Evaluation Of CpG Adjuvant For Efficacy Enhancement Of A Duck Tembusu Virus Disease Inactivated Vaccine

Duck Tembusu virus disease is an acute infectious disease caused by duck Tembusu Virus?DTMUV?,which brings a severe blow to the healthy development of the breeding industry.The inactivated vaccine has been widely used due to its high safety,but it is defective in immune protection and efficacy.The new adjuvant CpG ODN reportedly improves immune stimulation,and it has been used to preliminarily explore the effect of immune stimulation on waterfowl and achieved ideal results.However,the high cost of synthetic CpG ODN is not conducive to large-scale production and application.Inserting CpG motif into pUC18 vector has been proved to be feasible in some research,which can enhance the immune effect of vaccines.In this study,eighteen CpG ODNs were first designed and synthesized,and the stimulation index and mRNA expression levels of related immune genes were measured by duck peripheral blood lymphocyte proliferation test and RT-qPCR,respectively,to determine the optimal CpG ODN.On this basis,a pUC18-CpG recombinant plasmid containing 10 copies of this optimal CpG ODN was constructed and the immune activity was compared.The results showed that the three types of CpG ODNs with higher stimulation index were B-2,C-3 and P-4 through preliminary screening of cell proliferation test;RT-PCR detection revealed that IL-2 and IFN-??p<0.001?,IL-6 and IL-10?p<0.01?,and TLR21?p<0.05?mRNA expression levels were highest among the cells stimulated by P-4;pUC18-CpG had a slightly lower stimulation index than the P-4,and the mRNA expression of each cytokine was also slightly less than the P-4,but the differences were not significant?p>0.05?.The above results indicate that pUC18-CpG can replace the synthetic P-4 sequence for vaccine preparation.A total of 13 batches of vaccine were prepared in the laboratory with inactivated DTMUV-HB as antigen and pUC18-CpG recombinant plasmid as adjuvant?A:DTMUV+CpG,B:1/2 DTMUV+CpG,E:DTMUV,G:1/2 DTMUV?,and the animal evaluation experiment was conducted after the tests of stability,safety and sterility.Antibody titer levels and cytokine protein levels in each group were detected by HI assay and indirect ELISA,and the results of virus isolation after challenge were determined by RT-PCR.The results showed that,at a 1/4 dose,there was no significant difference in titer of HI antibody between the experimental groups?p>0.05?.When immunized at a 1/2 dose and a full dose,the titer of HI antibody in groups A and B was significantly higher than that in groups E and G.The change trend of antibody positive rate and GMT value was consistent with HI antibody.Three of the four detected proteins were up-regulated in the serum of groups A1 and B1,compared with the non-adjuvant groups.Especially at 14 dpi,the expression of IFN-?and IL-6 in group A1 was significantly more than that in group E1?p<0.01?,and that in group B1 was significantly higher than that in group G1?p<0.05?.Additionally,at24 dpi and 35 dpi,the expression of IL-2 in group A1 was significantly more than that in group E1?p<0.01;p<0.05?.Most importantly,the full dose and 1/2 dose subgroups in groups A and B were fully resistant to virus challenge.The PD₅₀ values of group A and group B were 4 and 4.5,respectively,which were much higher than 1.4 and 1.8 in group E and G.Therefore,CpG adjuvant can not only enhance humoral immune response,including promoting the production of early antibodies,improving antibody titer,and extending the duration of immunity.It can also enhance the cellular immune response and increase the concentration of serum cytokines.Besides,CpG adjuvant can provide complete protection against Duck Tembusu virus,which effectively enhances the immune effect of DTMUV-HB inactivated vaccine.Among them,the vaccine group with a full dose of 1/2 DTMUV+pUC18-CpG is the most cost-effective.