WO1997046582A1 - Vaccin meningococcique - Google Patents

Vaccin meningococcique Download PDF

Info

Publication number
WO1997046582A1
WO1997046582A1 PCT/GB1997/001518 GB9701518W WO9746582A1 WO 1997046582 A1 WO1997046582 A1 WO 1997046582A1 GB 9701518 W GB9701518 W GB 9701518W WO 9746582 A1 WO9746582 A1 WO 9746582A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
peptide
seq
antibody
ligand
Prior art date
Application number
PCT/GB1997/001518
Other languages
English (en)
Inventor
Peter Laing
Michael Darsley
Patrick Jason Tighe
Original Assignee
Peptide Therapeutics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peptide Therapeutics Limited filed Critical Peptide Therapeutics Limited
Priority to AU30386/97A priority Critical patent/AU3038697A/en
Publication of WO1997046582A1 publication Critical patent/WO1997046582A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an anti-meningococcal vaccine, particularly for group-B serotype meningococcus.
  • the invention provides antigenic peptide ligands which can act as an immunogen capable of eliciting an immune response to produce antibodies against the capsular polysaccharide of group-B meningococci (CPS-B) .
  • the immunogen may be in the form of a polypeptide or in the form of a conjugate of such a polypeptide coupled to a carrier molecule such as a protein or in the form of a peptide displayed upon a virus particle (ie. a virion) , for example a bacteriophage.
  • the immunogen may be used in treatment of or prophylaxis against group-B meningococcal infection.
  • the invention also provides anti-group-B-meningococcal antibodies for use in treatment and/or prophylaxis.
  • the invention relates to methods for producing such immunogens, antibodies and vaccines and relates to their use in methods of treatment and prophylaxis.
  • the capsular polysaccharide of group-B meningococci is a homopolymer of repeated residues of ⁇ - (2-8) -linked oligomers of sialic acid: more specifically it is a linear polymer of ⁇ (2'-8) linked ' -N-acetylneuraminic acid residues 18"20 (2 '-8 'NANA) .
  • polysaccharide capsule material is found only in group-B meningococci, E. coli Kl strain, which is also a human pathogen, Pastuerella haemolytica, a sheep pathogen and Moraxella nonliquefaciens a non-pathogenic commensal 21 .
  • E. coli Kl strain which is also a human pathogen, Pastuerella haemolytica, a sheep pathogen and Moraxella nonliquefaciens a non-pathogenic commensal 21 .
  • the polysaccharide When isolated from E. coli Kl the polysaccharide is referred to as "Colominic acid" .
  • Efforts have to a large extent turned away from the use of capsular polysaccharide alone as a potential vaccine material, and concentrated on other components of bacterium (such as fimbriae, outer membrane proteins (OMPs) etc.), without noticeable success in terms of potential vaccines.
  • OMPs outer membrane proteins
  • capsular polysaccharide material used as a vaccine, elicits protective immune responses.
  • capsular polysaccharide material used as a vaccine, elicits protective immune responses.
  • i munoglobulins which bind the group-B capsular polysaccharide can be shown to have bactericidal activity in vitro and protect against in vivo challenge with lethal doses of N. meningi tidis or E. coli Kl .
  • the capsular polysaccharide of group-B meningococci appears unsuitable for effective use as a vaccine material because it is non-immunogenic or very weakly immunogenic even when coupled to carrier proteins.
  • H. influenzae type B vaccines consists of a capsular polysaccharide conjugate to meningococcal outer membrane proteins that are used for their special immunogenic properties.
  • the infectious agent in an inactivated or attenuated form, or some part of that agent, as a basis for the vaccine.
  • the approach we have adopted uses instead an antibody which embodies a surface which is complementary to some site on the agent (in terms of shape, charge etc.) .
  • the antibody is used (metaphorically) as a mould to fashion peptide ligands which represent the corresponding site on the infectious agent.
  • the site, and the corresponding antibody comprise a neutralising site and a neutralising antibody respectively - such that attachment of the antibody to the infectious agent inactivates the infectious agent or toxic products of that agent.
  • the process of generating peptides representative of the infectious agent is accomplished using libraries of synthetic or phage-displayed random peptides.
  • Members of peptide libraries that fit the antibody well comprise candidates for incorporation into vaccines, for example, in the form of peptide/carrier protein conjugates, or in the form of phage-peptide constructs.
  • the invention provides a pharmaceutical compound which includes a chemical composition capable of adopting a structure essentially equivalent to the pharmacophoric pattern of a section of the capsular polysaccharide of Neisseria meningi tidis group B, optionally together with a pharmaceutically acceptable carrier or excipient for use as an anti-group B capsular polysaccharide immunogen.
  • the invention provides peptides which include an amino acid sequence capable of adopting a structure having an pharmacophoric pattern essentially equivalent to the pharmacophoric pattern of a section of the capsular polysaccharide of Neisseria meningi tidis group B.
  • the invention provides antigenic peptide ligands which cross react with antibodies against the capsular polysaccharide of group B meningococci (CPS-B) which peptides include 1 or more copies of a motif which consists of a ring aromatic moiety, a spacer moiety, and a moiety having negative charge.
  • CPS-B capsular polysaccharide of group B meningococci
  • Such peptides may have two or more of said motifs which may repeat sequentially or may repeat in an overlapping frame.
  • the invention provides an antigenic peptide which includes an amino acid sequence and structure essentially equivalent to the pharmacophore defined as follows: the pharmacophore includes at least 3 chemical features; a Ring Aromatic, a Negative Charge and a Hydrogen Bond Acceptor or Donor feature. In addition to these 3 key chemical features there is a further optional feature which is a possible additional hydrogen bond donor/acceptor.
  • the key features are further defined as follows-.
  • the hydrogen bond acceptor feature matches the following atom types or groups of atoms which are surface accessible; • sp or sp 2 nitrogens that have a lone pair and a charge less than or equal to zero
  • the hydrogen bond donor feature has the same chemical characteristics as the hydrogen bond acceptor except that it also includes basic nitrogen (there is no exclusion of electron-deficient pyridines and imidazoles) ; this feature matches the following atom types or groups of atoms;
  • the negative charge feature is defined as a negative charge not adjacent to a positive charge
  • the ring aromatic feature is defined as an aromatic moiety which may be replaced by amino acid residues having hydrophobic character such as the amino acid residues selected from the following group; methionine, alanine, leucine, isoleucine, valine, proline; and
  • the hydrogen bond acceptor is represented by a vector function consisting of two spheres; the smaller sphere (at least 1.6Angstroms radius up to 2.6 Angstroms) defines the centroid of the hydrogen bond acceptor on the ligand while the large sphere (at least 2.2Angstroms radius up to 2.6 Angstroms) defines the projected point of the hydrogen bond acceptor from the receptor; these two spheres are at least 3.0Angstroms apart;
  • the negative charge is represented by a sphere at least 1.6Angstroms radius (up to 2.6 Angstroms) ; and (iii) the ring aromatic is represented as two equal size spheres (at least 1.6Angstroms radius up to 2.0 Angstroms) whose centroids are 3.1Angstroms apart; one sphere corresponds to the position of an aromatic ring moiety and the other to the projected point of the electron pi stacking of the aromatic ring system; and wherein the tolerances on all distances between these features is +/- 0.5 Angstroms and the geometric angles +/- 20 Degrees and said distances and angles are shown in figure 1 or figure 2.
  • the invention provides antigenic peptides of 3 to 25 amino acids of the general formula: X-B-C-D-Y wherein
  • X is any group of amino acids of length P; D is any hydrophobic amino acid group; C is any amino acid;
  • Y is any group of amino acids of length Q
  • P and Q may be zero and P + Q is less than or equal to 22.
  • These peptides preferably have 6 to 20 amino acids, most preferably 9 to 15 amino acids.
  • P + Q is less than or equal to 17, most preferably P + Q is less than or equal to 8.
  • X contains His.
  • D is Tyr.
  • C is an uncharged amino acid; more preferably C is Ser or Thr, most preferably C is Thr.
  • B is an acidic residue, more preferably B is Asp or Glu, most preferably B is Glu.
  • Y contains His.
  • a preferred embodiment of the invention has the general formula
  • the invention provides antigenic peptides of 5 to 25 amino acids of general formula:
  • A is any amino acid
  • E is any amino acid
  • X is any group of amino acids of length P;
  • Y is any group of amino acids of length Q
  • P & Q may be zero and P + Q is less than or equal to 20 and B,C,D are as defined as above.
  • the peptide is 6 to 20 amino acids, most preferably the peptide is 9 to 15 amino acids.
  • B is Asp or Glu, most preferably B is Asp.
  • A is Met.
  • E is His.
  • P + Q is less than or equal to 15, most preferably P + Q is less than or equal to 10.
  • a preferred embodiment of the invention has the general formula X-Met-Asp-Arg-Tyr-His-Y [SEQ ID NO:33] .
  • Compounds and peptides of the invention may advantageously be immunogenic.
  • an immunogenic ligand which comprises a synthetic polypeptide selected from compounds of sequence SEQ ID N0:1:
  • GDNFESYACVDTPCS and analogue and homologue derivatives thereof by virtue of one or more amino acid addition, deletion, substitution; together with terminal functional derivatives thereof.
  • the immunogenic ligand may be the pentadecapeptide of SEQ ID NO:l
  • TIPLWFDDEIEVMIY or the pentadecapeptide of SEQ ID NO: 2 GDNFESYACVDTPCS and terminal functional derivatives thereof.
  • the immunogenic ligand is conjugated to a carrier.
  • the immunogenic ligand comprises a bacteriophage which encodes and is capable of expressing a synthetic polypeptide as defined above.
  • the invention provides an antibody produced by an immune response to an immunogenic ligand as defined above which antibody specifically recognises and binds the synthetic polypeptide of the ligand and also the group-B meningococcal Capsular Polysaccharide (CPS-B) .
  • an immunogenic ligand as defined above which antibody specifically recognises and binds the synthetic polypeptide of the ligand and also the group-B meningococcal Capsular Polysaccharide (CPS-B) .
  • the invention also provides a pharmaceutical composition containing as active ingredient at least one ligand or one antibody as defined above, and optionally including an adjuvant or excipient, for use in the treatment of infection by group-B meningococcal bacteria or for use in prophylactic prevention of infection by group-B meningococcal bacteria.
  • This invention also provides a method of treatment or prophylaxis of infection with group-B meningococcal bacteria, which comprises administering an effective amount of a composition, antigen, ligand or antibody as defined above.
  • Amino acids and amino acid residues herein represent D and L amino acids, their analogues or derivatives.
  • Figure 1 is a geometric representation of pharmacophore 1 in which position 1 indicates the ring aromatic, position 2 indicates the location of the negative charge, and position 3 indicates the location of either the hydrogen bond donor or the hydrogen bond acceptor;
  • Figure 2 is a geometric representation of pharmacophore 2 in which position 1 indicates the ring aromatic, position 2 indicates the location of the negative charge, and position 3 indicates the location of either the hydrogen bond donor or the hydrogen bond acceptor;
  • Figure 3 corresponds to the figure 1 pharmacophore wherein positions 1, 2 and 3 are represented by spheres based on the centroids of the chemical features;
  • Figure 4 corresponds to the figure 2 pharmacophore wherein positions 1, 2 and 3 are represented by spheres based on the centroids of the chemical features;
  • Figure 5 shows the pharmacophore of figure 3 superimposed with two palindromic molecular structures represented by the sequences YTE and DRY.
  • Figure 6 shows the pharmacophore of figure 4 superimposed with two palindromic molecular structures represented by the sequences DRY and YTE.
  • Figure 7 shows the pharmacophore of figure 3 superimposed with a molecular structure represented by the sequence MDRYH
  • Figure 8 shows the pharmacophore of figure 4 superimposed with a molecular structure represented by the sequence MDRYH;
  • Figure 9 shows the pharmacophore of figure 3 superimposed with a molecular structure of the dye remazol brilliant violet
  • Figure 10 shows the pharmacophore of figure 4 superimposed with a molecular structure of the dye remazol brilliant violet
  • Figure 11 shows the pharmacophore of figure 3 superimposed with a molecular structure of the capsular polysaccharide of group-B meningococci;
  • Figure 12 shows the pharmacophore of figure 4 superimposed with a molecular structure of the capsular polysaccharide of group-B meningococci;
  • Figure 13 corresponds to figure 1 but shows the presence of an additional hydrogen bond donor/acceptor
  • Figure 14 corresponds to figure 2 but shows the presence of an additional hydrogen bond donor/acceptor
  • Figure 15 corresponds to figure 3 but shows the presence of an additional hydrogen bond donor/acceptor
  • Figure 16 corresponds to figure 4 but shows the presence of an additional hydrogen bond donor/acceptor
  • Figures 17 through 21 correspond to figures 5, 7, 9 and 11 but show the presence of an additional hydrogen bond donor/acceptor,-
  • Figure 22 shows results of probing a primary synthetic peptide library with a human anti-group-B polysaccharide antibody (IgM NOV ) ;
  • Figure 23 shows a combinational pharmacophore based on pharmacophores 1 and 2 superimposed on eachother. The resulting more detailed pharmacophore has features of both pharmacophores 1 and 2.
  • Figure 24 shows the pharmacophore of figure 23 and the centroids are labelled.
  • Figure 25 shows the absolute positions of the centroids of the pharmacophore shown in figure 23.
  • FIGS 26 to 29 show the distance and angle constraints of the centroids shown in figures 24 and 25.
  • Figure 30 demonstrates that the antigenic core of the GDN... peptide (ESYACVDTPCS) maps to a combined version of the pharmacophore.
  • critical side chains map to all of the features of this pharmacophore: E and D to the negative ionizable features; Y to the aromatic/hydrophobic feature and the hydroxyl of the Y side-chain maps (in this instance) as a hydrogen bond donor.
  • the antigenic importance of other residues in the peptide that do not map to identified features of the pharmacophore is likely to be attributable to their effect on the conformation of this peptide, thereby affecting the disposition of these critical side-chains.
  • Figure 31 depicts the mapping of the combined pharmacophore to the antigenic core region of peptide TIP..., namely DEIEVMIY.
  • DI and E4 satisfy the negative ionizable features
  • Y satisfies the ring aromatic/hydrophobic feature, a backbone N-H providing the hydrogen-bonding element (donor in this instance) .
  • Figure 32 depicts the mapping of methylmalonyl coenzyme-A to the combined pharmacophore. Note that all of the features are satisfied.
  • Figure 33 shows the less active compound, D-luciferin, of IC50 36 ⁇ M fitting the combined pharmacophore. Note that in this mappinng, not all features are mapped, consistent with its lower activity.
  • Figure 34 depicts an alternative mapping of D-luciferin to the combined pharmacophore. As before, not all features are mapped. The ability of D-luciferin to map in two distinct modes to the pharmacophre may explain the fairly good antigenic activity of this relatively small compound.
  • Figure 35 shows that the polysaccharide is capable of satisfying several (4/5) structural features of the combined pharmacophore.
  • Other mappings were found to be possible (not shown#) .
  • the negative ionizables fit well with alternate carboxylates of the polysaccharide (and not with adjacent carboxylates as might be anticipated) while both of the hydrogen bonding features (having a shared point of origin) are also satisfied.
  • Other mappings of the pharmacophore (not shown) were found to be possible, including ones in which the aromatic/hydrophobic feature was occupied by the six-membered ring of the saccharide monomer.
  • this ring is not strongly hydrophobic in character, it may at least be compatible with occupying a hydrophobic site on the antibody.
  • the existence of a plurality of mappings of the combined pharmacophore to this structure may reflect an ability of the antibody to engage multiple aspects of the helical structure of the polysaccharide, meaning that antigens identified by the pharmacophore may be particularly effective immunogens.
  • Figure 38 shows inhibition of binding of human anti- polysaccahride antibodies IgM Nov and 64V to solid phase poysaccharide by solution phase haptenic antigens of the diadenosine polyphosphate homologous series.
  • Figure 39 shows graphs of the binding of IgM Nov to Ala scanned mutants of GDN...
  • Figure 40 shows the list of compounds fitting the combinatorial pharmacophore and the training set for the Combinatorial pharmacophore.
  • an antibody is used to select phage expressing the peptide from mixtures of phage clones expressing a great variety (up to 100 million) of unique peptide sequences, essentially as described by Scott and Smith (Science, 1990) .
  • the identity of the peptide ligands is then deduced by sequencing of the recombinant DNA encoding the inser .
  • suitable peptides can be generated most conveniently by screening of complex mixtures of peptides synthesised as pools, for example in the form of a solid phase array (Gao et al 1996 in press) as described below.
  • Panning of hexapeptide libraries on pill was performed using IgM Nov , a well-characterised human antibody against the capsular polysaccharide of the group-B meningococci which is both bactericidal and protective in the infant rat model.
  • the resulting clonal population of peptide-expressing phage selected by the antibody did not however exhibit any thematic or consensus motif among the peptides sequenced (see e.g. SEQ ID NOS:12-24) .
  • the selected clonal population of phage did not exhibit any evidence of specific binding to the selecting antibody on solid phase in ELISA.
  • Array libraries of synthetic peptides were constructed. Such libraries are constructed as an array of spots on a membrane - each spot conforming to a prescribed generic formula, e.g. XOXOXXXX [SEQ ID NO:3] - where 'X' represents a mixture of all 19 proteinogenic amino acid residues (excluding cysteine) and where 'O' represents a fixed position in the sequence wherein the identity of the amino acid residue at that position is known.
  • XOXOXXXX SEQ ID NO:3
  • 'X' represents a mixture of all 19 proteinogenic amino acid residues (excluding cysteine) and where 'O' represents a fixed position in the sequence wherein the identity of the amino acid residue at that position is known.
  • FIG. 25 shows the results of probing two secondary synthetic peptide libraries of generic formulae XYXEXXXX (upper panel) and XDXYXXXX (lower panel) with a human anti-group-B polysaccharide antibody. Not all signals were inhibited by preabsorption of the antibody with polysaccharide (Colominic acid) , the ones which were strongly inhibited are indicated.
  • the preferred linear peptides can be inferred from this data as -YT/sE- [H] (upper) where T/s represents a preference for threonine and a tolerance of serine.
  • the brackets around "H” represent unspecified position in the sequence.
  • the dashes represent occurrence internally in the sequence.
  • the other preferred peptide was MDXYH, where the presence of histidine at position 5 was preferred. This finding confirms the observations made with the primary libraries which explored positioning and spacing of the fixed 'O' residues.
  • These studies implicated -DXXH- [SEQ ID NO:27] as a positive motif, consistent with the XDXYHXXX [SEQ ID NO:31] peptide found in the secondary library study.
  • the identified motifs are unexpectedly short - given the requirement of IgM Nov to recognise higher oligomers of sialic acid (9-unit oligomers being optimal) . This may be because these peptides bind in an extended conformation, whereas the polysialic acid binds in a helical conformation (Brisson et al) . Alternatively, they may reflect the binding of the peptides to part only (i.e. a subsite) of the antigen-combining surface of the antibody.
  • linear peptide libraries may indicate that small linear peptides of the specified sequences are adequate to occupy the antigen-combing site of anti-group-B polysaccharide antibodies fully. Alternatively, they may indicate that the flexibility of linear peptides does not allow the techniques used to specify structural attributes of a larger antigenic structure, because appropriate conformers may be too low in abundance to allow this.
  • Linear peptides convey little structural information.
  • Such procedures have been used before to identify 'pharmacophores' that epitomise the ideal structural features of drugs, and which can be used as structural templates to search databases of molecular structures for novel drug leads.
  • such techniques have not previously been applied to antigens or antibodies, nor (to our knowledge) have peptide libraries been used to generate pharmacophores.
  • a pharmacophore is a constellation of physicochemical features (charge, hydrophobicity, etc.) defined by a map of their relative positions and absolute distances in three-dimensional space, which epitomises the structural attributes required for a compound to have a particular pharmacological activity.
  • the validity of a pharmacophoric hypothesis can be tested experimentally by demonstrating that exemplars of that hypothesis have the predicted attributes in assays of binding or biological activity.
  • Inter-feature spacing was set to 1.5 Angstroms.
  • the mapping coefficient was set to l, in order to force topologically similar molecules to map the generated hypothesis in similar ways. All other parameters were set to the manufacturer's default values for revision 3.0 of of the software (i.e. Catalyst) .
  • a hydrogen bond acceptor feature matches the following atom types or groups of atoms which are surface accessible;
  • a hydrogen bond donor feature has the same chemical characteristics as the hydrogen bond acceptor except that it also includes basic nitrogen. There is no exclusion of electron-deficient pyridines and imidazoles. This feature matches the following atom types or groups of atoms;
  • a negative charge feature is defined as a negative charge not adjacent to a positive charge.
  • a ring aromatic feature is defined as an aromatic moiety which may be replaced by amino acid residues having hydrophobic character.
  • Tyrosine may be categorised as
  • this feature matches the following group: methionine, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and tyrosine.
  • the hydrogen bond acceptor is represented by a vector function consisting of two spheres.
  • the smaller sphere (at least l.6Angstroms radius up to 2.6 Angstroms) defines the centroid of the hydrogen bond acceptor on the ligand while the large sphere (at least 2.2 Angstroms radius up to 2.6 Angstroms) defines the projected point of the hydrogen bond acceptor from the receptor. These two spheres are at least 3.0 Angstroms apart.
  • the hydrogen bond donor is represented by a two sphere vector function as above.
  • the ring aromatic is represented as two equal size spheres (at least 1.6 Angstroms radius up to 2.0 Angstoms) whose centroids 'are 3.1 Angstroms apart.
  • One sphere corresponds to the position of an aromatic ring moiety and the other to the projected point of the electron pi stacking of the aromatic ring system.
  • the negative charge is represented by a sphere at least 1.6Angstroms radius (up to 2.6 Angstoms) .
  • the tolerances on all distances between these features is +/- 0.5 Angstroms and the geometric angles +/- 20 Degrees.
  • Position 1 indicates the ring aromatic
  • position 2 indicates the first and second possible locations of the negative charge
  • Position 3 indicates the location of either the hydrogen bond donor or hydrogen bond acceptor.
  • Position 4 indicates the position of a possible additional hydrogen bond donor/acceptor (i.e. an optional extra hydrogen bond donor/acceptor feature) .
  • figure 5 demonstrates the fit of the two palindromic molecules of sequence YTE and DRY; figure 7 demonstrates the fit of the 5mer peptide of sequence MDRYH.
  • figure 6 demonstrates the fit of the same molecules in the alternative pharmacophoric model.
  • figures 19 and 20 show the same two molecules fitted to pharmacophore 1 which includes the optional additional fourth chemical feature of another hydrogen bond donor/acceptor.
  • the predictive accuracy of the pharmacophoric model of the antigen was evaluated by comparing the observed activity of peptide ligands versus that predicted by the model.
  • the synthetic peptides can be expressed on the surface of filamentous bacteriophage using standard techniques for phage display such as those described by Perham et al (WO92/07077) and Greenwood et al (Greenwood J. , Willis A. E. and Perham, R. N. 1991. J. Mol Biol. 220. 821-827) to produce peptide ligands in the correct structural orientation.
  • the constructs can then be used to evaluate the potential of the peptide ligands to mimic the capsular polysaccharide.
  • phage clones expressing the sequences TIPLWFDDEIEVMIY [SEQ ID NO:l] (referred to as “TIP") and GDNFESYACVDTPCS [SEQ ID NO:2] (referred to as “GDN --) were used to immunise rats, and the resulting sera were tested for anti-polysaccharide antibodies.
  • TIP TIPLWFDDEIEVMIY
  • GDN GDNFESYACVDTPCS
  • the phage clone expressing peptide GDN... was subjected to Ala-scan mutagenesis according to standard methods wherein the DNA encoding particular amino-acids in the peptide was substituted for a codon representing alanine. Phage were propagated in E.coli and purified by three rounds of precipitation with polyethylene glycol. The binding of unaltered and mutated phage clones was assesed in a solid phase electrochemiluminescent (ECL) assay using an Origen analyser (Igen) . Magnetic beads were coated with a polyclonal anti-phage (anti-Ml3) antibody and used to capture phage from solution.
  • ECL solid phase electrochemiluminescent
  • Igen Origen analyser
  • Peptide TIP... was subjected first to a 'window-scan' analysis whereby peptides were made on membrane (as above) representing subsequences comprising residues 1-8, 2-9, 3- 10 etc.of this 15mer peptide.
  • This analysis (not shown) identified two overlapping peptides of sequence DDEIEVMI and DEIEVMIY respectively to be antigenically active, localising the antigenicity of the 15mer peptide TIP... to a smaller 9-mer sequence (i.e. DDEIEVMIY) .
  • DDEIEVMIY 9-mer sequence
  • this sequence contains both tyrosine and the acidic residues D and E as important features, as for the peptides identified by the synthetic library studies and as in the case of the GDN... peptide (above) .
  • the substitution net of the 15mer phage- derived peptide TIP... demonstrates the existence of a core region which is highly optimised for antibody binding, namely DEIEVMIY, being surprisingly intolerant of substitution.
  • this peptide is richly provided with residues capable of fulfilling the requirements of the 'combined pharmacophore' description of the epitope (i.e. hydrophobic/aromatic and acidic residues) . It is likely therefore that various conformations or aspects of this peptide can mimic the polysaccharide epitope. This may explain why this peptide was selected during phage display as a favoured antigenic sequence by the antibody.
  • the two alternative pharmcophores were found to be identical except for the positioning of a negative ionizable feature. According to their descriptions, the negative ionizable feature could be in one of two alternative positions. However, another interpretation of these findings is that the antigen-binding requirements of the antibody could be satisfied better by a molecule which would provide the common features of these two hypotheses, but in addition, simultaneously satisfying both of the negative ionizable features suggested originally to be alternatives. In order to avoid generating a combined hypothesis which was too complex to search or too computationally laborious to generate, the optional hydrogen bond donor feature (common to both hypotheses) was discarded.
  • the two constituent hypotheses were superposed using the root-mean square fitting algorithm of Catalyst to generate the combined pharmacophore.
  • the common ring aromatic feature, the two negative ionizables (originating separately from the different constituent pharmacophores) , and the H-bond donor and acceptor features (having differing vectors - yet a common centroid point of origin) were combined to generate a new 5-feature hypothesis.
  • the combined hypothesis was used to search the Available Chemicals Directory, resulting in only 2 hits, one of which transpired to be the most active compound yet identified (i.e. methylmalonyl coenzyme-A at an IC50 of 7 ⁇ M) .
  • the strongly active compounds were: nicotinamide 1, N6 ethanoadenine dinucleotide phosphate, Pl,P5-di (adenosine-5 ' ) pentaphosphate, nicotinamide hypoxanthine dinucleotide phopshate and methylmalonyl coenzyme A (IC50 7 ⁇ M) .
  • This is a remarkably high hit rate (50%) compared to the rates expected for random screening of compounds ( ⁇ 1%) and therefore represents strong evidence for the validity of the more-detailed 'combined' form of the pharmacophoric hypothesis.
  • N6 ethenoadenine dinucleotide phosphate was markedly less active (IC50 of 150 ⁇ M) than its phosphorylaed counterpart (IC50, ll ⁇ M) emphasizing the importance of having two negative charges.
  • Figure 38 illustrates a comparison of the antigenic potency of this family of compounds in a hapten inhibition assay (the assay used to determine IC50 values for haptenic antigens) . It is evident from this figure that although all of the compounds (i.e. tri- up to hexa-phosphate) were fully antigenically active, there was a clear gradation of potency - increasing up to the pentaphosphate and being increased only marginally on moving up to the hexaphosphate homologue.
  • the pentaphosphate is the first compound capable of spanning the two negative ionizable features.
  • the tri and tetra phosphates were also active, although less so, despite being unable to satisfy simultaneously both negative ionizable features.
  • the fact that these latter compounds were active demonstrates that the satisfaction of one only of the two negative ionizable features is sufficient for antigenicity, provided that the other features of the hypothesis are satisfied.
  • a recombinant filamentous bacteriophage (fd) clone expressing the sequence GDNFESYACVDTPCS [SEQ ID NO:2] (GDN...) fused to the N-terminal of the gene-VIII protein was purified by caesium chloride gradient sedimentation and injected subcutaneously in 12.5 microgram doses into six Wistar rats. This procedure was repeated for each of the six rats a further two times at 2-week intervals, and serum samples were taken at intervals.
  • colominic acid alpha 2-8 linked polysialic acid, the capsular polysaccharide of E.
  • polystyrene ELISA plates coated with colominic acid were incubated first with serial dilutions of the sera and secondly with a species-specific anti-IgG alkaline-phosphatase conjugate, and finally developed with a chromogenic substrate for alkaline phosphatase.
  • the IgG class of the antibodies is particularly significant, since it demonstrates 'class-switching' of the immune response, a hallmark of immunological memory. It is also significant that this response was obtained in the absence of adjuvant, since it demonstrates that the phage construct is particularly immunogenic for this notoriously weak antigen (i.e. the group-B polysaccharide) .
  • Spurious signals can be generated in ELISA tests via the non-specific binding of antibodies to solid phase components other than the antigen (e.g. the polystyrene itself or the blocking agent used to minimise non-specific binding to the polystyrene) .
  • These signals may be distinguished from specific signals, because only the latter are inhibited by preabsorption with colominic acid in solution phase.
  • colominic acid in solution phase.
  • TIP TIP
  • a further peptide ligand according to the inventurn is
  • This peptide ligand has tyrosine (Y) and glutamic acid (E) residues brought into proximity by means of a disulphide bond.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention a trait à un vaccin méningococcique concernant en particulier le ménigocoque du sérotype du groupe B. L'invention concerne des ligands de peptide antigénique qui peuvent agir comme immunogène et induire une immunoréaction destinée à produire des anticorps dirigés contre le polysaccharide capsulaire de ménigocoques du groupe B (CPS-B).
PCT/GB1997/001518 1996-06-05 1997-06-05 Vaccin meningococcique WO1997046582A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30386/97A AU3038697A (en) 1996-06-05 1997-06-05 Meningococcal vaccine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9611673.6A GB9611673D0 (en) 1996-06-05 1996-06-05 Meningococcal vaccine
GB9611673.6 1996-06-05

Publications (1)

Publication Number Publication Date
WO1997046582A1 true WO1997046582A1 (fr) 1997-12-11

Family

ID=10794746

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/001518 WO1997046582A1 (fr) 1996-06-05 1997-06-05 Vaccin meningococcique

Country Status (3)

Country Link
AU (1) AU3038697A (fr)
GB (1) GB9611673D0 (fr)
WO (1) WO1997046582A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010372A1 (fr) * 1997-08-27 1999-03-04 Chiron Corporation Mimetiques moleculaires des epitopes b meningococciques
WO1999033969A1 (fr) * 1997-12-31 1999-07-08 Pincus Seth H Technique permettant d'isoler un peptide imunologiquement mimetique de glucides microbiens, et notamment des glucides des streptocoques du groupe b, et utilisation dudit peptide dans un vaccin
WO2000025814A2 (fr) * 1998-10-30 2000-05-11 University College London Constituant pour un vaccin
WO2001032692A2 (fr) * 1999-10-29 2001-05-10 Rice Peter A Mimetiques peptidiques d'epitopes gonococciques conserves, techniques et compositions les utilisant
EP1252182A1 (fr) * 2000-01-25 2002-10-30 The University Of Queensland PROTEINES COMPRENANT DES REGIONS CONSERVEES DE L'ANTIGENE DE SURFACE NEISSERIA MENINGITIDIS NhhA
EP1712915A1 (fr) * 2004-02-03 2006-10-18 Asahi Kasei Kabushiki Kaisha PROCEDE DE DETECTION D’ANALYTE EN UTLISANT UN CORDON MAGNETIQUE
US7189405B1 (en) 1999-10-29 2007-03-13 Rice Peter A Peptide mimics of conserved gonococcal epitopes and methods and compositions using them
WO2007110632A1 (fr) * 2006-03-28 2007-10-04 Liverpool School Of Tropical Medicine Vaccin bactérien
WO2009040529A1 (fr) * 2007-09-28 2009-04-02 Ulive Enterprises Limited Vaccin bactérien

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990006696A2 (fr) * 1988-12-19 1990-06-28 Praxis Biologics, Inc. Vaccin meningocoque de la proteine de la membrane externe de la classe 1
WO1992018150A1 (fr) * 1991-04-23 1992-10-29 Anergen, Inc. Conjugues cmh permettant de traiter l'auto-immunite
GB2282380A (en) * 1993-09-30 1995-04-05 Merck & Co Inc Conjugate of epitopes of HIV with a protein complex from Neisseria
WO1995026365A1 (fr) * 1994-03-28 1995-10-05 United Biomedical, Inc. Immunogenes a base de peptide synthetique pour le traitement de l'allergie
WO1996000238A1 (fr) * 1994-06-24 1996-01-04 Peptide Therapeutics Limited Epitopes de lymphocytes t d'allergene du pollen de ray-grass

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990006696A2 (fr) * 1988-12-19 1990-06-28 Praxis Biologics, Inc. Vaccin meningocoque de la proteine de la membrane externe de la classe 1
WO1992018150A1 (fr) * 1991-04-23 1992-10-29 Anergen, Inc. Conjugues cmh permettant de traiter l'auto-immunite
GB2282380A (en) * 1993-09-30 1995-04-05 Merck & Co Inc Conjugate of epitopes of HIV with a protein complex from Neisseria
WO1995026365A1 (fr) * 1994-03-28 1995-10-05 United Biomedical, Inc. Immunogenes a base de peptide synthetique pour le traitement de l'allergie
WO1996000238A1 (fr) * 1994-06-24 1996-01-04 Peptide Therapeutics Limited Epitopes de lymphocytes t d'allergene du pollen de ray-grass

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 119, no. 23, 6 December 1993, Columbus, Ohio, US; abstract no. 244157, CHO E.A.: "Substrate specificities of catalytic fragments of protein tyrosine phosphatases..." XP002039413 *
CHEMICAL ABSTRACTS, vol. 122, no. 13, 27 March 1995, Columbus, Ohio, US; abstract no. 161287, AGUILAR E.A.: "Molecular definition of the retention parameters of peptides separated by RP-HPLC" XP002039412 *
CHEMICAL ABSTRACTS, vol. 98, no. 9, 28 February 1983, Columbus, Ohio, US; abstract no. 65800, MEDVEDEV E.A.: "Effects of neuropeptides, modulators of memory, on background and induced electrocortical activity in rabbit cerebral hemispheres" XP002039375 *
DIAZ ROMERO E.A.: "Current status of meningococcal group B vaccine candidates: capsular or non-capsular", CLINICAL MICROBIOLOGY REVIEWS, vol. 7, no. 4, October 1994 (1994-10-01), pages 559 - 575, XP002039373 *
NEIROFIZIOLOGIYA, vol. 14, no. 6, 1982, pages 578 - 584 *
PEPT.RES., vol. 7, no. 4, 1994, pages 207 - 217 *
PROT.SCI, vol. 2, no. 6, 1993, pages 977 - 984 *
WESTERINK E.A.: "Peptide mimicry of the meningococcal group C capsular polysaccharide", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 92, April 1995 (1995-04-01), WASHINGTON US, pages 4021 - 4025, XP002039374 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6030619A (en) * 1997-08-27 2000-02-29 Chiron Corporation Molecular mimetics of meningococcal B epitopes
WO1999010372A1 (fr) * 1997-08-27 1999-03-04 Chiron Corporation Mimetiques moleculaires des epitopes b meningococciques
US6444787B1 (en) 1997-12-31 2002-09-03 Research Development Institute, Inc. Method of isolating a peptide which immunologically mimics microbial carbohydrates including group B streptococcal carbohydrates and the use thereof in a vaccine
WO1999033969A1 (fr) * 1997-12-31 1999-07-08 Pincus Seth H Technique permettant d'isoler un peptide imunologiquement mimetique de glucides microbiens, et notamment des glucides des streptocoques du groupe b, et utilisation dudit peptide dans un vaccin
WO2000025814A2 (fr) * 1998-10-30 2000-05-11 University College London Constituant pour un vaccin
WO2000025814A3 (fr) * 1998-10-30 2000-07-27 Univ London Constituant pour un vaccin
US7871628B2 (en) 1999-10-29 2011-01-18 University Of Massachusetts Peptide mimics of conserved gonococcal epitopes and methods and compositions using them
WO2001032692A3 (fr) * 1999-10-29 2002-03-07 Peter A Rice Mimetiques peptidiques d'epitopes gonococciques conserves, techniques et compositions les utilisant
US7189405B1 (en) 1999-10-29 2007-03-13 Rice Peter A Peptide mimics of conserved gonococcal epitopes and methods and compositions using them
CN101638433B (zh) * 1999-10-29 2014-10-29 彼得·A·赖斯 保守淋球菌表位的拟肽及应用它们的方法和组合物
WO2001032692A2 (fr) * 1999-10-29 2001-05-10 Rice Peter A Mimetiques peptidiques d'epitopes gonococciques conserves, techniques et compositions les utilisant
CN102417537A (zh) * 2000-01-25 2012-04-18 昆士兰大学 包含脑膜炎奈瑟氏球菌表面抗原NhhA的保守区域的蛋白质
EP1252182A1 (fr) * 2000-01-25 2002-10-30 The University Of Queensland PROTEINES COMPRENANT DES REGIONS CONSERVEES DE L'ANTIGENE DE SURFACE NEISSERIA MENINGITIDIS NhhA
EP1252182A4 (fr) * 2000-01-25 2005-03-02 Univ Queensland PROTEINES COMPRENANT DES REGIONS CONSERVEES DE L'ANTIGENE DE SURFACE NEISSERIA MENINGITIDIS NhhA
US8383790B2 (en) 2000-01-25 2013-02-26 The University Of Queensland Modified surface antigen
US8367070B2 (en) 2000-01-25 2013-02-05 The University Of Queensland Modified surface antigen
US7947291B2 (en) 2000-01-25 2011-05-24 The University Of Queensland Modified surface antigen
EP2395013A3 (fr) * 2000-01-25 2012-03-21 The University of Queensland Protéines comprenant des régions conservées d'antigène de surface NhhA de Neisseria meningitidis
EP1712915A1 (fr) * 2004-02-03 2006-10-18 Asahi Kasei Kabushiki Kaisha PROCEDE DE DETECTION D’ANALYTE EN UTLISANT UN CORDON MAGNETIQUE
EP1712915A4 (fr) * 2004-02-03 2009-07-22 Asahi Chemical Ind Procede de detection d'analyte en utlisant un cordon magnetique
WO2007110632A1 (fr) * 2006-03-28 2007-10-04 Liverpool School Of Tropical Medicine Vaccin bactérien
WO2009040529A1 (fr) * 2007-09-28 2009-04-02 Ulive Enterprises Limited Vaccin bactérien

Also Published As

Publication number Publication date
AU3038697A (en) 1998-01-05
GB9611673D0 (en) 1996-08-07

Similar Documents

Publication Publication Date Title
Fox et al. Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens.
Chong et al. A strategy for rational design of fully synthetic glycopeptide conjugate vaccines
Beachey et al. Epitope-specific protective immunogenicity of chemically synthesized 13-, 18-, and 23-residue peptide fragments of streptococcal M protein.
AU8091694A (en) Structured synthetic antigen libraries as diagnostics, vaccines and therapeutics
IE903899A1 (en) Vaccine composition with non-immunosuppressive T-cell¹epitope component
US6174528B1 (en) Synthetic peptides and vaccines comprising same
Beachey et al. Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein.
EP0896583A1 (fr) Nouveaux fragments peptidiques derives de ospc
WO1997046582A1 (fr) Vaccin meningococcique
JPH08501282A (ja) Ｃ型肝炎ウイルスに特異的な細胞障害性ｔ細胞を刺激するためのペプチド
CS68091A2 (en) Synthetic polypeptides
Kraus et al. Renal autoimmune epitope of group A streptococci specified by M protein tetrapeptide Ile-Arg-Leu-Arg.
US7122193B1 (en) Retro peptides, antibodies thereto and their uses for vaccination and in vitro diagnosis
Wiertz et al. T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.
JPH02503916A (ja) Ｈｉｖエンベロープ蛋白質に関連する合成ペプチド
CN105658661B (zh) 疫苗
US5000952A (en) Polypeptide pertussis toxin vaccine
Edwards et al. Antigenic analysis of gonococcal pili using monoclonal antibodies.
Du et al. Structural and immunological characterisation of heteroclitic peptide analogues corresponding to the 600–612 region of the HIV envelope gp41 glycoprotein
JPH05508838A (ja) Ｈｔｌｖ―ｉおよびｈｔｌｖ―ｉｉウイルスに対する抗体を検出するためのペプチドおよびアナログおよびその混合物
Garay et al. Study of various presentation forms for a peptide mimetic of Neisseria meningitidis serogroup B capsular polysaccharide
WO1991006562A1 (fr) Domaines immunologiques de l'antigene du virus delta de l'hepatite
WO2011103465A2 (fr) Compositions antigéniques comprenant la neurotoxine botulique et procédés associés
Delvig et al. Immune responses to linear epitopes on the PorB protein of Neisseria meningitidis in patients with systemic meningococcal disease
JP2002528517A (ja) ワクチン成分

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WA Withdrawal of international application
122 Ep: pct application non-entry in european phase
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA