Purification and immunological characterization of HPLC-purified pertussis toxin subunits
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
PELECHONG, STEPHENCOCKLE,HEATHERBOUX,AND MICHELKLEIN Connaught Centre for Biotechnology Research, 1755 Steeles Avenue West, Willowdale, Ont., Canada M2R 3T4 Received November 8, 1990 CHONG,P., COCKLE,S., BOUX,H., and KLEIN,M. 1991. Purification and immunological characterization of HPLC-purified pertussis toxin subunits. Biochem. Cell Biol. 69: 336-340. Pertussis toxin (PT), an oligomeric exotoxin of Bordetellapertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid singlestep procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (Sl), 70% (S2), >90% (S3), >90% (S4), and 50% (SS), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes. Key words: pertussis toxin, high-performance liquid chromatography, purification, pertussis vaccine. CHONG.P., COCKLE,S., BOUX,H., et KLEIN,M. 1991. Purification and immunological characterization of HPLC-purified pertussis toxin subunits. Biochem. Cell Biol. 69 : 336-340. La toxine de la coqueluche (PT), une exotoxine oligomtre de Bordetella pertussis, renferme cinq sous-unitts difftrentes et elle est considtrte comme un immunogtne essentiel dans les vaccins acellulaires et les vaccins renfermant pertussis contre la coqueluche. Utilisant la chromatographie liquide a haute performance en phase inverse, nous avons dtveloppt une technique rapide ne comportant qu'une seule &ape pour isoler les sous-unites de la PT. Tel que le montrent la SDS-PAGE et I'analyse des acides aminb, les valeurs de la rtcupiration des sous-unites individuelles sont : 75% (Sl), 70% (S2), > 90% (S3), >90% (S4) et 50% (S5). Les sous-unitts lyophilisies sont solubilisees dans 1'urCe et soumis a une dialyse par ttape pour enlever l'urie. Toutes les sous-unites sont inactives dans les essais de sensibilisation a I'histamine, de lymphocytose et d'htmagglutination. Cependant, la S1 purifite retient une activitt rtsiduelle de la NAD-glycohydrolase et de 1'ADP-ribosyltransferase.Le melange des cinq sous-unitis individuelles permet de gtntrer une holotoxine partiellement active. Toutes les sous-unit& sont immunogtnes chez les souris et les rats. Les antistrum monosptcifiques obtenus contre ces deux esptces animales sont capables de neutraliser l'agglomtration des cellules ovariennes du hamster chinois (CHO) par la PT, mais I'immunisation active des souris avec des sous-unitts individuelles ne les prottge pas dans I'essai challenge intractrebral. Ces prtprations des sous-unites retiennent donc des dtterminants neutralisants, mais ne renferment pas d'tpitopes protecteurs. Mots elks : toxine de la coqueluche, chromatographie liquide a haute performance, purification, vaccin (pertussis).
Introduction Pertussis toxin (PT) is an oligomeric exotoxin secreted by Bordetellapertussis, the microorganism that causes whooping cough. It contains five different subunits of molecular weight 26 000 (Sl), 22 000 (S2), 21 000 (S3), 12 000 (S4), and 11 000 (SS), noncovalently assembled in the molar proportions 1: 1: 1:2: 1, respectively (Tamura et al. 1982, 1983). Subunit S1 is an enzyme with ADP-ribosyltransferase activity, while subunits S2 t o S5 comprising the B oligomer are responsible for interactions with host cells (Tamura et al. 1983; Moss et al. 1986). The strongest intersubunit interactions define the dimeric units S2-S4 and S3-S4 within the B oligomer. Pertussis toxin displays a variety of biological activities, including promotion of lymphocytosis, sensitization t o histamine, stimulation of insulin secretion, and agglutination of erythrocytes. Immunization of mice with PT has been shown t o induce protection against intracerebral challenge with virulent B. pertussis, while antibodies t o P T are ABBREVIATIONS: PT, pertussis toxin; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; TBS, TRIS-buffered saline. ' ~ u t h o rto whom correspondence should be addressed. Printed in Canada / Imprim6 au Canada
prevalent in human convalescent and vaccination sera (see Manclark and Cowell 1984, Robinson et al. 1985, Weiss and Hewlett 1986, for reviews). PT is therefore regarded as an important component in the design of acellular pertussis vaccines, but it requires chemical treatment with aldehydes t o remove its toxicity. However, it has been shown that formalin-inactivated PT can revert t o its toxic form (Iwasa e t al. 1985). T o develop a safe and effective PT-based acellular pertussis vaccine, it is necessary t o understand the structure and function of the individual subunits. Ui and co-workers have approached this problem using chemical modification and reconstitution of hybrid toxins comprising normal and modified subunits (Nogimori e t al. 1985). A D P ribosyltransferase mediated activities of whole P T require a n intact cell-binding site o n either the S2-S4 or the S3-S4 dimer. However, mitogenicity is independent of the presence of S1, but requires binding sites on both dimers. The hemagglutination activity of P T can be mimicked by the isolated S2-S4 dimer alone. Sato's group reported that mouse monoclonal antibodies raised against S1, S2, and S3 inhibited several PT-induced activities, including lymphocytosis promotion, insulin secretion, and clustering of C H O cells (Sato et al. 1984, 1987). In addition, anti-Sl
CHONG ET AL.
monoclonal antibodies have been shown to protect mice against an intracerebral or aerosol challenge with live B. pertussis (Manclark and Cowell 1984). These data suggest that individual subunits of P T could function as immunogens in a pertussis subunit vaccine. I n this paper, we describe a rapid method for purifying P T subunits using reverse-phase HPLC, and examine the ability of individual subunits t o induce toxin-neutralizing antibodies in both rabbits and mice.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
Materials and methods Preparation of pertussis toxin and toxoid Pertussis toxin, subunit S1, and B oligomer were prepared from culture supernatants of B. pertussis strain 10536 by affinity chromatogravhy on fetuin-Sevharose or fetuin-agarose as described previously (chong and ~ l e i n1989; Cockle 1989). To obtain pertussis toxoid, 10 mg of pertussis toxin in 50 mL of PBS (10 mM potassium phosphate, 0.15 M NaCl, pH 7.4) were mixed with 0.4 mL of glutaraldehyde (25%; Fisher) at room temperature for 2 h, then 5 mL of 1 M of lysine-HC1, pH 7.0 was added for 30 min to quench the reaction. The mixture was dialysed 3 times against 10 mM potassium phosphate, 0.5 M NaCl, pH 7.4, containing 50 mM lysine, then 4 times against PBS, and stored at 4°C. Preparation of pertussis toxin subunits by HPLC Pertussis toxin subunits were separated and purified by reversephase HPLC on a Waters system using a 0.46 x 25 cm Vydac 214TP54 analytical column (Separations Group) (Cockle 1989). Solvents A and B contained 10 mM TFA in HPLC-grade water and 4:l HPLC-grade acetonitrile-water, respectively. The column was initially equilibrated with 35% acetonitrile at room temperature. Samples containing 501-200pg of PT in 1-2 mL of solution were fractionated by applying a linear gradient of 35-43% acetonitrile over 20 min at 0.75 mL/min. The effluent was monitored by UV absorption at 220 or 280 nm and peaks corresponding to the five individual PT subunits were collected and lyophilized. Subunits were redissolved in PBS (10 mM potassium phosphate, 0.15 M NaCl, pH 7.4) containing 8 M urea and refolded by step-wise dialysis for 4-6 h against 2 L of PBS buffer containing 4 M, 2 M, and no urea. Purity was assessed by SDSPAGE as described previously ( L a d 1970). Individual subunits were hydrolyzed in 6 M HCI at 110°C for 24 h and their amino acid compositions determined by cation-exchange chromatography with post-column ninhydrin derivatization. Assays for enzymic and biological activity The NAD-glycohydrolaseactivity of isolated S1 was determined as previously described (Cockle 1989). ADP-ribosylation of CHO cell membranes and the histamine sensitization assays were also performed as previously described (Chong and Klein 1989). CHO cell clustering and inhibition assays were carried out according to Gillenius et al. (1985). Leucocytosis and hemagglutination assays were carried out as described by Sato et al. (1987). Rabbit immunization Rabbits were immunized intramuscularly with PT (2 pg) in the absence of adjuvant, or with pertussis toxoid (25 pg) or HPLCpurified PT subunits (5-15 pg, with or without urea solubilization) emulsified in 200 pL of complete Freund's adjuvant. Each rabbit received 3 doses of the same immunogen over a 6-week period. Antisera were heat-treated at 56OC and screened for reactivity with PT by immunoblotting and ELISA. Toxin-neutralizing ability was tested in the CHO cell clustering assay. Protein immunoblots Immunoblot analysis was carried out as described by Towbin et al. (1979). Briefly, pertussis toxin (15-20 pg per lane) was separated into subunits by SDS-PAGE and electroblotted onto a nitrocellulose membrane at 36 V for 16-20 h at 4°C using 25 mM
337
Tris, 192 mM glycine, 20% methanol, pH 8.3, as transfer buffer. The membrane was washed with transfer buffer, then blocked with 5% BSA in TBS buffer (20 mM Tris-HC1,O. 15 mM NaCl, pH 7.5) for 2-4 h at room temperature. The nitrocellulose was cut into strips, each of which was incubated overnight at room temperature with rabbit antiserum raised against individual HPLC-purified PT subunits. Antisera against S1, S4, and S5 were diluted 1:2000 in TBS buffer containing 0.1 % BSA, whereas antisera against S2 and S3 were diluted 1:250. The strips were washed 3 times with 0.1% Tween-20 in TBS, then incubated for 4-6 h at room temperature with peroxidase-conjugated pig anti-rabbit-IgG (Dako Immunoglobulins, Denmark). The strips were again washed with TBS-Tween, then incubated for 15 min with peroxidase substrate (60 mg of 4-chloronaphthol in 20 mL of methanol added into 100 mL of TBS containing 0.6 mL of 3% hydrogen peroxide). The strips were finally washed with distilled water and air-dried.
Anti-PT specific ELISA The wells of a 96-well microtitre plate (Nunc) were coated overnight at 4°C with 0.1 pg of fetuin in 200 pL of coating buffer (35 mM NaHCO,, 20 mM Na,CO,, pH 9.6), then washed three times with 0.1% Tween-20 in PBS buffer. Standard PT (List Biologicals, 0.1 pg in 200 pL of PBS-Tween) was added to the wells, incubated for 30 min at room temperature, and the excess washed away with PBS-Tween. The wells were blocked with 0.1% BSA in PBS, then 2-fold serial dilutions of heat-treated rabbit antiPT-subunit serum (200 pL) were added to each well; incubation was for 30 min at room temperature and the excess was removed by washing with PBS-Tween. Peroxidase-conjugated pig antirabbit-IgG IgG (Dako Immunoglobulins, Denmark) diluted 1 : 10 000 in PBS containing 0.1% BSA was added and incubated for 1-2 h. After a final wash, bound peroxidase was determined using 4-chloronaphthol. Titres were expressed as the reciprocal of the highest dilution of antiserum that gave a 4-fold increase in absorbance at 492 nm with respect to controls containing the corresponding preimmune serum. Mouse protection test Groups of 10 mice were first immunized with 15 pg of individual urea-solubiied PT subunits emulsified in 200 pL of CFA. Two weeks later, each mouse was boosted with the same immunogen in incomplete Freund's adjuvant. Two weeks after the second injection, blood samples were collected and each mouse was challenged with B. pertussis 18323 according to the WHO protocol for the whole-cell pertussis vaccine potency test (World Health Organization 1982). The survival rate was determined 2 weeks after the challenge.
Results and discussion Purification of pertussis toxin subunits by HPLC Tamura et al. (1983) have described the isolation of P T subunits by multiple steps of ion-exchange chromatography in the presence of 5 M urea. We found this approach t o be unsatisfactory o n account of inconsistent yields and purity and the length of time required (5-7 days). We have previously demonstrated the single-step separation of P T subunits by reverse-phase HPLC (Cockle 1989), and therefore applied this technique t o the small-scale isolation of individual subunits. Out of a panel of six C4 and CI8 columns investigated, the best separation and resolution were achieved with a Vydac 214TP54 C4 column. However, peak shapes and retention times were significantly affected by p H and salt concentration, therefore, samples were pretreated with TFA t o p H 3 t o maintain reproducibility. The five subunits of P T from the Connaught B. pertussis vaccine strain 10536 were well separated in a 20-min run, the order of elution being S4, S2, S5, S1, and S3 (Fig. 1). By comparison, S1 from the Tohama strain eluted after S3, although
BIOCHEM. CELL BIOL. VOL. 69, 1991
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
338
IU
15
ZD
25
ELUTION TIME (min)
FIG. 1 . Chromatographic separation of PT subunits by reversephase HPLC using a 0.46 x 25 cm Vydac 214TP54 analytical column. The chromatographic conditions are described in Materials and methods; 10 pg of PT were used in this run. The peak assignments were verified by SDS-PAGE analysis as shown in Fig. 2.
the other subunits eluted in the same positions. Such difference in retention time indicates a significant difference in amino acid sequence between the two S1 molecules. Low molecular weight solutes and degradation fragments were eliminated in the initial void-volume peak. Subunits were identified by SDS-PAGE analysis, which indicated that S1, S2, S3, and S4 were free of other pertussis protein contaminants, while S5 was usually contaminated with S2 (Fig. 2). However, pure S5 could be obtained by a second pass through the same column. Partial degradation of S1 was observed following renaturation by dialysis into decreasing concentrations of urea. Subunits S1 and S5 have been found to be the most susceptible to protease degradation (Peppler et al. 1985). The identity of PT subunits was further substantiated by amino acid analysis (results not shown). Based on both SDS-PAGE and amino acid analyses, subunit recoveries were 75% for S1, 70% for S2, >90% for S3 and S4, and 50% for S5. We also found that PT subunits could be isolated from partially purified starting material by this technique, as all five subunits elute within a narrow solvent composition range (38-41% acetonitrile) that excludes most other pertussis proteins. Since the cycle time including column reequilibration was only 40 min, it was possible to obtain 1-3 mg of each purified subunit per day using an analytical column.
Biological activity of pertussis toxin subunits The lyophilized PT subunits were almost insoluble in phosphate buffer. To test for enzymic and biological activity, individual subunits were therefore dissolved in 8 M urea, and the solutions slowly dialysed to remove the urea. Ureasolubilized S1 retained 40% of the NAD-glycohydrolase activity of S1 isolated by fetuin-agarose affinity chromatography (Cockle 1989), and also displayed 5-10% residual ability to catalyse the ADP-ribosylation of CHO cell membranes. Thus it was clear that only partial refolding had been achieved.
FIG. 2. SDS-PAGE analysis of native PT (10 pg), and individual HPLC-purified subunits (about 3 pg). Lane 1 , native PT; lane 2, S4; lane 3, S2; lane 4, S5; lane 5, S1; lane 6, S3.
FIG. 3. Immunoblots of rabbit antisera raised against individual HPLC-purified PT subunits. Lane 1, PT; lane 2, antiS1; lane 3, anti-S2; lane 4, anti-S3; lane 5, anti-S4; lane 6, anti-S5.
All five subunits failed to induce CHO cell clustering or hemagglutination of erythrocytes, were incapable of sensitizing mice to histamine challenge, and provoked no lymphocytosis in mice at doses as high as 15 pg. The lack of biological function of individual subunits is consistent with other studies showing that the in vivo activities of PT require either intact holotoxin, B oligomer, or (in the case of hemagglutination) the S2-S4 dimer (Tamura et al. 1982; Nogimori et al. 1985; Burns et al. 1987). However, as with S1, other subunits may not have been correctly refolded by the present urea treatment and dialysis. When mixtures of all five subunits in 8 M urea were gradually dialysed to zero urea, a partially active holotoxin (about 5% with respect to the total protein) could be generated as judged by the CHO cell clustering assay.
Immune response to PT subunits in rabbits Immunization with denatured subunits Sera from rabbits immunized with lyophilized HPLCpurified subunits mixed with complete Freund's adjuvant were examined by immunoblot analysis to determine their subunit specificity and cross-reactivity. Both S1 and S4
CHONG ET
AL.
TABLE1. Immunological properties of PT and its HPLC-purified subunits in rabbits
Rabbit antisera titre'
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
Immunogens
Specific anti-PT ELISA
CHO cell PT-neutralization assay
Native PT Pertussis toxoid SIC B-oligomerc HPLC S1 HPLC S1, urea-treated HPLC S2 HPLC S2, urea-treated HPLC S3 HPLC S3, urea-treated HPLC S4 HPLC S4, urea-treated HPLC SS HPLC S5, urea-treated 'Titre values are expressed as the average of the reciprocal of the maximal dilution of the sera. b ~ h PT e toxicity neutralization assay was performed according to Gillenius et al. (1985). 'S1 and the B-oligomer were purified by fetuin-Sepharose CL-4B affinity chromatography (Chong and Klein 1989). dnd, not detected. individual HPLC-purified subunits were dissolved in PBS buffer containing 8 M urea, then dialysed against PBS buffer containing 4 M and 2 M urea, and finally against PBS buffer alone.
induced antibodies monospecific for the corresponding subunit (Fig. 3). Rabbits immunized with S2 or S3 produced antibodies that cross-reacted with S3 or S2, respectively. This is not surprising since S2 and S3 exhibit 70% amino acid homology, and have previously been shown to induce crossreactive polyclonal antibodies Frank and Parker 1984; Sato et al. 1987; Nicosia et al. 1986). Subunit S5 induced antibodies against S2 and S3 as well as against S5, presumably as a result of its known contamination with S2. This effect was evident at low serum dilution (1:250), but not at high dilution (1:5000) (as shown in Fig. 3). Rabbit antisera raised against S1, S2, and S3 failed to recognize native PT in an anti-PT specific ELISA, while antisera against S4 and S5 were only weakly reactive (Table 1). None of these antisera were able to inhibit PT-induced CHO cell clustering.
Immunization with urea-treated subunits In contrast to the above results, all PT subunits solubilized by the urea-dialysis method induced antibodies that not only reacted with native PT in the ELISA, but were also capable of neutralizing PT in the CHO cell clustering assay (Table 1). Neutralizing titres were, however, lower than those of antisera obtained from rabbits immunized with holotoxin, pertussis toxoid, or affinity-purified S1 or B oligomer. These results suggest that the urea-solubilized subunits were at least partially refolded so as to generate conformational epitopes resembling those of native PT. It is noteworthy that S4 and S5 induced the highest CHO cell PT-neutralizing titres among the five subunits. To the best of our knowledge, this is the first documented evidence for the presence of PT-neutralizing epitopes on these subunits. On the other hand, S2 and S3 were poor immunogens in rabbits, since three doses of 15 pg were required to induce PT-neutralizing antibodies, whereas one dose was sufficient for S1, S4, and S5.
TABLE 2. Immune response to HPLC-purified PT subunits in mice Subunit
Anti-PT titrea
MPT~
S1 S2 S3 S4 S5
16
0
8
3 2
2 4
0
2
3
Weutralization of PT-induced CHO cell clustering. Titres are expressed as the reciprocal of the highest effective dilution. b ~ mouse ~ protection ~ , test. Data are reported as the number of survivors out of 10 mice immunized.
Protective response to PT subunits in mice The ability of individual subunits to induce immunity in mice against live B. pertussis organisms was examined by the intracerebral challenge test. No significant protection was imparted by any subunit preparation (Table 2), in agreement with results on recombinant PT subunits from Escherichia coli reported by others (Locht et al. 1987; Nicosia et al. 1987). However, sera from immunized mice were capable of neutralizing PT in the CHO cell clustering assay. Evidently, the epitopes that give rise to protective antibodies are different from those that elicit antibodies inhibiting CHO cell clustering. It is quite possible that the protective epitopes exist only on fully or partially assembled groups of P T subunits. To examine this possibility, we are currently continuing protection studies using reconstituted oligomers from different HPLC-purified subunits. Acknowledgements The authors thank M. Flood and R. Robinson for technical help, and Dr. M. Blum of the Department of
340
BIOCHEM. CELL BIOL. VOL. 69, 1991
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
Biochemistry, University of Toronto, f o r carrying o u t t h e amino acid analysis. BURNS,D.L., KENIMER,J.G., and MANCLARK, C.R. 1987. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect. Immun. 55: 24-28. CHONG,P., and KLEIN, M. 1989. Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-Sepharose affinity chromatography. Biochem. Cell Biol. 67: 387-391. COCKLE,S. 1989. Identification of an active-site residue in subunit Sl of pertussis toxin by photocrosslinking to NAD. FEBS Lett. 249: 329-333. FRANK, D.W., and PARKER,C.D. 1984. Interaction of monoclonal antibodies with pertussis toxin and its subunits. Infect. Immun. 46: 195-201. GILLENIUS, P., JAATTMAA, E., ASKELOF,P., GRANDSTROM, M., and TIRU, M. 1985. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. J. Biol. Stand. 13: 61-67. IWASA,S., ISHIDA,S., ASAKAWA,S., and AWA, K. 1985. A curious histamine-sensitizingactivity shown by the newly developed Japanese acellular pertussis vaccine. Dev. Biol. Stand. 61: 453-460.
LAEMMLI,U.K. 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (London), 227: 680-685.
LOCHT, C., and KEITH, J.M. 1986. Pertussis toxin gene: nucleotide sequence and genetic organization. Science (Washington, D.C.), 232: 1258-1264. LOCHT, C., CIEPLAK.W., MARCHITTO,K.S., SATO, H., and KEITH,J.M. 1987. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli. Infect. Immun. 55: 2546-2553. MANCLARK, C.R., and COWELL,J.R. 1984. Pertussis. I n Bacterial vaccines. Edited by R. Germanier. Academic Press, New York. pp. 69-106. Moss, J., STANLEY,S.L., WATKINS,P.A., BURNS, D.R., MANCLARK, C.R., KASLOW,H.R., and HEWLETT,E.L. 1986. Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotide, phospholipids and detergents. Biochemistry, 25: 2720-2725. NICOSIA,A.. PERUGINI,M., FRANZINI,C., CASAGLI,M.C., BORRI,M.G., ANTONI,G., ALMONI,M., NERI, P., RATTI, G.,
and RAPPUOLI,R. 1986. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc. Natl. Acad. Sci. U.S.A. 83: 4631-4635. NICOSIA,A., BARTOLONI, A., PERUGINI,M., and RAPPUOLI,R. 1987. Expression and immunological properties of the five subunits of pertussis toxin. Infect. Immun. 55: 963-967. NOGIMORI, K., TAMURA,M., NAKAMURA, T., YAJIMA,M., ITO, K., and UI, M. 1985. Dual mechanisms involved in the development of diverse biological activities of islet-activating protein, as revealed by chemical modification of the toxin molecule. Dev. Biol. Stand. 61: 51-61. PEPPLER,M.S., JUDD,R.C., and MUNOZ,J.J. 1985. Effect of proteolytic enzymes, storage and reduction on the structure and biological activity of pertussigen, a toxin from Bordetella pertussis. Dev. Biol. Stand. 61: 75-87. ROBINSON,A., IRONS, L.I., and ASHWORTH,L.A.E. 1985. Pertussis vaccine: present status and future prospects. Vaccines, 3: 11-22. SATO,H., ITO, A., CHIBA,J., and SATO,Y. 1984. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infection. Infect. Immun. 46: 422-428. SATO, H., SATO,Y., ITO, A., and OHISHI,I. 1987. Effect of monoclonal antibody to pertussis toxin on toxin activity. Infect. Immun. 55: 909-915. TAMURA,M., NOGIMORI,K., YAJIMA,M., ITO,K., KATADA,T., UI, M., and ISHII,S. 1982. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry, 21: 5512-5516. TAMURA,M.,NOGIMORI, K., YAJIMA, M., ASE, K., and UI, M. 1983. The 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. TOWBIN,H., STAEHELIN,T., and GORDON,J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. WEISS, A., and HEWLETT,E. 1986. Virulence factors of Bordetella pertussis. Annu. Rev. Microbial. 40: 661-685. WORLD HEALTHORGANIZATION. 1982. WHO Test Manual. Manual of details of tests required on final vaccines used in the WHO expanded programme of immunization. World Health Organization, Geneva. pp. 74-82.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
PELECHONG, STEPHENCOCKLE,HEATHERBOUX,AND MICHELKLEIN Connaught Centre for Biotechnology Research, 1755 Steeles Avenue West, Willowdale, Ont., Canada M2R 3T4 Received November 8, 1990 CHONG,P., COCKLE,S., BOUX,H., and KLEIN,M. 1991. Purification and immunological characterization of HPLC-purified pertussis toxin subunits. Biochem. Cell Biol. 69: 336-340. Pertussis toxin (PT), an oligomeric exotoxin of Bordetellapertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid singlestep procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (Sl), 70% (S2), >90% (S3), >90% (S4), and 50% (SS), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes. Key words: pertussis toxin, high-performance liquid chromatography, purification, pertussis vaccine. CHONG.P., COCKLE,S., BOUX,H., et KLEIN,M. 1991. Purification and immunological characterization of HPLC-purified pertussis toxin subunits. Biochem. Cell Biol. 69 : 336-340. La toxine de la coqueluche (PT), une exotoxine oligomtre de Bordetella pertussis, renferme cinq sous-unitts difftrentes et elle est considtrte comme un immunogtne essentiel dans les vaccins acellulaires et les vaccins renfermant pertussis contre la coqueluche. Utilisant la chromatographie liquide a haute performance en phase inverse, nous avons dtveloppt une technique rapide ne comportant qu'une seule &ape pour isoler les sous-unites de la PT. Tel que le montrent la SDS-PAGE et I'analyse des acides aminb, les valeurs de la rtcupiration des sous-unites individuelles sont : 75% (Sl), 70% (S2), > 90% (S3), >90% (S4) et 50% (S5). Les sous-unitts lyophilisies sont solubilisees dans 1'urCe et soumis a une dialyse par ttape pour enlever l'urie. Toutes les sous-unites sont inactives dans les essais de sensibilisation a I'histamine, de lymphocytose et d'htmagglutination. Cependant, la S1 purifite retient une activitt rtsiduelle de la NAD-glycohydrolase et de 1'ADP-ribosyltransferase.Le melange des cinq sous-unitis individuelles permet de gtntrer une holotoxine partiellement active. Toutes les sous-unit& sont immunogtnes chez les souris et les rats. Les antistrum monosptcifiques obtenus contre ces deux esptces animales sont capables de neutraliser l'agglomtration des cellules ovariennes du hamster chinois (CHO) par la PT, mais I'immunisation active des souris avec des sous-unitts individuelles ne les prottge pas dans I'essai challenge intractrebral. Ces prtprations des sous-unites retiennent donc des dtterminants neutralisants, mais ne renferment pas d'tpitopes protecteurs. Mots elks : toxine de la coqueluche, chromatographie liquide a haute performance, purification, vaccin (pertussis).
Introduction Pertussis toxin (PT) is an oligomeric exotoxin secreted by Bordetellapertussis, the microorganism that causes whooping cough. It contains five different subunits of molecular weight 26 000 (Sl), 22 000 (S2), 21 000 (S3), 12 000 (S4), and 11 000 (SS), noncovalently assembled in the molar proportions 1: 1: 1:2: 1, respectively (Tamura et al. 1982, 1983). Subunit S1 is an enzyme with ADP-ribosyltransferase activity, while subunits S2 t o S5 comprising the B oligomer are responsible for interactions with host cells (Tamura et al. 1983; Moss et al. 1986). The strongest intersubunit interactions define the dimeric units S2-S4 and S3-S4 within the B oligomer. Pertussis toxin displays a variety of biological activities, including promotion of lymphocytosis, sensitization t o histamine, stimulation of insulin secretion, and agglutination of erythrocytes. Immunization of mice with PT has been shown t o induce protection against intracerebral challenge with virulent B. pertussis, while antibodies t o P T are ABBREVIATIONS: PT, pertussis toxin; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; TBS, TRIS-buffered saline. ' ~ u t h o rto whom correspondence should be addressed. Printed in Canada / Imprim6 au Canada
prevalent in human convalescent and vaccination sera (see Manclark and Cowell 1984, Robinson et al. 1985, Weiss and Hewlett 1986, for reviews). PT is therefore regarded as an important component in the design of acellular pertussis vaccines, but it requires chemical treatment with aldehydes t o remove its toxicity. However, it has been shown that formalin-inactivated PT can revert t o its toxic form (Iwasa e t al. 1985). T o develop a safe and effective PT-based acellular pertussis vaccine, it is necessary t o understand the structure and function of the individual subunits. Ui and co-workers have approached this problem using chemical modification and reconstitution of hybrid toxins comprising normal and modified subunits (Nogimori e t al. 1985). A D P ribosyltransferase mediated activities of whole P T require a n intact cell-binding site o n either the S2-S4 or the S3-S4 dimer. However, mitogenicity is independent of the presence of S1, but requires binding sites on both dimers. The hemagglutination activity of P T can be mimicked by the isolated S2-S4 dimer alone. Sato's group reported that mouse monoclonal antibodies raised against S1, S2, and S3 inhibited several PT-induced activities, including lymphocytosis promotion, insulin secretion, and clustering of C H O cells (Sato et al. 1984, 1987). In addition, anti-Sl
CHONG ET AL.
monoclonal antibodies have been shown to protect mice against an intracerebral or aerosol challenge with live B. pertussis (Manclark and Cowell 1984). These data suggest that individual subunits of P T could function as immunogens in a pertussis subunit vaccine. I n this paper, we describe a rapid method for purifying P T subunits using reverse-phase HPLC, and examine the ability of individual subunits t o induce toxin-neutralizing antibodies in both rabbits and mice.
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
Materials and methods Preparation of pertussis toxin and toxoid Pertussis toxin, subunit S1, and B oligomer were prepared from culture supernatants of B. pertussis strain 10536 by affinity chromatogravhy on fetuin-Sevharose or fetuin-agarose as described previously (chong and ~ l e i n1989; Cockle 1989). To obtain pertussis toxoid, 10 mg of pertussis toxin in 50 mL of PBS (10 mM potassium phosphate, 0.15 M NaCl, pH 7.4) were mixed with 0.4 mL of glutaraldehyde (25%; Fisher) at room temperature for 2 h, then 5 mL of 1 M of lysine-HC1, pH 7.0 was added for 30 min to quench the reaction. The mixture was dialysed 3 times against 10 mM potassium phosphate, 0.5 M NaCl, pH 7.4, containing 50 mM lysine, then 4 times against PBS, and stored at 4°C. Preparation of pertussis toxin subunits by HPLC Pertussis toxin subunits were separated and purified by reversephase HPLC on a Waters system using a 0.46 x 25 cm Vydac 214TP54 analytical column (Separations Group) (Cockle 1989). Solvents A and B contained 10 mM TFA in HPLC-grade water and 4:l HPLC-grade acetonitrile-water, respectively. The column was initially equilibrated with 35% acetonitrile at room temperature. Samples containing 501-200pg of PT in 1-2 mL of solution were fractionated by applying a linear gradient of 35-43% acetonitrile over 20 min at 0.75 mL/min. The effluent was monitored by UV absorption at 220 or 280 nm and peaks corresponding to the five individual PT subunits were collected and lyophilized. Subunits were redissolved in PBS (10 mM potassium phosphate, 0.15 M NaCl, pH 7.4) containing 8 M urea and refolded by step-wise dialysis for 4-6 h against 2 L of PBS buffer containing 4 M, 2 M, and no urea. Purity was assessed by SDSPAGE as described previously ( L a d 1970). Individual subunits were hydrolyzed in 6 M HCI at 110°C for 24 h and their amino acid compositions determined by cation-exchange chromatography with post-column ninhydrin derivatization. Assays for enzymic and biological activity The NAD-glycohydrolaseactivity of isolated S1 was determined as previously described (Cockle 1989). ADP-ribosylation of CHO cell membranes and the histamine sensitization assays were also performed as previously described (Chong and Klein 1989). CHO cell clustering and inhibition assays were carried out according to Gillenius et al. (1985). Leucocytosis and hemagglutination assays were carried out as described by Sato et al. (1987). Rabbit immunization Rabbits were immunized intramuscularly with PT (2 pg) in the absence of adjuvant, or with pertussis toxoid (25 pg) or HPLCpurified PT subunits (5-15 pg, with or without urea solubilization) emulsified in 200 pL of complete Freund's adjuvant. Each rabbit received 3 doses of the same immunogen over a 6-week period. Antisera were heat-treated at 56OC and screened for reactivity with PT by immunoblotting and ELISA. Toxin-neutralizing ability was tested in the CHO cell clustering assay. Protein immunoblots Immunoblot analysis was carried out as described by Towbin et al. (1979). Briefly, pertussis toxin (15-20 pg per lane) was separated into subunits by SDS-PAGE and electroblotted onto a nitrocellulose membrane at 36 V for 16-20 h at 4°C using 25 mM
337
Tris, 192 mM glycine, 20% methanol, pH 8.3, as transfer buffer. The membrane was washed with transfer buffer, then blocked with 5% BSA in TBS buffer (20 mM Tris-HC1,O. 15 mM NaCl, pH 7.5) for 2-4 h at room temperature. The nitrocellulose was cut into strips, each of which was incubated overnight at room temperature with rabbit antiserum raised against individual HPLC-purified PT subunits. Antisera against S1, S4, and S5 were diluted 1:2000 in TBS buffer containing 0.1 % BSA, whereas antisera against S2 and S3 were diluted 1:250. The strips were washed 3 times with 0.1% Tween-20 in TBS, then incubated for 4-6 h at room temperature with peroxidase-conjugated pig anti-rabbit-IgG (Dako Immunoglobulins, Denmark). The strips were again washed with TBS-Tween, then incubated for 15 min with peroxidase substrate (60 mg of 4-chloronaphthol in 20 mL of methanol added into 100 mL of TBS containing 0.6 mL of 3% hydrogen peroxide). The strips were finally washed with distilled water and air-dried.
Anti-PT specific ELISA The wells of a 96-well microtitre plate (Nunc) were coated overnight at 4°C with 0.1 pg of fetuin in 200 pL of coating buffer (35 mM NaHCO,, 20 mM Na,CO,, pH 9.6), then washed three times with 0.1% Tween-20 in PBS buffer. Standard PT (List Biologicals, 0.1 pg in 200 pL of PBS-Tween) was added to the wells, incubated for 30 min at room temperature, and the excess washed away with PBS-Tween. The wells were blocked with 0.1% BSA in PBS, then 2-fold serial dilutions of heat-treated rabbit antiPT-subunit serum (200 pL) were added to each well; incubation was for 30 min at room temperature and the excess was removed by washing with PBS-Tween. Peroxidase-conjugated pig antirabbit-IgG IgG (Dako Immunoglobulins, Denmark) diluted 1 : 10 000 in PBS containing 0.1% BSA was added and incubated for 1-2 h. After a final wash, bound peroxidase was determined using 4-chloronaphthol. Titres were expressed as the reciprocal of the highest dilution of antiserum that gave a 4-fold increase in absorbance at 492 nm with respect to controls containing the corresponding preimmune serum. Mouse protection test Groups of 10 mice were first immunized with 15 pg of individual urea-solubiied PT subunits emulsified in 200 pL of CFA. Two weeks later, each mouse was boosted with the same immunogen in incomplete Freund's adjuvant. Two weeks after the second injection, blood samples were collected and each mouse was challenged with B. pertussis 18323 according to the WHO protocol for the whole-cell pertussis vaccine potency test (World Health Organization 1982). The survival rate was determined 2 weeks after the challenge.
Results and discussion Purification of pertussis toxin subunits by HPLC Tamura et al. (1983) have described the isolation of P T subunits by multiple steps of ion-exchange chromatography in the presence of 5 M urea. We found this approach t o be unsatisfactory o n account of inconsistent yields and purity and the length of time required (5-7 days). We have previously demonstrated the single-step separation of P T subunits by reverse-phase HPLC (Cockle 1989), and therefore applied this technique t o the small-scale isolation of individual subunits. Out of a panel of six C4 and CI8 columns investigated, the best separation and resolution were achieved with a Vydac 214TP54 C4 column. However, peak shapes and retention times were significantly affected by p H and salt concentration, therefore, samples were pretreated with TFA t o p H 3 t o maintain reproducibility. The five subunits of P T from the Connaught B. pertussis vaccine strain 10536 were well separated in a 20-min run, the order of elution being S4, S2, S5, S1, and S3 (Fig. 1). By comparison, S1 from the Tohama strain eluted after S3, although
BIOCHEM. CELL BIOL. VOL. 69, 1991
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
338
IU
15
ZD
25
ELUTION TIME (min)
FIG. 1 . Chromatographic separation of PT subunits by reversephase HPLC using a 0.46 x 25 cm Vydac 214TP54 analytical column. The chromatographic conditions are described in Materials and methods; 10 pg of PT were used in this run. The peak assignments were verified by SDS-PAGE analysis as shown in Fig. 2.
the other subunits eluted in the same positions. Such difference in retention time indicates a significant difference in amino acid sequence between the two S1 molecules. Low molecular weight solutes and degradation fragments were eliminated in the initial void-volume peak. Subunits were identified by SDS-PAGE analysis, which indicated that S1, S2, S3, and S4 were free of other pertussis protein contaminants, while S5 was usually contaminated with S2 (Fig. 2). However, pure S5 could be obtained by a second pass through the same column. Partial degradation of S1 was observed following renaturation by dialysis into decreasing concentrations of urea. Subunits S1 and S5 have been found to be the most susceptible to protease degradation (Peppler et al. 1985). The identity of PT subunits was further substantiated by amino acid analysis (results not shown). Based on both SDS-PAGE and amino acid analyses, subunit recoveries were 75% for S1, 70% for S2, >90% for S3 and S4, and 50% for S5. We also found that PT subunits could be isolated from partially purified starting material by this technique, as all five subunits elute within a narrow solvent composition range (38-41% acetonitrile) that excludes most other pertussis proteins. Since the cycle time including column reequilibration was only 40 min, it was possible to obtain 1-3 mg of each purified subunit per day using an analytical column.
Biological activity of pertussis toxin subunits The lyophilized PT subunits were almost insoluble in phosphate buffer. To test for enzymic and biological activity, individual subunits were therefore dissolved in 8 M urea, and the solutions slowly dialysed to remove the urea. Ureasolubilized S1 retained 40% of the NAD-glycohydrolase activity of S1 isolated by fetuin-agarose affinity chromatography (Cockle 1989), and also displayed 5-10% residual ability to catalyse the ADP-ribosylation of CHO cell membranes. Thus it was clear that only partial refolding had been achieved.
FIG. 2. SDS-PAGE analysis of native PT (10 pg), and individual HPLC-purified subunits (about 3 pg). Lane 1 , native PT; lane 2, S4; lane 3, S2; lane 4, S5; lane 5, S1; lane 6, S3.
FIG. 3. Immunoblots of rabbit antisera raised against individual HPLC-purified PT subunits. Lane 1, PT; lane 2, antiS1; lane 3, anti-S2; lane 4, anti-S3; lane 5, anti-S4; lane 6, anti-S5.
All five subunits failed to induce CHO cell clustering or hemagglutination of erythrocytes, were incapable of sensitizing mice to histamine challenge, and provoked no lymphocytosis in mice at doses as high as 15 pg. The lack of biological function of individual subunits is consistent with other studies showing that the in vivo activities of PT require either intact holotoxin, B oligomer, or (in the case of hemagglutination) the S2-S4 dimer (Tamura et al. 1982; Nogimori et al. 1985; Burns et al. 1987). However, as with S1, other subunits may not have been correctly refolded by the present urea treatment and dialysis. When mixtures of all five subunits in 8 M urea were gradually dialysed to zero urea, a partially active holotoxin (about 5% with respect to the total protein) could be generated as judged by the CHO cell clustering assay.
Immune response to PT subunits in rabbits Immunization with denatured subunits Sera from rabbits immunized with lyophilized HPLCpurified subunits mixed with complete Freund's adjuvant were examined by immunoblot analysis to determine their subunit specificity and cross-reactivity. Both S1 and S4
CHONG ET
AL.
TABLE1. Immunological properties of PT and its HPLC-purified subunits in rabbits
Rabbit antisera titre'
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
Immunogens
Specific anti-PT ELISA
CHO cell PT-neutralization assay
Native PT Pertussis toxoid SIC B-oligomerc HPLC S1 HPLC S1, urea-treated HPLC S2 HPLC S2, urea-treated HPLC S3 HPLC S3, urea-treated HPLC S4 HPLC S4, urea-treated HPLC SS HPLC S5, urea-treated 'Titre values are expressed as the average of the reciprocal of the maximal dilution of the sera. b ~ h PT e toxicity neutralization assay was performed according to Gillenius et al. (1985). 'S1 and the B-oligomer were purified by fetuin-Sepharose CL-4B affinity chromatography (Chong and Klein 1989). dnd, not detected. individual HPLC-purified subunits were dissolved in PBS buffer containing 8 M urea, then dialysed against PBS buffer containing 4 M and 2 M urea, and finally against PBS buffer alone.
induced antibodies monospecific for the corresponding subunit (Fig. 3). Rabbits immunized with S2 or S3 produced antibodies that cross-reacted with S3 or S2, respectively. This is not surprising since S2 and S3 exhibit 70% amino acid homology, and have previously been shown to induce crossreactive polyclonal antibodies Frank and Parker 1984; Sato et al. 1987; Nicosia et al. 1986). Subunit S5 induced antibodies against S2 and S3 as well as against S5, presumably as a result of its known contamination with S2. This effect was evident at low serum dilution (1:250), but not at high dilution (1:5000) (as shown in Fig. 3). Rabbit antisera raised against S1, S2, and S3 failed to recognize native PT in an anti-PT specific ELISA, while antisera against S4 and S5 were only weakly reactive (Table 1). None of these antisera were able to inhibit PT-induced CHO cell clustering.
Immunization with urea-treated subunits In contrast to the above results, all PT subunits solubilized by the urea-dialysis method induced antibodies that not only reacted with native PT in the ELISA, but were also capable of neutralizing PT in the CHO cell clustering assay (Table 1). Neutralizing titres were, however, lower than those of antisera obtained from rabbits immunized with holotoxin, pertussis toxoid, or affinity-purified S1 or B oligomer. These results suggest that the urea-solubilized subunits were at least partially refolded so as to generate conformational epitopes resembling those of native PT. It is noteworthy that S4 and S5 induced the highest CHO cell PT-neutralizing titres among the five subunits. To the best of our knowledge, this is the first documented evidence for the presence of PT-neutralizing epitopes on these subunits. On the other hand, S2 and S3 were poor immunogens in rabbits, since three doses of 15 pg were required to induce PT-neutralizing antibodies, whereas one dose was sufficient for S1, S4, and S5.
TABLE 2. Immune response to HPLC-purified PT subunits in mice Subunit
Anti-PT titrea
MPT~
S1 S2 S3 S4 S5
16
0
8
3 2
2 4
0
2
3
Weutralization of PT-induced CHO cell clustering. Titres are expressed as the reciprocal of the highest effective dilution. b ~ mouse ~ protection ~ , test. Data are reported as the number of survivors out of 10 mice immunized.
Protective response to PT subunits in mice The ability of individual subunits to induce immunity in mice against live B. pertussis organisms was examined by the intracerebral challenge test. No significant protection was imparted by any subunit preparation (Table 2), in agreement with results on recombinant PT subunits from Escherichia coli reported by others (Locht et al. 1987; Nicosia et al. 1987). However, sera from immunized mice were capable of neutralizing PT in the CHO cell clustering assay. Evidently, the epitopes that give rise to protective antibodies are different from those that elicit antibodies inhibiting CHO cell clustering. It is quite possible that the protective epitopes exist only on fully or partially assembled groups of P T subunits. To examine this possibility, we are currently continuing protection studies using reconstituted oligomers from different HPLC-purified subunits. Acknowledgements The authors thank M. Flood and R. Robinson for technical help, and Dr. M. Blum of the Department of
340
BIOCHEM. CELL BIOL. VOL. 69, 1991
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Simon Fraser University on 11/20/14 For personal use only.
Biochemistry, University of Toronto, f o r carrying o u t t h e amino acid analysis. BURNS,D.L., KENIMER,J.G., and MANCLARK, C.R. 1987. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect. Immun. 55: 24-28. CHONG,P., and KLEIN, M. 1989. Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-Sepharose affinity chromatography. Biochem. Cell Biol. 67: 387-391. COCKLE,S. 1989. Identification of an active-site residue in subunit Sl of pertussis toxin by photocrosslinking to NAD. FEBS Lett. 249: 329-333. FRANK, D.W., and PARKER,C.D. 1984. Interaction of monoclonal antibodies with pertussis toxin and its subunits. Infect. Immun. 46: 195-201. GILLENIUS, P., JAATTMAA, E., ASKELOF,P., GRANDSTROM, M., and TIRU, M. 1985. The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells. J. Biol. Stand. 13: 61-67. IWASA,S., ISHIDA,S., ASAKAWA,S., and AWA, K. 1985. A curious histamine-sensitizingactivity shown by the newly developed Japanese acellular pertussis vaccine. Dev. Biol. Stand. 61: 453-460.
LAEMMLI,U.K. 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature (London), 227: 680-685.
LOCHT, C., and KEITH, J.M. 1986. Pertussis toxin gene: nucleotide sequence and genetic organization. Science (Washington, D.C.), 232: 1258-1264. LOCHT, C., CIEPLAK.W., MARCHITTO,K.S., SATO, H., and KEITH,J.M. 1987. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli. Infect. Immun. 55: 2546-2553. MANCLARK, C.R., and COWELL,J.R. 1984. Pertussis. I n Bacterial vaccines. Edited by R. Germanier. Academic Press, New York. pp. 69-106. Moss, J., STANLEY,S.L., WATKINS,P.A., BURNS, D.R., MANCLARK, C.R., KASLOW,H.R., and HEWLETT,E.L. 1986. Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotide, phospholipids and detergents. Biochemistry, 25: 2720-2725. NICOSIA,A.. PERUGINI,M., FRANZINI,C., CASAGLI,M.C., BORRI,M.G., ANTONI,G., ALMONI,M., NERI, P., RATTI, G.,
and RAPPUOLI,R. 1986. Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc. Natl. Acad. Sci. U.S.A. 83: 4631-4635. NICOSIA,A., BARTOLONI, A., PERUGINI,M., and RAPPUOLI,R. 1987. Expression and immunological properties of the five subunits of pertussis toxin. Infect. Immun. 55: 963-967. NOGIMORI, K., TAMURA,M., NAKAMURA, T., YAJIMA,M., ITO, K., and UI, M. 1985. Dual mechanisms involved in the development of diverse biological activities of islet-activating protein, as revealed by chemical modification of the toxin molecule. Dev. Biol. Stand. 61: 51-61. PEPPLER,M.S., JUDD,R.C., and MUNOZ,J.J. 1985. Effect of proteolytic enzymes, storage and reduction on the structure and biological activity of pertussigen, a toxin from Bordetella pertussis. Dev. Biol. Stand. 61: 75-87. ROBINSON,A., IRONS, L.I., and ASHWORTH,L.A.E. 1985. Pertussis vaccine: present status and future prospects. Vaccines, 3: 11-22. SATO,H., ITO, A., CHIBA,J., and SATO,Y. 1984. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infection. Infect. Immun. 46: 422-428. SATO, H., SATO,Y., ITO, A., and OHISHI,I. 1987. Effect of monoclonal antibody to pertussis toxin on toxin activity. Infect. Immun. 55: 909-915. TAMURA,M., NOGIMORI,K., YAJIMA,M., ITO,K., KATADA,T., UI, M., and ISHII,S. 1982. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry, 21: 5512-5516. TAMURA,M.,NOGIMORI, K., YAJIMA, M., ASE, K., and UI, M. 1983. The 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. TOWBIN,H., STAEHELIN,T., and GORDON,J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. WEISS, A., and HEWLETT,E. 1986. Virulence factors of Bordetella pertussis. Annu. Rev. Microbial. 40: 661-685. WORLD HEALTHORGANIZATION. 1982. WHO Test Manual. Manual of details of tests required on final vaccines used in the WHO expanded programme of immunization. World Health Organization, Geneva. pp. 74-82.
