Immunomodulatory Functions Of Recombinant Bordetella Avium OmpA-IgY Fc And Taishan Pinus Massoniana Pollen Polysaccharides On Chickens

Inactivated or killed vaccines have been widely applied to control infectious diseases.However,conventional formalin-or heat-inactivated vaccine formulations can alter the physio-chemical/structural properties of the antigens,thereby negatively affecting the development of protective immunity.Recombinant subunit vaccines can be used to effectively prevent bacterial diseases because of their resemblance to the native form as well as their rapid,consistent,and scalable production.Proteins or peptides generally show short serum half-life and limited antigenic stimulation because of conventional antigen capture by antigen presenting cells(APC)and the fast renal clearance.Therefore,it is important to develop a novel vaccine to prevent infectious diseases.Currently,Fc-fusion technologies,in which immunoglobulin Fc is genetically fused to an antigenic protein,have been developed to confer antibody-like properties to proteins and peptides.Mammalian IgG–Fc fusion exhibits improved antigen-induced immune responses by providing aggregates with high avidity for the IgG Fc receptor(FcR)and salvaging the antigenic portion from endosomal degradation.However,whether the linked chicken IgY Fc fragment shares similar characteristics to mammalian IgG Fc remains unclear.In this study,we established the linked chicken IgY Fc model,and used P.pastoris GS115 eukaryotic expression system to express a fusion protein containing the chicken IgY Fc and ompA of Bordetella avium,a common respiratory disease pathogen of avian species.Firstly,the effect of ompA-Fc fusion protein on the activity of chicken peritoneal macrophages was evaluated to evaluate the effect of the linked Fc on the non-specific immune response of chickens.Then,the fusion protein was used to immunize chickens to evaluate the effect of Fc on specific immune responses.In addition,in our previous studies,we found that Taishan Pinus massoniana pollen polysaccharides(TPPPS)is an effective adjuvant for the inactivated and subunit vaccines.TPPPS was also used as immune adjuvant to investigate its irritation on chicken peritoneal macrophages and immunomodulation on ompA–Fc-induced immunoresponses.This study was divided into the following three parts: 1.Expression and identification of the fused ompA-FcThe chicken IgY Fc and B.avium ompA genes were linked by overlapping PCR technology.The linked ompA–Fc gene fragment was cloned into the expression vector pPIC9 and verified by gene sequencing.The recombinant pPIC9-ompA–Fc plasmid was then transformed into P.pastoris.Upon induction with methanol at different induction times,a novel protein band corresponding to 47.4 kDa in the culture supernatant of the recombinant pPIC9-ompA–Fc transformant was determined through SDS-PAGE;however,this band did not appear in the culture supernatant of the control pPIC9(blank plasmid)transformant.The protein was detected in the supernatant after 48 h of cultivation,and the maximum protein yield(18 mg/L)was obtained at 72 h.After purification,only the target protein band with a molecular weight of 47.4 kDa was observed in the results of SDS-PAGE.Western blot analyses were performed with anti-His tag antibody,rat anti-omp polyclonal antibody,or rabbit anti-chicken IgG(HRP)to determine the immunogenicity of the fusion protein.After color development,a 47.4 kDa band was observed in the three independent assays;this band corresponded to the band detected in the SDS-PAGE.The results indicated the independent immunogenicity of the fused IgY Fc and ompA of B.avium.Moreover,at the start of ompA–Fc fusion,antigens expressed in P.pastoris were presented in a schematic and 3D structure of the fusion protein ompA–Fc and developed through homology modeling methods of SWISS-MODEL.In addition,the recombinant pPIC9-ompA plasmid constructed in our laboratory was used as control in the present study;the immunogenicity of the recombinant ompA expressed in P.pastoris was also verified 2.Influences of the linked Fc and TPPPS on the viability of peritoneal macrophagesChicken peritoneal macrophages were isolated with previous method.The viability of macrophages treated with the recombinant ompA and ompA–Fc were assayed by MTT to assess the effects of the recombinant proteins on the growth of chicken peritoneal macrophages.The cytotoxicity of TPPPS,which was used as the adjuvant in the following experiments,was examined.Internalization in the form of pinocytosis or phagocytosis is a key indicator of macrophage effector activity.NO and TNF-α secreted by macrophages also reflect the activity of the cells.The effect of TPPPS on the pinocytic activity of peritoneal macrophages was assayed via neutral red uptake.Immunofluorescent assay(IFA)was performed to analyze ompA phagocytosis.The expression of major histocompatibility complex class II(MHC-II)molecules on the surface of macrophages was detected by flow cytometry analysis.The results indicated that treatment with recombinant proteins did not change the viability of chicken peritoneal macrophages,whereas TPPPS dose-dependently promoted the proliferation and viability of peritoneal macrophages.Similarly,linked Fc and TPPPS adjuvant remarkably increased the antigen capture capacity of chicken peritoneal macrophages.The addition of TPPPS further enhanced MHC-II expression on fused ompA–Fc-treated macrophages.These data imply that the linked Fc and TPPPS adjuvant were likely also facilitate to the antigen procession of chicken macrophages.3.Immune-enhancing effects of TPPPS on the fused Fc-ompA subunit vaccineTPPPS was prepared in our laboratory by the method od water extraction and ethanol precipitation.The fusion protein ompA-Fc and ompA were mixed with TPPPPS.A total of 120 1-day-old specific pathogen-free white leghorn chickens were randomly placed into four sterilized isolators(groups I to IV),with 30 chickens each.The chickens were allowed to acclimatize for three days before the start of the experiments.Each chicken in groups I-IV was subcutaneously inoculated with 0.2 mL of recombinant ompA vaccines,ompA–Fc fusion protein vaccine,TPPPS adjuvant ompA–Fc fusion protein vaccines,and PBS,at 0,7,and 14 days post the first vaccination(dpv).Groups I-IV were named as ompA,ompA–Fc,ompA–Fc-TPPPS,and PBS,respectively.At 7,14,21,28,35,42,and 49 dpv,three chickens from each group were selected randomly to determine the serum antibody titers,serum IL-2,and IL-4 concentrations,CD4+ and CD8+ T lymphocytes counts in the peripheral blood,and T-lymphocyte transformation rate.At one week after the third vaccination(at 21 dpv),20 chickens from each group(except from the control group)were placed into a new isolator and challenged intranasally with 10 LD50 B.avium LL strain.Clinical manifestation and survival status of the chickens were recorded for seven days after challenge.The results showed that the ompA-Fc vaccine induced the production of anti-ompA antibody,the secretion of IL-2,IL-4,the increase of CD4+ T-lymphocytes counts and lymphocyte transformation rate in the peripheral blood.Moreover,the ompA-Fc vaccine provided a protection rate of 80 % after the B.avium challenge.Additionally,TPPPS,as the adjuvant,presents good immune-enhancing effects on the fused Fc-ompA subunit vaccine.Thus,TPPPS,combined with the linked Fc,synergistically facilitated the systemic immune response induced by antigen subunit.Overall,our findings provided a new perspective to improve the immune effect of subunit vaccines for poultry disease prevention.