Lipidated streptococcus pneumoniae antigen compositions, methods of preparation and use

Abstract

There are provided compositions and methods for prevention or treatment of Streptococcus pneumoniae (SP)-associated diseases. More specifically, there are provided recombinant lipidated fusion proteins comprising pneumococcal surface antigen A (PsaA), the recombinant lipidated fusion proteins comprising, from N-terminus to C-terminus, the N-terminal native lipid signal peptide of PsaA and the C-terminal structural gene for PsaA. Methods of inducing broad spectrum mucosal immunity against SP comprising administering a vaccine comprising recombinant lipidated fusion proteins are also described.

What is claimed is: 
     
         1 . A recombinant lipidated fusion protein comprising pneumococcal surface antigen A (PsaA), wherein the recombinant lipidated fusion protein comprises, from N-terminus to C-terminus, the N-terminal native lipid signal peptide of PsaA and the C-terminal structural gene for PsaA. 
     
     
         2 . The recombinant lipidated fusion protein of  claim 1 , wherein the recombinant lipidated fusion protein further comprises a tag or a detectable label at the C-terminus. 
     
     
         3 . The recombinant lipidated fusion protein of  claim 2  wherein the tag is an amino acid tag comprising 6 Histidine residues. 
     
     
         4 . The recombinant lipidated fusion protein of any one of  claims 1  to  3 , wherein the fusion protein is isolated or purified. 
     
     
         5 . The recombinant lipidated fusion protein of any one of  claims 1  to  4 , wherein the native lipid signal peptide has the amino acid sequence MKKLGTLLVLFLSAIILVAC (SEQ ID NO: 5). 
     
     
         6 . The recombinant lipidated fusion protein of any one of  claims 1  to  5 , wherein the recombinant lipidated fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 1 or 7, or a homolog, fragment, analog, or variant thereof. 
     
     
         7 . The recombinant lipidated fusion protein of any one of  claims 1  to  6 , wherein the recombinant lipidated fusion protein comprises an amino acid sequence at least about 80-95% identical to the amino acid sequence set forth in SEQ ID NO:1. 
     
     
         8 . The recombinant lipidated fusion protein of any one of  claims 1  to  7 , wherein the recombinant lipidated fusion protein is produced in  E. coli.    
     
     
         9 . The recombinant lipidated fusion protein of  claim 8 , wherein the recombinant lipidated fusion protein is produced by expression of a vector comprising the DNA having the nucleotide sequence set forth in SEQ ID NO: 6. 
     
     
         10 . The recombinant lipidated fusion protein of any one of  claims 1  to  9 , wherein the recombinant lipidated fusion protein comprises a homogeneous lipid structure. 
     
     
         11 . The recombinant lipidated fusion protein of  claim 10 , wherein the homogeneous lipid structure comprises a single major peak as analyzed by mass spectrometry or has the mass spectrometry spectrum shown in  FIG. 6D . 
     
     
         12 . The recombinant lipidated fusion protein of  claim 11 , wherein the single major peak has a m/z of about 1266. 
     
     
         13 . The recombinant lipidated fusion protein of any one of  claims 1  to  12 , wherein the recombinant lipidated fusion protein is capable of inducing a mucosal immune response against an  Streptococcus pneumoniae -associated disease in a subject. 
     
     
         14 . The recombinant lipidated fusion protein of  claim 13 , wherein a Th1 response and/or production of secretory IgA is induced in the subject. 
     
     
         15 . The recombinant lipidated fusion protein of  claim 14 , wherein the recombinant lipidated fusion protein is capable of inducing the mucosal immune response when administered in the absence of an adjuvant. 
     
     
         16 . The recombinant lipidated fusion protein of any one of  claims 13 - 15 , wherein the recombinant lipidated fusion protein is further capable of causing a mucosal immune response to be induced against one or more non-lipidated  Streptococcus pneumoniae  (SP) antigen administered concomitantly. 
     
     
         17 . The recombinant lipidated fusion protein of  claim 16 , wherein the one or more non-lipidated SP antigen is selected from pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal beta-galactosidase (BgaA), pneumococcal phosphorylcholine (ChoP), pneumococcal enolase (Eno), pneumococcal hyaluronate lyase (Hyl), pneumococcal autolysin A (LytA), pneumococcal neuraminidase (Nan), pneumococcal adhesion and virulence A (PavA), pneumococcal iron acquisition (PiaA), and pneumococcal surface association of Pht Proteins (PhtA, PhtB, PhtD, and PhtE). 
     
     
         18 . The recombinant lipidated fusion protein of any one of  claims 13  to  17 , wherein the mucosal immune response is not serotype-specific. 
     
     
         19 . The recombinant lipidated fusion protein of any one of  claims 13  to  18 , wherein the  Streptococcus pneumoniae -associated disease is pneumonia, meningitides, ear infection, sinus infection, or bacteremia. 
     
     
         20 . A method of producing the recombinant lipidated fusion protein according to any one of  claims 1  to  19 , the method comprising the steps of:
 (1) providing a host  E. coli  cell transformed with an expression vector that comprises a first nucleotide sequence encoding the N-terminal native lipid signal peptide of PsaA and a second nucleotide sequence encoding the C-terminal structural gene for PsaA; and 
 (2) cultivating the  E. coli  transformant to allow expression of the fusion protein comprising the N-terminal native lipid signal peptide of PsaA and the C-terminal structural gene for PsaA. 
 
     
     
         21 . The method of  claim 20 , wherein the host  E. coli  cell is from a strain that provides high-level protein expression. 
     
     
         22 . The method of  claim 21 , wherein the strain is selected from C43(DE3), (ECCC B96070445), C41(DE3) (ECCC B96070444), CO214(DE3), DK8(DE3)S (NCIMB 40885), and C2014(DE3) (NCIMB 40884). 
     
     
         23 . The method of any one of  claims 20  to  22 , wherein the  E. coli  transformant is cultivated in M9 medium. 
     
     
         24 . The method of any one of  claims 20  to  23 , wherein the method further comprises isolating the recombinant lipidated fusion protein from the  E. coli  after expression thereof. 
     
     
         25 . The method of any one of  claims 20  to  24 , wherein the expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 6. 
     
     
         26 . A composition comprising the recombinant lipidated fusion protein according to any one of  claims 1  to  19  or the recombinant lipidated fusion protein produced according to the method defined in any one of  claims 20  to  25  and a pharmaceutically acceptable diluent, carrier, or excipient. 
     
     
         27 . The composition of  claim 26 , wherein the composition further comprises one or more non-lipidated SP antigen. 
     
     
         28 . The composition of  claim 27 , wherein the one or more non-lipidated SP antigen is selected from pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal beta-galactosidase (BgaA), pneumococcal phosphorylcholine (ChoP), pneumococcal enolase (Eno), pneumococcal hyaluronate lyase (Hyl), pneumococcal autolysin A (LytA), pneumococcal neuraminidase (Nan), pneumococcal adhesion and virulence A (PavA), pneumococcal iron acquisition (PiaA), and pneumococcal surface association of Pht Proteins (PhtA, PhtB, PhtD, and PhtE). 
     
     
         29 . A vaccine for prevention or treatment of an SP-associated disease comprising the recombinant lipidated fusion protein according to any one of  claims 1  to  19 , or the recombinant lipidated fusion protein produced according to the method defined in any one of  claims 20  to  25 , and an adjuvant. 
     
     
         30 . The vaccine of  claim 29 , wherein the vaccine further comprises one or more non-lipidated SP antigen. 
     
     
         31 . The vaccine of  claim 30 , wherein the one or more non-lipidated SP antigen is selected from pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal beta-galactosidase (BgaA), pneumococcal phosphorylcholine (ChoP), pneumococcal enolase (Eno), pneumococcal hyaluronate lyase (Hyl), pneumococcal autolysin A (LytA), pneumococcal neuraminidase (Nan), pneumococcal adhesion and virulence A (PavA), pneumococcal iron acquisition (PiaA), and pneumococcal surface association of Pht Proteins (PhtA, PhtB, PhtD, and PhtE). 
     
     
         32 . An isolated antibody or fragment thereof specific for the recombinant lipidated fusion protein according to any one of  claims 1  to  19 , or the recombinant lipidated fusion protein produced according to the method defined in any one of  claims 20  to  25 . 
     
     
         33 . The isolated antibody or fragment of  claim 32 , wherein the antibody or fragment is a polyclonal antibody. 
     
     
         34 . The isolated antibody or fragment of  claim 32 , wherein the antibody or fragment is a monoclonal antibody. 
     
     
         35 . The isolated antibody or fragment of  claim 32 , wherein the antibody or fragment is humanized, human, or chimeric. 
     
     
         36 . The isolated antibody or fragment of any one of  claims 32  to  35 , wherein the antibody or fragment comprises a whole immunoglobulin molecule; a single-chain antibody; a single-chain variable fragment (scFv); a single domain antibody; an Fab fragment; an F(ab′)2 fragment; or a disulfide-linked Fv (di-scFv). 
     
     
         37 . The isolated antibody or fragment of any one of  claims 32  to  36 , wherein the antibody or fragment comprises a heavy chain immunoglobulin constant domain selected from human IgM, human IgG1, human IgG2, human IgG3, human IgG4, and human IgA1/2. 
     
     
         38 . The isolated antibody or fragment of any one of  claims 32  to  37 , wherein the antibody or fragment comprises a light chain immunoglobulin constant domain selected from human Ig kappa and human Ig lambda. 
     
     
         39 . The isolated antibody or fragment of any one of  claims 32  to  38 , wherein the antibody or fragment binds to an antigen with an affinity constant of at least about 10 7 -10 10  M. 
     
     
         40 . A composition comprising the isolated antibody or fragment of any one of  claims 32  to  39  and a pharmaceutically acceptable diluent, carrier, or excipient. 
     
     
         41 . The composition of  claim 40 , further comprising a second agent for preventing or treating an SP-associated disease. 
     
     
         42 . The composition of  claim 41 , wherein the second agent comprises one or more of: an antibody that binds to PspA; an antibody that binds to PspC; and an antibiotic. 
     
     
         43 . A method for preventing or treating an SP-associated disease comprising administering to a subject the composition according to any one of  claims 26 - 28  and  40 - 42 ; the vaccine according to any one of  claims 29 - 31 ; or the antibody or fragment according to any one of  claims 32 - 39 ; such that the SP-associated disease is prevented or treated in the subject. 
     
     
         44 . A method for preventing or treating an SP-associated disease comprising administering to a subject the recombinant lipidated fusion protein according to any one of  claims 1  to  19 , or the recombinant lipidated fusion protein produced according to the method defined in any one of  claims 20  to  25 , such that the SP-associated disease is prevented or treated in the subject. 
     
     
         45 . The method of  claim 43  or  44 , wherein mucosal immunity against SP is induced in the subject. 
     
     
         46 . The method of  claim 45 , wherein a Th1 response and/or production of secretory IgA is induced in the subject. 
     
     
         47 . The method of  claim 45  or  46 , wherein the mucosal immunity is not serotype-specific. 
     
     
         48 . The method of any one of  claims 43  to  47 , wherein mucosal immunity against the one or more non-lipidated SP antigen is induced. 
     
     
         49 . A method of inducing immunity against SP infection in a subject comprising administering to the subject the composition according to any one of  claims 26 - 28  and  40 - 42 ; the vaccine according to any one of  claims 29 - 31 ; or the antibody or fragment according to any one of  claims 32 - 39 ; such that SP infection is prevented or treated in the subject. 
     
     
         50 . A method of inducing immunity against SP infection in a subject comprising administering to the subject the recombinant lipidated fusion protein according to any one of  claims 1  to  19 , or the recombinant lipidated fusion protein produced according to the method defined in any one of  claims 20  to  25 , such that SP infection is prevented or treated in the subject. 
     
     
         51 . The method of any one of  claims 43  to  50 , wherein the composition, the vaccine, the antibody or fragment, or the recombinant lipidated fusion protein is administered intravenously, subcutaneously, intramuscularly, transmucosally, or orally. 
     
     
         52 . The method of any one of  claims 43  to  51 , wherein the composition, the vaccine, the antibody or fragment, or the recombinant lipidated fusion protein is administered in combination with a second agent for preventing or treating an SP infection or an SP-associated disease. 
     
     
         53 . The method of  claim 52 , wherein the composition, the vaccine, the antibody or fragment, or the recombinant lipidated fusion protein and the second agent are administered concomitantly or sequentially. 
     
     
         54 . Use of the the recombinant lipidated fusion protein according to any one of  claims 1  to  19 , or the recombinant lipidated fusion protein produced according to the method defined in any one of  claims 20  to  25 , in the manufacture of a vaccine for prevention or treatment of SP infection. 
     
     
         55 . A vaccine for prevention or treatment of SP infection comprising the composition according to any one of  claims 26  to  28 .