WO2002072624A2 - Analyses, methodes et elements pour le ciblage de streptocoques - Google Patents

Analyses, methodes et elements pour le ciblage de streptocoques Download PDF

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Publication number
WO2002072624A2
WO2002072624A2 PCT/GB2002/001200 GB0201200W WO02072624A2 WO 2002072624 A2 WO2002072624 A2 WO 2002072624A2 GB 0201200 W GB0201200 W GB 0201200W WO 02072624 A2 WO02072624 A2 WO 02072624A2
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Prior art keywords
sic
lysozyme
slpi
polypeptide
fragment
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PCT/GB2002/001200
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English (en)
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WO2002072624A3 (fr
Inventor
Peter Julius Lachmann
David Jeffrey Seilly
Barbara Ann Fernie-King
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Cambridge University Technical Services Limited
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Priority claimed from GB0106319A external-priority patent/GB0106319D0/en
Priority claimed from GB0123388A external-priority patent/GB0123388D0/en
Application filed by Cambridge University Technical Services Limited filed Critical Cambridge University Technical Services Limited
Priority to AU2002249342A priority Critical patent/AU2002249342A1/en
Publication of WO2002072624A2 publication Critical patent/WO2002072624A2/fr
Publication of WO2002072624A3 publication Critical patent/WO2002072624A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus

Definitions

  • the present invention is based on finding that a streptococcal protein termed "Streptococcal Inhibitor of Complement” (SIC) which is involved in streptococcus infection, interacts with and inhibits the enzymatic activity of lysozyme and interacts with secretory leucocyte proteinase inhibitor (SLPI) .
  • SIC Streptococcal Inhibitor of Complement
  • SLPI secretory leucocyte proteinase inhibitor
  • the invention in various aspects relates to disrupting or inhibiting the interaction between the proteins and the inhibition of lysozyme and of SLPI by SIC, primarily in an antibiotic or therapeutic context .
  • SIC Streptococcal Inhibitor of Complement
  • MAC complement membrane attack complex
  • the present inventors were initially interested in looking at interaction of SIC with intermediate forms of the terminal complement complex.
  • the interaction with complement that was found did not adequately explain why SIC should increase Streptococcal virulence.
  • SIC interacts with lysozyme.
  • SLPI SLPI activity, especially bactericidal activity of SLPI.
  • Figure 1 shows results of experiments investigating binding of SIC and recombinant SIC to partial and complete terminal complement complexes. The results are shown as means of OD for three wells with sera, minus means of three wells with no sera.
  • Figure 2 illustrates results showing lysis of guinea-pig erythrocytes by the complement membrane attack complex and inhibition by SIC.
  • Figure 2a shows lysis by pre-activated C56 complex and serum/EDTA as a source of C7, C8 and C9.
  • Figure 2b shows lysis by pre-activated C56 complex and purified C7, C8 and C9, with SIC added before addition of C7.
  • Figure 2c shows lysis by pre-activated C56 complex and purified C7, C8 and C9, with SIC added after addition of C7. Squares are results for SIC, circles for BSA (control) .
  • Figure 3 shows that SIC inhibits breakdown of S. suis cell walls by hen egg lysozyme.
  • the chart shows the number of colonies determined with different concentrations of SIC in ⁇ M, doubling dilutions from 32 ⁇ M SIC in HEL at 88 ⁇ M.
  • Figure 4 shows binding of SIC to lysoyme, detected by ELISA.
  • Figure 4a shows binding of SIC to hen egg lysozyme-coated plate. The plate was coated with hen egg lysozyme at
  • FIG. 4a shows binding of hen egg lysozyme to SIC-coated plate. The plate was coated with SIC at l ⁇ g/ml. Hen egg lysozyme was added at lO ⁇ g/ml and detected with rabbit anti- HEL 1/2000 and alkaline phosphatase conjugated goat anti- rabbit Ig 1/1200.
  • Figure 4c shows binding of human lysozyme to SIC-coated plate. The plate was coated with SIC at l ⁇ g/ml. Human lysozyme was added at 5 ⁇ g/ml and detected with sheep anti- human lysozyme 1/500 and alkaline phosphatase conjugated monoclonal anti-sheep Ig 1/1200.
  • Figure 5 shows an elution profile where a lysozyme-Sepharose column was loaded with 1ml SIC and eluted with sodium chloride gradient.
  • Figure 6 shows precipitation of SIC and hen egg lysozyme: constant concentration of SIC/variable concentration of lysozyme.
  • Figure 6A shows results of precipitation with total protein content assayed using the Lowry method.
  • Figure 6C shows the amount of residual HEL activity in supernatants .
  • Figure 7 - shows precipitation of SIC and hen egg lysozyme: constant concentration of HEL/variable concentration of SIC.
  • Figure 7A shows the amount of protein in the pellet as determined by the Lowry method following precipitation.
  • Figure 7B shows the amount of residual lysozyme activity left in the supernatant.
  • Figure 8 shows that SIC binds to SLPI by ELISA.
  • the plate was coated with SLPI at 2 ⁇ g/ml.
  • SIC was added at lO ⁇ g/ml and binding detected with rabbit anti-SIC and AP goat anti- rabbit Ig plus pNPP.
  • Figure 9 shows that SLPI binds to SIC by ELISA.
  • the plate was coated with SIC at 2 ⁇ g/ml.
  • rSLPI was added at lO ⁇ g/ml and binding detected with goat anti rSLPI and AP monoclonal anti-goat Ig.
  • Figure 10 shows that hen egg lysozyme and SLPI compete for binding sites on SIC.
  • the plate was coated with HEL at 2 ⁇ g/ml. Double dilutions of competitor protein were incubated with SIC at lO ⁇ g/ml, then added to the plate and bound was SIC detected.
  • Figure 11 shows results of experiments demonstrating binding of SLPI to SIC at a molar ratio of 3:1 and with an affinity of 100,000 I/M. Dilutions of SLPI between 200 ⁇ g/ml and 1.56 ⁇ g/ml at pH 7.3 were incubated with SIC at 50 ⁇ g/ml at 32°C. Aggregates were precipitated with 1.5M ammonium sulphate, spun down and the pellets and supernatants counted.
  • Figure 12 demonstrates that SLPI kills Ml Group A Streptococci (Ml GAS).
  • Ml GAS were incubated ' with double dilutions of SLPI from 40 ⁇ M. 10-fold dilutions were plated and the colonies counted after overnight incubation. Results are expressed as mean percentage surviving compared with bacteria incubated with buffer alone.
  • Figure 13 shows results of experiments demonstrating the SIC inhibits killing of Ml Group A Streptococci by SLPI. Double dilutions of SIC from 20 ⁇ M were pre-incubated with lO ⁇ M SLPI before the. addition of Ml GAS. 10-fold dilutions were plated and the colonies counted after overnight incubation. Results are expressed as percentage surviving compared with bacteria incubated with buffer or SIC alone. The increase survival in the presence of SIC alone has been shown experimentally to be due to presence of extra protein in the buffer. The results are mean of two.
  • Figure 14 shows SIC has no effect on the inhibition of trypsin activity by SLPI.
  • Double dilutions of SIC from 60 nM were incubated with SLPI at 30 nM. Trypsin to a concentration of 42 nM was added followed by the substrate solution. Controls containing buffer/trypsin only or SLPI/trypsin only are the far left hand points on each line respectively. Controls of buffer with substrate or buffer O 02/07262
  • Figure 15 shows that SIC does not bind to lactoferrin by ELISA.
  • the plate was coated with lactoferrin at 2 ⁇ g/ml.
  • SIC was added at 10 ⁇ g/ml and binding detected as before.
  • As a positive control wells were coated with SIC at 2 ⁇ g/ml.
  • the present invention is concerned with the inventors' newly identified interactions between SIC and lysozyme, and SIC and SLPI.
  • Various aspects of the present invention provide for the use of SIC, lysozyme, SLPI, SIC and lysozyme, or SIC and SLPI in screening methods and assays for agents which modulate SIC function and/or interaction between SIC and lysozyme and/or SIC and SLPI.
  • Inhibiting ability of SIC to interact with and bind lysozyme, and inhibiting its ability to inhibit lysozyme activity,- and inhibiting ability of SIC to interact with and bind SLPI, and inhibiting its ability to inhibit killing of GAS provide for antibacterial treatment against streptococal activity and infection.
  • the present invention in various aspects also provides for modulating, especially inhibiting, interfering with ' or interrupting SIC activity and/or SIC interaction with and inhibition of lysozyme and/or SLPI, using an appropriate agent .
  • Methods of obtaining agents able to modulate SIC function and/or interaction between SIC and lysozyme and/or SIC and SLPI include methods wherein a suitable end-point is used to assess interaction in the presence and absence of a test substance.
  • Assay systems may be used to determine lysozyme activity.
  • Assay systems may be used to determine.
  • SLPI activity e.g. killing of Ml Group A Streptococci.
  • the interaction between SIC and lysozyme and the interaction between SIC and SLPI provide novel therapeutic targets, e.g. via assays including scintillation proximity assays, direct protein-protein interactions in vi tro, peptide-protein interactions in vi tro, and so on.
  • Agents useful in accordance 'with the present invention may be identified by screening techniques which involve determining whether an agent under test binds or interacts with SIC or lysozyme or SLPI, and/or inhibits or disrupts the interaction of SIC protein or a suitable fragment thereof with lysozyme or SLPI, or a fragment thereof, or a suitable analogue, fragment or variant thereof (as discussed further below) .
  • Suitable fragments of SIC or lysozyme or SLPI include those which include residues which interact with the counterpart protein. Smaller fragments, and analogues and variants of this fragment may similarly be employed, e.g. as identified using techniques such as deletion analysis or alanine scanning.
  • the present invention provides a peptide fragment of SIC which is able to interact with lysozyme and/or inhibit interaction between SIC and lysozyme, and provides a peptide fragment of lysozyme which is able to interact with SIC and/or inhibit interaction between lysozyme and SIC. Also provided are a peptide fragment of SIC which is able to interact with SLPI and/or inhibit interaction between SIC and SLPI, and a peptide fragment of SLPI which is able to interact with SIC and/or inhibit interaction between SLPI and SIC.
  • Such peptide fragments are obtainable by means of deletion analysis and/or alanine scanning of the relevant protein - making an appropriate mutation in sequence, bringing together a mutated fragment of one of the proteins with the other or a fragment thereof and determining interaction.
  • the peptide is short, as discussed below, and may be a minimal portion that is able to interact with the relevant counterpart protein and/or inhibit the relevant interaction.
  • Preferred peptides according to embodiments of the present invention consist of or comprise one or more of the peptide sequences given above.
  • agents that can be used to affect SIC activity and/or disrupt the interaction of SIC and lysozyme and/or disrupt the interaction of SIC and SLPI are peptides based on the sequence motifs of SIC or lysozyme that interact with counterpart lysozyme or SIC or based on the sequence motifs or SIC or SLPI that interact with counterpart SLPI or SIC (as discussed already above) .
  • Such peptides tend to be short, and may be about 40 amino acids in length or less, preferably about 35 amino acids in length or less, more preferably about 30 amino acids in length, or less, more preferably about 25 amino acids or less, more preferably about 20 amino acids or less, more preferably about 15 amino acids or less, more preferably about 10 amino acids or less, or 9, 8, 7, 6, 5 or less in length.
  • Peptides of the invention may be about 10-20 amino acids, about 10-30 amino acids, about 20-30 amino acids or about 30-40 amino acids in length.
  • the present invention also encompasses peptides which are sequence variants or derivatives of a wild type SIC or lysozyme or SLPI sequence, but which retain ability to interact with lysozyme or SIC or SLPI (respectively, as the case may be) and/or ability to modulate interaction between SIC and lysozyme and/or SIC and SLPI.
  • a further aspect of the invention provides a SIC fragment • that inhibits lysozyme activity.
  • a further aspect of the invention provides a SIC fragment that inhibits SLPI activity.
  • variant peptide sequences and peptide and non- peptide analogues and mimetics may be employed, as discussed further below.
  • a substance which may be a single molecule or a composition including two or more components, which includes a peptide fragment of SIC or lysozyme or SLPI, a peptide consisting essentially of such a sequence, a peptide including a variant, derivative or analogue sequence, or a non-peptide analogue or mimetic which has the ability to modulate SIC activity, and/or interact with SIC or lysozyme, and/or interact with SIC or SLPI, and/or modulate, disrupt or interfere with interaction between SIC and lysozyme, and/or modulate, disrupt or interfere with interaction between SIC and SLPI, directly or indirectly via one or more factors.
  • Variants include peptides in which, individual amino acids can be substituted by other amino acids which are closely related as is understood in the art and indicated above.
  • Non-peptide mimetics of peptides are discussed further below.
  • a peptide according to the present invention and for use in various aspects of the present invention may include or consist essentially of a fragment of SIC or lysozyme or SLPI as disclosed. Where one or more additional amino acids are included, such amino acids may be from SIC or lysozyme or SLPI or may be heterologous or foreign to SIC or lysozyme or SLPI.
  • a peptide may also be included within a larger fusion protein, particularly where the peptide is fused to a non-SIC or non-lysozyme or non-SLPI (i.e. heterologous or foreign) sequence, such as a polypeptide or protein domain.
  • the invention also includes derivatives of the peptides, including the peptide linked to a coupling partner, e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand.
  • a coupling partner e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand.
  • a coupling partner e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand.
  • the carrier molecule is a 16 aa peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name "Pe
  • Assays may employ a streptococcal SIC polypeptide and a lysozyme polypeptide, or a streptrococcal SIC polypeptide and a SLPI polypeptide and these polypeptides may be as occur naturally (including any isoforms or species variants) , or may include one or more alterations introduced artificially.
  • SIC encoding sequence is available from GenBank Accession No. X92968, with hen egg lysozyme protein sequence at LZCH, human lysozyme protein sequence at 1LZ1 and SLPI nucleotide sequence at GenBank Accession no. NM 003064.
  • a fragment of SIC that binds lysozyme and/or SLPI, and/or a fragment of lysozyme and/or SLPI that binds SIC may be used.
  • a polypeptide or peptide may include an amino acid sequence which differs by one or more amino acid residues from the wild-type amino acid sequence, by one or more of addition, insertion, deletion and substitution of one or more amino acids.
  • variants, derivatives, alleles ' and mutants are included.
  • the amino acid sequence shares homology with a fragment of the relevant SIC or lysozyme or SLPI fragment sequence, preferably at least about 60%, or 70%, or 75%, or 80%, or 85%, 90% or 95% homology.
  • a peptide fragment of SIC or lysozyme or SLPI may include 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions with respect to the wild-type sequence.
  • a derivative of a peptide may be in certain embodiments the same length or shorter than the specific peptide.
  • the peptide sequence or a variant thereof may be included in a larger peptide, which may or may not include an additional portion of .SIC or lysozyme or SLPI. 1, 2, 3, 4 or 5 or more additional amino acids, adjacent to the relevant specific peptide fragment in SIC or lysozyme or
  • SLPI SLPI, or heterologous thereto may be included at one end or both ends of the peptide.
  • homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for "conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • Homology may be over the full-length of the relevant peptide or over a contiguous sequence of about 5, 10, 15, 20, 25, 30, 35, 50, 75, 100 or more amino acids, compared with the relevant wild-type amino acid sequence.
  • Homologous nucleic acid may be identified or its presence confirmed by means of hybridization experiments, for instance involving Southern blotting.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex.
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets .with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • suitable conditions include hybridization overnight at 65°C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.
  • Peptides such as fragments of SIC that inhibit binding of SIC and lysozyme and/or binding of SIC and SLPI, fragments of SIC and/or lysozyme and/or SLPI that bind one another and which may be used in assays for agents that disrupt the interaction, and so on, may be generated wholly or partly by chemical synthesis.
  • Peptides for use in various aspects and embodiments of the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M.
  • Bodanzsky and A. Bodanzsky The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California
  • they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • Another convenient way of producing a peptidyl molecule according to the present invention is to express nucleic acid encoding it, by use of nucleic acid in an expression system.
  • the present invention also provides in various aspects nucleic acid encoding the peptides of the invention, which may be used for production of the encoded peptide.
  • nucleic acid is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the genome (e.g. for lysozyme the human genome), except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as encompassing reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid sequences encoding a polypeptide or peptide in accordance with the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook and Russell “Molecular Cloning, A Laboratory Manual”, Third Edition, Cold Spring Harbor Laboratory Press, 2001, and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, 1994, or later edition thereof) , given the nucleic acid sequence and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding SIC or lysozyme or SLPI fragments may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the relevant sequence may be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preference in the host cells used to express the ' nucleic acid.
  • the sequences may be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium.
  • Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells.
  • the present invention also encompasses a method of making a polypeptide or peptide (as disclosed), the method including expression from nucleic acid encoding the polypeptide or peptide (generally nucleic acid according to the invention) .
  • This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide.
  • Polypeptides and peptides may also be expressed in in vi tro systems, such as reticulocyte lysate.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • a common, preferred bacterial host is E. coli .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al . , cited above.
  • a further aspect of the present invention provides a host cell containing heterologous nucleic acid as disclosed herein.
  • a still further aspect provides a method which includes introducing the nucleic acid into a host cell.
  • the introduction which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation”, may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • direct injection of the nucleic acid could be employed.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression, so that the encoded polypeptide (or peptide) is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g.
  • the present invention provides an assay method for a substance with ability to modulate, e.g. disrupt or interfere with interaction between (i) SIC and (ii) lysozyme and/or SLPI, the method including:
  • a test compound which disrupts, reduces, interferes with or wholly or partially abolishes interaction between said substances (e.g. including a fragment of either protein), and which may modulate SIC and/or lysozyme and/or SLPI activity, may thus be identified.
  • Agents which increase or potentiate interaction between the two substances may be identified using conditions which, in the absence of a positively-testing agent, prevent the substances interacting.
  • Another general aspect of the present invention provides an assay method for a substance able to interact with the relevant region of SIC or lysozyme or SLPI as the case may be, the method including:
  • test compound found to interact with the relevant portion of SIC may be tested for ability to modulate, e.g. disrupt or interfere with, SIC interaction with lysozyme and/or SLPI, and/or ability to affect lysozyme activity, SLPI activity and/or other activity mediated by SIC.
  • test compound found to interact with the relevant portion of lysozyme or SLPI may be tested for ability to modulate, e.g. disrupt or interfere with, SIC activity and/or SIC interaction with lysozyme and/or SLPI, and/or ability to affect lysozyme activity, SLPI activity and/or other activity mediated by SIC and/or lysozyme and/or SLPI.
  • Suitable screening methods are conventional in the art. They include techniques such as radioimmunosassay, scintillation proximetry assay and ELISA methods.
  • the precise format of an assay of the invention may be varied by those of skill in the art using routine skill and knowledge. For instance, in assaying for substances able to modulate an interaction between proteins, one of the relevant proteins or a fragment thereof, is immobilised whereupon the other is applied in the presence of the agents under test. For example, interaction between substances may be studied in vitro by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support.
  • Suitable detectable labels, especially for peptidyl substances include 35 S-methionine which may be incorporated into recombinantly produced peptides and polypeptides.
  • Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.
  • the protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se.
  • a preferred approach employs BIACORE and biotinylated beads.
  • a biotinylated protein fragment may be bound to streptavidin coated scintillant - impregnated beads (produced by Amersham) . Binding of radiolabelled peptide is then measured by determination of radioactivity induced scintillation as the radioactive peptide binds to the immobilized fragment. Agents which intercept this are thus inhibitors of the interaction.
  • An example of a scintillation proximity assay for high through-put screening is described by Gorman et al. (1996) J Biol Chem, 271: 6713- 6719.
  • an assay method comprises bringing into contact a SIC polypeptide or peptide that binds lysozyme and/or binds SLPI, a lysozyme polypeptide or peptide that has lysozyme enzymatic activity, and/or a SLPI polypeptide or peptide that has SLPI activity (e.g. ability to kill Ml Group A Streptococci) , and a test compound, and determining ability of the test compound to affect lysozyme activity and/or SLPI activity.
  • SIC inhibits lysozyme activity, so an agent that reduces that inhibition will manifest in such an assay an increase in lysozyme activity.
  • SIC inhibits SLPI activity, so an agent that reduces that inhibition will manifest in such an assay as an increase in SLPI activity.
  • Lysozyme activity can be measured by any of a variety of techniques at the disposal of the skilled person.
  • the standard assay for lysozyme activity is by digestion of a suspension of dried Micrococcus lysodeikticus cell walls in phosphate buffer. After incubation of the M. Lysoceikticus cell wall suspension at 300 ⁇ g/ml with doubling dilutions of lysozyme from 200 ng/ l for 2hr. at 37°C, the decrease in turbidity is read in the spectrophotometer at 450nm and the results plotted as a standard curve to show the amount of digestion. A range of dilutions of samples under investigation is made simultaneously and compared with the standard curve to find the concentration of active lysozyme present.
  • the amount of lysozyme present can be measured using a capture ELISA.
  • ELISA plates are coated with a polyclonal antiserum to lysozyme using any appropriate procedure, and a range of dilutions of a standard preparation of lysozyme is added to form a standard curve, together with a range of dilutions of the samples under examination.
  • Captured lysozyme can be detected for example using a biotinylated or horseradish peroxidase conjugated version of the capture antibody molecule, followed by appropriate secondary reagents/substrate, or a monoclonal anti-lysozyme and a conjugated second antibody molecule, " e.g. alkaline phosphatase conjugated anti-mouse Ig.
  • Lysozyme activity may be determined in the presence and absence of SIC to allow for an effect of a test compound on activity to be attributed to an effect on interaction between SIC and lysozyme, as disclosed.
  • SLPI activity may be assayed by ability to kill Ml Group A Streptococci, or more conveniently by its ability to kill a strain of E. coli, such as the laboratory strain DH5 ⁇ . Another option is to employ a bactericidal radial immunodiffusion assay.
  • Preliminary assays in vitro may be followed by, or run in parallel with, in v t ivo assays.
  • the person skilled in the art will design any appropriate control experiments with which to compare results obtained in test assays.
  • Performance of an assay method according to the present invention may be followed by isolation and/or manufacture and/or use of a compound, substance or molecule which tests positive for ability to affect SIC binding to and inhibition of lysozyme and/or SLPI.
  • a variety of molecules may be tested in assays of the invention.
  • Candidate molecules may be identified by various means. For instance, information may be obtained about residues which are important for SIC/lysozyme interaction and/or SIC/SLPI interaction using alanine scanning and deletion analysis of SIC and/or lysozyme and/or SLPI, and/or peptide fragments of these. When key residues are identified, computer sequence databases may be scanned for proteins including the same or similar pattern of residues, taking into account conservative variation in sequence (see below) as appropriate. Candidate molecules may then be used in one or more assays for interaction with SIC or lysozyme and/or SLPI and ability to alter interaction between SIC and lysozyme and between SIC and SLPI.
  • Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate an interaction with and/or activity of a polypeptide.
  • Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others.
  • the use of peptide libraries may be preferred in certain circumstances.
  • Antibodies directed to a site on SIC or lysozyme or SLPI form a further class of putative inhibitor compounds.
  • Candidate inhibitor antibodies may be characterised and ⁇ their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction.
  • Antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , (1992) Nature 357, 80-82) . Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
  • a mammal e.g. mouse, rat, rabbit, horse, goat, sheep or monkey
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used
  • an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not 'been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody molecule” should be construed as covering antibody fragments and derivatives comprising an antibody antigen binding domain.
  • Example antibody fragments capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • a hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs) , of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression.
  • the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
  • the reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
  • Antibodies may also be used in purifying and/or isolating a polypeptide or peptide according to the present invention, for instance following production of the polypeptide or peptide by expression from encoding nucleic acid therefor. Antibodies may be useful in a therapeutic context (which may include prophylaxis) to disrupt SIC/lysozyme interaction and/or SIC/SLPI activity with a view to inhibiting their activity.
  • candidate inhibitor compounds may be based on modelling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.
  • a compound found to have the ability to affect SIC activity, such as via interaction with lysozyme and/or with SLPI has therapeutic and other potential in a number of contexts, as discussed.
  • such a compound may be used in combination with any other active substance.
  • the assay of the invention when conducted in vivo, need not measure the degree of modulation of interaction between SIC and lysozyme and/or between SIC and SLPI (or appropriate fragment, variant or derivative thereof) or of modulation of activity caused by the compound being tested. Instead the therapeutic effect may be measured.
  • Such a modified assay is run in parallel with or subsequent to- the main assay of the invention in order to confirm that any such effect is as a result of the modulation of SIC function, such as inhibition of interaction between SIC and lysozyme or inhibition of interaction between SIC and SLPI, caused by said inhibitor compound and not merely a general toxic effect.
  • an agent identified using one or more primary screens ⁇ as having ability to interact with SIC and/or lysozyme and/or SLPI and/or modulate activity of SIC may be assessed further using one or more secondary screens.
  • the substance or agent may be investigated further.
  • it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for the purpose of treating a streptococcal infection, in particular a Group A Streptococcus infection by a sic+ strain.
  • the agent may be peptidyl, e.g. a peptide which includes a sequence as recited above, or may be a functional analogue of such a peptide.
  • the expression "functional analogue” relates to peptide variants or organic compounds having the same functional activity as the peptide in question, which may interfere with the interaction between SIC and lysozyme or the interaction between SIC and SLPI.
  • analogues include chemical compounds which are modelled to resemble the three dimensional structure of the relevant domain in the contact area, and in particular the arrangement of the key amino acid residues as they appear in the protein.
  • the present invention provides the use of the above substances in methods of designing or screening for mimetics of the substances.
  • the present invention provides a method of designing mimetics having the desired activity of modulating, especially inhibiting, SIC interaction with lysozyme and/or SIC interaction with SLPI, said method comprising:
  • Suitable modelling techniques are known in the art. This includes the design of so-called “mimetics” which involves the study of the functional interactions fluorogenic oligonucleotide the molecules and the design of compounds which contain functional groups arranged in such a manner that they could reproduced those interactions.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable .where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides may not be well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data ⁇ from a range of sources, e.g. spectroscopic techniques, X- ray diffraction data and NMR.
  • Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the present invention further provides the use of a peptide, derivative, active portion, analogue, variant or mimetic, thereof able to interact with SIC or lysozyme or SLPI and/or modulate, e.g. inhibit, interaction between SIC and lysozyme and/or interaction between SIC and SLPI and/or modulate, e.g. inhibit, SIC activity, in screening for a substance able to interact with SIC and/or lysozyme and/or SLPI, and/or modulate, e.g. inhibit, interaction between SIC and lysozyme, and/or interaction between SIC and SLPI, and/or inhibit SIC activity.
  • an agent or substance, e.g. inhibitor, according to the present invention is provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition may, however, include inert carrier materials or other pharmaceutically and physiologically acceptable excipients. As noted below, a composition according to the present invention may include in addition to an inhibitor compound as disclosed, one or more other molecules of therapeutic use, such as an anti-streptococcal.
  • the present invention extends in various aspects not only to a substance identified as a modulator of SIC and lysozyme interaction and/or SIC inhibition of lysozyme, and/or a modulator of SIC and SLPI interaction and/or SIC inhibition of SLPI, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for treating a streptococcal infection, which may include preventative treatment, use of such a substance in manufacture of a composition for administration, e.g. for treatment of a streptococcal infection or for use as an antibiotic, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a substance according to the present invention such as an inhibitor of SIC and lysozyme interaction or of SIC and SLPI interaction may be provided for use in a method of treatment of the human or animal body by therapy which affects a SIC- mediated activity of streptococcus .
  • the invention further provides a method which includes administering an agent which modulates, inhibits or blocks the interaction of SIC with lysozyme and/or the interaction of SIC with SLPI, such a method being useful in treatment where such modulation, inhibition or blocking is desirable.
  • administration is preferably in a "prophylactically effective amount" or a
  • therapeutically effective amount as the case may be, although prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isot ⁇ nicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isot ⁇ nicity and stability.
  • Suitable solutions using, for example, isotonic vehicles such as Sodium Chloride- Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Liposomes particularly cationic liposomes, may be used "in carrier formulations.
  • a composition may be administered alone or in combination with other treatment ' s such as antibiotics, either simultaneously or sequentially.
  • a polypeptide, peptide or other substance able to modulate or interfere with the interaction of the relevant polypeptide, peptide or other substance as disclosed herein, or a nucleic acid molecule encoding a peptidyl such molecule may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment.
  • a kit may include instructions for use.
  • SIC lysozyme
  • lysozyme is abundantly available, so normally it is envisaged that lysozyme or a fragment thereof that binds SIC will be used to purify SIC from a sample, or in an assay method for quantitation the amount of SIC present in a sample, but clearly SIC may be used the other way round in purifying lysozyme.
  • a method of purifying a SIC polypeptide comprising: bringing into contact (i) a sample that may contain or is suspected of containing a SIC polypeptide and (ii) a lysozyme polypeptide, under conditions for binding of the lysozyme polypeptide to a SIC polypeptide if the SIC polypeptide is present in the sample; separating from the sample lysozyme polypeptide bound to any SIC polypeptide; separating any SIC polypeptide from the lysozyme polypeptide.
  • the method becomes one of quantitating the amount of SIC polypeptide in a sample.
  • SIC polypeptide need not be separated from lysozyme as the amount bound to lysozyme can be determined, e.g. using an anti-SIC antibody molecule in an appropriate assay.
  • a method of determining the presence or absence of a SIC polypeptide in a sample comprising: bringing the sample into contact with a solid support to which is attached a lysozyme polypeptide that binds SIC polypeptide, whereby SIC polypeptide if present in the sample is bound by the lysozyme polypeptide and retained on the solid support; determining the presence or absence of SIC polypeptide retained on the solid support.
  • the presence or absence of SIC polypeptide retained on the solid support by virtue of its binding to lysozyme polypeptide immobilized on the support may be determined for instance using a specific binding member directed against SIC, such as a labeled antibody.
  • a method of purifying SIC polypeptide may have the analogous steps, with a step of separating SIC polypeptide from lysozyme polypeptide on the solid support being include instead of or in addition to the step of determining the presence or absence of the SIC polypeptide retained on the solid support.
  • SIC may be separated from lysozyme by elution, for example with a sodium chloride gradient.
  • Lysozyme (or SIC) may be immobilised oh beads or a column in accordance with standard techniques. Experimental exemplification of purification of SIC by means of a lysozyme column is included below.
  • the binding between SIC and SLPI may be utilised in a method of purifying or in determining the presence or absence of one of the binding partners in a sample, substituting use or detection of SLPI for lysozyme.
  • the inventors measured interaction of SIC with intermediate forms of the terminal complement complex by ELISA. Partially formed complexes generated in yeast-activated C7-, C8- or C9- deficient sera were added to ELISA plates coated with SIC, and binding was detected using polyclonal antibodies to C6.
  • the plate was coated with SIC or rSIC at 10 ⁇ g/ml O/N at 4 °C.
  • Yeast activated sera was added diluted 1/200, and incubated 1 hour at 37 °C.
  • Detection was with goat anti-C6 1/500, incubated for 1 hour at 37 °C, then alkaline phosphatase monoclonal anti-goat Ig 1/1500, incubated 2 hours at 37 °C, and PNPP.
  • MAC inhibition is really the primary role of SIC in vivo.
  • Group A Streps GAS are already resistant to lysis by the MAC as their membranes are protected by a thick cell wall and, in some cases, a capsule as well.
  • GAS Group A Streps
  • proteins all having one or more other functions attributed to them, have been reported to inhibit MAC formation in a similar way to SIC and with similar efficiency (e.g. clusterin) , yet the physiological significance of any of them is questionable in light of the fact that C8 is the major fluid-phase inhibitor of C5b-7 binding to membranes in vivo (Nemerow et al 1979) .
  • the inventors found that SIC binds lysozyme and inhibits its enzymatic activity.
  • SIC inhibits breakdown of streptococcus suis type II cell walls by hen egg lysozyme (HEL) .
  • The. effect of SIC upon lysozyme activity was tested by measuring lysozyme digestion of S . suis Type II cell walls.
  • S. suis is very susceptible to HEL digestion and does not produce SIC.
  • the minimum concentration of HEL required to kill 10 ⁇ l aliquots of 3 x 10 S . suis was titrated by incubating the bacteria with doubling dilutions of HEL from 10 mg/ml. Then the reaction mixture was spotted onto horse blood agar plates in a grid pattern and the number of colonies estimated after overnight incubation. Approximately 0.5 mg mg/ml was required to stop all growth.
  • Double dilutions of SIC from 0.5 mg/ml were made in a constant concentration (0.1 mg/ml) of HEL (all in 15 ⁇ l Tris/EDTA buffer) . Almost immediately, a precipitate could be seen forming in the first few tubes containing the highest concentrations of SIC. The tubes were incubated for a total of 16 hours at 4°C then 20 ⁇ l of the reaction
  • HEL was coupled to cyanogen bromide activated Sepharose CL4B at 8 mg/ml.
  • a 1 ml column was equilibrated in 50 mM Tris/10 mM EDTA pH 7.5, and loaded with 1 ml of SIC at 0.7 mg/ml in Tris/EDTA.
  • the column was washed with start buffer and eluted with a 20 column volume sodium chloride gradient to 2 M.
  • SIC bound to the column and eluted at 0.7 M sodium chloride in a .single peak as shown by SDS/PAGE. A typical elution profile is shown in. Figure 5. Quantitation Of Lysozyme Inhibition/Precipitation By SIC
  • the S . suis killing experiment showed that when SIC and hen egg lysozyme are mixed a visible precipitate forms almost immediately.
  • Different molar ratios of SIC and HEL were mixed, allowed to precipitate, the amount of protein in the precipitate measured and the residual amounts of SIC and HEL in the supernatants assayed.
  • Total protein in pellets was measured by the Lowry assay (Lowry, Rosebrough, Farr and Randall, 1951) , residual SIC was measured by quantitative ELISA and residual HEL activity was measured by functional assay.
  • the standard assay for lysozyme activity is by digestion of a suspension of dried Micrococcus lysodeikticus cell walls in phosphate buffer. After incubation of the M. lysodeikticus cell wall suspension at 300 ⁇ g/ml with doubling dilutions of lysozyme from 200 ng/ml for 2 hr. at
  • the decrease in turbidity is read in the spectrophotometer at 450 nm and the results plotted as a standard curve to show the amount of digestion.
  • the inventors used purified SIC to raise a polyclonal antiserum in a rabbit and an Ig fraction was purified from the whole antiserum. An aliquot of the Ig fraction was coupled to Biotin using biotinamido caproate-N-hydroxy- sulphosuccinimide ester. ELISA plates are to be coated with the unbiotinylated Ig fraction which captures SIC in samples added to the wells. After washing, the SIC can be detected with the biotinylated anti-SIC followed by alkaline phosphatase conjugated Extravidin and pNPP substrate. As little as 1 ng/ml can be measured by this ELISA.
  • Linear standard curves were constructed for both SIC and HEL and an average of the two was taken to approximately represent a pellet comprising a mixture of the two proteins.
  • Double dilutions of SIC from 2 mg/ml (64 ⁇ M) were combined with HEL at a constant 0.5 mg/ml (35 ⁇ M) in 100 ⁇ l volumes and allowed to precipitate overnight at 4°C. The precipitate was spun down, the pellet rinsed in PBS and the total protein content assayed using the Lowry method. The mass of the pellets in ⁇ g is shown in Figure 7A.
  • This assay shows the molar ratios involved in the binding interaction between SIC and HEL.
  • the inventors' experiments provide indication that there is more than one binding site for HEL on a SIC molecule, and there could be as many as eight.
  • Streptococcus pyogenes Type Ml - NCTC 8198 was obtained from the National Collection of Type Cultures,
  • SIC was purified from 1 litre overnight culture supernatants of type Ml GAS, by hydrophobic interaction chromatography. An Ig fraction of the rabbit anti-SIC was prepared using a Prosep A column.
  • ELISA In order to investigate the interaction of SIC with other major constituents of ASL, ELISA plates were coated with recombinant human SLPI or lactoferrin at 2 ⁇ g/ml. SIC at 10 ⁇ g/ml was added and bound protein was detected with rabbit anti-SIC followed by alkaline phosphatase conjugated goat anti-rabbit Ig and "Sigma Fast" pNPP substrate/buffer system.
  • the plates were coated with SIC at 2 ⁇ g/ml, recombinant human SLPI (rSLPI) at 10 ⁇ g/ml was added and bound protein was detected with goat anti-rSLPI followed by alkaline phosphatase conjugated monoclonal anti goat IgG.
  • rSLPI recombinant human SLPI
  • ELISA plates were coated with hen egg lysozyme (HEL) at 2 ⁇ g/ml. Double dilutions of SLPI or HEL from 20 ⁇ g/ml, (or human serum albumin as control from 30 ⁇ g/ml) were incubated with SIC at 10 ⁇ g/ml, then added to the plate. Bound SIC was detected as above.
  • HEL hen egg lysozyme
  • Iodinated SLPI was added to SIC in a series of different molar ratios and at different temperatures, allowed to bind, and the aggregates precipitated with ammonium sulphate. The aggregates were spun down and the pellets and supernatants counted. The ratio of bound/free was calculated and plotted.
  • SLPI serine proteinase inhibitor
  • the inventors decided to investigate whether SIC had any effect on the ability- of SLPI ' to inhibit trypsin activity using the substrate tosyl-glycyl-prolyl-lysine-4- nitranilide acetate (Chromozyme PL) .
  • SLPI is known to inhibit trypsin activity at a 1:1 molar ratio. Firstly, the assay conditions for the inhibition of trypsin by SLPI were optimised and a concentration of 30 nM SLPI to 42 nM trypsin gave almost complete inhibition of digestion of the substrate.
  • SIC binds to human recombinant SLPI by ELISA SIC bound extremely strongly to plates coated with recombinant human SLPI ( Figure 8) Similarly, SLPI bound extremely strongly to plates coated with SIC ( Figure 9) .
  • ELISA it appears that either a far greater amount of SLPI than HEL binds to SIC, or that SLPI binds with far greater affinity ( Figures 8 and 9) .
  • Results showed that the maximal binding of SLPI to SIC was somewhat variable but generally was around 3:1.
  • the binding affinity was in the order of 100,000 1/M at room temperature.
  • the binding affinity of HEL was around 5000 1/M at room temperature and 14,000 1/M at 4°C. This shows that the binding affinity of SLPI to SIC is approximately 20-times greater than that of HEL ( Figure 11) .
  • GAS are resistant to direct breakdown by lysozyme, inhibition of lysozyme activity in vivo could still be advantageous to the bacteria for a number of reasons.
  • the most common site of infection by GAS is the respiratory tract (naso-pharyngeal areas) .
  • SLPI secretory leukoproteinase inhibitor
  • the inventors have found that SIC binds to lysozyme and inhibits its activity. They have also found that SIC binds SLPI.
  • the Scatchard plots show a binding ratio of around 8:1 for HEL to SIC with an affinity of around 5000 1/M.
  • the binding ratio for secretory leucocyte proteinase inhibitor to SIC is 3:1 and is of greater affinity at around 100,000 1/M, i.e. about 20-times stronger.
  • the binding observed between SLPI and SIC by ELISA is in agreement with these results. No binding was observed between lactoferrin and SIC.
  • the inventors have also shown that Group A Streptococci are effectively killed by SLPI, and that SIC from the Ml strain of GAS efficiently inhibits the bactericidal activity of SLPI. However, SIC has no effect on the trypsin inhibitory property of SLPI .
  • SLPI is a low molecular weight protein of 11.7 kDa and consists of two nearly identical domains. One domain contains the antimicrobial activity, while the other contains the serine proteinase inhibitory activity (Hiemstra et al 1996) . It would appear therefore that SIC binds specifically to the "antimicrobial domain" of SLPI. It is thought that inhibition of elastase in the -lung is SLPI's main proteinase inhibitory role. The mechanism by which SLPI kills a variety of micro-organisms is at present unknown.
  • SIC does not break down SLPI or lysozyme, as demonstrated in experiments where coomassie stained SDS/PAGE gels of SIC incubated with SLPI, SIC incubated with human -lysozyme or SIC incubated with HEL showed no breakdown products.
  • SIC itself runs with an apparent molecular weight of around 45 kDa although its molecular weight is 31 kDa, with a slightly lower MW band appearing from SIC in gel tracks, representing a spontaneous breakdown product in which 33 amino acids have been lost from the N-terminus (Fernie-King et al. (2001) Immunol . 103 : • 390-398) ) .
  • SLPI competes with HEL for binding to SIC in a dose-dependent manner.
  • inhibition of SIC activity may be used to make it harder for a Streptococcal . infection to become established because the innate immune system would then be able to utilise its full potential to contain the bacterial infection, while giving the acquired immune system time to respond.

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Abstract

Selon la présente invention, 'l'inhibiteur streptococcique de complément' (SIC) joue un rôle dans l'infection par streptocoque, et il apparaît qu'il interagit avec l'activité enzymatique du lysozyme et inhibe cette activité enzymatique du lysozyme, de même qu'il interagit avec l'inhibiteur de la protéinase leucocytaire de sécrétion (SLPI). L'interaction entre les protéines et l'inhibition du lysozyme et du SLPI par le SIC peut être interrompue, principalement dans un contexte antibiotique ou thérapeutique.
PCT/GB2002/001200 2001-03-14 2002-03-14 Analyses, methodes et elements pour le ciblage de streptocoques WO2002072624A2 (fr)

Priority Applications (1)

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AU2002249342A AU2002249342A1 (en) 2001-03-14 2002-03-14 Assays, methods and means for targeting streptococcal inhibitor of complement (sic)

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WO2004074807A2 (fr) * 2003-02-19 2004-09-02 Hansa Medical Ab Analyse
WO2018076061A1 (fr) * 2016-10-26 2018-05-03 The University Of Melbourne Dosage et procédé de traitement

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074807A2 (fr) * 2003-02-19 2004-09-02 Hansa Medical Ab Analyse
WO2004074807A3 (fr) * 2003-02-19 2005-01-13 Hansa Medical Res Ab Analyse
WO2018076061A1 (fr) * 2016-10-26 2018-05-03 The University Of Melbourne Dosage et procédé de traitement

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