LAW_RS03650: a species-specific novel antigen of Lawsonia intracellularis revealed via pangenomic reverse vaccinology method and serologically validated.

Lawsonia Intracellularis (L. intracellularis) is an obligate intracellular bacterium that causes porcine proliferative enteropathy. In this study, a pan-RV (reverse vaccinology based on pangenome analysis) approach was applied to analyze the whole-genome sequences of 9 L. intracellularis strains downloaded from the National Center of Biotechnology Information (NCBI) server. Pangenome analysis revealed a closed pangenome and a core genome consisting of 1372 genes, while reverse vaccinology further revealed 16 candidate proteins with higher in silico immunogenicity parameters, including predicted antigenicity, "outer membrane protein" or "extracellular protein" by subcellular localization analysis and no more than 2 transmembrane regions by transmembrane helices prediction. Three of the 16 proteins that showed no homology with proteins of other species according to BLAST (Basic Local Alignment Search Tool) were selected for serological validation. The Western blotting results revealed that the 3 proteins did not cross-react with anti-Shigella and anti-Salmonella sera, with 1 protein (LAW_RS03650) showed antigenicity when it reacted with positive wild-type anti-L. intracellularis serum. The immunofluorescence of infected cells employing anti-LAW_RS03650 serum indicated a wide distribution of LAW_RS03650 protein in the host cells and L. intracellularis itself. This study identified a novel L. intracellularis antigen, LAW_RS03650, as a candidate for future recombinant vaccine development or species-specific serodiagnostic reagents.