Description of the related art Herpes simplex virus types 1 and 2 (HSl'-1 and HSV-2) are two closely related viruses that infect liumans. N4ost people over age 15 have antibodies to HSV-1 or -2 indicating that they have been infecte with and harbour these viruses. Both viruses produce orogenital lesions and can also infect the eye, skin and nervous system. HSV-I is mainly responsible for oral lesions while HSV-2 is mainly responsible for genital lesions.

Following primary infection. the virus can enter a latent state in neurological tissue from which it can periodically reactivate to produce recurrent infections. Reactivations are frequently asymptomatic so that virus can be shed and transmitted in the absence of overt clinical lesions.

The need for a simple and inexpensive serodiagnostic test capable of distinguishing HSV-1 from HSV-2 antibodies was recognised at the WHO recent conference (1996).

Serotyping of HSV will be important in trials of HSV vaccines in order to establish the immunological status of individuals before vaccination. It is hoped to prevent neonatal herpes by screening prenant women and their sexual partners for asymptomatic HSV-2 infections and, where appropriate, recommending precautions designed to prevent transmission.

Progress towards an HSV-2-specific serodiagnostic reagent was facilitated by the identification more than 10 years ago of an HSV-2 glycoprotein, designated 92K or (, G2. al.,1978,1984;Roizmanetal.,1984).Subsequently,theHSV-1counte rpart,(Marsdenet ; GI was identified. Determination and comparison of the DNA sequence of the gelles

andgG2showedthetwoproteinstohavedivergedconsiderably.Theenco dinggG1 existence of serotype-specific epitopes on gG1 and gG2 (Ackerman et al., 1986; Frame et al., 1986; Marsden et al., 1984, Roizman et al., 1984) raised the possibility of the use of gG2asserotype-specificdiagnosticreagents.gG1and Various serological tests for HSV-2 specific antibodies have been developed using lys2. These tests include EL, ISA. immunodot and western blotting assays, and serum blocliino assavs using serotype-specific monoclonal antibodies. For these purposes, gG2 has been obtained from a varietv of sources including HSV-2 infecte mammalian cells, transformed mammalian celles. insect cells infected with recombinant baculoviruses and E. scherichia coli using several expression vectors. E. coli has also been used to express fragments of gG2 containing type-specific regions, and slightly truncated, almost full ofgG2fusedtothemaltosebindingprotein.Antigensfromthesesource slengthfragments have been used either as crude extracts or following purification by a variety of procedures monoclonalantibody-affinityandlectin-affinitychromatography. includingion-exchange, serologicaltestsusinggG2.westernblottingisconsideredthemostA mongstthe reliable (WHO conference, Copenhagen, 1995). However, this method is cumbersome, relatively expensive and not suitable for general screening purposes in other than well- equipped diagnostic laboratories. There is a need for a simple. cheap and reliable whichcanbeusedasanELISA(Enzyme-LinkedImmunoSorbentserodiagno stictest Assay) in microtitre plates.

Summan of the invention It has now surprisingly been found that a 16aa (aa = amino acid) peptide, more preferably an 1$aa peptide, when subtended as multiple separate branches, each branch containing the peptide sequence, on a core, has a high specificity for HSV-2 antibodies and in particular gives few false positives or false negatives. Consequently, such a peptide structure is valable in testino for HSV-2 antibodies, especially in samples of body fluid patientandmostespeciallyinserumsamples.The16aapeptideisthatf romaminofroma acids 562-577 of gG2 and has the sequence SEQ ID NO: 1 set forth hereinafter. Further, it is possible to include a few additional amino acids at either or both ends of this peptide or to tmncate it slightly. The I Saa peptide I-'ds 561-578 of gG2, having SEQ setforthhereinafter,ismostpreferred.IDNO:55

The particularlysurprisingbecausethisisaregionoftheHSV-2is whichhashighhomologywiththeHSV-1glycoproteingG1,McGeochglyco proteingG2 (1987),yetetal. the peptides of arehighlyspecificforHSV-2antisera,givinginvention very few false positives with HSV-1 antisera.

In one aspect, the invention provides a multiply-displayed peptide structure of formula (1): [(X1)p - (16aa sequence) - (X2)q - Sp[n - core, wherein "16aa sequence" represents a sequence (SEQ ID nO: 68) Glu Glu Phe Glu GIN, Ala Glv Asp Glv Glu I'ro Pro Glu Asp Asp Asp 1 10 15 and wherein X 1 and X2, which may be the same or different, represent from I to 6 non- acidresidues,whichmaybethesameordifferentresidues;interferin gamino Sp represents a spacer group extending outwardly from the core; n is at least 4: p is 0 or 1: q is 0 or I: and the lino-age between the core and the spacer group may be chemical or physical.

The invention inclues the use of the peptide structure of the invention in diaonosis of antibodies to HSV-2. In particular, the invention provides a method of testing for antibodies to herpes type2,inwhichabindingreactionisperformedbetweenvirus the antibodies and a peptide structure of the invention, and the occurrence or extent oi binding is detected or measured respectivelv.

Further, the invention inclues a kit for testing the body fluid of a patient for the presence therein of antibodies, comprising (1) a multiply-displayed peptide structure of the invention; and (2) a labelle anti-human immunoglobulin antibody for detection or measurement of binding of the antibodies to the peptide structure.

The invention also inclues per Sev the peptides of formula (2): -(X2)q-(Sp)r,wherein"16aa(X1)p-16aasequence sequence", pandqareasX2, defined above and r is 0 or 1, and N- and C-terminal functional derivatives thereof.

Brief description of the drinjn_a5 showsdetectionofHSV-2andHSV-1antiserabythemethodoftheinventi on:Figure1 Panels A, =HSV-2;panelsB,DandF=HSV1.E

Figure 2 byELISAofserafromindividualsnotinfectedwithHSV.screening Description of embodimentspreferred The multiply-displayed peptide structure of the invention is preferably based on a core of branched lysines. Lysine is a dibasic amino acid, providing it with two sites for lysinecorestructuresarewellknown,havingbeendescribedbypeptid e-bonding.These andTametal.(1989),U.S.Patent5,229,490(Tam,1993),EuropeanPate ntTam(1988) voordeTechnischeWetenschappen)andPCTPublication339,695(Stich tung (MedicalResearchCouncil).Theymaybeusedinthisinvention.WO92/1 8528 Preferablv the structure is all-peptide and of formula (3) <BR> <BR> <BR> <BR> <BR> <BR> [(X)p-1Gaasequence-X-j-Sp]n-(Lvs)n-l-'Jm<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> wherein n is 2a, a beiiia a number from ? to D (i. e. n = 4, 8, 16 or 32), the lysine residues (Lys)n-1beingbranched,X3representsfrom1to4aminoacidresiduesw hichdenotedby ma\,, be the same or different, m is 0 or 1, and the other symbols are as defined above.

Preferably p is 0 or 1, q is 0 or 1, Sp is a spacer of length equivalent tu that of from 4 to 6 glycine residues (and most preferably Sp actually represents from 4 to 6 glycine residues) and m is 0 or 1. Most preferably the peptide structure of the invention is of formula (4): pepseq- (GIY) 4 \ lys pepseq- (Gly) 4/\\ Lvs- :'4) pepseq- (GIY) 4 \ LN, s/ pepseq- (Gly) 4 id residue, and"pepseq"is the sequence of the peptide (X) p-<BR> <BR> <BR> <BR> <BR> <BR> -(X2)q@asdefinedabove.16aasequence It essentialtouseabranchedlysinecore.Thecorecantakemanyforms.It not examplebeasolidinertmaterialontowhichtheremainderofthemolecu leismay,for physically absorbe or chemically bonded, normally by covalent bondit^. It could be, for example a liner or branche polymer, includino e. g. a polyester of trimethylolpropane or a weightpolymideorpolyacrylamideorapolymercontainingdendriticm olecular asinUSPatent4,507,466(Tomaliaetal.,1985).Itmaybeaprintedcirc uitbranches,e.g.

board or siliconchips,wherebywhentheantibodytobetestedbindstothepepti deof structure a change in electrical charge or current occtirs, which can be sensed. Preierably linkaoe to the core is through a covalent bond.

It should be evident from above that it is not necessary that the entire molecule of the peptide structure be compose of amino acids A non-peptide core or non-peptide spacer or non-peptide linkages between one branch and another can be included. However. peptideorwhollyaminoacidstructureisveryconvenientfromasynthe ticviewawholly point.

The spacer arm extends ouwardlv from the core of the structure and comprises anv compatible residue. Most preferably the spacer arm molecuìe comprises residues of amino acids. Preferably, the spacer arm comprises a majoritv of residues of glycine (which is an uncharged polar amino acid) or non-polar amino acids. The term"non-polar amino acid" as used herein inclues alanine. valine, leucine and isoleucine. The preferred length of the spacer arm is at least 4 lycine residues. but e ! en a short spacer. e.g. of length equivalent to a single glycine residue has a small effect. Little advantage is likely in having a lenoln equivalent to more than 6 glycine residues as there may be too much possibility of interference between such highly flexible branches, one with another.

The 16aa sequence SEQ ID NO : 68 can be extended at either its left-hand (N- terminal) or right-hand (C-terminal) end or both with a íew amil1o acid residues which do not interfere with its ability to bind to HSV-2 antibodies with high specificity. These can be those in the"natural"2G2 sequence. Thus, X1 may be Glu His Ara Gly Gly Pro (SEQ ID NO: 69), His Arg Gly Gly Pro (SEQ ID B1O: 70), Arg Gly Gly Pro (SEQ ID NO: 71), the 3aa sequence Gly Gly Pro. the 2aa sequence Glu Pro or the single aa Pro and X2 may <BR> <BR> <BR> be Ser Ala Thr Gly Leu Phe (SEQ ID NiO 72). Ser Ala Thr Gly Leu (SEQ ID NO: 73), Ser Ala Thr Glv (SEQ ID NO: 74), the 3aa sequence Ser Ala Thr. the 2aa sequence Ser Ala or the sinople aa Ser. X1 and X2 may altermatively comprise other amino acid (s). in which case they are preferably non-polar amino acids or glycine or serine. Most preferably. the 16aa sequence is extended by one aiiiiiio acid at either or both endos, especially by either or both of the ad jacent amino acids of the natural sequence which are an N-terminal Pro and a C- Thusthefollowing17aaand18aasequencesareespeciallypreferred:t erminalSer.

GluPheGluGlyAlaGlyAspGlyGluProProGluAspAspAspProGlu 10 15 (SEQ ID NO: 7-S) Glu Glu Phe Glu Gly Ala Gly Asp Gly Glu Pro Pro Glu Asp Asp Asp SCI 1 1510 (SEQ ID NO: 76) Pro Glu Glu Phe Glu Gly Ala Gly Asp Gly Glu Pro l'ro Glu Asp Asp Asp Ser I 10 15 NO:55).(SEQID Preliminary results indicate that peptide structures containing the C-or \- peptidesequencesgiveslightlylessgooddiagnosticresults,interm sterminallyextended of a few more false positives. than"peptide structure 55" containing the 18aa sequence SEQ ID NO: 55.

The whichthepeptidesequencesarederivedand,indeed,thepeptidefrom structures theyarewhollypeptide,canbemadebyanyofthewellknownif above,butsyntheticmethods the especiallythecontinuousflowversion,ismethod, particularlyconvenient.

The invention can be practised in any way appropriate to the detection of antibodies. especially on any appropriate fluid. especiallv a body fluid, likely to contain antibodies.

Although serum is the usual body fluid, other fluids which contain immuno rlobulins. e. y. ormilk,couldbeassayed.tears,saliva, In normal practice. the assay will be carried out heterogeneously by attaching the peptide structure of the invention to an insoluble carrier such as the plastic surface of a microtitre plate. althouah the invention is not limite to such assays. In a sandwich assay, the antiserum is incubated with the peptide structure for an appropriate period to allow binding and with an aiitibodv to the immunoglobulin. Thus, for example, assuming that the patient is a human and the antibody is of the IgG class, an anti-human IgG antibody, appropriately labelled. is added, the plate is washed and the amount oi label boulld to the <BR> <BR> <BR> <BR> <BR> plate (via the antibody and the peptide structure ! is measured. The invention can be applied to detecting IgM antibodies, using an anti-human IgM as the second antibody.

Alternatively, a competition assav can be performed in which a known amount of labelle antibody is permitted to compete with the serum or to displace it from its binding. Such however,islessappropriatefordetectingsmallconcentrationsofan tibody.Anyanassay,

usuallabels.e.g.radiolabelsandenzymelabels,canbeused,especia llyperoxidaseofthe and Anindirectlabel,suchasbiotin,canalsobeusedinconjunctionphosp hatase. appropriatedirectlylabelledbindingpartner,suchaslabelledstre ptavidinorwiththe avidin. Radiolabels assayedbyradioactivecountingandenzymelabelsbyabe orchemiluminescentsignal,forexample.Anamplificationcolorimet ric,fluorescent method of as"Ampak"(RegisteredTradeMark)ofNordiskDiagnosticssuch requiresLtd.,which an enzymelabel,canbeusedtofurthersensitisephosphatase the assay.

The amount of peptide structure to be used per assay, e. g. per well of a plate. will lessthen1µg,preferablylessthen0.1µgandpreferably0.05to0.5 µg.Thenormallybe most suitable amount in anv particular case will depend on the nature of the peptide structure involved.

In a kit for carrying out the assai. the essential components will in most cases be the peptide structure ofthe inuention and an anti-human immunoglobulin labelle antibody.

These are, of course, normally provided in the kit in separate containers.

The invention inclues the peptides of formula (2) per se, since they are useful as intermediates in making peptide structures of the invention. For example_ it mav be convenient to protide peptides lacking a spacer. i. e in uZhich r is 0. readv for attachment to the remainder of the molecule (core plus spacer). rhis is particularly helpful when it is more convenient to purchase the core plus spacer from a supplier, especially if tille core plus spacer is a non-amino acid polymer containing pendant groups, such as aminoethyl groups, readv to be attache to the C-terminus of the peptide by a peptide linkage. Alternatively the peptides may be synthesised with a spacer [r = I in formula (2)], ready to attach to a core. The preferred such peptides are as stated above, the 18aa peptide of sequence SEQ ID b ! O: 55 beino the most preferred.

Also included in the invention are derivatives of such peptides, especially havino aminoprotectinggroups,includingparticularlypeptidesinwhicham inoacidcarboxyand protectedbyt-butyloxycarbonylgroupsandtheN-terminusisprotect edbyside-chainsare 9-fluorenylmethoxycarbonyl (Fmoc). The invention further inclues peptides having a groupsuchasanester,especiallyapentafluorophenylester,"reacti ve"C-terminal peptideslackingthespacer(r=0)."Reactive"meanscapableofreacti ngwithespeciallyfor

free amino groups of the spacer to form an amide bond therewith. Other such reactivc derivatives are well known in the art and included within this invention.

The following Examples illustrate the invention.

EXAMPLES Materials and Ethos Sera and serology A total of 174 serum specimens, vere collecte from 118 individuals. Of these. 155 sera were collecte from 100 patients. Paired sera were collecte froc 55 of these patients.

A further 18 sera were collecte írom 18 children, with a variety of conditions uuelated to HSV. The HSV-specific antibody titres of sera collecte in Edinburgh were determined by complement fixation assay (Smith et al., 1967), while titres in sera collecte in Glasyo\\ were screened anti-HSVIgGELISAkit("Enzgnost"[RegisteredTradeMark].an Behring Diagnostics). The HSV-infected patients from whom sera were collecte xvere adults of average age 27 and approximately equal numbers of males and 1-empales.

Virus isolation and tapina All virus isolates xvere obtained from genital lesions with the single exception ol one that was isolated from the patient's lip. The viruses were types by indirect fluorescence usina a panel of type-specific monoclonal antibodies ("Syva Micro Trank" [Registered Trade Diagnostics).Behring Svnthesis of peptides A series of 67 18aalongoverlappingby10aa,thatspannedaminomostly acids 21-699 of the predicted open radin (y frame of gG2 were synthesised by continuos flow Fmoc chemistrv. Residues 1-20 were not synthesised, as these are thought to comprise the signal sequence that is cleaved from the maturing protein. The peptides were made as multiplv displayed peptide structures of the invention, each structure consisting of four copies of each amino acid sequence subtended on a branche lysine core (Tam. <BR> <BR> <BR> <BR> <P>1988), but each such aiiiiiio acid sequence being separated from the core by four (1lvcille *duels to increase sensitivity. Such peptide structures can be represented by the formula (5):

Glv Giv Gi, -'.'lys pepscq-Glv Gly Gly Gly'-\ prpseq-Gl-Gly Gly GI} \ pepseq-Gly Gly Gly Gly wherein"pepseq"denotes the same amino acid sequence in each occurrence within the formula and is selected fi-oiii the sequences of the peptides listed in one letter code in the following Table 1 and havin sequence identification nulllbers I to 67. They were made on a Shimadzu PSSM-S peptide synthesiser using standard Fmoc procedures described b-\,- the manufacturer and either HBTU [2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium TBTU[2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexa fluorophosphate]of tetrafluoroborate] as coupling reaoent according to the manufacturer's instructions.

Peptides were analysed by reverse phase HPLC and were judged to be at least 80% pure.

Table1 Peptides spanning HSV-2 glycoprotein G Peptide Amino acid residues Sequence of unique region Peptide Amino acid residues Sequence of uniq@ gG2/1 21-38 RGGSGVPGPINPPNSDVV gG2/35 361-378 KTPLPVSATAMA gG2/2 31-48 NPPNSDVVFPGGSPVAQY gG2/36 371-388 MAPSVDPSAEPT gG2/3 41-58 GGSPVAQYCYAYPRLDDP gG2/37 381-398 PTAPATTTPPDE gG2/4 51-68 AYPRLDDPGPLGSADAGR gG2/38 391-408 DEMATQAATVAV gG2/5 61-78 LGSADAGRQDLPRRVVRH gG2/39 401-418 AVTPEETAVASP gG2/6 71-88 LPRRVVRHELPGRSFLTG gG2/40 411-428 SPPATASVESSP gG2/7 81-98 LGRSFLTGGLVLLAPPVR gG2/41 421-438 SPLPAAAAATPG gG2/8 91-108 VLLAPPVRGFGAPNATYA gG2/42 431-448 PGAGHTNTSSAS gG2/9 101-118 GAPNATYAARVTYYRLTR gG2/43 441-458 ASAAKTPPTTPA gG2/10 111-128 VTYYRLTRACRQPILLRQ gG2/44 451-468 PAPTTPPPTSTH gG2/11 121-138 RQPILLRQYGGCRGGEPP gG2/45 461-478 THATPRPTTPGP gG2/12 131-148 GCRGGEPPSPKTCGSYTY gG2/46 471-488 GPQTTPPGPATP gG2/13 141-158 KTCGSYTYTYQGGGPPTR gG2/47 481-498 TPGPVGASAAPT gG2/14 151-168 QGGGPPTRVALVNASLLV gG2/48 491-508 PTADSPLTASPP gG2/15 161-178 LVNASLLVPIWDRAAETF gG2/49 501-518 PPATAPGPSAAN gG2/16 171-188 WDRAAETEEYQIELGGEL gG2/50 511-528 ANVSVAATTATP gG2/17 181-198 QIELGGELHVGLLWVEVG gG2/51 521-538 TPGTRGTARTPP gG2/18 191-208 GLLWVEVGGEGPGPTAPP gG2/52 531-548 PPTDPKTHPHGP gG2/19 201-218 GPGPTAPPQAARAEGGPC gG2/53 541-558 GPADAPPGSPAP gG2/20 211-228 APAEGGPCVPPVPAGRPW gG2/54 551-568 APPPPEHRGGPE gG2/21 221-238 PVPAGRPWRSVPPVWYSA gG2/55 561-578 PEEFEGAGDGEP gG2/22 231-248 VPPVWYSAPNPGFRGLRF gG2/56 571-588 EPPEDDDSATGL gG2/23 241-258 PGFRGLRFRERCLPRQTP gG2/57 581-598 GLAFRTPNPNKP gG2/24 251-268 RCLPPQTPAAPSDLPRVA gG2/58 591-608 KPPPARPGPIRP gG2/25 261-278 PSDLPRVAFAPQSLLVGI gG2/59 601-618 RPTLPPGILGPL gG2/26 271-288 PQSLLVGITGRTFIRMAR gG2/60 611-628 PLAPNTPRPPAQ gG2/27 281-298 RTFIRMARPTEDVGVLPP gG2/61 621-638 AQAPAKDMPSGP gG2/28 291-308 EDVGVLPPHWAPGALDDG gG2/62 631-648 GPTPQHIPLFWE gG2/29 301-318 APGALDDGPYAPFPPRPR gG2/63 641-658 WFLTASPALDIL gG2/30 311-328 APFPPRPRFRRALRTDPE gG2/64 651-668 ILFIISTTLHTA gG2/31 321-338 RALRTDPEGVDPDVRAPR gG2/65 661-678 TAAFVCLVALAA gG2/32 331-348 DPDVRAPRTGRRLMALTE gG2/66 671-688 AAQLWRGRAGRR gG2/33 341-358 RRLMALTEDTSSDSPTSA gG2/67 681-699 RRRYAHPSVRYV@ gG2/34 351-368 SSDSPTSAPEKTPLPVSA

LXAMYLE I assay(ELISA)Enzyme-linkedimmunoabsorbent Sera were tested for reactivitv with a truncated version of HSV-2 glycoprotein D (gD2t), as well as peptide structures containing the gG2 peptide sequences. Glycoproteins D of HSV-1 (gD1) and of HSV-2 (yD ?,) are hiohly homologous witll many epitopes that are antibodieselicitedbybothHSV-1andHSV-2.GlycoproteinDisalsoby an immunodominant protein. These two properties of °D make it a highly sensitive and useful ragent for diacynosis of HSV-specific antibodies. The truncated form contains the first 326 amino acids of gD, but lacks the carboxy-terminal transmembrane domain and is consequently secreted from cells. This property is convenient because the secreted protein can be more easily purifie from the cell culture medium than can whole gD from the cell membrane. but does not discriminate between HSV-1 and HSV-2 antibodies. The gD2t, purifie to homogeneity, following expression from DNA in Chinese Hamster Var celle was provided by SmithKline Beecham Biologicals (Rixensart, Belgium) at a concentration of 600 µg/ml. It was diluted in phosphate buffered saline (PBS) and used to coat microtitre wells (Immunolon l, Dynatech) with 0 5 ci or 0. 25µg in 50 µl.

The peptide structures (see Table 1) were dissolve in water whenever possible.

Peptide structures not soluble in water ère dissolve either in') 0% acetic acid (those shaving the sequences SEQ ID NO: 7, 8, 12, 14, 15, 21, 25, 26, 62) or by bubblinyl a small volume (<2ml) of ammonia vapour through the peptide structure water suspension, whereupon the peptide structure formed a clear solution (those having the sequences SEQ ID NO : l6,17 and 18). Solubilised peptide structures were then diluted in PBS to the concentration indicated in the text and 50µl added to wells. Both gD2t and the peptide structures were allowed to absorb to the plate at 4°C overnight. Antigen solution was removed fi-oiii the plate and unoccupied bindinQ sites on the plate blocked with PBS BSAfor1.5h-2h.Wellswerewashedsixtimeswith150mMNaClcontaining 1% containing 0.05%"Tween 20" ("NaCI-Tween") ("Tween"is a Registered Trade Mark) and were incubated with 50µl serum, diluted as indicated in the text, for 1.5h at room temperature on an orbital shaker. The semm was removed and wells were washed a further six times with "NaCl-Tween". For detection of bound antibody, the wells were incubated

with50µlofbiotinylatedsheepanti-humanIgG(Amersham,diluted1/ 1000)andinturn streptavidin-conjugatedhorseradishperoxidase(Amersham,dilute d1/1000),each50µlof for 1.5h at 37°C followed by six washes with NaCl-"Tween". Chromogenic substrate, o- (OPDA),(50µl)incitrate-phosphatebuffer(pH4.0)phenylenediami nedihydrochloride containing 0.01% hydrogen peroxide was added and aiter 10 minutes colour development \vas stopped bv addition of 50111 2N sulphuric acid. Plates were read on a"Titertek" Multiscan plate reader at 492 nm.

RESULTS ofallpeptidesagainstasubsetofthesera.Preliminaryscreening In this initial expriment all of the peptide structures were screened against a set of 24 sera. Four sera were from patients whose isolated virus was found to be type I and were antibody-positive, with CF titres ranging from 16 to 256. Fifteen sera were from patients rvllose isolated virus was found to be ype ? and were antibody-positive, with CF titres 16to256.Threeserawerefromindividualswhohadnolaboratoryeviden cerangingfrom infectionofHSV in andantibody-negative.Twoserawereantibody-virus-isolation- negative but from patients from whom virus was isolated: HSV-1 was cultured from one of the patients and HSV-2 was cultured from the other. The sera were also screened against gD2t (500ng per well) to confirm the presence of HSV-specific antibodies. Wells treated withoutanyantigenservedascontrols.Inthisinitialscreening,wel lswerewithPBS coated with I. Oktg of peptide and the absorbance values at 492 nm were recorde.

For a peptide to be specific for HSV-2, it should have the following properties. It should react with sera containing antibodies elicited by an HSV-2 infection. In addition, the peptide should not react with sera containing no HSV-specific antibodies or with sera containing antibodies elicited by an HSV-I infection in the absence of an HSV-2 infection.

In the absence of any prior data for the reactivity of the peptides, the data were analysed by two methods. In the first method, a cuit-off value of 0. 25 was chosen on the basis that it could serve as a suitable value for discriminating between HSV-antibody- positive and negative sera. Only the peptide structures containing the peptide sequences SEQ ID NO: 3, 7, 9,107 11, 12 13, 14, 17, 21, 26,55,57,62, and 66,67 produced absorbance values of >02 with all lifteen HSV-2 sera while only the peptide structures sequencesSEQIDNO:39,50,51,55,56,63and64gaveabsorbancecontain ingthe

values withallfiveantibody-negativeseraandonlythepeptidestructures& lt;0.25 containing the sequences S EQ ID NO: 38, 50 and 55 gave absorbance values of <0. 25 with all four HSV-1 sera. Thus, only"peptide structure 55" (havillo sequence SEQ ID NO: 55) met all of the above-defined criteria required of an HSV-2 antibodey-specific reagent. In the second method, the absorbance value of each serum with control, PBS-coated, wells was subtracted from the absorbance values with peptide-coated wells and a cut-off value of 0.1 was chosen. Only the peptide structures containing the sequences SEQ ID NO: 7, 9, 10, 11, 12, 13, 14,17, 23, 26, 39, 57, 62,66 and 67 produced absorbance values of >0. 1 with all fifteen HSV-2 sera while only the peptide structures containing the sequences SEQ ID NO: 50, 51, 55,56 and 64 gave absorbance values of <0.1 with all five antidoby-negative sera and only the peptide structures containing the sequences SEQ ID NO: 37, 38, 39 50, 55 and 64 gave absorbance values of <0.1 with all four HSV-1 sera.'Thus, none of the peptides met all criteria. However. of the four peptide structures containing sequences SEQ ID NO : 39, 50, 55 and 64 that gave absorbance values of <0.1 with all five antibodv- negative and all four HSV-1 sera, peptide struciures havin the sequences SEy 1D'v0: 39- 50 and 64 displayed reactivity with 5. 1 and 4 respectively of 15 HSV-2 sera, while "peptide structure 55"displaved reactivity with 14 of the 15 sera."Peptide structure 55" was therefore considered a likely candidate for type-specific serodiagnosis of HSV.

EXAMPLE 2 Optimisinei the amount of peptide and sera to be used Referring to Fixe. 1 of the drawings, wells were coated with four different amounts oh peptide structure 55 of the invention : 5µg (square), 1µg (diamond), 100ng (circle) and l Ong (triangle). Seven sera were diluted 20-fold followed by six further two-fold-dilutions.

Wells lacking peptide structure and/or senim served as controls (four square grid). Results for three HSV-2 sera of CF titres 16,256 and 16 and three HSV-1 sera of CF titres < 8, 256 and 16 are shown in Figure 1 (panels A. C, E = HSV-2; B, D and F = HSV-1. The HSV-2 sera all showed greater reactivity with the wells coated with peptide structure 55. in contrast with the HSV-1 scra which did not react with the peptide structure 5r abovc Ihe background levels seen vith the PBS control With all four concentrations of peptide structure 55 teste_ the reactivity of the sera was vert similar. A produced an absorbance of greater than 1 when diluted 1 : 40 or less and appeared to be reaching a plateau. In

seraCandEshowedlowerreactivityandtheshapeofthecurvessuggeste dcontrast.both that higher reactivities might be achieved with less dilute sera. To investigate whether even lower amounts of peptide could be used, wells were coated with different amounts ranging from 100ng to 5pg and tested for reactivity with sera A and E. Below 5ng per well there was a marked reduction in signal and no response below 100pg (data not shown).

EXAMPLE 3 Based on these observations all sera were then screened on wells coated with 100ng and 10ng of peptide structure 55 and on wells coated with oD2t: the amount of gD2t was reduced to 250 ng per well to reduce the signal to about that seen with 100 ng of the peptide Structure. Sera were tested at dilutions of 5-fold, 10-fold and 20-fold. Wells containing no peptide structure or protein were included for all dilutions of sera. valuesEstablishingcut-off The 21 sera that were used as HSV-negative controls comprised trom Edinburgh and Glasgow.Theywerejudgednegativebytheabsenceofanyclinicalsympt omsfrom associated andbytheirlackofreactivityinELISAorcomplementfixationHSV assays (see Methods in Example 1). The sera were diluted 5-fold and scrcened by ELISA on wells coated with gD2t (250ng) or peptide structure 55 (either 100ng or lOng) or no control).ThedataforgD2tand100ngofpeptidestructure55areshowni nantigen(PBS Figure 9. A cul-off value of corresponding to the mean plus 5 times the standard deviation (0.166 for gD2t and 0.167 for peptide structure 55) was used for subsequent analysis of other sera and is indicated by the dotted line in the figure.

Analvsis of té sera from HSV-positive patients All sera were screened against wells coated with both I OOng and l Ong of peptide 55, 250ng gD2t and no antigen (PBS control). Most sera were screened at three different dilutions: 5-fold l O-fold and 20-fold though some sera were screened usine only a 5-fol dilution. For each serum, the background absorbance observed without antigen was thevaluesobtainedforthedifferentantigens.Thesecorrectedvalue ssubtractedfrom constituted the data set For analvsis, the sera were grouped into 5 classes. Classes I and 2 comprise sera that were collected from patients presenting with clinical lesions: class l sera had a CF titre of less than 8 while class 2 sera had a CF titre of 8 or greater. Sera comprising cloasses 3

and 4 were collected between 7 and 2U days (class p) or greater than 20 days (class 4) after first presentation. Sera in class 5 ère from patients who had not presented with a primary lesion and form whom a previous serum had a CF titre of at least 8. Here, a primary lesion is defined as a clinically apparent lesion with a Cl-titre of less than 8 or a CF titre that rises 4-fold within 90 dans Sera diluted -',-fold and screened on wells coated with 100ng peptide gave the least number of false negatives. These results are summarised in Table 2 below. A positive pecordedwhentheabsorbancewasabove0.166(usingthemeansplus5tim esresultwas the standard deviation). TableTableNumber of sera positive/ total number tested (°7O) Class of seraHSV-2seraHSV-1 (seetext) gD2tpeptidegD2tpeptide 55(comparison)structure55(comparison)structure (invention)(invention) Cl,/96 (12) 0/26 (0) 4/16 (25) 2/16 (1-)) 1/3 (33) 0/3 (0) 11/14 (79) 6/14 (4p) C3 24/24 (100) 0/24 (0) 15/16 (94) 13/16 (81) C4 8/9 (89) 0/9 (0) 13/13 (100) 12/13 (92) C5 9/10 (90) 0/10 (0) 22/22 (100) 22/22 (100) Referring to Table 2, peptide structure 55 gave no false positives, with HSV-1 serum. Second, the reactivity of boih HSV-1 and HSV-2 sera with gD2t, and 1dS\Z-2 sera with peptide structure 55 xvas lower for those sera taken at the time of first presentation (class I and 2 sera), tllan the reactivity of subsequently sampled sera (clashs'), 4 and 5 sera).

Overall, 95% of HSV-1 sera (41/43) and 98% of'HSV-2 sera (50/51) from classes 3,4 and 5 reacted while92%ofHSV-2class3,4and5sera(47/51)reactedwithpeptidegD2t .

Third,ali(22/22)HSV-2class5reactedwithbothgD2tandpeptides tructure55. structure 55.

Thurs, peptide 55 was shown to be compietely type-specific for HSV-2 antibodies serum.especiallyfordetectingantibodiesafteranintervalofseven daysinhuman following initial presentation of the clinical lesions.

EXAMPLE 4 structuresweresynthesisedcorrespondingtoextensionsattheAddit ionalpeptide N-and C-terminus of"peptide structure 55"contailling the peptide sequence SEQ ID NO: 55 and made in the 4-branched form of formula (5) above, with the 4-glycine spacer. These were tested, along with gD2t, using the peptide structure or protein at (a) I µg or (b) 100 ng per well and in each case with the sera diluted 20-fold. Otherwise the asinExample3.ResultsasshowninTable3below.Peptidestructure55c onditionswere performed the best from the point of view of not giving false positives for HSV-1.

EXAMPLE5 Two peptide structures of the invention, peptide structure 55 and a peptide structure containing a peptide sequence extended by two aa of the"natural"sequence at each end, were commpared with a peptide structure in which the peptide sequence was extended at the N-terminal end but truncated at the C-terminal end bv 10 aa and 8 aa repectively. This comparison shows the criticality of the 16 aa core sequence of the present invention. All three peptides were made in the 4-branched, four glycine-spaced forez of fonnula (5). The) were all compared with the protein gD2t, as in Example 3, with 100 ng peptide structure or 250 Ho protein per well and a 5-fold dilution of the sera. It will be seen that the two peptide structures of the invention performed far better than that having the comparative peptide sequence SEQ ID NO: 86 from all points of view and that the peptide structures of the invention gave no or few false positives for HSV-1.

The following claims define some important aspects of the invention, but do not purport to include aspectforwhichprotectionmightbesoughtinconceivable subsequent continuing and foreign patent applications. and should not be construed as thegeneralityoftheinventiveconceptshereinbeforedescribed.det ractingfrom Table 3 SEQ Sera from Sera from individua individuals not ID infected with HSV infected with infecte@ NO. ("black") HSV-1 HSV-2 1 µg 100 ng 1 µga 100 ngb 1 µgc Total number of sera tested 3e 3 39 40 43 Peptides of formauia (5): "pepseq" sequence PEEFEGAGDGEPPEDDDS ("55") 55 0 0 0 0 28 GGPEEFEGAGDGEPPEDDDS 77 1 0 3 2 32 PEEFEGAGDGEPPEDDDSAT 78 0 0 0 0 25 GGPEEFEGAGDGEPPEDDDSAT 79 1 0 2 1 33 HRGGPEEFEGAGDGEPPEDDDS 80 1 0 2 1 32 PEEFEGAGDGEPPEDDDSATGL 81 1 0 3 1 32 HRGGPEEFEGAGDGEPPEDDDSATGL 82 1 0 3 0 31 EHRGGPEEFEGAGDGEPPEDDDS 83 1 1 4 1 31 PEEFEGAGDGEPPEDDDSATGLA 84 1 0 2 2 28 EHRGGPEEFEGAGDGEPPEDDDSATGLA 85 1 0 5 2 29 gD2t (for comparison) - 0 0 24 24 35 Table 4 SEQ Sera from individuals ID NO: not infected infected with infected with with HSV HSV-1 HSV-2 Total number of sera tested 3 41a 43b Peptides of formula (5) : "pepseq" sequence PEEFEGAGDGEPPEDDS ("55") 45 0 0 32 GGPEEFEGAGDGEPPEDDSAT 79 0 3 33 APPPPEHRGGPEEFEGAGDG 86 1 9 22 (for comparison) gD2t (for comparison) 0 27 35 - a. 15C1, 13C3, 6C4, 7C5. See Ex. 3. The 15 C1 sera gave only 5 reactions out of a total of 60 possible.<BR> b. 9C1, 11C3, 6C4, 17C5. See Ex. 3. The 9 C1 sera gave only 7 reactions out of a total of 36 possible.

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