EXPRESSION OF AN F1 V FUSION PROTEIN IN ATTENUATED SALMONELLA-TYPHIMURIUM AND PROTECTION OF MICE AGAINST PLAGUE/

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed . The nucleotide sequences encoding Yersinia pestis V antigen (lcrV) a nd the mature form of F1 antigen (caf1) were amplified by PCR with pri mers which included tails. At the 3' end of caf1 and the 5' end of lcr V, the tails encoded one of three six-or eight-amino acid linkers or t heir complementary sequences. The DNA overlap in each linker region wa s used to prime a second PCR to generate three F1/V fusions, which wer e cloned into pUCI8. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-lle- Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Ar g. As shown by Western blotting of bacterial cell lysates with anti-V and anti-Fl sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenic ity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Il e-Glu-Gly-Arg linker, was electroporated into the attenuated Salmonell a typhimurium strain SL3261 (aroA). Mice receiving two intravenous dos es of 5 x 10(6) cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG(1):IgG(2a) isotype ratios, and T cell res ponses specific for V and F1 antigens. Six weeks after vaccination, mi ce were challenged subcutaneously with 7.4 x 10(2) or 7.4 x 10(4) LD(5 0)s of Y. pestis strain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressin g Y. pestis antigens as fusion proteins to develop a live recombinant vaccine against plague. (C) 1997 Academic Press Limited.