Study On The Mechanism Of Enhancement Of Vaccines-induced Immune Responses By Sustained-release Injection Of Venenum Bufonis

Venenum Bufonis, one of important traditional Chinese material medicas, is effectiveto remove toxic agents, subdue swelling, alleviate pain and induce resuscitation，and isoften used to treat many diseases, such as hard furuncle, furunculosis, sore throat,sunstroke, asphyxia. It exhibits anti-tumor, cardiotonic and analgesia effect and canenhance the protective effect of vaccine. Although Venenum Bufonis showed good effectson the treatment of pig disease, such as fever and anorexia, the crude implantation wasnot only complicated for operation and accurate administration, but also did not meet therequirements in veterinary clinic. Therefore, the sustained-release injection of VenenumBufonis (SIVB) prepared with modern animal pharmacy technology was safet with thecharacteristic of long effect and convenient operation. Safety, effect and quality control isthe basic requirement for modern animal pharmacy. Based the safety of SIVB, itspharmacology was evaluated to establish the foundation for the further clinicalapplication of SIVB.According to previous reports, Venenum Bufonis has the adjuvant effect whichenhances the Th1, Th2or Th17immune response induced by vaccine. In order to studythe effect of SIVB on Th1, Th2or Th17immune response, OVA was used as an antigenmodel. ICR mice were subcutaneously injected with SIVB on day1and OVA on day1and day15. Two weeks after the second immunization, serum was collected for thedeterment of OVA-specific IgG, IgG1and IgG2a by ELSIA. After splenocytes wasprepared, MTS was used to analyze splenocytes proliferation assay, ELISA was used toanalyze the levels of IFNγ, IL-4and IL-17A in the supernatants of OVA-stimulatedsplenocytes. The results showed that SIVB significantly enhanced the levels ofOVA-specific IgG, IgG1and IgG2a in the serum, promoted the splenocyte proliferationresponse induced by ConA and LPS, and increased the production of IFNγ, IL-4andIL-17A in the supernatants. These results suggested that SIVB enhanced the Th1, Th2,and Th17immune response induced by OVA.In order to clarify the active components of SIVB, we also study the effect of TBGand CBG on the Th1, Th2and Th17immune response induced by OVA. ICR mice wereinjected with OVA, OVA plus TBG, OVA plus CBG on day1and day15. Two weeksafter the second immunization, serum was collected for the determent of OVA-specific IgG, IgG1and IgG2a by ELSIA. After splenocytes was prepared, MTS was used toanalyze splenocytes proliferation assay, ELISA was used to analyze the levels of IFNγ,IL-4and IL-17A in the supernatants of OVA-stimulated splenocytes, and FACS was usedto analyze the proportions of CD3+, CD3+CD4+and CD3+CD8+in the splenocyte. Theresults showed that TBG and CBG significantly enhanced the levels of OVA-specific IgGand IgG2a in the serum, promoted the splenocyte proliferation response induced byConA，LPS and OVA, increased the level of IFNγ and T-bet mRNA, and enhancedthe proportion of CD3+, CD3+CD4+and CD3+CD8+in the splenocytes. TBG has noeffect on OVA-Specific IgG1, IL-4and IL-17A, the mRNA level of GATA-3and RORγt,but CBG significantly decreased the level of OVA-Specific IgG1, IL-4and he mRNAlevel of GATA-3without the effect on IL-17A and the mRNA level of RORγt. Theseresults suggested that TBG and CBG enhanced the Th1immune response induced byOVA, which is related with the upregulation of T-bet mRNA.The above studies showed that SIVB, TBG and CBG enhanced Th1immuneresponse induced by OVA, suggesting that TBG and CBG may enhance the vaccineagainst intracellular pathogens. So we also studied the effect of SIVB, TBG and CBG onthe protective effect of formalin-inactivated Salmonella typhimurium (FIST) vaccine.ICR mice were subcutaneously injected with FIST, FIST plus TBG, CBG or SIVB on day1and day15. Two weeks after the second immunization, serum was collected for thedeterment of FIST-specific IgG, IgG1and IgG2a by ELSIA. After splenocytes wasprepared, MTS was used to analyze splenocytes proliferation assay, ELISA was used toanalyze the levels of IFNγ, IL-4and IL-17A in the supernatants of FIST-stimulatedsplenocytes. Two weeks after the last immunization, ICR mice were intragastricallyinfected with live Salmonella typhimurium to observe survival rates and spleenbacteria burdens. The results showed that SIVB, TBG and CBG augmented serumFIST-specific IgG and IgG2a, the splenocyte proliferation response and the production ofIFNγ and NO by FIST-restimulated splenocytes. In addition, SIVB enhanced serumFIST-specific IgG1and the production of IL-4and IL-17A by FIST-restimulatedsplenocytes. TBG and CBG decreased serum FIST-specific IgG1and the production ofIL-4by FIST-restimulated splenocytes, but didn’t have significant effect on theproduction of IL-17A. In in vivo protection studies showed that SIVB, TBG and CBGsignificantly decreased the bacterial burdens in the spleen and prolonged thesurvivaltime of FIST-immunized mice challenged with live Salmonella typhimurium. These results suggested that SIVB, TBG and CBG enhanced Th1immune responseinduced by FIST, and enhanced the anti-Salmonella typhimurium effect ofFIST-immunized mice.In order to clarify the role of IFNγ in the protective effect induced bySIVB+FIST, TBG+FIST and CBG+FIST, mice immunized with saline, FIST, SIVB,SIVB+FIST, CBG, CBG+FIST, TBG, and SIVB+FIST were treated with anintraperitoneal injection of anti-IFNγ mAb or rat IgG2a control mAb. ICR mice wereinjected with FIST, FIST plus TBG, CBG or SIVB on day1and day15. At the same time,ICR mice were injected intraperitoneally with800μg of anti-IFNγmAb or rat IgG2acontrol mAb on day1, day8, day15, and day22. The results showed that theinjection of an anti-IFNγ mAb notably altered the immunoglobulin subtype andcytokine profile. The enhancement of FIST-IgG2a and IFNγ by TBG, CBG or SIVBwas abrogated by in vivo injection of anti-IFNγ mAb. In vivo injection of anti-IFNγmAbs led to a reduction of the protective efficacy of SIVB, TBG and CBG onFIST-immunized mice. In addition, SIVB alone can decrease the spleen bacteriaburdens while TBG and CBG alone had no effect on spleen bacteria burdens. Theseresults suggested that SIVB, TBG and CBG enhanced the protective effect of FISTthrough the upregulation of IFNγ, which is the master of Th1immune response.These results suggested that SIVB can enhance the protective efficacy of vaccineand Th1immune response, suggesting that SIVB can be applied in clinic veterinary.Therefore, we investigate the safety and effect of SIVB on porcine. Porcineimmunized with swine fever vaccine, porcine Circovirus Type2vaccine and porcinereproductive and respiratory syndrome virus vaccine were injected with2ml of SIVBat Kaer acupoint. The results showed that after the administration of SIVB, porcinehad good general condition, items of body weight, hematologic index, andbiochemical index were all normal. Two weeks or three weeks after the administrationof SIVB, SIVB enhanced the levels of the antibodies of swine fever vaccine, porcineCircovirus Type2Vaccine and porcine reproductive and respiratory syndrome virusvaccine and the cytokines of IFNγ and IL-2in the serum. These results suggest SIVBhas good safety and can be used to enhance the immune response induced by swinefever vaccines.In conclusion, SIVB, TBG and CBG enhanced Th1immune response induced byOVA and FIST and the protective efficacy of FIST-immunized mice against Salmonella typhimurium. SIVB had high safety, and enhanced the levels of theantibodies of swine fever vaccine, porcine Circovirus Type2Vaccine and porcinereproductive and respiratory syndrome virus vaccine and the levels of IFNγ and IL-2in the serum. These results provided the evidences for the further clinical applicationof SIVB.