INFECTION AND IMMUNITY, June 1990, p. 1870-18780019-9567/90/061870-09$02.00/0Copyright C 1990, American Society for Microbiology

Epitope Analysis of the F4 (K88) Fimbrial Antigen Complex ofEnterotoxigenic Escherichia coli by Using Monoclonal AntibodiesFRED G. VAN ZIJDERVELD,l* JULIUS ANAKOTTA,2 RUUD A. M. BROUWERS,1 ANK M. VAN ZIJDERVELD,l

DOUWE BAKKER,3 AND FRITS K. DE GRAAF3

Departments of Bacteriology' and Immunology,2 Central Veterinary Institute, 8219 PH Lelystad, and Department ofMolecular Microbiology, Biological Laboratory, Vrije Universiteit, 1081 HV Amsterdam,3 The Netherlands

Received 27 December 1989/Accepted 2 April 1990

So far, three subtypes of the F4 (K88) fimbrial antigen of porcine enterotoxigenic Escherichia coli, F4ab,F4ac, and F4ad, have been distinguished by using polyclonal antisera in agglutination and precipitation tests.The a factor represents one or more common epitopes, whereas each of the b, c, and d factors represents oneor more subtype-specific epitopes. We further characterized the F4 antigen complex by using a panel of 40F4-specific monoclonal antibodies (MAbs). The specificity of all MAbs was proven by enzyme-linkedimmunosorbent assays, agglutination and radioimmunoprecipitation tests, and immunoelectron microscopy.The MAbs either reacted with all F4 subtypes, reacted with two subtypes, or were subtype specific. Epitopeanalysis by competition enzyme-linked immunosorbent assays revealed at least 11 epitope clusters on the F4antigen complex, designated al to a7, bl, b2, c, and d. The following antigenic formulas were found for the F4subtypes: F4ab, ala2a3a4aSa6blb2; F4ac, ala2a3(a4)a5a6a7c; and F4ad, ala2a3a4a7d. All MAbs weredirected against conformational epitopes located on the 27,500-dalton major fimbrial subunits. Consequencesfor the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed.

Fimbriae are long, nonflagellar, filamentous appendageson the bacterial cell surface. Fimbriae of Escherichia coli arecomposed of identical repeating protein subunits (majorfimbrial subunits). In addition, they usually carry variousminor protein components. At least four antigenically dif-ferent fimbrial antigens, F4 (K88), F5 (K99), F6 (987P), andF41, are important virulence factors of porcine enterotoxi-genic E. coli (ETEC) (6, 24). They enable ETEC strains tocolonize the small intestine by specific adhesion to themucosa. The production and secretion of enterotoxin(s) maythen cause disease characterized by diarrhea, dehydration,and death.

In the Netherlands, F4-positive (F4+) ETEC strains con-stitute the majority of ETEC strains isolated from diseasedpiglets, both neonatal and weaned. In contrast to F5, F6, andF41, several antigenic subtypes of F4 have been described.By using polyclonal antisera and monofactorial antiseraobtained by absorbing polyclonal antisera with bacteria ofheterologous subtypes, three subtypes are established inagglutination and precipitation tests: F4ab, F4ac, and F4ad(10, 25). The a factor represents one or more commonantigenic determinants, whereas the b, c, and d factors aresubtype specific. Almost all Dutch F4+ isolates are of theF4ac type. In vitro, F4+ strains do not express fimbriaewhen grown at 18°C (6).The genetic determinants of F4ab and F4ac have been

cloned (20, 27), and the F4ab genes in particular have beenstudied extensively (21, 22, 26). The genetic map of clonedF4ab DNA is given in Fig. 1. Genes C to H encode sixpolypeptides preceded by signal peptides (FaeC to FaeH).The G protein is the major fimbrial subunit; the D protein isan outer membrane protein that is necessary for transport offimbrial components through the outer membrane and servesas an anchor protein. The other polypeptides probably are

involved in the biosynthesis of the fimbriae; three of them,C, F, and H, are also minor components of the fimbrial

* Corresponding author.

structure (26; D. Bakker and F. K. de Graaf, unpublishedobservations). The major fimbrial subunit appears to beresponsible for the adhesive properties of the fimbriae (13,14).The primary structures of the major fimbrial subunits of

F4ab, F4ac, and F4ad have been determined from theirnucleotide sequences (3, 7, 8, 15) or by amino acid sequenc-ing (16); minor variations within one subtype have beendescribed (3). Antigenic determinants on the major fimbrialsubunits have been predicted (5, 17, 18). However, antiseraraised against synthetic peptides of some of these predictedantigenic determinants did not react with native F4 antigens,except for antisera against the segment of the F4ab and F4advariants consisting of amino acids 213 to 219 (18); whetherthis segment is also an immunogenic epitope of nativefimbriae is unknown.We have made panels of monoclonal antibodies (MAbs)

against the F4, F5, F6, and F41 antigens to (i) study thestructure and function of these fimbriae, (ii) replace poly-clonal antisera in diagnostic tests by MAbs, and (iii) evaluatethe protective value of MAbs against ETEC infections whenMAbs are used for the passive oral immunization of neo-nates. This paper describes the antigenic characterization ofthe F4 antigen complex by using a panel of 40 F4-specificMAbs in competition enzyme-linked immunosorbent assays(ELISAs). All MAbs were directed against the F4 majorfimbrial subunits.

MATERIALS AND METHODS

Bacterial strains and media. Fimbriae were purified fromthe E. coli strains G7 (08:K87:F4ab), 1072 (08:K87:F4ad),1087 (0149:K91:F4ac), and 1091 (0157:K-:F4ac). In addi-tion, all MAbs were tested against 7 other F4ab+ strains, 6F4ad+ strains, and 73 F4ac+ strains. The main characteris-tics of these strains are listed in Table 1. All F4ac+ strainswere isolated at the Central Veterinary Institute from dis-eased piglets. Each isolate originated from a different herd.Most of the F4ad+ strains were obtained from P. A. M.

1870

Vol. 58, No. 6

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

EPITOPE ANALYSIS OF ETEC F4 ANTIGEN COMPLEX BY MAbs

C D E F G H- -i

16.9 82.1 26.3 15.4 27.6 27.5

FIG. 1. Genetic organization of the F4ab gene cluster. Blocksindicate the sizes and positions of structural genes. The dark regionsin the left parts of the genes indicate signal peptides. The numbersrefer to the molecular masses of the gene products in kilodaltons.The major fimbrial subunit is indicated ( M ).

Guinde and W. H. Jansen (National Institute of PublicHealth and Environmental Protection, Bilthoven, The Neth-erlands), and most of the F4ab+ strains (29) were kindlydonated by C. J. Thorns (Central Veterinary Laboratory,Weybridge, Surrey, United Kingdom).

Five strains harboring different recombinant plasmids(Table 2) were used in ELISAs to localize the protein againstwhich MAbs were directed. Other details of the constructionof these plasmids have been described before (20, 21) or willbe described elsewhere.

Strains were grown in Minca medium, Minca-IsoVitaleXbroth (11), or Trypticase soy broth or on 5% sheep bloodagar and Minca-IsoVitaleX agar. Ultrasonic extracts frombacteria grown at 18°C (US18 extract) or 37°C (US37 extract)were prepared as described by Guinee and Jansen (10).

Purification of fimbriae. Bacterial cells of 30- to 40-literfermentor cultures of F4+ strains were collected by centrif-ugation and suspended to an A6. of approximately 150 in0.05 M sodium phosphate buffer containing 2 M urea.Fimbriae were detached from the bacteria by heating for 20mnin at 58°C, and the cells were pelleted by centrifugation.Fimbriae were purified from the supernatant as described byde Graaf and Roorda (2) or as described by Jacobs and deGraaf (12).The purity of the purified fimbrial preparation was as-

sessed by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) with 10% slab gels.

Antisera. Rabbit antisera against purified F4 antigens(RaF4ab, RaF4ac, and RaF4ad) were prepared as describedbefore for the F41 antigen (30). The specificity of the serawas assessed by slide agglutination tests (SATs), immuno-electrophoresis, and double immunodiffusion methods, asdescribed by Guinee and Jansen (10), and by ELISAs.

TABLE 1. OK types of bacterial strains used

F4 subtype OK type No. of strains

ab 0141:K85abc 5O?:K87 2O100:K100 1O100:K? 1

ac 0149:K91 3808:K87 150138:K81 10NTa 609:K204 1020:K? 10141:K85ac 10157:K- 1

ad 08:K87 308:K? 1032:K87 10147:K89 1

a NT, Not typed.

TABLE 2. Strains of E. coli harboring variousrecombinant plasmids

Genes present encoding proteins of:Strain

F4ab F4ac

M5(pFM205) C, D, E, F, G, HM5(pDB88-81) C, D, E, G, HHB1O1(pSV88-1) C, D, E, F, GM5(pDB88-101) G, H C, D, E, FM5(pDB88-103) C, D, E, F G, H

Subtype-specific antisera were obtained by absorbing seraagainst one subtype with whole bacterial cells of the heter-ologous subtypes. Agglutinating subtype-specific rabbit an-tisera, kindly donated by P. A. M. Guinee and W. H. Jansen,were used as reference antisera.MAbs. The immunization schedule of BALB/c mice, the

preparation of mouse hybridoma cell lines, the detection ofanti-F4 antibody-producing cell lines by ELISAs, the clon-ing procedure, and the production of MAbs against the F4variants were similar to those described previously forMAbs against the F41 antigen (30). MAb of each stable cellline was purified from ascitic fluid by ammonium sulfateprecipitation (35 to 40% saturation) followed by dialysisagainst 0.01 M phosphate-buffered saline, pH 7.2. Thepurified MAb preparations (8 mg of protein per ml inphosphate-buffered saline) were stored in aliquots of 1 nil at-70°C. The immunoglobulin isotype of each MAb wasdetermined in immunodiffusion tests with mouse isotype-specific antisera.SATs. Each MAb was used at a dilution of 1:20 in

phosphate-buffered saline in agglutination tests with all F4+strains grown at 37°C and randomly selected F4+ strainsgrown at 18°C. If no agglutination was observed, SATs werealso performed with lower dilutions (1:5 and 1:10).PEPSCAN for mapping of epitopes on F4. The PEPSCAN

method has been described in detail elsewhere (9, 19).Briefly, the total amino acid sequences of the major subunitsof all subtypes of F4 (7, 8, 15) were synthesized in overlap-ping peptides of seven amino acids on polyethylene rods,i.e., peptide 1 of F4ab consisted of amino acids 1 to 7 of theprimary sequence of its major subunit, peptide 2 consisted ofamino acids 2 to 8, peptide 3 consisted of amino acids 3 to 9,and so on. In this way, 257 peptides of F4ab, 255 peptides ofF4ac, and 257 peptides of F4ad were made to cover the totalsequences of the three variants. The peptides still coupled tothe polyethylene rods were then tested against selectedMAbs of different epitope specificity in an indirect ELISA.A known epitope of the VP1 protein of foot-and-mouthdisease virus and its corresponding MAb (19) were includedas appropriate controls on peptide synthesis and the subse-quent ELISA procedure.ELISAs. The ELISAs used in this study were similar to

those used in a study of the characterization of the F41antigen (30). For details of the buffers and substrate used,the length of incubation steps, and the conjugation of MAbsand polyclonal antisera to horseradish peroxidase (HRPO),we refer to that study. Optimal dilutions of HRPO-conju-gated MAbs were determined by checkerboard titrationagainst purified antigens of all subtypes. Briefly, the follow-ing ELISAs were used.

(i) Indirect ELISA for screening hybridoma culture super-natants and titer determination of purified MAbs. The wellsof microdilution plates were coated with 0.5 ,ug of purifiedF4ab, F4ac, or F4ad per well. After the plates were washed,

1871VOL. 58, 1990

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

1872 VAN ZIJDERVELD ET AL.

1:2 dilutions of hybridoma culture supernatants or serialtwofold dilutions of purified MAb preparations were added.The plates were incubated and washed, and HRPO-labeledrabbit anti-mouse immunoglobulins (Dakopatts, Copen-hagen, Denmark) were added. After the plates were washed,substrate solution was added, and the plates were read after2 to 3 h of incubation.

Titers of purified MAb preparations were expressed as thelogarithm of the reciprocal of the highest dilution giving an

A450 of 50% of the maximum obtainable absorbance value.(ii) Indirect ELISA for epitope mapping by the PEPSCAN

method. The polyethylene rods, each carrying a differentpeptide of seven amino acids, were placed in the wells ofmicrodilution plates with diluted MAb or polyclonal anti-sera. After incubation, the rods were washed and placed inmicrodilution plates containing HRPO-labeled goat anti-mouse or anti-rabbit immunoglobulins. After incubation andwashing, the rods were placed in microdilution trays withsubstrate solution, and these plates were read.

(iii) Direct-competition ELISA for epitope analysis. Non-conjugated MAbs (competition MAbs) were added in serialtwofold dilutions (range, 1:20 to 1:10,240) in volumes of 50 j.Ito the wells of plates coated with purified F4ab, F4ac, orF4ad antigen. After incubation for 30 min, 50 RI of theoptimal dilution of each of the HRPO-conjugated MAbs wasadded per well; in this way, the dilutions of each nonconju-gated MAb were allowed to compete with individual MAbconjugates for its epitope on all F4 subtypes. Incubation wascontinued for 1 h, and the substrate solution was added afterthe plates were washed.The titers of competition MAbs against a conjugated MAb

were expressed as the logarithm of the reciprocal of thehighest dilution giving an A450 of 50% of the absorbance ofwells to which only the conjugate was added.

Epitopes were defined on the basis of the assumption thattwo MAbs showing no competition at all recognize twodifferent epitopes. Two MAbs showing reciprocal competi-tion with high titers were assumed to recognize the sameepitope or two nearby epitopes. In general, no conclusionswere drawn from nonreciprocal competition, because it maybe caused by several possible factors (30), such as differ-ences in affinity. An epitope cluster was defined as a singleepitope or a group of adjacent or overlapping epitopes thatcannot be distinguished as separate epitopes by epitopeanalysis with competition ELISAs because of steric hin-drance. Epitope clusters present on two or more subtypeswere -designated as a clusters, whereas subtype-specificclusters were designated as b, c, or d clusters.The direct-competition ELISA with polyclonal RaF4 con-

jugates were used to detect whether single MAbs or combi-nations of MAbs were able to block the signal obtained bythe polyclonal RaF4-HRPO conjugates alone.

(iv) Double-antibody sandwich ELISA for screeningepitopes on the F4 antigen of field strains and recombinantDNA strains. Microdilution plates were coated with RaF4acimmunoglobulins. After the plates were washed, superna-tants of heated (20 min at 58°C) cultures or US37 and US18extracts were added. After the plates were incubated andwashed, conjugates of all MAbs, RaF4ab, RaF4ac, andRaF4ad were added in their optimal dilutions. After theplates were incubated and washed and the substrate solutionwas added, the plates were read.RIP assays for specificity control of MAbs. Radioimmuno-

precipitation (RIP) assays with crude antigens of strains G7,1077 (08:K?:F4ad), and 1087 were performed similarly tothose described previously for MAbs against the F41 antigen

(30). Briefly, bacteria were grown in broth containing 3H-labeled amino acids (TRK 550; Radiochemical Centre, Am-ersham, United Kingdom). The cultures were heated for 20min at 58°C, and the cells were pelleted by centrifugation.The supernatants to which sodium deoxycholate (1%), Tri-ton X-100 (1%), and SDS (0.1%) were added were used ascrude antigen preparations in RIP assays. Each MAb (1 ,ul)was added to 200 ,ul of crude antigen, and after incubation,50 RId of rabbit anti-mouse immunoglobulins was added. Theresulting precipitates were collected, washed, and used inSDS-PAGE as described before.IEM. Immunoelectron microscopy (IEM) was performed

as described before (30) either with colloidal gold-labeledMAbs or by an indirect assay with gold-labeled rabbitanti-mouse immunoglobulins. If present, at least two MAbsagainst each epitope cluster were tested. Appropriate con-trols with MAbs against F5, F6, and F41 antigens wereincorporated.

RESULTS

Production and characterization of MAbs. Fusions wereperformed with spleens of 17 mice. The cloning and selectionprocedures resulted in 40 stable hybridoma cell lines; 14lines were obtained from mice immunized with F4ab (CVIF4ab-1 to CVI F4ab-14), 10 from mice immunized with F4ac(CVI F4ac-1 to CVI F4ac-10), and 16 from mice immunizedwith F4ad (CVI F4ad-1 to CVI F4ad-16). The main charac-teristics of the purified MAbs (8 mg of protein per ml) arelisted in Table 3.

In SATs, all MAbs agglutinated F4+ strains of one or moresubtypes grown at 37°C and none of the randomly selectedF4+ strains grown at 18°C.Four of the F4ab MAbs (F4ab-1, -4, -5, and -7), five of the

F4ac MAbs (F4ac-1, -2, -3, -7, and -10), and eleven F4adMAbs (F4ad-4, -5, -7 to -9, and -11 to -16) reacted similarlyin SATs, RIP assays, and indirect ELISAs to all F4 variantsand hence were directed against one or more a epitopes.Each of these MAbs showed similar titers against the threedifferent F4 subtypes in the indirect ELISA. Each of theHRPO conjugates of these MAbs had similar optimal dilu-tions when tested against purified antigens of the threesubtypes, except for the F4ab-5 conjugate, which had opti-mal dilutions of 1:4,000 with F4ab and F4ac and 1:250 withF4ad. MAb F4ab-3 recognized all F4ab+ and F4ad+ strainsbut agglutinated only a part of the F4ac+ strains; its ELISAtiter against F4ac also differed depending on the strain usedfor antigen production.Ten MAbs (CVI F4ab-2, -8, -9, -10, -13, and -14; CVI

F4ac-5 and -8; and F4ad-6 and -10) were subtype specific inagglutination tests and hence were directed against one ormore b, c, or d epitopes; some of them had low titers in theindirect ELISA with heterologous types. HRPO conjugatesof these MAbs reacted only with antigen of the homologoussubtype.Four MAbs (F4ab-6, -11, and -12 and F4ac-4) reacted in a

similar way with the F4ab and F4ac antigen and did notrecognize the F4ad antigen.MAbs F4ad-1, -2, and -3 agglutinated all F4ac+ and F4ad+

strains and none of the F4ab+ strains. Their ELISA titerswere high against the F4ad antigen, intermediate against theF4ac antigen, and low against the F4ab antigen. A similarreaction pattern was observed for MAb F4ac-6, whichagglutinated only F4ab+ and F4ac+ strains and showed highELISA titers against F4ac, intermediate titers against F4ab,and low titers against F4ad. HRPO conjugates of MAbs

INFECT. IMMUN.

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

EPITOPE ANALYSIS OF ETEC F4 ANTIGEN COMPLEX BY MAbs 1873

TABLE 3. Characteristics of the 40 MAbs specific to the F4 antigen complex of E. coli

MAbMouse IsotypeReaction in SATs and RIPs against: ELISA titer against: Epitope clusterMAb

no. Isotype specificityno. ~~~~~F4ab F4ac F4ad F4ab F4ac F4adspcfit

1 IgGlb1 IgG2a2 IgGl3 IgGl3 IgGl4 IgGl5 IgGls IgG35 IgGl5 IgG35 IgG35 IgGl5 IgGl6 IgGl

7 IgG2a8 IgG2a9 IgG2b10 IgG2b11 IgGl11 IgGl11 IgA12 IgGl13 IgGl14 IgA

15 IgM16 IgG316 IgGl17 IgGl17 IgGl17 IgGl17 IgG2a17 IgG2b17 IgGl17 IgG2b17 IgGl17 IgGl17 IgGl17 IgGl17 IgG2a17 IgGl

+ +

+_

+ +I_Cb

+ +

+ +

+ +

+ +

+_

+_

+_

+ +

+ +

+_

+_

+ +

+ +

+ +

+ +

_ +

+ +

+ +

_ +

+ +

+ +

_ +

_ +

_ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ 6.1 5.8- 5.8 1.9+ 5.8 2.2c+ 5.5 5.8+ 5.5 6.1- 5.5 5.5+ 4.9 4.9- 4.0 0.0- 5.2 1.6- 4.0 0.0- 4.6 5.2- 5.5 5.2- 5.2 1.9- 5.2 0.0

-/we

5.51.65.55.25.20.05.20.00.00.00.00.01.30.0

5.8 5.5 5.55.2 4.9 5.25.5 5.2 5.24.9 4.9 0.01.3 5.2 0.03.4 5.8 1.63.7 4.0 3.41.9 5.5 1.93.4 4.0 3.14.6 4.3 4.6

+ 2.2 4.3 5.2+ 1.6 3.4 5.5+ 2.5 4.3 5.2+ 5.5 5.2 5.5+ 6.1 6.1 6.1+ 0.0 0.0 5.2+ 5.5 5.2 5.5+ 5.5 6.1 5.2+ 5.8 5.8 6.1+ 1.6 1.3 5.8+ 5.5 5.5 5.5+ 5.2 5.2 5.2+ 4.9 5.2 5.2+ 5.2 5.2 5.2+ 4.6 5.2 4.9+ 5.2 5.2 4.9

a ab, ac, and ad refer to antigens used for immunizing mice.b IgGl, Immunoglobulin Gl.c With MAb F4ab-3, two types of F4ac strains could be distinguished in SATs and RIPs; the strains were either negative in both tests or positive in both tests.

When F4ac antigen purified from a nonagglutinating strain was used in ELISA, the ELISA titer ofMAb F4ab-3 was 2.2; the ELISA titer of MAb F4ab-3 was 5.5when the MAb was tested on F4ac antigen purified from an agglutinating F4ac strain.

d F4ac-1 to F4ac-7 were made with purified F4ac antigen of strain 1091, and F4ac-8 to F4ac-10 were made with antigen of strain 1087.e Reactions: - in SAT, weak (w) in RIP assay.

F4ad-1 to -3 and F4ac-6 could be used in high dilutions in thedirect-competition ELISA against the homologous subtypeand, in general, against one of the other subtypes in lowdilutions; the conjugates did not recognize the subtype,against which unconjugated homologs had the lowest titers.

All attempts to make suitable HRPO conjugates of MAbsF4ac-7 and F4ac-9 failed. Their conjugates could only beused in very low dilutions against the F4ac antigen; theirtiters in the indirect ELISA suggest that they recognize allsubtypes, with a preference for F4ac.None of the MAbs showed titers in the indirect ELISA

with F41 as antigen.Specfficity control by RIP. The reaction patterns of all

MAbs against the three subtypes are shown in Table 3; theycorrelated with the findings of SATs. SDS-PAGE on 10%

slab gels of precipitates obtained with the MAbs showedsingle radiolabeled protein bands with apparent molecularmasses of approximately 27,500 daltons for all subtypes,similar to the molecular masses of 27,540 (F4ab), 27,239(F4ac), and 27,562 (F4ad) reported for the F4 major subunits(4, 7, 8, 15). A typical example of the results of the RIP assayis given in Fig. 2. On some autoradiograms, a second, minorband of a protein with a molecular mass of 1,000 to 1,500daltons more than those of the F4 bands was observed.

Epitope analysis by direct-competition ELISA. Each MAbin serial twofold dilutions was allowed to compete withconjugated MAbs for its epitope on the three F4 subtypes.MAbs were placed in epitope cluster specificity classesaccording to the criteria described in Materials and Methods.In addition, the reaction pattern of each MAb against all

CVI F4ab-laCVI F4ab-2CVI F4ab-3CVI F4ab-4CVI F4ab-5CVI F4ab-6CVI F4ab-7CVI F4ab-8CVI F4ab-9CVI F4ab-10CVI F4ab-11CVI F4ab-12CVI F4ab-13CVI F4ab-14

CVI F4ac-ldCVI F4ac-2CVI F4ac-3CVI F4ac-4CVI F4ac-5CVI F4ac-6CVI F4ac-7CVI F4ac-8CVI F4ac-9CVI F4ac-10

CVI F4ad-1CVI F4ad-2CVI F4ad-3CVI F4ad-4CVI F4ad-5CVI F4ad-6CVI F4ad-7CVI F4ad-8CVI F4ad-9CVI F4ad-10CVI F4ad-11CVI F4ad-12CVI F4ad-13CVI F4ad-14CVI F4ad-15CVI F4ad-16

a2bla4ala?aSa3blblblaSaSb2bl

ala3alaSca6a?ca?a2

a7a7a7ala2dala2aldalalalalalal

VOL. 58, 1990

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

1874 VAN ZIJDERVELD ET AL.

F-

E- 4D-

C-

a b C

D)- lb*.

C- II)

B- __... _

C- *'

B-W

A- *

AC AG AC AC F41 AC AC1 2 3 4 16 5 6

AC AC AC AC F41 AC AC1 2 3 4 16 5 6

AC AC AC AC F41 AC AC1 2 3 4 16 5 6

FIG. 2. SDS-PAGE of precipitates obtained with MAbs F4ac-1 to 6 and radiolabeled crude antigen of F4ab (a), F4ac (b), and F4ad (c).A to F, Molecular mass markers of 14.3, 30, 46, 69, 92.5, and 200 kilodaltons. The polyacrylamide concentration was 12.5 (a) or 10% (b andc).

other MAbs in the competition ELISA and, if necessary, theresults of other tests were also taken into account in decidingthe epitope specificity of each MAb.

In Fig. 3, the titers of all MAbs recognizing all subtypesare given against their conjugates. The titers shown in thisfigure are the averages of the titers found in competitionELISAs with the three subtypes. We concluded that theseMAbs were directed against four epitope clusters designatedal to a4. MAb F4ac-1 and especially Mab F4ac-3 did notblock conjugates of some of the other MAbs classified inepitope cluster specificity class al or had low titers. The

antigens used in the competition assay were F4ab from strainG7, F4ad from strain 1072, and F4ac from strain 1087. Whena competition assay was performed with F4ac antigen fromstrain 1091 (the antigen used for immunizing mice fromwhich MAbs F4ac-1 and -3 were obtained), F4ac-1 and -3 didblock all conjugates directed against epitope cluster al. Forthat reason, both MAbs were classified in epitope specificityclass al. Some variation probably exists in this cluster;however, we could not exclude the possibility that F4ac-1and -3 are directed against another epitope. It was alsodifficult to classify MAb F4ab-5; it may be classified as a

MAb numberAB ACACAD AD ADAD ADAD AD ADA4 1 3 4 7 9 11 12 13 14 15 11

I I I I I IT

IIIIJIH IIII LtI I I I I I I

kD ABACADAD AB ABAC AB1 1058 5 7 2 3

J i U EU El

i I KA

* EEJ lEo][I I-KA EaJlI I I n U

epitope cluster: al

J I v 'YL Y

a2 a? a3 a4FIG. 3. Titers of F4 MAbs recognizing all F4 variants against their conjugated homologs in the direct-competition ELISA.

titer

* >.2.8E3 >2.0Ea .1.0

>fi O

AE3 4AC- I

. AC-3AD-4AD- 7

E AD-9M AD-1I

AD-12

.0 AD-13AD-14

eAD-152 AD-16%

ABi1AC-10

*)AD-5@ AD- S

0"%AB-57

O AC-2

AB-3 I I

--

F

INFECT. IMMUN.

I

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

EPITOPE ANALYSIS OF ETEC F4 ANTIGEN COMPLEX BY MAbs 1875

MAb numberAB AB AB AB AB AB AB AB AB

al a2 a3 a4 6 11 12 13 2 91014EOEJO

[:

EJaE

titer ' O0AB -6

~AB-12j~

0 h.2.0 AA-13 OO0-_EENEOUJJ3 f1.0 AB2ClAB2lfi0 E AB

AB10AB-14 *..

LI

epitope cluster: al a2 a3 a4 a5 b2 bl

FIG. 4. Titers of F4ab MAbs in the direct-competition ELISAwith F4ab antigen. All MAbs against each of the epitope clusters alto a4 are represented by single squares. Partial shading of thesesquares represents the percentage of MAbs showing competition.

member of specificity class a3 or a2 with an affinity lowerthan those of the other members, or it may recognize anotherepitope. In contrast to members of epitope cluster specificityclass a2 and a3, the conjugate of F4ab-5 showed a loweroptimal dilution for F4ad antigen than for F4ab and F4ac.

Classifying the other MAbs was based on the results ofcompetition assays performed with their homologous anti-gens, because most of them reacted only with their homol-ogous antigen or their conjugates seemed to have a betteraffinity for their homologous antigen than for other subtypes.The results of the competition assay with F4ab antigen (Fig.4) revealed another three epitope clusters, designated a5, bl,and b2. Classifying F4ab MAbs on the basis of only thedirect-competition ELISA had resulted in one epitope clus-ter instead of the three epitope clusters a4, a5, and b2. Yetwe decided that there were three separate epitope clustersbecause of the results of the indirect ELISA and SATs(Table 3) and the results obtained with recombinant strains.The results of the competition assay with F4ad antigenclearly demonstrated another two clusters, called a7 and d

titer

* h.2.8aZ?:2.0E2>3b1.0O I o

MAb numberADAD AD ADAD

a1 a2 a3 &4 1 2 3 8 10

E a2 ID= ^EJEJEOEET.0 a2 Cl03-1: 1N: 3

*i AD- 3

S AD- 2AD-

yyvLyJLyvepitope cluster: al a2 a3 a4 a7 d

FIG. 5. Titers of F4ad MAbs in the direct-competition ELISAwith F4ad antigen. All MAbs against each of the epitope clusters alto a4 are represented by single squares. Partial shading of thesesquares represents the percentage of MAbs showing competition.

titer

* h-2.8E3 >2.0E3 ?1.0E ' O

MAb numberAC AC AC AC

al a2 a3 4 6 5 8

2 a1 *l M El Ea EC. a3ooo,AC- 4 M 13 1: 91 2

ACiEEflB:]EEMAC- *Ao5 E3EB gEJ JE

0AC

B.AC5ffllffl

epitope cluster: al a2 a3 a5 a6 c

FIG. 6. Titers of F4ac MAbs in the direct-competition ELISAwith F4ac antigen. All MAbs against each of the epitope clusters alto a3 are represented by single squares. Partial shading of thesesquares represents the percentage of MAbs showing competition.

(Fig. 5). The remaining F4ac MAbs were classified in epitopecluster specificity classes aS, a6, and c (Fig. 6). F4ac-4 wasprobably directed against the same epitope cluster as F4ab-6, -11, and -12, although F4ac-4 blocked the conjugates ofF4ab-6, -11, and -12 only in low dilutions when F4ab wasused as antigen, and F4ab-6, -11, and -12 showed low titersagainst the F4ac-4 conjugate when F4ac was used as antigen(not shown). Because of its reactions in other tests, MAbF4ac-6 was placed in a separate class, a6, although someinterference was noticed in the competition assay withMAbs of classes a5 and c. Competition assays with MAbs ofclasses aS, a6, and a7 on F4ac antigen (not shown) revealedthat these classes were probably unrelated, indicating thatthe MAbs recognized three different clusters and not threevariants of the same epitope cluster. F4ac-7 and F4ac-9 werenot classified because their conjugates could be used only onF4ac antigen in low dilutions; for instance, the F4ac-7conjugate was blocked by almost all other MAbs. These twoMAbs may be directed against one or two epitope clustersother than the ones already established.None of the MAbs blocked polyclonal RaF4 conjugates.

Complete blocking of polyclonal RaF4 conjugates could beachieved by combining at least three MAbs of differentepitope specificity.

Occurrence of epitopes on F4+ strains. In double-antibodysandwich ELISAs and SATs, all strains of each F4 subtypegrown at 37°C reacted similarly with individual MAbs, asshown in Table 3, except for F4ac strains with MAb F4ab-3.This MAb agglutinated all F4ab+ and F4ad+ strains but only26 of the 73 tested F4ac+ strains. Most of the F4ac+ strainsbelonged to three OK types (Table 1); all 0138:K81 strainsand 31 of the 0149:K91 strains were not recognized byF4ab-3, whereas 6 0149:K91 strains and all 15 08:K87strains showed positive tests with this MAb.

Locating epitopes on F4 proteins by using recombinant DNAstrains. The conjugates of all MAbs recognizing F4ab (Table3) reacted in the double-antibody sandwich ELISA withUS37 extracts of all strains listed in Table 2 except for theconjugates of F4ab-2, -3, -8, -9, -13, and -14, which did notreact with strain M5(pDB88-103). This demonstrates that theexpression of epitope clusters a4, bl, and b2 is dependentonly on amino acids located on the F4ab major fimbrialsubunit. The conjugates of the F4ac-specific MAbs, F4ac-5and -8, reacted only with antigen from strain M5(pDB88-103), which indicates that their epitope is restricted to theF4ac major fimbrial subunit or perhaps the H protein.

VOL. 58, 1990

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

1876 VAN ZIJDERVELD ET AL.

4

'I*0

*

9 4

a_

FIG. 7. IEM of strain 1087 with MAb F4ad-11. Magnification, x150,000. Bar, 100 nm.

Mapping of epitopes on the F4 major fimbrial subunit by thePEPSCAN method. MAbs F4ab-1, -2, and -3; F4ac-1, -2, -5,and -6; and F4ad-3, -4, -5, -6, and -10, representing epitopeclusters al, a2, a3, a4, a6, a7, bl, c, and d were tested in theindirect ELISA against the synthesized peptides. MAbsagainst epitope clusters aS and b2 were not available at thetime of testing. None of the tested F4 MAbs or polyclonalRaF4 antisera showed a significant reaction; the controlMAb against the VP1 protein of foot-and-mouth diseasevirus invariably showed positive reactions against its syn-thesized epitope.IEM. IEM was performed with one or two representatives

of each epitope specificity class. Their reaction patternagainst the three subtypes correlated completely with thosein the tests listed in Table 3. They were all directed againstfimbriae of approximately 2 to 3 nm in diameter, and the goldparticles were regularly distributed along the whole fimbrialstructure (Fig. 7) with small intervals, as was also seen byThiry et al. with our F4ad-9 MAb (28). Fimbriae often had astrong tendency to aggregate or to collapse against thebacterial cell wall in IEM.

DISCUSSION

We produced a panel of 40 MAbs directed against the F4antigen complex of E. coli. The specificity of the MAbs wasproven by ELISAs, SATs, RIP assays, and IEM. MAbseither reacted with all F4 subtypes (F4ab, F4ac, and F4ad) orwith two subtypes or were subtype specific. These findingsagree with those of another study of F4 MAbs (4). On thebasis of the results of competition ELISAs and the behaviorof MAbs in other tests, at least 11 epitope clusters, called al

to a7, bl, b2, c, and d, on the F4 antigen were defined. Thecommon a factor found with conventional serologic testscould be subdivided into at least seven epitope clustersshared by two or more subtypes. Although the number ofF4ad+ and F4ab+ strains used in this study was low, wepropose the following antigenic formulas for the three sub-types of F4: F4ab, ala2a3a4aSa6blb2; F4ac, ala2a3(a4)a5a6a7c; and F4ad, ala2a3a4a7d.The number of epitope clusters found on each F4 subtype

is relatively large compared with the five epitope clustersfound on F41 (30) and compared with the number of clustersfound on F5 and F6, which have fewer epitope clusters(F. G. van Zijderveld, unpublished observations). Becauseall strains of each F4 subtype had similar reaction patternswith our MAbs, the epitopes present on a particular subtypeseemed to be conserved, except for the epitope recognizedby MAb F4ab-3 (epitope cluster a4). In SATs or RIP assays,this MAb distinguished two types of F4ac strains (a4+ anda4-).The reaction patterns of MAbs in RIP assays, SATs, and

IEM agreed completely; the results of the indirect ELISAalso corresponded well with those of SATs and RIP assays,although low titers against a particular subtype were oftenfound for MAbs that were negative in SATs and RIP assays.Because none of the MAbs reacted with F41 antigen, theselow titers seemed to be specific, indicating that small parts of(for instance) subtype-specific epitopes not detectable byless sensitive techniques such as RIP assays and SATs arepresent on the other subtypes. When MAbs showed titers of.3.4 in the indirect ELISA, they also yielded positive SATsand RIP assays. In general, MAbs directed against two F4subtypes had the highest affinities to the F4 subtype used for

INFECT. IMMUN.

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

EPITOPE ANALYSIS OF ETEC F4 ANTIGEN COMPLEX BY MAbs

immunization, indicating that their epitopes on both sub-types were partially identical.The ELISAs with recombinant DNA strains demonstrated

that epitope clusters a4, bl, and b2 were located on the F4abmajor fimbrial subunit(s) and that epitope cluster c was

located on the F4ac major fimbrial subunit(s) or perhaps theH protein. Gaastra and Amstrup-Pedersen (5) also showedthat amino acids 80 to 264 of the major fimbrial subunits are

important for the expression of subtype-specific epitopes.On the basis of these findings combined with the identicaldistribution patterns of MAbs of all epitope clusters alongthe fimbriae in IEM, we concluded that all MAbs were

directed against epitopes on the major fimbrial subunits. Incontrast to the findings of others (4), none of our MAbsreacted with denatured antigen in immunoblotting proce-

dures (results not shown); this was also shown by Thiry et al.(28) with our MAb F4ac-3. These findings indicate that theMAbs probably are not directed against linear (continuous)epitopes. Attempts to map epitopes on the F4 major subunitsby using the PEPSCAN method failed. This method isespecially suited for locating amino acids contributing tolinear epitopes. Thus, it is probable that all epitopes recog-

nized by our MAbs are conformational epitopes dependenton the configuration of the subunit or the quaternary struc-ture of the whole fimbrial filament. The lack of MAbs againstlinear epitopes in this study probably is not due to theselection procedures of hybridoma supernatants because, ifpresent, MAbs against linear epitopes accessible on nativefimbriae should have been noticed in the ELISA. Anotherstudy (18), in which immunization with synthetic peptidesfrom the sequence of F4 major fimbrial subunits failed toinduce antibodies reacting with native fimbriae, also indi-cated that F4 epitopes are conformational epitopes. Furtherstudies with recombinant DNA strains (D. Bakker et al.,Microb. Pathog., in press), in which a part of the nucleotidesequence of the major fimbrial subunit of F4ab was replacedby heterologous sequences encoding viral epitopes, revealedparts of the F4 subunit essential to the expression of some

epitopes recognized by our MAbs.The results of the RIP assay, as shown in Fig. 2, suggest

that F4 fimbriae are composed of a repeating single 27,500-dalton subunit; however, minor components that shouldcoprecipitate may not have been detected because theirconcentrations were too low. Sometimes a second band of a

protein with a molecular mass of 1,000 to 1,500 daltonsgreater than that of the major fimbrial subunit was observed.The H protein is the most likely candidate for this protein.

Previous work on the F41 antigen of ETEC, which isclosely related to F4 (1, 23), showed that MAbs against onlyone of the five epitope clusters of F41 inhibited the in vitroadhesion of F41 (F. G. van Zijderveld, submitted for publi-cation). Further studies with the F4 MAbs are in progress toevaluate their antiadhesive capacities in in vitro tests andtheir protective capacities in challenge experiments whengiven orally to neonatal piglets.For the replacement of polyclonal antisera by MAbs in

diagnostic tests such as SATs and ELISAs to directly detectthe F4 antigen in clinical specimens, almost all MAbs or

combinations of MAbs are suitable, because the epitopesseem to be highly conserved, despite the existing antigenicvariation found by conventional serologic tests. In particu-lar, MAbs against epitope clusters al, a2, and a3, with hightiters in the different tests, seem to be the MAbs of choice forthis purpose. Subtype-specific MAbs can be used for furthertyping of F4 strains in SATs. We successfully use MAbF4ad-11 as a coating antibody and a combination of MAb

F4ac-5 and F4ab-1 as a conjugate in ELISAs to directlydetect F4+ ETEC in feces. The results of these ELISAscorrelate well with those of conventional bacteriologicalexamination followed by SATs.

ACKNOWLEDGMENTSWe thank Rob H. Meloen and Wouter C. Puyk for performing the

PEPSCAN and Jan M. A. Pol and Frans Wagenaar for advice onIEM.

LITERATURE CITED1. Anderson, D. G., and S. L. Moseley. 1988. Escherichia coli F41

adhesin: genetic organization, nucleotide sequence, and homol-ogy with the K88 determinant. J. Bacteriol. 170:4890-4896.

2. de Graaf, F. K., and I. Roorda. 1982. Production, purification,and characterization of the fimbrial adhesive antigen F41 iso-lated from calf enteropathogenic Escherichia coli strain B41M.Infect. Immun. 36:751-758.

3. Dykes, C. W., I. J. Halliday, M. J. Read, A. N. Hobden, and S.Harford. 1985. Nucleotide sequences of four variants of the K88gene of porcine enterotoxigenic Escherichia coli. Infect. Im-mun. 50:279-283.

4. Foged, N. T., P. Klemm, F. Elling, S. E. Jorsal, and J. Zeuthen.1986. Monoclonal antibodies to K88ab, K88ac and K88adfimbriae from enterotoxigenic Escherichia coli. Microb. Pathog.1:57-69.

5. Gaastra, W., and P. Amstrup-Pedersen. 1986. Serological vari-ants of the K88 antigen, p. 95-102. In D. L. Lark (ed.),Protein-carbohydrate interactions in biological systems. Aca-demic Press, Inc. (London), Ltd., London.

6. Gaastra, W., and F. K. de Graaf. 1982. Host-specific fimbrialadhesins of noninvasive enterotoxigenic Escherichia colistrains. Microbiol. Rev. 46:129-161.

7. Gaastra, W., P. Klemm, and F. K. de Graaf. 1983. The nucleo-tide sequence of the K88ad protein subunit of porcine entero-toxigenic Escherichia coli. FEMS Microbiol. Lett. 18:177-183.

8. Gaastra, W., F. R. Mooi, A. R. Stuitje, and F. K. de Graaf. 1981.The nucleotide sequence of the gene encoding the K88abprotein subunit of porcine enterotoxigenic Escherichia coli.FEMS Microbiol. Lett. 12:41-46.

9. Geysen, H. M., R. H. Meloen, and S. J. Barteling. 1984. Use ofpeptide synthesis to probe viral antigens for epitopes to aresolution of a single amino acid. Proc. Natl. Acad. Sci. USA81:3998-4002.

10. Guinee, P. A. M., and W. H. Jansen. 1979. Behavior ofEscherichia coli K antigens K88ab, K88ac, and K88ad inimmunoelectrophoresis, double diffusion, and hemagglutina-tion. Infect. Immun. 23:700-705.

11. Guinee, P. A. M., J. Veldkamp, and W. H. Jansen. 1977.Improved Minca medium for the detection of K99 antigen in calfenterotoxigenic strains of Escherichia coli. Infect. Immun.15:676-678.

12. Jacobs, A. A. C., and F. K. de Graaf. 1985. Production of K88,K99, and F41 fibrillae in relationship to growth phase, and arapid procedure for adhesin purification. FEMS Microbiol.Lett. 26:15-19.

13. Jacobs, A. A. C., B. Roosendaal, J. F. L. van Breemen, and F. K.de Graaf. 1987. Role of phenylalanine 150 in the receptor-binding domain of the K88 fibrillar subunit. J. Bacteriol. 169:4907-4911.

14. Jacobs, A. A. C., J. Venema, R. Leeven, H. van Pelt-Heerschap,and F. K. de Graaf. 1987. Inhibition of adhesive activity of K88fibrillae by peptides derived from the K88 adhesin. J. Bacteriol.169:735-741.

15. Josephsen, J., F. Hansen, F. K. de Graaf, and W. Gaastra. 1984.The nucleotide sequence of the protein subunit of the K88acfimbriae of porcine enterotoxigenic Escherichia coli. FEMSMicrobiol. Lett. 25:301-306.

16. Klemm, P. 1981. The complete amino acid sequence of the K88antigen, a fimbrial protein from Escherichia coli. Eur. J. Bio-chem. 117:617-627.

17. Klemm, P., and L. Mikkelsen. 1982. Prediction of antigenic

VOL. 58, 1990 1877

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

1878 VAN ZIJDERVELD ET AL.

determinants and secondary structures of the K88 and CFA1fimbrial proteins from enteropathogenic Escherichia coli. In-fect. Immun. 38:41-45.

18. Krogfelt, K. A., M. Meldal, and P. Klemm. 1987. K88 fimbrialantigens: identification of antigenic determinants by the use ofsynthetic peptides. Microb. Pathog. 2:465-472.

19. Meloen, R. H., W. C. Puyk, D. J. A. Meijer, H. Lankhof,W. P. A. Posthumus, and W. M. M. Schaper. 1987. Antigenicityand immunogenicity of synthetic peptides of foot-and-mouthdisease virus. J. Gen. Virol. 68:305-314.

20. Mooi, F. R., F. K. de Graaf, and J. D. A. van Embden. 1979.Cloning, mapping and expression of the genetic determinantthat encodes for the K88ab antigen. Nucleic Acids Res. 6:849-865.

21. Mooi, F. R., N. Harms, D. Bakker, and F. K. de Graaf. 1981.Organization and expression of genes involved in the productionof the K88ab antigen. Infect. Immun. 32:1155-1163.

22. Mooi, F. R., A. Wijfjes, and F. K. de Graaf. 1983. Identificationand characterization of precursors in the biosynthesis of theK88ab fimbria of Escherichia coli. J. Bacteriol. 154:41-49.

23. Moseley, S. L., G. Dougan, R. A. Schneider, and H. W. Moon.1986. Cloning of chromosomal DNA encoding the F41 adhesinof enterotoxigenic Escherichia coli and genetic homology be-tween adhesins F41 and K88. J. Bacteriol. 167:799-804.

INFECT. IMMUN.

24. 0rskov, I., and F. 0rskov. 1983. Serology of Escherichia colifimbriae. Prog. Allergy 33:80-105.

25. 0rskov, I., F. 0rskov, W. J. Sojka, and W. Wittig. 1964. Kantigens K88ab (L) and K88ac (L) in E. coli. Acta Pathol.Microbiol. Scand. 62:439-447.

26. Oudega, B., M. de Graaf, L. de Boer, D. Bakker, C. E. M.Vader, F. R. Mooi, and F. K. de Graaf. 1989. Detection andidentification of FaeC as a minor component of K88 fibrillae ofEscherichia coli. Mol. Microbiol. 3:645-652.

27. Shipley, P. L., G. Dougan, and S. Falkow. 1981. Identificationand cloning of the genetic determinant that encodes for theK88ac adherence antigen. J. Bacteriol. 145:920-925.

28. Thiry, G., A. Clippe, T. Scarcez, and J. Petre. 1989. Cloning ofDNA sequences encoding foreign peptides and their expressionin the K88 pili. Appl. Environ. Microbiol. 55:984-993.

29. Thorns, C. J., C. D. H. Boarer, and J. A. Morris. 1987.Production and evaluation of monoclonal antibodies directedagainst the K88 fimbrial adhesin produced by Escherichia colienterotoxigenic for piglets. Res. Vet. Sci. 43:233-238.

30. van Zijderveld, F. G., F. Westenbrink, J. Anakotta, R. A. M.Brouwers, and A. M. van Zijderveld. 1989. Characterization ofthe F41 fimbrial antigen of enterotoxigenic Escherichia coli byusing monoclonal antibodies. Infect. Immun. 57:1192-1199.

on May 10, 2018 by guest

http://iai.asm.org/

Dow

nloaded from