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

Abstract

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 × PR8 and A/H7N1 × 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 × 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.

Generation of candidate human influenza vaccine strains in cell culture – rehearsing the European response to an H7N1 pandemic threat

“…RNA segments for the six internal genes of PR8 virus obtained from NIBSC and used annually to generate standard vaccine reassortants were cloned into plasmids suitable for virus recovery by reverse genetics. The modified H7 HA and the N1 NA were rescued into the background of NIBSC PR8 using a reverse genetics method involving the transfection of WHO‐approved Vero cells and co‐culture in SPF chick embryo cells (CECs) 23 . The resulting recombinant virus was termed RD3.…”

“…Although an H7 subtype reverse genetics vaccine for use in chickens has previously been reported, 22 and a recombinant low pathogenicity H7 N7 virus vaccine was generated and tested in mice, 42 the H7 N1 vaccine virus produced in our cell culture system is suitable for use in the human population. Recently, another H7 N1 vaccine virus has been described that was produced by reverse genetics in substrates suitable for human use 23 . However, safety testing and further characterization beyond initial recovery of this virus was not described.…”

“…Therefore, it is prudent to generate vaccine seeds for both divergent lineages of H7 avian influenza viruses as either may pose a pandemic threat. The recent publication from 23 describes a vaccine virus bearing a North American lineage H7 antigen, A/Rhea/NC/39482/93. However, data from 43 suggest that antisera against H7 viruses of the Eurasian lineages may confer some protection against North American H7 viruses, although the relationship was not reciprocal.…”

“…Since the first outbreak of H5N1 influenza in Hong Kong in 1997 2,3 several candidate pandemic vaccine H5 viruses have been developed by reverse genetics, [15][16][17][18][19][20][21][22][23] although the difficulties of translating this research into a product that can be tested in humans should not be underestimated. 24 In each case, the multi-basic cleavage site has been removed from the hemagglutinin (HA) gene of the candidate vaccine strain to attenuate the virus.…”

Mammalian Models for the Study of H7 Virus Pathogenesis and Transmission

“…In recent years, there has been renewed interest in producing egg-free inactivated influenza vaccines, especially for viruses with pandemic potential. H7N1 vaccines have been generated in cell culture (Cox et al 2009), and those that have undergone further evaluation have been found to elicit protective antibody responses against homologous and heterologous viruses in both mice and ferrets (Legastelois et al 2007; Whiteley et al 2007). Non-infectious recombinant virus-like particles (VLPs) have been generated with both Eurasian and North American lineage H7 HAs (either alone or co-expressed with other subtypes) and have been evaluated for their ability to induce serum antibody responses and confer protection against viral challenge in both mice and ferrets (Smith et al 2013; Szecsi et al 2006; Tretyakova et al 2013).…”