中文摘要

流行性感冒病毒，简称流感病毒，属正粘病毒科（Orthomyxoviridae），其为有包膜的单负链RNA病毒，其基因组分为8个节段。根据流感病毒核蛋白（NP）和基质蛋白（M）抗原性的差异可划分为甲（A）、乙（B）和丙（C）三型。乙型流行性感冒病毒虽然没有像甲型流感病毒一样引起过人群内的大流行，但是能通过抗原漂移，产生新的病毒，每年能引起世界范围内的季节性流行，严重威胁人类的健康和生命安全。因此，季节性流感疫苗由A（H1N1）、A（H3N2）和乙型病毒共同组成生产毒株。

目前已上市的季节性流感疫苗主要有三类：裂解疫苗、亚单位疫苗和减毒活疫苗。流感减毒活疫苗采取滴鼻方式进行接种，模拟病毒自然感染的途径在呼吸道粘膜诱导产生免疫应答，免疫保护性好。目前上市的流感减毒活疫苗，都以鸡胚作为基质生产。使用鸡胚生产活疫苗，由于缺乏病毒灭活的手段，存在传播鸡源病原体的风险。此外，鸡胚较长的供应周期制约着疫苗生产，尤其是突然爆发高致病性流感时，疫苗生产难于满足应对流感大流行的需要。Vero细胞是WHO推荐用于生产人用生物制品的细胞基质，可采用发酵罐大规模培养病毒。相对于鸡胚培养，细胞培养病毒抗原变异小，更接近原始分离病毒。但Vero细胞不是培养流感病毒最敏感的基质，不同毒株间产毒量差异较大，甚或不能生长复制。目前全球尚无以Vero细胞为基质生产的季节性流感病毒减毒活疫苗面市。我们经过10多年的研究，在国际上率先成功选育出一株乙型流感病毒Vero细胞冷适应株B/Yunnan/23/2011 Vca（BVca）。

本研究以BVca作为母本毒株，与WHO推荐的乙型流感病毒流行株B/Brisbane/60/2008 NYMC BX-35（BX-35）遗传重配，以解决Vero细胞乙型流感病毒减毒疫苗生产毒株制备的瓶颈技术。试验研究中，使用Vero细胞冷适应株BVca与流行株BX-35共感染Vero细胞，在重配病毒中加入抗BVca血清中和带有母本株表面抗原HA和NA的子代病毒，经蚀斑纯化获得带有BX-35的表面抗原的乙型流感病毒Vero细胞冷适应株BX-35Vca。该病毒在Vero细胞上25°C培养，感染性滴度不低于1×10⁷ TCID₅₀/ml，具有温度敏感性（ts）、冷适应性（ca）和减毒（att）表型。在Vero细胞连续传代20代，HA、NA氨基酸没有发生变异。制备的减毒活疫苗滴鼻免疫小鼠能够抵抗致死剂量的同系乙型流感病毒的感染。本研究首次证实BVca可作为母本毒株，采用遗传重配技术制备Vero细胞乙型流感病毒减毒疫苗生产用毒株。

Abstract

Influenza virus is a virus that can cause acute respiratory disease, highly contagious, mainly through air droplets, which can occur suddenly in a short term, spread rapidly across the globe each year and cause seasonal epidemics, from time to time there will be a pandemic, although currently small number of antiviral drugs have a therapeutic effect,  still one of the infectious diseases of human influenza is not yet effectively controled. Vaccination is the prevention and control of influenza epidemics of the most economical and effective way. Although Influenza B virus currently not causeing a pandemic, by antigenic drift variant forms have different epidemic strains could cause seasonal epidemics worldwide every year, a serious threat to human health, cause significant economic losses. So it is an important component of the seasonal flu vaccine. The current seasonal flu vaccine are mainly three types: inactivated influenza vaccine (IIV), live attenuated influenza vaccines (LAIV) and recombinant subunit vaccines. LAIV take intranasal inoculation, viral infection analog form to induce the body to produce an immune response, compared with IIV, LAIVs have broader immunogenic response, can induce mucosal secretory IgA and IgG antibodies in serum, they can also irritate cellular immune responses to generate cross-protection among different subtypes of influenza.

Currently, most commercial vaccines against influenza viruses, are mainly produced in chicken embryos. While this method has been used since 1940s, there are several well-recognized limitations of this system in influenza vaccine production. Firstly, the global capacity of eggs for vaccine production is sufficient to protect only a small percentage of the worldwide population. Secondly, the egg adaptation selects variants which are antigenically distinct from viruses grown from the same source in mammalian cells, which could let egg-derived vaccine less effective against circulating strains. Thirdly, the current cycle of the seasonal influenza vaccine production requires detailed planning up to 6 months before vaccine manufacture to ensure an adequate supply of embryonated eggs, thus, the vaccine manufacturers are challenged by a tight production schedule and have very limited time to optimize the production conditions to improve process yields. Vero cell (African green kidney cells)  line is highly recommended by WHO for manufacture of biological products which can be used fermenter to achieve large-scale production. However, Vero cells are not sensitive influenza virus, only few influenza virus strains yield in Vero cells are optimistic. There is no licensed Vero cell based-LAIV yet.

Getting a influenza strain which has temperature sensitivity (ts), cold-adapted (ca) feature, attenuation (att) phenotype and  high-yielding in Vero cell is crucial for manufacturing Vero cell based LAIV. Only one cold adapted donor strain in Vero cells, A/Singapore/1/57ca was reported. Previous studies In our lab, an cold adapted virus strain B/Yunnan/23/2011Vca (BVca) in Vero cells was developed by serial passagesing at 25°C in Vero cell. In this study, we generated an influenza B reassortant viruses that carried HA and NA genes of Victoria lineage B/Brisbane/60/2008 NYMC BX-35 (BX-35) which was WHO recommended vaccine strains during 2007-2016 In the northern hemisphere. Then we identified the phenotype of the reassortment strain BX-35Vca. Safety and immunogenicity were assessed simultaneously.

After these two virus co-infect Vero cells, antiserum was added 3 times to neutralize MDV. By plaque purification we got a reassortment strain BX-35Vca, lgTCID₅₀/ml in Vero cells at 25°C is not less 7. Hemagglutination Inhibition (HI) assay and single immunodiffusion identified the lineage of BX-35Vca was same as BX-35. The lgTCID₅₀ of BX-35Vca at 25, 33, 39°C was 6.33±0.47、7.2±0.26、3.92±0.5 respectively which  proved ca, ts phenotype of reassortment virus. Proliferation of BX-35Vca in the respiratory tract in mice and ferret was significantly inhibited, upper respiratory tract viral load was 100 times higher than lower respiratory tract in both animal models, showed att phenotype. Gene Sequencing and blasting show the six internal genen of BX-35Vca are from BVca, HA and NA gene are from BX-35, no mutatuin were found in HA and NA gene during passaging of BX-35Vca. After Single immunization of 10⁶ TCID₅₀ BX-35Vca, mice HI titer could reach the level of > 1:40, booster immunization could exciting higher HI titers and promoting the secretion of mucosal sIgA. Immunization with BX-35Vca provided complete protection against Victoria lineage BX-35 and B/Malaysia/2506/2004 challenge with inoculum of 10 MLD_50. Meanwhile, the replication of the attacking virus in vivo was complete inhibited. In summary, this study proved BVca could be used as a donor strain for Vero cells based live attenuated influenza virus vaccine .