University of Groningen

Molecular aspects of antibody-antigen interactionsSchellekens, Gerardus Antonius

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:1996

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Schellekens, G. A. (1996). Molecular aspects of antibody-antigen interactions: size reduction of a herpessimplex virus neutralizing antibody and its antigen. s.n.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 27-05-2022

49

Chapter 3

Antibody and Antigen Binding Peptides Usinga Phage Displayed Random Peptide Library

Part of this chapter was published as: Gerard A. Schellekens, Edwin Lasonder, Matty Feijlbrief,Danny G.A.M. Koedijk, Jan Wouter Drijfhout, Albert-Jan Scheffer, Sytske Welling-Wester andGjalt W. Welling (1994) Identification of the core residues of the epitope of a monoclonalantibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a randompeptide library. The European Journal of Immunology 24, 3188-3193.

Chapter 3

50

3.1 Antigen-binding peptides from a surface displayed random peptide libraryusing human myoglobin as a target

3.1.1 Summary

The possibility to select peptides from a phage displayed random peptide librarywith a non-antibody target was investigated. For this purpose human myoglobin, anearly marker in myocardial infarction, was chosen as a model protein. Humanmyoglobin was isolated and biotinylated and used for the enrichment of bindingpeptide displaying phage in three selection rounds. Five clones were selected andthe DNA inserts were sequenced. Four peptides, 15 amino acid residues in length,were synthesized on the basis of the phage deduced sequences. One of the 15 aminoacid residue sequences could not be obtained in full length with solid phase peptidesynthesis. None of the peptides synthesized showed a detectable affinity for humanmyoglobin in affinity chromatography.

3.1.2 Introduction

Peptides are ideal molecules to use as a ligand for the detection of biomolecules inbiosensors. They are small, stable, and easily obtained through solid phase peptidesynthesis [Van den Heuvel et al., 1993]. Recently new methods have evolved for theselection of peptides binding a target molecule by means of screening large randompeptide libraries (RPL). These libraries can be either derived by means ofcombinatorial organic synthesis by the split synthesis strategy [Houghten et al,1991; Lam et al., 1991] or by the expression of random stretches of synthetic DNAon the surface of filamentous bacteriophage as fusion product with coat protein pIII[Parmley & Smith, 1988]. Both methods have been reviewed extensively [Scott,1992; Lane & Stephen, 1993; Houghten, 1993; Hoess, 1993; Gallop et al., 1994;Gordon et al., 1994].

Here we describe the use of a phage displayed random peptide library with an insertlength of 15 residues [Devlin et al., 1990] for the identification of peptides bindingto human myoglobin (hMb), a 17.8 kD protein involved in the oxygen transport inmuscle. No naturally occurring ligand, other than oxygen, with affinity towards hMbhas been identified. Peptides with affinity towards hMb could be useful in the earlydiagnosis of myocardial infarction. A suitable optical- or electro-chemical biosensorfor the detection of myoglobin in the blood of a patient within minutes afterhospitalization could influence the decision on further treatment.

Chapter 3

51

3.1.3 Methods

Isolation and biotinylation of human myoglobin

Human myoglobin was isolated from skeletal muscle basically as described byWittenberg & Wittenberg [1981]. Biopsy material taken from dorsal muscle wasfrozen at -20 °C. After thawing, the material was cleared from connective tissue andhomogenized in three volumes TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8)using a Sorvall omnimixer. This suspension was cleared by centrifugation (10 min15,000 g; 5 °C) and adjusted to pH 8.0 with 25% ammonia. The supernatant wasfractionated using ammoniumsulfate precipitation. The fraction precipitatingbetween 65-100% saturation was resuspended in a minimal volume of buffer (20mM Tris-HCl; 1 mM EDTA, pH 8.0). This sample was fractionated by size exclusionHPLC on a Superose-12 column (Pharmacia) in 200 µl portions. Chromatographywas monitored by measuring the absorbance at 280 and 418 nm. Fractionscontaining hMb as determined by SDS-PAGE were pooled and dialyzed against 20mM Tris-HCl; 1 mM EDTA, pH 8.7. Further purification was achieved by anion-exchange HPLC. From the pooled and dialyzed fractions, 3-4 ml was loaded onto aMonoQ column (Pharmacia), equilibrated with the same buffer. The sample waseluted with a gradient of 0-170 mM NaCl during 15 min. The myoglobin containingfractions (A418 , SDS-PAGE) were pooled and dialyzed against MilliQ andlyophilized. The final yield was 120 mg protein from 68 g of muscle tissue. Thepurity of the sample, as determined with SDS-PAGE, was comparable withcommercially available horse myoglobin (Sigma, M0630, 95-100 % pure) used as areference protein. Purified myoglobin was biotinylated by covalent coupling ofbiotin through N-hydroxysuccinimide ester (NHS) chemistry [Bayer et al., 1979].Biotin-NHS (Boehringer Mannheim) was dissolved in dimethylformamide (18mg/ml). Myoglobin (2 mg in 5 ml PBS) was incubated with 100 µl Biotin-NHSsolution for four h at room temperature. The mixture was then dialyzed extensivelyagainst TBS (50 mM Tris-HCl; 150 mM NaCl, pH 7.5) .

Selection and DNA sequencing of phage clones

A phage peptide library was used as the peptide pool. This library, constructed byDevlin and co-workers [Devlin et al., 1990], displays 2 x 107 different peptides as afusion with the minor coat protein pIII on the surface of bacteriophage M13. Threerounds of selection were performed by means of panning using streptavidin-coated

Chapter 3

52

polystyrene petri dishes (∅ 60 mm) prepared as described [Parmley & Smith, 1988].Purified and biotinylated hMb (5 µl of a 0.4 mg/ml solution) was incubated with 5 x1011 phage in 1 ml TBS during 16 h at 4 °C. This mixture was then incubated for 1 hon the streptavidin-coated dish at room temperature. After this, the solution wasremoved and the plate was washed 10 times over a period of 1 h with TBS/0.25%Tween-20. Bound phage were then eluted by incubation with 800 µl elution buffer(0.1 M HCl adjusted to pH 2.2 with glycine and containing 1 mg/ml BSA) for 10 min,which was then neutralized with 45 µl 2 M Tris. The recovery of phage wasdetermined by plating out serial dilutions of the eluted phage along with E. coli (XL-1 Blue, Stratagene) in top-agar solid media on LB agar plates containing 12.5 µg/mltetracycline. Amplification of phage after the first and second round of selection wasperformed by plating out as described above at a multiplicity of infection < 1 and adensity of about 400 PFU per cm2 in a top-agar solid medium. After 10-12 h growthat 37 °C the phage were eluted from the top-agar by adding to the plates 5 mlTBS/0.01% gelatin followed by incubation at 4 °C with gentle shaking for 4 h. Thephage suspension was removed and after removal of residual bacteria bycentrifugation (10 min. 10,000 g; 4 °C) phage were precipitated using polyethyleneglycol (PEG-6000) [Sambrook et al., 1989] and resuspended in TBS/0.01% gelatin.This suspension was titrated as described above to determine the volume to be usedin the following selection round. In total three rounds of selection were performed.Phage were eluted after the third round of selection and plated out at low density.Five plaques were selected at random and plated out for plaque purification. Thepresence of an insert was verified by DNA amplification of the insert region asdescribed [Devlin et al., 1990]. For isolation of single stranded DNA, plaques weretransferred to 5 ml LB together with 100 µl E.coli in log phase and grown for 6-8 h at37 °C. Bacteria were removed by centrifugation. Phage were precipitated as aboveand single stranded templates were obtained by phenol/chloroform extraction andethanol precipitation. A FITC-labelled primer with the sequence ACAGACAGCC-CTCATAGTTAGCG, hybridizing at a site 109 nucleotides downstream the site ofinsertion, was used as a primer in automated DNA sequencing (A.L.F. Pharmacia).

Peptide synthesis and affinity chromatography

Peptides were synthesized as amides as described previously [Lasonder & Welling,1994] or 0.1 mmol Rapp-resin (TentaGel S RAM) was used for the synthesis ofpeptides containing a lysine (peptides 1 and 4). This resin swells in water and can beused for affinity chromatography directly. Peptides were coupled to an affinity

Chapter 3

53

matrix for affinity chromatography. The peptides tested (3 mg) were coupled to theactivated groups of Affigel-10 (1 ml; Biorad) and remaining activated groups wereblocked by ethanolamine as described [Lasonder et al., 1994]. Coupling efficiencywas determined by reversed phase HPLC [Welling, et al., 1990]. A blank column wasprepared by blocking all activated groups with 1 M ethanolamine pH 8.0. Forchromatography the columns prepared were equilibrated in 20 mM Tris-HCl; 150mM NaCl, pH 7.4. Human myoglobin, 50 µg in 1 ml, was loaded onto the columnand then eluted with the same buffer. Chromatography was monitored at 280 nmusing an Uvicord (Pharmacia LKB).

3.1.4 Results and Discussion

Selection and DNA sequencing of phage clones

The five clones selected after three rounds of phage enrichment all contained aninsert in the cloning region. DNA sequencing revealed the deduced amino acidsequence of the displayed peptides that can be found in Table 1.

Table 1. Deduced amino acid sequence of the displayed peptides of 5 phageclones

selected after three rounds of biopanning with hMb as a target.

clone number amino acid sequence1 I S P H P P K P I L G H P M S2 S P M P T M L L A N L A T M R3 F Y H L E E L Q S P Y Y A P M4 L M L L Q A S P G L H H Y P K5 L S L W S G V S M T T Q L R V

Peptide synthesis and affinity chromatography

The peptides 1, 3 and 4 were synthesized in full length. The synthesis of peptide 2was stopped after coupling of Fmoc amino acid 11 (from the C-terminus). Couplingof this amino acid was not complete, resulting in a positive Kaiser test even after 3attempts. Peptide number 5 turned out to be a mixture in RP-HPLC and was notanalyzed in affinity chromatography. Variants of peptides 3 and 4 were synthesized(3b and 4b) to make coupling of the peptides at the opposite orientation possible

Chapter 3

54

(peptides are C-terminally coupled to the Rapp-resin). Table 2 summarizes thepeptides that were synthesized.

Table 2. Amino acid sequence of the peptides synthesized.

peptide amino acid sequence1 A Ea) I S P H P P Rb) P I L G H P Nlec) S K*2 *T M L L A N L A T Nlec) R3 *F Y H L E E L Q S P Y Y A P Nlec)

4 A Ea) L Nlec) L L Q A S P G L H H Y P K*3b F Y H L E E L Q S P Y Y A P Nlec) K*4b *L Nlec) L L Q A S P G L H H Y P

a) A E was added as the original N-terminus of the pIII coat protein.b) the internal K in peptide one was substituted by R.c) M in the original sequence was substituted by Nle (norleucine) for synthetic purposes.*) indicates the residue at which the peptide was coupled to the resin.

No retardation of hMb due to interaction with peptides 1 and 4 coupled to thesynthesis resin, nor peptides 2 and 3 coupled to Affigel 10 was observed. Reversal ofthe orientation did not improve binding of peptide 3 and 4. Consequently the affinityof the selected peptides for hMb is very low, i.e. the binding constant (Ka) is smallerthan 104 M-1 [Anonymous, 1994].

In the first report about a peptide selected from a phage library using a non-antibodytarget [Devlin et al., 1990], a streptavidin binding motif, His-Pro-Gln, was identified.The same pattern was identified using the 'split synthesis' strategy [Lam, et al.,1991]. However, no effort was made to quantify the affinity of the peptides forstreptavidin. The binding constant for the interaction is probably very low becauseno retardation of streptavidin is observed in affinity chromatography using a peptidewith the identified motif as a ligand (G.W. Welling, unpublished results).Crystallographic studies of streptavidin co-crystallized with a HPQ peptide [Weberet al., 1992] have shown that these peptides do not bind streptavidin at the biotinbinding-site and therefore are not truly biotin mimics.

Peptides with affinity towards the lectin concanavalin A (con A), a carbohydrate-binding protein were identified from phage libraries by two independent groups[Oldenburg, et al., 1992; Scott, et al., 1992]. The same binding pattern, Tyr-Pro-Tyr,was identified, but the reported affinity of the peptides differed 20-fold; Oldenburgreported a Ka of 2.2 x 104 M-1 as determined by equilibrium dialysis, Scott estimatedKa to be 1.25 x 103 M-1 by comparison of IC50 values.

Chapter 3

55

Peptide ligands with affinity towards the integrin gpIIb/IIIa, a cell adhesion proteinthat mediates platelet aggregation through binding of fibrinogen, were identifiedfrom a 'conformationally constrained' peptide library [O'Neil, et al., 1992]. Thehexapeptides displayed on the phage were flanked by cysteine residues andtherefore capable of forming cyclic disulfides. The pattern Lys/Arg-Gly-Asp wasidentified and confirmed the already known binding motif found in a number ofsnake venoms. The relative binding strength of the conformationally constrainedpeptides was 10-20 fold better than that of the analogues where the Cys residueswere replaced by Ser. No estimation was given for the affinity of the peptidestowards gpIIb/IIIa.

The general conclusion from the above examples can be that when attempts aremade to quantify the affinity of the peptides for their target molecules, theseaffinities are relatively low. This could be one of the reasons why we failed toidentify peptides with affinity towards hMb i.e. the affinity of the peptides is toosmall to retard hMb in affinity chromatography. Another reason could be that hMb isnot a suitable protein target because of its lack of a binding site. In all cases thatreport successful identification of peptide ligands after screening peptide libraries,the target molecules have a binding domain for either a protein/peptide ligand orother biomolecules (carbohydrate, biotin). The globular hMb molecule lacks such abinding cleft, groove or pocket and therefore has no 'natural surroundings' forbinding peptides. So, in either case the peptides can be regarded as functionally non-binding in a biosensor.

Although peptides selected from random peptide libraries with non-antibody targetsare not (yet) suitable to be used as a ligand in affinity chromatography or in abiosensor, they can be useful to identify enzyme inhibitors or peptide mimics ofligands interacting with receptor molecules. These inhibitors or peptide mimics canbe used as lead compounds in the development of pharmaceuticals where not theaffinity but the specificity for the target molecules is of primary interest.

Chapter 3

56

3.2 Antibody-binding peptides from a surface displayed random peptidelibrary using a monoclonal antibody as a target

3.2.1 Summary

Random peptide libraries (RPLs) displayed on the surface of a filamentousbacteriophage can be used to identify peptide ligands that interact with targetmolecules. We have used a 15-amino acid residue RPL displayed on bacteriophageM13 to identify the core residues within the epitope of a monoclonal antibody(mAb) A16 which interacts with a continuous epitope restricted to amino acidresidues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virustype 1 (gD-1). The single peptide sequence obtained after three rounds of selectioncontained identical residues at three positions compared to the authentic gD-1sequence. Synthetic peptides were prepared based on the sequence of the originalepitope and the phage-derived epitope. The binding constants (Ka) with mAb A16were determined using surface plasmon resonance (SPR) biosensor technology. TheRPL-derived peptide and peptide 9-19 of gD-1 had approximately the same affinityfor mAb A16. This suggests that those residues within the epitope that are essentialfor binding were identified. The synthesis of shorter versions of the RPL-derivedpeptide restricted the binding region to seven amino acid residues. These resultsshow that minimal information retrieved from the screening of an RPL combinedwith peptide synthesis can characterize the epitope of a mAb with high resolution.Immunization of mice with the phage derived peptide protected against a challengewith a lethal dose of herpes simplex virus type 1 (HSV-1) equally well as the gD-1derived peptide.

3.2.2 Introduction

An important aspect of the characterization of a monoclonal antibody is thedetermination of its binding site on the antigen. When protein antigens areconsidered, these binding sites or epitopes are usually divided into either continuous(or conformation-independent) or discontinuous (or conformation-dependent) ones.An epitope is considered continuous if it remains intact after denaturation of theprotein, and it is assumed that they are composed of amino acid residues adjacent toeach other in the protein sequence. Discontinuous epitopes are destroyed afterdenaturation of the protein, presumably they comprise residues of different parts ofthe polypeptide chain, brought in close proximity by folding of the protein into itsnative structure [Muggeridge et al., 1988]. Antibodies against gD-1 are grouped

Chapter 3

57

according to their reactivity with gD-1. At least seven groups, representing separateantigenic domains on gD-1 have been identified [Eisenberg et al., 1982]. Group VIImAbs are reactive with denatured gD. Studies with overlapping synthetic peptidesshow that group VII antibodies interact with a continuous epitope within residues 10through 19 of gD-1 [Eisenberg et al., 1985; Bosch et al., 1987]. Group VII mAbs arevirus type common and able to neutralize viral infectivity [Cohen et al., 1984].

Various methods are in use to determine the fine structure of continuous epitopes[Van Regenmortel, 1989]. These methods for epitope mapping include the use ofnaturally occurring variants of the antigen. This method is restricted to studies ofhomologous proteins of different species such as myoglobin, cytochrome C andlysozyme [Benjamin et al., 1984]. When epitopes on virus proteins are considered,sequence analysis of variants escaping neutralization, obtained after culturing thevirus in the presence of the mAb, can identify residues critical for binding theantibody [Minson et al., 1986; Highlander et al., 1987]. Other methods to locateepitopes include the use of overlapping synthetic peptides [Welling-Wester et al.,1991], and the use of site directed mutagenesis to obtain substitution variants ofcloned antigens [Muggeridge et al., 1990; Chiang et al., 1994].

Recently new methods have been described in which a large set of random peptidesserves as a pool for selecting peptide ligands. These peptide libraries can be derivedby means of solid-phase peptide synthesis [Houghten et al., 1991; Lam et al., 1991]or by insertion of random stretches of synthetic DNA expressed on the surface offilamentous bacteriophage as fusion products with coat protein pIII [Devlin et al.,1990; Cwirla et al., 1990]. In principle, screening of random peptide libraries cangive information about the localization of the epitope by looking for sequencesimilarities with the original antigen. Moreover, individual amino acids involved inbinding the antibody may be identified in this way.

Here, we describe the application of a 15 residue random peptide library inbacteriophage M13, for screening with mAb A16 [Scheffer et al., 1984], a group VIIantibody [Welling-Wester et al., 1994] as a target. Affinity measurements of thenative and phage-derived peptide with the mAb were performed using a biosensorbased on the surface plasmon resonance (SPR) principle. This optical techniquedetects refractive index changes close to the surface of a sensor chip. Ligandmolecules can be covalently coupled to the carboxylated dextran layer covering thesensor chip. Samples containing the analyte are transported over the sensor chip in acontinuous flow. Changes in adsorbed mass due to binding of the analyte to the

Chapter 3

58

immobilized ligand are proportional to the change in refractive index. The responsefrom the instrument expressed as resonance units (RU) plotted as a function of timeis called a sensorgram. With this system, commercially available under the nameBIAcore (Pharmacia Biosensor AB), interactions can be monitored in real-timewithout modification of the antibody or antigen. A more detailed description of theSPR principle and its applications is given by Malmqvist [1993]. Furthermore, theability to elicit protective immunity upon immunization with the mimotope wasinvestigated. Mice were immunized with a mimotope conjugate and challenged withHSV-1.

3.2.3 Methods

Monoclonal antibody A16

The production and immunological characterization of mAb A16 has been describedelsewhere [Scheffer et al., 1984]. A16 was purified chromatographically from mouseascites fluid using protein A-Sepharose [Harlow & Lane, 1988].

Phage selection

A 15-residue surface expressed random peptide library was used as the peptide pool.This library, constructed by Devlin and co-workers [Devlin et al., 1990], has acomplexity of 2 x 107 different clones and an insert frequency of 55%. Three roundsof selection were performed by means of panning using streptavidin-coatedpolystyrene petri dishes (∅ 60 mm) prepared as described by Parmley and Smith[1988]. Purified mAb (1 µl of a 1 mg/ml solution) was incubated with 5 x 1011 phagein 1 ml TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 16 h at 4 °C. Biotinylatedgoat anti-mouse IgG was added (1 µg) and incubated for another 2 h at 4 °C. Thismixture was then incubated for 1 h on the streptavidin-coated dish at roomtemperature. After this the solution was removed and the plate was washed ten timesover a period of 1 h with TBS/0.25% Tween-20. Bound phage were then eluted byincubation with 800 µl elution buffer (0.1 M HCl adjusted to pH 2.2 with glycine andcontaining 1 mg/ml BSA) for 10 min, which was then neutralized with 45 µl 2 MTris. The recovery of phage was determined by plating out serial dilutions of theeluted phage along with E. coli XL-1 Blue (Stratagene) in the top agar on LB agarplates containing 12.5 µg/ml tetracycline. Amplification of the phage after the firstand second round of selection was performed by plating out as described above at a

Chapter 3

59

multiplicity of infection < 1 and a density of about 400 PFU per cm2 in the top agar.After 10-12 h growth at 37 °C the phage were eluted from the top agar by adding tothe plates 5 ml TBS/0.01% gelatin followed by incubation at 4 °C with gentleshaking for 4 h. The phage suspension was removed and after removal of residualbacteria by centrifugation (10,000 g for 10 min at 4 °C) phage were precipitatedusing polyethylene glycol (PEG-6000) [Sambrook et al., 1989] and resuspended inTBS/0.01% gelatin. This suspension was titrated as described above to determine thevolume to be used in the following selection round. Altogether, three rounds ofselection were performed.

DNA sequencing

Phage were eluted after the third round of selection and plated out at low density.Five plaques were selected at random and plated out for plaque purification. Thepresence of an insert was verified by DNA amplification of the insert region asdescribed [Devlin et al., 1990]. For isolation of single-stranded DNA, plaques weretransferred to 5 ml LB together with 100 µl E.coli in log phase and grown for 6-8 h at37 °C. Bacteria were removed by centrifugation. Phage was precipitated as aboveand single-stranded templates were obtained by phenol/chloroform extraction andethanol precipitation. A FITC-labelled oligonucleotide with the sequence 5'-ACAGACAGCCCTCATAGTTA-GCG-3', hybridizing to a site 109 nucleotidesdownstream the site of insertion, was used as a primer in an automated sequencingprocedure (A.L.F., Pharmacia).

Peptide synthesis

Peptides were synthesized by a solid phase strategy on an automated multiplepeptide synthesizer (Abimed AMS 422) [Gausepohl et al., 1990]. Tentagel S AC[Sheppard & Williams, 1982; Rapp et al., 1990] was used as a resin (40-60 mg perpeptide, 10 µmol Fmoc amino acid loading). Repetitive couplings of the appropriateprotected Fmoc amino acids were performed by adding a mixture of 90 µl 0.67 MPyBOP [Martinez et al., 1985] in NMP, 20 µl NMM in NMP (2/1 v/v) and 100 µl ofan 0.60 M solution of the appropriate Fmoc amino acid [Fields & Noble 1990] inNMP (sixfold excess) to the reaction vessel. Fmoc-deprotection was performed byadding 0.8 ml piperidine/DMA (1/4; v/v) three times to the reaction vessel. Afterdeprotection of the last Fmoc amino acid the resin was washed extensively.Cleavage of the peptides from the resin and removal of side chain protecting groups

Chapter 3

60

was performed with TFA/water (19/1 v/v; 2 h). The peptides were precipitated twiceby adding ether/pentane (1/1 v/v). After drying, the peptides were dissolved in waterand freeze-dried. The purity of the peptides was determined by reversed phaseHPLC. The peptides were at least 90% pure, as deduced from their elution patternand the relative absorption at 214 nm, and were used for affinity measurementswithout further purification. The peptides used for immunization of mice wereelongated during synthesis at the N terminus with an aminohexane (Z) and twolysine residues (K K) that served as a spacer arm for coupling to a carrier protein.The sequence was identical to amino acid residues 7-18 of gD-1(K K Z-A S L K M A D P N R F R; gD-pep 7-18) and the aligned sequence of thephage derived peptide (K K Z-H N G R F S D A L R F T; phage-pep 7-18). Thesepeptides were coupled to BSA by glutaraldehyde [Geerligs et al., 1988].

Affinity determination using SPR

A biosensor based on the principle of SPR [Karlson et al., 1991; Fägerstam et al.,1992] was used to quantify the interaction of the peptides with the mAb A16. Withthe BIAcore system (Pharmacia Biosensors AB) quantitative interaction analysis ofbiomolecules can be performed in real-time. Immobilization of the peptides to thesensor surface and kinetic measurements were performed as described [Altschuh etal., 1992]. The association rate constants (ka) and dissociation rate constants (kd)were both determined from the association phase at different concentrations of mAb.Ten concentrations were used, ranging from 4.3 nM to 43 nM. Assuming a bindingstoichiometry of 1, the kinetic parameters were determined from the sensorgramsusing the software supplied by the manufacturer.

Inhibition assay using SPR

In order to determine the minimal length of the phage derived peptide still able tobind mAb A16, two sets of overlapping peptides were used: either shortened at the Nterminus or at the C terminus. These peptides were used in an inhibition assay usingBIAcore. The ability of a peptide to inhibit binding was determined by pre-incubation of mAb A16 (40 nM) with a molar excess of peptide (1 µM) in HBS (10mM Hepes, 0.15 M NaCl, 3.4 mM EDTA, 0.05 % P20 detergent, pH 7.4) containing3% DMSO during 5 h. This mixture was injected into the flowcell where the phagederived peptide was immobilized to the amount of 139 RU. Peptides were designated

Chapter 3

61

positive if no residual mAb A16 was detected i.e. if no binding curve appeared in thesensorgram.

Protection against a lethal HSV-1 challenge

Mice were immunized in order to investigate whether the phage derived peptide wasable to elicit protective immunity against HSV-1 infection. Female 6-week-oldBalb/c mice were used for immunization. Mice were immunized with 60 µg ofpeptide conjugated to BSA for the first injection and 40 µg of coupled peptide forthe three booster injections at 3-week intervals. Two groups of control animals wereinjected with PBS and 2.7 x 108 PFU of heat-inactivated HSV-1, respectively.Freund's complete adjuvant was used in the first injection and incomplete adjuvantin the following injections. Injections were given i.p. Seven days after the lastinjection, the mice were challenged with a lethal dose of HSV-1 virus (1.9 x 109 PFUper animal). The differences in survival ratios were considered to be significant if pvalues were < 0.05 in statistical analysis using the binomial theorem.

3.2.4 Results and Discussion

Selection of phage and DNA sequencing

A distinct increase of recovery of eluted phage from the streptavidin coated dishesindicated enrichment of phage displaying peptides with affinity towards mAb A16.The amount of input phage was 5 x 1011 in al three rounds of selection. Theenrichment factors (input phage/eluted phage) were 6 x 106, 200 and 55 in roundsone, two and three, respectively. No increase in recovery was found in a controlexperiment in which mAb A16 had been omitted during the panning procedure. Thisindicated that phage with affinity towards the secondary antibody or streptavidinhad not been enriched for. The five plaques randomly selected from the plated eluateafter the third round of selection all contained phage with an insert. DNA sequenceanalysis revealed that the five plaques contained identical phage clones. Thesynthetic peptides based on the amino acid sequence of gD 9-19 (gD-pep 9-19) andthe phage-derived peptide (phage-pep 5-19) are shown in Table 3. When comparedto residues 9-19 of gD-1, the amino acid sequence of the insert region containsidentical residues at positions 13, 16 and 17. No other sequence similarity of two ormore amino acids could be identified within the amino acid sequence of gD.

Chapter 3

62

Table 3. Sequence of phage-pep 5-19, gD-pep 9-19 and binding of N- and C-terminally shortened variants of phage-pep 5-19.

peptide variant inhibitinga)

binding

b) (phage-pep 5-19)(gD-pep 9-19)

++++++---++-----+

a) peptides are marked (+) if they were able to inhibit mAb A16 binding tophage-pep that was immobilized to the sensorchip surface.b) numbering of residues is based on the numbering of mature gD [Watson etal., 1982] i.e. the similar pattern DXXRF is aligned with this sequence.c) shortest peptide deduced to inhibit binding of mAb A16 to phage-pep 5 -19.

Affinity measurements with SPR based biosensor

The peptides gD-pep 9-19 and phage-pep 5-19 were coupled to the dextran layer ofa sensor chip to the amount of 139 and 38 RU, respectively. The kinetic parameterska, kd and the binding constant (Ka) for the interaction of mAb A16 with gD-pep 9-19 and phage-pep 5-19 were determined and are listed in Table 4. No significantdifferences in rate constants and binding constants between gD-pep and phage-pepwere measured.

Chapter 3

64

From inhibition studies with two series of peptides, either C- or N-terminallyshortened, it was deduced that the seven residue peptide FSDALRF was the shortestpeptide which might bind mAb A16 and indeed the assay showed that this peptidecould inhibit binding of mAb A16 to the immobilized phage-pep 5-19. Directaffinity measurements with this peptide were not possible. The resulting signal wastoo low for accurate determination of the relative binding constant.

Protection against a lethal HSV-1 challenge

Groups of BALB/c mice were immunized with phage-derived peptide conjugates(phage-pep 7-18), the gD-1 sequence (gD-pep 7-18) and heat inactivated HSV-1virus. Subsequently, the mice were challenged with a lethal dose of HSV-1. Theresults are summarized in table 5.

Table 5. Protection of mice against a lethal HSV-1 challenge by immunizationwith the phage derived peptide.

antigen survived total survivalratio

phage-pep 7-18 6 9 0.67gD-pep 7-18 6 10 0.60inactivated HSV-1 6 6 1.0PBS 2 10 0.20

Of the control group of ten mice, injected with PBS buffer, two survived after thechallenge. Of the groups injected with coupled phage-pep 7-18 or gD-pep 7-18, sixout of nine and six out of ten survived, respectively. Of the control group immunizedwith heat-inactivated HSV-1, all six mice survived. The survival ratio of the groupsinjected with phage-pep 7-18, gD-pep 7-18 and the inactivated virus showedstatistically significant differences to the control group injected with PBS. Thedifference between the survival ratios of the group immunized with gD-pep 7-18 andthe group immunized with phage-pep 7-18 was not significant, suggesting thesepeptides protected equally well against the HSV-1 challenge.

In this study we selected a high affinity peptide epitope from a large pool of randomsequences using surface display. Our finding that this peptide has only three aminoacid residues in common with the known epitope suggests that the residues Asp-13,Arg-16 and Phe-17 are indispensable constituents of the epitope of mAb A16.Analysis of mutants escaping neutralization has previously identified Arg-16 as acritical residue in binding mAb LP-14, also a group VII antibody [Minson et al.,

Chapter 3

65

1986]. The identification of three residues making up the core site for antibodyrecognition is consistent with the observation that only a few of the 15 to 20 residuesthat are seen in close contact with the antibody molecule in X-ray diffraction studiesare critical for binding. Nuss et al. [1993] examined the 19 amino acid residues ofinfluenza virus neuraminidase which are in contact with mAb NC41. Only five ofthese contact residues were identified as being crucial for antibody recognition.

Results of other studies in which RPL were used for epitope mapping differ in thenumber of peptides selected, the affinities of the phage derived peptides for the mAband the similarity patterns identified. Cwirla et al. [1990] used an antibody specificfor the sequence YGGF at the N terminus of ß-endorphin as a target for selectionfrom a hexapeptide library on phage. Only moderate affinity peptides with the motifsYGXX and YGGX were identified. In a later study [Barret et al., 1992] higheraffinity peptides were identified using the same mAb. Christian et al. [1992]identified two 'mimotopes' with moderate affinities for the anti-humanimmunodeficiency virus type 1 gp120 monoclonal antibody. No similarity with theknown decapeptide ligand was found. Stephen and Lane [1992] reported the finestructure of the epitope recognized by an anti-p53 (tumor suppression protein)monoclonal antibody, using a hexapeptide library. The RHSVV pattern foundenabled them to predict cross-reactivity with transcription factor TFIIIA fromXenopus. Recently Böttger and Lane [1994] found the S(X)LNP-motif as the epitopefor an anti-keratin 8 antibody LE41 but failed to select specific peptide sequencesfor six other mAbs. Lane and Stephen [1993] suggest that results are most clear-cutwhen each residue in the epitope sequence makes a distinct contribution to antibodybinding. This situation appears to occur in two cases reported [Stephen & Lane,1992; Dower, 1992]. However, our results suggest that epitopes which allowvariance within the sequence (amino acid residues 14 and 15 within the motif 13-DXXRF-17) can be elucidated using phage display.

Several explanations can be given why after the third selection round one sequencewas found to be dominantly enriched for. The simplest explanation is that just one,or very few, sequences are represented in the RPL that bind the antibody A16 withsufficient affinity. This explanation is supported by the fact that only a smallfraction of all possible 15-residue peptide sequences is represented in the library.The absence of low affinity binding phage after three rounds of selection can beexplained by competition effects during the selection rounds [Miceli et al., 1994].This effect could be extreme in our case where the displayed peptide has a relatively

Chapter 3

66

high affinity for the mAb. Another cause could be that the criterion for selection wasnot merely the primary structure of the peptide but that conformational features werealso selected for. Structural studies using nuclear magnetic resonance on N-terminalgD peptides indicate that moderately stable secondary structures are present in gDpeptide 7-23 [Williamson et al., 1986a] and gD peptide 11-32 [Williamson et al.,1986b]. For another group VII monoclonal antibody, LP14 [Minson et al., 1986],secondary structure seems to play a role in the interaction with a peptide antigen[Bosch et al., 1987]. This supports our assumption that although the epitope isrestricted to a relatively small sequence, some conformational constraint is neededfor high affinity binding. As is suggested by Hoess [1993], long inserts in an RPLmay be conformationally more constrained than small ones, thus favoring selectionof high affinity peptides. When the spatial requirements of the epitope for bindingthe antibody are less strict, random libraries of small peptides, either synthetic ordisplayed on the phage surface may be more suitable, because, on practical grounds,a larger fraction of all possible peptides is represented.

The inhibition assay identified a seven residue peptide as the shortest 'mimotope'able to inhibit binding of mAb A16. The requirement of the two additional aminoacid residues Phe and Ser at the amino terminus of the Asp-Xxx-Xxx-Arg-Phepattern suggests that these residues are involved in stabilizing the preferentialconformation for binding the antibody. The low signal increase after coupling theshortest peptide (FSDALRF) covalently to the sensor chip surface suggests that thecoupled peptide is not readily accessible for binding. Another possibility may bethat in the peptide some conformation is present that might be adversely affected bythe immobilization. Such an effect could be less prominent in longer peptides.Possibly a spacer arm of more than two amino acid residues is needed for completeavailability. The spacer arm could be needed to neutralize sterical hindrance fromthe dextran moieties surrounding the immobilized peptide.

Surprisingly immunization with the phage derived peptide mimotope peptideprotected mice equally well against a lethal challenge of HSV-1 virus as the peptidebased on the native gD-1 sequence. Clearly, at least in this case, merely the coreresidues within a major antigenic site are necessary for a protective B cell response.This knowledge could be useful in designing peptide or recombinant subunitvaccine candidates.

We gratefully acknowledge Dr. James Devlin for his generous gift of phage stockfrom the random peptide library, and Dr. Marcel Ruiters and Dr. Peter Terpstra

Chapter 3

67

from the Department of Biomedical Technology of the University of Groningen foroligonucleotide synthesis, DNA sequencing and the use of the BIAcore instrument.We thank Dr. Gijsbert Jansen for useful suggestions on the statistical analysis of thevirus challenge experiment. This research was supported by the TechnologyFoundation (STW) grant nr. GGN99.1924.

Chapter 3

68

3.3 References

Altschuh, D., Dubs, M.C., Weiss, E., Zeder-Lutz, G. & Van Regenmortel, M.H.V.(1992) Determination of kinetic constants for the interaction between a monoclonalantibody and peptides using surface plasmon resonance. Biochemistry 31, 6298-6304.

Anonymous (1994) Affinity chromatography, principles and methods (PharmaciaLKB Biotechnology, Uppsala Sweden) p15.

Barrett, R.W., Cwirla, S.E., Ackerman, M.S., Olsen, A.M., Peters, E.A. & Dower, W.J.(1992) Selective enrichment and characterization of high affinity ligands fromcollections of random peptides on filamentous phage. Analytical Biochemistry 204,357-364.

Bayer, E.A., Skutelsky, E. & Wilcher, M. (1979) The avidin-biotin complex inaffinity cytochemistry. Methods in Enzymology 62, 308-315.

Benjamin, D.C., Berzofsky, J.A., East, I.J., Gurd, F.R.N., Hannum, C., Leach, S.J.,Margoliash, E., Michael, J.G., Miller, A., Prager, E.M., Reichlin, M., Sercarz, E.E.,Smith-Gill, S.J., Todd, P.A., & Wilson, A.C. (1984) The antigenic structure ofproteins: a reappraisal. Annual Review of Immunology 2, 67-101.

Bosch, D.L., Geerligs, H.J., Weijer, W.J., Feijlbrief, M., Welling, G.W. & Welling-Wester, S. (1987) Structural properties and reactivity of N-terminal syntheticpeptides of herpes simplex virus type 1 glycoprotein D by using antipeptideantibodies and group VII monoclonal antibodies. Journal of Virology 61, 3607-3611.

Böttger, V. & Lane, E.B. (1994) A monoclonal antibody epitope on keratin 8identified using a phage peptide library. Journal of Molecular Biology 235, 61-67.

Chiang, H.Y., Cohen, G.H. & Eisenberg, R.J. (1994) Identification of functionalregions of herpes simplex virus glycoprotein gD by using linker-insertionmutagenesis. Journal of Virology 68, 2529-2543.

Christian, R.B., Zuckerman, R.N., Kerr, J.M., Wang, L. & Malcolm, B.A. (1992)Simplified method for construction, assessment and rapid screening of peptidelibraries in bacteriophage. Journal of Molecular Biology 227, 711-718.

Cohen, G.H., Dietzschold, B., Ponce de Leon, M., Long, D., Golub, E.A., Varrichio,A., Pereira, L. & Eisenberg, R.J. (1984) Localization and synthesis of an antigenicdeterminant of herpes simplex virus glycoprotein D that stimulates the production ofneutralizing antibody. Journal of Virology 49, 102-108.

Chapter 3

69

Cwirla, S.E., Peters, E.A., Barret, R.W. & Dower, W.J. (1990) Peptides on phage: Avast library of peptides for identifying ligands. Proceedings of the NationalAcademy of Science USA 87, 6378-6382.

Devlin, J.J., Panganiban, L.C. & Devlin, P.E. (1990) Random peptide libraries: asource of specific protein binding molecules. Science 249, 404-406.

Dower, W.J. (1992) Phage power. Current Biology 2, 251-253.

Eisenberg, R.J., Long, D., Pereira, L., Hampar, B., Zweig, M. & Cohen, G. (1982)Effect of monoclonal antibodies on limited proteolysis of native glycoprotein D ofherpes simplex virus type 1. Journal of Virology 41, 478-488.

Eisenberg, R.J., Long, D., Ponce de Leon, M., Matthews, J.T., Spear, P.G., Gibson,M.G., Lasky, L.A., Berman, P., Golub, E. & Cohen, G.H. (1985) Localization ofepitopes of herpes simplex virus type 1 glycoprotein D. Journal of Virology 53, 634-644.

Fägerstam, L.G., Frostell-Carlsson, A., Karlsson, R., Persson, B. & Rönnberg, I.(1992) Biospecific interaction analysis using surface plasmon resonance detectionapplied to kinetic, binding site and concentration analysis. Journal ofChromatography 597, 397-410.

Fields, G. & Noble, R. (1990) Solid phase peptide synthesis utilizing 9-fluorenyl-methoxycarbonyl amino acids. International Journal of Peptide and ProteinResearch 35, 161-214.

Gallop, M.A., Barret, R.W., Dower, W.J., Fodor, S.P.A. & Gordon, E.M. (1994)Applications of combinatorial Technologies to drug discovery. 1. Background andpeptide combinatorial libraries. Journal of Medicinal Chemistry 37, 1233-1251.

Gausepohl, H., Kraft, M., Boulin, C. & Frank, L.W. (1990) A multiple reactionsystem for automated simultaneous peptide synthesis. Peptides 206-207.

Geerligs, H.J., Weijer, W.J., Bloemhof, W. & Welling, G.W. (1988) The influence ofpH and ionic strength on the coating of peptides of herpes simplex virus type 1 in anenzyme-linked immunosorbent assay. Journal of Immunological Methods 106, 239.

Gordon, E.M., Barret, R.W., Dower, W.J., Fodor, S.P.A. & Gallop, M.A. (1994)Applications of combinatorial Technologies to drug discovery. 2. Combinatorialorganic synthesis, library screening strategies, and future directions. Journal ofMedicinal Chemistry 37, 1385-1401.

Harlow, E. & Lane, D. (1988) Antibodies: A laboratory manual (Cold Spring HarborLab., Cold Spring Harbor).

Chapter 3

70

Highlander, S.L., Sutherland, S.L., Gage, P.J., Johnnson, D.C., Levine, M. & Glorioso,J.C. (1987) Neutralizing monoclonal antibodies specific for herpes simplex virusglycoprotein D inhibit virus penetration. Journal of Virology 61, 3356-3364.

Hoess, R.H. (1993) Phage display of peptides and protein domains. Current Opinionin Structural Biology 3, 572-579.

Houghten, R.A., Pinella, C., Blondelle, S.E., Appel, J.R., Dooley, C.T. & Cuervo, J.H.(1991) Generation and use of synthetic peptide combinatorial libraries for basicresearch and drug discovery. Nature 354, 84-86.

Houghten, R.A. (1993) Peptide libraries: criteria and trends. Trends in Genetics 9,235-239.

Karlson, R., Michaelsson, A. & Mattson, L. (1991) Kinetic analysis of monoclonalantibody-antigen interactions with a new biosensor based analytical system. Journalof Immunological Methods 145, 229-240.

Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M. & Knapp, R.J.(1991) A new type of synthetic peptide library for identifying ligand-bindingactivity. Nature 354, 82-84.

Lane, D.P. & Stephen, C.W. (1993) Epitope mapping using bacteriophage peptidelibraries. Current Opinion in Immunology 5, 268-271.

Lasonder, E., Bloemhoff, W. & Welling, G.W. (1994) Interaction of lysozyme withsynthetic anti-lysozyme D1.3 antibody fragments studied by affinitychromatography and surface plasmon resonance. Journal of Chromatography A 676,91-98.

Lasonder, E. & Welling, G.W. (1994) Biphasic behavior of a peptide-antibodycomplex in optical biosensors. Submitted for publication.

Malmqvist, M. (1993) Surface plasmon resonance for detection and measurementsof antibody-antigen affinity and kinetics. Current Opinion in Immunology 5, 282-286.

Martinez, J., Bali, J.P., Rodriquez, M., Castroi, J., Laur, J. & Lignon, M.F. (1985)Synthesis and biological activities of some pseudo-peptide analogues oftetragastrin: The importance of the peptide backbone. Journal of MedicinalChemistry 28, 1874.

Miceli, R.M., DeGraaf, M.E. & Fischer, H.D. (1994) Two-stage selection ofsequences from a random phage display library delineates both core residues andpermitted structural range within an epitope. Journal of Immunological Methods167, 279-287.

Chapter 3

71

Minson, A.C., Hodgman, T.C., Digard, P., Hancock, D.C., Bell, S.E. & Buckmaster,E.A. (1986) An analysis of the biological properties of monoclonal antibodiesagainst glycoprotein D of herpes simplex virus and identification of amino acidsubstitution that confer resistance to neutralization. Journal of General Virology 67,1001-1013.

Muggeridge, M.I., Isola, V.J., Byrn, R.A., Tucker, T.J., Minson, A.C., Glorioso, J.C.,Eisenberg, R.J. & Cohen G.H. (1988) Antigenic analysis of a major neutralizationsite of herpes simplex virus glycoprotein D using deletion mutants and monoclonalantibody-resistant mutants. Journal of Virology 62, 3274-3280.

Muggeridge, M., Roberts, S.R., Isola, V.J., Cohen, G.H. & Eisenberg, R.J. (1990) InImmunochemistry of Viruses II. The Basis of Serodiagnostics and Vaccines (VanRegenmortel, M.H.V. and Neuratht, A.R., Eds, Elsevier Publishers B.V. Amsterdam).

Nuss, J.M., Bossart Whitaker, P. & Air, G.M. (1993) Identification of critical contactresidues in the NC41 epitope of a subtype N9 influenza virus neuraminidase.Proteins: Structure, Function, and Genetics 15, 121-132.

Oldenburg, K.R., Loganathan, D., Goldstein I.J., Schultz, P.G. & Gallop, M.A. (1992)Peptide ligands for a sugar-binding protein isolated from a random peptide library.Proceedings of the National Academy of Science USA 89, 5393-5397.

O'Neil, K.T., Hoess, R.H., Jackson, S.A., Ramachandran, N.S., Mousa, S.A. &DeGrado, W.F. (1992) Identification of novel peptide antagonist for GPIIb/IIIa froma conformationally constrained phage peptide library. Proteins: Structure, Function,and Genetics 14, 509-515.

Parmley, S.F. & Smith, G.P. (1988) Antibody-selectable filamentous fd phagevectors: affinity purification of target genes. Gene 73, 305-318.

Rapp, W., Zhang, L. & Bayer, E. (1990) Continuous flow peptide synthesis onPSPOE-graft-copolymers. Innovation and Perspectives in Solid Phase PeptideSynthesis, 205-210.

Sambrook, J., Fritsch, E.F. & Maniatis, R. (1989) Molecular cloning: a laboratorymanual. 2nd edition. (Cold Spring Harbor Lab., Cold Spring Harbor, NY).

Scheffer, A.J., Koedijk, D.G.A.M., Abee, T. & Osterhaus, A.D.M.E. (1984)Monoclonal antibodies against herpes simplex viruses. Developments in BiologicalStandardization 57, 269-274.

Scott, J.K., Loganathan, D., Easley, R.B., Gong, X. & Goldstein, I.J. (1992) A familyof concanavalin A-binding peptides from a hexapeptide epitope library. Proceedingsof the National Academy of Science USA 89, 5398-5402.

Chapter 3

72

Scott, J.K. (1992) Discovering peptide ligands using epitope libraries. Trends inBiochemical Sciences 17, 241-245.

Sheppard, R.C. & Williams, B.J. (1982) Acid-labile resin linkage for use in solidphase peptide synthesis. International Journal of Peptide and Protein Research 20,451-454.

Stephen, C.W. & Lane, D.P. (1992) Mutant conformation of p53: precise epitopemapping using a filamentous phage epitope library. Journal of Molecular Biology225, 577-583.

Van den Heuvel, D.J., Kooyman, R.P.H., Drijfhout, J.W. & Welling, G.W. (1993)Synthetic peptides as receptors in affinity sensors: a feasibility study. AnalyticalBiochemistry 215, 223-230.

Van Regenmortel, M.V.H. (1989) Structural and functional approaches to the studyof protein antigenicity. Immunology Today 10, 266-271.

Watson, R.H., Weis, J.H., Salstrom, J.S. & Enquist, L.W. (1982) Herpes simplex virustype-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli.Science 218, 381-384.

Weber, P.C., Pantoliano, M.W. & Thompson, L.D. (1992) Crystal structure andligand-binding studies of a screened peptide complexed with streptavidin.Biochemistry 31, 9350-9354.

Welling, G.W. , Geurts, T., Van Gorkum, J., Damhof, R.A., Drijfhout, J.W.,Bloemhoff, W. & Welling-Wester, S. (1990) Synthetic antibody fragment as ligandin immunoaffinity chromatography. Journal of Chromatography 512, 337-343.

Welling-Wester, S., Scheffer, A.J. & Welling, G.W. (1991) B and T cell epitopes ofglycoprotein D of herpes simplex virus type 1. FEMS Microbiology Immunology 76,59-68.

Welling-Wester, S., Feijlbrief, M., Koedijk, D.G.A.M., Drijfhout, J.W., Weijer, W.J.,Scheffer, A.J. & Welling, G.W. (1994) Analogues of peptide 9-21 of glycoprotein Dof herpes simplex virus and their binding to group VII monoclonal antibodies.Archives of Virology 138, 331-340.

Williamson, M.P., Hall, M.J. & Handa, B.K. (1986a) Secondary structure of a herpessimplex virus glycoprotein D antigenic domain. International Journal of Peptideand Protein Research 27, 562-568.

Williamson, M.P., Hall, M.J. & Handa, B.K. (1986b) H-NMR assignment andsecondary structure of a herpes simplex virus glycoprotein D-1 antigenic domain.European Journal of Biochemistry 119, 527-536.

Chapter 3

73

Wittenberg, J.B. & Wittenberg, B.A. (1981) Preparations of myoglobins. Methods inEnzymology 76, 29-39.