Immunoinformatics Approach to Design a Novel Epitope-Based Oral Vaccine Against Helicobacter pylori

Abstract

Helicobacter pylori is an infectious agent that colonizes the gastric mucosa of half of the population worldwide. This bacterium has been recognized as belonging to group 1 carcinogen by the World Health Organization for the role in development of gastritis, peptic ulcers, and cancer. Due to the increase in resistance to antibiotics used in the anti-H. pylori therapy, the development of an effective vaccine is an alternative of great interest, which remains a challenge. Therefore, a rational, strategic, and efficient vaccine design against H. pylori is necessary where the use of the most current bioinformatics tools could help achieve it. In this study, immunoinformatics approach was used to design a novel multiepitope oral vaccine against H. pylori. Our multiepitope vaccine is composed of cholera toxin subunit B (CTB) that is used as a mucosal adjuvant to enhance vaccine immunogenicity for oral immunization. CTB fused to 11 epitopes predicted of pathogenic (UreB_170-189, VacA_459-478, CagA_1103-1122, GGT_106-126, NapA_30-44, and OipA_211-230) and colonization (HpaA_33-52, FlaA_487-506, FecA_437-456, BabA_129-149, and SabA_540-559) proteins from H. pylori. CKS9 peptide (CKSTHPLSC) targets epithelial microfold cells to enhance vaccine uptake from the gut barrier. All sequences were joined to each other by proper linkers. The vaccine was modeled and validated to achieve a high-quality three-dimensional structure. The vaccine design was evaluated as nonallergenic, antigenic, soluble, and with an appropriate molecular weight and isoelectric point. Our results suggest that our newly designed vaccine could serve as a promising anti-H. pylori vaccine candidate.

Keywords: Helicobacter pylori; in silico; multiepitope; oral vaccine; reverse vaccinology.

FIG. 1.

Analysis of conservation of selected proteins. Entire protein sequences among the 85 annotated genomes of Helicobacter pylori were aligned using CLC Main workbench, and conservation scores were represented in a 2D line chart. (a) CagA, (b) SabA, (c) BabA, (d) VacA, and (e) FecA. 2D, two-dimensional.

FIG. 2.

Analysis of conservation of selected proteins. Entire protein sequences among the 85 annotated genomes of Helicobacter pylori were aligned using CLC Main workbench, and conservation scores were represented in a 2D line chart. (a) UreB, (b) GGT, (c) NapA, (d) FlaA, (e) OipA, and (f) HpaA.

FIG. 3.

Conservation of selected epitopes among strains of Helicobacter pylori. Individual proteins among the 85 annotated genomes were aligned using CLC Main workbench, and consensus sequence for each epitope was obtained and demonstrated in the black boxes. The percentage of conservation for each residue is shown in a bar chart. (a) NapA_30–49, (b) CagA_1103–1122, (c) FlaA_487–506, (d) OipA_211–230, (e) GGT_106–126, (f) HpaA_33–52, (g) BabA_129–149, (h) FecA_437–456, (i) SabA_540–559, (j) VacA_459–478, and (k) UreB_170–189.

FIG. 4.

Schematic diagram of vaccine construct consists of adjuvant in N-terminal joined to 11 epitopes fused together by proper linkers (GPGPG or KK) and CKS9 M cell homing peptide in C-terminal. CTB, cholera toxin subunit B; GPGPG, Gly-Pro-Gly-Pro-Gly; KK, Lys-Lys.

FIG. 5.

The secondary structure prediction of vaccine by PSIPRED. The protein vaccine consists of 35% α helix (H, cylinder), 14% β strand (E, arrow), and 49% coil (C, line) secondary structural elements. The bar chart represents the percentage of confidence.

FIG. 6.

The z-score plot of unrefined and refined 3D structure of vaccine by ProSA-web. (a) The z-score of the starting model is −4.03, (b) The z-score of model after refinement steps is −4.9. The z-score indicates overall model quality and is depicted as a black spot. The z-score plot contains the z-scores of all experimentally determined protein chains in current protein data bank (PDB) from NMR spectroscopy (charcoal) and X-ray crystallography (silver). 3D, three-dimensional.

FIG. 7.

Comparison of initial and refined 3D protein structure conformations of vaccine by Molecular Operating Environment software. The initial and refined models are colored in silver and charcoal, respectively.

FIG. 8.

Validation of vaccine 3D model using Ramachandran plot. The Ramachandran plots of (a) the unrefined model and (b) the refined model. The most-favored (A, B, and L) and additional allowed (a, b, l, and p) regions were demonstrated with charcoal and silver gray colors, respectively. The generously allowed regions (∼a, ∼b, ∼l, and ∼p) are indicated in silver, and the disallowed regions are in white color. Glycine residues are shown in black triangles, and other residues of protein are shown in black squares.

FIG. 9.

Comparison of the individual codon usage between host and optimized sequences. The host and optimized sequences are shown in charcoal and black lines, respectively.