PASSIVE-IMMUNITY TO YERSINIAE MEDIATED BY ANTI-RECOMBINANT V-ANTIGEN AND PROTEIN A-V-ANTIGEN FUSION PEPTIDE

LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberc ulosis, and Yersinia enterocolitica, has been implicated as a regulato r of the low-calcium response, virulence factor, and protective antige n. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yieldi ng major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globul in raised against this mixture of cleavage products provided significa nt protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to e ncode a fusion of lcrV and the structural gene for protein A (i.e., al l but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-a ssociated region of protein A). The resulting fusion peptide, termed P AV, could be purified to homogeneity in one step by immunoglobulin G a ffinity chromatography and was stable thereafter. Rabbit polyclonal ga mma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudo tuberculosis (P < 0.005) but was ineffective against Y. enterocolitica . Protection failed after absorption with excess PAV, cloned whole V a ntigen, or a large (31.5-kDa) truncated derivative of the latter but w as retained (P < 0.005) upon similar absorption with a smaller (19.3-k Da) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275.