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

Buettner FF, Konze SA, Maas A, Gerlach GF - Proteome Sci (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, Institute for Microbiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. falkbuettner@gmx.de.

ABSTRACT

Background: Protection 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.

Results: We 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.

Conclusions: A 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.

No MeSH data available.


Related in: MedlinePlus

Immunoblot analysis for the identification of cross-reactive proteins. "Detergent-wash" proteins were separated by 2-D and subsequently Western blotted. "Detergent-wash" proteins from serotype 7 were probed using immune sera of pigs immunized with the subunit vaccine (A) and the subunit vaccine was probed with convalescent sera from pigs upon experimental infection with serotype 7 (B). Immunogenic spots were assigned to spots on preparative Coomassie stained gels (Additional file 4, Figure S2) and identified by mass spectrometry. The numbers on each spot that has been identified allow the finding of the respective spot on preparative gels (Additional file 4, Figure S2).
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Figure 2: Immunoblot analysis for the identification of cross-reactive proteins. "Detergent-wash" proteins were separated by 2-D and subsequently Western blotted. "Detergent-wash" proteins from serotype 7 were probed using immune sera of pigs immunized with the subunit vaccine (A) and the subunit vaccine was probed with convalescent sera from pigs upon experimental infection with serotype 7 (B). Immunogenic spots were assigned to spots on preparative Coomassie stained gels (Additional file 4, Figure S2) and identified by mass spectrometry. The numbers on each spot that has been identified allow the finding of the respective spot on preparative gels (Additional file 4, Figure S2).

Mentions: For the identification of cross-reactive antibodies induced by vaccination with the subunit vaccine we performed immunoblotting of serotype 7 "detergent-wash" proteins and used sera of pigs immunized with the subunit vaccine (Figure 2A). These sera detected seven different proteins from serotype 7 (ApxIIA, D15, UshA, FrpB, TufB, OmpA1, and OmpA2).


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

Buettner FF, Konze SA, Maas A, Gerlach GF - Proteome Sci (2011)

Immunoblot analysis for the identification of cross-reactive proteins. "Detergent-wash" proteins were separated by 2-D and subsequently Western blotted. "Detergent-wash" proteins from serotype 7 were probed using immune sera of pigs immunized with the subunit vaccine (A) and the subunit vaccine was probed with convalescent sera from pigs upon experimental infection with serotype 7 (B). Immunogenic spots were assigned to spots on preparative Coomassie stained gels (Additional file 4, Figure S2) and identified by mass spectrometry. The numbers on each spot that has been identified allow the finding of the respective spot on preparative gels (Additional file 4, Figure S2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3107771&req=5

Figure 2: Immunoblot analysis for the identification of cross-reactive proteins. "Detergent-wash" proteins were separated by 2-D and subsequently Western blotted. "Detergent-wash" proteins from serotype 7 were probed using immune sera of pigs immunized with the subunit vaccine (A) and the subunit vaccine was probed with convalescent sera from pigs upon experimental infection with serotype 7 (B). Immunogenic spots were assigned to spots on preparative Coomassie stained gels (Additional file 4, Figure S2) and identified by mass spectrometry. The numbers on each spot that has been identified allow the finding of the respective spot on preparative gels (Additional file 4, Figure S2).
Mentions: For the identification of cross-reactive antibodies induced by vaccination with the subunit vaccine we performed immunoblotting of serotype 7 "detergent-wash" proteins and used sera of pigs immunized with the subunit vaccine (Figure 2A). These sera detected seven different proteins from serotype 7 (ApxIIA, D15, UshA, FrpB, TufB, OmpA1, and OmpA2).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, Institute for Microbiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. falkbuettner@gmx.de.

ABSTRACT

Background: Protection 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.

Results: We 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.

Conclusions: A 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.

No MeSH data available.


Related in: MedlinePlus