The Study On Genetic Reassortment Of Vero Cell-based Cold-adapted Influenza B Virus

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/l/57ca was reported. Previous studies In our lab, an cold adapted virus strain B/Yunnan/23/2011 Vca (BVca) in Vero cells was developed by serial passagesing at 25℃ 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-35 Vca, IgTCID50/ml in Vero cells at 25℃ 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℃ 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-35 Vca 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-35 Vca are from BVca, HA and NA gene are from BX-35, no mutatuin were found in HA and NA gene during passaging of BX-35 Vca. After Single immunization of 106 TCED50 BX-35 Vca, 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 MLD50. 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.