IMMUNOGENICITY OF VIRAL B-CELL EPITOPES INSERTED INTO 2 SURFACE LOOPSOF THE ESCHERICHIA-COLI K12 LAMB PROTEIN AND EXPRESSED IN AN ATTENUATED AROA STRAIN OF SALMONELLA-TYPHIMURIUM

We previously developed a general procedure which allows the genetic c oupling of a chosen foreign linear epitope in different 'permissive' s ites of a carrier protein. By using the outer membrane protein LamB of Escherichia coli K12 as a carrier, we were able to express a number o f different foreign epitopes at the bacterial surface. In the present work, taking advantage of the recent determination of the crystal stru cture of LamB, we inserted two model B-cell epitopes i.e. - the C3 epi tope from poliovirus (residues 93 to 103 of VP1) and the preS2 epitope from hepatitis B virus, (residues 132 to 145) - at the tip of the mos t distal and largest surface exposed region of LamB (after residues 38 6, into loop L9). We also used two previously constructed LamB hybrids , corresponding to the insertion of the C3B or preSB epitope into perm issive site 153 (lying in the middle of the fourth surface loop of Lam B), to construct two LamB proteins corresponding to the simultaneous i nsertion of the two different epitopes (with one epitope per site). Th e LamB hybrids were placed under the control of the anaerobically indu cible pnirB promoter and expressed in a LamB-negative derivative of th e aroA attenuated strain of S. typhimurium, SL3261. In vitro, the reco mbinant proteins were expressed at a high level (up to 10% of whole ce ll proteins) and in vivo the recombinant plasmids were stably maintain ed. For both epitopes, genetic coupling at site 386 appeared to be mor e favorable for the induction of anti-epitope antibodies than coupling at site 153. Moreover, the LamB hybrid corresponding to the simultane ous insertion of the preSB epitope at site 153 and of the C3B epitope at site 386 allowed the induction of both anti-poliovirus and anti-hep atitis B antibodies. (C) 1998 Elsevier Science Ltd. All rights reserve d.