title: Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice.
abstract: A long-held dogma posits that strong presentation to the immune system of the dominant influenza virus glycoprotein antigens neuraminidase (NA) and hemagglutinin (HA) is paramount for inducing protective immunity against influenza virus infection. We have deliberately violated this dogma by constructing a recombinant influenza virus strain of A/PR8/34 (H1N1) in which expression of NA and HA genes was suppressed. We down-regulated NA and HA expression by recoding the respective genes with suboptimal codon pair bias, thereby introducing hundreds of nucleotide changes while preserving their codon use and protein sequence. The variants PR8-NA(Min), PR8-HA(Min), and PR8-(NA+HA)(Min) (Min, minimal expression) were used to assess the contribution of reduced glycoprotein expression to growth in tissue culture and pathogenesis in BALB/c mice. All three variants proliferated in Madin-Darby canine kidney cells to nearly the degree as WT PR8. In mice, however, they expressed explicit attenuation phenotypes, as revealed by their LD50 values: PR8, 32 plaque-forming units (PFU); HA(Min), 1.7 × 10(3) PFU; NA(Min), 2.4 × 10(5) PFU; (NA+HA)(Min), ≥3.16 × 10(6) PFU. Remarkably, (NA+HA)(Min) was attenuated >100,000-fold, with NA(Min) the major contributor to attenuation. In vaccinated mice (NA+HA)(Min) was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD50) of 2.4 PFU. Moreover, at a PD50 of only 147 or 237, (NA+HA)(Min) conferred protection against heterologous lethal challenges with two mouse-adapted H3N2 viruses. We conclude that the suppression of HA and NA is a unique strategy in live vaccine development.
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Yang
authors.forename: Steven
authors.initials: S
authors.lastname: Skiena
authors.forename: Bruce
authors.initials: B
authors.lastname: Futcher
authors.forename: Steffen
authors.initials: S
authors.lastname: Mueller
authors.forename: Eckard
authors.initials: E
authors.lastname: Wimmer
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D056738:Cross Protection; D004285:Dogs; D015536:Down-Regulation / Q000235:genetics*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D053118:Influenza A Virus, H1N1 Subtype; D007928:Lethal Dose 50; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000378:metabolism*; D009976:Orthomyxoviridae Infections / Q000276:immunology / Q000517:prevention & control*; D014765:Viral Vaccines / Q000378:metabolism*
chemical_list: C543345:H1N1 virus hemagglutinin; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014765:Viral Vaccines; D009439:Neuraminidase
keywords: Protective dose 50; chemical synthesis; computer-aided design; hetero-subtypic immunity; margin of safety