人参茎叶皂甙提高口蹄疫和新城疫油佐剂疫苗免疫效果的研究
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摘要
疫苗是畜牧生产中控制动物传染病的重要工具,但在临床实际中存在疫苗免疫失败和油佐剂疫苗注射困难等现象。我们前期的研究表明,GSLS和油佐剂有协同作用,能提高疫苗的免疫效果。本论文在此基础上,进一步研究GSLS和油佐剂协同作用增强疫苗免疫效果的合适比例,观察GSLS与油佐剂组合增强猪FMD疫苗和鸡ND疫苗的免疫效果,并对油佐剂疫苗添加GSLS后的黏滞度进行了观察。研究结果可为应用GSLS改进动物疫苗提供参考。
1.FMD疫苗中添加不同剂量GSLS对小鼠免疫反应的影响
目的：观察GSLS对小鼠注射FMD油佐剂疫苗免疫佐剂作用,筛选GSLS对FMD由乳剂疫苗免疫佐剂作用的合适浓度。方法：将56只雌性ICR小鼠随机分为7组,组1小鼠皮下注射0.1ml的FMDV抗原+0.1ml生理盐水,组2-7每组小鼠腹部皮下分别注射含不同浓度GSLS(0、2、4、6、8和10μg/0.2ml)的FMD由乳剂疫苗,小鼠免疫2次,间隔3周。二免后2周采血,用双抗夹心ELISA方法检测血清中FMDV特异性IgG水平。结果：疫苗中添加4、6、8和10μg的GSLS能显著提高小鼠血清中FMDV特异性IgG水平(P<0.05),其中以4μg组的FMDV特异性IgG水平最高。结论：FMD油乳剂疫苗中添加GSLS可显著提高小鼠的FMDV特异性IgG水平,提高的幅度呈剂量依赖关系,以4μg组的幅度最高。
2. GSLS对低剂量抗原FMD油乳剂疫苗免疫效果的影响
目的：比较FMD全抗原剂量油乳剂疫苗和含GSLS的低剂量抗原油乳剂疫苗对小鼠的免疫效果。方法：将72只ICR小鼠随机分成9组,每组8只小鼠。各组动物皮下注射的实验疫苗组成如下：(1)100μl的FMDV抗原+100μl生理盐水；(2)100μl抗原+100μl油佐剂Oil#10；(3)100μl抗原+100μl油佐剂MC52；(4)～(6)100μl,50μl或25μl抗原+100μl油佐剂Oil#10+GSLS(4μg);(7)～(9)100μl,50μl或25μl抗原+100μl油佐剂MC52+GSLS(4μg)。免疫2次,间隔3周,每次注射的体积为200μl。二免后2周采血,并取脾脏制备脾脏淋巴细胞悬液。用双抗夹心ELISA检测血清中FMDV特异性IgG水平,用MTT法检测经ConA、LPS和FMDV刺激后的淋巴细胞增殖指数,并用RT-PCR方法检测脾脏淋巴细胞经FMDV刺激后的IFN-y和IL-4以及转录因子T-bet和GATA-3中mRNA表达水平。结果：含GSLS的油佐剂疫苗诱导的特异性IgG效价显著高于不含GSLS的油佐剂疫苗(P<0.05)；含GSLS的半剂量抗原的油乳剂疫苗诱导的血清特异性IgG效价高于不含GSLS的全剂量抗原油乳剂疫苗,但无统计学差异显著(P>0.05)；含GSLS的四分之一剂量抗原的油乳剂疫苗诱导的血清特异性IgG效价低于不含GSLS的全剂量抗原油乳剂疫苗,但无统计学差异(P>0.05)。此外,油乳剂疫苗中添加GSLS后,增强了小鼠脾脏淋巴细胞受Con A、 LPS和FMDV刺激产生增殖能力(P<0.05),提高脾脏淋巴细胞受FMDV刺激后IFN-γ、IL-4、T-bet和GATA-3的(?)nRNA表达水平(P<0.05)。结论：油佐剂疫苗中添加GSLS能显著增强小鼠的体液和细胞免疫应答,使仅含一半抗原剂量疫苗诱导的IgG水平到含全剂量抗原诱导的IgG水平。
3.FMD油佐剂疫苗中添加不同剂量GSLS对猪的免疫效果
目的：观察FMD油佐剂疫苗添加GSLS寸猪的免疫效果。方法：试验一：将48只猪随机分成6组,每头猪颈部分别皮下注射含GSLS为0μg、10μg、20μg、40μg、80μg和160μg的FMD疫苗,免疫2次,间隔3周,每次注射疫苗剂量2ml。在免疫前,免疫后第3、6周前腔静脉采血,用正向间接血凝法检测血清中IHA水平,并分离淋巴细胞,用MTT法检测淋巴细胞增殖。试验二：将24头猪随机分成4组,每头猪颈部皮下分别注射含不同佐剂FMD疫苗(Oil#10,MC52, Oil#10+40μg GSLS, MC52+40μg GSLS),免疫1次,注射体积为2ml。于免疫前,免疫后第3、6周前腔静脉采血,检测血清中IHA效价,IgG和IgG亚类水平。结果：试验一：免疫后3、6周,每个剂量的FMD疫苗中添加40μg GSLS能显著提高猪特异性IHA效价(P<0.05),外周血淋巴细胞受FMDV刺激后产生的增殖能力显著升高(P<0.05),而受Con A刺激后产生的淋巴细胞增殖能力高于对照组,但无统计学差异(P>0.05)。试验二：与不添加GSLS的对照组相比,添加GSLS的油佐剂疫苗组在免疫后3周,IHA效价升高不显著(P>0.05),IgG、IgG1和IgG2水平显著升高(P<0.05)；免疫后6周,血清中IHA效价显著升高(P<0.05),IgG、IgG1和IgG2水平均显著上升(P<0.05)。结论：每个剂量FMD油佐剂疫苗中添加40μg GSLS能显著增强猪的体液免疫和细胞免疫。
4.不同剂量GSLS对鸡新城疫油佐剂疫苗免疫的影响
目的：观察鸡ND疫苗中添加GSLS对鸡免疫反应的影响。方法：将60只1日龄梅岭土鸡分成5组,每组12只,12日龄时,在每只鸡的胸大肌内分别注射含不同剂量的GSLS (0、2、4、6和8μg)鸡ND油佐剂疫苗,免疫为0.2ml。于免疫前,免疫后1、2和3周经心脏采血收集血样,用血凝抑制试验测定血清HI效价,3周后摘取鸡脾脏,制备脾脏淋巴细胞悬液,采用MTT法检测脾脏淋巴细胞增殖。结果：每剂量疫苗中添加6μg的GSLS,血清HI效价在免疫后1周升高不显著,在免疫后第2周和第3周,显著升高(P<0.05)；免疫后3周,脾脏淋巴细胞对LPS和NDV刺激的增殖反应显著高于对照组(P<0.05),各组脾脏淋巴细胞对ConA刺激的增殖反应之间没有明显差异(P>0.05)；ND疫苗中添加GSLS对鸡的体重不产生显著差异(P>0.05)；各组鸡的疫苗注射部位没有发现明显的局部副作用。结论：ND疫苗中添加GSLS能显著增强鸡的体液免疫和细胞免疫应答水平,并对免疫鸡不产生可见的副作用。
5.油佐剂疫苗中添加GSLS对疫苗黏滞性的影响
目的：观察油佐剂疫苗中添加GSLS对疫苗黏滞性的影响。方法：将GSLS(30g)溶解在60ml二甲基亚砜中,制备361mg/ml的GSLS储存液(83m1),根据用量将GSLS储存液添加在MC52白油中并搅拌均匀,然后与FMDV抗原混合,采用均质乳化机制备分别制备油相/水相比为1比1(或2比1),吐温浓度分别为0.5%、1.0%、1.5%、2.0%、2.5%和3.0%的油包水型FMD油乳剂疫苗,采用玻璃粘度计测定各组疫苗的黏滞性。结果：当吐温浓度为1.0%和1.5%时,添加4μg/剂量GSLS油佐剂疫苗(1比1)流动时间显著短于对照组(P<0.05)；当吐温浓度为为1.5%和2.0%时,添加4μg/剂量GSLS能显著降低油佐剂疫苗(2比1)流动时间(P<0.05)。结论：油乳剂疫苗中添加GSLS能降低疫苗的黏滞性。
综上所述,FMD油乳剂疫苗中添加GSLS可显著提高小鼠血清特异性IgG水平,提高的幅度呈剂量依赖关系,以4μg组的幅度最高；FMD油佐剂疫苗中添加GSLS(?)显著增强小鼠血清FMDV特异性IgG水平和淋巴细胞增殖转化指数,增强使仅含一半抗原剂量疫苗诱导的IgG水平到含全剂量抗原诱导的IgG水平,说明GSLS增强小鼠对低剂量抗原疫苗的免疫应答；FMD疫苗中添加40μg的GSLS能显著增强猪血清IHA,IgG和IgG亚类水平,增强淋巴细胞增殖指数,表明GSLS对猪FMD疫苗具有较好的免疫佐剂作用；ND疫苗中添加6μgGSLS能显著增强鸡血清HI效价和淋巴细胞增殖转化指数,并且没有对免疫鸡产生明显可见的副作用,说明GSLS能通过增强鸡的体液免疫应答和细胞免疫应答来发挥免疫佐剂作用；当吐温浓度为1.0%和1.5%时,添加4gg/剂量GSLS由佐剂疫苗(油/水=1：1)流动时间显著短于对照组(P<0.05)；当吐温浓度为为1.5%和2.0%时,添加4Pg/剂量GSLS能显著降低油佐剂疫苗(油/水=2：1)流动时间(P<0.05),说明油乳剂疫苗中添加GSLS能降低疫苗的黏滞性。
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 mice
Objective: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 vaccine
Objective: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 pigs
Objective: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 chickens
Objective: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 viscosity
Objective: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.
1.FMD疫苗中添加不同剂量GSLS对小鼠免疫反应的影响
目的：观察GSLS对小鼠注射FMD油佐剂疫苗免疫佐剂作用,筛选GSLS对FMD由乳剂疫苗免疫佐剂作用的合适浓度。方法：将56只雌性ICR小鼠随机分为7组,组1小鼠皮下注射0.1ml的FMDV抗原+0.1ml生理盐水,组2-7每组小鼠腹部皮下分别注射含不同浓度GSLS(0、2、4、6、8和10μg/0.2ml)的FMD由乳剂疫苗,小鼠免疫2次,间隔3周。二免后2周采血,用双抗夹心ELISA方法检测血清中FMDV特异性IgG水平。结果：疫苗中添加4、6、8和10μg的GSLS能显著提高小鼠血清中FMDV特异性IgG水平(P<0.05),其中以4μg组的FMDV特异性IgG水平最高。结论：FMD油乳剂疫苗中添加GSLS可显著提高小鼠的FMDV特异性IgG水平,提高的幅度呈剂量依赖关系,以4μg组的幅度最高。
2. GSLS对低剂量抗原FMD油乳剂疫苗免疫效果的影响
目的：比较FMD全抗原剂量油乳剂疫苗和含GSLS的低剂量抗原油乳剂疫苗对小鼠的免疫效果。方法：将72只ICR小鼠随机分成9组,每组8只小鼠。各组动物皮下注射的实验疫苗组成如下：(1)100μl的FMDV抗原+100μl生理盐水；(2)100μl抗原+100μl油佐剂Oil#10；(3)100μl抗原+100μl油佐剂MC52；(4)～(6)100μl,50μl或25μl抗原+100μl油佐剂Oil#10+GSLS(4μg);(7)～(9)100μl,50μl或25μl抗原+100μl油佐剂MC52+GSLS(4μg)。免疫2次,间隔3周,每次注射的体积为200μl。二免后2周采血,并取脾脏制备脾脏淋巴细胞悬液。用双抗夹心ELISA检测血清中FMDV特异性IgG水平,用MTT法检测经ConA、LPS和FMDV刺激后的淋巴细胞增殖指数,并用RT-PCR方法检测脾脏淋巴细胞经FMDV刺激后的IFN-y和IL-4以及转录因子T-bet和GATA-3中mRNA表达水平。结果：含GSLS的油佐剂疫苗诱导的特异性IgG效价显著高于不含GSLS的油佐剂疫苗(P<0.05)；含GSLS的半剂量抗原的油乳剂疫苗诱导的血清特异性IgG效价高于不含GSLS的全剂量抗原油乳剂疫苗,但无统计学差异显著(P>0.05)；含GSLS的四分之一剂量抗原的油乳剂疫苗诱导的血清特异性IgG效价低于不含GSLS的全剂量抗原油乳剂疫苗,但无统计学差异(P>0.05)。此外,油乳剂疫苗中添加GSLS后,增强了小鼠脾脏淋巴细胞受Con A、 LPS和FMDV刺激产生增殖能力(P<0.05),提高脾脏淋巴细胞受FMDV刺激后IFN-γ、IL-4、T-bet和GATA-3的(?)nRNA表达水平(P<0.05)。结论：油佐剂疫苗中添加GSLS能显著增强小鼠的体液和细胞免疫应答,使仅含一半抗原剂量疫苗诱导的IgG水平到含全剂量抗原诱导的IgG水平。
3.FMD油佐剂疫苗中添加不同剂量GSLS对猪的免疫效果
目的：观察FMD油佐剂疫苗添加GSLS寸猪的免疫效果。方法：试验一：将48只猪随机分成6组,每头猪颈部分别皮下注射含GSLS为0μg、10μg、20μg、40μg、80μg和160μg的FMD疫苗,免疫2次,间隔3周,每次注射疫苗剂量2ml。在免疫前,免疫后第3、6周前腔静脉采血,用正向间接血凝法检测血清中IHA水平,并分离淋巴细胞,用MTT法检测淋巴细胞增殖。试验二：将24头猪随机分成4组,每头猪颈部皮下分别注射含不同佐剂FMD疫苗(Oil#10,MC52, Oil#10+40μg GSLS, MC52+40μg GSLS),免疫1次,注射体积为2ml。于免疫前,免疫后第3、6周前腔静脉采血,检测血清中IHA效价,IgG和IgG亚类水平。结果：试验一：免疫后3、6周,每个剂量的FMD疫苗中添加40μg GSLS能显著提高猪特异性IHA效价(P<0.05),外周血淋巴细胞受FMDV刺激后产生的增殖能力显著升高(P<0.05),而受Con A刺激后产生的淋巴细胞增殖能力高于对照组,但无统计学差异(P>0.05)。试验二：与不添加GSLS的对照组相比,添加GSLS的油佐剂疫苗组在免疫后3周,IHA效价升高不显著(P>0.05),IgG、IgG1和IgG2水平显著升高(P<0.05)；免疫后6周,血清中IHA效价显著升高(P<0.05),IgG、IgG1和IgG2水平均显著上升(P<0.05)。结论：每个剂量FMD油佐剂疫苗中添加40μg GSLS能显著增强猪的体液免疫和细胞免疫。
4.不同剂量GSLS对鸡新城疫油佐剂疫苗免疫的影响
目的：观察鸡ND疫苗中添加GSLS对鸡免疫反应的影响。方法：将60只1日龄梅岭土鸡分成5组,每组12只,12日龄时,在每只鸡的胸大肌内分别注射含不同剂量的GSLS (0、2、4、6和8μg)鸡ND油佐剂疫苗,免疫为0.2ml。于免疫前,免疫后1、2和3周经心脏采血收集血样,用血凝抑制试验测定血清HI效价,3周后摘取鸡脾脏,制备脾脏淋巴细胞悬液,采用MTT法检测脾脏淋巴细胞增殖。结果：每剂量疫苗中添加6μg的GSLS,血清HI效价在免疫后1周升高不显著,在免疫后第2周和第3周,显著升高(P<0.05)；免疫后3周,脾脏淋巴细胞对LPS和NDV刺激的增殖反应显著高于对照组(P<0.05),各组脾脏淋巴细胞对ConA刺激的增殖反应之间没有明显差异(P>0.05)；ND疫苗中添加GSLS对鸡的体重不产生显著差异(P>0.05)；各组鸡的疫苗注射部位没有发现明显的局部副作用。结论：ND疫苗中添加GSLS能显著增强鸡的体液免疫和细胞免疫应答水平,并对免疫鸡不产生可见的副作用。
5.油佐剂疫苗中添加GSLS对疫苗黏滞性的影响
目的：观察油佐剂疫苗中添加GSLS对疫苗黏滞性的影响。方法：将GSLS(30g)溶解在60ml二甲基亚砜中,制备361mg/ml的GSLS储存液(83m1),根据用量将GSLS储存液添加在MC52白油中并搅拌均匀,然后与FMDV抗原混合,采用均质乳化机制备分别制备油相/水相比为1比1(或2比1),吐温浓度分别为0.5%、1.0%、1.5%、2.0%、2.5%和3.0%的油包水型FMD油乳剂疫苗,采用玻璃粘度计测定各组疫苗的黏滞性。结果：当吐温浓度为1.0%和1.5%时,添加4μg/剂量GSLS油佐剂疫苗(1比1)流动时间显著短于对照组(P<0.05)；当吐温浓度为为1.5%和2.0%时,添加4μg/剂量GSLS能显著降低油佐剂疫苗(2比1)流动时间(P<0.05)。结论：油乳剂疫苗中添加GSLS能降低疫苗的黏滞性。
综上所述,FMD油乳剂疫苗中添加GSLS可显著提高小鼠血清特异性IgG水平,提高的幅度呈剂量依赖关系,以4μg组的幅度最高；FMD油佐剂疫苗中添加GSLS(?)显著增强小鼠血清FMDV特异性IgG水平和淋巴细胞增殖转化指数,增强使仅含一半抗原剂量疫苗诱导的IgG水平到含全剂量抗原诱导的IgG水平,说明GSLS增强小鼠对低剂量抗原疫苗的免疫应答；FMD疫苗中添加40μg的GSLS能显著增强猪血清IHA,IgG和IgG亚类水平,增强淋巴细胞增殖指数,表明GSLS对猪FMD疫苗具有较好的免疫佐剂作用；ND疫苗中添加6μgGSLS能显著增强鸡血清HI效价和淋巴细胞增殖转化指数,并且没有对免疫鸡产生明显可见的副作用,说明GSLS能通过增强鸡的体液免疫应答和细胞免疫应答来发挥免疫佐剂作用；当吐温浓度为1.0%和1.5%时,添加4gg/剂量GSLS由佐剂疫苗(油/水=1：1)流动时间显著短于对照组(P<0.05)；当吐温浓度为为1.5%和2.0%时,添加4Pg/剂量GSLS能显著降低油佐剂疫苗(油/水=2：1)流动时间(P<0.05),说明油乳剂疫苗中添加GSLS能降低疫苗的黏滞性。
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 mice
Objective: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 vaccine
Objective: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 pigs
Objective: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 chickens
Objective: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 viscosity
Objective: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.
引文
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