Despite numerous actions to prevent disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) remains a major
cause of porcine pleuropneumonia, resulting in economic losses to the swine industry worldwide. In this paper, we
describe the utilization of a reverse vaccinology approach for the selection and in vitro testing of serovar-independent
A. pleuropneumoniae immunogens. Potential immunogens were identifed in the complete genomes of three A. pleuropneumoniae
strains belonging to diferent serovars using the following parameters: predicted outer-membrane subcellular
localization; ≤ 1 trans-membrane helices; presence of a signal peptide in the protein sequence; presence in all
known A. pleuropneumoniae genomes; homology with other well characterized factors with relevant data regarding
immunogenicity/protective potential. Using this approach, we selected the proteins ApfA and VacJ to be expressed
and further characterized, both in silico and in vitro. Additionally, we analysed outer membrane vesicles (OMVs) of A.
pleuropneumoniae MIDG2331 as potential immunogens, and compared deletions in degS and nlpI for increasing yields
of OMVs compared to the parental strain. Our results indicated that ApfA and VacJ are highly conserved proteins, naturally
expressed during infection by all A. pleuropneumoniae serovars tested. Furthermore, OMVs, ApfA and VacJ were
shown to possess a high immunogenic potential in vitro. These fndings favour the immunogen selection protocol
used, and suggest that OMVs, along with ApfA and VacJ, could represent efective immunogens for the prevention of
A. pleuropneumoniae infections in a serovar-independent manner. This hypothesis is nonetheless predictive in nature,
and in vivo testing in a relevant animal model will be necessary to verify its validity.