EP1017828A1 - Proteinase aus streptococcus pneumoniae die den menschlichen komplementbestandteil c3 abbaut - Google Patents

Proteinase aus streptococcus pneumoniae die den menschlichen komplementbestandteil c3 abbaut

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Publication number
EP1017828A1
EP1017828A1 EP98948557A EP98948557A EP1017828A1 EP 1017828 A1 EP1017828 A1 EP 1017828A1 EP 98948557 A EP98948557 A EP 98948557A EP 98948557 A EP98948557 A EP 98948557A EP 1017828 A1 EP1017828 A1 EP 1017828A1
Authority
EP
European Patent Office
Prior art keywords
protein
seq
nucleic acid
isolated
pneumoniae
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98948557A
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English (en)
French (fr)
Inventor
Margaret K. Hostetter
David J. Finkel
Qi Cheng
Bruce A. Green
Amy W. Masi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wyeth Holdings LLC
University of Minnesota
Original Assignee
University of Minnesota
American Cyanamid Co
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Filing date
Publication date
Application filed by University of Minnesota, American Cyanamid Co filed Critical University of Minnesota
Publication of EP1017828A1 publication Critical patent/EP1017828A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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

Definitions

  • This invention relates to Streptococcus pneumoniae and in particular this invention relates to the identification of an S. pneumoniae protein that is capable of degrading human complement protein, C3.
  • S. pneumoniae Respiratory infection with the bacterium Streptococcus pneumoniae leads to an estimated 500,000 cases of pneumonia and 47,000 deaths annually.
  • Those persons at highest risk of bacteremic pneumococcal infection are infants under two years of age, individuals with a compromised immune system and the elderly.
  • S. pneumoniae is the leading cause of bacterial pneumonia and meningitis.
  • S. pneumoniae is the major bacterial cause of ear infections in children of all ages. Both children and the elderly share defects in the synthesis of protective antibodies to pneumococcal capsular polysaccharide after either bacterial colonization, local or systemic infection, or vaccination with purified polysaccharides.
  • Conjugate vaccines consist of pneumococcal capsular polysaccharides coupled to protein carriers or adjuvants in an attempt to boost the antibody response.
  • conjugate vaccines there are other potential problems with conjugate vaccines currently in clinical trials.
  • pneumococcal serotypes that are most prevalent in the United States are different from the serotypes that are most common in places such as Israel, Western Europe, South Africa, or Scandinavia. Therefore, vaccines that may be useful in one geographic locale may not be useful in another.
  • the potential need to modify currently available capsular polysaccharide vaccines or to develop protein conjugates for capsular vaccines to suit geographic serotype variability entails prohibitive financial and technical complications.
  • the search for immunogenic, surface-exposed proteins that are conserved worldwide among a variety of virulent serotypes is of prime importance to the prevention of pneumococcal infection and to the formulation of broadly protective pneumococcal vaccines.
  • pneumococcal proteins have been proposed for conjugation to pneumococcal capsular polysaccharide or as single immunogens to stimulate immunity against S. pneumoniae.
  • Surface proteins that are reported to be involved in adhesion of S. pneumoniae to epithelial cells of the respiratory tract include PsaA, PspC/CBPl 12, and IgAl proteinase (Sampson et al. Infect. Immun. 1994;62:319-324, Sheffield et al. Microb. Pathogen. 1992; 13: 261-9, and Wani, et al. Infect. Immun. 1996; 64:3967-3974).
  • cytosolic pneumococcal proteins such as pneumolysin, autolysin, neuraminidase, or hyaluronidase are proposed as vaccine antigens because antibodies could potentially block the toxic effects of these proteins in patients infected with S. pneumoniae.
  • these proteins are typically not located on the surface of S. pneumoniae, rather they are secreted or released from the bacterium as the cells lyse and die (Lee et al. Vaccine 1994; 12:875-8 and Berry et al. Infect. Immun. 1994; 62:1101-1108). While use of these cytosolic proteins as immunogens might ameliorate late consequences of S. pneumoniae infection, antibodies to these proteins would neither promote pneumococcal death nor prevent initial or subsequent pneumococcal colonization.
  • PspA pneumococcal surface protein A
  • PspA is a heterogeneous protein of about 70-140 kDa.
  • the PspA structure includes an alpha helix at the amino terminus, followed by a proline-rich sequence, and terminates in a series of 11 choline-binding repeats at the carboxy-terminus.
  • PspA is not structurally conserved among a variety of pneumococcal serotypes, and its function is entirely unknown (Yother et al. J. Bacteriol. 1992;174:601-9 and Yother J. Bacteriol.
  • the third component of complement, C3, and the associated proteins of the alternative complement pathway constitute the first line of host defense against S. pneumoniae infection.
  • complement proteins cannot penetrate the rigid cell wall of S. pneumoniae
  • deposition of opsonic C3b on the pneumococcal surface is the principal mediator of pneumococcal clearance.
  • Interactions of pneumococci with plasma C3 are known to occur during pneumococcal bacteremia, when the covalent binding of C3b, the opsonically active fragment of C3, initiates phagocytic recognition and ingestion (Johnston et al. J. Exp. Med 1969;129:1275-1290, Hasin HE, J Immunol.
  • This invention relates to the identification and use of a family of human complement C3-degrading proteins (proteinases) expressed by S. pneumoniae.
  • the proteins preferably have a molecular weight of about 15 kD to about 25 kD, as determined, for example, on a 10% SDS polyacrylamide gel.
  • the invention includes a number of proteins isolatable from different C3 -degrading strains of S. pneumoniae.
  • the invention relates to an isolated protein having at least
  • the protein is isolated from S. pneumoniae or alternatively the protein is a recombinant protein.
  • the isolated protein degrades human complement protein C3.
  • a preferred protein of this invention is an isolated protein having an amino acid sequence that includes SEQ ID NO:2, and more preferably, is SEQ ID NO:2.
  • isolated refers to a naturally occurring species that has been removed from its natural environment, as well as to synthetic species.
  • protein as used herein includes one or more functional units, which encompasses one or more peptides or polypeptides.
  • the invention also relates to isolated peptides or polypeptides from the
  • the invention provides peptides or polypeptides of at least 15 sequential amino acids from an isolated protein that has at least 80% sequence identity with SEQ ID NO:2, and more preferably, peptides or polypeptides of at least 15 sequential amino acids of SEQ ID NO:2.
  • the peptides or polypeptides are capable of degrading human complement protein C3.
  • Preferred embodiments of the invention include an isolated protein comprising amino acids of about 1 to about 58 of SEQ ID NO:2 and an isolated nucleic acid fragment comprising nucleotides of about 1 to about 174 of SEQ ID NO:l or its complementary strand.
  • the isolated nucleic acid fragment comprises nucleotides of about 150 to about 174 of SEQ ID NO:l or its complementary strand.
  • the invention in another aspect, relates to an isolated protein that degrades human complement protein C3, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions.
  • the invention also relates to an immune system stimulating composition (preferably, a vaccine) comprising an effective amount of an immune system stimulating peptide or polypeptide comprising at least 15 sequential amino acids derived from a protein, wherein the protein has at least 80% sequence identity with SEQ ID NO:2 and is capable of degrading human complement protein C3.
  • the protein is isolated from S. pneumoniae.
  • the immune system stimulating composition or vaccine further comprises at least one other immune system stimulating peptide, polypeptide or protein isolated from S. pneumoniae.
  • the invention further relates to an antibody capable of binding (typically, specifically binding) to a protein comprising at least 80% sequence identity with SEQ ID NO:2 and capable of degrading human complement protein C3.
  • the antibody is a monoclonal antibody and in another embodiment, the antibody is a polyclonal antibody.
  • the antibody is an antibody fragment.
  • the antibody or antibody fragments can be obtained from a mouse, a rat, a goat, a chicken, a human, or a rabbit.
  • the antibody is capable of binding to at least a portion of a protein, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:l or its complementary strand under highly stringent hybridization conditions.
  • the invention also relates to an isolated nucleic acid fragment (polynucleotide) capable of hybridizing to SEQ ID NO:l or its complimentary strand under highly stringent hybridization conditions.
  • highly stringent hybridization conditions include, for example, 6XSSC, 5X Denhardt, 0.5% SDS, and 100 ⁇ g/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65°C and washed in 2X SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at 65°C for about 15 minutes followed by at least one wash in 0.2XSSC, 0.1% SDS at room temperature for at least 3-5 minutes.
  • the nucleic acid fragment is isolated from S.
  • the nucleic acid fragment encodes at least a portion of a protein.
  • the protein degrades human complement protein C3.
  • the nucleic acid fragment encodes a peptide or polypeptide that does not degrade human complement C3.
  • the nucleic acid fragment is in a nucleic acid vector and the vector can be an expression vector capable of producing at least a portion of a protein.
  • Cells containing the nucleic acid fragment are also contemplated in this invention.
  • the cell is a bacterium or a eukaryotic cell.
  • the invention further relates to an isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO:l, or its complementary strand.
  • the invention further relates to an RNA fragment transcribed by a double-stranded DNA sequence comprising SEQ ID NO:l.
  • the invention in another aspect of this invention, relates to a method for producing an immune response to S. pneumoniae in a mammal (particularly a human) including the steps of: administering a composition comprising a therapeutically effective amount of at least a portion of a protein to an animal, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:l, or its complementary strand, under highly stringent hybridization conditions to produce an immune response to the protein.
  • the immune response can be a B cell response, a T cell response, an epithelial response or an endothelial response.
  • the composition is a vaccine composition.
  • the protein is at least 15 amino acids in length and also preferably the composition further comprises at least one other immune system stimulating peptide, polypeptide or protein from S. pneumoniae.
  • the protein comprises at least 15 amino acids of SEQ ID NO:2.
  • the invention further relates to an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae that is capable of degrading human complement C3 and to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation comprising the step of: contacting a Streptococcus pneumoniae bacterium with antibody capable of binding to a protein with at least 80% amino acid sequence identity to SEQ ID NO:2.
  • the invention also relates to a method for inhibiting C3 -mediated inflammation and rejection in xenotransplantation comprising the step of expressing on the surface of an organ of an animal used in xenotransplantation a protein with the amino acid sequence of SEQ ID NO:2.
  • This method is particularly advantageous for causing, for example, the kidneys of pigs to express the protein described herein and thereby to inhibit C3 mediated inflammation after xenotransplantation.
  • the invention also relates to an isolated nucleic acid molecule that contains a region of at least 15 nucleotides which hybridize under highly stringent hybridization conditions to at least a portion of a nucleic acid sequence of SEQ ID NO.l or its complementary strand.
  • an isolated nucleic acid molecule is capable of hybridizing under highly stringent hybridization conditions to at least one region of SEQ ID NO.1 or its complementary strand.
  • the at least one region includes nucleotides 1 - 174 or 320-492 of SEQ ID NO: 1.
  • the invention relates to an isolated nucleic acid molecule that contains a region of at least 15 nucleotides which hybridize under highly stringent hybridization conditions to at least a portion of a nucleic acid sequence of SEQ ID NO.4 or its complementary strand.
  • an isolated nucleic acid molecule is capable of hybridizing under highly stringent conditions to at least one region of SEQ ID NO.4 or its complementary strand.
  • the at least one region includes nucleotides 507-681 or 827-999 of SEQ ID NO:4.
  • At least a portion of the nucleic acid molecule of SEQ ID NO:4 encodes at least a portion of a protein.
  • the protein has a predicted amino acid sequence as shown in SEQ ID NO:5, and has a molecular weight of about 75 kDa to about 85 kDa as determined, for example, by SDS- PAGE.
  • the invention also relates to isolated DNA fragments or primers having the nucleic acid sequences as shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 AND SEQ ID NO:9.
  • the invention relates to an immune system-stimulating composition or vaccine containing a therapeutically effective amount of at least a portion of a protein, wherein nucleic acid encoding the protein is capable of hybridizing to the sequence of SEQ ID NO:4, or its complementary strand, under highly stringent hybridization conditions.
  • pneumoniae in a mammal includes the steps of: administering a composition comprising a therapeutically effective amount of at least a portion of a protein to a mammal, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:4, or its complementary strand, under stringent hybridization conditions to obtain an immune response to the protein.
  • the invention relates to a vaccine or immune system stimulating composition containing an amount of at least a portion of a protein that is effective to immunize or treat a mammalian subject against S. pneumoniae infection or colonization and a pharmaceutically acceptable carrier.
  • the protein is derived from a nucleic acid molecule that hybridizes under highly stringent conditions to a nucleic acid sequence as shown in SEQ ID NO:4, or its complementary strand, coding for the protein.
  • the protein is provided in an amount effective to provide a therapeutic effect to the mammalian subject, especially a human subject.
  • Figure 1 provides the nucleic acid sequence of the translated portion of a
  • Figure 2 provides the amino acid sequence of a C3-degrading proteinase of this invention (SEQ ID NO:2).
  • Figure 3 diagrams the amino acid sequence of a C3-degrading proteinase positioned with the nucleic acid sequence encoding a C3-degrading proteinase according to this invention (SEQ ID NOS:2-3).
  • Figure 4 provides the nucleic acid sequence for a predicted 79 kDa amino acid sequence (SEQ ID NO:4).
  • Figure 5 provides the predicted 79 kDa amino acid sequence (SEQ ID NO:5).
  • Figure 6 shows sequence alignments of SEQ ID NO:l and SEQ ID NO:4.
  • Figure 7 shows sequence alignment of SEQ ID NO:2 with corresponding amino acids 169-33 lof SEQ ID NO:5.
  • the present invention relates to the identification and isolation of a human complement C3 degrading proteinase with a molecular weight of about 20 kDa ( ⁇ 5 kDa) on a 10% SDS-PAGE gel and nucleic acid encoding a C3 degrading proteinase. It has been observed that exponentially growing cultures of pneumococci from several serotypes were able to first degrade the ⁇ -chain then degrade the ⁇ chain of C3 without producing defined C3 cleavage fragments (Angel, et al. J Infect. Dis. 170:600-608, 1994).
  • This pattern of degradation without cleavage differs substantially from that of other microbial products such as the elastase enzyme of Pseudomonas aeruginosa and the cysteine proteinase of Entamoeba histolytica.
  • the term "degrade” is used herein to refer to the removal of amino acids from proteinaceous molecules, generating peptides or polypeptides.
  • the proteins of this invention degrade C3 without producing specific cleavage fragments as observed on a polyacrylamide gel.
  • a C3 -degrading proteinase of about 20 kDa was isolated from a library of insertionally interrupted pneumococcal genes by identifying those clones that had decreased C3 degrading activity as compared to wild type S. pneumoniae.
  • An exemplary assay for assessing C3 -degrading activity of clones is provided in Example 1.
  • Clones with decreased C3-degrading activity were identified and a 546 bp Smal insert was selected, based on the sequence of the clones that had demonstrated decreased C3 -degrading activity. This Smal fragment was used to probe a ⁇ .pneumoniae library made from strain CP1200. Positive clones from the S.
  • SEQ ID NO: 10 which has sequence identify with a portion of PspA, was used to confirm, by differential hybridization, that the gene encoding the C3 -degrading protinease was distinct from the gene encoding PspA.
  • a complete open reading frame of a 20 kDa protein spans an area of 492 base pairs (SEQ ID NO:l) predicting a protein of molecular weight of about 20 kDa (+/- 5 kDa) or about 163 amino acids (SEQ ID NO:2).
  • An exemplary gene sequence encoding a C3 -degrading protein is provided in Figure 1 as SEQ ID NO:l and an amino acid sequence of the protein is provided in Figure 2 as SEQ ID NO:2.
  • Figure 3 combines a preferred gene sequence with a corresponding preferred translated protein as SEQ ID NO:3.
  • the amino acid sequence of the protein was determined to be unrelated to other proteins in the GenBank or Swiss Prot databases.
  • the predicted protein encompasses a proline-rich sequence characteristic of membrane domains in prokaryotes, particularly between amino acids 80-108 suggesting that the protein is expressed at the surface.
  • the amino acid sequence exhibits no apparent choline-binding repeats.
  • Electrophoresis of pneumococcal lysates and supernatants from cultures of CP 1200 on SDS-PAGE gels impregnated with C3 identified a lytic band at about 20 kDa ( ⁇ 5 kDa) in both supernatants and lysates, confirming that a protein of a size predicted by SEQ ID NO:2 had C3-degrading activity (see Example 2).
  • the gene encoding the 20 kDa C3-degrading proteinase is conserved in at least two dozen pneumococcal isolates representing five serotypes (serotypes 1, 3, 4, 14, and 19F).
  • the gene encoding the C3 degrading protein of this invention was identified using a plasmid library made with pneumococcal genomic DNA fragments from strain CP1200.
  • a plasmid library was constructed with Sau 3A digested pneumococcal genomic DNA fragments (0.5 -4.0 kb) from pneumococcal strain CP 1200 (obtained from D.A. Morrison, University of Illinois, Champagne-Urbana, Illinois and described in Havarstein LF, et al. Proc. Natl. Acad. Sci.
  • E. coli transformants revealed that all of them contained recombinant plasmids. Plasmid library DNA can be extracted from the E. coli transformants and used to transform the CP 1200 parent pneumococcal strain using insertional mutagenesis by homologous recombination.
  • the pneumococcal strain CP 1200 cells can be made competent using a pH shift with HC1 procedure in CTM medium.
  • the competent cells are frozen at
  • the isolated protein of this invention can be incubated with human complement C3 for 4 hours at 37°C in the presence of PBS to detect C3 degradation. Control samples without the isolated pneumococcal protein are used as controls for comparative purposes.
  • the protein of this invention has an apparent molecular weight on a 10% SDS-polyacrylamide gel of about 20 kDa ( ⁇ 5 kDa) and preferably has a molecular weight of about 15 kDa to about 25 kDa.
  • An exemplary protein sequence is provided by SEQ ID NO: 2.
  • conserved mutations do not detract from this invention nor do variations in amino acid sequence identity of less than about 80 % amino acid sequence identity and preferably less than about 90% amino acid sequence identity where the protein is capable of degrading human complement protein C3, and particularly where the protein is isolated or originally obtained from an S. pneumoniae bacterium. Fragments of the protein are also within the scope of the present invention, particularly if they are capable of degrading human complement protein C3.
  • nucleic acid sequence variability is expected among pneumococcal strains and serotypes as is some amino acid variability.
  • conserved amino acid substitutions are known in the art and include, for example, amino acid substitutions using other members from the same class to which the amino acid belongs.
  • the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, and tryptophan.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Such alterations are not expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point.
  • Particularly preferred conservative substitutions include, but are not limited to, Lys for Arg and vice verse to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free - OH is maintained; and Gin for Asn to maintain a free NH 2 .
  • a preferred protein of this invention includes a protein with the amino acid sequence of SEQ ID NO:2.
  • Other proteins include those degrading human complement protein C3 and having nucleic acid encoding the protein that hybridizes to SEQ ID NO:l under highly stringent hybridization conditions such as 6XSSC, 5X Denhardt, 0.5% SDS (sodium dodecyl sulfate), and 100 ⁇ g/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65 °C and washed in 2X SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at, 65 °C for about 15 minutes followed by at least one wash in 0.2XSSC, 0.1% SDS at room temperature for at least 3-5 minutes are also contemplated in this invention.
  • an SSC solution contains sodium chloride, sodium citrate, and water to prepare a stock solution.
  • Peptides or polypeptides of the protein can also be used.
  • a preferred protein of this invention comprises amino acids of about 1 to
  • the proteins of this invention can be isolated or prepared as recombinant proteins. That is, nucleic acid encoding the protein, or a portion of the protein, can be incorporated into an expression vector or incorporated into a chromosome of a cell to express the protein in the cell.
  • the protein can be purified from a bacterium or another cell, preferably a eukaryotic cell and more preferably an animal cell. Alternatively, the protein can be isolated from a cell expressing the protein, such as a S. pneumoniae cell.
  • Peptides or polypeptides are also considered in this invention.
  • the peptides or polypeptides are preferably at least 15 amino acids in length and preferred peptides or polypeptides have at least 15 sequential amino acids from SEQ ID NO:2.
  • Nucleic acid encoding the 20 kDa protein is also part of this invention.
  • SEQ ID NO:l is a preferred nucleic acid fragment encoding a C3-degrading proteinase. Those of ordinary skill in the art will recognize that some substitution will not alter the C3-degrading proteinase sequence to an extent that the character or nature of the C3 -degrading proteinase is substantially altered. For example, nucleic acid with an identity of at least 80% to SEQ ID NO:l is contemplated in this invention.
  • a method for determining whether a particular nucleic acid sequence falls within the scope of this invention is to consider whether or not a particular nucleic acid sequence encodes a C3-degrading proteinase and has a nucleic acid identity of at least 80% as compared with SEQ ID NO:l.
  • Other nucleic acid sequences encoding the C3 proteinase include nucleic acid encoding the C3 proteinase where the C3 proteinase has the same sequence or at least a 90% sequence identity with SEQ ID NO:2 but which includes degeneracy with respect to the nucleic acid sequence.
  • a degenerate codon means that a different three letter codon is used to specify the same amino acid. For example, it is well known in the art that the following RNA codons (and therefore, the corresponding DNA codons, with a T substituted for a U) can be used interchangeably to code for each specific amino acid:
  • nucleic acid sequences include nucleic acid fragments of at least 15, and preferably, at least 30 nucleic acids in length from SEQ ID NO:l or other nucleic acid fragments of at least 15, and preferably at least 30 nucleic acids in length where these fragments hybridize to SEQ ID NO:l under highly stringent hybridization conditions such as 6XSSC, 5X Denhardt, 0.5% SDS, and 100 ⁇ g/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65°C and washed in 2X SSC, 0.1 % SDS one time at room temperature for about 10 minutes followed by one time at, 65 °C for about 15 minutes followed by at least one wash in 0.2XSSC, 0.1 % SDS at room temperature for at least 3-5 minutes.
  • nucleic acid fragments of this invention can encode all, none (i.e., fragments that cannot be transcribed, fragments that include regulatory portions of the gene, or the like) or a portion of SEQ ID NO:2 or SEQ ID NO:5 and preferably containing a contiguous nucleic acid fragment that encodes at least nine amino acids from SEQ ID NO:2 or SEQ ID NO:5. Because nucleic acid fragments encoding a portion of the C3 proteinase are contemplated in this invention, it will be understood that not all of the nucleic acid fragments will encode a protein or peptide or polypeptide with C3 degrading activity.
  • nucleic acid of this invention can be mutated to remove or otherwise inactivate the C3 degrading activity of this protein. Therefore, fragments without C3 degrading activity that meet the hybridization requirements described above are also contemplated. Methods for mutating or otherwise altering nucleic acid sequences are well described in the art and the production of an immunogenic, but enzymatically inactive protein can be tested for therapeutic utility.
  • the nucleic acid fragments of this invention can be incorporated into nucleic acid vectors or stably incorporated into host genomes to produce recombinant protein including recombinant chimeric protein.
  • the C3-degrading protein is encoded by a gene in a vector and the vector is in a cell.
  • the cell is a prokaryotic cell such as a bacterium.
  • the genes and gene fragments can exist as the fusion of all or a portion of the gene with another gene and the C3-degrading protein can exist as a fusion protein of one or more proteins where the fusion protein is expressed as a single protein.
  • nucleic acid vectors of this invention include a number of commercially available expression plasmids or viral vectors. The use of these vectors is well within the scope of what is ordinary skill in the art. Exemplary vectors are employed in the examples, but should not be construed as limiting on the scope of this invention.
  • This invention also relates to antibodies capable of binding (typically specifically binding) to a protein of about 20 kDa, and preferably a protein of about 15 kDa to about 25 kDa, from S. pneumoniae and preferably where the protein is capable of degrading human complement C3.
  • Polyclonal antibody can be prepared to a portion of the protein or to all of the protein.
  • monoclonal antibodies can be prepared to all or to a peptide or polypeptide (fragment) of the about 20 kDa C3 degrading protein of this invention.
  • the antibodies can be human derived, rat derived, mouse derived, goat derived, chicken derived, or rabbit derived. Protein-binding antibody fragments and chimeric fragments are also known and are within the scope of this invention.
  • the invention also relates to the use of immune stimulating compositions.
  • immune stimulating or “immune system stimulating” composition refers to protein, peptide or polypeptide compositions according to the invention that activate at least one cell type of the immune system in a subject, such as a mammal.
  • the immune stimulating composition provides an immunizing response or prophylactic benefit in a normal, i.e., uninfected subject, typically a vaccine.
  • any measurable immune response is beneficial to the subject in a therapy application or protocol.
  • Preferred activated cells of the immune system include phagocytic cells such as neutrophils or macrophages, T cells, B cells, epithelial cells and endothelial cells.
  • Immune stimulating compositions comprising the peptides, polypeptides or proteins of the invention can be used to produce antibody in an animal such as a rat, mouse, goat, chicken, rabbit, or a human or an animal model for studying S. pneumoniae infection.
  • Preferred immune stimulating compositions include an immune stimulating amount, e.g, a therapeutically effective amount, of at least one peptide or polypeptide including at least 15 amino acids from the C3 degrading proteinase.
  • the term "vaccine” refers to a composition for immunization. This process can include the administration of a protein, peptide, polypeptide, antigen, nucleic acid sequence or complementary sequence, e.g., anti-sense, or antibody, or suspensions thereof, wherein upon administration, the molecule will produce active immunity and provide protection against an S. pneumoniae infection or colonization.
  • vaccines are prepared as i ⁇ jectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The vaccine preparation may optionally be emulsified, or encapsulated in liposomes.
  • the immune stimulating composition (such as a vaccine) can further include other proteins in a pharmaceutically acceptable buffer or carrier, such as PBS (phosphate buffer saline) or another buffer recognized in the art as suitable and safe for introduction of proteins into a host to stimulate the immune system.
  • the immune stimulating compositions can also include other immune system stimulating proteins such as adjuvants or immune stimulating proteins, peptides or polypeptides from S. pneumoniae or other organisms.
  • a cocktail of peptides or polypeptides may be most useful for controlling S. pneumoniae infection.
  • one or more fragments of the proteins of this invention are used in a vaccine preparation to protect against or limit S. pneumoniae colonization or the pathogenic consequences of S. pneumoniae colonization.
  • a “therapeutically effective amount,” as used herein, refers to that amount that is effective for production of a desired result. This amount varies depending upon the health and physical condition of a subject's immune system, i.e., to synthesize antibodies, the degree of protection desired, the formulation prepared and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the active immune stimulating ingredients are often mixed with excipients or diluents that are pharmaceutically acceptable as carriers and compatible with the active ingredient.
  • pharmaceutically acceptable carrier refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the immune stimulating composition 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 immune stimulating compostion.
  • adjuvants or carriers examples include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-( 1 ' -2 ' -dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion.
  • This invention also relates to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation comprising contacting a Streptococcus pneumonie bacterium with a protein, such as an antibody or another protein that is capable of binding to an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae.
  • a protein such as an antibody or another protein that is capable of binding to an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae.
  • the protein capable of binding to an isolated protein of about 15 kDa to about 25 kDa can be an antibody or a fragment thereof or the protein can be a chimeric protein that includes the antibody binding domain, such as a variable domain, from antibody that is capable of specifically recognizing an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae having C3 degrading activity.
  • the isolated S. pneumoniae protein of this invention can be isolated, and optionally purified, and the isolated protein or immunogenic fragments thereof can be used to produce an immunologic response, including, in one example, an antibody response in a human or an experimental animal.
  • Peptide or polypeptide fragments of the protein without C3 degrading ability can be tested for their ability to limit the effects of S. pneumoniae infection.
  • the protein of this invention can be modified, such as through mutation to interrupt or inactivate the C3 degrading activity of the protein.
  • Antibody capable of inhibiting the C3 -degrading activity of the protein of this invention may be used as a strategy for preventing C3 degradation and for promoting clearance of S. pneumoniae through the opsonic pathway.
  • Isolated protein can be used in assays to detect antibody to S. pneumoniae or as part of a vaccine or a multi-valent or multiple protein, peptide or polypeptide-containing vaccine for S. pneumoniae therapy.
  • treatment refers to prophylaxis and/or therapy of either normal mammalian subjects or mammalian subjects colonized with, diagnosed with, or exhibiting characteristics or symptoms of various S. pneumoniae infections.
  • therapy refers to providing a therapeutic effect to a mammalian subject such that the subject exhibits few or no symptoms of a pneumococcal infection or other related disease.
  • Such treatment can be accomplished by administration of nucleic acid molecules (sense or antisense), proteins, peptides or polypeptides or antibodies of the instant invention.
  • the proteins of this invention can be surface expressed on vertebrate cells and used to degrade C3, for example, where complement deposition (or activation) becomes a problem, such as in xenotransplantation or in complement-mediated glomerulonephritis.
  • complement deposition or activation
  • the entire pneumococcal protein, a recombinant protein, or a portion of either can be incorporated into xenotransplant cells and expressed as a surface protein or as a secreted protein to prevent or minimize complement deposition (and/or complement-mediated inflammation).
  • Another specific aspect of the present invention relates to using a vaccine vector expressing an isolated protein and peptides or polypeptides therefrom. Accordingly, in a further aspect this invention provides a method of inducing an immune response in a mammal, which comprises providing to a mammal a vaccine vector expressing at least one, or a mixture of an isolated protein and/or peptide or polypeptide of the invention.
  • the protein and peptides or polypeptides of the present invention can be delivered to the mammal using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the protein and/or peptides or polypeptides as a foreign polypeptide.
  • bacteria that colonizes the gastrointestinal tract such as Salmonella, Shigella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are discuessed by J. Holmgren et al., Immunobiol., 184, 157-179 (1992) and J. McGhee et al., Vaccine, 10, 75-88 (1992).
  • An additional embodiment of the present invention relates to a method of inducing an immune response in a subject, e.g,. mammal, comprising administering to the subject an amount of a DNA molecule encoding an isolated protein and/or peptide or polypeptide therefrom of this invention, optionally with a transfection-facilitating agent, where the protein and/or peptides or polypeptides retain immunogenicity and, when incorporated into an immune stimulating composition, e.g, vaccine, and administered to a human, provides protection without inducing enhanced disease upon subsequent infection of the human with S. pneumoniae pathogen.
  • Transfection-facilitating agents are known in the art.
  • antisense sequence of the gene encoding the 20kDa protein may be used as a vaccine or as a therapeutic treatment for pneumococcal infection.
  • Antisense DNA is defined as a non- coding sequence that is complementary, i.e., a complementary strand, to all or a portion of SEQ ID NO: 1.
  • ATGTCAAGC-3* is 3'-TACAGTTCG-5 ⁇ Delivery of antisense sequence or oligonuclotides into an animal may result in the production of antibody by the animal or in the incorporation of the sequence into living bacteria or other cells whereby transcription and/or translation of all or a portion of the 20 kDa gene product is inhibited.
  • an antisense nucleic acid sequence can be accomplished, for example, by loading the antisense nucleic acid into a suitable carrier, such as a liposome, for introduction into pneumococci or infected cells.
  • a suitable carrier such as a liposome
  • an antisense nucleic acid sequence having eight or more nucleotides is capable of binding to the bacterial nucleic acid or bacterial messenger RNA.
  • the antisense nucleic acid sequence typically contains at least about 15 nucleotides, preferably at least about 30 nucleotides or more nucleotides to provide necessary stability of a hybridization product of bacterial nucleic acid or bacterial messenger RNA.
  • Introduction of the sequences preferably inhibit the transcription or translation of at least one endongenous S. pneumoniae nucleic acid sequence.
  • Methods for loading antisense nucleic acid is known in the art as exemplified by U.S. Patent 4,242,046.
  • the present invention also provides nucleic acid having an open reading frame of 2163 bases (SEQ ID NO:4) that encompasses the open reading frame of a nucleic acid sequence (SEQ ID NO:l) that encodes a protein that has a molecular weight of about 20 kDa (SEQ ID NO:2).
  • the 20 kDa protein, described herein, is further characterized as a C3-degrading protein.
  • the larger open reading frame e.g., 2163 bp (SEQ ID NO:4), encodes for a putative protein of about 79 kDa (SEQ ID NO:5).
  • the cells were incubated at 37°C for 4 hrs.
  • One hundred ⁇ l of the mixture was added to ELISA plates and incubated overnight at 4°C.
  • the plates were washed three times with wash buffer and the wells were filled with 0.05% Tween 20 in PBS with five minute incubations between the washes.
  • One hundred ⁇ l of antibody to C3 polyclonal horse-radish peroxidase-conjugated goat antibody specific to human C3-IgG fraction, ICN Cappel, Costa Mesa, CA
  • the ELISA plate was incubated at 37°C for about 30 minutes to 1 hr in the dark and washed with wash buffer as above.
  • the assay was developed using 12 mg of OPD in 30 ml of 0.1M sodium citrate buffer with 12 ⁇ l of 30% hydrogen peroxide. Assay results were determined by optical density readings at 490 nm on an ELISA plate reader. Each clone was tested four times. Nineteen clones were selected that had less than 40% C3 degradation as compared to nonmutated controls. These 19 clones were screened 6 times by the assay described above and from these results 6 clones were selected with less than 30% C3 -degrading activity as compared to controls. These 6 clones were screened eleven times each and the two clones with the lowest C3-degrading activity were selected for further study.
  • a partial sequence of one of the clones was received and a Smal fragment of 546bp was labeled with P by random primer labeling (kit available from Stratagene, La Jolla, CA).
  • the 546bp Smal fragment from SEQ ID NO:l was hybridized to EcoRl and Kpnl digests of numerous pneumococcal strains on Southern blots. This same fragment was also used to screen a library of Sau3 A fragments of genomic DNA from S. pneumoniae strain CP1200.
  • a 3.5 kb insert was identified from the CP1200 library. The insert was sequenced and an open reading frame of 492 base pairs, including the stop codon, was identified. The open reading frame coded for a protein of 163 amino acids and a predicted molecular weight of about 18,500 daltons.
  • PCR primers were constructed to amplify the open reading frame; the 5' PCR primer incorporated a BamHI site; the 3' primer incorporated a PstI site.
  • the amplified insert was ligated in frame to a His-Tagged E. coli expression vector pQE30 (Qiagen, San Diego, CA).
  • the resulting plasmid was used to transform E. coli strain BL21 (Novagen, Madison, WI) containing the lac repressor plasmid pREP4 (Qiagen).
  • E. coli cultures were induced to express the His-Tagged protein and the protein was column purified with Ni-NTA resin (Qiagen). The purified protein was confirmed by SDS-PAGE gel.
  • Example 2 Identification of a 20kDa C3-Degrading Proteinase
  • C3 prepared according to Tack et al., Meth. Enzymol. 80:64-101, 1984
  • SDS sodium dodecyl sulfate
  • Pneumococcal supernatants were obtained from cultures of S. pneumoniae strain CP1200 grown to exponential phase in Todd Hewitt broth; pneumococcal lysates were obtained by incubating 5 x 10 cells with 5% SDS for 30 minutes at room temperature.
  • the lysate was concentrated 10 fold using a Centricon filtration device with a 10,000 mw cutoff (Amicon, Beverly, MA). The samples were not heated before electrophoresis. Samples of supernatants and lysates were added to the 15% C3-containing SDS- PAGE gels and electrophoresis was carried out at 4°C at 150 V until the dye front ran out. The gel was washed successively with 50 ml of 2.5% Triton X- 100 in water (2 times, 10 minutes), 2.5 % Triton X-100 in 50 mM Tris-HCl, pH 7.4 (2 times, 10 minutes), and 50 mm Tris-HCl, pH 7.4 (2 times, 10 minutes) to remove SDS.
  • C3 proteinase activities in the pneumococcal lysates were observed after a 1.5 hour incubation at 37°C, while C3 proteinase activities in the Pn supernatant were observed after an overnight incubation. Therefore, C3 proteinase activities appeared to be mainly cell associated.
  • Example 3 The gene encoding the 20 kD protein is conserved in a number of S. pneumoniae strains.
  • DNA was obtained from a variety of S. pneumoniae strains (Clinical isolates of Type 1, Type 3, LOO2 and LOO3 (type 3), Type 4, Type 14 and Laboratory isolates CP1200, WU2, R6X, 6303,109,110, JY1119, JY182, and JY53) and SEQ ID NO: 3 was used as a probe to detect the presence of nucleic acid encoding the 20 kD protein in DNA from these strains. Isolated chromosomal DNA was digested with EcoRI and separated by electrophoresis.
  • the DNA was transferred to a solid support and hybridized to end-labeled SEQ ID NO:3 under the hybridization and washing conditions of 6X SSC, 5X Denhart's, 0.5% SDS, 100 ⁇ g/ml denatured fragmented salmon sperm DNA hybridized at 65 °C overnight and washed in 2X SSC, 1 time at room temperature for 10 minutes and in 2X SSC, 0.1% SDS 1 time at 65°C for 15 minutes followed by two washes in 0.2X SSC, 0.1% SDS for 3 minutes each at room temperature.
  • the DNA encoding the 20 kDa C3-degrading protein appears to be part of a larger open reading frame of 2163 bp that putatively encodes a 79 kDa protein.
  • genomic DNA Five ug samples of genomic DNA were obtained from 11 strains ofS. pneumoniae. Each sample was digested with the restriction enzyme Kpnl . The samples were subsequently loaded onto an agarose gel and resolved by electrophoresis. The samples contained in the gel were subsequently transferred to a Hybond-N+ membrane available from Amersham (Upsalla, Sweden) by capillary transfer. A 540bp Smal fragment from an 5F1 isolate was random prime labeled with P 32 using a T7 QuickPrime kit (Pharmacia, Piscathaway, NJ) and purified from non-incorporated nucleotides using NucTrap column (Stragene, La Jolla, CA) and hybridized. The hybridization conditions were 6XSSC, 5X Denhardt, 0.5% SDS, and
  • Two DNA primers were prepared from SEQ ID NO:l and utilized to amplify the 20Kda gene sequence from S. pneumoniae (serotype 3) genomic DNA.
  • the first primer a 5'-primer, SEQ ID NO:6, spans the ATG start codon of the 20Kda gene, inserts a Ncol site, and had an Ala residue inserted after the ATG start codon to maintain a correct reading frame.
  • the second primer, a 3'- primer, SEQ ID NO: 7 spans the termination codon of the 20 Kda gene and inserts a BamHI site.
  • the two primers were synthesized on an Applied Biosystems 380A DNA synthesizer (Foster City, CA) using reagents purchased from Glen Research
  • Thermocycler (ABI) according to the manufacturer's directions.
  • the identified PCR product was ligated into the TA tailed PCR cloning vector PCR2.1 , available from Invitrogen, Carlsbad, CA, and used to transform OneShot Topi OF' competent cells (Invitrogen).
  • Kanamycin resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. An approximately 500bp insert fragment was identified and subsequently excised with restriction enzymes Ncol and BamHI . The 500 bp fragment was purified from a low melting agarose gel, and subsequently ligated into the Ncol- BamHl sites of the T7 promoted expression vector pET 28a, available from Novagen ( Madison, WI).
  • the ligation mixture was subsequently transformed into ToplOF'cells (Invitrogen), and the kanamycin resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis.
  • a recombinant plasmid (pLP505) was subsequently tranformed into BL21 (Novagen) cells and grown in SOB media supplemented with 30ug/ml kanamycin. Cells were grown to an O.D. 600 of 0.6, and were subsequently induced with 0.4mM IPTG (Boehringer Mannheim, Indianapolis, Indiana) for 2- 4 hours.
  • Whole cell lysates were prepared and electrophoresed on a 14% SDS- PAGE gel. The gel was stained with Coomassie and the expression product was detected. The coomassie stained gel revealed a band between the 28 kDa and the 18 kDa molecular weight markers, and was determined to be approximately 20 kDa.
  • the DNA sequence of the insert in the recombinant pLP505 plasmid was obtained using the ABI 370A DNA sequencer.
  • the DNA sequence was aligned with the DNA sequence of SEQ ID NO:l, using the Pustell DNA matrix plot feature of MacVector (Oxford Molecular Group, Campbell, CA). Alignment of the DNA sequence obtained from the pLP505 plasmid, SEQ ID NO:l, and the S.
  • SEQ ID NO:4 encodes for a predicted amino acid sequence as shown in SEQ ID NO:5.
  • the S. pneumoniae (serotype 4) genome sequence was obtained from The Institute for Genomic Research at www.tigr.org and/or through NCBI at www.ncbi.nlm.nih.gov, using the ClustalW feature of MacVector, (Oxford Molecular Group, Campbell CA).
  • a sequence comparison was made between the 20 kDa amino acid sequence (SEQ ID NO:2) and the predicted 79 kDa amino acid sequence (SEQ ID NO:5). It was observed that amino acids 1-58 and amino acids 90-132 of SEQ ID NO:2 have substantial sequence identity with amino acids 170-227 and amino acids 258-300 of SEQ ID NO:5, respectively. Proteins and peptides or polypeptides, containing these particular regions are preferred embodiments of the invention.
  • SEQ ID NOS:8 and 9 Based upon the available genomic DNA (serotype 4) sequence, two primers flanking the 2163bp ORF were designed and subsequently synthesized using the ABI 380A DNA synthesizer (SEQ ID NOS:8 and 9).
  • SEQ ID NO:8 was an S. pneumoniae 5 '-primer having an inserted Ncol site and a "Glu" residue added after the ATG start codon to maintain a correct reading frame.
  • SEQ ID NO:9 was an S. pneumoniae 3'-primer having an inserted Hindlll site. 5'-AGA GCT CCT CCC ATG GAA GAT CCG AAT TAT CAG-3'; (SEQ ID NO:8)

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EP0941335A2 (de) 1996-10-31 1999-09-15 Human Genome Sciences Polynukleotide und sequenzen aus streptococcus pneumoniae
US6676943B1 (en) 1997-04-24 2004-01-13 Regents Of The University Of Minnesota Human complement C3-degrading protein from Streptococcus pneumoniae
US6936252B2 (en) 1998-07-27 2005-08-30 Microbial Technics Limited Streptococcus pneumoniae proteins and nucleic acid molecules
EP1790730A3 (de) * 1998-07-27 2007-09-12 Sanofi Pasteur Limited Pneumokokkenproteine und Nukleinsäuremoleküle
IL142017A0 (en) * 1998-09-24 2002-03-10 Univ Minnesota Human complement c3-degrading polypeptide from streptococcus pneumoniae
JP4689044B2 (ja) 1998-12-21 2011-05-25 メディミューン，インコーポレーテッド ワクチン用の肺炎連鎖球菌タンパク質と免疫原断片
US7128918B1 (en) 1998-12-23 2006-10-31 Id Biomedical Corporation Streptococcus antigens
EP1950302B1 (de) * 1998-12-23 2012-12-05 ID Biomedical Corporation of Quebec Streptokokken-Antigene
KR100802198B1 (ko) * 1998-12-23 2008-02-11 샤이어 바이오켐 인코포레이티드 신규한 스트렙토코커스 항원
AU2005209689B2 (en) * 1998-12-23 2008-07-17 Id Biomedical Corporation Of Quebec Novel streptococcus antigens
US7074415B2 (en) 2000-06-20 2006-07-11 Id Biomedical Corporation Streptococcus antigens
WO2001098334A2 (en) * 2000-06-20 2001-12-27 Shire Biochem Inc. Streptococcus antigens
GB0022742D0 (en) 2000-09-15 2000-11-01 Smithkline Beecham Biolog Vaccine
ES2359625T3 (es) * 2000-12-28 2011-05-25 Wyeth Llc Proteina protectora recombinante de.
AU2002351623A1 (en) 2001-12-20 2003-07-09 Shire Biochem Inc. Streptococcus antigens
NZ595193A (en) 2005-10-21 2013-09-27 Catalyst Biosciences Inc Modified Proteases That Inhibit Complement Activation
TWI457133B (zh) 2005-12-13 2014-10-21 Glaxosmithkline Biolog Sa 新穎組合物
GB0607088D0 (en) 2006-04-07 2006-05-17 Glaxosmithkline Biolog Sa Vaccine
CA2808919C (en) 2005-12-22 2016-04-19 Glaxosmithkline Biologicals S.A. Streptococcus pneumoniae capsular saccharide vaccine
EP2687228B1 (de) 2007-06-26 2017-07-19 GlaxoSmithKline Biologicals S.A. Impfstoff mit streptococcus pneumoniae-kapsulären polysaccharid-konjugaten
KR20110009157A (ko) 2008-04-16 2011-01-27 글락소스미스클라인 바이오로지칼즈 에스.에이. 백신
GB201003924D0 (en) 2010-03-09 2010-04-21 Glaxosmithkline Biolog Sa Immunogenic composition
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GB201518684D0 (en) 2015-10-21 2015-12-02 Glaxosmithkline Biolog Sa Vaccine

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