MOLECULAR AND IMMUNOLOGICAL CHARACTERIZATION OF SOLUBLE AGGREGATED A VICTORIA/3/75 (H3N2) INFLUENZA HEMAGGLUTININ EXPRESSED IN INSECT CELLS/

A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutin in (HAOs) was cloned into the polyhedrin promoter-based pVL1392 transf er vector, and a recombinant baculovirus was isolated. 5 to 10 mu g/ml of secreted HA were obtained following infection of Spodoptera frugip erda-9 cells. Gel filtration revealed the presence in the cell superna tant of immunoreactive HA molecules with varying M(r). The high M(r) f raction (aHA0s) could be purified by Matrex Cellufine Sulphate and Len til-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subun its which were not cleaved into HA1 and HA2 polypeptides; aHA0s was hi ghly susceptible to trypsin treatment and reacted with two low pH-spec ific monoclonal antibodies, suggesting that aHA0s consists of monomeri c subunits. When the expression medium was adjusted to pH 8.5, no aHA0 s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s d eveloped high, aHA0s-specific antibody titres. Despite these high titr es, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-ada pted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observ ed. Moreover, binding to the low pH-treated HA of different drift vari ants, isolated between 1968 and 1989, occurred.