title: Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure.
abstract: Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892; lenouenc@niaid.nih.gov Eckard.Wimmer@stonybrook.edu.
authors.forename: Cyril
authors.initials: C
authors.lastname: Le Nouën
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Thomas
authors.initials: T
authors.lastname: McCarty
authors.affiliation: Pacific Biosciences Inc., Menlo Park, CA 94025.
authors.forename: Michael
authors.initials: M
authors.lastname: Brown
authors.affiliation: Pacific Biosciences Inc., Menlo Park, CA 94025.
authors.forename: Melissa Laird
authors.initials: ML
authors.lastname: Smith
authors.affiliation: Pacific Biosciences Inc., Menlo Park, CA 94025.
authors.forename: Roberto
authors.initials: R
authors.lastname: Lleras
authors.affiliation: Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Dolan
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Masfique
authors.initials: M
authors.lastname: Mehedi
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Yang
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Cindy
authors.initials: C
authors.lastname: Luongo
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Bo
authors.initials: B
authors.lastname: Liang
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Shirin
authors.initials: S
authors.lastname: Munir
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: DiNapoli
authors.affiliation: Codagenix Inc., Stony Brook, NY 11790.
authors.forename: Steffen
authors.initials: S
authors.lastname: Mueller
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794 lenouenc@niaid.nih.gov Eckard.Wimmer@stonybrook.edu.
authors.forename: Eckard
authors.initials: E
authors.lastname: Wimmer
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
authors.affiliation: RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892.
authors.forename: Ursula J
authors.initials: UJ
authors.lastname: Buchholz
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops / Q000235:genetics; D003062:Codon / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D012328:RNA Viruses / Q000235:genetics*; D018113:Respiratory Syncytial Virus, Human; D014613:Vaccines, Attenuated / Q000235:genetics; D014614:Vaccines, Synthetic / Q000235:genetics; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
chemical_list: D003062:Codon; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D014765:Viral Vaccines
keywords: codon pair deoptimization; genetic stability; live attenuated vaccine; negative-strand RNA virus; respiratory syncytial virus