Development Of Swine FMD Inactivated Vaccine Immunopotentiator CVC1302 And Its Action Mechanism

Foot-and-mouth disease(FMD)is an acute severe infectious disease caused by foot-and-mouth disease virus(FMDV).Vaccine immunization is a major method to prevent and control FMD in our country.Commercial vaccines of FMD vaccine have many problems,such as slow antibody production,insufficient antibody level and short duration of antibody.FMD will basically reach to free from disease by immunity until to 2020,according to the national medium-long-term control program on animal plague.It is urgently required to improve the immune efficacy for commercial vaccines.Toll-like receptor(TLR),nucleotide binding oligomerization domain-like receptors(NLR),belonging to natural immune receptor family,can activate the natural immune response and acquired immune response and is a kind of excellent candidate material for immunopotentiator selection.There have been commercial products in human vaccine.It is also a hotspot in veterinary vaccine research.Therefore an immunopotentiator with high-performance was developed based on TLRs and NLRs agonists in this study.Pigs were immunized with vaccine composed of inactivated FMDV and TLRs and NLRs agonists in single or combination manner.The immune efficacy was evaluated according to serum LPB-ELISA antibody titer,and thus the best prescription,dose and usage were decided.The immunopotentiator,named CVC1302 was successfully developed.Its immunoenhancement effects on swine FMD inactivated vaccine were evaluated taking antibody duration,cellular immunity,humoral immunity and challenge protection.In addition,the safety,acute toxicity and cumulative toxicity of CVC1302 were further studied on piglets.Besides,under oxidative stress state the effects of CVC1302 on FMD inactivated vaccine were also observed in mice PCV2 infection model in order to validate the safety and immune enhancement effect.Finally,mechanism for CVC1302 on FMD inactivated vaccine were explored using indexs including activation of APC in injection site and draining lymph nodes,B cells and Tfh cells,levels of related transcription factors and the ratio of long-lived plasma cells in bone marrow.Our study included four parts as follows:Experiment 1.Preparation and efficacy evaluation of immuopotentiator CVC 1302 on swine FMD inactivated vaccinePiglets were immunized with FMD inactivated vaccine together with nine agonists for innate immune response in single or combinatorial manner,respectively.Blood samples were collected on 28 days after vaccination for LPB-ELISA antibody detection to determine optimal prescription,optimal dosage and usage.The optimal immunopotentiator,named CVC1302 was successfully developed.Furthermore,we studied on the cellular immunity,humoral immunity and challenge protection and antibody duration.Results indicated immunopotentiator obtained in this studty could improve LPB-ELISA antibody level by 2-4 times for FMD inactivated vaccine.FMD-CVC1302 group had long-lasting antibody titers(>7.81og2),lasting for at least 6 months.The LPB-ELISA and SN antibody titers for three doses in FMD-CVC1302 groups were significantly higher than those in FMD-vaccine groups at the same doses(p<0.05)before virus challenge.The FMDV-specific RNA copy number in FMD-CVC1302 group was obviously lower than that in FMD-vaccine group at 3 days post virus challenge.The PD50 value was 15.85 for the FMD-CVC1302 group,which was obviously higher than that for FMD-vaccine group(10.96).These results indicated that CVC1302 could enhance the immune efficacy and protective ability for FMD inactivated vaccine in pigs.Experiment 2.Safety test and clinic application for swine FMD inactivated vaccine immunopotentiator CVC1302In order to further evaluate the safety of CVC1302 as vaccine manufacturing materials or vaccine additives.Piglets were intramuscular injected with FMD vaccine with or without CVC1302 in single dose inoculation,single dose repeated inoculation,and overdose inoculation.The muscle and histology observation following inoculation,average daily gain,pathological changes and tissue slice at the injection site was recorded.To further clarify the toxicity of CVC1302,acute toxicity test(intramuscular injection 1×,10×,100×solution)and cumulative toxicity test(injection 1×,10×,50× solution,7 injections,3 days apart)were carried out with piglets innoculation.Clinical feature,relative daily gain,appearance and section observation of the main immunological organs,and organ index were evaluated following injection.CVC1302 was applied to various commercial vaccines of foot-and-mouth disease to evaluate the immune efficacy of vaccines immunized in different farms with different breeding backgrounds.Results of safety tese showed that CVC1302 was safe for piglets in any dose.Vaccine was well absorbed at the injection site.The acute toxicity of CVC1302 showed slight toxicity at 10 times or higher dose.The accumulative toxicity test of CVC1302 showed that the low dose after seven immunizations had no effect on growth performance and organs index of piglets.Further clinic application tests showed that CVC1302 had an immune enhancing effect on all kinds of FMD disease commercial vaccines,significantly increasing antibody qualification rate by 20%-50%and the average antibody titer of LPB-ELISA by 2-6 times.Results indicated that CVC1302 was safe and useful for piglets and could be used in large-scale clinical application and show significant immune enhancement.Experiment 3.Evaluation of the immune efficacy of swine FMD inactivated vaccine with immunopotentiator CVC1302 under PCV2 infected stateThis section was aimed to investigate the effect of immunopotentiator CVC1302 on antibody production and oxidative stress in mice infected with PCV2.Mice were challenged with PCV2 via intraperitoneal injection to establish oxidative stress models and injected with FMD vaccine or FMD-CVC1302 at 7 dpc.Oxidative stress indexes,such as MDA,SOD,CAT,GSH,GSSG and the ratio of GSH/GSSG were analyzed at 3 dpi and the PCV2 copies in mice spleen were detected weekly after immunizated.LPB-ELISA antibody titer was detected at 28dpi in serum samples.Results showed that infected with PCV2 could cause the oxidative stress in mice.CVC1302 could significantly upregulated the SOD activites,CAT and GSH levels and the ratio of GSH/GSSG.The oxidative stress indexes were no significantly discrepancy with PBS group.The FMD inactivated vaccine-adjuvanted with CVC1302 can stimulate mice to produce higher LPB-ELISA antibody levels,Th1 type cytokines as well as the lower viral load than FMD vaccine.To summarize,CVC1302 can not only induce high levels of antibodies,but also regulate the body's immune status.Experiment 4.Mechanism Study for long-term antibodies response produced by swine FMD inactivated vaccine with immunopotentiator CVC1302In order to study the action mechanism of CVC1302,FMD inactivated antigen with or without CVC 1302 aqueous solution and inactivated FMD vaccine with or without CVC1302 were injected in mice,respectively.Two weeks after vaccination,the numbers of APC activation and chemokine transcription and expression level at injection site and lymph node were dectected.After 28dpi,FMDV specific LBP-ELISA titers and IgGl and IgG2a antibody levels in serum were dectected in FMD vaccine with or without CVC1302 immunization groups,IFN-? transcription and expression in lymph node,Flow cytometry germinal center B cell,number of Tfh cells,real-time PCR for chemokines and immunohistochemistry of germinal center of lymph node,number of long-lived plasma cells in bone marrow were dectected too.Results showed that mRNA and expression levels of related transcription factors activated by antigen-presenting cells were activated within one day,the number of APC cells in the injection site and draining lymph nodes could be significantly increased,the peak of the injection site was reached 3 days after immunization,drainage of lymph nodes,5 days after immunization peak following the injection of antigen and CVC1302 aqueous solution.Compared with FMD inactivated vaccines,the injection site and APC activation level also improved significantly,the IFN-? expression level of lymph node is obviously improved,the number of B cells and Tfh cells and germinal center number increase significantly increased in lymph nodes.The levels of various chemokines of B cells transformed into plasma cells were also significantly increased,which in turn led to an increase in the number of plasma cells in bone marrow.The amount of long-term plasma cell-in FMD-CVC1302 group was significantly higher than FMD-vaccine groups at 28 dpi,90 dpi and 150 dpi(p<0.001).Results showed that CVC1302 could start an efficient immune response,the lymph nodes generate high levels of B cells,and increased the number of germinal center,the action of various chemokines,make B cells into plasma cells,and in turn,increase the number of plasma cells in bone marrow to maintain long-lasting antibody duration.