Protection of Mice Against a Lethal Influenza Challenge by Immunization With Yeast-Derived Recombinant Influenza Neuraminidase

Eur J Biochem. 1997 Jul 1;247(1):332-8. doi: 10.1111/j.1432-1033.1997.00332.x.

Abstract

The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues. N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment. Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Female
  • Immunization
  • Influenza Vaccines / immunology*
  • Mice
  • Mice, Inbred BALB C
  • Neuraminidase / biosynthesis
  • Neuraminidase / chemistry
  • Neuraminidase / immunology*
  • Orthomyxoviridae Infections / prevention & control*
  • Pichia / genetics
  • Vaccines, Synthetic / biosynthesis
  • Vaccines, Synthetic / immunology*

Substances

  • Influenza Vaccines
  • Vaccines, Synthetic
  • Neuraminidase