Improvement Of Immune Responses To Foot-and-Mouth Disease And Newcastle Disease Oil-emulsified Vaccines By Ginseng Stem-leaf Saponins

Immunization with vaccines is an important strategy for the control of infectious disease in animal husbandry practice. However, poor immune responses to vaccines and difficult injection of oil emulsion vaccines have been frequently reported in clinical veterinary. Previous investigations in our lab showed that ginseng stem-leaf saponins (GSLS) and oil adjuvant synergistically improved immune responses to vaccines. This study was designed to investigate the optimal concentration of GSLS and oil adjuvant synergistically enhancing the immune responses to vaccines, the effect of GSLS and oil adjuvant synergistically improving efficacy of FMD vaccines in pigs and ND vaccines in chicken, and the effect of GSLS on vaccine viscosity. The results will supply references for GSLS improving the efficacy of animal vaccines.1. The effect of different dose of GSLS on immune responses to oil-emulsified FMD vaccine in miceObjective:To investigate the adjuvant effect of GSLS on the immune response to oil-emulsified FMD vaccine and find the optimal concentration of GSLS in oil-emulsified FMD vaccine for immunization in mice. Method:56mice were randomly divided into7groups with8mice in each. The animals in group1were subcutaneously (s.c.) immunized twice at3-week interval with200μl of FMD vaccine containing antigen (100μl) plus0.89%saline (100μl). The mice in group2to7were immunized with the same amount of antigen in oil adjuvant (Oil#10) with GSLS (0,2,4,6,8and10μg). Blood samples were collected2weeks after the booster immunization for detection of FMDV-specific IgG with a sandwich ELISA. Results:serum FMDV-specific IgG levels were significantly higher in mice immunized with200μl of vaccines containing GSLS (4,6,8and10μg) than in mice immunized with the vaccine without GSLS (P<0.05). The vaccine containing4μg of GSLS per dose elicited the highest IgG responses. Conclusion:Co-administration of GSLS in oil-emulsified FMD vaccine significantly enhanced the IgG responses in mice. The range of increased immune responses was dose dependent and the highest range was observed in group supplemented with4μg of GSLS.2. The effect of GSLS on immune responses to reduced amount dose of FMDV antigen in oil emulsion vaccineObjective:To determine the immune responses to reduced dose of FMDV antigen in oil emulsion vaccine supplement with GSLS and the immune responses to full dose of FMDV antigen in oil-emulsified FMD vaccine in mice. Method:72mice were randomly divided into9groups with8mice in each. The animals were injected twice at3week intervals with one of following FMD vaccines (200μl):(1)1dose of antigen (100μl)+0.89%saline (100μl);(2)100μl of antigen+oil adjuvant (Oil#10)(100μl);(3)100μl of antigen+oil adjuvant (MC52)(100μl);(4),(5) and (6)100μl,50μl or25μl of antigen+oil adjuvant (Oil#10,100μl)+GSLS(4μg);(7),(8) and (9)100μl,50μl or25μl of antigen+oil adjuvant (MC52,100μl)+GSLS(4μg). Blood samples were collected2weeks after booster immunization for measurement of FMDV-speciific IgG titers with a sandwich ELISA. The Splenocytes were prepared2weeks after last immunization for the detection of Splenocyte proliferation responses to ConA, LPS and FMDV by the MTT method and mRNA expression of cytokines IFN-y/IL-4and transcription factors T-bet/GATA-3by the RT-PCR method. Results:The IgG titers in groups supplement of GSLS in oil-emulsified FMD vaccines were significantly higher than in groups without GSLS (P<0.05); compared with mice immunized with full dose of FMDV antigen oil emulsion vaccine without GSLS, the IgG titers in mice immunized with50%dose of FMDV antigen oil emulsion vaccine plus GSLS were higher, but no statistically significant difference (P>0.05). The IgG titers in group received of injection of25%dose of FMDV antigen oil emulsion vaccine plus GSLS were lower than in group received of injection of full dose of FMDV antigen oil-emulsified vaccine without GSLS, but no statistically significant difference (P>0.05). The proliferative responses to ConA, LPS and FMDV stimulation were significantly higher in groups of oil-emulsified FMD vaccines plus GSLS than in control groups (P<0.05). Supplement of GSLS in oil-emulsified FMD vaccines significantly increased the mRNA expression of cytokines IFN-y/IL-4and transcription factors T-bet/GATA-3in splenocytes (P<0.05). Conclusion:Supplement of GSLS in oil emulsion FMD vaccine enhanced the humoral immune responses and cell mediated immune responses in mice. The IgG responses elicited by GSLS in50%dose of antigen oil emulsion FMD vaccines were equal to that in full dose of antigen oil emulsion FMD vaccines without GSLS.3. The effect of different concentration of GSLS on the immune responses to oil-emulsified FMD vaccine in pigsObjective:To investigate the effect of GSLS on the immune responses to oil-emulsified FMD vaccine in pigs. Method:In exp1,48pigs were randomly divided into6groups with8pigs in each. The pigs were intramuscularly (i.m.) immunized in neck twice at3weeks intervals with2ml of FMD vaccine containing1ml antigen and1ml Oil#10with GSLS (0,10,20,40,80or160μg). Blood samples were collected before,3and6weeks immunization for measurement of FMDV-specific IHA titers. The peripheral blood lymphocytes were prepared for the detection of lymphocyte proliferation by the MTT method. In exp2,24pigs were randomly divided into4groups with6pigs in each. The pigs were intramuscularly (i.m.) immunized once in the neck with2ml of FMD vaccine containing1ml antigen and1ml of oil adjuvant (Oil#10or MC52) with or without GSLS (40μg). Blood samples were collected before,3and6weeks after immunization for measurement of IHA, FMDV-specific IgG and the IgG subclasses. Results:In exp1, while oil-emulsified vaccines supplemented with GSLS elicited higher serum IHA titers than that without GSLS, only the vaccine supplemented with40μg of GSLS per dose induced significantly higher IHA titer than the vaccine without GSLS(P<0.05). Co-administration of40μg of GSLS GSLS in#10-emulsified FMD vaccine significantly increased the lymphocyte proliferative responses to FMDV (P<0.05). The lymphocyte proliferative response to ConA in pigs immunized with FMD vaccine plus GSLS was higher than in control group, but no statistically significant difference (P>0.05). In exp2, oil-emulsified vaccines supplemented with GSLS elicited numerically higher serum IHA titers than that without GSLS (P>0.05) at3weeks post vaccination. Serum IgG, IgG1and IgG2levels were significantly higher in pigs injected with FMD vaccines adjuvanted with GSLS-MC52at3weeks post vaccination (P<0.05). At6weeks post vaccination, the vaccine adjuvanted with GSLS-Oil#10or GSLS-MC52elicited significantly higher serum IHA titers, IgG, IgGl and IgG2levels than the vaccines without GSLS (P<0.05). Conclusion: Supplement of40μg of GSLS in oil-emulsified FMD vaccines improved the humoral immune responses and cell mediated immune responses in pigs.4. The effect of different concentration of GSLS on the immune responses to oil-emulsified ND vaccine in chickensObjective:To evaluate the adjuvant effect of GSLS on the immune responses against Newcastle disease (ND) in chickens. Method:Method:Sixty birds were randomly divided into5groups with12birds in each. The animals were intramuscularly (i.m) immunized once with0.2ml ND oil emulsion vaccine containing GSLS (0,2,4,6and8μg) at days12. Blood samples were collected from the heart or vein of all chickens before the immunization (0week) and1,2and3week post the immunization for the measurement of HI titers by the hemagglutination inhibition test method.3week after immunization, splenocytes were collected from birds immunization of ND oil emulsion vaccine containing GSLS (0and6μg) for lymphocyte proliferation test. Results:6μg of GSLS elicited significantly higher serum HI titers at2and3weeks post immunization than in control group, but no statistically significant difference between experimental group and control group at1week post immunization(P>0.05); at3week post immunization, chickens immunized with ND oil emulsion vaccine plus GSLS (6μg/0.2ml) promoted significantly higher splenocyte proliferative response to LPS and NDV antigen when compared to the chickens injected with ND oil emulsion vaccine alone (P<0.05), but there was no statistically significant difference in splenocyte proliferative response to Con A between experimental group and control group(P>0.05); No significant changes were found in body weight between the groups with or without GSLS at all time points(P>0.05); No adverse reactions at injection sites were observed in the chickens immunized with ND oil emulsion vaccines with or without GSLS. Conclusion:Supplement of GSLS in ND oil emulsion vaccine significantly increased the humoral and cell mediated immune responses of birds to NDV vaccination. Use of GSLS in ND emulsion vaccines has no visible adverse reactions in chickens.5. Effects of GSLS on the vaccine viscosityObjective:To investigate the effects of GSLS on the viscosity of vaccines. Method:GSLS powder was dissolved in dimethyl sulfoxide to form a stock solution (361mg/ml). To produce FMD emulsion vaccine(W/O) containing GSLS, oil adjuvant (MC52as oil phase containing6%span80) with or without GSLS was emulsified in FMDV antigen (aqueous phase, containing0.5,1.0,1.5,2.0,2.5and3.0%tween80) at a ratio of1:1or2:1(oil phase/aqueous phase, v/v) by using a dispersing device. A glass tube of viscosity calculation was used for measuring the viscosity of vaccines. Results:Compared with control groups co-administration of GSLS (4μg) significantly reduced the viscosity in vaccines containing polysorbate-80of1.0%and1.5%(oil phase/aqueous phase=1:1), respectively (P<0.05). The viscosity in vaccines containing polysorbate-80of1.5%and2.0%(oil phase/aqueous phase=2:1) was significantly lower than in control groups, respectively (P<0.05). Conclusion:Co-administration of GSLS in oil-emulsified vaccine significantly reduced the viscosity of vaccines.In conclusion, Co-administration of GSLS in oil-emulsified FMD vaccine significantly enhanced the IgG responses in mice. The range of increased immune responses was dose dependent and the highest range was observed in group supplemented with4μg of GSLS; supplement of GSLS in oil emulsion FMD vaccine enhanced the serum IgG titeres and splenocyte proliferation. The IgG responses elicited by GSLS in50%dose of antigen oil emulsion FMD vaccines were equal to that in full dose of antigen oil emulsion FMD vaccines without GSLS. Adjuvant GSLS in oil emulsion FMD vaccines improved the humoral immune responses and cell mediated immune responses in mice;40μg of GSLS per dose in FMD oil emulsion vaccines significantly increased the serum IHA titers, IgG and IgG subclass, as well as lymphocyte proliferation in pigs, indicating the adjuvant effect of GSLS on FMD vaccine in pigs; Supplement of GSLS in ND oil emulsion vaccine significantly increased the serum HI titers and splenocyte proliferation, indicating the adjuvant effect of GSLS by enhancing the humoral and cell mediated immune responses in chickens; co-administration of GSLS (4μg) in oil emulsion vaccines significantly reduced the flowing time of vaccines containing different concentration of polysorbate-80(1.0%and1.5%for W/O=1:1;1.5%and2.0%for W/O=1:2)(P<0.05), indicating that Co-administration of GSLS in oil-emulsified vaccine significantly reduced the viscosity of vaccines.