牛O型口蹄疫高倍浓缩灭活疫苗的研制
摘要
口蹄疫(Foot and mouth disease, FMD)是由口蹄疫病毒(FMDV)引起的主要危害牛、猪、羊等偶蹄动物的烈性传染病,发病率高,传染性强,并能形成国际大流行,国际兽疫局(OIE)将其排在A类家畜传染病的首位。世界各国政府都高度重视本病的防治工作。疫苗免疫是防制本病的重要手段之一,近一个世纪以来,许多国家为了预防和控制口蹄疫的发生和流行,对疫苗的研究一直是该领域的研究热点。
我国目前防制家畜口蹄疫病主要以使用抗原(病毒培养液)不经过浓缩的普通灭活疫苗为主,一般每头剂量3ml仅含3个PD_(50)。因普通疫苗的抗原未经浓缩处理抗原含量偏低,同时存在一定量的细胞碎片等非抗原蛋白成份,所以保护力低、免疫期相对较短,并有一定的副反应。
本研究通过对口蹄疫病毒抗原的高倍浓缩等一系列手段制备出符合国际标准的新型疫苗并中试放大,摸索出一套新的生产工艺。通过对疫苗毒株的各种生物学特性尤其是病毒毒力和免疫原性、病毒液的前处理和高倍超滤浓缩工艺、浓缩后病毒含量检测方法和标准的建立(运用ELISA、TCID_(50)等手段建立新的检测方法并与乳鼠LD_(50)试验平行比较)、应用国际通行标准(OIE)对疫苗进行PD_(50)测定并与普通疫苗的效力相比较等研究试验,最终研制成功的三批疫苗在实验室和田间试验中均达到了预先规定的各项标准:1.提高了安全性:动物注射疫苗后安全而无副作用。2.降低了使用剂量:2ml/头剂(普通疫苗使用量为3ml)。3.提高了免疫效力:每头剂达到6个PD_(50)以上(普通疫苗每头剂含3个PD_(50))。4.延长了免疫持续期:达到6～7个月(普通疫苗为4个月左右)。
目前,该疫苗已通过农业部的新兽药证书的评审,预期在我国口蹄疫疫病的防治中将发挥重要作用。
Foot and Mouth Disease (FMD), a major threat to cloven-hoofed animals such as bovine, swine and sheep, is a highly contagious disease caused by Foot and Mouth Disease Virus (FMDV). Since it has a high incidence and can easily cause an epidemic outbreak and enormous loss of economy, Office International des Epizooties (OIE) ranks it in the first in all contagious diseases for domestic animals. The governments of countries all over the world have focused much attention on the prevention and control of this disease.Since vaccination was one of major means to prevent domestic animals from this disease, many countries paid much attention to the development of FMDV vaccines for almost a century in order to prevent and control the outbreak and prevalence of FMD.Thus far, the means mainly adopted in China to prevent domestic animals from the infection of FMDV is the use of the crude, non-concentrated preparation from the viral culture prior to chemical inactivation. In general, the guideline of a FMDV vaccine requires that 3 milliliters per dose per animal contain 3 PD_(50) or that the protection rate after the viral challenge should reach at 4 protected out of 5 tested animals or greater. Since the concentration of viral antigens in the non-concentrated crude viral preparation is relatively low and it contains a trace amount of nonantigenic proteins from the host cell debris, this makes the vaccines usually in a weak protection, a relatively short duration for immune response and side-effects.The purpose of this study is to, through a series of means including a concentration process of the FMDV antigens, produce a higher standard vaccine to meet the international criteria and subsequently scale up the production, thus exploring a set of new technologies for the production. The aim of such new vaccine mainly focuses on improving the efficacy of the present vaccine and achieving the criteria as follows:1. The increasing of the safety: The inoculation of the vaccine is safer to animals with minimal side-effects.2. The reduction of the volume per dose: Each dose of the vaccine is 2 milliliters per animal instead of 3 milliliters from the pre-improved vaccine.3. The improvement of the efficacy: Each dose of the vaccine contains more than 6 PD_(50), rather than 3 PD_(50) from the pre-improved vaccines.4. The longer duration of the immune response: The vaccine can improve the duration from 4 to 4.5 months to 6 to 7 months.Through examining the biological characteristics of the vaccine strain especially for its the virulence and immunogenecity, evaluating the pre-process for the viral culture and the concentration technology, establishing the testing standard protocol for the viral antigen content after concentration (the results from the established new testing protocol obtained from ELISA and TCID_(50) were consistent with the results obtained from the suckling mouse LD_(50) protocol), using international standard to determine the PD_(50) and comparing the efficacy with the pre-improved vaccines, three batches of tested vaccines all met every preset criterion in both laboratory and field test with a 100% success. To date, this vaccine has been passed the assessment and evaluation by the Ministry of Agriculture of the People's Republic of China and will be certificated for the license soon.
我国目前防制家畜口蹄疫病主要以使用抗原(病毒培养液)不经过浓缩的普通灭活疫苗为主,一般每头剂量3ml仅含3个PD_(50)。因普通疫苗的抗原未经浓缩处理抗原含量偏低,同时存在一定量的细胞碎片等非抗原蛋白成份,所以保护力低、免疫期相对较短,并有一定的副反应。
本研究通过对口蹄疫病毒抗原的高倍浓缩等一系列手段制备出符合国际标准的新型疫苗并中试放大,摸索出一套新的生产工艺。通过对疫苗毒株的各种生物学特性尤其是病毒毒力和免疫原性、病毒液的前处理和高倍超滤浓缩工艺、浓缩后病毒含量检测方法和标准的建立(运用ELISA、TCID_(50)等手段建立新的检测方法并与乳鼠LD_(50)试验平行比较)、应用国际通行标准(OIE)对疫苗进行PD_(50)测定并与普通疫苗的效力相比较等研究试验,最终研制成功的三批疫苗在实验室和田间试验中均达到了预先规定的各项标准:1.提高了安全性:动物注射疫苗后安全而无副作用。2.降低了使用剂量:2ml/头剂(普通疫苗使用量为3ml)。3.提高了免疫效力:每头剂达到6个PD_(50)以上(普通疫苗每头剂含3个PD_(50))。4.延长了免疫持续期:达到6～7个月(普通疫苗为4个月左右)。
目前,该疫苗已通过农业部的新兽药证书的评审,预期在我国口蹄疫疫病的防治中将发挥重要作用。
Foot and Mouth Disease (FMD), a major threat to cloven-hoofed animals such as bovine, swine and sheep, is a highly contagious disease caused by Foot and Mouth Disease Virus (FMDV). Since it has a high incidence and can easily cause an epidemic outbreak and enormous loss of economy, Office International des Epizooties (OIE) ranks it in the first in all contagious diseases for domestic animals. The governments of countries all over the world have focused much attention on the prevention and control of this disease.Since vaccination was one of major means to prevent domestic animals from this disease, many countries paid much attention to the development of FMDV vaccines for almost a century in order to prevent and control the outbreak and prevalence of FMD.Thus far, the means mainly adopted in China to prevent domestic animals from the infection of FMDV is the use of the crude, non-concentrated preparation from the viral culture prior to chemical inactivation. In general, the guideline of a FMDV vaccine requires that 3 milliliters per dose per animal contain 3 PD_(50) or that the protection rate after the viral challenge should reach at 4 protected out of 5 tested animals or greater. Since the concentration of viral antigens in the non-concentrated crude viral preparation is relatively low and it contains a trace amount of nonantigenic proteins from the host cell debris, this makes the vaccines usually in a weak protection, a relatively short duration for immune response and side-effects.The purpose of this study is to, through a series of means including a concentration process of the FMDV antigens, produce a higher standard vaccine to meet the international criteria and subsequently scale up the production, thus exploring a set of new technologies for the production. The aim of such new vaccine mainly focuses on improving the efficacy of the present vaccine and achieving the criteria as follows:1. The increasing of the safety: The inoculation of the vaccine is safer to animals with minimal side-effects.2. The reduction of the volume per dose: Each dose of the vaccine is 2 milliliters per animal instead of 3 milliliters from the pre-improved vaccine.3. The improvement of the efficacy: Each dose of the vaccine contains more than 6 PD_(50), rather than 3 PD_(50) from the pre-improved vaccines.4. The longer duration of the immune response: The vaccine can improve the duration from 4 to 4.5 months to 6 to 7 months.Through examining the biological characteristics of the vaccine strain especially for its the virulence and immunogenecity, evaluating the pre-process for the viral culture and the concentration technology, establishing the testing standard protocol for the viral antigen content after concentration (the results from the established new testing protocol obtained from ELISA and TCID_(50) were consistent with the results obtained from the suckling mouse LD_(50) protocol), using international standard to determine the PD_(50) and comparing the efficacy with the pre-improved vaccines, three batches of tested vaccines all met every preset criterion in both laboratory and field test with a 100% success. To date, this vaccine has been passed the assessment and evaluation by the Ministry of Agriculture of the People's Republic of China and will be certificated for the license soon.
引文
[1] 国家“九五”攻关项目“热带亚热带畜禽主要疫病监测与控制研究(96-120-10)”子专题“口蹄疫病原监测及流行病学分析”.云南省热带亚热带动物病毒病重点实验室.昆明.2001(内部资料);
[2] 国际兽疫局.哺乳动物、禽和蜜蜂A和B类疾病—诊断试验和疫苗标准手册[M].农业部畜牧兽医局,1996,51-58;
[3] 殷震,刘景华主编.动物病毒学,北京:科学出版社,1985.395～414;
[4] 牛O型口蹄疫灭活苗制苗毒种的稳定性及其免疫原性监测 《中国兽医科技》1995,25(4):5-7 张永光等;
[5] 口蹄疫免疫学研究进展 《中国兽医科技》1995,25(4):21-22 张立昌,刘尚高;
[6] 牛O、A型口蹄疫双价灭活疫苗研究-A型毒种的选择 《中国兽医科技》1995,25(12):6-8 张永光,刘在新,方玉珍等;
[7] 口蹄疫-一个困扰全球的问题 湖北畜牧兽医 1995年,第4期,14-16 杨宜生;
[8] A型口蹄疫病毒vp1免疫活性肽基因串联片段的化学合成及克隆表达 复旦学报(自然科学版) 第36卷第5期496 易建中,黄海斌,杨志军等;
[9] 6株A型口蹄疫病毒结构蛋白VP1(1D)基因的核苷酸序列分析 病毒学报 第14卷 刘在新,赵启祖,刘卫;
[10] 口蹄疫研究进展 《中国兽医科技》1999,29(3):17-22 柳纪省;
[11] 四川畜牧兽医学院学报 1998,12(1) 46-51 何永强,周继勇,于涟;
[12] 口蹄疫研究进展 中国农业科学 1999,32(6):93-100 江鹏斐,赵启祖,谢庆阁;
[13] 口蹄疫流行毒株与疫苗毒株的生物学特性 Chinese journal of veterinary science and technology 2002, 19(2) 43;
[14] 家畜口蹄疫研究进展 中国预防兽医学报 第23卷第4期 2001年7月 雷连成,陈伟;
[15] 刘在新,赵启祖,张显升等.口蹄疫病毒非结构蛋白P3区的基因序列及分析.中国病毒学.2002,17(2):153-157;
[16] 张显升,赵启祖,刘在新等.口蹄疫病毒株China/99 P3区一级结构及与参考毒株的比较分析[J].畜牧兽医学报,2002,33(6):606—610;
[17] 1990-2001年世界口蹄疫流行情况及其防制对策China animal quarantine 2002,19(2)43 袁文铎,苏永生,李春阵;
[18] Capstick, P. B., Telling, R. C., Chapman, W. G., Stewart, D. L., 1962. growth of a cloned strain of hamster kidney cells in suspended cultures and their susceptibility to the virus of foot-and-mouth disease.Nature 195,1163-1164,OIE MANUL, 2003;
[19] Knowles, N. J., 1990b A Method for Direct Nucleotide Sequencing of Foot-and-mouth Disease Virus RNA for Epidemiological Studies Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-mouth Disease, Lindholm, Denmark, 25-29 June 1990. FAO, Rome;
[20] Knowles, N. J., Ansell, D. M., Samuel, A. R.,. 1998. Molecular Comparison of Recent Foot-and-Mouth Disease Type Amiruses from West Africa with Historical and Reference Virus Strains. Report of the Session of the ResearchGroup of the European Commission for the Control of Foot-and-Mouth Disease, Aldershot, United Kingdom, 14-18September 1998(Appendix 1). FAO Rome, pp. 20-31;
[21] 《口蹄疫实验研究操作方法》中国农业科学院兰州兽医研究所 1965年;
[22] 《牛O型口蹄疫灭活疫苗质量标准》(新标准).农业部畜牧兽医局,2003
[23] 《中华人民共和国兽用生物制品规程》(2000版)总则、附录部分;
[24] 朱彩珠,卢永干等.应用反转录-聚合酶链反应检测口蹄疫病毒.病毒学报,1998,3:272-277.
[25] ELISA在FMD诊断检疫中的应用Ⅰ、Ⅱ 朱彩珠 卢永干等;
[26] 张永光等“牛O、A口蹄疫双价灭活苗研究”《中国兽医科技》1996,26(5):3-4;
[27] 《家畜口蹄疫及其防制》农业部畜牧兽医司编;
[28] 《O型口蹄疫(内蒙古分离株)油乳剂灭活苗材料研究材料汇总》 内蒙古自治区口蹄疫研究课题组;
[29] Lochar, F., Suryanarayana, V. V. S., Tratschin, J. D.. 1995. Rapid detection and characterization of foot-and-mouth disease virus by restriction enzyme and nucleotide sequence analysis of PCRproducts. J. Cli. Immunal. 33 (2), 440-444, OIEMANUAL: VOLUME 2, 1990. 1/12-11/12;
[30] 张永光等“牛O、A口蹄疫双价灭活苗研究”《中国兽医科技》1996,26(5):3-4;
[31] 中国农业科学院兰州兽医研究所《科学研究年报》(家畜口蹄疫和猪传染性水泡病专集 1979-1985)1987.6;
[32] T. R. Doel 2003, FMD vaccines, virus Research 91 (2003)81-99
[2] 国际兽疫局.哺乳动物、禽和蜜蜂A和B类疾病—诊断试验和疫苗标准手册[M].农业部畜牧兽医局,1996,51-58;
[3] 殷震,刘景华主编.动物病毒学,北京:科学出版社,1985.395～414;
[4] 牛O型口蹄疫灭活苗制苗毒种的稳定性及其免疫原性监测 《中国兽医科技》1995,25(4):5-7 张永光等;
[5] 口蹄疫免疫学研究进展 《中国兽医科技》1995,25(4):21-22 张立昌,刘尚高;
[6] 牛O、A型口蹄疫双价灭活疫苗研究-A型毒种的选择 《中国兽医科技》1995,25(12):6-8 张永光,刘在新,方玉珍等;
[7] 口蹄疫-一个困扰全球的问题 湖北畜牧兽医 1995年,第4期,14-16 杨宜生;
[8] A型口蹄疫病毒vp1免疫活性肽基因串联片段的化学合成及克隆表达 复旦学报(自然科学版) 第36卷第5期496 易建中,黄海斌,杨志军等;
[9] 6株A型口蹄疫病毒结构蛋白VP1(1D)基因的核苷酸序列分析 病毒学报 第14卷 刘在新,赵启祖,刘卫;
[10] 口蹄疫研究进展 《中国兽医科技》1999,29(3):17-22 柳纪省;
[11] 四川畜牧兽医学院学报 1998,12(1) 46-51 何永强,周继勇,于涟;
[12] 口蹄疫研究进展 中国农业科学 1999,32(6):93-100 江鹏斐,赵启祖,谢庆阁;
[13] 口蹄疫流行毒株与疫苗毒株的生物学特性 Chinese journal of veterinary science and technology 2002, 19(2) 43;
[14] 家畜口蹄疫研究进展 中国预防兽医学报 第23卷第4期 2001年7月 雷连成,陈伟;
[15] 刘在新,赵启祖,张显升等.口蹄疫病毒非结构蛋白P3区的基因序列及分析.中国病毒学.2002,17(2):153-157;
[16] 张显升,赵启祖,刘在新等.口蹄疫病毒株China/99 P3区一级结构及与参考毒株的比较分析[J].畜牧兽医学报,2002,33(6):606—610;
[17] 1990-2001年世界口蹄疫流行情况及其防制对策China animal quarantine 2002,19(2)43 袁文铎,苏永生,李春阵;
[18] Capstick, P. B., Telling, R. C., Chapman, W. G., Stewart, D. L., 1962. growth of a cloned strain of hamster kidney cells in suspended cultures and their susceptibility to the virus of foot-and-mouth disease.Nature 195,1163-1164,OIE MANUL, 2003;
[19] Knowles, N. J., 1990b A Method for Direct Nucleotide Sequencing of Foot-and-mouth Disease Virus RNA for Epidemiological Studies Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-mouth Disease, Lindholm, Denmark, 25-29 June 1990. FAO, Rome;
[20] Knowles, N. J., Ansell, D. M., Samuel, A. R.,. 1998. Molecular Comparison of Recent Foot-and-Mouth Disease Type Amiruses from West Africa with Historical and Reference Virus Strains. Report of the Session of the ResearchGroup of the European Commission for the Control of Foot-and-Mouth Disease, Aldershot, United Kingdom, 14-18September 1998(Appendix 1). FAO Rome, pp. 20-31;
[21] 《口蹄疫实验研究操作方法》中国农业科学院兰州兽医研究所 1965年;
[22] 《牛O型口蹄疫灭活疫苗质量标准》(新标准).农业部畜牧兽医局,2003
[23] 《中华人民共和国兽用生物制品规程》(2000版)总则、附录部分;
[24] 朱彩珠,卢永干等.应用反转录-聚合酶链反应检测口蹄疫病毒.病毒学报,1998,3:272-277.
[25] ELISA在FMD诊断检疫中的应用Ⅰ、Ⅱ 朱彩珠 卢永干等;
[26] 张永光等“牛O、A口蹄疫双价灭活苗研究”《中国兽医科技》1996,26(5):3-4;
[27] 《家畜口蹄疫及其防制》农业部畜牧兽医司编;
[28] 《O型口蹄疫(内蒙古分离株)油乳剂灭活苗材料研究材料汇总》 内蒙古自治区口蹄疫研究课题组;
[29] Lochar, F., Suryanarayana, V. V. S., Tratschin, J. D.. 1995. Rapid detection and characterization of foot-and-mouth disease virus by restriction enzyme and nucleotide sequence analysis of PCRproducts. J. Cli. Immunal. 33 (2), 440-444, OIEMANUAL: VOLUME 2, 1990. 1/12-11/12;
[30] 张永光等“牛O、A口蹄疫双价灭活苗研究”《中国兽医科技》1996,26(5):3-4;
[31] 中国农业科学院兰州兽医研究所《科学研究年报》(家畜口蹄疫和猪传染性水泡病专集 1979-1985)1987.6;
[32] T. R. Doel 2003, FMD vaccines, virus Research 91 (2003)81-99