Vero细胞基质流感疫苗研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-0238

摘要/Abstract

摘要：

流感防控是人类至今面临的严峻挑战之一，流感疫苗接种是预防流感的关键。由于传统的流感疫苗生产工艺依赖鸡胚生产而存在局限性，规模化流感疫苗生产基质逐渐从鸡胚转为哺乳动物细胞，目前用于流感疫苗研发的常见细胞系有MDCK、Vero和PER.C6。其中Vero细胞低代次无成瘤性且已用于多种人用病毒性疫苗生产，是WHO推荐的广泛用于人和动物病毒性疫苗生产的首选细胞系，但Vero细胞基质流感疫苗在生产中仍面临挑战。现就流感疫苗研究、细胞基质流感疫苗的综合比较及Vero细胞在流感疫苗生产中所面临的困难予以综述，旨在为细胞基质流感疫苗研发提供参考。

关键词: 流感, Vero细胞, 疫苗, 悬浮培养

Abstract:

The prevention of influenza is one of the most severe challenges faced by mankind. Influenza vaccine inoculation is the key of preventing influenza. There are limitations in the traditional production process of influenza vaccine due to its dependence on chicken embryo, and the production of large-scale influenza vaccine is gradually transferred from chicken embryo to mammalian cells. Currently, MDCK, Vero and PER.C6 cell lines can be used for influenza vaccine development. Vero cells have non-tumogenesis within a certain range of generations and been used in the production of a variety of human viral vaccines. It is the cell line recommended by WHO that is widely used in the production of human and animal virial vaccines, however, there is still challenges in the Vero cell-based influenza vaccine production. This paper reviews the research of influenza vaccine, the comprehensive comparison of cell-based influenza vaccine and the difficulties faced by Vero cells in the production of influenza vaccine, aiming to provide reference for the development of cell-based influenza vaccine.

Key words: influenza, Vero cell, vaccine, suspension culture

马芳芳, 康碧静, 马春英, 刘振斌, 杨迪, 乔自林, 王明明, 马忠仁, 王家敏. Vero细胞基质流感疫苗研究进展[J]. 生物技术通报, 2022, 38(12): 137-143.

MA Fang-fang, KANG Bi-jing, MA Chun-ying, LIU Zhen-bin, YANG Di, QIAO Zi-lin, WANG Ming-ming, MA Zhong-ren, WANG Jia-min. Research Progress in Vero Cell-based Influenza Vaccine[J]. Biotechnology Bulletin, 2022, 38(12): 137-143.

图/表 6

参考文献 33

[1]	Long JS, Mistry B, Haslam SM, et al. Host and viral determinants of influenza A virus species specificity[J]. Nat Rev Microbiol, 2019, 17(2):67-81. doi: 10.1038/s41579-018-0115-z pmid: 30487536
[2]	叶淑英. 流感大流行带来的启示[J]. 科教文汇, 2012(7):131-146.
	Ye SY. The inspiration from pandemic influenza[J]. The Science Education Article Collects, 2012(7):131-146.
[3]	Pushko P, Tretyakova I. Influenza virus like particles(VLPs):opportunities for H7N9 vaccine development[J]. Viruses, 2020, 12(5):518. doi: 10.3390/v12050518 URL
[4]	Manini I, Trombetta CM, Lazzeri G, et al. Egg-independent influenza vaccines and vaccine candidates[J]. Vaccines, 2017, 5(3):18. doi: 10.3390/vaccines5030018 URL
[5]	Grohskopf LA, Sokolow LZ, Broder KR, et al. Prevention and control of seasonal influenza with vaccines:recommendations of the advisory committee on immunization practices-United States, 2018-19 influenza season[J]. MMWR Recomm Rep, 2018, 67(3):1-20. doi: 10.15585/mmwr.rr6703a1 pmid: 30141464
[6]	Hegde NR. Cell culture-based influenza vaccines:a necessary and indispensable investment for the future[J]. Hum Vaccin Immunother, 2015, 11(5):1223-1234. doi: 10.1080/21645515.2015.1016666 URL
[7]	World Health Organization. Cell culture as a substrate for the production of influenza vaccines:memorandum from a WHO meeting[J]. Bull World Health Organ, 1995, 73(4):431-435.
[8]	Chen JD, Wang JH, Zhang JP, et al. Advances in development and application of influenza vaccines[J]. Front Immunol, 2021, 12:711997. doi: 10.3389/fimmu.2021.711997 URL
[9]	Gresset-Bourgeois V, Leventhal PS, Pepin S, et al. Quadrivalent inactivated influenza vaccine(VaxigripTetra^TM)[J]. Expert Rev Vaccines, 2018, 17(1):1-11. doi: 10.1080/14760584.2018.1407650 pmid: 29157068
[10]	Shin D, Park KJ, Lee H, et al. Comparison of immunogenicity of cell-and egg-passaged viruses for manufacturing MDCK cell culture-based influenza vaccines[J]. Virus Res, 2015, 204:40-46. doi: 10.1016/j.virusres.2015.04.005 pmid: 25892718
[11]	Pérez Rubio A, Eiros JM. Cell culture-derived flu vaccine:present and future[J]. Hum Vaccin Immunother, 2018, 14(8):1874-1882. doi: 10.1080/21645515.2018.1460297 URL
[12]	Hein MD, Arora P, Marichal-Gallardo P, et al. Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles[J]. BMC Biol, 2021, 19(1):91. doi: 10.1186/s12915-021-01020-5 pmid: 33941189
[13]	Yasumura Y, Kawakita Y. Studies on SV40 in tissue culture - preliminary step for cancer research in vitro[J]. Nihon Rinsho, 1963, 21:1201-1215.
[14]	Ugiyadi M, Tan MI, Giri-Rachman EA, et al. The expression of essential components for human influenza virus internalisation in Vero and MDCK cells[J]. Cytotechnology, 2014, 66(3):515-523. doi: 10.1007/s10616-013-9602-2 pmid: 23912187
[15]	罗剑, 李秀玲. 基于细胞培养的流感疫苗研究进展[J]. 中国新药杂志, 2019, 28(21):2594-2599.
	Luo J, Li XL. Progress of cell culture-derived influenza vaccines[J]. Chin J New Drugs, 2019, 28(21):2594-2599.
[16]	Huang D, Peng WJ, Ye Q, et al. Serum-free suspension culture of MDCK cells for production of influenza H1N1 vaccines[J]. PLoS One, 2015, 10(11):e0141686. doi: 10.1371/journal.pone.0141686 URL
[17]	Barrett PN, Mundt W, Kistner O, et al. Vero cell platform in vaccine production:moving towards cell culture-based viral vaccines[J]. Expert Rev Vaccines, 2009, 8(5):607-618. doi: 10.1586/erv.09.19 pmid: 19397417
[18]	Ozaki H, Govorkova EA, Li CH, et al. Generation of high-yielding influenza A viruses in African green monkey kidney(Vero)cells by reverse genetics[J]. J Virol, 2004, 78(4):1851-1857. doi: 10.1128/JVI.78.4.1851-1857.2004 URL
[19]	Legastelois I, Garcia-Sastre A, Palese P, et al. Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells[J]. Influenza Other Respir Viruses, 2007, 1(3):95-104. doi: 10.1111/j.1750-2659.2007.00015.x URL
[20]	沈娟, 孙明波, 马磊, 等. 遗传重配获得H5N1流感病毒Vero细胞适应株[J]. 中华实验和临床病毒学杂志, 2010, 24(2):119-121.
	Shen J, Sun MB, Ma L, et al. Preparation of vero cell-adapted influenza H5N1 virus strain by genetic reassortment[J]. Chin J Exp Clin Virol, 2010, 24(2):119-121.
[21]	Hu WB, Zhang H, Han QL, et al. A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial passages[J]. Vaccine, 2015, 33(2):374-381. doi: 10.1016/j.vaccine.2014.11.007 pmid: 25448099
[22]	王嘉琪, 董育红, 姜菊玲, 等. 基于PK15细胞的猪圆环病毒2型全悬浮培养工艺[J]. 中国农业科学, 2021, 54(6):1280-1287.
	Wang JQ, Dong YH, Jiang JL, et al. Based on PK15 cell line for PCV2 fully suspension culture process[J]. Sci Agric Sin, 2021, 54(6):1280-1287.
[23]	Litwin J. The growth of vero cells as suspended aggregates in serum-free medium[M]//Spier RE, Griffiths JB, MacDonald C. Animal Cell Technology. Amsterdam:Elsevier, 1992:414-417.
[24]	Paillet C, Forno G, Kratje R, et al. Suspension-Vero cell cultures as a platform for viral vaccine production[J]. Vaccine, 2009, 27(46):6464-6467.
[25]	Rourou S, Ben Zakkour M, Kallel H. Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media[J]. Vaccine, 2019, 37(47):6987-6995. doi: S0264-410X(19)30749-2 pmid: 31201054
[26]	Shen CF, Guilbault C, Li XL, et al. Development of suspension adapted Vero cell culture process technology for production of viral vaccines[J]. Vaccine, 2019, 37(47):6996-7002. doi: S0264-410X(19)30881-3 pmid: 31288997
[27]	Lee DK, Park J, Seo DW. Suspension culture of Vero cells for the production of adenovirus type 5[J]. Clin Exp Vaccine Res, 2020, 9(1):48-55. doi: 10.7774/cevr.2020.9.1.48 URL
[28]	刘鹏, 钏鸿云, 阮朝列, 等. 悬浮驯化Vero细胞制备及其在大流行流感病毒株培养的初步应用[J]. 中国病毒病杂志, 2021, 11(3):182-186.
	Liu P, Chuan HY, Ruan CL, et al. Preparation of suspension-adapted Vero cells and its preliminary application in culture of pandemic influenza virus strains[J]. Chin J Viral Dis, 2021, 11(3):182-186.
[29]	Huang D, Peng WJ, Ye Q, et al. Serum-free suspension culture of MDCK cells for production of influenza H1N1 vaccines[J]. PLoS One, 2015, 10(11):e0141686. doi: 10.1371/journal.pone.0141686 URL
[30]	Tossolini I, López-Díaz FJ, Kratje R, et al. Characterization of cellular states of CHO-K1 suspension cell culture through cell cycle and RNA-sequencing profiling[J]. J Biotechnol, 2018, 286:56-67. doi: S0168-1656(18)30642-4 pmid: 30243609
[31]	Grieger JC, Soltys SM, Samulski RJ. Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector[J]. Mol Ther, 2016, 24(2):287-297. doi: S1525-0016(16)30334-3 pmid: 26437810
[32]	Montomoli E, Khadang B, Piccirella S, et al. Cell culture-derived influenza vaccines from Vero cells:a new horizon for vaccine production[J]. Expert Rev Vaccines, 2012, 11(5):587-594. doi: 10.1586/erv.12.24 pmid: 22827244
[33]	Sparrow E, Wood JG, Chadwick C, et al. Global production capacity of seasonal and pandemic influenza vaccines in 2019[J]. Vaccine, 2021, 39(3):512-520. doi: 10.1016/j.vaccine.2020.12.018 pmid: 33341308

商品名称 Name of product	制造商 Manufacturer	疫苗类型 Vaccine type	佐剂 Adjuvant	接种量Pre-sentation/mL	适用人群 Applicable people	每种疫苗每剂所含HA/病毒数HA or virus count for each vaccine virus（per dose）	汞（硫柳汞）含量 Mercury（from thimerosal）
Afluria	Seqirus	四价灭活疫苗	无	0.25	6-35 个月	7.5 μg/0.25 mL 15 μg/0.5 mL	-
				0.5	≥3 岁		-
				5.0	≥6 个月（needle/syringe） 18-64 岁（jet injector）		24.5 μg/0.5 mL
Fluarix	GlaxoSmithKline	四价灭活疫苗	无	0.5	≥6 个月	15 μg/0.5 mL	-
FluLaval	GlaxoSmithKline	四价灭活疫苗	无	0.5	≥6 个月	15 μg/0.5 mL	-
Fluzone	Sanofi Pasteur	四价灭活疫苗	无	0.5	≥6 个月	15 μg/0.5 mL	-
				0.5	≥6 个月
				5.0	≥6 个月
Fluzone High-Dose	Sanofi Pasteur	高剂量四价灭活疫苗	无	0.7	≥65 岁	60 μg/0.7 mL	-
Fluad	Seqirus	四价灭活疫苗	MF59	0.5	≥65 岁	15 μg/0.5 mL	-
Fluad	Seqirus	三价灭活疫苗	MF59	0.5	≥65 岁	15 μg/0.5 mL	-

商品名称 Name of product	制造商 Manufacturer	疫苗类型 Vaccine type	佐剂 Adjuvant	接种量Pre-sentation/mL	适用人群 Applicable people	每种疫苗每剂所含HA/病毒数HA or virus count for each vaccine virus（per dose）	汞（硫柳汞）含量 Mercury（from thimerosal）
Afluria	Seqirus	四价灭活疫苗	无	0.25	6-35 个月	7.5 μg/0.25 mL 15 μg/0.5 mL	-
				0.5	≥3 岁		-
				5.0	≥6 个月（needle/syringe） 18-64 岁（jet injector）		24.5 μg/0.5 mL
Fluarix	GlaxoSmithKline	四价灭活疫苗	无	0.5	≥6 个月	15 μg/0.5 mL	-
FluLaval	GlaxoSmithKline	四价灭活疫苗	无	0.5	≥6 个月	15 μg/0.5 mL	-
Fluzone	Sanofi Pasteur	四价灭活疫苗	无	0.5	≥6 个月	15 μg/0.5 mL	-
				0.5	≥6 个月
				5.0	≥6 个月
Fluzone High-Dose	Sanofi Pasteur	高剂量四价灭活疫苗	无	0.7	≥65 岁	60 μg/0.7 mL	-
Fluad	Seqirus	四价灭活疫苗	MF59	0.5	≥65 岁	15 μg/0.5 mL	-
Fluad	Seqirus	三价灭活疫苗	MF59	0.5	≥65 岁	15 μg/0.5 mL	-

生产厂家 Manufacturer	疫苗类型 Vaccine type	毒株 Strain	细胞基质 Cell substrate	培养方式 Cultivating way	商品名称 Name of product
Solvay	三价亚单位疫苗	A/H1N1、A/H3N2、一种乙型毒株	MDCK 细胞	无血清微载体贴壁培养	Influvac
Novartis	单价灭活疫苗	A/H1N1	MDCK细胞	-	Celtura
Novartis	三价亚单位疫苗	A/H1N1、A/H3N2、一种乙型毒株	MDCK细胞	无血清悬浮培养	Flucelvax
Seqirus	四价流感疫苗	A/H1N1，A/H3N2，B Yamagata，B Victoria	MDCK细胞	-	Flucelvax
Baxter	单价灭活疫苗	A/ H1N1或A/H5N1	Vero 细胞	微载体无血清培养	Celvapan
Baxter	三价灭活疫苗	A/H1N1、A/H3N2、一种乙型毒株	Vero 细胞	微载体无血清培养	Preflucel
Sanofi Pasteur	单价灭活疫苗	H7N1	PER.C6细胞	-	-

生产厂家 Manufacturer	疫苗类型 Vaccine type	毒株 Strain	细胞基质 Cell substrate	培养方式 Cultivating way	商品名称 Name of product
Solvay	三价亚单位疫苗	A/H1N1、A/H3N2、一种乙型毒株	MDCK 细胞	无血清微载体贴壁培养	Influvac
Novartis	单价灭活疫苗	A/H1N1	MDCK细胞	-	Celtura
Novartis	三价亚单位疫苗	A/H1N1、A/H3N2、一种乙型毒株	MDCK细胞	无血清悬浮培养	Flucelvax
Seqirus	四价流感疫苗	A/H1N1，A/H3N2，B Yamagata，B Victoria	MDCK细胞	-	Flucelvax
Baxter	单价灭活疫苗	A/ H1N1或A/H5N1	Vero 细胞	微载体无血清培养	Celvapan
Baxter	三价灭活疫苗	A/H1N1、A/H3N2、一种乙型毒株	Vero 细胞	微载体无血清培养	Preflucel
Sanofi Pasteur	单价灭活疫苗	H7N1	PER.C6细胞	-	-

细胞名称 Name of cell	培养方式 Cultivating way	有无血清 Presence of serum	毒株 Strain	最高增殖密度 Maximum proliferation density/（Cells·mL^-1）	血凝滴度 Degree of HA/ （lgHAU·100 μL^-1）	病毒感染性滴度 Titer of viral infectivity/ （lgHAU·100 μL^-1）
MDCK细胞	微载体Cytodex 1悬浮培养	有	H5N1（A /Vietnam /1194 /2004-NIB RG-14）	0.8 × 10⁷	2.9	8.64-9.22
Vero 细胞	微载体 Cytodex 1悬浮培养	有	H1N1（A/PR/8 /34）	-	2.6	7.83-7.88
Vero 细胞	微载体 Cytodex3 悬浮培养	无	A/Vietnam/1203 /2004和 A/Indonesia /05 /2005	2.73 × 10⁶	2.70-3.01	8.6-9.0
MDCK细胞	全悬浮培养	无	H1N1	5.97×10⁶	3.88	10.34
MDCK细胞	半灌注全悬浮培养	-	H1N1（A/PR/8/34）	6 × 10⁷	4.5	10