Characterization of immunodominant and potentially protective epitopes of Mannheimia haemolytica serotype 1 outer membrane lipoprotein PlpE

Abstract

Mannheimia haemolytica serotype 1 (S1) is the most common bacterial isolate found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. In a recently published work, members of our laboratory showed that recombinant PlpE (rPlpE) is highly immunogenic when injected subcutaneously into cattle and that the acquired immunity markedly enhanced resistance to experimental challenge (A. W. Confer, S. Ayalew, R. J. Panciera, M. Montelongo, L. C. Whitworth, and J. D. Hammer, Vaccine 21:2821-2829, 2003). The objective of this work was to identify epitopes of PlpE that are responsible for inducing the immune response. Western blot analysis of a series of rPlpE with nested deletions on both termini with bovine anti-PlpE hyperimmune sera showed that the immunodominant region is located close to the N terminus of PlpE. Fine epitope mapping, in which an array of overlapping 13-mer synthetic peptides attached to a derivatized cellulose membrane was probed with various affinity-purified anti-PlpE antibodies, identified eight highly reactive regions, of which region 2 (R2) was identified as the specific epitope. The R2 region is comprised of eight imperfect repeats of a hexapeptide (QAQNAP) and is located between residues 26 and 76. Complement-mediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killing M. haemolytica.

FIG. 1.

Twenty and 40 μl of whole-cell lysate (lanes 1 and 2), the same volumes of outer membrane proteins (lanes 3 and 4), and 0.5 and 1.0 μg of rPlpE (lanes 5 and 6) were separated by SDS-12.5% PAGE. M, molecular mass markers. One gel was stained with Coomassie brilliant blue (A), and the second was transferred onto nitrocellulose for a Western blot with hyperimmune calf serum immunized with rPlpE (B).

FIG. 2.

Predicted features of the deduced amino acid sequence of PlpE showing regions that are potentially antigenic, hydrophilic, and surface exposed and secondary structures.

FIG. 3.

Nested deletion mutants were tested for binding capacity of anti-rPlpE antibodies from bovine and murine sources. 1, rPlpE; 2, rPlpEΔN28; 3, rPlpEΔC86; 4, rPlpEΔC96; 5, rPlpEΔC106; 6, rPlpEΔN76; 7, rPlpEΔN150. (A) Anti-PlpE bovine hyperimmune serum; (B) anti-His-tagged mouse monoclonal antibody; (C) Coomassie stain.

FIG. 4.

Overlapping 13-mer peptides spanning PlpE were sequentially probed with a variety of antibodies. (A) Goat anti-bovine-HRP; (B) rabbit anti-bovine-HRP; (C) colostrum-deprived calf serum (naive); (D) bovine anti-PlpE hyperimmune serum; (E) immunodominant epitope (R2) identified by subtracting the background, i.e., panels A, B, and C from panel D. The clusters of peaks also known as regions (R) are numbered from left to right as they appear in panel D (R1, peptides 5 to 7; R2, peptides 13 to 26; R3, peptides 31 to 34; R4, peptides 39 to 44; R5, peptides 53 to 55; R6, peptides 62 to 64; R7, peptides 68 to 71; R8, peptides 75 to 77). The arrow shows the immunodominant epitope region (R2) of the PlpE protein.

FIG. 5.

Complement-mediated bacterial killing activity of anti-PlpE antibodies purified by affinity columns with intact rPlpE, deletion derivatives of rPlpE as ligands, and whole serum from a calf that was immunized with rPlpE and from a colostrum-deprived newborn calf.