Immunodominant T-cell epitopes in different adjuvants vaccination mice.

Spleens were collected from mice on day 10 post-vaccination with the antigen UreB incorporated with adjuvants CpG, MDP, and MPLA; cultured in vitro; and stimulated with a panel of overlapping UreB 18 mer peptides to assess the responsiveness of CD4+ T-cells for interferon-γ (IFN-γ) using ICS. The percentages of CD4+ T-cells secreting IFN-γ against each peptide were determined using flow cytometry. Locations of the dominant peptides in different groups are indicated.

MHC-II peptidome and proteome measurements in adjuvant-treated antigen-presenting cells (APCs).

A20 cells were treated with CpG ODN, MDP, or MPLA incorporated with Helicobacter pylori antigens for 12 h. Most cells (108) were lysed for immunopeptidomics and the remaining cells (107) were used for proteomics. (A) Experimental flow chart. (B) Number of MHC peptides identified in the different adjuvant-treated groups (n=3). The numbers indicate mean values. (C) Length distribution of MHC peptides in different adjuvant-treated groups. (D) Sequence motifs of the MHC peptides identified in the adjuvant-treated groups. (E) Binding heatmaps of all eluted MHC peptides between 9–22 mer in adjuvant-treated groups were predicted and assigned to alleles using NetMHCIIpan. ns: not significantly different (p>0.05).

Profiling exogenous MHC-II peptides in adjuvant-treated antigen-presenting cells (APCs).

(A) Peptide locations across the Helicobacter pylori genome from MHC-II immunopeptidomes. (B) Rank plot of each protein abundance detected in the whole-proteome of bacterial ultrasonic supernatant antigens. Proteins identified in immunopeptidomes are annotated with their respective gene names. (C) MHC-II presentation potential of bacterial proteins. All reported H. pylori proteins were ranked according to the ratio between the number of peptides predicted to be presented by MHC-II alleles (rank≤2) and the total number of 13- to 17-mer. Proteins identified in immunopeptidomes are annotated with their respective gene names. (D) Heatmap of exogenous MHC peptides from different adjuvant groups. The identified sequences are shown. (E) Numbers of MHC peptides derived from bacteria and host were compared among different adjuvant groups. n=3. **p<0.01, ****p<0.0001.

Antigen phagocytosis of antigen-presenting cells (APCs) treated with different adjuvants.

(A) Comparison of bacterial protein abundance in APCs 12 h post-adjuvant stimulation from whole proteomes. (B) Abundances of bacterial proteins from immunopeptidome and proteome were compared among adjuvant groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

The effects of adjuvants on antigen presentation.

(A) Rank plot of host and bacterial protein abundances in the whole-proteome and (B) MHC–peptide abundances from immunopeptidomes of different adjuvant groups. Bacterial proteins are marked with red, and some of them are annotated with their respective gene names. Bacterial MHC peptides are annotated with their respective amino acid sequence. (C) Volcano plots comparing protein levels between PBS- and adjuvant-treated groups in the whole-proteome. Proteins involved in antigen processing, ubiquitination, proteasome & peptidase, and interferon (IFN) pathways are colored accordingly. Above the dashed line (p<0.01) means significant.

Binding affinity of MS-detected peptides was determined.

(A) IC50 of the presented and deficient peptides post-adjuvant stimulation from immunopeptidome binding to H2-IA and H2-IE were predicted by the NN align method using the IEDB website. The data of peptides from adjuvants MPLA and CpG are shown. High IC50 means low binding stability. (B) Distribution of proteins corresponding to bacterial MHC peptides from immunopeptidome. The numbers of peptides identified by MS for each protein are indicated. (C) Information of 10 synthetic peptides from Top4 presented and deficient proteins. ×: Presence of peptides in the corresponding group. −: Peptides missing in the corresponding group. (D) Mirror plots with fragment ion mass spectra to confirm the sequences of MHC peptides from immunopeptidome. Positive y-axis, MHC-II IP sequences; negative y-axis, synthetic peptides. (E) Competitive binding curve of synthetic peptides for MHC-II H2-IA allele. The binding curves of peptides presented in adjuvant groups are marked with red.

T-cell responses induced by MS-detected peptides with different binding stability were analyzed.

Five BALB/c mice were immunized with a pool of 10 synthetic peptides. On days 10 and 28 post-immunization, the splenocytes were isolated and stimulated with individual peptides for interferon-γ (IFN-γ) Elisopt assay. (A) Experimental flow chart. (B) and (C) Elispot results on days 10 and 28. OVA peptide and non-stimulated wells were used as negative controls. PMA stimulation was used as the positive control. Responses to the peptides in the adjuvant groups are marked in red. The dashed line represents the 3× median of the OVA peptide used as the threshold for a positive response. Boxes show quartiles, bars indicate medians, and whiskers show distributions. Elispot images of positive responses from one immunized mouse are shown. Numbers indicate spot counts. (D) Epitope-specific CD4+ T-cells from immunized mice spleens were expanded in vitro and the IFN-γ producing CD4+ T-cells were assessed using the peptides pool (left). The present peptide recA #23 and deficient peptides ureA #2 and ureA #3 after adjuvant treatment were titrated to restimulate the expanded cells (right). The IFN-γ responses of CD4+ T-cells were detected by FACS. The responses induced by the indicated peptides at 50 μM were considered 100%. All other responses were evaluated based on their relative strengths.