A new candidate epitope-based vaccine against PspA PhtD of Streptococcus pneumoniae: a computational experimental approach

Abstract

Introduction: Pneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections.

Methods: In this study using immunoinformatics approaches, we constructed one fusion construct (called PAD) by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge.

Results: The computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies.

Conclusion: For the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine.

Keywords: Streptococcus pneumoniae; actual animal experiments; fusion protein; immunodominant B and T cell epitopes; immunoinformatics; pneumococcal epitope-based vaccine; pneumococcal histidine triad protein D (PhtD); pneumococcal surface protein A (PspA).

Figure 1

Schematic flowchart of this study for designing and evaluation epitope-based constructs against S. pneumoniae. The overall approach of this study is represented in two computational and experimental phases. The used servers/softwares/techniques are mentioned in the right side of the schematic figure.

Figure 2

Vaccination schedule and timeline of experimental procedures. (A) BALB/c mice were divided into 5 groups PA, PD, PA+PD, PAD, and control (n = 5 per group). (B) The mice were immunized subcutaneously on days 0, 14, 28 and 42. The sera were collected before each injection and 2 weeks after the last injection and used for further analysis.

Figure 3

Schematic diagram of vaccine candidates. PAD, fusion peptide consisting of PspA and PhtD epitopes; PA, peptide consisting of epitopes of PspA protein; PD, Peptide consisting of epitopes of PhtD protein.

Figure 4

Validation of the final models PAD, PA, and PD after refinement. (A) The Ramachandran plots show that in the refined models PAD, PA, and PD, 92.5%, 95.6% and 90.3% of the residues are located in the favorable areas, respectively. The most favored, allowed, generously allowed and disallowed regions are shown in red, dark yellow, lighter yellow and white, respectively. (B) The ProSA Z-scores are found to be -5.33, -3.46, and -6.55 in the refined models PAD, PA, and PD, respectively. The z-scores of proteins determined by NMR or X ray are represented in dark or light blue, respectively. (C) In the ERRAT plots, the overall quality factors of the refined structures PAD, PA, and PD are 93.31, 95.54, and 92.13, respectively. The red and yellow colours show the problematic parts while the white colour shows the normal parts in the structure.

Figure 5

3D images of structural B cell epitopes of the PAD construct and 2D score chart. (A) The yellow and gray areas represent the structural epitopes and the other protein segments, respectively. (B) Yellow areas with values ​​above a threshold of 0.5 indicate potential B-cell epitopes.

Figure 6

Molecular docking of immunogenic peptides with HLA-DRB1_01:01 molecule (chains A and B). Schematic images of peptide-HLA complexes were shown using Chimera software. The ≤30-mer peptides from the selected epitope-rich regions are shown in red. The A and B chains of HLA molecule are indicated in light green and light blue colors, respectively. The results displayed that the peptides had a favorable interaction with the receptor groove. (A, B) The peptides are related to the selected immunogenic part of A region of PspA. (C-F) The peptides are the selected B regions of PspA. (G) The peptide is the selected C region of PspA. (H-J) The peptides are related to the selected immunogenic regions of C-terminal of PhtD.

Figure 7

In silico immune simulation of the PAD construct. (A) Immunoglobulin production in response to injection of the PAD construct (antigens and immunoglobulin subclasses are represented as black and colored peaks, respectively). (B) Evolution of the B cell population after 4 injections. (C) Evolution of the T-helper cell population. (D) Analysis of the levels of different cytokines induced by the construct. The cytokines were distinguished by different colours and the concentration was expressed as ng/ml.

Figure 8

Verification of presence of purified proteins by SDS-PAGE electrophoresis. The calculated MW for the peptide constructs PAD, PA and PD are 35.69, 24.34 and 11.97 kDa, respectively. The calculated MW of PAD and PA is lower than the MW observed on the gel, indicating these proteins move more slowly. Lane M: protein marker; Lanes 1 to 3: Proteins PD, PA and PAD, respectively.

Figure 9

Immunogenicity of the recombinant proteins. Left figures: The comparison of specific total IgG responses in test and control groups was done before each injection and 2 weeks after the last injection (days 0, 14, 28, 42, and 56). Mice (n = 5 per group) were vaccinated with PA or PD alone, PA+PD, PAD or Al adjuvant, and then antisera were examined against PA or PD separately. (A) Anti-PA responses in the groups PA, PA+PD and PAD were significantly increased after the second, third, or fourth immunization compared to the first immunization. (B) Anti-PD responses in the groups PD, PA+PD and PAD after each immunization showed significant increases compared to the preceding phase. Right figures: The responses of IgG isotypes against the peptide PA or PD in the tested mice groups (n = 5) were examined 2 weeks after the third injection. (C) Specific IgG1 and IgG2a subclasses against the PA peptide in the groups PA, PA+PD and PAD did not show significant differences from each other. (D) The IgG1 subclass against the PD peptide showed 0.6-fold increase compared to IgG2a subclass in the PAD group. Statistical analysis was done using two-way ANOVA. **p < 0.01.

Figure 10

Evaluation of the levels of cytokines and the bactericidal activity of antibodies. (A-C) Measurement of the cytokines IFN-γ, IL-4, and IL-17 in test and control mice (n = 5 per group) was performed 2 weeks after the 3th injection. (D) The complement-mediated bactericidal activity of anti-recombinant proteins antibodies was evaluated 2 weeks after the 4th injection (n = 3). The Y-axis is the reverse of the dilution of serum in which more than 50% of the bacteria are killed compared to the control. The activity of antibodies in the sera of PA, PAD and PA+PD groups was higher than that in the serum of PD group. There was no significant difference in the serum activity of PA, PAD and PA+PD groups. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 11

Assessment of mice survival rates and bacterial loads after pneumococcal challenge. (A-D) mice in the control group died one day after the challenge. The survival rate of mice up to 7 days after challenge in the PD group was 80% and in the PA, PA+PD or PAD groups was 100% (n = 3 per group). (E, F) Bacterial loads present in blood and spleen tissue of control and immunized mice (n = 2) were counted one day after the challenge. DLs in the blood were 7.0 Log10 (CFU/ml) for mice immunized with PD and 8.0 Log10 (CFU/ml) for the other three groups. DLs in the spleen of mice immunized with PD, PA, PA+PD and PAD were 3.0 Log10 (CFU/g).