Native-like cyclic peptide models of a viralantigenic site: finding a balance between rigidityand flexibilityMari-Luz Valero,1 Julio A. Camarero,1† Thomas Haack,1‡ Mauricio G. Mateu,2Esteban Domingo,2 Ernest Giralt1 and David Andreu1*1Departament de Quımica Organica, Universitat de Barcelona, Barcelona, Spain2Centro de Biologıa Molecular ‘Severo Ochoa’ (CSIC-UAM), Madrid, Spain

Antigenic site A of foot-and-mouth disease virus (serotype C) has been reproduced by means of cyclicversions of peptide A15, YTASARGDLAHLTTT, corresponding to residues 136–150 of envelope proteinVP1. A structural basis for the design of the cyclic peptides is provided by crystallographic data fromcomplexes between the Fab fragments of anti-site A monoclonal antibodies and A15, in which the boundpeptide is folded into a quasi-cyclic pattern. Head-to-tail cyclizations of A15 do not provide peptides ofsuperior antigenicity. Internal disulfide cyclization, however, leads to analogs which are recognized as one totwo orders of magnitude better than linear A15 in both ELISA and biosensor experiments. CD and NMRstudies show that the best antigen, CTASARGDLAHLTT-Ahx-C (disulfide), is very insensitive toenvironment-induced conformational change, suggesting that cyclization helps to stabilize a bioactive-likestructure. Copyright! 2000 John Wiley & Sons, Ltd.

Keywords: antigenic site A; conformational stability; cyclic disulfide peptide; epitope mimics; foot-and-mouth diseasevirus

Received 3 August 1999; accepted 27 August 1999

INTRODUCTIONFoot-and-mouth disease virus (FMDV) causes the econo-mically most important disease of farm animals (Bachrach,1968; Pereira, 1981). Regular vaccination and slaughter ofinfected and contact animals are used to control the disease,which nevertheless remains enzootic in many areas ofAfrica, Asia, Eastern Europe and South America. Severaldisadvantages to the use of conventional FMDV vaccinesand important hurdles for new vaccine design have beenreported (Brown, 1992; Pereira, 1997). This has stimulatedefforts to develop FMDV vaccines requiring no handling oradministration of virus particles. One approach in this

direction has relied on the use of synthetic peptidesreproducing relevant antigenic sites of FMDV (Bittle etal., 1982; Pfaff et al., 1982; Sutcliffe et al., 1983; DiMarchiet al., 1986). This strategy is particularly attractive in thecase of FMDV, given the relevance of the humoral responsein the control of the disease (McCullough et al., 1992).A major antigenic determinant of FMDV (termed

antigenic site A), involved in neutralization of infectivity,is located around positions 140–160 of the capsid proteinVP1 (Bachrach et al., 1975; Strohmaier et al., 1982). Theantigenic structure of this region is rather complex (Logan etal., 1993; Berinstein et al., 1995). For instance, for serotypeC (isolate S8c1), many overlapping epitopes, all involved inviral neutralization, are distinguishable within the VP1(138–150) sequence by means of monoclonal antibodies(mAbs) (Mateu et al., 1987, 1990). The observation ofmultiple epitopes within such a short region suggests that itmay easily adopt a number of conformations recognized byantibodies. Although these epitopes can in general berepresented by synthetic peptides (Mateu et al., 1989;Carreno et al., 1992), the fact that some anti-FMDV mAbsrecognize site A somewhat better in the virus than whenreproduced by linear peptides (Mateu et al., 1995a) makes itplausible to assume that adequate conformational modula-tion of site A peptides may result in improved antigenicity.A similar approach has been used successfully for otherviral (Schulze-Gahmen et al., 1986; Muller et al., 1990;Joisson et al., 1993; Van der Werf et al., 1994) and non-viral(Williams et al., 1991) antigens.X-ray crystallographic analysis of the FMDV capsid of

JOURNAL OF MOLECULAR RECOGNITIONJ. Mol. Recognit. 2000;13:5–13

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

* Correspondence to: D. Andreu, Departament de Quımica Organica,Universitat de Barcelona, Martı i Franques 1, E-08028 Barcelona, Spain.E-mail: andreu@admin.qo.ub.es† Current address: Rockefeller University, New York, USA.‡ Current address: Menarini Ricerche S.p.A., Pomezia, Italy.

Abbreviations used: Ahx, 6-aminohexanoic acid; Amh, (S)-2-amino-6-mercaptohexanoic acid; Boc, tert-butyloxycarbonyl; CS, conformational shift;DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; Dnp, 2,4-dinitrophenyl; EDAC, N-ethyl-N!-dimethylaminopropylcarbodiimide; FMDV,foot-and-mouth disease virus; Fmoc, 9-fluorenylmethyloxycarbonyl; HEPES,4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; HFIP, hexafluoroiso-propanol; HOSu, N-hydroxysuccinimide; mAb, monoclonal antibody;MALDI-TOF MS, matrix-assisted laser desorption time-of-flight massspectrometry; MBHA, p-methylbenzhydrylamine; NOE, nuclear Overhausereffect; PEG-PS, polyethyleneglycol-polystyrene; Pmc, 2,2,5,7,8-penta-methylchroman-6-sulfonyl; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate; tBu, tert-butyl; TFE, 2,2,2-trifluoroethanol;Tris, tris(hydroxymethyl)aminomethane; Trt, trityl; VP1, viral protein 1.

several serotypes revealed that the region containing site Ais a protruding, disordered loop (termed the G–H loop) onthe viral surface, for which only limited structural informa-tion is available (Acharya et al., 1989; Lea et al., 1994,1995). An important feature of this G–H loop, common forall FMDV serotypes, is the Arg–Gly–Asp (RGD) motif,involved in both host cell and antibody recognition (Fox etal., 1989). Reduction of a disulfide bond close to the base ofthe loop causes it to fold into a well-defined structure alongthe viral surface (Logan et al., 1993; Fox et al., 1989). Asimilar X-ray analysis of serotype C (Lea et al., 1994),which has a four-residue shorter G–H loop and lacks theneighboring disulfide bridge, found again no definedstructure for the loop. This limited definition of site A hasfor some time been a serious drawback in the design ofconformationally biased peptides that aim to mimic thenative structure. Thus, our earlier work in this directionrelied only on the distance (ca. 11 A) between the lastresidues at each end of the loop (Tyr-136 and Arg-153 inisolate C-S8c1), for which reliable structural data wereavailable at the time. Internal disulfide formation betweenthose positions (Fig. 1) produced cyclic analogs (Camareroet al., 1993; Andreu et al., 1996) with reactivity toward anti-FMDV mAbs at best equal to that of the correspondinglinear versions, showing that attempts to bias site A peptidestoward native-like conformations by cyclization were stillvery much open to improvement.A clearer understanding of the G–H loop structure and the

mechanisms by which antibodies neutralize FMDV wasprovided by the X-ray crystallographic analysis (Verdagueret al., 1995) of a complex between peptide A15 (YTA-SARGDLAHLTTT-amide), corresponding to VP1 (136–150) (isolate C-S8c1), and mAb SD6 (Mateu et al., 1987),which defines a continuous epitope within site A. Thecomplexed peptide adopts a folded conformation which

displays an N-terminal !-sheet and an open turn at the RGDmotif (Plate 1), followed by a short helical stretch, all fairlysimilar to the structure described for the ‘reduced’ form ofthe loop in serotype O1 (Logan et al., 1993). An interestingfeature of this structure was that binding to the antibodybrought into close proximity the N- and C-termini of thepeptide, inducing it into a quasi-cyclical arrangement (Plate1). More recently, another complex between peptide A15and mAb 4C4 (defining a different epitope than SD6) hasbeen resolved (Verdaguer et al., 1998). Superposition of thepeptide backbones from both complexes (Plate 1) showsremarkable similarities, showing that each structure in-corporates only minimal deviations from a consensus,biologically relevant conformation of the G-H loop. Thesestructural data not only reinforce our original assumptionthat appropriate cyclization would improve the antigenicityof site A peptides; they also provide more reliable guide-lines for the design of such peptides, a requirement for thedevelopment of effective peptide-based vaccines. In thisreport we describe several cyclic analogs of antigenic site A,some of them with considerably enhanced antigenicity overthe linear versions. Kinetic and structural studies with themost relevant peptides provide a basis for discussion of theeffects of cyclization on antigenicity.

EXPERIMENTALPeptides

Peptide A15 (YTASARGDLAHLTTT) was synthesized inthe C-terminal carboxamide form by Fmoc chemistryprotocols (Fields et al., 1992) on a PEG-PS resin (Baranyet al., 1992) functionalized with the 2,4-dimethoxy-4!-(carboxymethyloxy)benzhydryl-amine handle (Bernato-wicz et al., 1989). Fmoc-protected amino acids, includingside chain-protected Tyr(tBu), Thr(tBu), Ser(tBu),Arg(Pmc), Asp(OtBu) and His(Trt), were coupled by meansof TBTU/DIEA (5 and 10 equivalent, respectively, 60 min)in DMF, with double couplings for the last two residues.The protected peptide-resin (250 mg, 62 !mol) was treatedwith trifluoroacetic acid–phenol–water–triisopropylsilane(88:5:5:2 v/v) for 90 min at 25°C to give 50 !mol (80%)crude peptide after ether precipitation and centrifugation.The material was purified by reverse-phase HPLC (VydacC18, 2! 30 cm) using a 5–35% gradient of acetonitrile inwater (both with 0.05% trifluoroacetic acid) over 3 h to give20 !mol (40%) of HPLC-homogeneous (98%) product, withamino acid analysis and MALDI-TOF mass spectrumconsistent with theory.

Cyclic peptides. The synthesis of head-to-tail analogs hasbeen described (Valero et al., 1999). The cyclic disulfideswere obtained from linear precursors assembled by Bocchemistry (Barany and Merrifield, 1979) on p-MBHA resin,as previously described (Camarero et al., 1993). Thepeptides were obtained in free dithiol form upon thiolyticHis(Dnp) deprotection and HF acidolysis, purified andcharacterized by amino acid analysis and MALDI-TOFmass spectrometry. The dithiol peptides were next diluted to50 !M concentration in 20 mM Tris–HCl, pH 8, and air-oxidized. Oxidations were complete after 4 h (peptide 10) or

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

6 M.-L. VALERO ET AL.

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

Copyright © 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000; 13

Plat

e 1.

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8).

MODELS OF A VIRAL ANTIGENIC SITE

20 h (peptides 8, 9). The cyclic disulfides were pure ("95%by HPLC) after desalting and their identity was confirmedby electrospray mass spectrometry.

Competitive ELISA

Site A-specific mAbs SD6, 4C4, 7JD1, 7CA11, 6D11,7FC12 and 5A2 have been described (Mateu et al., 1987,1990). The reactivity of these mAbs with peptides wasassayed by competitive ELISA (Novella et al. 1993).Microtiter plates were coated with 5 pmol of peptide A21-keyhole limpet hemocyanin (KLH) conjugate in 100 !LPBS at 4°C overnight. This conjugate, prepared aspreviously described (Carreno et al., 1992), contained0.1 mg of peptide/mg KLH. After PBS washes, mixturesof a non-saturating amount of mAb and of increasingamounts of peptide (0.33, 1, 3, 9, 27, 81, 242 pmol) in100 !l PBS, preincubated at 25°C for 1.5 h, were added toeach well. After incubation for 1 h at 25°C and washingwith 0.1% BSA, 0.05% Tween 20 in PBS, the plate wasincubated 1 h at 25°C with PBS solution of goat anti-mouseimmunoglobulin G labeled with peroxidase. After theincubation, the plate was throughly washed as above, andthe enzymatic reaction was carried out using o-phenylene-diamine and H2O2 as substrates. Absorbance was read at492 nm, corrected for background (no mAb, no peptide) andused to calculate IC50 values (concentration of peptidecausing 50% inhibition of binding of a nonsaturatingamount of mAb to plate antigen). These have beennormalized to the relative IC50 values of Table 2 bydividing by the IC50 of peptide A15.

Surface plasmon resonance

Consumables for the BIAcore 1000 instrument (carboxy-methyl-coated sensor chips, buffers and amine coupling kit)were from Biosensor AB (Uppsala, Sweden). In order tominimize the electrostatic adsorption profile of peptideligands onto the carboxylated dextran matrix, differentpeptide concentrations and pHs were explored initially at aconstant 5 !l/min flow rate on an underivatized chip at

25°C. Optimal conditions were found to be 300 !g/ml, pH 5(10 mM sodium acetate) for peptides A15 and 10, 150 !g/mL, pH 5 for peptide A22SS, and 150 !g/ml, pH 6 (5 mMdisodium maleate) for peptide A21. Standard manufacturerprocedures for immobilization were used (Lofas andJohnson, 1990). The carboxyl matrix was activated with35 !l of EDAC-HOSu (0.2 and 0.05 M, respectively) prior topeptide injection. Remaining carboxyl groups were blockedby a 30 !l pulse of 1 M ethanolamine hydrochloride, pH 8.5,followed by 20 !l of 100 mM HCl to remove non-covalentlybound peptide. A flow of HBS buffer [0.01 M HEPES,0.15 M NaCl; 3 mM EDTA, pH 7.4; 0.005% (v/v) SurfactantP20] of 5 !l/min was maintained throughout the immobi-lization procedure. No other flow-rates were examined. Theamounts of peptide immobilized on the sensor surface wereequivalent to 850, 3000, 830 and 300 RU for peptides A21,A22SS, A15 and 10, respectively. The mAbs were diluted to400–1900 nM in 10 mM PBS and 30 !l of each solution wereinjected over the peptide surface at 5 !l/min. Dissociationwas monitored for 6 min after stopping the injection. Thesurfaces were regenerated with 100 mM HCl washes.Sensorgrams corresponding to five injections of each mAbwere acquired for every immobilized peptide and used toderive kinetic and equilibrium constants by means of themanufacturer’s software, as described (Karlsson et al.,1991). No significant changes in baseline or peptide bindingcapacity were observed after 20 cycles of surface regenera-tion, as well as no evidence of mass transport-limitedinteractions (Schuck and Minton, 1996).

Structural studies

Circular dichroism. Spectra were recorded at 5°C in aJasco model J720 spectropolarimeter flushed with nitrogen(3 l/min). Peptides (25 !M in 1 mM sodium phosphate, pH7.3) were placed in a 1.0 mm-pathlength cell in the presenceof varying amounts of hexafluoroisopropanol (HFIP).Acquisition was carried out at 10 nm/min using a 0.2 nmspectral bandwidth and a 4 s time constant. Data from threeconsecutive scans were averaged and processed. Helicalcontents of peptides were estimated from the mean resi-due ellipticities [!] at 222 nm by the algorithm of Chen

)*+,& '( -./%0 121,"1 3"3"14 /5 *60"#&6"1 4"0& 7 /5 !89:

Head-to-tail mimics Cyclic disulfide mimics

Peptide U X Z Peptide X Z

1 — — — 8 Cys —2 Gly — — 9 Ahm —3 Cys — — 10 Cys Ahx4 !Ala — —5 Ahx — —6 Gly Cys —7 Gly Cys Gly

MODELS OF A VIRAL ANTIGENIC SITE 7

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

et al. (1974), taking 39500 (1–2.57/n) °/cm2/dmol as thereference value for a fully helical peptide with n peptidebonds.

1H-NMR. Experiments were performed with 2 mM peptidesolutions in 1.5 mM phosphate, 0.01 mM NaN3 in eitherH2O–D2O (85:15 v/v) or H2O–d2-trifluoroethanol–D2O(40:50:10 v/v). Spectra were acquired in a Varian VXR-500 instrument operating at a proton frequency of500.013 MHz, as previously described (Haack et al.,1997). Sequential assignment was done by the standardtwo-step procedure using through-bond TOCSY (COSY)and through-space NOESY spectroscopies (Bothner-By etal., 1984). Structural analysis included NOE connectivities,3J" N coupling constants, H" conformational shifts (CSs)and amide temperature coefficients between 5 and 25 °C.

RESULTSDesign and synthesis of peptides

Crystallographic data on peptide A15-mAb complexes(Verdaguer et al., 1995, 1998) strongly suggested thatcyclic versions of the peptide might be able to bias itsstructure toward the bioactive conformation and thus resultin improved antigenicity. With this goal in mind, and usingthe structural guidelines provided by the crystallographicdata (Verdaguer et al., 1995, 1998), cyclic versions ofantigenic peptide A15 were designed (Fig. 1). The closeproximity between C- and N-termini of A15 when foldedinto a complex with antibodies (Plate 1) suggested eitherhead-to-tail or internal disulfide as potential cyclizationpatterns. The head-to-tail series included the strict cyclicversion of A15, cyclo(YTASARGDLAHLTTT) (1), andanalogs (2–7) with extra spacer residues such as Gly, !-Alaor Ahx, which would presumably allow a broader explora-tion of the conformational space available for mAb binding,as well as provide (in the Cys-containing versions) ananchoring residue for possible conjugation to carrierproteins.The internal disulfide analogs (8–10), meant to induce a

less stringent conformational restriction than the head-to-tail set, were designed by replacing the termini of peptideA15 (Tyr-136, Thr-150) by Cys (in 8). The group alsoincluded the substitution of 2-amino-6-mercaptohexanoicacid (Amh) (Adeva et al., 1995) for Cys (9) or the insertionof an extra Ahx residue (10). A conformational evaluationof the sequences employing the Antheprot program(Geourjon et al., 1991) suggested that the additionalresidues extended the !-sheet structure at the N-terminusof A15, without significant alteration of the overall topologyof the loop. Synthetic approaches to the head-to-tail set ofsite A mimics involved different cyclization strategieswhich have been described in detail elsewhere (Valero etal., 1999). The disulfide analogs were prepared from theircorresponding linear precursors. The side chain of Amh inpeptide 9 was protected as the p-methoxybenzyl derivative,which is only marginally stable against acid deprotection(Erickson and Merrifield, 1973), but acceptable for residuesat either the N-terminus or close to it (Heeb et al., 1994;Adeva et al., 1995). Deprotection and cleavage of thepeptide-resins followed by reverse-phase purification andair-oxidation (Andreu et al., 1994) of the dithiols led to thetarget disulfides, which were satisfactorily characterized byamino acid analysis and mass spectrometry.

Antigenicity of peptides

ELISA. The two sets (head-to-tail and disulfide) of cyclicpeptides were tested by competitive ELISA against a panelof seven mAbs representative of antigenic site A (Mateu etal., 1987, 1990) (Table 2). These mAbs can be classifiedinto two groups (Mateu et al., 1990) (Fig. 2): (i) those thatdefine epitopes at the N-terminal region of antigenic site A,including the RGD triplet (SD6, 5A2, 4C4); and (ii) thosedirected at the C-terminal region of site A (7FC12, 7JD1,7CA11). Sharp differences in antigenic behavior wereclearly observed among both groups of peptides. The head-to-tail set (peptides 1–7) competed only very feebly with thelinear (A15, A21) sequences for recognition by all mAbstested. Loss of reactivity was especially important forpeptides 1, 2, 4, 6 and 7, which were virtually inert under theexperimental conditions used. Peptides 3 and 5 showedsome (weak) interaction with mAbs (SD6, 5A2, 4C4 and6D11), mapping at the N-terminal region of antigenic site A,including the RGD triplet (Fig. 2). For mAbs 7JD1 and7CA11, the relative IC50s were 5–10-fold higher.The disulfide analogues (8–10), in contrast, were very

well recognized by each mAb (Table 2), giving consistentlylower relative IC50 values than their linear counterparts,with the sole exception of peptide 9 with mAb 7JD1.Furthermore, most mAbs reacted more efficiently with thecyclic disulfides than with linear peptide A21, which isconsidered to reproduce very faithfully the antigenicproperties of site A (Mateu et al., 1995b). Within thedisulfide group of peptides, the Ahx-containing analog 10behaved as the best antigen, with relative IC50 values 20-fold lower on average than the reference linear peptide A15and 5–10-fold lower than the other two disulfides. Analog 9,with the mixed Cys–Amh disulfide, was the poorestperformer within the group, but still slightly better (two-fold on average) than linear A15 peptide.

!"#$%& ;( !$.#/*$#' ).;,'.,;* %- )#.* ! %- 01234 T9* 3@AHANGUAOQI&"#$% &'#( )*+,*$'* %- 0123B #)%:&.* F>EG'AB #) )9%<$4 ! 7%V(*:#"#.) .9* )9%;.*). 6*6.#(* -%,$( .% 7* ;*&'.#W* <#.9 .9*'%;;*)6%$(#$/ "!74 ?&.'9*( ;*/#%$) #$(#'&.* .9* "#$#"&::%'&.#%$ %- *&'9 *6#.%6*4 0#::*( )+,&;*) #$(#'&.* 6%)#.#%$) <9*;*%$* %; "%;* &"#$% &'#( ),7).#.,.#%$) &--*'. .9* 7#$(#$/ %- .9*"!7 #$ #"",$%&))&8)4 !(&6.*( -;%" 1&.*, !" #$% HAMMXI4

8 M.-L. VALERO ET AL.

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

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

)*+,& <( ="6&0"1 >*0* 5/% 0.& >"44/1"*0"/6 ?.*4& /5 0.& "60&%*10"/6 +&0@&&6 ?&?0">& 7;' *6> 37+ -9A )*+,& <( *

[SD6] (nM) R1 (RU) kd1 (S"1) R2 (RU) kd2 (S"1)

460 "2.13! 10"3 "5.22! 10"4 1.77! 103 "2.66! 10"4700 6.72 0.0246 2.16! 103 9.36! 10"5930 13.3 0.0213 2.32! 103 1.12! 10"41400 17.4 0.0201 2.32! 103 1.24! 10"41860 129 3.35! 10"3 7.43! 103 "2.02! 10"5

a R and kD describe, respectively, the extent of complex formation (RU = response units) and the dissociation constant in a parallel model(AiBj = Ai# Bj) of dissociation. Subindexes 1 and 2 refer to the two types of dissociating complexes considered within the model.

)*+,& ;( B&,*0"C& DEFG C*,$&4 /5 121,"1 ?&?0">& 3/>&,4 /5 *60"#&6"1 4"0& 7 *#*"640 * 4&0 /5 %&?%&4&60*0"C& 37+4

Peptide antigen

Linear Head-to-tail Disulfide

mAb A15 A21 1 2 3 4 5 6 7 8 9 10

SD6 1 0.17 "25 "25 3.5 "25 2.5 "25 2.5 0.33 0.72 0.055A2 1 n.d. "9 "9 4.4 "9 4 "25 "25 0.29 0.42 0.054C4 1 0.24 "37 "37 7.5 37 4 "25 "25 0.09 0.13 0.026D11 1 0.13 "240 "240 7.1 110 9 "25 9 0.05 0.03 0.027FC12 1 0.36 "25 "24 3.5 24 "25 "25 "25 0.017 0.18 0.027JD1 1 1 "240 "240 21 100 "25 "25 "25 0.42 1.5 0.057CA11 1 0.05 "240 "240 20 240 20 "25 "25 0.27 0.14 0.19

n.d.: not determined.

MODELS OF A VIRAL ANTIGENIC SITE 9

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

Surface plasmon resonance. Confirmation of the enhancedantigenicity of peptide 10 was further sought by thisbiosensor technique. This cyclic disulfide peptide and threeadditional peptides [A15, A21 and A22SS, a disulfide oflarger size (Camarero et al., 1993) see Fig. 1] were assayedagainst two site A-specific mAbs: SD6, strongly neutraliz-ing and mapping on the VP1(138–147) region; and 4G3,only moderately neutralizing and mapping essentially on anadjoining area [VP1(146–159)] (Mateu et al., 1990) withinsite A (see Fig. 2). Very different patterns were observed forthe two mAbs with all the peptides tested: with 4G3 theinteractions were too fast (Fig. 3) to allow reliable kineticdeterminations; the dR/dt derivatives for both phases of thesensorgrams were too high. In contrast, peptide–SD6complexes had association and dissociation rates whichreadily allowed derivation of kinetic parameters. Data forA21-SD6 (Fig. 3, Table 2) are representative of the behaviorfound for all four peptides. Analysis of the dissociationphases of different sensorgrams above 500 nM fitted bestwith an AiBj = Ai# Bj model, corresponding to the paralleldissociation of two complexes. One of these seemed todissociate rather fast, as reflected by an average kd value of0.022/s, but it was poorly represented (0.3, 0.5 and 0.7% at700, 930 and 1400 nM, respectively). This complex mayhave been stabilized by non-specific interactions, assuggested by the increase in complexation with mAbconcentration, as well as by the high value of kd. Below500 nM, however, fitting to the parallel (AiBj = Ai# Bj)model produced negative kd values. At this low concentra-tion, a simple AB = A# Bmodel was the best description ofthe interaction.Table 3 summarizes data for all four peptides. Several

points are worth emphasizing. First, there are no substantialdifferences in kinetic or thermodynamic behavior betweenthe larger linear (A21) and cyclic disulfide (A22SS)peptides, in agreement with earlier ELISA results (Camar-ero et al., 1993). The biosensor data indicate that bothassociation and dissociation steps are slightly faster for thecyclic version, thus resulting in a similar KA for bothcomplexes. On the other hand, a smaller size of the antigen(A15 vs A21) induces a five-fold drop in KA, mostly due to asubstantial decrease in the association rate. This shows thatC-terminal extension of A15 by six residues has animportant effect on the interaction, in contrast with previousstudies, suggesting no direct involvement of the C-terminusin mAb binding. More interestingly, cyclization of this shortantigen via disulfide to give peptide 10 causes a remarkableincrease in affinity [KA(10)/KA(A15) = 6]. This is mostlydue to an improved association step, the fastest among allsite A antigens examined here.

Structural analysis of site A mimics

Circular dichroism. A preliminary assessment of theconformational traits underlying the significant variationsin antigenicity observed for the different peptides in thisstudy was obtained by analyzing the tendency of threerepresentative structures [A15 (linear), 1 (head-to-tail cycle)and 10 (cyclic disulfide)] to adopt a certain degree ofstructural organization. In aqueous solution, all threepeptides gave spectra characteristic of aperiodic conforma-tion, with no evidence of helical structure. Upon addition ofHFIP, ellipticity curves for all three peptides displayedminima at 209 and 222 nm and the typical "-helix shapes.Analysis of the estimated "-helix content at differentconcentrations (Fig. 4) showed a fairly standard behaviorfor the linear A15 peptide, i.e. a substantial increase inhelicity with added solvent. In contrast, both cyclic peptidesnot only had lower helix levels at any HFIP concentrationbut, more importantly, showed a remarkable lack ofconformational response to solvent variation.

NMR. The above CD data suggested that the improvedantigenicity of peptide 10 might be related to theinsensitivity to environment-induced conformationalchange. To further investigate this point, the conformationof 10 was analyzed by two-dimensional NMR in both waterand 50% water-trifluoroethanol (TFE). A mostly continuousseries of sequential " N(i,i# 1) and ! N(i,i# 1) NOEs wasobserved in 50% TFE (Fig. 5). NN(i,i# 1) NOEs were alsoclearly observed in 50% TFE but were less evident in water.Some medium-range NOEs suggestive of helical conforma-tion, such as a continuous series of " N(i,i# 1) from Gly-

)*+,& H( ="6&0"1 *6> *5I6"02 >*0* 5/% 0.& "60&%*10"/6+&0@&&6 37+ -9A *6> 4260.&0"1 *60"#&6 3/>&,4 /54"0& 7

Peptide kas (M/s) kdis (S"1) KA (M"1)

Linear A21 1.07! 104 1.1! 10"4 9.7! 107A22 SS 1.4! 104 1.3! 10"4 1.0! 108Linear A15 3.7! 103 1.95! 10"4 1.9! 107Peptide 10 2.62! 104 2.2! 10"4 1.2! 108

!"#$%& H( E%:W*$.>(*6*$(*$. F2 7*9&W#%; %- )8$.9*.#' 6*6.#(*"%(*:) %- )#.* !4 ">?*:#'&: '%$.*$.) %- :#$*&; 6*6.#(* !AO H<9#.*7&;)IB 9*&(>.%>.&#: '8':#' 6*6.#(* ' H/;&8 7&;)I &$( '8':#' (#),:K(*'G H7:&'_ 7&;)I &. YO !1 '%$'*$.;&.#%$ #$ A "1 69%)69&.*B 6? [4NB#$ .9* 6;*)*$'* %- .9* #$(#'&.*( 6*;'*$.&/*) %- ?0Z@4 @*;'*$.&/*)%- .9* 9*:#V <*;* '&:',:&.*( -;%" .9* "*&$ ;*)#(,* *::#6.#'#.8W&:,*) &. YYY $" &''%;(#$/ .% F9*$ !" #$% HAM[RI4

10 M.-L. VALERO ET AL.

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

142 to Thr-149, were detected in 50% TFE at lowtemperature. The corresponding NN(i,i# 2) connectivities,typical of helical conformation, were difficult to detect dueto a severe peak overlap.While NOE data did not provide sufficient evidence in

support of a predominant conformation, analysis of the H"conformational shifts was quite illustrative. The CS profiles(Fig. 6) had similar shapes and absolute values in bothsolvents, confirming the insensitivity to solvent-inducedconformational change observed in the CD experiments.While the Tyr-136–Asp-143 sequence did not show a clearconformational preference, a cluster of negative CSs fromAsp-143 to Leu-147 suggested a short helical patch in thisregion, in agreement with the NOEs observed for theseresidues in 50% TFE (Fig. 5).It is illustrative to compare the present results with those

from a previous NMR study (Haack et al., 1997) of A22SS,

a cyclic disulfide version of A21. While the CSs of bothpeptides correlate well with an open turn conformation ofthe RGD tripeptide (Haack et al., 1997) and are also verysimilar at the Asp-143–Leu-147 helical segment, the lengthof this region in 10 is insensitive to solvent changes,whereas in A22SS it extends to Thr-150 upon addition ofHFIP. Thus, peptide 10 seems to have achieved a level ofstructural organization that provides a conformationallysteady molecule under a variety of environments. Sincepeptide 10 is better recognized by site A mAbs than A21, itis reasonable to conclude that, although 10 may lack thestabilization provided by the three extra helical residues(148–150) of A22SS or A21, it more than makes up for thisby adopting a stable, much less plastic structure than thelarger peptides, and from which it can access the productiveconformations with very low energy cost. In summary, inthis particular case improved antigenicity would seem to beclosely related to a finely tuned balance between rigidity andflexibility.

DISCUSSIONPrevious results from our laboratories using larger cyclicdisulfide models of site A (Camarero et al., 1993) showedlevels of mAb recognition similar to the correspondinglinear versions. We concluded at the time that the closure ofsuch a large cycle did not provide sufficient conformationalrestriction to bias the structure toward a native-liketopology. Rather, the cyclic peptide remained flexibleenough to include, within a relatively broad conformationalrepertoire, a significant population of bioactive arrange-ments (Haack et al., 1997).If the ring size is substantially reduced, as is the case with

the peptides of the present study, more pronounced effectsof cyclization on antigenicity appear, for either better orworse. The negative effects are clearly illustrated by thehead-to-tail set of cycles (1–7), which show essentiallynegligible recognition by all seven antibodies tested. Theimposition of a fairly rigid amide bond between the two

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69%)69&.*B X4XA"1 `&`NB #$ ?YCU&Y>.;#],%;%*.9&$%:U2YC &. O°F4 5*:&.#W* `Ca #$.*$)#.#*) &;*;*6;*)*$.*( 78 .9* .9#'_$*)) %- .9* 7&;)4 CW*;:&66#$/ %; &"7#/,%,) `Ca) &;* #$(#'&.*( 78(#)'%$.#$,%,) :#$*)4

!"#$%& A( F%$-%;"&.#%$&: )9#-. 6;%K:*) %- '8':#' (#),:K(* 'G4F9*"#'&: )9#-. (#--*;*$'*) ;*:&.#W* .% ;&$(%" '%#: W&:,*) %- .9* F"6;%.%$) %- 6*6.#(* 'G4 "# W&:,*) #$ ?YCU2YC HGObAOB W^WI &$( ?YCU&Y>.;#],%;%*.9&$%:U2YC HRXbOXbAXB W^WI &;* #$(#'&.*( 78 )+,&;*)&$( '#;':*)B ;*)6*'.#W*:84 T9* ,$,),&: )#"#:&;#.8 #$ .9* "# 6;%K:*)&:%$/ "%). %- .9* )*+,*$'* &$( 6&;.#',:&;:8 .9* ">9*:#'&: !;/>ARNUL*,>AR[ )*/"*$. ),//*).) .9&. .9* 6*6.#(* #) ;*"&;_&7:8#$)*$)#.#W* .% *$W#;%$"*$.&: W&;#&.#%$)4

MODELS OF A VIRAL ANTIGENIC SITE 11

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

ends of the A15 peptide obviously induces a spatialarrangement which completely impedes access to thebioactive conformation, a situation which is not modifiedby insertion of different spacer residues. The structuralmodifications caused by this cyclization may have affectedthe global structure of the peptide; however, the sharpdifferences observed in comparison with either the linear orcyclic disulfide analogs (see below) make it more likely thatcyclization in this case alters the relative position of keyresidues in the binding event.The situation is strikingly reversed in the disulfide cycles

(8–10), for which the goal of keeping both ends of peptideA15 relatively close without hindering access to productiveconformations is clearly fulfilled. In comparison with A15,all three peptides show higher antigenicity scores towardsmore than half of the mAbs tested, with peptide 10providing clearly the best results. The significant enhance-ment in the antigenic response of this cyclic peptide overlinear A15 is noteworthy. Cyclizations have been described

to increase the antigenicity of peptides that in the linearform perform poorly relative to the native antigen (Joissonet al., 1993; Van der Werf et al., 1994; Richalet-Secordel etal., 1994a,b). Peptide 10 is remarkable in that it improves byone to two orders of magnitude the antigenicity of a linearpeptides such as A21, which is already a very good mimic ofsite A (Mateu et al., 1995b). The kinetic data for theinteraction of 10 with mAb SD6, a fairly representative anti-site A antibody (Mateu et al., 1995a; Verdaguer et al.,1998), indicate that this enhanced recognition is due to asubstantial improvement in the association rate of theantigen with the antibody. These data must be consideredalongside the structural information provided by CD andNMR, which show a remarkable imperviousness of peptide10 to environmental variations. This in turn suggests thatcyclization allows the peptide to fold into a rather rigidconformation, which provides the degree of structuralpreorganization needed to attain the conformations leadingto effective binding with minimal energy expenditure.

REFERENCES

!'9&;8& 5B 0;8 aB E.,&;. 2B 0%V =B 5%<:&$() 2cB D;%<$ 04 AMGM4T9* .9;**>(#"*$)#%$&: ).;,'.,;* %- -%%.>&$(>"%,.9 (#)*&)*W#;,) &. Y4M !d ;*)%:,.#%$4 '#"()! <<Jb [XMU[AQ4

!(*W& !B F&"&;*;% c!B =#;&:. aB !$(;*, 24 AMMO4 D%'>*>"*.98:7*$P8:>H*I>Y>&"#$%>Q>"*;'&6.%9*V&$%#' &'#(b 6;*>6&;&.#%$ &$( &66:#'&.#%$ .% .9* )8$.9*)#) %- & :&;/* '8':#'(#),:K(* 6*6.#(*4 +!")#,!&)-. /!""% <Ab NGGOUNGGG4

!$(;*, 2B !:7*;#'#% 0B E%:*e `!B 1,$)%$ 1FB 0*;;*; 1B D&;&$8 =4AMMR4 0%;"&.#%$ %- (#),:K(* 7%$() #$ )8$.9*.#' 6*6.#(*) &$(6;%.*#$)4 0!",% 0-$% 12-$% <Fb MAUAQM4

!$(;*, 2B F&"&;*;% c!B 3&:*;% 1LB 5%#/ JB ?&&'_ TB =#;&:. aB3*;(&/,*; `B 0#.& ZB 1&.*, 1=B 2%"#$/% a4 AMMQ4 E8$.9*.#'B).;,'.,;&: &$( #"",$%'9*"#'&: &66;%&'9*) .% &$.#/*$>&$.#>7%(8 #$.*;&'.#%$ #$ -%%.>&$(>"%,.9 (#)*&)* W#;,)4 Z$ 3!45"2&!67 8,!926"):; *")(<"()! #.& 12-$-=:% 3)-<!!&2.=6 -> ",!?-()"!!.", @9!)2<#. 3!4"2&! *:94-62(9B \&,"&8& @T@B?%(/*) 5EB H*()I4 1&8]%<*; E'#*$.#K'b \#$/)<#$-%;(S G[OUG[[4

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12 M.-L. VALERO ET AL.

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13

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MODELS OF A VIRAL ANTIGENIC SITE 13

Copyright ! 2000 John Wiley & Sons, Ltd. J. Mol. Recognit. 2000;13:5–13