Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase.
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.
Martinet
W
W
Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Belgium.
Saelens
X
X
Deroo
T
T
Neirynck
S
S
Contreras
R
R
Min Jou
W
W
Fiers
W
W
D000818:Animals; D005260:Female; D007114:Immunization; D007252:Influenza Vaccines / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D009976:Orthomyxoviridae Infections / Q000517:prevention & control*; D010843:Pichia / Q000235:genetics; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000276:immunology*
D007252:Influenza Vaccines; D014614:Vaccines, Synthetic; D009439:Neuraminidase