Evaluation Of The Immune Efficacy Of SCD40L Adjuvant On Duck Tembusu Virus PrM-E DNA Vaccine

Antigen presenting cell（APC）CD40L is a combination of immunocostimulator and antigen presenting cell（APC）CD40,which provides a secondary signal for T and B lymphocyte activation and regulates cellular and humoral immune responses.In this study,duck CD40L extracellular domain fragment（du＿s CD40L）was used as an adjuvant to investigate its effect on the immune effect of Duck Tembusu virus（DTMUV）prM-E DNA vaccine.The main contents and results of the research are as follows:1.Preparation of du＿s CD40L polyclonal antibodyProkaryotic expression strain BL21（p ET28a-du＿s CD40L）was constructed successfully.After induction with IPTG at 0.4 mmol/L and 37°C for 4 h,the strain could express a specific inclusion body protein of about 20 k Da,which was consistent with the theoretical size of du＿s CD40L（20.6 k Da）.After purification by nickel,the serum of New Zealand rabbits was isolated after three times of co-immunization with du＿s CD40L prokaryotic expression protein and water adjuvant,which was called rabbit anti-du＿s CD40L polyclonal antibody.2.In vitro biological activity analysis of du＿s CD40LThe plasmid p VAX1-TPA-du＿s CD40L was successfully constructed and transfected into Duck embryo fibroblasts（DEF）cells to obtain du＿s CD40L protein.Peripheral blood mononuclear cell（PBMC）proliferation was determined by CCK-8 and 1:16 dilution（P<0.001）in concentration-dependent way.The results of RT-q PCR showed that du＿s CD40L could up-regulate the m RNA levels of APC,T and B cell related markers in duck PBMC,and enhance the activation of T helper 1（Th1）and T helper 2（Th2）cells.The results indicated that du＿s CD40L can promote immune cell response and has immunomodulatory effect.3.Construction of DTMUV prM-E DNA vaccine containing du＿s CD40L adjuvantThe recombinant p VAX1-prM-E-du＿s CD40L plasmid was constructed by fusion of prM and E fragments with du＿s CD40L fragments.p VAX1-prM-E and p VAX1-prM-E-du＿s CD40L were transfected into DEF cells.Both E and du＿s CD40L proteins were detected by Western blot（WB）and Indirect immunoinfluscent assay（IFA）.p VAX1-prM-E and p VAX1-prM-E-du＿s CD40L were injected intramuscularly,and the spleen was collected 7 days later.The expression of E protein could be detected by immunohistochemistry.In conclusion,the DNA vaccine plasmid constructed in this study can be expressed in vitro and in vivo.4.du＿s CD40L enhances the immune response of DTMUV prM-E DNA vaccineThe experimental animals were divided into three groups:Group A（Inactivated vaccine）,Group B（Control）,Group C（prM-E）,Group D（prM-E-s CD40L）.Group A was immunized twice,and group B,C and D were immunized three times with an interval of 3weeks.The group prM-E-s CD40L had higher Ig G titers than group prM-E（P<0.01）,and the group prM-E-s CD40L had higher Ig G titers than group prM-E（P<0.01）.The group prM-E-s CD40L had higher Ig G titers than group prM-E（P<0.01）.The group prM-E-s CD40L had higher Ig G titers than group prM-E（P<0.01）.Neutralizing antibody assays revealed that at 5 w and 8 w,the prM-E-s CD40L group produced higher titers of neutralizing antibodies than the prM-E group（P<0.01）.The results of cell proliferation assay showed that the proliferation rate of prM-E-s CD40L group was significantly higher than that of prM-E group at 5 w（P<0.01）.ELISA assay of IFN-γlevel in serum showed that du＿s CD40L significantly increased the expression of IFN-γinduced by DTMUV prM-E DNA vaccine（P<0.05）.These results suggest that du＿s CD40L can enhance the humoral and cellular immune responses of DTMUV prM-E DNA vaccine.5.du＿s CD40L can improve the immune protection of DTMUV prM-E DNA vaccineThe Ducks were challenged by intramuscular injection(DTMUV 10⁸TCID₅₀)11weeks after the first immunization.Serum,spleen,liver and brain were collected at 7 days post infection（7 dpi）.The results of TCID₅₀showed that compared with prM-E group,DNA vaccine containing du＿s CD40L adjuvant could significantly reduce the virus titer in serum（P<0.05）,spleen（P<0.01）and brain（P<0.05）.In the prM-E-s CD40L group,no pathological changes were found in the spleen,liver and brain tissues,while in the prM-E group,eosinophilic granular material and severe vacuolation were histopathology in the liver cells.These results suggest that du＿s CD40L as an adjuvant can enhance the vaccine-induced resistance to DTMUV.