Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium

摘要

We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different 'permissive' sites 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 of different foreign epitopes at the bacterial surface. In the present work, taking advantage of the recent determination of the crystal structure of LamB, we inserted two model B-cell epitopes i.e. - the C3 epitope 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 most distal and largest surface exposed region of Lamb (after residues 386, into loop L9). We also used two previously constructed Lamb hybrids, corresponding to the insertion of the C3B or preSB epitope into permissive site 153 (lying in the middle of the fourth surface loop of LamB), to construct two Lamb proteins corresponding to the simultaneous insertion of the two different epitopes (with one epitope per site). The Lamb hybrids were placed under the control of the anaerobically inducible pnirB promoter and expressed in a LamB-negative derivative of the aroA attenuated strain of S. typhimurium, SL3261. In vitro, the recombinant proteins were expressed at a high level (up to 10% of whole cell proteins) and in vivo the recombinant plasmids were stably maintained. For both epitopes, genetic coupling at site 386 appeared to be more favorable for the induction of anti-epitope antibodies than coupling at site 153. Moreover, the Lamb hybrid corresponding to the simultaneous 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-hepatitis B antibodies.