Giuseppe
Emeritus
[Source: Proceedings of the National Academy of Sciences of the United States of America, full text: (LINK). Abstract, edited.]
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Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice
Chen Yang<SUP>a</SUP>, Steven Skiena<SUP>b</SUP>, Bruce Futcher<SUP>a</SUP>, Steffen Mueller<SUP>a</SUP>,<SUP>1</SUP>, and Eckard Wimmer<SUP>a</SUP>,<SUP>1</SUP>
<SUP></SUP>
Author Affiliations: Departments of <SUP>a</SUP>Molecular Genetics and Microbiology and <SUP>b</SUP>Computer Science, Stony Brook University, Stony Brook, NY 11794
Contributed by Eckard Wimmer, April 24, 2013 (sent for review February 22, 2013)
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<SUP>Min</SUP>, PR8-HA<SUP>Min</SUP>, and PR8-(NA+HA)<SUP>Min</SUP> (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<SUB>50</SUB> values: PR8, 32 plaque-forming units (PFU); HA<SUP>Min</SUP>, 1.7 ? 10<SUP>3</SUP> PFU; NA<SUP>Min</SUP>, 2.4 ? 10<SUP>5</SUP> PFU; (NA+HA)<SUP>Min</SUP>, ≥3.16 ? 10<SUP>6</SUP> PFU. Remarkably, (NA+HA)<SUP>Min</SUP> was attenuated >100,000-fold, with NA<SUP>Min</SUP> the major contributor to attenuation. In vaccinated mice (NA+HA)<SUP>Min</SUP> was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD<SUB>50</SUB>) of 2.4 PFU. Moreover, at a PD<SUB>50</SUB> of only 147 or 237, (NA+HA)<SUP>Min</SUP> 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.
chemical synthesis - computer-aided design - margin of safety - Protective dose 50 - hetero-subtypic immunity
Footnotes
<SUP>1</SUP>To whom correspondence may be addressed. E-mail: steffen.mueller@stonybrook.edu or Eckard.Wimmer@stonybrook.edu.
Author contributions: C.Y., S.S., B.F., S.M., and E.W. designed research; C.Y. and S.M. performed research; C.Y., S.S., B.F., S.M., and E.W. analyzed data; and C.Y., S.M., and E.W. wrote the paper.
Conflict of interest statement: The authors declare a conflict of interest. Stony Brook University has a patent pending covering the strategy described in this paper; S.S., B.F., S.M., and E.W. are coinventors named in the patent.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1307473110/-/DCSupplemental.
Freely available online through the PNAS open access option.
-Chen Yang<SUP>a</SUP>, Steven Skiena<SUP>b</SUP>, Bruce Futcher<SUP>a</SUP>, Steffen Mueller<SUP>a</SUP>,<SUP>1</SUP>, and Eckard Wimmer<SUP>a</SUP>,<SUP>1</SUP>
<SUP></SUP>
Author Affiliations: Departments of <SUP>a</SUP>Molecular Genetics and Microbiology and <SUP>b</SUP>Computer Science, Stony Brook University, Stony Brook, NY 11794
Contributed by Eckard Wimmer, April 24, 2013 (sent for review February 22, 2013)
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<SUP>Min</SUP>, PR8-HA<SUP>Min</SUP>, and PR8-(NA+HA)<SUP>Min</SUP> (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<SUB>50</SUB> values: PR8, 32 plaque-forming units (PFU); HA<SUP>Min</SUP>, 1.7 ? 10<SUP>3</SUP> PFU; NA<SUP>Min</SUP>, 2.4 ? 10<SUP>5</SUP> PFU; (NA+HA)<SUP>Min</SUP>, ≥3.16 ? 10<SUP>6</SUP> PFU. Remarkably, (NA+HA)<SUP>Min</SUP> was attenuated >100,000-fold, with NA<SUP>Min</SUP> the major contributor to attenuation. In vaccinated mice (NA+HA)<SUP>Min</SUP> was highly effective in providing long-lasting protective immunity against lethal WT challenge at a median protective dose (PD<SUB>50</SUB>) of 2.4 PFU. Moreover, at a PD<SUB>50</SUB> of only 147 or 237, (NA+HA)<SUP>Min</SUP> 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.
chemical synthesis - computer-aided design - margin of safety - Protective dose 50 - hetero-subtypic immunity
Footnotes
<SUP>1</SUP>To whom correspondence may be addressed. E-mail: steffen.mueller@stonybrook.edu or Eckard.Wimmer@stonybrook.edu.
Author contributions: C.Y., S.S., B.F., S.M., and E.W. designed research; C.Y. and S.M. performed research; C.Y., S.S., B.F., S.M., and E.W. analyzed data; and C.Y., S.M., and E.W. wrote the paper.
Conflict of interest statement: The authors declare a conflict of interest. Stony Brook University has a patent pending covering the strategy described in this paper; S.S., B.F., S.M., and E.W. are coinventors named in the patent.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1307473110/-/DCSupplemental.
Freely available online through the PNAS open access option.
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