title: Evaluation of the attenuation, immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus, in ferrets.
abstract: Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA+NA)(Min)), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA+NA)(Min) virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10(6)TCID50/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA+NA)(Min) virus at doses ranging from 10(3) to 10(5) TCID50 led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA+NA)(Min) virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Broadbent
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Celia P
authors.initials: CP
authors.lastname: Santos
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Amanda
authors.initials: A
authors.lastname: Anafu
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, United States.
authors.forename: Eckard
authors.initials: E
authors.lastname: Wimmer
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, United States.
authors.forename: Steffen
authors.initials: S
authors.lastname: Mueller
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, United States. Electronic address: ksubbarao@niaid.nih.gov.
authors.forename: Kanta
authors.initials: K
authors.lastname: Subbarao
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D003062:Codon; D004285:Dogs; D005289:Ferrets; D005818:Genetic Engineering; D006385:Hemagglutination Inhibition Tests; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000502:physiology; D007252:Influenza Vaccines / Q000276:immunology*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D009976:Orthomyxoviridae Infections / Q000517:prevention & control; D016865:Reassortant Viruses / Q000235:genetics / Q000502:physiology; D014613:Vaccines, Attenuated / Q000276:immunology; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D003062:Codon; D007252:Influenza Vaccines; D014613:Vaccines, Attenuated
keywords: Codon pair bias de-optimization; Influenza; Vaccine