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. Complementmediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killing M. haemolytica.Bovine respiratory disease arises from the interaction of numerous contributing factors, including physical stresses associated with weaning, shipment, inclement weather, and overcrowding coupled with viral and bacterial infections (8,31,32,59,63). The result in severe cases is colonization of the lungs with pathogenic bacteria resulting in severe pneumonia. Pasteurella multocida, Haemophilus somnus, and Mannheimia (formerly Pasteurella) haemolytica are associated with bovine pneumonia. However, M. haemolytica serotype 1 (S1) is by far the most important bacterial pathogen in the development of the often-fatal fibrinous pleuropneumonia in beef cattle known as pneumonic pasteurellosis or shipping fever (31, 32).Immunity against M. haemolytica is thought to be primarily through production of serum antibodies that neutralize the secreted leukotoxin (LKT) and antibodies against surface antigens (45). The mechanism of activity of antisurface antibodies and the specific surface antigens involved in anti-M. haemolytica immunity are not known; however, complementmediated bacterial lysis and bacterial phagocytosis and killing are thought to be important in defense against M. haemolytica infection (45). Complement-mediated bactericidal activity against M. haemolytica and phagocytosis of M. haemolytica by bovine neutrophils has been demonstrated with bovine immune serum (12,17,40,46).Litt...

Expeditious screening of candidate proteins for microbial vaccines

“…2; Table 1). Others have suggested that these proteins have potential as vaccine candidates against M. haemolytica (Confer and Ayalew, 2013;Ayalew et al, 2004). The moderate ranking of these proven immunogens in our assay is attributable to their high quantitation values and immunoreactivities as indicated by the relative colorimetric units (RCU) for the anti-Strep-II ELISA (Table 1) and Western blots (Fig.…”

“…The selected protein candidates included two known immunogenic outer membrane proteins, PlpE (MHA_1514) and OmpA (MHA_1054) previously identified as potential vaccine candidates against M. haemolytica (Confer and Ayalew, 2013;Ayalew et al, 2004) and 28 other proteins of known, predicted and unknown functions. The EasyXpress Linear Template Kit (Qiagen, Toronto, ON) was used to generate translationally active PCR products suitable for cell-free biosynthesis of protein using an Escherichia coli-based coupled in vitro transcription-translation (IVTT) system from Qiagen (EasyXpress Protein Synthesis kit).…”

Immunoproteomic analyses of outer membrane proteins ofMannheimia haemolyticaand identification of potential vaccine candidates

“…M. haemolytica S1 strain 89010807N, isolated from the lung of a feedlot calf with pneumonic pasteurellosis 14 was used in this study. Frozen cultures of this strain were routinely streaked onto brain heart infusion (BHI) agar supplemented with 5% sheep blood and grown at 37°C in a 5% CO 2 environment for 18 h 21. Starter cultures were prepared by transferring single colonies into tubes containing BHI broth and grown at 37°C in a shaking incubator to mid log phase.…”

Immunogenicity of Mannheimia haemolytica Recombinant Outer Membrane Proteins Serotype 1-Specific Antigen, OmpA, OmpP2, and OmpD15

“…Statistically defined endpoint titers were calculated by the method of Frey et al (17), a practical method for establishing a statistically valid cutoff. Sera were also assayed for anti-M. haemolytica IgG antibodies to OMPs by single-dilution ELISA as previously described (3,11).…”

“…Cattle vaccinated with commercial M. haemolytica vaccines to which 100 g of recombinant M. haemolytica S1 PlpE (rPlpE) was added had significantly greater resistance against experimental challenge with either S1 or S6 than did cattle vaccinated with the commercial vaccine alone (11,12). The major epitope region of M. haemolytica S1 PlpE, designated region 2 (R2), consists of 8 imperfect hexapeptide repeats of QAQNAP located near the N-terminal region (3,37).…”