Study On The Immune Adjuvant Effect Of Neisseria Meningitidis Ag473 Lipoprotein On PCV2 Cap Protein

Porcine circovirus virus disease(PCVD)is a general term for a series of disease syndroms caused mainly by porcine circovirus type 2(PCV2),leading to significant economical loss to the pig industry worldwide.Presently,control of the disease relies mainly on vaccination.The current vaccines include inactivated virus vaccines and virus-like particle(VLP)subunit vaccines.However,the virus replicates poorly in vaccine production cell liness,and the VLPs expressed in E.coli or insect cells require chromatographic columns for purification.Elastin-like polypeptides(ELPs)undergo temperature-sensitive phase transition,allowing their fusion protein purification by simple inverse transition cycling(ITC).Self-aggregating peptides such as ELK16 can form active inclusion bodies in E.coli,allowing their fusion protein purification by centrifugation or filtration.Bacterial Lipoproteins such as Ag473 of Neisseria meningitides are important pathogen-associated molecular patterns that can stimulate innate immune cells to release cytokines with immunoadjuvant effects.In this study,ELP and ELK16 were used as the purification tags for expression and purification of PCV2 recombinant Cap protein and lipoprotein Ag473 of Neisseria meningiditis,aiming at simplifying the PCV2 subunit vaccine preparation and developing a novel molecular adjuvant.First,the ELK16-coding sequence was fused to Ag473 gene or PCV2 Cap gene,and transformed into E.coli for fusion protein expression.The fusion proteins ELK16-Cap,ELK16-Ag473 and ELK16-Ag473-Cap were purified by centrifugation in the presence of TriTon X-100,with a purity of 95%,94%or 91%,and a yield of 92,45 or 60mg/L.Twenty four mice were divided into 4 groups,and immunized with ELK16-Cap plus IFA,ELK16-Cap plus ELK16-Ag473,ELK16-Ag473-Cap or PBS as the control.The serum samples were collected at day 7,14,21 and 28 after primary immunization and the Cap-specific antibodies were detected by ELISA.The results showed that,on day 7 post immunization,only ELK16-Ag473 plus ELK16-Cap group was positive for Cap-specific antibodies.From day 14 post immunization,the antibody levels of the three immunization groups increased gradually,and the highest antibody level was detected in the ELK16-Ag473 plus ELK 16-Cap immunization group.On day 14 post boosting immunization,serm samples were collected from the immunized mice for cytokine detection.Compared to the PBS control group,the serum concentrations of TNF-?,IL-12 and IFN-y of ELK16-Cap plus IF,A,ELK16-Ag473-Cap or ELK16-Ag473 plus ELK16-Cap immunization group were increased by 3.6,6.1 and 1.8-folds,4.9,8.8 and 7.7 folds or 2.6,8.3 and 9.6 folds,respectively.These data indicate that both mixed and fused Ag473 have adjuvant effects on humoral and cellular responses against PCV2 Cap protein in mice.Then,the coding sequences for the neutralizing epitopes of PCV2a,PCV2d and PCV2e(4Cap)were fused to the Cap-coding sequence of PCV2b,and expressed as a His-tagged protein.In addition,the recombinant Ag473 was also expressed as a His-tagged protein.Thirty mice were divided into 5 groups,and immunized with PBS,4Cap-His,4Cap-His plus ELP-Ag473,4Cap-His plus ELK16-Ag473 or 4Cap-His plus IFA.On different days post immunization,serum samples were collected for Cap-specific antibody detection by ELISA.On day 14 post boosting immunization,splenic lymphocytes were collected and cultured for cytokine diction.On day 14 post boosting immunization,three mice from each group were challenged with 103 TCID50 PCV2b,and serm samples were collected for viremia detection on day 3 or 7 post challenge.The results showed that only 4Cap-His plus ELK16-Ag473 and 4Cap-His plus ELP-Ag473 immunization groups were positive for Cap-specific antibodies.On day 14 post primary immunization,the antibody levels of 4Cap-His plus ELK16-Ag473 and 4Cap-His plus ELP-Ag473 immunization groups were significantly increased compared to 4Cap-His plus IFA immunization group,whereas 4Cap-His immunization group was still negative for Cap-specific antibodies.From day 21 post primary immunization,the antibody levels of all four immunization groups were significantly increased,with the highest antibody level in Cap-His plus ELP-Ag473 immunization group.Compared to the mock immunization group,serum concentrations of TNF-?,IL-2 and FN-y of all four immunization groups were significantly increased after 4Cap-His stimulation,which were lower than that after Con A stimulation.The viremia of all four immunization groups were significantly decreased on day 7 post challenge,with the highest decrease in 4Cap-His plus ELK16-Ag473 immunization group,followed by 4Cap-His plus ELP-Ag473,4Cap-His plus IFA and 4Cap-His immunization group.These data indicate that both ELP and ELK16 tags had overt influence on Ag473 adjuvant activity,and recombinant Ag473 had strong adjuvant effect on humoral and cellular responses against PCV2 Cap protein in mice.