Identification of potential antigenic peptides of Brucella through proteome and peptidome

Abstract

Background: Brucellosis, caused by Brucella spp., is a major zoonotic public health threat. Although several Brucella vaccines have been demonstrated for use in animals, Brucella spp. can cause human infection and to date, there are no human-use vaccines licensed by any agency. Recently, methods in vaccine informatics have made major breakthroughs in peptide-based epitopes, opening up a new avenue of vaccine development.

Objectives: The purpose of this article was to identify potential antigenic peptides in Brucella by proteome and peptidome analyses.

Methods: Mouse infection models were first established by injection with Brucella and spleen protein profiles were then analysed. Subsequently, the major histocompatibility complex class I or II (major histocompatibility complex [MHC]-I/II)-binding peptides in blood samples were collected by immunoprecipitation and peptides derived from Brucella proteins were identified through liquid chromatography-mass spectrometry (LC-MS/MS). These peptides were then evaluated in a variety of ways, such as in terms of conservation in Brucella and synchronicity in predicted peptides (similarity and coverage), which allowed us to more effectively measure their antigenic potential.

Results: The expression of the inflammatory cytokines IL1B and IFN-γ was significantly altered in the spleen of infected mice and some Brucella proteins, such as Muri, AcpP and GroES, were also detected. Meanwhile, in blood, 35 peptides were identified and most showed high conservation, highlighting their potential as antigen epitopes for vaccine development. In particular, we identified four proteins containing both MHC-I- and MHC-II-binding peptides including AtpA, AtpD, DnaK and BAbS19_II02030. They were also compared with the predicted peptides to estimate their reliability.

Conclusions: The peptides we screened could bind to MHC molecules. After being stimulated with antigen T epitopes, Memory T cells can stimulate T cell activation and promote immune responses. Our results indicated that the peptides we identified may be good candidate targets for the design of subunit vaccines and these results pave the way for the study of safer vaccines against Brucella.

Keywords: Brucella abortus; MHC; mass spectrometry; proteomics.

FIGURE 1

Schematic diagram of this study . Mice were injected with Brucella abortus intraperitoneally and then the spleen cells and PBMCs were isolated 6 days after infection. Proteins from spleen were extracted and assessed by LC–MS/MS. MHC‐binding peptides in PBMCs were extracted by immunocoprecipitation and further detected through proteome analysis.

FIGURE 2

Differential expression analysis of murine proteins in the spleens of infected mice. (a) Proteomics results from infected and uninfected mice were compared. Fold changes indicate the degree of changes in the infected group compared with the control group. | log2FoldChange | ≥ 1 was used as the threshold, and the adjusted p value (FDR) < 0.05 was regarded as significant. Each dot represents a gene, red dots represent an upregulated gene, green dots represent a downregulated gene and black dots represent nonsignificant genes. In particular, the blue dots represent immune‐related genes, and the brown dots represent histocompatibility antigen related genes. (b) Each cell represents the gene expression level relative to another group. This value comes from the mass spectrometer, where the count of one protein in each sample is divided by the average of another group and the logarithm is taken. The level of inflammatory cytokines (c) and MHC‐related proteins (d) identified in infected and controlled mice

FIGURE 3

GO analysis and KEGG pathway enrichment of DEGs. (a–b) Secondary‐level GO analysis, including upregulated genes (a) and downregulated genes (b). Each column represents a term, and its length represents the number of DEGs contained under each term. The green terms are BP ontologies, and orange terms are molecular function ontologies. (c, d) KEGG pathway enrichments, including upregulated genes (c) and downregulated genes (d). Each bubble represents a pathway, its size being proportional to the number of genes, and the colour represents the level of statistical significance. The abscissa is the gene ratio, that is, the proportion of genes in DEGs that are enriched in each pathway. Abbreviations: Ont, ontology; BP, biological process; MF, molecular function

FIGURE 4

Conservation of MHC‐I‐binding peptides (a) and MHC‐II‐binding peptides (b) in Brucella spp. The horizontal axis represents each peptide, and the vertical axis represents their conservation. Overlapping sequences were merged to show only the longer sequences from each.

FIGURE 5

Comparison of MHC‐binding peptide sequences experimentally identified with those predicted by bioinformatics methods. The ordinate represents the MHC‐binding capacity of different amino acids at this site, where the icon above the abscissa indicates that this amino acid at this site will promote MHC binding, while the icon below the abscissa indicates that this amino acid at this site will inhibit MHC binding. (a) Identified MHC‐I‐binding peptides. (b) Identified MHC‐II‐binding peptides. (c) MHC‐I‐predictive peptides. (d) MHC‐II‐predictive peptides