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流感病毒与流感疫苗

流感病毒与流感疫苗. 流感病毒. 流 感. - 由流感病毒引发的急性呼吸道传染病 . - 侵袭世界上所有性别、年龄和种族 . - 在全世界，每年有 5-15% 的人群会得流感，儿童、老年人和慢性病患者最易受到 流感病毒的侵袭 . - 缺乏终身免疫力 . - 缺少有效的治疗方法，预防接种仍是最 主要的手段. 流感病毒的命名. A/Brisbane /59 /2007 (H1N1). 型别. 分离地点. 毒株序号. 亚型. 分离年代. 流感病毒的抗原性变异. 人与动物之间. 世界大流行. 每年都发生. 影响每年流感疫苗的生产.

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流感病毒与流感疫苗

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  1. 流感病毒与流感疫苗

  2. 流感病毒

  3. 流 感 • -由流感病毒引发的急性呼吸道传染病. • -侵袭世界上所有性别、年龄和种族. • -在全世界，每年有5-15%的人群会得流感，儿童、老年人和慢性病患者最易受到 流感病毒的侵袭. • -缺乏终身免疫力. • -缺少有效的治疗方法，预防接种仍是最 主要的手段.

  4. 流感病毒的命名 A/Brisbane/59/2007(H1N1) 型别 分离地点 毒株序号 亚型 分离年代

  5. 流感病毒的抗原性变异 人与动物之间 世界大流行 每年都发生 影响每年流感疫苗的生产

  6. Past Antigene Shifts H1 N1 Spanish Influenza >40 million deaths H2N2 Asian Flu > 2 million deaths H3N2 Hong Kong flu 1 million deaths H1BN1 Swine flu No pandemic H5N1 Bird Flu Pandemic averted?

  7. 1918-1919的流感大流行 • 全世界有5亿人被传染 • 2000-4000千万人死亡,其中20-40岁占50% • 美国死亡人数大于50万

  8. 流感病毒的抗原性漂移 • 产生新型的流感变异株 • 人类缺乏免疫能力 • 引起新的流感传播

  9. 流感爆发对全球的影响(WHO estimates) • 死亡2-7百万人 • 上千万人被传染 • 损失数十亿

  10. 流感疫苗 • 三价疫苗: H1N1, H3N2 和B,每人份含抗原各15ug • 包含 :甲醛 ,裂解剂 • WHO每年指定的毒株

  11. WHO 流感疫苗毒株筛选程序 采接医生 来自82个国家114个实验室数千名采 样医生监测到的毒株变异情况汇总 国家流感中心 （114个实验室，分部在82个国家） 定期将结果报送到流感预测研究合作中心 （伦敦、亚特兰大、墨尔本和东京） 世界卫生组织 （WHO-日内瓦） 每年2月份在日内瓦召开全球 流感专家会议确认疫苗当年使用毒株 疫苗生产厂家 应用于人群预防接种

  12. 流感疫苗毒种 • 毒种传代 应按病毒株的培育条件进行传代，以保持其特性，疫苗所用的毒株代次应在许可范围内。疫苗只能由工作种子批传一代。 • 毒种保存 原始种子批、主种子批、工作种子批经分装密封后，均应保存于-60℃以下。

  13. 毒种批的建立 • 疫苗生产应以病毒种子批系统为基础进行三级管理，即原始种子批、主种子批和工作种子批。工作种子批用于生产。

  14. 种批的检定 • 鉴别试验 • 无菌试验 • 病毒血凝滴度 • 病毒滴定

  15. 流感疫苗生产工艺流程 • 9-11日龄鸡胚→接种病毒后放置恒温室培养→冷胚→单价病毒尿囊液收获→低速离心→超滤浓缩→第一次区带离心→第二次区带离心→裂解→超滤洗滤→除菌灭活→单型病毒液血凝素含量测定→三型单价病毒液合并→半成品→半成品检定→分装→成品→成品检定→包装→入库

  16. 生产用鸡胚要求 • 选用血管清晰，活动健壮的鸡胚 • 凡有以下情况鸡胚不能用于生产: • 未受精卵，仅见卵黄形，无鸡胚迹象。 • 胚胎活动呆滞或不能主动地运动，血管模糊扩张或折断沉落。 • 倒置胚、横胚。

  17. 病 毒 接 种 • 选择9-11日龄、血管清晰、活动健壮的鸡胚，照检后画出气室边界和胎位，在胚胎面与气室交界边缘约1MM处避开血管做一标记，此即为注射点。 • 在标记好的注射部位用酒精由内至外消毒，并用扎卵器在标记部位扎一孔，勿损伤壳膜。然后再用酒精消毒。 • 用5ML注射器抽取流感病毒稀释液后用镊子将针头插好，用排气管排净注射器内空气，按顺序接种，每胚0.2ML。

  18. 流感病毒的培养 • 将接种好的鸡胚用熔化石蜡封口，然后置33-35℃恒温室培养。 • 收获时间根据不同病毒而异，甲型流感病毒一般需34℃培养48小时进行收获，而乙型流感病毒需34℃培养72小时后进行收获。 • 收获前，鸡胚须置4 ℃过夜，目的是将鸡胚冻死使血液凝固，避免收获时流出红细胞并同尿囊液里的病毒发生凝集，造成病毒滴度下降。

  19. 病 毒 液 的 收 获 • 用酒精消毒气室部卵壳，用烙蛋器在消毒部位烙一焦圈，用眼科镊子将气室部卵壳取下。 • 左手用消毒镊子将鸡胚和卵黄压向一侧，右手持连续负压的多孔吸头通过绒毛尿囊膜进入尿囊腔，吸取尿囊液，勿将血球及卵黄吸入，鸡胚内容物异常者废弃。

  20. 单型病毒液检定项目 • 无菌试验 • 血凝试验 • 细菌内毒素含量测定

  21. 流感病毒的纯化 • 低速离心 • 超滤浓缩 • 第一次区带离心 • 第二次区带离心 • 裂解 • 超滤洗滤除菌灭活

  22. 区带离心

  23. 裂解

  24. 电泳

  25. Eggs held in incubators  9-11 days Inoculation with specific influenza virus Incubation for 48-96 hours at 33°-36 °C Pre-chilling in refrigerators Harvest of allantoic fluid

  26. Egg Based Flu Vaccine Process (3) Composition of allantoic fluid Mostly ovalbumin and lipids

  27. Egg Based Flu Vaccine Process (4) Allantoic fluid harvesting Inactivation Eg : Formaldehyde Chemical Treatment Zonal centrifugation Clarification Formulation/Filling Purification/ Concentration

  28. Flu Market • Should more than double by 2010 • Price per dose has risen by 25 %in the last months Increased capacity More coverage elderly More coverage children Improved pricing New markets 1.2 B US $ To 1.5 B US $ 2.75 B US $ To 3.4 B US $ 2010 2005 Source GSK Internet

  29. Egg Based Process & Associated Risk Lead time 6 months Production time ; 6 months • Embryonated eggs: 6 months from order to delivery • 1 egg = 1 trivalent dose • Limited flexibility and reliability • Could be killed by virulent avian flu • How to respond to unanticipated demand : production failure, pandemic, strain changes

  30. Potential Advantages of Cell Culture Based Influenza • Allows greater flexibility for surge capacity • Provides opportunities for year around vaccines production • Requires less manufacturing space • Circumvents possible problems presented by highly virulent influenza strains ( e.g lethality to chicken embryos) • Tolerated by people with egg allergies

  31. Potential Advantages of Cell Culture Based Influenza (Ctn’ed) • Use readily available raw material • Use close bioreactors instead of millions of eggs • Scalable, flexible, high volume process • Characterizable, potential to use animal free components • Already used with therapeutics ( around 30) and inactivated polio

  32. Cell Based Flu Vaccines ( Solvay ) Infection Zonal Centrifugation CellCulture Production Clarification Centrigation UF Virus Propagation Inactivation with Formaldehyde DNAse treatment Purification UF Detergent treatment Formulation Detergent Removal Source FDA VRBPAC

  33. Cell Based Flu Vaccines ( Solvay ) Strain 1 Blending Strain 2 Trivalent Vaccine 15 µg HA /strain/dose Strain 3

  34. Flu Cell Based Vaccine (Chiron ) Detergent Addition 0.45 µm B-Propiolactone Inactivation Chemical treatment 0.2 µm Adsorption Trivalent blending 0.2 µm 0.2 µm Source VRBPAC meeting/ FDA site

  35. Critical Aspects/ Scaling Up Issues • Low yields of virus production in cell culture (bothSystem) • Huge capital investment for cell culture equipment (fermentors) • Low hemagglutinin concentration of in the cell culture supernatant • very large harvesting volumes • The MDCK cell line used for virus propagation has a lower acceptability than the Vero cell line • tumoregenicity will be scrutinised

  36. Players in Non Egg Based Vaccine Solvay Sanofi Pasteur Baxter Chiron GSK ID Biomedical MedImmuneNobilon Acambis Novavax PowderMed Protein Science Considered cells : MDCK, Vero, and PERC.6 Universal Recombinant VLP Particles DNA vaccine Baculovirus/ Insect cells

  37. Particular Issues Caused by CellBased Process • Need to look at 3 main safety aspects • Cells : removal of whole cell debris and no oncogenicity • DNA : no oncogenicity, process is capable of removal/ Inactivation • Adventitious agents : no infection , oncogenicity, process is capable of removal/ inactivation

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