Identification and characterization of serovar-independent immunogens in Actinobacillus pleuropneumoniae

Abstract

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 identified in the complete genomes of three A. pleuropneumoniae strains belonging to different 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 findings favour the immunogen selection protocol used, and suggest that OMVs, along with ApfA and VacJ, could represent effective 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.

Figure 1

1D SDS-PAGE analysis of purified ApfA and VacJ proteins. Tags were removed from the expressed recombinant proteins by thrombin cleavage. Subsequently, proteins + tags were loaded on an ÄKTAxpress chromatography system and purified by size exclusion chromatography (SEC). A ApfA SEC; B VacJ SEC; Input: Proteins + cleaved tags before SEC; Fractions 1–2: Isolated fractions of purified ApfA and VacJ proteins after SEC. ApfA and VacJ proteins are indicated by black arrows. The size of selected bands of the protein marker in kilodaltons (kDa) is shown. ApfA theoretical molecular weight: 20 kDa; VacJ theoretical molecular weight: 27 kDa; Human thrombin molecular weight: 37 kDa.

Figure 2

Transmission electron microscopy (TEM) analysis of A. pleuropneumoniae outer membrane vesicles (OMVs). TEM images show OMVs isolated from A. pleuropneumoniae MIDG2331 wild-type (A) and ΔdegS (B), ΔnlpI (C) mutants. Red arrows indicate OMVs.

Figure 3

TRPS analysis of OMVs. TRPS analysis of OMVs from A. pleuropneumoniae MIDG2331 wt (A), ΔdegS (B) and ΔnlpI (C) mutants. Size distribution and concentration of OMV samples are shown on individual graphics and resumed on the table on the right side of the figure.

Figure 4

IgG response to ApfA and VacJ proteins. IgG response in convalescent pigs against ApfA (A) and VacJ (B) proteins. IgG response is shown as area under the curve (AUC), calculated from sera dilution curves of an ELISA assay (1:500 to 1:1 093 500 dilutions) against 100 ng/well of protein. Data sets were transformed to log₁₀ and submitted to D’Agostino & Pearson omnibus normality test to verify the assumption of Gaussian distribution of the data (alpha = 0.05). After conversion of the data sets to AUC units, differences between mean AUC values of each group were analyzed by Ordinary one-way ANOVA, followed by Holm-Sidak post hoc test when overall statistical significance was determined by ANOVA (P < 0.05). Asterisks indicate statistical difference between individual groups and controls by Holm-Sidak (P < 0.05).

Figure 5

IgG response to OMVs. IgG response to A. pleuropneumoniae 2331 ΔnlpI OMVs was assessed by Western blot analysis using pig sera pooled from the same group of pigs, before (A) and after a challenge with live A. pleuropneumoniae HK 361 cells (B). Bands size in kilodaltons (kDa) of the protein marked used are provided on the right side of the figure.