CLONING AND EXPRESSION OF COLONIZATION FACTOR ANTIGEN-I (CFA I) EPITOPES OF ENTEROTOXIGENIC ESCHERICHIA-COLI (ETEC) IN SALMONELLA FLAGELLIN/

Oligonucleotides coding for linear epitopes of the fimbrial colonizati on factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) were cloned and expressed in a deleted form of the Salmonella muenche n flagellin fliC (H1-d) gene. Four synthetic oligonucleotide pairs cod ing for regions corresponding to amino acids 1 to 15 (region I), amino acids 11 to 25 (region II), amino acids 32 to 45 (region III) and ami no acids 88 to 102 (region IV) were synthesized and cloned in the Salm onella flagellin-coding gene. All four hybrid flagellins were exported to the bacterial surface where they produced flagella, but only three constructs were fully motile, Sera recovered from mice immunized with intraperitoneal injections of purified flagella containing region II (FlaII) or region IV (FlaIV) showed high titres against dissociated so lid-phase-bound CFA/I subunits. Hybrid flagellins containing region I (FlaI) or region III (FlaIII) elicited a weak immune response as measu red in enzyme-linked immunosorbent assay (ELISA) with dissociated CFA/ I subunits, None of the sera prepared with purified hybrid flagella we re able to agglutinate or inhibit haemagglutination promoted by CFA/I- positive strains. Moreover, inhibition ELISA tests indicated that anti sera directed against region I, 11, III or IV cloned in flagellin were not able to recognize surface-exposed regions on the intact CFA/I fim briae.