Mutant forms of cholera holotoxin as an adjuvant

Abstract

Mutant cholera holotoxins having single or double amino acid substitutions or insertions have reduced toxicity compared to the wild-type cholera holotoxin. The mutant cholera holotoxins are useful as adjuvants in antigenic 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 holotoxin (CT), wherein said subunit A comprises a mutant subunit A with at least one mutation selected from the group consisting of 
 (a) at least a single amino acid insertion in the amino acid position 49 in the A subunit,    (b) at least a double amino acid insertion in the amino acid positions 35 and 36 in the A subunit,    (c) at least a single amino acid substitution in the amino acid position 30; and    (d) at least a double amino acid insertion in the amino acid positions 31 and 32 in the A subunit,    wherein the mutant CT-CRM has reduced toxicity compared to said wild-type CT.    
     
     
         2 - 3 . (canceled)  
     
     
         4 . The CT-CRM according to  claim 1 , further comprising at least one additional mutation in the A subunit of the cholera holotoxin at an amino acid position other than that of any of (a) through (d).  
     
     
         5 . The CT-CRM according to  claim 4 , wherein said 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 amino acid 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.  
     
     
         6 - 65 . (canceled)  
     
     
         66 . The CT-CRM according to  claim 1 , wherein the holotoxin has a mutant subunit A in which the amino acid histidine is inserted in the amino acid position 49 in the A subunit, between the wild-type amino acid positions 48 and 49.  
     
     
         67 . The CT-CRM according to  claim 1 , wherein the holotoxin has a mutant subunit A in which the amino acids glycine and proline are inserted in the amino acid positions 35 and 36 in the A subunit between wild-type amino acid positions 34 and 35.  
     
     
         68 . The CT-CRM according to  claim 1 , wherein the holotoxin has a mutant subunit A in which the amino acid tyrosine in the amino acid position 30 in the A subunit is substituted with a tryptophan.  
     
     
         69 . The CT-CRM according to  claim 1 , wherein the holotoxin has a mutant subunit A in which amino acids alanine and histidine are inserted in the amino acid positions 31 and 32 in the A subunit between wild-type amino acid positions 30 and 31.  
     
     
         70 . The CT-CRM according to  claim 69 , wherein the amino acid tyrosine in the amino acid position 30 in the A subunit is substituted with a tryptophan.  
     
     
         71 . 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.  
     
     
         72 . The composition according to  claim 71 , wherein said immunogenic, mutant cholera holotoxin (CT-CRM) comprises an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein the amino acid histidine is inserted in the amino acid position 49 in the A subunit, between wild-type amino acid positions 48 and 49.  
     
     
         73 . The composition according to  claim 71 , wherein said immunogenic, mutant cholera holotoxin (CT-CRM) comprises an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein the amino acids glycine and proline are inserted in the amino acid positions 35 and 36 in the A subunit between wild-type amino acid positions 34 and 35.  
     
     
         74 . The composition according to  claim 71 , wherein said immunogenic, mutant cholera holotoxin (CT-CRM) comprises an amino acid sequence of subunit A of the wild-type cholera toxin (CT), wherein the amino acid tyrosine in the amino acid position 30 in the A subunit is substituted with a tryptophan, and wherein amino acids alanine and histidine are inserted in the amino acid positions 31 and 32 in the A subunit between wild-type amino acid positions 30 and 31.  
     
     
         75 . The composition according to  claim 71 , further comprising an antigen selected from the group consisting of a pathogenic bacterium, a pathogenic virus, a pathogenic fungus and a 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.  
     
     
         76 . The composition according to  claim 75 , wherein the bacterial antigen is selected from a bacterial species consisting of typable and non-typable  Haemophius influenzae, Haemophilus somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, 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 epidermis, Clostridium tetani, Leptospira interrogans, Borrelia burdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleauropneumoniae and Mycoplasma galliseptium.    
     
     
         77 . The composition according to  claim 76 , wherein the  Haemophilus influenza  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 ).  
     
     
         78 . The composition according to  claim 76 , wherein the  Helicobacter Pylori  antigen is the  Helicobacter pylori  urease protein.  
     
     
         79 . The composition according to  claim 77 , 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).  
     
     
         80 . The composition according to  claim 75 , 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, 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.  
     
     
         81 . The composition according to  claim 80 , wherein the respiratory syncytial virus is the respiratory syncytial virus fusion protein.  
     
     
         82 . The composition according to  claim 80 , wherein the herpes simplex virus (HSV) antigen is the herpes simplex virus (HSV) type 2 glycoprotein D (gD2).  
     
     
         83 . The composition according to  claim 75 , wherein the fungal antigen is derived from a fungus selected from the group of pathogenic fungi consisting of  Aspergillis, Blastomyces, Candida, Coccidiodes, Cryptococcus  and  Histoplasma.    
     
     
         84 . The composition according to  claim 75 , wherein the parasite antigen is from a parasite selected from the group of pathogenic parasites consisting of  Leishmania major, Ascaris, Trichuris, Giardia, Schistoma, Cryptosporidium, Trichomonas, Toxoplasma gondii  and  Pneumocystis carinii.    
     
     
         85 . The composition according to  claim 75 , wherein the 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.  
     
     
         86 . The composition according to  claim 75 , wherein said antigen is a polypeptide, peptide or fragment derived from amyloid precursor protein, or an allergen.  
     
     
         87 . The composition according to  claim 86 , wherein said amyloid precursor protein is the Aβ peptide, which is a 42 amino acid fragment of amyloid precursor protein, or a fragment of the Aβ peptide.  
     
     
         88 . The composition according to  claim 71 , further comprising a diluent, excipient or carrier.  
     
     
         89 . The composition according to  claim 71 , further comprising a second adjuvant in addition to the mutant cholera holotoxin.  
     
     
         90 . A method for enhancing the immune response of a vertebrate host to an antigen, said method comprising administering to the host 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.  
     
     
         91 . An isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin of  claim 1 .  
     
     
         92 . The nucleic acid sequence according to  claim 91 , wherein said mutant cholera holotoxin (CT-CRM) comprises a single amino acid insertion wherein the amino acid residue histidine is inserted in the amino acid position 49 in the A subunit between wild-type amino acid positions 48 and 49.  
     
     
         93 . The nucleic acid sequence according to  claim 91 , wherein said mutant cholera holotoxin (CT-CRM) comprises a double amino acid insertion wherein the amino acid residues glycine and proline are inserted in the amino acid positions 35 and 36 in the A subunit between wild-type amino acid positions 34 and 35.  
     
     
         94 . The nucleic acid sequence according to  claim 91 , wherein said mutant cholera holotoxin (CT-CRM) comprises a single amino acid substitution and a double amino acid insertion wherein the amino acid tyrosine in the amino acid position 30 in the A subunit is substituted with a tryptophan, and wherein amino acids alanine and histidine are inserted in the amino acid positions 31 and 32 in the A subunit between wild-type amino acid positions 30 and 31.  
     
     
         95 . A nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding the immunogenic, mutant cholera holotoxin of  claim 91 , wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of the mutant holotoxin in a host cell.  
     
     
         96 . The molecule according to  claim 95 , wherein said regulatory sequence is an inducible promoter.  
     
     
         97 . The molecule according to  claim 96 , wherein said promoter is the arabinose inducible promoter.  
     
     
         98 . The molecule according to  claim 95 , wherein said molecule is a viral or non-viral vector.  
     
     
         99 . The molecule according to  claim 98 , wherein said non-viral vector is a DNA plasmid.  
     
     
         100 . A host cell transformed, transduced, infected or transfected with a nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin of  claim 1 , wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of the mutant holotoxin in a host cell.  
     
     
         101 . A method of producing an immunogenic, mutant cholera holotoxin, wherein the cholera holotoxin has reduced toxicity compared to a wild-type cholera holotoxin comprising culturing a host cell transformed, transduced, infected or transfected with a nucleic acid molecule comprising an isolated and purified nucleic acid sequence encoding an immunogenic, mutant cholera holotoxin of  claim 1 , wherein the sequence encoding the immunogenic, mutant cholera holotoxin is operatively linked to regulatory sequences enabling expression of the mutant holotoxin in a host cell under conditions which permit the expression of said immunogenic, mutant cholera holotoxin by the host cell.