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

Abstract

We previously identified Mannheimia haemolytica outer membrane proteins (OMPs) that may be important immunogens by using immunoproteomic analyses. Genes for serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15 were cloned and expressed, and recombinant proteins were purified. Objective 1 of this study was to demonstrate immunogenicity of the four recombinant OMPs in mice and cattle. Objective 2 was to determine if the addition of individual recombinant OMPs or combinations of them would modify immune responsiveness of mice to the recombinant chimeric protein SAC89, containing the main epitope from M. haemolytica outer membrane lipoprotein PlpE and the neutralizing epitope of M. haemolytica leukotoxin. Mice vaccinated with recombinant OmpA (rOmpA), rSSA-1, rOmpD15, and rOmpP2 developed significant antibody responses to M. haemolytica outer membranes and to the homologous recombinant OMP. Cattle vaccinated with rOmpA and rSSA-1 developed significant antibodies to M. haemolytica outer membranes by day 28, whereas cattle vaccinated with rOmpD15 and rOmpP2 developed only minimal responses. Sera from cattle vaccinated with each of the recombinant proteins stimulated complement-mediated killing of the bacterium. Concurrent vaccination with SAC89 plus any of the four rOMPs singly resulted in increased endpoint anti-SAC89 titers, and for the SAC89/rSSA-1 vaccinees, the response was increased significantly. In contrast, the SAC89/P2/SSA-1 and SAC89/OmpA/P2/D15/SSA-1 combination vaccines resulted in significant decreases in anti-SAC89 antibodies compared to SAC89 vaccination alone. In conclusion, under the conditions of these experiments, vaccination of mice and cattle with rOmpA and rSSA-1 stimulated high antibody responses and may have protective vaccine potential.The major cause of severe bacterial pneumonia in cattle is Mannheimia haemolytica serotype 1 (S1), and current vaccines against M. haemolytica are only moderately efficacious (9, 36). Shewen and Wilkie (43) demonstrated that immunity against M. haemolytica requires antibodies against leukotoxin (LKT), which causes necrosis, apoptosis, or activation of ruminant leukocytes, as well as antibodies against bacterial cell surface antigens. The important surface immunogens needed to stimulate protective immunity against M. haemolytica appear to be outer membrane proteins (OMPs) (13,33,34,37,38).Our laboratory demonstrated that high antibody responses to a major 45-kDa outer membrane lipoprotein, PlpE, correlated with resistance against experimental challenge, and PlpE proteins were nearly identical among serotype 1 and serotype 6 isolates (2). 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 loc...

Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

“…These include the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA. Especially OmpA and the serotype 1-specific antigen have previously been shown to be highly immunogenic in mice and cattle and are thus considered to be potential M. haemolytica vaccine candidates (Ayalew et al, 2011). Interestingly, OmpA from M. haemolytica and P. multocida share a sequence identity of 55%.…”

Proteomic Analysis and Immunogenicity of Mannheimia haemolytica Vesicles

“…An immunoproteomic study of M. haemolytica OMPs conducted in our laboratory identified 57 OMPs that may have the potential to be developed into vaccines (4). The immunogenicity of recombinant forms of several M. haemolytica OMPs, including PlpE, OmpA, PlpF, OmpP2, serotype 1-specific antigen (SSA-1), and OmpD15, has been studied (5)(6)(7)(8)(9)(10). Vaccination of calves with recombinant PlpE partially protects cattle against challenge with virulent M. haemolytica and significantly enhances the efficacy of commercial vaccines (7)(8)(9).…”

Proteomic and bioinformatic analyses of putative Mannheimia haemolytica secretome by liquid chromatography and tandem mass spectrometry

“…Shewen and Wilkie (1988) demonstrated that immunity against M. haemolytica requires leukotoxin (LKT) neutralizing antibodies as well as antibodies against bacterial cell surface antigens. Studies in our and others' laboratories demonstrated that high antibody responses to OMPs, ranging from 16 to 86 kDa, correlated with resistance to challenge with virulent M. haemolytica S1, and vaccination of cattle with OMP-enriched cellular fractions from M. haemolytica S1 enhanced resistance of cattle against experimental challenge (Ayalew et al, 2010(Ayalew et al, , 2011aConfer et al, 2003Confer et al, , 2006Morton et al, 1995;Mosier et al, 1989;Orouji et al, 2012). In this regard, we used immunoproteomics, which uses a combination of 2D-gel electrophoresis (2-DE) and Western blotting with bovine convalescent sera, and identified 132 immunoreactive proteins in outer membrane preparations of M. haemolytica using LC-MS/MS.…”

“…M. haemolytica proteins whose immunogenicity have been established in ours and other laboratories include LKT (Gentry et al, 1985;Lo et al, 1987;Shewen and Wilkie, 1988), PlpE (Ayalew et al, 2004;Confer et al, 2003;Confer et al, 2006), PlpE-LKT chimera , serotype 1 specific antigen, OmpA, OmpD15, OmpP2 (Ayalew et al, 2011a,b), PlpF (Ayalew et al, 2011a, b), outer membrane vesicles (Ayalew et al, 2013), cholera toxin subunit B-PlpE-LKT chimera ), sialoglycoprotease (Shewen et al, 2003), GS60 (Lee et al, 2008;Orouji et al, 2012), adhesins (Kisiela and Czuprynski, 2009;Klima et al, 2014), and iron-regulated OMPs (Deneer and Potter, 1989;Potter et al, 1999). Representative M. haemolytica proteins whose immunogenicity has been shown in our earlier works and other investigators are given in Table 7.…”