-α, IL-10, IL-6, IFN-β, IL-18, IL-23, and IL-2 cytokines induced by the HP13138PB vaccine were analyzed. Different cytokines were distinguished by different colors. Cytokine concentrations were expressed as ng/ml.

Figure 10

IFN-γ⁺ T lymphocytes detection with enzyme-linked immunospot assay (ELISPOT). The HP13138PB vaccine was used to stimulate the peripheral blood mononuclear cells (PBMCs) collected from health control (HC), individuals with latent tuberculosis infection (LTBI), and active tuberculosis (ATB) patients in vitro. The spot-forming cells (SFCs) of IFN-γ⁺ T lymphocytes were determined with a human ELISPOT kit. The data were analyzed with the Unpaired t-test or Mann Whitney test according to the normality. Data were shown as mean + SEM (n = 21, 18, and 24 in HCs, ATB patients, and LTBI volunteers, respectively). P<0.05 was considered significantly different. SEM, standard error of the mean.

Figure 11

The levels of cytokines induced by the HP13138PB vaccines in peripheral blood mononuclear cells (PBMCs) from humans. The levels of interleukin-4 (IL-4, (A), IL-6 (B), IL-10 (C), IL-17A (D), tumor necrosis factor-α (TNF-α, E), interferon-γ (IFN-γ, F), and IL-2 (G) cytokines were detected by a human Th1/Th2/Th17 cytokine detection kit. The PBMCs collected from health control (HC, n=21), individuals with latent tuberculosis infection (LTBI, n=24), and patients with active tuberculosis (ATB, n=18) were stimulated with the HP13138PB vaccine in vitro. Furthermore, the PBMCs collected from HCs were stimulated with an AIM medium as a negative control. The differences were compared with the one-way analysis of variance (ANOVA) or Kruskal-Wallis test according to the data normality and homogeneity of variances. All data were shown as mean + SEM. P<0.05 was considered significantly different. SEM, standard error of the mean.