U.S. patent application number 11/876418 was filed with the patent office on 2008-09-04 for mutant forms of cholera holotoxin as an adjuvant.
This patent application is currently assigned to Wyeth Holdings Corporation. Invention is credited to Bruce A. Green, Randall K. Holmes, Michael G. Jobling, Duzhang Zhu.
Application Number | 20080213303 11/876418 |
Document ID | / |
Family ID | 23142433 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080213303 |
Kind Code |
A1 |
Green; Bruce A. ; et
al. |
September 4, 2008 |
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.
Inventors: |
Green; Bruce A.; (New City,
NY) ; Holmes; Randall K.; (Golden, CO) ;
Jobling; Michael G.; (Aurora, CO) ; Zhu; Duzhang;
(Pomona, NY) |
Correspondence
Address: |
HOWSON AND HOWSON
SUITE 210, 501 OFFICE CENTER DRIVE
FT WASHINGTON
PA
19034
US
|
Assignee: |
Wyeth Holdings Corporation
Madison
NJ
The Regents of the University of Colorado, A Body Corporate
Health Science Center
Aurora
CO
|
Family ID: |
23142433 |
Appl. No.: |
11/876418 |
Filed: |
October 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10478307 |
Dec 4, 2003 |
7285281 |
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PCT/US02/20978 |
Jun 5, 2002 |
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11876418 |
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60296537 |
Jun 7, 2001 |
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Current U.S.
Class: |
424/200.1 ;
435/325; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
Y02A 50/489 20180101;
Y02A 50/423 20180101; C07K 14/28 20130101; Y02A 50/30 20180101;
A61K 2039/55544 20130101; A61P 33/02 20180101; A61P 35/00 20180101;
Y02A 50/41 20180101; A61K 39/00 20130101; A61P 31/00 20180101; A61P
31/12 20180101; Y02A 50/464 20180101; A61P 31/04 20180101; A61K
39/39 20130101; A61P 31/10 20180101 |
Class at
Publication: |
424/200.1 ;
530/350; 536/23.7; 435/325; 435/69.3 |
International
Class: |
A61K 39/106 20060101
A61K039/106; C07K 14/195 20060101 C07K014/195; C12N 15/31 20060101
C12N015/31; C12N 5/10 20060101 C12N005/10; C12P 21/00 20060101
C12P021/00 |
Claims
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.sub.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 A.beta. peptide, which is a 42
amino acid fragment of amyloid precursor protein, or a fragment of
the A.beta. 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.sub.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 A.beta. peptide, which is a 42
amino acid fragment of amyloid precursor protein, or a fragment of
the A.beta. 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.sub.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 A.beta. peptide, which is a 42
amino acid fragment of amyloid precursor protein, or a fragment of
the A.beta. 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.sub.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 A.beta. peptide, which is a 42
amino acid fragment of amyloid precursor protein, or a fragment of
the A.beta. 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.sub.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 A.beta. peptide, which is a 42
amino acid fragment of amyloid precursor protein, or a fragment of
the A.beta. 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.
Description
CROSS-REFERENCE TO OTHER APPLICATIONS
[0001] This application claims the benefit of the priority of U.S.
provisional patent application No. 60/296,537, filed Jun. 7,
2001.
BACKGROUND OF THE INVENTION
[0002] The body's immune system activates a variety of mechanisms
for attacking pathogens (Janeway, Jr, C A. and Travers P., eds., in
Immunobiology, "The Immune System in Health and Disease," Second
Edition, Current Biology Ltd., London, Great Britain (1996)).
However, not all of these mechanisms are necessarily activated
after immunization. Protective immunity induced by immunization is
dependent on the capacity of an immunogenic composition to elicit
the appropriate immune response to resist or eliminate the
pathogen. Depending on the pathogen, this may require a
cell-mediated and/or humoral immune response.
[0003] Many antigens are poorly immunogenic or non-immunogenic when
administered by themselves. Strong adaptive immune responses to
antigens almost always require that the antigens be administered
together with an adjuvant, a substance that enhances the immune
response (Audbert, F. M. and Lise, L. D. 1993 Immunology Today, 14:
281-284).
[0004] The need for effective immunization procedures is
particularly acute with respect to infectious organisms that cause
acute infections at, or gain entrance to the body through, the
gastrointestinal, pulmonary, nasopharyngeal or genitourinary
surfaces. These areas are bathed in mucus, which contains
immunoglobulins consisting largely of secretory immunoglobulin IgA
(Hanson, L. A., 1961 Intl. Arch. Allergy Appl. Immunol., 18,
241-267; Tomasi T. B., and Zigelbaum, S., 1963 J. Clin. Invest.,
42, 1552-1560, Tomasi, T. B., et al., 1965 J. Exptl. Med., 121,
101-124). This immunoglobulin is derived from large numbers of
IgA-producing plasma cells, which infiltrate the lamina propria
regions underlying the mucosal membranes (Brandtzaeg, P., and
Baklein, K, 1976 Scand. J. Gastroenterol., 11 (Suppl. 36), 1-45;
and Brandtzaeg, P., 1984 "Immune Functions of Human Nasal Mucosa
and Tonsils in Health and Disease" page 28 et seq. in Immunology of
the Lung and Upper Respiratory Tract, Bienstock, J., ed.,
McGraw-Hill, New York, N.Y.). The secretory immunoglobulin IgA is
specifically transported to the luminal surface through the action
of the secretory component (Solari, R., and Kraehenbuhi, J-P, 1985
Immunol. Today, 6, 17-20).
[0005] Parenteral immunization regimens are usually ineffective in
inducing secretory IgA responses. Secretory immunity is most often
achieved through the direct immunization of mucosally associated
lymphoid tissues. Following their induction at one mucosal site,
the precursors of IgA-producing plasma cells extravasate and
disseminate to diverse mucosal tissues where final differentiation
to high-rate IgA synthesis occurs (Crabbe, P. A., et al., 1969 J.
Exptl. Med., 130, 723-744; Bazin, H., et al., 1970 J. Immunol.,
105, 1049-1051; Craig, S. W., and Cebra, J. J., 1971 J. Exptl.
Med., 134, 188-200). Extensive studies have demonstrated the
feasibility of mucosal immunization to induce this common mucosal
immune system (Mestecky, J., et al., 1978 J. Clin. Invest., 61,
731-737), but with rare exceptions the large doses of antigen
required to achieve effective immunization have made this approach
impractical for purified antigens.
[0006] Among the strategies investigated to overcome this problem
is the use of mucosal adjuvants. A number of adjuvants that enhance
the immune response of antigens are known in the prior art (Elson,
C. O., and Ealding, W., 1984 J. Immunol., 132, 2736-2741). These
adjuvants, when mixed with an antigen, render the antigen
particulate, helping retain the antigen in the body for longer
periods of time, thereby promoting increased macrophage uptake and
enhancing immune response. However, untoward reactions elicited by
many adjuvants or their ineffectiveness in inducing mucosal
immunity have necessitated the development of better adjuvants for
delivery of immunogenic compositions. Unfortunately, adjuvant
development to date has been largely an empirical exercise
(Janeway, Jr., et al, cited above at pages 12-25 to 12-35). Thus, a
rational and a more direct approach is needed to develop effective
adjuvants for delivery of antigenic compositions.
[0007] It has been reported that the toxin secreted by the
Gram-negative bacterium Vibrio cholerae (V. cholerae), the
causative agent of the gastrointestinal disease cholera, is
extremely potent as an adjuvant. Cholera toxin (CT) has been
reported as a 382 amino acid sequence (SEQ ID NO: 1) (Mekalanos, J.
J., et al., 1983 Nature, 306, 551-557), which has an 18 amino acid
signal (amino acids 1 to 18 of SEQ ID NO: 1). The cholera toxin
holotoxin molecule is a hexaheteromeric complex that consists of a
peptide subunit designated CT-A (SEQ ID NO: 2 or amino acids 19 to
258 of SEQ ID NO: 1), which is responsible for the enzymatic
activity of the toxin, and five identical peptide subunits, each
designated CT-B (each having a 21 amino acid signal (amino acids
259 to 379 of SEQ ID NO: 1), followed by the CT-B peptide subunit
(amino acids 280-382 of SEQ ID NO: 1)), which is involved in the
binding of the toxin to the intestinal epithelial cells as well as
other cells which contain ganglioside GM.sub.1 on their surface
(Gill, D. M., 1976 Biochem., 15, 1242-1248; Cuatrecasas, P., 1973
Biochem., 12, 3558-3566). CT produced by V. cholerae has the CT-A
subunit proteolytically cleaved within the single disulfide-linked
loop between the cysteines at amino acid positions 187 and 199 of
the mature CT-A (SEQ ID NO: 2) to produce an enzymatically active
A1 polypeptide (Kassis, S., et al., 1982 J. Biol. Chem., 257,
12148-12152), and a smaller polypeptide A2, which links fragment A1
to the CT-B pentamer (Mekalanos, J. J., et al., 1979 J. Biol.
Chem., 254, 5855-5861). Toxicity results when the enzymatically
active fragment CT-A1, upon entry into enterocytes, ADP-ribosylates
a regulatory G-protein (Gs.alpha.). This leads to constitutive
activation of adenylate cyclase, increased intracellular
concentration of cAMP, and secretion of fluid and electrolytes into
the lumen of the small intestine, thereby causing toxicity (Gill,
D. M., and Meren, R., 1978 Proc. Natl. Acad. Sci., USA, 75,
3050-3054). In vitro, ADP-ribosyl transferase activity of CT is
stimulated by the presence of accessory proteins called ARFs, small
GTP-binding proteins known to be involved in vesicle trafficking
within the eukaryotic cell (Welsh, C. F., et al., "ADP-Ribosylation
Factors: A Family of Guanine Nucleotide-Binding Proteins that
Activate Cholera Toxin and Regulate Vesicular Transport", pages
257-280 in Handbook of Natural Toxins: Bacterial Toxins and
Virulence Factors in Disease Vol. 8 (Moss, J., et al., eds., Marcel
Dekker, Inc., New York, N.Y. (1995)).
[0008] Co-administration of CT with an unrelated antigen has been
reported to result in the induction of concurrent circulating and
mucosal antibody responses to that antigen (Mekalanos, J. J., et
al., 1983 Nature, 306, 551-557). To minimize the occurrence of
undesirable symptoms such as diarrhea caused by wild-type CT in
humans, it would be preferable to use as an adjuvant a form of the
CT holotoxin that has substantially reduced toxicity. Mutants of CT
have been suggested as a means for achieving a more useful
adjuvant. One way to rationally design mutant cholera toxin
holotoxins (CT-CRMs) with substantially reduced toxicity is to
identify and alter amino acid residues in the toxin molecule that
are completely consented in the family of cholera (CT) and related
heat-labile enterotoxins (LT-I, LT-IIa and LT-IIb) of E. coli.
Another rational way to generate mutant CT-CRMs with substantially
reduced toxicity is to alter amino acid residues in the holotoxin
molecule that have been identified as being important for
NAD-binding based on the structural alignment of the CT backbone
with the backbone of related toxins possessing ADP-ribosyl
transferase enzyme activity such as diphtheria toxin (DT) and
pertussis toxin (PT) (Holmes, R. K., "Heat-labile enterotoxins
(Escherichia coli)" in Guidebook to Protein Toxins and their Use in
Cell Biology, Montecucco, C. and Rappnoli, R., Eds., Oxford Univ.
Press, Oxford, England (1997); and Holmes, R-- K et al, "Cholera
toxins and related enterotoxins of Gram-negative bacteria", pp.
225-256 in Handbook of Natural Toxins: Bacterial Toxins and
Virulence Factors in Disease, vol. 8, Moss. J., et al, Eds., Marcel
Dekker, Inc., New York, N.Y. 1995).
[0009] Recently, one such rationally-designed,
genetically-detoxified mutant of CT was disclosed wherein a single
nonconservative amino acid substitution (glutamic acid to
histidine) was introduced by altering the amino acid at position 29
in the A subunit (designated CT-CRM.sub.E29H). The resulting mutant
cholera holotoxin demonstrated substantially reduced enzymatic
toxicity, but with superior adjuvanting and immunogenic properties
(International Patent Publication No. WO 00/18434, incorporated in
its entirety by reference).
[0010] Thus, there is a need to identify and/or rationally design
additional mutant forms of the CT holotoxin that have substantially
reduced toxicity, yet possess the same or enhanced adjuvanting
properties as the wild-type CT holotoxin.
SUMMARY OF THE INVENTION
[0011] In one aspect this invention provides novel mutant,
immunogenic forms of cholera holotoxin (CT-CRMs) having
significantly reduced toxicity compared to a wild-type CT, but
which retain their ability as powerful stimulators of the immune
system. Specifically, the invention pertains to four mutant cholera
holotoxins (CT-CRMs), desirably generated by site-directed
mutagenesis and having substantially reduced toxicity compared to a
wild-type CT, but with no loss in adjuvanting properties.
[0012] In one embodiment, a novel CT-CRM of this invention
comprises the amino acid sequence of CT subunit A or a fragment
thereof wherein the amino acid residue in the amino acid position
16 of the A subunit is substituted with another amino acid which
substitution results in a substantial reduction in toxicity. In a
preferred embodiment of the invention, the amino acid isoleucine at
amino acid position 16 of the A subunit is substituted with an
alanine. For determination of the amino acid position, the sequence
of CT-A is exemplified in SEQ ID NO: 2. However, other variants and
fragments of CT-A may also be employed.
[0013] In another embodiment, a novel CT-CRM of this invention
comprises the amino acid sequence of CT subunit A or a fragment
thereof, wherein the amino acid residue in the amino acid position
72 of the A subunit is substituted with another amino acid which
substitution results in a substantial reduction in toxicity. In a
preferred embodiment of the invention, the amino acid valine at the
amino acid position 72 of the A subunit is substituted with a
tyrosine.
[0014] In another embodiment, a novel immunogenic, mutant CT-CRM of
this invention has substantially reduced CT toxicity and comprises
the amino acid sequence of subunit A of CT or a fragment thereof,
wherein both amino acid residues in the amino acid positions 16 and
68 in the A subunit are substituted with amino acids different from
that present in amino acid positions 16 and 68 of wild-type CT,
which substitutions result in a substantial reduction in toxicity.
In a preferred embodiment of this aspect of the invention, the
amino acid alanine is substituted for isoleucine at the amino acid
position 16 in the A subunit, and the amino acid tyrosine is
substituted for serine at amino acid position 68 in the A
subunit.
[0015] In yet another embodiment, a novel immunogenic, mutant
CT-CRM of this invention has substantially reduced CT toxicity and
comprises the amino acid sequence of subunit A of CT or a fragment
thereof, wherein both amino acid residues in the amino acid
positions 68 and 72 in the A subunit are substituted with amino
acids different from that present in amino acid positions 68 and 72
of wild-type CT, which substitutions result in a substantial
reduction in toxicity. In a preferred embodiment of this aspect of
the invention, the amino acid tyrosine is substituted for serine at
amino acid position 68 of the A subunit, and the amino acid
tryosine is substituted for valine at amino acid position 72 of the
A subunit.
[0016] In another aspect, the invention provides a method for
producing the novel CT-CRMs described above by employing
site-directed mutagenesis of the DNA encoding the A subunit in the
wild-type CT using conventional techniques, such that the
mutagenized CT now has substantially reduced toxicity without
compromising the toxin's ability to stimulate an immune
response.
[0017] In yet another aspect of the invention, there is provided an
immunogenic composition comprising a selected antigen, a mutant
CT-CRM as described above as an adjuvant to enhance the immune
response in a vertebrate host to the antigen, and a
pharmaceutically acceptable diluent, excipient or carrier.
Preferably, the CT-CRM is useful for the generation or enhancement
of systemic and/or mucosal antigenic immune responses in a
vertebrate host to the selected antigen. The selected antigen may
be a polypeptide, peptide or fragment derived from a pathogenic
virus, bacterium, fungus or parasite. The selected antigen may be a
polypeptide, peptide or fragment derived from a cancer cell or
tumor cell. The selected antigen may be a polypeptide, peptide or
fragment derived from an allergen so as to interfere with the
production of IgE so as to moderate allergic responses to the
allergen. The selected antigen may be a polypeptide, peptide or
fragment derived from a molecular portion thereof which represents
those produced by a host (a self molecule) in an undesired manner,
amount or location, such as those from amyloid precursor protein,
so as to prevent or treat disease characterized by amyloid
deposition in a vertebrate host.
[0018] In still another aspect, this invention provides a method
for using these CT-CRMs as adjuvants in immunogenic compositions or
methods for increasing the ability of an antigenic composition
containing a selected antigen as described above to elicit an
immune response in vertebrate host by including an effective
adjuvanting amount of one or more of the novel detoxified mutant
cholera holotoxins (CT-CRMs) described above.
[0019] In yet a further aspect of the invention there are provided
DNA sequences encoding the novel immunogenic, mutant CT-CRMs with
substantially reduced toxicity as described above. Preferably, the
DNA sequence(s) encodes for both the mutant A subunit with reduced
toxicity and subunit B. Alternatively, the DNA sequence may encode
only the mutant A subunit with reduced toxicity, where the mutant
CT-A is fused with an additional binding domain, or is co-expressed
with LT-B and allowed to co-assemble.
[0020] In a further aspect of the invention, there is provided a
plasmid containing isolated and purified DNA sequence comprising a
DNA sequence encoding an immunogenic, detoxified, mutant cholera
holotoxin as described herein, and wherein such a DNA sequence is
operatively linked to regulatory sequences which direct expression
of the CT-CRM in a host cell. Preferably the regulatory sequences
comprise an arabinose inducible promoter. In one embodiment of this
aspect, the invention relates to a plasmid, designated pLP903, that
contains an isolated and purified DNA sequence comprising a DNA
sequence encoding an immunogenic mutant CT-CRM with substantially
reduced toxicity wherein the amino acid alanine is substituted for
isoleucine at amino acid position 16 in the A subunit. In a second
embodiment of this aspect, the invention relates to a plasmid,
designated pLP905, that contains an isolated and purified DNA
sequence comprising a DNA sequence encoding an immunogenic mutant
CT-CRM with substantially reduced toxicity wherein the amino acid
tyrosine is substituted for valine at the amino acid position 72 in
the A subunit. In a third embodiment of his aspect, the invention
relates to a plasmid, designated pLP904, that contains an isolated
and purified DNA sequence comprising a DNA sequence encoding an
immunogenic, mutant CT-CRM with substantially reduced toxicity
wherein the amino acid alanine is substituted for isoleucine at
amino acid position 16, and amino acid tyrosine is substituted for
serine at amino acid position 68 in the A subunit. In yet an
additional embodiment of this aspect, the invention relates to a
plasmid, designated pLP906, that contain an isolated and purified
DNA sequence comprising a DNA sequence encoding an immunogenic,
mutant CT-CRM with substantially reduced toxicity wherein the amino
acid tyrosine is substituted for serine at the amino acid position
68, and amino acid tyrosine is substituted for valine at amino acid
position 72 in the A subunit.
[0021] In a further aspect of the invention, there is provided a
suitable host cell line transformed, infected, transduced or
transfected with a plasmid as described herein. The immunogenic,
detoxified, mutant cholera holotoxins are produced by transforming,
infecting, transducing or transfecting a suitable host cell with
one of the plasmids described above and culturing the host cell
under culture conditions which permit the expression by the host
cell of said recombinant immunogenic, mutant cholera holotoxin
protein with substantially reduced toxicity.
[0022] These and other aspects of the invention will be apparent to
one of skill in the art upon reading of the following detailed
description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Mutant forms of cholera holotoxin that exhibit reduced
toxicity, but which retain their superior adjuvanting properties,
and the utility of these mutant forms of CTs as adjuvants in
immunogenic compositions are described herein.
[0024] A. Mutant, Detoxified Cholera Toxin Holotoxins
[0025] Novel mutant, detoxified immunogenic forms of cholera
holotoxin (CT-CRMs) of this invention are characterized by
significantly reduced toxicity compared to a wild-type CT. However,
such CT-CRMs retain their ability as powerful stimulators of the
immune system. The CT-CRMs of this invention are characterized by
one or several amino acid substitutions in the mature CT-A subunit
of cholera toxin. The various mutant CT-A subunits of this
invention also retained their ability to assemble with CT-B
subunits to form mutant CT holotoxins that resembled wild-type CT
in adjuvanticity, but which exhibited substantially reduced
toxicity compared to the wild-type CT. The CT-CRMs of this
invention may employ mutant or altered CT-A subunits associated
with wild-type CT-B subunits to create a functional holotoxin.
Alternatively, the CT-CRMs of this invention may comprise the
altered or mutated CT-A subunits associated with altered or mutated
CT-B subunits.
[0026] For determination of the amino acid position numbers
describing the locations of the amino acid substitutions in the
CT-CRMs of this invention, the sequence of mature CT-A is
exemplified as SEQ ID NO: 2, i.e., amino acids 19-258 of SEQ ID NO:
1, a wild-type CT sequence. The nucleotide sequence encoding the A
subunit of the cholera holotoxin is set forth in International
patent publication No. WO 93/13202. Similarly, a suitable mature
CT-B sequence may be illustrated by amino acids 280-382 of SEQ ID
NO: 1. However, other variants, biotypes and fragments of CT-A and
CT-B of V. cholerae may also be employed as sequences containing
the amino acid substitutions described herein. See, for example,
the ELTOR biotype of C. Shi et al, 1993 Sheng Wu Hua Hsueh Tsa
Chih, 9(4):395-399; NCBI database locus No. AAC34728, and other
sources of variants of V. cholerae toxin.
[0027] Preferably, the amino acid substitutions resulting in the
CT-CRMs of this invention are the result of replacing one amino
acid with another amino acid having similar structural and/or
chemical properties, i.e. conservative amino acid replacements.
"Conservative" amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. For example, non-polar hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, tryptophan, and
methionine; polar/neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine;
positively charged (basic) amino acids include arginine, lysine,
and histidine; and negatively charged (acidic) amino acids include
aspartic acid and glutaric acid. This invention is exemplified by
CT-CRMs having a single amino acid substitution at either amino
acid position 16 or at amino acid position 72 or double amino acid
substitution at amino acid positions 16 and 68 or 68 and 72, as
summarized in Table 1.
TABLE-US-00001 TABLE 1 Single and Double CT-CRM Mutants Amino Acid
Substitution Native Mutant Abbreviation 16 Isoleucine.sub.16
Alanine.sub.16 CT-CRM.sub.I16A 72 Valine.sub.72 Tyrosine.sub.72
CT-CRM.sub.V72Y 16 and 68 Isoleucine.sub.16 Alanine.sub.16
CT-CRM.sub.I16A,S68Y Serine.sub.58 Tyrosine.sub.68 68 and 72
Serine.sub.68 Tyrosine.sub.68 CT-CRM.sub.S68Y,V72Y Valine.sub.72
Tyrosine.sub.72
[0028] Thus, in one embodiment, a novel CT-CRM of this invention
comprises the amino acid sequence of CT subunit A or a fragment
thereof, wherein the amino acid residue in the amino acid position
16 of the A subunit is substituted with another amino acid which
substitution results in a substantial reduction in toxicity. In a
preferred embodiment of the invention, the amino acid isoleucine at
amino acid position 16 of the A subunit is substituted with an
alanine. This CT-CRM.sub.I16A demonstrates superior adjuvanting
properties.
[0029] In another embodiment, a novel CT-CRM of this invention
comprises the amino acid sequence of CT subunit A or a fragment
thereof, wherein the amino acid residue in the amino acid position
72 of the A subunit is substituted with another amino acid, which
substitution results in a substantial reduction in toxicity. In a
preferred embodiment of the invention, the amino acid valine at the
amino acid position 72 of the A subunit is substituted with a
tyrosine, resulting in CT-CRM.sub.V72Y. This CT-CRM.sub.V72Y
demonstrates superior adjuvanting properties.
[0030] In another embodiment, a novel immunogenic, mutant CT-CRM of
his invention has substantially reduced CT toxicity and comprises
the amino acid sequence of subunit A of CT or a fragment thereof,
wherein both amino acid residues in the amino acid positions 16 and
68 in the A subunit are substituted with amino acids different from
that present in amino acid positions 16 and 68 of wild-type CT,
which substitutions result in a substantial reduction in toxicity.
In a preferred embodiment of this aspect of the invention, the
amino acid alanine is substituted for isoleucine at the amino acid
position 16 in the A subunit, and the amino acid tyrosine is
substituted for serine at amino acid position 68 in the A subunit,
resulting in CT-CRM.sub.I16A, S68Y, which demonstrates superior
adjuvanting properties
[0031] In yet another embodiment, a novel immunogenic, mutant
CT-CRM of this invention has substantially reduced CT toxicity and
comprises the amino acid sequence of subunit A of CT or a fragment
thereof, wherein both amino acid residues in the amino acid
positions 68 and 72 in the A subunit are substituted with amino
acids different from that present in amino acid positions 68 and 72
of wild-type CT, which substitutions result in a substantial
reduction in toxicity. In a preferred embodiment of this aspect of
the invention, the amino acid tyrosine is substituted for serine at
amino acid position 68 of the A subunit, and the amino acid
tyrosine is substituted for valine at amino acid position 72 of the
A subunit. This CT-CRM.sub.S68Y, V72Y demonstrates superior
adjuvanting properties.
[0032] The phenotypic effects of the novel CT-CRMs of Table 1 on
the structure and function of CT were assessed. The mutant A
subunits generated by site directed mutagenesis of the CT-encoding
gene were also able to assemble into immunoreactive holotoxin in
the presence of subunit B as determined by non-denaturing gel
electrophoresis assay (see Table 3, Example 2). Each mutant
holotoxin was also tested in a Y-1 adrenal tumor cell assay to
determine its residual toxicity compared to wild-type CT holotoxin
(see Tables 4 and 5, Example 3). The results presented in Table 4
demonstrate that the mutant CT-CRMs had substantially reduced
toxicity when compared with wild-type cholera holotoxin. The
residual toxicities of the CT-CRMs with single and double amino
acid substitutions were substantially reduced in comparison to that
of the wild-type CT.
[0033] Each of the mutant CT-CRMs was also compared to wild-type CT
in an ADP-ribosyltransferase activity assay (See Example 4). The
results, which were generally in agreement with the toxicity data
generated in the Y-1 adrenal cell assay, indicated that the
ADP-ribosyltransferase activity of the various CT-CRMs was
substantially diminished when compared to wild-type CT (Table 6).
The mutant with the largest ADP-ribosyl-transferase activity
appeared to be the double mutant CT-CRM.sub.I16A,S68Y. This
activity was approximately only 3.3% of wild-type CT. The enzyme
activity of CT-CRMs, CT-CRM.sub.V72Y, CT-CRM.sub.I16A, and
CT-CRM.sub.S68Y,V72Y, were 1.1%, 2.4% and 1.2% respectively of the
activity of the wild-type CT.
[0034] Still other CT-CRMs of this invention may contain at least
the single or double mutations described specifically above and at
least one additional mutation at a position other than at one or
more of the amino acid residues 16, 68, or 72 as set forth above.
International patent publication No. WO 93/13202, which is hereby
incorporated b, reference, describes a series of mutations in the
CT-A subunit that serve to reduce the toxicity of the cholera
holotoxin. These mutations include malting substitutions for the
arginine at amino acid 7, the aspartic acid at position 9, the
arginine at position 11, the glutaric acid at position 29, the
histidine at position 44, the valine at position 53, the arginine
at position 54, the serine at position 61, the serine at position
63, the histidine at position 70, the valine at position 97, the
tyrosine at position 104, the proline at position 106, the
histidine at position 107, the glutamic acid at position 110, the
glutamic acid at position 112, the serine at position 114, the
tryptophan at position 127, the arginine at position 146 and the
arginine at position 192. International patent publication No. WO
98/42375, which is hereby incorporated by reference, describes
making a substitution for the serine at amino acid 109 in the A
subunit, which serves to reduce the toxicity of the cholera
holotoxin.
[0035] Other useful CT-CRM mutant proteins useful in this invention
include a full-length holotoxin with one or more of the specific
mutations provided above, a polypeptide or a fragment thereof
containing the mutagenized residues described above and which
protein, polypeptide or fragment retains the adjuvanticity of
wild-type CT from which it is derived, but is characterized by
reduced toxicity.
[0036] Immunologically active fragments of these CT-CRMs with
reduced enzymatic activity may also be useful in the methods and
compositions of this invention. Fragments ordinarily will contain
at least at least about 25 contiguous amino acids of the CT-CRM
proteins containing the site of mutagenesis noted above. More
typically a CT-CRM fragment contains at least about 75 contiguous
amino acids. Another fragment of a CT-CRM contains at least about
100 contiguous amino acids. Still another embodiment of a CT-CRM
subunit A contains at least about 150 contiguous amino acids in
length.
[0037] A fragment of the CT-CRMs described herein is useful in the
methods and compositions described below if it generates or
enhances the immune response to selected antigens in the vertebrate
host, Fragments include truncations of the carboy-terminal region
of the CT-CRMs. For example, a CT-CRM truncated so that it contains
only a CT-A mutant subunit is a desirable fragment. Similarly, CT-A
subunits truncated at about residues 240 or 250 are desirable
fragments. Still other fragments CT-CRMs of this invention may be
selected. Additional fragments of the CT-CRM holotoxin may contain
less than five repetitions of the CT-B subunits or truncated CT-B
subunits. The foregoing fragments may also contain one or more of
the specific mutations described above.
[0038] Other suitable CT-CRM proteins may include those in which
one or more of the amino acid residues includes a substituted
group. Still another suitable CT-CRM holotoxin protein is one in
which the CT-CRM polypeptide is fused with another compound, such
as a compound to increase the half-life of the polypeptide (for
example, polyethylene glycol). Another suitable CT-CRM protein is
one in which additional amino acids are fused to the polypeptide,
such as a leader or secretory sequence, or a sequence which is
employed to enhance the immunogenicity of the CT-CRM protein. Still
other modifications of the CT-CRMs include the above-mentioned
deletion of the CT-A signal or leader sequence at the N terminus of
CT, i.e., amino acids 1-18 of SEQ ID NO: 1 and/or the deletion of
the CT-B signal or leader sequence at amino acids 259-279 of SEQ ID
NO: 1, and/or the deletion of other regions that do not effect
immunogenicity. Similarly, a modification of the CT-CRMs described
herein includes include replacing either signal or leader sequence
with another signal or leader sequence. See, e.g., U.S. Pat. No.
5,780,601, incorporated by reference herein.
[0039] Still another example of suitable CT-CRM proteins are those
in which optional amino acids (e.g., -Gly-Ser-) or other amino acid
or chemical compound spacers may be included at the termini of the
polypeptide for the purpose of linking multiple holotoxin proteins
together or to a carrier. For example, useful CT-CRMs may include
one or more of the above-described CT-CRMs coupled to a carrier
protein. Alternatively, a useful CT-CRM ray be present in a fusion
protein containing multiple CT-CRMs, optionally coupled to carrier
protein.
[0040] For these embodiments, the carrier protein is desirably a
protein or other molecule that can enhance the immunogenicity of
the selected CT-CRM. Such a carrier may be a larger molecule that
also has an adjuvanting effect. Exemplary conventional protein
carriers include, without limitation, E. coli DnaK protein,
galactokinase (Gall which catalyzes the first step of galactose
metabolism in bacteria), ubiquitin, .alpha.-mating factor,
.beta.-galactosidase, and influenza NS-1 protein. Toxoids (i.e.,
the sequence which encodes the naturally occurring toxin, with
sufficient modifications to eliminate its toxic activity) such as
diphtheria toxoid and tetanus toxoid, their respective toxins, and
any mutant forms of these proteins, such as CRM.sub.197 (a
non-toxic form of diphtheria toxin, see U.S. Pat. No. 5,614,382),
may also be employed as carriers. Other carriers include exotoxin A
of Pseudomonas aeruginosa, heat labile toxins of E. coli and
rotaviral particles (including rotavirus and VP6 particles).
Alternatively, a fragment or epitope of the carrier protein or
other immunogenic protein may be used. For example, a hapten may be
coupled to a T cell epitope of a bacterial toxin. See U.S. Pat. No.
5,785,973. Similarly a variety of bacterial heat shock proteins,
e.g., mycobacterial hsp-70 may be used. Glutathione-S-transferase
(GST) is another useful carrier. One of skill in the art can
readily select an appropriate carrier for use in this context. The
fusion proteins may be formed by standard techniques for coupling
proteinaceous materials. Fusions may be expressed from fused gene
constructs prepared by recombinant DNA techniques as described
below.
[0041] Other suitable CT-CRMs described herein can differ from the
specifically exemplified CT-CRMs by modifications that do not
revive enzymatic toxicity, and do not diminish adjuventicity, or by
combinations of such attributes. For example, conservative amino
acid changes may be made, which, although they alter the primary
sequence of the CT-CRM protein, do not normally alter its function.
In making such changes, the hydropathic index of amino acids can be
considered. The importance of the hydropathic amino acid index in
conferring interactive biologic function on a polypeptide is
generally understood in the art (Kyte & Doolittle, 1982 J. Mol.
Biol., 157(1):105-132). It is known that certain amino acids can be
substituted for other amino acids having a similar hydropathic
index or score and still result in a polypeptide with similar
biological activity. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. Those indices are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9), alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0042] It is believed that the relative hydropathic character of
the amino acid residue determines the secondary and tertiary
structure of the resultant polypeptide, which in turn defines the
interaction of the polypeptide with other molecules, such as
enzymes, substrates, receptors, antibodies, antigens, and the like.
It is known in the art that an amino acid can be substituted by
another amino acid having a similar hydropathic index and still
obtain a functionally equivalent polypeptide. In such changes, the
substitution of amino acids whose hydropathic indices are within
+/-2 is preferred, those within +/-1 are particularly preferred,
and those within +/-0.5 are even more particularly preferred.
[0043] Substitution of like amino acids can also be made on the
basis of hydrophilicity, particularly where the biologically
functional equivalent polypeptide or peptide thereby created is
intended for use in immunological embodiments. U.S. Pat. No.
4,554,101, incorporated herein by reference, states that the
greatest local average hydrophilicity of a polypeptide, as governed
by the hydrophilicity of its adjacent amino acids, correlates with
its immunogenicity and antigenicity, i.e. with a biological
property of the polypeptide.
[0044] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); proline (-0.5.+-.1); threonine (-0.4); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity
values are within is preferred; those within .+-.1 are particularly
preferred; and those within .+-.0.5 are even more particularly
preferred.
[0045] As outlined above, amino acid substitutions are generally
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions which take
various of the foregoing characteristics into consideration are
well known to those of skill in the art and include: arginine and
lysine; glutamate and aspartate; serine and threonine; glutamine
and asparagine; and valine, leucine and isoleucine.
[0046] In addition, modifications, which do not normally alter the
primary sequence of the CT-CRM protein, include in vivo or in vitro
chemical derivatization of polypeptides, e.g., acetylation,
methylation, or carboxylation. Also included as CT-CRMs of this
invention are these proteins modified by glycosylation, e.g., those
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing or in further processing steps;
or by exposing the polypeptide to enzymes which affect
glycosylation, such as mammalian glycosylating or deglycosylating
enzymes. Also embraced as CT-CRMs are the above-identified
mutagenized sequences, which have phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or
phosphothreonine.
[0047] Also included as CT-CRMs of this invention are the above
sequences that have been modified using ordinary molecular
biological techniques so as to improve their resistance to
proteolytic degradation or to optimize solubility properties. Among
such CT-CRMs are included those containing residues other than
naturally occurring L-amino acids, e.g., D-amino acids or
non-naturally occurring synthetic amino acids. Among other known
modifications which may be present in CT-CRMs of the present
invention are, without limitation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cystine, formation of
pyroglutamate, formylation, gamma-carboxylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination.
[0048] The mutant CT-CRMs of this invention are thus holotoxins and
exhibit reduced toxicity or are substantially less toxic than
wild-type CT. As used herein, the terms and phrases "the holotoxin
has reduced toxicity" or "substantially less toxic" or the like
mean that the CT-CRM mutant of this invention, such as the four
CT-CRM mutants described herein (CT-CRM.sub.I16A, CT-CRM.sub.V72Y,
CT-CRM.sub.I16A, S68Y, and CT-CRM.sub.S68Y, V72Y), exhibits a
substantially lower toxicity per unit of purified toxin protein
compared to the wild-type CT. This "reduced toxicity" enables each
mutant to be used as an adjuvant in an immunogenic composition
without causing significant side effects, particularly those known
to be associated with CT, e.g., diarrhea. As described in more
detail below, the mutant CT-CRMs of this invention display
significantly lower levels of toxicity than the wild-type CT in the
Y-1 mouse adrenal cell assay, and a significantly reduced
ADP-ribosyltransferase activity when compared to wild-type CT.
[0049] The immunogenic mutant CT-CRMs according to the present
invention exhibit a balance of reduced toxicity and retained
adjuvanticity, such that the resulting mutant CT protein functions
as an adjuvant while being tolerated safely by the vertebrate host
to which it is introduced. As indicated in the examples below,
results in murine model assay systems indicate that the mutant
CT-CRMs disclosed herein were able to significantly augment mucosal
and systemic immune responses following intranasal administration
of disparate antigens. Furthermore, even in the presence of
pre-existing anti-CT immune responses, the mutant CT-CRs were able
to serve as efficient mucosal adjuvants. The studies which support
these characteristics of the CT-CRMs of this invention are
summarized below and more specifically stated in the Examples.
[0050] To evaluate the efficacy of the mutant CT-CRMs as mucosal
adjuvants for compositions containing bacterial or viral antigens
that have been identified as candidates for inclusion in
immunogenic compositions, three disparate model antigen systems
were examined: (1) the recombinant P4 outer membrane protein (also
known as protein "e" (rP4)) of the nontypable Haemophilus
influenzae bacterium (NTHi), (see U.S. Pat. No. 5,601,831), (2) the
native UspA2 outer membrane protein of the Moraxella catarrhalis
bacterium (International Patent Publication No. WO 98/28333), and
(3) the native fusion glycoprotein (F protein) of respiratory
syncytial virus (RSV) (see U.S. Pat. No. 5,223,254). The mutant
CT-CRMs were compared with each other, and to CT-CRM.sub.E29H and
the wild-type CT as an adjuvant for the NTHi rP4. In a first study,
the adjuvanting ability of the mutant CT-CRM.sub.I16A to enhance
the induction of systemic and mucosal antibodies to rP4 were
assessed and compared with that of wild-type CT and
CT-CRM.sub.E29H. The results indicated that the CT-CRM.sub.I16A,
like the wild-type CT and CT-CRM.sub.E29H, augmented the capacity
of rP4 protein to elicit systemic and humoral immune responses (see
Tables 8 and 9). For example, six weeks after tertiary IN
immunization, the anti-rP4 IgG antibody titers of mice immunized
with rP4 protein formulated with either CT-CRM.sub.I16A or
CT-CRM.sub.E29H were 40 times greater than that of mice immunized
with the recombinant proteins in PBS alone. The antibody titers
(IgG) of mice administered the recombinant protein plus wild-type
CT holotoxin at a concentration of 1 .mu.g were elevated 67-fold in
comparison to antibody titers in mice administered recombinant rP4
alone in saline six weeks after the primary IN immunization. The
antibody titers of mice immunized with 1 .mu.g of the mutant,
CT-CRM.sub.E29H were elevated 48-fold over antibody titers in nice
immunized with rP4 alone. In comparison, the antibody titers of
mice immunized with 1 .mu.g and 0.1 .mu.g of the mutant,
CT-CRM.sub.I16A, were increased 15-fold and 27-fold respectively
over the anti-rP4 antibody titers in mice immunized with rP4 alone
in saline.
[0051] An examination of the protein-specific antibodies in the
mucosal secretions two weeks after tertiary immunization further
indicated that the CT-CRM.sub.I16A facilitated the generation of
local immune responses against the rP4 protein. Moreover, the
anti-rP4 antibody titers were comparable to those induced by
wild-type CT adjuvanted immunogenic composition (Table 9).
[0052] To test and compare the adjuvanting effects of mutant
CT-CRMs in formulations containing 1 .mu.g recombinant rP4 and 1
.mu.g of one of the mutant CT-CRMs (CT-CRM.sub.I16A,
CT-CRM.sub.I16A,S68Y, CT-CRM.sub.V72Y, and CT-CRM.sub.S68Y,V72Y)
with a formulation containing 1 .mu.g rP4 and 1 .mu.g
CT-CRM.sub.E29H, or 1 .mu.g rP4 alone in saline. The various
compositions were delivered intranasally to female BALB/c mice, and
the anti-rP4 IgG and IgA titers measured at weeks 3 and 5, and at
week 5, day 6. The data suggest that CT-CRMs, CT-CRM.sub.I16A and
CT-CRM.sub.V72Y are as potent as CT-CRM.sub.E29H in inducing
systemic as well as mucosal anti-rP4 antibody response (Tables 10
and 11). The serum IgG titers of anti-rP4 antibody induced by the
formulation containing rP4 and CT-CRM.sub.I16A at week 5, day 6 was
22-fold greater than that induced by rP4 alone and half of the IgG
levels induced by CT-CRM.sub.E29H. However, serum IgG titers of
anti-rP4 antibody induced by the formulation containing rP4 and
CT-CRM.sub.V72Y was 1.2-fold more than that induced by
CT-CRM.sub.E29H and 53-fold greater than that induced by rP4 alone.
Although the rP4-specific IgG titers induced by
CT-CRM.sub.I16A,S68Y and CT-CRM.sub.S68Y,V72Y were only
approximately one-fifth of that induced by CT-CRM.sub.I16A and
CT-CRM.sub.V72Y, these levels were still significantly higher than
that induced by rP4 alone in saline.
[0053] The protein-specific IgA antibody titers in the sera of mice
immunized with CT-CRMs, CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y,
CT-CRM.sub.V72Y and CT-CRM.sub.S68Y,V72Y were 6 to 23-fold greater
than those of mice immunized IN with the rP4 alone.
[0054] The protein-specific IgA antibody titers in the
bronchioalveolar wash, nasal wash, saliva and vaginal wash of mice
immunized with CT-CRMs, CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y,
CT-CRM.sub.V72Y and CT-CRM.sub.S68Y,V72Y were comparable to the IgA
levels in the mucosal wash pools of mice immunized with
CT-CPM.sub.E29H, but significantly greater than those of mice
immunized with rP4 alone. (See Table II).
[0055] In the above study, anti-rP4 antibody titers in the serum of
each individual mouse in the six groups were also assessed.
Specifically, 41 days following IN administration, IgA and IgG
including IgG subclass IgG1, IgG2a, IgG2b and IgG3 endpoint titers
were determined by ELISA. The results indicate that IgA and IgG
subclass titers in each individual mouse receiving the formulation
containing rP4 and any one of the four mutant CT-CRMs were
significantly higher than the IgA and IgG titers in animals
receiving only the rP4 antigen in saline. (See Tables 12-17). The
results further indicate that the IgA and IgG titers in animals
receiving rP4 and one of the mutant CT-CRMs, CT-CRM.sub.I16A,
CT-CRM.sub.I16A,S68Y, and CT-CRM.sub.V72Y, were comparable to the
IgA and IgG titers detected in mice receiving rP4 plus
CT-CRM.sub.E29H.
[0056] The capacity of the CT-CRMs of the present invention to
augment systemic and mucosal immune responses against respiratory
syncytial virus (RSV) glycoproteins was examined using the purified
native fusion (F) protein. Previously, it was demonstrated that
BALB/c mice immunized IN with F protein adjuvanted with either CT
or CT-CRM.sub.E29H generated systemic and local IgG and IgA titers
(Tebbey et al, cited above). This study also indicated that
pre-existing anti-CT antibodies did not have a negative impact on
the level of local or systemic anti-F protein IgA and IgG
antibodies. Indeed, the study indicated that pre-existing anti-CT
antibodies were beneficial for the generation of an augmented
anti-F protein antibody response. Additionally, the data also
suggested a mechanism involving the neutralization of infectious
virus by either mucosal or humoral Immunoglobins that are
stimulated in response to the IN immunization protocol containing
F/CT-CRM.sub.E29H. In the present study, purified F protein (3
.mu.g/dose) alone in saline or in a formulation containing 0.1 or 1
.mu.g of the wild-type CT or 0.1 or 1 .mu.g of one of the mutant
CT-CRMs (CT-CRM.sub.E29H, CT-CRM.sub.I16A, CT-CPM.sub.I16A,S68Y,
CT-CRM.sub.V72Y and CT-CRM.sub.S68Y, V72Y) was administered IN to
BALB/c mice. The protein-specific IgG and IgA antibody titers in
the bronchioalveolar wash, nasal wash and vaginal wash of mice were
determined. The protein-specific IgG and IgA antibody titers in the
bronchioalveolar wash, nasal wash and vaginal wash of mice
immunized with 1 .mu.g of CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y,
CT-CRM.sub.V72Y or CT-CRM.sub.S68Y,V72Y were comparable to the IgG
and IgA levels in the mucosal wash pools of mice immunized with
wild-type CT or CT-CRM.sub.E29H. (See Table 19). The mucosal
protein-specific IgG levels in mice immunized with 0.1 .mu.g of
CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y, CT-CRM.sub.V72Y or
CT-CRM.sub.S68Y,V72Y though significantly higher than the levels
detected in mice immunized with F-protein in saline, were
nevertheless one-third to one-tenth less than the levels detected
in mice immunized with 1 .mu.g of the CT-CRMs. In contrast,
significantly elevated levels of IgA in mucosal washes were only
observed in mice immunized with 1 .mu.g of the mutant CT-CRMs.
[0057] The capacity of mutant CT-CRMs to augment systemic and
mucosal immune responses in mice against the native UspA2 outer
membrane protein of MA catarrhalis was examined. Purified UspA2 (5
.mu.g/dose) alone in 10 .mu.l saline or in a 10 .mu.l formulation
containing 0.1 .mu.g/dose of a mutant CT-CRM (CT-CRM.sub.E29H,
CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y, CT-CRM.sub.V72Y or
CT-CRM.sub.S68Y,V72Y) was administered IN at days 0, 7 and 14.
Protein-specific IgG and IgA levels in the serum and in mucosal
lavages were examined at day 28. Statistically significant levels
of IgG and IgA were detected only in the serum of animals immunized
with CT-CRM.sub.E29H, CT-CRM.sub.I16A,S68Y and CT-CRM.sub.V72Y.
Significant levels of IgG were also detected in the bronchial wash
of animals immunized with CT-CRM.sub.E29H, CT-CRM.sub.I16A,S68Y and
CT-CRM.sub.V72Y.
[0058] B. Nucleic Acid Molecules Encoding CT-CRMs
[0059] Another aspect of this invention includes isolated,
synthetic or recombinant nucleic acid molecules and sequences
encoding the above-described CT-CRMs having the specified site
directed mutations or fragments that may further contain one or
more of those mutations.
[0060] An isolated nucleotide molecule comprising a nucleic acid
sequence encoding a CT-CRM protein may be preferably under the
control of regulatory sequences that direct expression of the
CT-CRM in a host cell. As described herein, such nucleic acid
molecules may be used to express the CT-CRM protein in vitro or to
permit expression of the CT-CRM protein in vivo in a human.
[0061] As used herein, the term "isolated nucleotide molecule or
sequence" refers to a nucleic acid segment or fragment which is
free from contamination with other biological components that may
be associated with the molecule or sequence in its natural
environment. For example, one embodiment of an isolated nucleotide
molecule or sequence of this invention is a sequence separated from
sequences which Plank it in a naturally occurring state, e.g., a
DNA fragment which has been removed from the sequences which are
normally adjacent to the fragment, such as the sequences adjacent
to the fragment in a genome in which it naturally occurs. Further,
the nucleotide sequences and molecules of this invention have been
altered to encode a CT-CRM protein of this invention. Thus, the
term "isolated nucleic acid molecule or sequence" also applies to
nucleic acid sequences or molecules that have been substantially
purified from other components that naturally accompany the
unmutagenized nucleic acid, e.g., RNA or DNA or proteins, in the
cell. An isolated nucleotide molecule or sequence of this invention
also encompasses sequence and molecules that have been prepared by
other conventional methods, such as recombinant methods, synthetic
methods, e.g., mutagenesis, or combinations of such methods. The
nucleotide sequences or molecules of this invention should not be
construed as being limited solely to the specific nucleotide
sequences presented herein, but rather should be construed to
include any and all nucleotide sequences which share homology
(i.e., have sequence identity) with the nucleotide sequences
presented herein.
[0062] The terms "substantial homology" or "substantial
similarity," when referring to a nucleic acid or fragment thereof,
indicates that, when optimally aligned with appropriate nucleotide
insertions or deletions with another nucleic acid (or its
complementary strand), there is nucleotide sequence identity in at
least about 70% of the nucleotide bases, as measured by any
well-known algorithm of sequence identity, such as FASTA, a program
in GCG Version 6.1. The term "homologous" as used herein, refers to
the sequence similarity between two polymeric molecules, e.g.,
between two nucleic acid molecules, e.g., two DNA molecules or two
RNA molecules, or between two polypeptide molecules. When a
nucleotide or amino acid position in both of the two molecules is
occupied by the same monomeric nucleotide or amino acid, e.g., if a
position in each of two DNA molecules is occupied by adenine, then
they are homologous at that position. The homology between two
sequences is a direct function of the number of matching or
homologous positions, e.g., if half (e.g., five positions in a
polymer ten subunits in length) of the positions in two compound
sequences are homologous then the two sequences are 50% homologous.
If 90% of the positions, e.g., 9 of 10, are matched or homologous,
the two sequences share 90% homology. By way of example, the DNA
sequences 3'ATTGCC5' and 3'TATGCG5' share 50% homology. By the term
"substantially homologous" as used herein, is meant DNA or RNA
which is about 70% homologous, more preferably about 80% homologous
and most preferably about 90% homologous to the desired nucleic
acid.
[0063] The invention is also directed to an isolated nucleotide
molecule comprising a nucleic acid sequence that is at least 70%,
80% or 90% homologous to a nucleic acid sequence encoding a CT-CRM
protein of this invention that has reduced enzymatic toxicity
compared to wild-type CT protein and that retains adjuvanticity of
the wild-type CT. Furthermore, due to the degeneracy of the genetic
code, any three-nucleotide codon that encodes a mutated amino acid
residue of CT-CRM, described herein is within the scope of the
invention.
[0064] Where, as discussed herein, CT-CRMs, mutant CT-A subunits or
mutant CT-B subunits, and/or DNA sequences encoding them, or other
sequences useful in nucleic acid molecules or compositions
described herein are defined by their percent homologies or
identities to identified sequences, the algorithms used to
calculate the percent homologies or percent identities include the
following: the Smith-Waterman algorithm (S. F. Collins et al, 1988,
Comput. Appl. Biosci., 4:67-72; J. F. Collins et al, Molecular
Sequence Comparison and Alignment, (M. J. Bishop et al, eds.) In
Practical Approach Series: Nucleic Acid and Protein Sequence
Analysis XVIII, IRL Press: Oxford, England, UK (1987) pp. 417), and
the BLAST and FASTA programs (E. G. Shpaer et al, 1996, Genomics,
38:179-191). These references are incorporated herein by
reference.
[0065] By describing two DNAs as being "operably linked" as used
herein, is meant that a single-stranded or double-stranded DNA
comprises each of the two DNAs and that the two DNAs are arranged
within the DNA in such a manner that at least one of the DNA
sequences is able to exert a physiological effect by which it is
characterized upon the other.
[0066] Preferably, for use in producing a CT-CRM protein of this
invention or administering it for in vivo production in a cell,
each CT-CRM protein encoding sequence and necessary regulatory
sequences are present in a separate viral or non-viral recombinant
vector (including non-viral methods of delivery of a nucleic acid
molecule into a cell). Alternatively, two or more of these nucleic
acid sequences encoding duplicate copies of a CT-CRM protein or
encoding multiple different CT-CRMs of this invention may be
contained in a polycistronic transcript, i.e., a single molecule
designed to express multiple gene products.
[0067] The invention further relates to vectors, particularly
plasmids, containing isolated and purified DNA sequences comprising
DNA sequences that encode an immunogenic mutant cholera holotoxin.
Desirable embodiments include plasmids containing DNA sequences
encoding CT-CRMs having a single amino acid substitution at amino
acid position 16 or 72 or double amino acid substitution at amino
acid position 16 and 68 or 68 and 72 respectively. By the term
"vector" as used herein, is meant a DNA molecule derived from viral
or non-viral, e.g., bacterial, species that has been designed to
encode an exogenous or heterologous nucleic acid sequence. Thus,
the term includes conventional bacterial plasmids. Such plasmids or
vectors can include plasmid sequences from viruses or phages. Such
vectors include chromosomal, episomal and virus-derived vectors,
e.g., vectors derived from bacterial plasmids, bacteriophages,
yeast episomes, yeast chromosomal elements, and viruses. Vectors
may also be derived from combinations thereof, such as those
derived from plasmid and bacteriophage genetic elements, cosmids,
and phagemids. The term also includes non-replicating viruses that
transfer a gene from one cell to another. The term should also be
construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for
example, polylysine compounds and the like.
[0068] The nucleic acid molecules of the invention include
non-viral vectors or methods for delivery of the sequence encoding
the CT-CRM protein to a host cell according to this invention. A
variety of non-viral vectors are known in the art and may include,
without limitation, plasmids, bacterial vectors, bacteriophage
vectors, "naked" DNA and DNA condensed with cationic lipids or
polymers.
[0069] Examples of bacterial vectors include, but are not limited
to, sequences derived from bacille Calmette Guerin (BCG),
Salmonella, Shigella, E. coli, and Listeria, among others. Suitable
plasmid vectors include, for example, pBR322, pBR325, pACYC177,
pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pK37, pKC101,
pAC105, pVA51, pKH47, pUB110, pMB9, pBR325, Col E1, pSC101, pBR313,
pML21, RSF2124, pCR1, RP4, pBAD18, and pBR328.
[0070] Examples of suitable inducible Escherichia coli expression
vectors include pTrc (Amann et al., 1988 Gene, 69:301-315), the
arabinose expression vectors (e.g., pBAD18, Guzman et al, 1995 J.
Bacteriol., 177:4121-4130), and pETIId (Studier et al., 1990
Methods in Enzymology, 18.5:60-89). Target gene expression from the
pTrc vector relies on host RNA polymerase transcription from a
hybrid trp-lac fusion promoter. Target gene expression from the
pETIId vector relies on transcription from a T7 gn10-lac fusion
promoter mediated by a coexpressed viral RNA polymerase T7 gn1.
This viral polymerase is supplied by host strains BL21 (DE3) or HMS
I 74(DE3) from a resident prophage harboring a T7 gn1 gene under
the transcriptional control of the lacUV5 promoter. The pBAD system
relies on the inducible arabinose promoter that is regulated by the
araC gene. The promoter is induced in the presence of
arabinose.
[0071] As one example, a plasmid, designated pLP903, contains an
isolated and purified DNA sequence comprising a DNA sequence
encoding an immunogenic mutant CT-CRM with substantially reduced
toxicity wherein the amino acid alanine is substituted for
isoleucine at amino acid position 16 in the A subunit. A second
plasmid, designated pLP905, contains an isolated and purified DNA
sequence comprising a DNA sequence encoding an immunogenic mutant
CT-CRM with substantially reduced toxicity wherein the amino acid
tyrosine is substituted for valine at the amino acid position 72 in
the A subunit. Another exemplary plasmid is designated pLP904. This
plasmid contains an isolated and purified DNA sequence comprising a
DNA sequence encoding an immunogenic, mutant CT-CRM with
substantially reduced toxicity wherein the amino acid alanine is
substituted for isoleucine at amino acid position 16, and amino
acid tyrosine is substituted for serine at amino acid position 68
in the A subunit. Another plasmid exemplified in this invention is
designated pLP906. It contains an isolated and purified DNA
sequence comprising a DNA sequence encoding an immunogenic, mutant
CT-CRM with substantially reduced toxicity wherein the amino acid
tyrosine is substituted for serine at the amino acid position 68,
and amino acid tyrosine is substituted for valine at amino acid
position 72 in the A subunit.
[0072] Another type of useful vector is a single or double-stranded
bacteriophage vector. For example, a suitable cloning vector
includes, but is not limited to the vectors such as bacteriophage
.lamda., vector system, .lamda.gt11, .mu.gt .mu.WES.tB, Charon 4,
.lamda.gt-WES-.lamda.B, Charon 28, Charon 4A,
.lamda.gt-1-.lamda.BC, .lamda.gt-1-.lamda.B, M13mp7, M13mp8, or
M13mp9, among others.
[0073] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in a yeast
such as S. cerevisiae include pYepSec I (Baldari, et al., 1987
Protein Eng., 1(5):433-437), pMFa (Kurjan and Herskowitz, 1982
Cell, 30(3):933-943), pJRY88 (Schultz et al., 1987 Gene,
61(2):123-133), and pYES2 (Invitrogen Corporation, San Diego,
Calif.).
[0074] Alternatively, baculovirus expression vectors are used.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 or Sf21 cells) include the pAc
series (Smith et al., 1983 Biotechnol., 24:434-443) and the pVL
series (Luckow and Summers, 1989 Virol., 170(1):31-39). In yet
another embodiment, a mammalian expression vector is used for
expression in mammalian cells. Examples of mammalian expression
vectors include pCDM8 (Seed, 1987 Nature, 329:840-842) and pMT2PC
(Kaufman et al., 1987 EMBO J., 6(1): 187-93). When used in
mammalian cells, the expression vector's control functions are
often provided by viral regulatory elements.
[0075] One type of recombinant vector is a recombinant single or
double-stranded RNA or DNA viral vector. A variety of viral vector
systems are known in the art. Examples of such vectors include,
without limitation, recombinant adenoviral vectors, herpes simplex
virus (HSV)-based vectors, adeno-associated viral (AAV) vectors,
hybrid adenoviral/AAV vectors, recombinant retroviruses or
lentiviruses, recombinant poxvirus vectors, recombinant vaccinia
virus vectors, SV-40 vectors, insect viruses such as baculoviruses,
and the like that are constructed to carry or express a selected
nucleic acid composition of interest.
[0076] Retrovirus vectors that can be employed include those
described in EP 0 415 731; International Patent Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; and WO 93/25234; U.S. Pat. No.
5,219,740; International Patent Publication Nos. WO 93/11230 and WO
93/10218; Vile and Hart, 1993 Cancer Res. 53:3860-3864; Vile and
Hart, 1993 Cancer Res. 53:962-967; Ram et al., 1993 Cancer Res.
53:83-88; Takamiya et al., 1992 J. Neurosci. Res. 33:493-503; Baba
et al., 1993 Neurosurg. 79:729-735; U.S. Pat. No. 4,777,127; GB
Patent No. 2,200,651; and EP 0 345 242. Examples of suitable
recombinant retroviruses include those described in International
Patent Publication No. WO 91/02805.
[0077] Alphavirus-based vectors may also be used as the nucleic
acid molecule encoding the CT-CRM protein. Such vectors can be
constructed from a wide variety of viruses, including, for example,
Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC
VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and
Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250;
ATCC VR 1249; ATCC VR-532). Representative examples of such vector
systems include those described in U.S. Pat. Nos. 5,091,309;
5,217,879; and 5,185,440; and International Patent Publication Nos.
WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO
95/07994.
[0078] Examples of adenoviral vectors include those described by
Berkner, 1988 Biotechniques 6:616-627; Rosenfeld et al., 1991
Science 252:431-434; International Patent Publication No. WO
93/19191; Kolls et al., 1994 PNAS 91:215-219; Kass-Eisler et al.,
1993 PNAS 90:11498-11502; Guzman et al., 1993 Circulation
88:2838-2848; Guzman et al., 1993 Cir. Res. 73:1202-1207; Zabner et
al., 1993 Cell 75:207-216; Li et al., 1993 Hum. Gene Ther.
4:403-409; Cailaud et al., 1993 Eur. J. Neurosci. 5:1287-1291;
Vincent et al., 1993 Nat. Genet. 5:130-134; Jaffe et al., 1992 Nat.
Genet. 1:372-378; and Levrero et al., 1991 Gene 101:195-202.
Exemplary adenoviral vectors include those described in
International Patent Publication Nos. WO 94/12649; WO 93/03769; WO
93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Other
adenoviral vectors include those derived from chimpanzee
adenoviruses, such as those described in U.S. Pat. No.
6,083,716.
[0079] Another viral vector is based on a parvovirus such as an
adeno-associated virus (AAV). Representative examples include the
AAV vectors described in International Patent Publication No. WO
93/09239, Samulski et al., 1989 J. Virol. 63:3822-3828; Mendelson
et al., 1988 Virol. 166:154-165; and Flotte et al., 1993 PNAS
90:10613-10617. Other particularly desirable AAV vectors include
those based upon AAV1; see, International Patent Publication No. WO
00/28061, published May 18, 2000. Other desirable AAV vectors
include those which are pseudotyped, i.e., contain a minigene
composed of AAV 5' ITRS, a transgene, and AAV 3' ITRs packaged in a
capsid of an AAV serotype heterologous to the AAV ITRs. Methods of
producing such pseudotyped AAV vectors are described in detail in
International Patent Publication No. WO01/83692.
[0080] In an embodiment in which the nucleic acid molecule of the
invention is "naked DNA", it may be combined with polymers
including traditional polymers and non-traditional polymers such as
cyclodextrin-containing polymers and protective, interactive
noncondensing polymers, among others. The "naked" DNA and DNA
condensed with cationic lipids or polymers are typically delivered
to the cells using chemical methods. A number of chemical methods
are known in the art for cell delivery and include using lipids,
polymers, or proteins to complex with DNA, optionally condensing
the same into particles, and delivering to the cells. Another
non-viral chemical method includes using cations to condense DNA,
which is then placed in a liposome and used according to the
present invention. See, C. Henry, 2001 Chemical and Engineering
News, 79(48):35-41.
[0081] The nucleic acid molecule encoding the CT-CRM of this
invention is introduced directly into the cells either as "naked"
DNA (U.S. Pat. No. 5,580,859) or formulated in compositions with
agents, which facilitate immunization, such as bupivacaine and
other local anesthetics (U.S. Pat. No. 6,127,170).
[0082] All components of the viral and non-viral vectors above may
be readily selected from among known materials in the art and
available from the pharmaceutical industry. Selection of the vector
components and regulatory sequences are not considered a limitation
on this invention. Each nucleic acid sequence encoding a CT-CRM
protein according to this invention is preferably under the control
of regulatory sequences that direct the replication and generation
of the product of each nucleic acid sequence in a mammalian or
vertebrate cell. By the term "promoter/regulatory sequence" is
meant a DNA sequence required for expression of a nucleic acid
operably linked to the promoter/regulatory sequence. In some
instances, the promoter/regulatory sequence may function in a
tissue specific manner. For example, the promoter/regulatory
sequence is only capable of driving expression in a cell of a
particular tissue type. In some instances, this sequence may be the
core promoter sequence and in other instances, this sequence may
also include an enhancer sequence and other regulatory elements
that are required for expression in a tissue-specific manner.
[0083] Preferably, the nucleic acid molecule encoding a CT-CRM
protein of this invention and/or the recombinant vector further
comprises regulatory sequences. For example, such regulatory
sequences comprise a promoter that drives expression of the CT-CRM
protein. Preferably the promoter/regulatory sequence is positioned
at the 5' end of the coding sequence such that it drives expression
of the CT-CRM protein in a cell.
[0084] Suitable promoters may be readily selected from among
constitutive promoters, inducible promoters, tissue-specific
promoters and others. Examples of constitutive promoters that are
non-specific in activity and employed in the nucleic acid molecules
encoding the CT-CRM protein of this invention include, without
limitation, the retroviral Rous sarcoma virus (RSV) promoter, the
retroviral LTR promoter (optionally with the RSV enhancer), the
cytomegalovirus (CMV) promoter (optionally with the CMV enhancer)
(see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40
promoter, the dihydrofolate reductase promoter, the .beta.-actin
promoter, the phosphoglycerol kinase (PGK) promoter, and the
EF1.alpha. promoter (Invitrogen).
[0085] Inducible promoters that are regulated by exogenously
supplied compounds, include, without limitation, the arabinose
promoter, the zinc-inducible sheep metallothionine (MT) promoter,
the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV)
promoter, the T7 polymerase promoter system (WO 98/10088); the
ecdysone insect promoter (No et al, 1996 Proc. Natl. Acad. Sci.
USA, 93:3346-3351), the tetracycline-repressible system (Gossen et
al, 1992 Proc. Natl. Acad. Sci. USA, 89:5547-5551), the
tetracycline-inducible system (Gossen et al, 1995 Science,
268:1766-1769, see also Harvey et al, 1998 Curr. Opin. Chem. Biol.,
2:512-518), the RU486-inducible system (Wang et al, 1997 Nat.
Biotech., 15:239-243 and Wang et al, 1997 Gene Ther., 4:432-441)
and the rapamycin-inducible system (Magari et al, 1997 J. Clin.
Invest, 100: 2865-2872). A particularly preferred promoter for use
in expression systems for CT-CRMs is an arabinose inducible
promoter.
[0086] Other types of inducible promoters that may be useful in
this context are those regulated by a specific physiological state,
e.g., temperature or acute phase or in replicating cells only.
Useful tissue-specific promoters include the promoters from genes
encoding skeletal .beta.-actin, myosin light chain 2A, dystrophin,
muscle creatine kinase, as well as synthetic muscle promoters with
activities higher than naturally-occurring promoters (see Li et
al., 1999 Nat. Biotech., 17:241-245). Examples of promoters that
are tissue-specific are known for liver (albumin, Miyatake et al.
1997 J. Virol., 71:5124-32; hepatitis B virus core promoter, Sandig
et al., 1996 Gene Ther., 3: 1002-9; alpha-fetoprotein (AFP),
Arbuthnot et al., 1996 Hum. Gene Ther., 7:1503-14), bone
(osteocalcin, Stein et al., 1997 Mol. Biol. Rep., 24:185-96; bone
sialoprotein, Chen et al., 1996 J. Bone Miner. Res., 11:654-64),
lymphocytes (CD2, Hansal et al., 1988 J. Immunol., 161:1063-8;
immunoglobulin heavy chain; T cell receptor .alpha. chain),
neuronal (neuron-specific enolase NSE) promoter, Andersen et al.
1993 Cell. Mol. Neurobiol., 13:503-15; neurofilament light-chain
gene, Piccioli et al., 1991 Proc. Natl. Acad. Sci. USA, 88:5611-5,
the neuron-specific B ngf gene, Piccioli et al., 1995 Neuron,
15:373-84); among others. See, e.g. International Patent
Publication No. WO00/55335 for additional lists of known promoters
useful in this context.
[0087] Additional regulatory sequences for inclusion in a nucleic
acid sequence, molecule or vector of this invention include,
without limitation, an enhancer sequence, a polyadenylation
sequence, a splice donor sequence and a splice acceptor sequence, a
site for transcription initiation and termination positioned at the
beginning and end, respectively, of the polypeptide to be
translated, a ribosome binding site for translation in the
transcribed region, an epitope tag, a nuclear localization
sequence, an IRES element, a Goldberg-Hogness "TATA" element, a
restriction enzyme cleavage site, a selectable marker and the like.
Enhancer sequences include, e.g., the 72 bp tandem repeat of SV40
DNA or the retroviral long terminal repeats or LTRs, etc. and are
employed to increase transcriptional efficiency. Selection of
promoters and other common vector elements are conventional and may
such sequences are available with which to design the nucleotide
molecules and vectors useful in this invention. See, e.g., Sambrook
et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory, New York, (1989) and references cited therein at, for
example, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York (1989). One of skill in the art may readily select from among
such known regulatory sequences to prepare molecules of this
invention. The selection of such regulatory sequences is not a
limitation of this invention.
[0088] C. Methods for Making the CT-CRM Proteins and Nucleotide
Molecules of this Invention
[0089] In view of the demonstrated utility of mutant CT-CRMs as
adjuvants for antigenic compositions, production of suitable
quantities of mutant CT-CRMs is desirable. The preparation or
synthesis of the nucleotide sequences and CT-CRMs, as well as
compositions containing the nucleotide molecules or CT-CRM protein
of this invention disclosed herein is well within the ability of
the person having ordinary skill in the art using available
material. The synthesis methods are not a limitation of this
invention. The examples below detail presently preferred
embodiments of synthesis of sequences encoding the CT-CRMs of this
invention.
[0090] The CT-CRMs and nucleotide molecules and sequences of this
invention may be produced by chemical synthesis methods,
recombinant genetic engineering methods, site directed mutagenesis,
among others, and combinations of such methods. For example, the
nucleotide sequences/CT-CRMs of the invention may be prepared
conventionally by resort to known chemical synthesis techniques,
e.g., solid-phase chemical synthesis, such as described by
Merrifield, 1963 J. Amer. Chem. Soc., 85:2149-2154; J. Stuart and
J. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company,
Rockford, Ill. (1984); Matteucci et al., 1981 J. Am. Chem. Soc.,
103:3185; Alvarado-Urbina et al., 1980 Science, 214:270; and Sinha,
N. D. et al., 1984 Nucl. Acids Res., 13:4539, among others. See,
also, e.g., PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.,
T. E. Creighton, W.H. Freeman and Company, New York, 1993; Wold,
F., "Posttranslational Protein Modifications: Perspectives and
Prospects", pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al., 1990 Meth. Enzymol., 182:626-646, and Rattan et
al., 1992 Ann. N.Y. Acad. Sci., 663:48-62.
[0091] Alternatively, compositions of this invention may be
constructed recombinantly using conventional molecular biology
techniques, site-directed mutagenesis, genetic engineering or
polymerase chain reaction, such as, by cloning and expressing a
nucleotide molecule encoding a CT-CRM protein with optional other
immunogens and optional carrier proteins within a host
microorganism, etc. utilizing the information provided herein (See,
e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d
Edit., Cold Spring Harbor Laboratory, New York (1989); Ausubel et
al. Current Protocols in Molecular Biology, John Wiley & Sons,
New York (1997)). Coding sequences for the CT-CRMs and optional
immunogens can be prepared synthetically (W. P. C. Stemmer et al)
1995 Gene, 164:49).
[0092] In general, recombinant DNA techniques involve obtaining by
synthesis or isolation a DNA sequence that encodes the CT-CRM
protein as described above, and introducing it into an appropriate
vector/host cell expression system where it is expressed,
preferably under the control of an arabinose inducible promoter.
Any of the methods described for the insertion of DNA into an
expression vector may be used to ligate a promoter and other
regulatory control elements into specific sites within the selected
recombinant vector. Suitable host cells are then transformed,
infected, transduced or transfected with such vectors or plasmids
by conventional techniques.
[0093] A variety of host cell-vector (plasmid) systems may be used
to express the immunogenic mutant cholera holotoxin. The vector
system, which preferably includes the arabinose inducible promoter,
is compatible with the host cell used. The DNA encoding the mutant
CT-CRMs are inserted into an expression system, and the promoter
(preferably the arabinose inducible promoter), and other control
elements are ligated into specific sites within the vector so that
when the vector is inserted into a host cell (by transformation,
transduction or transfection, depending on the host cell-vector
system used) the DNA encoding the CT-CRM is expressed by the host
cell.
[0094] The vector may be selected from one of the viral vectors or
non-viral vectors described above but must be compatible with the
host cell used. The recombinant DNA vector may be introduced into
appropriate host cells (bacteria, virus, yeast, mammalian cells or
the like) by transformation, transduction or transfection
(depending upon the vector/host cell system). Host-vector systems
include but are not limited to bacteria transformed with
bacteriophage DNA, plasmid DNA or cosmid DNA; microorganisms such
as yeast containing yeast vectors; mammalian cell systems infected
with virus (e.g., vaccinia virus, adenovirus, etc.); and insect
cell systems infected with virus (e.g., baculovirus).
[0095] Systems for cloning and expressing the CT-CRMs and other
compositions of this invention using the synthetic nucleic acid
molecules include the use of various microorganisms and cells that
are well known in recombinant technology. The host cell may be
selected from any biological organism, including prokaryotic (e.g.,
bacterial) cells and eukaryotic cells, including, mammalian, insect
cells, yeast cells. Preferably, the cells employed in the various
methods and compositions of this invention are bacterial cells,
Suitable bacterial cells include, for example, various strains of
E. coli, Bacillus, and Streptomyces. Yeast cells such as
Saccharomyces and Pichia, and insect cells such as Sf9 and Sf21
cells are also useful host cells for production purposes. Mammalian
cells including but not limited to Chinese hamster ovary cells
(CHO), chick embryo fibroblasts, baby hamster kidney cells, NIH3T3,
PER C6, NSO, VERO or COS cells are also suitable host cells, as
well as other conventional and non-conventional organisms and
plants.
[0096] The selection of other suitable host cells and methods for
transformation, culture, amplification, screening and product
production and purification can be performed by one of skill in the
art by reference to known techniques. See, e.g., Gething and
Sambrook, 1981 Nature, 293:620-625, among others.
[0097] Typically, the host cell is maintained under culture
conditions for a period of time sufficient for expression. Culture
conditions are well known in the art and include ionic composition
and concentration, temperature, pH and the like. Typically,
transfected cells are maintained under culture conditions in a
culture medium. Suitable media for various cell types are well
known in the art. In a preferred embodiment, temperature is from
about 20.degree. C. to about 50.degree. C., more preferably from
about 30.degree. C. to about 40.degree. C. and, even more
preferably about 37.degree. C.
[0098] The pH is preferably from about a value of 6.0 to a value of
about 8.0, more preferably from about a value of about 6.8 to a
value of about 7.8 and, most preferably about 7.4. Osmolality is
preferably from about 200 milliosmols per liter (mosm/L) to about
400 mosm/l and, more preferably from about 290 mosm/L to about 310
mosm/L. Other biological conditions needed for transfection and
expression of an encoded protein are well known in the art.
[0099] Recombinant CT-CRM protein is recovered or collected either
from the host cells or membranes thereof or from the medium in
which those cells are cultured. Recovery comprises isolating and
purifying the recombinant CT-CRM protein, Isolation and
purification techniques for polypeptides are well known in the art
and include such procedures as precipitation, filtration,
chromatography, electrophoresis and the like.
[0100] When produced by conventional recombinant means, CT-CRMs of
this invention may be isolated and purified from the cell or medium
thereof by conventional methods, including chromatography (e.g.,
ion exchange, affinity, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
techniques for the purification or proteins. Several techniques
exist for purification of heterologous protein from prokaryotic
cells. See, U.S. Pat. Nos. 4,518,526; 4,599,197; and 4,734,362. The
purified preparation however produced should be substantially free
of host toxins, which might be harmful to humans. In particular,
when expressed in gram negative bacterial host cells such as E.
coli, the purified peptide or protein should be substantially free
of endotoxin contamination. See, e.g., Sambrook et al., Molecular
Cloning. A Laboratory Manual., 2d Edit., Cold Spring Harbor
Laboratory, New York (1989).
[0101] The CT-CRMs used in methods and compositions of the
invention are not limited to products of any of the specific
exemplary processes listed herein. In fact, the protein may be
prepared by the methods in the texts cited immediately above or by
methods of the texts cited elsewhere in this specification. It is
within the skill of the art to isolate and produce recombinantly or
synthetically protein compositions for such use.
[0102] The four exemplary CT-CRMs of Table 1, two bearing a single
amino acid substitution and two bearing double amino acid
substitutions were generated as described in detail in Example 1
using some of the methods described above. Specifically, a set of
mutant CT clones (CT-CRMs) were generated in E. coli by standard
site-directed mutagenesis protocols on plasmids encoding the known
CT holotoxin molecules.
[0103] It has previously been shown that the resulting yield of
purified CT-CRM.sub.E29H holotoxin was approximately 50 .mu.g per
liter of culture medium (see International patent publication No.
WO 00/18434). Initial attempts to increase CT-CRM.sub.E29H yield
via modifications to the original plasmid, showed little or no
effect. A moderate increase in yield was achieved through
co-expression of the plasmid pIIB29H, and derivatives, with Vibrio
cholerae DsbA and E. coli RpoH. Co-expression and purification
modifications increased the yield of CT-CRM.sub.E29H to
approximately 2 mg/liter.
[0104] In order to increase the expression of CT-CRMs of the
present invention, the lactose inducible promoter in the plasmids
was replaced with an arabinose inducible promoter (Invitrogen
Corporation, Carlsbad, Calif.), which was operatively linked to the
DNA sequence encoding the CT-CRMs, During cloning it was determined
that plasmid pIIB29H contained a ctxA gene encoding CT subunit A
from Vibrio cholerae strain 569B, linked to a ctxB gene encoding CT
subunit B from Vibrio cholerae strain 2125. Cross alignment of
these genes indicated seven base substitutions between the two ctxB
genes and a single base change between the ctxA genes. Several of
these base substitutions led to amino acid changes in the mature
subunits. Of special note is the substitution between the ctxA
genes which leads to an amino acid change within the A-2 portion,
or the holotoxin assembly domain of the A subunit. It was not known
whether the heterogeneity between these genes had a negative impact
on toxin expression or holotoxin assembly. However, it was thought
preferable from an evolutionary standpoint that both toxin subunit
genes originate from the same source. As such, both the ctxA and
ctxB genes used in the construction of the arabinose inducible
system originated from Vibrio cholerae strain 569B. The
construction of plasmids pLP903, pLP904, pLP905, pLP906, is
described in Example 1. The immunogenic mutant cholera holotoxin is
produced by transforming, infecting, transducing or transfecting a
host cell with a plasmid described above, and culturing the host
cell under conditions that permit the expression of said
recombinant immunogenic detoxified protein by the host cell.
Production of CT-CRMs from pLP903, pLP904, pLP905 and pLP906 is
approximately 10 mg of purified material per liter of culture.
[0105] The resulting CT-CRM protein or nucleic acid molecule may be
formulated into an immunogenic composition with any number of
selected antigens and screened for adjuvant efficacy by in vivo
assays, such as those described in the examples below.
[0106] D. Immunogenic Compositions
[0107] An effective immunogenic composition according to the
invention is one comprising a mutant cholera holotoxin of this
invention. Preferably the mutant cholera holotoxin CT-CRM has
reduced toxicity compared to a wild-type cholera holotoxin. This
"reduced toxicity" enables each mutant to be used as an adjuvant in
an immunogenic composition without causing significant side
effects, particularly those known to be associated with wild-type
CT, e.g., diarrhea. More preferably, the CT-CRM in the immunogenic
composition of this invention has a single amino acid substitution
at the amino acid position 16 or 72 in the A subunit of the
holotoxin, or a double amino acid substitution at amino acid
positions 16 and 68 or 68 and 72 of the A subunit of the cholera
holotoxin. In one embodiment, the CT-CRM may have one or more
additional modifications as described above. In another embodiment,
the composition comprises a selected antigen and a suitable
effective adjuvanting amount of the CT-CRM, wherein said holotoxin
significantly enhances the immune response in a vertebrate host to
said antigen. The compositions of the present invention modulate
the immune response by improving the vertebrate host's antibody
response and cell-mediated immune responses to the administration
of a composition comprising a selected antigen as described
above.
[0108] As used herein, the term "effective adjuvanting amount"
means a dose of one of the CT-CRM mutants of this invention that is
effective in eliciting an increased immune response in a vertebrate
host. In a more specific definition, the term "effective
adjuvanting amount" means a dose of one of the four CT-CRM mutants
described herein (CT-CRM.sub.I16A, CT-CRM.sub.V72Y,
CT-CRM.sub.I16A, S68Y, CT-CRM.sub.S68Y, V72Y), effective in
eliciting an increased immune response in a vertebrate host.
Specifically, the CT-CRMs disclosed herein augment mucosal and
systemic immune responses following intranasal administration of
disparate antigens. Furthermore, even in the presence of
pre-existing anti-CT immune responses, the mutant CT-CRMs were able
to serve as efficient mucosal adjuvants. The immunogenic mutant
CT-CRMs according to the present invention exhibit a balance of
reduced toxicity and retained adjuvanticity, such that the
resulting mutant CT protein functions as an adjuvant while being
tolerated safely by the vertebrate host to which it is introduced.
The particular "effective adjuvanting dosage or amount" will depend
upon the age, weight and medical condition of the host, as well as
on the method of administration. Suitable doses are readily
determined by persons skilled in the art.
[0109] The immunogenic compositions containing as an adjuvant the
mutant cholera holotoxins of this invention also contain at least
one antigen selected from among a wide variety of antigens. The
antigen(s) may comprise a whole cell or virus, or one or more
saccharides, proteins, protein subunits, polypeptide, peptide or
fragments, poly- or oligonucleotides, or other macromolecular
components. If desired, the antigenic compositions may contain more
than one antigen from the same or different pathogenic
microorganisms.
[0110] Thus, in one embodiment, the immunogenic compositions of
this invention comprise as the selected antigen a polypeptide,
peptide or fragment derived from a pathogenic bacterium Desirable
bacterial immunogenic compositions including the CT-CRM mutants as
an adjuvant include those directed to the prevention and/or
treatment of disease(s) caused by, without limitation, Haemophilus
influenzae (both typable and nontypable), Haemophilus somnus,
Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus
pyogenes, Streptococcus agalactiae, Streptococcus faecalis,
Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoea,
Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci,
Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi,
Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli,
Shigella, Vibrio cholerae, Corynebacterium diphtheria,
Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium
intracellulare complex, Proteus mirabilis, Proteus vulgaris,
Staphylococcus aureus, Staphylococcus epidermidis, Clostridium
tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella
haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae
and Mycoplasma gallisepticum.
[0111] In another embodiment, the immunogenic compositions of this
invention comprise as the selected antigen a polypeptide, peptide
or fragment derived from a pathogenic virus, Desirable viral
immunogenic compositions including the CT-CRM mutants as an
adjuvant include those directed to the prevention and/or treatment
of disease caused by, without limitation, Respiratory syncytial
virus, Parainfluenza virus types 1-3, Human metapneumovirus,
Influenza virus, Herpes simplex virus, Human cytomegalovirus, Human
immunodeficiency virus, Simian 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 various Encephalitis viruses.
[0112] In another embodiment, the immunogenic compositions of this
invention comprise as the selected antigen a polypeptide, peptide
or fragment derived from a pathogenic fungus. Desirable immunogenic
compositions against fungal pathogens including the CT-CRM mutants
as an adjuvant include those directed to the prevention and/or
treatment of disease(s) caused by, without limitation, Aspergillis,
Blastomyces, Candida, Coccidiodes, Cryptococcus and
Histoplasma.
[0113] In still another embodiment, the immunogenic compositions of
this invention comprise as the selected antigen a polypeptide,
peptide or fragment derived from a pathogenic parasite. Desirable
immunogenic compositions against parasites including the CT-CRM
mutants as an adjuvant include those directed to the prevention
and/or treatment of disease(s) caused by, without limitation,
Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma,
Cryptosporidium, Trichomonas, Toxoplasma gondii and Pneumocystis
carinii.
[0114] Desirable immunogenic compositions directed against
non-infectious diseases including the CT-CRM mutants as an adjuvant
are also within the scope of this invention. Such immunogenic
compositions include those directed to vertebrate antigens,
particularly compositions directed against antigens for the
prevention and/or treatment of disease(s), without limitation, such
as allergy, autoimmune disease, Alzheimer disease and cancer.
[0115] For example, the immunogenic composition of this invention
may contain a polypeptide, peptide or fragment derived from a
cancer cell or tumor cell. Desirable immunogenic compositions for
eliciting a therapeutic or prophylactic anti-cancer effect in a
vertebrate host, which contain the CT-CRM mutants of this
invention, include those utilizing a cancer antigen or
tumor-associated antigen including, without limitation, prostate
specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125,
MAGE-3, hormones, hormone analogs and so forth.
[0116] Other immunogenic compositions of this invention are
desirable for moderating responses to allergens in a vertebrate
host. Such compositions contain the CT-CRM mutant(s) of this
invention and a polypeptide, peptide or fragment derived from an
allergen or fragment thereof. Examples of such allergens are
described in the U.S. Pat. No. 5,830,877 and International patent
publication No. WO 99/51259, which are hereby incorporated by
reference, and include pollen, insect venoms, animal dander, fungal
spores and drugs (such as penicillin). The immunogenic compositions
interfere with the production of IgE antibodies, a known cause of
allergic reactions, so as to moderate allergic responses to the
allergen.
[0117] In still another embodiment, the immunogenic compositions of
this invention contain as the selected antigen a polypeptide,
peptide or fragment derived from a molecular portion of an antigen,
which represents those produced by a host (a self molecule) in an
undesired manner, amount or location, such as those from amyloid
precursor protein so as to prevent or treat disease characterized
by amyloid deposition in a vertebrate host. Desirable compositions
for moderating responses to self molecules in a vertebrate host,
which contain CT-CRM mutants of this invention, include those
containing a self molecule or fragment thereof. Examples of such
self molecules include .beta.-chain insulin involved in diabetes,
the G17 molecule involved in gastroesophageal reflux disease, and
antigens which downregulate autoimmune responses in diseases such
as multiple sclerosis, lupus and rheumatoid arthritis.
[0118] Still other immunogenic compositions of this invention are
desirable for preventing or treating disease characterized by
amyloid deposition in a vertebrate host. Such compositions contain
the CT-CRM mutant(s) of this invention as well as portions of
amyloid precursor protein (APP). This disease is referred to
variously as Alzheimer's disease, amyloidosis or amyloidogenic
disease. The .beta.-amyloid peptide (also referred to as A.beta.
peptide) is a 42 amino acid fragment of APP, which is generated by
processing of APP by the .beta. and .gamma. secretase enzymes, and
has the following sequence: Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn
Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala (SEQ ID
NO: 3). In some patients, the amyloid deposit takes the form of an
aggregated A.beta. peptide. Surprisingly, it has now been found
that administration of isolated A.beta. peptide induces an immune
response against the A.beta. peptide component of an amyloid
deposit in a vertebrate host (International patent publication No.
WO 99/27944). Thus, embodiments of this invention include the
CT-CRM mutants of this invention plus A.beta. peptide, as well as
fragments of A.beta. peptide and antibodies to A.beta. peptides or
fragments thereof. One such fragment of A.beta. peptide is the 28
amino acid peptide having the following sequence (U.S. Pat. No.
4,666,829): Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His
Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO:
4).
[0119] Such immunogenic compositions further comprise an
immunologically acceptable diluent or a pharmaceutically acceptable
carrier, such as sterile water or sterile isotonic saline. The
antigenic compositions may also be mixed with such diluents or
carriers in a conventional manner. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with administration to humans or other vertebrate
hosts. The appropriate carrier will be evident to those skilled in
the art and will depend in large part upon the route of
administration.
[0120] The immunogenic compositions may also include, but are not
limited to, suspensions, solutions, emulsions in oily or aqueous
vehicles, pastes, and implantable sustained-release or
biodegradable formulations. Such formulations may further comprise
one or more additional ingredients including, but not limited to,
suspending, stabilizing, or dispersing agents. In one embodiment of
a formulation for parenteral administration, the active ingredient
is provided in dry (i.e., powder or granular) form for
reconstitution with a suitable vehicle (e.g., sterile pyrogen-free
water) prior to parenteral administration of the reconstituted
composition. Other parenterally-administrable formulations, which
are useful, include those, which comprise the active ingredient in
microcrystalline form, in a liposomal preparation, or as a
component of a biodegradable polymer system Compositions for
sustained release or implantation may comprise pharmaceutically
acceptable polymeric or hydrophobic materials such as an emulsion,
an ion exchange resin, a sparingly soluble polymer, or a sparingly
soluble salt.
[0121] Still additional components that may be present in the
protein immunogenic compositions of this invention are adjuvants in
addition to the CT-CRMs, preservatives, chemical stabilizers, or
other antigenic proteins. Typically, stabilizers, adjuvants, and
preservatives are optimized to determine the best formulation for
efficacy in the target human or animal. Suitable exemplary
preservatives include chlorobutanol, potassium sorbate, sorbic
acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,
glycerin, phenol, and parachlorophenol. Suitable stabilizing
ingredients that may be used include, for example, casamino acids,
sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate,
lactose, lactalbumin hydrolysate, and dried milk.
[0122] The antigenic compositions of this invention may comprise
further adjuvants in addition to the mutant CT-CRMs. A conventional
non-CT-CRM adjuvant used to enhance an immune response include,
without limitation, MPL.TM. (3-O-deacylated monophosphoryl lipid A;
Corixa, Hamilton, Mont.), which is described in U.S. Pat. No.
4,912,094, which is hereby incorporated by reference. Also suitable
for use as adjuvants are synthetic lipid A analogs or aminoalkyl
glucosamine phosphate compounds (AGP), or derivatives or analogs
thereof, which are available from Corixa (Hamilton, Mont.), and
which are described in U.S. Pat. No. 6,113,918, which is hereby
incorporated by reference. One such AGP is
2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl
2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-t-
etradecanoyoxytetradecanoyl-amino]-b-D-glucopyranoside, which is
also known as 529 (formerly known as RC529). This 529 adjuvant is
formulated as an aqueous form or as a stable emulsion.
[0123] Still other non-CT-CRM adjuvants include mineral oil and
water emulsions, aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, etc., Amphigen, Avridine, L121/squalene,
D-lactide-polylactide/glycoside, pluronic polyols, muramyl
dipeptide, killed Bordetella, saponins, such as Stimulon.TM. QS-21
(Antigenics, Framingham, Mass.), described in U.S. Pat. No.
5,057,540, which is hereby incorporated by reference, and particles
generated therefrom such as ISCOMS (immunostimulating complexes),
Mycobacterium tuberculosis, bacterial lipopolysaccharides,
synthetic polynucleotides such as oligonucleotides containing a CpG
motif (U.S. Pat. No. 6,207,646, which is hereby incorporated by
reference), a pertussis toxin (PT), or an E. coli heat-labile toxin
(LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129; see, e.g.,
International Patent Publication Nos. WO 93/13302 and WO 92/19265,
incorporated herein by reference.
[0124] Various cytokines and lymphokines are also suitable for
inclusion in the immunogenic compositions of this invention. One
such cytokine is granulocyte-macrophage colony stimulating factor
(GM-CSF), which has a nucleotide sequence as described in U.S. Pat.
No. 5,078,996, which is hereby incorporated by reference. A plasmid
containing GM-CSF cDNA has been transformed into E. coli and has
been deposited with the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209, under
Accession No. 39900. The cytokine Interleukin-12 (IL-12) is another
adjuvant that is described in U.S. Pat. No. 5,723,127, which is
hereby incorporated by reference (available from Genetics
Institute, Inc., Cambridge, Mass.). Other cytokines or lymphokines
have been shown to have immune modulating activity, including, but
not limited to, the interleukins 1-.alpha., 1-.beta., 2, 4, 5, 6,
7, 8, 10, 13, 14, 15, 16, 17 and 18, the interferons-.alpha.,
.beta. and .gamma., granulocyte colony stimulating factor, and the
tumor necrosis factors .alpha. and .beta., and are suitable for use
as adjuvants.
[0125] Still other suitable optional components of the immunogenic
compositions of this invention include, but are not limited to:
surface active substances (e.g., hexadecylamine, octadecylamine,
octadecyl amino acid esters, lysolecithin,
dimethyldioctadecylammonium bromide), methoxyhexadecylgylcerol, and
pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC,
carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine,
tuftsin; oil emulsions; and mineral gels, e.g., aluminum phosphate,
etc. and immune stimulating complexes. The CT-CRM and antigen may
also be incorporated into liposomes, or conjugated to
polysaccharides, lipopolysaccharides and/or other polymers for use
in an immunogenic composition.
[0126] Immunogenic compositions of this invention including the
CT-CRM mutant(s), or DNA sequences and molecules encoding the
desired CT-CRM of this invention, are also useful as polynucleotide
compositions (also known as DNA immunogenic compositions) or
administered with polynucleotides encoding the selected antigen.
For example, it has been previously demonstrated that BALB/c mice
administered a formulation of plasmid DNA (pDNTA) encoding the full
length glycoprotein D of herpes simplex virus (HSV) type 2 (gD2),
along with CT-CRM.sub.E29H by the intradermal route generated a
higher average cellular response than those that received plasmid
DNA encoding HSV gD2 by itself by the intradermal route. In
addition, the average serum antibody titers for mice, which
received the plasmid DNA HSV gD2 composition along with
CT-CRM.sub.E29H was approximately the same as that seen in mice
that received the plasmid DNA HSV gD2 composition without adjuvant.
Similarly, the plasmid DNA HSV gD2 composition adjuvanted with
CT-CRM.sub.E29H also generated a gD2-specific antibody response in
vaginal wash samples at levels that were comparable to those seen
following the delivery of the non-adjuvanted composition by
intradermal or intramuscular routes. Mice immunized with the
plasmid DNA HSV gD2 composition adjuvanted with CT-CRM.sub.E29H or
CT and delivered by the intradermal route also generated
substantially higher levels of gamma interferon and IL-5 than mice
that received the plasmid DNA HSV-gD2 composition without adjuvant.
Thus, the CT-CRMs enhance proliferative and gamma interferon
responses when administered with a plasmid DNA composition against
HSV.
[0127] In addition to a carrier as described above, immunogenic
compositions composed of polynucleotide molecules desirably contain
optional polynucleotide facilitating agents or "co-agents", such as
a local anesthetic, a peptide, a lipid including cationic lipids, a
liposome or lipidic particle, a polycation such as polylysine, a
branched, three-dimensional polycation such as a dendrimer, a
carbohydrate, a cationic amphiphile, a detergent, a benzylammonium
surfactant, or another compound that facilitates polynucleotide
transfer to cells. Such a facilitating agent includes bupivacaine
(see U.S. Pat. No. 5,593,972, which is hereby incorporated by
reference). Other non-exclusive examples of such facilitating
agents or co-agents useful in this invention are described in U.S.
Pat. Nos. 5,703,055; 5,739,118; 5,837,533; International Patent
Publication No. WO96/10038, published Apr. 4, 1996, and
International Patent Publication No WO94/16737, published Aug. 8,
1994, which are each incorporated herein by reference.
[0128] Most preferably, the local anesthetic is present in an
amount that forms one or more complexes with the nucleic acid
molecules. When the local anesthetic is mixed with nucleic acid
molecules or plasmids of this invention, it forms a variety of
small complexes or particles that pack the DNA and are homogeneous.
Thus, in one embodiment of the immunogenic compositions of this
invention, the complexes are formed by mixing the local anesthetic
and at least one plasmid of this invention. Any single complex
resulting from this mixture may contain a variety of combinations
of the different plasmids. Alternatively, in another embodiment of
the compositions of this invention, the local anesthetic may be
pre-mixed with each plasmid separately, and then the separate
mixtures combined in a single composition to ensure the desired
ratio of the plasmids is present in a single immunogenic
composition, if all plasmids are to be administered in a single
bolus administration. Alternatively, the local anesthetic and each
plasmid may be mixed separately and administered separately to
obtain the desired ratio. Where, hereafter, the term "complex" or
"one or more complexes" or "complexes" is used to define this
embodiment of the immunogenic composition, it is understood that
the term encompasses one or more complexes with each complex
containing a mixture of the CT-CRM-encoding plasmids and
antigen-encoding plasmids, or a mixture of complexes formed
discretely, wherein each complex contains only one type of plasmid,
or a one or a mixture of complexes wherein each complex contains a
polycistronic DNA. Preferably, the complexes are between about 50
to about 150 nm in diameter. When the facilitating agent used is a
local anesthetic, preferably bupivacaine, an amount of from about
0.1 weight percent to about 1.0 weight percent based on the total
weight of the polynucleotide composition is preferred. See, also,
International Patent Publication No. WO99/21591, which is hereby
incorporated by reference, and which teaches the incorporation of
benzylammonium surfactants as co-agents, preferably administered in
an amount of between about 0.001-0.03 weight %. According to the
present invention, the amount of local anesthetic is present in a
ratio to said nucleic acid molecules of 0.01-2.5% w/v local
anesthetic to 1-10 .mu.g/ml nucleic acid. Another such range is
0.05-1.25% w/v local anesthetic to 100 .mu.g/ml to 1 ml/ml nucleic
acid.
[0129] As used, such a polynucleotide immunogenic composition
expresses the CT-CRM and antigens on a transient basis in vivo; no
genetic material is inserted or integrated into the chromosomes of
the host. This use is thus distinguished from gene therapy, where
the goal is to insert or integrate the genetic material of interest
into the chromosome. An assay is used to confirm that the
polynucleotides administered by immunization do not rise to a
transformed phenotype in the host (U.S. Pat. No. 6,168,918).
[0130] The immunogenic compositions may also contain other
additives suitable for the selected mode of administration of the
composition. The composition of the invention may also involve
lyophilized polynucleotides, which can be used with other
pharmaceutically acceptable excipients for developing powder,
liquid or suspension dosage forms. See, e.g., Remington: The
Science and Practice of Pharmacy, Vol. 2, 19.sup.th edition (1995),
e.g., Chapter 95 Aerosols; and International Patent Publication No.
WO99/45966, the teachings of which are hereby incorporated by
reference. Routes of administration for these compositions may be
combined, if desired, or adjusted.
[0131] These nucleic acid molecule-containing immunogenic
compositions can contain additives suitable for administration via
any conventional route of administration. In some preferred
embodiments, the immunogenic composition of the invention is
prepared for administration to human subjects in the form of, for
example, liquids, powders, aerosols, tablets, capsules,
enteric-coated tablets or capsules, or suppositories.
[0132] The immunogenic compositions of the present invention
(whether protein-containing or nucleic acid molecule-containing
compositions), as described above, are not limited by the selection
of the conventional, physiologically acceptable, carriers,
adjuvants, or other ingredients useful in pharmaceutical
preparations of the types described above. The preparation of these
pharmaceutically acceptable compositions, from the above-described
components, having appropriate pH isotonicity, stability and other
conventional characteristics is within the skill of the art.
[0133] E. Methods of Use of the Compositions of this Invention
[0134] The immunogenic compositions of this invention that comprise
the CT-CRM alone or a combination of the CT-CRM and a selected
antigen, are administered to a human or to a non-human vertebrate
by a variety of routes to enhance the immune response to an
antigen, preferably a disease-causing antigen, as identified above.
The compositions of the present invention modulate the immune
response L-5 improving the vertebrate host's antibody response and
cell-mediated immunity after administration of a composition
comprising a selected antigen as described above, and an effective
adjuvanting amount of a mutant CT-CRM where the mutant CT-CRM has
substantially reduced toxicity compared to a wild-type CT, and
wherein the reduced toxicity is a result of a single amino acid
substitution, a double amino acid substitution, or amino acid
insertions.
[0135] In one embodiment, the immunogenic composition containing
the CT-CRM (either as a protein or encoded by a nucleic acid
molecule) is administered prior to administration of a composition
comprising the selected antigen (either as a protein or as a
nucleic acid). In another embodiment, the immunogenic composition
is administered simultaneously with the antigen, whether it is
administered in a composition containing both antigen and CT-CRM or
as a separate composition from that of the antigen-containing
composition. In still a further embodiment, the composition
containing the CT-CRM is administered after the composition
containing the antigen. It is preferable, although not required,
that the antigen and the mutant CT-CRM be administered at the same
time.
[0136] The immunogenic composition containing the CT-CRM may be
administered as a protein or as a nucleic acid molecule encoding
the protein, as described above. The immunogenic composition
containing the CT-CRM may be administered as a protein in
combination with a selected antigen administered as a protein.
Alternatively, as described above, the CT-CRM immunogenic
composition may be administered as a protein with a nucleic acid
molecule encoding the antigen, as described above. Still another
alternative involves administering both the CT-CRM and the antigen
as nucleic acid sequences encoding these proteins.
[0137] Any suitable route of administration may be employed to
administer the immunogenic composition containing the CT-CRM. The
route may be the same or different from a route selected to
administer a composition containing the selected antigen, if the
CT-CRM and antigen are administered in separate compositions or in
different forms, e.g., protein or nucleic acids. Suitable routes of
administration include, but are not limited to, intranasal, oral,
vagina, rectal, parenteral, intradermal, transdermal (see, e.g.,
International patent publication No. WO 98/20734, which is hereby
incorporated by reference), intramuscular, intraperitoneal,
subcutaneous, intravenous and intraarterial. The appropriate route
is selected depending on the nature of the immunogenic composition
used, and an evaluation of the age, weight, sex and general health
of the patient and the antigens present in the immunogenic
composition, and similar factors by an attending physician.
[0138] In general, selection of the appropriate "effective amount"
or dosage for the CT-CRM and/or antigen components of the
immunogenic composition(s) of the present invention will also be
based upon the protein or nucleic acid form of the CT-CRM and
antigen, the identity of the antigen in the immunogenic
composition(s) employed, as well as the physical condition of the
subject, most especially including the general health, age and
weight of the immunized subject. The method and routes of
administration and the presence of additional components in the
immunogenic compositions may also affect the dosages and amounts of
the CT-CRM and antigen. Such selection and upward or downward
adjustment of the effective dose is within the skill of the art.
The amount of CT-CRM and antigen required to induce an immune
response, preferably a protective response, or produce an exogenous
effect in the patient without significant adverse side effects
varies depending upon these factors. Suitable doses are readily
determined by persons skilled in the art.
[0139] As an example, in one embodiment, for the compositions
containing protein components, e.g., a CT-CRM variant protein
and/or antigen as described above, each dose may comprise between
about 1 .mu.g to about 20 mg of the protein per mL of a sterile
solution. Other dosage ranges may also be contemplated by one of
skill in the art. Initial doses may be optionally followed by
repeated boosts, where desirable. In another example, the amounts
of nucleotide molecules in the DNA and vector compositions may be
selected and adjusted by one of skill in the art. In one
embodiment, each dose will comprise between about 50 .mu.g to about
1 mg of CT-CRM-encoding or antigen-encoding nucleic acid, e.g., DNA
plasmid, per mL of a sterile solution.
[0140] The number of doses and the dosage regimen for the
composition are also readily determined by persons skilled in the
art. Protection may be conferred by a single dose of the
immunogenic composition containing the CT-CRM, or may require the
administration of several doses with or without the selected
antigen, in addition to booster doses at later times to maintain
protection. In some instances, the adjuvant property of the mutant
CT-CRM may reduce the number of doses containing antigen that are
needed or may reduce the time course of the dosage regimen. The
levels of immunity can be monitored to determine the need, if any,
for boosters.
[0141] In order that this invention may be better understood, the
following examples are set forth. The examples are for the purpose
of illustration only and are not to be construed as limiting the
scope of the invention.
[0142] All references cited herein are hereby incorporated by
reference.
EXAMPLE 1
Expression of CT Mutants
[0143] A. Bacterial Strains, Plasmids and Growth Condition
[0144] E. coli TG1 (Amersham-Pharmacia Biotech, Piscataway, N.J.),
and TX1, a nalidixic acid-resistant derivative of TG1, carrying
FTc, lacIq from XL1 blue (Stratagene, LaJolla, Calif.; and
CJ236(FTc, lacIq) (Bio-Rad, Hercules, Calif.) were used as hosts
for cloning recombinant plasmids and expression of mutated
proteins. Plasmid-containing strains were maintained on LB agar
plates with antibiotics as required (ampicillin, 50 .mu.g/ml;
kanamycin 25 .mu.g/m; tetracycline 10 .mu.g/ml). A complete CT
operon from V. cholerae 0395 was subcloned into the phagemid vector
pSKII-, under the control of the lac promoter, to create the IPTG
inducible plasmid designated pMGJ67 (Jobling, M. G., and Holmes, R.
K., 1992 Infect. Immun., 60, 4915-4924).
[0145] B. Mutagenesis of ctxA Gene
[0146] The method of Kunkel, T. A., 1985 Proc. Natl. Acad. Sci.,
USA, 82, 488-492 was used to select for oligonucleotide-derived
mutants created in plasmid pMGJ67. The oligonucleotides used to
generate the mutant CT-CRMs and the various amino acid
substitutions in the mutant CT-CRMs are listed in Table 2.
TABLE-US-00002 TABLE 2 Sequence of Oligonucleotides Introduced into
ctxA SEQ ID Substitution Oligonucleotide Sequence.sup.a NO. I16A
CCTCCTGATGAAGSYCAAGCAGTCAGG 5 S68Y GTTTGAGATCTGCCCACT 6 V72Y
GTTTGACCCACTAAGTGGGC 7 S6SY + V72Y GTTTGAGATATGCCCACTTATATGGTCAAC 8
.sup.aAltered bases are underlined. S represents G or C; Y
represents C or T.
[0147] Briefly, the CT-CRM.sub.I16A mutant was made directly in
pMGJ142 using the QuickChange mutagenesis kit as described by the
supplier (Stratagene Inc., LaJolla, Calif.). The double mutant
plasmid containing the CT-CRM.sub.S68Y, V72Y substitutions was made
by PCR using the mutagenic primer disclosed in Table 2 to create a
megaprimer followed by cloning of the mutated ctxA-encoding
XbaI-ClaI fragment into pMGJ142. The CT-CRM.sub.I16A,S68Y double
mutant was made by PCR of the I16A containing clone using the
mutagenic primer to create a megaprimer followed by cloning of the
mutated ctxA-encoding XbaI-ClaI fragment into pMGJ142. The
CT-CRM.sub.V72Y and CT-CRM.sub.I16A,S68Y mutants were made by
reversion of the CT-CRM.sub.S68Y,V72Y double mutant back to
wild-type at amino acid position 68 using the QuickChange
mutagenesis kit. Each single-stranded oligonucleotide was
phosphorylated and used to direct second strand synthesis on a
uracil-containing single-stranded DNA template rescued from the E.
coli dut ung strain CJ236 (F'Tc, pMGJ67). Following ligation and
transformation of ung.sup.+ strain TX1, single-stranded DNA was
rescued from Amp.sup.R transformants and sequenced by the dideoxy
chain termination method (Kunkel, cited above).
[0148] C. Construction of Arabinose Promoted CT-CRM Expression
Vectors.
[0149] Previous experience with CT-CRM.sub.E29H (International
patent publication No. WO 00/18434) has shown that maximal
production in E. coli could be achieved by substituting synthetic
Shine-Delgarno sequences upstream of the ctxA gene and placing the
operon under the control of the arabinose promoter system CT
operons containing site directed mutations in the A subunit were
made as previously described (supra). CT-CRMs were originally under
the control of a .beta.lac promoter and expression levels in E.
coli were low. PCR was used to modify the region 5' to the ATG of
the CT-A subunit and insert an NheI site at the 5' end. The
corresponding 3' primer added a HindIII site at the 3' end of the
CT-B gene.
TABLE-US-00003 Primer sequences used were: (SEQ. ID NO. 9) CT
forward: 5' TTTTTTGGGCTAGCATGGAGGAAAAGATGAGC; and (SEQ. ID NO. 10)
CT reverse: 5' CGAGGTCGAAGCTTGCATGTTTGGGC.
[0150] PCR was performed on each mutant CT-CRM operon and the PCR
products were ligated into pCR2.1-Topo (Invitrogen) according to
the manufacturer's directions and transformed into Top10F' cells.
Recombinant E. coli were plated onto SOB agar containing Kanamycin
(25 .mu.g/ml) and X-gal (40 .mu.g/ml). Plasmids from white colonies
were screened for inserts by digestion with EcoRI. Plasmids
containing inserts of the correct size were digested with NheI and
HindIII according to the manufacturer's directions and the DNA
fragments containing the CT operons isolated from low melting point
agarose. Plasmid pBAD18-Cm (Invitrogen) was digested with
NheI-HindIII and the linear DNA isolated from low melting point
agarose. Digested pBAD18 and the CT operons were ligated at
12.degree. C. and transformed into Top10F E. coli. Plasmids from
chloramphenicol-resistant colonies were screened for inserts by
restriction analysis, and representative clones were sequenced to
confirm the presence of the site directed mutations. Plasmids were
transformed into DH5.alpha. for expression of CT-CRMs. The plasmids
encoding the mutant CT-CRMs bearing a single amino acid
substitution at positions 16 and 72 are designated as pLP903 and
pLP905 respectively, and the plasmids encoding the mutant CT-CRMs
bearing double amino acid substitution at positions 16 and 68 and
at positions 68 and 72 are designated as pLP904 and pLP906
respectively. The plasmids contain the polycistron of V. cholerae
genes ctxA and ctxB which encode the CT.
[0151] D. Expression of CT-CRMs in E. coli.
[0152] E. coli DH5.alpha. cells containing plasmids pLP903, pLP904,
pLP905, or pLP906, cells expressing CT-CRMs CT-CRM.sub.I16A,
CT-CRM.sub.S68Y,I16A, CT-CRM.sub.V72Y, and CT-CRM.sub.S68Y, V72Y
respectively, were grown in phosphate buffered Hy-Soy media
containing chloramphenicol (25 .mu.g/ml) and glycerol (0.5%) at
37.degree. C. with aeration. When cultures reached an OD.sub.600 of
approximately 4.5-5.5, they were induced by addition of L-arabinose
to a final concentration of 0.5%. Cultures were incubated at
37.degree. C. with aeration for three hours post-induction and then
the cells collected by centrifugation. Cell pellets were stored at
-20.degree. C.
[0153] E. Preparation and Purification of CT-CRMs.
[0154] Cell pellets were thawed at room temperature and resuspended
in 10 mM NaPO.sub.4 and 1 mM EDTA (pH 7.0) at 9% of the original
culture volume. Cell suspensions were mechanically disrupted in a
microfluidizer and centrifuged for 10 minutes at 8,500.times.g.
Cell lysates were further clarified at 160,000.times.g for one
hour. The clarified cell lysate was loaded, at a flow rate of 2
ml/min, onto a carboxymethyl (CM)-sepharose.TM. column (300 ml
CM-sepharosen.TM. per 10 l of culture) (Amersham, Pharmacia)
equilibrated with 10 mM NaPO.sub.4 (pH 7.0). The column was washed
with >10 volumes of 10 mM NaPO.sub.4 (pH 7.0) at a flow rate of
5 ml/min. CT-CRM.sub.E29H holotoxin was eluted with four column
volumes of 10 mM NaPO.sub.4 (pH 8.3). Purified CT-CRMs were buffer
exchanged by dialysis into PBS and stored at 4.degree. C. The
presence of intact holotoxin and the respective subunits was
determined by native polyacrylamide gel electrophoresis (PAGE) and
SDS-PAGE, respectively. Native PAGE indicated the presence of a
purified molecule of 86 kDa (data not shown), the expected
molecular weight for intact cholera holotoxin (Tebbey et al., 2000
Vaccine, 18 (24): 2723-2734.
[0155] In addition, SDS-PAGE showed two bands that aligned with the
CT-A (27 kDa) and CT-B (12 kDa) subunits that comprise the intact
holotoxin (data not shown).
EXAMPLE 2
Non-Denaturing Polyacrylamide Gel Electrophoresis
[0156] Mutant CT-CRMS, CT-CRM.sub.I16A,S68Y and CT-CRM.sub.v72Y,
were analyzed by non-denaturing page electrophoresis to determine
the percentage of the CT-CRMs present after purification as intact
holotoxin. Purified CT-CRMs, 15 .mu.l each (at various protein
concentrations), were run through a 6% polymerized non-denaturing
polyacrylamide gel. Three different concentrations (300, 600 and
1200 ng) of CT-B were used as a standard. After electrophoresis the
gel was stained with Coomassie blue. The gel was then scanned using
a densitometer, and the percentage of the holotoxin was calculated
from the densitometer readings of the CT-CRMs and CT-B standard.
The data indicated that 97.8% of CT-CRM.sub.I16A,S68Y and 99.4% of
CT-CRM.sub.V72Y were present as intact holotoxins (Table 3).
TABLE-US-00004 TABLE 3 Native Gel Assay for Intact Holotoxin CT-CRM
% of holotoxin CT-CRM.sub.I16A 97.8 CT-CRM.sub.V72Y 99.4
CT-CRM.sub.I16A,S68Y Not done CT-CRM.sub.S68Y,V72Y Not done
EXAMPLE 3
Y-1 Adrenal Cell Assay for Residual Toxicity of CT-CRMS
[0157] Mutant CT-CRMs were compared with wild-type CT for toxicity
in the mouse Y-1 adrenal tumor cell assay, which is used in vitro
to measure toxicity of enterotoxins in the cholera toxin/heat
labile enterotoxin family. The assay depends upon binding of the
toxin to cell surface receptors, and the subsequent entry of the A1
subunit of the toxin into the cytoplasm of the cell.
[0158] Native cholera toxin isolated from V. cholerae is
proteolytically nicked at the CT-A1-CT-A2 junction, resulting in
the A1 and A2 subunits of cholera toxin being held together by only
a disulfide bond. This makes the A1 and A2 subunits unstable and
easily dissociable from each other. The A1 subunit of the nicked CT
dissociates from the AZ subunit upon binding cell surface receptor,
and enters the cell, where it ADP-ribosylates the regulatory
G-protein (Gs.alpha.), leading to its toxic effects as described in
the background above. In contrast, enterotoxins produced in E. coli
(either CT or LT) are unnicked, and thus, have the A1-A2 peptides
still joined. Consequently, the CT produced in V. cholerae are
significantly more toxic in the Y-1 adrenal cell assays than the CT
produced in a heterologous bacterial cell such as E. coli.
[0159] In a first Y-1 adrenal cell assay, mutant CT-CRMs were
compared to nicked wild-type CT from V. cholerae for toxicity. In
this assay, Y-1 adrenal cells (ATCC CCL-79) were seeded in 96-well
flat-bottom plates at a concentration of 10 Cells per well.
Thereafter, three-fold serial dilutions of purified (.about.90%
purity as determined by Coomassie staining) CT-CRMs were added to
the tumor cells and incubated at 37.degree. C. (5% CO.sub.2) for 18
hours. The cells were then examined by light microscopy for
evidence of toxicity (cell rounding). The endpoint titer was
defined as the minimum concentration of toxin required for greater
than 50% cell rounding. The percent of residual toxicity was then
calculated using the endpoint titer of wild-type nicked CT from V.
cholerae (100% toxicity) divided by the titer elicited by CT-CRMs
multiplied by 100. The data set forth in Table 4 indicate that the
residual toxicity of the four purified mutant holotoxins,
CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y, CT-CRM.sub.v72Y, and
CT-CRM.sub.S68Y,V72Y tested using the Y-1 adrenal cell assay was
only 0.37%.
TABLE-US-00005 TABLE 4 Y-1 Adrenal Cell Assay CT-CRM % Residual
Toxicity CT-CRM.sub.I16A 0.37 CT-CRM.sub.I16A,S68Y 0.37
CT-CRM.sub.V72Y 0.37 CT-CRM.sub.S68Y,V72Y 0.37
[0160] In a second independent study, crude periplasmic extracts of
E. coli cells (TG1) expressing elevated levels of mutant CT-CRMs,
were compared against unnicked wild-type CT holotoxin expressed in
E. coli for residual toxicity in Y-1 adrenal cell assay. Y-1
adrenal cells were incubated in multi-well dishes in an RPMI medium
containing 10% fetal calf serum in the presence of crude E. coli
cell lysate. Cell toxicity was monitored as before. In this study,
one toxic unit was defined as the smallest amount of toxin or
supernatant that caused rounding of 75-100% of the cells in a well
after overnight incubation. The results of this study are presented
in Table 5 below.
TABLE-US-00006 TABLE 5 Y-1 Adrenal Cell Assay CT-CRM % Residual
Toxicity CT-CRM.sub.I16A 5 CT-CRM.sub.V72Y 100 CT-CRM.sub.S68Y,V72Y
5 CT-CRM.sub.I16A,S68Y Not Determined
[0161] The results of this study indicated that while the
toxicities of CT-CRM.sub.I16A and CT-CRM.sub.S68Y,V72Y were
substantially reduced (5%), the CT-CRM.sub.V72Y was as toxic as the
wild-type CT. Without being bound by theory, the variant results in
second study (Table 5) may be attributable to the fact that
periplasmic crude E. coli cell lysates used in the second study
contained unnicked mutant CT-CRMs, and to the fact that the
toxicity was measured as a percentage of the toxicity of wild-type,
unnicked CT produced by E. coli. In contrast, the unnicked
wild-type CT from E. coli has a 50% cell rounding dose of 6250
pg/ml in the Y1 cell assay (data not shown). In the first study,
the residual cytotoxicity of the mutant CT-CRMs is expressed as a
percentage of the toxicity of wild-type, nicked CT produced by K
cholerae, wherein the nicked holotoxin has a 50% cell rounding dose
of 125 pg/ml in the Y1 cell assay. Consequently, the residual
toxicity reported in the second study is 50 fold higher than that
obtained in the first study.
EXAMPLE 4
The ADP-Ribosyltransferase Assay
[0162] NAD.sup.+:agmatine ADP-ribosyltransferase activity was
measured as the release of [carbonyl-.sup.14C] nicotinamide from
radiolabeled NAD.sup.+. Briefly, CT and CT-CRMs were trypsin
activated and incubated for 30 minutes at 30.degree. C. with 50 mM
glycine/20 mM dithiothreitol in TEAN buffer (Tris/EDTA/sodium
azide/sodium chloride) (pH 8.0). Thereafter, the following
materials were added to the reaction: 0.1 .mu.g of soybean trypsin
inhibitor, 50 mM potassium phosphate, 10 mM agmatine, 20 mM
dithiothreitol, 10 mM magnesium chloride, 100 .mu.M GTP, 3 mM
dimyristoylphosphatidyl-choline, 0.2% cholate, 0.03 mg of
ovalbumin, 100 .mu.M [adenine-U-.sup.14C]NAD (DuPont NEN.TM.,
Boston, Mass.) and water to a final volume of 300 .mu.l. After
incubation for 90 minutes at 3.degree. C., 100 .mu.l samples were
applied to columns (0.64.times.5 cm) of AG1-X2 (Bio-Rad) that were
washed five times with 1.0 ml of distilled/deionized H.sub.2O.
Eluates containing [.sup.14C]ADP-ribosylagmatine were collected for
radioassay. Mean recovery of .sup.14C in the eluate is expressed as
percentage of that applied to column. The results are presented in
Table 6.
TABLE-US-00007 TABLE 6 NAD: Agmatine ADP-Ribosyltransferase
Activity ADP-ribosylagmatine formed % ADP-ribosylation CT/CT-CRM
(nmol/hr/.mu.g protein) activity CT, 10 .mu.g 52.5 100
CT-CRM.sub.I16A 3.3 2.4 CT-CRM.sub.I16A,S68Y 3.4 3.3
CT-CRM.sub.V72Y 2.7 1.1 CR-CRM.sub.S68Y,V72Y 2.9 1.2
[0163] ADP-ribosyltransferase activity was also independently
determined using diethylamino (benzylidine-amino) guanidine
(DEABAG) as a substrate. In this assay, 25 .mu.l aliquots of mutant
CT-CRMS from purified cell lysates, activated for 30 minutes at
30.degree. C. with 1/50 w/w trypsin, were incubated with 200 .mu.l
2 mM DEABAG in 0.1M K.sub.2P0.sub.4, pH 7.5, 10 .mu.M NAD, 4 mM DTT
for two hours. The reaction was stopped by adding 800 .mu.l of a
slurry buffer containing 400 mg DOWEX AG50-X8 resin, to bind
unreacted substrate. ADP-ribosylated DEABAG in the supernatant was
quantitated by florescence emission in a DyNA Quant fluorimeter
calibrated with DEABAG. With the exception of the mutant
CT-CRM.sub.V72Y, ADP ribosyl-transferase activities of the mutant
CT-CRMs were substantially reduced over that of wild-type (Table
7). The high level of ADP-ribosyl-transferase activity seen with
CT-CRM.sub.V72Y may be attributable to the fact that in this study
the ADP ribosyl-transferase activity of mutant CT-CRMs was measured
using a different substrate in a different assay protocol.
TABLE-US-00008 TABLE 7 ADP-ribosyltransferase Activity of CT-CRMs
using Diethylamino (benzylidine-amino) Guanidine (DEABAG) CT/CT-CRM
% ADP-ribosylation Activity CT 100 CT-CRM.sub.S68Y 11
CT-CRM.sub.V72Y 68 CT-CRM.sub.S68Y,V72Y 3 CT-CRM.sub.I16A,S68Y Not
done
EXAMPLE 5
Immune Responses of Balb/c Mice Immunized with Recombinant P4 Outer
Membrane Protein (RP4) of Nontypable Haemophilus influenzae (NTHI)
Alone or in Conjunction with CT-CRMS
[0164] In a first experiment, the ability of the mutant
CT-CRM.sub.I16A to enhance the induction of systemic and mucosal
antibodies to recombinant P4 outer membrane protein, (rP4) were
assessed. Serum and mucosal anti-P4 antibody titers induced by
mutant CT-CRM.sub.I16A, were assessed and compared with that of
wild-type CT and mutant, CT-CRM.sub.E29H (WO 00/18434). In this
study, Balb/c mice were immunized intranasally (IN) at weeks 0, 3
and 5 and at week 5, day 6 with a formulation containing 1 .mu.g of
recombinant P4 protein in saline or 1 .mu.g of P4 together with 1
.mu.g of wild-type CT, 1 .mu.g of CT-CRM.sub.E29H or 0.1, 1, or 10
.mu.g of CT-CRM.sub.I16A.
[0165] The results indicate that the CT-CRM.sub.I16A, like the
wild-type CT and CT-CRM.sub.E29H, augmented the capacity of rP4
protein to elicit systemic and mucosal immune responses (Table 8).
For example, six weeks after primary IN immunization the anti-rP4
IgG antibody titers of mice immunized with rP4 protein formulated
with either CT-CRM.sub.I16A or CT-CRM.sub.E29H were 40 times
greater than that of mice immunized with the recombinant proteins
in PBS alone. The antibody titers (IgG) of mice administered the
recombinant proteins plus wild-type CT holotoxin at a concentration
of 1 .mu.g were elevated 67-fold in comparison to antibody titers
in mice administered recombinant rP4 alone in saline. The antibody
titers of mice immunized with 1 .mu.g of the mutant CT-CRM,
CT-CRM.sub.E29H were elevated 55-fold over antibody titers in mice
immunized with rP4 alone. In comparison, the antibody titers of
mice immunized with 1 .mu.g and 0.1 .mu.g of the mutant CT-CRM,
CT-CRM.sub.I16A, were increased 15-fold and 27-fold respectively
over the anti-rP4 antibody titers in mice immunized with rP4 alone
in saline.
TABLE-US-00009 TABLE 8 Serum Antibody Responses to Recombinant P4
Protein Week 0 Week 3 Week 5 Week 5, Day 6 Adjuvant Amount IgA IgG
IgA IgG IgA IgG IgA IgG Saline 148 386 199 394 140 444 CT 1.0 .mu.g
184 1,348 203 1,949 1,570 29,939 CT-CRM.sub.E29H 1.0 .mu.g 108 615
286 1,057 1,492 21,251 CT-CRM.sub.I16A 1.0 .mu.g <100 426 114
2,071 1,096 6,708 CT-CRM.sub.I16A 0.1 .mu.g 261 1,463 403 2,629
1,105 10,541 CT-CRM.sub.I16A 10.0 .mu.g 151 502 121 788 108 1,460
183 2,043
[0166] An examination of the protein-specific antibodies in the
mucosal secretions of the Balb/c mice immunized IN at weeks 0, 3,
and 5 was made two weeks after tertiary immunization. Mucosal
samples were collected at week 5, day 6 from vaginal wash (VW), the
nasal wash (NW); the bronchioalveolar lavage (BAL) and from saliva
(SAL). These results shown in Table 9 further indicated the
CT-CRM.sub.I16A facilitated the generation of local immune
responses against the rP4 protein. Moreover, the anti-rP4 antibody
titers were comparable to those induced bed the wild-type CT
adjuvanted immunogenic composition.
TABLE-US-00010 TABLE 9 Mucosal Antibody Responses to rP4 Protein.
VW NW BAL SAL Adjuvant Amt IgA IgG IgA IgG IgA IgG IgA IgG Saline
24 <10 <10 <10 <10 <10 20 <10 CT 1.0 .mu.g 125 74
38 <10 158 152 152 14 CT-CRM.sub.E29H 1.0 .mu.g 254 12 58 <10
523 364 454 27 CT-CRM.sub.I16A 1.0 .mu.g 154 16 39 <10 330 38
654 32 CT-CRM.sub.I16A 0.1 .mu.g 422 26 60 <10 125 53 1027 74
CT-CRM.sub.I16A 10.0 .mu.g 19 <10 <10 <10 13 16 34
<10
[0167] In a second experiment, five BALB/c mice per group were
immunized IN on days 0, 21 and 35 with a 15 .mu.l dose containing 1
.mu.g rP4 alone or 1 .mu.g rP4 plus 1 .mu.g of one of the mutant
CT-CRMs. CT-CRM.sub.E29H, CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y,
CT-CRM.sub.V72Y or CT-CRM.sub.S68Y,V72Y as an adjuvant as indicated
in Table 10. The anti-rP4 IgA and IgG antibody titers were
determined by ELISA on pooled samples collected at weeks 0, 3, 5
and week 5, day 6 and the results shown in Table 10. The results
indicate that serum anti-rP4, IgA and IgG titers were substantially
increased in mice that were administered the antigen along with one
of the mutant CT-CRMS. The mucosal antibody responses to rP4 were
also measured one week after the last immunization (week 5, day
6).
TABLE-US-00011 TABLE 10 Adjuvant Effects of Mutant Cholera Toxins
on the Immune Response to NTHi rP4 Protein Delivered Intranasally
to Female BALB/c Mice.sup.a Anti-NTHi rP4 ELISA Endpoint Titers on
Pooled Sera.sup.c Week 0 Week 3 Week 5 Week 5, day 6 Adjuvant.sup.b
IgA IgG IgA IgG IgA IgG IgA IgG Saline 123 593 <100 715 227
1,953 224 6,458 CT-CRM.sub.E29H 116 934 1,808 135,824 7,365 282,099
CT-CRM.sub.I16A 269 423 788 17,465 2,609 143,313
CT-CRM.sub.I16A,S68Y 256 545 821 6,062 1,998 438,553
CT-CRM.sub.V72Y 294 878 1,725 40,443 5,239 343,711
CT-CRM.sub.S68Y,V72Y 172 429 333 4,353 1,333 55,571 .sup.a1 .mu.g
NTHi rP4 was delivered IN to female BALB/c mice in a 15 .mu.l
volume at weeks 0, 3 and 5. .sup.bNTHi rP4 compositions were
formulated with saline or 1 .mu.g of various mutant cholera toxins.
.sup.cSera were collected at weeks 0, 3, 5 and week 5, day 6;
pooled samples represent an n = 5.
[0168] Table 11 sets forth the IgA and IgG titers from nasal,
bronchioalveolar and vaginal washes, and saliva respectively. These
results also indicate that mucosal anti-rP4, IgA and IgG titers
were also substantially elevated in mice administered rP4 antigen
together with one of the mutant CT-CRMs in comparison to mice
administered rP4 in saline.
TABLE-US-00012 TABLE 11 Anti-NTHi rP4 ELISA Endpoint Titers on
Mucosal Wash Pools.sup.a Vaginal Lung Wash.sup.c Nasal Wash.sup.c
Saliva.sup.c Wash.sup.c Adjuvant.sup.b IgA IgG IgA IgG IgA IgG IgA
IgG Saline <10 65 <10 <10 12 <10 <10 <10
CT-CRM.sub.E29H 1,131 1,815 196 70 977 1,906 255 149
CT-CRM.sub.I16A 360 708 166 37 733 96 1,066 414
CT-CRM.sub.I16A,S68Y 449 376 164 19 1,221 177 1,521 261
CT-CRM.sub.V72Y 3,850 4,226 500 300 2,753 281 1,066 212
CT-CRM.sub.S68Y,V72Y 84 164 111 35 843 103 251 109 .sup.a1 .mu.g
NTHi rP4 was delivered IN to female BALB/c mice in a 15 .mu.l
volume at weeks 0, 3 and 5. .sup.bNTHi rP4 compositions were
formulated with saline or 1 .mu.g of various mutant cholera toxins.
.sup.cMucosal samples were collected on week 6; pooled samples
represent an n = 5.
[0169] On week 5, day 6, following IN administration, IgA and IgG
including IgG subclass IgG1, IgG2a, IgG2b and IgG3 endpoint titers
in the serum of each individual mouse in the six groups were also
determined by ELISA. (Tables 12-17). In the data reported in Tables
12-17, statistical analyses were performed using JMP, SAS
Institute, Inc.; one-way analysis of variance was significant at
the p<0.0001 level; and multiple comparisons were performed
using Tukey-Kramer HSD, alpha=0.05.
TABLE-US-00013 TABLE 12 IgA Anti-NTHi rP4 ELISA Endpoint Titers in
each Individual Mouse at Week 5, Day 6. Adjuvant 1 2 3 4 5 GMT
StDev SEM Saline 217 63 44 48 35 63 76 34 CT-CRM.sub.E29H 6,791
2,196 1,740 1,526 4,626 2,835 2,276 1,018 CT-CRM.sub.I16A 2,075
2,186 1,208 2,847 1,975 1,985 585 262 CT-CRM.sub.I16A,s68Y 1,739
920 739 1,289 5,694 1,541 2,058 921 CT-CRM.sub.V72Y 6,523 1,634
1,290 4,089 10,521 .sup. 3,584.sup..dagger. 3,826 1,711
CT-CRM.sub.s68Y,V72Y 1,646 878 310 2,724 116 676* 1,069 478 *Values
differ significantly from the saline group .sup..dagger.Value
differs significantly from the CT-CRM.sub.s68Y,V72Y group.
TABLE-US-00014 TABLE 13 IgG Anti-NTHi rP4 ELISA Endpoint Titers in
Each Individual Mouse at Week 5, Day 6. Adjuvant 1 2 3 4 5 GMT
StDev SEM Saline 78,369 2,287 3,505 1,518 1,223 4,105* 34,105
15,252 CT-CRM.sub.E29H 440,101 280,030 17,291 111,803 193,831
135,797* 161,927 72,416 CT-CRM.sub.I16A 69,986 92,347 68,406
193,467 62,196 88,141* 54,968 24,582 CT-CRM.sub.I16A,s68Y 57,721
59,832 17,521 57,172 29,725 40,034* 19,471 8,708 CT-CRM.sub.V72Y
161,796 143,545 72,764 187,118 363,958 163,008* 108,260 48,415
CT-CRM.sub.s68Y,V72Y 80,770 71,053 14,366 73,904 2,658 27,662*
36,953 16,526 *Values differ significantly from the saline
group
TABLE-US-00015 TABLE 14 IgG1 Anti-NTHi rP4 ELISA Endpoint Titers in
Each Individual Mouse on Week 5, Day 6 Sera. Adjuvant 1 2 3 4 5 GMT
StDev SEM Saline 2,533 33 33 33 33 79 1,118 500 CT-CRM.sub.E29H
11,462 3,029 1,801 8,517 1,172 3,623* 4,547 2034 CT-CRM.sub.I16A
4,732 10,033 3,546 14,399 6,836 6,980* 4,385 1961
CT-CRM.sub.I16A,s68Y 2,945 1,036 1,016 2,945 343 1,256* 1,208 540
CT-CRM.sub.V72Y 9,123 13,942 545 7,939 35,887 7,230* 13,410 5997
CT-CRM.sub.s68Y,V72Y 28,434 13,553 394 7,579 33 2,070* 11,725 5244
*Values differ significantly from the saline group
TABLE-US-00016 TABLE 15 IgG2a Anti-NTHi rP4 ELISA Endpoint Titers
in Each Individual Mouse Week 5, Day 6 Adjuvant 1 2 3 4 5 GMT StDev
SEM Saline 10,611 317 431 230 217 591 4,613 2063 CT-CRM.sub.E29H
230,644 189,940 3,489 43,336 132,159 61,439* 95,830 42858
CT-CRM.sub.I16A 24,886 43,309 42,582 89,228 23,210 39,408* 26,663
11924 CT-CRM.sub.I16A,s68Y 39,555 37,700 6,947 29,050 17,604
22,122* 13,814 6178 CT-CRM.sub.V72Y 125,810 75,952 41,286 108,108
210,157 97,834* 63,522 28409 CT-CRM.sub.s68Y,V72Y 10,136 27,702
6,289 30,058 1,462 9,505* 12,950 5792 *Values differ significantly
from the saline group
TABLE-US-00017 TABLE 16 IgG2b Anti-NTHi rP4 ELISA Endpoint Titers
in Each Individual Mouse on Week 5, Day 6 Sera Adjuvant 1 2 3 4 5
GMT StDev SEM Saline 5,473 132 153 102 66 237 2,397 1072
CT-CRM.sub.E29H 85,041 70,413 529 8,597 36,760 15,852 37,108 16596
CT-CRM.sub.I16A 10,554 15,798 7,562 27,484 7,885 12,227* 8,302 3713
CT-CRM.sub.I16A,s68Y 13,545 9,942 2,731 8,724 6,257 7,253* 4,048
1810 CT-CRM.sub.V72Y 25,782 23,712 14,433 25,347 74,216 27,798*
23,664 10583 CT-CRM.sub.s68Y,V72Y 5,096 14,958 3,205 16,081 582
4,697* 7,074 3164 *Values differ significantly from the saline
group
TABLE-US-00018 TABLE 17 IgG3 Anti-NTHi rP4 ELISA Endpoint Titers in
Each Individual Mouse on Week 5, Day 6 Sera Adjuvant 1 2 3 4 5 GMT
StDev SEM Saline 992 33 33 33 33 65 429 192 CT-CRM.sub.E29H 311 36
23 344 1,246 162 500 224 CT-CRM.sub.I16A 2,256 62 390 290 150 299
918 411 CT-CRM.sub.I16A,s68Y 120 1,953 33 612 64 198 816 365
CT-CRM.sub.V72Y 479 929 25 1,124 462 357 432 193
CT-CRM.sub.s68Y,V72Y 202 69 3 92 33 42 76 34
EXAMPLE 6
The Immune Responses of Balb/c Mice Immunized with the Purified
Native Fusion (F) Glycoprotein of Respiratory Syncytial Virus
(RSV)
[0170] The capacity of the CT-CRMs of the present invention to
augment systemic and mucosal immune responses against respiratory
syncytial virus (RSV) glycoproteins was examined using the purified
native fusion (F) protein. Previously it was demonstrated that
BALB/c mice immunized IN with F protein adjuvanted with either CT
or CT-CRM.sub.E29H generated systemic and local IgG and IgA titers
(Tebbey et al., cited above). That study also indicated that
pre-existing anti-CT antibodies did not have a negative impact on
the level of local or systemic anti-F protein IgA and IgG
antibodies. Indeed, the study indicated that pre-existing anti-CT
antibodies were beneficial for the generation of an augmented
anti-F protein antibody response. Additionally, the data also
suggested a mechanism involving the neutralization of infectious
virus by either mucosal or humoral immunoglobulins that were
stimulated in response to the IN immunization protocol containing
F/CT-CRM.sub.E29H.
[0171] BALB/c mice (5 per group) were immunized (IN, 0.01 ml) at
weeks 0 and 3 with native purified F protein (3 .mu.g/dose) alone
in saline or in a formulation containing 0.1 or 1 .mu.g of one of
the wild-type CT (Sigma) or 0.1 or 1 .mu.g of one of the
genetically detoxified mutant CT-CRMs (CT-CRM.sub.E29H,
CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y, CT-CRM.sub.V72Y and
CT-CRM.sub.S68Y,V72Y). Sera were collected 2 weeks post secondary
immunization. The titration of the protein-specific IgG, IgA and
IgG subclass, serum neutralizing, antibodies in the
bronchioalveolar wash, nasal wash and vaginal wash was performed in
duplicate on HEp-2 cell monolayers in 96-well microplates.
Furthermore, a subgroup of immunized mice was challenged with live
virus to determine the protective capacity of the immunogenic
formulations. The results are presented in Table 20. The numbers
are geometric mean endpoint anti-F protein IgG, subclass and IgA
antibody titers (.+-.1 standard deviation).
[0172] Analysis of serum antibodies post-secondary immunization
showed that immunization with any of the cholera toxin-derived
adjuvants significantly induced immune responses to RSV F protein
(Table 18). The use of each of the cholera toxin mutants
CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y, CT-CRM.sub.V72Y,
CT-CRM.sub.S68Y,V72Y and CT-CRM.sub.E29H, at concentrations of 0.1
or 1.0 .mu.g/dose significantly (p<0.05) induced serum
antibodies (total IgG, IgG1, IgG2a and IgA) to RSV F protein. The
magnitude of the total IgG immune response to RSV F protein was
increased approximately 25-fold by inclusion of the cholera
toxin-derived adjuvants when compared to the response achieved by
animals administered a composition containing F/PBS.
[0173] Statistical significance of the data reported in Table 18
below is as follows. For total IgG: p<0.05: F/PBS vs. All,
p>0.05: F/CT vs. F/CT-CRM.sub.E29H vs. F/CT-CPM.sub.I16A vs.
F/CT-CRM.sub.I16A,S68Y vs. F/CT-CRM.sub.S68Y,V72Y vs.
FCT-CPM.sub.S68Y,V72Y (at both 0.1 and 1.0 .mu.g/dose). For IgG1:
p<0.05: F/PBS vs. All. p>0.05: F/CT vs. F/CT-CRM.sub.E29H vs.
F/CT-CRM.sub.I16A vs. F/CT-CRM.sub.I16A,S68Y vs. F/CT-CRM.sub.V72Y
vs. F/CT-CRM.sub.S68Y,V72Y (at both 0.1 and 1.0 .mu.g/dose). For
IgG2a: p<0.05: F/PBS vs. All. F/CT (0.1 .mu.g) vs.
F/CT-CRM.sub.I16A (0.1 .mu.g). p>0.05: At 1.0 .mu.g dose, F/CT
vs. F/CT-CRM.sub.E29H vs. F/CT-CRM.sub.I16A vs.
F/CT-CRM.sub.I16A,S68Y vs. F/CT-CRM.sub.V72Y vs.
F/CT-CRM.sub.S68Y,V72Y. At 0.1 .mu.g dose. F/CT vs.
F/CT-CRM.sub.E29H vs. F/CT-CRM.sub.I16A,S68Y vs. F/CT-CRM.sub.V72Y
vs. F/CT-CRM.sub.S68Y,V72Y.
[0174] No significant differences were observed in total anti-FE
IgG or IgG1 titers between each of the new mutant toxins
(CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y, CT-CRM.sub.V72Y, and
CT-CRM.sub.S68Y,V72Y) and either CT.sub.E29H or wild-type CT.
Evidence of statistical differences was observed between specific
groups upon analysis of IgG2a and IgA titers. However, these
comparisons did not reveal any consistent trends regarding the
immunological performance of one mutant versus another.
TABLE-US-00019 TABLE 18 The Humoral Immune Responses of BALB/c Mice
after Intranasal Immunization with F Protein and Mutant CT-CRMs
Antigen Adjuvant (.mu.g) IgG IgG1 IgG2a IgA F protein None 4.1 .+-.
0.6 3.1 .+-. 0.4 1.9 .+-. 0.3 1.9 .+-. 0.4 F protein
CT-CRM.sub.E29H(1) 6.3 .+-. 0.7 5.8 .+-. 0.8 4.9 .+-. 0.7 4.3 .+-.
0.6 F protein CT-CRM.sub.E29H(0.1) 5.6 .+-. 0.2 5.4 .+-. 0.5 4.4
.+-. 0.2 4.0 .+-. 0.2 F protein CT-CRM.sub.I16A(1) 5.6 .+-. 1.6 5.2
.+-. 1.4 4.7 .+-. 1.1 4.3 .+-. 0.2 F protein CT-CRM.sub.I16A(0.1)
5.5 .+-. 0.3 5.0 .+-. 0.1 3.9 .+-. 0.4 3.5 .+-. 0.5 F protein
CT-CRM.sub.V72Y(1) 6.4 .+-. 0.2 5.8 .+-. 0.3 5.3 .+-. 0.3 4.5 .+-.
0.3 F protein CT-CRM.sub.V72Y(0.1) 5.6 .+-. 0.2 5.2 .+-. 0.4 4.1
.+-. 0.4 3.5 .+-. 0.2 F protein CT-CRM.sub.I16A,V72Y(1) 6.4 .+-.
0.1 5.7 .+-. 0.2 5.3 .+-. 0.3 4.6 .+-. 0.4 F protein
CT-CRM.sub.I16A,V72Y(0.1) 5.5 .+-. 0.04 5.5 .+-. 0.3 4.8 .+-. 0.2
4.1 .+-. 0.3 F protein CT-CRM.sub.S68Y,V72Y(1) 6.5 .+-. 0.1 5.6
.+-. 0.2 4.9 .+-. 0.3 4.8 .+-. 0.2 F protein
CT-CRM.sub.S684,V72Y(0.1) 5.8 .+-. 0.3 5.6 .+-. 0.2 4.2 .+-. 0.3
4.6 .+-. 0.2 F Protein CT(1) 6.7 .+-. 0.3 6.0 .+-. 0.3 5.7 .+-. 0.2
5.3 .+-. 0.5 F protein CT(0.1) 5.8 .+-. 0.1 5.7 .+-. 0.2 5.0 .+-.
0.4 4.9 .+-. 0.2
[0175] In another experiment, groups of 5 BALB/c mice were
immunized (TN, 0.01 ml) at weeks 0 and 3 with native F protein (3
.mu.g/dose). The F protein was admixed with 1 or 0.1 .mu.g of
genetically detoxified mutants or wild-type CT. Anti-F protein
antibody responses were also analyzed in pooled mucosal wash
samples of bronchioalveolar lavage (BAL), nasal wash (NW) and
vaginal wash (VW), collected 2 weeks post-secondary immunization
(Table 19). The data represent endpoint anti-F protein IgG and IgA
antibody titers of pooled samples. As expected, no induction of
antibody in mucosal washes from F/PBS immunized mice was observed.
However, the potent mucosal adjuvant capacity of each mutant
cholera holotoxin was readily apparent. Although no statistical
analyses were performed on these pooled samples, some trends
surfaced. For example, mice that received F/CT-CRM.sub.V72Y (1.0
.mu.g) displayed elevated IgG and IgA in each of the BAL, NW and VW
samples taken. In comparison mutant toxins CT-CRM.sub.I16A,
CT-CRM.sub.I16A,S68Y and CT-CRM.sub.S68Y,V72Y appeared to be
comparable to CT-CRM.sub.E29H in adjuvanting local immune responses
to RSV F protein.
TABLE-US-00020 TABLE 19 The Mucosal Immune Responses of BALB/c Mice
after Intranasal Immunization with F Protein and Genetically
Detoxified Mutants BAL NW VW Antigen Adjuvant (.mu.g) IgG IgA IgG
IgA IgG IgA F protein None <25 <25 <25 <25 <25
<25 F protein CT-CRM.sub.E29H(1) 1569 211 320 793 265 1629 F
protein CT-CRM.sub.E29H(0.1) 549 <25 45 136 99 202 F protein
CT-CRM.sub.I16A(1) 1349 43 415 287 325 1427 F protein
CT-CRM.sub.I16A(0.1) 376 <25 103 187 217 350 F protein
CT-CRM.sub.V72Y(1) 1177 121 314 280 222 2289 F protein
CT-CRM.sub.V72Y(0.1) 144 <25 51 71 48 311 F protein
CT-CRM.sub.I16A,V72Y(1) 2093 458 392 627 739 9683 F protein
CT-CRM.sub.I16A,V72Y(0.1) 499 39 133 785 134 500 F protein
CT-CRM.sub.S68Y,V72Y(1) 1248 79 1181 510 204 1374 F protein
CT-CRM.sub.S68Y,V72Y(0.1) 522 25 109 98 81 770 F protein CT(1) 3271
142 1593 710 619 4136 F protein CT(0.1) 6436 1037 395 362 1185
1100
[0176] In another experiment, BALB/c mice were immunized (IN, 0.01
.mu.l) at weeks 0 and 3 with native F protein (3 .mu.g/dose). The F
protein was admixed with 1 or 0.1 .mu.g of each genetically
detoxified mutant or wild-type CT. At week 5, mice were challenged
with the A2 strain of RSV and lungs harvested 4 days later to
quantitate virus infectivity. Each of the mutant cholera holotoxins
induced a protective immune response to RSV challenge as measured
by viral lung load (Table 20). Data are presented as the mean virus
recovered (log.sub.10)/g tissue. Neutralizing antibodies were
assayed in the presence of 5% guinea pig serum as a source of
complement (C') in bleeds taken two weeks post-secondary
immunization. Data show the mean titer (log.sub.10) which showed a
60% reduction in pfu/well compared to control wells.
[0177] The statistical analyses of the data from the virus
infectivity assays is reported as p<0.05: F/PBS vs. all;
p>0.05: F/CT-E29H vs. F/CT vs. F/CRM/.sub.I16A vs.
F/CT-CRM.sub.V72Y vs. F/CT-CRM.sub.I16A,V72Y vs.
F/CT-CRM.sub.S68Y,V72Y at both 0.1 and 1.0 .mu.g/dose. Serum
neutralizing responses: p<0.05: F/PBS vs. all except
F/CT-CRM.sub.I16A,S68Y (0.1 .mu.g). F/CT(1.0) vs. F/CT-CRM.sub.I16A
(0.1). F/CT-CRM.sub.I16A,S68Y (0.11) vs. F/CT-CRM.sub.I16A,S68Y
(1.0) F/CT (9.0), F/CT-CRM.sub.I16A (1.0), F/CT-CRM.sub.E29H (1.0),
F/CT-CRM.sub.V72Y (0.1).
[0178] Lungs from mice immunized with F/PBS were clearly populated
with RSV (log.sub.10 3.4 pfu/g tissue). In contrast, those mice
immunized with F protein co-formulated with mutant cholera
holotoxins displayed no detectable virus. A somewhat similar
pattern was observed for serum neutralizing antibodies (Table 20).
Those mice immunized with F/PBS displayed complement-assisted
neutralizing antibodies that were significantly reduced compared to
all mice that had received mutant cholera holotoxins as an adjuvant
except F/CT-CRM.sub.I16A,S68Y at 0.1 .mu.g per dose. Whereas
evidence of neutralizing activity was observed in the absence of
complement, no statistical differences were observed (Table 20).
Collectively these data suggest that the inclusion of the mutant
cholera holotoxins CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y,
CT-CRM.sub.V72Y, and CT-CRM.sub.S68Y,V72Y contributes substantially
to the magnitude of the functional immune responses to RSV F
protein.
TABLE-US-00021 TABLE 20 Functional Immune Responses Elicited by
Immunization with Purified F Protein and Mutant CT-CRMs Geometric
GMT pfu/g Mean Serum (Log.sub.10) (Log.sub.10) Antigen Adjuvant
(.mu.g) Lung Tissue +C' -C' F protein None 3.38 .+-. 0.72 1.1 .+-.
0.2 <1.3 F protein CT-CRM.sub.E29H(1) <1.5 .+-. 0.03 3.0 .+-.
0.6 1.6 .+-. 0.6 F protein CT-CRM.sub.E29H(0.1) <1.5 .+-. 0.1
2.7 .+-. 0.5 <1.3 F protein CT-CRM.sub.I16A(1) <1.6 .+-. 0.03
3.1 .+-. 0.3 1.5 .+-. 0.7 F protein CT-CRM.sub.I16A(0.1) <1.5
.+-. 0.03 2.3 .+-. 0.7 <1.3 F protein CT-CRM.sub.V72Y(1) <1.6
.+-. 0.05 2.9 .+-. 0.5 1.6 .+-. 0.6 F protein CT-CRM.sub.V72Y(0.1)
<1.5 .+-. 0.03 1.8 .+-. 0.4 1.1 .+-. 0.1 F protein
CT-CRM.sub.I16A,V72Y(1) <1.5 .+-. 0.02 2.7 .+-. 0.7 1.4 .+-. 0.6
F protein CT-CRM.sub.I16A,V72Y(0.1) <1.5 .+-. 0.05 3.1 .+-. 0.2
1.5 .+-. 0.6 F protein CT-CRM.sub.S68Y,V72Y(1) <1.6 .+-. 0.05
2.5 .+-. 0.6 1.4 .+-. 0.6 F protein CT-CRM.sub.S684,V72Y(0.1)
<1.5 .+-. 0.5 2.5 .+-. 0.5 <1.3 F protein CT(1) <1.5 .+-.
.07 3.5 .+-. 0.4 1.4 .+-. 0.6 F protein CT(0.1) <1.6 .+-. 0.06
2.6 .+-. 0.5 1.5 .+-. 0.6
[0179] In yet additional experiments, naive BALB/c mice (8-10 weeks
of age, 5/group) were immunized (1N, 10 .mu.l) at weeks 0 and 3
with native purified fusion (F) protein purified from the 248/404
strain of RSV. The protein (3 .mu.g/dose) was prepared in mixture
with 1.0 or 0.1 .mu.g of the indicated CT-CRM. Control mice were
immunized with F protein admixed with CT-CRM.sub.E29H alone, with
wild-type CT, or with PBS. Serum (geometric mean titer .+-.1
standard deviation) and bronchioalveolar (BAW), nasal (NW) and
vaginal (VW) wash fluids were collected two weeks after secondary
immunization for the determination of end-point anti-F protein
total and subclass IgG and IgA titers by ELISA. The mucosal wash
samples were pooled for the determination of endpoint titers.
[0180] The results from two experiments are presented in Tables 21
and 22.
TABLE-US-00022 TABLE 21 Geometric Serum Ig Titers of BALB/c Mice
Immunized with F Protein Formulated with the Mutant CT-CRMs Anti-F
Protein Ig Titers (Log.sub.10) Antigen Adjuvant (.mu.g) IgG IgG1
IgG2a IgA F protein None 4.0 .+-. 0.7 2.7 .+-. 1.0 2.3 .+-. 0.7 2.3
.+-. 0.7 F protein CT-CRM.sub.I16A(1) 5.4 .+-. 0.3 5.1 .+-. 0.2 5.1
.+-. 0.3 4.3 .+-. 0.3 F protein CT-CRM.sub.I16A(0.1) 5.1 .+-. 0.4
4.6 .+-. 0.4 4.0 .+-. 0.5 3.9 .+-. 0.5 F protein
CT-CRM.sub.I16A,S68Y(1) 5.7 .+-. 0.2 5.4 .+-. 0.3 5.3 .+-. 0.3 4.7
.+-. 0.3 F protein CT-CRM.sub.I16A,S68Y(0.1) 5.2 .+-. 0.3 5.5 .+-.
0.3 4.2 .+-. 0.2 4.0 .+-. 0.2 F protein CT-CRM.sub.V72Y(1) 5.6 .+-.
0.1 5.4 .+-. 0.2 5.0 .+-. 0.2 4.7 .+-. 0.1 F protein
CT-CRM.sub.V72Y(0.1) 5.3 .+-. 0.3 4.8 .+-. 0.2 4.7 .+-. 0.2 4.6
.+-. 0.2 F protein CT-CRM.sub.S68Y,V72Y(1) 5.7 .+-. 0.2 5.5 .+-.
0.3 4.3 .+-. 0.3 4.4 .+-. 0.2 F protein CT-CRM.sub.S68Y,V72Y(0.1)
5.1 .+-. 0.2 4.5 .+-. 0.4 4.0 .+-. 0.3 4.1 .+-. 0.3 F protein
CT-CRM.sub.E29H(1) 5.4 .+-. 0.3 5.4 .+-. 0.1 5.5 .+-. 0.6 4.6 .+-.
0.3 F protein CT-CRM.sub.E29H(0.1) 5.3 .+-. 0.4 5.4 .+-. 0.1 4.3
.+-. 0.4 4.3 .+-. 0.1 F protein CT(1) 5.4 .+-. 0.5 5.0 .+-. 0.5 4.4
.+-. 0.8 4.6 .+-. 1.0 F protein CT(0.1) 4.6 .+-. 0.3 4.5 .+-. 0.4
3.5 .+-. 0.2 4.3 .+-. 0.3
TABLE-US-00023 TABLE 22 The Ig Titers of Pooled Mucosal Wash
Samples from BALB/c Mice Immunized with F Protein Formulated with
Mutant CT-CRMs Anti-F Protein Ig Titers BAW NW VW Antigen Adjuvant
(.mu.g) IgG IgA IgG IgA IgG IgA F protein None <25 <25 <25
157 <25 44 F protein CT-CRM.sub.I16A(1) 1,177 60 1,062 1,319 270
1,773 F protein CT-CRM.sub.I16A(0.1) 340 75 280 228 57 8,008 F
protein CT-CRM.sub.I16A,S68Y(1) 6,029 917 656 1,543 1,200 7,660 F
protein CT-CRM.sub.I16A,S68Y(0.1) 2,318 1,028 273 415 669 5,904 F
protein CT-CRM.sub.V72Y(1) 5,879 497 6,327 1,940 1,325 4,632 F
protein CT-CRM.sub.V72Y(0.1) 4,696 2,954 764 1,002 1,726 684 F
protein CT-CRM.sub.S68Y,V72Y(1) 2,179 364 444 4,153 907 849 F
protein CT-CRM.sub.S68Y,V72Y(0.1) 1,030 125 289 646 440 201 F
protein CT-CRM.sub.E29H(1) 1,972 217 616 437 327 43,466 F protein
CT-CRM.sub.E29H(0.1) 1,893 222 1,993 1,013 845 5,489 F protein CT
(1) 2,189 434 269 474 1,308 994 F protein CT(0.1) 1,791 308 316
1,997 315 358
[0181] When the CT-CRM mutants of this invention were used as
mucosal adjuvants at the 1.0 .mu.g dose, results similar to the use
of mutant CT-CRM.sub.E29H or wild-type CT were obtained (Table 21).
Noteworthy differences from the anti-F protein IgG or IgA titers
elicited following immunization with F protein admixed with
CT-CRM.sub.E29H or wild-type CT were not observed. Because pooled
samples were used to determine Ig titers in mucosal wash fluids,
statistical analyses could not be performed. Nonetheless, the
titers elicited by the mutant CT-CRMs of this invention were
comparable to those induced by F protein admixed with
CT-CRM.sub.E29H or wild-type CT (Table 22).
[0182] Thus, all CT-CRM mutants of this invention had adjuvant
activity for F protein.
EXAMPLE 7
The Immune Responses of Balb/C Mice Immunized with the UspA2 Outer
Membrane Protein of M. Catarrhalis
[0183] In this study, the capacity of mutant CT-CRMs to augment
systemic and mucosal immune responses against the native UspA2
outer membrane protein of M. catarrhalis was examined. Purified
UspA2 (5 .mu.g/dose) alone in 10 .mu.l saline or in a 10 .mu.l
formulation containing 0.1 .mu.g/dose of a mutant CT-CRM
(CT-CRM.sub.E29H, CT-CRM.sub.I16A, CT-CRM.sub.I16A,S68Y,
CT-CRM.sub.V72Y or CT-CRM.sub.S68Y, V72Y) was administered to
Balb/c mice IN on days 0, 7 and 14. Protein-specific IgG and IgA
levels in the serum and in mucosal lavages were examined on day 28.
The resulting serum and mucosal IgG titers are shown in Table 23,
All mutant CT-CRMs, except CT-CRM.sub.I16A, elicited enhanced serum
IgG antibody response. The levels of 1-G and IgA in bronchial,
nasal and vaginal washes were measured No IgA was detected in any
of the washes, and IgG was detected only in a few washes.
TABLE-US-00024 TABLE 23 Titers of sera to UspA2 elicited in mice by
UspA2 administered intranasally with different CT-CRMs. Mucosal
Serum Antibodies IgG antibodies CT Mutants IgG (log.sub.10titer
ISD) IgA Lung Nose Vagina None 530 (2.724 .+-. 0.38) <10 <10
<10 <10 CT-CRM.sub.E29H 17,378 (4.24 .+-. 0.47) 37 35 <10
<10 CT-CRM.sub.I16A 548 (2.739 .+-. 0.48) <10 <10 <10
<10 CT-CRM.sub.I16A,S68Y 7943 (3.90 .+-. 1.15) 45 23 <10 15
CT-CRM.sub.V72Y 9550 (3.98 .+-. 0.82) 42 45 <10 19 CT- 1072
(3.03 .+-. 0.89) <10 <10 <10 <10 CRM.sub.S68Y,V72Y
EXAMPLE 8
Adjuvanticity of Mutant Cholera Toxin Holotoxins
[0184] To create a comprehensive panel of mutant CT-CRMs with
different characteristics of toxicity, functionality and
immunogenicity, the above-described CT-CRM mutants were analyzed as
mucosal adjuvants, and the toxicity and enzymatic activity profiles
of each of the mutants were determined. As summarized in Table 24,
all of the mutant CT-CRMs have significantly reduced toxicity and
enzyme activity compared to wild-type CT. The following data was
generated from two studies performed to evaluate these genetically
detoxified mutant CTs for their capacity to adjuvant immune
responses to native UspA2 protein from M. catarrhalis.
[0185] The experiments were performed as follows: BALB/c mice (6-8
weeks old, 5 mice/group) were immunized at weeks 0, 2 and 4 with 5
.mu.g of purified native UspA2 protein in PBS or co-formulated with
1 .mu.g of wild-type CT, or CT-CRM.sub.E29H, or CT-CRM.sub.I16A, or
CT-CRM.sub.V72Y, or CT-CRM.sub.I16A,S68Y, or CT-CRM.sub.S68Y,V72Y
per immunization. A total volume of 10 .mu.l was administered
intranasally (5 .mu.l per nostril). Mice were bled at weeks 0, 2,
4, or 6 in order to assay serum antibody responses. Two weeks after
the last immunization (week 6), mice were sacrificed for the
analysis of mucosal antibody responses. Significant differences
between groups were determined by the Tukey-Kramer HSD multiple
comparisons test using JMP.RTM. statistical discovery software (SAS
Institute Inc., Cary, N.C.).
[0186] Adjuvanticity of the CT-CRMs can be summarized as follows,
Analysis of serum antibodies at week 6 showed that immunization
with UspA2 protein formulated with any of the CT-CRM mutants, at a
concentration of 1 .mu.g/dose, significantly induced IgG antibody
responses to UspA2 protein. The magnitude of the total IgG antibody
response to UspA2 protein was increased approximately 17-38 fold by
inclusion of the CT-derived mutants (excluding
CT-CRM.sub.I16A,S68Y) (Table 25). No significant differences were
observed in total anti-UspA2 IgG titers between the mutant toxins,
CT-CRM.sub.I16A, CT-CRM.sub.V72Y, and CT-CPM.sub.S68,V72Y and
CT-CRM.sub.E29H, even though they all elicited significantly higher
IgG titers than UspA2 protein alone by Tukey-Kramer HSD test (Table
25). The use of each of the CT-CRM mutants also enhanced serum IgG
subclass antibodies (IgG1, IgG2a and IgG2b) to UspA2 protein (Table
27). The ratio of IgG1 and IgG2a or IgG2b titers was approximately
1.0, indicating a balanced Th1/Th2 type of immune response.
[0187] Anti-UspA2 protein antibody responses were also analyzed in
pooled mucosal wash samples (Table 27). As expected, no induction
of antibody in bronchioalveolar lavage (BAL), nasal washes (NW),
vaginal washes (VW) or saliva from UspA2/PBS immunized mice was
observed. However, the potent mucosal adjuvant capacity of
CT-CRM.sub.I16A, CT-CRM.sub.V72Y, CT-CRM.sub.S68Y,V72Y, and
CT-CRM.sub.I16A,S68Y was clearly shown. There were UspA2 specific
mucosal IgA antibodies detected in most of the mucosal samples.
Although no statistical analysis can be performed on these pooled
samples, some trends appeared. For example, mice that received
CT-CRM.sub.V72Y displayed elevated UspA2 specific IgA antibodies in
each of the NW, VW and saliva samples tested.
[0188] CT-CRM.sub.I16A, CT-CRM.sub.V72Y, and CT-CRM.sub.S68Y,V72Y
are potent mucosal adjuvants for M. catarrhalis UspA2 protein. The
serum antibody data showed that all the CT-CRMs except
CT-CRM.sub.I16A,S68Y at 1 .mu.g dose are equally as capable in
adjuvanting immune responses to UspA2 protein as is CT-CRM.sub.E29H
(Tables 25 and 26). The mucosal wash data appears to suggest that
all the mutant CT-CRMs retain potent mucosal adjuvant properties
(Table 27). Furthermore, they all have significantly lower residual
toxicity and enzyme activity than wild-type CT, as shown in Table
24. Therefore, CT-CRM.sub.I16A, CT-CRM.sub.V72Y,
CT-CRM.sub.S68Y,V72Y and CT-CRM.sub.I16A,S68Y are additional
effective mucosal adjuvants.
TABLE-US-00025 TABLE 24 Characterization of Mutant Cholera Toxins
ADP- Y-1 cell Ribosyl- Homogeneity Holotoxin toxicity transferase
Mutant CT (%) (%) (%) activity (%) CT-CRM.sub.I16A >90 Not done
0.37 3.3 CT-CRM.sub.I16A,S68Y 75.8 97.8 0.37 2.4 CT-CRM.sub.V72Y
95.5 99.4 0.37 1.1 CT-CRM.sub.S68Y,V72Y 78.9 Not done 0.37 1.2
[0189] Groups of five female BALB/c mice were immunized
intranasally at weeks 0, 2, and 4 with 10 .mu.L containing 5 .mu.g
nUspA2 adjuvanted with 1 .mu.g CT (Sigma) or CT mutants, Endpoint
antibody titers were determined from sera collected at week 5 day
6. Data are presented as the geometric mean (.+-.1 SD) of the
reciprocal dilution resulting in an OD.sub.405 of 0.1. Statistical
analysis was by Tukey-Kramer. The results are shown in Table
25.
TABLE-US-00026 TABLE 25 The serum anti-nUspA2 responses of BALB/c
mice after intranasal immunization with nUspA2 adjuvanted with
mutant CTs Mean log 10 Antibody Titers Antigen (.+-.1SD) Group (5
.mu.g) Adjuvant (1 .mu.g) IgG IgA AG414 nUspA2 PBS <2.00
<2.00 AH415 nUspA2 CT 4.08 .+-. 0.20* 2.47 .+-. 0.33 AH416
nUspA2 CT-CRM.sub.E29H 3.37 .+-. 0.37* 2.04 .+-. 0.09 AH417 nUspA2
CT-CRM.sub.I16A 3.23 .+-. 0.21* 2.00 .+-. 0.02 AH418 nUspA2
CT-CRM.sub.I16A,S68Y .sup. 2.63 .+-. 0.12*.sup..PHI. <2.00 AH419
nUspA2 CT-CRM.sub.V72Y 3.59 .+-. 0.27* 2.11 .+-. 0.15 AH420 nUspA2
CT-CRM.sub.S68Y,V72Y 3.41 .+-. 0.22* <2.00 *Significantly higher
than the nUspA2/PBS group .sup..PHI.Significantly lower than all
other adjuvanted groups
[0190] Groups of five female BALB/c mice were immunized
intranasally at weeks 0, 2, and 4 with 10 .mu.L containing 5 .mu.g
nUspA2 adjuvanted with 1 .mu.g CT (Sigma) or CT mutants. Endpoint
antibody titers were determined from sera collected at week 5 day
6. Data are presented as the geometric mean (.+-.1 SD) of the
reciprocal dilution resulting in an OD.sub.405 of 0.1. Statistical
analysis was by Tukey-Kramer. The results are shown in Table
26.
TABLE-US-00027 TABLE 26 The serum anti-nUspA2 responses of BALB/c
mice after intranasal immunization with nUspA2 adjuvanted with
mutant CT-CRMs Antigen Adjuvant Mean log 10 Antibody Titers (.+-.
1SD) Group (5 .mu.g) (1 .mu.g) IgG1 IgG2a IgG2b AG414 nUspA2 PBS
<2.00 <2.00 <2.00 AH415 nUspA2 CT 3.27 .+-. 0.12* 3.39
.+-. 0.34* 3.03 .+-. 0.17* AH416 nUspA2 CT-CRM.sub.E29H 2.60 .+-.
0.08* 2.82 .+-. 0.32* 2.68 .+-. 0.25* AH417 nUspA2 CT-CRM.sub.I16A
2.30 .+-. 0.20 2.91 .+-. 0.24* 2.54 .+-. 0.21* AH418 nUspA2
CT-CRM.sub.I16A,S68Y 2.36 .+-. 0.09* 2.65 .+-. 0.21* 2.44 .+-.
0.24* AH419 nUspA2 CT-CRM.sub.V72Y 3.07 .+-. 0.28* 3.30 .+-. 0.35*
2.83 .+-. 0.19* AH420 nUspA2 CT-CRM.sub.S68Y,V72Y 2.80 .+-. 0.25*
2.86 .+-. 0.43* 2.52 .+-. 0.18* *Significantly higher than the
nUspA2/PBS group
[0191] Groups of five female BALB/c mice were immunized
intranasally at weeks 0, 2, and 4 with 10 .mu.L containing 5 .mu.g
nUspA2 adjuvanted with 11 g CT (Sigma) or CT mutants. Endpoint
antibody titers were determined from pooled mucosal wash samples
collected at week 6. Data are presented as the reciprocal dilution
resulting in an OD.sub.405 of 0.1. The results are shown in Table
25.
TABLE-US-00028 TABLE 27 The mucosal anti-nUspA2 responses of BALB/c
mice after intranasal immunization with nUspA2 adjuvanted with
mutant CTs Bronch Nasal Vaginal Antigen Adjuvant Wash Wash Wash
Saliva Group (5 .mu.g) (1 .mu.g) IgG IgA IgG IgA IgG IgA IgG IgA
AG414 nUspA2 PBS <10 <10 <10 <10 <10 <10 <10
<10 AG415 nUspA2 CT <10 <10 <10 34 <10 35 <10 33
AG416 nUspA2 CT-CRM.sub.E29H <10 <10 <10 22 <10 <10
<10 <10 AG417 nUspA2 CT-CRM.sub.I16A <10 <10 <10
<10 <10 17 <10 22 AG418 nUspA2 CT- <10 <10 <10 23
<10 12 <10 17 CRM.sub.I16A,S68Y AG419 nUspA2 CT-CRM.sub.V72Y
<10 16 <10 15 <10 58 <10 46 AG420 nUspA2 CT- <10
<10 <10 14 <10 41 <10 43 CRM.sub.S68Y,V72Y
[0192] All publications and references cited in this specification
are incorporated herein by reference. While the invention has been
described with reference to a particularly preferred embodiment, it
will be appreciated that modifications can be made without
departing from the spirit of the invention. Such modifications are
intended to fall within the scope of the appended claim.
Sequence CWU 1
1
101382PRTVibrio cholerae 1Met Val Lys Ile Ile Phe Val Phe Phe Ile
Phe Leu Ser Ser Phe Ser1 5 10 15Tyr Ala Asn Asp Asp Lys Leu Tyr Arg
Ala Asp Ser Arg Pro Pro Asp 20 25 30Glu Ile Lys Gln Ser Gly Gly Leu
Met Pro Arg Gly Gln Ser Glu Tyr 35 40 45Phe Asp Arg Gly Thr Gln Met
Asn Ile Asn Leu Tyr Asp His Ala Arg 50 55 60Gly Thr Gln Thr Gly Phe
Val Arg His Asp Asp Gly Tyr Val Ser Thr65 70 75 80Ser Ile Ser Leu
Arg Ser Ala His Leu Val Gly Gln Thr Ile Leu Ser 85 90 95Gly His Ser
Thr Tyr Tyr Ile Tyr Val Ile Ala Thr Ala Pro Asn Met 100 105 110Phe
Asn Val Asn Asp Val Leu Gly Ala Tyr Ser Pro His Pro Asp Glu 115 120
125Gln Glu Val Ser Ala Leu Gly Gly Ile Pro Tyr Ser Gln Ile Tyr Gly
130 135 140Trp Tyr Arg Val His Phe Gly Val Leu Asp Glu Gln Leu His
Arg Asn145 150 155 160Arg Gly Tyr Arg Asp Arg Tyr Tyr Ser Asn Leu
Asp Ile Ala Pro Ala 165 170 175Ala Asp Gly Tyr Gly Leu Ala Gly Phe
Pro Pro Glu His Arg Ala Trp 180 185 190Arg Glu Glu Pro Trp Ile His
His Ala Pro Pro Gly Cys Gly Asn Ala 195 200 205Pro Arg Ser Ser Met
Ser Asn Thr Cys Asp Glu Lys Thr Gln Ser Leu 210 215 220Gly Val Lys
Phe Leu Asp Glu Tyr Gln Ser Lys Val Lys Arg Gln Ile225 230 235
240Phe Ser Gly Tyr Gln Ser Asp Ile Asp Thr His Asn Arg Ile Lys Asp
245 250 255Glu Leu Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val
Leu Leu 260 265 270Ser Ser Ala Tyr Ala His Gly Thr Pro Gln Asn Ile
Thr Asp Leu Cys 275 280 285Ala Glu Ser His Asn Thr Gln Ile Tyr Thr
Leu Asn Asp Lys Ile Phe 290 295 300Ser Tyr Thr Glu Ser Leu Ala Gly
Lys Arg Glu Met Ala Ile Ile Thr305 310 315 320Phe Lys Asn Gly Ala
Ile Phe Gln Val Glu Val Pro Ser Ser Gln His 325 330 335Ile Asp Ser
Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg 340 345 350Ile
Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn 355 360
365Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn 370 375
3802240PRTVibrio cholerae 2Asn Asp Asp Lys Leu Tyr Arg Ala Asp Ser
Arg Pro Pro Asp Glu Ile1 5 10 15Lys Gln Ser Gly Gly Leu Met Pro Arg
Gly Gln Ser Glu Tyr Phe Asp 20 25 30Arg Gly Thr Gln Met Asn Ile Asn
Leu Tyr Asp His Ala Arg Gly Thr 35 40 45Gln Thr Gly Phe Val Arg His
Asp Asp Gly Tyr Val Ser Thr Ser Ile 50 55 60Ser Leu Arg Ser Ala His
Leu Val Gly Gln Thr Ile Leu Ser Gly His65 70 75 80Ser Thr Tyr Tyr
Ile Tyr Val Ile Ala Thr Ala Pro Asn Met Phe Asn 85 90 95Val Asn Asp
Val Leu Gly Ala Tyr Ser Pro His Pro Asp Glu Gln Glu 100 105 110Val
Ser Ala Leu Gly Gly Ile Pro Tyr Ser Gln Ile Tyr Gly Trp Tyr 115 120
125Arg Val His Phe Gly Val Leu Asp Glu Gln Leu His Arg Asn Arg Gly
130 135 140Tyr Arg Asp Arg Tyr Tyr Ser Asn Leu Asp Ile Ala Pro Ala
Ala Asp145 150 155 160Gly Tyr Gly Leu Ala Gly Phe Pro Pro Glu His
Arg Ala Trp Arg Glu 165 170 175Glu Pro Trp Ile His His Ala Pro Pro
Gly Cys Gly Asn Ala Pro Arg 180 185 190Ser Ser Met Ser Asn Thr Cys
Asp Glu Lys Thr Gln Ser Leu Gly Val 195 200 205Lys Phe Leu Asp Glu
Tyr Gln Ser Lys Val Lys Arg Gln Ile Phe Ser 210 215 220Gly Tyr Gln
Ser Asp Ile Asp Thr His Asn Arg Ile Lys Asp Glu Leu225 230 235
240342PRTartificial sequencebeta-amyloid peptide 3Asp Ala Glu Phe
Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe
Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu
Met Val Gly Gly Val Val Ile Ala 35 40428PRTartificial
sequencealpha-beta-amyloid peptide 4Asp Ala Glu Phe Arg His Asp Ser
Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp
Val Gly Ser Asn Lys 20 25527DNAArtificial sequenceoligonucleotide
5cctcctgatg aagsycaagc agtcagg 27618DNAArtificial
sequenceogigonucleotide 6gtttgagatc tgcccact 18720DNAArtificial
sequenceoligonucleotide 7gtttgaccca ctaagtgggc 20830DNAArtificial
sequenceoligonucleotide 8gtttgagata tgcccactta tatggtcaac
30932DNAArtificial sequenceprimer sequence 9ttttttgggc tagcatggag
gaaaagatga gc 321026DNAArtificial sequenceprimer sequence
10cgaggtcgaa gcttgcatgt ttgggc 26
* * * * *