Characterization of the protective capacity and immunogenicity of the 69-kD outer membrane protein of Bordetella pertussis

Immunization with the 69-kD outer membrane protein (OMP) of Bordetella pertussis protected neonatal mice against lethal respiratory challenge with B. pertussis 18323. Active immunization elicited a serum IgG anti- 69-kD OMP response at the time of challenge, with IgG anti-69-kD OMP antibodies detected in bronchoalveolar lavage fluid after challenge. Intravenous administration of BPE8, a monoclonal IgG1 anti-69-kD OMP, also protected young mice against B. pertussis challenge. Intravenously injected BPE8 was detected in the lungs of mice at the time of aerosol challenge, suggesting that the presence of specific antibody in the lungs may mediate protection. Thus the 69-kD OMP of B. pertussis is a protective antigen in mice that elicits specific serum antibody that can transude to the lung. The 69-kD OMP was detected in a preparation of a Takeda acellular vaccine by immunoblot analysis and a serum antibody response to the 69-kD OMP was observed in 18-mo-old children boosted with this preparation of Japanese acellular vaccine. Our results demonstrate that the B. pertussis 69-kD OMP is a protective antigen in animals, is immunogenic in humans, and is present in a preparation of acellular pertussis vaccine that is widely used in Japan. These findings indicate that the 69-kD OMP should be seriously considered as a candidate for inclusion in new formulations of antigenically defined acellular pertussis vaccines.

Murine serum and bronchoalveolar lavage fluid were analyzed for specific antibody to the 69-kD OMP by an ELISA (15). Microtiter plates (Immunolon I; Dynatech Laboratories, Chantilly, VA) were coated with 5 kg/ml of the 69-kD OMP in 0.1 M carbonate buffer, pH 9, and incubated overnight . The plates were washed and incubated with dilutions of mouse serum or bronchoalveolar lavage fluid in PBS containing 0.05% Tween and 0 .2% sodium azide for 3 h. After washing, the plates were incubated for 2 h with alkaline-phosphatase-conjugated goat anti-mouse total Ig, or with alkaline phosphatase-conjugated goat anti-mouse IgM, IgG, or IgA (Southern Biotechnology, Inc., Birmingham, AL) . The sensitivity and isotype specificity of the alkaline phosphatase conjugates were established using a panel of mAbs generously provided by Dr. John Cebra, University of Pennsylvania, Philadelphia, PA. The plates were read 30 min after addition of Sigma 104 phosphatase substrate (Sigma Chemical Co., St. Louis, MO), using a BioTek EL 312 reader (Biotek Instruments, Winooski, VT) . Total anti-69-kD OMP titers are expressed as the reciprocal of the endpoint dilution, calculated by extrapolation to zero from the linear part of the titration curve . The relative amounts of IgM, IgG, or IgA anti-69-kD OMP are reported as the OD4o5 of a 1 :100 dilution of serum or a 1 :4 dilution of bronchoalveolar lavage fluid.
Human sera were analyzed for specific antibody to the 69-kD OMP by ELISA as described above with the following modifications . Plates coated with 2 wg/m169-kD OMP were incubated with dilutions of human sera, washed, and were incubated overnight with alkaline phosphatase-conjugated goat anti-human IgG (Cappel Laboratories, Malvern, PA) before addition of substrate . Antibody unitage was calculated by a parallel line method (16).
Electrophoretic Anaosis. SDS-gel electrophoresis was performed by the method ofLaemmli (17). Electrophoresis of 69-kD OMP or Takeda pertussis vaccine and transfer to nitrocellulose (18) was done as previously described (5) . For immunoblotting, dilutions of sera were incubated with individual strips of nitrocellulose for 2 h at room temperature before incubation with alkaline phosphatase-conjugated goat anti-human Ig for 2 hours (Southern Biotechnology, Inc., Birmingham, AL), and were developed with the Protoblot substrate system (Promega, Inc., Madison, WI) .

Results
Protection Mediated by Active Immunization with the 69-kD OMP Immunization of adult mice with our preparation of 69-kD OMP elicited polyclonal antisera that reacted only with a single band of 69-kD when immunoblotted to tricine-urea-solubilized antigens of B. pertussis separated by SDS-electrophoresis (data not shown).
Immunization with the 69-kD OMP protected young mice from lethal respiratory challenge with B. pertussis 18323. All mice immunized on days 5 and 12 pp with 16 hg 69-kD OMP survived a respiratory challenge with B. pertussis 18323 on day 19 pp (Fig. 1); these mice exhibited a mild leukocytosis that subsequently returned to baseline after day 21 (data not shown) and gained weight at a rate similar to uninfected controls (Fig. 1). Mice immunized with pertussis toxoid, as a positive control, exhibited no leukocytosis and were protected from death ( Fig. 1). In contrast, mice immunized with tetanus toxoid and then challenged with an aerosol of B. pertussis failed to gain weight and exhibited a pronounced leukocytosis ; all ofthese mice were dead within 19 d after challenge (Fig. 1). As the dose of 69-kD OMP per immunization decreased from 16 to 1 ug, the proportion ofmice with severe leukocytosis (>70 x 103 WBC/I,1) increased and the percentage of survivors declined (Table I). However, -50% of the mice injected with 1 p,g 69-kD OMP survived B. pertussis respiratory challenge .
Since vaccine components are frequently chemically inactivated, a preparation ofthe 69-kD OMP was treated wtih glutaraldehyde in a manner similar to that used to inactivate pertussis toxin. This glutaraldehyde treated preparation was also pro-   tective. Of the mice immunized with 16 Ag of glutaraldehyde-fixed 69-kD OMP, 92% survived respiratory challenge with B. pertussis (Table I) .
Detection ofSpecific Antibody. Serum and bronchoalveolar lavage titers of anti-69-kD antibody were measured in mice that had been immunized days 5 and 12 pp with 8 Etg 69-kD OMP and challenged on day 19 with an aerosol of B. pertussis 18323 (Table II). A low serum IgM as well as an IgG anti-69-kD antibody response was detected in immunized mice that were not challenged .  (Table  III), demonstrating that the antibody had reached the site of infection in the lungs. In contrast to the protection observed with BPE8, 80% of the mice injected with a second IgG1 anti-69-kD OMP mAb, BPD8, died within 23 d after challenge, with all but 3 of 10 mice displaying leukocyte counts of >70 x 10 3 WBC/11 (Fig . 2) . BPD8 was also detected by ELISA in the bronchoalveolar lavage fluids of mice 24 h after intravenous injection (footnote to Table III) . Thus, the inability of BPD8 to protect cannot be attributed to its failure to transude to the lungs from the circulation . Passive administration of a mAb to an unrelated toxin, 18 .1 .7, an IgG1 anti-tetanus toxin (14), also failed to protect against B. pertussis aerosol challenge. Detection of the 69-kD OMP in an Acellular Pertussis Vaccine. Immunoblot analysis, using the anti-69-kD OMP mAb BPE3 (5), revealed the presence of 69-kD OMP in a preparation of a Takeda acellular pertussis vaccine (Fig . 3 A). Densitometry analysis of silver-stained gels reveals that the 69-kD OMP comprises as much as 5%, by weight, of the total protein in this preparation . The fact that this vaccine contained the 69-kD OMP offered the opportunity to examine the human serum antibody response to this protein . Immunoblot analysis was done on paired sera from 10 children who had received whole cell pertussis vaccine at 2, 4, and 6 mo of age and then were boosted at 18 mo with the above preparation of acellular pertussis vaccine . These children were bled immediately before receiving acellular pertussis vaccine, as well as 1 mo after the 18-mo booster dose . 6 of the 10 paired samples had a marked increase in reactivity with purified 69-kD OMP in postimmunization serum taken 1 mo after boosting . Immunoblot analysis of two of these paired sera is shown in Fig . 3 B . The IgG anti-69-kD OMP serum antibody response, measured in four of the six paired sera by ELISA, is shown in Table IV. All four individuals demonstrated 10-100-fold rises in IgG anti-69-kD OMP in response to immunization with the Takeda acellular vaccine containing 69-kD OMP.

Discussion
We have demonstrated that the 69-kD OMP is a protective antigen in an animal model of B. pertussis respiratory infection . Neonatal mice immunized with the 69-kD OMP or a glutaraldehyde-treated preparation of 69-kD OMP before respiratory challenge were protected from death and did not exhibit the severe leukocytosis and weight loss observed in unprotected controls . Protection was dose dependent, as only 50% of the mice immunized with 44 ltg 69-kD OMP survived B. pertussis respiratory challenge . That antibody-mediated protection was due to immunological reactivity to the 69-kD OMP and not due to a contaminant in the preparation was established as polyclonal antisera raised to the 69-kD preparation reacted with a single The appearance of anti-69-kD OMP in the lungs of immunized and challenged mice could be due to transudation of antibody from the serum as well as to local production of protective antibody. We have previously shown that intraperitoneal immunization of neonatal mice at days 5 and 12 pp with pertussis toxoid disseminates a primed population of antigen-specific B lymphocytes to the respiratory mucosa (19) ; this memory population may be stimulated to produce protective antibody in the lung upon respiratory challenge with B. pertussis. The mAb BPE8 passively protected mice challenged with an aerosol of B. pertussis against leukocytosis and death, while BPD8 did not. Both BPE8 and BPD8 could be detected in the lungs of mice 24 h after intravenous injection, at the time of challenge. Transudation of IgG1 antibody from the systemic circulation to the lung has previously been demonstrated by Toews et al . (20) . Since both BPE8 and BPD8 are IgG1 mAbs that recognize the 69-kD OMP (5) and both were found in the lung at the time of challenge, the ability of BPE8 to protect suggests that this antibody recognizes a protective epitope on the 69-kD OMP distinct from the epitope recognized by BPD8 . Alternatively, the observed differential protection could be due to the differences in the affinity of each mAb for the 69-kD OMP IgGI antibody to the 69-kD OMP is thus sufficient to prevent leukocytosis and death in neonatal mice challenged with B. pertussis, presumably by transudation from the serum to the lung, although active immunization with the 69-kD OMP may elicit mechanisms of protective immunity in addition to serum antibody.
The mAb BB05, which was raised to the 68-kD protein of B. bronchiseptica, and which reacts by immunoblot with the 69-kD protein purified from B. pertussis (5), has been shown to passively protect mice from atrophic rhinitis and death when challenged with an aerosol of B. bronchiseptica (21). Active immunization of pregnant sows with the 68-kD OMP purified from B. bronchiseptica has also been shown to prevent atrophic rhinitis in young offspring challenged intranasally with B. bronchiseptica (22). Presumably these piglets were protected by specific antibody in mother's milk, as there is no transplacental transfer of Ig in ungulates (23). Thus both the 69-kD OMP purified from B. pertussis and an immunologically related protein isolated from B. bronchiseptica have been shown to be protective antigens .
We show here that significant amounts of the 69-kD OMP are detectable in a T-type acellular vaccine prepared by Takeda . T-type vaccines are sucrose gradientpurified preparations of B. pertussis extracts that have been demonstrated to contain a mixture of antigens, including pertussis toxin, filamentous hemagglutinin, and fimbriae (24,25). Since purified FHA is easily degraded and commonly runs as several bands on an acrylamide gel, including some near 69-kD, the presence of69-kD OMP in a Ttype acellular pertussis vaccine has until now gone undetected . Using mAbs specific for these antigens we have demonstrated the presence of the 69-kD OMP as well as FHA in the vaccine by immunoblotting.
In the preliminary studies described here, an antibody response to the 69-kD OMP was detected by immunoblot analysis in 18-mo-old children boosted with the Takeda acellular pertussis vaccine. One to two log increases in the concentration of serum anti-69-kD OMP antibodies were also detected by ELISA. Serum antibody responses to the 69-kD OMP have also been detected in children within 3-9 mo after completing a course of vaccination with whole cell vaccine (26), as well as in convalescent serum and saliva (27). In addition, De Magistris et al. have demonstrated that several human T cell clones, derived from a volunteer convalescent from pertussis, specifically proliferated in response to stimulation with 69-kD OMP purified from B. pertussis (28). The 69-kD OMP antigen, therefore, elicits both B cell-mediated and T cell-mediated immunity in humans.
The majority of the doses of acellular vaccine administered in Japan are T-type, containing inactivated pertussis toxin, filamentous hemagglutinin, and surface proteins that induce agglutinating antibodies (4). The ability of a whole cell pertussis vaccine to elicit agglutinating antibody has previously been correlated with clinical protection (29). However, it remains to be established if serum antibodies to the 69-kD OMP will provide a correlate of protective immunity for acellular pertussis vaccines .
We have demonstrated that the 69-kD OMP induces agglutinating antibodies (5), elicits specific antibody detected in the serum and lungs of mice, and is protective in a murine model ofB. pertussis infection. A passively administered anti-69-kD OMP mAb, detected in the lungs at the time of challenge, was also found to protect mice. We further show that the 69-kD OMP elicits an antibody response in humans, and is present in an acellular pertussis vaccine preparation. The 69-kD OMP has recently been cloned and sequenced (6) and a protocol suitable for the large scale purification of antigen has recently been developed (30). Thus, the availability of sufficient quantities of purified antigen will allow for the analysis of the role and mechanism of the 69-kD OMP in eliciting protective immunity in humans.

Summary
Immunization with the 69-kD outer membrane protein (OMP) of Bordetella pertussis protected neonatal mice against lethal respiratory challenge with B. pertussis 18323 . Active immunization elicited a serum IgG anti-69-kD OMP response at the time of challenge, with IgG anti-69-kD OMP antibodies detected in bronchoalveolar lavage fluid after challenge . Intravenous administration ofBPE8, a monoclonal IgG1 anti-69-kD OMP, also protected young mice against B. pertussis challenge. Intravenously injected BPE8 was detected in the lungs of mice at the time of aerosol challenge, suggesting that the presence of specific antibody in the lungs may mediate protection. Thus the 69-kD OMP of B. pertussis is a protective antigen in mice that elicits specific serum antibody that can transude to the lung.
The 69-kD OMP was detected in a preparation of a Takeda acellular vaccine by immunoblot analysis and a serum antibody response to the 69-kD OMP was observed in 18-mo-old children boosted with this preparation ofJapanese acellular vaccine. Our results demonstrate that the B. pertussis 69-kD OMP is a protective antigen in animals, is immunogenic in humans, and is present in a preparation ofacellular pertussis vaccine that is widely used inJapan . These findings indicate that the 69-kD OMP should be seriously considered as a candidate for inclusion in new formulations of antigenically defined acellular pertussis vaccines .