Proteomic and immunoproteomic characterization of a DIVA subunit vaccine against Actinobacillus pleuropneumoniae

Abstract

BackgroundProtection of pigs by vaccination against Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is hampered by the presence of 15 different serotypes. A DIVA subunit vaccine comprised of detergent-released proteins from A. pleuropneumoniae serotypes 1, 2 and 5 has been developed and shown to protect pigs from clinical symptoms upon homologous and heterologous challenge. This vaccine has not been characterized in-depth so far. Thus we performed i) mass spectrometry in order to identify the exact protein content of the vaccine and ii) cross-serotype 2-D immunoblotting in order to discover cross-reactive antigens. By these approaches we expected to gain results enabling us to argue about the reasons for the efficacy of the analyzed vaccine.ResultsWe identified 75 different proteins in the vaccine. Using the PSORTb algorithm these proteins were classified according to their cellular localization. Highly enriched proteins are outer membrane-associated lipoproteins like OmlA and TbpB, integral outer membrane proteins like FrpB, TbpA, OmpA1, OmpA2, HgbA and OmpP2, and secreted Apx toxins. The subunit vaccine also contained large amounts of the ApxIVA toxin so far thought to be expressed only during infection. Applying two-dimensional difference gel electrophoresis (2-D DIGE) we showed different isoforms and variations in expression levels of several proteins among the strains used for vaccine production. For detection of cross-reactive antigens we used detergent released proteins of serotype 7. Sera of pigs vaccinated with the detergent-released proteins of serotypes 1, 2, and 5 detected seven different proteins of serotype 7, and convalescent sera of pigs surviving experimental infection with serotype 7 reacted with 13 different proteins of the detergent-released proteins of A. pleuropneumoniae serotypes 1, 2, and 5.ConclusionsA detergent extraction-based subunit vaccine of A. pleuropneumoniae was characterized by mass spectrometry. It contained a large variety of immunogenic and virulence associated proteins, among them the ApxIVA toxin. The identification of differences in expression as well as isoform variation between the serotypes implied the importance of combining proteins of different serotypes for vaccine generation. This finding was supported by immunoblotting showing the induction of cross-reactive antibodies against several surface associated proteins in immunized animals.

Simultaneous Detection of Antibodies against Apx Toxins ApxI, ApxII, ApxIII, and ApxIV in Pigs with Known and Unknown Actinobacillus pleuropneumoniae Exposure Using a Multiplexing Liquid Array Platform

“…Slight seroconversion against ApxII was detected in pigs vaccinated with serovar 5 and against both ApxII and ApxIII in pigs vaccinated with serovar 7. In a previous study, convalescent-phase serum samples of pigs infected with serovar 7, which is thought to exclusively express the apxII gene, allowed detection of toxins ApxI and ApxIII of a subunit vaccine in which ApxII was not present (45). These additional observations further confirm the cross-reactivity between the A. pleuropneumoniae Apx toxins.…”

“…Therefore, ApxIV antigen has been used to differentiate infected from vaccinated animals (DIVA), as it is immunogenic, specific to A. pleuropneumoniae, and encoded by all serovars (44). However, recently, an ApxIVA protein was identified for the first time from an in vitro growth of A. pleuropneumoniae as part of a subunit vaccine (45). Furthermore, in line with a previous study using ELISA and CFTs (3), a poor or absent seroconversion to ApxII and ApxIII, or ApxI, respectively, was reported for vaccinated pigs by FMIA.…”

A Multivalent Adsorption Apparatus Explains the Broad Host Range of Phage phi92: a Comprehensive Genomic and Structural Analysis

“…Protein identification was performed by ultraperformance liquid chromatography (UPLC)-coupled quadrupole time of flight (Q-TOF) tandem mass spectroscopy (MS/MS) (nanoACQUITY UPLC system coupled to Q-TOF Ultima; Waters) as described previously (11). Processing of spectra and searching of the phi92 protein database generated from its genome (accession number FR775895 [this study]) were performed using the program ProteinLynxTM Global Server (Version 2.1; Waters).…”

“…The protein pellet was washed twice with 80% acetone, dried by aeration with nitrogen, and dissolved in 30 mM Tris-HCl (pH 8.5)-7 M urea-2 M thiourea-4% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}. Electrophoresis was performed on Immobiline DryStrips (GE Healthcare) (24 cm in length) with a pI gradient of from 3 to 11 for the first-dimension focusing and 10% polyacrylamide gels for the second dimension according to a procedure described previously (11).…”

Biology and Diseases of Swine

“…Vaccination of seronegative animals prior to introduction with killed whole-cell, cell-free antigens, or subunit type along with maintaining optimal ambient temperature, ventilation, and humidity may reduce morbidity and mortality (Gottschalk, 2012a;Buettner et al, 2011;Oishi et al, 1995). Oral immunization with live or inactivated A. pleuropneumoniae serotype 9 has been shown to provide partial clinical protection from aerosol challenge (Hensel et al, 1995).…”