Oral Immunization with a Multivalent Epitope-Based Vaccine, Based on NAP, Urease, HSP60, and HpaA, Provides Therapeutic Effect on H. pylori Infection in Mongolian gerbils

Abstract

Epitope-based vaccine is a promising strategy for therapeutic vaccination against Helicobacter pylori (H. pylori) infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori, remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP), heat shock protein 60 (HSP60) and H. pylori adhesin A (HpaA) was constructed based on mucosal adjuvant cholera toxin B subunit (CTB), Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes (UreA_27-53, UreA_183-203, HpaA_132-141, and HSP60_189-203), and also the epitope-rich regions of urease B subunit (UreB_158-251 and UreB_321-385) predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori-infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes (UreB_158-172, UreB_181-195, UreB_211-225, UreB_349-363, HpaA_132-141, and HSP60_189-203). In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4⁺ T cell (Th cell) response, IgG, and secretory IgA (sIgA) antibodies to H. pylori. These results indic ate that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.

Keywords: HSP60; Helicobacter pylori; HpaA; NAP; multivalent epitope-based vaccine; therapeutic vaccine; urease.

Figure 1

The experimental design of therapeutic vaccination. Briefly, Mongolian gerbils were infected with H. pylori SS1. We tested whether H. pylori-infected Mongolian gerbils are successful at the 10th week. H. pylori-infected Mongolian gerbils were administered orally with 100 μg of CWAE, CTB-UE, Urease in 500 μl aluminum hydroxide adjuvant on 4 occasions (weeks 11, 12, 13, and 14), and 500 μl PBS was also given orally on 4 occasions as control. Two weeks after the final immunization (At 16th weeks), Mongolian gerbils were sacrificed for various testing items.

Figure 2

The structural diagrams of multivalent epitope-based vaccine CWAE and urease epitope-based vaccine CTB-UE. CWAE and CTB-UE share some same B or Th cell epitopes (UreB_229–25, UreA_183–203, and UreB_327–334) which are marked in red fonts. (A) The CWAE vaccine is composed of two molecular adjuvants (CTB and NAP), tandem copies of the selected B and Th cell epitopes (UreA_27–53, UreA_183–203, HpaA_132–141, and HSP60_189–203), and epitope-rich regions UreB_158–251 containing four known Th or B cell epitopes (UreB_158–172, UreB_181–195, UreB_211–225, UreB_229–251) and UreB_321–385 containing three Th or B cell epitopes (UreB_327–334, UreB_349–363, UreB_373–385). In order to avoid generating new epitopes at linkage sites, these linkers (DPRVPSS, KK, GGG, and GS) were designed to separate different epitopes or adjuvants. (B) The CTB-UE vaccine contains molecular adjuvants CTB and tandem copies of four different epitopes which are Th cell epitopes (UreA_74–90 and UreB_229–251) and B cell epitopes (UreA_183–203 and UreB_327–334) from H. pylori urease. In addition, the linkers (DPRVPSS, KK, and GS) were designed to retain the immunologic competence of each Th or B epitope and avoid the generation of new epitopes at linkage sites among epitopes.

Figure 3

CWAE expression, purification, and antigen characteristics. (A) Protein expression and purification of CWAE. (Lane 1) protein marker, (lanes 3 and 5) the inclusion bodies of E. coli BL21(DE3)/pETCWAE, (lanes 2 and 4) the soluble proteins of E. coli BL21(DE3)/pETCWAE, (lane 6 and 7) the purified CWAE proteins. (B) Protein expression and purification of CTB-UE. (Lane 1) protein marker, (lanes 2, 3, and 5) the inclusion bodies of E. coli BL21(DE3)/pETCUE, (lane 4) the soluble proteins of E. coli BL21(DE3)/pETCUE, (lane 6, 7, and 8) the purified CTB-UE proteins. (C) Immunogenicity and immunoreactivity of CWAE analyzed by Western blot. (i) CWAE and CTB-UE reaction with Rabbit anti-H. pylori polyclonal antibody (Rabbit anti-Hp PcAb). (Lane M) protein marker, (lane 1) CWAE proteins, (lane 2) CTB-UE proteins. (ii) The H. pylori antigens (UreA, UreB, HpaA, Hsp60 and NAP) reaction with antiserum induced by CWAE vaccine. (Lane M) protein marker; (lane 1, 60 KD) Hsp60; (lane 2, 64 KD) UreB; (lane 3, 30 KD) UreA; (lane 4, 31 KD) HpaA; (lane 5, 14 KD) NAP. (D) The adjuvanticity of CTB component in CWAE and CTB-UE vaccine analyzed by GM1-ELSIA. In order to confirm the CTB component in CWAE or CTB-UE with the ability to bind GM1 gangliosides, GM1-ELISA was performed. ELISA plates were coated with 1 μg/well of GM1 ganglioside or BSA. The recombinant proteins CWAE, CTB-UE, CTB, and UreB with a concentration of 100 μg/ml were used to evaluate their capability of binding GM1. Data are mean ± SD. p < 0.05 was considered as statistically significant. ^**p < 0.01; ^***p < 0.001; ns, not significant.

Figure 4

Assessment of antibodies specific for large antigens (H. pylori urease, UreA, UreB, Hsp60, NAP, and HpaA). The SPF BALB/c mice were immunized with CWAE, CTB-UE, or CTB by subcutaneous multi-point injection. (A) Detection of antibodies specific for H. pylori urease. ELISA plates were coated with 0.5 μg/well of native H. pylori urease. p <0.05 was considered as statistically significant. ^***p < 0.001, ns, not significant. (B) Detection of antibodies specific for UreA. ELISA plates were coated with 0.5 μg/well of UreA. p < 0.05 was considered as statistically significant. ^***p < 0.001, ns, not significant. (C) Detection of antibodies specific for UreB. ELISA plates were coated with 0.5 μg/well of UreB. p < 0.05 was considered as statistically significant. ^***p < 0.001, ns, not significant. (D) Detection of antibodies specific for Hsp60. ELISA plates were coated with 0.5 μg/well of Hsp60. p < 0.05 was considered as statistically significant. ^***p < 0.001, ns, not significant. (E) Detection of antibodies specific for NAP. ELISA plates were coated with 0.5 μg/well of NAP. p < 0.05 was considered as statistically significant. ^***p < 0.001, ns not significant. (F) Detection of antibodies specific for HpaA. ELISA plates were coated with 0.5 μg/well of HpaA. p < 0.05 was considered as statistically significant. ^***p < 0.001, ns, not significant.

Figure 5

Detection of epitope-specific antibodies. The epitope peptides UreA_183–203, UreB_321–339, UreB_158–172, UreB_181–195, UreB_211–225, UreB_349–363, HpaA_132–141, and HSP60_189–203 were synthetized. Data are mean ± S.D. p < 0.05 was considered as statistically significant. ^***p < 0.001, ns, not significant. (A) Measurement of antibodies specific for the UreA_183–203 peptide. ELISA plates were coated with 1 μg/well of synthetic UreA_183–203 peptides. (B) Measurement of antibodies specific for the UreB_321–339 peptide. ELISA plates were coated with 1 μg/well of synthetic UreB_321–339 peptides. (C) Measurement of antibodies specific for the UreB_158–172 peptide. ELISA plates were coated with 1 μg/well of synthetic UreB_158–172 peptides. (D) Measurement of antibodies specific for the UreB_181–195 peptide. ELISA plates were coated with 1 μg/well of synthetic UreB_181–195 peptides. (E) Measurement of antibodies specific for the UreB_211–225 peptide. ELISA plates were coated with 1 μg/well of synthetic UreB_211–225 peptides. (F) Measurement of antibodies specific for the UreB_349–363 peptide. ELISA plates were coated with 1 μg/well of synthetic UreB_349–363 peptides. (G) Measurement of antibodies specific for the HpaA_132–141 peptide. ELISA plates were coated with 1 μg/well of synthetic HpaA_132–141 peptides. (H) Measurement of antibodies specific for the HSP60_189–203 peptide. ELISA plates were coated with 1 μg/well of synthetic HSP60_189–203 peptides. (I) Inhibition of H. pylori urease activity by specific antibodies. Natural H. pylori urease was preincubated with a serial dilution of IgG from mice immunized with CWAE, CTB-UE, or rCTB. The optical density of the mixture was determined at 550 nm by the indicator of phenol red. The data are expressed as percentage inhibition.

Figure 6

Evaluation of therapeutic effect by quantitative culture and rapid urease test. The H. pylori-infected Mongolian gerbils were orally immunized with CWAE, CTB-UE, Urease, or PBS. Data are mean ± S.D. p <0.05 was considered as statistically significant. ^*p <0.05, ^**p <0.01, ^***p <0.001, ns, not significant. (A) Quantitative culture of H. pylori in the stomach after oral therapeutic immunization. The number of bacteria (CFU) per stomach was determined for individual mice in each group by quantitative culture. (B) The H. pylori urease activity in the stomach after oral therapeutic immunization. The H. pylori urease activity was measured by rapid urease test.

Figure 7

Histological analysis of stomach tissue. The H. pylori-infected Mongolian gerbils were orally immunized with CWAE, CTB-UE, Urease, or PBS. Data are mean ± S.D. p <0.05 was considered as statistically significant. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ns, not significant. (A) Assessment on therapeutic efficacy based on gastritis scores. The Inflammation score from Mongolian gerbils after immunization with CWAE is least. (B) Histopathological analysis after therapeutic vaccination. H. pylori-infected Mongolian gerbils after immunization with PBS showed severe inflammatory infiltrates (HE stain, 100×). However, H. pylori-infected Mongolian gerbils after immunization with CWAE, CTB-UE or Urease (Ure) showed mild inflammatory infiltrate. (C) Observation of H. pylori by IHC staining. The H. pylori-infected Mongolian gerbils after immunization with Urease (Ure) or PBS showed positively stained H. pylori within a glandular lumen. H. pylori colonizing in the stomach were denoted by black arrows.

Figure 8

Antibody responses after oral therapeutic immunization. ELISA plates were coated with 0.5 μg/well of native H. pylori lysates. Data are mean ± S.D. p < 0.05 was considered as statistically significant. ^*p <0.05, ^**p < 0.01, ^***p < 0.001, ns, not significant. (A) Detection of IgG against H. pylori lysates in serum. The different sera was diluted 1:800. (B) Detection of IgA against H. pylori lysates in serum. The different sera was diluted 1:800. (C) Detection sIgA against H. pylori lysates in the gastric mucus, intestinal mucus or feces. The supernatants from the homogenized stomach tissue, intestinal tissue or feces were diluted 1:5 in PBS for analysis of sIgA.

Figure 9

Lymphocyte responses and cytokine production after therapeutic vaccination. Data are mean ± S.D. p < 0.05 was considered as statistically significant. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ns, not significant. (A) Proliferation of splenic lymphocytes after stimulation with H. pylori antigens. Splenic lymphocytes were separated from H. pylori-infected Mongolian gerbils after therapeutic immunization with CWAE, CTB-UE, Urease, or PBS, and were incubated with NAP, Urease or H. pylori lysates (5 μg/ml). (B–D) Detection of IL-4, IFN-γ, and IL-17 cytokine production after therapeutic vaccination. The concentrations of cytokines in the supernatants of lymphocytes cultures were determined by ELISA. Splenic lymphocytes from H. pylori-infected Mongolian gerbils after therapeutic immunization with CWAE, CTB-UE, Urease, or PBS were stimulated with H. pylori lysates for 72 h, and cytokine production was detected by ELSA.