Preparation of genetically engineered A/H5N1 and A/H7N1 pandemic vaccine viruses by reverse genetics in a mixture of Vero and chicken embryo cells

BACKGROUND In case of influenza pandemic, a robust, easy and clean technique to prepare reassortants would be necessary. OBJECTIVES Using reverse genetics, we prepared two vaccine reassortants (A/H5N1 x PR8 and A/H7N1 x PR8) exhibiting the envelope glycoproteins from non-pathogenic avian viruses, A/Turkey/Wisconsin/68 (A/H5N9) and A/Rhea/New Caledonia/39482/93 (A/H7N1) and the internal proteins of the attenuated human virus A/Puerto Rico/8/34 (H1N1). METHODS The transfection was accomplished using a mixture of Vero and chicken embryo cells both of which are currently being used for vaccine manufacturing. RESULTS This process was reproducible, resulting in consistent recovery of influenza viruses in 6 days. Because it is mainly the A/H5N1 strain that has recently crossed the human barrier, it is the A/PR8 x A/H5N1 reassortant (RG5) that was further amplified, either in embryonated hen eggs or Vero cells, to produce vaccine pre-master seed stocks that met quality control specifications. Safety testing in chickens and ferrets was performed to assess the non-virulence of the reassortant, and finally analysis using chicken and ferret sera immunized with the RG5 virus showed that the vaccine candidate elicited an antibody response cross-reactive with the Hong Kong 1997 and 2003 H5N1 strains but not the Vietnam/2004 viruses. CONCLUSIONS The seeds obtained could be used as part of a pandemic vaccine strain 'library' available in case of propagation in humans of a new highly pathogenic avian strain.