WO2001040472A2 - Streptococcus pneumoniae antigens - Google Patents

Streptococcus pneumoniae antigens Download PDF

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Publication number
WO2001040472A2
WO2001040472A2 PCT/GB2000/004602 GB0004602W WO0140472A2 WO 2001040472 A2 WO2001040472 A2 WO 2001040472A2 GB 0004602 W GB0004602 W GB 0004602W WO 0140472 A2 WO0140472 A2 WO 0140472A2
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WIPO (PCT)
Prior art keywords
protein
pneumoniae
antigenic fragment
nucleic acid
sds
Prior art date
Application number
PCT/GB2000/004602
Other languages
French (fr)
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WO2001040472A3 (en
Inventor
Alan William Cripps
Jennelle Maree Kyd
Maha Jomaa
Original Assignee
Cortecs (Om) Pty Limited
Chapman, Paul, William
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Publication date
Application filed by Cortecs (Om) Pty Limited, Chapman, Paul, William filed Critical Cortecs (Om) Pty Limited
Priority to AU15405/01A priority Critical patent/AU1540501A/en
Priority to JP2001542537A priority patent/JP2003515341A/en
Priority to EP00977771A priority patent/EP1240329A2/en
Priority to CA002392895A priority patent/CA2392895A1/en
Publication of WO2001040472A2 publication Critical patent/WO2001040472A2/en
Publication of WO2001040472A3 publication Critical patent/WO2001040472A3/en

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    • 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
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci

Definitions

  • the present application relates to a protein which is derived from Streptococcus pneumoniae, in particular to a cell wall protein.
  • the invention also relates to the use of this protein in medicine, particularly in the prophylaxis and diagnosis of
  • Streptococcus pneumoniae is a major causative pathogen in the respiratory tract. Infections caused by this pathogen include otitis media, lower respiratory tract infections, bacteremia and meningitis.
  • the development of an effective vaccine against S. pneumoniae involves identification of highly conserved protective antigens and an understanding of the immune response.
  • the present inventors have isolated and purified a protein from the cell wall of S. pnuemoniae. This protein is of use as a vaccine and also in the diagnosis of S. pneumoniae infection.
  • a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
  • the protein of the present invention is isolatable from S. pneumoniae and may be provided in substantially pure form. For example, it may be provided in a form which is substantially free of other proteins.
  • the protein of the invention is useful as antigenic material.
  • Such material can be "antigenic” and/or “immunogenic”.
  • antigenic is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject.
  • immunogenic is taken to mean that the protein is capable of eliciting a protective immune response in a subject.
  • the protein may be capable of not only generating an antibody response but, in addition, non-antibody based immune responses.
  • homologues or derivatives of the protein or polypeptide of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material.
  • proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention.
  • a program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score Both types of analysis are contemplated in the present invention.
  • a protein sequence may be regarded as substantially homologous to another protein sequence if a significant number of the constituent amino acids exhibit homology when using the one of the algorithms mentioned above. At least 40%, 50,%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids, may be homologous or identical.
  • the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
  • the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein.
  • the key issue, once again, is that the fragment retains the antigenic/immunogenic properties of the protein from which it is derived.
  • the invention provides a protein comprising an antigenic fragment as defined above.
  • the molecular weight of the protein has been measured by SDS- PAGE under reducing conditions.
  • Lie molecular weight values obtained by this method are accurate only to a degree of about ⁇ 20% and are dependent upon the particular gel and molecular weight markers which are used.
  • the protein may be prepared by expressing the appropriate nucleic acid.
  • an isolated or recombinant nucleic acid encoding a protein of the first aspect of the invention or antigenic fragment thereof or a nucleic acid complementary thereto.
  • the nucleic acid which may be DNA
  • a vector which may be a plasmid, cosmid or a phage.
  • the vector may be incorporated into the genome of a host organism, which may be either a prokaryotic or a eukaryotic organism.
  • the nucleic acid or vector may be suitable for expressing the protein of the present invention in a subject to be treated, i.e. it may be in the form of a so-called DNA vaccine.
  • Methods and agents suitable for the preparation of a DNA vaccine are well known to the skilled person.
  • the protein or fragments thereof may be useful in a method for the prophylaxis of S. pneumoniae infection, the method comprising administering to a patient in need of such treatment an effective amount of the protein or fragment thereof.
  • the invention also provides a method of vaccinating a subject against S. pneumoniae which comprises the step of administering to the subject an effective amount of protein or antigenic fragment as defined above.
  • the protein is also useful for diagnosing S pneumoniae infection.
  • a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, or nucleic acid coding therefor for use in medicine, particularly in the prophylaxis or diagnosis of infection by S. pneumoniae, wherein the protein has a molecular weight of about 34 kDa as determined by SDS- PAGE under reducing conditions and has the following amino terminal sequence:
  • the protein of the present invention is also of use in the diagnosis of such infection.
  • a further aspect provides a method of detecting and/or diagnosing S. pneumoniae which comprises:
  • a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, or nucleic acid coding therefor in the preparation of an agent for the prophylaxis or diagnosis of infection by S. pneumoniae, wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
  • the protein isolated by the inventors has been shown to be antigenic and they can therefore be used as vaccines or agents for the diagnosis of infection by S. pneumoniae.
  • the invention also provides a pharmaceutical or vaccine composition
  • a pharmaceutical or vaccine composition comprising the protein of the present invention together with a pharmaceutically acceptable excipient.
  • the pharmaceutical or vaccine composition may also comprise an adjuvant.
  • adjuvants well known in the art include inorganic gels such as aluminium hydroxide or water-in-oil emulsions such as incomplete Freund's adjuvant.
  • Other useful adjuvants will be well known to the skilled man.
  • the protein may be administered by a variety of routes including enteral, for example oral, nasal, buccal, topical or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
  • enteral for example oral, nasal, buccal, topical or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
  • compositions and the excipients it contains will, of course, depend upon the chosen route of administration.
  • oral formulations may be in the form of syrups, elixirs, tablets or capsules, which may be enterically coated to protect the protein from degradation in the stomach.
  • Nasal or transdermal formulations will usually be sprays or patches respectively.
  • Formulations for injection may be solutions or suspensions in distilled water or another pharmaceutically acceptable solvent or suspending agent.
  • a suitable dose for vaccination may be from about 5 to 100 ⁇ g when administered parenterally.
  • the dosage may be 10-100 fold higher for nasal and oral administration, depending on the formulation, adjuvant, delivery system, patient profile, etc.
  • the protein isolated by the inventors is antigenic and therefore the invention also provides an antibody which binds specifically to the protein.
  • the antibody may be a monoclonal or a polyclonal antibody. Techniques for the preparation of both monoclonal and polyclonal antibodies are well known to those skilled in the art.
  • the present invention includes derivatives thereof which are capable of binding to proteins etc as described herein.
  • the present invention includes antibody fragments and synthetic constructs, and the term "antibody” as used herein is intended to include these. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
  • Antibody fragments include, for example, Fab, F(ab') and Fv fragments. Fab fragments (These are discussed in Roitt et al [supra] ). Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V h and N regions, which contributes to the stability of the molecule.
  • Other synthetic constructs that can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains.
  • Synthetic constructs include chimaeric molecules.
  • humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention.
  • An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimaeric antibodies are discussed for example by Morrison et al in PNAS, 81, 6851-6855 (1984) and by Takeda et al in Nature. 314, 452-454 (1985).
  • Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding.
  • the moiety may be a label (e.g. a fluorescent or radioactive label, or latex or an equivalent solid physical label such as an erythrocyte).
  • it may be a pharmaceutically active agent.
  • Antibodies, or derivatives thereof, find use in detection diagnosis of S. pneumoniae.
  • the present invention provides a method for the detection/diagnosis of S. pneumoniae which comprises the step of bringing into contact a sample to be tested and antibodies capable of binding to one or more proteins of the invention, or to fragments thereof.
  • binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain (Nord et al,).
  • binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain (Nord et al,).
  • small protein domains capable of specific binding to different target proteins can be selected using combinatorial approaches.
  • FIGURE 1 is a flow chart with a schematic summary of the protein purification procedure used in the present invention.
  • FIGURE 2 is the electroelution profile from S. pneumoniae cell wall extract analysed on SDS-PAGE.
  • Lane 1 coomassie stain of crude extract separated by SDS-PAGE; Lane 3: molecular mass standards;
  • Lanes 2 and 4-11 proteins recovered by electroelution.
  • FIGURE 3 is a profile from anion exchange chromatography.
  • FIGURE 4 shows purified S. pneumoniae proteins of molecular masses 14, 16, 34 and
  • FIGURE 5 is a histogram showing pulmonary clearance following immunisation with S. pneumoniae protein of 16 kDa.
  • FIGURE 6 is a histogram showing pulmonary clearance following immunisation with S. pneumoniae protein of 34 kDa.
  • FIGURE 7 is a histogram showing pulmonary clearance following immunisation with S. pneumoniae protein of 57 kDa.
  • FIGURE 8 is a gel showing the results of a two-dimensional gel analysis of S. pneumoniae protein of 34 kDa.
  • FIGURE 9 is gel showing the results of a Western blot analysis of S. pneumoniae protein of 34 kDa..
  • Lane 1 homologous cell wall extract
  • Lane 2 Dimensional strip slot
  • Lane 3 molecular weight standards in Lane 3.
  • Bacteria Serogroup 3 Streptococcus pneumoniae (ATCC 49619) was used to obtain antigens investigated in this study and used in homologous bacterial challenge in the animal studies. Bacterial strains were grown overnight on blood agar at 37°C and 5% CO 2 or cultured in tryptic soya broth (Oxoid Ltd, Basingstoke, Hampshire, UK) overnight in a shaker incubator at 37°C.
  • a loop full of S. pneumoniae was inoculated into 10 mL of sterile tryptone soya broth and cultured overnight in a 37°C shaker incubator. 2 x 5 mL aliquots were subcultured into 2 x 500 mL volumes of sterile tryptone soya broth and cultured overnight in a 37°C shaker incubator. Aseptically, a loop of bacterial suspension was removed from each culture, streaked onto blood agar and incubated overnight at 37°C in CO 2 as a growth and contaminant check.
  • the bacterial culture was centrifuged at 18000 x g for 20 minutes at 4°C using a
  • Beckman J-2TM centrifuge The pellet was washed twice in phosphate buffered saline (PBS) by centrifugation, then resuspended in 10 mL PBS and 200 ⁇ l 10% (w/v) sodium deoxycholate and sti ⁇ ed at room temperature for 1 hour.
  • the suspension was centrifuged at 27000 x g for 15 minutes at 4°C, the supernatant was recovered and sti ⁇ ed while gradually adding ammonium sulphate to a final concentration of 70% (w/v).
  • the suspension was centrifuged at 27000 x g for 15 minutes at 4°C, the pellet redissolved in 10 mL 10 mM sodium phosphate, pH 7.0.
  • the resuspended pellet was dialysed against 3 x IL changes of 10 mM sodium phosphate, pH 7.0 at 4°C, leaving a minimum of 2 hours between changes.
  • the dialysed protein suspension was centrifuged for 20 minutes at 15000 rpm at 4°C, the supernatant was kept and a protein assay performed.
  • the protein suspension was concentrated by lyophilisation and a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis performed.
  • the Protean II xi cellTM (Bio-Rad) was used to separate proteins according to their molecular weights.
  • a discontinuous gel consisting of 12% (w/v) acrylamide/BIS separating gel and a 4% (w/v) acrylamide/BIS stacking (upper) gel was prepared from a 30% (w/v) stock solution of acrylamide/BIS (N,N'-methylenebisacrylamide) in Tris buffer.
  • the polyacrylamide gel was polymerised using ammonium persulfate and
  • TEMED TEMED.
  • the lyophilised protein extract was suspended 1 :1 (v/v) in sample buffer (0.5M Tris HC1 pH 6.8; 10% v/v) glycerol; 10% (w/v) SDS; 0.05% (w/v) bromophenol blue; 0.05% (v/v) ⁇ -mercaptoethanol), boiled for 5 minutes and then approximately 1 mL of this was loaded onto the top of the gel. Electrophoresis was performed at a constant cu ⁇ ent of 16mA per gel until the dye front passed through the stacker and then increased to 24 mA for electrophoresis through the resolving gel. The average running time was between 4 and 5 hours.
  • the separated proteins were then recovered by electroelution using the BIORADTM flat bed electroeluter for 1 hour at 200V and a maximum of 0.2mA into 30 individual tubes. Protein composition of the recovered fractions was assessed by analytical SDS-PAGE and either Coomassie or Silver staining of proteins. Analytical SDS-PAGE was performed using a Mini- protean II TM cell (Bio-Rad) at a constant 200V for about 45 minutes. Protein concentrations were determined using the Pierce Micro BCATM protein assay and comparison with albumin standards. SDS Removal from Purified Proteins
  • Samples containing SDS were treated with a 200 ⁇ L volume of lOOmM potassium phosphate per lmL of sample and left on ice for 60 minutes. The sample was centrifuged at 10000 x g for 20 minutes at 4°C in a microcentrifuge. The supernatant was recovered and desalted by overnight dialysis against nanopure water.
  • the extracted proteins were additionally purified by anion exchange chromatography and separated according to their molecular charge interactions.
  • the column (Q5 column, Bio-Rad) was equilibrated with a low salt buffer (20mM Tris-HCl, pH 8.45) at a flow rate of lmL/min for 10 minutes. Lyophilised cell wall extracts were resuspended in the same buffer to a concentration of 5mg per mL and loaded onto the column. Proteins were eluted using an increasing salt gradient by gradually increasing the proportion of 20mM Tris-HCl, 500mM sodium chloride, pH 8.6 passed through the column. Fractions were recovered, lyophilised and assessed by analytical SDS- PAGE. Fractions from multiple runs were pooled and proteins were further purified by preparative SDS-PAGE and electroelution as previously described.
  • the elution profile from anion exchange chromatography is shown in Figure 3.
  • the first peak represents elution of unbound proteins.
  • the subsequent two major peaks contained most of the proteins that were eluted with increasing salt concentration.
  • the proteins in these peaks were further purified by SDS-PAGE.
  • the N-terminal sequence of the proteins was determined from an excised band from an analytical SDS-PAGE. Analyses were performed by the Biomolecular Resource Unit, The John Curtin School of Medical Science (Australian Capital Te ⁇ itory, Australia).
  • the information obtained from the partial amino acid sequence was searched through the GenBank databases to determine homology to known protein sequences. It was found that the 12-14 kDa protein has a 100% sequence homology match with that of a 12 kDa protein from S. pneumoniae. The 16kDa protein has been found to be an isomer of the 12-14 kDa protein.
  • the 34kDa protein appears to be a novel protein as a BLAST search of publicly available databases revealed no significant similarities.
  • mice 6-10 weeks old were housed and maintained in a pathogen free environment with free access to sterilised food and water.
  • mice were initially immunised on day 0 by Peyer' s patches inoculation and boosted by intratracheal administration 14 days later. On day 21, these mice were challenged with live S. pneumoniae. Peyer's Patch Immunisation
  • mice were sedated by a subcutaneous injection of 0.25 L ketamine/xylazine at a dosage of 5 mg/ml ketamine hydrochloride; 2mg/ml xylazine hydrochloride.
  • the small intestine was exposed through a mid-line abdominal incision and the protein injected subserosal into each Peyer's patch.
  • the immunisation protein was prepared by emulsifying 2.5 ⁇ g/ ⁇ L protein in a 1 :1 ratio with incomplete Freund's adjuvant (Sigma Immunochemicals, St Louis, MI, USA) and a total concentration of lO ⁇ g protein administered to each animal.
  • mice received an intratracheal boost.
  • the mice were sedated by intravenous injection with 20 mg saffan per kg of body weight.
  • lO ⁇ g protein in PbS in a total volume of 20 ⁇ L was delivered via the trachea into the lungs with a 22.1/2G catheter.
  • mice received a live bacterial challenge.
  • the mice were sedated with saffan as described above, and an inoculum of 1 x 10 7 CFU in 20 ⁇ L of live S. pneumoniae was introduced into the lungs via the trachea as for the intratracheal boost.
  • Five hours following the challenge the mice were euthanased by an intraperitoneal injection of 0.2mL of sodium pentobarbital.
  • Blood was collected by heart puncture and the separated serum stored below-20°C prior to analysis.
  • the trachea was exposed and the lungs were lavaged by insertion and removal of 0.5mL sterile PBS.
  • the recovered fluid (BAL) was assessed for bacterial recovery by plating 10 fold serial dilutions onto blood agar for CFU determination. An aliquot was removed for cytospin slide preparation, staining and differential cell counts.
  • the BAL was then centrifuged for 10 min at 1000 rpm at 4°C and the supernatant stored below -20°C until required. The pellet was resuspended in PBS and methylene blue and the total number of white cells in the BAL were counted.
  • the lungs were removed following lavage, placed in 2mL sterile PBS and homogenised.
  • the lung homogenate was assessed by plating 10-fold serial dilutions onto blood agar for CFU determination. Results are presented only for the proteins which showed significant degrees of pulmonary clearance from the lungs.
  • Serum from mice immunised with either the 34 kDa protein or the 12-14 kDa protein was assessed for the presence of antibody to the immunising protein by ELIS A.
  • Polysorb microtitre wells were coated with 10 ⁇ g per ml of purified protein, either 34 kDa protein or 12-14 kDa protein, in coating buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6) by incubation with 50 ⁇ l per well overnight at 4 °C. The plates were washed 5 times in washing buffer (PBS containing 0.05% Tween-20). The wells were blocked with 100 ⁇ l of 5% Skim Milk in PBS/Tween-20 buffer for 60 min at room temperature. The plates were washed 5 times as above and 50 ⁇ l serum dilutions commencing at 1/5 were added to the wells and incubated at room temperature for 90 minutes.
  • washing buffer PBS containing 0.05% Tween-20
  • the wells were incubated with anti-mouse IgG- horseradish peroxidase conjugated antisera for 90 minutes.
  • the wells were washed and developed with TMB (tetra methylbenzidine in dimethyl sulphoxide) in phospate citrate buffer, pH 5 containing hydrogen peroxide.
  • the reaction was stopped with 0.5 M H SO 4 and the optical density of the wells read at 450 nm.
  • the concentration of immunoglobulin in the test wells was determined by calculation from known concentrations of IgG assayed as standards on the same microtitre plate.
  • protean IEF Cell was used for the first dimension isoelectric focusing and for the second dimensional electrophoretic protein analysis.
  • the first dimension electrophoresis was performed using 7cm IPG strip which was actively rehydrated overnight for 17 hours at 50V. 60uL (lug/uL) of cell wall extract and a total volume of 140uL of rehydration solution ( 8.3M urea, 2% CHAPS, 50mM Dithiothreilol, 0.1% Bio-Lytes, and 0.001% Bromocresol blue), was applied to the sample well.
  • the gel strips were overlayed onto 12% SDS- PAGE gels and a 4% stacker (as described earlier) and ran at 5mA for 1 hour and 15mA for 1.5 hours per gel strip.
  • the molecular weight of the 34 kDa protein was calculated to be 34.1 kDa.
  • Figure 8 An identified band similar to the one in figure 2 of a 34.1 kDa protein was excised from the gel.
  • Figure 9 34 kDa protein using specific antisera from the mice (immunisations shown previously) was detected in the homologous cell wall extract (lane 1), and the same band was also seen in the 2 Dimensional strip slot in lane 2, compared to the molecular weight standards in Lane 3.

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Abstract

The invention relates to an antigenic protein derived from Streptococcus pneumoniae. The protein is antigenic and is useful in the preparation of vaccines and the diagnosis of S. pneumoniae infection.

Description

STREPTOCOCCUS PNEUMONIAE ANTIGENS
The present application relates to a protein which is derived from Streptococcus pneumoniae, in particular to a cell wall protein. The invention also relates to the use of this protein in medicine, particularly in the prophylaxis and diagnosis of
Streptococcus pneumoniae infection.
Respiratory diseases remain a major cause of morbidity and mortality throughout the world. Streptococcus pneumoniae is a major causative pathogen in the respiratory tract. Infections caused by this pathogen include otitis media, lower respiratory tract infections, bacteremia and meningitis.
The burden of disease caused by these pathogens is highly significant and contributes significantly to national health budgets. Although there is a vaccine available for Streptococcus pneumoniae, this vaccine is not highly efficacious in children under two years. Current therapy relies on antibiotic treatment of the infection. Many suffering from infections caused by Streptococcus pneumoniae live in developing countries, where some communities have very limited access to adequate medical treatment. Thus, antibiotic treatment may not be available. In the developed world, where antibiotics are available, there has been a significant emergence of antibiotic resistance in these bacteria.
The development of an effective vaccine against S. pneumoniae is therefore a desirable objective. In particular, it is desirable to develop a vaccine which can be used in young children.
The development of an effective vaccine against S. pneumoniae involves identification of highly conserved protective antigens and an understanding of the immune response. The present inventors have isolated and purified a protein from the cell wall of S. pnuemoniae. This protein is of use as a vaccine and also in the diagnosis of S. pneumoniae infection.
Therefore, in a first aspect of the present invention, there is provided a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
V X X V G I N T X S X X Q S
(Val X X Val Gly He Asn Thr X Ser X X Gin Ser) (SEQ ID NO: 1);
wherein X represents an unknown amino acid residue.
The protein of the present invention is isolatable from S. pneumoniae and may be provided in substantially pure form. For example, it may be provided in a form which is substantially free of other proteins.
As discussed herein, the protein of the invention is useful as antigenic material. Such material can be "antigenic" and/or "immunogenic". Generally, "antigenic" is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject. "Immunogenic" is taken to mean that the protein is capable of eliciting a protective immune response in a subject. Thus, in the latter case, the protein may be capable of not only generating an antibody response but, in addition, non-antibody based immune responses.
The skilled person will appreciate that homologues or derivatives of the protein or polypeptide of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material. Thus, for instance proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar "type". For instance replacing one hydrophobic amino acid with another. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score Both types of analysis are contemplated in the present invention.
In the case of homologues, the degree of identity with a protein as described herein is less important than that the homologue should retain its antigenicity and/or immunogenicity to S. pneumoniae. However, for the purposes of the present invention, a protein sequence may be regarded as substantially homologous to another protein sequence if a significant number of the constituent amino acids exhibit homology when using the one of the algorithms mentioned above. At least 40%, 50,%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids, may be homologous or identical.
It is well known that is possible to screen an antigenic or immunogenic protein or polypeptide to identify epitopic regions, i.e. those regions which are responsible for the protein or polypeptide' s antigenicity or immunogenicity. Methods well known to the skilled person can be used to test fragments and/or homologues and/or derivatives for antigenicity. Thus, the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention, the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein. The key issue, once again, is that the fragment retains the antigenic/immunogenic properties of the protein from which it is derived. The invention provides a protein comprising an antigenic fragment as defined above.
In this specification, the molecular weight of the protein has been measured by SDS- PAGE under reducing conditions. As one skilled in the art will appreciate, Lie molecular weight values obtained by this method are accurate only to a degree of about ± 20% and are dependent upon the particular gel and molecular weight markers which are used.
In a further aspect of the invention, there is provided a process for the preparation of an isolated and purified protein, the process comprising the following steps:
(a) preparing cultures of S. pneumoniae, growing the cultures under appropriate conditions and harvesting them, followed by washing with centrifugation to yield a washed cell pellet;
(b) resuspending the washed cells in an appropriate buffer followed by disruption of the cells;
(c) centrifuging to remove cell debris and obtaining the supernatant containing soluble cell proteins;
(d) subjecting the solution obtained to anion exchange chromatography with a sodium chloride gradient elution, and pooling the fractions corresponding to each separate peak;
(e) suspending the protein fractions in a buffer comprising 0.5M Tris HC1 pH 6.8; 10%(v/v) glycerol; 10% (w/v) SDS; 0.05% (w/v) bromophenol blue; and 0.05% (v/v) β-mercaptoethanol; boiling the mixture and then purifying by SDS-PAGE using a 12% (w/v) acrylamide/BIS separating gel with a 4% (w/v) acrylamide/BIS stacking gel, run at 16 mA in the stacking gel and 24 mA in the resolving gel;
(f) selecting a fraction containing a protein having a molecular weight of 34kDa and isolating the protein from the selected fraction.
Alternatively, the protein may be prepared by expressing the appropriate nucleic acid.
Therefore, in a further aspect of the present invention, there is provided an isolated or recombinant nucleic acid encoding a protein of the first aspect of the invention or antigenic fragment thereof or a nucleic acid complementary thereto.
For the purposes of expression, the nucleic acid, which may be DNA, may be inserted into a vector, which may be a plasmid, cosmid or a phage. The vector may be incorporated into the genome of a host organism, which may be either a prokaryotic or a eukaryotic organism.
The nucleic acid or vector may be suitable for expressing the protein of the present invention in a subject to be treated, i.e. it may be in the form of a so-called DNA vaccine. Methods and agents suitable for the preparation of a DNA vaccine are well known to the skilled person.
The protein or fragments thereof may be useful in a method for the prophylaxis of S. pneumoniae infection, the method comprising administering to a patient in need of such treatment an effective amount of the protein or fragment thereof. The invention also provides a method of vaccinating a subject against S. pneumoniae which comprises the step of administering to the subject an effective amount of protein or antigenic fragment as defined above. The protein is also useful for diagnosing S pneumoniae infection.
Therefore, in a further aspect of the present invention, there is provided a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, or nucleic acid coding therefor, for use in medicine, particularly in the prophylaxis or diagnosis of infection by S. pneumoniae, wherein the protein has a molecular weight of about 34 kDa as determined by SDS- PAGE under reducing conditions and has the following amino terminal sequence:
V X X V G I N T X S X X Q S
(Val X X Val Gly He Asn Thr X Ser X X Gin Ser) (SEQ ID NO: 1)
wherein X represents an unknown amino acid residue.
In addition to the prophylaxis of infection by S. pneumoniae, the protein of the present invention is also of use in the diagnosis of such infection. Thus, a further aspect provides a method of detecting and/or diagnosing S. pneumoniae which comprises:
(a) bringing into contact with a sample to be tested a protein as defined above or a homologue thereof or an antigenic fragment of the protein or homologue; and
(b) detecting the presence of antibodies to S pneumoniae.
In a further aspect of the invention, there is provided the use of a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, or nucleic acid coding therefor in the preparation of an agent for the prophylaxis or diagnosis of infection by S. pneumoniae, wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
V X X V G I N T X S X X Q S (Val X X Val Gly He Asn Thr X Ser X X Gin Ser) (SEQ ID NO: 1)
wherein X represents an unknown amino acid residue.
As already mentioned, the protein isolated by the inventors has been shown to be antigenic and they can therefore be used as vaccines or agents for the diagnosis of infection by S. pneumoniae.
Thus, the invention also provides a pharmaceutical or vaccine composition comprising the protein of the present invention together with a pharmaceutically acceptable excipient.
The pharmaceutical or vaccine composition may also comprise an adjuvant. Examples of adjuvants well known in the art include inorganic gels such as aluminium hydroxide or water-in-oil emulsions such as incomplete Freund's adjuvant. Other useful adjuvants will be well known to the skilled man.
The protein may be administered by a variety of routes including enteral, for example oral, nasal, buccal, topical or anal administration or parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.
The form taken by the composition and the excipients it contains will, of course, depend upon the chosen route of administration. For example, oral formulations may be in the form of syrups, elixirs, tablets or capsules, which may be enterically coated to protect the protein from degradation in the stomach. Nasal or transdermal formulations will usually be sprays or patches respectively. Formulations for injection may be solutions or suspensions in distilled water or another pharmaceutically acceptable solvent or suspending agent.
The appropriate dosage of the protein of the present invention to be administered to a patient will be determined by a clinician. However, as a guide, a suitable dose for vaccination may be from about 5 to 100 μg when administered parenterally. However, the dosage may be 10-100 fold higher for nasal and oral administration, depending on the formulation, adjuvant, delivery system, patient profile, etc.
As mentioned above, the protein isolated by the inventors is antigenic and therefore the invention also provides an antibody which binds specifically to the protein. The antibody may be a monoclonal or a polyclonal antibody. Techniques for the preparation of both monoclonal and polyclonal antibodies are well known to those skilled in the art.
In addition to whole antibodies, the present invention includes derivatives thereof which are capable of binding to proteins etc as described herein. Thus the present invention includes antibody fragments and synthetic constructs, and the term "antibody" as used herein is intended to include these. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).
Antibody fragments include, for example, Fab, F(ab') and Fv fragments. Fab fragments (These are discussed in Roitt et al [supra] ). Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining Vh and N regions, which contributes to the stability of the molecule. Other synthetic constructs that can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains. Synthetic constructs include chimaeric molecules. Thus, for example, humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimaeric antibodies are discussed for example by Morrison et al in PNAS, 81, 6851-6855 (1984) and by Takeda et al in Nature. 314, 452-454 (1985).
Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label, or latex or an equivalent solid physical label such as an erythrocyte). Alternatively, it may be a pharmaceutically active agent.
Antibodies, or derivatives thereof, find use in detection diagnosis of S. pneumoniae.
Thus, in another aspect, the present invention provides a method for the detection/diagnosis of S. pneumoniae which comprises the step of bringing into contact a sample to be tested and antibodies capable of binding to one or more proteins of the invention, or to fragments thereof.
In addition, so-called "Affibodies" may be utilised. These are binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain (Nord et al,). Thus, small protein domains, capable of specific binding to different target proteins can be selected using combinatorial approaches.
Preferred features of each aspect of the invention as for each other aspect, mutatis mutandis. The disclosure of the prior art documents mentioned herein is incorporated to the fullest extent permitted by law. The invention will now be described in greater detail with reference to the following examples and to the drawings in which:
FIGURE 1 is a flow chart with a schematic summary of the protein purification procedure used in the present invention.
FIGURE 2 is the electroelution profile from S. pneumoniae cell wall extract analysed on SDS-PAGE.
Lane 1 : coomassie stain of crude extract separated by SDS-PAGE; Lane 3: molecular mass standards;
Lanes 2 and 4-11 : proteins recovered by electroelution.
FIGURE 3 is a profile from anion exchange chromatography.
FIGURE 4 shows purified S. pneumoniae proteins of molecular masses 14, 16, 34 and
57 kDa
FIGURE 5 is a histogram showing pulmonary clearance following immunisation with S. pneumoniae protein of 16 kDa.
FIGURE 6 is a histogram showing pulmonary clearance following immunisation with S. pneumoniae protein of 34 kDa.
FIGURE 7 is a histogram showing pulmonary clearance following immunisation with S. pneumoniae protein of 57 kDa.
FIGURE 8 is a gel showing the results of a two-dimensional gel analysis of S. pneumoniae protein of 34 kDa. FIGURE 9 is gel showing the results of a Western blot analysis of S. pneumoniae protein of 34 kDa.. Lane 1 : homologous cell wall extract Lane 2: Dimensional strip slot Lane 3: molecular weight standards in Lane 3.
Example 1 - Isolation and Purification of Antigens
Bacteria Serogroup 3 Streptococcus pneumoniae (ATCC 49619) was used to obtain antigens investigated in this study and used in homologous bacterial challenge in the animal studies. Bacterial strains were grown overnight on blood agar at 37°C and 5% CO2 or cultured in tryptic soya broth (Oxoid Ltd, Basingstoke, Hampshire, UK) overnight in a shaker incubator at 37°C.
Protein Purification
Extraction of Cell Wall Proteins
Aseptically, a loop full of S. pneumoniae was inoculated into 10 mL of sterile tryptone soya broth and cultured overnight in a 37°C shaker incubator. 2 x 5 mL aliquots were subcultured into 2 x 500 mL volumes of sterile tryptone soya broth and cultured overnight in a 37°C shaker incubator. Aseptically, a loop of bacterial suspension was removed from each culture, streaked onto blood agar and incubated overnight at 37°C in CO2 as a growth and contaminant check.
The bacterial culture was centrifuged at 18000 x g for 20 minutes at 4°C using a
Beckman J-2™ centrifuge. The pellet was washed twice in phosphate buffered saline (PBS) by centrifugation, then resuspended in 10 mL PBS and 200 μl 10% (w/v) sodium deoxycholate and stiπed at room temperature for 1 hour. The suspension was centrifuged at 27000 x g for 15 minutes at 4°C, the supernatant was recovered and stiπed while gradually adding ammonium sulphate to a final concentration of 70% (w/v). The suspension was centrifuged at 27000 x g for 15 minutes at 4°C, the pellet redissolved in 10 mL 10 mM sodium phosphate, pH 7.0. The resuspended pellet was dialysed against 3 x IL changes of 10 mM sodium phosphate, pH 7.0 at 4°C, leaving a minimum of 2 hours between changes. The dialysed protein suspension was centrifuged for 20 minutes at 15000 rpm at 4°C, the supernatant was kept and a protein assay performed. The protein suspension was concentrated by lyophilisation and a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis performed.
SDS-PAGE
The Protean II xi cell™ (Bio-Rad) was used to separate proteins according to their molecular weights. A discontinuous gel consisting of 12% (w/v) acrylamide/BIS separating gel and a 4% (w/v) acrylamide/BIS stacking (upper) gel was prepared from a 30% (w/v) stock solution of acrylamide/BIS (N,N'-methylenebisacrylamide) in Tris buffer. The polyacrylamide gel was polymerised using ammonium persulfate and
TEMED. The lyophilised protein extract was suspended 1 :1 (v/v) in sample buffer (0.5M Tris HC1 pH 6.8; 10% v/v) glycerol; 10% (w/v) SDS; 0.05% (w/v) bromophenol blue; 0.05% (v/v) β-mercaptoethanol), boiled for 5 minutes and then approximately 1 mL of this was loaded onto the top of the gel. Electrophoresis was performed at a constant cuπent of 16mA per gel until the dye front passed through the stacker and then increased to 24 mA for electrophoresis through the resolving gel. The average running time was between 4 and 5 hours. The separated proteins were then recovered by electroelution using the BIORAD™ flat bed electroeluter for 1 hour at 200V and a maximum of 0.2mA into 30 individual tubes. Protein composition of the recovered fractions was assessed by analytical SDS-PAGE and either Coomassie or Silver staining of proteins. Analytical SDS-PAGE was performed using a Mini- protean II ™ cell (Bio-Rad) at a constant 200V for about 45 minutes. Protein concentrations were determined using the Pierce Micro BCA™ protein assay and comparison with albumin standards. SDS Removal from Purified Proteins
Samples containing SDS were treated with a 200μL volume of lOOmM potassium phosphate per lmL of sample and left on ice for 60 minutes. The sample was centrifuged at 10000 x g for 20 minutes at 4°C in a microcentrifuge. The supernatant was recovered and desalted by overnight dialysis against nanopure water.
Liquid Chromatography Separation Anion Exchange Liquid Chromatography The extracted proteins were additionally purified by anion exchange chromatography and separated according to their molecular charge interactions. The column (Q5 column, Bio-Rad) was equilibrated with a low salt buffer (20mM Tris-HCl, pH 8.45) at a flow rate of lmL/min for 10 minutes. Lyophilised cell wall extracts were resuspended in the same buffer to a concentration of 5mg per mL and loaded onto the column. Proteins were eluted using an increasing salt gradient by gradually increasing the proportion of 20mM Tris-HCl, 500mM sodium chloride, pH 8.6 passed through the column. Fractions were recovered, lyophilised and assessed by analytical SDS- PAGE. Fractions from multiple runs were pooled and proteins were further purified by preparative SDS-PAGE and electroelution as previously described.
Results
The methods described above successfully purified ten proteins of different molecular weights which were able to be assessed in animal immunisation studies as described in Example 3 below. The most active proteins purified had molecular masses of 12- 14kDa, 16kDa, 34kDa and 57kDa. In total, 23 different proteins were separated in yields ranging from 20 to 500μg in a 6L culture with a total protein concentration of cell wall extract of from 25-30mg. Figure 2 shows the profile of the cell wall extract and the different proteins separated by electroelution from the crude protein extract. Not all proteins eluted from the gel as a single protein band; some fractions were composed of 2 or 3 different proteins.
Elution profile for Cell Wall Proteins Using Anion Exchange Chromatography
The elution profile from anion exchange chromatography is shown in Figure 3. The first peak represents elution of unbound proteins. The subsequent two major peaks contained most of the proteins that were eluted with increasing salt concentration. The proteins in these peaks were further purified by SDS-PAGE.
Example 2 - N-terminal Sequence Analysis
The N-terminal sequence of the proteins was determined from an excised band from an analytical SDS-PAGE. Analyses were performed by the Biomolecular Resource Unit, The John Curtin School of Medical Science (Australian Capital Teπitory, Australia).
Table 1 - Amino Acid Sequence Analysis Results of the Purified Proteins
Figure imgf000015_0001
To assist in the characterisation of the proteins, the information obtained from the partial amino acid sequence was searched through the GenBank databases to determine homology to known protein sequences. It was found that the 12-14 kDa protein has a 100% sequence homology match with that of a 12 kDa protein from S. pneumoniae. The 16kDa protein has been found to be an isomer of the 12-14 kDa protein.
According to a study by Koberg et al, (Microbiology, 143(1), 55-61 (Jan 1997)), two monoclonal antibodies against Streptococcus pneumoniae reacted with a highly conserved epitope on eubacterial L7/L12 ribosomal proteins. A high degree of amino acid sequence homology was found across 66 eubacteria, representing 27 different species. The approximate 12-14kDa protein had a 100% sequence match with the 12 kDa protein from this study (Kolberg et al).
The 34kDa protein appears to be a novel protein as a BLAST search of publicly available databases revealed no significant similarities.
Example 3 - Mouse Lung Clearance Model Animals
Balb/c mice, 6-10 weeks old were housed and maintained in a pathogen free environment with free access to sterilised food and water.
Preparation of Live Bacteria Bacteria were grown overnight on blood agar plates at 37°C and 5% CO . The bacteria were harvested and washed twice in sterile PBS by centrifugation at 10000 x g at room temperature. The bacterial concentration was determined by optical density at 405 nm and calculated from a regression curve, the accuracy of the concentration for viable bacterial count was confirmed by titration and overnight culture.
Immunisation Regime
Mice were initially immunised on day 0 by Peyer' s patches inoculation and boosted by intratracheal administration 14 days later. On day 21, these mice were challenged with live S. pneumoniae. Peyer's Patch Immunisation
The mice were sedated by a subcutaneous injection of 0.25 L ketamine/xylazine at a dosage of 5 mg/ml ketamine hydrochloride; 2mg/ml xylazine hydrochloride. The small intestine was exposed through a mid-line abdominal incision and the protein injected subserosal into each Peyer's patch. The immunisation protein was prepared by emulsifying 2.5μg/μL protein in a 1 :1 ratio with incomplete Freund's adjuvant (Sigma Immunochemicals, St Louis, MI, USA) and a total concentration of lOμg protein administered to each animal.
Intratracheal Inoculation of Mice
On day 14, mice received an intratracheal boost. The mice were sedated by intravenous injection with 20 mg saffan per kg of body weight. lOμg protein in PbS in a total volume of 20μL was delivered via the trachea into the lungs with a 22.1/2G catheter.
Pulmonary Challenge
On day 21, the mice received a live bacterial challenge. The mice were sedated with saffan as described above, and an inoculum of 1 x 107 CFU in 20μL of live S. pneumoniae was introduced into the lungs via the trachea as for the intratracheal boost. Five hours following the challenge, the mice were euthanased by an intraperitoneal injection of 0.2mL of sodium pentobarbital.
Blood was collected by heart puncture and the separated serum stored below-20°C prior to analysis. The trachea was exposed and the lungs were lavaged by insertion and removal of 0.5mL sterile PBS. The recovered fluid (BAL) was assessed for bacterial recovery by plating 10 fold serial dilutions onto blood agar for CFU determination. An aliquot was removed for cytospin slide preparation, staining and differential cell counts. The BAL was then centrifuged for 10 min at 1000 rpm at 4°C and the supernatant stored below -20°C until required. The pellet was resuspended in PBS and methylene blue and the total number of white cells in the BAL were counted. The lungs were removed following lavage, placed in 2mL sterile PBS and homogenised. The lung homogenate was assessed by plating 10-fold serial dilutions onto blood agar for CFU determination. Results are presented only for the proteins which showed significant degrees of pulmonary clearance from the lungs.
Results
Three proteins assessed in immunisation and bacterial challenge showed significant degrees of pulmonary clearance from the lungs. These were proteins with molecular masses of 16, 34 and 57 kDa and identified in Table 1 above. The results of the bacterial clearance and comparison with the recovery in non-immune mice challenged at the same time are shown in Table 2 below and graphically represented in Figures 5 to 7. A fourth protein of significance was the 12-14kDa protein. This protein has been previously identified through monoclonal antibody assay (see above) as being present in a large number of bacteria. However, there is no evidence in the literature that it has been tested as a vaccine antigen.
Table 2 - Pulmonary Clearance Following Immunisation With Purified Proteins
Figure imgf000018_0001
Example 4 - Serum Antibody
Serum from mice immunised with either the 34 kDa protein or the 12-14 kDa protein was assessed for the presence of antibody to the immunising protein by ELIS A.
Method
Polysorb microtitre wells were coated with 10 μg per ml of purified protein, either 34 kDa protein or 12-14 kDa protein, in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) by incubation with 50 μl per well overnight at 4 °C. The plates were washed 5 times in washing buffer (PBS containing 0.05% Tween-20). The wells were blocked with 100 μl of 5% Skim Milk in PBS/Tween-20 buffer for 60 min at room temperature. The plates were washed 5 times as above and 50 μl serum dilutions commencing at 1/5 were added to the wells and incubated at room temperature for 90 minutes. Following washing, the wells were incubated with anti-mouse IgG- horseradish peroxidase conjugated antisera for 90 minutes. The wells were washed and developed with TMB (tetra methylbenzidine in dimethyl sulphoxide) in phospate citrate buffer, pH 5 containing hydrogen peroxide. The reaction was stopped with 0.5 M H SO4 and the optical density of the wells read at 450 nm. The concentration of immunoglobulin in the test wells was determined by calculation from known concentrations of IgG assayed as standards on the same microtitre plate.
Results
Group Serum IgG (ng/ml)
Nonlmmune 0
34 kDa protein (10 μg) 2474
Group Serum IgG (ng/ml)
Nonlmmune 0
12-14 kDa protein (10 μg) 39
34 kDa protein (10 μg) 130 Example 5 - Preparation of 34 kDa protein for sequencing
The protean IEF Cell was used for the first dimension isoelectric focusing and for the second dimensional electrophoretic protein analysis.
Method
The first dimension electrophoresis was performed using 7cm IPG strip which was actively rehydrated overnight for 17 hours at 50V. 60uL (lug/uL) of cell wall extract and a total volume of 140uL of rehydration solution ( 8.3M urea, 2% CHAPS, 50mM Dithiothreilol, 0.1% Bio-Lytes, and 0.001% Bromocresol blue), was applied to the sample well. In the second dimension, the gel strips were overlayed onto 12% SDS- PAGE gels and a 4% stacker (as described earlier) and ran at 5mA for 1 hour and 15mA for 1.5 hours per gel strip. One of the gels was coomassie stained and is shown in Figure 8, and the other was transfeπed to a membrane for a western blot analysis as described in the methods. The _esults of the western blot are shown in Figure 9.
Results
Using the 2 Dimensional electrophoresis, the molecular weight of the 34 kDa protein was calculated to be 34.1 kDa.
Figure 8: An identified band similar to the one in figure 2 of a 34.1 kDa protein was excised from the gel. Figure 9: 34 kDa protein using specific antisera from the mice (immunisations shown previously) was detected in the homologous cell wall extract (lane 1), and the same band was also seen in the 2 Dimensional strip slot in lane 2, compared to the molecular weight standards in Lane 3.

Claims

1. A protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
V X X V G I N T X S X X Q S
(Val X X Val Gly He Asn Thr X Ser X X Gin Ser) (SEQ ID NO: 1);
wherein X represents an unknown amino acid residue.
A process for the preparation of an isolated and purified protein the process comprising the following steps:
(a) preparing cultures of S. pneumoniae, growing the cultures under appropriate conditions and harvesting them, followed by washing with centrifugation to yield a washed cell pellet;
(b) resuspending the washed cells in an appropriate buffer followed by disruption of the cells;
(c) centrifuging to remove cell debris and obtaining the supernatant containing soluble cell proteins;
(d) subjecting the solution obtained to anion exchange chromatography with a sodium chloride gradient elution, and pooling the fractions coπesponding to each separate peak; (e) suspending the protein fractions in a buffer comprising 0.5M Tris HC1 pH 6.8; 10%(v/v) glycerol; 10% (w/\) SDS; 0.05% (w/v) bromophenol blue; and 0.05% (v/v) β-mercaptoethanol; boiling the mixture and then purifying by SDS-PAGE using a 12% (w/v) acrylamide/BIS separating gel with a 4% (w/v) acrylamide/BIS stacking gel, run at 16 mA in the stacking gel and 24 mA in the resolving gel;
(f) selecting a fraction containing a protein having a molecular weight of 34kDa and isolating the protein from the selected fraction.
3. Nucleic acid encoding a protein or antigenic fragment thereof as defined in claim 1 or nucleic acid complementary thereto.
4. A vector containing nucleic acid as claimed in claim 3.
5. An organism transformed with nucleic acid as claimed in claim 3.
6. A process for the preparation of a protein as claimed in claim 1, the process comprising expressing nucleic acid as claimed in claim 3.
7. A protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, or nucleic acid coding therefor, for use in medicine, particularly in the prophylaxis or diagnosis of infection by S. pneumoniae, wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
V X X V G I N T X S X X Q S
(Val X X Val Gly He Asn Thr X Ser X X Gin Ser) (SEQ ID NO: 1) wherein X represents an unknown amino acid residue.
8. A method of detecting and/or diagnosing S. pneumoniae which comprises:
(a) bringing into contact with a sample to be tested a protein or antigenic fragment as defined in claim 1 ; and
(b) detecting the presence of antibodies to S. pneumoniae.
9. The use of a protein derived from Streptococcus pneumoniae or a homologue thereof or an antigenic fragment of the protein or homologue, or nucleic acid coding therefor, in the preparation of an agent for the prophylaxis or diagnosis of infection by S. pneumoniae, wherein the protein has a molecular weight of about 34 kDa as determined by SDS-PAGE under reducing conditions and has the following amino terminal sequence:
V X X V G I N T X S X X Q S
(Val X X Val Gly He Asn Thr X Ser X X Gin Ser) (SEQ ID NO: 1)
wherein X represents an unknown amino acid residue.
10. A pharmaceutical or vaccine composition comprising a protein or antigenic fragment as defined in claim 1 together with a pharmaceutically acceptable excipient.
11. A pharmaceutical or vaccine composition as claimed in claim 10 which further comprises an adjuvant.
12. An antibody which binds specifically to a protein or antigenic fragment as defined in claim 1.
13. A method of vaccinating a subject against S. pneumoniae which comprises the step of administering to the subject an effective amount of protein or antigenic fragment as defined in claim 1.
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GB9928678D0 (en) 2000-02-02
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CA2392895A1 (en) 2001-06-07
EP1240329A2 (en) 2002-09-18
JP2003515341A (en) 2003-05-07

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