猪传染性胸膜肺炎二价(血清7型+血清8型)灭活疫苗的制备及对小鼠免疫效果的评价

Preparation of bivalent inactivated vaccine of porcine contagious pleuropneumonia(serotype APP7 and APP8)and evaluation of immune efficacy on mice

为制备猪传染性胸膜肺炎(PCP)二价(血清7型H170株+8型HB1株)灭活疫苗,并评价其对小鼠的免疫效果,本研究以2株猪传染性胸膜肺炎放线杆菌(APP)(编号/血清型分别为H170/APP7、HB1/APP8)为疫苗株,复苏培养后调整活菌浓度分别为1.0×10^(9) cfu/mL、2.0×10^(9) cfu/mL、4.0×10^(9) cfu/mL,甲醛灭活后相同浓度的两种菌液等体积混合,再与等体积ISA 201 VG矿物油佐剂乳化后制成PCP二价灭活疫苗,分别命名为A-1、A-2和A-3。以昆明鼠为实验动物模型,将A-1、A-2、A-3和PCP商用二价灭活疫苗均以0.2 mL/只分别经颈背部皮下多点注射免疫小鼠(A~D组),对照组小鼠(E组)注射等体积灭菌生理盐水。于首免后21 d和二免后14 d收集各组小鼠血清,采用间接ELISA测定各组小鼠血清中IgG抗体水平和相关细胞因子(IL-4、IL-10、IFN-γ、TNF-β、MCP-1)含量;同时采用血清杀菌试验测定各组小鼠血清中功能性抗体水平。分别于首免后21 d和二免后14 d剖杀各组小鼠,制备脾淋巴细胞悬液,采用噻唑蓝(MTT)比色法测定各组小鼠脾淋巴细胞增殖指数(SI),采用流式细胞术测定其外周血中CD4^(+)与CD8^(+)T淋巴细胞亚群百分比。为了评价该二价灭活疫苗的攻毒保护效果,于二免后14 d分别采用CVCC265、CVCC266标准菌株以腹腔注射的方式对各组小鼠攻菌(5 LD50/只),实验设未免疫攻菌组(F组),攻菌后观察各组小鼠的临床症状,统计免疫保护率。攻菌后3 d和7 d采集小鼠脏器,采用平板计数法测定各组织荷菌数,分析攻毒菌株在小鼠体内的定植情况,通过制作肺、肝、脾、肾脏切片观察其组织病理学变化。结果显示,与E组相比,A~D组小鼠血清中IgG抗体含量及相关细胞因子水平均显著升高(P<0.05),且均能诱导小鼠血清中功能性抗体水平的持续升高,二免后14 d的免疫小鼠血清均具有良好的杀菌效果;A~D组小鼠的SI、外周血CD4^(+)与CD8^(+)T细胞亚群百分比均显著升高(P<0.05)。攻菌试验结果显示,A~D组小鼠对CVCC265菌株攻击的免疫保护率依次为60%、60%、80%、80%,对CVCC266菌株攻击的免疫保护率依次为60%、60%、80%、60%。攻菌后7 d,与F组相比,A~C组小鼠各脏器(肺、肝、肾、脾)细菌定植量显著降低且低于D组(P<0.05);A、B组小鼠各脏器病理变化均较轻微,C组各脏器则无明显病理变化,E组小鼠各脏器均无明显病理变化,而F组小鼠各脏器均有明显病理变化。上述结果表明,该PCP二价灭活疫苗可诱导小鼠机体产生良好的体液免疫和细胞免疫反应,有效抵抗血清7型、8型APP标准菌株的攻击,其中高浓度组(4.0×10^(9) cfu/mL)免疫效果更佳,可作为PCP二价(7型+8型)灭活疫苗的候选疫苗。本研究首次制备了新型PCP二价灭活疫苗并初步评价其免疫效果,为PCP多价疫苗的开发提供了技术储备。

To prepare a bivalent inactivated vaccine of porcine contagious pleuropneumonia(PCP)and evaluate its immune efficacy on mice,two Actinobacillus pleuropneumoniae(APP)strains H170(serotype APP7)and HB1(serotype APP8)were selected as vaccine candidates.After bacterial recovery and growth,the live bacteria concentrations of 1.0×10^(9)cfu/mL,2.0×10^(9) cfu/mL,4.0×10^(9)cfu/mL were adjusted,and formaldehyde was chosen to inactivate and ISA 201 VG mineral oil to prepare PCP bivalent inactivated vaccine,named as A-1,A-2,A-3,respectively.Kunming mice were used as the experimental animal model to evaluate the vaccine's immune efficacy.The mice were divided into A-D groups and immunized with A-1,A-2,A-3 and commercial vaccine of PCP,respectively,at a dose of 0.2mL/mouse by subcutaneous multi-point injection at the back of neck.Mice in group E were injected with sterile saline.At 21 days post first immunization and 14 days post second immunization,the IgG antibody and related cytokines(IL-4,IL-10,IFN-γ,TNF-β,MCP-1)in serum of all immune groups were detected by indirect ELISA.The functional antibody levels in serum were detected by serum sterilization test.Mice were killed to prepare splenic lymphocyte suspension and the MTT colorimetric method was used to determine spleen lymphocyte stimulation index(SI).The percentage of CD4^(+)and CD8^(+)peripheral blood T lymphocyte subsets were determined by flow cytometry.In order to further verify immune efficacy of the vaccine,at 14 days post second immunization,mice in each group were challenged with CVCC265 and CVCC266 standard strains by intraperitoneal injection,at a dose of 5LD50.At the same time,the challenge control group was set up and named F.After a challenge by bacteria,the clinical symptoms of each group were observed and the immune protection rate was determined.At 3 and 7 days post challenge,the colonization of the challenge strains in mice was determined by plate counting method.Pathological sections of the lung,liver,spleen and kidney were taken to observe the histopathological changes.The results showed that the IgG content in mice of A-D groups was significantly higher than that in the negative control group of E(P<0.05)and the functional antibody levels increased continuously and had high bactericidal activity at 14 days post second immunization.The related cytokines levels,SI and the percentage of CD4^(+)and CD8^(+)in mice of A-D groups were significantly higher than negative control group(P<0.05).In the challenge bacteria test,the immune protection rates of mice in groups A-D against CVCC265 were 60%,60%,80%,and 80%,respectively,and the immune protection rates against CVCC266 were 60%,60%,80%,and 60%,respectively.After 7 days post challenge,compared with group F,the bacterial colonization in each organ(lung,liver,spleen and kidney)of mice in groups A-C were significantly reduced and lower than that of the immune group D(P<0.05).Compared to group D,the organ pathological changes of the group A and B were slight,while group C displayed no obvious pathological changes.There were no obvious pathological changes in each organ of mice in group E,but there were obvious pathological changes in each organ of mice in group F.The results showed that immunization with A-1,A-2,or A-3 can produce good humoral and cellular immunity in mice.At the same time,they could effectively resist the challenge of serotype 7,serotype 8 standard strains.Among them,A-3 has the best immune efficacy and can be used as a candidate vaccine for PCP bivalent(serotype 7 and 8)inactivated vaccine.In this study,a new type of PCP bivalent inactivated vaccine was prepared and its immune efficacy was preliminarily evaluated,aiming to provide technical reserve for the expansion and development of PCP multivalent vaccine.