Study On Genetic Reassortment Of Vero Cell-based Cold-adapted Influenza Virus Of H3N2 Subtype

Influenza virus is a globally important respiratory pathogen which causes a high degree of morbidity and mortality annually. Vaccination is still the most effective method of prophylaxis. There are three types of currently licensed influenza vaccines including inactivated, live attenuated, and recombinant HA vaccines. Split influenza virus vaccine and subunit vaccine are used in domestic. Live attenuated influenza vaccines(LAIVs) infect intranasally which is similar to natural infection, elicit mucosal and cellular immunity besides humoral immunity, express safe and effective immune effect. The formation of cold adapted LAIVs mainly depends on the segmented genome of influenza virus, which can be reassorted by genetic reassortment to acquire an reassortment strain inherited surface antigen gene from epidemic strain and phenotype from attenuated donor strain. America and Russia did most research in this field. In 2003, the FDA approved the commercialization of Flumist(?), the first LAIVs employs cold adapted donor strains A/Ann Arbor/6/60 (H2N2) and B/Ann Arbor/1/66 provided by Russia. For now, LAIVs were produced in embryonated chicken eggs. Egg-based vaccines have risks of egg contamination and spread of contagious disease of poultry. Meanwhile, the limited capacity of the egg-dependent vaccine supply could be problematic in terms of securing enough doses when facing a pandemic situation, especially in the event of a pandemic originating from a highly pathogenic avian virus. Given all this, World Health Organization(WHO) recommended to substitute continuous mammalian cell lines for embryonated chicken eggs to produce influenza vaccines. Vero cells are approved by WHO as a safe substrate for vaccine production for human use, but show insensitive to influenza virus and instable yield. At present, there is no Vero cell-based attenuated seasonal influenza vaccine and it is crucial for the research and development of attenuated LAIVs to breed a cold-adapted donor strain in Vero cells. Only one cold adapted donor strain in Vero cells, A/Singapore/1/57ca was reported. In our lab, one cold adapted virus strain A/Yunnan/1/2005Vca(H3N2) in Vero cells was developed by gradient cooling and cultured for 72 serial passages at 25℃ and its lgTCIDso/ml maintained 7.8-8.2. After launched several tests, cold-adaptation(ca), temperature sensitivity (ts) and attenuation (att) phenotype has been demonstrated. At the same time, this strain showed good immunogenicity which could induce cellular immunity and humoral immunity effectively in ferrets and mice models, provided protection against lethal challenge of subtype of the strain, indicates A/Yunnan/1/2005 Vca(H3N2) might be an ideal candidate donor strain for Vero cell cold-adapted LAIVs. At the present study, reassortment was proceeded with influenza virus strain A/Yunnan/1/2005Vca (H3N2) which was bred in our lab and epidemic strain for vaccine production A/Solomon Islands/3/2006(H1N1) recommended by WHO, sought to establish a rapid and stable genetic reassortment technology. Then we launched a research for the identification of biology phenotypic characteristic, safety and immunogenicity of the reassortment strain reA/SI/2006(H1N1) Vca, in order to prove the donor strain can be a vaccine strain for Vero cell-based LAIVs.By using these two virus co-infect three substrates including Vero cells, MDCK cells and chicken embryo and adding neutral antibody of the subtype of the donor strain and cultivating at 25℃, we finally acquired an influenza virus strain reA/SI/2006(HINl)Vca in Vero cells at 33℃ and in MDCK cells at 25℃ which could replicate in Vero cells at 25 ℃. By carrying out Hemagglutination Inhibition (HI) assay and single immunodiffusion to identify the subtype of the reassortment strain, it has been demonstrated that the coincidence of antigenicity between reassortment strain and epidemic strain.Inaddition sequencing analysis manifests the surface antigen gene HA and NA of the reassortment strain derived from epidemic strain while the six internal genes belong to donor strain A/Yunnan/1/2005Vca(H3N2). In optimized culture condition, inoculate reassortment strain reA/SI/2006(H1N1)Vca to Vero cells for 18 consecutive passages at 25℃, virus titre maintained 7.5 1gTCID50/mL above and phenotypes stayed stable.Experiment showed the tissue culture infective dose of reassortant reA/SI/2006(HIN1) at 25℃,33℃,39℃ were 6.9,7.8,3.5 1gTCID50/mL, which have proved to possess ca, ts phenotype. Mice were infected intranasally with 106 TCID50 influenza viruses, the viral load in turbinates and lung of mice were measured in 7 continuous days after immunization. In the first five days, the viral load of the reassortant strain reA/SI/2006(HIN1) showed at least 3 1gTCIDso/g lower than epidemic strain in both upper and lower respiratory tract, indicates the attenuated virus phenotype. Later, mice were immunized second time with the same dose, leaded effective stimulation in mice and yielded hemagglutination inhibition antibody and neutral antibody against the epidemic strain A/SI/2006(HIN1) and also promoted the secretion of mucosal sIgA. After the inoculation of reA/SI/2006(HINl)Vca, the virus provided complete protection against hypotypic (A/JN/2009H1N1) challenge with 50MLD50. Meanwhile, the replication of the attacking virus in vivo was complete inhibited. This study proved that reA/SI/2006(H1N1)Vca is a reassortant virus obtained by genetic reassortment, inherited surface antigen gene of epidemic strain, and can be inoculated in Vero cells with attenuated phenotypic characteristics. Therefore, A/Yunnan/1/2005Vca(H3N2) can be used as a donor strain for Vero cell cold-adapted live attenuated influenza virus vaccine.