INFECTION AND IMMUNITY, Dec. 1991, p. 4732-4734
Vol. 59, No. 12
0019-9567/91/124732-03$02.00/0 Copyright C 1991, American Society for Microbiology
Properties of the B Oligomer of Pertussis Toxin LUCIANO NENCIONI,* MARIAGRAZIA PIZZA, GIANFRANCO VOLPINI, MARIA TERESA DE MAGISTRIS, FRANCO GIOVANNONI, AND RINO RAPPUOLI
Sclavo Research Center, Siena, Italy Received 9 July 1991/Accepted 12 September 1991
The B oligomer of pertussis toxin was purified from culture supernatants of Bordetella pertussis strains which do not secrete the Si subunit. The purified B oligomer is devoid of toxicity for CHO cells and other in vivo properties of the toxin, such as leukocytosis and histamine sensitization, but it retains the abilities to agglutinate erythrocytes and to induce the proliferation of T lymphocytes. The B oligomer is also able to induce protective immunity in mice but is less potent than molecules containing the S1 subunit also.
into the holotoxin and secrete into the culture medium only the B oligomer (13) (Table 1). The B oligomers produced by these strains were tested for their biological and immunological properties in various in vitro and in vivo experimental systems. These properties were compared with those of wild-type PT and with those of genetically inactivated double mutant PT-9K/129G, which is being used as a component of new acellular pertussis vaccines (9, 14). The results obtained showed that the B oligomers, like double mutant PT-9K/129G, did not possess any of the toxic properties of the enzymatically active S1 subunit of PT. In fact, the B oligomers secreted by the four engineered strains of B. pertussis did not show any detectable ADP-ribosyltransferase activity (11) and could not induce morphological changes in CHO cells (7), even at a concentration of 5 p,g/ml (Table 1). On the contrary, in the same assay 5 pg of wild-type PT per ml was sufficient to cause a visible clustering effect. As expected, in a radioimmunoassay, the purified B oligomers were not recognized by monoclonal antibody 1B7, specific for the Sl subunit (16). On the other hand, they were recognized at a lower affinity, as compared with both PT and double mutant PT-9K/129G, by a polyclonal antibody raised against the holotoxin (12). The only in vitro activities of PT which were entirely retained by the B oligomers were hemagglutination and mitogenicity for T cells (5, 6, 15, 20). In fact, the B oligomers could agglutinate glutaraldehyde-fixed chicken erythrocytes (17) and induce the proliferation of human peripheral blood mononuclear cells at the same concentrations as those of wild-type PT and PT-9K/129G (Table 1). In vivo, all the B oligomers were unable to promote leukocytosis in mice (8) and to sensitize them to the histamine challenge (8) up to a dose of 50 ,ug per mouse, a dose which is 100 times higher than the lethal median dose of wild-type PT. To investigate the immunogenicity of the B oligomers, we treated groups of six guinea pigs with two injections, 4 weeks apart, of 0.6, 3, or 15 ,ug of B oligomer, PT-13L/26I/129G, and genetically detoxified PT double mutant PT-9K/129G. A representative example is shown in Fig. 1. Undiluted sera from animals bled 4 weeks after the first immunization with PT-13L/261/129G showed in an enzyme-linked immunosorbent assay (ELISA) (9) A405 values which were 9- to 22-fold higher than those prior to immunization. A further increase
Pertussis toxin (PT) is a bacterial toxin composed of the A protomer and the B oligomer (19). The B oligomer is
composed of four noncovalently linked subunits, S2, S3, S4, and S5, present in a 1:1:2:1 ratio. The A protomer contains the enzymatically active S1 subunit. The Si subunit possesses ADP-ribosyltransferase activity and is responsible for the toxicity of PT, while the B oligomer binds to cellular receptors on eukaryotic cells and mediates the internalization of the toxic S1 subunit. We have previously described the construction of genetically engineered strains of Bordetella pertussis which produce mutated forms of PT containing two amino acid substitutions in the sequence of the S1 subunit (12). These PT mutants have lost all the toxic properties typical of PT but are still capable of agglutinating chicken erythrocytes and inducing the proliferation of human T cells (9, 12). On the basis of these results, we previously suggested that hemagglutination and proliferation are properties of the B oligomer and are not affected by genetic manipulation of the Si subunit. Studies based on the chemical modification of the PT subunits (2, 10) or on B oligomers purified from wild-type toxin (3, 5, 6, 18) have been performed to establish whether the activities of PT are due to the S1 subunit-mediated ADP-ribosylation of GTP-binding proteins or whether some of them are the results of the cell-binding property of the B oligomer. Moreover, antibodies elicited in mice with a B oligomer purified from wild-type PT (1, 3) could neutralize the toxin-induced clustering of Chinese hamster ovary (CHO) cells (4). Such a B oligomer could also prevent the induction of leukocytosis in vivo in mice (1) and protect them against B. pertussis infection in a model of aerosol challenge (18). However, these studies were not conclusive, since the chemical treatment may not completely and irreversibly inactivate PT and the purified B oligomer may still contain as much as 1% active S1 subunit. Thus, to perform a definitive evaluation of the properties of the B oligomer, it has been necessary to develop a molecule completely devoid of the S1 subunit. We have constructed four strains of B. pertussis encoding the S1 subunit with single or multiple amino acid substitutions; these strains do not assemble the S1 subunit
*
Corresponding author. 4732
NOTES
VOL. 59, 1991
4733
TABLE 1. Properties of the B oligomera Presence (~ml of S1 (Ag/llll) subunit
Yieldi
molecule PTPT molecule
PT
PT-9K/129G: Arg-9--*Lys, Glu-129-*Gly PT-80/9G: Tyr-8--+Asp, Arg-9-*Lys PT-SOE: Phe-50--+Glu PT-88E/89S: Ile-88--*Glu, Tyr-89--÷Ser PT-13L/261/129G: Arg13--Leu, Trp-26-*Ile,
ADP-ribo-
ofsunit sylation syg/lato (>~g/ml)
CHO toxicity
(>±g/ml)
4 4
+ +
5 x 10-3 >10
5 x 10-6 >5
0.4
-
>10
0.4 0.4
-
0.4
-
T cell Leukocytosis- Histamine- Hemagglu- mitogepromoting sensitizing tination nicity activity activity (p.g/ (gm 10 (10 (p.gml) )
actl/mouse) (Rg/mouse)
cp)
Affinity constant mol) for:
Mooclonal antibody 1B7
(liters/
Ga mma globulins glbin
1.8 x 108 4.8 x 1010
0.04 >50
0.02 >50
0.30 0.25
0.4 0.35
>5
>50
>50
0.30
0.35
0.0
2.7 x 109
>10 >10
>5
>5
>50 >50
>50 >50
0.25 0.35
0.4 0.3
0.0 0.0
1.3 x 109 3.1 x 109
>10
>5
>50
>50
0.20
0.35
0.0
ND
1.7 x 108 2.5 x 1010
Glu-129lestGly I
All assays were performed as described previously (9); numerical values represent the lowest effective protein concentrations. ND, not determined.
in the ELISA values was observed in sera obtained 2 weeks after the booster injection. These ELISA values were comparable to those obtained after immunization with PT-9K/ 129G. Nevertheless, the increase in antibody titers did not correlate with neutralizing activity in the CHO cell assay (4). In fact, the sera resulting from a single injection of 0.6, 3, or 15 ,ug of PT-13L/261/129G did not neutralize the clustering effect of wild-type PT in CHO cells. In marked contrast, antibodies obtained from animals given equivalent doses of double mutant PT-9K/129G were able to neutralize toxin activity at a dilution of 1/20, 1/20, or 1/80, respectively. Good neutralizing activity was observed with a second immunization with the B oligomer, but this activity was still lower than that induced by PT-9K/129G (Fig. 1). PT-13L/261/129G was able to protect mice, in a dose-
dependent fashion, against intracerebral challenge with virulent B. pertussis (12), an assay whose results correlate with the protective activity of the cellular vaccine. However, B oligomer PT-13L/26I/129G was less potent than double mutant PT-9K/129G, containing the Si subunit also (Table 2). In conclusion, the development of pertussis strains which secrete only the B oligomer of PT has provided us with unique reagents which allow definitive determination of the properties of PT. We have confirmed that the toxicity of PT is entirely dependent on the enzymatically active S1 subunit and have provided conclusive evidence that the abilities of PT to agglutinate erythrocytes and to induce the proliferation of T lymphocytes belong to the B oligomer alone and are, consequently, independent of the enzymatic mechanism of PT.
4. LU I~-
3.
F C,
10 a
z
2a ILU z
C,,
1
0
0
-j
oi
PRE IMMUNE
0.6
3
15
PT-9K/129G
--
0.6 -
3
15
PT-13L/261/129G
-_
FIG. 1. ELISA values and toxin-neutralizing titers of antibodies raised in guinea pigs by one or two subcutaneous injections of different doses of PT-9K/129G and PT-13L/261/129G. Antitoxin antibody titers are expressed as A405 values (ABS-MAX). Neutralizing titers (NT) are expressed as reciprocals of the highest serum dilutions resulting in 100% inhibition of the clustering effect induced in CHO cells by 120 pg of wild-type PT tested in triplicate.
4734
NOTES
INFECT. IMMUN.
TABLE 2. Vaccine potencies determined by mouse survival following intracerebral infection with virulent B. pertussis No. of
Vaccine
Dosea
survivors
National Institutes of Health standard cellular vaccine (lot 9)c
0.05 0.01 0.002
14 8 5
PT-9K/129G
15 3 0.6
13 9 4
PT-13L/261/129G
15 3 0.6
8 5 0
a Doses of the National Institutes of Health vaccine are in milliliters; doses of the other, plain vaccines are in micrograms per mouse. b A total of 16 mice were tested. c This vaccine contains 8 IU/ml.
We thank H. Sato for kindly providing monoclonal antibody 1B7, G. Corsi for graphic work, and A. Mori and C. Mallia for editing. REFERENCES 1. Arciniega, J. L., D. L. Burns, E. Garcia-Ortigza, and C. R. Manclark. 1987. Immune response to the B oligomer of pertussis toxin. Infect. Immun. 55:1132-1136. 2. Burns, D. L., J. G. Kenimer, and C. R. Manclark. 1987. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect. Immun. 55:24-28. 3. Burns, D. L., and C. R. Manclark. 1986. Adenine nucleotides promote dissociation of pertussis toxin subunits. J. Biol. Chem. 261:4324-4327. 4. Gillenius, P., E. Jaatmaa, P. Askelof, M. Granstrom, and M. Tiru. 1985. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. J. Biol. Stand. 13:61-66. 5. Gray, L. S., K. S. Huber, M. C. Gray, E. L. Hewlett, and V. H. Englehard. 1989. Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein. J. Immunol. 142:1631-1638. 6. Hausman, S. Z., D. S. Burns, V. C. Sickler, and C. R. Manclark. 1989. Immune response to dimeric subunits of the pertussis toxin B oligomer. Infect. Immun. 57:1760-1764. 7. Hewlett, E. L., K. T. Sauer, G. A. Myers, J. L. Cowell, and R. L. Guerrant. 1983. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect. Immun. 40:1198-1203. 8. Munoz, J. J., H. Arai, R. K. Bergman, and P. L. Sadowski. 1981. Biological activities of crystalline pertussigen from Bordetella
pertussis. Infect. Immun. 33:820-826. 9. Nencioni, L., M. G. Pizza, M. Bugnoli, M. T. De Magistris, A. Di Tommaso, F. Giovannoni, R. Manetti, I. Marsili, G. Matteucci, D. Nucci, R. Olivieri, P. Pileri, R. Presentini, L. Villa, J. G. Kreeftenberg, S. Silvestri, A. Tagliabue, and R. Rappuoli. 1990. Characterization of genetically inactivated pertussis toxin mutants: candidates for a new vaccine against whooping cough. Infect. Immun. 58:1308-1315. 10. Nogimori, K., M. Tamura, M. Yajima, K. Ito, T. Nakamura, and N. Kajikawa. 1984. Dual mechanisms involved in development of diverse biological activities of islet-activating protein, pertussis toxin, as revealed by chemical modifications of lysine residues in the toxin molecule. Biochim. Biophys. Acta 801: 232-239. 11. Pizza, M., A. Bartoloni, A. Prugnola, S. Silvestri, and R. Rappuoli. 1988. Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity. Proc. Natl. Acad. Sci. USA 85:7521-7525. 12. Pizza, M., A. Covacci, A. Bartoloni, M. Perugini, L. Nencioni, M. T. De Magistris, L. Villa, D. Nucci, R. Manetti, M. Bugnoli, F. Giovannoni, R. Olivieri, J. T. Barbieri, H. Sato, and R. Rappuoli. 1989. Mutants of pertussis toxin suitable for vaccine development. Science 246:497-500. 13. Pizza, M., A. Covacci, M. Bugnoli, R. Manetti, and R. Rappuoli. 1990. The S1 subunit is important for pertussis toxin secretion. J. Biol. Chem. 265:17759-17763. 14. Podda, A., L. Nencioni, M. T. De Magistris, A. Di Tommaso, P. Bossu, S. Nuti, P. Pileri, S. Peppoloni, M. Bugnoli, P. Ruggiero, I. Marsili, A. D'Errico, A. Tagliabue, and R. Rappuoli. 1990. Metabolic, humoral, and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT-9K/129G. J. Exp. Med. 172:861-868. 15. Randall, L. L., and S. J. S. Hardy. 1984. Export of protein in bacteria. Microbiol. Rev. 48:290-298. 16. Sato, H., A. Ito, J. Chiba, and Y. Sato. 1984. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infection. Infect. Immun. 46:422-428. 17. Sato, Y., J. L. Cowell, H. Sato, D. J. Burstyn, and R. Charles. 1983. Separation and purification of the hemagglutinins from Bordetella pertussis. Infect. Immun. 41:313-320. 18. Shahin, R. D., M. H. Witvliet, and C. R. Manclark. 1990. Mechanism of pertussis toxin B oligomer-mediated protection against Bordetella pertussis respiratory infection. Infect. Immun. 58:4063-4068. 19. Tamura, M., K. Nogimori, S. Murai, M. Yajima, K. Ito, T. Katada, M. Ui, and S. Ishii. 1982. Subunit structure of isletactivating protein, pertussis toxin, in conformity with the A-B model. Biochemistry 21:5516-5522. 20. Tamura, M., K. Nogimori, M. Yajima, K. Ase, and M. Ui. 1983. A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells. J. Biol. Chem. 258:6756-6761.
Vol. 59, No. 12
0019-9567/91/124732-03$02.00/0 Copyright C 1991, American Society for Microbiology
Properties of the B Oligomer of Pertussis Toxin LUCIANO NENCIONI,* MARIAGRAZIA PIZZA, GIANFRANCO VOLPINI, MARIA TERESA DE MAGISTRIS, FRANCO GIOVANNONI, AND RINO RAPPUOLI
Sclavo Research Center, Siena, Italy Received 9 July 1991/Accepted 12 September 1991
The B oligomer of pertussis toxin was purified from culture supernatants of Bordetella pertussis strains which do not secrete the Si subunit. The purified B oligomer is devoid of toxicity for CHO cells and other in vivo properties of the toxin, such as leukocytosis and histamine sensitization, but it retains the abilities to agglutinate erythrocytes and to induce the proliferation of T lymphocytes. The B oligomer is also able to induce protective immunity in mice but is less potent than molecules containing the S1 subunit also.
into the holotoxin and secrete into the culture medium only the B oligomer (13) (Table 1). The B oligomers produced by these strains were tested for their biological and immunological properties in various in vitro and in vivo experimental systems. These properties were compared with those of wild-type PT and with those of genetically inactivated double mutant PT-9K/129G, which is being used as a component of new acellular pertussis vaccines (9, 14). The results obtained showed that the B oligomers, like double mutant PT-9K/129G, did not possess any of the toxic properties of the enzymatically active S1 subunit of PT. In fact, the B oligomers secreted by the four engineered strains of B. pertussis did not show any detectable ADP-ribosyltransferase activity (11) and could not induce morphological changes in CHO cells (7), even at a concentration of 5 p,g/ml (Table 1). On the contrary, in the same assay 5 pg of wild-type PT per ml was sufficient to cause a visible clustering effect. As expected, in a radioimmunoassay, the purified B oligomers were not recognized by monoclonal antibody 1B7, specific for the Sl subunit (16). On the other hand, they were recognized at a lower affinity, as compared with both PT and double mutant PT-9K/129G, by a polyclonal antibody raised against the holotoxin (12). The only in vitro activities of PT which were entirely retained by the B oligomers were hemagglutination and mitogenicity for T cells (5, 6, 15, 20). In fact, the B oligomers could agglutinate glutaraldehyde-fixed chicken erythrocytes (17) and induce the proliferation of human peripheral blood mononuclear cells at the same concentrations as those of wild-type PT and PT-9K/129G (Table 1). In vivo, all the B oligomers were unable to promote leukocytosis in mice (8) and to sensitize them to the histamine challenge (8) up to a dose of 50 ,ug per mouse, a dose which is 100 times higher than the lethal median dose of wild-type PT. To investigate the immunogenicity of the B oligomers, we treated groups of six guinea pigs with two injections, 4 weeks apart, of 0.6, 3, or 15 ,ug of B oligomer, PT-13L/26I/129G, and genetically detoxified PT double mutant PT-9K/129G. A representative example is shown in Fig. 1. Undiluted sera from animals bled 4 weeks after the first immunization with PT-13L/261/129G showed in an enzyme-linked immunosorbent assay (ELISA) (9) A405 values which were 9- to 22-fold higher than those prior to immunization. A further increase
Pertussis toxin (PT) is a bacterial toxin composed of the A protomer and the B oligomer (19). The B oligomer is
composed of four noncovalently linked subunits, S2, S3, S4, and S5, present in a 1:1:2:1 ratio. The A protomer contains the enzymatically active S1 subunit. The Si subunit possesses ADP-ribosyltransferase activity and is responsible for the toxicity of PT, while the B oligomer binds to cellular receptors on eukaryotic cells and mediates the internalization of the toxic S1 subunit. We have previously described the construction of genetically engineered strains of Bordetella pertussis which produce mutated forms of PT containing two amino acid substitutions in the sequence of the S1 subunit (12). These PT mutants have lost all the toxic properties typical of PT but are still capable of agglutinating chicken erythrocytes and inducing the proliferation of human T cells (9, 12). On the basis of these results, we previously suggested that hemagglutination and proliferation are properties of the B oligomer and are not affected by genetic manipulation of the Si subunit. Studies based on the chemical modification of the PT subunits (2, 10) or on B oligomers purified from wild-type toxin (3, 5, 6, 18) have been performed to establish whether the activities of PT are due to the S1 subunit-mediated ADP-ribosylation of GTP-binding proteins or whether some of them are the results of the cell-binding property of the B oligomer. Moreover, antibodies elicited in mice with a B oligomer purified from wild-type PT (1, 3) could neutralize the toxin-induced clustering of Chinese hamster ovary (CHO) cells (4). Such a B oligomer could also prevent the induction of leukocytosis in vivo in mice (1) and protect them against B. pertussis infection in a model of aerosol challenge (18). However, these studies were not conclusive, since the chemical treatment may not completely and irreversibly inactivate PT and the purified B oligomer may still contain as much as 1% active S1 subunit. Thus, to perform a definitive evaluation of the properties of the B oligomer, it has been necessary to develop a molecule completely devoid of the S1 subunit. We have constructed four strains of B. pertussis encoding the S1 subunit with single or multiple amino acid substitutions; these strains do not assemble the S1 subunit
*
Corresponding author. 4732
NOTES
VOL. 59, 1991
4733
TABLE 1. Properties of the B oligomera Presence (~ml of S1 (Ag/llll) subunit
Yieldi
molecule PTPT molecule
PT
PT-9K/129G: Arg-9--*Lys, Glu-129-*Gly PT-80/9G: Tyr-8--+Asp, Arg-9-*Lys PT-SOE: Phe-50--+Glu PT-88E/89S: Ile-88--*Glu, Tyr-89--÷Ser PT-13L/261/129G: Arg13--Leu, Trp-26-*Ile,
ADP-ribo-
ofsunit sylation syg/lato (>~g/ml)
CHO toxicity
(>±g/ml)
4 4
+ +
5 x 10-3 >10
5 x 10-6 >5
0.4
-
>10
0.4 0.4
-
0.4
-
T cell Leukocytosis- Histamine- Hemagglu- mitogepromoting sensitizing tination nicity activity activity (p.g/ (gm 10 (10 (p.gml) )
actl/mouse) (Rg/mouse)
cp)
Affinity constant mol) for:
Mooclonal antibody 1B7
(liters/
Ga mma globulins glbin
1.8 x 108 4.8 x 1010
0.04 >50
0.02 >50
0.30 0.25
0.4 0.35
>5
>50
>50
0.30
0.35
0.0
2.7 x 109
>10 >10
>5
>5
>50 >50
>50 >50
0.25 0.35
0.4 0.3
0.0 0.0
1.3 x 109 3.1 x 109
>10
>5
>50
>50
0.20
0.35
0.0
ND
1.7 x 108 2.5 x 1010
Glu-129lestGly I
All assays were performed as described previously (9); numerical values represent the lowest effective protein concentrations. ND, not determined.
in the ELISA values was observed in sera obtained 2 weeks after the booster injection. These ELISA values were comparable to those obtained after immunization with PT-9K/ 129G. Nevertheless, the increase in antibody titers did not correlate with neutralizing activity in the CHO cell assay (4). In fact, the sera resulting from a single injection of 0.6, 3, or 15 ,ug of PT-13L/261/129G did not neutralize the clustering effect of wild-type PT in CHO cells. In marked contrast, antibodies obtained from animals given equivalent doses of double mutant PT-9K/129G were able to neutralize toxin activity at a dilution of 1/20, 1/20, or 1/80, respectively. Good neutralizing activity was observed with a second immunization with the B oligomer, but this activity was still lower than that induced by PT-9K/129G (Fig. 1). PT-13L/261/129G was able to protect mice, in a dose-
dependent fashion, against intracerebral challenge with virulent B. pertussis (12), an assay whose results correlate with the protective activity of the cellular vaccine. However, B oligomer PT-13L/26I/129G was less potent than double mutant PT-9K/129G, containing the Si subunit also (Table 2). In conclusion, the development of pertussis strains which secrete only the B oligomer of PT has provided us with unique reagents which allow definitive determination of the properties of PT. We have confirmed that the toxicity of PT is entirely dependent on the enzymatically active S1 subunit and have provided conclusive evidence that the abilities of PT to agglutinate erythrocytes and to induce the proliferation of T lymphocytes belong to the B oligomer alone and are, consequently, independent of the enzymatic mechanism of PT.
4. LU I~-
3.
F C,
10 a
z
2a ILU z
C,,
1
0
0
-j
oi
PRE IMMUNE
0.6
3
15
PT-9K/129G
--
0.6 -
3
15
PT-13L/261/129G
-_
FIG. 1. ELISA values and toxin-neutralizing titers of antibodies raised in guinea pigs by one or two subcutaneous injections of different doses of PT-9K/129G and PT-13L/261/129G. Antitoxin antibody titers are expressed as A405 values (ABS-MAX). Neutralizing titers (NT) are expressed as reciprocals of the highest serum dilutions resulting in 100% inhibition of the clustering effect induced in CHO cells by 120 pg of wild-type PT tested in triplicate.
4734
NOTES
INFECT. IMMUN.
TABLE 2. Vaccine potencies determined by mouse survival following intracerebral infection with virulent B. pertussis No. of
Vaccine
Dosea
survivors
National Institutes of Health standard cellular vaccine (lot 9)c
0.05 0.01 0.002
14 8 5
PT-9K/129G
15 3 0.6
13 9 4
PT-13L/261/129G
15 3 0.6
8 5 0
a Doses of the National Institutes of Health vaccine are in milliliters; doses of the other, plain vaccines are in micrograms per mouse. b A total of 16 mice were tested. c This vaccine contains 8 IU/ml.
We thank H. Sato for kindly providing monoclonal antibody 1B7, G. Corsi for graphic work, and A. Mori and C. Mallia for editing. REFERENCES 1. Arciniega, J. L., D. L. Burns, E. Garcia-Ortigza, and C. R. Manclark. 1987. Immune response to the B oligomer of pertussis toxin. Infect. Immun. 55:1132-1136. 2. Burns, D. L., J. G. Kenimer, and C. R. Manclark. 1987. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect. Immun. 55:24-28. 3. Burns, D. L., and C. R. Manclark. 1986. Adenine nucleotides promote dissociation of pertussis toxin subunits. J. Biol. Chem. 261:4324-4327. 4. Gillenius, P., E. Jaatmaa, P. Askelof, M. Granstrom, and M. Tiru. 1985. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. J. Biol. Stand. 13:61-66. 5. Gray, L. S., K. S. Huber, M. C. Gray, E. L. Hewlett, and V. H. Englehard. 1989. Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein. J. Immunol. 142:1631-1638. 6. Hausman, S. Z., D. S. Burns, V. C. Sickler, and C. R. Manclark. 1989. Immune response to dimeric subunits of the pertussis toxin B oligomer. Infect. Immun. 57:1760-1764. 7. Hewlett, E. L., K. T. Sauer, G. A. Myers, J. L. Cowell, and R. L. Guerrant. 1983. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect. Immun. 40:1198-1203. 8. Munoz, J. J., H. Arai, R. K. Bergman, and P. L. Sadowski. 1981. Biological activities of crystalline pertussigen from Bordetella
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