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Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses
Porntippa Lekcharoensuka, Corresponding author contact information, E-mail the corresponding author,
Witthawat Wiriyaratb,
Nuntawan Petcharata,
Chalermpol Lekcharoensuka,
Prasert Auewarakulc,
Juergen A. Richtd
a Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
b Faculty of Veterinary Medicine, Mahidol University, Nakornpathom 73140, Thailand
c Faculty of Medicine, Mahidol University, Siriraj Hospital, Bangkok 10700, Thailand
d Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, KS 66506-5601, USA
Received 12 September 2011. Revised 8 December 2011. Accepted 22 December 2011. Available online 8 January 2012.
http://dx.doi.org/10.1016/j.vaccine.2011.12.109, How to Cite or Link Using DOI
Abstract
Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin?Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 29 HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.
Highlights
► The novel 8 plasmid reverse genetics of influenza A virus, rg1930, was generated. ► Each gene contains the introduced genetic marker for easy monitoring. ► The 6 internal genes (backbone) of rg1930 accommodated various HA and NA subtypes. ► The rg1930 derived viruses grew better than those with widely used backbone (rgPR8). ► Efficacy and safety of rg1930- and rgPR8-based vaccines are similar.
http://www.sciencedirect.com/science/article/pii/S0264410X11020706
Porntippa Lekcharoensuka, Corresponding author contact information, E-mail the corresponding author,
Witthawat Wiriyaratb,
Nuntawan Petcharata,
Chalermpol Lekcharoensuka,
Prasert Auewarakulc,
Juergen A. Richtd
a Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
b Faculty of Veterinary Medicine, Mahidol University, Nakornpathom 73140, Thailand
c Faculty of Medicine, Mahidol University, Siriraj Hospital, Bangkok 10700, Thailand
d Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, KS 66506-5601, USA
Received 12 September 2011. Revised 8 December 2011. Accepted 22 December 2011. Available online 8 January 2012.
http://dx.doi.org/10.1016/j.vaccine.2011.12.109, How to Cite or Link Using DOI
Abstract
Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin?Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 29 HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.
Highlights
► The novel 8 plasmid reverse genetics of influenza A virus, rg1930, was generated. ► Each gene contains the introduced genetic marker for easy monitoring. ► The 6 internal genes (backbone) of rg1930 accommodated various HA and NA subtypes. ► The rg1930 derived viruses grew better than those with widely used backbone (rgPR8). ► Efficacy and safety of rg1930- and rgPR8-based vaccines are similar.
http://www.sciencedirect.com/science/article/pii/S0264410X11020706