In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Preparation and characterization of immunostimulating complexes (ISCOMs) of Japanese encephalitis virus.

ISCOMs (immunostimulating complexes) were prepared from envelope glycoprotein (Egp) of Japanese encephalitis (JE) virus. ISCOMs showed a single band of the viral Egp in SDS-PAGE, which reacted with polyclonal and monoclonal antibody (MAb) raised against Egp. Comparison between the epitopes exposed on JE virion and JE ISCOMs, by antigen capture ELISA, utilizing a panel of domain-specific MAbs, revealed identical epitopes exposed on the Egp incorporated in ISCOMs and the whole virion. Electron micrographs of ISCOMs showed spherical cage-like structures of 35 nm. ISCOMs with Egp were good immunogenes, which stimulated high titres of neutralizing antibodies, both in mice and rabbits.