Mutant forms of cholera holotoxin as an adjuvant

Abstract

Mutant cholera holotoxins comprising a cholera toxin subunit A having single amino acid substitutions in the amino acid positions 16 or 72 or a double amino acid substitution in the amino acid positions 16 and 68 or 68 and 72 have reduced toxicity compared to the wild-type cholera holotoxin. The mutant cholera holotoxins are useful as adjuvants in immunogenic compositions to enhance the immune response in a vertebrate host to a selected antigen from a pathogenic bacterium, virus, fungus, or parasite, a cancer cell, a tumor cell, an allergen, or a self-molecule.

1 . An immunogenic, mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 68 and an amino acid substitution in the wild-type CT subunit A amino acid position 72, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT. 
     
     
         2 . The CT-CRM according to  claim 1 , wherein the amino acid serine in the amino acid position 68 in the A subunit is substituted with a tyrosine, and wherein the amino acid valine in the amino acid position 72 in the A subunit is substituted with a tyrosine. 
     
     
         3 . An immunogenic composition comprising a mutant cholera holotoxin (CT-CRM) of  claim 1 , wherein the mutant holotoxin enhances the immune response in a vertebrate host to an antigen. 
     
     
         4 . The composition according to  claim 3 , further comprising an antigen derived from the member of the group consisting of a pathogenic bacterium, pathogenic virus, pathogenic fungus, pathogenic parasite, a cancer cell, a tumor cell, an allergen and a self-molecule, and a protein, polypeptide, peptide or fragment derived from said antigen. 
     
     
         5 . The composition according to  claim 4 , wherein the bacterial antigen is selected from the bacterial species consisting of typable and non-typable  Haemophilus influenzae, Haemophilis somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neiserria gonorrhoea, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diptherieae, Mycobacterium tuberculosis, Mycobacterium avium - Mycabacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae  and  Mycoplasma galliseptium.    
     
     
         6 . The composition according to  claim 4 , wherein the  Haemophilus influenzae  antigen is selected from the group consisting of the  Haemophilus influenzae  P4 outer membrane protein, the  Haemophilus influenzae  P6 outer membrane protein and  Haemophilus influenzae  adherence and penetration protein (Hap s ). 
     
     
         7 . The composition according to  claim 6 , wherein the  Helicobacter Pylori  antigen is the  Helicobacter pylori  urease protein. 
     
     
         8 . The composition according to  claim 6 , wherein the  Neisseria meningitidis  antigen is selected from the group consisting of the  Neisseria meningitidis  Group B recombinant class 1 pilin (rpilin) and the  Neisseria meningitidis  Group B class 1 outer membrane protein (PorA). 
     
     
         9 . The composition according to  claim 4 , wherein the viral antigen is selected from the viral species consisting of Respiratory syncytial virus, Parainfluenza virus types, 1,2,3, Human metapneumovirus, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, poliovirus, rotavirus, caliciviruses, measles virus, mumps virus, Rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, avian pneumovirus (formerly turkey rhinotracheitis virus), Hendra virus, Nipah virus, coronavirus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, feline infectious peritonitis virus, avian infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and the encephalitis viruses. 
     
     
         10 . The composition according to  claim 9 , wherein the respiratory syncytial virus antigen is the respiratory syncytial virus fusion protein. 
     
     
         11 . The composition according to  claim 9 , wherein the herpes simplex virus (HSV) antigen is the herpes simplex virus (HSV) type 2 glycoprotein D (gD2). 
     
     
         12 . The composition according to  claim 4 , wherein the fungal antigen is from a fungus selected from the group of pathogenic fungi consisting of  Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus  and  Histoplasma.    
     
     
         13 . The composition according to  claim 4 , wherein the parasite antigen is from a parasite selected from the group of pathogenic parasites consisting of  Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii  and  Pneumocystis carinii.    
     
     
         14 . The composition according to  claim 4 , wherein said cancer or tumor cell antigen is selected from the group consisting of prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125, MAGE-3, a hormone, and a hormone analog. 
     
     
         15 . The composition according to  claim 4 , wherein said antigen is a polypeptide, peptide or fragment derived from amyloid precursor protein, or an allergen. 
     
     
         16 . The composition according to  claim 15 , wherein the amyloid precursor protein antigen is the Aβ peptide, which is a 42 amino acid fragment of amyloid precursor protein, or a fragment of the Aβ peptide. 
     
     
         17 . The composition according to  claim 3 , further comprising a diluent, excipient or carrier. 
     
     
         18 . The composition according to  claim 3 , further comprising a second adjuvant in addition to the mutant cholera holotoxin. 
     
     
         19 . A method for enhancing the immune response of a vertebrate host to an antigen, said method comprising administering to the host the composition according to  claim 4 . 
     
     
         20 . An isolated and purified DNA sequence encoding an immunogenic, mutant cholera holotoxin of  claim 1 . 
     
     
         21 . A nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin according to  claim 1 , and wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of said mutant holotoxin in a host cell. 
     
     
         22 . The molecule according to  claim 21 , wherein said regulatory sequence is an inducible promoter. 
     
     
         23 . The molecule according to  claim 21 , wherein said promoter is the arabinose inducible promoter. 
     
     
         24 . The molecule according to  claim 21 , wherein said molecule is a viral or non-viral vector. 
     
     
         25 . The molecule according to  claim 24 , wherein said non-viral vector is a DNA plasmid. 
     
     
         26 . A host cell transformed, transduced, infected or transfected with the nucleic acid molecule according to  claim 21 . 
     
     
         27 . A method of producing an immunogenic mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 68 and an amino acid substitution in the wild-type CT subunit A amino acid position 72, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT,
 comprising transforming, infecting, transducing or transfecting a host cell with the nucleic acid molecule according to  claim 21 , and culturing the host cell under conditions which permit expression of said recombinant immunogenic detoxified protein by the host cell.   
     
     
         28 . An immunogenic, mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 16 and an amino acid substitution in the wild-type CT subunit A amino acid position 68, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT. 
     
     
         29 . The CT-CRM according to  claim 28 , wherein the amino acid isoleucine in the amino acid position 16 in the A subunit is substituted with an alanine, and wherein the amino acid serine in the amino acid position 68 in the A subunit is substituted with a tyrosine. 
     
     
         30 . An immunogenic composition comprising a mutant cholera holotoxin (CT-CRM) of  claim 28 , wherein the mutant holotoxin enhances the immune response in a vertebrate host to an antigen. 
     
     
         31 . The composition according to  claim 30 , further comprising an antigen derived from the member of the group consisting of a pathogenic bacterium, pathogenic virus, pathogenic fungus, pathogenic parasite, a cancer cell, a tumor cell, an allergen and a self-molecule, and a protein, polypeptide, peptide or fragment derived from said antigen. 
     
     
         32 . The composition according to  claim 31 , wherein the bacterial antigen is selected from the bacterial species consisting of typable and non-typable  Haemophilus influenzae, Haemophilis somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neiserria gonorrhoea, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diptherieae, Mycobacterium tuberculosis, Mycobacterium avium - Mycabacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae  and  Mycoplasma galliseptium.    
     
     
         33 . The composition according to  claim 31 , wherein the  Haemophilus influenzae  antigen is selected from the group consisting of the  Haemophilus influenzae  P4 outer membrane protein, the  Haemophilus influenzae  P6 outer membrane protein and  Haemophilus influenzae  adherence and penetration protein (Hap s ). 
     
     
         34 . The composition according to  claim 33 , wherein the  Helicobacter Pylori  antigen is the  Helicobacter pylori  urease protein. 
     
     
         35 . The composition according to  claim 33 , wherein the  Neisseria meningitidis  antigen is selected from the group consisting of the  Neisseria meningitidis  Group B recombinant class 1 pilin (rpilin) and the  Neisseria meningitidis  Group B class 1 outer membrane protein (PorA). 
     
     
         36 . The composition according to  claim 31 , wherein the viral antigen is selected from the viral species consisting of Respiratory syncytial virus, Parainfluenza virus types, 1,2,3, Human metapneumovirus, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, poliovirus, rotavirus, caliciviruses, measles virus, mumps virus, Rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, avian pneumovirus (formerly turkey rhinotracheitis virus), Hendra virus, Nipah virus, coronavirus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, feline infectious peritonitis virus, avian infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and the encephalitis viruses. 
     
     
         37 . The composition according to  claim 36 , wherein the respiratory syncytial virus antigen is the respiratory syncytial virus fusion protein. 
     
     
         38 . The composition according to  claim 36 , wherein the herpes simplex virus (HSV) antigen is the herpes simplex virus (HSV) type 2 glycoprotein D (gD2). 
     
     
         39 . The composition according to  claim 31 , wherein the fungal antigen is from a fungus selected from the group of pathogenic fungi consisting of  Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus  and  Histoplasma.    
     
     
         40 . The composition according to  claim 31 , wherein the parasite antigen is from a parasite selected from the group of pathogenic parasites consisting of  Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii  and  Pneumocystis carinii.    
     
     
         41 . The composition according to  claim 31 , wherein said cancer or tumor cell antigen is selected from the group consisting of prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125, MAGE-3, a hormone, and a hormone analog. 
     
     
         42 . The composition according to  claim 31 , wherein said antigen is a polypeptide, peptide or fragment derived from amyloid precursor protein, or an allergen. 
     
     
         43 . The composition according to  claim 42 , wherein the amyloid precursor protein antigen is the Aβ peptide, which is a 42 amino acid fragment of amyloid precursor protein, or a fragment of the Aβ peptide. 
     
     
         44 . The composition according to  claim 30 , further comprising a diluent, excipient or carrier. 
     
     
         45 . The composition according to  claim 30 , further comprising a second adjuvant in addition to the mutant cholera holotoxin. 
     
     
         46 . A method for enhancing the immune response of a vertebrate host to an antigen, said method comprising administering to the host the composition according to  claim 31 . 
     
     
         47 . An isolated and purified DNA sequence encoding an immunogenic, mutant cholera holotoxin of  claim 28 . 
     
     
         48 . A nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin according to  claim 28 , and wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of said mutant holotoxin in a host cell. 
     
     
         49 . The molecule according to  claim 48 , wherein said regulatory sequence is an inducible promoter. 
     
     
         50 . The molecule according to  claim 48 , wherein said promoter is the arabinose inducible promoter. 
     
     
         51 . The molecule according to  claim 48 , wherein said molecule is a viral or non-viral vector. 
     
     
         52 . The molecule according to  claim 51 , wherein said non-viral vector is a DNA plasmid. 
     
     
         53 . A host cell transformed, transduced, infected or transfected with the nucleic acid molecule according to  claim 48 . 
     
     
         54 . A method of producing an immunogenic mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 16 and an amino acid substitution in the wild-type CT subunit A amino acid position 68, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT,
 comprising transforming, infecting, transducing or transfecting a host cell with the nucleic acid molecule according to  claim 48 , and culturing the host cell under conditions which permit expression of said recombinant immunogenic detoxified protein by the host cell.   
     
     
         55 . An immunogenic, mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 16, an amino acid substitution in the wild-type CT subunit A amino acid position 68 and an amino acid substitution in the wild-type CT subunit A amino acid position 72, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT. 
     
     
         56 . The CT-CRM according to  claim 55 , wherein the amino acid isoleucine in the amino acid position 16 in the A subunit is substituted with an alanine, wherein the amino acid serine in the amino acid position 68 in the A subunit is substituted with a tyrosine, and wherein the amino acid valine in the amino acid position 72 in the A subunit is substituted with a tyrosine. 
     
     
         57 . An immunogenic composition comprising a mutant cholera holotoxin (CT-CRM) of  claim 1 , wherein the mutant holotoxin enhances the immune response in a vertebrate host to an antigen. 
     
     
         58 . The composition according to  claim 57 , further comprising an antigen derived from the member of the group consisting of a pathogenic bacterium, pathogenic virus, pathogenic fungus, pathogenic parasite, a cancer cell, a tumor cell, an allergen and a self-molecule, and a protein, polypeptide, peptide or fragment derived from said antigen. 
     
     
         59 . The composition according to  claim 58 , wherein the bacterial antigen is selected from the bacterial species consisting of typable and non-typable  Haemophilus influenzae, Haemophilis somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neiserria gonorrhoea, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diptherieae, Mycobacterium tuberculosis, Mycobacterium avium - Mycabacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae  and  Mycoplasma galliseptium.    
     
     
         60 . The composition according to  claim 58 , wherein the  Haemophilus influenzae  antigen is selected from the group consisting of the  Haemophilus influenzae  P4 outer membrane protein, the  Haemophilus influenzae  P6 outer membrane protein and  Haemophilus influenzae  adherence and penetration protein (Hap s ). 
     
     
         61 . The composition according to  claim 60 , wherein the  Helicobacter Pylori  antigen is the  Helicobacter pylori  urease protein. 
     
     
         62 . The composition according to  claim 60 , wherein the  Neisseria meningitidis  antigen is selected from the group consisting of the  Neisseria meningitidis  Group B recombinant class 1 pilin (rpilin) and the  Neisseria meningitidis  Group B class 1 outer membrane protein (PorA). 
     
     
         63 . The composition according to  claim 58 , wherein the viral antigen is selected from the viral species consisting of Respiratory syncytial virus, Parainfluenza virus types, 1,2,3, Human metapneumovirus, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, poliovirus, rotavirus, caliciviruses, measles virus, mumps virus, Rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, avian pneumovirus (formerly turkey rhinotracheitis virus), Hendra virus, Nipah virus, coronavirus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, feline infectious peritonitis virus, avian infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and the encephalitis viruses. 
     
     
         64 . The composition according to  claim 63 , wherein the respiratory syncytial virus antigen is the respiratory syncytial virus fusion protein. 
     
     
         65 . The composition according to  claim 63 , wherein the herpes simplex virus (HSV) antigen is the herpes simplex virus (HSV) type 2 glycoprotein D (gD2). 
     
     
         66 . The composition according to  claim 58 , wherein the fungal antigen is from a fungus selected from the group of pathogenic fungi consisting of  Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus  and  Histoplasma.    
     
     
         67 . The composition according to  claim 58 , wherein the parasite antigen is from a parasite selected from the group of pathogenic parasites consisting of  Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii  and  Pneumocystis carinii.    
     
     
         68 . The composition according to  claim 58 , wherein said cancer or tumor cell antigen is selected from the group consisting of prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125, MAGE-3, a hormone, and a hormone analog. 
     
     
         69 . The composition according to  claim 58 , wherein said antigen is a polypeptide, peptide or fragment derived from amyloid precursor protein, or an allergen. 
     
     
         70 . The composition according to  claim 69 , wherein the amyloid precursor protein antigen is the Aβ peptide, which is a 42 amino acid fragment of amyloid precursor protein, or a fragment of the Aβ peptide. 
     
     
         71 . The composition according to  claim 57 , further comprising a diluent, excipient or carrier. 
     
     
         72 . The composition according to  claim 57 , further comprising a second adjuvant in addition to the mutant cholera holotoxin. 
     
     
         73 . A method for enhancing the immune response of a vertebrate host to an antigen, said method comprising administering to the host the composition according to  claim 58 . 
     
     
         74 . An isolated and purified DNA sequence encoding an immunogenic, mutant cholera holotoxin of  claim 55 . 
     
     
         75 . A nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin according to  claim 55 , and wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of said mutant holotoxin in a host cell. 
     
     
         76 . The molecule according to  claim 75 , wherein said regulatory sequence is an inducible promoter. 
     
     
         77 . The molecule according to  claim 75 , wherein said promoter is the arabinose inducible promoter. 
     
     
         78 . The molecule according to  claim 75 , wherein said molecule is a viral or non-viral vector. 
     
     
         79 . The molecule according to  claim 78 , wherein said non-viral vector is a DNA plasmid. 
     
     
         80 . A host cell transformed, transduced, infected or transfected with the nucleic acid molecule according to  claim 75 . 
     
     
         81 . A method of producing an immunogenic mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 16, an amino acid substitution in the wild-type CT subunit A amino acid position 68 and an amino acid substitution in the wild-type CT subunit A amino acid position 72, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT,
 comprising transforming, infecting, transducing or transfecting a host cell with the nucleic acid molecule according to  claim 75 , and culturing the host cell under conditions which permit expression of said recombinant immunogenic detoxified protein by the host cell.   
     
     
         82 . An immunogenic, mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 16 or position 72, further comprising at least one additional mutation in the A subunit of the cholera holotoxin at an amino acid position other than the amino acid positions 16, 68 and 72 in the A subunit, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT. 
     
     
         83 . An immunogenic, mutant cholera holotoxin (CT-CRM) according to  claim 82 , wherein the one additional mutation is a substitution for a subunit A amino acid selected from the group consisting of the arginine at amino acid position 7, the aspartic acid at amino acid position 9, the arginine at amino acid position 11, the glutamic acid at position 29, the histidine at amino acid position 44, the valine at amino acid position 53, the arginine at amino acid position 54, the serine at amino acid position 61, the serine at amino acid position 63, the histidine at amino acid position 70, the valine at amino acid position 97, the tyrosine at amino acid position 104, the proline at amino acid position 106, the histidine at amino acid position 107, the serine at amino acid position 109, the glutamic acid at amino acid position 110, the glutamic acid at amino acid position 112, the serine at amino acid position 114, the tryptophan at amino acid position 127, the arginine at amino acid position 146, and the arginine at amino acid position 192 
     
     
         84 . An immunogenic composition comprising a mutant cholera holotoxin (CT-CRM) of  claim 82 , wherein the mutant holotoxin enhances the immune response in a vertebrate host to an antigen. 
     
     
         85 . The composition according to  claim 84 , further comprising an antigen derived from the member of the group consisting of a pathogenic bacterium, pathogenic virus, pathogenic fungus, pathogenic parasite, a cancer cell, a tumor cell, an allergen and a self-molecule, and a protein, polypeptide, peptide or fragment derived from said antigen. 
     
     
         86 . The composition according to  claim 85 , wherein the bacterial antigen is selected from the bacterial species consisting of typable and non-typable  Haemophilus influenzae, Haemophilis somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neiserria gonorrhoea, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia colt, Shigella, Vibrio cholerae, Corynebacterium diptherieae, Mycobacterium tuberculosis, Mycobacterium avium - Mycabacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae  and  Mycoplasma galliseptium.    
     
     
         87 . The composition according to  claim 85 , wherein the  Haemophilus influenzae  antigen is selected from the group consisting of the  Haemophilus influenzae  P4 outer membrane protein, the  Haemophilus influenzae  P6 outer membrane protein and  Haemophilus influenzae  adherence and penetration protein (Hap s ). 
     
     
         88 . The composition according to  claim 87 , wherein the  Helicobacter Pylori  antigen is the  Helicobacter pylori  urease protein. 
     
     
         89 . The composition according to  claim 87 , wherein the  Neisseria meningitidis  antigen is selected from the group consisting of the  Neisseria meningitidis  Group B recombinant class 1 pilin (rpilin) and the  Neisseria meningitidis  Group B class 1 outer membrane protein (PorA). 
     
     
         90 . The composition according to  claim 85 , wherein the viral antigen is selected from the viral species consisting of Respiratory syncytial virus, Parainfluenza virus types, 1,2,3, Human metapneumovirus, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Human immunodeficiency virus, Hepatitis A, virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, poliovirus, rotavirus, caliciviruses, measles virus, mumps virus, Rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, avian pneumovirus (formerly turkey rhinotracheitis virus), Hendra virus, Nipah virus, coronavirus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, feline infectious peritonitis virus, avian infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and the encephalitis viruses. 
     
     
         91 . The composition according to  claim 90 , wherein the respiratory syncytial virus antigen is the respiratory syncytial virus fusion protein. 
     
     
         92 . The composition according to  claim 90 , wherein the herpes simplex virus (HSV) antigen is the herpes simplex virus (HSV) type 2 glycoprotein D (gD2). 
     
     
         93 . The composition according to  claim 85 , wherein the fungal antigen is from a fungus selected from the group of pathogenic fungi consisting of  Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus  and  Histoplasma.    
     
     
         94 . The composition according to  claim 85 , wherein the parasite antigen is from a parasite selected from the group of pathogenic parasites consisting of  Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii  and  Pneumocystis carinii.    
     
     
         95 . The composition according to  claim 85 , wherein said cancer or tumor cell antigen is selected from the group consisting of prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125, MAGE-3, a hormone, and a hormone analog. 
     
     
         96 . The composition according to  claim 85 , wherein said antigen is a polypeptide, peptide or fragment derived from amyloid precursor protein, or an allergen. 
     
     
         97 . The composition according to  claim 96 , wherein the amyloid precursor protein antigen is the Aβ peptide, which is a 42 amino acid fragment of amyloid precursor protein, or a fragment of the Aβ peptide. 
     
     
         98 . The composition according to  claim 84 , further comprising a diluent, excipient or carrier. 
     
     
         99 . The composition according to  claim 84 , further comprising a second adjuvant in addition to the mutant cholera holotoxin. 
     
     
         100 . A method for enhancing the immune response of a vertebrate host to an antigen, said method comprising administering to the host the composition according to  claim 85 . 
     
     
         101 . An isolated and purified DNA sequence encoding an immunogenic, mutant cholera holotoxin of  claim 83 . 
     
     
         102 . A nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin according to  claim 83 , and wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of said mutant holotoxin in a host cell. 
     
     
         103 . The molecule according to  claim 102 , wherein said regulatory sequence is an inducible promoter. 
     
     
         104 . The molecule according to  claim 102 , wherein said promoter is the arabinose inducible promoter. 
     
     
         105 . The molecule according to  claim 102 , wherein said molecule is a viral or non-viral vector. 
     
     
         106 . The molecule according to  claim 105 , wherein said non-viral vector is a DNA plasmid. 
     
     
         107 . A host cell transformed, transduced, infected or transfected with the nucleic acid molecule according to  claim 102 . 
     
     
         108 . A method of producing an immunogenic mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 16 or position 72, further comprising at least one additional mutation in the A subunit of the cholera holotoxin at an amino acid position other than the amino acid positions 16, 68 and 72 in the A subunit, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT,
 comprising transforming, infecting, transducing or transfecting a host cell with the nucleic acid molecule according to  claim 102 , and culturing the host cell under conditions which permit expression of said recombinant immunogenic detoxified protein by the host cell.   
     
     
         109 . An immunogenic, mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 72, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT. 
     
     
         110 . An immunogenic composition comprising a mutant cholera holotoxin (CT-CRM) of  claim 109 , wherein the mutant holotoxin enhances the immune response in a vertebrate host to an antigen. 
     
     
         111 . The composition according to  claim 110 , further comprising an antigen derived from the member of the group consisting of a pathogenic bacterium, pathogenic virus, pathogenic fungus, pathogenic parasite, a cancer cell, a tumor cell, an allergen and a self-molecule, and a protein, polypeptide, peptide or fragment derived from said antigen. 
     
     
         112 . The composition according to  claim 111 , wherein the bacterial antigen is selected from the bacterial species consisting of typable and non-typable  Haemophilus influenzae, Haemophilis somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neiserria gonorrhoea, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diptherieae, Mycobacterium tuberculosis, Mycobacterium avium - Mycabacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae  and  Mycoplasma galliseptium.    
     
     
         113 . The composition according to  claim 111 , wherein the  Haemophilus influenzae  antigen is selected from the group consisting of the  Haemophilus influenzae  P4 outer membrane protein, the  Haemophilus influenzae  P6 outer membrane protein and  Haemophilus influenzae  adherence and penetration protein (Hap s ). 
     
     
         114 . The composition according to  claim 113 , wherein the  Helicobacter Pylori  antigen is the  Helicobacter pylori  urease protein. 
     
     
         115 . The composition according to  claim 113 , wherein the  Neisseria meningitidis  antigen is selected from the group consisting of the  Neisseria meningitidis  Group B recombinant class 1 pilin (rpilin) and the  Neisseria meningitidis  Group B class 1 outer membrane protein (PorA). 
     
     
         116 . The composition according to  claim 111 , wherein the viral antigen is selected from the viral species consisting of Respiratory syncytial virus, Parainfluenza virus types, 1,2,3, Human metapneumovirus, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Human immunodeficiency virus, Hepatitis A, virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, poliovirus, rotavirus, caliciviruses, measles virus, mumps virus, Rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, avian pneumovirus (formerly turkey rhinotracheitis virus), Hendra virus, Nipah virus, coronavirus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, feline infectious peritonitis virus, avian infectious bursal disease virus, Newcastle disease virus, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and the encephalitis viruses. 
     
     
         117 . The composition according to  claim 116 , wherein the respiratory syncytial virus antigen is the respiratory syncytial virus fusion protein. 
     
     
         118 . The composition according to  claim 116 , wherein the herpes simplex virus (HSV) antigen is the herpes simplex virus (HSV) type 2 glycoprotein D (gD2). 
     
     
         119 . The composition according to  claim 111 , wherein the fungal antigen is from a fungus selected from the group of pathogenic fungi consisting of  Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus  and  Histoplasma.    
     
     
         120 . The composition according to  claim 111 , wherein the parasite antigen is from a parasite selected from the group of pathogenic parasites consisting of  Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii  and  Pneumocystis carinii.    
     
     
         121 . The composition according to  claim 111 , wherein said cancer or tumor cell antigen is selected from the group consisting of prostate specific antigen, carcinoembryonic antigen, MUC-1, Her2, CA-125, MAGE-3, a hormone, and a hormone analog. 
     
     
         122 . The composition according to  claim 111 , wherein said antigen is a polypeptide, peptide or fragment derived from amyloid precursor protein, or an allergen. 
     
     
         123 . The composition according to  claim 112 , wherein the amyloid precursor protein antigen is the Aβ peptide, which is a 42 amino acid fragment of amyloid precursor protein, or a fragment of the Aβ peptide. 
     
     
         124 . The composition according to  claim 113 , further comprising a diluent, excipient or carrier. 
     
     
         125 . The composition according to  claim 114 , further comprising a second adjuvant in addition to the mutant cholera holotoxin. 
     
     
         126 . A method for enhancing the immune response of a vertebrate host to an antigen, said method comprising administering to the host the composition according to  claim 110 . 
     
     
         127 . An isolated and purified DNA sequence encoding an immunogenic, mutant cholera holotoxin of  claim 109 . 
     
     
         128 . A nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin according to  claim 108 , and wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of said mutant holotoxin in a host cell. 
     
     
         129 . The molecule according to  claim 128 , wherein said regulatory sequence is an inducible promoter. 
     
     
         130 . The molecule according to  claim 128 , wherein said promoter is the arabinose inducible promoter. 
     
     
         131 . The molecule according to  claim 128 , wherein said molecule is a viral or non-viral vector. 
     
     
         132 . The molecule according to  claim 131 , wherein said non-viral vector is a DNA plasmid. 
     
     
         133 . A host cell transformed, transduced, infected or transfected with the nucleic acid molecule according to  claim 128 . 
     
     
         134 . A method of producing an immunogenic mutant cholera holotoxin (CT-CRM) comprising an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein said subunit A comprises an amino acid substitution in the wild-type CT subunit A amino acid position 72, and wherein said mutant CT-CRM has reduced toxicity compared to said wild-type CT,
 comprising transforming, infecting, transducing or transfecting a host cell with the nucleic acid molecule according to  claim 128 , and culturing the host cell under conditions which permit expression of said recombinant immunogenic detoxified protein by the host cell.