Construction and Characterization of Multivalent ETEC Vaccine Antigens

The E. coli strains and plasmids used in this study are listed in Table 1. ETEC strains deposited at Johns Hopkins University, Washington University and the E. coli Reference Strain Center at University of Gothenburg (Sweden), and two recombinant E. coli strains expressing CS1 and CS2 adhesins (gifts from Dr. J. Scott at Emory University) [42 (link),43 (link)] were used for CFA adhesin extraction and in antibody adherence inhibition assays. CFA and CS fimbriae and non-fimbrial outer membrane proteins were generally referred to as CFA adhesins in this study, and LT, STa, and derived toxoids described were of human-type. Recombinant strains 9175 (CFA/I/II/IV MEFA) [39 (link)] and 9164 (3xSTa_A14Q-tmLT_{S63K/R192G/L211A}) [37 (link)] were used as templates first to construct the CFA/I/II/VI-STa_A14Q-dmLT MEFA gene. Recombinant strain 9318 (3xSTa_N12S-dmLT_R192G/L211A) was included for the construction of CFA/I/II/VI-STa_N12S-dmLT MEFA, after 3xSTa_N12S-dmLT was identified as the optimal LT-STa toxoid fusion in inducing anti-STa antibody response [38 (link)]. E. coli BL21 (GE Healthcare, Piscataway, NJ) and vector pET28α (Novagen, Madison, WI) were used to express the CFA/I/II/VI-STa_-toxoid-dmLT MEFA proteins.