Vol. 59, No. 1
INFECTION AND IMMUNITY, Jan. 1991, p. 172-180
0019-9567/91/010172-09$02.00/0 Copyright © 1991, American Society for Microbiology
A Monoclonal Antibody against a Pasteurella multocida Outer Membrane Protein Protects Rabbits and Mice against Pasteurellosis YUE-SHOUNG LU,* WAYNE C. LAI, STEVEN P. PAKES, AND L. C. NIE
Division of Comparative Medicine, Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 Received 13 June 1990/Accepted 2 October 1990
Monoclonal antibodies (MAbs) directed against the 37.5-kDa outer membrane protein were produced by fusing myeloma cells with spleen cells obtained from mice immunized with a pathogenic strain of Pasteurella multocida isolated from a rabbit. Desirable MAbs were selected by enzyme-linked immunosorbent assay, whole-cell radioimmunoprecipitation (WC-RIP), and Western blot (immunoblot) analysis. WC-RIP and Western blot analyses, using MAb 1608 adsorbed with intact P. multocida cells and the eluted MAb, demonstrated that the antigen recognized by this MAb is exposed on the cell surface, is antibody accessible, and has an estimated molecular mass of 37.5 kDa. Treatment of outer membrane vesicles of P. multocida with proteinase K totally abrogated the MAb 1608 activity, indicating that this MAb binds to a protein antigenic determinant. Furthermore, MAb 1608 was nonreactive to purified lipopolysaccharide in Western blot analysis. Passive transfer studies showed that nine rabbits inoculated intranasally with MAb 1608 and homologously challenged intranasally had significantly reduced mortality, severity of pneumonia, prevalence of P. multocida colonization in nonrespiratory organs, and numbers of P. multocida in nasal cavities compared with the controls. Furthermore, the number of P. multocida in lungs was reduced 84,750-fold. Similarly, passive transfer experiments indicated that MAb 1608 protected mice against homologous and heterologous challenges with P. multocida strains bearing the antigenic determinant recognized by MAb 1608. However, no protection was afforded by MAb 1608 when mice were challenged with a P. multocida strain lacking the antigenic determinant recognized by MAb 1608. This study establishes that the 37.5-kDa outer membrane protein is the target for a protective MAb. an
portant bacterial disease in rabbits. Infected rabbits
Pasteurellosis caused by Pasteurella multocida is
To define and characterize these specific surface outer membrane antigens, we produced and characterized murine monoclonal antibodies (MAbs) to a virulent strain of P. multocida of rabbit origin. Characterization of MAbs in terms of chemical characteristics of the binding site, specificity of the epitope, cell surface exposure, accessibility of the epitope, and protection in rabbits and mice provides preliminary information for the identification of the protective immunogens of P. multocida. The results of the study showed that MAb 1608, directed to the 37.5-kDa outer membrane protein, protects rabbits and mice.
unsuitable for research involving respiratory, genital, and sensory systems because of colonization by the organism and lesions in these systems (17). One approach to control and eliminate pasteurellosis in rabbits is to develop an effective and safe vaccine. Live mutant P. multocida vaccines and P. multocida subunit vaccines have been developed and evaluated for their efficacy in preventing the disease. Rabbits immunized with a streptomycin-dependent live mutant vaccine (17), KSCN extracts (25), or outer membranes of P. multocida (17a) developed resistance against homologous challenge. Further, rabbit immune serum against the KSCN extract of P. multocida passively protected rabbits against homologous challenge (18). Since rabbits mounted immune responses to these vaccines and the immune serum passively protected rabbits against the homologous challenge (18), it is concluded that the antigens identified by these immune sera are immunogenic in rabbits and are potential vaccine candidates. At least 15 protein immunogens were identified by radioimmunoprecipitation (RIP) and Western blot (immunoblot) analysis with various rabbit immune sera (15). However, only a subset of these immunogens which are exposed on the cell surface and are antibody accessible has a great potential to be protective immunogens. We have identified five such P. multocida outer membrane proteins with molecular masses of 27, 37.5, 49.5, 58.7, and 64.4 kDa by whole-cell RIP (WC-RIP). The 37.5-kDa protein appears to be the major outer membrane protein (15). *
MATERIALS AND METHODS Bacterial strains and culture media. P. multocida UT-1, isolated from a rabbit with suppurative rhinitis, was used in most of the experiments. The P. multocida organisms were grown on blood agar plates inoculated with lyophilized stock and then in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) at 37°C. All P. multocida cultures were harvested in the log phase of growth and used for experiments. Other P. multocida rabbit strains used in adsorption and mouse challenge experiments were strain 81-291 (serotype NT:3,7), isolated from a rabbit with tympanitis; strain 81-556 (serotype NT:10,12), from a rabbit with pneumonia; strain 84-439 (serotype NT:3,10), from a rabbit with rhinitis; strain 85-763 (serotype A:3,10), from a rabbit with rhinitis; and strain 86-313 (serotype D:3,10), from a rabbit with pyometritis. Preparation of P. multocida outer membrane vesicles. Outer membrane vesicles were prepared by a modification of the method of McDade and Johnston (20). The protein was examined qualitatively by sodium dodecyl sulfate-polyacryl-
Corresponding author. 172
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amide gel electrophoresis (SDS-PAGE), and the concentration was determined by the modified Lowry procedure (2). These outer membrane vesicles were used as antigens in immunizing mice and in enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Animals. Pasteurella-free New Zealand White female rabbits, weighing 2.7 to 3.6 kg, were purchased from HazletonDutchland Laboratories, Denver, Pa. All rabbits were determined to be free of P. multocida infection when there were two negative P. multocida cultures from the nasal cavity and serum immunoglobulin G (IgG) antibody was negative against P. multocida by ELISA (18). Six- to eight-week-old BALB/c female mice (Jackson Laboratories, Bar Harbor, Maine) free of murine microbial pathogens, including viruses, bacteria, mycoplasma, and parasites, were used for production of MAbs and protection studies. The murine pathogens tested were ectromelia virus, GD VII virus, lymphocytic choriomeningitis virus, minute virus of mice, mouse adenovirus, mouse hepatitis virus, mouse rotavirus, murine cytomegalovirus, polyomavirus, reovirus 3, Sendai virus, Mycoplasma arthritidis, Mycoplasma pulmonis, Salmonella spp, P. multocida, Bordetella bronchiseptica, Syphacia obvelata, Aspiculuris tetraptera, Spironucleus muris, and mites. MAb production. MAb 1608 was produced from a mouse immunized (twice at 2-week intervals) intraperitoneally with 50 jig of purified P. multocida outer membrane mixed with complete Freund's adjuvant. Twenty-five micrograms of P. multocida outer membrane in phosphate-buffered saline (PBS) was injected intravenously 3 days prior to a fusion. Immunized mice which produced ELISA antibodies directed against outer membrane vesicles of P. multocida were used for hybridoma production as described previously (26), with modification. SP2/0-nonsecreting mouse myeloma cells were grown in Dulbecco modified Eagle medium (DMEM; GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% heatinactivated fetal calf serum, 2 mM L-glutamine, and 50 U of penicillin-streptomycin (GIBCO) per ml. Immune spleen cells (108) from an immunized BALB/c mouse were mixed with 2 x 107 SP2/0 cells and centrifuged (40C) at 270 x g. The cell pellet was then warmed at 37°C and mixed with DMEM containing 35% polyethylene glycol (molecular weight, 1,000; BDH Chemicals, Poole, England) and 10% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) for 1.5 min. The cell suspension was centrifuged at 270 x g, and the supernatant was discarded. The final cell pellet was resuspended in DMEM-HY medium (26) and plated out into 96-well tissue culture plates (Costar, Data Packaging Corp., Cambridge, Mass.) at 50 ,u.l per well. The plates were incubated in a 37°C incubator containing 5% CO2. On day 2, a 50-,lI portion of a twofold concentration of Littlefield HAT selection medium (14) was added to each well. On day 4, an additional 100-,ul portion of HAT medium was added to each well. Approximately 7 to 10 days after fusion, hybridoma colonies became visible. After day 7, all wells were fed DMEM-HT-glycine medium (26). These colonies were expanded into 2 wells in a 96-well plate. If growth continued, cells from 2 wells of a 96-well plate were expanded into 1 well of a 24-well plate. Culture supernatants from the 24-well plate were then analyzed for antibody activities directed against P. multocida outer membrane antigens by ELISA and Western blot. Hybridoma cells producing the desired antibodies were cloned by limiting dilution (6) and retested by the same screening procedures. ELISA. Hybridoma culture supernatants were screened
A PROTECTIVE MAb AGAINST P. MULTOCIDA
by ELISA for antibodies directed against outer membrane antigens of P. multocida, using outer membrane vesicles (1 jig of protein per well) as the antigen source. A 0.15-ml volume of antigen in carbonate buffer (pH 9.6) was coated onto a 96-well microtiter plate (Dynatech Laboratories, Alexandria, Va.) overnight at 4°C. The wells were washed three times with PBS-T (0.01 M PBS containing 0.05% Tween 20 [pH 7.2]) to remove the unbound antigens, incubated with PBS-T containing 1% bovine serum albumin (BSA) for 1 h at room temperature to block the nonspecific binding sites on the wells, and washed three times with PBS-T. A 100-,ul portion of the test material (hybridoma culture supernatant) was added to each well. The plates were incubated at room temperature for 90 min and washed three times with PBS-T. A 0.15-ml volume of diluted conjugate (alkaline phosphatase-conjugated goat anti-mouse immunoglobulins [IgG, IgA, and IgM] diluted 1:500 in PBS-T; Cappel Laboratories, West Chester, Pa.) was added, and the plate was incubated at room temperature for 90 min. The plate was washed three times with PBS-T, and 0.10 ml of p-nitrophenyl phosphate substrate (1 mg/ml in 10% diethanolamine buffer [pH 9.8] containing 0.01% MgCl2) was added. The plate was wrapped with aluminum foil and incubated at 37°C for 30 min. The reaction was terminated by adding 3 M NaOH. The A405 of each well was read on a Microelisa reader (Dynatech). Each plate contained appropriate controls, including a known positive mouse serum, culture supernatant from the SP2/0 cell line (medium control), and wells without antigen, antibody, or conjugate. Wells in which the A405 was twice that of the negative control were considered positive. Characterization of MAb 1608. MAb 1608 cloned three times by limiting dilution was characterized by WC-RIP, Western blot analysis, immunodiffusion analysis of isotypes, and protection against P. multocida challenge in rabbits and mice. Western blot immunoanalysis of MAb 1608. Hybridoma culture supernatants that were ELISA positive were further screened by Western blot analysis (18), using P. multocida outer membrane vesicles as antigens. The membrane antigens were separated by SDS-PAGE and transferred to nitrocellulose paper (NCP) by the methanol-Tris glycine method (27). After transfer, the NCP strips were incubated with PBS-T containing 2% BSA for 1 h at room temperature to block the nonspecific binding sites. The NCP strips were then incubated with hybridoma culture supernatant (1:2 dilution in PBS-T) overnight at 4°C and washed three times with PBS-T at the end of incubation. The NCP was next reacted with a 1:1,000 dilution (in PBS-T) of horseradish peroxidase-conjugated goat anti-mouse IgG (heavy and light chains specific; Cappel) for 8 h at 4°C, washed three times (15 min each) with PBS-T, and incubated with a 1:1,000 dilution of a horseradish peroxidase conjugate of rabbit anti-goat IgG (Cappel) overnight at 4°C. The strips were then washed three times in PBS-T and immersed in 50 mM Tris buffer (pH 7.4) containing 4-chloro-1-naphthol (0.5 mg/ml) and hydrogen peroxidase (0.01%) for color development. The color reaction was terminated by flooding the NCP with distilled water. WC-RIP. Selected hybridoma culture supernatants positive by ELISA and Western blot analysis were further characterized by WC-RIP to confirm the antibody specificity. Briefly, mixtures containing 125I-labeled P. multocida cells (2 x 108 cpm/ml), hybridoma culture supernatant, SP2/0 culture supernatant (medium control), or PBS in Eppendorf tubes were incubated on a rocking platform for 1
LU ET AL.
h at 4°C. The mixtures were centrifuged, and the pellets were washed with PBS twice. The final pellets containing cellsantibodies were solubilized by incubating the pellets with solubilization buffer (10 mM Tris [pH 7.8], 10 mM EDTA, 150 mM NaCl, 1% [vol/vol] Triton X-100, 0.2% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] SDS) at 39°C for 1 h, with mixing every 15 min. The supernatant was collected after centrifugation of the mixture at 45,000 x g for 1 h at 16°C. A prewashed staphylococcal protein A suspension was added. The mixture was incubated at 4°C for 1 h and centrifuged at 12,000 x g for 1 min at 25°C. The pellet containing the staphylococcal protein A-antigen-antibody complex was washed five times in solubilization buffer. The final pellet was suspended in digestion buffer (62.5 mM Tris [pH 6.8], 2% [wt/vol] SDS, 10% [vol/vol] glycerol), boiled for 5 min, and centrifuged. The supernatants were frozen at -20°C until analyzed by SDS-PAGE. SDS-PAGE. The SDS-PAGE procedure of Laemmli (13) was used. The samples were run by discontinuous SDSPAGE in 1.5-mm-thick slab gels. A 4% (wt/vol) polyacrylamide stacking gel and a 10% (wt/vol) polyacrylamide separating gel were used to separate the distinct proteins into bands. Electrophoresis was conducted at a 30-mA constant current per gel at 4°C until the pyronin Y tracking dye entered the separating gel; the constant current was then increased to 45 mA per gel. The protein contents of samples were measured by the method of Bradford (2) with Bio-Rad reagent (Bio-Rad Laboratories, Richmond, Calif.). Samples were boiled with digestion buffer containing 5% (vol/vol) 2-mercaptoethanol prior to being loaded onto the gel. Labeled proteins on the gel were fixed, stained with Coomassie blue, destained, dried, and identified by autoradiography (9). Adsorption and elution of MAb 1608. Adsorption was accomplished by mixing 500 RI of MAb 1608 with 500 ,ul of washed P. multocida live cells (108 CFU) at 4°C for 1 h and centrifuging at 12,000 x g for 5 min. The supernatant was removed and added to 500 ,u of fresh P. multocida cells. The mixture was incubated at 4°C for 1 h and centrifuged as described above. This procedure was repeated three times to remove total antibodies. The cell suspension was kept at 4°C to ensure that cells remained intact. The final supernatant was designated adsorbed MAb 1608. The eluted MAb 1608 was obtained by incubating MAb 1608 with intact P. multocida cells and collecting the sediment after centrifugation. The sediment was resuspended in a fresh aliquot (500 ,ul) of MAb 1608. The suspension was incubated at 4°C for 1 h and centrifuged. The same procedure was repeated three times to obtain the iRaximum amount of adsorbed antibody on cell surfaces. The final pellet containing bacterium-antibody complexes was washed with PBS and resuspended in 0.2 M glycine-HCl (pH 2.8). The supernatant was removed and adjusted to pH 7.2 with 1 mM Tris-HCl (pH 8.0). BSA (fraction V; Sigma) was added to 0.1%, and the eluted MAb 1608 was stored at -20°C. Isotype determination. The isotype of MAb 1608 was determined by the Ouchterlony immunodiffusion method (23). The purified MAb 1608 was used as antigen, and goat anti-mouse heavy-chain immunoglobulins (ICN Biomedicals, Inc., Costa Mesa, Calif.) served as antibody. Proteinase K digestion of P. multocida outer membrane proteins. P. multocida outer membranes extracted with lithium chloride buffer were digested with proteinase K (12) for 30 min to characterize the antigen-binding site of MAb 1608. Preparation of LPS of P. multocida. Lipopolysaccharide (LPS) was extracted from P. multocida (strain UT-1) by
using a hot phenol-water procedure described previously (24).
Purification of MAbs. MAbs collected from hybridoma cells growing in DMEM-HT-glycine medium (26) without antibiotics were purified on a protein A-Sepharose 4B col-
(5). Immunoprotective studies in rabbits. Passive transfer studies in rabbits were performed to determine the protective efficacy of MAb 1608. Twenty-four rabbits (Table 1) were inoculated intranasally (i.n.) with affinity-purified MAb 1608, SP2/0 supernatants (medium control), or MAb 6G6 against M. pulmonis (1 mg of protein per time per rabbit) at 2 h prior to and 12 and 24 h after i.n. challenge with 2 x 107 CFU of the virulent homologous P. multocida strain. Each rabbit was necropsied after death or at day 14 postchallenge and
evaluated microbiologically and pathologically (19). At necropsy, bilateral nasal swabs, liver, spleen, uterus, and lungs, and left and right tympanic bulla swabs were collected from each rabbit. The nasal swabs were vortexed in 1 ml of PBS to make a suspension. The suspension was then serially diluted 10-fold and inoculated onto blood agar plates in triplicate at each dilution to determine the number of CFU per nasal swab. Parts of the lungs from each rabbit were homogenized in PBS to make a 10% (wt/vol) suspension. The lung suspensions were diluted 10-fold and plated on blood agar plates to determine the number of CFU per gram of lungs. A small piece of the liver, spleen, and uterus and left and right tympanic bulla swabs were inoculated onto blood agar plates to determine the presence or absence of P. multocida (19). For pathologic evaluation, the nasal turbinates, lungs (all lobes), and tympanic bullae were prepared for routine histopathologic examination (19). Microscopic lesions of nasal turbinates (three sections), lungs (five sections), and tympanic bullae (two sections) from each rabbit were scored as 0 (normal), 1 (mild), 2 (moderate), or 3 (severe). To compare scores, the sum of lesion scores for each organ from individual rabbits within a group was divided by the sum of the maximum possible scores to determine the group lesion index (GLI). A GLI of 1.0 indicates the most severe lesions possible for a group. Immunoprotective studies in mice. A kinetic study was performed to determine the optimal time of immunoprotection afforded by MAb 1608. Sixty-eight female BALB/c mice were divided into two groups (see Table 3). Mice in group 1 were anesthetized with methoxyflurane, inoculated i.n. with 16 jig of affinity-purified MAb 1608 protein in 50 RI of PBS, and challenged i.n. two h later with 2 x 106 homologous virulent P. multocida cells. Mice in group 2 were treated identically except that they received SP2/0 culture supernatants, which contained no antibody. Five to eight mice from group 1 and 2 were killed by methoxyflurane overdose at 0, 4, 8, 24, or 48 h postchallenge. Lungs were removed aseptically and homogenized in PBS to make a 10% (wt/vol) suspension. P. multocida in lungs was quantitated by plating a serial 10-fold dilution of the suspension on TSAY plates (in triplicate for each dilution; 18) and incubating the plates at 37°C for 48 h. Typical P. multocida colonies were counted, and selected colonies were confirmed by standard biochemical characteristics and Western blot or dot blot analysis, using MAb 1608 as the primary antibody. The protective ability of affinity-purified MAb 1608 was determined by passive immunization of mice with the MAb followed by challenge with P. multocida. Challenge organisms included the homologous strain (UT-1), heterologous strains (85-763 and 81-556) bearing the antigenic determinant recognized by MAb 1608, and a heterologous strain (86-313)
VOL. 59, 1991
A PROTECTIVE MAb AGAINST P. MULTOCIDA
C D E
45K urnik;. _*
FIG. 1. WC-RIP analysis of the cell surface-exposed P. multocida outer membrane protein recognized by MAb 1608. The WCRIP method was used as described in the text; immune precipitates obtained by WC-RIP were analyzed by SDS-PAGE with a 10%o (wt/vol) polyacrylamide separating gel. Lanes: A, "25I-labeled outer membrane protein precipitated by 500 tl of hybridoma culture supernatant containing MAb 1608; B, "25I-labeled outer membrane protein precipitated by 500 ,ul of SP2/0 culture supernatant (medium control); C, "25I-labeled outer membrane proteins precipitated by 200 ,ul of mouse immune serum against P. multocida outer membranes. Molecular mass markers are on the left. K, Kilodaltons.
lacking the 37.5-kDa antigenic determinant. In each experiment, BALB/c female mice were divided into two groups (see Table 4). Mice from groups 1 and 2 were anesthetized with methoxyflurane, inoculated i.n. with 50 ,ul of affinitypurified MAb containing 16 ,ug of protein or with SP2/0 culture supernatants, respectively (MAb and SP2/0 grown in a medium without antibiotics), allowed to recover from the anesthesia for 2 h, challenged i.n. with live P. multocida (2 x 106 CFU), and killed by methoxyflurane overdose 48 h postchallenge. Lungs were collected and homogenized to determine numbers of P. multocida as described above. Statistical analysis. Analysis of variance was applied to determine significant differences in numbers of P. multocida in lungs and in nasal cavities among the groups of rabbits. The chi-square test was used to determine significant differences in mortality and prevalence of P. multocida colonization in nonrespiratory organs among the groups of rabbits. Student's t test was applied to determine significant differences in the numbers of P. multocida in lungs of mice (28). RESULTS Molecular specificity of MAb 1608. We used WC-RIP and Western blot analysis to determine the molecular mass of the antigen reactive to MAb 1608, which is of the IgG2a isotype. MAb 1608 reacted with an antigen with an estimated molecular mass of 37.5 kDa (Fig. 1, lane A), whereas the SP2/0
FIG. 2. Western blot analysis of the cell surface-exposed outer membrane protein recognized by MAb 1608. Outer membrane vesicles of P. multocida (strain UT-1) were used as antigen; electrophoretic blots were incubated with hybridoma culture supematant containing MAb 1608 and then reacted with horseradish peroxidase-conjugated antibodies as described in the text. Lanes: A, outer membrane protein detected by unadsorbed MAb 1608; B, outer membrane protein detected by adsorbed MAb 1608; C, outer membrane protein detected by eluted MAb 1608; D, outer membrane protein detected by SP2/0 culture supernatant (medium control); E, outer membrane proteins detected by mouse immune serum against P. multocida outer membranes. Adsorbed and eluted MAb 1608 were prepared by incubating the MAb with homologous P. multocida (strain UT-1) as described in the text. Molecular mass markers are on the left. K, Kilodaltons.
supernatant was nonreactive (lane B) in WC-RIP. As expected, the ELISA-positive mouse serum, obtained from the mouse immunized with purified outer membranes, had a strong antibody activity against the same 37.5-kDa antigen (lane C). Since the WC-RIP (8) detects only the cell surfaceexposed and antibody accessible immunogens, the 37.5-kDa outer membrane antigen probably resides on the cell surface and is antibody accessible. To confirm this, we analyzed unadsorbed, adsorbed, and eluted MAb 1608 by Western blot analysis. Adsorbed and eluted MAb 1608 were obtained by mixing MAb 1608 with intact homologous P. multocida (strain UT-1). Antibody activity was evident for unadsorbed (Fig. 2, lane A) and eluted (lane C) MAb 1608 but not for the adsorbed MAb 1608 (lane B). To confirm the specificity of adsorption, we performed the same experiment except that heterologous strains of P. multocida bearing (strains 84-439 and 85-763) or lacking (strain 81-291) the antigenic determinant recognized by MAb 1608 were used for adsorption. MAb 1608 reactivity was removed completely (Fig. 3, B and H) when MAb 1608 was adsorbed with P. multocida strains bearing the antigenic determinant but not with the isolate without the antigenic determinant (lane E). Consistent with these results, antibody activity directed against the 37.5-kDa antigen was detected for eluted MAb 1608 that was adsorbed
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INFECT. IMMUN. D
2114FIG. 3. Western blot analysis of MAb 1608 specificity against the cell-surface exposed outer membrane protein. Outer membrane vesicles of P. multocida (strain UT-1) were used as antigen; electrophoretic blots were incubated with hybridoma culture supernatant containing MAb 1608 and then reacted with horseradish peroxidase-conjugated antibodies as described in the text. Hybridoma culture supernatants containing MAb 1608 were incubated with heterologous P. multocida strains bearing (strains 84-439 and 85763) or lacking (strain 81-291) the antigenic determinant recognized by MAb 1608 to prepare adsorbed and eluted MAb 1608 as described in the text. Lanes: A, outer membrane protein detected by unadsorbed MAb 1608; B, outer membrane protein detected by adsorbed MAb 1608 which was adsorbed with strain 84-439; C, outer membrane protein detected by eluted MAb 1608 which was adsorbed with strain 84-439; D, outer membrane protein detected by unadsorbed MAb 1608; E, outer membrane protein detected by adsorbed MAb 1608 which was adsorbed with strain 81-291; F, outer membrane protein detected by eluted MAb 1608 which was adsorbed with strain 81-291; G, outer membrane protein detected by unadsorbed MAb 1608; H, outer membrane protein detected by unadsorbed MAb 1608 which was adsorbed with strain 85-763; I, outer membrane protein detected by eluted MAb 1608 which was adsorbed with strain 85-763; J, outer membrane proteins detected by mouse immune serum against P. multocida outer membranes; K, outer membrane protein detected by SP2/0 culture supernatant (medium control). Molecular mass markers (in kilodaltons) are on the left.
with the MAb 1608-positive isolates (lanes C and I) but not for that adsorbed with the MAb 1608-negative isolate (lane F). Antigen characteristics. The outer membrane of P. multocida extracted with lithium chloride, the proteinase K-digested outer membrane of P. multocida, and the purified LPS of the homologous strain of P. multocida were separated by SDS-PAGE, transferred to nitrocellulose paper, and reacted with MAb 1608 in a Western blot. The antigenbinding site for MAb 1608 was abrogated completely after proteinase K treatment, suggesting that MAb 1608 binds to a protein determinant (Fig. 4, lane B). Consistent with this result, MAb 1608 failed to bind to the purified LPS of P. multocida (lane C). MAb 1608-mediated protection of rabbits against P. multocida infection. Clinical, pathological, and microbiological
FIG. 4. Identity of the antigenic determinant (epitope) reactive with MAb 1608 revealed by Western blot analysis. Various P. multocida (strain UT-1) preparations were used as antigen; electrophoretic blots were incubated with hybridoma culture supernatant containing MAb 1608 and then reacted with horseradish peroxidaseconjugated antibodies as described in the text. Lanes: A, outer membrane protein detected by MAb 1608, using P. multocida outer membrane vesicles as antigen; B, outer membrane protein detected by MAb 1608, using proteinase K-digested (30 min) outer membrane vesicles as antigen; C, outer membrane protein detected by MAb 1608, using purified homologous P. multocida LPS as antigen. Molecular mass markers are on the left. K, Kilodaltons.
criteria were used to determine the protective efficacy of MAb 1608 against homologous challenge in rabbits. (i) Clinical criteria. Mean survival time (days) and mortality were compared among the groups (Table 1). The mean survival time was increased significantly (P < 0.002) from 6.7 days (group 1) to 11.8 days (group 2). The mortality rate was reduced significantly (P < 0.025) from 100% (group 1) to 33% (group 2). (ii) Pathological criteria. The prevalence and severity of lesions in lungs, nasal cavities, and tympanic bullae were compared among the groups (Table 1). The prevalences of pneumonia were 100% (nine of nine), 67% (six of nine), and 100% (six of six) for groups 1, 2, and 3, respectively, and the differences were not significant among the groups. The severity of pneumonia as measured by GLI was reduced significantly (P < 0.001) from 0.98 (group 1) to 0.32 (group 2). No significant difference (P > 0.1) in the GLIs for pneumonia was noted between groups 1 and 3. The prevalences of rhinitis were 56% (five of nine), 11% (one of nine), and 33% (two of six) for groups 1, 2, and 3, respectively, and the differences were not significant among the groups. Similarly, the mean GLIs for rhinitis in groups 1, 2, and 3 were not significantly different. The prevalences of tympanitis were 0% (zero of nine), 11% (one of nine), and 17% (one of six) for groups 1, 2, and 3, respectively, and the differences were not significant among the groups. Similarly, the mean GLIs for tympanitis were not significantly different among the groups. Gross evaluation of other tissues indicated that no lesions were present in any of the experimental groups. (iii) Microbiological criteria. The prevalence and number of P. multocida in nasal cavities and lungs and prevalence of P. multocida in nonrespiratory organs were compared
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TABLE 1. GLIs, survival time, and mortality of rabbits treated with MAb 1608 and challenged with homologous P. multocida UT-1 Group
SP2/0 MAb 1608 MAb 6G6
1 2 3
GLPb(mean ± SEM)Suvaltm nSurvval time
No. of rabbits
9 9 6
0.98 ± 0.02
0.14 ± 0.07 0.06 ± 0.06 0.07 ± 0.05
0 ± 0 0.03 ± 0.03 0.03 ± 0.03
6.7 + 0.7 11.8e ± 1.1 7.3 ± 2.1
0.32±d 0.14 0.75 ± 0.14
9/9 (100) 3/9 (33}f 4/6 (67)
a Each rabbit was inoculated i.n. with affinity-purified MAb 1608, SP2/0 supernatants, or MAb 6G6 (1 mg per rabbit per time) at 2 h prior to and 12 and 24 h after i.n. challenge with 2 x 10' CFU of the virulent homologous P. multocida strain and killed at day 14 postchallenge. MAb 1608 is directed against the 37.5-kDa P. multocida outer membrane protein, and MAb 6G6 is directed against M. pulmonis. SP2/0 supernatants contain no antibody. MAb 1608 and MAb 6G6 are of the IgG2a isotype. SP2/0 served as a medium control, and MAb 6G6 served as an antibody specificity control. b The GLI represents the severity of the lesions of the group. A GLI of 1.0 is the most severe possible. The GLI was calculated as described in the text. c Number of rabbits that died/number of rabbits in the group; values in parentheses are percentages. d Mean GLI significantly less (P < 0.001) than that for group 1. e Mean survival time significantly longer (P < 0.002) than that of group 1. f Mortality significantly less (P < 0.025) than that of group 1.
the groups (Table 2). The challenge virulent P. multocida organisms colonized the nasal cavities of all rabbits as determined at necropsy. The number of P. multocida organisms in the nasal cavity, however, was reduced significantly (P < 0.005) from 1.31 x 106 (group 1) to 8.81 x 103 (group 2), a 148-fold reduction. The prevalences of P. multocida in lungs were 100% (nine of nine), 44% (four of nine), and 100% (six of six) for groups 1, 2, and 3, respectively, and a significant (P < 0.05) difference was evident between groups 1 and 2. The number of P. multocida organisms in the lungs was reduced from 2.89 x 107 to 3.41 x 102, a 84,750-fold reduction (P < 0.001). P. multocida organisms were isolated with various frequencies from nonrespiratory organs including the liver, spleen, uterus, left tympanic bulla, and right tympanic bulla. The combined P. multocida prevalences in nonrespiratory organs were 67% (30/45), 24% (11/45), and 73% (22/30) for groups 1, 2, and 3, respectively. Rabbits treated with MAb 1608 and challenged had a reduction of P. multocida prevalence from 67% (group 1) to 24% (group 2), a significant (P < 0.001) 43% reduction. Rabbits which were inoculated with MAb 6G6 (against M. pulmonis) and challenged developed severe pneumonia, high mortality, high prevalence of P. multocida colonization in nonrespiratory organs, and high numbers of P. multocida among
organisms in the nasal cavity and lungs (Tables 1 and 2). These results were similar to those observed in group 1 rabbits treated with SP2/0 culture supematant (medium control), indicating MAb 6G6 is nonprotective in rabbits against P. multocida challenge. These results demonstrate that MAb 1608 is a specific protective MAb in rabbits. Immunoprotection of mice with MAb 1608. Since MAb 1608 protects rabbits against homologous challenge, we would like to know if MAB 1608 also protects mice against heterologous challenges with strains of P. multocida bearing the antigenic determinant or lacking the antigenic determinant.
A kinetic study showed that optimal protection, as defined by the lowest number of P. multocida organisms in lungs, was achieved at 48 h after i.n. homologous challenge (Table 3). Consequently, mice inoculated with MAb 1608 and challenged were killed at 48 h postchallenge to determine the protective efficacy of MAb 1608 against various P. multocida strains. Passive immunization of mice with purified MAb 1608 significantly reduced (P < 0.001) the number of P. multocida in lungs of mice which were challenged with the homologous UT-1 strain (Table 4, experiment 1). Significant protection (P < 0.002 and P < 0.005) was also observed in mice immu-
TABLE 2. Isolation of P. multocida from nasal cavities, lungs, and nonrespiratory organs of rabbits treated with MAb 1608 and challenged with homologous P. multocida UT-1 P. multocida recovered at necropsy from:
1 2 3
SP2/0 MAb 1608 MAb 6G6
9 9 6
Nasal cavity _______________________________________ Prevalenceb Mean + SEMC CFU/swabd
9/9 (100) 9/9 (100) 6/6 (100)
6.117 ± 0.233 3.943 ± 0.566f 5.472 ± 0.455
1.31 x 106 8.77 x 103 2.97 x 105
Lungs _______________________________________ Prevalence Mean + SEM CFU/g
9/9 (100) 4/9 (44) 6/6 (100)
7.461 ± 0.227 2.533 ± 1.101k 6.103 ± 0.981
2.89 3.41 1.27
x x x
107 102 106
Nonrespiratory organs (no. positive/total no.
of samples [%J)C
30/45 (67) 11/45 (24)h 22/30 (73)
a Each rabbit was inoculated i.n. with affinity-purified MAb 1608, SP2/0 supernatants, or MAb 6G6 (1 mg per rabbit per time) at 2 h prior to and 12 and 24 h after i.n. challenge with 2 x 107 CFU of the virulent homologous P. multocida strain and killed at day 14 postchallenge. MAb 1608 is directed against the 37.5-kDa P. multocida outer membrane protein, and MAb 6G6 is directed against M. pulmonis. SP2/0 supernatants contain no antibody. MAb 1608 and MAb 6G6 are of the IgG2a isotype. SP2/0 served as a medium control, and MAb 6G6 served as an antibody specificity control. b Number positive/number sampled; values in parentheses are percentages. cRepresents mean of the log of geometric mean CFU per nasal swab or per gram of lung tissue. The geometric mean CFU per nasal swab or per gram of lung in each rabbit was calculated and converted to the logarithmic number. The mean and standard error of the mean of the log of the geometric mean of the group was then calculated. d The group mean in logarithmic units was converted to the geometric mean CFU per nasal swab or per gram of lung tissue. e At necropsy, a small piece of the liver, spleen, and uterus and swabs collected from the left and right tympanic bullae were inoculated onto blood agar plates for isolation of P. multocida. f Mean CFU significantly less (P < 0.005) than for group 1. 8 Mean CFU significantly less (P < 0.001) than for group 1. h Prevalence significantly less (P < 0.001) than for group 1.
LU ET AL.
TABLE 3. Kinetic studies of protective MAb 1608 in mice challenged with homologous P. multocida (UT-1 strain) Group
Log CFU at indicated time (h) after challenge (mean ± SEM)b Treatmenta
MAb 1680 SP2/0
Mean ratio (between groups) P valued
4.578 ± 0.425 (5C) 4.480 ± 0.174 (5) 1
3.799 ± 0.229 (7) 5.226 ± 0.103 (7) 27
3.659 ± 0.422 (8) 5.001 ± 0.440 (8) 23
2.735 ± 0.431 (8) 4.121 ± 0.639 (8) 24
1.552 ± 0.519 (6) 5.695 ± 0.677 (6) 13,900