The Experimental Study Of An Influenza H5N1 Pandemic Vaccine Made In Vero Cells

Influenza viruses are pathogens that cause influenza, which is highly infectious and highly epidemic, even to a worldwide pandemic, each pandemic had caused enormous losses of human and material resources. At present H5N1 avian influenza vaccine is mainly developed and produced by using embryos eggs. However, when the H5N1 avian flu outbreaks in the short time, it is difficult to provide sufficient embryos to produce vaccines. In addition, the embryonated chicken eggs may be with a foreign-derived factor, which also affected the quality of vaccines. Vero cell is WHO resigned cell strain to produce vaccine and can be cultured at large scale by the bioreactor. So it is important to obtain high-growth influenza H5N1 virus in Vero cells for the influenza vaccine production with Vero cells.In this experiment, we prepared a Vero cell-adapted influenza H5N1 virus strain by Genetic Reassortment and produced influenza H5N1 vaccine using Vero cell as a substrate. Embryonated specific pathogen-free (SPF) hen's eggs and Vero cells were co-infected with Vero cell-adapted influenza virus A/Yunnan/1/2005 (H3N2)va and A/Anhui/1/2005(H5N1) via reverse genetics. The reassortant was screened with goat antibody against strain A/Yunnan/1/2005(H3N2)va and identified for subtype by hemagglutination-inhibition (HI) assays and gene analysis of HA and NA. Results A Vero cell-adapted influenza H5N1 virus strain was obtained. The Vero cell-adapted influenza virus of epidemic strain may be reassortmented between Vero cell-adapted and epidemic strains.At the same time, other relevant biological experiments of the reassortant has been done. The infection titer was 1×10 PFU/ml in plaque experiment by using MDCK cells. The reaction of immunofluorescence experiment is positive. Complete virus particles would be observed by Electron microscope. In signal immunodifusion test, there were obvious settling circles in agarose gel plate of standard H5N1 anti-serum. And the reassortant had no toxicity both for embryonated chicken eggs and mice.The reassortant had been cultured using miacro-carrior method and the culture conditions optimized. As DMEM/F12 was basic media, we had a relatively high yields of virus by miacro-carrior culture with serum. The reassorted monovalent vaccine was made by inactivating virus with formaldehyde and had a good immunogenicity. And there was no significant difference in serum antibody titers of monovalent inactivated vaccines before and after reassortment (F=0.857, P> 0.05). The reassortant also has a higher yield via microcarrier culture.We could get the better anti-serum by immuning female goat, and selected suitable purification method of the anti-serum.