Analysis of the Yersinia pestis V protein for the presence of linear antibody epitopes

Abstract

The V protein expressed by pathogenic Yersinia pestis is an important virulence factor and protective immunogen. The presence of linear B-cell epitopes in the V protein was investigated by using a series of 17 overlapping linear peptides. Groups of 10 mice were immunized intraperitoneally with 30 microg of each peptide on days 0, 30, and 60. Although the V protein-specific antibody response to the peptides varied, most of the peptides elicited high antibody titers. The immunized mice were challenged subcutaneously with 60 50% lethal doses (LD50) (1 LD50 = 1.9 CFU) of a virulent Y. pestis strain, CO92. None of the peptide-immunized mice survived challenge. The animals immunized with the V protein were completely protected against challenge. The immunogenicity of some of the V peptides was increased by conjugating them to keyhole limpet hemocyanin. Only one peptide (encompassing amino acids 1 to 30) conjugate demonstrated some protection; the others were not protective. In additional experiments, V peptides that reacted well with sera from mice surviving Y. pestis infection were combined and used to immunize mice. Although the combined peptides appeared to be very immunogenic, they were not protective. Therefore, the protective B-lymphocyte epitope(s) in the V protein is most likely to be conformational.

FIG. 1

Heterologous immune response to the V antigen elicited by the individual V peptides. Groups of Hsd:ND4 mice (10 mice/group) were immunized with 30 μg of peptide (30-mer) on days 0, 30, and 60. After the second (day 42) and third (day 72) immunizations, mice were bled, the sera from the individual animals were pooled, and the presence of V-specific antibodies was determined by ELISA. aa, amino acids.

FIG. 2

Homologous peptide-specific response to each V peptide as measured by a peptide ELISA. Each peptide was adsorbed to the wells of a microtiter plate at 10 μg/ml, and the pooled sera from peptide-immunized, V-immunized, and Hsd:ND4 mice given adjuvant alone were analyzed for reactivity with the V peptide panel. aa, amino acids.

FIG. 3

Effect of conjugation to KLH on the immunogenicity of nonimmunogenic peptides. Hsd:ND4 mice were immunized with either 30 μg of free peptide or 50 μg of conjugated peptide on days 0, 30, and 60. Additional animals served as controls and were immunized with the carrier molecule KLH, Plague Vaccine USP, or the recombinant V protein. The mice were bled on day 70, and the sera were analyzed by ELISA for the presence of V-specific antibodies. aa, amino acids.

FIG. 4

Analysis of sera, pooled from representative infected Hsd:ND4 mice, for antibodies capable of reacting with each of 17 overlapping peptides. Microtiter plates were coated with 500 ng of each peptide, and then each peptide was analyzed for reactivity with pooled sera from mice surviving infection with wild-type Y. pestis, mice immunized with the V protein, and normal mice. aa, amino acids.