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WO1998003543A1 - Identification de sequences peptidiques courtes representant des epitopes de la glycoproteine g de hsv-2, au moyen d'une banque d'affichage de peptides phages - Google Patents

Identification de sequences peptidiques courtes representant des epitopes de la glycoproteine g de hsv-2, au moyen d'une banque d'affichage de peptides phages Download PDF

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Publication number
WO1998003543A1
WO1998003543A1 PCT/GB1997/001990 GB9701990W WO9803543A1 WO 1998003543 A1 WO1998003543 A1 WO 1998003543A1 GB 9701990 W GB9701990 W GB 9701990W WO 9803543 A1 WO9803543 A1 WO 9803543A1
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hsv
polypeptide
antibodies
phage
sequence
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PCT/GB1997/001990
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Anna Marie Grabowska
William Lucien Irving
Peter Laing
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Peptide Therapeutics Limited
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Priority to AU36301/97A priority Critical patent/AU3630197A/en
Publication of WO1998003543A1 publication Critical patent/WO1998003543A1/fr

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    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/02Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • C12N15/1037Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • G01N33/56994Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in epitope analysis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/16011Herpesviridae
    • C12N2710/16611Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01DNA viruses
    • G01N2333/03Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035Herpes simplex virus I or II

Definitions

  • Phage peptide libraries typically comprise more than 10 7 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.
  • Herpes simplex virus type 2 (HSV-2) is the mam cause of recurrent genital herpes (1)
  • HSV-2 Herpes simplex virus type 2
  • the vast majority of individuals infected with HSV-2 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.
  • 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.
  • gG-2-based assays for the detection of HSV-2 antibodies using Helix poz ⁇ atia-pu ⁇ fled gG-2 as antigen in immunoblot and ELISA formats have been described (6, 7, 13) .
  • difficulties in large-scale production of gG2 of sufficient purity have precluded the widespread availability of such assays .
  • 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.
  • the present invention provides a polypeptide consisting of 3 to 38 amino acid residues, having the sequence of ⁇ EQ ID:1 or a portion thereof; and analogous polypeptide derivatives by virtue of point mutation, amino acid substitution, deletion or addition;
  • 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 :
  • (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 :
  • (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 antibody.
  • the invention also provides a polypeptide which is the sequence SEQ ID: 2 consisting of A 1 to G 20 of SEQ ID:1 (PT71) .
  • the invention also provides a polypeptide which is the sequence SEQ ID: 3 consisting of P 4 to P 23 of SEQ ID:1 (PT 487) .
  • the invention also provides a polypeptide conjugated to a B ⁇ otm-NH 2 terminus by a lysme (K) group.
  • the invention also provides a polypeptide which is the sequence SEQ ID: 42 consisting of A 1 to F 14 to SEQ ID: 1 (PT444) .
  • 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.
  • this region having some HSV-2 unique portions but also some homology to HSV-l portions.
  • 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.
  • 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 ⁇ )
  • 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 .
  • 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.
  • 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- NH 2
  • 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'PPPPEHRGGPEEF 14
  • PT445 SEQ ID:43: KTPPTTPAPTTPPPTSTHAT
  • Anti-gG2 monoclonal antibodies 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.
  • the library used was a gift from Dr. G. Smith (Missouri, USA) containing approximately 10 8 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.
  • 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.
  • IPTG iso-propyl-thio-galactose
  • the K91Kan strain of E. coli a ⁇ -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.
  • 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.
  • TBS Tris- buffered saline
  • 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.
  • 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)
  • elution buffer 0.2M glycme, 0.1M HC1, 0.1% BSA, 0. lmg/ml phenol red, pH 2.2
  • 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.
  • 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.
  • PEG-precipitated phage from round 2 were used.
  • 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 MgCl 2 , 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 were a gift from Peptide Therapeutics Ltd. (Cambridge, UK) . They were synthesised by standard f-moc methodology.
  • 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.
  • 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 H 2 S0 4 .
  • 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.
  • Clustal W 1.
  • Multi Sequence Alignment http://biology.ncsa.uiuc.edu/BW/BW.cgi
  • PAE ammo acids
  • 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.
  • 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.
  • 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.
  • PFT second motif
  • Peptides were added at a range of concentrations from 500 ⁇ g/ml to 7.5 ⁇ g/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.
  • PFT Another apparent motif, 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) .
  • 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) .
  • 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.
  • 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 10 8 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.
  • 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 ant ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • peptides 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.
  • 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 ant ⁇ -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.
  • 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.
  • 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 t ⁇ tiated N-succmimidyl propionate (Amersham catalogue) .
  • the antigenic peptide 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.
  • 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.
  • 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
  • Carriers 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 .
  • synthetic peptides e.g. collinearly synthesised with HSV-2 peptides
  • 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 .
  • 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 ni ⁇ bs IIS, I' 1 and Ii5, the se(]i ⁇ enccs 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(
  • I I56 lillRGGPTiP. (gG2, amino acids 556- 562, 8n ⁇ er variant of P 171) S > £S_ ip • ⁇ Mo3?

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Abstract

L'invention concerne un polypeptide, qui comporte 3 à 38 restes d'acides aminés et possède la séquence SEQ ID:1 ou une portion de celle-ci, ainsi que des dérivés polypeptidiques analogues obtenus par mutation ponctuelle, substitution, délétion ou addition d'acides aminés, la séquence SEQ ID:1 étant la suivante: A?1PPP4PE6H7R8GGPEEF14EGAGDG20 EPP23¿EDDDSATGLAFRTPN38, dans laquelle est compris le reste histidine H7, et où E6 peut être substitué par ordre de préférence par D?6⊃T6, R8¿ peut être substitué par A8, cette séquence étant reconnue par des sérums humains positif anti-gG2 obtenus à partir de patients présentant une infection à HSV-2, et n'étant pas reconnue par des sérums négatifs anti-gG2 provenant de patients présentant une infection à HSV-1. On décrit encore des anticorps dirigés contre ce polypeptide, ainsi que des utilisations prophylactiques, thérapeutiques et diagnostiques de ce polypeptide en rapport avec l'infection à HSV-2.
PCT/GB1997/001990 1996-07-24 1997-07-24 Identification de sequences peptidiques courtes representant des epitopes de la glycoproteine g de hsv-2, au moyen d'une banque d'affichage de peptides phages WO1998003543A1 (fr)

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PCT/GB1997/002003 WO1998003544A1 (fr) 1996-07-24 1997-07-24 IDENTIFICATION DE SEQUENCES PEPTIDIQUES COURTES REPRESENTANT DES EPITOPES DE LA GLYCOPROTEINE G DE HSV-2, AU MOYEN D'UNE BANQUE D'AFFICHAGE DE PEPTIDES PHAGES UTILE DANS UN DOSAGE ELISA ANTI-gG2

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Cited By (10)

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GB2323360A (en) * 1997-01-14 1998-09-23 Medical Res Council Peptide structures for use in detecting herpes simplex virus type 2
WO2001081421A3 (fr) * 2000-04-21 2002-04-04 Tripep Ab Peptides de synthese qui se fixent au coeur du virus de l'hepatite b et aux antigenes e
US6660842B1 (en) 1994-04-28 2003-12-09 Tripep Ab Ligand/receptor specificity exchangers that redirect antibodies to receptors on a pathogen
US6933366B2 (en) 1996-12-27 2005-08-23 Tripep Ab Specificity exchangers that redirect antibodies to bacterial adhesion receptors
JP2007224043A (ja) * 2007-04-18 2007-09-06 Medical Res Council ペプチド構造物およびそれらの単純ヘルペスウイルス2型の診断における使用
US7267940B2 (en) * 2003-03-04 2007-09-11 Bio-Rad Laboratories, Inc. HSV-2 type-specific immunoassays using glycoprotein G2 peptides
JP2007526919A (ja) * 2004-03-05 2007-09-20 バイオ−ラッド ラボラトリーズ,インコーポレイティド 糖タンパク質g2ペプチドを用いたhsv−2型特異的免疫検定
US7318926B2 (en) 2003-02-06 2008-01-15 Tripep Ab Glycosylated specificity exchangers
US7335359B2 (en) 2003-02-06 2008-02-26 Tripep Ab Glycosylated specificity exchangers
US10654899B2 (en) 2016-02-01 2020-05-19 Simplexia Ab Truncated glycoprotein G of herpes simplex virus 2

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WO1990013652A1 (fr) * 1989-05-12 1990-11-15 Triton Diagnostics, Inc. Proteines et polypeptides de la glycoproteine g du virus de type 2 de l'herpes simplex
WO1991017443A1 (fr) * 1990-05-04 1991-11-14 E.I. Du Pont De Nemours And Company Procede servant a determiner des anticorps specifiques au type du virus de l'herpes simple des types 1 et 2

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WO1991017443A1 (fr) * 1990-05-04 1991-11-14 E.I. Du Pont De Nemours And Company Procede servant a determiner des anticorps specifiques au type du virus de l'herpes simple des types 1 et 2

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M. LEVI ET AL.: "Peptide sequences of glycoprotein G-2 discriminate between herpes simplex virus type 2 (HSV-2) and HSV-1 antibodies", CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, vol. 3, no. 3, May 1996 (1996-05-01), pages 265 - 269, XP002042984 *
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6660842B1 (en) 1994-04-28 2003-12-09 Tripep Ab Ligand/receptor specificity exchangers that redirect antibodies to receptors on a pathogen
US7019111B2 (en) 1994-04-28 2006-03-28 Tripep Ab Glycosylated ligand/receptor specificity exchangers specific for bacterial adhesion receptors
US6933366B2 (en) 1996-12-27 2005-08-23 Tripep Ab Specificity exchangers that redirect antibodies to bacterial adhesion receptors
GB2323360A (en) * 1997-01-14 1998-09-23 Medical Res Council Peptide structures for use in detecting herpes simplex virus type 2
GB2323360B (en) * 1997-01-14 1999-06-23 Medical Res Council Peptide structures and their use in diagnosis of herpes simplex virus type 2
WO1999038886A1 (fr) * 1997-01-14 1999-08-05 Medical Research Council Structures peptidiques et leur application en matiere de diagnostic de la presence du virus de l'herpes simplex de type 2
US5965357A (en) * 1997-01-14 1999-10-12 Medical Research Council Peptides structures and their use in diagnosis of herpes simplex virus type 2
AU756368B2 (en) * 1997-01-14 2003-01-09 Medical Research Council Peptide structures and their use in diagnosis of herpes simplex virus type 2
WO2001081421A3 (fr) * 2000-04-21 2002-04-04 Tripep Ab Peptides de synthese qui se fixent au coeur du virus de l'hepatite b et aux antigenes e
US6417324B1 (en) 2000-04-21 2002-07-09 Tripep Ab Synthetic peptides that bind to the hepatitis B virus core and e antigens
US7335359B2 (en) 2003-02-06 2008-02-26 Tripep Ab Glycosylated specificity exchangers
US7318926B2 (en) 2003-02-06 2008-01-15 Tripep Ab Glycosylated specificity exchangers
US7332166B2 (en) 2003-02-06 2008-02-19 Tripep Ab Glycosylated specificity exchangers
US7534435B2 (en) 2003-02-06 2009-05-19 Tripep Ab Glycosylated specificity exchangers
US8303956B2 (en) 2003-02-06 2012-11-06 Chrontech Pharma Ab Glycosylated specificity exchangers
US8658179B2 (en) 2003-02-06 2014-02-25 Chrontech Pharma Ab Glycosylated specificity exchangers
US9079962B2 (en) 2003-02-06 2015-07-14 Tripep Ab Glycosylated specificity exchangers
US7267940B2 (en) * 2003-03-04 2007-09-11 Bio-Rad Laboratories, Inc. HSV-2 type-specific immunoassays using glycoprotein G2 peptides
JP2007526919A (ja) * 2004-03-05 2007-09-20 バイオ−ラッド ラボラトリーズ,インコーポレイティド 糖タンパク質g2ペプチドを用いたhsv−2型特異的免疫検定
JP2007224043A (ja) * 2007-04-18 2007-09-06 Medical Res Council ペプチド構造物およびそれらの単純ヘルペスウイルス2型の診断における使用
US10654899B2 (en) 2016-02-01 2020-05-19 Simplexia Ab Truncated glycoprotein G of herpes simplex virus 2

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