Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin
Article first published online: 25 DEC 2001
European Journal of Biochemistry
Volume 260, Issue 1, pages 166–175, February (II) 1999
How to Cite
Saelens, X., Vanlandschoot, P., Martinet, W., Maras, M., Neirynck, S., Contreras, R., Fiers, W. and Jou, W. M. (1999), Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin. European Journal of Biochemistry, 260: 166–175. doi: 10.1046/j.1432-1327.1999.00150.x
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- (Received 17 August 1998, accepted 24 November 1998)
- influenza hemagglutinin;
- recombinant vaccine;
- Pichia pastoris;
- protective immunity
The A/Victoria/3/75 (H3N2-subtype ) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiaeα-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10–12 glycosyl residues.
Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.