乙型流感病毒Vero细胞高产适应株传代稳定性研究
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摘要
由于能快速、大规模生产以应对随时可能暴发的流感,细胞流感疫苗已成为当今流感疫苗的主要研发方向,而选育流感病毒Vero细胞高产适应株对于细胞流感疫苗的研发至关重要。本文旨在研究流感病毒Vero细胞高产适应株BX-51BVa(By)在Vero细胞上的传代稳定性,以证明该毒株是否可以作为制备Vero细胞流感疫苗的种子批。以Vero细胞高产株BX-51BVa(By)在Vero细胞上连续传代50次,测定其血凝效价和感染性滴度,并通过型别鉴定及基因序列测定考察传代过程中生物学性状的稳定性。结果显示BX-51BVa在Vero细胞连续传代50次,血凝效价维持在256～512,感染性滴度稳定在1×107TCID50/mL左右,型别未发生改变,HA和NA氨基酸序列与流行株BX-51B相比没有发生变化。本研究发现B型流感病毒重配株能够稳定地在Vero细胞连续传代,可以作为制备Vero细胞流感疫苗的种子批。
Cellular influenza vaccines(CIVs)are being developed because they can be produced rapidly and in large quantities to cope with a possible outbreak of influenza. Selecting high-yield adaptive strains of the influenza virus in Vero cells is very important for CIV development. We studied the passage stability of the high-yield strain BX-51 BVa(By)of an influenza virus in Vero cells to ascertain if this strain could be used as a seed batch for preparing an influenza vaccine in Vero cells. The Vero cell-adapted BX-51 BVa(By)was passaged continuously for 50 generations in Vero cells to determine its hemagglutination titer and infectivity titer. The stability of biological characteristics during passage was examined by type identification and gene sequencing. The hemagglutination titer was maintained at 256～512. The infectious titer was stable at about 1× 107 TCID50/mL,and the type did not change. The sequence of HA and NA amino acids did not change compared with the prevalent strain(BX-51 B). These data suggest that influenza B virus reassortant strains can be passaged continuously in Vero cells and used as a seed lot for the preparation of influenza vaccines in Vero cells.
Cellular influenza vaccines(CIVs)are being developed because they can be produced rapidly and in large quantities to cope with a possible outbreak of influenza. Selecting high-yield adaptive strains of the influenza virus in Vero cells is very important for CIV development. We studied the passage stability of the high-yield strain BX-51 BVa(By)of an influenza virus in Vero cells to ascertain if this strain could be used as a seed batch for preparing an influenza vaccine in Vero cells. The Vero cell-adapted BX-51 BVa(By)was passaged continuously for 50 generations in Vero cells to determine its hemagglutination titer and infectivity titer. The stability of biological characteristics during passage was examined by type identification and gene sequencing. The hemagglutination titer was maintained at 256～512. The infectious titer was stable at about 1× 107 TCID50/mL,and the type did not change. The sequence of HA and NA amino acids did not change compared with the prevalent strain(BX-51 B). These data suggest that influenza B virus reassortant strains can be passaged continuously in Vero cells and used as a seed lot for the preparation of influenza vaccines in Vero cells.
引文
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[9] Neri-Bazan R M,Garcia-Machorro J,Mendez-Luna D,Tolentino-Lopez L E,Martinez-Ramos F,Padilla-M I I,Aguilar-Faisal L,Soriano-Ursua M A,Trujillo-Ferr-ara J G,Fragoso-Vazquez M J,Barron B L,Correa-Ba-surto J. Design,in silico studies,synthesis and in vitro evaluation of oseltamivir derivatives as inhibitors of neur-aminidase from influenza A virus H1N1[J]. Eur J Med Chem,2017,128:154-167.
[10]Yang F,Ma L,Zhou J,Wu Y N,Gao J X,Song S H,Geng X L,Guo Q,Li Z F,Li W D,Liao G Y,Li Y.Development and identification of a new Vero cell-based live attenuated influenza B vaccine by a modified classi-cal reassortment method[J]. Expert Rev Vaccines,2017,16(8):855-863.
[11]Yu W,Yang F,Yang J,Ma L,Cun Y N,Song S H,Liao G Y. Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment[J]. J Med Virol,2016,88(11):1914-1921.
[2]邵铭,宋绍辉,徐康维,刘书珍,廖国阳,李长贵.重配技术构建高产H1N1型流感疫苗病毒株[J].微生物学免疫学进展,2012,40(4):21-24.
[3] Zhou J,Yang F,Yang J,Ma L,Cun Y N,Song S H,Liao G Y. Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains[J]. Hum Vaccin Immunother,2017,13(1):111-116.
[4] Barrett P N,Terpening S J,Snow D,Cobb R R,Kist-ner O. Vero cell technology for rapid development of in-activated whole virus vaccines for emerging viral diseases[J]. Expert Rev Vaccines,2017,16(9):883-894.
[5] Hu W,Zhang H,Han Q,Li L,Chen Y,Xia N,Chen Z,Shu Y,Xu K,Sun B. A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial pas-sages[J]. Vaccine,2015,33(2):374-381.
[6] Long Y F,Ma L,Liu Z,Song S H,Geng X L,Yang F,Guo Q,Li Z F,Li W D,Liao G Y. Preparation and evaluation of a novel,live,attenuated influenza H1N1vaccine strain produced by a modified classical reassort-ment method[J]. Hum Vaccin Immunother,2017,24:1-8.
[7]徐国峰,耿兴良,宋绍辉,马磊,廖国阳,李卫东. Benzo-nase酶去除流感疫苗中Vero细胞DNA条件的优化[J].中国生物制品学杂志,2017,30(5):540-545.
[8] Lauster D,Glanz M,Bardua M,Ludwig K,Hellmund M,Hoffmann U,Hamann A,Bottcher C,Haag R,Hackenberger C P R,Herrmann A. Multivalent peptide-nanoparticle conjugates for influenza-virus inhibition[J].Angew Chem Int Ed Engl,2017,56(21):5931-5936.
[9] Neri-Bazan R M,Garcia-Machorro J,Mendez-Luna D,Tolentino-Lopez L E,Martinez-Ramos F,Padilla-M I I,Aguilar-Faisal L,Soriano-Ursua M A,Trujillo-Ferr-ara J G,Fragoso-Vazquez M J,Barron B L,Correa-Ba-surto J. Design,in silico studies,synthesis and in vitro evaluation of oseltamivir derivatives as inhibitors of neur-aminidase from influenza A virus H1N1[J]. Eur J Med Chem,2017,128:154-167.
[10]Yang F,Ma L,Zhou J,Wu Y N,Gao J X,Song S H,Geng X L,Guo Q,Li Z F,Li W D,Liao G Y,Li Y.Development and identification of a new Vero cell-based live attenuated influenza B vaccine by a modified classi-cal reassortment method[J]. Expert Rev Vaccines,2017,16(8):855-863.
[11]Yu W,Yang F,Yang J,Ma L,Cun Y N,Song S H,Liao G Y. Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment[J]. J Med Virol,2016,88(11):1914-1921.