Vol. 58, No. 10

INFECTION AND IMMUNITY, OCt. 1990, p. 3369-3374 0019-9567/90/103369-06$02.00/0 Copyright © 1990, American Society for Microbiology

Protective Activities in Mice of Monoclonal Antibodies against Pertussis Toxin HIROKO SATO'* AND YUJI SATO2 Departments of Applied Immunology' and Bacteriology,2 National Institute of Health,

Kamiosaki, Shinagawa-Ku, Tokyo 141, Japan Received 19 April 1990/Accepted 25 July 1990

Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subunits, designated Si through S5. Immunoprotective activity of 20 different mouse monoclonal antibodies (MAbs) against pertussis toxin, 10 anti-Si, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, were investigated by aerosol and intracerebral challenges with virulent B. pertussis organisms in mice. Four anti-Si, named 1B7, 1D7, 3F11, and 1OD6, and three anti-S23 antibodies, named 11E6, 1OB5, and 1OC9, showed the highest, and almost complete, protectivity against the aerosol challenge. Mouse protectivity against the intracerebral challenge was significant for these four anti-Si MAbs but not for any of the three anti-S23 MAbs. Four anti-Si and two anti-S4 MAbs did not protect the mice against either challenge. The other seven MAbs also showed dose-dependent moderate but signfficant protection against the aerosol challenge. In the aerosol challenge system, bacterial numbers and amounts of PT detected in the lung and the number of peripheral leukocytes were lower in the mice given the protective MAbs. All mice surviving 5 weeks after the infection produced high titers of antibodies against PT, ifiamentous hemagglutinin (FHA), and agglutinogens from the challenge organisms. A combination of the protective MAbs 1B7 and 11E6 strongly suppressed the disease and mortality of the mice at smaller amounts than with the anti-PT polyclonal antibody. Although combinations of one of the protective MAb and anti-FHA or anti-agglutinogen 2 also showed extremely high mouse protection without development of symptoms of the disease, antibody titers of the survivors against PT, FHA, and agglutinogens were significantly low. The foregoing results suggest that some important protective epitopes should be in Si and S2 and/or S3, although there are both differences and similarities in the protective roles between anti-Si and anti-S23 antibodies and also in the pathogenic mechanisms between aerosol and intracerebral infections. Furthermore, it was suggested that although not only FHA and agglutinogen 2 but also PT have roles as attachment factors, the processes of infection and protection are different between mice immunized with antibody against FHA or agglutinogen 2 and that against PT because the latter mice are also able to neutralize toxicity of PT diffused into the mice.

tic activity for the disease (12, 14). The anti-S23 MAb, 11E6, also protected mice against the aerosol challenge but not against the i.c. challenge (14). Although several other laboratories have published papers on MAbs against PT (1, 2, 5, 7, 20), there has been no report focusing on the immunoprotectivity of MAbs against different subunits. No other group has reported on the protectivity of anti-B oligomer antibody against challenge with virulent B. pertussis organisms. In this study, we investigated the mouse protectivity of 20 different anti-PT MAbs, 10 anti-Si, 1 anti-S2, 2 anti-S3, 4 anti-S23, and 3 anti-S4 antibodies, in aerosol challenge and i.c. challenge systems with B. pertussis to expand and confirm previous results and to determine the location of the most important epitopes for immunoprotectivity. The role of specific antibodies against different protective epitopes in immunity of mice to pertussis infection and disease was investigated by a combination of antibodies not only against PT but also against filamentous hemagglutinin (FHA) and agglutinogen 2 (Agg 2), which were also considered to be effective protective antigens. The other purpose of this study was to provide reference antibody preparations by the mixing of several MAbs suitable for assay of PT and of anti-PT antibody and for quality control or evaluation of the vaccine. To determine a reasonable composition and the constant proportions of the antibodies as a reference, it is essential to know the protective properties and activity of each MAb.

The importance of pertussis toxin (PT) as a protective antigen of pertussis vaccine has been demonstrated by the results of a field trial with pure pertussis toxoid in Sweden (21) as well as the results of mouse protection experiments in the laboratory (8, 9, 11, 13, 17). PT has many different biological and toxic activities caused by its complicated hexameric structure consisting of five different subunits, designated Si through S5. The toxicity of PT is considered to be generated by the A (active) protomer, Si, with ADPribosyltransferase activity in cooperation with the B (binding) oligomer composed of two dimers, S24 and S34, and one S5 and to be related to some of the undesirable side effects. Which antibodies against which subunits or epitopes are important in the prevention of pertussis disease? Which antibodies should be produced by the vaccination? To improve the PI antigen in the vaccine for a safer and more potent antigen, the protective epitopes on PT should be dissected and these questions should be answered. We have been studying immunoprotective and antitoxic properties of five monoclonal antibodies (MAbs) against PT, two anti-Si and one anti-S2, S23, and S4 antibody each (12, 14, 15). One of the anti-Si MAbs, 1B7, showed highly potent mouse protectivity against either aerosol or intracerebral (i.c.) challenge with Bordetella pertussis as well as therapeu-

*

Corresponding author. 3369

3370

SATO AND SATO

INFECT. IMMUN.

MATERIALS AND METHODS

a-SI

Preparation of MAbs. A hybridoma of spleen cells of a BALB/c mouse and myeloma SP2/0 was generated twice previously (12, 15) and kept in liquid nitrogen after screening for antibody production but without cloning. The frozen hybridoma was taken out of the nitrogen to select the hybridoma cells which produce different MAbs from those already characterized (12, 15). The selection was carried out by enzyme-linked immunosorbent assay (ELISA) by using PT, 8 M urea-treated PT, Si, and S234 as coating antigens and by the neutralization test for CHO cell clustering activity of PT (3). After cloning of the new hybridoma by repeated limiting dilution as described in the previous paper (15), 15 MAbs were selected for this study. The isotype of all the MAbs was immunoglobulin Gl (IgGi). Binding activity of the MAbs to PT and its subunits. The binding activity in ELISA was calculated from the values relative to the activity of anti-PT polyclonal antibody (PAb), M5 (1,050 ELISA U/ml) or M6 (5,500 ELISA U/ml), as reference antibody, and expressed in ELISA U/ml. M5 and M6 were antibodies fractionated with 50% saturation of ammonium sulfate from the pooled sera of mice hyperimmunized with pertussis toxoid which was detoxified with Formalin and precipitated with aluminum hydroxide gel. The titer of the reference antibodies were estimated by a parallel line assay by using previous (M3 and M4) and original (Mi) reference antibodies. An ELISA unit was defined as the reciprocal of the dilution of the serum, Ml, that gave 0.5 A405 after 30 min of color development at room temperature, and ELISA titer of Mi was set as 2,700 ELISA U/ml. The binding specificity of the MAbs was determined by ELISA by using each subunit purified by conventional chromatography (19) or high-pressure liquid chromatography (23) as coating antigens and also by immunoblotting analysis for which PT preparations incubated at 50°C for 30 min with 1% sodium dodecyl sulfate but without any reducing reagent were electrophoresed on a 0.1% sodium dodecyl sulfate-15% polyacrylamide gel with a 5% stacking gel as reported previously (15). The binding patterns of the MAbs and PAb, M6, on each subunit are shown in Fig. 1. Although two MAbs, llD9 and 1OC9, were not included in Fig. 1, their binding specificity was proved to be anti-Si and anti-S23, respectively, by other blotting experiments. The five MAbs, iB7, 3F10, 9G8, liE6, and iH2, were characterized and reported before (12, 15). The other new MAbs have also been characterized concerning their biological properties

(unpublished data). Preparation of PAbs against FHA and Agg 2. Antibodies against FHA were obtained by immunization of mice (ddY SPF female) as reported previously (13). An ELISA titer of a reference anti-FHA antibody was determined in the same way as anti-PT reference antibodies. Antibody against Agg 2 was obtained by immunization of two rabbits (SPF males, 10 months of age) which did not have either antibodies against PT and FHA or agglutinins to B. pertussis strains 18323 (serotype 1,2,3), Tohama (serotype 1,2), and Yamaguchi (serotype 1,3) before the immunization. The purified preparation of fimbriae (Agg 2) was kindly donated by L. I. Irons (Center for Applied Microbiology and Research). The antigen emulsified with Freund incomplete adjuvant

was

given

to the rabbits intradermally: 50 jig of the antigen was injected twice at a 3-day interval for primary immunization, and 1 month later, the rabbits were boosted with 62 ,ug of the same antigen. The rabbits were bled three times from 2 weeks after the booster for 2 weeks. The sera were pooled,

1 B7 3F10

.

t

ElE E2E 1D7 3Fl1 4D10 8G4

I

I

a-S2 9G8 8'''' a-S 3 7E10 a-S23

I.

7G11

llE6

G9A a-S4

I.

..

l

IOB5 6H2 6G8

PAb

M6

FIG. 1. Reaction of MAbs with each subunit of PT in immunoblots. PAb, M6, used as positive control shows 5 immunoprecipitin bands corresponding to Si to S5 and a dye front band. MAbs against Si (a-Sl), S2 (a-S2), S3 (a-S3), both S2 and S3 (a-S23), and S4 (a-S4) are observed.

inactivated at 56°C for 30 min, and fractionated with 50% saturation of ammonium sulfate. Agglutinin titer to strain 18323 was 8,000 U/ml. The agglutination unit was expressed as the reciprocal of the highest dilution of the antibody showing agglutination. Since antibody titers against PT and FHA were less than 25 and 9 ELISA U/ml, respectively, the effect of these antibodies on the protection experiments was considered negligible. Assay of antibody titer. Antibodies against PT or FHA were measured by the ELISA system, and ELISA units per milliliter were estimated from the dose-response curves by the reference line assay method with anti-PT, M6, or anti-

PAbs, respectively, as reference, as reported previously (13). The agglutinin titer was estimated by agglutination with B. pertussis, strain 18323, in a microplate (13). Mouse protection test against aerosol challenge. Mouse protection tests were carried out as described previously (16, 18). Suckling mice (6 days old) were immunized passively with 0.2 ml of the antibody by intraperitoneal injection 2 h before aerosol challenge with B. pertussis strain 18323. Aerosol challenge was performed by using improved aerosol inhalation equipment to permit the reproducible and uniform infection of 500 mice simultaneously in a relatively short time. Two hundred and fifty suckling mice (removed tempowere placed in a rarily from their mothers for the exposure) transparent aerosol chamber (120 x 120 x 40 cm) with 50 compartments. The suckling mice were exposed for 30 min to an aerosol generated from the bacterial suspension (109/ ml) at a nebulizer pressure of 1.5 kg/cm2 by using filterFHA

mouse

sterilized air.

Leukocytes and body weight were measured every week for 5 weeks, and then the survivors were bled for assay of the antibodies against pertussis antigens, PT, FHA, and

PROTECTIVE ACTIVITIES OF MAbs AGAINST PERTUSSIS TOXIN

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TABLE 1. Protective effect of the MAbs" against aerosol (Expt A) and i.c. (Expt B) challenge with B. pertussis, strain 18323 Expt A (aerosol challenge) Expt B % Antibody at: Leukocytes/nlb % Survival (S/T)" Body weightc at: Antibody titer' to: survivald (S/T) 2wk 5wk 2wk 5wk 2wk 5wk PT FHA Agg anti-Sl 1B7 32 (23-43) 20 (16-25) 79 240 148 83 (25/30) 7.9 (7.1-8.8) 23 (22-25) 100 (20/20) 100 (20/20) 3F10 96 (41-224) 152 (112-207) 5.8 (5.1-6.7) 8 (5-12) 30 (6/20) 5

Protective activities in mice of monoclonal antibodies against pertussis toxin.

Pertussis toxin (PT) protein, which is the most important protective antigen of Bordetella pertussis, has a hexameric structure composed of five subun...
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