大流行流感裂解疫苗的研制及关键技术的研究
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摘要
当前,高致病性人禽流感H5N1已在15个国家和地区发生,死亡率高,特别是印尼家族性感染被美国科学家证实为人传染人事例后,使得大流行流感防控形势异常严峻。WHO预言,下一次流感大流行不是有和无的问题,而是早和晚的问题。因此,做好大流行流感疫苗研发和关键技术平台的研究,已成为世界各国应对流感大流行采取的重大举措。
疫苗免疫接种是控制高致病性人禽流感最有效而经济的手段之一,但面临着高致病性人禽流感病毒H5N1毒株变异、鸡胚生产疫苗原料来源受限、注射接种繁复等重大问题。因此WHO要求全球加快大流行流感疫苗研发和关键技术储备研究进程,为应对流感大流行做好充分的准备。
目前,高致病性人禽流感病毒H5N1感染主要分布在亚洲,特别是印尼、越南和中国,对其分离的毒株进行血清学、致病性和基因结构等分析表明,高致病性人禽流感病毒H5N1具有高度变异的特性,就中国分离的毒株存在南北之分。因此,WHO要求各国开展大流行流感原型疫苗的研究。目前大流行流感疫苗的研究重点主要集中在灭活全病毒疫苗、裂解疫苗、减毒喷鼻疫苗、亚单位疫苗等。其中裂解疫苗具有不可多得的优势:与全病毒灭活疫苗仅可用于成年人的特点比较,可广泛用于各类人群,尤其是作为禽流感病毒的高危人群--老人和12岁以下的儿童;不存在减毒疫苗毒力恢复的缺点,更安全;较亚单位疫苗具有更强的免疫原性和制备简单等优势。目前,大流行流感裂解疫苗已成为世界著名疫苗企业和研究机构介入的重点,并取得了阶段性成果,为流感大流行作好了充分的准备。
基于大流行流感裂解疫苗应对流感大流行具有不可多得的优势,我们在国内率先成功研发了大流行流感裂解疫苗,解决了我国“无”和应急的问题。同时围绕大流行流感疫苗关键技术平台:针对高致病性人禽流感病毒H5N1不断变异,需要运用RG技术来快速制备疫苗株的问题、鸡胚生产原料来源和质量的问题、注射免疫不能产生全面免疫应答和接种复杂的问题进行了研究,其研究进展如下:
大流行流感裂解疫苗的研制:我们从美国CDC引进大流行流感疫苗株VNH5N1-PR8/CDC-RG(H5N1),该疫苗株是利用反向遗传学技术,将高致病性人禽流感病毒A/Viet Nam/1203/2004 (H5N1)的HA、NA基因定点突变后和PR8的6个内部基因进行重组,得到大流行流感疫苗减毒株。
开展了大流行流感裂解疫苗种子批库的研究。通过SPF鸡胚传代建立了大容量符合疫苗生产的原始种子批、主种子批和工作种子批库。分别对大流行流感疫苗各种子批进行了血凝滴度的检测、病毒型别的检定、EID50、无菌实验、支原体检测、外原因子检测及动物实验,实验证实建立的原始种子批、主种子批和工作种子批库具有安全、有效和稳定,符合WHO规定大流行流感疫苗生产的要求。
开展了大流行流感裂解疫苗原液生产的研究。通过健康鸡胚培养、收获、灭活、裂解大流行流感裂解疫苗病毒,制备疫苗生产裂解病毒抗原。实验表明病毒培养温度为33℃、培养时间为52h,病毒灭活采用1:4000的甲醛20～25℃灭活72h,病毒的纯化使用凝胶过滤Sepharose 4 Fast Flow,病毒裂解选择浓度为0.5%的Triton X-100,室温作用2.0 h,建立了疫苗生产的最佳生产工艺,制备了大流行流感裂解疫苗原液。
开展了注射型大流行流感裂解疫苗成品和有效性的研究。选用铝盐佐剂,采用动物模型和抗原吸附率检测进行铝佐剂浓度的确定,确定的铝佐剂浓度为1.2mg/ml。选用3.75、7.5、15.0、30.0、45.0、60.0、75.0、90.0μg/0.5ml 8个疫苗剂量组,分别肌肉免疫小鼠和豚鼠,对疫苗免疫原性进行了初步研究。通过血凝抑制实验来筛选疫苗的有效剂量,结果表明加铝盐佐剂的7.5、15.0、30.0μg/0.5ml是最佳的疫苗剂量组。我们同时对小鼠和豚鼠免疫一针和两针时的免疫效果进行了统计分析,表明免疫两针效果明显优于一针。因此,确定的大流行流感疫苗临床试验为7.5、15.0、30.0μg/0.5ml成品剂量组,免疫两针,为临床研究提供了依据。
开展了注射型大流行流感裂解疫苗安全性和稳定性的研究。通过动物过敏实验、异常毒性实验、急性毒性实验、热源质实验,结果表明大流行流感裂解疫苗无毒性,使用安全。将大流行流感裂解疫苗成品置2～8℃、室温(22～25℃)、37℃,通过解吸附单扩实验验证抗原含量,免疫Balb/c小鼠测定疫苗效力(中和抗体),通过pH仪检测pH值,外观的观察来鉴定疫苗稳定性,并通过疫苗加速实验,证明制备的大流行流感裂解疫苗可在2～8℃保存两年以上。
开展了注射型大流行流感裂解疫苗Ⅰ期临床研究,通过双盲实验完成了疫苗安全性、有效性研究,实验证明制备的疫苗安全、有效。
大流行流感疫苗关键技术的研究:围绕大流行流感疫苗研发中的疫苗毒株、培养介质和免疫途径的关键技术问题进行了研究,为应对流感大流行疫苗快速研发做好准备。
一是针对高致病性人禽流感病毒H5N1高度变异的问题,开展了RG技术用于疫苗株的拯救。因为高致病性人禽流感病毒H5N1高度变异,需要不断更换疫苗株,而快速、及时对流行株获得大流行流感疫苗株将是有效控制流感爆发流行的首要环节之一。我们通过流感病毒8质粒拯救系统,在BSL-3培养A/Anhui/1/2005 (H5N1)病毒,提取病毒RNA,利用基因重配技术将该病毒的HA(定点突变)、NA构建到双向表达质粒pHW2000上,将构建好的2质粒和PR8 6质粒共转染COS-1细胞,获得重组的PR8/A/Anhui/1/2005疫苗株病毒。运用RT-PCR、血凝实验、电镜、间接免疫荧光实验检测拯救的重组疫苗株病毒。实验结果表明,拯救PR8/A/Anhui/1/2005疫苗株病毒基因序列正确,血凝滴度达到1:320以上;电镜下病毒形态正常,成丝状和囊状。CPE证实病毒毒力明显降低,符合大流行流感疫苗株的要求,在国内率先建立了反向遗传学拯救流感疫苗株的关键技术平台。
二是针对大流行流感疫苗生产原料来源的问题,开展了Vero细胞替代鸡胚生产疫苗的研究。构建了pCDNA3.1-ST3GalⅢ表达载体,转染、G418加压筛选Vero细胞,获得稳定高效表达高致病性人禽流感病毒受体的Vero细胞株。运用改造好的Vero细胞株培养PR8/A/Anhui/1/2005疫苗株病毒,通过细胞病变和血凝实验结果证明,PR8/A/Anhui/1/2005疫苗株病毒感染Vero细胞病毒复制滴度提高,TCID50达到106.5-7,较普通的Vero细胞提高了100-150倍,初步建立了Vero细胞生产大流行流感疫苗的细胞系。我们将Eng53/v-aNS基因置换了8质粒系统的NS基因,重组病毒理论上可在Vero细胞上大量复制,但我们的实验结果却与预期值有差异,进一步实验仍在进行中。
三是喷鼻型大流行流感疫苗的研究,喷鼻大流行流感疫苗具有快速、简便大规模接种、免疫保护谱广的特点。将大流行流感裂解疫苗分别使用不同的佐剂,采用不同的免疫途径,分别检测唾液、肺灌洗液中分泌型IgA,血液中的IgG、IgA,以及运用HI实验来确定喷鼻疫苗最佳剂型和最佳免疫途径。结果表明,蛋白体佐剂与VNH5N1-PR8/CDC-RG(H5N1)制备的喷鼻型大流行流感裂解疫苗具用良好的免疫原性和安全性,其分泌型IgA是各类佐剂中最高的,其中肺灌洗液中IgA滴度为1:640,HI实验结果也证明,运用蛋白体佐剂喷鼻免疫大流行流感裂解疫苗将是一个具有良好开发前景的举措,。
结论:为加快适应和满足我国流感大流行疫苗研发和关键技术储备能力的需求。我们在应急上,已完成针对越南株大流行流感裂解原型疫苗的研究;围绕疫苗研发可持续发展战略上,针对高致病性人禽流感病毒H5N1株变异问题,建立了快速制备疫苗株的技术平台、针对疫苗生产原料来源和质量难以控制的特点,初步建立了Vero细胞替代鸡胚生产疫苗的技术平台、针对注射疫苗不能产生局部免疫和交叉免疫保护的特点,制备了喷鼻型大流行流感疫苗,可同时激发黏膜免疫和系统免疫,实现了对高致病性人禽流感病毒疫苗动态互补的研发创新体系,为流感大流行做好了充分的准备。
Recent outbreaks of highly pathogenic avian influenza(HPAI H5N1) in 15 countries and regions and associated human infections in Indonesia have led to a heightened level of awareness and preparation for a possible influenza pandemic.WHO has predicted that a new influenza pandemic might occur in the near future. Effective vaccines against H5N1 virus are, therefore, urgently needed.
Vaccination is considered one of the most-effective preventive measures for the control of influenza pandemics.However,Vrius variation,egg-based production and intramuscular vaccination with needles are barriers for production of vaccine.With the recommendation of WHO,we need find the effective strategies for developing vaccine against influenza pandemic.
Currently,HPVI type A virus(H5N1) mainly scatter in Asia,especially in Indonesia,Vietnam and China.The wild type virus isolated from human and various animals display the high variation by serology,pathogenicity and gene structural analysis. The viruses isolated in China belong to two clades.So we need to develop the prototype vaccine,now,include the inactive whole virion vaccine,the split vaccine,the live attenuated vaccine and the subunit vaccine.Among of the total,the split vaccine has the rare and good quality.It can be used in different old person,including the old and the children below 12 year old.it is more safe than live attenuated vaccine and can induced more strong immne response than subunit vaccine. Now,influenza split vaccines have been prepared and are undergoing clinical evaluation in several countries.
In China,we have develop the influenza split vaccine for the first time,to solve the problem of meeting an emergency and nought.We also develop the new strategies for vaccine,include reverse genetics for generation vaccine strain,Vero cell instead of eggs to production and alternative routes of administration.
R&D of Pandemic Influenza Split Vaccine
We purchased the virus vaccine strain-VNH5N1-PR8/CDC-RG(H5N1)which was recombinated from A/Viet Nam/1203/2004 (HA,NA) and PR8 from CDC in American.
We have successfully established the seed lots for the vaccine strain including the master seed lot and the working seed lot.The HA titer, identity, EID50, Sterility test, mycoplasma and extraneous agents detection were carried out seperately,and prove that the virus seed lots are safe and stabile, accordant with the regulations of WHO.
Fellow the establishment of seed lots,we have selected the best condition for virus culture,harvest,inactivation,split and purification.The tempreature and time for the virus culture is 33℃for 52h.To use 1:4000 formaldehyde to inactive virus for 72h, and select the Sepharose 4 Fast Flow to purify the virus and 0.5% Triton X-100 to split virus about 2h.At last,we prepared the stock solution of vaccine.
One approach for increasing the number of available split vaccine doses without a compensatory increase in egg production is to enhance the immunogenicity of the antigen; addition of an effective, safe adjuvant could result in a more immunogenic vaccines and also enable injection of a smaller quantity of antigen to elicit an effective immune response,so we select 1.2mg/ml AL as adjuvant and 3.75,7.5,15.0,30.0,45.0,60.0, 75.0,90.0μg/0.5ml dose form to intramuscular vaccinate the mice and guinea pigs one dose and two dose.The HI test showed that 7.5,15.0,30.0μg/0.5ml dose form will be selected into the clinical trial,and immunizd two dose.
The safety and stability of the pandemic vaccine are also important. Allergy test,single dose toxicity test,abnormal toxicity test,acute-term toxicity test were carried out seperately to test the safety of vaccine.We placed the vaccine in different conditon, 2～8℃,22～25℃,37℃to detect the HI titer,HA content,pH value and appearance to determine the vaccine stability.Our split vaccin can kept in 2～8℃for two years by the vaccine accelerate test.
The vaccine prepared with this virus was assessed in a double-blind Phase I clinical trial, the results of which showed that, although antibody responses indicative of immune protection were achieved by administration of the vaccine with AL adjuvant.
R&D of Key Technology for Pandemic influenza vaccine
The technology for pandemic influenza vaccine include generation the virus vaccine strain,selection of cultural medium and alternative routes of administration.
RG(reverse genetics): New vaccines that exploit reverse genetics technology are being developed,and the knowledge that the pathogenicity of avian influenza viruses is primarily determined by HA variation.The virus vaccine strain can be rescued by this technique in time when the influenza pandemic break out.There is no vaccine strain reference laboratory in China,we want to establish it using the 8 plasmids system. Briefly, in our system (pHW2000), six plasmids that encode exact copies of the six individual RNA segments of the PR8, together with two plasmids that express the two viral proteins (HA,NA) of A/Anhui/1/2005 (H5N1), are introduced into cultured COS-1cells, resulting in the generation of new vaccine strain.RT-PCR,HA test,electron microscope,IFA were carried out to detect the recombinant virus.The result showed that the gene sequence,HA titer(1:320),virus morphous and virulence accordant with the virus vaccine strain.
Cell-culture-based vaccine:Given that chicken embryonated eggs, which are currently used for inactivated-vaccine production, would be in a short supply during a pandemic, the development of Vero cell-culture-based H5 vaccines is an attractive alternative approach. Modification of the Vero cell that that can be easily infected by influenza virus or modification of vaccine strain that grow well in cell cultures are two useful strategies.We constructed pCDNA3.1-ST3GalⅢexpressing vector,transfected Vero cell,got the new Vero cell line which can express the 2-3 type receptor efficiently.The vaccine strain, PR8/A/Anhui/1/2005 can replicated in modified Vero cell,the TCID50 is 106.5-7,is higher than usual Vero cell.We exchanged NS gene of PR8 into NS gene of Eng53/v-a to get the modified virus,but we did not obtain the reasonable result.
Alternative routes of administration: We investigated the aerosolized vaccine for influenza pandemic which can reduced the antigen quantity and be used widely. Split vaccines combined with adjuvants-such as AL as well as outer membrane proteins of Neisseria meningitidis, referred to as proteosomes have been delivered directly to the nasal mucosa in animal models.We detected the secreting type IgA in saliva and lung lavage,and we also detedted the antibody in serum.The IgA titer in lung lavage is 1:640.All the results displayed the the aerosolized vaccine with protosome is the good selection for pandemic vaccine.
Conclusion
In order to respond to the influenza pandemic in China,we have finished the influenza split prototype vaccine to meet an emergeny outbreak of influenza.We also established the key technology for developing of vaccine:reverse genetics can slove the problem of virus variation and the vaccine strain can be generated in short of period; the development of Vero cell-culture-based vaccine can provide the stabile system to overcome the obstacle of a short supply of eggs during a pandemic; aerosolized vaccine for influenza pandemic can induced the systemic immunity and mucosal immunity at the same time,and also induced cross immunity among different virus strain.These findings indicate that the technology in this study is a better way for vaccine development of pandemic influenza.
疫苗免疫接种是控制高致病性人禽流感最有效而经济的手段之一,但面临着高致病性人禽流感病毒H5N1毒株变异、鸡胚生产疫苗原料来源受限、注射接种繁复等重大问题。因此WHO要求全球加快大流行流感疫苗研发和关键技术储备研究进程,为应对流感大流行做好充分的准备。
目前,高致病性人禽流感病毒H5N1感染主要分布在亚洲,特别是印尼、越南和中国,对其分离的毒株进行血清学、致病性和基因结构等分析表明,高致病性人禽流感病毒H5N1具有高度变异的特性,就中国分离的毒株存在南北之分。因此,WHO要求各国开展大流行流感原型疫苗的研究。目前大流行流感疫苗的研究重点主要集中在灭活全病毒疫苗、裂解疫苗、减毒喷鼻疫苗、亚单位疫苗等。其中裂解疫苗具有不可多得的优势:与全病毒灭活疫苗仅可用于成年人的特点比较,可广泛用于各类人群,尤其是作为禽流感病毒的高危人群--老人和12岁以下的儿童;不存在减毒疫苗毒力恢复的缺点,更安全;较亚单位疫苗具有更强的免疫原性和制备简单等优势。目前,大流行流感裂解疫苗已成为世界著名疫苗企业和研究机构介入的重点,并取得了阶段性成果,为流感大流行作好了充分的准备。
基于大流行流感裂解疫苗应对流感大流行具有不可多得的优势,我们在国内率先成功研发了大流行流感裂解疫苗,解决了我国“无”和应急的问题。同时围绕大流行流感疫苗关键技术平台:针对高致病性人禽流感病毒H5N1不断变异,需要运用RG技术来快速制备疫苗株的问题、鸡胚生产原料来源和质量的问题、注射免疫不能产生全面免疫应答和接种复杂的问题进行了研究,其研究进展如下:
大流行流感裂解疫苗的研制:我们从美国CDC引进大流行流感疫苗株VNH5N1-PR8/CDC-RG(H5N1),该疫苗株是利用反向遗传学技术,将高致病性人禽流感病毒A/Viet Nam/1203/2004 (H5N1)的HA、NA基因定点突变后和PR8的6个内部基因进行重组,得到大流行流感疫苗减毒株。
开展了大流行流感裂解疫苗种子批库的研究。通过SPF鸡胚传代建立了大容量符合疫苗生产的原始种子批、主种子批和工作种子批库。分别对大流行流感疫苗各种子批进行了血凝滴度的检测、病毒型别的检定、EID50、无菌实验、支原体检测、外原因子检测及动物实验,实验证实建立的原始种子批、主种子批和工作种子批库具有安全、有效和稳定,符合WHO规定大流行流感疫苗生产的要求。
开展了大流行流感裂解疫苗原液生产的研究。通过健康鸡胚培养、收获、灭活、裂解大流行流感裂解疫苗病毒,制备疫苗生产裂解病毒抗原。实验表明病毒培养温度为33℃、培养时间为52h,病毒灭活采用1:4000的甲醛20～25℃灭活72h,病毒的纯化使用凝胶过滤Sepharose 4 Fast Flow,病毒裂解选择浓度为0.5%的Triton X-100,室温作用2.0 h,建立了疫苗生产的最佳生产工艺,制备了大流行流感裂解疫苗原液。
开展了注射型大流行流感裂解疫苗成品和有效性的研究。选用铝盐佐剂,采用动物模型和抗原吸附率检测进行铝佐剂浓度的确定,确定的铝佐剂浓度为1.2mg/ml。选用3.75、7.5、15.0、30.0、45.0、60.0、75.0、90.0μg/0.5ml 8个疫苗剂量组,分别肌肉免疫小鼠和豚鼠,对疫苗免疫原性进行了初步研究。通过血凝抑制实验来筛选疫苗的有效剂量,结果表明加铝盐佐剂的7.5、15.0、30.0μg/0.5ml是最佳的疫苗剂量组。我们同时对小鼠和豚鼠免疫一针和两针时的免疫效果进行了统计分析,表明免疫两针效果明显优于一针。因此,确定的大流行流感疫苗临床试验为7.5、15.0、30.0μg/0.5ml成品剂量组,免疫两针,为临床研究提供了依据。
开展了注射型大流行流感裂解疫苗安全性和稳定性的研究。通过动物过敏实验、异常毒性实验、急性毒性实验、热源质实验,结果表明大流行流感裂解疫苗无毒性,使用安全。将大流行流感裂解疫苗成品置2～8℃、室温(22～25℃)、37℃,通过解吸附单扩实验验证抗原含量,免疫Balb/c小鼠测定疫苗效力(中和抗体),通过pH仪检测pH值,外观的观察来鉴定疫苗稳定性,并通过疫苗加速实验,证明制备的大流行流感裂解疫苗可在2～8℃保存两年以上。
开展了注射型大流行流感裂解疫苗Ⅰ期临床研究,通过双盲实验完成了疫苗安全性、有效性研究,实验证明制备的疫苗安全、有效。
大流行流感疫苗关键技术的研究:围绕大流行流感疫苗研发中的疫苗毒株、培养介质和免疫途径的关键技术问题进行了研究,为应对流感大流行疫苗快速研发做好准备。
一是针对高致病性人禽流感病毒H5N1高度变异的问题,开展了RG技术用于疫苗株的拯救。因为高致病性人禽流感病毒H5N1高度变异,需要不断更换疫苗株,而快速、及时对流行株获得大流行流感疫苗株将是有效控制流感爆发流行的首要环节之一。我们通过流感病毒8质粒拯救系统,在BSL-3培养A/Anhui/1/2005 (H5N1)病毒,提取病毒RNA,利用基因重配技术将该病毒的HA(定点突变)、NA构建到双向表达质粒pHW2000上,将构建好的2质粒和PR8 6质粒共转染COS-1细胞,获得重组的PR8/A/Anhui/1/2005疫苗株病毒。运用RT-PCR、血凝实验、电镜、间接免疫荧光实验检测拯救的重组疫苗株病毒。实验结果表明,拯救PR8/A/Anhui/1/2005疫苗株病毒基因序列正确,血凝滴度达到1:320以上;电镜下病毒形态正常,成丝状和囊状。CPE证实病毒毒力明显降低,符合大流行流感疫苗株的要求,在国内率先建立了反向遗传学拯救流感疫苗株的关键技术平台。
二是针对大流行流感疫苗生产原料来源的问题,开展了Vero细胞替代鸡胚生产疫苗的研究。构建了pCDNA3.1-ST3GalⅢ表达载体,转染、G418加压筛选Vero细胞,获得稳定高效表达高致病性人禽流感病毒受体的Vero细胞株。运用改造好的Vero细胞株培养PR8/A/Anhui/1/2005疫苗株病毒,通过细胞病变和血凝实验结果证明,PR8/A/Anhui/1/2005疫苗株病毒感染Vero细胞病毒复制滴度提高,TCID50达到106.5-7,较普通的Vero细胞提高了100-150倍,初步建立了Vero细胞生产大流行流感疫苗的细胞系。我们将Eng53/v-aNS基因置换了8质粒系统的NS基因,重组病毒理论上可在Vero细胞上大量复制,但我们的实验结果却与预期值有差异,进一步实验仍在进行中。
三是喷鼻型大流行流感疫苗的研究,喷鼻大流行流感疫苗具有快速、简便大规模接种、免疫保护谱广的特点。将大流行流感裂解疫苗分别使用不同的佐剂,采用不同的免疫途径,分别检测唾液、肺灌洗液中分泌型IgA,血液中的IgG、IgA,以及运用HI实验来确定喷鼻疫苗最佳剂型和最佳免疫途径。结果表明,蛋白体佐剂与VNH5N1-PR8/CDC-RG(H5N1)制备的喷鼻型大流行流感裂解疫苗具用良好的免疫原性和安全性,其分泌型IgA是各类佐剂中最高的,其中肺灌洗液中IgA滴度为1:640,HI实验结果也证明,运用蛋白体佐剂喷鼻免疫大流行流感裂解疫苗将是一个具有良好开发前景的举措,。
结论:为加快适应和满足我国流感大流行疫苗研发和关键技术储备能力的需求。我们在应急上,已完成针对越南株大流行流感裂解原型疫苗的研究;围绕疫苗研发可持续发展战略上,针对高致病性人禽流感病毒H5N1株变异问题,建立了快速制备疫苗株的技术平台、针对疫苗生产原料来源和质量难以控制的特点,初步建立了Vero细胞替代鸡胚生产疫苗的技术平台、针对注射疫苗不能产生局部免疫和交叉免疫保护的特点,制备了喷鼻型大流行流感疫苗,可同时激发黏膜免疫和系统免疫,实现了对高致病性人禽流感病毒疫苗动态互补的研发创新体系,为流感大流行做好了充分的准备。
Recent outbreaks of highly pathogenic avian influenza(HPAI H5N1) in 15 countries and regions and associated human infections in Indonesia have led to a heightened level of awareness and preparation for a possible influenza pandemic.WHO has predicted that a new influenza pandemic might occur in the near future. Effective vaccines against H5N1 virus are, therefore, urgently needed.
Vaccination is considered one of the most-effective preventive measures for the control of influenza pandemics.However,Vrius variation,egg-based production and intramuscular vaccination with needles are barriers for production of vaccine.With the recommendation of WHO,we need find the effective strategies for developing vaccine against influenza pandemic.
Currently,HPVI type A virus(H5N1) mainly scatter in Asia,especially in Indonesia,Vietnam and China.The wild type virus isolated from human and various animals display the high variation by serology,pathogenicity and gene structural analysis. The viruses isolated in China belong to two clades.So we need to develop the prototype vaccine,now,include the inactive whole virion vaccine,the split vaccine,the live attenuated vaccine and the subunit vaccine.Among of the total,the split vaccine has the rare and good quality.It can be used in different old person,including the old and the children below 12 year old.it is more safe than live attenuated vaccine and can induced more strong immne response than subunit vaccine. Now,influenza split vaccines have been prepared and are undergoing clinical evaluation in several countries.
In China,we have develop the influenza split vaccine for the first time,to solve the problem of meeting an emergency and nought.We also develop the new strategies for vaccine,include reverse genetics for generation vaccine strain,Vero cell instead of eggs to production and alternative routes of administration.
R&D of Pandemic Influenza Split Vaccine
We purchased the virus vaccine strain-VNH5N1-PR8/CDC-RG(H5N1)which was recombinated from A/Viet Nam/1203/2004 (HA,NA) and PR8 from CDC in American.
We have successfully established the seed lots for the vaccine strain including the master seed lot and the working seed lot.The HA titer, identity, EID50, Sterility test, mycoplasma and extraneous agents detection were carried out seperately,and prove that the virus seed lots are safe and stabile, accordant with the regulations of WHO.
Fellow the establishment of seed lots,we have selected the best condition for virus culture,harvest,inactivation,split and purification.The tempreature and time for the virus culture is 33℃for 52h.To use 1:4000 formaldehyde to inactive virus for 72h, and select the Sepharose 4 Fast Flow to purify the virus and 0.5% Triton X-100 to split virus about 2h.At last,we prepared the stock solution of vaccine.
One approach for increasing the number of available split vaccine doses without a compensatory increase in egg production is to enhance the immunogenicity of the antigen; addition of an effective, safe adjuvant could result in a more immunogenic vaccines and also enable injection of a smaller quantity of antigen to elicit an effective immune response,so we select 1.2mg/ml AL as adjuvant and 3.75,7.5,15.0,30.0,45.0,60.0, 75.0,90.0μg/0.5ml dose form to intramuscular vaccinate the mice and guinea pigs one dose and two dose.The HI test showed that 7.5,15.0,30.0μg/0.5ml dose form will be selected into the clinical trial,and immunizd two dose.
The safety and stability of the pandemic vaccine are also important. Allergy test,single dose toxicity test,abnormal toxicity test,acute-term toxicity test were carried out seperately to test the safety of vaccine.We placed the vaccine in different conditon, 2～8℃,22～25℃,37℃to detect the HI titer,HA content,pH value and appearance to determine the vaccine stability.Our split vaccin can kept in 2～8℃for two years by the vaccine accelerate test.
The vaccine prepared with this virus was assessed in a double-blind Phase I clinical trial, the results of which showed that, although antibody responses indicative of immune protection were achieved by administration of the vaccine with AL adjuvant.
R&D of Key Technology for Pandemic influenza vaccine
The technology for pandemic influenza vaccine include generation the virus vaccine strain,selection of cultural medium and alternative routes of administration.
RG(reverse genetics): New vaccines that exploit reverse genetics technology are being developed,and the knowledge that the pathogenicity of avian influenza viruses is primarily determined by HA variation.The virus vaccine strain can be rescued by this technique in time when the influenza pandemic break out.There is no vaccine strain reference laboratory in China,we want to establish it using the 8 plasmids system. Briefly, in our system (pHW2000), six plasmids that encode exact copies of the six individual RNA segments of the PR8, together with two plasmids that express the two viral proteins (HA,NA) of A/Anhui/1/2005 (H5N1), are introduced into cultured COS-1cells, resulting in the generation of new vaccine strain.RT-PCR,HA test,electron microscope,IFA were carried out to detect the recombinant virus.The result showed that the gene sequence,HA titer(1:320),virus morphous and virulence accordant with the virus vaccine strain.
Cell-culture-based vaccine:Given that chicken embryonated eggs, which are currently used for inactivated-vaccine production, would be in a short supply during a pandemic, the development of Vero cell-culture-based H5 vaccines is an attractive alternative approach. Modification of the Vero cell that that can be easily infected by influenza virus or modification of vaccine strain that grow well in cell cultures are two useful strategies.We constructed pCDNA3.1-ST3GalⅢexpressing vector,transfected Vero cell,got the new Vero cell line which can express the 2-3 type receptor efficiently.The vaccine strain, PR8/A/Anhui/1/2005 can replicated in modified Vero cell,the TCID50 is 106.5-7,is higher than usual Vero cell.We exchanged NS gene of PR8 into NS gene of Eng53/v-a to get the modified virus,but we did not obtain the reasonable result.
Alternative routes of administration: We investigated the aerosolized vaccine for influenza pandemic which can reduced the antigen quantity and be used widely. Split vaccines combined with adjuvants-such as AL as well as outer membrane proteins of Neisseria meningitidis, referred to as proteosomes have been delivered directly to the nasal mucosa in animal models.We detected the secreting type IgA in saliva and lung lavage,and we also detedted the antibody in serum.The IgA titer in lung lavage is 1:640.All the results displayed the the aerosolized vaccine with protosome is the good selection for pandemic vaccine.
Conclusion
In order to respond to the influenza pandemic in China,we have finished the influenza split prototype vaccine to meet an emergeny outbreak of influenza.We also established the key technology for developing of vaccine:reverse genetics can slove the problem of virus variation and the vaccine strain can be generated in short of period; the development of Vero cell-culture-based vaccine can provide the stabile system to overcome the obstacle of a short supply of eggs during a pandemic; aerosolized vaccine for influenza pandemic can induced the systemic immunity and mucosal immunity at the same time,and also induced cross immunity among different virus strain.These findings indicate that the technology in this study is a better way for vaccine development of pandemic influenza.
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