Immune Boosting Effect Of Total Ginseng Stem And Leaf Saponin And Monosaponin Rb₁ On Inactivated BVD Vaccine

Bovine viral diarrhea（BVD）is a major cause of economic losses in the global cattle industry.Infection of cattle with this virus leads to reduced performance,immunosuppression,growth retardation,decreased milk production and other complications.Currently,effective means of prevention and control include vaccination and culling of persistently infected animals.Vaccine immunization,the most widely used and effective means internationally,has bottlenecks in its immune effect,and the search for immune boosters with strong immune enhancement effects and safe use has become a new research direction.Ginseng is the root of Ginseng,a plant in the family of Wujia,which is a tonic herb that not only has the effect of nourishing and calming the mind,but also has the effect of strengthening the immune system.Therefore,this study applied network pharmacology to analyze the information of active ingredients and BVD targets contained in ginseng,selected relevant active ingredients as immune enhancers to prepare inactivated BVD vaccines,verified the core disease targets analyzed by network pharmacology by immunogenicity analysis,and laid the foundation for the use and development of animal vaccine adjuvants.To determine the interaction between ginseng active ingredients and BVD diseases,this study used TCMSP database to search ginseng active ingredients,OMIM database,Gene Cards database and TTD database to obtain disease targets,Cytoscape software to obtain network diagrams of ginseng active ingredients and disease targets,identify co-target information,draw Wayne diagrams,establish protein interaction PPI networks,and finally identify ginseng active ingredients and disease core targets by GO functional enrichment analysis and KEGG enrichment analysis.The results showed that there are 87 active ingredients in ginseng,with a total of 2571 BVD disease targets,73 intersectional targets,and 15 core targets for PPI network analysis:AKT1,PTGS2,IL4,CASP3,IFNY,FOS,F2,TP53,ESR1,HMOX1,PPARG,MMP9,VEGFA,STAT3,and NOS3;IL-4 and IFN-γare mainly responsible for transmitting information,activating and regulating immune cells,mediating T and B cell activation,proliferation and differentiation and playing an important role in the inflammatory response;FOS,TP53,ESR1,NOS3 and HMOX1belong to cancer pathway genes;AKT1,CASP3,STAT3,PPARG,MMP9,PTGS2and VEGFA are involved in regulating cell proliferation and apoptosis;F2 is a coagulation factor responsible for regulating the rapid increase in cytokine levels after viral infection resulting in loss of coagulation,impaired blood supply and organ failure.The results of GO functional enrichment analysis showed that the molecular functions of the intersection genes mainly involved:inflammation regulation,immune response activation,cell apoptosis,oxidative stress,cell secretion regulation,protein phosphorylation and other functions;KEGG pathway enrichment analysis showed that the main enrichment pathways of crossover genes were T cell receptor signaling pathway,inflammatory response,apoptosis,oxidative stress and lipid metabolism.To prepare an inactivated BVDV vaccine,two ginseng components were selected as adjuvants for the study based on the screening of ginseng active ingredients in network pharmacology:total saponins from ginseng stems and leaves and ginsenoside monomer Rb₁.Firstly,the safe doses of the two saponins were investigated through hemolysis;Secondly,stimulating MDBK cells to investigate the maximum safe concentration and cell activity of each component on the cells;The antigen used for vaccine preparation was obtained by inactivating the virus with BEI,and GSLS and Rb₁were added to the antigen in proportion.After emulsification,the vaccine was prepared and tested for physical properties and safety.The results showed that when GSLS<1 mg/m L and Rb₁<0.72 mg/m L,compared with distilled water,there was basically no hemolysis;The maximum safety concentration of GSLS and Rb₁is 750μg/m L;The optimal concentration and time for GSLS to stimulate MDBK cells in vitro using the CCK-8 method is 25μg/m L,24 h;Rb₁could enhance the activity of MDBK cells in a dose-dependent manner,and the effect was better at 10μg/m L and 24 h.When the inactivation condition of BEI was 3 m M and 20～24 h,the inactivation effect was better,and no lesion was produced after three generations of blind transmission of MDBK cells after inoculation;seven groups of inactivated BVD vaccines were prepared,and the quality tests of each vaccine were in accordance with the standard of Chinese veterinary pharmacopoeia.To evaluate the immune effect of the inactivated vaccine strain BVDV-JL3,targets related to the immune pathway were selected to verify the results of the network pharmacology analysis:seven groups of prepared vaccines were immunized by subcutaneous injection,and serum antibody potency,splenic lymphocyte proliferation,cytokine content and CD4+and CD8+T lymphocyte subpopulation content were determined by serum neutralization test and flow cytometry,and finally the immune enhancing effect of the vaccine was evaluated by viraemia.The results showed that,compared with the blank control group,all vaccine groups could produce neutralizing antibodies at 14 d after immunization（P<0.01）,and the serum neutralizing antibody potency was highest in the 28-d dose group;among them,the total saponin group had the highest neutralizing antibody potency in the vaccine group containing high-dose ginseng stem and leaf saponin;the monomer group had the highest potency in the vaccine group containing low-dose ginseng monomer Rb₁,and the serum antibody potency of the vaccine group containing low-dose ginseng monomer Rb₁was not significantly different from that of the vaccine group containing high-dose ginseng stem and leaf saponin（P>0.05）.The serum antibody potency of the low-dose ginseng monomer Rb₁vaccine group was not significantly different from that of the high-dose ginsenoside vaccine group（P>0.05）,but both were higher than that of the ISA206 vaccine group.The results of the splenic lymphocyte proliferation test showed that 28 days after the first vaccination,the stimulation index SI was significantly higher in the GSLS inactivated vaccine group and Rb₁inactivated vaccine group than in the control group after stimulation by LPS and Con A（P<0.05）,and the immune enhancement effect was better in the GSLS-50μg/each and Rb₁-50μg/each vaccine groups;in the cytokine expression level,the GSLS inactivated vaccine group and In the cytokine expression level,both GSLS inactivated vaccine group and Rb₁inactivated vaccine group could significantly stimulate the expression level of cytokines（IFN-γ,IL-4）（P<0.01）,but not IL-2 and IL-6（P>0.05）;meanwhile,the stimulation level of cytokines in each dose vaccine group decreased in a dose-dependent manner,and the immune effect of the vaccine weakened with increasing dose;CD3⁺CD4⁺T lymphocyte subpopulation The CD3⁺CD4⁺T lymphocyte subpopulation content showed that the highest CD3⁺CD4⁺lymphocyte content was found in the Rb₁-50μg/vaccine group and the highest CD3⁺CD4⁺T lymphocyte content was found in the GSLS-50μg/vaccine group compared with the control group;the CD3⁺CD8⁺T lymphocyte subpopulation content showed that the highest CD3⁺CD8⁺T lymphocyte subpopulation content was found in the GSLS-50μg/vaccine group compared with the control group at 28 d after the first vaccination.The CD3⁺CD8⁺T-lymphocyte subpopulations were higher in the GSLS-containing and Rb₁-containing vaccine groups than in the ISA206 vaccine group at 28 d after the first vaccination compared with the control group（P<0.01）;the results of the attack test showed that the active ingredients of ginseng could effectively reduce the pathological damage caused by the strong strain of BVDV in mice and lower the viral load in all organs,with GSLS-50μg/each and Rb₁-50μg/each vaccine groups having the highest CD3⁺CD4⁺T-lymphocyte subpopulations.The best results were obtained in the groups of GSLS-50μg/per mouse and Rb₁-50μg/per mouse.In conclusion,both total ginseng stem and leaf saponin and ginsenoside monomer Rb₁have immune enhancing effects on inactivated BVD vaccines.They have potential to be used as immune enhancers for novel animal vaccines,and also provide reference value for further research of BVDV vaccines.