General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombin ant immunogen as fused to a composite tag comprising one domain suitable fo r affinity purification and a hydrophobic tag designed for direct incorpora tion through hydrophobic interaction of the affinity-purified immunogen int o an adjuvant system, in this case immunostimulating complexes (iscoms). Th ree different hydrophobic tags were evaluated: (i) a tag denoted IW contain ing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (i i) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-depen dent in such a way that an amphiphatic alpha-helix would be formed at low p H. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf1 55/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating i scom incorporation. This was further supported by electron microscopy analy sis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient MS-specific antibody responses upon i mmunization of mice, confirming successful incorporation into iscoms. The i mplications of these results for the design and production of subunit vacci nes are discussed. (C) 1999 Elsevier Science B.V. All rights reserved.