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Anti-R-LPS mAb used were mAb A68/25B03/. D08 (lgGI) ... B06 (IgG3) and A68/10A06/BI 1 (lgM). ... l h with peroxidase-conjugated protein A (Sigma).


Res. Microbiol.

1993, 144, 475-484

Monoclonal antibodies to Brucella rough lipopolysaccharide: characterization and evaluation of their protective effect against B. abortus A. Cloeckaert (=) (*), I. Jacques 0), R.A. Bowden (0, G. Dubray (I) and J.N. Limet (2) ¢o Institut National de la Recherche Agronomique, Centre de Recherches de Tours, Laboratoire de Pathologic infectieuse et hnmunologie, 37380 Nouzilty (France), and (2) Facult~s Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, 5009 Namur (Belgium)

SUMMARY We characterized 4 monoclonal antibodies (mAb) specific for rough lipopolysaccharide (R-LPS) of Bruce#a. mAb were selected by enzyme-linked immunosorbent assay IELISA) on whole R. abortus 45/20 rough cells and R-LPS from B. melitensis B115 rough cells. Specificity ~as confirmed by immunoblot analysis using R-LPS and smooth LPS (S-LPS) preparations. Anti-R-LPS revealed the low molecular mass R-LPS molecules below 20.1 kDa in the R-LPS and S-LPS preparations as well as the typical A and M patterns in high molecular mass S-LPS molecules (between 21.5 and 66 kDa) in the S-LPS preparations. An O-polysaccharide-specific mAb revealed only high molecular mass S-LPS molecules in the S-LPS preparation. In ELISA the anti-R-LPS mAb bound better on rough than on smooth B. abortus 544 whole cells, and this was confirmed by immunoelectron microscopy. Protective activity of anti-R-LPS mAb of different isotypes was tested on mice and compared with an S-LPS-specific mAb. Only the IgG3 mAb reduced significantly the splenic infection but did not reach the level of protection conferred by the S-LPS-specific mAb.

Key-words: LPS, Brucella abortus, Brucella melitensis, IgG, mAb; Mouse, Protection, Epitopes.

INTRODUCTION The smooth lipopolysaccharides (S-LPS) of Brucella spp. and other Gram-negative bacteria

are complex molecules comprising three structurally and serologicaUy distinct regions, i.e. a glycolipid moiety called lipid A, a core oligosaccharide and the O-polysaccharide lOPS) chain.

Submitted March II, 1993, accepted July 28, 1993. {*) Corresponding author.

The Brucella 0 chain contains two distinct epitopes, designated A and M (Bundle et aL, 1989). Their relative amounts vary among the smooth Brucella strains, and these epitopes are absent on the rough strains, which lack OPS (Alton et aL, 1988). mAb to Brucella S-LPS have been reported with specificity for the A, the M and the common epitopes, some of which cross-react


A. C L O E C K A E R T E T A L .

with Yersinia enterocolitica 0:9 S-LPS (Bundle et al., 1989; Cloeckaert et al., 1992; GarinBastuji et al., 1990; Limet et al., 1989; Palmer and Douglas, 1989; Vizcaino et al., 1991). These mAb confer protection on mice by reducing the number of Brucella in the spleen and liver (Cloeckaert et al., 1992; Limet et al., 1989, 1987; Montaraz et al., 1986; Phillips et al., 1989). Brucella rough LPS (R-LPS) lacking OPS and expressed predominantly in rough strains of Brucella has been immunochemically characterized (Moreno et al., 1979), and antibody response against it has been observed in B. ovis infections (Riezu-Boj et al., 1986). We obtained mAb specific for R-LPS by fusion with the NSO myeloma of spleen cells of mice infected by the rough B. a b o r t u s 45/20 and B. melitensis B115 strains (Cloeckaert et al., 1990). Our purpose here was to characterize the anti-R-LPS mAb in terms of molecular and cellular binding and to compare their protective activity in mice against B. a b o r t u s with an anti-S-LPS mAb already shown to be protective (CIoeckaert et al., 1992; Limet et al., 1989; Jacques et al., 1992; Limet et al., 1987; CIoeckaert et al., 1991).



(Difco Laboratories, Detroit, MI). Cells were harvested by gentle agitation in sterile distilled water. Purity and phase (smooth, S, or rough, R) were checked using standard procedures (Alton et aL, 1988). Dilutions were made in phosphate-buffered saline (PBS) containing 0.5 % iv/v) Tween-80 to avoid autoagglutination, and the number of cells was determined by optical density measurements at 600 nm in a spectrophotometer (OD=0.165 for 1 0 9 cells/ml, with I-cm light path). Cells were killed by addition of 20 ptl/ml to 10 ~°cells/ml of a 5 °70 (v/v) peracetic acid solution and incubated overnight at room temperature. After three washes in sterile distilled water, sterility was checked by plating 0.2 ml of each cell suspension on petri dishes containing the culture medium, LPS fractions

S-LPS fractions of B. melitensis 16M and B. abortus 99 were prepared by the phenol-water method (Leong et al., 1970). The R-LPS fraction of B. mefitensis B115 was obtained by the phenol-waterchloroform-petroleum-ether method (Galanos et al., 1969). Proteinase-K-digested S-LPS fractions of B. rnelitensis 16M and B. abortus 544 were prepared as described previously (Garin-Bastuji et al., 1990).

OPS of B. abortus 99 and B. melitensis 16M were extracted by autoclaving S cells in 2 070 iv/v) acetic acid/10 070(w/v) NaCI (Jacques et al., 1991) and purified by high-performance liquid chromatography (Zygmunt et al., 1988).

Brucella strains Monoclonal antibodies

B. abortus 544 (S) and B. abortus 544 (R), B. abortus 99 (S), B. abortus 45/20 (R), B. mefitensis 16M (S) and B. melitensis B! 15 (R) were obtained

from the Institut National de la Recherche Agronomique, Nouziily, France. Whole Brucella S and R cells used for binding studies

Cultures were grown at 37°C for 48 h on trypticase soy agar (BioM6rieux, Marcy-I'Etoile, France) slants supplemented with 0.1% (w/v) yeast extract



= = = = = = =

colony-forming u:fit. lipopolysaccharide. monoclonal antibody. outer m e m b r a n e protein. O polysaccharide. phosphate-buffered saline. PBS+Twccn-20.

Anti-R-LPS mAb used were mAb A68/25B03/ D08 (lgGI), A68/03F03/D05 (lgG2b), A68/29C05/ B06 (IgG3) and A68/10A06/BI 1 (lgM). These mAb were derived fi'om fusion of spleen cells from B. abortus-45/20-infected BALB/c mice with the NSO myeloma (CIoeckaert el al., 1990). Hybridomas were cultured in 200-mi flasks (Falcon) at 37°C with 6.5 070 CO 2 in Dulbecco modified Eagle's medium supplemented with 10 °70 foetal calf serum, 2 °70 hypoxanthine-thymidine (HT supplement), 2 070nonessential amino acid supplement, 2 mM sodium pyru-


= = =


= =

rough LPS. smooth LPS. sodium dodecyl sulphate/polyacrylamide gel el¢ctrophoresis. Tris-buffered saline. TBS+Tween-20.


vate, 4 mM glutamine, 200 IU/ml penicillin and 200 tzg/ml streptomycin (all from Gibco). Hybridoma supernates were taken after confluent growth and iysis of the cells and were centrifuged (i,500 g) to remove cell debris. Screening for specificity was done by ELISA using rough B. abortus 45/20 cells and purified B. meliwnsis Bll5 R-LPS as antigens (Cloeckaert et al., 1990). The anti-S-LPS mAb O4F9 (IgG2a) and 12Gl2 (IgGI), obtained as described previously (Limet et al., 1987; Cloeckaert et ai., 1990), were shown to be specific for OPS. ELISA Anti-R-LPS and anti-S-LPS (12G12) hybridoma supernates or purified anti-S-LPS (04F9) immunoglobulin (Ig) fraction were assayed for antibody activity by solid-phase ELISA against antigens coated by overnight incubation at 37°C. Antigens were S-LPS (phenol phase or proteinase-K-digested) (4 izg/ml), R-LPS (I0 ~tg/ml), OPS (4 v~g/ml) and killed S and R B. abortus 544 cells. Brucella cells (OD60o = 1.5) were immobilized on microtitre plates (Greiner, Stuttgart, Germany) by capture with rabbit anti-Brucella immunoglobulins (Cloeckaert et al., 1990) at 10 v.g/ml. For binding studies on whole bacteria, 20-fold concentrated hybridoma supernates or Ig fraction were serially diluted (1/10 to 1/590,490) in PBS 0.02 M pH 7.2 containing 0.05 070Tween-20 (PBS-T). Binding of mAb to the cells was detected with peroxidase-conjugated goat anti-mouse immunoglobulins (Jackson, West Grove, PA) diluted in PBS-T. ABTS (2,2'-azino-di-(3-ethylbenzthiazoline sulphonate) was used as a coloured reagent in the substrate solution as described (Alton et a/., 1988). Light absorbance w,~s measured at 414 nm after l-h incubation with the substrate solution. For binding studies on LPS and OPS, hybridoma supernates were serially diluted as above, starting from !/3 dilution.

Immunoblot techniques The S-LPS and R-LPS antigens were separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (Garin-Bastuji et ai., 1990). After electrophoresis, the antigens were transferred to nitrocellulose at 0.8 mA/cm 2 for 1 h. Unoccupied sites on the nitrocellulose membranes were blocked by a 30-min incubation in Tris-buffered saline (TBS) (0.15 M NaCI, 10 mM Tris hydrochloride pH 7.5) containing 3 070(w/v) skim milk, at room temperature with agitation. The membranes were then successively incubated overnight at room temperature with hybridoma supernates diluted 1/2 in TBS containing 0.05 070 (v/v) Tween-20 (Tw-TBS) and ! 070 (w/v)


skim milk, for l h with rabbit anti-mouse immunoglobulin serum (Nordic, The Netherlands) and l h with peroxidase-conjugated protein A (Sigma) diluted 1/250 and l/l,000, respectively, in the same buffer. Washings between incubations were performed with Tw-TBS. Finally, after 3 washings in Tw-TBS and 1 in TBS, the blots were developed by incubation at room temperature in a solution of TBS containing 0.06 070 (w/v) 4-chloro-l-naphthol and 5 mM H202. The reaction was stopped by washing in distilled water. Membranes were air-dried and photographed.

Immunoelectron microscopy lmmunoelectron microscopy was performed as described previously (Cloeckaert et al., 1990). Binding of the mAb (3-fold diluted hybridoma supernates) was detected with sheep anti-mouse biotinylated immunoglobulins (Amersham, UK) and gold-labelled streptavidin (15 nm) (Amersham, UK).

Mice protection test mAb were screened for protection of mice as described previously (Limet et al., 1987). mAb preparations were controlled for endotoxin contamination by the chromogenie Limulus amoebocyte lysate test (Toxicolor, Seikagaku, Japan), following the recommendations of the manufacturer. Endotoxin contamination was in the order of l0 pg/ml as compared with a standard of Escherichia coil Ol I l LPS. AntiR-LPS mAb (20-fold concentrated hybridoma superhates) and anti-S-LPS mAb 04F9 (4 t~g) were injected subcutaneously (0.1 ml) to groups of six CD-I mice l day before the standard intravenous challenge with B. abortus 544 smooth strain (2 x 105 CFU). Spleen counts were done 7 and 21 days after challenge. The experiment included one negative control group which received saline, and as a positive control we included the anti-S-LPS mAb. The statistical significance was determined after variance analysis by one-side comparison of means with the negative control mean (F-test) (Lison, 1968).

RESULTS mAb specificity by E L I S A The anti-Brucella R-LPS mAb showed weak or absence o f reactivity against S-LPS (phenol phase or proteinase-K-treated S-LPS preparat i o n s ) o r O P S o f B. m e l i t e n s i s 16M (M dominant) (fig. l) and B. a b o r t u s 99



(A dominant) (not shown), but bound at high dilution on B. melitensis Bi 15 R-LPS (fig. IA). They thus recognize epitopes present in the core lipid A moiety. The mAb 12G!2, specific for OPS, did not react with the R-LPS preparation (fig. 1).


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