Characterization of an internal permissive site in the cholera toxin B-subunit and insertion of epitopes from human immunodeficiency virus-1, hepatitis B virus and enterotoxigenic Escherichia coli.

We previously described the construction of novel hybrid proteins based on the B-subunit of cholera toxin (CTB) [Bäckström et al., Gene 149 (1994) 211-217], in which a neutralizing B-cell epitope from the third variable (V3) loop in the envelope glycoprotein gp120 from human immunodeficiency virus type 1 (HIV-1) was inserted within a surface-exposed region between amino acids (aa) 55 and 64. The resulting protein retained properties of native CTB and could induce strong anti-CTB antibody (Ab) responses, but the inserted gp120 epitope was only modestly immunogenic. In this study, the potential use of this internal permissive site in CTB for the insertion of heterologous epitopes has been further investigated. Six additional plasmids were constructed encoding HIV::CTB hybrid proteins with ten to fourteen aa from the V3 loop of gp120 genetically inserted at different positions between aa 52 and 65, with deletions of different CTB aa. Plasmids encoding proteins with peptides inserted between aa 53 and 64 in CTB gave rise to stable proteins which reacted with CTB-specific monoclonal antibodies (mAb) and bound to GM1 gangliosides (GM1), indicating that insertions between these positions do not drastically alter the conformation or the receptor-binding properties of native CTB. Plasmids were also constructed encoding CTB hybrid proteins which had either an 11-aa peptide from hepatitis B virus (HBV) pre-S(2) or one of two peptides related to the heat-stable toxin (STa) of enterotoxigenic Escherichia coli inserted between aa 55 and 64 of CTB. This resulted in the production of HBV::CTB or ST::CTB hybrid proteins and illustrated that the internal permissive site can be used for insertion of peptides of varying aa composition. The reactivity of the inserted epitopes with epitope-specific mAb in GM1-ELISA and immunoblots varied greatly between hybrid proteins and depended on the position in CTB and the aa composition of the inserted peptides. Despite these differences, all the HIV::CTB, ST::CTB and HBV::CTB hybrid proteins could induce low, but significant, levels of serum Ab in mice against gp120, STa or pre-S(2), in addition to strong serum Ab responses against CTB. The Ab response against the internally inserted gp120 peptide was similar to that against the same peptide fused to the N-terminus of CTB, indicating that internally placed peptides had similar immunogenicity to the same peptides added terminally.