Vol. 58, No. 12
INFECTION AND IMMUNITY, Dec. 1990, p. 3980-3987
0019-9567/90/123980-08$02.00/0 Copyright ©) 1990, American Society for Microbiology
Identification of Seven Surface-Exposed Brucella Outer Membrane Proteins by Use of Monoclonal Antibodies: Immunogold Labeling for Electron Microscopy and Enzyme-Linked Immunosorbent Assay A. CLOECKAERT,l* P. DE WERGIFOSSE,l G. DUBRAY,2 AND J. N. LIMET' Unit of Experimental Medicine, Catholic University of Louvain, 75 avenue Hippocrate, B-1200 Brussels, Belgium,' and Institut National de la Recherche Agronomique, Station de Pathologie de la Reproduction, 37380 Nouzilly, France2 Received
5
July 1990/Accepted 12 September 1990
A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide 0 side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.
Brucellae are facultative intracellular bacteria which develop mainly in the reticuloendothelial system and occasionally in other target organs, such as joints and placenta. The Brucella cell envelope is a three-layered structure in which an inner or cytoplasmic membrane, a periplasmic space, and an outer membrane can be differentiated (10). Brucella cell walls consist of a peptidoglycan layer strongly associated with the outer membrane. The outer membrane contains lipopolysaccharide (LPS), proteins, and phospholipids. Outer membrane proteins (OMPs) can be extracted from the Brucella cell by Zwittergent or sodium dodecyl sulfate (SDS) without using lysozyme (if the bacteria have not been inactivated before extraction) or with lysozyme (if the cells have been inactivated by Formalin or heat) (7, 23, 38). The major Brucella OMPs are group 2 porin proteins (5, 23, 33, 39), also called 36- to 38-kDa proteins (8); group 3 proteins, also called 25- to 27-kDa proteins; and a lipoprotein covalently linked to peptidoglycan (13). Group 1 minor proteins have a molecular mass of 88 to 94 kDa (38, 39). Killed bacterial vaccines (21, 22, 26, 28, 31) or chemical fractions extracted from bacterial cells (6, 7, 19, 31, 32, 42) can confer good immunity to mice, and this immunity can be transferred by either immune sera (2, 26, 28, 30, 36) or immune splenic cells (20, 27-29). Two fractions are of particular interest: the Brucella abortus SDS-insoluble (SDS-I) cell wall fraction (6-8), which contains the two major OMPs of 25 to 27 and 36 to 38 kDa strongly associated with peptidoglycan, and the smooth LPS fraction (S-LPS). SDS-I fractions or cell walls of B. melitensis contain another
*
major band of 31 to 34 kDa, which is a minor band in B. abortus strains (G. Dubray, Ph.D. thesis, Universite de Paris-Sud, 1987). Immunization with isolated major bands of the SDS-I fraction do not induce such good protection as the entire fraction does (6, 7). S-LPS contaminating the SDS-I fractions of smooth (S) Brucella strains may play an important role in protection, since SDS-I fractions isolated from rough (R) Brucella strains and showing the same protein pattern in SDS-polyacrylamide gel electrophoresis (PAGE) are much less protective than those isolated from S strains (6; Dubray, Ph.D. thesis). The protective role of antibodies against S-LPS was emphasized by the fact that anti S-LPS monoclonal antibodies (MAbs) confer good protection in passive-immunization experiments in mice (18, 22). Two immunoglobulin M (IgM) MAbs specific for the Brucella porins, and produced by Montaraz et al. (22), failed to confer protection in mice. These anti-porin MAbs did not bind on the cell surface of B. abortus. Our purpose was to produce a panel of MAbs against the Brucella OMPs to identify the proteins and the epitopes exposed on the surface of the bacteria. MATERIALS AND METHODS Bacterial strains. Bacterial strains were obtained from the Institut National de Recherches Veterinaires, Brussels, Belgium (B. abortus B3, B19, 99, 2308, and 45/20) or from the Station de Pathologie de la Reproduction, Nouzilly, Tours, France (B. abortus 544 and B. melitensis H38 and B115). Whole bacterial S and R cells used for binding studies. Cultures were grown at 37°C for 48 h on Trypticase soy agar (BioMdrieux, Marcy-l'Etoile, France) slants supplemented with 0.1% (wt/vol) yeast extract (Difco Laboratories, De-
Corresponding author. 3980
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SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS
troit, Mich.). S cells were harvested by gentle agitation in sterile distilled water. The purity and phase (S or R) were checked by using standard procedures (1). Dilutions were made in phosphate-buffered saline containing 0.5% (vol/vol) Tween 80, and the number of cells was determined by optical density measurements at 600 nm in a spectrophotometer (optical density = 0.165 for 109 cells per ml for a 1-cm light path). Cells were killed by addition of a 5% (vol/vol) peracetic acid solution (Aldrich-Chimie, Strasbourg, France) at 20 ,uI/ml to cells present at 10'0/ml and incubated overnight at room temperature. Before use, the fact that cell killing had occurred was checked by taking 1 ml of each cell suspension and, after three washes in sterile distilled water, spreading 0.2 ml on petri dishes containing the culture medium. Bacterial fractions. Cell walls and SDS-I fractions were extracted as previously described (8). Briefly, cells were inactivated by heating at 65°C for 1 h and broken with glass beads in a Braun MSK Homogenizer or a Dyno-Mill apparatus (W. A. Bachofen, Basel, Switzerland). Crude cell walls were recovered by centrifugation (at 53,000 x g and 4°C for 1.5 h). Cell walls from B. melitensis B115(R) (CW B115R) and B. abortus 99(S) (CW 99S) were obtained by treating crude cell walls with 1% Triton X-100 in 0.2 M NaCl-0.01 M MgCl2 for 30 min at 20°C, washing six times in distilled water, and lyophilizing. SDS-I fractions from B. melitensis H38(R) and B. abortus B19(S) were obtained by treating crude cell walls in 4% SDS solution in the Laemmli sample buffer for electrophoresis. Insoluble fractions were recovered by centrifugation, washed six times in distilled water, and then lyophilized. Cell extracts of B. melitensis B115(R) were obtained by sonication. Cells were inactivated by heat at 65°C for 1 h, washed three times in 0.9% NaCl, and sonicated for 15 min in 1 mM EDTA-30 mM Tris (pH 8). The sonicated cells were then centrifuged for 10 min at 4,000 x g, and the supernatant was recovered. LPS fractions. S-LPS fractions of B. abortus B3 (S-LPS of A > M specificity) and B. melitensis 16M (S-LPS of M > A specificity) were prepared by the phenol-water method (16). The rough LPS fraction of B. melitensis B115(R) (R-LPS) was obtained by the phenol-water-chloroform-petroleum ether method of Galanos et al. (11). Mice, immunizations, and hybridomas. BALB/c mice were immunized by 3 weekly intraperitoneal injections of 100 ,ug of SDS-I fraction or cell walls or 109 heat-killed B. abortus 45/20 cells. After 3 months, mice were boosted intraperitoneally with 1 mg of SDS-I fraction or cell walls or with 109 heat-killed B. abortus 45/20 cells. BALB/c mice were also infected by intraperitoneal injection of 109 B. abortus 45/ 20(R), B. melitensis B115(R), or B. abortus B3(S) cells. After 4 months, mice were boosted intraperitoneally with 109 heat-killed B. abortus 45/20(R), B. melitensis B115(R), or B. abortus 99(S) cells. Four days after the boost injection, spleen cells were fused with cells of the NSO nonsecreting myeloma cell line at a ratio of 5:1. After fusion, cells were suspended in selective hypoxanthine-aminopterin-thymidine-containing medium and seeded in 96-well microtiter plates at 5 x 104 splenocytes per well. Anti-Brucella hybridomas were screened by the enzyme-linked immunosorbent assay (ELISA) and cloned by the limiting-dilution technique. Ascitic fluids were prepared in BALB/c mice by the method of Limet et al. (18). Reagents. The following reagents were used: glycinebuffered saline (GBS) (0.17 M NaCl, 0.1 M glycine, 6 mM NaN3 [pH 9.2]); GBS-EDTA-Tw (GBS plus 50 mM EDTA and 0.1% Tween 80 [final pH 9.2]); citrate-phosphate buffer
3981
(0.051 M Na2HPO4, 0.024 M citric acid [pH 5]); NaCl-Tw (0.15 M NaCl, 0.01% Tween 20); Tris-buffered saline (TBS) (0.15 M NaCl, 10 mM Tris hydrochloride [pH 7.5]); Tw-TBS (TBS containing 0.05% [vol/vol] Tween 20); TBS-3% BSA (TBS containing 3% [wt/vol] bovine serum albumin [BSA]); Tw-TBS-1% BSA (Tw-TBS containing 1% [wt/vol] BSA); PBS-Tw (phosphate-buffered saline [PBS; pH 7.5] containing 0.05% Tween 20); and PBS-3% BSA (PBS containing 3% [wt/vol] BSA). Antisera. Rabbit anti-mouse immunoglobulin antiserum was produced by repeated intradermal injections of 100 ,ug of mouse IgG. Rabbits were injected and bled every 2 weeks for several months. The best bleedings were pooled. Rabbit anti-Brucella antiserum was produced by repeated intradermal injections of 109 B. abortus 45/20(R) or B. abortus 99(S) killed cells. The immunoglobulin fraction was obtained by (NH4)2SO4 precipitation. Peroxidase conjugation. Protein A (Sigma, St. Louis, Mo.) was conjugated with horseradish peroxidase (Sigma) by a modification of the method of Nakane and Kawaoi (24) as described by Dubray and Limet (9). The protein/enzyme ratio was 1:2. ELISA. Supernatants of hybridoma cultures or ascitic fluids were assayed for antibody activity by solid-phase ELISA against antigens in fivefold-diluted GBS in water (40). The antigens used to coat the plates consisted of SDS-I fractions or cell walls (20 ,xg/ml), S-LPS (4 ,ug/ml), R-LPS (10 ,ug/ml), and whole bacterial cells of B. abortus or B. melitensis (A6w = 1.5). Whole bacterial cells were immobilized on microtiter plates (Greiner Labortechnic-Stuttgart) by using the intermediate of rabbit anti-Brucella immunoglobulin at a coating concentration of 10 ,ug/ml. Hybridoma supernatants were serially diluted (1/10 to 1/590,490) in GBS-EDTA-Tw. Binding of the antibodies to SDS-I fractions or cell walls was visualized by using rabbit anti-mouse immunoglobulin antiserum diluted in GBS-EDTA-Tw and peroxidase-conjugated protein A diluted in GBS-EDTA-Tw containing 2% fetal calf serum. Binding to whole bacterial cells was visualized by using peroxidase-conjugated goat anti-mouse immunoglobulins (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) diluted in GBS-EDTA-Tw containing 2% fetal calf serum. Excess reagents between the different incubations were removed by five washings with NaCl-Tw. O-Phenylenediamine (0.4%, wt/vol) and 2 mM H202 in citrate-phosphate buffer were used to visualize peroxidase activity. The ELISA titers were estimated as the highest dilution giving a difference in A492 -A620 of more than twice the mean of the corresponding blank values. Immunogold labeling. A 5-pl sample of cell suspension in water (1010 cells per ml) was deposited on carbon-Formvarcoated 200-mesh copper grids. After being air dried (about 30 min), grids were incubated in the following reagents: PBS-3% BSA (30 min at 37°C), ascitic fluid containing the MAb diluted 1/50 in PBS-Tw (2 h at 37°C), goat anti-mouse biotinylated immunoglobulin (Amersham) diluted 1/200 in PBS-Tw (1 h at room temperature), and 15-nm gold-labeled streptavidin (Amersham) diluted 1/20 in PBS-Tw. Washings between incubation periods were performed with NaCl-Tw. After four washings in NaCl-Tw and four washings in distilled water, grids were observed in a transmission electron microscope (Philips CM10). Immunoblot techniques. The protein antigens were separated by the SDS-PAGE method, originally described by Laemmli (15), in a 2001 vertical electrophoresis unit (LKBProdukter AB, Bromma, Sweden). After electrophoresis, the proteins were transferred to nitrocellulose by using a
_
INFECT. IMMUN.
CLOECKAERT ET AL.
3982
TABLE 1. Number of OMP-specific hybridomas obtained by immunization with different Brucella fractions or whole cells OMPs (kDa)
Live 45/20(R)
10 16.5 19 25-27 31-34 36-38 89 R-LPS
[killed 45/20(R)] 3 3 2 11 0 5 0 22 0
S-LPS a b
No. of hybridomas obtained with cells or fractions used for first immunization [antigen used for boosting] Killed 45/20(R) Live B115(R) Live B3(S) SDS-I H38(R) SDS-I H38(R) CW B115(R) [CW B115(R)]a [killed 45/20(R)] [killed B115(R)] [killed 99(S)] [SDS-I H38(R)] [SDS-I B19(S)]
CW, Cell walls. ND, Not determined.
0 3 0 0 0 0 0
0 26 7 17 0 0 1 3 23
NDb ND
0 0 0 2 0 0 0 0 >50
Transblot apparatus (Biolyon, Dardilly, France) at 12 V for 2 h. Unoccupied sites on the nitrocellulose membranes were blocked by a 30-min incubation in TBS-3% BSA at room temperature with agitation. The membranes were then successively incubated overnight at room temperature with hybridoma supernatants diluted 1/2 in Tw-TBS-1% BSA, 1 h with rabbit anti-mouse immunoglobulin antiserum, and 1 h with peroxidase-conjugated protein A, diluted 1/250 and 1/1,000, respectively, in the same buffer. Washings between incubation periods were performed with Tw-TBS. After two washings in Tw-TBS and two washings in TBS, the blots were developed by incubation at room temperature in a solution of TBS containing 0.06% (wt/vol) 4-chloro-1-naphthol (Bio-Rad, Richmond, Calif.) and 5 mM H202. The reaction was stopped by washing in distilled water.
89 kDa :),
36-38 kDa
31-34 kDa
1
P
l9 kDa )
16.5 kDa )
Ilif
10 kDa 0R-LPS )'.
_
_
*IS
_.
3
0 4 0 1 0 0 1 ND ND
RESULTS Isolation of Brucella OMP-specific hybridomas. Tissue culture supernatants from hybridomas were initially screened for the presence of antibody by ELISA with the antigen used for immunization, i.e., live or killed whole R cells, SDS-I fractions, or cell walls of B. abortus or B. melitensis strains. Reactivity against S-LPS or R-LPS was also monitored by ELISA. Most anti-OMP hybridomas produced antibodies against the major OMPs of 25 to 27 and 36 to 38 kDa (Table 1). By infecting mice with the B. abortus 45/20(R) strain, we obtained antibodies mostly against R-LPS (22 of 46, or 48%) and the 25- to 27-kDa OMP (11 of 46, or 24%). By infecting
I 92 kDa 67 kDa ;
i
I
'i
.i-fI :.
AM:.
_L
43 IDa
_%W.
30 kDa
^.
-
..
r-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2
0 0 0 2 0 17 0 ND ND
MAb isotypes. MAb isotypes were determined by direct latex agglutination immunoassay of the culture supernatants. Latex particles were coated with rat monoclonal anti-mouse IgGl, IgG2a, IgG2b, IgG3, and IgM and polyclonal antimouse IgA by a method similar to those described by Limet et al. (17).
_ _ *_ =L _
25-27 kDa I0
0 0 0 8 1 0 0 ND ND
21 kDa
6
FIG. 1. Western immunoblot of OMPs of B. melitensis B115(R). Lane 1 to 10 show immunoblots of sonicated cell extracts of B. melitensis B115(R) with anti-R-LPS MAb A68/29C05/B06 (lane 1), anti-10-kDa MAb A68/08E07/B11 (lane 2), anti-16.5-kDa MAb A68/ 04G01/C06 (lane 3), anti-19-kDa MAb A68/25H10/A05 (lane 4), anti-25- to 27-kDa MAb A59/05F01/CO9 (lane 5), anti-31- to 34-kDa MAb A59/1OF09/G1O (lane 6), anti-36- to 38-kDa MAb A63/05A07/ A08 (lane 7), anti-89-kDa MAb A53/1OB02/A01 (lane 8), serum from B. melitensis B115(R)-infected mice (lane 9), and serum from B. abortus 45/20(R)-infected mice (lane 10).
14.4 kDa
*4 1
2
3
4
5
FIG. 2. Immunoblot of SDS-I fraction of B. abortus B19(S) with anti-36- to 38-kDa MAb A68/25G05/A05. Lanes: 1, Coomassie blue-stained molecular mass standards; 2, Coomassie blue-stained SDS-I fraction not heated in SDS; 3, Coomassie blue-stained SDS-I fraction heated for 10 min at 100°C in SDS; 4, immunoblot of SDS-I fraction not heated in SDS with MAb A68/25G05/A05; 5, same as lane 4 but heated for 10 min at 100°C in SDS.
VOL. 58, 1990
SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS
_
92 kDa 67
*
kDa
43 kDa
%
30 kDa
21 kDa 14.4
kDa
1
2
3
4
FIG. 3. Immunoblot of SDS-I fraction of B. abortus B19(S) with anti-36- to 38-kDa MAb A63/03H02/BO1 produced by using the SDS-I fraction as the immunogen (lane 3) and with anti-36- to 38-kDa MAb A68/25G05/AO5 produced by using whole cells as the immunogen (lane 4). The color development reaction was stopped at the same time. Lane 1, Coomassie blue-stained molecular mass standards; lane 2, Coomassie blue-stained SDS-I fraction of B. abortus B19(S).
mice with the B. melitensis B115(R) strain, we obtained antibodies that were specific mostly for the 16.5-kDa OMP (26 of 77, or 34%) and S-LPS (23/77, or 30%). Only two anti-OMP MAbs were obtained after infection of mice with S brucellae. OMP specificity by Western immunoblot analysis. To determine the specificity of the anti-Brucella MAbs, we performed Western immunoblotting with cell walls, SDS-I fractions, and cell extracts of B. abortus or B. melitensis
3983
strains separated by SDS-PAGE (Fig. 1). Multiple bands (doublets or triplets) were observed with all the anti-25- to 27-, -31- to 34-, and -36- to 38-kDa MAbs. MAb A59/1OF09/ G10 also revealed less stained and more diffuse bands of molecular mass lower than 31 kDa. These bands were not observed with the SDS-I fraction containing large amounts of 25- to 27-kDa OMP. With some of the anti-36- to 38-kDa MAbs, a major high-molecular-mass band (>92 kDa) was also revealed when cell walls were not heated in SDS before electrophoresis (Fig. 2). This band disappeared when cell walls were heated for 10 min at 100°C. The anti-25- to 27- and -36- to 38-kDa MAbs produced by using SDS-I fractions as the immunogen reacted more strongly with the denatured antigens after SDS-PAGE than did those which bind to the cell surface and were obtained from infected mice or mice immunized with cell walls or whole bacterial cells (Fig. 3). MAbs binding to the Brucella cell surface. The ability of MAbs to bind to the cell surface of intact B. abortus and B. melitensis R and S strains was checked by ELISA. Binding of the anti-OMP MAbs was compared with binding of the MAbs directed against R-LPS and S-LPS. The strains used were B. abortus 544(R) and 544(S) and B. melitensis H38(R) and H38(S). Most (90%) of the MAbs produced by using the SDS-I fraction as the immunizing antigen did not bind to the cell surface (data not shown). Most of the other MAbs against anti-OMPs showed a better binding on the R cells, i.e., higher titers and maximal absorbances (Table 2). MAbs which bound to the cell surface of the S strains used did not bind as well as an anti-LPS MAb did. Surface exposure of OMPs was visualized by immunogold labeling (Fig. 4), with the MAbs showing the greatest binding by ELISA. The labeling obtained with the anti-10-, -16.5-, and 89-kDa MAbs on B. abortus 544(R) was comparable to results shown in Fig. 4A. The labeling of B. abortus 544(S) with the anti-10- and 16.5-kDa MAbs was comparable to results shown in Fig. 4B. Results are summarized in Table 3. As visualized on the R strains used, surface-exposed OMPs are the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs. Binding on the S B. abortus strains was
TABLE 2. ELISA binding of the anti-Brucella OMP MAbs on R and S Brucella cells B. abortus 544(R)
Specificity, immunogen, and clone
(isotyp)
(isotype)
Titer
Titer
10 kDa, live B. abortus 45/20(R), A68/07G11/C10
(IgG2a) 16.5 kDa, live B. abortus 45/20(R), A68/04G01/ C06 (IgG2a) 19 kDa, live B. melitensis B115(R), A76/18B02/ D06 (IgG2a) 25 to 27 kDa, live B. abortus 45/20(R), A68/04B10/F05 (IgG2a) 31 to 34 kDa, SDS-I fraction from H38(R), A59/ 10F09/G1O (IgG2a) 36 to 38 kDa, live B. abortus 45/20(R), A68/25G05/A05 (IgG2a) 89 kDa, cell wall from B115(R), A53/1OB02/AO1 (IgGl) R-LPS, live B. abortus 45/20(R), A68/03F03/DO5 (IgG2b) S-LPS, live B. melitensis B115(R), A76/12G12/ F12 (IgGl) Negative control ascitic fluid (nonrelated MAb)
B. abortus 544(S)
Max Tier Max Titer absorbance absorbance
B. melitensis H38(R)
TtrMax
absorbance
B. melitensis H38(S) Max Titer
absorbance
810
1.049
30
0.435
2,430
1.146
270
0.434
810
1.270
30
0.713
2,430
1.376
270
0.786
270
0.665
270
1.265
270
0.462
270
1.640
65,610
1.474
810
1.367
65,610
1.516
270
0.711
270
0.857
30
0.619
590,490
1.137
7,290
1.421
196,830
1.324
2,430
1.571
196,830
1.181
90
0.730
270
0.590
10
0.314
2,430
0.497
30
0.243
7,290
2.011
270
1.025
7,290
1.068
90
0.761
90
0.424
590,490
2.256
90
0.393
590,490
2.364
90
0.304
30
0.422
30
0.341
30
0.135
aMaximal absorbance after subtraction of the blank value.
3984
CLOECKAERT ET AL.
INFECT. IMMUN.
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FIG. 4. Immunoelectron microscopy of Brucella OMPs. The figure shows labeling of B. abortus 544(R) by the anti-19-kDa MAb
A76/18B02/D06 (A), anti-25- to 27-kDa MAb A68/04B10/F05 (C), anti-31- to 34-kDa MAb A59/1OF09/G10 (E), and anti-36- to 38-kDa MAb A68/25G05/A05 (G), and labeling of B. abortus 544(S) by the same MAbs in the same order (B, D, F, and H, respectively). Magnification, x 57,000.
VOL. 58, 1990
SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS
3985
TABLE 3. Electron microscopy binding of the anti-Brucella OMP MAbs on R and S Brucella cells Labeling of:
Specificity, immunogen, and clone (isotype) 10 kDa, live B. abortus 45/20(R), A68/07G11/C10 (IgG2a) 16.5 kDa, live B. abortus 45/20(R), A68/04G01/C06 (IgG2a) 19 kDa, live B. melitensis B115(R), A76/18B02/D06 (IgG2a) 25 to 27 kDa, live B. abortus 45/20(R), A68/04B10/F05 (IgG2a) 31 to 34 kDa, SDS-I fraction from H38(R), A59/1OF09/G10 (IgG2a) 36 to 38 kDa, live B. abortus 45/20(R), A68/25G05/A05 (IgG2a) 89 kDa, cell wall from B115(R), A53/1OB02/A01 (IgGl) R-LPS, live B. abortus 45/20(R), A68/03F03/D05 (IgG2b) S-LPS, live B. melitensis B115(R), A76/12G12/F12 (IgGl) Negative control ascitic fluid (nonrelated MAb) a ND, Not determined.
observed for all MAbs except for the anti-31- to 34-kDa MAb and the anti-89-kDa MAb. However, the anti-31- to 34-kDa MAb bound on both the S and R B. melitensis strains used. The anti-S-LPS MAb did not bind on the R strains.
DISCUSSION We report the production of a panel of MAbs specific for seven Brucella OMPs. As shown by Western immunoblotting, most antibodies were specific for the major OMPs: the 25- to 27-kDa OMP (8) and the 36- to 38-kDa OMP, which is a porin (5). The other MAbs were specific for the 10-, 16.5-, 19-, 31- to 34-, and 89-kDa OMP. By infecting mice with B. abortus 45/20(R), we obtained antibodies directed mainly against R-LPS and the 25- to 27- and 36- to 38-kDa OMPs. These antigens are the most abundant in the cell wall as described in the literature (8, 33, 38, 39), but not necessarily the most immunogenic of the B. abortus rough strains as shown by the immunoblots with the sera of B. abortus 45/20-infected mice. The immunogenicity of OMPs seems to be related to the S-R variation in brucellae. The immunogenicity of Braun's outer membrane lipoprotein was also greatly influenced by S-R variation and the presence of a capsule in Escherichia. coli: elevated titers were obtained with either live or heat-killed R cells (4). After infection with B. melitensis B115(R), most of the antibodies found were specific for the 16.5-kDa minor OMP and for S-LPS. The fact that MAbs specific for S-LPS were obtained by infection with this R strain has yet to be explained. In Western immunoblottings after SDS-PAGE, the anti25- to 27- and 36- to 38-kDa MAbs generated by immunization with SDS-I fractions reacted more strongly with the denatured antigens than did those which bind to the cell surface and were obtained after immunization with cell walls or whole bacterial cells. This could be explained by the fact that the MAbs which bind to the cell surface recognize conformational epitopes while the others recognize mainly denatured sequential epitopes or epitopes that are inaccessible to antibodies in cell walls. Some anti-36- to 38-kDa MAbs which bind to the cell surface, like MAb A68/25G05/ A05, probably recognize a conformational epitope. Indeed, immunoblot analysis with this MAb revealed a high-molecular-mass band (>92 kDa) in an SDS-I fraction that had not been heated in SDS, which disappeared when the SDS-I fraction was heated at 1000C. This high-molecular-mass band probably corresponds to the porin trimer described by Moriyon and Berman (23). The anti-25- to 27-, 31- to 34-, and 36- to 38-kDa MAbs revealed multiple bands in sonicated cell extracts. The multiple band pattern of the group 2 or 36-
B.
abortus
B. abortus
B. melitensis
544(R)
B. melitensis
544(S)
H38(R)
+ + + + + + + +
+ + + +
NDa ND ND ND
H38(S) ND ND ND ND + ND ND ND +
-
-
+
+
ND ND ND -
+ +
to 38-kDa OMP has already been described by Douglas et al.
(5). They postulated that the multiple bands can represent modified forms of a single protein and that since the protein was obtained after lysozyme digestion of peptidoglycan, it seems possible that different bands contain peptidoglycan fragments of different sizes or different amounts of adherent LPS molecules or both. The same explanation can be given for the 25- to 27- and 31- to 34-kDa OMPs since these proteins are also released from cell walls only by lysozyme treatment. The multiple bands pattern of the 25- to 27-kDa OMP can also be explained by the fact that this OMP appears in gel as a glycoprotein (7) after silver staining (periodic oxydation) and that it fails to migrate to one distinct position on the electrophoresis gel, probably owing to the heterogeneity of the oligosaccharide components. This type of behavior has been encountered for Myxococcus xanthus (12) and other microorganisms such as Dictyostelium discoideum (35). As visualized by electron microscopy and as indicated by ELISA binding of MAbs, the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface of intact R Brucella strains. In ELISA all the anti-OMP MAbs except the anti-19-kDa MAb showed a better binding on the R Brucella strains than on the corresponding S strains. Immunoelectron microscopy showed that all MAbs used, except the anti-31- to 34-kDa MAb and the anti-89-kDa MAb, bound on the R and S Brucella strains. The anti-31- to 34-kDa MAb bound on B. melitensis R and S cells but not on B. abortus S cells, for which this OMP is a minor protein (Dubray, Ph.D. thesis). Surface exposure of the 89-kDa OMP was observed only for the R B. abortus strain used. S-LPS probably hinders the binding of these MAbs on epitopes of the OMPs close to the S-LPS. Such steric hindrance has already been described for Coxiella burnetii (14), members of the family Enterobacteriacae (3, 37, 41), and Neisseria gonorrhoeae and N. meningitidis (25, 34). All MAbs will be used in passive protection experiments in mice to determine whether Brucella OMPs are potentially useful vaccine antigens. Protection experiments have already been performed by Montaraz et al. (22) with O-polysaccharide-specific MAbs and two IgM MAbs specific for the porn of B. abortus. The latter MAbs failed to confer protection. These MAbs, however, did not bind on the cell surface of B. abortus 2308. The same results were observed in ELISA with all our anti-porin MAbs on this strain (data not shown). On other less virulent strains such as B. abortus 99 and B19, mainly the anti-25- to 27- and to a less extent the
3986
CLOECKAERT ET AL.
anti-36- to 38-kDa MAbs bound to the cell surface, but at much higher concentrations. Binding of anti-LPS MAbs is, however, very efficient. Therefore, anti-OMP MAbs will probably be less efficacious than anti-LPS MAbs for opsonizing brucellae and promoting their ingestion and killing by macrophages. ACKNOWLEDGMENTS We are most grateful to J. M. Verger, R. Wijffels, P. Michel, and M. Grayon for culture and control of bacterial strains. We thank G. Bezard, J. C. Nicolle, A. Van Rompaye, and J. Van Broeck for expert technical assistance. A. Cloeckaert is supported by the Institut National pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture. The work was supported by Biotechnology Action Programme BAP-012-B (GDF) from the European Economic Community.
LITERATURE CITED 1. Alton, G. G., L. M. Jones, R. D. Angus, and J. M. Verger. 1988. Techniques for the brucellosis laboratory. Institut National de la Recherche Agronomique, Paris. 2. Bascoul, S., A. Cannat, M. F. Huguet, and A. Serre. 1978. Studies on the immune protection to murine experimental brucellosis conferred by Brucella fractions. I. Positive role of immune serum. Immunology 35:213-221. 3. Bentley, A. T., and P. E. Klebba. 1988. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface. J. Bacteriol. 170:1063-1068. 4. Braun, V. 1975. Covalent lipoprotein from the outer membrane of Escherichia coli. Biochim. Biophys. Acta 415:335-377. 5. Douglas, J. T., E. Y. Rosenberg, H. Nikaido, D. R. Verstraete, and A. J. Winter. 1984. Porins of Brucella species. Infect. Immun. 44:16-21. 6. Dubray, G. 1987. Protective antigens in brucellosis. Ann. Inst. Pasteur Microbiol. 138:84-86. 7. Dubray, G., and G. Bezard. 1980. Isolation of three protective cell-wall antigens of Brucella abortus in experimental brucellosis in mice. Ann. Rech. Vet. 11:367-373. 8. Dubray, G., and C. Charriault. 1983. Evidence of three major polypeptide species and two major polysaccharide species in the Brucella outer membrane. Ann. Rech. Vet. 14:311-318. 9. Dubray, G., and J. Limet. 1987. Evidence of heterogeneity of lipopolysaccharides among Brucella biovars in relation to A and M specificities. Ann. Inst. Pasteur Microbiol. 138:27-37. 10. Dubray, G., and M. Plommet. 1976. Structure et constituants des Brucella. Caracterisation des fractions et proprietes biologiques. Dev. Biol. Stand. 31:68-91. 11. Galanos, C., 0. Luderitz, and 0. Westphal. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9:245-249. 12. Gill, J., E. Steliwag, and M. Dworkin. 1985. Monoclonal antibodies against cell-surface antigens of developing cells of Myxococcus xanthus. Ann. Inst. Pasteur Microbiol. 136A:11-18. 13. Gomez-Miguel, M. J., and I. Moriyon. 1986. Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp. Infect. Immun. 53:678-684. 14. Hackstadt, T. 1988. Steric hindrance of antibody binding of surface proteins of Coxiella burnetii by phase I lipopolysaccharide. Infect. Immun. 56:802-807. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 16. Leong, D., R. Diaz, K. Milner, J. Rudbach, and J. B. Wilson. 1970. Some structural and biological properties of Brucella endotoxin. Infect. Immun. 1:174-182. 17. Limet, J. N., A. Berbinschi, A. Cloeckaert, C. L. Cambiaso, and P. L. Masson. 1988. Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine. J. Med. Microbiol. 26:37-45.
INFECT. IMMUN. 18. Limet, J. N., A. M. Plommet, G. Dubray, and M. Plommet. 1987. Immunity conferred to mice by anti-LPS monoclonal antibodies in murine brucellosis. Ann. Inst. Pasteur Immunol. 138:417424. 19. Lopez-Merino, A., J. Asselineau, A. Serre, J. Roux, S. Bascoul, and C. Lacave. 1976. Immunization by an insoluble fraction extracted from Brucella melitensis: immunological and chemical characterization of the active substances. Infect. Immun. 13: 311-321. 20. Madraso, E. D., and C. Cheers. 1978. Polyadenylic acid-polyuridylic acid (polyA-U) and experimental brucellosis. 2. Macrophages as a target cells of polyA-U in experimental brucellosis. Immunology 35:77-84. 21. Montaraz, J. A., and A. J. Winter. 1986. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice. Infect. Immun. 53:245-251. 22. Montaraz, J. A., A. J. Winter, D. M. Hunter, B. A. Sowa, A. M. Wu, and L. G. Adams. 1986. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies. Infect. Immun. 51:961-963. 23. Moriyon, I., and D. T. Berman. 1983. Isolation, purification, and partial characterization of Brucella abortus matrix protein. Infect. Immun. 39:394-402. 24. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labelled antibody, a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. 25. Paques, M., J. S. Teppema, E. C. Beuvery, H. Abdillahi, J. T. Poolman, and A. J. VerkleiJ. 1989. Accessibility of gonococcal and meningococcal surface antigens: immunogold labeling for quantitative electron microscopy. Infect. Immun. 57:582-589. 26. Pardon, P. 1977. Resistance against a subcutaneous Brucella challenge of mice immunized with living or dead Brucella or by transfer of immune serum. Ann. Inst. Pasteur Immunol. 128C: 1025-1037. 27. Pavlov, H., M. Hogart, I. F. C. Mckenzie, and C. Cheers. 1982. In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection. Cell. Immunol. 71:127-138. 28. Plommet, M. 1987. Brucellosis and immunity: humoral and cellular components in mice. Ann. Inst. Pasteur Microbiol. 138:105-110. 29. Plommet, M., and A. M. Plommet. 1987. Anti-Brucella cell mediated immunity in mice vaccinated with a cell wall fraction. Ann. Rech. Vet. 18:429-437. 30. Plommet, M., and A. M. Plommet. 1983. Immune serummediated effects on brucellosis evolution in mice. Infect. Immun. 41:97-105. 31. Plommet, M., A. Serre, and R. Fensterbank. 1987. Vaccines, vaccination in brucellosis. Ann. Inst. Pasteur Microbiol. 138: 117-121. 32. Rasooly, G., A. L. Olitzki, and D. Sulitzeanu. 1966. Immunization against Brucella with killed vaccines. Immunizing activity in mice of Brucella cell-wall and of fractions derived from them. Isr. J. Med. Sci. 2:569-584. 33. Santos, J. M., D. R. Verstraete, V. Y. Perera, and A. J. Winter. 1984. Outer membrane proteins from rough strains of four Brucella species. Infect. Immun. 46:188-194. 34. Shafer, W. M. 1988. Lipopolysaccharide masking of gonococcal outer-membrane proteins modulates binding of bactericidal cathepsin G to gonococci. J. Gen. Microbiol. 134:539-545. 35. Springer, W. R., and S. H. Barondes. 1983. Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum. J. Biol. Chem. 258:4698-4701. 36. Sulitzeanu, D., L. Jones, and A. W. Stableforth. 1955. Protective activity of monospecific anti-Brucella sera in mice. Nature
(London) 175:1040-1041. 37. Van der Ley, P., 0. Kuipers, J. Tommassen, and B. Lugtenberg. 1986. 0-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae. Microb. Pathog. 1:43-49. 38. Verstraete, D. R., M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter. 1982. Outer membrane proteins of Brucella abortus: isolation and characterization. Infect. Immun. 35:979-989.
VOL. 58, 1990 39.
Verstraete,
SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS D.
R., and A. J. Winter.
1984.
Comnparison
of
sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains. Infect. Immun. 46:182-187. 40.
VolIer, A.,
D. E.
Bidwell,
and A. Bartlett.
1979. The enzyme
linked immunosorbent assay (ELISA). Dynatech Europe, Guernsey, United Kingdom.
3987
41. Voorhout, W. F., J. J. M. Leunissen-Bijvelt, J. L. M. Leunissen, and A. J. Verkleij. 1986. Steric hindrance in immunolabelling. J. Microsc. 141:303-310. 42. Winter, A. J., G. E. Rowe, J. R. Duncan, M. J. Eis, J. Widom, B. Ganem, and B. Morein. 1988. Effectiveness of natural and synthetic complexes of porin and O-polysaccharide as vaccines against Brucella abortus in mice. Infect. Immun. 56:2808-2817.
INFECTION AND IMMUNITY, Dec. 1990, p. 3980-3987
0019-9567/90/123980-08$02.00/0 Copyright ©) 1990, American Society for Microbiology
Identification of Seven Surface-Exposed Brucella Outer Membrane Proteins by Use of Monoclonal Antibodies: Immunogold Labeling for Electron Microscopy and Enzyme-Linked Immunosorbent Assay A. CLOECKAERT,l* P. DE WERGIFOSSE,l G. DUBRAY,2 AND J. N. LIMET' Unit of Experimental Medicine, Catholic University of Louvain, 75 avenue Hippocrate, B-1200 Brussels, Belgium,' and Institut National de la Recherche Agronomique, Station de Pathologie de la Reproduction, 37380 Nouzilly, France2 Received
5
July 1990/Accepted 12 September 1990
A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide 0 side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.
Brucellae are facultative intracellular bacteria which develop mainly in the reticuloendothelial system and occasionally in other target organs, such as joints and placenta. The Brucella cell envelope is a three-layered structure in which an inner or cytoplasmic membrane, a periplasmic space, and an outer membrane can be differentiated (10). Brucella cell walls consist of a peptidoglycan layer strongly associated with the outer membrane. The outer membrane contains lipopolysaccharide (LPS), proteins, and phospholipids. Outer membrane proteins (OMPs) can be extracted from the Brucella cell by Zwittergent or sodium dodecyl sulfate (SDS) without using lysozyme (if the bacteria have not been inactivated before extraction) or with lysozyme (if the cells have been inactivated by Formalin or heat) (7, 23, 38). The major Brucella OMPs are group 2 porin proteins (5, 23, 33, 39), also called 36- to 38-kDa proteins (8); group 3 proteins, also called 25- to 27-kDa proteins; and a lipoprotein covalently linked to peptidoglycan (13). Group 1 minor proteins have a molecular mass of 88 to 94 kDa (38, 39). Killed bacterial vaccines (21, 22, 26, 28, 31) or chemical fractions extracted from bacterial cells (6, 7, 19, 31, 32, 42) can confer good immunity to mice, and this immunity can be transferred by either immune sera (2, 26, 28, 30, 36) or immune splenic cells (20, 27-29). Two fractions are of particular interest: the Brucella abortus SDS-insoluble (SDS-I) cell wall fraction (6-8), which contains the two major OMPs of 25 to 27 and 36 to 38 kDa strongly associated with peptidoglycan, and the smooth LPS fraction (S-LPS). SDS-I fractions or cell walls of B. melitensis contain another
*
major band of 31 to 34 kDa, which is a minor band in B. abortus strains (G. Dubray, Ph.D. thesis, Universite de Paris-Sud, 1987). Immunization with isolated major bands of the SDS-I fraction do not induce such good protection as the entire fraction does (6, 7). S-LPS contaminating the SDS-I fractions of smooth (S) Brucella strains may play an important role in protection, since SDS-I fractions isolated from rough (R) Brucella strains and showing the same protein pattern in SDS-polyacrylamide gel electrophoresis (PAGE) are much less protective than those isolated from S strains (6; Dubray, Ph.D. thesis). The protective role of antibodies against S-LPS was emphasized by the fact that anti S-LPS monoclonal antibodies (MAbs) confer good protection in passive-immunization experiments in mice (18, 22). Two immunoglobulin M (IgM) MAbs specific for the Brucella porins, and produced by Montaraz et al. (22), failed to confer protection in mice. These anti-porin MAbs did not bind on the cell surface of B. abortus. Our purpose was to produce a panel of MAbs against the Brucella OMPs to identify the proteins and the epitopes exposed on the surface of the bacteria. MATERIALS AND METHODS Bacterial strains. Bacterial strains were obtained from the Institut National de Recherches Veterinaires, Brussels, Belgium (B. abortus B3, B19, 99, 2308, and 45/20) or from the Station de Pathologie de la Reproduction, Nouzilly, Tours, France (B. abortus 544 and B. melitensis H38 and B115). Whole bacterial S and R cells used for binding studies. Cultures were grown at 37°C for 48 h on Trypticase soy agar (BioMdrieux, Marcy-l'Etoile, France) slants supplemented with 0.1% (wt/vol) yeast extract (Difco Laboratories, De-
Corresponding author. 3980
VOL. 58, 1990
SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS
troit, Mich.). S cells were harvested by gentle agitation in sterile distilled water. The purity and phase (S or R) were checked by using standard procedures (1). Dilutions were made in phosphate-buffered saline containing 0.5% (vol/vol) Tween 80, and the number of cells was determined by optical density measurements at 600 nm in a spectrophotometer (optical density = 0.165 for 109 cells per ml for a 1-cm light path). Cells were killed by addition of a 5% (vol/vol) peracetic acid solution (Aldrich-Chimie, Strasbourg, France) at 20 ,uI/ml to cells present at 10'0/ml and incubated overnight at room temperature. Before use, the fact that cell killing had occurred was checked by taking 1 ml of each cell suspension and, after three washes in sterile distilled water, spreading 0.2 ml on petri dishes containing the culture medium. Bacterial fractions. Cell walls and SDS-I fractions were extracted as previously described (8). Briefly, cells were inactivated by heating at 65°C for 1 h and broken with glass beads in a Braun MSK Homogenizer or a Dyno-Mill apparatus (W. A. Bachofen, Basel, Switzerland). Crude cell walls were recovered by centrifugation (at 53,000 x g and 4°C for 1.5 h). Cell walls from B. melitensis B115(R) (CW B115R) and B. abortus 99(S) (CW 99S) were obtained by treating crude cell walls with 1% Triton X-100 in 0.2 M NaCl-0.01 M MgCl2 for 30 min at 20°C, washing six times in distilled water, and lyophilizing. SDS-I fractions from B. melitensis H38(R) and B. abortus B19(S) were obtained by treating crude cell walls in 4% SDS solution in the Laemmli sample buffer for electrophoresis. Insoluble fractions were recovered by centrifugation, washed six times in distilled water, and then lyophilized. Cell extracts of B. melitensis B115(R) were obtained by sonication. Cells were inactivated by heat at 65°C for 1 h, washed three times in 0.9% NaCl, and sonicated for 15 min in 1 mM EDTA-30 mM Tris (pH 8). The sonicated cells were then centrifuged for 10 min at 4,000 x g, and the supernatant was recovered. LPS fractions. S-LPS fractions of B. abortus B3 (S-LPS of A > M specificity) and B. melitensis 16M (S-LPS of M > A specificity) were prepared by the phenol-water method (16). The rough LPS fraction of B. melitensis B115(R) (R-LPS) was obtained by the phenol-water-chloroform-petroleum ether method of Galanos et al. (11). Mice, immunizations, and hybridomas. BALB/c mice were immunized by 3 weekly intraperitoneal injections of 100 ,ug of SDS-I fraction or cell walls or 109 heat-killed B. abortus 45/20 cells. After 3 months, mice were boosted intraperitoneally with 1 mg of SDS-I fraction or cell walls or with 109 heat-killed B. abortus 45/20 cells. BALB/c mice were also infected by intraperitoneal injection of 109 B. abortus 45/ 20(R), B. melitensis B115(R), or B. abortus B3(S) cells. After 4 months, mice were boosted intraperitoneally with 109 heat-killed B. abortus 45/20(R), B. melitensis B115(R), or B. abortus 99(S) cells. Four days after the boost injection, spleen cells were fused with cells of the NSO nonsecreting myeloma cell line at a ratio of 5:1. After fusion, cells were suspended in selective hypoxanthine-aminopterin-thymidine-containing medium and seeded in 96-well microtiter plates at 5 x 104 splenocytes per well. Anti-Brucella hybridomas were screened by the enzyme-linked immunosorbent assay (ELISA) and cloned by the limiting-dilution technique. Ascitic fluids were prepared in BALB/c mice by the method of Limet et al. (18). Reagents. The following reagents were used: glycinebuffered saline (GBS) (0.17 M NaCl, 0.1 M glycine, 6 mM NaN3 [pH 9.2]); GBS-EDTA-Tw (GBS plus 50 mM EDTA and 0.1% Tween 80 [final pH 9.2]); citrate-phosphate buffer
3981
(0.051 M Na2HPO4, 0.024 M citric acid [pH 5]); NaCl-Tw (0.15 M NaCl, 0.01% Tween 20); Tris-buffered saline (TBS) (0.15 M NaCl, 10 mM Tris hydrochloride [pH 7.5]); Tw-TBS (TBS containing 0.05% [vol/vol] Tween 20); TBS-3% BSA (TBS containing 3% [wt/vol] bovine serum albumin [BSA]); Tw-TBS-1% BSA (Tw-TBS containing 1% [wt/vol] BSA); PBS-Tw (phosphate-buffered saline [PBS; pH 7.5] containing 0.05% Tween 20); and PBS-3% BSA (PBS containing 3% [wt/vol] BSA). Antisera. Rabbit anti-mouse immunoglobulin antiserum was produced by repeated intradermal injections of 100 ,ug of mouse IgG. Rabbits were injected and bled every 2 weeks for several months. The best bleedings were pooled. Rabbit anti-Brucella antiserum was produced by repeated intradermal injections of 109 B. abortus 45/20(R) or B. abortus 99(S) killed cells. The immunoglobulin fraction was obtained by (NH4)2SO4 precipitation. Peroxidase conjugation. Protein A (Sigma, St. Louis, Mo.) was conjugated with horseradish peroxidase (Sigma) by a modification of the method of Nakane and Kawaoi (24) as described by Dubray and Limet (9). The protein/enzyme ratio was 1:2. ELISA. Supernatants of hybridoma cultures or ascitic fluids were assayed for antibody activity by solid-phase ELISA against antigens in fivefold-diluted GBS in water (40). The antigens used to coat the plates consisted of SDS-I fractions or cell walls (20 ,xg/ml), S-LPS (4 ,ug/ml), R-LPS (10 ,ug/ml), and whole bacterial cells of B. abortus or B. melitensis (A6w = 1.5). Whole bacterial cells were immobilized on microtiter plates (Greiner Labortechnic-Stuttgart) by using the intermediate of rabbit anti-Brucella immunoglobulin at a coating concentration of 10 ,ug/ml. Hybridoma supernatants were serially diluted (1/10 to 1/590,490) in GBS-EDTA-Tw. Binding of the antibodies to SDS-I fractions or cell walls was visualized by using rabbit anti-mouse immunoglobulin antiserum diluted in GBS-EDTA-Tw and peroxidase-conjugated protein A diluted in GBS-EDTA-Tw containing 2% fetal calf serum. Binding to whole bacterial cells was visualized by using peroxidase-conjugated goat anti-mouse immunoglobulins (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) diluted in GBS-EDTA-Tw containing 2% fetal calf serum. Excess reagents between the different incubations were removed by five washings with NaCl-Tw. O-Phenylenediamine (0.4%, wt/vol) and 2 mM H202 in citrate-phosphate buffer were used to visualize peroxidase activity. The ELISA titers were estimated as the highest dilution giving a difference in A492 -A620 of more than twice the mean of the corresponding blank values. Immunogold labeling. A 5-pl sample of cell suspension in water (1010 cells per ml) was deposited on carbon-Formvarcoated 200-mesh copper grids. After being air dried (about 30 min), grids were incubated in the following reagents: PBS-3% BSA (30 min at 37°C), ascitic fluid containing the MAb diluted 1/50 in PBS-Tw (2 h at 37°C), goat anti-mouse biotinylated immunoglobulin (Amersham) diluted 1/200 in PBS-Tw (1 h at room temperature), and 15-nm gold-labeled streptavidin (Amersham) diluted 1/20 in PBS-Tw. Washings between incubation periods were performed with NaCl-Tw. After four washings in NaCl-Tw and four washings in distilled water, grids were observed in a transmission electron microscope (Philips CM10). Immunoblot techniques. The protein antigens were separated by the SDS-PAGE method, originally described by Laemmli (15), in a 2001 vertical electrophoresis unit (LKBProdukter AB, Bromma, Sweden). After electrophoresis, the proteins were transferred to nitrocellulose by using a
_
INFECT. IMMUN.
CLOECKAERT ET AL.
3982
TABLE 1. Number of OMP-specific hybridomas obtained by immunization with different Brucella fractions or whole cells OMPs (kDa)
Live 45/20(R)
10 16.5 19 25-27 31-34 36-38 89 R-LPS
[killed 45/20(R)] 3 3 2 11 0 5 0 22 0
S-LPS a b
No. of hybridomas obtained with cells or fractions used for first immunization [antigen used for boosting] Killed 45/20(R) Live B115(R) Live B3(S) SDS-I H38(R) SDS-I H38(R) CW B115(R) [CW B115(R)]a [killed 45/20(R)] [killed B115(R)] [killed 99(S)] [SDS-I H38(R)] [SDS-I B19(S)]
CW, Cell walls. ND, Not determined.
0 3 0 0 0 0 0
0 26 7 17 0 0 1 3 23
NDb ND
0 0 0 2 0 0 0 0 >50
Transblot apparatus (Biolyon, Dardilly, France) at 12 V for 2 h. Unoccupied sites on the nitrocellulose membranes were blocked by a 30-min incubation in TBS-3% BSA at room temperature with agitation. The membranes were then successively incubated overnight at room temperature with hybridoma supernatants diluted 1/2 in Tw-TBS-1% BSA, 1 h with rabbit anti-mouse immunoglobulin antiserum, and 1 h with peroxidase-conjugated protein A, diluted 1/250 and 1/1,000, respectively, in the same buffer. Washings between incubation periods were performed with Tw-TBS. After two washings in Tw-TBS and two washings in TBS, the blots were developed by incubation at room temperature in a solution of TBS containing 0.06% (wt/vol) 4-chloro-1-naphthol (Bio-Rad, Richmond, Calif.) and 5 mM H202. The reaction was stopped by washing in distilled water.
89 kDa :),
36-38 kDa
31-34 kDa
1
P
l9 kDa )
16.5 kDa )
Ilif
10 kDa 0R-LPS )'.
_
_
*IS
_.
3
0 4 0 1 0 0 1 ND ND
RESULTS Isolation of Brucella OMP-specific hybridomas. Tissue culture supernatants from hybridomas were initially screened for the presence of antibody by ELISA with the antigen used for immunization, i.e., live or killed whole R cells, SDS-I fractions, or cell walls of B. abortus or B. melitensis strains. Reactivity against S-LPS or R-LPS was also monitored by ELISA. Most anti-OMP hybridomas produced antibodies against the major OMPs of 25 to 27 and 36 to 38 kDa (Table 1). By infecting mice with the B. abortus 45/20(R) strain, we obtained antibodies mostly against R-LPS (22 of 46, or 48%) and the 25- to 27-kDa OMP (11 of 46, or 24%). By infecting
I 92 kDa 67 kDa ;
i
I
'i
.i-fI :.
AM:.
_L
43 IDa
_%W.
30 kDa
^.
-
..
r-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2
0 0 0 2 0 17 0 ND ND
MAb isotypes. MAb isotypes were determined by direct latex agglutination immunoassay of the culture supernatants. Latex particles were coated with rat monoclonal anti-mouse IgGl, IgG2a, IgG2b, IgG3, and IgM and polyclonal antimouse IgA by a method similar to those described by Limet et al. (17).
_ _ *_ =L _
25-27 kDa I0
0 0 0 8 1 0 0 ND ND
21 kDa
6
FIG. 1. Western immunoblot of OMPs of B. melitensis B115(R). Lane 1 to 10 show immunoblots of sonicated cell extracts of B. melitensis B115(R) with anti-R-LPS MAb A68/29C05/B06 (lane 1), anti-10-kDa MAb A68/08E07/B11 (lane 2), anti-16.5-kDa MAb A68/ 04G01/C06 (lane 3), anti-19-kDa MAb A68/25H10/A05 (lane 4), anti-25- to 27-kDa MAb A59/05F01/CO9 (lane 5), anti-31- to 34-kDa MAb A59/1OF09/G1O (lane 6), anti-36- to 38-kDa MAb A63/05A07/ A08 (lane 7), anti-89-kDa MAb A53/1OB02/A01 (lane 8), serum from B. melitensis B115(R)-infected mice (lane 9), and serum from B. abortus 45/20(R)-infected mice (lane 10).
14.4 kDa
*4 1
2
3
4
5
FIG. 2. Immunoblot of SDS-I fraction of B. abortus B19(S) with anti-36- to 38-kDa MAb A68/25G05/A05. Lanes: 1, Coomassie blue-stained molecular mass standards; 2, Coomassie blue-stained SDS-I fraction not heated in SDS; 3, Coomassie blue-stained SDS-I fraction heated for 10 min at 100°C in SDS; 4, immunoblot of SDS-I fraction not heated in SDS with MAb A68/25G05/A05; 5, same as lane 4 but heated for 10 min at 100°C in SDS.
VOL. 58, 1990
SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS
_
92 kDa 67
*
kDa
43 kDa
%
30 kDa
21 kDa 14.4
kDa
1
2
3
4
FIG. 3. Immunoblot of SDS-I fraction of B. abortus B19(S) with anti-36- to 38-kDa MAb A63/03H02/BO1 produced by using the SDS-I fraction as the immunogen (lane 3) and with anti-36- to 38-kDa MAb A68/25G05/AO5 produced by using whole cells as the immunogen (lane 4). The color development reaction was stopped at the same time. Lane 1, Coomassie blue-stained molecular mass standards; lane 2, Coomassie blue-stained SDS-I fraction of B. abortus B19(S).
mice with the B. melitensis B115(R) strain, we obtained antibodies that were specific mostly for the 16.5-kDa OMP (26 of 77, or 34%) and S-LPS (23/77, or 30%). Only two anti-OMP MAbs were obtained after infection of mice with S brucellae. OMP specificity by Western immunoblot analysis. To determine the specificity of the anti-Brucella MAbs, we performed Western immunoblotting with cell walls, SDS-I fractions, and cell extracts of B. abortus or B. melitensis
3983
strains separated by SDS-PAGE (Fig. 1). Multiple bands (doublets or triplets) were observed with all the anti-25- to 27-, -31- to 34-, and -36- to 38-kDa MAbs. MAb A59/1OF09/ G10 also revealed less stained and more diffuse bands of molecular mass lower than 31 kDa. These bands were not observed with the SDS-I fraction containing large amounts of 25- to 27-kDa OMP. With some of the anti-36- to 38-kDa MAbs, a major high-molecular-mass band (>92 kDa) was also revealed when cell walls were not heated in SDS before electrophoresis (Fig. 2). This band disappeared when cell walls were heated for 10 min at 100°C. The anti-25- to 27- and -36- to 38-kDa MAbs produced by using SDS-I fractions as the immunogen reacted more strongly with the denatured antigens after SDS-PAGE than did those which bind to the cell surface and were obtained from infected mice or mice immunized with cell walls or whole bacterial cells (Fig. 3). MAbs binding to the Brucella cell surface. The ability of MAbs to bind to the cell surface of intact B. abortus and B. melitensis R and S strains was checked by ELISA. Binding of the anti-OMP MAbs was compared with binding of the MAbs directed against R-LPS and S-LPS. The strains used were B. abortus 544(R) and 544(S) and B. melitensis H38(R) and H38(S). Most (90%) of the MAbs produced by using the SDS-I fraction as the immunizing antigen did not bind to the cell surface (data not shown). Most of the other MAbs against anti-OMPs showed a better binding on the R cells, i.e., higher titers and maximal absorbances (Table 2). MAbs which bound to the cell surface of the S strains used did not bind as well as an anti-LPS MAb did. Surface exposure of OMPs was visualized by immunogold labeling (Fig. 4), with the MAbs showing the greatest binding by ELISA. The labeling obtained with the anti-10-, -16.5-, and 89-kDa MAbs on B. abortus 544(R) was comparable to results shown in Fig. 4A. The labeling of B. abortus 544(S) with the anti-10- and 16.5-kDa MAbs was comparable to results shown in Fig. 4B. Results are summarized in Table 3. As visualized on the R strains used, surface-exposed OMPs are the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs. Binding on the S B. abortus strains was
TABLE 2. ELISA binding of the anti-Brucella OMP MAbs on R and S Brucella cells B. abortus 544(R)
Specificity, immunogen, and clone
(isotyp)
(isotype)
Titer
Titer
10 kDa, live B. abortus 45/20(R), A68/07G11/C10
(IgG2a) 16.5 kDa, live B. abortus 45/20(R), A68/04G01/ C06 (IgG2a) 19 kDa, live B. melitensis B115(R), A76/18B02/ D06 (IgG2a) 25 to 27 kDa, live B. abortus 45/20(R), A68/04B10/F05 (IgG2a) 31 to 34 kDa, SDS-I fraction from H38(R), A59/ 10F09/G1O (IgG2a) 36 to 38 kDa, live B. abortus 45/20(R), A68/25G05/A05 (IgG2a) 89 kDa, cell wall from B115(R), A53/1OB02/AO1 (IgGl) R-LPS, live B. abortus 45/20(R), A68/03F03/DO5 (IgG2b) S-LPS, live B. melitensis B115(R), A76/12G12/ F12 (IgGl) Negative control ascitic fluid (nonrelated MAb)
B. abortus 544(S)
Max Tier Max Titer absorbance absorbance
B. melitensis H38(R)
TtrMax
absorbance
B. melitensis H38(S) Max Titer
absorbance
810
1.049
30
0.435
2,430
1.146
270
0.434
810
1.270
30
0.713
2,430
1.376
270
0.786
270
0.665
270
1.265
270
0.462
270
1.640
65,610
1.474
810
1.367
65,610
1.516
270
0.711
270
0.857
30
0.619
590,490
1.137
7,290
1.421
196,830
1.324
2,430
1.571
196,830
1.181
90
0.730
270
0.590
10
0.314
2,430
0.497
30
0.243
7,290
2.011
270
1.025
7,290
1.068
90
0.761
90
0.424
590,490
2.256
90
0.393
590,490
2.364
90
0.304
30
0.422
30
0.341
30
0.135
aMaximal absorbance after subtraction of the blank value.
3984
CLOECKAERT ET AL.
INFECT. IMMUN.
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FIG. 4. Immunoelectron microscopy of Brucella OMPs. The figure shows labeling of B. abortus 544(R) by the anti-19-kDa MAb
A76/18B02/D06 (A), anti-25- to 27-kDa MAb A68/04B10/F05 (C), anti-31- to 34-kDa MAb A59/1OF09/G10 (E), and anti-36- to 38-kDa MAb A68/25G05/A05 (G), and labeling of B. abortus 544(S) by the same MAbs in the same order (B, D, F, and H, respectively). Magnification, x 57,000.
VOL. 58, 1990
SURFACE-EXPOSED BRUCELLA OUTER MEMBRANE PROTEINS
3985
TABLE 3. Electron microscopy binding of the anti-Brucella OMP MAbs on R and S Brucella cells Labeling of:
Specificity, immunogen, and clone (isotype) 10 kDa, live B. abortus 45/20(R), A68/07G11/C10 (IgG2a) 16.5 kDa, live B. abortus 45/20(R), A68/04G01/C06 (IgG2a) 19 kDa, live B. melitensis B115(R), A76/18B02/D06 (IgG2a) 25 to 27 kDa, live B. abortus 45/20(R), A68/04B10/F05 (IgG2a) 31 to 34 kDa, SDS-I fraction from H38(R), A59/1OF09/G10 (IgG2a) 36 to 38 kDa, live B. abortus 45/20(R), A68/25G05/A05 (IgG2a) 89 kDa, cell wall from B115(R), A53/1OB02/A01 (IgGl) R-LPS, live B. abortus 45/20(R), A68/03F03/D05 (IgG2b) S-LPS, live B. melitensis B115(R), A76/12G12/F12 (IgGl) Negative control ascitic fluid (nonrelated MAb) a ND, Not determined.
observed for all MAbs except for the anti-31- to 34-kDa MAb and the anti-89-kDa MAb. However, the anti-31- to 34-kDa MAb bound on both the S and R B. melitensis strains used. The anti-S-LPS MAb did not bind on the R strains.
DISCUSSION We report the production of a panel of MAbs specific for seven Brucella OMPs. As shown by Western immunoblotting, most antibodies were specific for the major OMPs: the 25- to 27-kDa OMP (8) and the 36- to 38-kDa OMP, which is a porin (5). The other MAbs were specific for the 10-, 16.5-, 19-, 31- to 34-, and 89-kDa OMP. By infecting mice with B. abortus 45/20(R), we obtained antibodies directed mainly against R-LPS and the 25- to 27- and 36- to 38-kDa OMPs. These antigens are the most abundant in the cell wall as described in the literature (8, 33, 38, 39), but not necessarily the most immunogenic of the B. abortus rough strains as shown by the immunoblots with the sera of B. abortus 45/20-infected mice. The immunogenicity of OMPs seems to be related to the S-R variation in brucellae. The immunogenicity of Braun's outer membrane lipoprotein was also greatly influenced by S-R variation and the presence of a capsule in Escherichia. coli: elevated titers were obtained with either live or heat-killed R cells (4). After infection with B. melitensis B115(R), most of the antibodies found were specific for the 16.5-kDa minor OMP and for S-LPS. The fact that MAbs specific for S-LPS were obtained by infection with this R strain has yet to be explained. In Western immunoblottings after SDS-PAGE, the anti25- to 27- and 36- to 38-kDa MAbs generated by immunization with SDS-I fractions reacted more strongly with the denatured antigens than did those which bind to the cell surface and were obtained after immunization with cell walls or whole bacterial cells. This could be explained by the fact that the MAbs which bind to the cell surface recognize conformational epitopes while the others recognize mainly denatured sequential epitopes or epitopes that are inaccessible to antibodies in cell walls. Some anti-36- to 38-kDa MAbs which bind to the cell surface, like MAb A68/25G05/ A05, probably recognize a conformational epitope. Indeed, immunoblot analysis with this MAb revealed a high-molecular-mass band (>92 kDa) in an SDS-I fraction that had not been heated in SDS, which disappeared when the SDS-I fraction was heated at 1000C. This high-molecular-mass band probably corresponds to the porin trimer described by Moriyon and Berman (23). The anti-25- to 27-, 31- to 34-, and 36- to 38-kDa MAbs revealed multiple bands in sonicated cell extracts. The multiple band pattern of the group 2 or 36-
B.
abortus
B. abortus
B. melitensis
544(R)
B. melitensis
544(S)
H38(R)
+ + + + + + + +
+ + + +
NDa ND ND ND
H38(S) ND ND ND ND + ND ND ND +
-
-
+
+
ND ND ND -
+ +
to 38-kDa OMP has already been described by Douglas et al.
(5). They postulated that the multiple bands can represent modified forms of a single protein and that since the protein was obtained after lysozyme digestion of peptidoglycan, it seems possible that different bands contain peptidoglycan fragments of different sizes or different amounts of adherent LPS molecules or both. The same explanation can be given for the 25- to 27- and 31- to 34-kDa OMPs since these proteins are also released from cell walls only by lysozyme treatment. The multiple bands pattern of the 25- to 27-kDa OMP can also be explained by the fact that this OMP appears in gel as a glycoprotein (7) after silver staining (periodic oxydation) and that it fails to migrate to one distinct position on the electrophoresis gel, probably owing to the heterogeneity of the oligosaccharide components. This type of behavior has been encountered for Myxococcus xanthus (12) and other microorganisms such as Dictyostelium discoideum (35). As visualized by electron microscopy and as indicated by ELISA binding of MAbs, the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface of intact R Brucella strains. In ELISA all the anti-OMP MAbs except the anti-19-kDa MAb showed a better binding on the R Brucella strains than on the corresponding S strains. Immunoelectron microscopy showed that all MAbs used, except the anti-31- to 34-kDa MAb and the anti-89-kDa MAb, bound on the R and S Brucella strains. The anti-31- to 34-kDa MAb bound on B. melitensis R and S cells but not on B. abortus S cells, for which this OMP is a minor protein (Dubray, Ph.D. thesis). Surface exposure of the 89-kDa OMP was observed only for the R B. abortus strain used. S-LPS probably hinders the binding of these MAbs on epitopes of the OMPs close to the S-LPS. Such steric hindrance has already been described for Coxiella burnetii (14), members of the family Enterobacteriacae (3, 37, 41), and Neisseria gonorrhoeae and N. meningitidis (25, 34). All MAbs will be used in passive protection experiments in mice to determine whether Brucella OMPs are potentially useful vaccine antigens. Protection experiments have already been performed by Montaraz et al. (22) with O-polysaccharide-specific MAbs and two IgM MAbs specific for the porn of B. abortus. The latter MAbs failed to confer protection. These MAbs, however, did not bind on the cell surface of B. abortus 2308. The same results were observed in ELISA with all our anti-porin MAbs on this strain (data not shown). On other less virulent strains such as B. abortus 99 and B19, mainly the anti-25- to 27- and to a less extent the
3986
CLOECKAERT ET AL.
anti-36- to 38-kDa MAbs bound to the cell surface, but at much higher concentrations. Binding of anti-LPS MAbs is, however, very efficient. Therefore, anti-OMP MAbs will probably be less efficacious than anti-LPS MAbs for opsonizing brucellae and promoting their ingestion and killing by macrophages. ACKNOWLEDGMENTS We are most grateful to J. M. Verger, R. Wijffels, P. Michel, and M. Grayon for culture and control of bacterial strains. We thank G. Bezard, J. C. Nicolle, A. Van Rompaye, and J. Van Broeck for expert technical assistance. A. Cloeckaert is supported by the Institut National pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture. The work was supported by Biotechnology Action Programme BAP-012-B (GDF) from the European Economic Community.
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INFECT. IMMUN. 18. Limet, J. N., A. M. Plommet, G. Dubray, and M. Plommet. 1987. Immunity conferred to mice by anti-LPS monoclonal antibodies in murine brucellosis. Ann. Inst. Pasteur Immunol. 138:417424. 19. Lopez-Merino, A., J. Asselineau, A. Serre, J. Roux, S. Bascoul, and C. Lacave. 1976. Immunization by an insoluble fraction extracted from Brucella melitensis: immunological and chemical characterization of the active substances. Infect. Immun. 13: 311-321. 20. Madraso, E. D., and C. Cheers. 1978. Polyadenylic acid-polyuridylic acid (polyA-U) and experimental brucellosis. 2. Macrophages as a target cells of polyA-U in experimental brucellosis. Immunology 35:77-84. 21. Montaraz, J. A., and A. J. Winter. 1986. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice. Infect. Immun. 53:245-251. 22. Montaraz, J. A., A. J. Winter, D. M. Hunter, B. A. Sowa, A. M. Wu, and L. G. Adams. 1986. Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies. Infect. Immun. 51:961-963. 23. Moriyon, I., and D. T. Berman. 1983. Isolation, purification, and partial characterization of Brucella abortus matrix protein. Infect. Immun. 39:394-402. 24. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labelled antibody, a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. 25. Paques, M., J. S. Teppema, E. C. Beuvery, H. Abdillahi, J. T. Poolman, and A. J. VerkleiJ. 1989. Accessibility of gonococcal and meningococcal surface antigens: immunogold labeling for quantitative electron microscopy. Infect. Immun. 57:582-589. 26. Pardon, P. 1977. Resistance against a subcutaneous Brucella challenge of mice immunized with living or dead Brucella or by transfer of immune serum. Ann. Inst. Pasteur Immunol. 128C: 1025-1037. 27. Pavlov, H., M. Hogart, I. F. C. Mckenzie, and C. Cheers. 1982. In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection. Cell. Immunol. 71:127-138. 28. Plommet, M. 1987. Brucellosis and immunity: humoral and cellular components in mice. Ann. Inst. Pasteur Microbiol. 138:105-110. 29. Plommet, M., and A. M. Plommet. 1987. Anti-Brucella cell mediated immunity in mice vaccinated with a cell wall fraction. Ann. Rech. Vet. 18:429-437. 30. Plommet, M., and A. M. Plommet. 1983. Immune serummediated effects on brucellosis evolution in mice. Infect. Immun. 41:97-105. 31. Plommet, M., A. Serre, and R. Fensterbank. 1987. Vaccines, vaccination in brucellosis. Ann. Inst. Pasteur Microbiol. 138: 117-121. 32. Rasooly, G., A. L. Olitzki, and D. Sulitzeanu. 1966. Immunization against Brucella with killed vaccines. Immunizing activity in mice of Brucella cell-wall and of fractions derived from them. Isr. J. Med. Sci. 2:569-584. 33. Santos, J. M., D. R. Verstraete, V. Y. Perera, and A. J. Winter. 1984. Outer membrane proteins from rough strains of four Brucella species. Infect. Immun. 46:188-194. 34. Shafer, W. M. 1988. Lipopolysaccharide masking of gonococcal outer-membrane proteins modulates binding of bactericidal cathepsin G to gonococci. J. Gen. Microbiol. 134:539-545. 35. Springer, W. R., and S. H. Barondes. 1983. Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum. J. Biol. Chem. 258:4698-4701. 36. Sulitzeanu, D., L. Jones, and A. W. Stableforth. 1955. Protective activity of monospecific anti-Brucella sera in mice. Nature
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Verstraete,
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R., and A. J. Winter.
1984.
Comnparison
of
sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains. Infect. Immun. 46:182-187. 40.
VolIer, A.,
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and A. Bartlett.
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