INFECTION AND IMMUNITY, June 1993, p. 2408-24180019-9567/93/062408-11$02.00/0Copyright C) 1993, American Society for Microbiology

Identification of B-Cell Epitopes on the S4 Subunitof Pertussis Toxin

PER H. IBSEN,'* ARNE HOLM,2 JESPER W. PETERSEN,' CARL E. OLSEN,2 AND IVER HERON'

Bacterial Vaccine Department and Research Center for Medical Biotechnology, Statens Seruminstitut,DK-2300 Copenhagen S, 1 and Chemical Institute, Royal Veterinary and Agricultural University,

DK-1870 Frederiksberg C,2 Denmark

Received 9 December 1992/Accepted 15 March 1993

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT)by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlappingpeptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PIantibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii)analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization ofmice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were testedin an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISAby the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32,33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by themonoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (Bi to B6)and three potential T-cell epitopes (Ti to T3) gave rise to very high antibody responses towards the homologousconjugates. With the exception of the anti-Tl/diphtheria toxoid antisera, all anti-peptide conjugate antiseracross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however,showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominantin the S4 subunit of native PT.

Pertussis toxin (PT) is regarded as one of the majorvirulence factors produced by Bordetella pertussis, thecausative bacterium of whooping cough (24, 36, 57). PT is ahexamer composed of five different subunits, Si through S5(52). The Si subunit (the A promoter) harbors the enzymaticactivity of PT, whereas S2, S3, S4, and S5 (the B oligomer)mediate the binding of the toxin to the cell surface (53). PTexhibits a wide array of biological effects such as histaminesensitization, leukocytosis-promoting activity, adjuvantic-ity, hypoglycemia, and lethal toxicity (26). Inactivation ofPT by either genetic manipulation or chemical treatment hasbeen shown to eliminate the toxicity (21, 26-28, 37, 39, 47)and to confer protection on immunized mice against chal-lenge with virulent B. pertussis cells (21, 26, 28, 37). Re-cently, a trial performed in Sweden revealed that activeimmunization of children with detoxified PT following atwo-dose schedule could prevent disease among part of thevaccines (1). The potential risk of incomplete detoxificationof PT or reversion to toxicity following chemical treatment,however, is a serious topic (39, 49).Although the anti-Si antibody response following pertus-

sis infection or vaccination of humans with the commonwhole-cell pertussis vaccine was found to be predominant(54), immunization of mice with the B oligomer inducedPT-neutralizing antibodies (2, 15) and protection againstrespiratory infection with B. pertussis (48). In addition,monoclonal anti-PT antibodies (PT MAbs) recognizing Si,S2, S3, and S4 are able to neutralize PT (14, 19, 40, 44, 55)and to prevent lethal infection of mice with virulent B.pertussis cells (40, 44).

The development of a completely or partially synthetic

* Corresponding author.

pertussis vaccine might be an alternative to the forthcominggeneration of acellular vaccines composed of purified anti-gens such as filamentous hemagglutinin and detoxified PT. Asynthetic vaccine could include synthetic peptides coveringB- and T-cell epitopes that induce a protective immuneresponse against pertussis. In recent years, the identificationof B- as well as T-cell epitopes on the PT subunits Si, S2,and S3 by the synthetic peptide approach has been described(3, 4, 9, 17, 31, 41, 43). In our laboratory, two major and oneminor human T-cell epitope as well as four murine T-cellepitopes on the S4 subunit have been identified (34, 35).The main purpose of the present study was to identify

B-cell epitopes on the S4 subunit of PT. Two series ofoverlapping synthetic peptides covering the entire S4 se-quence (20, 30) were screened in an enzyme-linked immu-nosorbent assay (ELISA) by various anti-PT antisera and apanel of PT MAbs recognizing either the S4 or both the S3and the S4 subunits. The results of the ELISA suggested thepresence of six B-cell epitope-containing domains. In addi-tion, nine peptides selected by predictive algorithms wereconjugated to diphtheria toxoid (DTd) and administered tomice. Eight of the anti-peptide conjugate antisera recognizednative PT in ELISA, but none of the antisera showed anyPT-neutralizing activity.

MATERUILS AND METHODS

Selection of peptides. Two different approaches for selec-tion of peptides were used. (i) The first approach was

multipeptide synthesis covering the entire S4 sequence (Ta-bles 1 and 2). Fifty 12-residue (12r) peptides (designated12r/1 to 50, each overlapping with 10 amino acids) and 1125/26-residue (25r) peptides (designated 25r/1 to 11, eachoverlapping with 15 to 16 amino acids) were synthesized and

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TABLE 1. Sequence of overlapping 12r synthetic peptidesPeptide Residues Sequence Peptide Residues Sequence

1 1-12 DVPYVL V KT NMV 26 51-62 C FGKDL KBRP G SS2 3-14 PYVL VKT NM VVT 27 53--64 G KD LK R PGS SP M3 5-16 VL VKT NM VVT SV 28 55-66 D L KR PGS SP M EV4 7-18 V KTNIMLVVT SVAM 29 57-68 KRBP G SSP M EVM L5 9-20 TN MVV T SVAM K PA 30 59-70 PG S SPM EV M LR A6 11-22 MXVVTSV A MK PYE 31 61-72 S SPM E VM LRAV F7 13-24 V T SVAIMLK PY EVT 32 63-74 PM EV M LR AVF MQ8 15-26 SVA M KPY E VT PT 33 65-76 E V MLBRAVFM QQBR9 17-28 AMKPY E VT PT RM 34 67-78 MLRA V F MQQ RP L10 19-30 K PY E VT PTBRM LV 35 69-80 RBAV F MQQ RP LR M11 21-32 Y EV TPTR ML VCG 36 71-82 V FMQQ RP L RMF L12 23-34 V T PTBRM L VCG IA 37 73-84 MQ QR P LRMFL GP A13 25-36 PTRM L VC GI AAK 38 75-86 QBRP LBRMF LG P KQ14 27-38 RIMLLVC G IAAKLG 39 77-88 P LBRMF LG P KQ LT15 29-40 L VC GI A AKLGAA 40 79-90 R MF LG P KQ LTF E16 31-42 C GI A AKLG AAAS 41 81-92 F LG P KQ LTF EG K17 33-44 IA AKLGAAA S SPA 42 83-94 G P KQL TF EG KP A18 35-46 AKL GAAA S SPDA 43 85-96 K QLTFE G KPAL E19 37-48 L GAAASBP D AHV 44 87-98 L TF E G KPAL EL I20 39-50 AAA S SPDAH VPF 45 89-100 FE G KPAL EL IR M21 41-52 ABSSP DAHV P FCF 46 91-102 GKPA L ELI R MV E22 43-54 S PDAHVP F CF GK 47 93-104 P AL EL IR MVEC S23 45-56 D A HV P F C FGDL 48 95-106 LEBLIBRM VEC SG K24 47-58 HVPF C FG KD L KR 49 97-108 LIBRM V EC SG K QD25 49-60 P FC FG K DLKRP G 50 99-110 RBMVE C SGKQ D CP A

tested. (ii) The second approach involved predicted epitopes(Table 2). Six peptides covering sequences 18 to 28, 40 to 46,53 to 65, 73 to 80, 84 to 92, and 101 to 110 (designated Bi toB6) of the S4 subunit were selected as potential B-cellepitopes by a computer program for protein structure pre-diction entitled Surfaceplot (Synthetic Peptides Incorpo-rated, Edmonton, Alberta, Canada). The surface profile(Fig. 1) is based on hydrophilicity, accessibility, and flexi-bility parameters (32). In addition, three peptides coveringthe sequences 59 to 72,72 to 88, and 93 to 106 (designated Tito T3), although originally predicted as potential T-cellepitopes by the method of Margalit et al. (22), were synthe-sized and tested as potential B-cell epitopes.

Peptide synthesis. All peptides were synthesized withfluorenylmethyloxycarbonyl L-amino acid pentafluorophe-nyl esters except Thr and Ser, which were used as the3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yI esters. Tert-butylfor Ser, Thr, Asp, and Glu, tert-butyloxycarbonyl for Lys,and 4-methoxy-2,3,6-trimethylbenzenesulfonyI for Arg wereused as side chain-protecting groups. Tert-butyl was used asthe side chain-protecting group for Cys with the 12r pep-tides, while triphenylmethyl was used in all other cases.Synthesis of the 50 12r peptides was performed on kiesel-guhr-supported polydimethylacrylamide resin (PepSyn KA)in a parallel multiple-column peptide synthesizer developedin our laboratory (16, 23). However, for peptides 5, 17, 37,and 50 for which proline would become the C terminus, thechain was extended to 13 residues with Ala at the C terminusto avoid diketopiperazine formation. The 11 25r peptides aswell as the 9 predicted peptide sequences were synthesizedon PepSyn KA by the continuous-flow method (10) on a fullyautomated peptide synthesizer, as described previously (25).T'he photochemical coupling agent 4-benzoylbenzoic acid(99% pure; Aldrich) (33) was coupled as the 3,4-dihydro-4-oxo-1,2,3,-benzotriazin-3-OH ester to the N-terminaloa-amino group during peptide synthesis (dimethylforma-mide, threefold in excess, 2 h). The peptides were cleaved

from the resin with either trifluoroacetic acid (TFA)-H20(95:5, vol/vol) or TFA-H20-ethanedithiole-thioanisole (90:5:3:2, vol/vol/vol/vol) at room temperature for 2 or 18 h whenthe peptide contained L-Arg (4-methoxy-2,3,6-trimethylben-zenesulfonyl) and then washed with TFA-H20 (95:5, vol!vol). The combined TFA washes were concentrated invacuo, and the peptide was precipitated and washed in ether,dried, purified by gel filtration on a Sephadex G-15 column(Pharmacia, Uppsala, Sweden), and lyophilized. Fluorenyl-methyloxycarbonyl amino acid pentafluorophenyl esters andPepSyn KA were purchased from Milligen. Dimethylforma-mide was destilled at reduced pressure and analyzed for freeamines by the addition of 3,4-dihydro-4-oxo-1,2,3-benzotri-azin-3-OH prior to use. Amino acid analysis was performedby the PICOTAG system (Waters). High-performance liquidchromatography (HPLC) was run on a Waters HPLC systemwith a C18 reversed-phase column (Hamilton PRP-3; flowrate, 1.5 ml/min for analytical preparations) with a bufferconsisting of 0.1% TFA and a buffer consisting of 0.1% TFAand 10% water in acetonitrile. The 12r peptides were ana-lyzed by fast atom bombardment mass spectrometry on aJeol AX5OSw instrument, while all other peptides wereanalyzed by liquid secondary ion mass spectrometry on aVG analytical 70SE instrument.

After synthesis, peptide Bi was purified by HPLC andobtained in >90% purity by HPLC analysis. The gel-filtratedpeptides B2, B4, and B6 were >80 to 95% pure by HPLCand were used directly for measurements. The 11 gel-filtrated 25r peptides were all >80 to 95% pure by HPLC andwere used directly in the studies with the exception of 25r15,which was purified by HPLC, resulting in a >90% pureproduct. All peptides showed a satisfactory amino acidanalysis, and liquid secondary ion mass spectra confirmedthe calculated molecular weights in all cases. The gel-filtrated peptides Ti to T3 were obtained as crude productsof >90 to 95% purity, and amino acid analysis of the peptidesproved satisfactory. The 50 12r peptides were analyzed by

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TABLE 2. Sequences of predicted and overlapping25r synthetic peptides

Peptide Residues Sequence

25r11 -10-15a AMTHLSPALADVPYVLVKTNMVVTS

25r/2 1-26 DVPYVLVKTNMVVTSVAMKPYEVTPT

25r/3 11-35 MVVTSVAMKPYEVTPTRMLVCGIAABlb 18-28 -------MKPYEVTPTRM-------

25r/4 21-45 YEVTPTRMLVCGIAAKLGAAASSPD

25r/5 31-55 CGIAAKLGAAASSPDAHVPFCFGKDB2 40-46 ---------AASSPDA---------

25r/6 41-65 ASSPDAHVPFCFGKDLKRPGSSPMEB3 53-65 ------------GKDLKRPGSSPME

25r/7 51-75 CFGKDLKRPGSSPMEVMLRAVFMQQTlC 59-72 --------PGSSPMEVMLRAVF---

25r/8 61-85 SSPMEVMLRAVFMQQRPLRMFLGPKB4 73-80 ------------MQQRPLRM-----

25r/9 71-95 VFMQQRPLRMFLGPKQLTFEGKPALT2 72-88 -FMQQRPLRMFLGPKQLT-------

25r/10 81-105 FLGPKQLTFEGKPALELIRMVECSGB5 84-92 ---PKQLTFEGK-------------

25r/11 86-110 QLTFEGKPALELIRMVECSGKQDCPAT3 93-106 -------PALELIRMVECSGK-----B6 101-110 ---------------VECSGKQDCPA

a The first 10 amino acids of this peptide are leader peptide residues.b Peptides B1 to B6 are computer-predicted B-cell epitopes.c Peptides Ti to T3 are computer-predicted T-cell epitopes.

HPLC, indicating a purity as follows, given as a percentage,with the peptide numbers (Table 1) given in parentheses: 90to 100 (8, 12, 17, 18, 28, 30, 31, 32, 33, 35, 36, 39, 44); 80 to89 (1, 6, 9, 10, 11, 15, 16, 21, 24, 25, 26, 27, 34, 37, 38, 40, 47,49); 70 to 79 (2, 4, 5, 7, 14, 19, 22, 23, 29, 41, 42, 43, 48); 60to 69 (3, 13); 50 to 59 (20, 45, 50). In most cases a singlemajor component was observed, but compound 46 gave twomajor (30 and 40%) and two minor peaks. Peptides 1, 4, 7,10, 14, 18, 21, 24, 28, 31, 35, 38, 42, 44, 47, and 50 wereselected at random for fast atom bombardment mass spec-tometry. The calculated molecular weights were confirmedin all cases, but Cys-containing peptides showed a variableamount of uncleaved side chain-protecting tert-butyl.

4-Benzoylbenzoic acid 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl ester. 4-Benzoylbenzoic acid (1.13 g) was dissolved indry tetrahydrofuran (15 ml) and cooled to -15°C. Dicyclo-hexylcarbodiimide (1.03 g) dissolved in tetrahydrofuran (2ml) was added under stirring, and the mixture was left for 5min at -15°C. Solid 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-OH (0.815 g) was added under stirring, and the mixturewas incubated at -10°C with stirring for 30 min and thenstored overnight at 4°C. The precipitated dicyclohexylureawas removed by filtration through a sintered glass funnel andwashed with a small amount of tetrahydrofuran. The solu-tion of tetrahydrofuran was evaporated in vacuo, leaving anoil to which 10 ml of dry diethyl ether was added. Theresulting crystalline material was isolated by filtration on asintered glass funnel, washed in ether, and dried to give the3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl ester (1.63 g) in

a)

ci)c)

Cl)

1 10 20 30 40 50 60 70 80 90 100 110

Residue numberFIG. 1. Prediction of potential B-cell epitopes on the S4 se-

quence of PT by Surfaceplot analysis (peptides Bi to B6). Thepositions of these as well as the predicted T-cell epitopes (Ti to T3)and the 11 25r peptides (not named) are marked by horizontal bars.Residues are numbered from the N- to the C-terminal end.

88% yield relative to 4-benzoylbenzoic acid (mp 166 to168°C). The result of the elemental analysis was satisfactory.The infrared spectrum was devoid of an azide band.

Conjugation to carrier protein. Three milliliters of DTd(4.88 mg of protein nitrogen per ml; Statens Seruminstitut,Copenhagen, Denmark) was dialyzed against water (500 ml,twice in 48 h) and lyophilized. The N-4-benzoylbenzoylpep-tide (-1 mg; approximately 5 x 104 mol) was mixed withDTd (4 mg; 8.3 x 10-8 mol) in a Pyrex test tube to which 0.1ml of 0.1 M KH2PO4 (pH 6.2) was added. The photochemicalcoupling (50) was carried out for 1 h in a box equipped witheight 350-nm lamps (Philips TL K 40 W/09N). The test tubewas cooled in an ice bath every 15 min, and a constant-airbath temperature surrounding the sample was maintained byan electric fan mounted in the bottom of the box. Followingphotolysis, the conjugate was dissolved in 8 M urea (5 ml),dialyzed against 0.1 M NH4HCO3 (500 ml, 24 h)-0.025 MNH4HCO3 (500 ml, twice in 48 h), and lyophilized.

Antigens. Hydrogen peroxide-detoxified pertussis toxin(46) was a generous gift from North American Vaccines,Beltsville, Md. The acellular Japanese pertussis vaccineJNIH-7 (Japanese National Institute of Health) containinghighly purified formalin-detoxified PT was kindly providedby M. Tiru, Statens Bakteriologiska Laboratorium, Stock-holm, Sweden. The preparations of purified PT, B oligomer,S2-S4/S3-S4 dimers, and S4 subunit (7) were generouslyprovided by C. Capiau, SmithKline Beecham, Rixensart,Belgium.

Subunit S4 was purified by haptoglobin-Sepharose 4B andDEAE-Sepharose CL-6B chromatographies after exposureof PT to 8 M urea.

Antisera. (i) Peptide conjugate antisera. A group of femaleCF1 mice (17 to 22 g) was divided into subgroups of five (Ti,T2, B2, B5, and B6), six (T3, B3, and B4), and ten (B1) miceeach and immunized intraperitoneally (i.p.) with 0.1 mg ofpeptide conjugate adsorbed to aluminium hydroxide (1 mg/ml; Superfos, Vedbaek, Denmark) on days 0, 14, 28, and 35.Seven days after the last injection, the serum was collected.

(ii) Rabbit anti-B. pertussis antiserum. A pool of rabbitanti-B. pertussis whole-cell antiserum was kindly providedby Kate Runeberg-Nyman, National Public Health Institute,Helsinki, Finland.

(iii) Mouse anti-PT. Twenty female CF1 mice (17 to 22 g)

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were each immunized i.p. with 0.5 ,ug of PT on days 0, 14,

28, and 40. Aluminum hydroxide was used as adjuvant (1

mg/ml). One week following the fourth injection, the serum

was collected and pooled.

(iv) Mouse anti-dimer/S4. Three female BALB/c-CFl mice

(17 to 22 g) were immunized i.p. with 10 ,ug of S2-S4 dimer

and 2 ,ug of S3-S4 dimer adsorbed to aluminum hydroxide (1

mg/ml) on days 0, 14, 35, 36, and 37. In addition, the animals

were injected with S ,ug of 54 on days 67 and 85. One week

after the last injection, the serum was collected and pooled.

(v) Mouse anti-B oligomer/dimer. Five female CF1 mice (17

to 22 g) were immunized i.p. with 5 p.g of B oligomer and 2

,ug of S3-S4 dimer on days 0, 9, 15, 22, and 36. Aluminum

hydroxide (1 mg/ml) was used as adjuvant. One week after

the last injection, the serum was collected and pooled.

(vi) Mouse anti-pertussis toxoid. (i) A group of 20 female

C57BL/6j mice (4 to 8 weeks of age) was immunized i.p. four

times at intervals of 2 weeks with 1 ,ug of hydrogen peroxide-

detoxified PT adsorbed to aluminum hydroxide (1 mg/ml) per

dose. Two weeks following the fourth injection, the serum

was collected and pooled. (ii) Another group of 20 female

CF1 mice (17 to 22 g) was immunized i.p. with one-half of a

human dose (0.25 ml) of the Japanese pertussis vaccine

(JNIH-7) consisting of 12 ,ug of protein nitrogen per ml of

formalin-detoxified PT on days 0, 14, 28, and 55. One week

after the last injection, the serum was collected and pooled.

PT MAbs. Three PT MAbs recognizing 54 (designated

56.2, 58.1, and 58.3) as well as six PT MAbs reacting with

both S3 and 54 (designated 18.3, 20.6, 47.2, 47.4, 58.2, and

58.5) were tested for cross-reactivity with the S4 peptides in

an ELISA. The specificities of these MAbs were determined

by an ELISA employing isolated 54 and immunoblotting of

PT (data not shown). Five of these MAbs (20.6, 47.2, 47.4,

58.2, and 58.5) exhibit PT-neutralizing activities in the leu-

kocytosis-promoting activity test and in the Chinese hamster

ovary (CHO) cell assay (data not shown).

The fusion procedure for the MAbs was essentially as

described by Kohler and Milstein (18), using X-63 Ag 8.6.5.3

myeloma cells for fusion. The designations given to all MAbs

refer to the fusion numbers listed below. Fusions 18 and 20

were as described earlier by Schou et al. (44). For fusion 47,

BALB/c mice were immunized i.p. with 0.12 ,ug of PT on

days 0 and 8 and with 10 ,ug of PT on day 30. Eventually, the

mice were boosted with 3 ,ug of PT on days 55, 56, and 57.

Aluminum hydroxide was used as adjuvant (1 mg/ml). For

fusion 56, BALB/c mice were immunized i.p. with 20 ,ug of

B oligomer adsorbed to aluminum hydroxide on days 0, 14,

21, and 38. Fusion 58 was as described for the mouse

anti-dimer/S4 antiserum.

ELISA. Throughout these assays 0.1-ml volumes were

applied, and the ELISA plates were washed five times

between each step in phosphate-buffered saline (PBS; pH

7.4) containing 1% (vol/vol) Triton X-100.

(i) Free peptides. Polystyrene Maxisorp microtiter plates

(Nunc, Roskilde, Denmark) were coated with free 25r pep-

tides (20 ,ug/ml) in a 50 mM carbonate buffer (pH 9.6)

overnight at room temperature. Coating of the 12r, Ti to T3,

and B1 to B6 peptides was performed as stated above but

with 0.5 M sodium chloride added to the buffer. Peptides not

recognized by any of the antisera or PT MAbs when coated

at pH 9.6 were also tested following coating in 50 mM

sodium citrate buffer (pH 4.0; + 0.5 M NaCl) and/or 50 mM

sodium phosphate buffer (pH 7.0; + 0.5 M NaCl). Next, the

plates were blocked in PBS plus 5% (wt/vol) low-fat milk

(M-PBS) at 37°C for 2 h. All antisera and PT MAbs were

diluted in M-PBS and incubated for 2 h at room temperature.

Horseradish peroxidase-conjugated goat anti-murine immu-noglobulins 0 plus M (Pierce Chemical Co., Rockford, Ill.)diluted 1:1,000 in M-PBS was applied to each well, and theincubation was continued for 1 h at room temperature.O-phenylenediamine was added, and hydrogen peroxide wasused as the substrate. After 30 min the color developmentwas stopped with 0.15 ml of 1 N sulfuric acid per well, andthe A490 was read on an Immunoreader NJ-2000 (Technunc,Roskilde, Denmark).The PT MAbs were tested as hybridoma cell culture

supernatants diluted 1:5. To be able to compare the antipep-tide reactivity of the individual antisera, the latter werediluted as described below before testing to compensate fordifferent anti-PT ELISA titers (data not shown) in theseantisera. The antisera were diluted 1:20 (mouse anti-PT andrabbit anti-B. pertussis), 1:50 (mouse anti-formalin-detoxi-fled PT), and 1:100 (mouse anti-dimer/S4, mouse anti-Boligomer/dimer, and mouse anti-hydrogen peroxide-detoxi-fied PT) prior to testing. The PT titer of the rabbit anti-B.pertussis antiserum, however, was too low to be compen-sated for by predilution. Each antiserum and PT MAb wastested at least twice.A hyperimmune mouse anti-diphtheria/tetanus toxoid

(DTd/TTFd) antiserum constituted the negative control. Tomonitor the potential nonspecific binding of the individualantiserum and PT MAb, all samples were assayed in ablocked, uncoated well. The reactivity of the antibodiestowards the peptides (expressed in optical density [OD]values) was corrected for nonspecific binding, if any, bysubtraction. The highest level of nonspecific binding ob-served was 0.8 0D490.

(ii) Peptide conjugate, PT, B oligomer, and S4. The assayfor peptide conjugate, PT, B oligomer, and S4 was per-formed as described for the free peptides with the followingmodifications. (i) The coating concentrations of the peptideconjugates, PT, B oligomer, and S4 were 2.0, 0.3, 0.3, and0.1 ,ug/ml, respectively. Dilution and incubation were donein 50 mM carbonate buffer (pH 9.6) at 4°C overnight. (ii) Noseparate blocking step was included. (iii) All antibodies andthe horseradish peroxidase conjugate were diluted in PBS(pH 7.4) containing 1%o (vol/vol) Triton X-100 and 1%(wt/vol) bovine serum albumin. The anti-peptide conjugateantisera were tested in 10-fold dilution steps following fourimmunizations. In the peptide conjugate ELISA, a pool ofpreimmune CF1 mouse serum and a pool of mouse anti-DTd/TTJd antiserum were used as negative and positive controls,respectively. The mouse anti-formalin-detoxified PT anti-serum constituted the positive control in the PT, B oligomer,and S4 ELISA.

(iii) PT and B oligomer inhibition. Microtiter plates werecoated with either PT (0.12 ,ug/ml) or B oligomer (0.3 ,ug/ml)as described above. The anti-peptide conjugate antiserawere pooled in groups (see below) and prediluted 1:10 (PTELISA) and 1:100/1:1,000 (B oligomer ELISA) in buffer 1(PBS [pH 7.4], 1% [vol/vol] Triton X-100, 1% [wt/hol] bovineserum albumin) and in buffer 2 (as buffer 1 but containingeither PT [20 ,ug/ml; PT ELISA] or B oligomer [20 ,ug/ml;B-oligomer ELISA]). The positive (mouse anti-PT) andnegative (mouse anti-DTd/TT7d) control antisera weretreated likewise. All mixtures were incubated for 2 h at roomtemperature. Next, the samples were applied to the coatedplates, diluted serially in twofold steps, and allowed toincubate for another 2 h at room temperature. Addition ofhorseradish peroxidase conjugate, color development, andreading were performed as described above. The differencebetween the PT/B oligomer titers of the individual pools of

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antiserum diluted in buffer 1 (no inhibition) and buffer 2(potential inhibition) was measured and expressed as percentreduction of the titers. Only sera showing titer reductions of275% were regarded as inhibited. The pools of anti-peptideconjugate antisera consisted of serum from animals showingPT/B-oligomer cross-reactivity only. The experiment wasperformed twice.CHO cell inhibition assay. The CHO cell inhibition assay

was mainly performed as described by Gillenius et al. (13).The ability of the anti-peptide conjugate antisera to inhibitthe clustering effect of PT on CHO cells was assessed byincubating twofold serial dilutions of antisera (prediluted1:10) mixed with PT (0.5 ng/ml) for 2 h at 37°C. Then 50 p,l ofCHO cells (2 x 105 cells per ml) was added, and theincubation was continued for 48 to 72 h. Following this stepthe cells were dried, fixed with methanol, and stained withdrops of sterile filtered Giemsa stain. Finally, the cells werewashed in deionized water, and the results were scored as 0(no clustering), 1 (weak clustering), 2 (medium clustering),or 3 (maximum clustering).

Leukocytosis-promoting activity inhibition test. Pairs offemale CF1 mice (17 to 22 g) were injected intravenouslywith 0.2 ml of PT (2 ,ug/ml), and 3 days later the circulatingleukocytes were counted in a Coulter counter (CoulterElectronics, Ltd., Luton, United Kingdom). The PT-neutral-izing effect of the anti-peptide conjugate antisera was testedby i.p. injection of 1 ml of prediluted antiserum (1:10) at thesame time PT was administered. The positive and negativecontrols consisted of mouse anti-PT and mouse anti-DTd/TTd, respectively.

RESULTS

Screening of overlapping 12r peptides. The reactivity of thedifferent anti-PT antisera towards free 12r peptides inELISA is shown in Fig. 2. The antiserum raised againstnative PT showed a moderate level of binding to peptides12r/18, 20, 46, 48, and 49 and a weak binding to peptidesi2r/11, 27, 29, 31, 35, 36, 42, 43, 45, and 50. In general, thespecificity of the antiserum against hydrogen peroxide-de-toxified PT was comparable to that of mouse anti-PT but thebinding to the peptides appeared to be at a higher level.Some differences, however, were seen as peptides 12r/25,27, and 30 and 12r/40 and 41, which were barely recognizedby mouse anti-PT, reacted more strongly with mouse anti-hydrogen peroxide-detoxified PT.The testing of another pertussis toxoid antiserum raised

against formalin-detoxified PT did not reveal binding to anyof the 12r peptides (data not shown).A slightly different picture was obtained by testing the

antiserum from mice immunized with the B-oligomer/dimerS3-S4 as peptides 12r/35 to 37 were recognized. In addition,this antiserum showed moderate binding to peptides 12r/20and 21, 12r/31, and 12r/46 and 48.The specificity of the mouse anti-dimer/S4 antiserum

resembled that of mouse anti-B-oligomer/dimer, but in thiscase the sequence covered by peptides 12r/35 and 37 was notrecognized.Only three peptides (12r/10 and 11 and 12r/45) were

recognized by rabbit anti-B. pertussis antiserum. The bind-ing to peptide 12r/45, however, was poor.None of the PT MAbs reacted with the 12r peptides tested.Screening of overlapping 25r peptides. The results of test-

ing 11 25r peptides covering the entire S4 sequence in anELISA are shown in Fig. 3. Mouse anti-PT antiserumshowed weak binding to peptides 25r/9 and 10 which span

the carboxy-terminal end of the sequence. This area was alsorecognized by mouse anti-PT antiserum through the testingof peptides 12r/42 to 50. No binding to the 25r peptidescovering the same region as peptides 12r/18 and 20 wasdetected.

In contrast to the screening of the 12r peptides, the mouseanti-formalin-detoxified PT antiserum reacted strongly withthe 25r/5 peptide and, to a lesser extent, with peptides 25r/7and 8.

Testing of mouse anti-hydrogen peroxide-detoxified PTantiserum revealed a strong binding to peptides 25r/4, 6, 7,10, and 11. Parts of the sequences covered by these peptideswere also recognized as 12r peptides.Whereas the level of recognition of the 12r peptides by the

mouse anti-B-oligomer/dimer antiserum was low, the bind-ing to some of the 25r peptides was found to be strong. Asseen with mouse anti-hydrogen peroxide-detoxified PT, pep-tides 25r/10 and 11 were recognized. In addition, 25r/5, 6,and 9 were bound by this antiserum.Mouse anti-dimer/S4 antiserum differed fromn mouse anti-

B-oligomer/dimer mainly in two ways: first, the binding topeptides 25r/6 and 7 was very strong; second, peptide 25r/11was left unrecognized by mouse anti-dimer/S4.The recognition of the 25r peptides by rabbit anti-B.

pertussis was poor; the binding to 25r/3 and 4, however,corresponds to the recognition of peptides 12r/10 and 11.None of the 25r peptides were recognized by the panel of

PT MAbs.Screening of the predicted peptides. A total of six peptides

covering sequences predicted to contain B-cell epitopes (B1to B6) were tested in an ELISA as described above. PeptideB1 was recognized by the rabbit anti-B. pertussis antiserumand revealed an absorption value of 0.95. This peptide spansalmost the same region as 12r/10 and 11 and part of 25r/3.

Likewise, three predicted T-cell epitopes (Ti to T3) weretested in an ELISA, and two of these, T2 and T3, were solelyselected by the mouse anti-B-oligomer/dimer antiserum,showing absorption values of 0.9 and 0.7, respectively. Thebinding of this antiserum to T2 corresponds well to thebinding to 25r/9 and 12r/35, 36, and 37, as the binding to T3corresponds to the detection of 25r/10 and 11 and 12r/46 and48.Again, the PT MAbs were tested, but found negative.Immunization with the predicted peptides conjugated to

DTd. The ability of the computer-predicted peptides toinduce formation of antibodies cross-reacting with PT, Boligomer, and S4 in ELISA was assessed by immunizingmice with peptides conjugated to DTd. The results of theseimmunizations are given in Table 3. All peptide conjugatesgave rise to mean antibody titers exceeding 106 against thehomologous conjugates. With the exception of the T1/DTd-and T3/DTd-immunized mice, all anti-peptide conjugate an-tisera reacted with the relevant unconjugated peptide, show-ing mean titers ranging from 2,620 (anti-B5/DTd) to 64,000(anti-T2/DTd). The cross-reactivity of the anti-peptide con-jugate antibodies to PT was investigated at three levels bymeasuring the ELISA titers to native PT, B oligomer, and S4(Table 3). As expected, the anti-T1/DTd antisera did not bindto PT, B oligomer, or S4. Apart from a low PT titer, theanti-B6/DTd antisera showed no and only negligible bindingto S4 and the B oligomer, respectively. Surprisingly, theanti-T3/DTd antisera, which did not bind to the free peptide,reacted with PT as well as the B oligomer and S4, althoughthe titers were noted to be moderate. The remaining antisera(B1/DTd through B5/DTd and T2/DTd) all revealed a re-sponse towards PT, B oligomer, and S4. With mean titers

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5 10 15 20 25 30 3S 4.0 4.5 50

2

Ma-B/D

o. .w 1,00 1..11w-4-Cl1 5 10 15S 20 25 30 9S5 4 4X5 50

Ma-D/S4

1S1 0 .10 Oa am MCI90 3S 401 .5 50

Ra-Bp

°.

1 5 10 1 5 20 25 30 35 40 45 S0

Peptide (12r)FIG. 2. Screening of free, overlapping 12r peptides by various anti-PT antisera in an ELISA. Each peptide was tested at least twice.

Recognition of the peptides is expressed as average OD490 value. For dilution details, see Materials and Methods. Abbreviations of antisera:Ma-PT, mouse anti-PT; Ma-PTd/H, mouse anti-hydrogen peroxide-detoxified PT; Ma-B/D, mouse anti-B oligomer/dimer; Ma-D/S4, mouse

anti-dimer/S4; Ra-Bp, rabbit anti-B. pertussis cells. *Not determined.

ranging from 47,360 to 82,000, the anti-T2/DTd antisera werefound to be far superior in cross-reactivity against native anddenatured PT.The recognition of PT by the anti-peptide conjugate anti-

sera in an ELISA was confirmed by immunoblotting of PTfollowing sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) (data not shown).PI/B oligomer inhibition ELISA. The ability of the anti-

peptide conjugate antisera to bind to PT and the B oligomer

in solution was tested in an inhibition ELISA. In this assay,the pooled antisera from each group were allowed to prein-cubate with either PT or the B oligomer before being appliedto the conventional ELISA in which the sera were screenedfor recognition of the surface-bound antigens. The binding ofthe anti-peptide conjugate antibodies to soluble PT/B oli-gomer is reflected in reduced titers towards coated antigen inthe conventional ELISA.The results of the inhibition ELISA are shown in Table 4.

Ma-PT

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3.5

3 Ma-PT2.5

2

1.5

0.5

1 2 3 4 5 6 7 8 9 10 11

a5

3 Ma-PTd/H2.5

2

1.5

0.5

01 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

Peptide (25r)FIG. 3. Screening of free, overlapping 25r peptides by various anti-PT antisera in an ELISA. Each peptide was tested at least twice.

Recognition of the peptides is expressed as average OD490 value. For dilution details, see Materials and Methods. Abbreviations of antiseraare given in the legend to Fig. 2. Ma-PTd/F, mouse anti-formalin-detoxified PT.

According to this assay, none of the antipeptide antiserawere able to bind soluble PT. In contrast, the antiserarevealed different levels of binding to the B oligomer insolution. The reactivity of the anti-T2/DTd antiserum to-wards surface-bound B oligomer was completely abolished,as shown by the 100% reduction in the ELISA titer. In theremaining groups a reduction in the ELISA titers exceeding80% was observed with the anti-B2/,B3/,B4/DTd antisera,whereas the anti-B1/,B5/,B6/,TlI,T3/DTd antisera showedno or low titer reduction.CHO cell and leukocytosis-promoting LP activity inhibition

assays. The PT-neutralizing effect of the anti-peptide conju-gate antisera was assessed in the CHO cell and leukocytosis-promoting activity assays as described above. None of theantisera, however, showed any PT-neutralizing activity as

measured by these tests (data not shown).

DISCUSSION

PT is regarded as a major virulence factor of B. pertussisand is, in a detoxified form, a principal or sole component ofthe new generation of acellular pertussis vaccines. The risk

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TABLE 3. ELISA titers' of anti-peptide conjugate antisera

Titer of given coated antigen

Antiserum" Homologouspeptide Peptide PT B oligomer S4

conjugate

Anti-Bl/DTd >106 16,300 1,700 3,610 1,235Anti-B2/DTd > 106 3,025 330 550 88Anti-B3/DTd > 106 8,867 480 1,575 2,125Anti-B4/DTd > lo6 27,733 1,200 2,333 3,940Anti-B5/DTd > 106 2,620 260 4,420 350Anti-B6/DTd >106 4,600 260 40 <50Anti-T1/DTd >106 <100 64 <20 <50Anti-T2/DTd >106 64,000 62,200 82,000 47,360Anti-T3/DTd > 106 33 540 533 633Anti-PTd/F NDd ND >20,000 >12,800 4,800Anti-DTdiTTd _104 20 100 20 <50Preimmune -10l <20 ND ND ND

a Mean titers expressed as a reciprocal of the endpoint dilution showingOD490 values between 0.8 and 1.0.

b Sera from 5 to 10 animals as described in Materials and Methods.c Anti-PTd-F, mouse anti-formalin-detoxified PT.d ND, not determined.

of reversion to toxicity following chemical detoxification ofPT, however, remains a problem. In addition, chemicaltreatment might modify the antigenic properties of PT (29).One way of circumventing these problems might be thedevelopment of a pertussis vaccine based on syntheticpeptides. Askelof et al (4) have described two B-cell epitopeson the Si subunit of PT which were able to induce toxin-neutralizing immunity in mice. Schmidt et al. (41, 42) dem-onstrated inhibition of PT-mediated hemagglutination ofgoose erythrocytes by anti-S2/S3 peptide antibodies andinhibition of the binding of PT to fetuin by anti-S2 peptideantibodies. None of these anti-S2 antibodies, however, wereable to inhibit the clustering effect of PT on CHO cells (42).The purpose of the present study was to identify B-cell

epitopes on the S4 subunit of PT. The S4 subunit was chosenon the basis of published data on PT-neutralizing and mouse-protective murine anti-S4 MAbs (40, 55) and because severalPT-neutralizing and mouse-protective MAbs recognizingboth S3 and S4 have been raised in our laboratory.The search for B-cell epitopes on S4 was addressed in two

TABLE 4. Inhibition of antipeptide antibody binding toPT and B oligomer in ELISA

% Inhibition' of antibody binding to:Antiseruma

PT B oligomer

Anti-B1/DTd c 50Anti-B2/DTd 88Anti-B3/DTd 86Anti-B4/DTd 86Anti-B5/DTd 75Anti-B6/DTdAnti-T1/DTdAnti-T2/DTd 100Anti-T3/DTd 50Anti-PT 100 100Anti-DTd/TTd

a Pools of anti-peptide conjugate antisera as described in Materials andMethods.

b Expressed as percent reduction in ELISA titers following preincubationof antisera with soluble antigen.c-, <25% inhibition.

ways: (i) screening of free, overlapping synthetic peptides inan ELISA by anti-PT antibodies, and (ii) immunization ofmice with computer-predicted peptides coupled to DTd andsubsequent screening of anti-peptide conjugate antisera forPT cross-reactivity in an ELISA. The advantage of the firstapproach resides in the thorough testing of the entire S4sequence made feasible by the limited number of aminoacids present in this subunit. To enhance the possibility ofidentifying B-cell epitopes, we also chose to immunize micewith peptide conjugates. The use of predictive algorithms forselection of potential B-cell epitopes is widespread (12, 38,41, 43, 45, 51, 56).

Obviously, the minimum number of amino acids of asynthetic peptide required for recognition by antibodiesraised against the native protein depends on the compositionand conformation of the epitope. The binding of antiproteinantibodies to short peptides (8 to 15 residues), most probablymimicking continuous epitopes on the native protein, hasbeen reported (38, 41, 43). Any absence of such cross-reactivity might be due to the presence of discontinuousepitopes consisting of residues separated in the primarysequence (5, 6, 8). To address this problem, we screened twodifferent-sized series of overlapping peptides consisting of 12and 25 to 26 residues, respectively, in an ELISA by a panelof PT MAbs and antisera raised against native, detoxified,and denatured PT. None of the PT MAbs were able to reactwith the peptides in the ELISA, suggesting that the epitopesrecognized by these MAbs are of a discontinuous nature asis suggested for other PT MAbs (19). Although no sequencehomology between the S3 and S4 subunits has been re-ported, certain of the PT MAbs tested in this study recog-nized both of these subunits. This finding might be explainedby the existence of discontinuous epitopes in native PTcomposed of separate S3 and S4 epitopes which are bothrecognized by the relevant MAb despite separation of the S3and S4 subunits following SDS-PAGE and immunoblotting.

Testing of the free S4 peptides in ELISA with the variousPT antisera suggests the existence of at least six B-cellepitope-containing domains on the S4 subunit of PT.Roughly, these domains appear to cover the following resi-dues: 1, 18-32 (25r/3 and 4, B1, and 12r/10 and 11); 2, 33 to46 (25r/4 and 12r/18); 3, 39 to 52 (25r/5 and 6 and 12r/20 and21); 4, 51 to 65 (25r/6 and 7 and 12r/25 to 31); 5, 71 to 84(25r/9, 12r/35 to 37, and T2); and 6, 91 to 106 (25r/10 and 11,12r/45 to 50, and T3). Interestingly, previous studies made inour laboratory revealed that peptides 25r/4 and 5, 25r/8, and25r/10 and 11 also contain murine T-cell epitopes (35).With few exceptions, the results of the ELISA coated with

12r peptides tend to support the data from the 25r peptideELISA. The reasons for these few discrepancies are notknown, but differences in antibody specificity, epitope struc-ture, peptide size, and conformation of the peptides follow-ing adsorption to the ELISA plates might be importantfactors.The magnitude and specificity of the peptide recognition

between the individual antisera varied considerably. Miceimmunized with purified, native PT showed a very poorbinding to the peptides. Likewise, a poor peptide recognitionwas seen with the rabbit anti-B. pertussis antiserum which,however, was noted to reveal a much reduced PT titer inELISA compared with the other antisera tested (data notshown). This finding indicates that the majority of the anti-S4antibodies in mouse anti-PT and rabbit anti-B. pertussis aredependent on the conformation of native S4 in PT. Theheterogeneous peptide reactivity of the mouse anti-PT andrabbit anti-B. pertussis antisera is probably due to the use of

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different animal species or to minor differences in the struc-ture of purified and cell-associated PT. Mice immunized withdenatured PT (mouse anti-B-oligomer/dimer and mouse anti-dimer/S4) revealed higher levels of peptide cross-reactinganti-PT antibodies. Apparently, the denaturation process ofPT into B oligomer, S2-S4/S3-S4 dimers, and isolated S4subunit results in profound conformational changes of the S4subunit which might expose linear as well as conformationalepitopes hidden or absent in native PT. Detoxification of PTby either hydrogen peroxide or formalin also seems toinduce structural changes in PT favoring formation of S4antibodies cross-reacting with the peptides. Obviously, thedetoxification procedures have different effects on the result-ing specificity of the two anti-pertussis toxoid antiseratested. However, native PT might itself also be able tomodulate the immune response.

In general, the binding of the antibodies to the 25r peptidesappeared to be stronger than the binding to the 12r peptides.The reason for this observation is not evident, but the lownumber of residues might impose limitations on the ability ofthe 12r peptides to adsorb to the polystyrene wells orperhaps reduce the strength of the binding between theantibodies and the epitope covered (in part) by the peptide.Our use of several pH values during the coating step as wellas the addition of NaCl to the coating buffer should, how-ever, increase the adsorption of the 12r peptides (11).Immunization of mice with the six Surfaceplot-predicted

peptides coupled to DTd elicited formation of anti-peptideconjugate antibodies cross-reacting with PT at differentlevels in the ELISA. This also applied to the anti-T2/DTdand anti-T3/DTd antisera. The induction of anti-peptideconjugate antisera cross-reacting with PT in the ELISAappears to support the results of the ELISA screening of freepeptides by anti-PT antisera.The anti-T1/DTd antiserum, however, did not react and

the anti-T3/DTd antibodies reacted only poorly with the freepeptide in ELISA. These findings may have resulted fromminor conformational differences between the coated andDTd-associated peptides. Interestingly, the T2 peptide,which induced PT titers exceeding 60,000, covers two pre-dicted S4 epitopes (B4 and B5) which individually gave riseto much lower PT titers. Although most of the residues in T3appear to cover a sequence in the primary structure notpredicted by the Surfaceplot, immunization of mice withT3/DTd resulted in the formation of PT cross-reactive anti-bodies. As suggested by Cheetham et al. (8), the inclusion ofhydrophobic residues, which are considered unimportant bymost predictive algorithms, might also be of importance tothe ability of a peptide to mimic epitope structures.

Despite the observed recognition of coated PT in ELISA,none of the antipeptide antisera appeared to bind PT insolution as measured in the inhibition ELISA. This discrep-ancy might be explained by partial denaturation of PT duringbinding to the polystyrene surface, thereby inducing confor-mational changes in the native structure and thus giving riseto increased affinity for antibody binding in this configura-tion. Binding, however, of some of the antipeptide antibod-ies to soluble B oligomer, which is probably conformation-ally different from PT, was demonstrated. The latter findingalso seems to correlate with the higher levels of peptiderecognition in ELISA shown by the antisera raised againstdenatured PT as opposed to antisera against the toxin (Fig. 2and 3).The ability of the antipeptide antisera to neutralize PT was

assessed in the CHO cell assay and the leukocytosis-promot-ing activity test, but no PT-neutralizing activity was ob-

served. This finding is in accordance with the results of thePT inhibition ELISA.

In summary, several B-cell epitope-containing domains onthe S4 subunit of PT were identified in the present study. Theresults of the peptide conjugate immunizations as well as thepoor peptide recognition by the PT MAbs and the anti-nativePT antisera indicate that epitopes of a discontinuous natureare predominant in this subunit of PT. Experiments includ-ing immunization of mice with 25r peptide conjugates, whichmight resemble native epitope structures more naturally, arein progress in our laboratory.

ACKNOWLEDGMENTS

We thank Ingrid Balle, Charlotte Bisgaard Holm, and Vita Skovfor expert technical assistance and Karin Suhr for skillful secretarialassistance.

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