Using Vita-PAMP To Improve Inactivated Pseudorabies Vaccine Immune Efficacy Research

The innate immune system distinguishes self and non-self through pattern recognition receptors(PPRs)that recognize pathogen-related molecular patterns(PAMPs)unique to pathogens,thereby activating antigen-presenting cells and adaptive immune responses.viability-associated PAMPs(vita-PAMPs)are a type of PAMPs that only exist in active microorganisms.They are a sign of microbial viability.After being recognized by natural immune cells,they can mobilize a high degree of inflammation and activate the body’s adaptive immune response.Pseudorabies(PR)is an acute,multi-host,highly contagious infectious disease caused by Pseudorabies virus(PRV),which brings huge losses to the swine industry.In recent years,the effect of pseudorabies virus mutation traditional vaccines is obviously insufficient.Attenuated vaccines are not suitable for special swine herds.Adding immunopotentiators to improve inactivated vaccines is a research hotspot.Purpose: In this project,representatives of vita-PAMPs immunopotentiators(c-di-AMP or c-di-GMP)were added to formaldehyde-inactivated PRV Bartha-K61 strains to immunize mice.Mice serum specific PRV g B Ig G antibody level,draining lymph nodes recruit antigen presenting cells(APC)and T cell activation,and the differentiation and germination of T follicular helper cell(Tfh),the effect of central B cell activation(GC B)and the level of long-lived plasma cell production is to explore the effect of c-di-AMP and c-di-GMP on the immune efficacy of inactivated vaccines in a mouse pseudorabies artificial infection model.Methods: BALB/c mice aged 6-8 weeks were randomly divided into five groups with 8 mice in each group.The mice were infected with the classic Bartha-K61 strain live virus of different titers by subcutaneous injection,and the survival rate of mice in each group were calculated after 7 days.BALB/c mice aged6-8 weeks were randomly divided into six groups with 8 mice in each group.A safe dose was selected to infect mice with different immunization pathways,and the best route was ensured by comparing the early immune differential molecules of live virus and inactivated virus.(ELISA kit detects mice serum TNF-α,IL-1β、IFN-β and IL-6 levels,ELISA kit detects mice serum PRV g B Ig G antibody levels and lymphocyte proliferation index)Screen the best immune route.Using formaldehyde and BEI to inactivate PRV Bartha-K61 strain virus respectively to explore the effects of different inactivation methods on the early immune factors.After clarifying the optimal infection dose,immune route and early difference factors,the artificial infection model of pseudorabies in mice was successfully established,and the immune enhancement efficacy of c-di-AMP and c-di-GMP was evaluated.BALB/c mice aged 6-8 weeks were randomly divided into five groups,each with16 mice,namely: PRV Bartha-K61 strain live virus group,formaldehyde inactivated virus vaccine group,formaldehyde inactivated virus plus c-di-AMP group,formaldehyde inactivated virus plus c-di-GMP group and blank control group.1.On the 5th day after immunization,the superficial cervical lymph nodes were taken,lymph node cells were separated,and the number of recruits for c DC(CD11b+CD11c+),Mo(CD11b+CD11cLy6G+Ly6C-)and M ?(CD11b+CD11c-F4 80+)was detected by flow cytometry.2.On the7 th day after the mice immunization,blood was collected from the ocular venous plexus to separate the serum,and the serum TNF-α、IL-1β、IFN-β and IL-6 levels were detected by the ELISA kit;the superficial cervical lymph nodes were taken,and the lymph node cells were separated.Flow cytometry was used to detect the activation of T cells(CD3+CD4+T cell and CD3+CD8+T cell);3.On the 14 th day after immunization,the superficial cervical lymph nodes were taken,lymph node cells were separated,and Tfh(CXCR5+PD-1+CD4+)activation status was detected by flow cytometry.4.On the 28 th day after the mice immunization,the superficial cervical lymph nodes were taken,the lymph node cells were separated,and flow cytometry was used to detect the activation of GC B cells(CD38-GL-7+B220+)and the production level of long-lived plasma cells(B220-CD138+).5.On the 7d,14 d,28d,42 d,60d and 90 d after immunization,blood was collected from the ocular venous plexus to separate serum,and the serum PRV g B Ig G antibody level and antibody duration level were detected by ELISA kit;Results: 1.5×104 TCID50 is the safe infection dose for 6-8 weeks old mice;subcutaneous injection is the best method of infection;live virus group infected mice 7 days after the mice serum TNF-α、IL-1β、 IFN-β and IL-6,mice serum PRV g B Ig G antibody level,lymphocyte proliferation index(SI)was significantly higher than the inactivated virus group(p<0.001,p<0.01,p<0.01;p<0.05;p<0.001);There is no significant difference in cytokines and SI between different inactivation methods(p>0.05).2.c-di-AMP and c-di-GMP are used as immunopotentiators to significantly increase the level of early differential factors produced by the inactivated pseudorabies virus vaccine to stimulate the body’s immune response.3.c-di-AMP and c-di-GMP are used as immunopotentiators to significantly increase the number of draining lymph nodes APCs recruited and T cell activation of the Pseudorabies inactivated virus vaccine.4.c-di-AMP and c-di-GMP as immunopotentiators can significantly increase the level of Tfh differentiation after the pseudorabies inactivated virus vaccine enters the body,thereby activating the strong activity response of GC B cells to promote the production of longlived plasma cells.Conclusion: This project has successfully established a mice pseudorabies artificial infection model,and it is clear that after live or inactivated pseudorabies virus immunize mouse,there are immune factors related to and significantly different from vita-PAMPs in the early stage of the body,which are the follow-up vitaPAMPs category evaluation and screening of immunopotentiators provide a research platform.Use mouse models to screen out suitable immunopotentiators of pseudorabies inactivated vaccine vita-PAMPs,and evaluate the immune efficacy from the aspects of early immune factors,cellular immunity and humoral immunity,and prove that c-di-AMP and c-di-GMP are used as vita-PAMPs immunopotentiators can improve the immune efficacy of inactivated pseudorabies vaccine,and c-di-AMP has a stronger immune enhancement effect.Provide ideas for the development of safer and more efficient inactivated vaccines and the improvement of the effectiveness of inactivated vaccines.