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Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells

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Summary

A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutinin (HA0s) was cloned into the polyhedrin promoter-based pVL1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 µg/ml of secreted HA were obtained following infection ofSpodoptera frugiperda-9 cells. Gel filtration revealed the presence in the cell supernatant of immunoreactive HA molecules with varyingM r. The highM r fraction (aHA0s) could be purified by Matrex Cellufine Sulphate and Lentil-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subunits which were not cleaved into HA1 and HA2 polypeptides; aHA0s was highly susceptible to trypsin treatment and reacted with two low pH-specific monoclonal antibodies, suggesting that aHA0s consists of monomeric subunits. When the expression medium was adjusted to pH 8.5, no aHA0s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s developed high, aHA0s-specific antibody titres. Despite these high titres, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-adapted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observed. Moreover, binding to the low pH-treated HA of different drift variants, isolated between 1968 and 1989, occurred.

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Authors and Affiliations

  1. Laboratory of Molecular Biology and Flemish Institute for Biotechnology, University of Ghent, Ghent, Belgium

    P. Vanlandschoot, E. Beirnaert, S. Neirynck, X. Saelens, W. Min Jou & W. Fiers

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  1. P. Vanlandschoot
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  2. E. Beirnaert
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  3. S. Neirynck
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  4. X. Saelens
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  5. W. Min Jou
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  6. W. Fiers
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Vanlandschoot, P., Beirnaert, E., Neirynck, S. et al. Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells. Archives of Virology 141, 1715–1726 (1996). https://doi.org/10.1007/BF01718294

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  • DOI: https://doi.org/10.1007/BF01718294

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