Vero细胞制备H5N1流感大流行疫苗的实验研究
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摘要
流感病毒是引起流行性感冒的病原体,具有高度传染性,极易发生流行,甚至达到世界范围的大流行,每次大流行都造成了巨大的人力资源和物力资源的损失。目前H5N1人用禽流感疫苗主要是以鸡胚为培养基质研发生产,然而当H5N1禽流感暴发时,难于在短期内提供足够的鸡胚来生产疫苗。此外,鸡胚中可能带有的外源性因子也影响疫苗的质量。非洲绿猴肾细胞Vero是WHO推荐的疫苗生产细胞,可采用微载体发酵罐进行大规模培养。获得流感病毒Vero细胞高产适应株对于流感疫苗的生产具有重要意义。
本实验以传统遗传重配技术获得H5N1流感病毒Vero细胞适应株,制备Vero细胞H5N1流感疫苗。以流感病毒Vero细胞适应株A/Yunnan/1/2005(H3N2)va为母株与反向遗传学技术改造的禽流感病毒疫苗株A/Anhui/1/2005(H5N1)共同感染SPF鸡胚或Vero细胞,用抗A/Yunnan/1/2005(H3N2)va羊抗体筛选,血凝抑制试验和基因测序鉴定病毒型别。结果表明：通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,获得了1株高产的H5N1流感病毒Vero细胞适应株。同时进行了重配毒株的一些生物学试验：病毒感染性滴度为1×107.2PFU/ml、流感特异性免疫荧光阳性、电子显微镜下可观察到完整的病毒颗粒、与H5N1标准抗血清琼脂糖板上形成明显的沉淀圈；且该毒株对鸡胚和小鼠不具有致毒性。
对该重配病毒株进行微载体培养,优化培养条件。其中,以DMEM/F12为基础培养基,进行的有血清微载体培养,可获得较高的病毒产量。经甲醛灭活配制的重配单价疫苗具有良好的免疫原性,重配前后的单价灭活苗免疫小鼠的抗血清血抑效价差异无统计学意义(F=0.857,P>0.05)。该重配病毒免疫雌性山羊后获得较好的抗血清,且对纯化该抗血清的方法进行了探索。
Influenza viruses are pathogens that cause influenza, which is highly infectious and highly epidemic, even to a worldwide pandemic, each pandemic had caused enormous losses of human and material resources. At present H5N1 avian influenza vaccine is mainly developed and produced by using embryos eggs. However, when the H5N1 avian flu outbreaks in the short time, it is difficult to provide sufficient embryos to produce vaccines. In addition, the embryonated chicken eggs may be with a foreign-derived factor, which also affected the quality of vaccines. Vero cell is WHO resigned cell strain to produce vaccine and can be cultured at large scale by the bioreactor. So it is important to obtain high-growth influenza H5N1 virus in Vero cells for the influenza vaccine production with Vero cells.
In this experiment, we prepared a Vero cell-adapted influenza H5N1 virus strain by Genetic Reassortment and produced influenza H5N1 vaccine using Vero cell as a substrate. Embryonated specific pathogen-free (SPF) hen's eggs and Vero cells were co-infected with Vero cell-adapted influenza virus A/Yunnan/1/2005 (H3N2)va and A/Anhui/1/2005(H5N1) via reverse genetics. The reassortant was screened with goat antibody against strain A/Yunnan/1/2005(H3N2)va and identified for subtype by hemagglutination-inhibition (HI) assays and gene analysis of HA and NA. Results A Vero cell-adapted influenza H5N1 virus strain was obtained. The Vero cell-adapted influenza virus of epidemic strain may be reassortmented between Vero cell-adapted and epidemic strains.At the same time, other relevant biological experiments of the reassortant has been done. The infection titer was 1×10 PFU/ml in plaque experiment by using MDCK cells. The reaction of immunofluorescence experiment is positive. Complete virus particles would be observed by Electron microscope. In signal immunodifusion test, there were obvious settling circles in agarose gel plate of standard H5N1 anti-serum. And the reassortant had no toxicity both for embryonated chicken eggs and mice.
The reassortant had been cultured using miacro-carrior method and the culture conditions optimized. As DMEM/F12 was basic media, we had a relatively high yields of virus by miacro-carrior culture with serum. The reassorted monovalent vaccine was made by inactivating virus with formaldehyde and had a good immunogenicity. And there was no significant difference in serum antibody titers of monovalent inactivated vaccines before and after reassortment (F=0.857, P> 0.05). The reassortant also has a higher yield via microcarrier culture.We could get the better anti-serum by immuning female goat, and selected suitable purification method of the anti-serum.
本实验以传统遗传重配技术获得H5N1流感病毒Vero细胞适应株,制备Vero细胞H5N1流感疫苗。以流感病毒Vero细胞适应株A/Yunnan/1/2005(H3N2)va为母株与反向遗传学技术改造的禽流感病毒疫苗株A/Anhui/1/2005(H5N1)共同感染SPF鸡胚或Vero细胞,用抗A/Yunnan/1/2005(H3N2)va羊抗体筛选,血凝抑制试验和基因测序鉴定病毒型别。结果表明：通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,获得了1株高产的H5N1流感病毒Vero细胞适应株。同时进行了重配毒株的一些生物学试验：病毒感染性滴度为1×107.2PFU/ml、流感特异性免疫荧光阳性、电子显微镜下可观察到完整的病毒颗粒、与H5N1标准抗血清琼脂糖板上形成明显的沉淀圈；且该毒株对鸡胚和小鼠不具有致毒性。
对该重配病毒株进行微载体培养,优化培养条件。其中,以DMEM/F12为基础培养基,进行的有血清微载体培养,可获得较高的病毒产量。经甲醛灭活配制的重配单价疫苗具有良好的免疫原性,重配前后的单价灭活苗免疫小鼠的抗血清血抑效价差异无统计学意义(F=0.857,P>0.05)。该重配病毒免疫雌性山羊后获得较好的抗血清,且对纯化该抗血清的方法进行了探索。
Influenza viruses are pathogens that cause influenza, which is highly infectious and highly epidemic, even to a worldwide pandemic, each pandemic had caused enormous losses of human and material resources. At present H5N1 avian influenza vaccine is mainly developed and produced by using embryos eggs. However, when the H5N1 avian flu outbreaks in the short time, it is difficult to provide sufficient embryos to produce vaccines. In addition, the embryonated chicken eggs may be with a foreign-derived factor, which also affected the quality of vaccines. Vero cell is WHO resigned cell strain to produce vaccine and can be cultured at large scale by the bioreactor. So it is important to obtain high-growth influenza H5N1 virus in Vero cells for the influenza vaccine production with Vero cells.
In this experiment, we prepared a Vero cell-adapted influenza H5N1 virus strain by Genetic Reassortment and produced influenza H5N1 vaccine using Vero cell as a substrate. Embryonated specific pathogen-free (SPF) hen's eggs and Vero cells were co-infected with Vero cell-adapted influenza virus A/Yunnan/1/2005 (H3N2)va and A/Anhui/1/2005(H5N1) via reverse genetics. The reassortant was screened with goat antibody against strain A/Yunnan/1/2005(H3N2)va and identified for subtype by hemagglutination-inhibition (HI) assays and gene analysis of HA and NA. Results A Vero cell-adapted influenza H5N1 virus strain was obtained. The Vero cell-adapted influenza virus of epidemic strain may be reassortmented between Vero cell-adapted and epidemic strains.At the same time, other relevant biological experiments of the reassortant has been done. The infection titer was 1×10 PFU/ml in plaque experiment by using MDCK cells. The reaction of immunofluorescence experiment is positive. Complete virus particles would be observed by Electron microscope. In signal immunodifusion test, there were obvious settling circles in agarose gel plate of standard H5N1 anti-serum. And the reassortant had no toxicity both for embryonated chicken eggs and mice.
The reassortant had been cultured using miacro-carrior method and the culture conditions optimized. As DMEM/F12 was basic media, we had a relatively high yields of virus by miacro-carrior culture with serum. The reassorted monovalent vaccine was made by inactivating virus with formaldehyde and had a good immunogenicity. And there was no significant difference in serum antibody titers of monovalent inactivated vaccines before and after reassortment (F=0.857, P> 0.05). The reassortant also has a higher yield via microcarrier culture.We could get the better anti-serum by immuning female goat, and selected suitable purification method of the anti-serum.
引文
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