The influence of adjuvant on UreB protection against Helicobacter pylori through the diversity of CD4+ T-cell epitope repertoire

Abstract

Adjuvants are widely used to enhance the effects of vaccines against pathogen infections. Interestingly, different adjuvants and vaccination routes usually induce dissimilar immune responses, and can even have completely opposite effects. The mechanism remains unclear. In this study, urease B subunit (UreB), an antigen of Helicobacter pylori (H. pylori) that can induce protective immune responses, was used as a model to vaccinate mice. We investigated the effects of different adjuvants and routes on consequent T cell epitope-specific targeting and protection against H. pylori infection. Comparison of the protective effects of UreB, administered either subcutaneously (sc) or intranasally (in), with the adjuvants AddaVax (sc), Complete Freund's adjuvant (CFA; sc), or CpG oligonucleotide (CpG; sc or in), indicated that only CFA (sc) and CpG (in) were protective. Protective vaccines induced T cells targeting epitopes that differed from that targeted by control vaccination. Subsequent peptide vaccination demonstrated that only two of the identified epitopes were protective: UreB_373-385 and UreB_317-329. Overall, we found that both adjuvant and vaccination route affected the T cell response repertoire to antigen epitopes. The data obtained in this study contribute to improved characterization of the relationship between adjuvants, routes of vaccination, and epitope-specific T cell response repertoires.

Keywords: adjuvants; epitopes; immunodominant; protection; vaccine.

Figure 1. Immune responses in groups vaccinated using different adjuvants and by various routes

Mice were immunized with UreB antigen in combination with different adjuvants and by various routes. Then, mice were challenged with H. pylori. Four weeks after the last challenge, mice were sacrificed, their stomachs harvested for evaluation of bacterial colonization, and their spleens collected for expansion of antigen-specific T cells. (A) Colonization of the gastric mucosa with H. pylori was evaluated in the different vaccination groups. (B) Splenic lymphocytes were isolated from PBS/UreB immunized mice post-challenge with H. pylori and cultured in vitro in the presence of UreB. On day 7, CD4+ T cells secreting IFN-γ were assessed using a UreB peptide pool. Representative flow cytometry plots demonstrating antigen-specific T cell responses from the CpG sc vaccination group are shown. (C) IFN-γ-producing CD4+ T cells from all immunized mice were assayed. All experiments were repeated three or more times. Data are expressed as means ± S. D. (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 (compared with PBS controls, unless indicated otherwise).

Figure 2. Mapping of UreB immunodominant epitopes

Four weeks after H. pylori challenge, spleens were harvested from all mouse vaccination groups. Antigen-specific T cells were expanded in vitro and screened for specific IFN-γ responses to 93 overlapping 18mer peptides using ICS assays. Antigens, adjuvants, vaccination routes, and the locations of the identified 18mer peptides are indicated. The results are representative of three independent experiments. Data are expressed as means ± S. D. (n = 5).

Figure 3. Identification of 13mer epitopes within dominant 18mer epitopes

Four weeks after H. pylori challenge, UreB-specific T cells expanded from the spleens of immunized mice were screened for IFN-γ responses to 13mer peptides in ICS assays. (A) Overlapping 13mer peptides (filled bars) within the UreB_313–330 18mer identified as dominant in the CpG sc group (open bar) were tested, along with a DMSO control (open bar). (B) 13mer peptides within UreB_373–390 identified in the CpG in group were tested, and the 13mer peptides titrated to compare their ability to activate T cell responses. (C) 13mer peptides within UreB_403–420 and UreB_409–426 identified in the CpG (sc) group were tested and titrated to compare their activities. (D, E) 13mer peptides within UreB_481–498 and UreB_487–504 18mers identified in the AddaVax (sc) and without adjuvant control (sc) groups were tested. All experiments were repeated four or more times. Data are expressed as means ± S. D. (n = 5).

Figure 4. MHC restriction and natural processing and presentation of immunodominant epitopes

Splenic antigen-specific T cells from immunized mice were expanded in vitro after H. pylori challenge. (A) To determine which MHC molecule bound to the epitopes, specific MHC antibodies were used to block MHC molecules, followed by evaluation of T cell responses. Results of DMSO control experiments and T cell responses to peptides in the absence of antibodies are shown as open bars. (B) Natural processing and presentation of epitopes by DCs. Results from DMSO control experiments and APCs pulsed with DMSO are shown as open bars. Antigen, adjuvants, and vaccination routes are shown. The results are representative of four or more times independent experiments. Data are expressed as means ± S. D. (n = 5).

Figure 5. Effects of identified immunodominant epitopes in protection against H. pylori infection

Mice were immunized by the intranasal route with the identified epitopes combined with CpG adjuvant and then challenged with H. pylori. (A) Evaluation of H. pylori colonization in the gastric mucosa of mice immunized with peptides. The effects of the whole protein antigen with different adjuvants and vaccination routes and the effects of corresponding epitopes are summarized to the right of the panel. (B) Levels of serum anti-UreB IgG, anti-peptide IgG, gastric mucosal anti-UreB sIgA, and anti-peptide sIgA antibodies were determined. (C) The epitope-specific T cell responses of mice immunized with peptides were analyzed. All experiments were repeated three or more times. Data are expressed as means ± S. D. (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Epitope-specific T cell responses in groups vaccinated with UreB combined with CFA sc and CpG sc

(A) The effects of vaccination with UreB combined with CFA (sc) and CpG (sc) and their corresponding epitopes are summarized. (B) Lymphocytes collected from mice immunized with UreB combined with CFA (sc) or UreB combined with CpG (sc) after H. pylori challenge were expanded in vitro, followed by identification of T cells responses to the dominant epitopes by FACS. (C) Four weeks after H. pylori infection, UreB_317–329 and UreB_409–421 peptide-pulsed DCs were used to stimulate purified CD4+ T cells from mice immunized with UreB combined with CFA (sc) or CpG (sc) for ex vivo ELISPOT assays. The numbers of epitope-specific T cells/5 × 10⁵ CD4+ T cells are presented. (D) UreB_317–329 and UreB_409–421 peptide-specific CD4+T cells from CFA (sc) and CpG (sc) vaccination groups were determined using ELISPOT assays on day10 after immunization. The numbers of epitope-specific T cells/5 × 10⁵ CD4+ T cells are presented. All experiments were repeated three or more times. Data are expressed as means ± S. D. (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. Effects of adjuvants on epitope-specific T cells responses

(A) Summary of dominant epitopes from mice immunized with UreB combined with AddaVax (sc) and UreB combined with CpG (sc). (B) BM-DCs were pulsed with UreB antigen for 1 h in the presence of AddaVax. Then, these DCs were used to stimulate expanded splenic epitope-specific T cells and ICS assays performed. (C) DCs pulsed with UreB antigen in the presence of CpG were used to stimulate epitope-specific T cells and ICS assays performed. An increased percentage of epitope-specific T cell responses post CpG administration was demonstrated. All experiments were repeated three times. Data are expressed as means ± S. D. (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.