INFECTION AND IMMUNITY, July 1992, p. 2718-2725 0019-9567/92/072718-08$02.00/0 Copyright ©) 1992, American Society for Microbiology

Vol. 60, No. 7

Immunological Characterization of the Lipooligosaccharide B Band of Bordetella pertussis DENIS MARTIN,1 MARK S. PEPPLER,2 AND BERNARD R. BRODEUR"* National Laboratory for Immunology, Laboratory Centre for Disease Control, Ottawa, Canada KIA OL2,1 and Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Canada T6G 2H72 Received 14 February 1992/Accepted 1 May 1992

Two structurally and immunologically different components of Bordetella pertussis endotoxin can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining: a major A band and a faster-migrating minor B band. Certain mutant strains of B. pertussis express only the B band, while the wild-type strains produce both lipooligosaccharides (LOS). Two monoclonal antibodies (MAbs) directed against the minor LOS B band were generated, allowing the study of this surface molecule on different strains of Bordetella. These two MAbs, designated BL-8 and BL-9, reacted strongly with phenol-water-purified LOS obtained from a B. pertussis LOS B mutant strain. Sodium periodate treatment of the purified LOS prevented binding of the MAbs, indicating the carbohydrate nature of the epitope(s). Western immunoblotting experiments revealed that the epitope(s) recognized by these MAbs is conserved on all B. pertussis and Bordetella bronchiseptica Vir- (avirulent) variant strains tested but is not present on Bordetella parapertussis and B. bronchiseptica Vir+ (virulent) wild-type strains. Further studies showed that although present in the lipopolysaccharide B band expressed by Vir- strains, the epitope(s) recognized by the MAbs is not accessible on the surface of intact B. bronchiseptica cells. For B. pertussis, the density and accessibility of this epitope(s) are dependent on the virulence-associated or LOS phenotype expressed by the strain. Our data demonstrate that the expression and accessibility of the epitope(s) are significantly greater on the LOS B variant strains and LOS AB Vir- strains compared with fresh B. pertussis clinical isolates. For these latter strains, which are Vir+, this epitope(s) was barely detectable on the surface of intact bacteria, despite Western blot analyses that revealed specific reactions between the MAbs and the LOS B band. The two LOS B-specific MAbs had no bacteriolytic activity against a LOS AB wild-type strain, while the control MAb BL-2, which is specific for the B. pertussis LOS A band, significantly reduced the number of living bacteria in the same assay. Moderate lytic activity against a mutant strain expressing only the LOS B band was observed for MAb BL-8 but not for MAb BL-9 or BL-2. These data demonstrate that the type, amount, and surface exposure of the LOS are related to the phenotype expressed by a specific B. pertussis strain. In addition, the LOS B MAbs also reveal the antigenic conservation of carbohydrate epitopes among B. pertussis and B. bronchiseptica strains.

Several Bordetella pertussis virulence determinants, such the pertussis toxin, adenylate cyclase toxin, and filamentous hemagglutinin, are regulated by a single genetic locus named vir, or bvg (5, 38). The virulence determinants of the other two virulent species, Bordetella parapertussis and Bordetella bronchiseptica, are also under the control of a similar regulatory locus (26). Strains which are unable to express these virulence factors are also unable to cause whooping cough (37). The virulent phenotype (Vir+) is unstable and can give rise to phase variants, a genetic event which occurs at a frequency of i0' to 10-6, such that variants no longer stably express any of the virulenceassociated genes in any environment (32, 35). A second type of variation, called phenotypic modulation, also results in the reversible loss of certain virulence factors and is dependent on changes in the environment such as temperature and the presence of magnesium sulfate or nicotinic acid (25). Modifications of surface components such as outer membrane (OM) proteins and lipooligosaccharide (LOS) have also been described for these variant strains (6). The B. pertussis LOS possesses a chemical structure different from that of the well-known lipopolysaccharides (LPS) of smooth-type members of the family Enterobac-

teriaceae. Specifically, the pertussis endotoxin contains a lipid A moiety covalently linked to an oligosaccharide core but lacks the long polysaccharide 0-specific chains (2, 3, 10, 17). The LOS of B. pertussis exhibits biological activities similar to those of endotoxins from other gram-negative bacteria, including pyrogenicity, toxicity, induction of the Shwartzman reaction, induction of tumor necrosis factor production, and adjuvant properties (7, 17, 21, 27, 34, 36). Previous analysis of pertussis endotoxin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting identified two distinct LOS components, a slowly migrating major band and a fast-migrating minor band, designated A and B bands, respectively (28). Subsequently, on the basis of their LOS profiles, B. pertussis strains were classified into two physiologically and serologically distinct phenotypes: the LOS wild-type phenotype possessing both the A and B bands and the LOS variant phenotype possessing only the B band. Caroff et al. (10) have suggested that LOS B of variant LOS B strains is identical to the LOS B component of endotoxin from LOS AB wild-type strains. In fact, they have demonstrated that the carbohydrate regions of LOS B variant strains lack three amino sugars present in LOS A of the wild-type strains. In a previous report, we described seven monoclonal antibodies (MAbs) specific for a surface-accessible epitope(s) present on the B. pertussis LOS A band (4). In the

as

*

Corresponding author. 2718

VOL. 60, 1992

presence of complement, five of these MAbs were highly bacteriolytic against B. pertussis strains. Recently, we also reported the generation of a human MAb, called HBp2, which reacted with all the B. pertussis wild-type isolates tested (8). This human MAb was also directed against a surface-accessible epitope present on the LOS A band and was highly bacteriolytic against B. pertussis. We have studied the B. pertussis LOSs of several phenotypic variant strains with LOS A- and LOS B-specific MAbs. Our results show that a conserved carbohydrate epitope(s) is present on all the B. pertussis strains and on the B. bronchiseptica Vir(avirulent) variant strains. Moreover, a relationship between the amount of LOS B and the phenotype expressed by each strain was observed with Vir- B. pertussis strains expressing a higher concentration of LOS B at their surface than the virulent strains (Vir+).

MATERIALS AND METHODS Bacterial strains. B. pertussis wild-type strains ATCC 9340 and ATCC 9797 (18323) as well as the LOS B variant strains ATCC 11615 and NCTC 10901 were obtained from the American Type Culture Collection (Rockville, Md.) or the National Collection of Type Cultures (Central Public Health Laboratory, London, United Kingdom). B. pertussis wildtype Tohama 1 and 3779 strains were originally obtained from John J. Munoz, Rocky Mountain Laboratories, Hamilton, Mont. The LOS B variant strains 134 and D3148 were obtained from A. Wardlaw, Department of Microbiology, University of Glasgow, Glasgow, United Kingdom, and J. M. Dolby, Clinical Research Centre, Harrow, Middlesex, United Kingdom, respectively. The phenotypic variants Vir+ Gna-, Vir- Gna-, and Vir- Gna+ were obtained from the three B. pertussis strains, 134, Tohama 1, and 3779, and were described previously (29). The Gna+ organisms were plated on fresh nutrient agar to confirm their phenotypes. Two B. pertussis revertant LOS AB strains designated 11615R Vir+ and 10901R Vir+ were selected from their corresponding LOS B variant strains after multiple passages through HeLa cell monolayers and were screened for hemolytic colonies on Bordet-Gengou agar plates (BGA). Sixtyfour clinical isolates of B. pertussis and 13 B. parapertussis isolates were obtained from P. Lebel, Sainte-Justine Hospital, Montreal, Quebec, Canada, and the Quebec Public Health Laboratory, Sainte-Anne-de-Bellevue, Quebec, Canada. B. bronchiseptica strains 110H Vir+ and Vir- were obtained from D. Bemis, University of Tennessee, Knoxville, Tenn. The isogenic Vir+ and Vir- B. bronchiseptica strains 214 and 22067 were provided by John J. Munoz. Other gram-negative and gram-positive bacteria were isolated from the respiratory tracts of patients at Sainte-Justine Hospital or obtained from a collection held by the Laboratory Centre for Disease Control, Ottawa, Canada. All B. pertussis strains were cultivated at 36°C on BGA, and other bacterial strains were cultured by routine procedures. OM and LOS preparations. Lithium chloride extraction of OM from the bacteria was performed as described elsewhere (9). Protein concentrations were determined by the Lowry method adapted to membrane fractions (19). Purified LOS from B. pertussis 134 was obtained by the phenol-water extraction procedure described by Inzana (14). Immunization of mice and fusion procedure. BALB/c mice, 6 to 10 weeks of age, were immunized subcutaneously with 0.1 ml of phosphate-buffered saline (PBS) containing 15 ,ug of OM from the B. pertussis 134 LOS B variant strain and 25 jg of Quil A (CedarLane Laboratories, Hornby, Ontario,

MAbs AGAINST B. PERTUSSIS LOS B

2719

Canada), which was used as an adjuvant. A second booster injection was administered 3 weeks later. A final inoculation of 15 ,ug of OM without Quil A was given intraperitoneally 4 days before the fusion procedure. Hybridomas were produced by fusion of spleen cells recovered from immunized animals with the nonsecreting SP2/O myeloma cells as described elsewhere (12). Hybrid clone supernatants were tested for antibody production by enzyme-linked immunosorbent assay (ELISA), as previously described (12), by using OM from the B. pertussis 134 LOS B variant strain as the coating antigen (7.5 ,ug of protein per ml). Specific hybrids were cloned by sequential limiting dilutions, expanded, and frozen in liquid nitrogen. The class, subclass, and light-chain type were determined by ELISA with commercially available reagents (Fisher Biotech, Ottawa, Canada). A previously isolated MAb, designated BL-2, which is directed against the LOS A band, was used as a positive control. Sodium periodate and proteinase K treatment of the OM. Sodium periodate oxidation, as described elsewhere (22), was used to determine whether a MAb reacted specifically with a carbohydrate antigenic determinant present on the LOS. Protein digestion of B. pertussis OM was performed as described previously (23). Antibody accessibility radioimmunobinding assay. Adsorption of MAbs on the surface of live bacteria was performed as previously described by Martin et al. (22). Bacteria were grown for 2 days on BGA, suspended in Dulbecco modified Eagle medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% (vol/vol) calf serum (HyClone Laboratories, Inc., Logan, Utah), and adjusted to a concentration of 5 x 109 CFU/ml. Each bacterial suspension was sequentially incubated with undiluted hybridoma culture supernatants containing the MAb and 0.25 ,uCi of 1251_ labelled goat anti-mouse immunoglobulin G (IgG) (Du Pont Company, Wilmington, Del.). The amount of cell-bound immunoglobulins was evaluated by using a gamma counter. Western immunoblotting procedure. For Western blot experiments, the bacterial preparations containing the LOSs were prepared by following one of two methods. In the first method, the bacterial concentrations were carefully adjusted to obtain a semiquantitative evaluation of the amount of LOS. BGA-grown organisms were collected, suspended in PBS (pH 7.2), and spectrophotometrically adjusted to an optical density of 2.5 (A = 490 nm). Fifty microliters of a 10% (wt/vol) solution of SDS (Bio-Rad Laboratories, Richmond, Calif.) was added to 500 ,ul of the bacterial suspension. Each preparation was boiled for 10 min and centrifuged for 10 min in a microcentrifuge to remove the cellular debris. This step was repeated once. In the second method, purified LOS was obtained after enzymatic digestion of whole bacterial cells. BGA-grown organisms were treated with 1 mg of proteinase K (Sigma Chemical Co., St. Louis, Mo.) per ml for 3 h at 56°C with agitation. These bacterial preparations were then centrifuged at 3,000 x g for 10 min to remove the undigested cellular fragments. Two volumes of cold acetone (BDH Inc., Toronto, Ontario, Canada) was added to the supernatants and incubated at -20°C for 5 min. The resulting precipitate was collected by centrifugation at 3,000 x g for 10 min, and the pellet was suspended in distilled water. All bacterial preparations were resolved by electrophoresis by using the discontinuous buffer system of Laemmli (16) with 18% (wt/vol) gels, and Western blot analysis were performed as previously described (23). The silver stain method of Tsai and Frasch was used to visualize the LOS (33). The gels

2720

MARTIN ET AL.

were also counterstained with 0.2% Coomassie brilliant blue R-250 (Bio-Rad) to detect the presence of protein. Dot enzyme immunoassay. A dot enzyme immunoassay was used for the rapid screening of MAbs against a large number of bacterial strains as described elsewhere (20). Briefly, the microbial lawn was removed from the surface of

the agar plate and suspended in Tris-buffered saline, pH 7.5. Each bacterial suspension was adjusted spectrophotometrically (A = 490 nm; optical density, 0.25), which corresponded to approximately 1 x 108 CFU/ml. Fifty microliters of each suspension was filtered through the nitrocellulose membrane by gravity by using a Bio-Dot microfiltration apparatus (Bio-Rad). Each well was then washed with 0.02% (vol/vol) Tween-Tris-buffered saline and blocked with 0.5% (wt/vol) bovine serum albumin (Sigma). The nitrocellulose membrane was removed from the apparatus and sequentially incubated with undiluted hybridoma culture supernatant, peroxidase-conjugated goat anti-mouse IgG and IgM (Cappel, Organon Teknika Corp., West Chester, Pa.), and devel-

oped. Bactericidal assay. The bactericidal activity of ascitic fluid was tested in vitro as described previously (4). Briefly, all ascites were heat inactivated at 56°C for 20 min. Bacteria (50 RI), adjusted to 2 x 104 CFU/ml, were dispensed into

Eppendorf tubes containing 410 ,ul of PBS with 0.15 mM CaCl2, 0.5 mM MgCl2, 0.1% bovine serum albumin, 40 ,ul of guinea pig serum as a source of complement, and 10 pu1 of heat-inactivated ascites. Duplicate antigen-antibody mixtures were incubated either in the absence of complement or with heat-inactivated guinea pig serum. After incubation for 1 h at 37°C, 100 ,u1 of each mixture was plated onto BGA plates. The plates were incubated for 3 to 5 days at 37°C, after which time the bacterial colonies were counted. RESULTS Description of the MAbs. Two MAbs, BL-8 and BL-9, were obtained from separate fusion experiments and were determined to be IgG2b(K) and IgA, respectively. The mice used for these fusions were immunized with OM obtained from B. pertussis LOS B variant strain 134. As determined by ELISA, the reactivity of these two MAbs with OM of strain 134 was lost after sodium periodate treatment but was unaffected by treatment with proteinase K. Moreover, these MAbs reacted strongly with phenol-water-purified LOS from LOS B variant strain 134. These results suggested that both MAbs were directed against a carbohydrate epitope(s) present in the LOS of B. pertussis 134. Control MAb BL-2, and IgG3(K) which recognizes the B. pertussis LOS A band, has been described in detail previously (4). In contrast to the LOS B-specific MAbs, MAb BL-2 showed no reactivity with the LOS B of the variant strain 134. Reactivities of the MAbs against a panel of bacterial strains by immunodot assay. An immunodot assay was used to verify the reactivities of the two LOS B-reacting MAbs against four B. pertussis LOS AB laboratory strains and their phenotypic variants, 64 clinical isolates, 4 LOS B variant strains, and 2 revertant strains. These MAbs were also tested against 13 B. parapertussis isolates, 3 isogenic Vir+ and Vir- B. bronchiseptica strains, and 22 gramnegative and gram-positive bacterial strains (Table 1). MAb BL-2 strongly recognized all LOS AB laboratory strains, revertant strains, and the 64 clinical isolates of B. pertussis but not the LOS B variant strains. This MAb also reacted with the three B. bronchiseptica Vir+ strains but not with their corresponding Vir- isogenic strains. In contrast, MAbs

INFECT. IMMUN.

BL-8 and BL-9 strongly reacted only with the four B. pertussis LOS B variant strains, the Vir- phenotypic variants derived from the LOS AB strains Tohama 1 and 3779, and LOS AB strain 9797. These two MAbs also displayed weak reactivities against the LOS AB strain 9340, as well as the two revertant strains, but did not recognize any B. pertussis clinical isolates or the three B. bronchiseptica strains. Contrary to results with MAb BL-9, we observed that MAb BL-8 reacted weakly with the B. pertussis phenotypic variant Vir+ Gna- Tohama 1 and 3779 strains. No MAbs reacted with B. parapertussis or with the other bacterial strains tested. Western blot analysis of the reactivities of the MAbs with Bordetella strains. SDS-treated whole-cell preparations of B. pertussis Tohama 1, 3779, and 134 and their isogenic phenotypic variants were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The reactivities of MAbs BL-2 and BL-8 were evaluated to clearly determine their specificities for B. pertussis LOS A and B bands. Results are presented in Fig. 1. The reactivities of these MAbs with three Vir+ and Vir- strains of B. bronchiseptica were also determined, as shown in Fig. 2. The number of bacteria in each preparation was adjusted in order to compare the relative amounts of LOS. MAb BL-2 recognized the LOS A band of the two LOS AB strains Tohama 1 and 3779 as well as their variants but did not react with the LOS B variant strain 134 and its two isogenic variants (Fig. 1B). MAb BL-2 also recognized an LPS band present on both the Vir+ and Vir- phenotypic variant strains of B. bronchiseptica (Fig. 2B), with stronger reactions always noted against the three Vir+ variant strains. In contrast, MAb BL-8 recognized the LOS B band expressed by the three B. pertussis strains and their corresponding isogenic phenotypic variants (Fig. 1C). However, in this case weaker reactivity of MAb BL-8 for the Vir+ phenotypic variants of strains Tohama 1 and 3779 in comparison to the Vir- variants was observed. Silver staining of duplicate gels indicated that the Vir+ phenotypic variant strains expressed less of the LOS B band even when an equivalent number of bacterial cells were used. As indicated by the Western blot and silver stain results, there is not a comparable difference in the amount of LOS B for the different phenotypic variants of the LOS B strain 134. MAb BL-8 also reacted with an LPS band present in the preparations obtained from the Vir- variant strains of B. bronchiseptica (Fig. 2C). Compared with the LPS band recognized by MAb BL-2, this band has a slightly lower molecular weight. Results similar to the ones already described for MAb BL-8 were also recorded for MAb BL-9

(data not shown). The reactivities of these three MAbs with proteinase K-treated bacterial preparations of additional strains were evaluated, and the results are summarized in Table 1. As expected, MAb BL-2 reacted with the LOS A band of all B. pertussis strains tested, with the exception of the four LOS B variant strains which do not have a LOS A band. In contrast, MAbs BL-8 and BL-9 recognized the LOS B band expressed by all B. pertussis strains tested. No reactivities in the Western blot assay were noted between the B. parapertussis strain and these three MAbs. Surface accessibility of the LOS epitopes. A radioimmunobinding assay was used to determine whether the LOS epitopes recognized by the MAbs were exposed and accessible at the surface of live bacterial cells. As shown in Fig. 3A, MAb BL-2 bound very strongly to its target epitope on the surface of the B. pertussis LOS AB Tohama 1 and 3779 strains and their variants. Only low binding values, under

MAbs AGAINST B. PERTUSSIS LOS B

VOL. 60, 1992

2721

TABLE 1. Reactivities of the LOS-specific MAbs with Bordetella strains as evaluated by dot immunoassay, Western immunoblotting, and radioimmunobinding assay Reactivity of MAb in:

LOS AB laboratory strains 9340 9797 Tohama 1 and 3779 Vir+ GnaVir- GnaVir- Gna+ LOS B variant strains 134 Vir+ GnaVir- GnaVir- Gna+ D3148 10901 11615 Revertant LOS AB strains 10901R 11615R B. pertussisd B. parapertussisd B. bronchiseptica Vir+ (3) Vir- (3) Other bacterial strainsf (21)

Western immunoblotting"

Dot immunoassaya

Strains

Radioimmunobinding assay'

BL-2

BL-8

BL-9

B B

++ ++

+

+

+

+

B B B

B B B

++ ++ ++

++ ++

+

-

B B B B B B

B B B B B B

-

++ ++ ++ ++ ++ ++

+ + + + +

A A A(4) -(1)

B B B(4) -(1)

B B B(4) -(1)

++ ++ + +(4) -(3)

+ +

+ +

0/64 0/13

+ + 0/64 0/13

±(4) -(3)

+(3) _(3)

-

-

-

0

0

0

A A 0

B 0

NDe ND 0

+ _ +0 0

BL-2

BL-8

BL-9

BL-2

BL-8

BL-9

+ +

+ +

+ +

A A

B B

+ + +

+ + +

+ +

A A A

-

+ + + + + +

+ + + + + +

+ +

-

+ + 64/64 0/13 +

-

+ + +

+

_ -

0

a +, Strong reaction; +, weak reaction; -, no reaction. b A, identification of the LOS A band; B, identification of the LOS B band. Numbers in parentheses indicate the numbers of strains tested. c + +, More than 10,000 cpm; +, between 5,000 and 10,000 cpm; ±, between 500 and 5,000 cpm; -, less than 500 cpm. Numbers in parentheses indicate the numbers of strains tested. d Clinical isolates. e ND, not tested. f Clinical isolates or bacterial strains (ATCC number) are Alcaligenes faecalis (8750), Branhamella catarrhalis, Citrobacter freundii, Edwardsiella tarda (15947), Enterobacter aerogenes (13048), Enterobacter cloacae (23355), Escherichia coli, Haemophilus influenzae type b (Eagan strain), Klebsiella pneumoniae (13883), Neisseria catarrhalis (8176), Neisseria perflava (14799), Neisseria subflava (19243), Proteus rettgeri (25932), Proteus vulgaris (13315), Pseudomonas aeruginosa, Salmonella typhimurium (14028), Serratia marcescens (8100), Shigella flexneri (12022), Shigella sonnei (9290), Streptococcus faecalis, and Streptococcus pneumoniae group A.

1,000 cpm, were recorded when this MAb was incubated with the LOS B strain 134 and its variants. Binding values of approximately 5,000 cpm were noted when this MAb was incubated with Vir+ B. bronchiseptica strains, but only background values were recorded for their corresponding Vir- isogenic strains (Fig. 3B). In contrast, the epitope recognized by MAb BL-8 is readily accessible on the surface of the LOS B strain 134 and its Vir- phenotypic variants as well as on the surface of the Vir- phenotypic variants of the LOS AB strains Tohama 1 and 3779 (Fig. 3A). Lower values, between 2,000 and 9,000 cpm, were observed when MAb BL-8 was incubated with the Vir+ wild-type strains Tohama 1 and 3779. Since the radiolabelled antiserum used in this study is known to have a weak affinity for mouse immunoglobulins of the IgA class, lower binding values were always recorded for MAb BL-9. Even with that limitation, readings higher than 5,000 cpm were noted when MAb BL-9 was incubated with B. pertussis LOS B strain 134 and its phenotypic variants, indicating that a specific epitope was accessible on the surface of these bacteria. In contrast to MAb BL-2, MAbs BL-8 and BL-9 did not bind the surface of either Vir+ or Vir- B. bronchiseptica strains (Fig. 3B). Additional strains were tested by using the accessibility assay, and the results are presented in Table 1. In general, the reactivities of the MAbs with these strains were similar

to those previously observed. Moreover, only very weak near the background level, were recorded when the three MAbs were reacted with B. parapertussis strains.

values,

Bacteriolytic activity. The bacteriolytic activities of the three LOS-specific MAbs against two B. pertussis strains, the LOS AB strain 9340 and the LOS B variant strain 134, were evaluated (Table 2). In the presence of complement, MAb BL-2 efficiently killed strain 9340 but did not reduce the number of viable bacteria of the LOS B variant strain. Reductions between 46 and 55%, and less than 32%, of the CFU per milliliter were observed when BL-8 was incubated with strains 134 and 9340, respectively. MAb BL-9 did not exhibit any bacteriolytic activity. MAb LO-1, which specifically recognized the LOS of Haemophilus influenzae type b, was used as a negative control. DISCUSSION Serological analyses of the antigenic determinants present on the B. pertussis LOS have generated results that are often confusing and sometimes contradictory. For example, several studies have demonstrated both intraphase and interphase variations among LOS serotypes (1, 3, 15, 28, 31), while Le Dur et al. (17) reported no differences in the endotoxin composition from any serological phase of B.

2722

S.i

MARTIN ET AL.

A

A

INFECT. IMMUN.

B~~~~~~~~~~~~~~.-..

B

B A

A

..;

1 2 3

4 5

6 7 8

1

9

C

2

3 4

5

6

C B

FIG. 1. Silver stained SDS-PAGE gels and Western immunoblots showing the reactivities of the MAbs with the LOS A and B bands of B. pertussis strains. The bacterial cell concentrations of the preparations were adjusted, applied to SDS-18% PAGE gels, and transferred onto nitrocellulose membranes. The membranes were then sequentially incubated with undiluted culture supernatants containing the MAb and alkaline phosphatase-conjugated second antibody and developed. (A) Silver-stained SDS-PAGE gel; (B and C) duplicate gels transferred to nitrocellulose membranes and reacted with MAbs BL-2 and BL-8, respectively. The following bacterial strains were used: 3779 Vir- Gna+ (lane 1), 3779 VirGna- (lane 2), 3779 Vir+ Gna- (lane 3), Tohama 1 Vir- Gna+ (lane 4), Tohama 1 Vir- Gna- (lane 5), Tohama 1 Vir+ Gna- (lane 6), 134 Vir- Gna+ (lane 7), 134 Vir- Gna- (lane 8), and 134 Vir+ Gna- (lane 9). A and B, positions of LOS A and LOS B bands, respectively.

pertussis. Recently we have developed MAbs, rather than conventional polyvalent antisera, in order to precisely identify the B-cell epitopes present on the surface of bacterial cells. We have described several human and murine MAbs which are directed against carbohydrate epitope(s) on the B. pertussis LOS A (4, 8). In this article, we present the characterization of two MAbs that are specific for the LOS B band. Three immunological assays were used to study the epitope(s) identified by the LOS B-specific MAbs BL-8 and BL-9. First, a dot enzyme immunoassay was used to rapidly evaluate the reactivities of the MAbs against a large panel of strains (4, 8, 20, 22). In the second assay, each of the bacterial cell components was resolved by using SDS-PAGE followed by Western immunoblotting. Under these conditions, the reactivities of the MAbs were completely independent of the cell surface exposure of the epitopes and of their relationship with other cell surface components. The third test, the radioimmunobinding assay, was designed to identify only those epitopes present on the surface of intact bacterial cells. By using this latter assay, we have previously been able to discriminate between epitopes of Haemophilus influenzae type b OM protein that were deeply embedded inside the membrane versus those easily accessible on the surface of intact bacteria (24, 30). The LOS-specific MAbs and the three immunological tests were used to study the relationships between the type, amount, and exposure of

FIG. 2. Silver-stained SDS-PAGE gels and Western immunoblots showing the reactivities of the MAbs with the LOS A and B

bands of B. bronchiseptica strains. The bacterial cell concentrations of the preparations were adjusted, applied to SDS-18% PAGE gels, and transferred onto nitrocellulose membranes. The membranes were then sequentially incubated with undiluted culture supernatants containing the MAb and alkaline phosphatase-conjugated second antibody and developed. (A) Silver-stained SDS-PAGE gel; (B and C) duplicate gels transferred to nitrocellulose membranes and reacted with MAbs BL-2 and BL-8, respectively. The following bacterial strains were used: 22067 Vir- (lane 1), 22067 Vir+ (lane 2), 214 Vir- (lane 3), 214 Vir+ (lane 4), 110H Vir- (lane 5), and 110H Vir+ (lane 6). A and B, positions of LOS A and LOS B bands, respectively.

LOS expressed by a B. pertussis strain and its isogenic phenotype variants. It has been shown previously that all B. pertussis strains express an LOS B band (28). We thus anticipated that, in the dot immunoassay, the B band-specific MAbs would recognize all the B. pertussis strains regardless of the phase or the LOS phenotype. To our surprise, we found that MAbs BL-8 and BL-9 reacted strongly only with the LOS B variant strains and certain laboratory LOS AB strains and did not recognize any B. pertussis clinical strains (Table 1). In particular, they did not clearly identify the Vir+ LOS AB wild-type Tohama 1 and 3779 strains, while they reacted very strongly with their isogenic Vir- phenotypic variant strains (Table 1). In spite of these observations, all of these bacterial preparations did express the LOS B band as shown by silver-stained SDS-PAGE gels, and MAbs BL-8 and BL-9 recognized the LOS B band in all B. pertussis strains, as shown by Western immunoblotting. What these studies demonstrated, however, was that the Vir+ phenotypic variants expressed less LOS B than their Vir- isogenic strains (Fig. 1A). These latter results indicated that most wild-type B. pertussis strains, like the clinical isolates which have a reduced amount of LOS B at their surface, will not react or will react weakly with the LOS B-specific MAbs in the dot assay. This explains the observed discrepancies between the results obtained by the dot immunoassay and the Western immunoblots. The third immunological test, the radioimmu-

VOL. 60, 1992

MAbs AGAINST B. PERTUSSIS LOS B

A)

TABLE 2. Bacteriolytic activity of the MAbs

* BL-2 O BL-8 0 BL-9

25000 -

0.

c11S

2723

MAb activity against: MAb

Isotype class

B.

and subclass

pertussis LOS AB strain 9340

CFU/mla 20000

% Killing

B. pertussis LOS B variant strain 134

CFU/mla

% Killing

-

15000

-

10000

-

BL-2 BL-8 BL-9

5

LO-1b

1

C

5000 -

_

+

X .6

Bordetella

B)

CL

16s

a

pertussis strains

0. -

mS

C0C

+

I o

I

0

+

+

t

N ,CD D

m%l

33 545 655 675

+ 15 ± 106 ± 49 ± 290

>95