猪链球菌三价灭活苗的制备及对小鼠免疫效果的评价

Preparation of trivalent inactivated vaccine of Streptococcus suis and evaluation of immune efficacy on mice

为制备猪链球菌(SS)三价灭活苗并评价其对小鼠的免疫效果,本研究利用筛选出的3株SS菌株(代号/血清型分别为HF2/SS2、BB15-4/SS3、HBgu18-4/SS4)作为疫苗株,甲醛为灭活剂、ISA 201VG矿物油为佐剂制备灭活前分别为1.0×10^(9)cfu/mL、2.0×10^(9)cfu/mL、4.0×10^(9)cfu/mL 3种活菌浓度的SS三价灭活苗,分别命名为V-a、V-b和V-c。以昆明鼠为实验动物模型,将V-a、V-b、V-c、商用疫苗均以0.2 mL/只的剂量采取颈背部皮下多点注射的方式依次免疫A~D组小鼠后进行以下免疫效果评价:采集首免后14 d与二免后7 d的小鼠血清,每组3只,通过间接ELISA方法检测各组小鼠血清中IgG抗体效价;以血清杀菌试验检测血清中功能性抗体水平。血清中IgG抗体检测结果显示,在各个免疫阶段,A~D组小鼠的IgG抗体含量始终保持相同水平且均显著高于阴性对照组(P<0.05),二免后7 d,A~D组小鼠的IgG抗体水平显著上升,抗体效价均为1:12800;血清杀菌试验结果显示,二免后7 d,A~D组均能诱导小鼠血清中功能性抗体水平持续升高且无显著差异(P>0.05),具有高度杀菌活性。利用噻唑蓝溴化四唑溶液(MTT)测定小鼠脾淋巴细胞增殖指数(SI);采用双抗夹心ELISA方法检测小鼠血清相关细胞因子含量(IL-4、IL-10、IFN-γ、TNF-β、MCP-1);利用流式细胞术测定CD4^(+)与CD8^(+)外周血T淋巴细胞亚群的百分比。结果显示,二免后7 d,A~D组均能刺激小鼠血清中相关细胞因子水平、SI及CD4^(+)与CD8^(+)外周血T细胞亚群百分比持续上升且均显著高于阴性对照组(P<0.05)。二免7 d后,均以BZ1、BB15-4和HBgu18-4菌株攻毒各免疫组和攻毒对照组小鼠,阴性对照组接种等体积灭菌生理盐水,每组15只,观察7 d,计算小鼠死亡率;在小鼠攻毒3 d、7 d后,采用平板计数法测定攻毒菌株在小鼠体内的定植情况;并于攻毒7 d后取各组小鼠的肺、肝、脾、肾脏制作组织病理切片,观察各组织病理变化。结果显示,腹腔攻毒后7 d,A~D组及攻毒对照组小鼠免疫保护率依次为86.67%、100%、100%、80%和0;A~C免疫组小鼠各脏器的细菌定植量均显著降低且低于免疫组D(P<0.05);与D组相比,A~C组小鼠各器官病理变化均较为轻微。本研究结果表明,V-a、V-b和V-c免疫小鼠后均能产生良好的体液免疫和细胞免疫反应,其中V-b和V-c的免疫效力最优,均可有效抵抗SS2、SS3、SS4强毒株的攻击,可作为SS三价(血清2、3、4型)灭活苗的候选疫苗。本研究首次制备了新型SS三价灭活苗,并初步进行了免疫效果评价,为SS疫苗研发提供技术储备。

To prepare a trivalent inactivated vaccine of Streptococcus suis(SS)and evaluate its immune efficacy on mice,three SS strains HF2(serotype SS2),BB15-4(serotype SS3)and HBgu18-4(serotype SS4))were selected as vaccine candidates,and inactivated by formaldehyde and prepared at concentrations of 1.0×10^(9)cfu/mL,2.0×10^(9)cfu/mL,4.0×10^(9)cfu/mL,named as V-a,V-b and V-c.Kunming mice were used as the experimental animal model,the mice were divided into A-D groups and immunized with V-a,V-b,V-c and commercial vaccine,respectively,at a dose of 0.2mL/mouse by subcutaneous multi-point injection of the back of neck.The sera of 3 mice in each group were collected at 14 days post first immunization and 7 days post second immunization,and the IgG antibody titer was detected by indirect ELISA.The functional antibody levels in serum were detected by serum sterilization test.The results showed that the IgG content in mice of A-D groups remained the same level and significantly higher than that in the negative control group(P<0.05),the IgG titers of mice in A-D groups significantly increased at 7 days post second immunization,reaching at 1:12800.The results of serum sterilization test showed that the functional antibody levels in sera of A-D groups increased continuously and had highly bactericidal activity,and no significant differences were observed among the four groups(P>0.05).The mouse spleen lymphocyte proliferation index(SI)was determined by thiazole blue bromide solution(MTT),the concentration of related cytokine(IL-4,IL-10,IFN-γ,TNF-β and MCP-1)was detected by a dual anti-sandwich ELISA method,and the percentage of CD4^(+)and CD8^(+)was determined by flow cytometry.The results showed that,at 7 days post second immunization,the relevant cytokine levels in mice sera of A-D groups were stimulated,and the SI and percentage of CD4^(+)and CD8^(+)peripheral T cell subgroups increased continuously and were significantly higher than those of control group(P<0.05).After 7 days post second immunization,15 mice in each group were challenged with BZ1,BB15-4 and HBgu18-4 strains,respectively,the control group was treated with sterilized saline of equal volume,and the mortality was measured after 7 days.At 3 and 7 days post challenge,the colonization of attacking strains in mice was determined by plate counting method.At 7 days post challenge,pathological sections of the lung,liver,spleen and kidney were taken to observe the histopathological changes.The results showed that the immune protection rates of A-D and control groups were 86.67%,100%,100%,80% and 0,respectively.The bacterial colonization in all organs of mice in groups A-C was significantly reduced and lower than that of the immune group D(P<0.05).The organ pathological changes of the groups A-C were mild compared to group D.The results showed that immunization with Va,V-b and V-c can stimulate good humoral and cellular immunity in mice.Among them,V-b and V-c have the best immune efficacy,which can effectively protect the mice from the attacks of SS2,SS3 and SS4 virulent strains,and can serve as candidate vaccines for SS trivalent(serotype 2,3 and 4)inactivated vaccines.In this study,a new type of SS trivalent inactivated vaccine was prepared for the first time,and the immune efficacy was preliminarily evaluated to provide technical reserves for the development of SS vaccine.