Identification of vaccine candidates against rhodococcus equi by combining pangenome analysis with a reverse vaccinology approach.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that can cause life-threatening infections. The rapid evolution of multidrug-resistant R. equi and the fact that there is no currently licensed effective vaccine against R. equi warrant the need for vaccine development. Reverse vaccinology (RV), which involves screening a pathogen's entire genome and proteome using various web-based prediction tools, is considered one of the most effective approaches for identifying vaccine candidates. Here, we performed a pangenome analysis to determine the core proteins of R. equi. We then used the RV approach to examine the subcellular localization, host and gut flora homology, antigenicity, transmembrane helices, physicochemical properties, and immunogenicity of the core proteins to select potential vaccine candidates. The vaccine candidates were then subjected to epitope mapping to predict the exposed antigenic epitopes that possess the ability to bind with major histocompatibility complex I/II (MHC I/II) molecules. These vaccine candidates and epitopes will form a library of elements for the development of a polyvalent or universal vaccine against R. equi. Sixteen R. equi complete proteomes were found to contain 6,238 protein families, and the core proteins consisted of 3,969 protein families (∼63.63% of the pangenome), reflecting a low degree of intraspecies genomic variability. From the pool of core proteins, 483 nonhost homologous membrane and extracellular proteins were screened, and 12 vaccine candidates were finally identified according to their antigenicity, physicochemical properties and other factors. These included four cell wall/membrane/envelope biogenesis proteins; four amino acid transport and metabolism proteins; one cell cycle control, cell division and chromosome partitioning protein; one carbohydrate transport and metabolism protein; one secondary metabolite biosynthesis, transport and catabolism protein; and one defense mechanism protein. All 12 vaccine candidates have an experimentally validated 3D structure available in the protein data bank (PDB). Epitope mapping of the candidates showed that 16 MHC I epitopes and 13 MHC II epitopes with the strongest immunogenicity were exposed on the protein surface, indicating that they could be used to develop a polypeptide vaccine. Thus, we utilized an analytical strategy that combines pangenome analysis and RV to generate a peptide antigen library that simplifies the development of multivalent or universal vaccines against R. equi and can be applied to the development of other vaccines.