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) 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.[1]References
- Expression of an F1/V fusion protein in attenuated Salmonella typhimurium and protection of mice against plague. Leary, S.E., Griffin, K.F., Garmory, H.S., Williamson, E.D., Titball, R.W. Microb. Pathog. (1997) [Pubmed]
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