WO1998003543A1

WO1998003543A1 - Identification of short peptide sequences representing epitopes of glycoprotein g of hsv-2 using a phage peptide display library

Info

Publication number: WO1998003543A1
Application number: PCT/GB1997/001990
Authority: WO
Inventors: Anna Marie Grabowska; William Lucien Irving; Peter Laing
Original assignee: Peptide Therapeutics Limited
Priority date: 1996-07-24
Filing date: 1997-07-24
Publication date: 1998-01-29
Also published as: AU3630997A; AU3630197A; GB9615533D0; WO1998003544A1

Abstract

The invention provides a polypeptide consisting of 3 to 38 amino acid residues, having the sequence of SEG ID:1 or a portion thereof; and analogous polypeptide derivatives by virtue of point mutation, amino acid substitution, deletion or addition; SEQ ID:1 = A?1PPP4PE6H7R8GGPEEF14EGAGDG20 EPP23¿EDDDSATGLAFRTPN38 wherein the sequence includes histidine residue H7; and wherein E6 may be substituted in order of preference by D6 > T6; and wherein R8 may be substituted by A8; which sequence is recognised by anti-gG2 positive human sera from patients with HSV-2 infection and is not recognised by anti-gG2 negative sera from patients with HSV-1 infection; and antibodies raised thereto; and prophylactic and therapeutic and diagnostic uses thereof relating to HSV-2 infection.

Description

IDENTIFICATION OF SHORT PEPTTDE SEQUENCES REPRESENTING EPITOPES OF GLYCOPROTEIN G OF HSV-2 USING A PHAGE PEPTTDE DISPLAY LIBRARY

Introduction

Identification of antigenic regions within viral proteins subserves a number of functions. Detailed knowledge of epitopes which induce protective immune responses may allow generation of prophylactic subunit vaccines; synthetically- derived antigenic epitopes may be used in diagnostic assays to detect virus-specifIC antibodies; mapping of epitopes within a whole protein may provide important clues as to the three-dimensional structure of that protein, and may enhance understanding of the mechanisms of immune escape adopted by the virus .

The recently described phage peptide display library technology (16,17) is a powerful tool for the identification of individual epitopes recognised by antibodies . Phage peptide libraries typically comprise more than 10⁷ different phage clones, each expressing a different peptide, encoded in the single-stranded DNA genome as an insert in one of their coat proteins. Phage clones displaying peptides able to mimic the epitope recognised by a particular antibody are selected from the library by the antibody, and the sequences of the inserted peptides deduced from the DNA sequences of the phage clones. This approach has the maior advantages that (l) no prior knowledge of the primary sequence of the target antigen is necessary, di) epitopes represented within the antigen either by a linear sequence of amino acids (linear epitope) or by the spatial juxtaposition of ammo acids distant from each other within the primary sequence (conformational epitope) are both identifiable, and (m) peptidic mimotopes of epitopes derived from non- proteinaceous molecules such as lipids and carbohydrate moieties can also be generated (12, 14).

Herpes simplex virus type 2 (HSV-2) is the mam cause of recurrent genital herpes (1) The vast majority of individuals infected with HSV-2, however, give no clinical history of disease, and yet these asymptomatic individuals will shed virus from epithelial surfaces at intervals, and are therefore an infection risk for their sexual partners (9,11). Establishment of serological assays which can distinguish between antibodies to HSV-1 and HSV-2 is difficult due to the considerable shared antigenicity of the two viruses. Nevertheless, such assays would find wide application in the development of rational programmes designed to reduce transmission of infection, eg to identify sexual partners who are discordant for HSV 2 infection as part of a strategy to reduce the incidence of neonatal herpes simplex infection, a disease of high morbidity and mortality (2, 4, 11) .

The glycoprotem G (gG) molecule of HSV-2 has a large insert (over 500 amino acids) compared with its counterpart m HSV- 1 (15) , and has therefore attracted much attention as a likely source of type-specific antigens. Indeed, gG-2-based assays for the detection of HSV-2 antibodies using Helix pozπatia-puπfled gG-2 as antigen in immunoblot and ELISA formats have been described (6, 7, 13) . However, difficulties in large-scale production of gG2 of sufficient purity have precluded the widespread availability of such assays . Following the work of McGeoch et al ^αs> , WO 90/13652 (Triton Biosciences Inc.) disclosed proteins and polypeptides from the unique DNA sequence of about 1461 base pairs (coding for about 486 ammo acids) of the HSV-2 envelope glycoprote G gene, which sequence was not found in the HSV-1 gene. This was suggested to provide epitopic regions type specific for HSV-2 and not for HSV-1.

An alternative approach to the use of whole gG2 would be to construct an assay using synthetic peptides representing key gG2 epitopes as antigen. We describe the use of a phage library expressing random 15-mer peptides to identify a variety of peptide sequences recognised by 3 monoclonal antι-gG2 antibodies. Proof is provided that at least some of these peptides are also recognised by human sera known to contain antι-HSV-2 antibodies, thus validating this approach towards the development of a cheap and widely applicable assay for the detection of human antι-gG2 antibodies.

The present invention provides a polypeptide consisting of 3 to 38 amino acid residues, having the sequence of ΞEQ ID:1 or a portion thereof; and analogous polypeptide derivatives by virtue of point mutation, amino acid substitution, deletion or addition;

SEQ ID:1 A¹PPP⁴PE⁶H⁷R^βGGPEEF^X4EGAGDG²⁰ EPP²³EDDDSATGLAFRTPN^3β

wherein the sequence includes histidine residue H⁷; and wherein E⁶ may be substituted in order of preference by D⁶>T⁶,- and wherein R⁸ may be substituted by A^β; which sequence is recognised by antι-gG2 positive human sera from patients with HSV-2 infection and is not recognised by anti- gG2 negative sera from patients with HSV-1 infection.

The invention also provides a polypeptide which is antigenic .

The invention also provides a polypeptide which is immunogenic and is capable of inducing antibodies in an immunised host against type-specific HSV-2 gG.

The invention also provides pharmaceutical composition containing as an active ingredient an immunogenic polypeptide .

The invention also provides a vaccine composition containing as an active ingredient an immunogenic polypeptide together with a physiologically acceptable adjuvant and/or carrier and/or diluent.

The invention also provides an antibody to the polypeptide obtainable by immunisation of a host with the immunogenic polypeptide .

The invention also provides a recomb ant DNA molecule comprising a DNA sequence encoding a polypeptide.

The invention also provides a filamentous bacteriophage including, in at least a proportion of its major coat protein sub-units, multiple display of a polypeptide.

The invention also provides a vaccine composition comprising a bacteriophage together with a physiologically acceptable adjuvant and/or carrier and/or diluent. The invention also provides a substantially pure non- glycosylated .polypeptide.

The invention also provides a method of testing for the presence of type-speci ic HSV-2 gG2 antibodies a fluid, which comprises contacting the fluid with one or more polypeptide (s) and testing whether or not antibodies bind to the polypeptide (s) .

The invention also provides a method of testing for the presence of type-specific HSV-2 gG2 antibodies in a fluid, which comprises contacting the fluid (1) with a labelled form of one or more polypeptide (s) and (11) with antibodies, whereby antigen in the fluid competes with polypeptide (s) in binding to the antibodies.

The invention also provides a test kit for testing for the presence of HSV-2 type specific antibodies a fluid, which comprises :

(I) a solid phase on which is immobilised one or more polypeptide (s) ; and

(II) means for detecting binding of antibodies to polypeptide (s) .

The invention also provides a test kit for testing for the presence of HSV-2 type specific antibodies in a fluid, which comprises :

(1) a solid phase on which is immobilised one or more polypeptide (s) m labelled form;

(li) antibodies; and

(II ) means for detecting competitive binding of antibodies to polypeptide (s) . The invention also provides a method of diagnosis of HSV-2 infection which comprises employing the test method.

The invention also provides a method of diagnosis of HSV-2 infection which comprises employing the test kit in the test method.

The invention also provides a method of treatment of HSV-2 infection which comprises administration to an infected patient of an immunologically therapeutically effective amount of the vaccine composition.

The invention also provides a method of treatment of HSV-2 infection which comprises administration to an infected patient of an immunologically therapeutically effective amount of the antibody.

The invention also provides a method of prevention of HSV-2 infection which comprises administration to a patient a prophylactically effective amount of the vaccine composition.

The invention also provides a method of prevention of HSV-2 infection which comprises administration to a patient a prophylactically effective amount of the antibody.

The invention also provides a polypeptide which is the sequence SEQ ID: 2 consisting of A¹ to G²⁰ of SEQ ID:1 (PT71) .

The invention also provides a polypeptide which is the sequence SEQ ID: 3 consisting of P⁴ to P²³ of SEQ ID:1 (PT 487) . The invention also provides a polypeptide conjugated to a Bιotm-NH₂ terminus by a lysme (K) group.

The invention also provides a polypeptide which is the sequence SEQ ID: 42 consisting of A¹ to F¹⁴ to SEQ ID: 1 (PT444) .

Surprisingly, the sequence SEQ ID:1 according to the invention corresponds to a region downstream of that targeted in WO 90/13652, this region having some HSV-2 unique portions but also some homology to HSV-l portions. Despite this HSV-l homology polypept des from this region and antibodies raised thereto can exhibit good HSV-2 type specificity. The selection of the claimed region of the native sequence to provide polypeptides, and their serological and antigenic specificity, is unexpected in view of the teaching of the prior art towards using unique HSV-2 regions. It is particularly interesting and surprising that truncated versions of SEQ ID: 1 which are shorter than SEQ ID:2 (PT71), such as SEQ ID:42. (PT444), by virtue of omitting a downstream portion having homology with HSV-l are able to show type-specific HSV-2 activity which appears to be less HSV-2 -specifIC than the longer version (PT71) which includes a region having HSV-l homology. On the basis of prior art teaching one might expect the inclusion of parts of a sequence not unique to HSV-2 might lead to a reduction in the ability to distinguish between HSV-2 and HSV-l in a diagnostic test.

From the foregoing, PT71 SEQ ID: 2 is currently the most preferred polypeptide for use m a diagnostic, by virtue of its considerable ability to distinguish HSV-2 from HSV-l. The invention is described with reference to the accompanying .drawings which:

Figure 1 shows a) Inhibition of binding of H5 to gG2 by phage clones

Two phage clones ( 2.10 ♦ and 3.15 ■ ) selected by mAb H5 are able to inhibit binding of H5 to gG2 ; no inhibition is seen with wild-type phage (M13 ▲ )

b) Inhibition of binding of E5 to gG2 by phage clones Phage clone (12.18 ■ ) selected by mAb E5 is able to inhibit binding of E5 to gG2 , while no inhibition is seen with wild-type phage (M13 A ) . Inhibition by phage clone 12.17 (♦) is weak at the concentrations shown here, but at higher phage concentrations, inhibition of up to 70% was achieved .

c) Inhibition of binding of Fll to gG2 by phage clones

Two phage clones ( 8.22 ♦ and 9.4 ■ ) selected by mAb Fll are able to inhibit binding of Fll to gG2 ; no inhibition is seen with wild-type phage (M13 ▲ )

Figure 2 shows a) Inhibition of binding of H5 to gG2 by synthetic peptides

Peptides Chl6685 (•) and PT73 (▲) , with sequences derived from phage clone inserts 2.10 and 3.15 respectively, and PT71(^) , with sequence derived from gG2 , were able to inhibit binding of H5 to gG2 at all concentrations tested, but inhibition was seen with PT72 (■) , a scrambled version of PT71 only at 500ug/ml. Reduced inhibition was seen w th peptide PT74 (X) , which ammo acids derived from pVIll at the N-terminal side of the insert were omitted, compared with PT73 (A) However, PT75(Z) which lacked the PFT motif present in PT73 was still able to inhibit The 8mer peptide PT156 (0) , a .shortened version of PT71 was also able to inhibit binding of H5 to gG2. Details of the sequences of these peptides are given in Table 2.

b) Inhibition of binding of E5 to gG2 by synthetic peptides

Peptides Chl6688 (♦) and Chl6689 (■) , with sequences derived from phage clone inserts 12.18 and 12.17 respectively and PT71 (A), with sequence derived from gG2 , were able to inhibit binding of E5 to gG2 , but no such inhibition was seen with PT166 (0) Details of the sequences of these peptides are given in Table 2.

c) Inhibition of binding of Fll to gG2 by synthetic peptides

Peptide Chl6687 (■) with sequence derived from phage clone insert 8.22 and PT173 (A) , with sequence derived from gG2 , were able to inhibit binding of Fll to gG2. No inhibition was seen using Chl6686 (♦) , with sequence derived from phage clone insert 9.4 Details of the sequences of these peptides are given in Table 2.

Figure 3 shows

Reactivity of human sera with peptides

The reactivity of human sera with four different peptides is illustrated :PT71, derived from gG2 native sequence containing epitopes recognised by mAbs H5 and E5 (Fig. 3a) ,

PT75, derived from phage insert selected by mAb H5 (Fig. 3b) , Chl6687 derived from phage insert selected by mAb Fll (Fig. 3c), and PT173, derived from gG2 native sequence, containing epitope recognised by mAb Fll (Fig. 3d) . The sera were used at a dilution of 1:25 and fall into 4 groups based on the presence of antibodies to HSV-l and HSV-2 proteins detectable by Western Blotting: 1) antibodies to neither HSV-l nor HSV-2; 2) antibodies to HSV-l only; 3) antibodies to. HSV-2 only; 4) antibodies to both HSV-l and HSV-2.

Figure 4 shows 92 human sera were tested for their reactivity with both PT71 and gG2. The results are shown as a graph of reactivity with PT71 against reactivity with gG2. There was a correlation coefficient of 0.61 using a Pearson correlation test, giving a probability of p<0.0001 that these results correlate by chance.

Figure 5 shows the results of an experiment in which 10 positive and 5 negative sera were used to stain the peptides whose sequences are given in the table, which had been synthesised on membrane. Positive sera are defined here as sera which are reactive with gG2 in ELISA and were taken from patients who were culture positive for HSV- 2 at the time the serum sample was taken; negative sera are those which do not react with gG2 in ELISA and were taken from patients who were culture positive for HSV-l at the time the serum sample was taken.

Figure 6 shows graphs of reactivity with biotinylated peptides SEQ ID:2 (PT71) and SEQ ID:3 (PT487) , and a control peptide SEQ ID: 44 (PT482) . The test used a panel of " positive" and " negative" sera in ELISA against streptavidin-coated plates, with or without the biotinylated peptide attached. The results are shown as OD with buffer alone or with peptide. Thus, one is looking not at the magnitude of the OD alone, but at the difference in OD when the peptide is added to the plate. This therefore takes into account any reactivity with streptavidin alone. SEQ ID: 44 is PT482 and has the sequence PPEHRGGPEEFEGAGDGEPP-K-Biotin- NH₂

Figure 7 shows Western blot results of four experiments showing the ability of polypeptides of the invention (PT71 and PT444) to distinguish between HSV-l and HSV-2 positive, +/-, -/+ and negative serum samples, as compared to a gG2 fragment (PT 445) and Gg2.

PT444: SEQ ID: 42: A'PPPPEHRGGPEEF¹⁴ PT445: SEQ ID:43: KTPPTTPAPTTPPPTSTHAT

Materials and Methods

Monoclonal antibodies

Anti-gG2 monoclonal antibodies (mAbs), O2E10.A3.H5, 01B9.E5,

P4A10.F11 (abbreviated to H5 , E5 and Fll respectively throughout) , in the form of culture supernatants were used. All mAbs are positive against gG2 in ELISA. H5 was used at a dilution of 1:100, and E5 , Fll at a dilution of 1:200, as this was found to be optimal m ELISA against gG2.

Phage peptide display library

The library used was a gift from Dr. G. Smith (Missouri, USA) containing approximately 10⁸ different phage clones based on the filamentous phage fd-tet which is composed of the genome of the filamentous phage fd and a segment of the transposon TnlO, coding for tetracycline resistance, thus allowing the selection of infected host bacteria by plating out in the presence of tetracycline. In addition to a wild- type gene VIII encoding the major coat protein pVIII, the phage in this library were engineered to express a recombinant form of gene VIII containing a degenerate DNA insert encoding random 15-mer peptides (Smith, personal communication) and are, therefore, type 88 vectors (18) . The recombinant gene VIII is under the control of a tac promoter; the ratio of the peptide-displaying to wild-type pVIII can, therefore, be altered by varying the concentration of iso-propyl-thio-galactose (IPTG) added to the host bacterial culture.

Bacteria

The K91Kan strain of E. coli , a λ-derivative of K-38 was used throughout. It is Hfr Cavalli and has chromosomal genotype thi . Bacteria were cultured in LB medium (Sigma), with the addition of kana ycin (50 g/ml) , tetracycline (20 g/ml) or IPTG (ImM) where appropriate.

Infection of bacteria

Infections were carried out by incubating phage for 30 mins at room temperature (RT) with an equal volume of K91Kan, grown to log-phase in LB containing kanamycin. LB containing an inducer tetracycline concentration of 1 g/ml was added and the bacteria were incubated for a further 45 minutes at 37°C.

Preparation of polyethylene glycol (PEG) -precipitated phage

Phage were purified from the culture supernatants of infected bacteria by addition of l/5th of the volume of 20% PEG/2.5M NaCl, followed by incubation for lhr at 4°C. The precipitated phage were pelleted, resuspended in Tris- buffered saline (TBS) , and the PEG precipitation was repeated. Phage from a culture supernatant volume of 5ml were usually esuspended a final volume of 150 1 of TBS.

The optical density was then read at 269nm and the concentration of the phage preparations were standardised to 150ug/ml, assuming that an O.D. of 1 is equivalent to a concentration of 3.8mg/rnl.

Biopann ng

Three rounds of biopanning were carried out with each mAb During the first round, ELISA wells (Nunc Maxisorp) were used as the solid phase; they were coated with aliquots of mAb over night at RT in a humid atmosphere, washed in TBS, blocked in TBS-1% BSA, then washed m TBS-0.05% BSA. One aliquot of the library containing 10¹⁰ phage in 50 1 TBS- 0.05% BSA was added to the antibody-coated well, for lhr at RT. Unbound phage were removed and the wells were washed 4 times in TBS-0.05% BSA and 4 times in TBS 50 1 of elution buffer (0.2M glycme, 0.1M HC1, 0.1% BSA, 0. lmg/ml phenol red, pH 2.2) were added for 10-20 seconds, then removed and neutralised by addition of Tris-HCl pH8.8 (Sigma T5753) The phage eluted from each antibody were used to infect log phase K91Kan, then grown over night LB containing tetracycline. They were purified by PEG-precipitation.

The second and third rounds of biopanning were carried out using a 20 1 aliquot of Goat anti-mouse coated dynabeads (Dynal) as the solid phase. The beads were washed 4 times in TBS, incubated with a 50 1 aliquot of the mAb, then washed and blocked. During round 2, a 50 1 aliquot of the PEG precipitated phage from round 1 was incubated with the mAb-coated beads, then washed. Bound phage were eluted, amplified and purified by PEG-precipitation as m round 1. During round 3, PEG-precipitated phage from round 2 were used. Again,, bound phage were eluted, amplified and purified by PEG-precipitation. Phage eluted during the third round of biopanning were used to infect bacteria which were then plated out at a low concentration on LB-agar tetracycline plates to allow individual phage clones to be isolated.

ELISA to identify positive phage clones

ELISA wells (Nunc Maxisorp) were coated by incubating overnight with Rabbit anti-fd antibodies (Sigma) diluted 1:1000 in coating buffer (carbonate-bicarbonate buffer, pH 9.6) . After each incubation the wells were washed with PBS- 0.05% Tween 20. The plates were blocked by addition of PBS- 0.05% Tween 20-1% BSA (blocking buffer) Individual phage clones were grown overnight LB containing tetracycline and IPTG, to maximise expression of the recombinant form of gene VIII containing the peptide insert. The rabbit anti-fd coated wells were incubated in turn for 1 hour at RT with supernatant from such cultures, the test mAb diluted m blocking buffer (dilutions as described above) and alkaline phosphate conjugated Goat anti-mouse IgG (Sigma A1682) diluted to 1:1000 in blocking buffer. pNPP at lmg/ml n diethanolamme buffer (10% diethanolamme, pH 9.8, 0.5mM MgCl₂, 0.02% sodium azide) was used as a substrate for the alkaline phosphatase and the O.D. of each well was read at 405nm.

Sequencing ssDNA was prepared from 1.5ml overnight cultures by PEG precipitation followed by phenol -chloroform extraction and ethanol precipitation . Sequencing was carried out using a Sequenase Version 2.0 T7 DNA polymerase kit (Amersham) according to the manufacturer s instructions. The oligonucleotide AGCAGAAGCCTGAAGAGAGTC (SEQ ID: 4), complementary to the genomic DNA of the phage 3' of the insert, was used as a primer.

Peptides

Peptides were a gift from Peptide Therapeutics Ltd. (Cambridge, UK) . They were synthesised by standard f-moc methodology.

Inhibition ELISAs

Wells were coated with Helix Poma tia lectin-purifled gG2 at a dilution of 1.500 coating buffer. After blocking, peptides or phage were added simultaneously with the mAb diluted blocking buffer. The mAbs were diluted by a factor of 1:2 compared with the concentration used m the ELISA above. Binding of the mAb was detected using the same procedure as the ELISA above.

gG2 and Peptide ELISAs

ELISA wells (Nunc Maxisorp) were coated by incubating over night with peptides at 5ug/ml n PBS. After each incubation the wells were washed with PBS-0.05% Tween 20. The plates were blocked by addition of a 1:10 dilution of Boehrmger Mannheim ECL blocking solution (Cat. No. 1500 694) in PBS. Incubation buffer was a 1:20 dilution of this reagent PBS. Wells were incubated in turn with serum diluted 1:25 and horse radish peroxidase conjugated Rabbit F(ab)₂ anti- human IgG (Da.ko P0406) diluted to 1:1000 m PBS-10%NGS. Sigma Fast OPD tablets (Sigma P9187) were used as a substrate for the peroxidase and the O.D. of each well was read at 490nm after stopping the reaction with 2M H₂S0₄.

Human sera

24 patient sera were collected at the Virology Department, Centre for Infectious diseases and Microbiology, Westmead Hospital, Sydney, Australia. These had previously been characterised by Western Blotting (7) for the presence of IgG reactive with HSV-l and HSV-2 proteins, and fall into 4 groups of 6 sera based on those reactivities : no antibodies to either HSV-l or -2 (group 1, antibodies to HSV-l only (group 2); antibodies to HSV-2 only (group 3); antibodies to both HSV-l and -2 (group 4) .

Results

Selection of phage clones. 3 mAbs (H5, E5, Fll) with specificity for gG2 were used to screen the library of phage containing random 15-mer peptide inserts. After three rounds of biopanning, individual phage clones were isolated and screened by ELISA to identify those which bound strongly to the antibody of interest, and those which gave a clear positive signal were sequenced. The sequences of the phage clone inserts are given m Table 1. Note that SEQ ID: 14 " native" is PT71; the same as claimed in claim 21 SEQ ID:2. Identification of motifs amongst the sequences of the phage clone inserts and within the native sequence of gG2. Motifs could be identified amongst the phage clones for mAbs H5 , E5, and Fll using Clustal W (1.4) for Multi Sequence Alignment (http://biology.ncsa.uiuc.edu/BW/BW.cgi), followed by minor manual adjustment. For mAb H5 , it can be seen that the motif ([D/E]HRS) tended to appear at the N-termmal side of the 15-mer insert. We postulated that adjacent ammo acids derived from the natural protein VIII sequence may have contributed to the antibody binding site, and therefore have included these ammo acids (PAE) in the alignment. The sequence of gG2 was then scanned using Clustal W - Multi Sequence Alignment program

(http://biology.ncsa.uiuc.edu/BW.BW.cgi) to identify regions with sequence similarity to these motifs (native sequence, Table 1) .

Inhibition of binding of the mAbs to gG2 by phage clones.

If the inserts present in phage clones selected by the mAbs truly contained epitopes or mimotopes of the native antigen, then such clones should inhibit binding of the relevant mAb to gG2. To test this hypothesis, two representative phage clones for each mAb were used in an inhibition assay. Each phage clone was used at a range of concentrations. For each mAb, wild-type phage M13 was used as a negative control to ensure that inhibition of binding of the mAb to gG2 was due to the phage insert rather than the mere physical presence of the phage. The percentage inhibition, compared with wells to which no phage were added, was calculated. The results are shown in Figure l(a-c) . For each mAb, both phage clones tested were able to inhibit binding of the mAb to gG2 although the degree of inhibition varied for different clones. Inhibition of E5 by 12.17 was particularly low at the range of concentrations shown Fig. lb, but when it was used at higher concentrations, up to 2.5mg/ml, inhibition of. as much as 70% was observed. In comparison, little inhibition was observed using the wild-type phage M13 over the same range of concentrations.

Inhibition of binding of the mAbs to gG2 by peptides representing phage inserts or the primary amino acid sequence of gG2. Further proof that the epitopes of gG2 recognised by each of the mAbs were indeed represented by the phage clone inserts was sought by testing a number of synthetic peptides for their ability to inhibit binding of the mAbs to gG2. The sequences of the peptides used are given in Table 2. For mAbs H5 , Fll and E5, two peptides, with sequences derived from the inserts of phage selected by that mAb, and one peptide derived from the native sequence of gG2 with most similarity to the motif common to phage selected by the mAb (native sequence, table 1) were tested. At least one irrelevant peptide was included in each assay as a negative control.

For mAb H5 three further peptides were used : d) PT74 , to test the hypothesis that phage ammo acids at the N-terminal side of the insert were contributing to the antibody-bmding site, di) PT75, to investigate the importance of a second motif (PFT) apparently common to some of the phage selected by this antibody, though not selected by ClustalW as a motif, and (in) PT156, to localise the sequence of importance withm gG2.

Peptides were added at a range of concentrations from 500μg/ml to 7.5 μg/ml . The percentage inhibition, compared with wells to which no peptide was added, was calculated. Binding of mAb H5 to gG2 was inhibited by both peptides PT73 and Chl6685 with sequences derived from phage clone inserts 3.15 and 2.10 respectively, and by the peptide PT71 derived from the sequence of gG2 (Fig. 2a) . The inhibition of binding of H5 to gG2 was clearly dependent on the sequence of the peptides as PT72, a scrambled version PT71, did not have this effect. In phage clone 3.15, from which the sequence of PT73 was derived, the amino acids at the N- termmal side of the insert were clearly necessary for the formation of the epitope recognised by H5 ; binding of H5 to gG2 was not inhibited by peptide PT74 which was identical to PT73 except that instead of the 5 phage ammo acids (PAEGD) at the N-terminal side of the insert, 5 phage ammo acids (MLSFA) from the C terminal were added. However, this was not the case for phage clone 2.10 as a peptide with sequence derived from its insert only (Chl6685) was able to inhibit H5 binding as effectively as PT73. Another apparent motif, PFT, common to a number of the phage clones selected by H5 was not essential as these ammo acids could be deleted, as m peptide PT75, without preventing the peptide' s ability to inhibit binding of H5 to gG2 (Fig. 2a) . The region of gG2 which is involved in binding H5 was further localised by the use of peptide PT156, an 8mer peptide derived from PT71, which was also able to inhibit binding of H5 to gG2 (Fig. 2a) .

Similarly, binding of E5 to gG2 could be inhibited by both of the peptides Chl6688 and Chl6689, derived from phage 12.18 and 12.17 respectively, and by PT71, derived from native gG2 sequence (Fig. 2b); binding of Fll to gG2 could be inhibited by the peptide Chl6687, derived from phage 8.17, and PT173 , derived from native gG2 but not by Chl6686 derived from phage 9 4 (Fig 2c) .

Cross-inhibition of mAbs by peptides. The above experiments indicated that peptides with sequences derived from the insert of phage clones selected by a particular mAb were able to inhibit binding of that mAb to native gG2. Similarly, peptides with sequences derived from the known primary sequence of gG2 were also able to inhibit H5, E5, and Fll binding to gG2. All of these inhibitory peptides were then tested at a single concentration (250ug/ml) for their ability to inhibit binding of the other mAbs to gG2 (Table 3) .

With one exception, peptide sequences selected by one mAb did not inhibit binding of heterologous mAbs to gG2 , a result to be expected if the 3 mAbs did indeed recognise separate epitopes within gG2. The exception was peptide Chl6689, derived from phage clone 12.17 selected by mAb E5. This peptide also inhibited H5 , though not Fll Peptide PT71, which inhibited both H5 and E5 has sequence derived from gG2 and contains the motif recognised by both mAbs.

Binding of human sera to peptides. When the peptides were bound directly to wells of an ELISA plate only peptides PT71, PT75, Chl6687 and PT173 were reactive with their associated mAbs (results not shown) . A panel of 24 human sera, whose anti -HSV-l and -2 reactivity had previously been determined by Western blotting were tested for their ability to bind to these peptides. The results are illustrated in Fig. 3. The binding of all sera lacking any anti -HSV reactivity (group 1) or containing only antι-HSV-1 antibodies (group 2) to all 4 peptides was very low. In contrast 9/12 sera known to contain antι-HSV-2 antibodies were reactive with PT71 (Fig. 3a) , 7/12 with Chl6687 (Fig. 3c) and 8/12.with PT173 (Fig. 3d) . Clearly, sera from group 3 (with both antι-HSV-1 and -2 reactivity) showed the greatest reactivity with these peptides. None of the sera were reactive with PT75.

Discussion

Using the phage library technology, we have identified peptides which are able to mimic 3 epitopes of gG2. The epitopes are defined by 3 mAbs, H5 , E5, Fll which were used to select phage from a library of approximately 10⁸ different phage expressing random 15mer peptides as a part of the major coat protein. A number of filamentous phage libraries expressing random peptides have been described, varying in terms of the size of the peptide insert, the coat protein used to display the peptide, and in the presence or absence of constraints on the flexibility of the inserted peptides (5) . Each library has its particular advantages and disadvantages. We chose to use an unconstrained 15-mer library expressed protein VIII. The increased length of this insert may allow development of internal secondary structure, so increasing the possibility that the insert, when synthesised as an isolated peptide, will adopt the same conformation as the inserted peptide. A potential disadvantage of this effect is that any secondary structure within the insert could impair recognition of a sequence motif common to selected phage clones, as the relevant ammo acid residues within the inserts mediating binding to antibody will not necessarily be contiguous in the insert sequences. However, as our primary aim was not to identify the specific am o acid - antibody contact residues, but rather to identify peptide sequences capable of binding to antι-gG2 monoclonal antibodies, this was deemed not to be a problem.

Positive phage clones recognised by each mAb were identified by ELISA and assayed for their ability to inhibit binding of the relevant mAb to gG2 to verify that the interaction between the mAb and phage was occurring through the antigen- specific domain of the antibody. One would expect a given test mAb to select multiple phage clones whose inserts are structurally similar to each other, and to the epitope against which the mAb was raised. Comparison of the ammo acid sequences of the inserts of a number of selected phage clones may, therefore, lead to recognition of a motif of commonly recurring residues. This information can then be used to scan the native sequence of the target antigen (if known) m order to determine whether the motif is present a linear format within that sequence. Such an analysis of the sequences of positive phage clones for three of the mAbs revealed common motifs, different for each mAb, suggesting that they recognise distinct epitopes That the mAbs recognise distinct epitopes is further supported by the fact that none of the phage identified by any individual mAb was recognised in ELISA by any of the other mAbs (data not shown), and that, in general, the mAbs were not inhibited by peptides associated with other mAbs.

The first epitope is defined by mAb H5. A motif common to the majority of the phage clones selected by this mAb (EHRSP) could be identified within the native gG2 sequence, and two synthetic peptides containing this sequence (PT71, PT156) , one only 8 ammo acids long, as well as peptides with the sequence of two phage clone inserts (PT73, PT75, Chl6685) , could inhibit binding of H5 to gG2. Ammo acids from outside the 15mer insert were found to contribute to the epitope in at least one of the phage clones (3.15) recognised by th s mAb, as a peptide in which these ammo acids were not included (PT74) was unable to inhibit binding of H5 to gG2. That these am o acids were important n a number of the phage clones selected by H5 was suggested by the fact that the motif common to the majority of the clones was usually found at the N- erminal end of the insert. However, these ammo acids did not appear to be essential in the case of the phage clone 2.10, as a peptide synthesised with the sequence of its insert alone (Chl6685) was able to mimic the epitope in the inhibition ELISA. The insert of one phage clone (2.4) recognised by H5 had a sequence apparently unrelated to that of the remaining clones, even though it was consistently positive in the ELISA with H5 but not with an irrelevant antibody, nor with Fll or E5. This may, therefore, be a mimotope which is able to mimic the shape and charge distribution of the native epitope (3) .

The epitope defined by E5 is apparently adjacent to that defined by H5 since the motif common to phage clones selected by E5 is found m the region of gG2 present m peptide PT71. However, this is a distinct motif as neither PT73 or Chl6685, nor PT156, a shortened version of PT71, inhibit binding of E5 to gG2. Interestingly, Chl6689 a peptide with the sequence of the insert of one of the phage clones selected by E5 did inhibit binding of H5 , as well as E5 , to gG2 , and this peptide has a region (EHP) with sequence similarity to the motif of clones selected by H5. However, the E5 peptide Chl6688 which has the three ammo acids EHR does not bind to H5, and Chl6689 also inhibits binding of H7 to gG2 , so it is not clear at present whether the interactions of Chl6689 with H5 and H7 are specific.

The epitope defined by Fll comes from a different region of gG2. A shorter motif (TPL) was found to be common to phage clones selected by this mAb and a region of gG2 including ammo acids 359 - 378 containing this motif (PT173), as well as two peptides with the sequence of phage clones selected by Fll (Chl6686, Chl6687) , inhibited binding of Fll to gG2.

Thus, via use of the phage peptide display library technique, we have successfully defined a number of peptides (PT71, PT73, PT173, PT156, Chl6685, Chl6688, Chl6689, Chl6687, PT173) capable of binding to HSV type-specific monoclonal antibodies. These peptides therefore act as representations of the epitopes seen by those mAbs within native gG2. Their precise secondary structures may indeed be exact replicas of the native epitopes such that the mAbs bind to exactly identical ammo acids within the peptides as within gG2. Alternatively, the peptides may be true mimotopes, adopting the shape and charge characteristics of the epitope, but being composed of dissimilar residues. The value of having identified these peptides lies in their potential use as antigens capable of distinguishing between anti-gGl and antι-gG2 antibodies.

The gG2 epitopes we have described were defined by murine mAbs. In order to determine whether these epitopes are also antigenic in humans infected with HSV-2, it was necessary to bind the peptide mimics to the solid phase in an ELISA. However, whilst the majority of the peptides tested were able to inhibit binding of their associated mAbs to gG2 , only a subset of these peptides (PT71, PT75, Chl6687 and PT173) retained reactivity with their cognate mAb when bound to the solid phase. Presumably, m solution, the peptides are free to adopt an appropriate conformation which will allow reactivity with the mAb but wnen bound to the solid phase, their conformation is restricted and the epitope may be lost.

Those peptides which retained their antigenicity were tested in ELISA for their reactivity with a well-characterised panel of human sera. Each of the peptides PT71, Chl6687 and PT173 showed reactivity with some of those sera which were known to react with HSV-2 proteins in Western blots, but with none of the sera which were reactive with HSV-l proteins only or with neither HSV-l nor HSV-2. Interestingly, for each of these peptides the strongest reactivity was seen with sera containing both anti-HSV-l and -2 antibodies. It is likely, given the the epidemiology of HSV-l and HSV-2 infections, that this group of patients had been exposed to HSV-l first, followed by HSV-2, and therefore, it is interesting to speculate that the stronger reactivity in this group against type 2 specific epitopes may be a result of the generation of type-common T helper cells during the first infection.

In addition, 3/6 of the sera with only antι-HSV-2 antibodies showed clear reactivity against PT71. It is not surprising that not all these sera react with a single peptide. The pattern of recognition of multiple epitopes within a large and complex protein such as gG2 by different individuals is likely to be heterogeneous. Some sera this group were also reactive with peptides representing Fll epitopes, although the reactivity with these peptides was less impressive. The data presented m Fig. 3 confirm the type- specificity of the epitopes we have described, and also indicate that these epitopes are recognised by the human immune system. This raises the possibility of generating a peptide-based assay for the detection of HSV-2 type-specific antibody in human sera which may find wide clinical application (2, 4, 11).

Presentation of Peptides

For diagnostic use it may be advantageous to label the peptide, for example, with biotm using ammo-hexanoic acid biot incorporated during synthesis, or using N- hydroxysuccinimido biotm to derivatise free ammo groups (such as the N-terminus) , or any lysyl side chains) . It may also be convenient to use other labelling reagents such as acridmium esters or europium chelates which are used in a number of commercial assay systems. Radioactive labelling might also be useful, e.g. by the appending of a tyrosme residue to the C- or N-termmus of the pept.ide to allow introduction of iodine atoms via oxidation of ¹²⁵I iodide ion in the presence of chloramme-T according to widely used methods for radioimmunoassay . Similarly radioactive iodine could be incorporated via the Bolton-Hunter reagent (an N- hydroxysuccmnimide ester) according to methods described m the Amersham catalogue. Tritium would also be a convenient label - incorporated during synthesis with one or more radioactive ammo acid, or post-synthetically using ammo- directed reagents such as tπtiated N-succmimidyl propionate (Amersham catalogue) .

For solid phase assays such as ELISA, it may also be advantageous to increase the valency of the antigenic peptide by coupling it, for example to branched lysme cores according to methods described by James Tarn of Rockerfeller University. his could also be achieved via attachment of the peptide to poly-L or poly-D lysme (or poly-L or poly-D glutamic acid, or these polymers with aspartic acid in place of glutamic) using homo to heterobifunctional cross-linking agents such as glutaraldehyde or carbodiimides, according to methods described in the Pierce (Rockford Illinois) Chemical Company catalogue Ammo-acid copolymers containing an abundance of any of the three residues individually or combination (Asp, Glu, Lys) or analogues of these residues containing carboxylate or ammo acid side chains (e.g. ornithme m place of Lys) might also be used Such polymers could be of random or ordered sequence, and might usefully contain other ammo acids such as alanme, beta alanme, epsilon ammo caproic acid or glycme as spacers to facilitate the optimal degree of substitution of the peptide without contributing spurious additional epitopes to the construct. In particular the randomness of the sequence of the ammo acid copolymer core would contrive to avoid the generation of spurious antigenic reactions with human sera, since the abundance of any individual motif generated in the random copolymer would be effectively diluted among numerous other random sequences. Antigenically irrelevant carrier proteins (e.g. human serum albumin) could also be used for the purpose of increasing the valency of the antigenic peptide, using similar cross-linkmg chemistries.

In solid phase assays it may also be advantageous to attach the peptide indirectly to the solid phase to minimise adverse effects of direct adsorption to the solid phase, which might include adverse influence on accessibility of the peptide, or critical subgroups (e.g. side chains) therein. Such adverse effects might also include conformational effects e.g. interference by the solid phase in the attainment of antigenically relevant conformations by the peptide. Any of the methods described above to increase the valency of the peptide could also be used to facilitate the attachment of the peptide to the solid phase in a solid phase assay.

Carriers (other than phage) might also be used with any of the peptides to generate immunogenic constructs capable of eliciting antibodies or cellular (e.g. T-cell) immune responses against the peptides and against HSV-2. Such carriers would most advantageously be non-human in origin - thereby enhancing the ability of the human immuno system to response to the peptides (e.g. by providing a carrier function such as T-cell epitopes) . Exemplary carriers would be tetanus and diphtheria toxoids, hepatitis-B virus cores, keyhole limpet haemocyanin, virus particles (such as bacteriophage) . Carriers might also be synthetic - such as poly-L lysine, poly D-lysine, branched lysine (multiple antigenic peptide constructs referred to above) etc. Carriers might also comprise synthetic peptides (e.g. collinearly synthesised with HSV-2 peptides) comprising known or candidate T-cell epitopes of HSV-2 or any other pathogen or molecule .

The peptides may also be used to purify antibodies from infected sera for the purpose of standardisation of the diagnostic test or for the purpose of passive immunotherapy of infected individuals . References

1 Corey, L. , H. G. Adams, Z. A. Brown, and K. K. Holmes. 1983. Genital herpes simplex virus infections: clinical manifestations, course and complications. Ann. Int. Med. 98:958-972.

2 Frenkel, L. M. , E. M. Garratty, J. P. Shen, N. Wheeler, O. Clark, Y. J. Bryson. 1993. Clinical reactivation of herpes simplex virus type 2 infection in seropositive pregnant women with no history of genital herpes. Ann. Int. Med. 118:414-418.

3 Geysen, H. M. , S. J. Rodda, T. M. Mason. 1996. The delineation of peptides able to mimic assembled epitopes . pl31-149 In R. Porter and J. Wheelan (ed.). Synthetic peptides as antigens. Ciba Foundation Symposium Vol. 119. John Wiley and Sons, New York.

4 Gibbs, R. S. and P. B. Mead. 1992. Preventing neonatal herpes- current strategies (Editorial). N. Engl. J Med. 326:946 - 947.

5 Grabowska A., W. L. Irving. 1996. Epitope mapping using phage peptide display libraries : implications for diagnosis and vaccine development. PHLS Microbiology Digest 13:132-137.

6 Ho, D. W. T., P. R. Field, E. Sjogren-Jansson, S. Jeansson, A. L. Cunningham. 1992. Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG2) . J. Virol. Meth. 36:249-264.

7 Ho, D. W. T., P. R. Field, W. L. Irving, D. R. Packham, A. L. Cunningham. 1993. Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blotting (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections. J. Cl n. Micro. 31:3157-3164.

8 Hoess. R., U. Brinkmann, T. Handel, I. Pastan. 1993. Identification of a peptide which binds to the carbohydrate- specific monoclonal antibody B3. Gene 128:43-49.

9 Johnson, R. E., A. J. Nahmias, L. S. Magder, F. K. Lee, C. A. Brooks, C. B. Snowden. 1989. A seroepidemiologic survey of the prevalence of herpes simplex virus type 2 infection in the United States. N. Engl. J. Med. 321:7-12.

10 Koutsky, L. A., C. E. Stevens, K. K. Holmes, R. L. Ashley, N. B. Kiviat, C. W. Critchlow, L. Corey, 1992. Underdiagnosis of genital herpes by current clinical and viral-isolation procedures. N. Engl. J. Med. 326:1533-1539

11 Kulhanjian, J. A., V. Soroush, D. S. Au, R. N. Bronzan, L. L. Yasukawa, L. E. Weylman, A. M. Arvin, C. G. Prober. 1992. Identification of women at unsuspected risk of primary infection with herpes simplex virus type 2 during pregnancy. N. Engl. J. Med. 326:916-920.

12 Laing, P., P. Tighe, E. Kwiatkowski, J. Milligan, M. Price, H. Sewell. 1995. Selection of peptide ligands for the antimucm core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine. J. Clin. Path. 48 :M136-M141.

13 Lee F. K., R. M. Colβman, L. Pereira, P. D. Bailey, M. Tatsuno, A. J. Nahmias. 1985. Detection of herpes simplex virus type 2 -specific antibody with glycoprotein G. J. Clin. Micro. 22:641-644.

14 Luzzago, A., F. Felici, A. Tramontane A. Pessi, R. Cortese. 1993. Mimicking of discontinuous epitopes by phage- displayed peptides, I Epitope mapping of human H ferπtm using a phage library of constrained peptides. Gene 128, 51- 57. 15 McGeoch, D. J., H. W. M. Moss, D. McNab, M. C. Frame. 1987. DNA sequence and genetic content of the Hiis-DIII 1 region in the short unique component of the herpes simplex virus, type 2 genome: identification of the gene encoding glycoprotein G, and evolutionary comparisons. J. Gen. Virol. 68:19-38.

16 Parmley, S. F. and G. P. Smith. 1988. Antibody selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305-318.

17 Smith, G. P. & J. K. Scott. 1993. Libraries of peptides and proteins displayed on filamentous phage. Meth. Enzymol. 217:228-257

18 Smith, G. P. 1993. Surface display and peptide libraries. Gene 128:1-2

Table I Sequences of (lie iπscils ol |ilι:ι e clones iccogniscd l>y iΛbs and native sequence .villi liomoloyy lo (lie p age clones

niΛb I iaμe clone Sequence of inscif ^£-..3 ID: r-/° J

115 1 IS r S F r p v i c; ι> L I. 5 3 1 S I T N I^' I' I. V S II 1. H _. 321 ^'I^' C, S V Y S I' I ⁽i I. I. li V

25 I. I. T K I P I' N V Y I I. w

2 II X I' I' I- I S Λ V (i G V 1⁾ 3

3 1 Λ 1^» 1' I- ^'1 S Λ V (i (i V I⁾ ιo

210 M n n i) r i; K I- I^» Γ I I

3 <; R Λ R I⁾ Λ I i Z 24

I I L S I (j S I 3

Nalivc Λ P I" P P l^; II R ⁽i ⁽i P I. !^• I- I- ⁽i Λ ⁽i I⁾ (i

1.5 12 17 Λ I S I. P P I P N M Y O ( i 15 1221 Λ Cl ⁽J Y S P 1 Λ I-^' II I' P 122X 1 S I P I I Y P I I' 1 I I I 7 12 10 I⁾ P (ι 1 Λ ⁽i V P I. R II Λ l s? 1220 Y Ci Λ R P 1 I, I. Y S R Λ S .9 12 18 S P I, P li P ^■: II R Λ I. V P 10 1 .4 Y M Q P D P P P i Λ p n Y -LI 123 R M P I. P N II Y li P P - T 21

Table I (conld.)

C Sets. ID NI.

I ll 8 I / 822 2.4 810 2_3 8 13 I. P P h P R S V It 8 14 P F P Λ I. 1^; I S 27

81 V S Y I (i Λ 28

9 I ^l)Λ li 1 p li S I- y 5

R S I I I w 31 <) . i Λ r i v r P R R 1 P Y Λ P I _-2

Native P !^• K I P I P V S Λ I Λ M Λ P S V I) P S, _S

l'oi each mΛb, the secpienccs ol the niseils ol phage clones selected by that mΛb aie given, using the slaiulaid single leltei aniino acid ( ode. Foi the clones selected hy 115, the 3 amino acids at the N teiniinal side ol the inscil aie also shown, in biacKel , foi I he sequences ending with (D/ ) I IRS Foi niΛbs IIS, I' 1 and Ii5, the se(]iιenccs weie aligned using Clustal W (14) foi Multi Sequence Alignment (h(lp://biology ncsa nine edu/BW/l.W cgi), followed by manual adjuslmenl ^'I lie molds found hy alignment wet c iciiiu m Clustal W against the gG2 se(|iιence to identify the native sequence most similai to the motif

able 2 Sequences of peptides

niΛb Peptide Sequence

115 Ch 16685 MDDD IFRFP I I1RSLP (phage 2.10 insert) SCB. i? ' *J. II

PT73 TSPFTPVIGPU.IIRSPΛΓ.GD (phage 3.15 insert will, amino acids derived horn pVIII al the N leiminal side of the insert)

PT71 ΛPPPPl_HRGGP!iliFr.GΛGI>G(gG2, amino acids 551 -570)S£ls? iD.Ni t 5ⁱ=E^> φ : Klo3-+

1 72 RAGPIiGPPGIiPGliΛPFIiPGII (sciamhled version of P 171) - negative control S ES. 35

PI74 Ml.SFΛTSPF^'l VIGPUillRS (phage 3.15 inscit with amino acids deiived fioin pVIII at (he C-teiininal side of the inseil)

PT75 MI.SFAPVlGPI.LillRSPAFGD(phage .l5 inserl without PFT motif) 5tS.iD.Mo 37 ^ lL>^;M°3(_s.

I I56 lillRGGPTiP. (gG2, amino acids 556- 562, 8nιer variant of P 171) S_>£S_ ip ^•■ Mo3?

-ζ5 C i 688 SPFPIiPPPiilIRΛLVP(phagcl2.l8 inscil) SE& iD i N^O

C l668 ATSI.I TF.IIPNMYQG (phage 12.17 inseit) SEQ ,T : r4° tS

I 7I ΛPPPPIillRGGPIiliFI.GΛGDG (g⁽i2, amino acids 551 - 570) Sr-ftD (O " >H

PT16b RMΛRPriinVGVI.PPIIWΛPGΛ - negative control >_r§> t ϊ> •" \1° 39

Fll CI1I6686 DY TPQTSLFLPPIiSF (phage 9.4 inseil) ≤ E-f_5> l 1 • ° 3 o

Clιl6687 ΛLSSQGGMSPliPTPL (phage 8.22 inscil) Se® v >. ^J ^^

I I73 PI.KTPU'VSΛTAMAPSVDPS (gG2, amino acids 35 - 378)

■ N*° 33

The sequences of the peptides used aie given, using the standaid single-lellei code for amino acids, fioni the ( .' leiininus to the N leiininus. 'The derivation of the sequence is give in brackets.

Table 3 Crcss-ir.nib t-.on cf mAbs by pepticies representing ct er eoitCDes . Peotiάes^' were ail useα at a smcie concentration of

250ug/m

Table 4 Sequences of the inserts of the phage clones used as immunogens

Phage clone Sequence of insert

2.10 M D D D T Ξ R F P T H R S L ?

2.11 X P P F T S A V G G V D H R S 2.19 5 T T N T ? L 7 S H 1 Ξ H R S

Native (SEQ ID: 14) =,..-. ? ? ? ? Ξ H R G G ? G----

Table 5 Number of mice surviving 14 days after intraperitoneal challenge with HSV-2

Immunising dose Unabsorbed Polvmixin B absorbed Total of phage (μg)

100 3/3 3/3 6/6

75 3/3 3/3 6/6

50 1/3 2 / 3 3/6

10 1/3 1/3 2/6

Claims

1. A polypeptide consisting of 3 to 38 ammo acid residues, having the sequence of SEQ ID:1 or a portion thereof; and analogous polypeptide derivatives by virtue of point mutation, amino acid substitution, deletion or addition;

SEQ ID : 1 A^PP'PE'ΗVGGPEEF^EGAGDG²⁰

E P P²³ EDDDS ATGLAFRTPN³⁸

wherein the sequence includes histidine residue H^~; and wherein E⁶ may be substituted in order of preference by

D⁶>T⁶; and wherein R^θ may be substituted by A⁸, which sequence is recognised by antι-gG2 positive human sera from patients with HSV-2 infection and is not recognised by antι-gG2 negative sera from patients with HSV-l infection.

2. A polypeptide according to claim 1 which is antigenic

3. A polypeptide according to claim 2 which is immunogenic and is capable of inducing antibodies in an immunised host against type-specific HSV-2 gG.

4. A pharmaceutical composition containing as an active ingredient an immunogenic polypeptide according to claim 3.

5. A vaccine composition containing as an active ingredient an immunogenic polypeptide according to claim 3 together with a physiologically acceptable adjuvant and/or carrier and/or diluent.

6. An antibody to the polypeptide of claim 1 obtainable by immunisation of a host with the immunogenic polypeptide of claim 3.

7. A recombinant DNA molecule comprising a DNA sequence encoding a polypeptide according to claim 1.

8. A filamentous bacteriophage including, in at least a proportion of its major coat protein sub-units, multiple display of a polypeptide according to claim 1.

9. A vaccine composition comprising a bacteriophage according to claim 8 together with a physiologically acceptable adjuvant and/or carrier and/or diluent.

10. A substantially pure non-glycosylated polypeptide according to claim 1, 2 or 3.

11. A method of testing for the presence of type- specific HSV-2 gG2 antibodies m a fluid, which comprises contacting the fluid with one or more polypeptide (s) according to claim 1, 2, 3 or 10 and testing whether or not antibodies bind to the polypeptide (s) .

12. A method of testing for the presence of type- specific HSV-2 gG2 antibodies in a fluid, which comprises contacting the fluid (i) with a labelled form of one or more polypeptide (s) according to claim 1, 2, 3 or 10 and (li) with antibodies according to claim 6, whereby antigen n the fluid competes with polypeptide (s) in binding to the antibodies .

13. A test kit for testing for the presence of HSV-2 type specific antibodies in a fluid, which comprises: (ι) a solid phase on which is immobilised one or more polypeptide (s) according to claim 1, 2 , 3 or 10, and (n) means for detecting binding of antibodies to polypeptide (s) .

14. A test kit for testing for the presence of HSV-2 type specific antibodies in a fluid, which comprises: (l) a solid phase on which is immobilised one or more polypeptide (s) according to claim 1, 2, 3 or 10 m labelled form;

(n) antibodies according to claim 6, and

(m) means for detecting competitive binding of antibodies to polypeptide (s) .

15. A method of diagnosis of HSV-2 infection which comprises employing the test method of claim 11 or 12.

16. A method of diagnosis of HSV-2 infection which comprises employing the test kit of claim 13 or 14 in the test method of claim 11 or 12.

17. A method of treatment of HSV-2 infection which comprises administration to an infected patient of an immunologically therapeutically effective amount of a vaccine composition according to claim 5 or 9

18. A method of treatment of HSV-2 infection which comprises administration to an infected patient of an immunologically therapeutically effective amount of an ant tbody according to claim 6.

- X IS . A method of prevention of HSV-2 infection which comprises administration to a patient a prophylactically effective amount of a vaccine composition according to claim 5 or 9.

20. A method of prevention of HSV-2 infection which comprises administration to a patient a prophylactically effective amount of an antibody according to claim 6.

21. A polypeptide which is the sequence SEQ ID: 2 consisting of A¹ to G²⁰ of SEQ ID : 1 (PT71) .

22. A polypeptide which is the sequence SEQ ID: 3 consisting of P⁴ to P²³ of SEQ ID:1.

23. A polypeptide according to claims 1 to 3 , 10, 21 or 22 conjugated to a Biotin-NH₂ terminus by a lysine (K) group .

24. A polypeptide which is the sequence SEQ ID: 42 consisting of A¹ to F¹⁴ to SEQ ID: 1 (PT444) .