In Vivo Validation of Novel Synthetic tbp1 Peptide-Based Vaccine Candidates against Haemophilus influenzae Strains in BALB/c Mice

Abstract

Haemophilus influenzae is a Gram-negative bacterium characterized as a small, nonmotile, facultative anaerobic coccobacillus. It is a common cause of a variety of invasive and non-invasive infections. Among six serotypes (a-f), H. influenzae type b (Hib) is the most familiar and predominant mostly in children and immunocompromised individuals. Following Hib vaccination, infections due to other serotypes have increased in number, and currently, there is no suitable effective vaccine to induce cross-strain protective antibody responses. The current study was aimed to validate the capability of two 20-mer highly conserved synthetic tbp1 (transferrin-binding protein 1) peptide-based vaccine candidates (tbp1-E₁ and tbp1-E₂) predicted using in silico approaches to induce immune responses against H. influenzae strains. Cytokine induction ability, immune simulations, and molecular dynamics (MD) simulations were performed to confirm the candidacy of epitopic docked complexes. Synthetic peptide vaccine formulations in combination with two different adjuvants, BGs (Bacterial Ghosts) and CFA/IFA (complete/incomplete Freund's adjuvant), were used in BALB/c mouse groups in three booster shots at two-week intervals. An indirect ELISA was performed to determine endpoint antibody titers using the Student's t-distribution method. The results revealed that the synergistic use of both peptides in combination with BG adjuvants produced better results. Significant differences in absorbance values were observed in comparison to the rest of the peptide-adjuvant combinations. The findings of this study indicate that these tbp1 peptide-based vaccine candidates may present a preliminary set of peptides for the development of an effective cross-strain vaccine against H. influenzae in the future due to their highly conserved nature.

Keywords: BALB/c mice; H. influenzae; IgG antibodies; adjuvants; indirect ELISA; peptide antigens; tbp1 peptides.

Figure 1

Abstract diagram showing the basic methodology followed.

Figure 2

Molecular dynamics simulation of peptide–DRB01010 docked complex, showing (a) eigenvalue; (b) variance; (c) B-factor; (d) deformability; (e) covariance matrix, which indicates coupling between pairs of residues (red), and uncorrelated (white) or anti-correlated (blue) motions; and (f) elastic network analysis, which defines which pairs of atoms are connected by springs.

Figure 3

The immune simulation results were generated with the online tool C-Immism. The graphs show (a) immunological reactions in terms of IgG titers (primary, secondary, and tertiary); (b) induction of cytokines and interleukins, where the inset plot shows danger signal together with leukocyte growth factor IL-2; (c) cytotoxic T-cell population; (d,e) B-cell population; (f) helper-T-cell population; (g) natural killer cells; (h,i) dendritic-cell and T-cell populations per state. (Legends: Act = active; Intern = internalized the Ag; Pres II = presenting on MHC II; Dup = in the mitotic cycle; Anergic = anergic; Resting = not active.)

Figure 4

Spatial representation of the dock molecule (ligands and H2 allele receptor). (a,b) Active pocket residues on beta chain of receptor molecule, showing the tbp1-E₁ (a) and tbp1-E₂ (b) ligand binding sites. (c,d) Two-dimensional structure of docking ligand and receptor molecule.

Figure 5

IgG antibody response in BALB/c mice against tbp1 peptides and adjuvants, inoculated via subcutaneous route. OD values of peptides vs. controls are depicted on the Y-axis. Absorbance was measured at 450 nm. Data collected from 6 mice per group are expressed as ± standard error of the mean (SEM) at a serum dilution of 1:16,000. (a) Endpoint IgG antibody titers in sera of mice primed subcutaneously with peptide + CFA/BG and boosted with peptide + IFA/BG are shown; the titer is represented as reciprocal of serum dilutions corresponding to 1:16,000. (b) Statistical significance was determined with the Mann–Whitney U test, and p-value < 0.05 was taken as significant. The asterisks indicate significant statistical difference. p-Values are indicated as follows: * p = 0.02, ** p = 0.002, and “ns” p = 0.1 when compared with negative control group D3.