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.


OBJECTIVE
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.
Legastelois
Isabelle
I
Research and Development, sanofi pasteur, 1541 Avenue Marcel Mérieux, Marcy L'Etoile, France. isabelle.legastelois@sanofipasteur.com
Garcia-Sastre
Adolfo
A
Palese
Peter
P
Tumpey
Terrence M
TM
Maines
Taronna R
TR
Katz
Jacqueline M
JM
Vogel
Frederick R
FR
Moste
Catherine
C
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D003429:Cross Reactions; D004196:Disease Outbreaks / Q000517:prevention & control; D005289:Ferrets; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology; D009980:Influenza A virus / Q000235:genetics* / Q000276:immunology*; D007252:Influenza Vaccines / Q000009:adverse effects / Q000235:genetics* / Q000276:immunology*; D007251:Influenza, Human / Q000517:prevention & control*; D016865:Reassortant Viruses / Q000235:genetics* / Q000276:immunology*; D014709:Vero Cells
D000914:Antibodies, Viral; D007252:Influenza Vaccines