Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin

The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered f or expression in Pichia pastoris as a soluble secreted molecule. The HA cDN A lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae cl-mating factor secretion signal and placed u nder control of the methanol-inducible P. postoris alcohol oxidase I (AOX1) promoter. Growth of transformants an methanal-containing medium resulted i n the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Re markably, the pH of the induction medium had an important effect on the exp ression level, the highest level being obtained at pH 8.0, The gel filtrati on profile and the reactivity against a panel of different HA-confirmation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P, pastoris type of glycosylatio n, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete p rotection against a challenge with 10 LD50 of mouse-adapted homologous viru s (X47), whereas all control mice succumbed. Heterologous challenge with X3 1 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher sur vival rates in the immunized group compared with the control group, These r esults, 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.