WO1998003544A1

WO1998003544A1 - IDENTIFICATION OF SHORT PEPTIDE SEQUENCES REPRESENTING EPITOPES OF GLYCOPROTEIN G OF HSV-2 USING A PHAGE PEPTIDE DISPLAY LIBRARY FOR USE IN AN ANTI-gG2 ELISA

Info

Publication number: WO1998003544A1
Application number: PCT/GB1997/002003
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: AU3630197A; GB9615533D0; AU3630997A; WO1998003543A1

Abstract

The invention provides a polypeptide consisting of 3 to 20 amino acid residues, or a peptido mimetic analogue thereof wherein the analogue is at least partly non-peptide in nature and the analogue has a length equivalent to that determined by 3 to 20 amino acid residues; which is a sequence not naturally occurring in the native sequence of the HSV-2 gG protein; and which has the following sequence SEQ ID:1 (X)n-α-β-η-(Y)m or SEQ ID:2 (X)n-δ-ε-ζ-(Y)m wherein n=0 or an integer; m=0 or an integer; n+m ≤17; α is E or may be substituted in order of preference by D⊃T⊃S⊃Q⊃N; β is H; η is R or may be susbtituted in order of preference by R⊃A or L⊃T or P; δ is T or may be substituted in order of preference by E⊃L; ε is P; ζ is L or may be substituted in order of preference by B or Y; and X or Y are positively charged, negatively charged or neutral amino acids or peptido mimetic analogues thereof; and antibodies raised thereto; and prophylactic and therapeutic and diagnostic uses thereof relating to HSV-2 infection.

Description

Identification of short peptide sequences representing epitopes of glycoprotem G of HSV-2 using a phage peptide display library for use in an anti-gG2 ELISA.

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 subumt 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 m the single-stranded DNA genome as an insert m 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 major advantages that (i) no prior knowledge of the primary sequence of the target antigen is necessary, (ii) epitopes represented within the antigen either by a linear sequence of amino acids (linear epitope) or by the spatial juxtaposition of amino acids distant from each other within the primary sequence (conformational epitope) are both identifiable, and (lii) 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 main 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 sned 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 f nd 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 in 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 pomatia-punfled gG-2 as antigen in immunoblot and ELISA formats have been described (6, 7, 13). However, difficulties m large-scale production of gG2 of sufficient purity have precluded the widespread availability of such assays. 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 axso recognised by human sera known to contain anti-HSV-2 antibodies, thus validating this approach towards the development of a cheap and widely applicable assay for the detection of human anti-gG2 antibodies .

The present invention provides a polypeptide consisting of

3 to 20 amino acid residues, or a peptido mimetic analogue thereof wherein the analogue is at least partly non- peptide in nature and the analogue has a length equivalent to that determined by 3 to 20 amino acid residues; which is a sequence not naturally occurring in the native sequence of the HSV-2 gG protein; and which has the following sequence SEQ ID:1 or SEQ ID:2

SEQ ID:1 (X)_n-α-β-γ- (Y)_m

SEQ ID:2 (X) _n-δ-ε-φ- (Y) _m

wherein n=0 or an integer; m=0 or an integer; n+m < 17; is E or may be substituted in order of preference by

D>T>S>Q>N; β is H γ is R or may be substituted in order of preference by R>A or L>T or P; δ is T or may be substituted in order of preference by

E>L; ε is P; φ is L or may be substituted in order of preference by B or Y; and X or Y are positively charged, negatively charged or neutral amino acids or peptido mimetic analogues thereof. The invention also provides a polypeptide wherein; in order of preference if present; X adjacent α s P>L>T; X adjacent δ is P;

Y adjacent γ is S>G;

Y adjacent φ is A or S or L.

The invention also provides a polypeptide wherein SEQ ID:1 is modified as SEQ ID: 3

SEQ ID:3 (X) _P-α-β-γ-Y -Y,-Y -E- (Y) ₀

wherein n=0 or an integer, p=0 or an integer, and n+p < 14, with the effect that a resiαue E is spaced three amino acids downstream of γ.

The invention also provides a polypeptide which contains a motif sequence

The invention also provides a polypeptide selected from the group consisting of the sequences having SEQ ID: Nos as follows;

SEQ ID NOS: 6, 9, 11.

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

The invention also provides a pharmaceutical composition containing as an active ingredient an lmmunogenic polypeptide . The invention also provides a vaccine composition containing as an active ingredient an lmmunogenic 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 lmmunogenic polypeptide.

The invention also provides a recomb ant DNA molecule comprising a DNA sequence encoding the 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-specific HSV-2 gG2 antibodies in 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:

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

(n) antibodies; and

(m) 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, or employing the test kit.

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

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

The invention also provides a polypeptide conjugated to a Bιotm-NH₂ terminus by a lysine (K) group.

The invention is described with reference to the accompanying drawings in 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 £5 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 ▲ ) . 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 (A), 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 with peptide PT74 (X) , in which ammo acids derived from pVIII at the N-termmal 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-1 and HSV-2 proteins detectable by Western Blotting: 1) antibodies to neither HSV-1 nor HSV-2; 2) antibodies to HSV-1 only; 3) antibodies to HSV-2 only; 4) antibodies to both HSV-1 and HSV-2. Figure 4 shows 90 human sera were tested at a 1:25 dilution in ELISA for their reactivity with 5 peptides and at a 1:250 dilution for their reactivity with gG2. The peptides were as follows:

PT168 GCRGGEPPSPKTCGSYTYTY control SEQ ID: 40 PT445 KTPPTTPAPTTPPPTSTHAT SEQ ID: 41

PT166 RMARPTEDVGVLPPHWAPGA control SEQ ID: 39 PT173 PEKTPLPVSATAMAPSVDPS SEQ ID: 33

Chl6687 ALSSQGGMSPEPTPL SEQ ID: 24

Figure 5 shows the results of a lethal challenge with HSV- 2 in mice protected by immunisation with polypeptides of the invention.

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 in 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 recombmant gene VIII is under the control of a tac promoter; the ratio of the peptide- display g 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 Σ. coli, a λ- derivative of K-38 was used throughout. It is Hfr Cavalli and has chromosomal genotype t i . Bacteria were cultured in LB medium (Sigma) , with the addition of kanamycin (50μg/ml) , tetracycline (20μg/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 lμg/ml was added and the bacteria were mcuoated 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 resuspended in a final volume of 150μl 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/ml.

Biopanning

Three rounds of biopann g 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 in TBS-0.05% BSA. One aliquot of the library containing 10^;o phage in 50μl 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. 50μl of elution buffer (0.2M glycine, 0.1M HC1, 0.1% BSA, O.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 in LB containing tetracycline. They were purified by PEG- precipitation.

The second and third rounds of biopannmg were carried out using a 20μl aliquot of Goat anti-mouse coated dynabeads (Dynal) as the solid phase. The beads were washed 4 times in TBS, incubated with a 50μl aliquot of the mAb, then washed and blocked. During round 2, a 50μl 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 in 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 biopannmg 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 in 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 in 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 in diethanolamme buffer (10% diethanolamme, pH 9.8, 0.5πuM 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 oligonudeotide 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 in coating buffer. After blocking, peptioes or phage were added simultaneously with the mAb diluted in blocking buffer. The mAbs were diluted by a factor of 1:2 compared with the concentration used in the ELISA above. Binding of the mAb was detected using the same procedure as in the ELISA above.

gG2 and Peptide ELISAs

ELISA wells (Nunc Maxisorp) were coated by incubating over night with peptides at 5ug/ml in 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 Boehnnger Mannheim ECL blocking solution (Cat. No. 1500 694) in PBS. Incubation buffer was a 1:20 dilution of this reagent in PBS. Wells were incubated in turn with serum diluted 1:25 and horse radish peroxidase conjugated Rabbit F(ab)₂ anti-human IgG (Dako P0406) diluted to 1:1000 in 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 H₂S0„.

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-1 and HSV-2 proteins, and fall into 4 groups of 6 sera based on those reactivities : no antibodies to either HSV-1 or -2 (group 1; antibodies to HSV-1 only (group 2); antibodies to HSV-2 only (group 3); antibodies to both HSV-1 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 biopannmg, 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 in Table 1.

Identification of motifs amongst the sequences of the phage clone inserts and within the native sequence of gG2.

Motifs (indicated in Table 1) 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-terminal side of the 15-mer insert. We postulated that adjacent amino acids derived from the natural protein VIII sequence may have contributed to the antibody binding site, and therefore have included these am o 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 1 (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 in 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 : (1) PT74, to test the hypothesis that phage ammo acids at the N- terminal side of the insert were contributing to the antibody-binding site, (11) 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 (m) PT156, to localise the sequence of importance within 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 am o 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 amino acids (PAEGD) at the N- termmal side of the insert, 5 phage amino 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 amino acids could be deleted, as in 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 aole 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 antι-HSV-1 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 anti- 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 in 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 amino 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 amino acid - antibody contact residues, but rather to identify peptide sequences capable of binding to anti-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 amino 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) in order to determine whether the motif is present in 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 amino acids long, as well as peptides with the sequence of two phage clone inserts (PT73, PT75, Chl6685) , could inhibit binding of H5 to gG2. Amino 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 this mAb, as a peptide in which these amino acids were not included (PT74) was unable to inhibit binding of H5 to gG2. That these am o acids were important in 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-termmal end of the insert. However, these amino acids did not appear to be essential m 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 in the region of gG2 present in 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 amino 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, PT73, 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 amino acids withm 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 munne 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, in solution, the peptides are free to adopt an appropriate conformation which will allow reactivity witn the mAb but when 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-1 proteins only or with neither HSV-1 nor HSV-2. Interestingly, for each of these peptides the strongest reactivity was seen with sera containing both anti-HSV-1 and -2 antibodies. It is likely, given the the epidemiology of HSV-1 and HSV-2 infections, that this group of patients had been exposed to HSV-1 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 anti-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 in this group were also reactive with peptides representing Fll epitopes, although the reactivity with these peptides was less impressive. The data presented in 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).

Immunogenicity of Phage Clones

Materials and Methods

Phage clones

Phage clones were isolated from a phage peptide display library, based on the filamentous phage fd, consisting of approximately 10⁸ different phage clones (ref our paper) . 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 vectors¹⁸. 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.

Three clones were used : 2.10, 2.11, 3.19. The sequences of the peptides displayed by these phage clones are given in Table 4. In addition, the wild-type phage, f88-4 from which the library was derived, was used.

Preparation of PEG-precipitated phage for use as immunogens

Phage clones were prepared from a 500ml over night culture of infected bacteria grown in Terrific Broth with tetracycline (20ug/ml) to select for phage expression and IPTG (ImM) to maximise expression of the recombinant gene VIII carrying the peptide insert. f88-4 was grown in the absence of IPTG as no peptide is displayed by the recombinant gene VIII.

Bacteria were removed by centrifugation at 10,000g maximum in a Sorvall GSA rotor at 4°C. Phage were precipitated from the supernatant by addition of PEG 8000 and NaCl to final concentrations of 4% and 3% respectively, followed by a 45 mm incubation at RT with stirring. The phage were pelleted by centrifugation at 10,000g at 4°C. The supernatant was discarded and the phage pellet was resuspended in Tris-buffered saline (TBS) . Insoluble material was removed by a further centrifugation at 10,000g. The PEG precipitation was then repeated.

Phage concentration was assessed using the Sigma BCA assay, using ovalbumen as a standard, according to the manufacturer's instructions, and adjusted to a standard concentration of lmg/ml. Phage reactivity with the monoclonal antibody H5 was also assessed by ELISA (results not shown) , to check their antigenicity .

Polymixin B absorption of PEG precipitated phage

LPS was absorbed out of the PEG precipitated phage preparations by passing them through polymixin B columns (Pierce)

Immunisation

BALB/c mice were obtained from a closed colony at the Sheffield University animal facility. Immunisations were administered subcutaneously at two-week intervals. Pre- bleeds were obtained from the mice prior to immunisation and further bleeds were taken two weeks after each immunisation. Four weeks after the last immunisation, mice were challenged with 5xLD₅₀ of HSV-2. Survival of the mice was followed over the next 14 days.

gG2 ELISA

After each step wells were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20. Wells were coated with helix poma tia lectin purified gG2 at a dilution of 1:500 in carbonate-bicarbonate buffer, pH 9.6, blocked with 30% NGS in PBS, then incubated in turn with mouse sera at a 1:100 dilution and horseradish peroxidase conjugated rabbit anti-mouse Ig at 1:1000 in PBS-10% NGS. 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 H₂SO_< .

Results

In a first experiment, 8 groups of 3 mice were immunised with a pool of three phage clones, 2.10, 2.11 and 3.19. The phage were prepared individually by PEG precipitation and pooled, then half of the pool was absorbed against polymixin B while the other half was left untreated. The aim of the polymixin B absorption was to remove lipopolysaccharide (LPS) which may be present in the phage preparation, as it was possible that it may be toxic to mice. Both of these preparations were used separately as an immunogen at one of four doses (100, 75, 50 or lOμg) and each mouse received 2 immunisations. The number of mice surviving in each group, 14 days after lethal challenge, is shown in Table 5. There was no significant difference between the survival of the mice receiving polymixin B absorbed phage compared with those receiving unabsorbed phage at a particular dose of phage; therefore, these results are also shown combined in Table 4.5. At the two highest phage doses (lOOμg and 50μg) all of the mice survived, and with decreasing phage doses, fewer mice survived.

Sera from these mice were analysed for their reactivity with gG2 by ELISA. Antibodies to gG2 could be detected in a number of mice in each group. In general, there was an increasing level of antibody with increasing numbers of immunisations. There was a greater response in the mice immunised with unabsorbed phage, but for both immunogens, with the exception of the group given lOOμg of absorbed immunogen, a larger dose of immunogen gave rise to a higher level of anti-gG2 antibodies.

To find out whether the protection observed was specific, i.e. was dependent on the presence of the displayed peptide, a larger-scale experiment was carried out. Here, none of the phage preparations used were polymixin B treated as no toxic effects had been observed in experiment 1 in the mice given unabsorbed phage. The same three phage clones, 2.10, 2.11 and 3.19 were used as immunogen, either individually at lOOμg or 30μg or as a pool consisting of a total of lOOμg or 30μg of phage. In addition, control groups were immunised with lOOμg or 30μg of the wild-type phage f88-4 or TBS only. Groups of 10 mice were used and each mouse was immunised three times at two weekly intervals. Four weeks after the third immunisation, the mice were challenged with 6xLD50 of HSV- 2 peritoneally, then monitored daily.

Fig. 5 shows their survival over 17 days following challenge. At the end of the 17 day period, 20% of the mice in the control groups, immunised with wild-type phage or TBS, were still alive. In contrast, between 40 and 60% of the mice immunised with individual phage clones or lOOμg of pooled phage , and 80% of the mice given 30μg of the pool of phage, had survived. Discussion

Following immunisation of mice with phage clones displaying 15 amino acid peptides representing a single epitope of gG2, we have been able to detect antibodies to gG2 in the sera of the immunised mice. These mice had never been exposed to gG2, and no anti-gG2 antibodies were detected in serum obtained from the mice prior to immunisation. The level of the anti-gG2 antibody response observed was dependent on the phage dose administered, and increased after each immunisation. The peptides displayed by the phage are, therefore, not only antigenic, i.e. able to mimic this gG2 epitope, but are also immunogenic.

In addition, we have been able to show there was a biologically relevant response to these peptides. In the first experiment, subsequent challenge of the immunised mice showed that all of the mice that had received the highest doses of immunogen and a proportion of those which had received lower doses were protected against a lethal challenge of HSV-2. In the second experiment, although the survival rates were lower, the protection observed was shown to be dependent on the presence of the insert peptide, since wild-type phage, administered in the same way, gave rise to much lower levels of protection equivalent to that observed in mice immunised with buffer alone. This provides further evidence that the phage clones induce an immune response against gG2.

Whilst it has been shown before that phage expressing peptides derived from either viral proteins^19,20 or expressing mimotopes of epitopes from viral proteins, are able to induce an antibody response to the native protein²¹, it has not been demonstrated previously that they can induce protection against challenge with virus. Here, protection could be obtained with single phage clones. However, the highest levels of protection were obtained using a pool of 3 clones, perhaps because this allows stimulation of a greater variety of B cell clones.

It is particularly striking that phage displaying a single epitope are able to induce protection against a whole virus. We have achieved similar levels of protection to those achieved previously using a whole baculovirus expressed glycoprotein G²⁴. However, passive protection has been achieved before with certain mAbs directed against epitopes of HSV²⁵.

The peptides displayed by these phage are not derived from the native sequence of gG2. The phage clones were selected from a random phage peptide display library and display peptides with some similarity to the native sequence of gG2 over part of their length, approximately 5 amino acids (see Table 4) . It is not clear at present whether the remaining amino acids within the 15 amino acid insert are required to maintain the antigenicity or immunogenicity of the peptide; they may, for example, contribute to the folding of the peptide. Since these phage do not contain significant lengths of sequence derived from the native protein, it is likely that the mechanism involved in protection is antibody-mediated, rather than T-cell dependent. However, in experiment 1, good protection was observed both in mice given absorbed and unabsorbed phage although the former apparently had lower levels of antibody. It is possible that protection is dependent on the quality rather than the quantity of antibody produced, for example the isotype of the antibodies induced.

In these experiments, phage were administered without any additional adjuvant. The phage preparations used are likely to contain some bacterial contaminants; it will be of interest to find out whether these or components of the phage itself are able to provide an adjuvant effect. Willis et al.¹⁹, showed that the immune response to phage- displayed peptides was T-cell dependent. Presumably, this T-cell help is provided by components of the phage and may be involved in directing the immune response towards a Thl or Th2 type of response.

Presentation of Peptides

For diagnostic use it may be advantageous to label the peptide, for example, with biot using ammo-hexanoic acid biotin incorporated during synthesis, or using N- hydroxysuccinimido biot to derivatise free ammo groups (such as the N-termmus) , or any lysyl side chains) . It may also be convenient to use other labelling reagents such as acridinium 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 tyrosine residue to the C- or N-terminus of the peptide to allow introduction of iodine atoms via oxidation of ¹²⁵I iodide ion in the presence of chloramine-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 in 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 tritiated N-succinimidyl 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 lysine cores according to methods described by James Tam of Rockerfeller University. This could also be achieved via attacnment of the peptide to poly-L or poly-D lysine (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. Amino-acid copolymers containing an abundance of any of the three residues individually or in combination (Asp, Glu, Lys) or analogues of these residues containing carboxylate or amino acid side chains (e.g. ornithine in place of Lys) might also be used. Such polymers could be of random or ordered sequence, and might usefully contain other amino acids such as alanine, beta alanine, epsilon amino caproic acid or glycine 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 amino 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-linking 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. 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. Gibbs, R. S. and P. B. Mead. 1992. Preventing neonatal herpes- current strategies (Editorial). N. Engl. J. Med. 326:946 - 947. 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. 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. 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. Clin. Micro. 31:3157-3164. 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. 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. Koutsky, L. A., C. E. Stevens, K. K. Holmes, R. L. Ashley, N. B. Kiviat, C. . Critchlow, L. Corey, 1992. Underdiagnosis of genital herpes by current clinical and viral-isolation procedures. N. Engl. J. Med. 32.6:1533-1539. 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. Laing, P., P. Tighe, E. Kwiatkowski , J. Milligan, M. Price, H. Sewell. 1995. Selection of peptide ligands for the antimucin core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine. J. Clin. Path. 48:M136-M141. Lee F. K. , R. M. Coleman, 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. 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 ferritin using a phage library of constrained peptides. Gene 128, 51-57. McGeoch, D. J. , H. W. M. Moss, D. McNab, M. C. Frame. 1987. DNA sequence and genetic content of the HinDIII 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. Parmley, S. F. and G. P. Smith. 1988. Antibody selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305-318. Smith, G. P. & J. K. Scott. 1993. Libraries of peptides and proteins displayed on filamentous phage. Meth. Enzymol. 217:228-257. Smith, G. P. 1993. Surface display and peptide libraries. Gene 128:1-2 Willis AE, Perham RN, Wraith D (1993) I munological properties of foreign peptides in multiple display on a filamentous bacteriophage. Gene 128 : 79-83 Veronese FDM, Willis AE, Boyer-Thompson C, Appella E, Perham RN (1994) Structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage. The V3 loop of HIV-1 gpl20. Mol. Biol . 243 : 167-172 Meola A, Delmastro P, Monaci P, Luzzago A, Nicosia A, Felici F, Cortese R, Galfre G (1995) Derivation of vaccines from mimotopes. Immunologic properties of human hepatitis B virus surface antigen mimotopes displayed on filamentous phage. J. Immunol. 154 : 3162-3172 Greenwood J, Willis A, Perham RN (1991) Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum sporozoite protein as antigens. J. Mol. Biol. 220 : 821-827. Grabowska A, Laing P, Jeansson S, Sjogren-Jansson E, Cunningham A, Irving WL (submitted to J. Virol.) Identification of short peptide sequences representing epitopes of glycoprotein G of HSV-2 using a phage peptide display library for use in an anti-gG2 ELISA. Ghiasi H, Kaiwar R, Nesburn AB, Wechsler (1 92) Baculovirus-expressed glycoprotein g of herpes simplex virus type 1 partially protects vaccinated mice against lethal HSV-1 challenge. Virology 190 : 233-239 Mester JC, Rouse BT (1991) The mouse model and understanding immunity to HSV Rev. Inf. Dis. 13 : S935-945. McKenzie R, Straus SE (1996) Vaccine therapy for HSV infections: an historical perspective. Rev. Med. Virol. £ : 85-96.

Table I Sequences of (he inserts of phage clones recognised by mAbs and native sequence with homology to the phage clones Ab: 115 Sequence of insert |SI^:.Q ID: No. Phage clone

3.15 T S P F T P V I G P L E 3

3.19 S T T N T P L V S H L E 6

3.21 T G S V Y S P T G E L E 7

2.5 R E T K L P F N V Y T E 8

2.11 X P P F T S A V G G V 1) 9

A P P F T S A V G G V 10

M D D D 7 E R F P T I II R A R D 1 Q E II A T 12

I. I. L I G 13

A P P E 11 R G I: F E G A G D G "o 14

A T I. P P T E II P N Y (i 1

A G Ci Y S P T E II A I' II S P P 1

T S T P T E 11 T Y P E I I T 17

D P G T E II G V P I. R II S 18

Y G A R P P E A S 19

S P L P E P P P P 20

M Q P D P P P P L 21

T R M P L P N II Y E P P P R

22

Table 1 (conttl.) niAbiFH [SEQ ID: No.]

Phage clone

8.17 A S S Q G G M S P E P T P L 23

8.22 A L S S Q G G S E P T P L 24 g β.lO V S S R P T T H Y Y L P E P L 25

09

*5 813 T E S T L P P F P R S V 26

H

C 8.14 S T N P E P L P p P A E E L S 27

O) 8.16 Q K Y A P E T T P V S Y L G A 28

X rπ 9.1 H V L S S R P T T L Λ L P L F 29

H 9.4 D Y T P Q T S L E L P P E S F 30 m 9.5 T P Λ Q Λ Y P Λ L R S L I P W 31 ro

9.3 T A T T V T P R R T P Y A P I 32

Native [SEQ ID: No.33] "' P E K T P L P V S A T A M A P S V D P S₃₇₈

For each mAb, the sequences of the inserts of phage clones selected by that mAb are given, using the slandard single letter amino acid code. For the clones selected by H5, the 3 amino acids at the N-terminal side of the insert are also shown, in brackets, for the sequences ending with (D/E)

HRS. For mAbs H5, FI 1 and E5, the sequences were aligned using Clustal W (1.4) for Multi Sequence Alignment (http://biology.ncsa.uiuc.edu/BW/BW.cgi), followed by minor manual adjustment. The motifs found by alignment were rerun in Clustal W against the gG2 sequence to identify the native sequence most similar to the motif.

Table 2 Sequence of peptides mAb Peptide Sequence H5 Ch 16685 MDDDTERFPTH SLP (phage 2 10 inserl)

P 173 TSPFTPVIGPLEI 1RSPAEGD (phage 3 15 insert with amino acids derived from pVIII at the N terminal side of the inseit)

PT71 APPPPEI IRGGPEEFEGAGDG (gG2, ammo acids 551 -570)

PT72 RAGPEGPPGEPGEADFEPGII (scrambled veision of PT7I) - negative control O rø P I 74 MLSFATSPFTPVIGPl EI IRS (phage 3 15 insert with amino acids derived I mm pVIII at (lie C-tcrminal side ol the insei l)

(0

H PT75 MLSFAPVIGPLEHRSPAEGD (phage 3 15 mseit without PFT motif)

H

C

PT156 EIIRGGPEE (gG2, ammo acids 556-562, 8ιneι v.ii iant of P I 7I ) m o x m π] E5 Ch l6688 SPLPEPPPEI IRΛLVP (phage 12 18 inseil) 3 Ch l 6689 ΛTSEPP 1 EIIPNMYQG (phage 12 17 mseit) m PT71 APPPPEI IRGGPEEFEGAGDG (gG2, amino acids 551 -570) ro P T166 RMARPIΕDVGVLPPI1WAPGA - negative control

FI 1 CI1I6686 DYTPQTSLELPPESF (phage 9 4 insert) Chi 6687 ALSSQGGMSPEPTPL (phage 8 22 insert) PT173 PEKTPLPVSATAMAPSVDPS (gG2, ammo acids 359-378)

The sequences of the peptides used are given, using (lie standard single-letter code for amino acids, from the C-terminus to th The derivation of the sequence is given in brackets.

Table 3 Cross-inhibition of mAbs by peptides representing other epitopes. Peptides were all used at a sinsle concentration of 250ue/ml

Sequences of tlie inserts of the phage clones used as immunogens Sequence or insert

0) M D D D T E R F P T H R S L P SEQ ID: No. 11 c σ

(0 X P P F T S A V G G V D II R S SEQ ID: No.9 H H S T T N T P L V S H L E II R S SEQ ID: No.6 c

H m

551 Λ P P P P E II R G P E E F E G A G D G sw o

X m m

H

3D

C m r ro σ>

Table 5 Number of mice surviving 14 days after intraperitoneal challenεe with

HSV-2

Claims

1. A polypeptide consisting of 3 to 20 amino acid residues, or a peptido mimetic analogue thereof wherein the analogue is at least partly non-peptide in nature and the analogue has a length equivalent to that determined by

3 to 20 amino acid residues; which is a sequence not naturally occurring in the native sequence of the HSV-2 gG protein; and which has the following sequence SEQ ID:1 or SEQ ID: 2

SEQ ID:1 (X) _n-α-β-γ- (Y) _m

SEQ ID:2 (X) _n-δ-ε-φ- (Y) _m

wherein n=0 or an integer; m=0 or an integer; n+m < 17; α is E or may be substituted in order of preference by

E>L; ε is P; φ is L or may be substituted in order of preference by B or Y; and X or Y are positively charged, negatively charged or neutral amino acids or peptido mimetic analogues thereof.

2. A polypeptide wherein; in order of preference if present;

X adjacent α is P>L>T;

X adjacent δ is P; Y adjacent γ is S>G;

Y adjacent φ is A or S or L.

3. A polypeptide according to claim 1 or 2 wherein SEQ ID:1 is modified as SEQ ID: 3

SEQ ID: 3 (X) -α-β-γ-Y.-Y₂-Y₃-E- (Y) _p

wherein n=0 or an integer, p=0 or an integer, and n+p < 14, with the effect that a residue E is spaced three ammo acids downstream of γ.

4. A polypeptide according to claim 1, 2 or 3 which contains a motif sequence

α-β-γ is E-H-R or δ-ε-φ is T-P-L.

5. A polypeptide according to claim 1, 2 or 3 selected from the group consisting of the sequences having SEQ ID: Nos as follows;

SEQ ID NOS: 6, 9, 11.

6. A polypeptide according to claims 1 to 5 which is antigenic.

7. A polypeptide according to claim 6 which is immunogenic and is capable of inducing antibodies in an immunised host against type-specific HSV-2 gG.

8. A pharmaceutical composition containing as an active ingredient an immunogenic polypeptide according to claim 7.

9. A vaccine composition containing as an active ingredient an immunogenic polypeptide according to claim 7 together with a physiologically acceptable adjuvant and/or carrier and/or diluent.

10. An antibody to the polypeptide of any of claims 1 to 7 obtainable by immunisation of a host with the immunogenic polypeptide of claim 7.

11. A recombinant DNA molecule comprising a DNA sequence encoding a polypeptide according to any of claims 1 to 7.

12. A filamentous bacteriophage including, in at least a proportion of its major coat protein sub-units, multiple display of a polypeptide according to claims 1 to 7.

13. A vaccine composition comprising a bacteriophage according to claim 12 together with a physiologically acceptable adjuvant and/or carrier and/or diluent.

14. A substantially pure non-glycosylated polypeptide according to claim 1 to 7.

15. A method of testing for the presence of type-specific HSV-2 gG2 antibodies in a fluid, which comprises contacting the fluid with one or more polypeptide (s) according to claim 1 to 7 or 14 and testing whether or not antibodies bind to the polypeptide (s) .

16. A method of testing for the presence of type-specific HSV-2 gG2 antibodies in a fluid, which comprises contacting the fluid d) with a labelled form of one or more polypeptide (s) according to claim 1 to 7 or 14 and

(11) with antibodies according to claim 10, whereby antigen in the fluid competes w th polypeptide (s) in binding to the antibodies.

17. A test kit for testing for the presence of HSV-2 type specific antibodies in a fluid, which comprises: (I) a solid phase on which is immobilised one or more polypeptide (s) according to claim 1 to 7 or 14; and

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

18. A test kit for testing for the presence of HSV-2 type specific antibodies in a fluid, which comprises:

(I) a solid phase on which is immobilised one or more polypeptide (s) according to claim 1 to 7 or 14 in labelled form;

(n) antibodies according to claim 6; ana (m) means for detecting competitive binding of antibodies to polypeptide (s) .

19. A method of diagnosis of HSV-2 infection which comprises employing the test method of claim 15 or 16.

20. A method of diagnosis of HSV-2 infection which comprises employing the test kit of claim 17 or 18 in the test method of claim 15 or 16.

21. A method of treatment of HSV-2 infection which comprises administration to an infected patient of an lmmunologically therapeutically effective amount of a vaccine composition according to claim 9 or 13.

22. A method of treatment of HSV-2 infection which comprises administration to an infected patient of an lmmunologically therapeutically effective amount of an antibody according to claim 10.

23. 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 9 or 13.

24. A method of prevention of HSV-2 infection which comprises administration to a patient a prophylactically effective amount of an antibody according to claim 10.

25. A polypeptide according to claim 1 to 7 conjugated to a Biotin-NH₂ terminus by a lysine (K) group.