Vol. 58, No. 3

INFECTION AND IMMUNITY, Mar. 1990, p. 746-752

0019-9567/90/030746-07$02.00/0 Copyright C) 1990, American Society for Microbiology

Monoclonal Antibodies That Inhibit ADP-Ribosyltransferase but Not NAD-Glycohydrolase Activity of Pertussis Toxin HARVEY R. KASLOW,1* JOHN D. SCHLOTTERBECK,l AND JAMES G. KENIMER2 Department of Physiology and Biophysics, University of Southern California School of Medicine, 1333 San Pablo Street, Los Angeles, California 90033,1 and Laboratory of Cellular Physiology, Division of Bacterial Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 208922 Received 19 June 1989/Accepted 3 December 1989

Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identffied three classes of monoclonal antibodies, termed A, B, and C, that recognize the Si subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the Si subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or Si subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native Si proteins and fails to distinguish between neutralizing and nonneutralizing antibodies.

analogs for vaccines and allow for the identification of protective antibodies in serum. Understanding how antibodies neutralize pertussis toxin requires an understanding of the molecular mechanisms of the toxin. Pertussis toxin is a complex, multisubunit toxin composed of an A protomer consisting of a single catalytic Si subunit and a B oligomer containing one S2, one S3, two S4, and one S5 subunits. The B oligomer binds to specific receptors on target cells, delivering the Si subunit (37). The sequence of the pertussis toxin gene predicts that the mature, proteolytically processed S1 subunit contains two cysteine residues at positions 41 and 200 (25, 30). (There is general agreement that the first cysteine in the S1 subunit occurs at position 41 [25, 30]. One report indicates that the second cysteine is at position 200 [25], and the other indicates position 201 [30].) These cysteines form an intrasubunit disulfide bond which apparently must be reduced for the subunit to express enzymatic activity (15, 18, 19, 28, 36). Two assays are commonly used to measure the enzymatic activity of the S1 subunit: assay 1, an ADP-ribosyltransferase assay, quantitates the transfer of 32p from [32P]NAD to a target G protein; and assay 2, an NAD-glycohydrolase assay, detects the release of labeled nicotinamide from NAD in a reaction that can be considered the ADP-ribosylation of water. Nucleotides, such as ATP, and lipophilic substances, such as phospholipids or detergents [e.g., CHAPS (3-[(3-

The bacterium Bordetella pertussis is the causative agent of the disease whooping cough (for a review, see reference 38). A vaccine consisting of killed, whole-cell B. pertussis is currently used in the United States and is considered to effectively protect against disease (31). However, unpleasant side reactions and reports (regardless of validity) of an association with permanent neurologic damage threaten acceptance of the whole-cell vaccine in the United States (13, 27). As a result, there is a need for information that will allow for the rational design of acceptable alternative vaccines and the improvement of assays used to evaluate these vaccines. B. pertussis produces toxic virulence factors, one of which is a protein termed pertussis toxin (38). This observation has spurred efforts towards development of inactive, immunogenic toxin analogs, using site-directed mutagenesis (1, 3, 4, 11, 17). Because many of the actions of the toxin are thought to arise from its catalytic ADP-ribosyltransferase activity (18, 19), many of these studies center on elimination of the ability of the toxin to catalyze the ADP-ribosylation of guanine nucleotide-binding proteins (G proteins). Evidence suggests that some antibodies protect against B. pertussis by binding to pertussis toxin and blocking its ADP-ribosyltransferase activity (20, 34, 35). For example, a monoclonal antibody termed 1B7 which inhibits the ADPribosyltransferase activity of the toxin can protect mice from a challenge with live B. pertussis and blocks the ability of the toxin to cluster cultured CHO cells (34, 35). Bartoloni et al. (1) studied a group of independently derived neutralizing monoclonal antibodies (including 1B7 and also 3CX4 and 6D11C used in the study described below) and suggested that the antibodies shared a common predominant epitope. Thus, understanding the mechanism allowing such antibodies to neutralize the toxin may aid in the design of inactive toxin *

cholamidopropyl)-dimethylammonio]-1-propane sulfonate)],

promote the dissociation of the S1 subunit from the B oligomer, facilitate the reduction of the disulfide bond within the S1 subunit, and increase the NAD-glycohydrolase and ADP-ribosyltransferase activities of the toxin in vitro. Similar events probably activate the toxin in vivo (7, 16, 23, 29). Thus, there are several mechanisms by which an antibody specific for the S1 subunit could inhibit toxin activity. For example, the antibody could interfere with the binding of regulatory ligands, the dissociation process, the reduction of

Corresponding author. 746

ANTIBODY INHIBITION OF PERTUSSIS TOXIN ENZYME ACTIVITY

VOL. 58, 1990

the S1 disulfide bond, or the binding of toxin substrates such NAD or G proteins. In this report, we address these possibilities and discuss the significance of the results in terms of inactivation of the toxin for use in a vaccine. The results arise from studies of seven monoclonal antibodies that recognize the S1 subunit of pertussis toxin. Five of these antibodies were previously grouped into three classes (A, B, and C) on the basis of their ability to block the binding of each other to pertussis toxin. The three classes were found to have distinct functional characteristics (20). A class A antibody (3CX4) blocked the ability of pertussis toxin to induce clustering of CHO cells and also inhibited the ADP-ribosyltransferase activity of the toxin. The class B antibody (6FX1) blocked these actions but only at 40-fold higher concentrations. The class C antibody (X2X5) had essentially no effect on the ability of the toxin to cluster CHO cells and blocked ADP-ribosyltransferase activity only at a concentration nearly 700 times that required for inhibition by a class A antibody. Using synthetic 15-residue portions of the S1 sequence, Kim et al. (21) subsequently determined that the class C antibody (X2X5) recognizes an epitope residing between S1 amino acids 16 to 26. In contrast, a class A antibody (3CX4) did not recognize any of the synthetic peptides tested, consistent with the notion that class A antibodies recognize an epitope arising from discontinuous portions of the S1 sequence (1). This report also describes two additional monoclonal antibodies, termed 3F7 and G6X1, which were produced by immunizing mice with a synthetic peptide corresponding to amino acids 6 to 17 of the S1 subunit (J. G. Kenimer et al., manuscript in preparation). In the remainder of this report, we will sometimes refer to 3F7 and G6X1 as well as X2X5 as peptide-epitope antibodies because they recognize epitopes found on small peptides.

as

747

says were performed, using [nicotinamide-4-3H]NAD (approximately 50,000 cpm per 100 IlI) in the assay buffer described above for the ADP-ribosyltransferase assay. Incubations were for 60 to 90 min at 30°C. The [3H]nicotinamide generated from the reaction was isolated and counted as previously described (15). The NAD-glycohydrolase activity

MATERIALS AND METHODS

detected under these assay conditions was less than that previously reported (15) because (i) the reaction temperature was lower (30°C versus 37°C) and (ii) Lubrol-PX was substituted for CHAPS. These changes were made to make the NAD-glycohydrolase and ADP-ribosyltransferase assay conditions as similar as possible. Lubrol-PX was used because CHAPS severely inhibited the ADP-ribosyltransferase reaction (16, 29) and, compared with the absence of detergent, the addition of Lubrol-PX dramatically stimulated the ADP-ribosyltransferase activity (data not shown). Substituting Lubrol-PX for CHAPS in the NAD-glycohydrolase assay reduced activity by about one-half compared with equivalent conditions in the presence of CHAPS (data not shown). Sulfhydryl-alkylation reactions. These reactions were performed under subdued light without degassing solutions or using a nitrogen atmosphere as previously described (15). Pertussis holotoxin and purified Si subunit. Pertussis toxin was from List Biological Laboratories, Campbell, Calif., and was solubilized in 100 mM Tris hydrochloride (pH 8.0)-i mM EDTA-1% CHAPS. The S1 subunit of the toxin was purified as previously described (17). Previous experience (17) indicated that (i) reducing the disulfide bond in the S1 subunit increases the lability of its enzymatic activities and (ii) including NAD in buffers stabilizes the activity, particularly at temperatures above 0°C (i.e., 30 to 37°C). Thus, after reduction, .25 ,uM NAD was routinely included in buffers. Other experiments (not shown) demonstrated that the rate of NAD-glycohydrolase activity at 0 to 4°C was negligible. The presence of NAD in the buffer was accounted for in calculations of specific activities in NAD-glycohydrolase and

NAD-glycohydrolase and ADP-ribosyltransferase assays. The assays were performed in 100-pAl volumes in glass tubes (12 by 75 mm). The ADP-ribosyltransferase assay was performed by using modifications of previously described methods (16, 17). The assay buffer consisted of 100 mM Tris hydrochloride (pH 8.0)-i mM EDTA-0.1% Lubrol-PX-50 mM NaCl-25 puM [adenylate-32P]NAD (approximately 200,000 cpm per assay tube) with or without 100 ,uM ATP. Reaction incubations (100 pA) were at 30°C for 5 min. The acceptor protein in the ADP-ribosyltransferase assay was transducin in bovine rod outer segments (100 ,g of membrane protein per assay tube) (17, 39). The ADP-ribosyltransferase reactions were quenched by adding 50 ptl of 0.03% (wt/vol) sodium deoxycholate and 5 mM NAD. After the samples were incubated for 10 min on ice, 50 ,ud of 20% trichloroacetic acid was added. At this point, the samples were either iced for 15 min or frozen for processing at a later time. Cold water (2.0 ml) was then added, and the tubes were centrifuged for 30 to 60 min at 2,000 x g in a swinging-bucket rotor at 4°C. The supernatant was decanted, and the pellet was suspended in 100 ,ul of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (14, 22) (lacking bromphenol blue) and heated to 90 to 95°C for 3 min. The samples were vortexed and spotted onto a piece of Whatman 3MM paper, and the paper was washed for 20 min in 50% isopropanol-5% acetic acid-5% trichloroacetic acid. The papers were then washed twice in 50% isopropanol-5% acetic acid, rinsed with 50% isopropanol, dried, and analyzed with a scintillation counter. NAD-glycohydrolase as-

Monoclonal antibodies. The production and characterization of the monoclonal antibodies termed 3CX4, 6D11C, 3C4D, 6FX1, and X2X5 have been described previously (1, 20, 21). Two additional monoclonal antibodies termed CP73003F7 and CP7-300G6X1 (referred to in this report as 3F7 and G6X1, respectively) were produced as described elsewhere (5) by immunizing mice with a synthetic peptide with the sequence Thr-Val-Tyr-Arg-Tyr-Asp-Ser-Arg-ProPro-Glu-Asp, which corresponds to the predicted sequence from positions 6 to 17 of the S1 subunit of pertussis toxin (25, 30). Seven of these twelve amino acids match when the sequence is aligned with amino acids 4 to 15 of cholera toxin, and mutations in this region can essentially inactivate the toxin (4, 11). The monoclonal antibodies used in the reported experiments were purified from ascites fluid, using highpressure liquid chromatography as described elsewhere (20), except for 3F7. This antibody precipitates at cold temperatures and was purified by diluting 1 part ascites fluid with 2 parts Dulbecco phosphate-buffered saline, incubating the mixture on ice for 1 h, centrifuging at 10,000 rpm for 10 min, and suspending the pelleted antibody in Dulbecco phosphate-buffered saline. Dot blots. Unless noted otherwise, all procedures were at room temperature. Samples in 100 mM Tris hydrochloride (pH 8.0)-i mM EDTA-0.1% Lubrol-PX were applied to nitrocellulose prewetted with water, using a 96-well filtration apparatus. The wells were washed three times with 0.5 ml of this same buffer. The nitrocellulose was then removed from the manifold and incubated for 60 min in 75 ml of BLOTTO

ADP-ribosyltransferase assays.

748

KASLOW ET AL.

(50 mM NaXPO4 [pH 7.4]-0.9% NaCl [wt/vol]-5.0% nonfat dry milk [wt/vol]). The nitrocellulose was then cut into smaller pieces, incubated with antibodies in 10 ml of BLOTTO overnight, and rinsed briefly in water; the pieces were washed individually two times in 75 ml of BLOTTO, 10 min per wash. Each piece was then incubated individually for 90 min with 10 ml of BLOTTO containing 60 RI of goat anti-mouse immunoglobulin G peroxidase (Cappel). The pieces were washed separately three times for 15 min with 75 ml of BLOTTO and exposed to 60 ml of peroxidase solution (50 ml of 200 mM Tris hydrochloride [pH 7.5]-200 mM NaCl plus 10 ml of 30 mg of 4-chloro-1-napthol in ice-cold 100% methanol plus 30 pI of 30% hydrogen peroxide). Color was detected within 10 min, the reaction was stopped by rinsing with water, and the nitrocellulose was photographed. Other. The pHs of Tris hydrochloride buffers were determined at 30°C. Analysis of purified preparations for protein content was as described elsewhere (2, 26). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described previously (14, 22). Protein G-Sepharose was from Pharmacia, Inc. Lubrol-PX was from Pierce Chemical Co. Protocols for experiments. (i) Exposure of monoclonal antibodies to sulfhydryl-alkylated Si subunit (Table 1). The following additions were made to a glass tube (12 by 75 mm), and then the ADP-ribosyltransferase activity of the mixture was determined. For the first addition, 10 plI of TEL buffer (100 mM Tris hydrochloride [pH 8.0]-i mM EDTA-0.1% Lubrol-PX) with 1 mM dithiothreitol (DTT) and 25 puM NAD either with or without 25 ng of monoclonal antibody was added. For the second addition, 30 pul of buffer with or without 100 ng of alkylated Si subunit was prepared as follows. A 40-,u portion of 100 mM Tris hydrochloride (pH 8.0)-i mM EDTA-1.0% CHAPS-1 mM DTT, either with or without 3.2 p.g of purified Si subunit, was incubated for 15 min at 30°C and then 45 min on ice. A 50-pu portion of 22.5 mM iodoacetate in the same buffer without DTT was then added, and the tubes were incubated for 30 min at 30°C. The reaction was quenched by the addition of 5 pd of 100 mM DTT-250 p.M NAD in TEL buffer and incubated on ice for 60 min. The samples were then diluted to 960 pul with ice-cold TEL buffer with 1 mM DTT, and 25 p.M NAD was added. Portions (30 ,ul each) of these solutions were then added to tubes containing the first addition, and the tubes were incubated on ice for 2.5 h. For the third addition, 10 pI of buffer either with or without 10 ng of reduced Si subunit was prepared as follows. Purified Si subunit (800 ng) was diluted into 800 pl of TEL buffer with 1 mM DTT-25 p.M NAD added and incubated at 30°C for 15 min and then 45 min on ice. Portions (10 pI each) of these solutions were then added to tubes, and the tubes were incubated for 2 h on ice. For the fourth addition, 50 RI of buffer containing the other components of the ADP-ribosyltransferase assay was added. The samples were incubated and processed, and the incorporation of [32P]ADP-ribosome was assessed. (ii) Effect of monoclonal antibodies (Fig. 1). Portions (90 p.1 each) containing 0.5 ,ug of purified Si subunit in assay buffer (100 mM Tris hydrochloride [pH 8.01-1 mM EDTA-0.1% Lubrol-PX-3 mM DTT-50 mM NaCl-25 p.M NAD) containing [3H]NAD were placed in tubes (12 by 75 mm) and incubated for 5 min at 30°C and then 55 min on ice to reduce the Si subunit. An 80-,ug portion of monoclonal antibody in 10 plI of TEL buffer (100 mM Tris hydrochloride [pH 8.0]-1 mM EDTA-0.1% Lubrol-PX) was then added to each tube, and the tubes were vortexed and left on ice. After 60 min, a

INFECT. IMMUN.

10-pu portion (containing 50 ng of Si subunit) was removed and further diluted in buffer (lacking 3H), and the ADPribosyltransferase activity of 10 ng of Si subunit was determined. The Si subunit (450 ng) remaining in the tube (12 by 75 mm) was assayed for NAD-glycohydrolase activity by warming the tube to 30°C and measuring the rate of nicotinamide release catalyzed by the Si subunit. (iii) Immobilization of pertussis holotoxin by monoclonal antibodies (Fig. 2). For technical reasons, two separate experiments were performed, using the same protocol. Pertussis toxin (150 p.g) was diluted into 750 pI of 100 mM Tris hydrochloride (pH 8.0)-i mM EDTA-0.1% Lubrol-PX-50 mM NaCl (buffer A) and centrifuged at 10,000 x g for 30 min at 4°C to remove insoluble material. A 567-,u portion of the supernatant was then diluted into 3,000 pd of buffer A. Three 10-pd portions were then removed and stored on ice as a control; activities of other samples are expressed relative to the activities of these samples. The effects of antibodies were assayed in triplicate: a 10-pul portion of the remaining toxin was added to a tube containing 8.0 p.g of antibody in 20 RI of buffer A with 0.1 mg of bovine serum albumin added per ml. The tube was vortexed and incubated for 5 min at 30°C and then for 55 min on ice. A 10-pul (pelleted bed volume) portion of protein G-Sepharose in 80 pI of buffer A was then added, and the tubes were incubated for 5 min at 30°C and then for 55 min on ice with vortexing every 10 min. Beads were pelleted by centrifugation, and 100 RI of the supernatant (SUP) was assayed for NAD-glycohydrolase activity by adding ATP to 0.1 mM, DTT to 3 mM, and [3H]NAD to 25 p.M. The beads were then washed in 900 RI of buffer A with bovine serum albumin and pelleted, and all but 100 RI of the supernatant was removed. The beads (BEADS 1) were then suspended and assayed for NADglycohydrolase activity by adding ATP to 1 mM1, DTT to 3 mM, and [3H]NAD to 25 puM (assay buffer) and incubating the tube for 90 min at 30°C. A 900-pd portion of the assay buffer with nonradioactive NAD was added, the beads were pelleted, all but 100 pI of the supernatant was removed, and the [3H]nicotinamide content of the removed portion was determined. The beads were then washed again with 900 pul of assay buffer, and the NAD-glycohydrolase activity remaining on the beads (BEADS 2) was again assayed in the same way. The activity measured in any given sample is normalized to the control by correcting for losses of volume where appropriate. RESULTS As a first step towards defining the mechanism by which class A antibodies inhibit toxin effects, we determined that although the antibodies inhibited ADP-ribosyltransferase activity (20) (Fig. 1), the addition of the antibodies directly to an assay reaction did not inhibit the NAD-glycohydrolase activity of holotoxin (not shown) or of the isolated, reduced Si subunit (Fig. 1). Because the class A antibodies bind to the holotoxin molecule (20) yet allow for the expression of NAD-glycohydrolase activity, they can apparently bind to the Si subunit with its disulfide bond intact and not interfere with its subsequent reduction under the conditions of the NAD-glycohydrolase assay. Furthermore, because these antibodies inhibit the ADP-ribosyltransferase activity of reduced Si (Fig. 1), the class A antibodies bind directly to reduced Si as well. Thus, the disulfide bond of the Si subunit is not a key component of the class A neutralizing

epitope.

In contrast to the class A antibodies under these condi-

VOL. 58, 1990

~BEADS

ANTIBODY INHIBITION OF PERTUSSIS TOXIN ENZYME ACTIVITY

749

For technical reasons, the ADP-ribosyltransferase and NAD-glycohydrolase assays in the above experiments (Fig. 1) were not done under identical conditions. Thus, in order to unambiguously demonstrate that class A antibodies could bind Si subunits without inhibiting NAD-glycohydrolase activity, we incubated holotoxin, nonreduced Si, or reduced Si with these antibodies and then immobilized the antibodies (either free or complexed with toxin or Si) with protein G-Sepharose. The beads were pelleted, and the supernatant was assayed for NAD-glycohydrolase activity. The beads

1.50

I- 1.00 0

F 0.50 _

0.00 NONE

I3CX4

6DI1C

3C4D1 6PFX1 IX2X5

3F7

Q6X11

CLASS: A B Poptide Epitope FIG. 1. Effects of monoclonal antibodies on the NAD-glycohydrolase and ADP-ribosyltransferase activity of the reduced Si subunit of pertussis toxin. All measurements were performed in triplicate. Bars represent standard errors of the means. Activities are expressed relative to the activities from control tubes lacking monoclonal antibody (NONE). Control values (expressed as the mean of the triplicate determination plus or minus the standard error of the mean) were as follows: ADP-ribosyltransferase (ADP-R. Trans.) activity (10 ng of Si), 178 ± 6 pmollh; NAD-glycohydrolase (NADase) activity (450 ng of S1), 119 ± 7 pmol/h. X2X5 was characterized in a separate experiment in which control values were as follows: ADP-ribosyltransferase activity (10 ng of S1), 171 ± 5 pmol/h; NAD-glycohydrolase activity (450 ng of S1), 81 + 2 pmol/h.

tions, the class B antibody (6FX1) had only a modest effect on ADP-ribosyltransferase activity and inhibited NAD-glycohydrolase activity by about 50%. The peptide-epitope antibodies all had essentially no effect on either enzymatic activity of the Si subunit (Fig. 1).

SUP 1.26 _

0 M > 0.75 I X

8.

0

0

(.) .j

were washed and also assayed for activity; after this assay, the beads were washed a second time and reassayed. The results of the experiments involving holotoxin are shown in Fig. 2; equivalent results (not shown) were obtained by using nonreduced or reduced Si for all antibodies except 3F7 (see below). Class A antibodies (3CX4, 6D11C, and 3C4D) depleted the supernatant of NAD-glycohydrolase activity, and the activity was associated with the washed Sepharose beads. Because the activity was retained on the beads after a wash of the beads subsequent to the first assay, the activity did not arise from Si subunit released during the assay of the beads. The class B antibody (6FX1) immobilized the Si subunit, and the subunit retained NAD-glycohydrolase activity. The peptide-epitope antibodies X2X5 and G6X1 did not inhibit or immobilize reduced Si subunit, suggesting that the epitopes recognized by these antibodies are obscured in this reduced Si subunit preparation or that the antibodies bind with low affinity under the conditions of this assay. For the peptide-epitope antibody 3F7, in the experiment involving holotoxin, a substantial amount of immobilized activity was detected in the first assay of the beads; washing the beads after this assay removed the activity (Fig. 2). In contrast, in the experiments involving nonreduced or reduced Si (data not shown), 3F7 did not immobilize a

BEADS 2

I

0.26

I .

z

-0.25L NONE CLASS:

I3CX4

6D1iC 3C40

A

6FX1

B

3F7

G6XlI

Peptide Epitope

NONE

3CX4

A

IX2X5 Poptide Epitope

FIG. 2. Immobilization of pertussis holotoxin by monoclonal antibodies. Immobilization was detected by assaying for NAD-glycohydrolase activity. Values shown are means standard errors. Pertussis holotoxin was incubated with antibodies and exposed to protein G-Sepharose. The beads were pelleted by centrifugation, and the supematant (SUP) was assayed for NAD-glycohydrolase activity. The beads were then washed and assayed (BEADS 1) and then washed and assayed again (BEADS 2). For technical reasons, two separate experiments were performed using the same protocol. Negative activities arose from apparent imprecision in determining the blank of the NADglycohydrolase assay. The NAD-glycohydrolase activity of the control preparation (expressed per assay tube) in experiment (EXP.) 1 was 248 + 8 pmol/h; for experiment 2 it was 156 + 1 pmol/h. For experimental details see Materials and Methods.

750

INFECT. IMMUN.

KASLOW ET AL. ANTI IBOD)Y

a

;.4

I

,

SAMPLE

ANVI

tB3ODY

I

..X

:...I.

FIG. 3. Immunodetection of pertussis toxin applied to nitrocellulose. Pertussis holotoxin was diluted into 100 mM Tris hydrochloride (pH 8.0)-i mM EDTA-0.1% Lubrol-PX to a concentration of 3.75 ,ug/ml and either used directly (C [control]) or incubated at 90 to 95°C for 3 min (H [heated]). Portions (100 ,ul each) (375 ng) of toxin were applied with a 96-well filtration apparatus; the nitrocellulose was then washed, cut into strips, and exposed to monoclonal antibodies, and the antibodies were detected as described in Materials and Methods. Each strip contained duplicate portions of each type of toxin preparation.

substantial amount of activity. An explanation for these results is that the holotoxin bound via the B oligomer to a carbohydrate moiety on 3F7 and that during the first assay, ATP promoted the release of the Si subunit and activity was expressed. During the washing step, the freed Si subunit was removed and not detected in the second assay. The above experiments involving purified Si subunit failed to detect any effect of the addition of the peptideepitope antibodies. In order to verify that these particular lots of antibodies were active, we demonstrated their ability to recognize the toxin spotted onto nitrocellulose (Fig. 3). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we also verified that the protein G-Sepharose immobilized these antibodies (data not shown). In the experiment shown in Fig. 3, holotoxin was spotted either with or without a heating step to denature the preparation. Heating prevented subsequent recognition by the class A antibodies, indicating that the discontinuous epitope recognized by such antibodies is heat labile. In contrast, two of the three peptide-epitope antibodies (X2X5 and 3F7) recognized the heated preparation better than the nonheated one. Combined with the observation that these antibodies did not immobilize the NAD-glycohydrolase activity of toxin, nonreduced Si, or reduced Si, this result suggests that X2X5 and 3F7 recognize only denatured toxin. For the Si subunit of pertussis toxin, amino acids critical for activity can be functionally divided into at least two groups: those that interact with NAD and those that interact with the G protein target. The observation that antibodies that inhibit ADP-ribosyltransferase activity do not inhibit NAD-glycohydrolase activity (Fig. 1) leads to the hypothesis that class A antibodies inhibit pertussis toxin ADP-ribosyltransferase activity by interfering with the interaction of the Si subunit with its G protein substrate, not NAD. It has been proposed that NAD interacts with cysteine 41 of the pertussis toxin Si subunit (15, 17). This proposal arises from the observations that alkylation of cysteine 41 inactivates the NAD-glycohydrolase and ADP-ribosyltransferase activities of the Si subunit (6, 8, 15, 17) and that the presence of NAD in alkylation reactions protects cysteine 41 from alkylation (15, 17). These observations, coupled with the above hypothesis regarding the mechanism by which class A antibodies inhibit ADP-ribosyltransferase activity, suggested that alkylation of cysteine 41 might not destroy the

epitope recognized by class A antibodies, even though the alkylation inactivates the subunit. The following experiments indicate that this suggestion is correct. In the first experiment, we determined the amount of antibody required to inhibit 10 ng of reduced Si subunit, an amount of Si which gives a large signal in but does not saturate this ADP-ribosyltransferase assay (17). For the class A antibodies, 20 ng was sufficient to reduce activity by >90% (not shown). Assuming a molecular weight of 26,220 for the Si subunit (30), 10 ng is 0.38 pmol. The molecular weight of the antibodies appeared to be about 140,000 (deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, assuming two heavy and two light chains per molecule). If each antibody can bind two Si molecules, then 0.38 pmol of Si would be bound by 0.19 pmol of antibody, or 27 ng. Using this relationship in a second experiment (Table 1), we first exposed class A antibodies (25 ng) to either alkylated Si subunit (100 ng) or buffer. The antibodies were then exposed to reduced Si subunit (10 ng). Antibodies exposed only to buffer in the first incubation inhibited the activity of the subsequently added reduced Si TABLE 1. Inhibition of ADP-ribosyltransferase activity of pertussis toxin Si subunit by class A monoclonal antibodies is prevented by prior exposure of the antibodies to sulfhydryl-alkylated S1 subunit Activitya of buffer Antibodyb

Without reduced Sl'

None 3CX4 6D11C 3C4D

With alkylated S1 subunitc

Without alkylated Si subunitc

231 223 215 214

± ± ± ±

16 13 6 12

With reduced

2,140 ± 84 374 ± 45 319 ± 10 464 ± 67

Without reduced S1d

325 296 307 313

± 5 ± 10 ± 3

+ 17

With reduced

1,892 1,819 1,930 1,937

± ± ± ±

72 196 102 89

a Values are the means of triplicate determinations plus or minus standard errors of the counts per minute as obtained from the scintillation counter; no correction for a blank from the assay reaction or the scintillation counter was calculated. b First addition. c Second addition. dThird addition. For details concerning additions, see Materials and Methods.

VOL. 58, 1990

ANTIBODY INHIBITION OF PERTUSSIS TOXIN ENZYME ACTIVITY

subunit. In contrast, antibodies first exposed to an alkylated (and essentially inactive) Si subunit preparation failed to inhibit reduced Si subunits. A simple explanation for this result is that during the first incubation, the antibodies bound the alkylated Si preparation and thus could not subsequently bind and inhibit the reduced Si subunits. Thus, alkylation of cysteine 41 inactivates the subunit without destroying the epitope recognized by class A antibodies. DISCUSSION The data of this report bear on two issues: (i) inactivation of pertussis toxin for vaccine purposes and (ii) identification of neutralizing antibodies. (i) Previous work has demonstrated that proper sitedirected mutagenesis of Arg-9 and Glu-129 of the S1 subunit of pertussis toxin can decrease the enzymatic activities of the Si subunit of pertussis toxin without destroying the predominant neutralizing epitope (4, 24, 33). Glu-129 can be photolabeled by [14C]NAD (12), as are glutamates which interact with NAD in other toxins that are ADP-ribosyltransferases (9, 10). Arg-9 lies in a region of the Si subunit with sequence homology to cholera toxin (25, 30), which hints that it may interact with NAD, the common substrate of these two toxins. Thus, two residues that may form the NAD-binding site of pertussis toxin are not required to form the predominant epitope recognized by neutralizing antibodies. This report demonstrates that class A monoclonal antibodies which recognize this epitope do not block the NADglycohydrolase activity of the toxin (Fig. 1). This result extends the conclusion arising from these previous studies, namely, that none of the key residues in the NAD-binding site of pertussis toxin are crucial for this neutralizing epitope. This report presents additional data consistent with this conclusion: alkylation of cysteine 41, which may interact with NAD (15, 17), inactivates the toxin but apparently does not destroy this epitope (Table 1). If eliciting antibodies recognizing this epitope provides protection from B. pertussis and it is desirable to use antigens lacking ADP-ribosyltransferase activity, then alterations of amino acids that interact with NAD may provide inactive versions of pertussis toxin suitable for a vaccine.

(ii) This report also demonstrates that neutralizing class A antibodies do not recognize heat-treated pertussis toxin, whereas nonneutralizing peptide-epitope antibodies do not recognize native toxin. Because both class A and peptideepitope antibodies recognize the Si subunit by Western blot (immunoblot) analysis (20), it follows that this type of analysis presents either a heterogeneous mixture of renatured and denatured Si proteins, a partially renatured form of Si, or both. The undefined nature of the secondary and tertiary structure of the Si protein in a Western blot combined with the above observations suggests that Western blot analysis is not suitable for the quantitative analysis of antibodies blocking pertussis toxin action in polyclonal antisera. Similarly, because both class A and peptideepitope antibodies recognize pertussis toxin coated onto plastic 96-well plates by enzyme-linked immunosorbent assay (20), this method must also present a heterogeneous or undefined form of pertussis toxin and thus is also not suitable for such analysis. ACKNOWLEDGMENTS Gary Johnson generously provided bovine rod outer segments containing transducin. This work was supported in part by Public Health Service grant R01 AI-24320 from the National Institutes of Health to H.R.K.

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