WO1999052939A1 - Peptides - Google Patents
Peptides Download PDFInfo
- Publication number
- WO1999052939A1 WO1999052939A1 PCT/GB1999/001104 GB9901104W WO9952939A1 WO 1999052939 A1 WO1999052939 A1 WO 1999052939A1 GB 9901104 W GB9901104 W GB 9901104W WO 9952939 A1 WO9952939 A1 WO 9952939A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- protein
- polypeptide
- amino acids
- pyogenes
- Prior art date
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- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the invention relates to peptides and their use in vaccines against streptococcal infection.
- Streptococcus species responsible for causing disease states in humans and other animals.
- Infections caused by streptococci can vary from uncomplicated suppurative diseases like pharyngitis and skin infections to severe diseases such as sepsis and toxic shock syndrome. Rheumatic fever and glomerulonephritis are serious nonsuppurative sequelae following acute S. pyogenes infections.
- Other diseases caused by streptococci include scarlet fever, impetigo, erypsipelas, myositis, necrotizing fasciitis, septic arthritis, cellulitis, colonization and destruction of heart valves (endocarditis), neonatal infections, conjunctivitis, sinusitis, perotonitis, omphalitis, meningitis, abortion and chorioamnionitis, post-partum sepsis, upper respiratory tract disease in humans, pneumonia, otitis media, wound infections, abscesses, empyema, mastitis, urinary tract infections, osteomyelitis, strangles in horses, dental pathogens in humans, renal infections in humans.
- Prompt and aggressive treatment is essential in severe streptococcal infection.
- this treatment may encompass surgical debridement, antibiotic treatment, intravenous fluid, oxygen or ventilator support, dialysis, vasoconstrictives to elevate blood pressure, steroids and anti-thrombolytics. Even so, severe streptococcal infection can be fatal.
- the present invention provides a polypeptide of up to 50 amino acids in length, suitable for use as a vaccine against streptococcal infection, comprising:
- the present invention also provides a chimeric protein comprising a first polypeptide having the sequence (a), (b), (c), (d) above and a second polypeptide which is not naturally contiguous to the first polypeptide.
- the present invention provides a polynucleotide encoding a polypeptide of the present invention.
- the present invention also relates to expression vectors comprising a polynucleotide in the invention and regulatory sequences operably linked to said polynucleotide for expression of a polypeptide encoded by the polynucleotides and to host cells transfected with such expression vectors.
- the present invention also provides a pharmaceutical composition comprising a polypeptide of the invention together with a pharmaceutical composition comprising a polypeptide of the invention together with a pharmaceutically acceptable carrier and a vaccine composition comprising a polypeptide of the invention, an adjuvant in a pharmaceutically acceptable carrier or a polynucleotide of the invention together with a pharmaceutically acceptable carrier for the polynucleotide.
- the invention also relates to an antibody against a polypeptide according to the invention.
- the invention provides a polypeptide of up to 50 amino acids in length comprising:
- sequence (b) an amino acid sequence of an outer membrane protein of a streptococcal strain corresponding to sequence (a), said sequence having the ability to interfere with aggregation or adhesion of said streptococcal strain;
- API bacteria were incubated with PBS (O), papain (•), streptococcal cysteine proteinase ( ⁇ ) or CNBr (A). Following digestion bacteria were centrifuged, washed, resuspended and analyzed for sedimentation.
- C SDS-PAGE analysis of CNBr-solubilized material from API bacteria.
- the fragments generated by CNBr are indicated in the schematic representations of protein H and Ml protein.
- the NH 2 -terminal signal sequences (Ss) are indicated, and the proteins are associated with the bacterial cell wall through the COOH-terminal D domains.
- the sequences in the Ss, C and D domains show a high degree of homology.
- IgGFc-binding is located in the A-B domains of protein H and in the S domain of Ml protein. Numbers in the figure refer to amino acid residue positions.
- the protein H used herein is a truncated form (42-349) lacking the 27 COOH-terminal amino acid residues associated with the bacterial cell wall, whereas the Ml protein used covers residues 42-484
- FIG. 1 Schematic figure of protein H. The various protein H fragments are indicated below the figure together with the sequence for the aggregative protein H peptide (APP). Numbers refer to amino acid residues.
- Radiolabelled protein H has affinity for protein H-Sepharose.
- 125 I-labelled protein H (10 6 cpm) was applied to a protein H-Sepharose column. The column was extensively washed with PBSAT, eluted with human IgG (5 mg/ml) and, after a second wash with PBSAT, finally eluted with 3 M KSCN. The radioactivity of the fractions were plotted against the volume.
- Protein H (300 pmol) was crosslinked with DSS in the presence of 12 T-labelled APP (approximately 450 pmol). Samples were analyzed by SDS-PAGE, one gel was stained and one gel was dried and autoradiographed. Lane 1 : protein H without crosslinker; Lane 2: protein H with crosslinker; Lane 3: protein H crosslinked in the presence of ,2 T-APP; Lane 4: protein H crosslinked in the presence of l2 T-APP and excess amount of unlabelled APP (450 nmol); Lane 5: protein H crosslinked in the presence of l25 I-labelled B 1 domain of protein L.
- Figure 9 Results of ELISA using antiserum prepared by injecting APP conjugated to KLH together with adjuvant into rabbits.
- Figure 10 Nucleotide sequence of APP-peptide (33-mer) and homology to other M proteins.
- Figure 12 Effect of pre-adsorption with peptides on detection of antibodies induced by Spy-PH-YQE33 peptide. Sheep were primed and boosted with Protein H- derived peptide Spy-PH-YQE33 conjugated to KLH carrier protein. Post-immune sera
- a polypeptide of the invention is one of up to 50 amino acids in length, suitable for use as a vaccine against a streptococcal infection.
- the polypeptide may consist essentially of (a) the amino acids 150-168 of Protein H of S.pyogenes having the sequence QKQQQLETEKQISEASRKS.
- a polypeptide of the invention may also comprise (b) an amino acid sequence of an outer membrane protein of a streptococcal strain corresponding to sequence (a), a fragment of either sequence (a) or (b) of six or more amino acids in length; or (d) a sequence comprising a sequence (a), (b) or (c) modified by deletion, insertion, substitution or rearrangement.
- Streptococci have been classified into various groups under the Lancefield classification. This classification is based on group-specific antigens in the cell wall polysaccharide.
- Key Lancefield groups are Group A comprising Streptococcus pyogenes, Group B comprising Streptococcus agalactiae, Group C and G including Streptococcus equi and Streptococcus equisimilis, Group D including Enterococcus faecalis and Group Viridian including Streptococcus mutans and Streptococcus sanguis.
- a non-groupable species is Streptococcus pneumoniae. -7-
- bacteria may be further classified according to their serotype.
- serotype For Group A Streptococcus, serotyping is carried out by reference to additional surface antigens, the M proteins. These proteins divide the group A Streptococcus into more than 80 serotypes. Some M serotypes are associated with increased virulence and pathogenicity. In particular, Ml, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14,
- S.pyogenes can express a number of other outer membrane proteins including immunoglobulin binding proteins.
- Protein H is an example of an outer membrane protein expressed by Ml serotypes of S. pyogenes.
- Protein H. along with other IgG binding proteins expressed by S. pyogenes, is structurally related to M proteins which may also bind IgG, and now considered to be part of the same family.
- Nielson et al Biochemistry 1995 Vol. 34 No. 41 13688-13698 describes the structure of protein H and Ml protein of API strain of S. pyogenes and sets out the full sequence and domain structure of these proteins.
- streptococcal strains have been shown to aggregate, with a greater ability to aggregate being associated with an increase in the virulence of the bacteria.
- the examples set out below demonstrate the role of protein H in aggregation, through protein H - protein H interactions. This aggregative activity has been mapped to the sequence (a) above comprising residues 150-168 of protein H and referred to herein as
- APP is suitable for use as a vaccine against strepococci in view of its role in the aggregative properties associated with virulence of the bacteria.
- sequence of residues 145-177 of protein H encompassing the sequence (a), namely sequence YQEQLQKQQQLETEKQISEASRKSLSRDLEASR is of particular interest, and is referred to herein as a 33-mer.
- sequence (b) comprising a corresponding sequence of an outer membrane protein of a streptococcal strain. This includes those proteins expressed on the surface of Groups A, B, C, D, G and Viridian -8- streptococcus.
- sequence (b) is derived from an outer membrane protein of Group A Streptococcus, S.pyogenes.
- the amino acid sequence is derived from one of the more virulent serotypes of S.pyogenes namely Ml, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 17, 18, 19, 22, 24, 49, 55, 57 and 60.
- the outer membrane protein may for example comprise one of the
- M proteins of Group A Streptococcus examples include protein Arp and protein Sir22 expressed by some streptococcal strains which are associated with binding of plasma proteins.
- the relevant outer membrane proteins and sequences can be elucidated by using APP or the 33-mer and establishing the best possible alignment when looking at the sequences of outer membrane proteins of streptococci.
- sequences which correspond to sequence (a) which emphasises the suitability of the sequences as vaccines.
- Other corresponding sequences may include sequences within the C2 and C3 repeat domains of protein H having a high degree of homology with APP.
- a polypeptide of sequence (b) or (d) of the invention will be at least
- sequence (b) or (d) of the invention is at least 50% and more preferably at least 60%, 70% or 80% homologous to the sequence (a). More preferably, the sequence (b) or (d) comprises the sequence that is at least 90% and more preferably at least 95%, 97% or 98% homologous to the sequence (a) or the 33-mer identified above.
- the presence of a corresponding sequence may be established through hybridisation using the nucleotide sequences set out in Figure 10, as discussed in more detail below.
- the invention also relates to fragments of the sequences (a) and (b) of six or more amino acids in length, preferably 8 or more amino acids or 10 or more amino acids in length.
- This fragments may be up to 10 amino acids in length preferably up to 12, 16 or 18 amino acids in length.
- Amino acid substitutions, modifications deletions or rearrangements may be made to the sequences (a), (b) or (c) for example at from 1, 2, 3, 4, 5 up to 9 amino acids of sequence (a) preferably, at from 1, 2 or 3 amino acids.
- Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
- amino acid polypeptides of the invention may also be modified to include non naturally-occurring amino acids or to increase the stability of the compound in vivo. When the compounds are produced by synthetic means, such amino acids may be introduced during production. The compound may also be modified following either synthetic or recombinant production.
- Polypeptides of the invention may also be made synthetically using D-amino acids.
- the amino acids will be linked in a reverse sequence in the C to N orientation. This is conventional in the art for producing such peptides.
- side-chain modifications for amino acids are known in the art and may be made with the side-chains of polypeptides of the present invention. Such modifications include for example, modifications of the amino acid group by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 , amidination with methyl acetimidate, for acylation with acetic anhydride.
- the carboxy terminus and any other carboxy side-chains may be blocked in the form of an ester group, e.g. a C,. 6 alkyl ester.
- polypeptide of the invention may consist essentially of the sequences (a), (b), (c) or (d).
- polypeptides of the present invention are selected or modified to maintain their suitability as vaccine compositions.
- a polypeptide suitable for use as a vaccine composition in accordance with the invention is a polypeptide which is able to generate a protective immune response against a streptococcal infection.
- the polypeptides of the present invention can typically be selected by the ability to interfere with bacterial aggregation or adhesion to epithelial surfaces.
- the sequence (a) may be used as an antiaggregative agent.
- sequence (b) -1 1- may be used to inhibit aggregation of the streptococcal strain from which the sequence derives.
- the sequences may interfere generally with streptococcal aggregation and/or adhesion of the bacteria to epithelial cells.
- aggregation of the bacteria means that the aggregation seen in streptococci as exemplified in Example 1 and measured in absorbence studies.
- the peptide will reduce aggregation by 30% more preferably by at least 50%.
- the polypeptide may interfere with adhesion of the bacteria to epithelial cells as exemplified in Example 4.
- Fragments of sequences (a) and (b) may also be selected on the basis of their ability to interfere with bacterial aggregation or adhesion. Modifications, substitutions, deletions or rearrangements as outlined above may also be selected to maintain the ability of the polypeptide to interfere with bacterial aggregation.
- amino acids in length comprising: (a) the amino acids 150-168 of protein H of S. pyogenes having the sequence QKQQQLETEKQISEASRKS;
- sequence (b) a corresponding sequence to sequence (a) of an outer membrane protein of a streptococcus strain, said sequence having the ability to interfere with aggregation or adhesion of said streptococcal strain; (c) a fragment of sequence (a) or (b) of 6 or more amino acids, which retains the ability to interfere with streptococcal aggregation or adhesion; and
- polypeptides of the present application are thus also proposed for use to inhibit bacterial aggregation or adherence to epithelial cells and thus may be used to reduce virulence of the bacteria.
- Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated.
- a polypeptide of the invention may also be in a substantially -12- purified form, in which case the polypeptide of the invention will generally comprise 90%, e.g. more than 95% or more than 99% by weight of the polypeptide in the preparation produced by the isolation or purification procedure.
- Polypeptides of the invention may be made synthetically or recombinantly using techniques which are widely available in the art. Synthetic production generally involves stepwise addition of individual amino acid residues to a reaction vessel in which a polypeptide of a desired sequence is being made. Examples of recombinant techniques are described below.
- polypeptides of the present invention are up to 50 amino acids in length. More preferably, the polypeptide is of no more than 40 to 45 amino acids in length, and most preferably up to 30, 20 or 15 amino acids in length.
- the invention also relates to chimeric proteins comprising a first polypeptide in accordance with the invention and a second polypeptide which is not naturally contiguous to said first polypeptide.
- the polypeptide of the invention may comprise repeats of the sequences (a), (b), (c) or (d) or combinations thereof.
- the polypeptide may be cyclized.
- the polypeptide may comprise separate repeat regions linked through a linker sequence such as a polylysine bridge.
- the peptides may also be formulated as fusion proteins with a carrier, particularly when used as a vaccine with carriers such as keyhole limpet hemacyanin, diptheria toxoid, tetanus toxoid.
- the polypeptides of the invention may be formulated into pharmaceutical compositions.
- the compositions comprise the polypeptides together with a pharmaceutically acceptable carrier or diluent.
- Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, topical or parenteral (e.g. intramuscular or intravenous) administration.
- the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
- formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may -13- include suspending agents and thickening agents, and liposomes or other micro- particulate systems which are designed to target the polypeptide within the body.
- the peptides of the invention may also be used to generate an immune response to provide protection against later infections by Streptococcus.
- Means of presentation of the peptide immunogen(s) include, but are not restricted to: free peptides, as peptides conjugated to suitable carrier proteins such as keyhole limpet haemocyanin, bovine serum albumin, ovalbumin, inactived bacterial toxins such as tetanus and diptheria toxoids, bacterial or mammalian heat shock proteins; as fusion or chimeric proteins, expressed by recombinant bacteria or viruses comprising the peptide of choice and one or more peptides or proteins containing T- or
- B-cell epitopes or specific binding domains for molecules present on the lymphocyte surface; as peptides or fusion proteins expressed on the surface of, or secreted by, live bacterial or viral vectors.
- Peptides may also be entrapped within, or presented on the surface of, liposomes; be incorporated into biodegradable microspheres formulated from poly
- (D,L) lactic co-glycolic acid or other polymers or presented on the surface of micelles formulated from saponins such as Quil A, cholesterol and phospholipid suitable detergent.
- Multiple copies of the peptide may be administered to enhance the immune response as synthetic polypeptides containing between 2 and 20 copies of the peptide vaccinogen and/or single or multiple copies of additional T- or B-cell epitopes.
- between 2 and 20 copies of the peptide vaccinogen and/or multiple copies of additional T- and B-cell epitopes may be presented as chemically synthesised branched oligomers formed around a core matrix of lysine or another amino acid.
- Peptides used for vaccination may be chemically modified to facilitate conjugation to protein carriers and or to increase their immunogenicity. Suitable modifications include, but are not confined to, the addition of cysteine residues at each terminus to permit polymerisation via disulphide bond formation.
- Peptides may be modified to increase their immunogenicity by conjugation to lipids such as ⁇ - aminohexadecanoic acid at the N-terminus.
- vaccines which contain an immunogenic polypeptide(s) as -14- active ingredient(s), is known to one skilled in the art.
- such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
- the preparation may also be emulsified, or the protein encapsulated in liposomes.
- the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like in combinations thereof.
- the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and/or adjuvants which enhance the effectiveness of the vaccines.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents and/or adjuvants which enhance the effectiveness of the vaccines.
- adjuvants which may be effective include but are not limited to: alum or other aluminum salts, calcium salts; water-in-oil emulsions containing mineral oil, squalene or squalane; oil-in-water emulsions of squalene, squalene or oils in combination with surfactants such as Tween 80 or Span 85.
- emulsions may also include components such as N-acetyl- muramyl-L-alanyl-D-isoglutamine (MDP), or derivatives of synthetic sulpholippolysaccharides, or non-ionic block copolymers; compositions containing saponins such as Quil A and/or monophosphoryl lipid A (MPL); carbohydrate polymers such as ma nan or beta 1-3 glucose; natural or recombinant bacterial toxins such as cholera toxin or Escherichia coli labile toxin; natural or recombinant cytokines such as human interleukin-1 (IL-1), IL-2, IL-4 or IL-12.
- MDP N-acetyl- muramyl-L-alanyl-D-isoglutamine
- IL-12 human interleukin-1
- an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic peptide resulting from administration of this peptide in vaccines which are also comprised of the various adjuvants.
- the vaccines are conventionally administered parenterally by injection, for example, either subcutaneously or intramuscularly.
- Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or topical application to the nasal, rectal or vaginal mucosa, or through inhalation of a liquid or powder formulation.
- traditional binders and carriers may include for example, polyalkylene glycol triglycerides.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of -15- marmitol, lactose, starch, magnesium stearate, sodium saccarine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension.
- Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
- polypeptides of the invention may be formulated into the vaccines as natural or salt form.
- Pharmaceutically acceptable salts include the acid addition salts
- salt forms for the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide and such organic bases as isopropylamine and trimethylamine, 2-ethylamino ethanol, histidine and procaine.
- inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic.
- Salt forms for the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide and such organic bases as isopropylamine and trimethylamine, 2-ethylamino ethanol, histidine and procaine.
- Peptide vaccinogens may be given as single or repeat doses, with each does consisting of between 2 and 5000 micrograms, preferably between 10 and 1000 micrograms of the peptide. Preferably two or more repeat doses will be given.
- additional antigenic components derived from streptococcal proteins may be incorporated.
- streptococcal extracellular cysteine proteinase SCP
- SCP is thought to play an important role in the virulence of S. pyogenes
- S.pyogenes which are deficient in active SCP production have been shown to lose virtually all their virulence.
- Synthetic peptides such as those described in WO96/08569 representing conserved immuno-dominant epitopes within SCP may readily be incorporated in a suitable vaccine composition.
- streptococcal protein which may be useful in a vaccine formulation is the streptococcal inhibitor of complement-mediated lysis, namely protein SIC. Again, this protein plays a role in S. pyogenes pathogenicity and virulence and is described in
- the peptides of the invention may be made by recombinant techniques.
- the invention also provides nucleic acids encoding polypeptides of the invention.
- Particularly preferred are the polynucleotides set out in Figure 10 for the 33-mer sequence or fragments thereof encoding the 19-mer APP sequence (a).
- a polynucleotide of the invention is capable of hybridising selectively with the coding sequence of sequence (a) for example the sequence set out in Figure 10 for the 33-mer sequence or the fragment thereof encoding the 19-mer APP sequence (a), or to a sequence complementary to that coding sequence.
- Polynucleotides of the invention include variants of the sequence shown in Figure 10 which encode the amino acid sequences of APP or the 33-mer and variants thereof which encode the polypeptide having the sequence (b), (c) or (d) of the invention.
- a polynucleotide of the invention is a contiguous sequence of nucleotides which is capable of selectively hybridising to the polynucleotide sequence of the 33-mer or 19-mer as set out in Figure 10 or to the complement of that sequence.
- a polynucleotide of the invention hybridising to the sequence of Figure 10 encoding the 19-mer APP sequence (a) can hybridise at a level significantly above background.
- the signal level generated by the interaction between a polynucleotide of the invention and the polynucleotide sequence of Figure 10 encoding the 19-mer sequence (a) is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the polynucleotide sequence of Figure 10 encoding sequence (a).
- the intensity of interaction may be measured, for example by radiolabelling the probe e.g. with 32 P.
- Selective hybridisation is typically achieved using conditions of medium to high stringency, for example, 0.03 M sodium chloride and 0.03 M sodium citrate at from about 50 °C to about 60 °C.
- a nucleotide sequence capable of selectively hybridising to the DNA coding sequence of sequence (a) or to the sequence complementary to that coding sequence will be generally at least 50%, preferably at least 70 or 80% and more preferably at least 90 or 95% homologous to the polynucleotide of Figure 10 or its complement over a region of at least 30, preferably at least 40, for instance, at least 60, 80 or 90 contiguous nucleotides over the entire length of the polynucleotide sequence set out in Figure 10 -17- for the 33-mer sequence.
- Methods of measuring polynucleotide homology are well known in the art.
- the UWGCG package, which provides the BESTFIT program can be used to calculate homology, e.g.
- Polynucleotides of the invention can be incorporated into a recombinant replicable vector.
- the vector may be used to replicate the nucleic acid in a compatible host cell.
- the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
- the vector may be recovered from the host cell.
- Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example, insect SF9 cells.
- the polynucleotide of the invention in a vector is operably linked to a regulatory sequence which is capable of providing for the expression of the coding sequence of the host cell, i.e. the vector is an expression vector.
- the term operably linked refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner.
- a regulatory system operably linked to a coding sequence is located in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
- Such vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention.
- This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide expression by the vector of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide.
- the vector may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and -18- optionally a regulator of the promoter.
- the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
- Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
- Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
- yeast promoters include S. cerevisiae GAL4 and ADH promoters.
- Viral promoters include the SV40 large T antigen promoter, retroviral LTR promoters and adenovirus promoters. All these promoters are readily available in the art.
- the nucleotide sequences of the invention and expression vectors can also be used as vaccine formulations as outlined above.
- the vaccines may comprise naked nucleotide sequences or in combination with cationic lipids, polymers or targeting systems.
- the immunogenic polypeptides prepared as described above can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide of the invention. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffmity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
- Monoclonal antibodies directed against Streptococcal epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art.
- the general methodology for making monoclonal antibodies by hybridomas is well known.
- Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against polypeptides of the invention can be screened for various properties; i.e., for isotype and epitope affinity.
- Antibodies both monoclonal and polyclonal, which are directed against -19- polypeptides of the invention are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
- Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
- Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired.
- anti-idiotype antibodies may also be useful for treatment of Streptococci, as well as for an elucidation of the immunogenic regions of polypeptides of the invention.
- Example 1 Surface proteins promote aggregation of API bacteria
- Papain and a cysteine proteinase produced by S. pyogenes can remove proteins H and Ml from the surface of API bacteria.
- API bacteria were suspended in 0.01 M Tris-HCI, pH 8.0, to 1% (v/v) (2x10 9 cells/ml). The bacteria were incubated with papain (Sigma) and L-cystein (100 ⁇ g papain and 28 ⁇ l 1M L-cystein/ml cell solution) at 37°C for 1 h. lodoacetic acid (Sigma) was added to a final concentration of 10 mM to terminate the digestion. Bacteria were collected by centrif ⁇ gation at 3000 x g, washed -20- twice with PBS and submitted to sedimentation analysis.
- Binding of l2 T-labelled IgG to the cells was also performed.
- 0.5 ml API bacteria (2xl0 10 cells/ml PBS) were incubated with 5 ⁇ g of the activated enzyme for 3 h at 37°C.
- the enzyme was inactivated by the addition of iodoacetic acid to 6 mM and cells were washed twice with PBS and analyzed for sedimentation and binding of l25 I-labelled IgG.
- bands I and II were determined to Asn-Gly-Asp-Gly-Asn and Glu-Val-Ala-Gly-Arg, sequences that start at positions 42 and 82, respectively, in Ml protein, whereas the NH 2 -terminal sequence of band III (Glu-Gly-Ala-Lys-Ile) corresponds to a protein H fragment starting at position 42.
- the size of the fragments generated with CNBr correspond well with the positions of methionine residues in the Ml protein and protein H sequences.
- Radiolabelled protein H or Ml protein labelled with 125 I using the Bolton and Hunter reagent (Amersham, UK), was incubated with API bacteria in TH as before. Protein H was found to bind to the bacterial cells (Fig. 3 A).
- the AP6 strain of the M6 serotype is a non-protein H-expressing strain and neither protein H nor Ml protein showed affinity for these bacteria. Included in Figure 3A, as a positive control, are also the binding curves obtained with 125 I-labelled IgG. The binding of protein H to API bacteria could be inhibited with protein H efficiently (see below, Fig. 4B). Protein H and Ml protein were also applied in slots to PVDF membranes and probed with 125 I- labelled protein H or Ml protein. An interaction could be seen between the protein H molecules, whereas Ml protein bound neither protein H nor Ml protein itself (Fig. 3B).
- FIG. 4C shows a schematic representation of protein H, where additionally the fragments used in the experiments below are indicated.
- Radiolabelled protein H and fragment AB, but not fragment A showed affinity for API bacteria (Fig. 4A).
- the binding of radiolabelled protein H to API bacteria could also be blocked with fragment AB but not with fragment A, suggesting that the binding is located in the B region (Fig. 4B).
- a larger amount of AB was required for inhibition.
- fibronectin and albumin could not block the binding of protein H to API bacteria (data not shown), which also suggests that B represents the self-associating region.
- IgG-binding to protein H has been mapped to a region covering the NH 2 - terminal part of B and the COOH-terminal part of A.
- radiolabelled protein H was applied to a column of protein H-Sepharose, the bound radioactivity could not be eluted with an excess of unlabelled IgG (Fig. 5). In contrast, the radioactivity was readily eluted with 3 M KSCN.
- the protein H fragments were also tested for binding to protein H-Sepharose, and again only the AB fragment had affinity (not shown).
- This fragment includes also the 10 NH 2 -terminal amino acid residues from the Cl repeat (see Fig. 4C), which raised the possibility that this sequence and or the COOH-terminal part of B represents the self-associating region.
- a peptide covering this region was therefore synthesized (Fig. 4C) and designated APP; aggregative protein H peptide 150-168.
- APP aggregative protein H peptide 150-168.
- APP aggregative protein H peptide 150-168.
- ⁇ -helical coiled-coil dimers At the bacterial surface protein H and other members of the M protein family form ⁇ -helical coiled-coil dimers.
- the homobifunctional cross-linker disuccinimidyl -22- suberate (DSS) from Pierce in Me 2 SO 4 was added to a final concentration of ImM to protein solutions in PBS for 30 min. at 4°C.
- Example 4 The APP sequence is related to bacterial aggregation, adherence and resistance to phagocytosis To investigate whether the APP sequence also influences bacterial aggregation, the APP peptide was added to API cultures. APP, and proteins H and Ml, reduced the sedimentation rate (Table below), whereas a peptide from the COOH-terminal part of protein H and protein L-derived peptides were without effect.
- APP-related sequences were identified in several M and M-like proteins, including the Ml protein (Fig. 7).
- M serotypes in which APP-related sequences were identified three additional strains of M serotypes 4, 12 and 49 were tested and found to aggregate, an aggregation that could be blocked with protein H (data not shown).
- the proteins containing the APP-related sequences listed in Figure 7 are found in some of the most frequent and important Streptococcal strains and in particular S. Pyogenes serotypes.
- Fig. 7 The related serotypes set out in Fig. 7 represent 46 percent of clinical isolates of S. pyogenes in the UK during the period 1980-1990 Colman et al J. Med Microbiol 1993 Vol. 39 165-178.
- APP-related sequences are very common in clinical isolates implicating that aggregation contributes to the pathogenicity of S. Pyogenes, and other
- Adherence to mucosal surfaces is an important and early step in S. Pyogenes infections. We therefore analyzed the significance of APP-related sequences at the streptococcal surface for aggregation and adherence, using mutants of the API strain and the M6 protein-expressing strain JRS4 which is protein H deficient. -24-
- Bacteria were grown in TH at 37°C over night, collected by centrifugation, washed once in PBS and resuspended in MEM supplemented with 10 FCS. 2 x 10 7 bacteria were added per well and plates were incubated for 2h at 37°C. The wells were washed three times with PBS to remove non-adherent bacteria 0.1 ml trypsin (2.5 mg/ml in PBS) and 0.5 ml Triton X-100 (0.025% in PBS) were added to each well for lysis of the epithelial cells. To determine the number of viable bacteria appropriate dilutions were plated, on TH plates, in triplicate from each well.
- Each well in a 96-microwell plate (Nunc, Maxisorp) was coated overnight at +4°C with 200 ⁇ l of a 1 ⁇ g/ml solution of respectively protein, protein H, APP-peptide and protein L-peptide (Actigen, Cambridge UK) diluted in coupling buffer (0.016 M na 2 CO 3 , 0.035 M NaHCO 3 pH 9.6). After washing, in PBS, the wells were incubated with 200 ⁇ l preimmunserum or antiserum for 1 h at 37°C.
- the sera were serially -26- diluted, 1 :1000; 1 :2000; 1 :4000; 1 :8000; 1 :16000; 1 :32000; 1 :64000 and 1 :128000, in PBS + 0.05% Tween TM-20, 2% bovine serum albumin (BSA).
- BSA bovine serum albumin
- a colour reagent mixture consisting of 40 ml substrate buffer (0.1 M citric acid, 0.1 M Na 2 HPO 4 x 2H 2 O, pH 4.5), 2ml ABTS solution, and 0.8 ml of 30% H 2 O 2 was prepared.
- the plate was developed by adding 200 ⁇ l of colour reagent mixture to each well followed by incubation for 30 minutes at 37°C.
- the plates were read in an ELISA reader at 405 nm.
- KLH Keyhole Limpet Haemacyanin
- Microtitre plates (96 well) were coated with Spy-PH- YQE33 peptide by addition of 5 ⁇ g/ml peptide solution in 0.05 M bicarbonate buffer (pH 9.6) and incubation (60 min, 37°C). Plates were blocked (60 min, 37°C) with 1% bovine serum albumin (BSA) in PBS supplemented with 0.05% Tween-20 (PBS-Tween) to minimise background binding.
- BSA bovine serum albumin
- Sheep immune serum prepared as described above was diluted 1 :10000 in PBS-Tween and pre-incubated (60 min, 37°C) with 100 ⁇ g/ml Spy-PH- QKQ19 (QKQQQLETEKQLSEASRKSC-COOH) relevant peptide or lOO ⁇ g/ml Spy- LP-KEY17 (CKEY TDKLDKLDKESKDK-COOH) irrelevant peptide.
- Control serum was incubated in the absence of peptide. Pre-incubated sera was added to the plates and incubated for 60 min at 37°C.
- Donkey anti-sheep IgG conjugated to horse radish -27- peroxidase (1 :1000 dilution in PBS-Tween) was added to the plate (60 min, 37°C) followed by enzyme substrate solution containing 3,3',5,5'-tetramethyl-bemzidine in phosphate citrate buffer at pH 5.0 (10 min, RT).
- the colorimetric reaction was stopped by addition of 2 M HC1 and optical density determined at 450 nm. Plates were washed between each stage of the ELISA for a minimum of 3 times with PBS-Tween.
- Recombinant protein H was obtained by expression in E. coli as described in Berge et al., (1997) J. Biol. Chem. 272, 20774-20781. Protein H was purified from E. coli cell lysates in a single affinity chromatography step using a human IgG-Sepharose column. Bound protein H was eluted with 3 M KSCN and dialysed against PBS.
- Protein extracts were prepared from S. pyogenes by partial digestion of the cell wall with lysozyme and mutanolysin. Approximately 2 ml of cells grown to OD 600nm of 1.0 were pelleted by low speed centrifugation and resuspended in 100 ⁇ l of 10 mM Tris-HCl containing 5mg/ml of freshly added lysozyme (Sigma) and 100 U/ml of mutanolysin (Sigma).
- the proteins were transferred to PVDF membrane filters using the Novex XCell II blotting apparatus and the conditions recommended by the manufacturer. After transfer the PVDF filters were blocked using a 1 : 1 mixture of UHT virtually fat free milk and phosphate buffered saline containing 0.05% v/v Tween 20 (PBST) for 30 min at room temperature. The filters were incubated with the primary antibody serum (1 : 1000) in blocking buffer (1 part fat free milk to 3 parts PBST) for 1 h at room temperature and then washed for 3 x 5 min in blocking buffer. The filters were incubated with the anti-sheep IgG alkaline phosphate conjugate diluted 1 :5000 in blocking buffer for 30 min at room temperature. -28-
- TH Todd Hewitt broth
- pre- APP undiluted preimmunserum
- anti-APP undiluted antiserum
- pre-IgG undiluted antiserum
- anti-IgG 1 mg/ml Ig-fractions of preimmunserum
- anti-IgG 1 mg/ml Ig-fractions of preimmunserum
- anti-IgG 1 mg/ml Ig-fractions of preimmunserum
- anti-IgG antiserum
- a S. pyogenes strain API of Ml serotype were grown in Todd Hewitt culture medium overnight at 37°C in 5% CO 2 atmosphere. Bacteria were harvested by centrifugation at 3,800 x g for 10 minutes. The resulting bacterial pellet was washed twice and resuspended in 1 x phosphate buffered saline, PBS.
- mice that received only bacteria died within 24 hours, while mice that were given bacteria plus APP-peptide were still alive 4 weeks later.
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JP2000543495A JP2002511241A (en) | 1998-04-09 | 1999-04-09 | peptide |
EP99915909A EP1068229A1 (en) | 1998-04-09 | 1999-04-09 | Peptides |
AU34332/99A AU758555B2 (en) | 1998-04-09 | 1999-04-09 | Peptides |
CA002324981A CA2324981A1 (en) | 1998-04-09 | 1999-04-09 | Peptides |
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GBGB9807890.0A GB9807890D0 (en) | 1998-04-09 | 1998-04-09 | Peptides |
GB9807890.0 | 1998-04-09 |
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PCT/GB1999/001104 WO1999052939A1 (en) | 1998-04-09 | 1999-04-09 | Peptides |
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JP (1) | JP2002511241A (en) |
AU (1) | AU758555B2 (en) |
CA (1) | CA2324981A1 (en) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002050107A2 (en) * | 2000-12-21 | 2002-06-27 | Shire Biochem Inc. | Streptococcus pyogenes antigens and corresponding dna fragments |
WO2002066650A2 (en) * | 2001-02-21 | 2002-08-29 | Shire Biochem Inc. | Streptococcus pyogenes polypeptides and corresponding dna fragments |
US7255867B2 (en) * | 2002-11-15 | 2007-08-14 | Id Biomedical Corporation Of Quebec | Vaccine |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4454121A (en) * | 1982-07-27 | 1984-06-12 | The University Of Tennessee Research Corporation | Synthetic peptides corresponding to antigenic determinants of the M protein of Streptococcus pyogenes |
US4521334A (en) * | 1982-07-27 | 1985-06-04 | The University Of Tennesse Research Corporation | Synthetic polypeptide fragments |
US4784948A (en) * | 1983-08-10 | 1988-11-15 | The Rockefeller University | Production of streptococcal m protein immunogens and molecular probes |
EP0371199A1 (en) * | 1988-11-21 | 1990-06-06 | Sumitomo Pharmaceuticals Company, Limited | Novel protein H being capable of binding to IgG, gene coding for said protein H and a process for producing said protein H |
WO1991019740A1 (en) * | 1990-06-21 | 1991-12-26 | Hightech Receptor Ab | IgG-BINDING PROTEIN |
-
1998
- 1998-04-09 GB GBGB9807890.0A patent/GB9807890D0/en not_active Ceased
-
1999
- 1999-04-09 EP EP99915909A patent/EP1068229A1/en not_active Withdrawn
- 1999-04-09 CA CA002324981A patent/CA2324981A1/en not_active Abandoned
- 1999-04-09 JP JP2000543495A patent/JP2002511241A/en active Pending
- 1999-04-09 AU AU34332/99A patent/AU758555B2/en not_active Ceased
- 1999-04-09 WO PCT/GB1999/001104 patent/WO1999052939A1/en not_active Application Discontinuation
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4454121A (en) * | 1982-07-27 | 1984-06-12 | The University Of Tennessee Research Corporation | Synthetic peptides corresponding to antigenic determinants of the M protein of Streptococcus pyogenes |
US4521334A (en) * | 1982-07-27 | 1985-06-04 | The University Of Tennesse Research Corporation | Synthetic polypeptide fragments |
US4784948A (en) * | 1983-08-10 | 1988-11-15 | The Rockefeller University | Production of streptococcal m protein immunogens and molecular probes |
EP0371199A1 (en) * | 1988-11-21 | 1990-06-06 | Sumitomo Pharmaceuticals Company, Limited | Novel protein H being capable of binding to IgG, gene coding for said protein H and a process for producing said protein H |
WO1991019740A1 (en) * | 1990-06-21 | 1991-12-26 | Hightech Receptor Ab | IgG-BINDING PROTEIN |
Non-Patent Citations (9)
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002050107A2 (en) * | 2000-12-21 | 2002-06-27 | Shire Biochem Inc. | Streptococcus pyogenes antigens and corresponding dna fragments |
WO2002050107A3 (en) * | 2000-12-21 | 2003-04-10 | Shire Biochem Inc | Streptococcus pyogenes antigens and corresponding dna fragments |
US7595057B2 (en) | 2000-12-21 | 2009-09-29 | Id Biomedical Corporation | Streptococcus pyogenes antigens and corresponding DNA fragments |
US8298551B2 (en) | 2000-12-21 | 2012-10-30 | Id Biomedical Corporation Of Quebec | Streptococcus pyogenes antigens and corresponding DNA fragments |
US8821895B2 (en) | 2000-12-21 | 2014-09-02 | Id Biomedical Corporation Of Quebec | Streptococcus pyogenes antigens and corresponding DNA fragments |
US9340586B2 (en) | 2000-12-21 | 2016-05-17 | Id Biomedical Corporation Of Quebec | Streptococcus pyogenes antigens and corresponding DNA fragments |
WO2002066650A2 (en) * | 2001-02-21 | 2002-08-29 | Shire Biochem Inc. | Streptococcus pyogenes polypeptides and corresponding dna fragments |
WO2002066650A3 (en) * | 2001-02-21 | 2002-10-31 | Shire Biochem Inc | Streptococcus pyogenes polypeptides and corresponding dna fragments |
US7883706B2 (en) | 2001-02-21 | 2011-02-08 | Id Biomedical Corporation | Methods for using Streptococcus pyogenes polypeptides |
US7255867B2 (en) * | 2002-11-15 | 2007-08-14 | Id Biomedical Corporation Of Quebec | Vaccine |
Also Published As
Publication number | Publication date |
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CA2324981A1 (en) | 1999-10-21 |
GB9807890D0 (en) | 1998-06-10 |
AU758555B2 (en) | 2003-03-27 |
JP2002511241A (en) | 2002-04-16 |
EP1068229A1 (en) | 2001-01-17 |
AU3433299A (en) | 1999-11-01 |
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