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) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3' end of caf1 and the 5' end of lcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuated Salmonella typhimurium strain SL3261 (aroA). Mice receiving two intravenous doses of 5 x 10(6) cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG1:IgG2a isotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 x 10(2) or 7.4 x 10(4) LD50s of Y. pestis strain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressing Y. pestis antigens as fusion proteins to develop a live recombinant vaccine against plague.