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Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice

Yang, Chen, Skiena, Steven, Futcher, Bruce, Mueller, Steffen, Wimmer, Eckard
Proceedings of the National Academy of Sciences of the United States of America 2013 v.110 no.23 pp. 9481-9486
Orthomyxoviridae, antigens, dogs, gene expression regulation, genes, glycoproteins, hemagglutinins, immune system, immunity, kidney cells, live vaccines, mice, pathogenesis, phenotype, sialidase, tissue culture, vaccine development, viruses
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 LD ₅₀ 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 (PD ₅₀) of 2.4 PFU. Moreover, at a PD ₅₀ 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.