Identification and characterization of novel pneumococcal choline binding protein, CbpG, and diagnostic and therapeutic uses thereof

Abstract

The present invention provides isolated polypeptides comprising an amino acid sequence of a choline binding protein CbpG. This invention provides an isolated polypeptide comprising an amino acid sequence of a choline binding polypeptide CbpG or N-terminal CbpG truncate, including analogs, variants, mutants, derivatives and fragments thereof. This invention further provides an isolated immunogenic polypeptide comprising an amino acid sequence of a choline binding protein CbpG. This invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of a choline binding protein CbpG. This invention provides pharmaceutical compositions, vaccines, and diagnostic and therapeutic methods of use of the isolated polypeptides and nucleic acids. Assays for compounds which alter or inactivate the polypeptides of the present invention for use in therapy are also provided.

What is claimed is:  
     
         1 . An isolated streptococcal CbpG choline binding polypeptide comprising a choline binding domain which is homologous to the amino acid sequence set out as SEQ ID NO: 1.  
     
     
         2 . An isolated streptococcal CbpG choline binding polypeptide comprising an amino acid sequence that is homologous to the choline binding sequence set out as SEQ ID NO: 9.  
     
     
         3 . An isolated streptococcal CbpG choline binding polypeptide variant wherein one or more amino acids set out as SEQ ID NO: 2 is substituted by a conserved amino acid.  
     
     
         4 . An isolated streptococcal CbpG choline binding polypeptide comprising an immunogenic fragment of the amino acid sequence set out as SEQ ID NO: 2 or SEQ ID NO: 11.  
     
     
         5 . The CbpG choline binding polypeptide according to  claim 4  wherein the polypeptide inhibits attachment of  S. pneumoniae  to nasopharyngeal or pulmonary mucosal cells.  
     
     
         6 . An isolated streptococcal N-terminal CbpG truncate polypeptide variant comprising a contiguous amino acid sequence set out in SEQ ID NO: 11 wherein one or more amino acids of the truncate polypeptide is substituted by a conserved amino acid.  
     
     
         7 . The N-terminal CbpG truncate according to  claim 6  wherein the truncate polypeptide inhibits attachment of  S. pneumoniae  to nasopharyngeal or pulmonary mucosal cells.  
     
     
         8 . An isolated streptococcal N-terminal CbpG truncate polypeptide variant comprising amino acids 1 through 90 of SEQ ID NO: 2 wherein one or more amino acids of the truncate polypeptide is substituted by a conserved amino acid.  
     
     
         9 . The N-terminal truncate CbpG polypeptide according to  claim 8  wherein the truncate polypeptide inhibits attachment of  S. pneumoniae  to nasopharyngeal or pulmonary mucosal cells.  
     
     
         10 . An isolated streptococcal N-terminal CbpG polypeptide which comprises a fragment of SEQ ID NO: 2 wherein the polypeptide inhibits attachment of  S. pneumoniae  to nasopharyngeal or pulmonary mucosal cells.  
     
     
         11 . An isolated streptococcal N-terminal CbpG polypeptide derivative comprising the amino acid set out as SEQ ID NO: 2 or SEQ ID NO: 11 and at least one chemical moiety selected from a water soluble polymer or polyethylene glycol attached to the polypeptide.  
     
     
         12 . A vaccine comprising the CbpG choline binding polypeptide of  claim 5  and a pharmaceutically acceptable adjuvant.  
     
     
         13 . A pharmaceutical composition comprising a CbpG choline binding polypeptide of  claim 10  and a pharmaceutically acceptable carrier.  
     
     
         14 . A purified antibody to a streptococcal choline binding polypeptide CbpG.  
     
     
         15 . A monoclonal antibody to the streptococcal choline binding polypeptide of  claim 5 .  
     
     
         16 . An immortal cell line that produces a monoclonal antibody according to  claim 15 .  
     
     
         17 . The antibody of any of claims  14  or  15  labeled with a detectable label.  
     
     
         18 . The antibody of  claim 17  wherein the label is selected from the group consisting of an enzyme, a chemical which fluoresces, and a radioactive elements.  
     
     
         19 . A pharmaceutical composition comprising an antibody to a choline binding protein of  claim 4  and a pharmaceutically acceptable carrier.  
     
     
         20 . A pharmaceutical composition comprising a combination of at least two antibodies, wherein one of such antibodies is directed to the N-terminal CbpG truncate polypeptide according to  claim 9  and a pharmaceutically acceptable carrier.  
     
     
         21 . An isolated nucleic acid which encodes the streptococcal choline binding protein of  claim 4  or a fragment thereof.  
     
     
         22 . The isolated nucleic acid of  claim 21 , wherein the nucleic acid is selected from the group consisting of: 
 a. the DNA sequence of SEQ ID NO:3;    b. DNA sequences that hybridize to the DNA sequence of SEQ ID NO:3 under standard hybridization conditions;    c. degenerate variants thereof;    d. alleles thereof, and    e. hybridizable fragments thereof.    
     
     
         23 . The isolated nucleic acid of  claim 21 , wherein the nucleic acid is selected from the group consisting of: 
 a. the DNA sequence of SEQ ID NO: 12;    b. DNA sequences that hybridize to the DNA sequence of SEQ ID NO: 12 under standard hybridization conditions;    c. degenerate variants thereof;    d. alleles thereof; and    e. hybridizable fragments thereof.    
     
     
         24 . The nucleic acid of  claim 21  which is a DNA molecule having a nucleotide sequence selected from the group consisting of: 
 a. a DNA sequence encoding a polypeptide comprising the amino acid sequence set out in SEQ 1D NO:2;  
 b. a DNA sequence that hybridizes to the DNA sequence of (a) under moderately stringent hybridization conditions; and  
 c. a DNA sequence capable of encoding the amino acid sequence encoded by any of the foregoing DNA sequences of (a) or (b).  
 
     
     
         25 . A vector which comprises the nucleic acid of  claim 24  and a promoter.  
     
     
         26 . The vector of  claim 25 , wherein the promoter comprises a bacterial, yeast, insect or mammalian promoter.  
     
     
         27 . The vector of  claim 25 , wherein the vector is a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA.  
     
     
         28 . A host vector system for the production of a polypeptide which comprises the vector of  claim 26  in a suitable host cell.  
     
     
         29 . The host vector system of  claim 28 , wherein the suitable host cell comprises a prokaryotic or eukaryotic cell.  
     
     
         30  A unicellular host transformed with a recombinant DNA molecule of  claim 29 .  
     
     
         31 . A nucleic acid vaccine comprising the recombinant DNA molecule of  claim 29 .  
     
     
         32 . A method for detecting the presence of a streptococcal choline binding polypeptide of  claim 5 , wherein the streptococcal choline binding polypeptide is measured by: 
 a. contacting a sample in which the presence or activity of the choline binding polypeptide CbpG is suspected with an antibody to the choline binding polypeptide CbpG under conditions that allow binding of the choline binding polypeptide CbpG to the binding partner to occur; and    b. detecting whether binding has occurred between the choline binding polypeptide CbpG from the sample and the antibody; wherein the detection of binding indicates the presence or activity of the choline binding polypeptide CbpG in the sample.    
     
     
         33 . A method for detecting the presence of a bacterium having a gene encoding a choline binding polypeptide of  claim 5 , comprising: 
 a. contacting a sample in which the presence or activity of the bacterium is suspected with an oligonucleotide which hybridizes to the choline binding polypeptide CbpG gene under conditions that allow specific hybridization of the oligonucleotide to the gene to occur; and    b. detecting whether hybridization has occurred between the oligonucleotide and the gene;    wherein the detection of hybridization indicates that presence or activity of the bacterium in the sample.    
     
     
         34 . A method for preventing infection with a bacterium that expresses a streptococcal choline binding polypeptide comprising administering an immunogenically effective dose of a vaccine of  claim 12  to a subject.  
     
     
         35 . A method for preventing infection with a bacterium that expresses a streptococcal choline binding polypeptide comprising administering an immunogenically effective dose of a vaccine of  claim 12  to a subject.  
     
     
         36 . A method for treating infection with a bacterium that expresses a streptococcal choline binding polypeptide comprising administering a therapeutically effective dose of a pharmaceutical composition of  claim 13  to a subject.  
     
     
         37 . A method for treating infection with a bacterium that expresses a streptococcal choline binding polypeptide comprising administering a therapeutically effective dose of a pharmaceutical composition of  claim 13  to a subject.  
     
     
         38 . A method for treating infection with a bacterium that expresses a streptococcal choline binding polypeptide comprising administering pulmonarily an adhesion inhibitory agent wherein said agent is a choline binding polypeptide CbpG or fragment thereof.  
     
     
         39 . A method of inducing an immune response in a subject which has been exposed to or infected with a pneumococcal bacterium comprising administering to the subject an amount of the pharmaceutical composition of  claim 13 , thereby inducing an immune response.  
     
     
         40 . A method for preventing infection by a pneumococcal bacterium in a subject comprising administering to the subject an amount of a pharmaceutical composition of  claim 13  and a pharmaceutically acceptable carrier or diluent, thereby preventing infection by a pneumococcal bacterium.