among a panel of strains of H. somnus (45 strains), H. agni. (1 strain), and Histophilus ovis (3 strains) as described previously (10), an immunodot assay was ...
Vol. 61, No. 5
INFECTION AND IMMUNITY, May 1993, p. 2257-2259 0019-9567/93/052257-03$02.00/0 Copyright C) 1993, American Society for Microbiology
Antigenic Analysis of the Major Outer Membrane Protein of Haemophilus somnus with Monoclonal Antibodies YUICHI TAGAWA,* MAKOTO HARITANI,t HITOSHI ISHIKAWA, AND NOBORU YUASA National Institute of Animal Health, 3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan Received 25 November 1992/Accepted 2 March 1993
The major outer membrane protein of Haemophilus somnus possesses at least five distinct epitopes. Three surface-exposed epitopes on the major outer membrane protein include a conserved epitope with potential for development of a vaccine and a diagnostic test and two variable epitopes responsible for antigenic differences among strains; the remaining two epitopes are well preserved among strains but not exposed on the cell surface.
The outer membrane of Haemophilus somnus contains a major outer membrane protein (MOMP) which represents the most abundant outer membrane protein (OMP) in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (1). Recently, we purified the MOMP of H. somnus and demonstrated that the MOMP shows considerable similarity in the amino-terminal amino acid sequence to porin proteins of other gram-negative bacteria (11). The MOMP was immunogenic to animals and showed antigenic heterogeneity among H. somnus strains (11). In this study, we extended the previous work to elucidate the antigenic characteristics of the MOMP further by producing and using monoclonal antibodies (MAbs). MAbs to H. somnus 8025 were developed as described previously (10, 11). One set of BALB/c mice was immunized with the purified MOMP (fusion 1), and a second group of mice was immunized with live bacteria (fusion 2). Hybridoma culture supernatant fluids were screened by enzymelinked immunosorbent assay (ELISA) with an OMP preparation, purified MOMP, and purified lipopolysaccharide from strain 8025 as the antigens, as described previously (11). The specificities of MAbs were determined by immunoblot analysis with a whole-cell lysate of strain 8025 as the antigen, as described previously (11), and nonradioimmunoprecipitation analysis by the presolubilized immunoprecipitation system (9), with modifications. Protein labeling was performed by the bacterial protein biotinylation system as described previously (2). Labeled cells were solubilized in buffer consisting of 100 mM Tris-hydrochloride (pH 8.0) containing 100 mM NaCl, 10 mM EDTA, 1% (vol/vol) Triton X-100, 0.2% (wt/vol) sodium deoxycholate, and 0.1% (wt/ vol) SDS. Aliquots of solubilized, biotinylated cellular proteins (500 ,u) were incubated with the individual MAbs (500 RI) at 4°C overnight. The resultant antigen-antibody complexes were precipitated by addition of 150 ,ul of a 1:1 (vol/vol) slurry of protein A-Sepharose (Pharmacia) in 10 mM Tris-hydrochloride (pH 8.0)-140 mM NaCl-0.025% (wt/ vol) sodium azide. After incubation at room temperature for 3 h, the immunoprecipitates were washed five times with solubilization buffer and dissociated by boiling for 10 min in 25 VI of SDS sample buffer (5). The Sepharose was removed by centrifugation at 12,000 x g for 5 s, and the immunopre* Corresponding author. t Present address: Tohoku Branch, National Institute of Animal
Health, Shichinohe, Kamikita, Aomori 039-25, Japan.
cipitates were subjected to SDS-polyacrylamide gel electrophoresis and electrotransfer to a nitrocellulose membrane sheet by the same manner as for immunoblot analysis. Luminescent detection of biotinylated proteins on the blot was performed as described previously (6). Streptavidinperoxidase conjugate (Bethesda Research Laboratories) and a luminol substrate solution (ECL Western blotting [immunoblotting] detection reagents; Amersham) were used in accordance with manufacturer instructions. To determine whether the MAbs react with cell surface-exposed epitopes on the MOMP, we used immunogold probes to visualize antibody binding to the surface of whole bacteria as described previously (10). For studies of epitope conservation among a panel of strains of H. somnus (45 strains), H. agni (1 strain), and Histophilus ovis (3 strains) as described previously (10), an immunodot assay was used. The immunodot assay was done as described previously (4) but with modifications. Bacterial antigens boiled for 5 min were used, teleostean gelatin was used in place of bovine serum albumin, and HRP color developing reagent (containing 4-chloro1-naphthol; Bio-Rad Laboratories) was used. Cross-reactivity of MAbs with various gram-negative bacteria, including Haemophilus influenzae, H. parainfluenzae, H. haemoglobinophilus, Actinobacillus lignieresii, A. seminis, Pasteurella multocida, P. haemolytica, Brucella abortus, Moraxella bovis, Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, C. sputorum subsp. bubulus, Eschenichia coli, and Salmonella dublin, was examined by immunodot assay as described previously (10). From two cellular fusions, seven hybridomas producing anti-MOMP MAbs were obtained (Table 1). Fusion 1 produced MAbs 59-8-2, 34-2-1, 81-7-5, 52-21-4, 60-3-5, and 57-16-2 (MAb 57-16-2 has been reported previously [11]). Fusion 2 produced MAb 43-4. All MAbs reacted with the purified MOMP, but not with the purified lipopolysaccharide, in the ELISA. In immunoblot analysis with a whole-cell lysate of strain 8025 as the antigen, MAbs 59-8-2, 34-2-1, 81-7-5, 52-21-4, and 60-3-5 all reacted with a protein band with an apparent molecular mass of 40 kDa, a value that is similar to that for the MOMP of H. somnus; however, MAbs 57-16-2 and 43-4 failed to react with the MOMP in an immunoblot analysis (Fig. 1). Therefore, nonradioimmunoprecipitation analysis was performed to confirm the specificity of MAbs 57-16-2 and 43-4 for the MOMP. In this experiment, MAbs 57-16-2 and 43-4 precipitated the biotinlabeled MOMP (Fig. 2, lanes 5 and 7). In contrast, MAbs 2257
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TABLE 1. Immunological analysis of MAb reactivity by ELISA MAb
59-8-2 34-2-1 81-7-5 52-21-4 60-3-5 57-16-2 43-4
1 2 3 4 5 6 7 8 910
A492' with the following antigen:
OMP
MOMP
LPSb
1.92 0.70 1.78 2.18 2.10 2.62 2.87
0.85 1.03 1.42 0.53 0.56 0.75 0.53
0.05 0.08 0.04 0.04
0.06 0.02 0.04
a ELISA value taken 30 min after addition of substrate; the value for supernatants from a control mouse myeloma culture was subtracted from each value. b LPS, lipopolysaccharide.
59-8-2, 34-2-1, and 81-7-5 failed to precipitate the biotinlabeled MOMP (Fig. 2, lanes 1, 2, and 6), indicating that the MOMP epitopes recognized by these MAbs are not antibody accessible in the presolubilized immunoprecipitation system. In control experiments, MAbs 27-1, directed to a heat-modifiable OMP of H. somnus (10), and 1A5, directed to Bacillus cereus flagella, were used as a specificity control and an unrelated negative control, respectively. Protein precipitated with MAb 27-1 was resolved to two bands with apparent molecular masses of 28 and 37 kDa corresponding to a non-heat-modified form and a heat-modified form, respectively (Fig. 2, lane 8), whereas MAb lA5 failed to precipitate any proteins (Fig. 2, lane 9). From these results, we conclude that the MAbs developed in this study are specific for the MOMP. By using immunogold probes, we demonstrated that MAbs 52-21-4, 60-3-5, 57-16-2, and 43-4 bind to the surface of H. somnus bacteria (Table 2). Representative electron micrographs are shown in Fig. 3. In epitope conservation studies, the MAbs showed five distinct reactivity patterns with the panel of strains (Table 2). MAb 57-16-2 reacted with
1 234567 9467-
433020.1FIG. 1. Immunoblot analysis of MAb specificity. A whole-cell lysate of H. somnus 8025 was used as the antigen. Lanes: 1, 59-8-2; 2, 34-2-1; 3, 52-21-4; 4, 60-3-5; 5, 57-16-2; 6, 81-7-5; 7, 43-4. Molecular mass standards (in kilodaltons) are noted on the left.
946743-
3020.1-
ep
4. 4mbb
FIG. 2. Nonradioimmunoprecipitation analysis of the specificity of MAbs. Solubilized, biotinylated cellular proteins of H. somnus 8025 were used as the antigen. Lanes: 1, 59-8-2; 2, 34-2-1; 3, 52-21-4; 4, 60-3-5; 5, 57-16-2, 6, 81-7-5; 7, 43-4; 8, 27-1 (anti-H. somnus heat-modifiable OMP, as a specificity control); 9, 1A5 (anti-B. cereus flagella, as a negative control); 10, solubilized, biotinylated cellular proteins. Molecular mass standards (in kilodaltons) are noted on the left.
an epitope present on all of the strains tested, and MAbs 59-8-2, 34-2-1, and 81-7-5 recognized epitopes that were conserved in most of the strains tested. In contrast, MAbs 52-21-4, 60-3-5, and 43-4 reacted with limited numbers of the strains tested. In cross-reactivity studies, all of the MAbs but one showed no cross-reactivity with the 14 species and subspecies of gram-negative bacteria tested. MAb 57-16-2 was cross-reactive with H. haemoglobinophilus. From the results of characterization of the MAbs specific for the MOMP (Table 2), we demonstrated that there are at least five epitopes on the MOMP of H. somnus. Furthermore, the expression of at least three cell surface-exposed epitopes on the MOMP indicates that the MOMP is antibody accessible on the cell surface and may have an important role in immunity to the disease caused by H. somnus. Common surface-exposed epitopes must be identified on the MOMP if that protein is to be considered as a vaccine antigen. MAb 57-16-2 is of particular interest, since it reacts with all of the H. somnus, H. agni, and H. ovis strains tested and can bind to the surface of whole cells. The existence of an antibody-accessible and conserved epitope on the MOMP provides an attractive target for development of a new vaccine. Whether this MAb shows a protective effect is of great interest and will have to be evaluated in future studies. On the other hand, characterization of MAbs 52-21-4, 60-3-5, and 43-4 revealed that the MOMP possesses at ieast two surface-exposed epitopes shared by limited numbers of H. somnus, H. agni, and H. ovis strains tested. In human pathogens like H. influenzae and Neissenia gonorrhoeae, the MOMPs (porin proteins) exhibit some antigenic heterogeneity with regard to the antigenic determinants involving the potential protective effect in in vivo and in vitro model systems (3, 7, 8, 12). If the variable epitopes on the MOMP of H. somnus are immunodominant and involved in protective immunity to infection, they may result in production of antibodies having limited reactivity with heterologous strains, allowing other strains to cause recurrent infection. Thus, the antigenic differences in the MOMP molecules
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TABLE 2. Summary of reactivity of MAbs directed to the MOMP of H. somnus No. of strains reactive with
MAbW
Immunoelectron
Immunoblot
microscopy Immunoprecipitation Total H. H. ovis H.alnus 4gns analysis (n =49) (%)reutalyirslteut (n = 1) (n = 3) (n = 45) + 48 (98) 3 44b 1 + 48 (98) 3 1 44" + 44 (90) 3 1 40 + + + 16 (33) 0 1 15C + + + 16 (33) 0 1 15C + + 49 (100) 3 1 45 + + 20 (41) 0 0 20
MAb
59-8-2 34-2-1 81-7-5 52-21-4 60-3-5 57-16-2 43-4
Reactivity was tested by immunodot assay. The two MAbs reacted with the same strains. c The two MAbs reacted with the same strains.
a
b
among strains may be a potential drawback in using the MOMP as a vaccine component. MAbs 59-8-2, 34-2-1, and 81-7-5 recognized epitopes that are present on most of the strains tested. However, from the present results obtained with immunoelectron microscopy, the epitopes recognized by these MAbs appear to be inaccessible to antibodies in the native state in whole H. somnus cells, suggesting that these epitopes are not involved in protective immunity to infection. Cross-reactivity studies revealed that the MAbs directed to the MOMP are specific for H. somnus, H. agni, and H. ovis, except that MAb 57-16-2 reacted with H. haemoglobinophilus. H. haemoglobinophilus is an unusual pathogen in cattle and sheep. Therefore, from the standpoint of clinically relevant isolates, MAb 57-16-2 is highly specific for H. somnus, H. agni, and H. ovis. An MAb reacting with an accessible, surface-exposed epitope present on all strains of H. somnus, H. agni, and H. ovis could be useful for development of a diagnostic test for the diseases caused by these pathogens. In this study, we developed MAbs specific for the MOMP
and demonstrated both the conserved surface-exposed epitope with potential for development of a vaccine and a diagnostic test and the variable surface-exposed epitopes responsible for antigenic differences among strains on the MOMP molecule. Studies to evaluate the antigen determinants defined by the MAbs further as targets for protective immunity to infection are in progress.
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D
FIG. 3. Immunoelectron micrographs of H. somnus 8025 whole cells incubated with MAbs 34-2-1 (A), 81-7-5 (B), 52-21-4 (C), 57-16-2 (D), and 43-4 (E) and a P3U1 cell culture supernatant (as a negative control) (F) and then goat anti-mouse immunoglobulin G conjugated to colloidal gold spheres.
6. Leong, M. M. L., and G. R. Fox. 1990. Luminescent detection of immunodot and Western blots. Methods Enzymol. 184:442-451. 7. Munson, R. S., Jr., J. L. Shenep, S. J. Barenkamp, and D. M. Granoff. 1983. Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates. J. Clin. Invest. 72:677-684. 8. Murphy, T. F., and L. C. Bartos. 1988. Purification and analysis with monoclonal antibodies of P2, the major outer membrane protein of nontypable Haemophilus influenzae. Infect. Immun. 56:1084-1089. 9. Robertson, S. M., C. F. Frisch, P. A. Gulig, J. R. Kettman, K. H. Johnston, and E. J. Hansen. 1982. Monoclonal antibodies directed against a cell surface-exposed outer membrane protein of Haemophilus influenzae type b. Infect. Immun. 36:80-88. 10. Tagawa, Y., M. Haritani, H. Ishikawa, and N. Yuasa. 1993. Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus. Infect. Immun. 61:1750-1755. 11. Tagawa, Y., H. Ishikawa, and N. Yuasa. 1993. Purification and partial characterization of the major outer membrane protein of Haemophilus somnus. Infect. Immun. 61:91-96. 12. Visji, M., K. Zak, and J. E. Heckels. 1986. Monoclonal antibodies to gonococcal outer membrane protein IB: use in investigation of the potential protective effect of antibodies directed against conserved and type-specific epitopes. J. Gen. Microbiol. 132:1321-1329.
