Genomic characterization and immunoinformatics analysis of the Brucella melitensis vaccine strain BA0711

Abstract

B. melitensis BA0711 is a novel genetically engineered attenuated live vaccine strain constructed by the targeted deletion of the cold shock protein A (CspA) gene. While previous studies demonstrated its safety and protective efficacy in animals, its underlying comparative genomic characteristics, pathogenicity evolution, and theoretically predicted immunogenic potential from a genome-wide perspective remain to be systematically elucidated. In this study, a high-quality complete genome of B. melitensis BA0711 was obtained using a hybrid next-generation sequencing and third-generation sequencing strategy. The genome comprised two closed circular chromosomes totaling 3,310,449 bp, with no plasmids or exogenously acquired antimicrobial resistance genes detected. Phylogenetic, multilocus sequence typing, and whole-genome similarity analyses determined B. melitensis BA0711 as the ST8 lineage and revealed its closest genetic relationship with B. melitensis M28 among the reference strains analyzed. Whole-genome collinearity and pangenome analyses further revealed a highly conserved genomic backbone and macro-syntenic organization. Horizontal gene transfer analysis identified 263 putative horizontally transferred genes, including a pathogenicity-associated genomic island, GI_6, containing multiple core lipopolysaccharide biosynthesis and modification genes. Comparative profiling of core virulence factors confirmed the targeted absence of cspA and revealed lineage-associated amino-acid variation in several virulence-related proteins. Following host-homology screening, immunoinformatics analysis of 78 core virulence-associated proteins retained 5,212 non-host-homologous MHC-I strong-binding epitope records, 7,476 non-host-homologous MHC-II strong-binding epitope records, and 574 non-host-homologous linear B-cell epitope candidates. Cgs and BPE043 were prioritized as representative T-cell epitope-rich proteins. Sequential screening based on antigenicity, allergenicity, toxicity, and HLA-binding breadth further identified four representative peptides: SSAAISSAF, LSMFVAPTF, SLNSLSVDIARAIDH, and RRFIRQLPLYKVPLE. AlphaFold 3 modeling and complementary HPEPDOCK analysis supported structurally plausible presentation of these peptides within the corresponding canonical HLA-binding grooves. The targeted deletion of cspA was additionally confirmed by PCR. Our whole-genome comparative analysis elucidates the genomic architecture and evolutionary background of B. melitensis BA0711. The strain possesses a conserved ST8-associated genomic background and shows the closest genetic relationship with B. melitensis M28 among the analyzed reference strains. Systematic profiling of genome organization, putative horizontally transferred genes, and virulence-factor variation provides a molecular framework for understanding this vaccine strain. Moreover, immunoinformatics analysis prioritized Cgs and BPE043 as representative T-cell epitope-rich proteins and identified four peptide candidates with computationally plausible HLA-presentation modes. These in silico findings identify experimentally testable candidates for further investigation but require experimental validation of HLA binding and immunogenicity.