Be Aware of Immunogenic But not Protective Antigens: The Actinobacillus pleuropneumoniae PalA as an Example

Background: Identification of immunogenic antigens is an important step for the vaccine improvement. Previous studies indicated that Actinobacillus pleuropneumoniae PalA is homologous to a Haemophilus influenzae protective antigen Hi-PAL (P6) protein. However, PalA protein adversely affects the Apx toxinbased subunit vaccine. The role of PalA in the inactivated vaccine is not known, and the mechanism involved in the PalA-mediated interference has not been investigated. Objectives: The main objective of this study was to investigate the possible impacts of PalA on the protective immunity of A. pleuropneumoniae inactivated vaccine. Methods: Coding sequence of the mature peptide of PalA was amplified from A. pleuropneumoniae SLW01, and inserted into the prokaryotic expression plasmid pGEX-KG, so as to generate the recombinant PalA (rPalA) protein. The immunogenicity of rPalA was verified in rabbits. For the protection assay, mice were assigned into 4 groups, and were immunized with TSB, rPalA, bacterin (Bac) and bacterin + rPalA (BacPal), respectively. Humoral immune response was evaluated before each immunization and before challenge. Two weeks after three immunizations, mice were infected with virulent A. pleuropneumoniae 4074. The clinical signs, survival rates and lung bacteria loads were determined. Then a passive protection assay was performed using pooled sera from the active immunization assay. Results: rPalA was produced in E. coli and was confirmed to be immune-reactive. rPalA is able to elicit a strong humoral immune response in rabbit. Besides, polyclonal antibodies against rPalA is able to recognize the natural PalA in the outer membrane of A. pleuropneumoniae. The positive immunization assay showed that mice immunized with BacPal produced significantly less antibodies against Apx toxins, relative to that of animals immunized with Bac before challenge (P <0.01). After virulent challenge, all mice in the TSB and rPalA groups died within 48 hpi. The survival rates of the Bac and the BacPal groups were 100% and 75%, respectively. The average bacterial loads of the BacPal group was lower than that of the TSB and rPalA groups (P <0.01), but higher than that of the Bac group (P <0.01). The survival rates of mice received pooled anti-sera against TSB, rPalA, BacPal and Bac, were 0%, 0%, 37.5% and 100% after challenge, respectively. In addition, mice in the BacPal group showed moderate to severe lung damage, whereas mice in the Bac group showed relatively normal lung tissues during the histological examination. Conclusion: Our results demonstrate that A. pleuropneumoniae PalA is an immunogenic but not protective antigen, the existence of PalA suppresses the production of protective antibodies, and thus reduces the protective immunity of inactivated vaccine. Therefore, it should be taken into consideration of these immunogenic but not protective proteins during the development of highly effective vaccines in future.