U.S. patent application number 11/434934 was filed with the patent office on 2007-01-04 for chimeric protein comprising non-toxic pseudomonas exotoxin and type iv pilin sequences.
This patent application is currently assigned to The Gov. of the U.S.A as represented by the Sec. of The Dep. of Health and Huamn Services. Invention is credited to David J. Fitzgerald.
Application Number | 20070003578 11/434934 |
Document ID | / |
Family ID | 22978159 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070003578 |
Kind Code |
A1 |
Fitzgerald; David J. |
January 4, 2007 |
Chimeric protein comprising non-toxic pseudomonas exotoxin and type
IV pilin sequences
Abstract
The invention provides chimeric proteins comprising a non-toxic
Pseudomonas exotoxin A sequence and a Type IV pilin loop sequence,
wherein the Type IV loop sequence is inserted within the non-toxic
Pseudomonas exotoxin A. The invention also provides polynucleotides
encoding the chimeric proteins, and compositions comprising the
polynucleotides or the chimeric proteins. The invention also
provides methods for using the chimeric proteins, polynucleotides
and compositions of the invention.
Inventors: |
Fitzgerald; David J.;
(Rockville, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
The Gov. of the U.S.A as
represented by the Sec. of The Dep. of Health and Huamn
Services
Rockville
MD
|
Family ID: |
22978159 |
Appl. No.: |
11/434934 |
Filed: |
May 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10432412 |
May 21, 2003 |
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PCT/US01/49143 |
Dec 20, 2001 |
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11434934 |
May 15, 2006 |
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60257877 |
Dec 21, 2000 |
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Current U.S.
Class: |
424/236.1 ;
424/260.1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/21 20130101; A61P 37/04 20180101; A61P 11/00 20180101; A61K
39/00 20130101; A61P 31/00 20180101; A61P 9/00 20180101; A61P 31/04
20180101; A61K 2039/53 20130101; C07K 2319/00 20130101; A61P 29/00
20180101; A61P 27/02 20180101 |
Class at
Publication: |
424/236.1 ;
424/260.1 |
International
Class: |
A61K 39/108 20060101
A61K039/108 |
Claims
1. A chimeric protein comprising: a non-toxic Pseudomonas exotoxin
A sequence and a Type IV pilin loop sequence, the Type IV pilin
loop sequence being located within the non-toxic Pseudomonas
exotoxin A sequence, wherein the chimeric protein is capable of
reducing adherence of a microorganism expressing the Type IV pilin
loop sequence to epithelial cells, and further wherein the chimeric
protein, when introduced into a host, is capable of generating
polyclonal antisera that reduce adherence of the microorganism
expressing the Type IV pilin loop sequence to the epithelial
cells.
2. The chimeric protein of claim 1, wherein the chimeric protein,
when introduced into the host, is also capable of generating
polyclonal antisera that neutralize cytotoxicity of Pseudomonas
exotoxin A.
3. The chimeric protein of claim 1, wherein the non-toxic
Pseudomonas exotoxin A sequence comprises: (a) a translocation
domain sufficient to effect translocation of the chimeric protein
to a cell cytosol; and (b) an endoplasmic reticulum retention
domain that functions to translocate the chimeric protein from
endosome to endoplasmic reticulum.
4. The chimeric protein of claim 3, wherein the chimeric protein
further comprises a cell recognition domain that functions as a
ligand for a cell surface receptor and that mediates binding of the
chimeric protein to a cell.
5. The chimeric protein of claim 4, wherein the Type IV pilin loop
sequence is located between the translocation domain and the
endoplasmic reticulum retention domain.
6. The chimeric protein of claim 5, wherein the Type IV pilin loop
sequence comprises cysteine residues at both the N- and C-termini
of the Type IV pilin loop sequence.
7. The chimeric protein of claim 5, wherein the Type IV pilin loop
sequence is from bacteria or yeast.
8. The chimeric protein of claim 7, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa, Neisseria meningtidis,
Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam, or
Candida.
9. The chimeric protein of claim 8, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa.
10. The chimeric protein of claim 9, wherein the Type IV pilin loop
sequence is selected from the group consisting of SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, and SEQ ID NO:10.
11. The chimeric protein of claim 5, wherein the translocation
domain comprises amino acids 280 to 364 of domain II of Pseudomonas
exotoxin A.
12. The chimeric protein of claim 5, wherein the translocation
domain is domain II of Pseudomonas exotoxin A.
13. The chimeric protein of claim 5, wherein the endoplasmic
reticulum retention domain is domain III of Pseudomonas exotoxin A
except that amino acid Glu at position of 553 is deleted.
14. The chimeric protein of claim 1, wherein the chimeric protein
comprises more than one Type IV pilin loop sequence.
15. The chimeric protein of claim 5, wherein the cell recognition
domain is domain Ia of Pseudomonas exotoxin A.
16. The chimeric protein of claim 5, wherein the cell recognition
domain binds to .alpha.2-macroglobulin receptor, epidermal growth
factor receptor, transferrin receptor, interleukin-2 receptor,
interleukin-6 receptor, interleukin-8 receptor, Fc receptor,
poly-IgG receptor, asialoglycoprotein receptor, CD3, CD4, CD8,
chemokine receptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha
receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger
receptor, VEGF receptor, or cytokine receptor.
17. A chimeric protein comprising: (a) a non-toxic Pseudomonas
exotoxin A sequence comprising domain Ia, domain II, and domain
III; and (b) a Type IV pilin loop sequence, wherein the Type IV
pilin loop sequence is located between domain II and domain III of
the non-toxic Pseudomonas exotoxin A sequence.
18. The chimeric protein of claim 17, wherein the non-toxic
Pseudomonas exotoxin A sequence has the amino acid sequence of SEQ
ID NO:2 with .DELTA.E553.
19. The chimeric protein of claim 17, wherein the Type IV pilin
loop sequence is from Pseudomonas aeruginosa, Neisseria
meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurella
multocidam, or Candida.
20. The chimeric protein of claim 17, wherein the Type IV pilin
loop sequence is from Pseudomonas aeruginosa.
21. A polynucleotide encoding a chimeric protein, the chimeric
protein comprising: a non-toxic Pseudomonas exotoxin A sequence and
a Type IV pilin loop sequence, the Type IV pilin loop sequence
being located within the non-toxic Pseudomonas exotoxin A sequence,
wherein the chimeric protein is capable of reducing adherence of a
microorganism expressing the Type IV pilin loop sequence to
epithelial cells, and further wherein the chimeric protein, when
introduced into a host, is capable of generating polyclonal
antisera that prevent adherence of the microorganism expressing the
Type IV pilin loop sequence to the epithelial cells.
22. The polynucleotide of claim 21, wherein the chimeric protein,
when introduced into the host, is also capable of generating
polyclonal antisera that neutralize cytotoxicity of Pseudomonas
exotoxin A.
23. The polynucleotide of claim 21, wherein the non-toxic
Pseudomonas exotoxin A sequence comprises: (a) a translocation
domain sufficient to effect translocation of the chimeric protein
to a cell cytosol; and (b) an endoplasmic reticulum retention
domain that functions to translocate the chimeric protein from
endosome to endoplasmic reticulum.
24. The polynucleotide of claim 23, wherein the chimeric protein
further comprises a cell recognition domain that functions as a
ligand for a cell surface receptor and that mediates binding of the
chimeric protein to a cell.
25. The polynucleotide of claim 24, wherein the Type IV pilin loop
sequence is located between the translocation domain and the
endoplasmic reticulum retention domain.
26. The polynucleotide of claim 25, wherein the Type IV pilin loop
sequence comprises cysteine residues at both the N- and C-termini
of the Type IV pilin loop sequence.
27. The polynucleotide of claim 25, wherein the Type IV pilin loop
sequence is from bacteria or yeast.
28. The polynucleotide of claim 27, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa, Neisseria meningtidis,
Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam, or
Candida.
29. The polynucleotide of claim 28, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa.
30. The polynucleotide of claim 29, wherein the Type IV pilin loop
sequence is selected from the group consisting of SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, and SEQ ID NO:10.
31. The polynucleotide of claim 25, wherein the translocation
domain comprises amino acids 280 to 364 of domain II of Pseudomonas
exotoxin A.
32. The polynucleotide of claim 25, wherein the translocation
domain is domain II of Pseudomonas exotoxin A.
33. The polynucleotide of claim 25, wherein the endoplasmic
reticulum retention domain is domain III of Pseudomonas exotoxin A
except that amino acid Glu at position of 553 is deleted.
34. The polynucleotide of claim 25, wherein the cell recognition
domain is domain Ia of Pseudomonas exotoxin A.
35. The polynucleotide of claim 25, wherein the cell recognition
domain binds to .alpha.2-macroglobulin receptor, epidermal growth
factor receptor, transferring receptor, Fc receptor, poly-IgG
receptor, asialoglycoprotein receptor, CD3, CD4, CD8, chemokine
receptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLL
receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, VEGF
receptor, or cytokine receptor.
36. A polynucleotide encoding a chimeric protein, the chimeric
protein comprising: (a) a non-toxic Pseudomonas exotoxin A sequence
comprising domain Ia, domain II, and domain III; and (b) a Type IV
pilin loop sequence, wherein the Type IV pilin loop sequence is
located between domain II and domain III of the non-toxic
Pseudomonas exotoxin A sequence.
37. The polynucleotide of claim 36, wherein the non-toxic
Pseudomonas exotoxin A sequence has the amino acid sequence of SEQ
ID NO:2 with .DELTA.E553.
38. The polynucleotide of claim 36, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa, Neisseria meningtidis,
Neisseria gonorrhoeae, Vibro cholera, Pasteurella multocidam, or
Candida.
39. The polynucleotide of claim 36, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa.
40. An expression cassette comprising the polynucleotide of claim
21.
41. A cell comprising the expression cassette of claim 40.
42. A composition comprising a chimeric protein, the chimeric
protein comprising: a non-toxic Pseudomonas exotoxin A sequence and
a Type IV pilin loop sequence, the Type IV pilin loop sequence
being located within the non-toxic Pseudomonas exotoxin A sequence,
wherein the chimeric protein is capable of reducing adherence of a
microorganism expressing the Type IV pilin loop sequence to
epithelial cells, and further wherein the chimeric protein, when
introduced into a host, is capable of generating polyclonal
antisera that prevent adherence of the microorganism expressing the
Type IV pilin loop sequence to the epithelial cells.
43. The composition of claim 42, wherein the chimeric protein, when
introduced into the host, is also capable of generating polyclonal
antisera that neutralize cytotoxicity of Pseudomonas exotoxin
A.
44. The composition of claim 42, wherein the composition further
comprises a pharmacologically acceptable carrier.
45. The composition of claim 42, wherein the composition is
formulated as a nasal or oral spray.
46. The composition of claim 42, wherein the non-toxic Pseudomonas
exotoxin A sequence comprises: (a) a translocation domain
sufficient to effect translocation of the chimeric protein to a
cell cytosol; and (b) an endoplasmic reticulum retention domain
that functions to translocate the chimeric protein from endosome to
endoplasmic reticulum.
47. The composition of claim 46, wherein the chimeric protein
further comprises a cell recognition domain that functions as a
ligand for a cell surface receptor and that mediates binding of the
chimeric protein to a cell.
48. The composition of claim 47, wherein the Type IV pilin loop
sequence is from Pseudomonas aeruginosa.
49. A method for eliciting an immune response in a host, the method
comprising the step of administering to the host an immunologically
effective amount of a composition comprising a chimeric protein
comprising: a non-toxic Pseudomonas exotoxin A sequence and a Type
IV pilin loop sequence, the Type IV pilin loop sequence being
located within the non-toxic Pseudomonas exotoxin A sequence,
wherein the chimeric protein is capable of reducing adherence of a
microorganism expressing the Type IV pilin loop sequence to
epithelial cells, and further wherein the chimeric protein, when
introduced into the host, is capable of generating polyclonal
antisera that prevent adherence of the microorganism expressing the
Type IV pilin loop sequence to the epithelial cells.
50. The method of claim 49, wherein the chimeric protein, when
introduced into the host, is capable of generating polyclonal
antisera that neutralize cytotoxicity of Pseudomonas exotoxin
A.
51. The method of claim 49, wherein the host is a human.
52. The method of claim 49, wherein the non-toxic Pseudomonas
exotoxin A sequence comprises: (a) a translocation domain
sufficient to effect translocation of the chimeric protein to a
cell cytosol; and (b) an endoplasmic reticulum retention domain
that functions to translocate the chimeric protein from endosome to
endoplasmic reticulum.
53. The method of claim 52, wherein the chimeric protein further
comprises a cell recognition domain that functions as a ligand for
a cell surface receptor and that mediates binding of the chimeric
protein to a cell.
54. The method of claim 53, wherein the Type IV pilin loop sequence
is from Pseudomonas aeruginosa.
55. A method of eliciting an immune response in a host, the method
comprising the step of administering to the host an immunologically
effective amount of an expression cassette comprising a
polynucleotide encoding a chimeric protein comprising: a non-toxic
Pseudomonas exotoxin A sequence and a Type IV pilin loop sequence,
the Type IV pilin loop sequence being located within the non-toxic
Pseudomonas exotoxin A, wherein the chimeric protein is capable of
reducing adherence of a microorganism expressing the Type IV pilin
loop sequence to epithelial cells, and further wherein the chimeric
protein, when introduced into the host, is capable of generating
polyclonal antisera that reduce adherence of the microorganism
expressing the Type IV pilin loop sequence to the epithelial
cells.
56. The method of claim 55, wherein the chimeric protein, when
introduced into the host, is capable of generating polyclonal
antisera that neutralize cytotoxicity of Pseudomonas exotoxin
A.
57. The method of claim 55, wherein the host is a human.
58. The method of claim 55, wherein the non-toxic Pseudomonas
exotoxin A sequence comprises: (a) a translocation domain
sufficient to effect translocation of the chimeric protein to a
cell cytosol; and (b) an endoplasmic reticulum retention domain
that functions to translocate the chimeric protein from endosome to
endoplasmic reticulum.
59. The method of claim 58, wherein the chimeric protein further
comprises a cell recognition domain that functions as a ligand for
a cell surface receptor and that mediates binding of the chimeric
protein to a cell.
60. The method of claim 59, wherein the Type IV pilin loop sequence
is from Pseudomonas aeruginosa.
61. A method of generating antibodies specific for a Type IV pilin
loop sequence, comprising introducing into a host a composition
comprising a chimeric protein comprising a non-toxic Pseudomonas
exotoxin A sequence and a Type IV pilin loop sequence, the Type IV
pilin loop sequence being located within the non-toxic Pseudomonas
exotoxin A, wherein the chimeric protein is capable of reducing
adherence of a microorganism expressing the Type IV pilin loop
sequence to epithelial cells, and further wherein the chimeric
protein, when introduced into the host, is capable of generating
polyclonal antisera that reduce adherence of the microorganism
expressing the Type IV pilin loop sequence to epithelial cells.
62. The method of claim 61, wherein the chimeric protein, when
introduced into the host, is capable of generating polyclonal
antisera that neutralize cytotoxicity of Pseudomonas exotoxin
A.
63. The method of claim 61, wherein the host is a human.
64. The method of claim 61, wherein the non-toxic Pseudomonas
exotoxin A sequence comprises: (a) a translocation domain
sufficient to effect translocation of the chimeric protein to a
cell cytosol; and (b) an endoplasmic reticulum retention domain
that functions to translocate the chimeric protein from endosome to
endoplasmic reticulum.
65. The method of claim 64, wherein the chimeric protein further
comprises a cell recognition domain that functions as a ligand for
a cell surface receptor and that mediates binding of the chimeric
protein to a cell.
66. The method of claim 65, wherein the Type IV pilin loop sequence
is from Pseudomonas aeruginosa.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional
Application No. 60/257,877 filed Dec. 21, 2000, the contents of
which is incorporated herein by reference in their entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] Type IV pilin is the major subunit of the pilus or pili
which are filamentous structures covering many microorganisms
including bacteria and yeast. Among these microorganisms, many
pathogenic species express Type IV pilins, including, e.g., P.
aeruginosa, N. meningitides, N. gonorrhoeae, Vibro cholera, and
Pasteurella multocidam. The first step in infection with these
pathogenic microorganisms is adherence to target cells through the
pili. In particular, Type IV pilins of Pseudomonas aeruginosa bind
to asialoGM1 receptors on epithelial cells (Saiman et al., J. Clin.
Invest. 92(4):1875-80 (1993); Sheth et al., 11 (4):715-23 (1994);
Imundo et al., Proc. Natl. Acad. Sci. USA, 92(7):3019-23 (1995);
Hahn, Gene 192(1):99-108 (1997)). Thus, the pili of these
microorganisms are a major virulence factor, and result in
colonization by pathogenic microorganisms and infections in
humans.
[0004] For example, Pseudomonas aeruginosa causes between 10% and
20% infections in most hospitals. Pseudomonas infection is common
among patients with cystic fibrosis, burn wounds, organ
transplants, and intravenous-drug addiction. Pseudomonas infections
can lead to serious conditions, such as endophthalmitis,
endocarditis, meningitis, pneumonia, and septicemia. In particular,
colonization of cystic fibrosis (CF) individuals with Pseudomonas
aeruginosa represents a significant negative milestone in the
progression of this disease. Once colonized, patients are subject
to the damaging effects of various secreted virulence factors and
to the inflammatory response of the host immune system.
[0005] Type IV pili are composed of pilin polymers arranged in a
helical structure with five subunits per turn (Forest et al., Gene
192(1):165-9 (1997); Parge, Nature 378(6552):32-8 (1995)). The
portion of the pilin protein responsible for cell binding is found
near the C-terminus (amino acids 122-148) in a .beta.-turn loop
subtended from a disulfide bond (Campbell et al., Biochemistry
36(42):12791-801 (1997); Campbell et al., J. Mol. Biol.
267(2):382-402 (1997); Hazes et al., J. Mol. Biol. 299(4):1005-1017
(2000); McInnes et al., Biochemistry 32(49):13432-40 (1993)). For
P. aeruginosa, a 12 or 17 amino acid sequence (depending on the
strain) in this loop interacts with receptors on epithelial cells.
For CF individuals, the overproduction of the R domain of mutant
cystic fibrosis transmembrane conductance regulator (CFTR) can lead
to an increased level of asialoGM1 and, accordingly, an increased
binding of P. aeruginosa (Imundo et al., Proc. Natl. Acad. Sci. USA
92(7):3019-23 (1995); Saiman et al., J. Clin. Invest. 92(4):1875-80
(1993); Bryan et al., Am. J. Respir. Cell Mol. Biol. 19(2):269-77
(1998); Imundo et al., Proc. Natl. Acad. Sci. USA 92(7):3019-23
(1995); Saiman et al., J. Clin. Invest. 92(4):1875-80 (1993)).
Functional studies of pilin have indicated that only the last pilin
subunit (the tip) of a pilus interacts with epithelial cell
receptors (Lee et al., Mol. Microbiol. 11(4):705-13 (1994)).
[0006] To date, efforts to produce an effective anti-pilin vaccine
have not been very successful. In part, this limited success is
because the most immunogenic portion of the protein (the middle)
does not generate antibodies that interfere with adhesion.
Unfortunately, the C-terminal loop of pilin is not very
immunogenic, and high titer responses have only been reported with
the use of strategies that employ multiple display copies of the
loop sequence (Hahn et al., Behring. Inst. Mitt. (98):315-25
(1997)). For CF patients, strategies to inhibit Pseudomonas
colonization are considered an important element in reducing the
morbidity normally associated with the development of chronic
infections (Tang et al., Infect. Immun. 63(4):1278-85 (1995); Li et
al., Proc. Natl. Acad. Sci. USA 94(3):967-72 (1997); Tang et al.,
Infect. Immun. 63(4):1278-85 (1995) Doig, P. et al., Infect Immun
58(1):124-30 (1990); El-Zaim, H. S. et al. Infect Immun
66(11):5551-4 (1998)).
[0007] Accordingly, there is a need to develop compositions for
reducing or preventing infections by pathogenic microorganisms
including, in particular, Pseudomonas aeruginosa. Embodiments of
this invention address this and other needs.
SUMMARY OF THE INVENTION
[0008] Embodiments of the invention provide chimeric proteins
comprising a non-toxic Pseudomonas exotoxin A sequence and a Type
IV pilin loop sequence, wherein the Type IV pilin loop sequence is
located within the non-toxic Pseudomonas exotoxin A sequence. In
the present invention, a Type IV pilin loop sequence refers to the
sequence that forms an intrachain disulfide loop at the C-terminus
of the pilin. This loop interacts and binds to receptors on
epithelial cells. The present invention is based on, in part, the
discovery that the Type IV pilin loop sequence within the
Pseudomonas exotoxin A sequence is presented in near-native
conformation, and can react with receptors on epithelial cells. As
a result, the present chimeric protein comprises the Type IV pilin
loop sequence which competes for binding to these epithelial cells,
and which can reduce adherence of pathogenic microorganisms
expressing the Type IV pilin to the epithelial cells. Therefore,
the chimeric protein can be used on its own or in a composition to
directly reduce adherence of pathogenic microorganisms in a
host.
[0009] The present invention is also based on, in part, the
discovery that antisera generated against the chimeric proteins of
the invention are also useful in reducing adherence of pathogenic
microorganisms (expressing Type IV pilins) in a host. Since the
chimeric protein presents the Type IV pilin loop in near-native
conformation, the chimeric proteins of the invention, when
introduced into a host, generate polyclonal antisera that bind to
the pilin loop portion of the chimeric proteins. The antisera can
also bind to Type IV pilins on pathogenic microorganism, and thus
competitively inhibit binding of the pathogenic microorganisms to
epithelial cell receptors. Accordingly, the chimeric protein can be
used as a vaccine to generate antisera in a host which can result
in reduction of both adherence and colonization of pathogenic
microorganisms in the host.
[0010] Furthermore, since the chimeric protein presents the
non-toxic Pseudomonas exotoxin A sequence in near-native
conformation, the chimeric proteins of the invention, when
introduced into a host, generate polyclonal antisera that bind to
the non-toxic Pseudomonas exotoxin A as well as to the native
Pseudomonas exotoxin A. The native Pseudomonas exotoxin A which is
secreted by Pseudomonas aeruginosa is known to cause cell
cytotoxicity by entering into cells by receptor-mediated
endocytosis and then, after a series of intracellular processing
steps, translocate to the cell cytosol and ADP-ribosylate
elongation factor 2. This results in the inhibition of protein
synthesis and cell death. The antisera generated against the
present chimeric protein can bind exotoxin A released from
Pseudomonas and can neutralize cell cytotoxicity. Therefore, should
small numbers of Pseudomonas overcome the first line of defense
(antibodies against the pilin loop sequence preventing
colonization), the normal destructive power of the exotoxin A will
be neutralized by antibodies generated against the non-toxic
Pseudomonas exotoxin A sequence.
[0011] The chimeric proteins, the chimeric polynucleotides, and the
compositions of the present invention have many other utilities.
For example, the chimeric proteins and the compositions comprising
chimeric proteins can be used to in diagnostic tests, such as
immunoassays. Such diagnostic tests can be used to detect the
presence of microorganisms bearing a Type IV pilin loop sequence,
such as Pseudomonas aeruginosa, or to determine whether a host has
antisera against a Type IV pilin loop due to an infection. In
another example, the chimeric proteins and the compositions
comprising the chimeric proteins can also be used to purify
antibodies against, e.g., the Type IV pilin loop sequence. In
another example, the antibodies against the chimeric protein can be
used to clone and isolate other related Type IV pilin
sequences.
[0012] Accordingly, in one aspect of the invention, the invention
provides a chimeric protein comprising: a non-toxic Pseudomonas
exotoxin A sequence and a Type IV pilin loop sequence, the Type IV
pilin loop sequence being located within the non-toxic Pseudomonas
exotoxin A sequence, wherein the chimeric protein is capable of
reducing adherence of a microorganism expressing the Type IV pilin
loop sequence to epithelial cells, and further wherein the chimeric
protein, when introduced into a host, is capable of generating
polyclonal antisera that reduce adherence of the microorganism
expressing the Type IV pilin loop sequence to the epithelial
cells.
[0013] In another aspect, the invention provides a chimeric protein
comprising: (a) a non-toxic Pseudomonas exotoxin A sequence
comprising domain Ia, domain II, and domain III; and (b) a Type IV
pilin loop sequence, wherein the Type IV pilin loop sequence is
located between domain II and domain III of the non-toxic
Pseudomonas exotoxin A sequence.
[0014] In another aspect, the invention provides a polynucleotide
encoding a chimeric protein, the chimeric protein comprising: a
non-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loop
sequence, the Type IV pilin loop sequence being located within the
non-toxic Pseudomonas exotoxin A sequence, wherein the chimeric
protein is capable of reducing adherence of a microorganism
expressing the Type IV pilin loop sequence to epithelial cells, and
ftuther wherein the chimeric protein, when introduced into a host,
is capable of generating polyclonal antisera that prevent adherence
of the microorganism expressing the Type IV pilin loop sequence to
the epithelial cells.
[0015] In another aspect, the invention provides a polynucleotide
encoding a chimeric protein, the chimeric protein comprising: (a) a
non-toxic Pseudomonas exotoxin A sequence comprising domain Ia,
domain II, and domain III; and (b) a Type IV pilin loop sequence,
wherein the Type IV pilin loop sequence is located between domain
II and domain III of the non-toxic Pseudomonas exotoxin A
sequence.
[0016] In another aspect, the invention provides a composition
comprising a chimeric protein, the chimeric protein comprising: a
non-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loop
sequence, the Type IV pilin loop sequence being located within the
non-toxic Pseudomonas exotoxin A sequence, wherein the chimeric
protein is capable of reducing adherence of a microorganism
expressing the Type IV pilin loop sequence to epithelial cells, and
further wherein the chimeric protein, when introduced into a host,
is capable of generating polyclonal antisera that prevent adherence
of the microorganism expressing the Type IV pilin loop sequence to
the epithelial cells.
[0017] In another aspect, the invention provides a method for
eliciting an immune response in a host, the method comprising the
step of administering to the host an immunologically effective
amount of a composition comprising a chimeric protein comprising: a
non-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loop
sequence, the Type IV pilin loop sequence being located within the
non-toxic Pseudomonas exotoxin A sequence, wherein the chimeric
protein is capable of reducing adherence of a microorganism
expressing the Type IV pilin loop sequence to epithelial cells, and
further wherein the chimeric protein, when introduced into the
host, is capable of generating polyclonal antisera that prevent
adherence of the microorganism expressing the Type IV pilin loop
sequence to the epithelial cells.
[0018] In another aspect, the invention provides a method of
eliciting an immune response in a host, the method comprising the
step of administering to the host an immunologically effective
amount of an expression cassette comprising a polynucleotide
encoding a chimeric protein comprising: a non-toxic Pseudomonas
exotoxin A sequence and a Type IV pilin loop sequence, the Type IV
pilin loop sequence being located within the non-toxic Pseudomonas
exotoxin A, wherein the chimeric protein is capable of reducing
adherence of a microorganism expressing the Type IV pilin loop
sequence to epithelial cells, and further wherein the chimeric
protein, when introduced into the host, is capable of generating
polyclonal antisera that reduce adherence of the microorganism
expressing the Type IV pilin loop sequence to the epithelial
cells.
[0019] In another aspect, the invention provides a method of
generating antibodies specific for a Type IV pilin loop sequence,
comprising introducing into a host a composition comprising a
chimeric protein comprising a non-toxic Pseudomonas exotoxin A
sequence and a Type IV pilin loop sequence, the Type IV pilin loop
sequence being located within the non-toxic Pseudomonas exotoxin A,
wherein the chimeric protein is capable of reducing adherence of a
microorganism expressing the Type IV pilin loop sequence to
epithelial cells, and further wherein the chimeric protein, when
introduced into the host, is capable of generating polyclonal
antisera that reduce adherence of the microorganism expressing the
Type IV pilin loop sequence to epithelial cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A illustrates in cartoon form the replacement of
domain Ib with the C-terminal loop of pilin. The pilin insert
corresponds to the sequence of pilin reported for the PAK strain of
P. aeruginosa.
[0021] FIG. 1B illustrates in cartoon form the domain structure of
PE from Allured et al., Proc. Natl. Acad. Sci. 83:1320-1324 (1986).
PE64 lacks the loop region of domain Ib. PE64pil includes the
insertion of the pilin loop (residues 129-142) of the PAK strain of
P. aeruginosa. The deletion of glutamic acid 553 (indicated by a
dot) removes an active site residue (Lukac et al., Infect. Immuno.
56(12):3095-8 (1988)) and produces proteins PE64.DELTA.553 and
PE64.DELTA.553pil with no ADP-ribosylating activity. The Ib loop is
shown in light shading and the pilin loop in darker shading.
[0022] FIG. 2 illustrates SDS PAGE (Panel A and C) and Western blot
analysis (Panel B) of PE proteins and pilin. A. Lanes 1-4 show
substantially pure PE proteins (4-5 .mu.g of protein was loaded per
lane) after MonoQ chromatography. From left to right the proteins
loaded were: PE64, PE64pil, PE64.DELTA.553 and PE64.DELTA.553pil.
Purified PAK pilin was added to lane 5. B. Lanes 6-10 show the same
proteins as A but probed with a monoclonal antibody to the pilin
loop. Lane 11 is PE64.DELTA.553pil after gel filtration
chromatography. Standard proteins and their molecular masses in kDa
are indicated.
[0023] FIG. 3 illustrates the toxicity of PE64pil compared to PE64.
To assess the effect of introducing a third party loop into PE, we
compared the toxicity of PE64 (.box-solid.) with PE64pil
(.tangle-solidup.). Increasing concentrations of each protein was
added to L929 cells and, after an overnight incubation, inhibition
of protein synthesis was determined. Results are expressed as
percent control compared to cells receiving no toxin. Error bars
represent one SD of the mean from triplicate wells.
[0024] FIG. 4 illustrates the interaction of PE64pil and
PE64.DELTA.553pil with immobilized asialo-GM1. (A). Various
concentrations of PE64pil or PE64 were added to plates coated with
asialo-GM1 and binding was determined by reactivity with rabbit
anti-PE followed by a peroxidase labeled goat anti-rabbit IgG
antibody. Absorbance at 450 .mu.m was used to monitor binding. (B).
and (C). To investigate ganglioside specificity, a competition
assay was devised whereby soluble asialo-GM1 or monosialo-GM1 at 2
ug/ml was preincubated with PE64pil (B) or PE64.DELTA.553pil (C)
and the percent residual binding determined as described in panel
(A). For (B) and (C), graphs show the mean of a representative
triplicate experiment. Error bars represent one SD. N.A.=no
addition of competitor.
[0025] FIG. 5 illustrates adhesion of Ps. aeruginosa (PAK strain)
to A549 cells. Bacteria were added to cells at an MOI of 100 in the
presence or absence of potential inhibitors. Peptides were added to
a final concentration of 40 .mu.M, while proteins were added to a
concentration of 2 .mu.M. The graph indicates the percentage of
cell-bound bacteria compared to samples with no inhibitor. Error
bars represent one standard deviation from the mean of three
independent experiments.
[0026] FIG. 6 illustrates antibody titers post immunization with
PE64pil with and without adjuvant. Sera were collected from each of
four rabbits (numbered 87-90) at various times, diluted 1:100 and
then added to streptavidin-coated plates that had been loaded with
biotinylated pilin peptides. Rabbit IgG was detected by the
addition of a peroxidase conjugated goat anti-rabbit antibody.
Rabbits 87 and 88 received adjuvant while rabbits 89 and 90 did
not.
[0027] FIG. 7 illustrates antibody-mediated interference with
adhesion to A549 cells. (A). The PAK strain of Ps. aeruginosa was
incubated with 1:20 to 1:100 dilutions of prebleed or immune (taken
after the fourth injection of antigen) sera from rabbit #87.
Bacteria were then added to cells and the percent adhesion
determined by comparison with bacteria that had been incubated in
media alone. (B). A 1:20 dilution of sera from each rabbit,
prebleed and immune, was tested for antibody mediated interference.
(C). Various strains of Ps. aeruginosa were incubated with immune
sera (1:20) from one of the rabbits that received antigen alone
(rabbit #90) and one that received antigen plus adjuvant (rabbit
#88). For each panel of FIG. 7, the bar represents the number of
bacteria per cell determined by examining one hundred A549 cells.
The error bars represent one standard deviation from the mean of
three independent experiments.
[0028] FIG. 8 illustrates antibody-mediated neutralization of PE
toxicity. Immune sera (.tangle-solidup.) or prebleed sera
(.box-solid.) were diluted 1:20 and mixed with PE64 at 1.0 ug/ml.
Samples were then diluted to the concentration indicated and added
to L929 cells for an overnight incubation. Results are expressed as
percent control of protein synthesis compared to cells receiving no
toxin. Error bars represent one SD of the mean from triplicate
wells.
DEFINITIONS
[0029] "Pseudomonas exotoxin A" or "PE" is secreted by P.
aeruginosa as a 67 kDa protein composed of three prominent globular
domains (Ia, II, and III) and one small subdomain (Ib) connecting
domains II and III. (Allured et. al., Proc. Natl. Acad. Sci.
83:1320-1324 (1986).) Domain Ia of PE located at the N-terminus and
mediates cell binding. In nature, domain Ia binds to the low
density lipoprotein receptor-related protein ("LRP"), also known as
the .alpha.2-macroglobulin receptor (".alpha.2-MR"). (Kounnas et
al., J. Biol. Chem. 267:12420-23 (1992).) It spans amino acids
1-252. Domain II mediates translocation to the cytosol. It spans
amino acids 253-364. Domain lb has no known function. It spans
amino acids 365-399. Domain III is responsible for cytotoxicity and
includes an endoplasmic reticulum retention sequence. It mediates
ADP ribosylation of elongation factor 2 ("EF2"), which inactivates
protein synthesis. It spans amino acids 400-613. The native
Pseudomonas aeruginosa exotoxin A nucleic acid sequence and the
amino acid sequence are shown as SEQ ID NO:1 and SEQ ID NO:2,
respectively. SEQ ID NOS: 1 and 2 are the mature form of exotoxin
A, wherein the signal sequence has been cleaved off. As a virulence
factor, PE can kill PMNs, macrophages and other elements of the
immune system (Pollack et al., Infect. Immuno. 19(3):1092-6
(1978)).
[0030] As used herein, "Pseudomonas exotoxin A" or "PE" refer to
those having the functions described above and includes the native
Pseudomonas exotoxin A having the nucleic acid and amino acid
sequences (as shown as SEQ ID NO:1 and SEQ ID NO:2, respectively)
and also polymorphic variants, alleles, mutants and interspecies
homologs that: (1) have about 80% amino acid sequence identity,
preferably about 85-90% amino acid sequence identity to SEQ ID NO:2
over a window of about 25 amino acids, preferably over a window of
about 50-100 amino acids; (2) bind to antibodies raised against an
immunogen comprising an amino acid sequence of SEQ ID NO:2 and
conservatively modified variants thereof; or (3) specifically
hybridize (with a size of at least about 500, preferably at least
about 900 nucleotides) under stringent hybridization conditions to
a sequence SEQ ID NO:1 and conservatively modified variants
thereof. For example, genetically modified forms of PE are
described in, e.g., Pastan et al., U.S. Pat. No. 5,602,095; Pastan
et al., U.S. Pat. No. 5,512,658 and Pastan et al., U.S. Pat. No.
5,458,878. Allelic forms of PE are included in this definition.
See, e.g., Vasil et al., Infect. Immunol. 52:538-48 (1986).
[0031] "Non-toxic Pseudomonas exotoxin A" or "non-toxic PE" refers
to any Pseudomonas exotoxin A described herein (including modified
variants) that lacks ADP ribosylation activity. The ribosylating
activity of PE is located between about amino acids 400 and 600 of
PE. For example, deleting amino acid E553 (".DELTA.E553") from
domain III detoxifies the molecule. This detoxified PE is referred
to as "PE .DELTA.E553." In another example, substitution of
histidine residue of PE at 426 with a tyrosine residue also
inactivates the ADP-ribosylation of PE (see Kessler & Galloway,
J. Biol. Chem. 267:19107-11 (1992)). Other amino acids within
domain III can be modified by, e.g., deletion, substitution or
addition of amino acid residues, to eliminate ADP ribosylation
activity. Domain III of non-toxic PE is sometimes referred to
herein as "detoxified domain III."
[0032] The term "a non-toxic Pseudomonas exotoxin A sequence" is
used generically to refer to either a nucleic acid sequence or an
amino acid sequence of non-toxic Pseudomonas exotoxin A. As used
herein, a non-toxic Pseudomonas exotoxin A sequence may be a full
length sequence or portion(s) of the full length sequence.
Generally, a non-toxic Pseudomonas exotoxin A sequence has one or
more domains or portions of domains with certain biological
activities of a non-toxic Pseudomonas exotoxin A, such as a cell
recognition domain, a translocation domain, or an endoplasmic
reticulum retention domain. For example, a non-toxic Pseudomonas
exotoxin A sequence may include only domain II and detoxified
domain III. In another example, a non-toxic Pseudomonas exotoxin A
sequence may include only domain Ia, domain II, and detoxified
domain III. In another example, a non-toxic Pseudomonas exotoxin A
sequence may include all of domains Ia, Ib, II, and detoxified III.
Therefore, a non-toxic Pseudomonas exotoxin A sequence may be a
contiguous sequence of the native Pseudomonas exotoxin A, or it can
be a sequence comprised of non-contiguous subsequences of the
native Pseudomonas exotoxin A that lacks ADP ribosylation activity.
While a non-toxic Pseudomonas exotoxin A sequence may be smaller
contiguous or non-contiguous portion(s) of the native PE, the
numberings of the native PE amino acid and nucleic acid sequences
are used to refer to certain positions within the non-toxic
Pseudomonas exotoxin A sequence (e.g., deletion of Glu at position
553).
[0033] A "chimeric protein" or a "chimeric polynucleotide" is an
artificially constructed protein or polynucleotide comprising
heterologous amino acid sequences or heterologous nucleic acid
sequences, respectively.
[0034] The term "heterologous" when used with reference to a
protein or a nucleic acid indicates that the protein or the nucleic
acid comprises two or more sequences or subsequences which are not
found in the same relationship to each other in nature. For
instance, the nucleic acid is typically recombinantly produced,
having two or more sequences from unrelated genes arranged to make
a new functional nucleic acid. For example, in one embodiment, the
nucleic acid has a promoter from one gene arranged to direct the
expression of a coding sequence from a different gene. Thus, with
reference to the coding sequence, the promoter is heterologous.
Similarly, a sequence from a Pseudomonas exotoxin A is heterologous
with reference to a Type IV pilin loop sequence when the two
sequences are placed in a relationship other than the naturally
occurring relationship of the nucleic acids in the genome.
[0035] "Type IV pili" refers to filamentous structures covering
many gram-negative bacteria, yeast and other microorganisms. The
pili on the surface of a microorganism adhere to epithelial cells.
In particular, the pili of Pseudomonas or Candida bind to
epithelial cells through specific interaction with asialoGM1
receptors. Type IV pili are primarily composed of protein pilins,
which are polymers arranged in a helical bundle. For example, pili
of Pseudomonas aeruginosa have an average length of 2.5 .mu.m and
consist of a single protein with a molecular mass of around 15,000
(Paranchych et al., Am. Soc. Microbio. 343-351 (1990)).
[0036] The term "Type IV pilin" as used herein refer to pilins that
contain a conserved amino terminal hydrophobic domain beginning
with an amino-terminal phenylalanine that is methylated upon
processing and secretion of the pilin. Another characteristic
feature of Type IV pilins is that in the propilin form they contain
similar six- or seven-amino acid long leader peptides, which are
much shorter than typical signal sequences. Type IV pilins are
expressed by several bacterial genuses, including Neisseria,
Moraxella, Bacteroides, Pasteurella and Pseudomonas, E. coli, and
yeast such as Candida. Species within these genuses which express
Type IV pilins are, for example, P. aeruginosa, N. gonorrhoeae, N.
meningtidis, Pasteurella multocida, M. bovis, B. nodosus. As used
herein, the term "Type IV pilin" also includes the Tcp pilin of
Vibrio, (e.g., V. cholera), that is highly homologous to the Type
IV pilins of other genuses. Tcp pilin contains the characteristic
amino-terminal hydrophobic domain as well as having a modified
N-terminal amino acid that in this case may be a modified
methionine because the Tcp pilin gene encodes a methionine residue
at the position where all the others encode a phenylalanine.
Precursor TcpA contains a much longer leader sequence than typical
Type IV propilins but retains homology in the region surrounding
the processing site. Generally, a pilin protein comprises a region
at the N-terminus that is highly conserved, with the rest of the
protein containing moderately conserved and hypervariable regions
(Paranachych et al., supra). A characteristic feature of all pilins
is an intrachain disulfide loop at the C-terminus of the pilin.
[0037] The amino acid sequences and nucleic acid sequences of Type
IV pilins of various microorganisms are known in the art. See,
e.g., NCBI Database Accession No. M14849, J02609 for Pseudomonas
PAK strain; NCBI Database Accession No. AAC60462 for Pseudomonas
T2A strain; NCBI Database Accession No. M11323 for Pseudomonas PAO
strain; NCBI Database Accession No. P17837 for Pseudomonas CD
strain; NCBI Database Accession No. B31105 for Pseudomonas P1
strain; NCBI Database Accession No. Q53391 for Pseudomonas KB7
strain; NCBI Database Accession No. AAC60461 for Pseudomonas 577B
strain; NCBI Database Accession No. A33105 for Pseudomonas K122-4
strain; NCBI Database Accession Nos. Z49820, Z69262, and Z69261 for
N. meningtidis; NCBI Database Accession Nos. X66144 and AF043648
for N. gonorrhoeae; NCBI Database Accession Nos. U09807 and X64098
for V. cholera; NCBI Database Accession No. AF154834 for
Pasteurella multocida.
[0038] A "Type IV pilin loop sequence" refers to the sequence that
forms an intrachain disulfide loop at the C-terminus of the pilin.
This region is physically exposed at the tip of the pilus, and
interacts with epithelial cell receptors. A Type IV pilin loop
sequence as used herein can refer to a sequence between the two
cysteine residues that form an intrachain disulfide loop at the
C-terminus of the pilin (i.e., excluding the cysteine residues), or
a sequence that includes both cysteine residues and amino acids
between the two cysteine residues. Depending on whether the site of
insertion within non-toxic Pseudomonas exotoxin A sequences has
cysteine residues, the Type IV pilin loop sequence with or without
the flanking cysteine residues can be used to make chimeric
proteins of the invention. Examples of Type IV pilin loop sequence
are shown as SEQ ID NOS: 3 to 20.
[0039] The term "immunogenic fragment thereof" or "immunogenic
portion thereof" refers to a polypeptide comprising an epitope that
is recognized by cytotoxic T lymphocytes, helper T lymphocytes or B
cells.
[0040] "Polyclonal antisera" refers to sera comprising polyclonal
antibodies against an immunogen, which sera is obtained from a host
immunized with the immunogen (e.g., a chimeric protein of the
present invention).
[0041] Polyclonal antisera that "reduce adherence" of a
microorganism expressing a Type IV pilin loop sequence refer to
polyclonal antisera that reduce adherence of the microorganism by
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, compared
to a control. A control can be a prebleed or sera that is not
exposed to the chimeric proteins of the present invention.
[0042] The term polyclonal antisera that "neutralize cytotoxicity"
of Pseudomonas exotoxin A in the context of the present invention
refer to the ability of antisera to reduce the inhibition of
protein synthesis by Pseudomonas exotoxin A. Typically, polyclonal
antisera can reduce inhibition of protein synthesis by Pseudomonas
exotoxin A by at least about 30%, more typically at least about
50%, more typically at least about 80%, even more typically at
least about 90%, 95%, or 99% compared to a control. A control can
be a prebleed or sera that is not exposed to the chimeric proteins
of the present invention.
[0043] "Nucleic acid" or "polynucleotide" refers to
deoxyribonucleotides or ribonucleotides and polymers thereof in
either single- or double-stranded form. The term encompasses
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized
in a manner similar to the reference nucleotides. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0044] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0045] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0046] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0047] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0048] "Conservatively modified variants" apply to both amino acid
and nucleic acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refer to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0049] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid.
[0050] Conservative substitution tables providing functionally
similar amino acids are well known in the art. Such conservatively
modified variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention.
[0051] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0052] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0053] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C.
[0054] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency.
[0055] An "expression cassette" refers to a polynucleotide molecule
comprising expression control sequences operatively linked to
coding sequence(s).
[0056] A "vector" is a replicon in which another polynucleotide
segment is attached, so as to bring about the replication and/or
expression of the attached segment.
[0057] "Control sequence" refers to polynucleotide sequences which
are necessary to effect the expression of coding sequences to which
they are ligated. The nature of such control sequences differs
depending upon the host organism; in prokaryotes, such control
sequences generally include promoter, ribosomal binding site, and
terminators; in eukaryotes, generally, such control sequences
include promoters, terminators and, in some instances, enhancers.
The term "control sequences" is intended to include, at a minimum,
all components whose presence is necessary for expression, and may
also include additional components whose presence is advantageous,
for example, leader sequences.
[0058] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0059] A "ligand" is a compound that specifically binds to a target
molecule.
[0060] A "receptor" is compound that specifically binds to a
ligand.
[0061] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0062] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms variable light chain (V.sub.L) and variable heavy chain
(V.sub.H) refer to these light and heavy chains respectively.
[0063] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990)).
[0064] For preparation of monoclonal or polyclonal antibodies, any
technique known in the art can be used (see, e.g., Kohler &
Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology
Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies
and Cancer Therapy (1985)). Techniques for the production of single
chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to
produce antibodies to polypeptides of this invention. Also,
transgenic mice, or other organisms such as other mammals, may be
used to express humanized antibodies. Alternatively, phage display
technology can be used to identify antibodies and heteromeric Fab
fragments that specifically bind to selected antigens (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990); Marks et al.,
Biotechnology 10:779-783 (1992)).
[0065] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein at least two times the background and
do not substantially bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, polyclonal
antibodies raised to fusion proteins can be selected to obtain only
those polyclonal antibodies that are specifically immunoreactive
with fusion protein and not with individual components of the
fusion proteins. This selection may be achieved by subtracting out
antibodies that cross-react with the individual antigens. A variety
of immunoassay formats may be used to select antibodies
specifically immunoreactive with a particular protein. For example,
solid-phase ELISA immunoassays are routinely used to select
antibodies specifically immunoreactive with a protein (see, e.g.,
Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity). Typically a specific or
selective reaction will be at least twice background signal or
noise and more typically more than 10 to 100 times background.
[0066] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an individual antigen or a portion
thereof) or may comprise a variant of such a sequence.
Polynucleotide variants may contain one or more substitutions,
additions, deletions and/or insertions such that the biological
activity of the encoded chimeric protein is not diminished,
relative to a chimeric protein comprising native antigens. Variants
preferably exhibit at least about 70% identity, more preferably at
least about 80% identity and most preferably at least about 90%
identity to a polynucleotide sequence that encodes a native
polypeptide or a portion thereof.
[0067] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95%
identity over a specified region), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection. Such
sequences are then said to be "substantially identical." This
definition also refers to the compliment of a test sequence.
Optionally, the identity exists over a region that is at least
about 25 to about 50 amino acids or nucleotides in length, or
optionally over a region that is 75-100 amino acids or nucleotides
in length.
[0068] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0069] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 25 to 500, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0070] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. Using PILEUP, a reference sequence is
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps. PILEUP can be obtained from the GCG sequence analysis
software package, e.g. version 7.0 (Devereaux et al., Nuc. Acids
Res. 12:387-395 (1984)).
[0071] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0072] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0073] "Immunogen" refers to an entity that induces antibody
production in the host animal.
[0074] "Vaccine" refers to an agent or composition containing an
agent effective to confer a therapeutic degree of immunity on an
organism while causing only very low levels of morbidity or
mortality. Vaccines and methods for making vaccines are useful in
the study of the immune system and in preventing and treating
animal or human disease.
[0075] An "immunogenic amount" or "immunologically effective
amount" is an amount effective to elicit an immune response in a
subject.
[0076] "Substantially pure" or "isolated" means an object species
is the predominant species present (i.e., on a molar basis, more
abundant than any other individual macromolecular species in the
composition), and a substantially purified fraction is a
composition wherein the object species comprises at least about 50%
(on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition means that about 80% to
90% or more of the macromolecular species present in the
composition is the purified species of interest. The object species
is purified to essential homogeneity (contaminant species cannot be
detected in the composition by conventional detection methods) if
the composition consists essentially of a single macromolecular
species. Solvent species, small molecules (<500 Daltons),
stabilizers (e.g., BSA), and elemental ion species are not
considered macromolecular species for purposes of this
definition.
[0077] "Naturally-occurring" as applied to an object refers to the
fact that the object can be found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an
organism (including viruses) that can be isolated from a source in
nature and which has not been intentionally modified by man in the
laboratory is naturally-occurring.
[0078] A "host" refers to any animal including human or non-human
animals, such as rodents (e.g., mice or rats), primates, sheep,
pigs, guinea pigs, etc.
[0079] "Treatment" refers to prophylactic treatment or therapeutic
treatment.
[0080] A "prophylactic" treatment is a treatment administered to a
host who does not exhibit signs of a disease or exhibits only early
signs for the purpose of decreasing the risk of developing
pathology.
[0081] A "therapeutic" treatment is a treatment administered to a
host who exhibits signs of pathology for the purpose of diminishing
or eliminating those signs.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
I. Chimeric Proteins Comprising a Non-Toxic Pseudomonas Exotoxin a
Sequence and a Type IV Pilin Loop Sequence
[0082] In one aspect, the invention provides a chimeric protein
comprising: a non-toxic Pseudomonas exotoxin A sequence and a Type
IV pilin loop sequence, the Type IV pilin loop sequence being
located within the non-toxic Pseudomonas exotoxin A sequence,
wherein the chimeric protein is capable of reducing the adhesion or
adherence of a microorganism expressing the Type IV pilin loop
sequence to epithelial cells, and further wherein the chimeric
protein, when introduced into a host, is capable of generating
polyclonal antisera that reduce adherence of the microorganism
expressing the Type IV pilin loop sequence to the epithelial cells.
In some embodiments, the chimeric proteins of the invention, when
introduced into a host, are also capable of generating polyclonal
antisera that neutralize cytotoxicity of Pseudomonas exotoxin A. In
another aspect, the invention provides a chimeric protein
comprising: (a) a non-toxic Pseudomonas exotoxin A sequence
comprising domain Ia, domain II, and domain III; and (b) a Type IV
pilin loop sequence, wherein the Type IV pilin loop sequence is
located between domain II and domain III of the non-toxic
Pseudomonas exotoxin A sequence. In some embodiments, the chimeric
protein comprises a non-toxic Pseudomonas exotoxin A sequence
including domains Ia, II, and III in the native organization
structure, except that a Type IV pilin loop sequence, partially or
completely, replaces domain Ib and is located between domain II and
domain III. Alternatively or additionally, in some embodiments, the
chimeric protein comprises a Type IV pilin loop sequence in domain
II, replacing amino acids 265 to 287. The nature of non-toxic
Pseudomonas exotoxin A sequences, various domains of non-toxic
Pseudomonas exotoxin A sequences, Type IV pilin loop sequences, and
their physical relationship within chimeric proteins of the
invention are described in detail below.
[0083] A. Non-toxic Pseudomonas Exotoxin A Sequences
[0084] As described in the Definition section above, Pseudomonas
exotoxin A or PE is secreted by Pseudomonas aeruginosa and
comprises three prominent domains (Ia, II, and III) and one small
subdomain (Ib) connecting domains II and III. In nature, domain Ia
of PE, spanning amino acids 1-252, mediates cell binding. Domain
II, spanning amino acids 253-364, mediates translocation of the
protein to the cytosol. Domain Ib, spanning amino acids 365-399,
has no known function. Domain III, spanning amino acids 400-613, is
responsible for cytotoxicity and includes an endoplasmic reticulum
retention sequence. It also contains sequences that mediates ADP
ribosylation of elongation of factor 2 ("EF2"), which inactivates
protein synthesis and thus rendering PE to be toxic to cells. Thus,
domain Ia or its variant that mediates cell binding is referred to
as "a cell recognition domain." Domain II or its variant that
mediates translocation of the proteins to the cytosol is referred
to as "a translocation domain." Domain III or its variant that
functions in translocating the protein from the endosome to the
endoplasmic reticulum is referred to as "an endoplasmic reticulum
retention domain."
[0085] A non-toxic Pseudomonas exotoxin A sequence refers to any
Pseudomonas exotoxin A sequence that lacks ADP ribosylation
activity. Generally, a non-toxic Pseudomonas exotoxin A sequence
has one or more domains or portions of domains with certain
biological activities. For example, a non-toxic Pseudomonas
exotoxin A sequence may comprise a translocation domain (e.g.,
domain II of Pseudomonas exotoxin A) and an endoplasmic reticulum
domain (e.g., detoxified domain III of Pseudomonas exotoxin A
without ADP ribosylation activity). In another example, a non-toxic
Pseudomonas exotoxin A sequence may be constructed by eliminating
amino acids 1-252 yielding a construct referred to as "PE40". In
another example, a non-toxic Pseudomonas exotoxin A sequence may be
constructed by eliminating amino acids 1-279 yielding a construct
referred to as "PE37." (See Pastan et al., U.S. Pat. No.
5,602,095.).
[0086] Optionally, a cell recognition domain of Pseudomonas
exotoxin A (e.g., domain I) or other cell recognition domains
unrelated to Pseudomonas exotoxin A can be included in the present
chimeric proteins. A cell recognition domain can be linked,
directly or indirectly, to the rest of the chimeric protein. For
example, one can ligate sequences encoding a cell recognition
domain to the 5' end of non-toxic versions of PE40 or PE37
constructs, which further comprise a Type IV pilin loop
sequence.
[0087] 1. Translocation Domain
[0088] The chimeric proteins of the invention comprise a non-toxic
Pseudomonas exotoxin A sequence comprising a "PE translocation
domain." The PE translocation domain comprises an amino acid
sequence sufficient to effect translocation of chimeric proteins
that have been endocytosed by the cell into the cytosol. The amino
acid sequence is identical to, or substantially identical to, a
sequence selected from domain II of PE.
[0089] The amino acid sequence sufficient to effect translocation
can be derived from the translocation domain of native PE. This
domain spans amino acids 253-364. The translocation domain can
include the entire sequence of domain II. However, the entire
sequence is not necessary for translocation. For example, the amino
acid sequence can minimally contain, e.g., amino acids 280-344 of
domain II of PE. Sequences outside this region, i.e., amino acids
253-279 and/or 345-364, can be eliminated from the domain. This
domain can also be engineered with substitutions so long as
translocation activity is retained.
[0090] The translocation domain functions as follows. After binding
to a receptor on the cell surface, the chimeric proteins enter the
cell by endocytosis through clathrin-coated pits. Residues 265 and
287 are cysteines that form a disulfide loop. Once internalized
into endosomes having an acidic environment, the peptide is cleaved
by the protease furin between Arg279 and Gly280. Then, the
disulfide bond is reduced. A mutation at Arg279 inhibits
proteolytic cleavage and subsequent translocation to the cytosol.
Ogata et al., J. Biol. Chem. 265:20678-85 (1990). However, a
fragment of PE containing the sequence downstream of Arg279 (called
"PE37") retains substantial ability to translocate to the cytosol.
Siegall et al., J. Biol. Chem. 264:14256-61 (1989). Sequences in
domain II beyond amino acid 345 also can be deleted without
inhibiting translocation. Furthermore, amino acids at positions 339
and 343 appear to be necessary for translocation. Siegall et al.,
Biochemistry 30:7154-59 (1991).
[0091] Methods for determining the functionality of a translocation
domain are described below in the section on testing.
[0092] 2. ER Retention Domain
[0093] The chimeric protein of the invention can also comprise an
amino acid sequence encoding an "endoplasmic reticulum retention
domain" as part of a non-toxic exotoxin A sequence. The endoplasmic
reticulum ("ER") retention domain functions in translocating the
chimeric protein from the endosome to the endoplasmic reticulum,
from where it is transported to the cytosol. The ER retention
domain is located at the position of domain III in PE. The ER
retention domain comprises an amino acid sequence that has, at its
carboxy terminus, an ER retention sequence. The ER retention
sequence in native PE is REDLK (SEQ ID NO:21). Lysine can be
eliminated (i.e., REDL (SEQ ID NO:22)) without a decrease in
activity. REDLK (from SEQ ID NO:21) can be replaced with other ER
retention sequences, such as KDEL (SEQ ID NO:23), or polymers of
these sequences. See Ogata et al., J. Biol. Chem. 265:20678-85
(1990); Pastan et al., U.S. Pat. No. 5,458,878; Pastan et al.,
Annu. Rev. Biochem. 61:331-54 (1992).
[0094] Sequences up-stream of the ER retention sequence can be the
native PE domain III (preferably de-toxified), can be entirely
eliminated, or can be replaced by another amino acid sequence. If
replaced by another amino acid sequence, the sequence can, itself,
be highly immunogenic or can be slightly immunogenic. Activity of
this domain can be assessed by testing for translocation of the
protein into the target cell cytosol using the assays described
below.
[0095] In native PE, the ER retention sequence is located at the
carboxy terminus of domain III. Domain III has two functions in PE.
It exhibits ADP-ribosylating activity and directs endocytosed toxin
into the endoplasmic reticulum. Eliminating the ER retention
sequence from the chimeric protein does not alter the activity of
Pseudomonas exotoxin as a superantigen, but does inhibit its
utility to elicit an MHC Class I-dependent cell-mediated immune
response.
[0096] The ribosylating activity of PE is located between about
amino acids 400 and 600 of PE. In methods of vaccinating a host
using the chimeric proteins of this invention, it is preferable
that the protein be non-toxic. One method of doing so is by
eliminating ADP ribosylation activity. In this way, the chimeric
protein can function as a vector for Type IV pilin loop sequences
to be processed by the cell and presented on the cell surface with
MHC Class I molecules, rather than as a toxin. ADP ribosylation
activity can be eliminated by, for example, deleting amino acid
E553 (".DELTA.E553") of the native PE. See, e.g., Lukac et al.,
Infect. and Immun. 56:3095-3098 (1988). In another example,
substitution of histidine residue of PE at 426 with a tyrosine
residue also inactivates the ADP-ribosylation of PE (see Kessler
& Galloway, supra). Other amino acids in domain III can be
modified from the protein to eliminate ADP ribosylation activity.
An ER retention sequence is generally included at the
carboxy-terminus of the chimeric protein.
[0097] In one embodiment, the sequence of the ER retention domain
is substantially identical to the native amino acid sequences of
the domain III, or a fragment of it. In some embodiments, the ER
retention domain is domain III of PE.
[0098] In another embodiment, a cell recognition domain is inserted
into the amino acid sequence of the ER retention domain (e.g., into
domain III). For example, the cell recognition domain can be
inserted just up-stream of the ER retention sequence, so that the
ER retention sequence is connected directly or within ten amino
acids of the carboxy terminus of the cell recognition domain.
[0099] B. Cell Recognition Domain
[0100] Optionally, the chimeric protein of the invention can
comprise an amino acid sequence encoding a "cell recognition
domain." The cell recognition domain functions as a ligand for a
cell surface receptor. It mediates binding of the protein to a
cell. It can be used to target the chimeric protein to a cell which
will transport it to the cytosol for processing. A cell recognition
domain may not be necessarily included in the chimeric protein, as
a Type IV pilin loop sequence within the chimeric protein targets
receptors on epithelial cells.
[0101] The cell recognition domain functions to attach the chimeric
protein to a target cell, and it can be any suitable material,
e.g., a polypeptide known to a particular receptor in the target
cell. For example, the cell recognition domain generally has the
size of known polypeptide ligands, e.g., between about 10 amino
acids and about 1500 amino acids, or about 100 amino acids and
about 300 amino acids. Several methods are useful for identifying
functional cell recognition domains for use in chimeric proteins.
One method involves detecting binding between a chimeric protein
that comprises the cell recognition domain with the receptor or
with a cell bearing the receptor. Other methods involve detecting
entry of the chimeric protein into the cytosol, indicating that the
first step, cell binding, was successful. These methods are
described in detail below in the section on testing.
[0102] In one embodiment, the cell recognition domain is domain Ia
of PE, thereby targeting the chimeric protein to the .alpha.2-MR
domain. In other embodiments domain Ia can be substituted with
ligands that bind to cell surface receptors or antibodies or
antibody fragments directed to cell surface receptors. For example,
to target epithelial cells, a cell binding domain can be a ligand
for or antibodies against the EGF receptor, transferrin receptors,
interleukin-2 receptors, interleukin-6 receptors, interleukin-8
receptors, or Fc receptors, or poly-IgG receptors. To target liver
cells, a cell binding domain can be, e.g., a ligand for or
antibodies against asialoglycoprotein receptors. To target T cells,
a cell binding domain can be, e.g., a ligand for or antibodies
against CD3, CD4, CD8, or chemokine receptors. To target activated
T-cells and B-cells, a cell binding domain can be, e.g., a ligand
for or antibodies against CD25. To target dendritic cells, a cell
binding domain can be, e.g., ligands for or antibodies against
CD11B, CD11C, CD80, and CD86 MHC class I and II. To target
macrophages, a cell binding domain can be, e.g., ligands for or
antibodies against TNFalpha receptors, chemokine receptors, TOLL
receptors, M-CSF receptors, GM-CSF receptors, scavenger receptors,
and Fc receptors. To target endothelial cells, a cell binding
domain can be, e.g., a ligand for or antibodies against VEGF
receptors. Also, cytokine receptors which are found in many cell
types can be targeted. Pastan et al. Ann. Rev. Biochem. 61:331-54
(1992).
[0103] The cell recognition domain can be located at any suitable
position within the present chimeric proteins. For example, the
cell recognition domain can be located in the N-terminus of the
chimeric protein (e.g., position equivalent to domain Ia of
non-toxic PE). However, this domain can be moved out of the normal
organizational sequence of exotoxin A. More particularly, the cell
recognition domain can be inserted upstream of the ER retention
domain. Alternatively the cell recognition domain can be chemically
coupled to the rest of the chimeric protein. Also, the chimeric
protein can include a first cell recognition domain at the location
of the Ia domain and a second cell recognition domain upstream of
the ER retention domain. Such constructs can bind to more than one
cell type. See, e.g., Kreitman et al., Bioconjugate Chem. 3:63-68
(1992). For example, TGFa has been inserted into domain III just
before amino acid 604, i.e., about ten amino acids from the
carboxy-terminus. This chimeric protein binds to cells bearing EGF
receptor. Pastan et al., U.S. Pat. No. 5,602,095.
[0104] The cell recognition domain can be inserted or attached to
the rest of the chimeric proteins using any suitable methods. For
example, the domain can be attached to the rest of the chimeric
protein directly or indirectly using a linker. The linker can form
covalent bonds or high-affinity non-covalent bonds. Suitable
linkers are well known to those of ordinary skill in the art. In
another example, the cell recognition domain is expressed as a
single chimeric polypeptide from a nucleic acid sequence encoding
the single contiguous chimeric protein.
[0105] C. Type IV Pilin Loop Sequences
[0106] The chimeric protein also comprises a Type IV pilin loop
sequence within a non-toxic Pseudomonas exotoxin A sequence. The
Type IV pilin loop sequence is generally derived from a sequence
that forms an intrachain disulfide loop at the C-terminus of the
pilin protein. The Type IV pilin loop sequence allows the chimeric
protein to react with asialoGM1 receptors on epithelial cells. This
loop is dominated by main chain residues. Therefore, pilins from
several strains bind the same receptor despite sequence variation
and the difference in length (e.g., for certain Pseudomonas
strains, 12 and 17 amino acid loops (or 14 to 19 amino acids
including flanking cysteine residues)). A Type IV loop pilin
sequence comprises at least about 5 amino acid residues, typically
between about 10 to 100 amino acids, more typically about 12 to 70
amino acids, even more typically about 12 to 20 amino acids.
Embodiments of the invention can have one unit of the Type IV pilin
loop sequence or multiple repeating units (e.g., 2, 3, 4, etc.) of
the same or different Type IV pilin loop sequences. In some
embodiments, the chimeric proteins comprise more than one Type IV
pilin loop sequences at different locations.
[0107] A Type IV pilin loop sequence can be derived from any
microorganism that adhere to epithelial cells. For example, a Type
IV pilin sequence can be derived from bacteria or yeast, such as
Pseudomonas aeruginosa, Neisseria meningtidis, Neisseria
gonorrhoeae, Vibro cholera, Pasteurella multocidam or Candida.
Examples of a Type IV pilin sequence are shown as SEQ ID NOS: 3 to
20.
[0108] Type IV pilin sequences from different Pseudomonas
aeruginosa strains vary in terms of their sequence as well as their
length. Several Pseudomonas aeruginosa strains have a short pilin
loop consisting of 14 amino acids (from cysteine 129 to cysteine
142) as shown in Table 1 below. Other Pseudomonas aeruginosa
strains have a long pilin loop consisting of 19 amino acids (from
cysteine 133 to 151) as shown in Table 2 below. TABLE-US-00001
TABLE 1 P. aeruginosa strains Type IV pilin loop sequence (with a
short pilin (Cysteine 129 to Cysteine loop) 142) PAK CTSDQDEQFIPKGC
(SEQ ID NO: 3) T2A CTSTQDEMFIPKGC (SEQ ID NO: 4) PAO, 90063
CKSTQDPMFTPKGC (SEQ ID NO: 5) CD, PA103 CTSTQEEMFIPKGC (SEQ ID NO:
6) K122-4 CTSNADNKYLPKTC (SEQ ID NO: 7) KB7, 82932, 82935
CATTVDAKFRPNGC (SEQ ID NO: 8) 1071 CESTQDPMFTPKGC (SEQ ID NO:
9)
[0109] TABLE-US-00002 TABLE 2 P. aeruginosa strains (with a long
pilin Type IV pilin loop sequence loop) (Cysteine 133 to Cysteine
151) 577B CNITKTPTAWKPNYAPANC (SEQ ID NO: 10) 1244, 9D2, P1
CKITKTPTAWKPNYAPANC (SEQ ID NO: 11) SBI-N CGITGSPTNWKANYAPANC (SEQ
ID NO: 12)
[0110] Type IV pilin loop sequences from microorganisms other than
P. aeruginosa can also be included in the chimeric proteins of the
invention. Examples of Type IV pilin loop amino acid sequences from
other microorganisms are shown in Table 3 below. TABLE-US-00003
TABLE 3 Micro- organism Type IV pilin loop sequence Neisseria
CGLPVARDDTDSATDVKADTTDNINTKHLPSTC meningtidis (SEQ ID NO: 13)
(Z49820) Neisseria CGQPVTRGAGNAGKADDVTKAGNDNEKINTKHLPSTC
meningtidis (SEQ ID NO: 14) (Z69262) Neisseria
CGQPVTRAKADADAAGKDTTNIDTKHLPSTC meningtidis (SEQ ID NO: 15)
(Z69261) Neisseria CGQPVTRTGDNDDTVADAKDGKEIDTKHLPSTC gonorrhoeae
(SEQ ID NO: 16) (pilE; X66144) Neisseria
CGQPVKRDAGAKTGADDVKADGNNGINTKHLPSTC gonorrhoeae (SEQ ID NO: 17)
(pilE; AF043648) Vibrio CKTLVTSVGDMFPFINVKEGAFAAVADLGDFETSVADA
cholera ATGAGVIKSIAPGSANLNLTNITHVEKLC (U09807) (SEQ ID NO: 18)
Vibrio CKTLITSVGDMFPYIAIKAGGAVALADLGDFENSAAAAE cholera
TGVGVIKSIAPASKNLDLTNITHVEKLC (X64098) (SEQ ID NO: 19) Pasteurella
CNGGSEVFPAGFC multocida (SEQ ID NO: 20) (AF154834)
[0111] One of skill in the art will recognize that the above
described Type IV pilin sequences are merely exemplary and that
other Type IV pilin sequences can be readily inserted into the
chimeric proteins of the present invention. For example, Type IV
pilin loop sequences described in, e.g., U.S. Pat. No. 5,612,036
(Hodges et al.) can also be incorporated into the chimeric proteins
of the present invention.
[0112] The Type IV pilin loop sequence can be located at any
suitable position within the chimeric protein of the invention. In
one embodiment, the Type IV pilin sequence is inserted between the
translocation domain (e.g., domain II of non-toxic exotoxin A) and
the ER retention domain (e.g., domain III of non-toxic exotoxin A).
In another embodiment, the chimeric protein has the basic
organization structure of non-toxic Pseudomonas exotoxin A
including domain Ia, domain II, domain lb, and domain III, except
that domain lb is, partially or completely, replaced by the Type IV
pilin loop sequence. In native Pseudomonas exotoxin A, domain lb
spans amino acids 365 to 399. The native lb domain is structurally
characterized by a disulfide bond between two cysteines at
positions 372 and 379. Domain lb is not essential for cell binding,
translocation, ER retention or ADP ribosylation activity.
Therefore, it can be partially or entirely replaced by a Type IV
pilin loop sequence. For example, a Type IV pilin loop sequence can
be inserted between the two cysteines at positions 372 and 379,
replacing the 6 amino acid residues between the two cysteines. In.
another embodiment, the Type IV pilin loop sequence can be inserted
into the lb domain without removing any of the lb domain sequences.
In another embodiment, the Type IV pilin loop sequence can be
positioned in another location which forms a cysteine-cysteine
disulfide bonded loop, such as amino acids 265-287 of domain II of
non-toxic Pseudomonas exotoxin A. In some embodiments, more than
one Type IV pilin loop sequences can be inserted into different
locations within the chimeric protein.
[0113] Depending on whether the site of insertion within a
non-toxic Pseudomonas exotoxin A sequence has cysteine residues, a
Type IV pilin loop sequence with or without cysteine residues at
the N- and C-termini can be used. For example, if the site of
insert in the non-toxic Pseudomonas exotoxin A sequence does not
have cysteine residues, then a Type IV pilin loop sequence with
cysteine residues at its termini (e.g., 14 amino acids shown in SEQ
ID NO:3) can be inserted. In another example, if a Type IV pilin
loop sequence is inserted in the cysteine-cysteine loop of the
native lb domain, replacing the six amino acids between the
cysteine residues, then a Type IV pilin loop sequence can be a
sequence without terminal cysteines (e.g., 12 amino acids between
the two cysteines shown in SEQ ID NO:3). Therefore, a
cysteine-cysteine loop can be preferably formed within the chimeric
protein of the invention. When the Type IV pilin loop sequence
within the chimeric protein is presented as a cysteine-cysteine
disulfide bonded loop, the Type IV pilin loop structure may stick
out from the rest of the chimeric protein, where it is available to
interact with, e.g., asialoGM1 receptors or with immune system
components.
II. Chimeric Polynucleotides and Expression of Polynucleotides
[0114] A. Polynucleotides Encoding the Chimeric Proteins
[0115] In another aspect, the invention provides polynucleotides
encoding the chimeric proteins of the invention. Suitable amino
acid sequences of non-toxic Pseudomonas exotoxin A sequences (e.g.,
comprising a translocation domain and an ER retention domain), cell
recognition domains, and Type IV pilin loop sequences and their
physical locations within the present chimeric proteins are
described in detail above. Any polynucleotides that encode these
amino acid sequences are within the scope of the present
invention.
[0116] 1. Identification of Non-Toxic Pseudomonas Exotoxin A
Sequences Polynucleotides that encode non-toxic Pseudomonas
exotoxin A amino acid sequences may be identified, prepared and
manipulated using any of a variety of well established techniques.
A nucleotide encoding native Pseudomonas exotoxin A is shown as SEQ
ID NO:1. The practitioner can use this sequence to prepare
non-toxic Pseudomonas exotoxin A sequences using various cloning
and in vitro amplification methodologies known in the art. PCR
methods are described in, for example, U.S. Pat. No. 4,683,195;
Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987);
and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989);
Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995).
These primers can be used, e.g., to amplify either the full length
sequence, partial sequences or a probe of one to several hundred
nucleotides, which is then used to screen cDNA or genomic libraries
for related nucleic acid sequence homologs. Polynucleotides can
also be isolated by screening genomic or cDNA libraries (e.g.,
Pseudomonas aeruginosa) with probes selected from the sequences of
the desired polynucleotide under stringent hybridization
conditions.
[0117] As an illustration, to clone a Pseudomonas exotoxin A
sequence comprising all of the domains (domain Ia, domain II,
domain lb, and domain III), the following primers can be used:
Forward--GGCCCATATGCACCTGATACCCCAT (SEQ ID NO:24); and
Reverse--GAATTCAGTTACTTCAGGTCCTCG (SEQ ID NO:25). To clone a
Pseudomonas exotoxin A sequence comprising domain II, domain Ib,
and domain III, the following primers can be used:
Forward--GGCCCATATGGAGGGCGGCAGCCTGGCC (SEQ ID NO:26); and
Reverse--GAATTCAGTTACTTCAGGTCCTCG (SEQ ID NO:27).
[0118] Other Pseudomonas exotoxin A constructs that can be used in
the embodiments of the invention are also described in, e.g., U.S.
Pat. No. 5,602,095 (Pastan et al.). As described in the '095
patent, eliminating nucleotides encoding amino acids 1-252 yields a
construct referred to as "PE40." Eliminating nucleotides encoding
amino acids 1-279 yields a construct referred to as "PE37."
Non-toxic versions of these constructs (which lack domain Ia of
native exotoxin A) are particularly useful for ligating them to
sequences encoding heterologous cell recognition domains to the 5'
end of these constructs. These constructs can optionally encode an
amino-terminal methionine.
[0119] In addition, Pseudomonas exotoxin A can be further modified
using site-directed mutagenesis or other techniques known in the
art, to alter the molecule for a particular desired application.
Means to alter Pseudomonas exotoxin A in a manner that does not
substantially affect the functional advantages provided by the PE
molecules described herein can also be used and such resulting
molecules are intended to be covered herein.
[0120] Non-toxic Pseudomonas exotoxin A sequences can be generated
from these Pseudomonas exotoxin A sequences by modifying portions
of domain III so that they lack ADP ribosylation activity. The
ribosylating activity of PE is located between about amino acids
400 and 600 of native Pseudomonas exotoxin A. For example, deleting
amino acid E553 (".DELTA.E553") from domain III detoxifies the
molecule. This detoxified PE is referred to as "PE .DELTA.E553."
Other amino acids within domain III can be modified by, e.g.,
deletion, substitution or addition of amino acid residues, to
eliminate ADP ribosylation activity. For example, substitution of
histidine residue of PE at 426 with a tyrosine residue also
inactivates the ADP-ribosylation of PE (see Kessler & Galloway,
supra).
[0121] In some embodiments, non-toxic Pseudomonas exotoxin A
sequences can be further modified to accommodate cloning sites for
insertion of a Type IV pilin loop sequence. For example, a cloning
site for the Type IV pilin sequence can be introduced between the
nucleotides encoding the cysteines of domain lb of non-toxic
Pseudomonas exotoxin A. For example, a nucleotide sequence encoding
a portion of the lb domain between the cysteine-encoding residues
can be removed and replaced with a nucleotide sequence encoding an
amino acid. sequence and that includes a PstI cloning site. This
example is described in detail in the Example section.
Alternatively, a longer portion of domain lb or entire domain lb
can be removed and replaced with an amino acid sequence and that
includes cloning site(s).
[0122] The construct can also be engineered to encode a secretory
sequence at the amino terminus of the protein. Such constructs are
useful for producing the chimeric proteins in mammalian cells. In
vitro, such constructs simplify isolation of the chimeric proteins.
In vivo, the constructs are useful as polynucleotide vaccines;
cells that incorporate the construct will express the protein and
secrete it where it can interact with the immune system.
[0123] 2. Identification Type IV Pilin Loop Sequences
[0124] Polynucleotides that encode Type IV pilin loop amino acid
sequences may be identified, prepared and manipulated using any of
a variety of well-established techniques. Type IV pilin nucleotide
and amino acid sequences from various microorganisms are well-known
in the art. See, e.g., NCBI Database Accession No. M14849 J02609
for Pseudomonas PAK strain; NCBI Database Accession No. AAC60462
for Pseudomonas T2A strain; NCBI Database Accession No. M11323 for
Pseudomonas PAO strain; NCBI Database Accession No. P17837 for
Pseudomonas CD strain; NCBI Database Accession No. B31105 for
Pseudomonas P1 strain; NCBI Database Accession No. Q53391 for
Pseudomonas KB7 strain; NCBI Database Accession No. AAC60461 for
Pseudomonas 577B strain; NCBI Database Accession No. A33105 for
Pseudomonas K122-4 strain; NCBI Database Accession Nos. Z49820,
Z69262, and Z69261 for N. meningtidis; NCBI Database Accession Nos.
X66144 and AF043648 for N. gonorrhoeae; NCBI Database Accession
Nos. U09807 and X64098 for V. cholera; NCBI Database Accession No.
AF154834 for Pasteurella multocida. The practitioners can clone and
identify other pilin nucleotides and amino acid sequences from
other microorganisms using various cloning and in vitro
amplification methodologies known in the art. For example, to clone
other pilin loop Pseudomonas strains from a library, primers for
amplification from the highly conserved 5' end of the pilin gene
and the 3' end of the neighboring gene (Nicotinate-nucleotide
pyrophosphorylase) in the Pseudomonas genome can be used. Exemplary
primers PCR (listed in the 5' to 3' direction) for sequencing the
pilin genes are as follows: pilATG (26 nc)
GAGATATTCATGAAAGCTCAAAAAGG (SEQ ID NO:28); and nadB4 (20 nc)
ATCTCCATCGGCACCCTGAC (SEQ ID NO:29); or nadB 1 (21 nc)
TGGAAGTGGAAGTGGAGAACC (SEQ ID NO:30).
[0125] From these Type IV pilin polynucleotides, the portion that
forms the C-terminal intrachain disulfide loop (i.e., Type IV pilin
loop) can be readily identified visually. Examples of Type IV pilin
loop amino acids are shown as SEQ ID NO:3 to 20 in Tables 1-3
above. Any degenerate nucleotides encoding these and other Type IV
pilin loop amino acids can be used to construct chimeric
polynucleotides of the invention. In some embodiments, to
facilitate insertion of Type IV pilin loop sequence into a
non-toxic Pseudomonas exotoxin A sequence, 5' and/or 3' ends of
Type IV pilin loop nucleotide sequence can be modified to
incorporate cohesive ends for cloning sites (e.g., PstI).
[0126] As described above, typically, a Type IV pilin loop sequence
is inserted into domain lb, or can partially or fully replace
domain lb of non-toxic Pseudomonas exotoxin A. In some embodiments,
a Type IV pilin loop sequence can be inserted into other suitable
locations within a non-toxic Pseudomonas exotoxin A sequence. For
example, a Type IV pilin loop sequence can be inserted in another
location of non-toxic Pseudomonas exotoxin A which forms a
cysteine-cysteine disulfide bonded loop, such as amino acids
265-287 of domain II of non-toxic Pseudomonas exotoxin A. Other
suitable locations for insertion can be readily tested using
functional tests described herein. In some embodiments, more than
one Type IV pilin loop sequences can be inserted into chimeric
polynucleotides of the invention (e.g., a first pilin loop sequence
in domain Ib and a second pilin loop sequence in domain II).
[0127] 3. Identification of Cell Recognition Domain
[0128] Polynucleotides encoding various cell recognition domains
are well-known in the art. As described above, in one embodiment,
the cell recognition domain is domain Ia of PE, thereby targeting
the chimeric protein to the .alpha.2-MR domain. In this embodiment,
the cell recognition domain can be readily included in the chimeric
polynucleotides using SEQ ID NO:1 as described above. In other
embodiments domain Ia can be substituted with ligands that bind to
cell surface receptors or antibodies or antibody fragments directed
to cell surface receptors. Suitable ligands and antibodies or
antibody fragments are described above in section IB above.
Suitable locations for insertion of cell recognition domain into
chimeric proteins and chimeric polynucleotides are also described
above in section IB.
[0129] The cell recognition domain can be inserted or attached to
the rest of the chimeric proteins using any suitable methods. For
example, the domain can be attached to the rest of the chimeric
protein directly or indirectly using a linker. The linker can form
covalent bonds or high-affinity non-covalent bonds. Suitable
linkers are well known to those of ordinary skill in the art. In
another example, the cell recognition domain is expressed as a
single chimeric polypeptide from a nucleic acid sequence encoding
the single contiguous chimeric protein.
[0130] B. Expression Cassettes and Vectors
[0131] Embodiments of the invention also provide expression
cassettes and vectors for expressing the present chimeric proteins.
Expression cassettes are recombinant polynucleotide molecules
comprising expression control sequences operatively linked to a
polynucleotide encoding the chimeric protein. Expression vectors
comprise these expression cassettes in addition to other sequences
necessary for replication in cells.
[0132] Expression vectors can be adapted for function in
prokaryotes or eukaryotes by inclusion of appropriate promoters,
replication sequences, markers, etc. for transcription and
translation of mRNA. The construction of expression vectors and the
expression of genes in transfected cells involves the use of
molecular cloning techniques also well known in the art. Sambrook
et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., (1989) and Current Protocols
in Molecular Biology, F. M. Ausubel et al., eds., (Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc.). Useful promoters for such
purposes include a metallothionein promoter, a constitutive
adenovirus major late promoter, a dexamethasone-inducible MMTV
promoter, a SV40 promoter, a MRP polIII promoter, a constitutive
MPSV promoter, a tetracycline-inducible CMV promoter (such as the
human immediate-early CMV promoter), and a constitutive CMV
promoter. A plasmid useful for gene therapy can comprise other
functional elements, such as selectable markers, identification
regions, and other genes.
[0133] Expression vectors useful in this invention depend on their
intended use. Such expression vectors must contain expression and
replication signals compatible with the host cell. Expression
vectors useful for expressing the chimeric proteins include viral
vectors such as retroviruses, adenoviruses and adeno-associated
viruses, plasmid vectors, cosmids, and the like. Viral and plasmid
vectors are preferred for transfecting mammalian cells. The
expression vector pcDNA1 (Invitrogen, San Diego, Calif.), in which
the expression control sequence comprises the CMV promoter,
provides good rates of transfection and expression.
Adeno-associated viral vectors are useful in the gene therapy
methods of this invention.
[0134] A variety of means are available for delivering
polynucleotides to cells including, for example, direct uptake of
the molecule by a cell from solution, facilitated uptake through
lipofection (e.g., liposomes or immunoliposomes), particle-mediated
transfection, and intracellular expression from an expression
cassette having an expression control sequence operably linked to a
nucleotide sequence that encodes the inhibitory polynucleotide. See
also Inouye et al., U.S. Pat. No. 5,272,065; Methods in Enzymology,
vol. 185, Academic Press, Inc., San Diego, Calif. (D. V. Goeddel,
ed.) (1990) or M. Krieger, Gene Transfer and Expression--A
Laboratory Manual, Stockton Press, New York, N.Y., (1990).
Recombinant DNA expression plasmids can also be used to prepare the
polynucleotides of the invention for delivery by means other than
by gene therapy, although it may be more economical to make short
oligonucleotides by in vitro chemical synthesis.
[0135] The construct can also contain a tag to simplify isolation
of the protein. For example, a polyhistidine tag of, e.g., six
histidine residues, can be incorporated at the amino terminal end
of the protein. The polyhistidine tag allows convenient isolation
of the protein in a single step by nickel-chelate
chromatography.
[0136] C. Recombinant Cells
[0137] The invention also provides recombinant cells comprising an
expression cassette or vectors for expression of the nucleotide
sequences encoding a chimeric protein of this invention. Host cells
can be selected for high levels of expression in order to purify
the protein. The cells can be prokaryotic cells, such as E. coli,
or eukaryotic cells. Useful eukaryotic cells include yeast and
mammalian cells. The cell can be, e.g., a recombinant cell in
culture or a cell in vivo.
[0138] E. coli has been successfully used to produce the chimeric
proteins of the present invention. The protein can fold and
disulfide bonds can form in this cell.
[0139] D. Chimeric Protein Purification and Preparation
[0140] Once a recombinant chimeric protein is expressed, it can be
identified by assays based on the physical or functional properties
of the product, including radioactive labeling of the product
followed by analysis by gel electrophoresis, radioimmunoassay,
ELISA, bioassays, etc.
[0141] Once the encoded protein is identified, it may be isolated
and purified by standard methods including chromatography (e.g.,
high performance liquid chromatography, ion exchange, affinity, and
sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins. See, generally, R. Scopes, Protein Purification,
Springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol.
182: Guide to Protein Purification, Academic Press, Inc. N.Y.
(1990). The actual conditions used will depend, in part, on factors
such as net charge, hydrophobicity, hydrophilicity, etc., and will
be apparent to those having skill in the art.
[0142] After biological expression or purification, the chimeric
proteins may possess a conformation substantially different than
the native conformations of the constituent proteins. In this case,
it is helpful to denature and reduce the chimeric protein and then
to cause the protein to re-fold into the preferred conformation.
Methods of reducing and denaturing polypeptides and inducing
re-folding are well known to those of skill in the art (see
Debinski et al., J. Biol. Chem. 268:14065-14070 (1993); Kreitman
& Pastan, Bioconjug. Chem. 4:581-585 (1993); and Buchner et
al., Anal. Biochem. 205:263-270 (1992)). Debinski et al., for
example, describe the denaturation and reduction of inclusion body
polypeptides in guanidine-DTE. The polypeptide is then refolded in
a redox buffer containing oxidized glutathione and L-arginine.
[0143] E. Testing Functional Properties of the Chimeric Protein
[0144] The functional properties of the chimeric protein as a whole
or each component thereof are using various routine assays. For
example, the chimeric proteins are tested in terms of cell
recognition, cytosolic translocation, Type IV pilin adhesion, and
immunogenicity. The entire chimeric protein can be tested, or the
function of various domains can be tested by substituting them for
native domains of the wild-type exotoxin A.
[0145] 1. Receptor Binding/Cell Recognition
[0146] To determine whether the cell binding domain present in the
chimeric protein functions properly, the ability of the chimeric
protein to bind to the target receptor (either isolated or on the
cell surface) is tested using various methods known in the art.
[0147] In one method, binding of the chimeric protein to a target
is performed by affinity chromatography. For example, the chimeric
protein is attached to a matrix in an affinity column, and binding
of the receptor to the matrix detected. Alternatively, the target
receptor is attached to a matrix in an affinity column, and binding
of the chimeric protein to the matrix is detected.
[0148] Binding of the chimeric protein to receptors on cells can be
tested by, for example, labeling the chimeric protein and detecting
its binding to cells by, e.g., fluorescent cell sorting,
autoradiography, etc.
[0149] In some embodiments, toxic version of chimeric proteins
(which has ADP ribosylation activity) can be used to test whether
the cell binding domain of the chimeric proteins binds to its
target receptor. For example, the toxic version of chimeric
proteins can be incubated with either cells that express the target
receptors or cells that do not express the target receptors, and
cytotoxic effects of the toxic version of chimeric proteins can be
determined (e.g., by measuring inhibition of [.sup.3H]leucine
incorporation).
[0150] If antibodies have been identified that bind to the ligand
from which the cell recognition domain is derived, they are also
useful to detect the existence of the cell recognition domain in
the chimeric protein by immunoassay, or by competition assay for
the cognate receptor.
[0151] In above testing methods, typically a specific or selective
reaction of the chimeric protein to a target will be at least twice
background signal or noise and more typically more than 10 to 100
times background.
[0152] These methods are described in detail in, e.g., Kreitman et
al., Proc. Natl. Acad. Sci. U.S.A 87:8291-5 (1990); Siegall et al.,
Semin. Cancer Biol. 1:345-50 (1990); Siegall et al., Cancer Res.
50:7786-8 (1990); FitzGerald et al., J. Cell Biol. 126(6):1533-41
(1995).
[0153] 2. Translocation to the Cytosol
[0154] To determine whether the translocation domain and the ER
retention domain of the chimeric protein properly functions, the
ability of the chimeric protein to gain access to the cytosol is
tested.
[0155] a) Presence in the Cytosol
[0156] In one method, access to the cytosol is determined by
detecting the physical presence of the chimeric protein in the
cytosol. For example, the chimeric protein can be labeled and the
chimeric protein exposed to the cell. Then, the cytosolic fraction
is isolated and the amount of label in the fraction determined.
Detecting label in the fraction indicates that the chimera has
gained access to the cytosol. This result can be compared with a
control, e.g., background noise or signal. If the detectable label
in the cytosolic fraction is at least twice background signal or
noise and more typically more than 10 to 100 times background,
then, this result indicates that the chimeric protein has gained
access to the cytosol.
[0157] b) ADP Ribosylation Activity
[0158] In another method, the ability of the translocation domain
and ER retention domain to effect translocation to the cytosol can
be tested with a construct containing a domain III having ADP
ribosylation activity. Briefly, cells are seeded in tissue culture
plates and exposed to the toxic version of the chimeric protein
containing the modified translocation domain or ER retention
sequence. ADP ribosylation activity is determined as a function of
inhibition of protein synthesis by, e.g., monitoring the
incorporation of .sup.3H-leucine. This method is further described
in detail in FitzGerald et al., J. Bio. Chem. 273:9951-9958 (1998).
The incorporation of .sup.3H-leucine in cells exposed the toxic
version of the chimeric protein can be compared to that of a
non-toxic counterpart or to background noise. If the incorporation
of .sup.3H-leucine in cells exposed the toxic version is reduced by
at least twice, more typically more than 10 to 100 times that of
the non-toxic counterpart (or compared to background noise), then
it can be said that the chimeric protein has properly gained entry
to the cytosol.
[0159] 3. Type IV Pilin Loop Adhesion
[0160] If the Type IV pilin sequence within the chimeric protein
has a structure that is exposed to a solvent and has near-native
conformation, the Type IV pilin loop sequence within the chimeric
protein should bind to, e.g., asialoGM1 receptors or other
receptors on epithelial cells and also compete with microorganisms
expressing the Type IV pilin loop sequence for binding to these
receptors. Therefore, whether or not the Type IV pilin loop
sequence is properly functioning within the chimeric protein is
tested by measuring its ability to adhere to epithelial cells or
its ability to block adherence of microorganisms expressing a Type
IV pilin loop sequence (e.g., P. aeruginosa) to epithelial cells.
These assays can be readily designed by one of skill in the
art.
[0161] As an example, if a Type IV pilin loop sequence is derived
from P. aeruginosa or Candida, an adhesion assay can be performed
with a substrate coated with asialoGM1. Various concentrations of
the chimeric protein comprising Type IV pilin sequence can be
assayed for reactivity with immobilized asialoGM1. To determine
specificity of this reactivity between the chimeric protein and
asialoGM1, a competition assay can be performed. For example,
soluble asialoGM1 can be added to interfere the chimeric protein
binding to immobilized asialoGM1. This method is described in
detail in the example section IIB3. This binding result can be
compared to a control (e.g., the same chimeric protein without the
pilin loop insert or with a scrambled pilin loop sequence insert).
If the amount of binding of the chimeric protein to immobilized
asialo GM1 is at least twice, typically about 10 to 100 times
greater than the control, then it can be said that the pilin loop
insert in the chimeric protein is functioning properly.
[0162] In another example, one can test the ability of the chimeric
protein to block binding of microorganisms expressing the Type IV
pilin loop sequence to epithelial cells. The selection of
epithelial cells depends on which microorganism Type IV pilin loop
sequence within the chimeric protein is derived from. For instance,
if the Type IV pilin loop sequence within the chimeric protein is
derived from V. cholera, then intestinal epithelial cells can be
used binding assays. If the Type IV pilin loop sequence within the
chimeric protein is derived from N. gonorrhoeae, then epithelial
cells of genital urinary system can be used for binding assays. If
the Type IV pilin loop sequence within the chimeric protein is
derived from P. aeruginosa, then lung epithelial cells can be used
for binding assays.
[0163] As an illustration, various Pseudomonas aeruginosa strains
that express Type IV pilin can be added different to the human lung
epithelial cell line, A549, which will result in the binding of
Pseudomonas aeruginosa to these cells. Then, the chimeric protein
can be added. If the Type IV pilin sequence within the chimeric
protein is present in near-native conformation, the chimeric
protein would compete with Pseudomonas aeruginosa binding and would
result in reduction of Pseudomonas aeruginosa adherence to the
epithelial cells. This method is described in detail in the example
section III below. The result from this competition assay can be
compared to the result obtained with a control (e.g., the same
chimeric protein except without the pilin loop insert or the same
chimeric protein with a scrambled pilin loop sequence insert). If
the chimeric protein can reduce Pseudomonas binding at least twice
or typically about 10 to 100 times better than the control, then it
can be said that the pilin loop insert in the chimeric protein is
functioning properly.
[0164] 4. Immunogenicity
[0165] To determine whether the chimeric protein retains its
immunogenicity respect to both parts of the chimeric protein (i.e.,
a Type IV pilin loop sequence and a non-toxic Pseudomonas exotoxin
A sequence), properties of the antisera raised against the chimeric
protein are tested.
[0166] a) Immunogenicity of Type IV Pilin Sequence
[0167] Immunogenicity of a Type IV pilin sequence within the
chimeric protein is tested by adhesion test using the antisera
raised against the chimeric protein. An animal, such as a mouse or
a rabbit, can be immunized with a composition comprising the
chimeric protein as described below in Example section IVA. The
post immunization antisera from the animal can be obtained and
prepared to determine if the antisera can inhibit binding of
microorganisms expressing the Type IV pilin sequence to the
epithelial cells. For example, Pseudomonas aeruginosa can be added
to the epithelial cells, and the amount of Pseudomonas binding to
the epithelial cells is determined. Then, the post immunization
antisera can be added to the epithelial cells to determine if
antisera reduce binding of Pseudomonas aeruginosa to the epithelial
cells. This assay is described in detail in Example section IVB. If
the pilin loop sequence within the chimeric protein is present in
near native conformation, then it is expected that antisera raised
against the chimeric protein (at a suitable dilution, e.g., 1:10 or
1:100) can reduce Pseudomonas binding by at least about 20%,
typically at least about 30%, more typically at least about
50%.
[0168] b) Toxin Neutralizing Response
[0169] Immunogenicity of a non-toxic Pseudomonas exotoxin A
sequence within the chimeric protein is tested by using antisera
raised against the chimeric protein. Specifically, post
immunization antisera is tested for its ability to neutralize
cytotoxicity of Pseudomonas exotoxin A. For example, one can test
the inhibition of protein synthesis of purified Pseudomonas
exotoxin A on eukaryotic cells in culture. When Pseudomonas
exotoxin A is added to eukaryotic cells, it reduces or prevents
protein synthesis in cells, causing cell cytotoxicity. To determine
if antisera can reduce or inactivate cell cytotoxicity of
Pseudomonas exotoxin A, Pseudomonas exotoxin A can be incubated
with antisera containing antibodies directed against the chimeric
protein. This incubated mixture is added to cells in culture. Then,
the effect of antisera on the protein synthesis in the cells can be
measured (e.g., monitoring the incorporation of [.sup.3H] leucine).
This assay is described in Example section IVC below and also in
Ogata et al., J. Biol. Chem. 265(33):20678-85 (1990). If the
non-toxic exotoxin A sequence within the chimeric protein is
present in near-native conformation, then it is expected that
antisera raised against the chimeric protein (at a suitable
dilution, e.g., 1:10 or 1:100) can reduce cytotoxicity of
Pseudomonas exotoxin A by at least about 30%, typically at least
about 50%, more typically at least about 70%, 80%, 90%, 95%, or 99%
compared to a control (e.g., addition of purified Pseudomonas
exotoxin A without antisera).
III. Compositions Comprising Chimeric Proteins or
Polynucleotides
[0170] The invention also provides formulations of one or more
chimeric polypeptide or polynucleotide compositions disclosed
herein in pharmaceutically-acceptable solutions for administration
to a cell or an animal, either alone or in combination with other
components.
[0171] A. Compositions Comprising Chimeric Proteins
[0172] The chimeric protein of the invention can be administered
directly to a subject as a pharmaceutical composition.
Administration is by any of the routes normally used for
introducing a chimeric protein into ultimate contact with the
tissue to be treated, preferably the mucosal membrane and
epithelial cells. The compositions comprising chimeric proteins are
administered in any suitable manner, preferably with
pharmaceutically acceptable carriers. Suitable methods of
administering such modulators are available and well known to those
of skill in the art. Although more than one route can be used to
administer a particular composition, a particular route can often
provide a more immediate and more effective reaction than another
route.
[0173] Pharmaceutical compositions comprising the chimeric proteins
of the invention may be formulated in conventional manner using one
or more physiologically acceptable carriers, diluents, excipients
or auxiliaries which facilitate processing of the polypeptides into
preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
[0174] Pharmaceutically acceptable carriers, diluents, or
excipients are determined in part by the particular composition
being administered, as well as by the particular method used to
administer the composition. Accordingly, there are a wide variety
of suitable formulations of pharmaceutical compositions of the
present invention. For example, pharmaceutical compositions can be
formulated for topical administration, systemic formulations,
injections, transmucosal administration, oral administration,
inhalation/nasal administration, rectal or vaginal administrations.
Suitable formulations for various administration methods are
described in, e.g., Remington's Pharmaceutical Sciences, 17.sup.th
ed. 1985.
[0175] Briefly, for topical administration, the proteins may be
formulated as solutions, gels, ointments, creams, suspensions, etc.
Systemic formulations include those designed for administration by
injection, e.g. subcutaneous, intravenous, intramuscular,
intrathecal or intraperitoneal injection, as well as those designed
for transdermal, transmucosal, oral or pulmonary administration.
For injection, the proteins may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hank's
solution, Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. For oral
administration, a composition can be readily formulated by
combining the chimeric proteins with pharmaceutically acceptable
carriers to enable the chimeric proteins to be formulated as
tablets, pills, capsules, liquids, gels, syrups, slurries,
suspensions and the like. For administration by inhalation, the
chimeric proteins for use according to the present invention are
conveniently delivered in the form of an aerosol spray from
pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
The proteins may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0176] Other suitable formulations and administration methods will
be readily apparent to one of skill in the art and can be applied
to the present invention.
[0177] B. Compositions Comprising Chimeric Polynucleotides
[0178] The invention also provides compositions comprising the
polynucleotides encoding the chimeric proteins (sometimes referred
to as "chimeric nucleic acids" or "chimeric polynucleotides").
These nucleic acids can be inserted into any of a number of
well-known vectors for the transfection of target cells or host
tissues. For example, nucleic acids are delivered as DNA plasmids,
naked nucleic acid, and nucleic acid complexed with a delivery
vehicle such as a liposome. Viral vector delivery systems include
DNA and RNA viruses, which have either episomal or integrated
genomes after delivery to the cell. For a review of gene therapy
procedures, see Anderson, Science 256:808-813 (1992); Nabel &
Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH
11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,
Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154
(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36
(1995); Kremer & Perricaudet, British Medical Bulletin
51(1):31-44 (1995); Haddada et al., in Current Topics in
Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu
et al., Gene Therapy 1:13-26 (1994).
[0179] Methods of non-viral delivery of nucleic acids include
lipofection, microinjection, biolistics, virosomes, liposomes,
immunoliposomes, polycation or lipid:nucleic acid conjugates, naked
DNA, artificial virions, and agent-enhanced uptake of DNA.
Lipofection is described in, e.g., U.S. Pat. No. 5,049,386, U.S.
Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355) and lipofection
reagents are sold commercially (e.g., Transfectam.TM. and
Lipofectin.TM.). Cationic and neutral lipids that are suitable for
efficient receptor-recognition lipofection of polynucleotides
include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be
to cells (ex vivo administration) or target tissues (in vivo
administration).
[0180] C. Vaccines
[0181] In some preferred embodiments of the present invention,
vaccines are provided. The vaccines will generally comprise one or
more pharmaceutical compositions, such as those discussed above, in
combination with an immunostimulant. An immunostimulant may be any
substance that enhances or potentiates an immune response (antibody
and/or cell-mediated) to an exogenous antigen. Examples of
immunostimulants include adjuvants, biodegradable microspheres
(e.g., polylactic galactide) and liposomes (into which the compound
is incorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877).
Vaccine preparation is generally described in, for example, Powell
& Newman, eds., Vaccine Design (the subunit and adjuvant
approach) (1995). Pharmaceutical compositions and vaccines within
the scope of the present invention may also contain other
compounds, which may be biologically active or inactive.
[0182] Any of a variety of immunostimulants may be employed in the
vaccines of this invention. For example, an adjuvant may be
included. Most adjuvants contain a substance designed to protect
the antigen from rapid catabolism, such as aluminum hydroxide or
mineral oil, and a stimulator of immune responses, such as lipid A.
Suitable adjuvants are commercially available as, for example,
Freund's Incomplete Adjuvant and Complete Adjuvant (Difco
Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and
Company, Inc., Rahway, N.J.); AS-2 and derivatives thereof
(SmithKline Beecham, Philadelphia, Pa.); CWS, TDM, Leif, aluminum
salts such as aluminum hydroxide gel (alum) or aluminum phosphate;
salts of calcium, iron or zinc; an insoluble suspension of acylated
tyrosine; acylated sugars; cationically or anionically derivatized
polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or
interleukin-2, -7, or -12, may also be used as adjuvants.
[0183] Any suitable carrier known in the art can be employed in the
vaccines of the invention, and the type of carrier will vary
depending on the mode of administration. The vaccines can be
formulated for any appropriate manner of administration, including
for example, topical, oral, nasal, intravenous, intracranial,
intraperitoneal, subcutaneous or intramuscular administration.
These formulations and administration methods are described above,
and will not be repeated in this section.
[0184] Pharmaceutical compositions and vaccines of the present
invention may be presented in unit-dose or multi-dose containers,
such as sealed vials. Such containers are preferably hermetically
sealed to preserve sterility of the formulation until use. In
general, formulations can be stored as suspensions, solutions or
emulsions in oily or aqueous vehicles. Alternatively, a
pharmaceutical composition or vaccine may be stored in a
freeze-dried condition requiring only the addition of a sterile
liquid carrier immediately prior to use.
[0185] D. Effective Dose
[0186] Determination of an effective amount of the chimeric protein
for inducing an immune response in a subject is well within the
capabilities of those skilled in the art, especially in light of
the detailed disclosure provided herein.
[0187] An effective dose can be estimated initially from in vitro
assays. For example, a dose can be formulated in animal models to
achieve an induction of an immune response using techniques that
are well known in the art. One having ordinary skill in the art
could readily optimize administration to humans based on animal
data. Dosage amount and interval may be adjusted individually. For
example, when used as a vaccine, the polypeptides and/or
polynucleotides of the invention may be administered in about 1 to
3 doses for a 1-36 week period. Preferably, 3 doses are
administered, at intervals of about 3-4 months, and booster
vaccinations may be given periodically thereafter. Alternate
protocols may be appropriate for individual patients. A suitable
dose is an amount of polypeptide or DNA that, when administered as
described above, is capable of raising an immune response in an
immunized patient sufficient to protect the patient from infections
by microorganisms expressing Type IV pilin sequence for at least
1-2 years. In general, the amount of polypeptide or nucleic acid
present in a dose (or produced in situ by the DNA in a dose) ranges
from about 1 pg to about 5 mg per kg host, typically from about 10
pg to about 1 mg, and preferably from about 100 pg to about 1
.mu.g. Suitable dose range will vary with the size of the patient,
but will typically range from about 0.1 mL to about 5 mL.
IV. Methods of Eliciting an Immune Response
[0188] The chimeric proteins of the invention are useful in
eliciting an immune response in a host. Eliciting a humoral immune
response is useful in the production of antibodies that
specifically recognize the Type IV pilin loop sequence or the
non-toxic exotoxin A sequence and in immunization against
microorganisms that bear the Type IV pilin sequence.
[0189] A. Prophylactic and Therapeutic Treatments
[0190] The chimeric proteins can include the Type IV pilin loop
sequences from various pathogenic microorganisms, including
Pseudomonas aeruginosa, Neisseria meningitides, Neisseria
gonorrhoeae, Vibro cholera, etc. Accordingly, this invention
provides prophylactic and therapeutic treatments for diseases
involving the pathological activity of pathogens bearing the Type
IV pilin loop sequences. The methods involve immunizing a subject
with non-toxic Pseudomonas exotoxin A based chimeric proteins
bearing the Type IV pilin sequence. The resulting immune responses
mount an attack against the pathogens, themselves. For example, if
the pathology results from bacterial or yeast infection, the immune
system mounts a response against the pathogens.
[0191] B. Humoral Immune Response
[0192] The chimeric proteins are useful in eliciting the production
of antibodies against the Type IV loop pilin sequence and the
non-toxic Pseudomonas exotoxin A sequence by a subject. The
chimeric proteins are attractive immunogens for making antibodies
against the Type IV pilin loop sequences that naturally occur
within a cysteine-cysteine loop: Because they contain the Type IV
pilin loop sequences within a cysteine-cysteine loop, they present
the Type IV pilin loop sequence to the immune system in near-native
conformation. The resulting antibodies generally recognize the
native antigen better than those raised against linearized versions
of the Type IV pilin sequence.
[0193] Methods for producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a
purified polypeptide, a polypeptide coupled to an appropriate
carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a
polypeptide incorporated into an immunization vector, such as a
recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed
with an adjuvant. Animals are immunized with the mixture. An
animal's immune response to the immunogenic preparation is
monitored by taking test bleeds and determining the titer of
reactivity to the polypeptide of interest. When appropriately high
titers of antibody to the immunogen are obtained, blood is
collected from the animal and antisera are prepared. Further
fractionation of the antisera to enrich for antibodies reactive to
the polypeptide is performed where desired. See, e.g., Coligan,
Current Protocols in Immunology Wiley/Greene, NY (1991); and Harlow
and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Press,
NY (1989).
[0194] In various embodiments, the antibodies ultimately produced
can be monoclonal antibodies, humanized antibodies, chimeric
antibodies or antibody fragments.
[0195] Monoclonal antibodies are prepared from cells secreting the
desired antibody. These antibodies are screened for binding to
polypeptides comprising the epitope, or screened for agonistic or
antagonistic activity, e.g., activity mediated through the agent
comprising the non-native epitope. In some instances, it is
desirable to prepare monoclonal antibodies from various mammalian
hosts, such as mice, rodents, primates, humans, etc. Description of
techniques for preparing such monoclonal antibodies are found in,
e.g., Stites et al. (eds.) Basic and Clinical Immunology (4th ed.)
Lange Medical Publications, Los Altos, Calif., and references cited
therein; Harlow and Lane, Supra; Goding (1986) Monoclonal
Antibodies: Principles and Practice (2d ed.) Academic Press, New
York, N.Y.; and Kohler and Milstein (1975) Nature 256: 495-497.
[0196] In another embodiment, the antibodies are humanized
immunoglobulins. Humanized antibodies are made by linking the CDR
regions of non-human antibodies to human constant regions by
recombinant DNA techniques. See Queen et al., U.S. Pat. No.
5,585,089.
[0197] In another embodiment of the invention, fragments of
antibodies against the Type IV pilin loop sequence are provided.
Typically, these fragments exhibit specific binding to the Type IV
pilin loop sequence similar to that of a complete immunoglobulin.
Antibody fragments include separate heavy chains, light chains,
Fab, Fab'F(ab').sub.2 and Fv. Fragments are produced by recombinant
DNA techniques, or by enzymatic or chemical separation of intact
immunoglobulins.
[0198] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al., Science 246: 1275-1281 (1989); and Ward et al., Nature 341:
544-546 (1989).
[0199] An approach for isolating DNA sequences which encode a human
monoclonal antibody or a binding fragment thereof is by screening a
DNA library from human B cells according to the general protocol
outlined by Huse et al., Science 246:1275-1281 (1989) and then
cloning and amplifying the sequences which encode the antibody (or
binding fragment) of the desired specificity. The protocol
described by Huse is rendered more efficient in combination with
phage display technology. See, e.g., Dower et al., WO 91/17271 and
McCafferty et al., WO 92/01047. Phage display technology can also
be used to mutagenize CDR regions of antibodies previously shown to
have affinity for the polypeptides of this invention or their
ligands. Antibodies having improved binding affinity are
selected.
[0200] The antibodies of this invention are useful for affinity
chromatography in isolating agents bearing the Type IV pilin
sequence. Columns are prepared, e.g., with the antibodies linked to
a solid support, e.g., particles, such as agarose, Sephadex, or the
like, where a cell lysate is passed through the column, washed, and
treated with increasing concentrations of a mild denaturant,
whereby purified agents are released.
[0201] As described in the Example section, sera from immunized
rabbits had two reactivities: one that blocks adhesion and one that
neutralizes exotoxin A. Therefore, by introducing the chimeric
protein as a composition (e.g., a vaccine) into a subject,
antibodies that prevent colonization of microorganisms bearing Type
IV pilin sequences (e.g., Pseudomonas aeruginosa) can be provided
in the subject. In particular for Pseudomonas aeruginosa, should
small numbers of these bacteria overcome this defense, the normal
destructive power of the exotoxin A will be also neutralized by the
antisera.
[0202] C. IgA-mediated Secretory Immune Response
[0203] Mucosal membranes are primary entryways for many infectious
pathogens, including those bearing the Type IV pilin sequences.
Mucosal membranes include, e.g., the mouth, nose, throat, lung,
vagina, rectum and colon. As a defense against entry, the body
secretes secretory IgA on the surfaces of mucosal epithelial
membranes against pathogens. Furthermore, antigens presented at one
mucosal surface can trigger responses at other mucosal surfaces due
to trafficking of antibody-secreting cells between these mucosae.
The structure of secretory IgA has been suggested to be crucial for
its sustained residence and effective function at the luminal
surface of a mucosa. As used herein, "secretory IgA" or "sIgA"
refers to a polymeric molecule comprising two IgA immunoglobulins
joined by a J chain and further bound to a secretory component.
While mucosal administration of antigens can generate an IgG
response, parenteral administration of immunogens rarely produce
strong sIgA responses.
[0204] Pseudomonas exotoxin binds to receptors on mucosal
membranes. Therefore, the chimeric proteins comprising non-toxic
exotoxin A sequences are an attractive vector for bringing the type
IV pilin loop sequence to a mucosal surface. There, the chimeric
proteins elicit an IgA-mediated immune response against the
chimeric proteins. Accordingly, this invention provides the
non-toxic Pseudomonas exotoxin A-based chimeric proteins comprising
a Type IV pilin loop sequence from a pathogen that gains entry
through mucosal membranes. The cell recognition domain can be
targeted to any mucosal surface receptor. These chimeric proteins
are useful for eliciting an IgA-mediated secretory immune response
against immunogens that gain entry to the body through mucosal
surfaces. The chimeric proteins used for this purpose should have
ligands that bind to receptors on mucosal membranes as their cell
recognition domains. For example, epidermal growth factor binds to
the epidermal growth factor receptor on mucosal surfaces.
[0205] The chimeric proteins can be applied to the mucosal surface
by any of the typical means, including pharmaceutical compositions
in the form of liquids or solids, e.g., sprays, ointments,
suppositories or erodible polymers impregnated with the immunogen.
Administration can involve applying the immunogen to a plurality of
different mucosal surfaces in a series of immunizations, e.g., as
booster immunizations. A booster inoculation can also be
administered parenterally, e.g., subcutaneously. The chimeric
protein can be administered in doses of about 1 .mu.g to 1000
.mu.g, e.g., about 10 .mu.g to 100 .mu.g.
[0206] The IgA response is strongest on mucosal surfaces exposed to
the immunogen. Therefore, in one embodiment, the immunogen is
applied to a mucosal surface that is likely to be a site of
exposure to the particular pathogen. Accordingly, depending on the
site of exposure to the particular pathogen, the chimeric proteins
can be administered to the lung, nasal mucosa, vaginal, anal or
oral mucosal surfaces, or they can be given as an oral medication.
For example, for cystic fibrosis patients, the chimeric proteins
can be administered to the lung.
[0207] Mucosal administration of the chimeric protein of this
invention result in strong memory responses, both for IgA and IgG.
Therefore, in vaccination with them, it is useful to provide
booster doses either mucosally or parenterally. The memory response
can be elicited by administering a booster dose more than a year
after the initial dose. For example, a booster dose can be
administered about 12, about 16, about 20 or about 24 months after
the initial dose.
[0208] The potential value of a Pseudomonas vaccine relates in part
to its ability to protect individuals broadly from the strains that
are present in the environment. Based on the length of the pilin
loop insert, there are two groupings for Ps. aeruginosa: one group
with a 12 amino acid sequence and one with a 17 amino acid insert.
Both loops apparently bind asialo-GM1 and are thought to exhibit
similar structures. Reflecting this, we note that our vaccine
protein, containing a 12 amino acid loop from the PAK strain, was
able to generate antibodies that were reactive not only for strains
with the shorter loop but also for the SBI-N strain, which
displayed the longer loop. Our studies have also provided
additional sequence data for pilin and pilin loop sequences. We
report here two pilin loop sequences (those for Ps. aeruginosa
strain 1071 and Ps. aeruginosa strain SBI-N) that have not
previously been entered in databases (Tables 1 and 2).
[0209] Chronic pulmonary colonization by Ps. aeruginosa is
associated with a decline in the clinical course of CF patients.
Frequently, antibiotic therapy, even via pulmonary delivery, fails
to eradicate Ps. aeruginosa infections in these patients
(Steinkamp, G., B. et al. Pediatr Pulmonol 6(2):91-8(1989)).
Controlling Ps. aeruginosa infections, or better yet, preventing
them, has thus become a critical unmet medical need in the care of
CF patients ((Bauernfeind, A. et al. Behring Inst Mitt (98):256-61
(1997)). To address this, a number of vaccine approaches have been
explored, many focused on outer membrane constituents
(Matthews-Greer, J. M., et al.; J Infect Dis 155(6):1282-91 (1987);
Owen, P. Biochem Soc Trans 20(1):1-6 (1992); Sawa, et al.; Nat Med
5(4):392-8(1999), some on toxins (Chen, T. Y., et al. J Biomed Sci
6(5):357-63 (1999); Denis-Mize, K. S., et al.; FEMS Immunol Med
Microbiol 27(2):147-54 (2000); Gilleland, H. E., et al.; J Med
Microbiol 38(2):79-86 (1993); Matsumoto, et al.; J Med Microbiol
47(4):303-8 (1988)). and some on a combination approach (Cryz, S.
J., et al.; Antibiot Chemother 39:249-55 (1987); Cryz, S. J., et
al. Infect Immun 52(1):161-5 (1986); Cryz, S. J., et al.; Infect
Immun 55(7):1547-51 (1987); and Cryz, S. J., et al. J Infect Dis
154(4):682-8 (1986) (Johansen, H. K., et al.; APMIS
102(7):545-53(1994).
[0210] The compositions of the present invention in some
embodiments are used to treat persons at risk of infection and
particularly, Pseudomonas aeruginosa infection. These persons
include, in particular, hospitalized patients having cystic
fibrosis, burn wounds, organ transplants, compromised immune
function, or intravenous-drug addition.
[0211] Previously, we compared the subcutaneous route with mucosal
delivery of toxin-V3 loop proteins (Mrsny, R. et al., Vaccine
17(11-12):1425-33 (1999). Results of mucosal vaccination indicated
that a robust anti-V3 loop response could be achieved with high
titer responses of both serum IgG and secretory IgA antibodies.
Because the toxin-pilin chimeric protein is a candidate vaccine to
prevent Pseudomonas colonization in CF, one embodiment provides a
vaccine delivered to target mucosal antibody responses at airway
epithelia.
EXAMPLES
I. Construction of Plasmids
[0212] Four plasmids, pPE64, pPE64.DELTA.553, pPE64pil,
pPE64.DELTA.553pil, were constructed. Plasmid pPE64 encodes native
the Pseudomonas exotoxin A, except the plasmid encoded a slightly
smaller version of PE that lacked much of domain lb and has a novel
PstI site in domain lb as described in detail below. Plasmid
pPE64.DELTA.553 encodes the a non-toxic version of plasmid pPE64,
whereby the plasmid pPE64 was modified by subcloning to introduce
the enzymatically inactive domain III of PE (i.e., Glu at amino
acid position 553 is deleted). To generate a PE-based pilin
chimeric protein, an oligonucleotide duplex that encoded amino
acids 129-142 from the PAK strain of pilin was synthesized. Then
plasmid pPE64pil is constructed based on plasmid pPE64, wherein a
pilin loop sequence from P. aeruginosa PAK strain was inserted into
the PstI site of plasmid pPE64. Plasmid pPE64.DELTA.553pil is
constructed based on plasmid pPE64.DELTA.553, wherein a pilin loop
sequence from P. aeruginosa PAK strain was inserted into the PstI
site of plasmid pPE64.DELTA.553. All of these vectors were
constructed without a bacterial secretion sequence which allowed
recombinant proteins to be expressed as inclusion bodies.
[0213] Specifically, plasmids pPE64 and pPE64.DELTA.553 are
constructed as follows. Plasmid pMOA1A2VK352 (Ogata et al., J.
Biol. Chem. 267, 25396-401 (1992)), encoding PE, was digested with
Sfi1 and ApaI (residues 1143 and 1275, respectively) and then
re-ligated with a duplex containing a novel PstI site. The coding
strand of the duplex had the following sequence: 5'-tggccctgac
cctggccgcc gccgagagcg agcgcttcgt ccggcagggc accggcaacg acgaggccgg
cgcggcaaac ctgcagggcc-3'. The resulting plasmid encoded a slightly
smaller version of PE and lacked much of domain lb. The PstI site
was then used to introduce duplexes encoding pilin loop sequences
flanked by cysteine residues. To make non-toxic proteins, vectors
were modified by the subcloning in an enzymatically inactive domain
III from pVC45.DELTA.E553. An additional subcloning, from pJH4
(Hwang et. al., Cell 48:129-136 (1987)), was needed to produce a
vector that lacked a signal sequence. Construction of plasmids
pPE64 and pPE64.DELTA.553 are also described in FitzGerald et al.,
J. Biol. Chem. 273(16):9951-8 (1998).
[0214] Plasmids pPE64pil and pPE64.DELTA.553pil with a pilin loop
sequence insert were constructed based on plasmids pPE64 and
pPE64.DELTA.553, respectively. A 54 bp sense oligonucleotide with
cohesive ends for PstI and encoding the 12 amino acid pilin loop of
the PAK strain, was annealed with a 54 bp antisense oligonucleotide
in 10 mM Tris/HCl, 50 mM NaCl pH 7.4. The sense and antisense
oligonucleotides had the following sequences: Sense
5'-TTGTACTAGTGATCAGGATGAACAGTTTATTCCGAAAGGTTGTTCACGTATGCA-3';
Antisense
5'-TACGTGAACAACCTTTCGGAATAAACTGTTCATCCTGATCACTAGTACAATGCA-3'.
Annealing was accomplished by heating to 94.degree. C. for 5 min
followed by cooling to 25.degree. C. over a period of 40 min.
Plasmids pPE64 and pPE64.DELTA.5532), encoding enzymatically active
and inactive PE respectively, were digested with PstI at residue
1470. (FitzGerald, D. J., et al., J Biol Chem 273(16):9951-8
(1998). Ligation with the phosphorylated pilin oligoduplex
destroyed the PstI site and introduced a unique SpeI site. A
XhoI/SpeI double digest was used to check for the correct
orientation of the insert. Ligation of the pilin oligoduplex into
the PstI-cut vector was followed by several characterization steps
to confirm the presence of the pilin insert in the correct
orientation. Final constructs were verified by dideoxy double
strand sequencing.
II. Expression and Characterization of Proteins
[0215] A. Expression and Purification
[0216] Using the T7 expression system described by Studier et al.,
(Methods Enzymol. 185:60-89 (1990)), four PE-related proteins were
expressed E. coli. These included PE64, PE64.DELTA.553, PE64pil and
PE64.DELTA.553pil.
[0217] Chimeric proteins were expressed and isolated as inclusion
bodies as described in Buchner et al., Anal. Biochem. 205(2):263-70
(1992). Each protein was expressed separately and purified to near
homogeneity. Briefly, strain BL21(.lamda.DE3) was transformed with
plasmids harboring a T7 promoter upstream of the initial ATG of the
toxin-expressing vectors. Cultures were grown in Superbroth (KD
Medical, Bethesda, Md.) with ampicillin (50 ug/ml) and then induced
for protein expression by the addition of IPTG (1 mM). After two
hours of further culture, bacterial cells were harvested by
centrifugation. Following cell lysis, expressed proteins were
recovered in inclusion bodies.
[0218] Proteins were solubilized with Guanidine HCl (6.0 M), 2 mM
EDTA pH 8.0 plus dithioerythreitol (65 mM). Solubilized proteins
were then refolded by dilution into a redox shuffling buffer
(Buchner et al., Anal. Biochem. 205(2):263-70 (1992). Refolded
proteins were dialyzed against a 20 mM Tris, 100 mM urea pH 7.4,
adsorbed on Q Sepharose (Amersham Pharmacia Biotech), washed with
150 mM NaCl, 20 mM Tris, 1 mM EDTA pH 6.5 and eluted with 280 mM
NaCl, 20 mM Tris, 1 mM EDTA. Eluted proteins were diluted 5-fold
and then adsorbed onto a MonoQ column (HR 10/10, Amersham Pharmacia
Biotech) and further purified by the application of a linear salt
gradient (0-0.4 M NaCl in Tris EDTA, pH 7.4). PE proteins eluted
between 0.2 and 0.25 M NaCl. Final purification was achieved using
a gel filtration column (Superdex 200, Amersham Pharmacia Biotech)
in PBS, pH 7.4.
[0219] B. Characterization of Proteins
[0220] 1. Western Blot Analysis
[0221] The PK99H mouse monoclonal antibody and purified pilin
proteins were obtained from Dr. Randall Irvin, University of
Alberta, Canada. Antimouse IgG and antirabbit IgG antibodies were
used to detect primary antibodies in Western blots and ELISAs
(available from Jackson Immuno Research Lab, West Grove, Pa.).
[0222] Proteins were initially analyzed by SDS-PAGE (FIG. 2 A).
Substantially pure proteins were obtained using the purification
scheme outlined above. In Western blot analysis the PE64pil and
PE64.DELTA.553pil proteins reacted with PK99H, a monoclonal
antibody to the C-terminal loop of pilin (FIG. 2 B). The same
antibody also reacted with soluble preparations of the same
proteins, indicating that the pilin insert was exposed on the
surface of the PE-pilin chimeric protein. PE proteins without
inserts did not react with the PK99H antibody (FIG. 2 B).
[0223] 2. Cytotoxicity Assay
[0224] To investigate the influence of the pilin insert on toxin
structure and function, the two enzymatically active proteins, PE64
and PE64pil, were compared in a cytotoxicity assay. Cytotoxicity
assay methods described in Ogata et al., J. Biol. Chem.
265(33):20678-85 (1990) was used. Concentrations of PE64 or PE64pil
ranging from 0.002-20 ng/ml were added to L929 cells for an
overnight incubation. Cytotoxicity was then determined by measuring
the inhibition of cellular protein synthesis (e.g., monitoring the
incorporation of .sup.3H-leucine). Data indicated that PE64 and
PE64pil exhibited similar toxicities with IC.sub.50 values in the
range of 0.1 ng/ml for both proteins (FIG. 3). This result
suggested that the insert of 14 amino acids did not unduly perturb
toxin function and, by inference, toxin structure.
[0225] 3. Reactivity with Immobilized Asialo-GM1
[0226] Previous results had indicated that synthetic peptides
derived from the C-terminus of pilin could block the binding of
pili to epithelial cells (Irvin et al., Infect. Immun.
57(12):3720-6 (1989); Yu, L. et al., Mol Microbiol 19(5):1107-16
(1996)). Blocking was attributed to peptide binding to asialo-GM1
on the surface of epithelial cells. To test the functionality of
the pilin insert in the PE64 proteins, various concentrations of
PE64pil were assayed for reactivity with immobilized
asialo-GM1.
[0227] 96-well plates were coated with asialo-GM1 or monosialo-GM1
(Sigma Chem Co, St Louis, Mo.) that had been solubilized in
methanol. A 100 .mu.l solution of ganglioside (5 .mu.g/ml) was
added to each well and evaporated at 4.degree. C. until dry. Wells
were washed 3 times with PBS and blocked with Fish-gelatin-PBS
(BioFX, Randallstown, USA) for 16 h at 4.degree. C. Test proteins
in blocking buffer were added at various concentrations. After
incubation for 1 h at 22.degree. C., the supernatant was removed
and bound protein was detected using heat-inactivated anti
PE64.DELTA.553pil serum (1:100) as the primary antibody. For
competition studies, proteins at 0.2 ug/ml were incubated with 2
ug/ml of asialo-GM1 or monosialo-GM1 for 30 min at room
temperature. Samples were then added to asialo-GM1 coated plates as
above.
[0228] Increasing concentrations of PE64pil from 0.1-2.0 ug/ml
reacted specifically with immobilized asialo-GM1 (FIG. 4A). PE64
was used as a control and exhibited only a low level of binding
(FIG. 4A). Additional studies were carried out to confirm the
ganglioside specificity of both PE64pil and PE64.DELTA.553pil.
Soluble asialo-GM1 reduced the binding of PE64pil and
PE64.DELTA.553pil to immobilized asialo-GM1 while the addition of
monosialo-GM1 did not (FIGS. 4B and 4C). Neither ganglioside
interfered with the low level binding of PE64 and PE64.DELTA.553
(FIGS. 4B and 4C). Taken together, these results confirmed not only
the presence of reactive pilin sequences but revealed a
gain-of-function for the PE64pil proteins.
III. Adhesion Assays
[0229] A. Pseudomonas Strains
[0230] The following strains of Pseudomonas were used in adhesion
and other assays: PAK, PAO1, SBN-1, 1071, M2, 82932, 82935 and
90063. Pseudomonas strains used for adherence studies were grown on
LB agar and then in M9 minimal medium (KD Medical, Bethesda, Md.)
supplemented with 0.4% glucose at 30.degree. C. without shaking.
Cultures in late log phase were routinely used for adhesion
assays.
[0231] B. Cell Cultures
[0232] A549 (ATCC, CCL-185), L929 (ATCC, CCL-1), WI 38, Vero and
CHO cells were maintained in DMEM/F12 or RMPI 1640 supplemented
with 10% fetal bovine serum (FBS), 2.5 mM glutamine, standard
Penicillin/Streptomycin (100 U/100 ug/ml, GibcoBRL, Grand Island,
USA) (further designed as complete medium) in 5% CO.sub.2 at
37.degree. C. Cells were fed every 2 to 3 days and passaged every 5
to 7 days. For assays, cells were seeded into 24-well or 96-well
plates and grown to confluence.
[0233] C. Quantification of Bacterial Adherence
[0234] To quantify the association of Pseudomonas with A549 cells,
we followed the adhesion assay described by Chi et al., Infect.
Immun. 59(3):822-8 (1991). Briefly, A549 cells were grown in a 24
well plates (antibiotic free medium), to a density of approximately
2.times.10.sup.4 cells per well. Cells were washed three times in
HBSS without serum and were overlayed with 0.5 ml of DMEM/F12
complete medium without FBS. A MOI of 20 was achieved by adding 10
.mu.l of an appropriate bacterial dilution. Plates were incubated
for 1 or 2 h at 37.degree. C., 5% CO.sub.2.
[0235] To remove unbound bacteria, cells were gently washed three
times with HBSS. Cells were then fixed for 1 h in 3.7%
paraformaldehyde, 200 mM HEPES, pH 7.2. Cells were washed twice
with saline and stained with 10% Giemsa for 10 min. Samples were
washed three times with water and examined under light microscopy
at 400.times. magnification. Adherent bacteria were quantified by
counting the cell-associated bacteria of one hundred A549
cells.
[0236] D. Results
[0237] Pilin-mediated adhesion to epithelial cells allows P.
aeruginosa to initiate an infection. Agents that block adherence
will therefore reduce the bacterial burden. The following three
peptides were synthesized: a long C-terminal peptide (peptide 1:
acetyl-KCTSDQDEQFIPKGCSK-NH.sub.2) corresponding to amino acids
128-142 of the PAK strain (this peptide was oxidized to allow the
formation of a disulfide bond), a core peptide (peptide 2:
acetyl-DEQFIPK-NH.sub.2) corresponding to amino acids 134-140 and a
scrambled peptide (peptide 3: acetyl-QIDPEFK-NH.sub.2) having the
same amino acid composition as the core but in a jumbled sequence.
To enhance stability, the N-termini of these synthetic peptides
were acetylated while the C-termini were amidated. These peptides
were custom synthesized by Sigma Genosys. The same peptides were
also synthesized with a biotin label.
[0238] To test these peptides functionally, an adhesion assay was
devised whereby washed bacteria of P. aeruginosa PAK strain were
added to the human lung epithelial cell line, A549. Specifically,
cultures of confluent A549 cells were incubated 60 min at
37.degree. C. with 40 .mu.M peptide 1, 40 .mu.M peptide 2, 40 .mu.M
peptide 3, 2 nmol/ml PAK-pilin protein, 2 nmol/ml PE64, 2 nmol/ml
PE64.DELTA.553, 2 nmol/ml PE64pil, 2 nmol/ml PE64.DELTA.553pil and
4 nmol/ml bovine albumin. Washed once with prewarmed DMEM and P.
aeruginosa PAK strain was added at a MOI of approximately 50 in
DMEM, 2% FBS. Bacteria were centrifuged onto the cells (700 g, 5
min) and incubated 60 min, 37.degree. C. 5% CO.sub.2. Adherence was
determined as described above.
[0239] The results were as follows. Adherence to A549 cells was
reduced by approximately 50% in the presence of 40 .mu.M of the
long or the core pilin peptide (see FIG. 5). The scrambled peptide
did not interfere with adherence.
[0240] Because the PE-pilin proteins had exhibited binding activity
to asialo-GM1, these were also tested. At approximately the same
molar concentration as the synthetic peptides, PE64pil and
PE64.DELTA.553pil also blocked bacterial adherence. Effects were
due to the presence of the insert, because toxin molecules without
insert failed to compete for adherence.
IV. Immune Response to PE64.DELTA.553pil
[0241] A. Production of Polyclonal Antibodies
[0242] To test the ability of the toxin-pilin protein to generate
relevant antibody responses, four rabbits were injected with the
PE64.DELTA.553pil protein. Two rabbits (numbered 87 and 88)
received the protein plus adjuvant (complete Freunds for the first
injection followed by incomplete Freunds for subsequent injections)
and two (numbered 89 and 90) received the protein alone. Two
hundred micrograms of protein per injection was given
subcutaneously for a total of four cycles spaced approximately 2
weeks apart. About 12 ml serum was isolated biweekly from each
rabbit. The sera were heat inactivated to 20 min, 56.degree. C. and
dilutions thereof were used for assays without further
purification. Anti-pilin titers were determined using an ELISA
assay where biotinylated pilin peptides were immobilized on
strepavidin coated plates. Over the period of immunization,
anti-pilin titers increased in all four animals (FIG. 6). However,
the speed and extent of the response were greater in the two
rabbits that received antigen plus adjuvant. To avoid
complement-mediated bacterial killing, immune sera were heat
inactivated. This treatment did not significantly alter antibody
titers in the ELISA assay (data not shown).
[0243] B. Inhibition of Adhesion by Post Immunization Sera
[0244] To assess antibody mediated inhibition of adherence,
anti-PE64.DELTA.553pil rabbit sera were incubated at dilutions from
1:20 to 1:100 with 4.times.10.sup.5 bacteria at 22.degree. C. for
30 min. Bacteria were then centrifuged, resuspended in DMEM without
supplements and added to confluent monolayers of A549 cells at a
MOI of 20 for 1-2 hrs. Adherence was determined as described above.
Immune sera taken after the fourth injection were compared to
prebleed samples taken from the same rabbits.
[0245] 1. Inhibition of P. aeruginosa (AK Strain)
[0246] Sera taken 2 weeks after the last injection were assayed for
blocking activity in the bacterial adherence assay. Compared to
prebleeds, immune sera at various dilutions blocked adherence of
the PAK strain of Ps. aeruginosa (FIG. 7A). Reduction of adherence
ranged from 60% at a dilution of 1:100 to 90% at a dilution of
1:20. At a dilution of 1:20, blocking activity was comparable
without regard to the presence of adjuvant in the antigen
preparation (FIG. 7B).
[0247] 2. Inhibition of P. aeruginosa (Various Strains)
[0248] Inhibition of PAK strain adhesion confirmed that rabbits
responded to the specific pilin sequence that was administered in
the vaccine. However, because the C-terminal loop of pilin exhibits
considerable sequence variation, it was important to determine the
reactivity of the immune sera for other strains of Ps. aeruginosa.
Strains PAO1, 1071, SBI-N, 82935, 82932, 90063 1244 and M2 were
tested for adherence to A549 cells under similar conditions as the
PAK strain. The specific cell binding of all strains were reduced
in adhesion when heat inactivated immune rabbit sera were mixed
with bacteria at a 1:20 dilution (FIG. 7C). The reduction in
adhesion among the different strains was more or less in the range
of the PAK strain (about 90% reduction).
[0249] While it was unlikely that each of the above strains
expressed the same loop sequence as the PAK strain, it was of
interest to analyze variations at this portion of the pilin gene.
Pilin sequences were determined by generating PCR clones of each
strain's pilin gene and sequencing these. Primers for amplification
were from the 5' end of the pilin gene and the 3' end of the
neighboring gene (Nicotinate-nucleotide pyrophosphorylase) in the
Pseudomonas genome (to be described in greater detail elsewhere).
Results revealed the following: most strains exhibited a 12 amino
acid loop while one, SBI-N, had a 17 amino acid loop. Strains 82932
and 82935 had the same loop sequence as KB7 (accession No, Q53391)
and 90063 had a loop that matched PAO1 (accession No, A25023).
Strains 1071 and SBI-N exhibited loops with novel sequences (See
Tables 1 and 2). Strain M2, a mouse isolate, was not sequenced.
[0250] B. Toxin Neutralizing Response
[0251] The inhibition of protein synthesis of purified PE64 and
PE64pil on eukaryotic cells in culture was determined as described
in Ogata et al., J. Biol. Chem. 265(33):20678-85 (1990). For
inactivating cytotoxic activity, the PE64pil proteins were
incubated 30 min at 22.degree. C. with rabbit sera, containing anti
PE64.DELTA.553pil antibodies, prior they were added to L929 or A549
cells in 24 well tissue culture dishes.
[0252] Rabbit antisera were evaluated for toxin neutralizing
activity. All four of the immunized rabbits at a 1:20 dilution of
sera neutralized 1.0 .mu.g/ml of toxin completely (FIG. 8). From
these results, it was concluded that PE-pilin vaccine can generate
antibodies of two reactivities: one that blocks adhesion and one
that neutralizes the exotoxin.
[0253] The present invention provides novel materials and methods
for chimeric proteins comprising a non-toxic Pseudomonas exotoxin A
and a Type IV pilin loop sequence. While specific examples have
been provided, the above description is illustrative and not
restrictive. Any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
Furthermore, many variations of the invention will become apparent
to those skilled in the art upon review of the specification. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
[0254] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their invention.
Sequence CWU 1
1
36 1 1839 DNA Pseudomonas aeruginosa CDS (1)..(1839) mature form of
Exotoxin A 1 gcc gaa gaa gct ttc gac ctc tgg aac gaa tgc gcc aaa
gcc tgc gtg 48 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys
Ala Cys Val 1 5 10 15 ctc gac ctc aag gac ggc gtg cgt tcc agc cgc
atg agc gtc gac ccg 96 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg
Met Ser Val Asp Pro 20 25 30 gcc atc gcc gac acc aac ggc cag ggc
gtg ctg cac tac tcc atg gtc 144 Ala Ile Ala Asp Thr Asn Gly Gln Gly
Val Leu His Tyr Ser Met Val 35 40 45 ctg gag ggc ggc aac gac gcg
ctc aag ctg gcc atc gac aac gcc ctc 192 Leu Glu Gly Gly Asn Asp Ala
Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60 agc atc acc agc gac
ggc ctg acc atc cgc ctc gaa ggc ggc gtc gag 240 Ser Ile Thr Ser Asp
Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65 70 75 80 ccg aac aag
ccg gtg cgc tac agc tac acg cgc cag gcg cgc ggc agt 288 Pro Asn Lys
Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 tgg
tcg ctg aac tgg ctg gta ccg atc ggc cac gag aag ccc tcg aac 336 Trp
Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105
110 atc aag gtg ttc atc cac gaa ctg aac gcc ggc aac cag ctc agc cac
384 Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His
115 120 125 atg tcg ccg atc tac acc atc gag atg ggc gac gag ttg ctg
gcg aag 432 Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu
Ala Lys 130 135 140 ctg gcg cgc gat gcc acc ttc ttc gtc agg gcg cac
gag agc aac gag 480 Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His
Glu Ser Asn Glu 145 150 155 160 atg cag ccg acg ctc gcc atc agc cat
gcc ggg gtc agc gtg gtc atg 528 Met Gln Pro Thr Leu Ala Ile Ser His
Ala Gly Val Ser Val Val Met 165 170 175 gcc cag acc cag ccg cgc cgg
gaa aag cgc tgg agc gaa tgg gcc agc 576 Ala Gln Thr Gln Pro Arg Arg
Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 ggc aag gtg ttg tgc
ctg ctc gac ccg ctg gac ggg gtc tac aac tac 624 Gly Lys Val Leu Cys
Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205 ctc gcc cag
caa cgc tgc aac ctc gac gat acc tgg gaa ggc aag atc 672 Leu Ala Gln
Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220 tac
cgg gtg ctc gcc ggc aac ccg gcg aag cat gac ctg gac atc aaa 720 Tyr
Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys 225 230
235 240 ccc acg gtc atc agt cat cgc ctg cac ttt ccc gag ggc ggc agc
ctg 768 Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser
Leu 245 250 255 gcc gcg ctg acc gcg cac cag gct tgc cac ctg ccg ctg
gag act ttc 816 Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu
Glu Thr Phe 260 265 270 acc cgt cat cgc cag ccg cgc ggc tgg gaa caa
ctg gag cag tgc ggc 864 Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln
Leu Glu Gln Cys Gly 275 280 285 tat ccg gtg cag cgg ctg gtc gcc ctc
tac ctg gcg gcg cgg ctg tcg 912 Tyr Pro Val Gln Arg Leu Val Ala Leu
Tyr Leu Ala Ala Arg Leu Ser 290 295 300 tgg aac cag gtc gac cag gtg
atc cgc aac gcc ctg gcc agc ccc ggc 960 Trp Asn Gln Val Asp Gln Val
Ile Arg Asn Ala Leu Ala Ser Pro Gly 305 310 315 320 agc ggc ggc gac
ctg ggc gaa gcg atc cgc gag cag ccg gag cag gcc 1008 Ser Gly Gly
Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala 325 330 335 cgt
ctg gcc ctg acc ctg gcc gcc gcc gag agc gag cgc ttc gtc cgg 1056
Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg 340
345 350 cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc gac gtg
gtg 1104 Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp
Val Val 355 360 365 agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg
ggc ccg gcg gac 1152 Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys
Ala Gly Pro Ala Asp 370 375 380 agc ggc gac gcc ctg ctg gag cgc aac
tat ccc act ggc gcg gag ttc 1200 Ser Gly Asp Ala Leu Leu Glu Arg
Asn Tyr Pro Thr Gly Ala Glu Phe 385 390 395 400 ctc ggc gac ggc ggc
gac gtc agc ttc agc acc cgc ggc acg cag aac 1248 Leu Gly Asp Gly
Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn 405 410 415 tgg acg
gtg gag cgg ctg ctc cag gcg cac cgc caa ctg gag gag cgc 1296 Trp
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg 420 425
430 ggc tat gtg ttc gtc ggc tac cac ggc acc ttc ctc gaa gcg gcg caa
1344 Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala
Gln 435 440 445 agc atc gtc ttc ggc ggg gtg cgc gcg cgc agc cag gac
ctc gac gcg 1392 Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln
Asp Leu Asp Ala 450 455 460 atc tgg cgc ggt ttc tat atc gcc ggc gat
ccg gcg ctg gcc tac ggc 1440 Ile Trp Arg Gly Phe Tyr Ile Ala Gly
Asp Pro Ala Leu Ala Tyr Gly 465 470 475 480 tac gcc cag gac cag gaa
ccc gac gca cgc ggc cgg atc cgc aac ggt 1488 Tyr Ala Gln Asp Gln
Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly 485 490 495 gcc ctg ctg
cgg gtc tat gtg ccg cgc tcg agc ctg ccg ggc ttc tac 1536 Ala Leu
Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr 500 505 510
cgc acc agc ctg acc ctg gcc gcg ccg gag gcg gcg ggc gag gtc gaa
1584 Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val
Glu 515 520 525 cgg ctg atc ggc cat ccg ctg ccg ctg cgc ctg gac gcc
atc acc ggc 1632 Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp
Ala Ile Thr Gly 530 535 540 ccc gag gag gaa ggc ggg cgc ctg gag acc
att ctc ggc tgg ccg ctg 1680 Pro Glu Glu Glu Gly Gly Arg Leu Glu
Thr Ile Leu Gly Trp Pro Leu 545 550 555 560 gcc gag cgc acc gtg gtg
att ccc tcg gcg atc ccc acc gac ccg cgc 1728 Ala Glu Arg Thr Val
Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg 565 570 575 aac gtc ggc
ggc gac ctc gac ccg tcc agc atc ccc gac aag gaa cag 1776 Asn Val
Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln 580 585 590
gcg atc agc gcc ctg ccg gac tac gcc agc cag ccc ggc aaa ccg ccg
1824 Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro
Pro 595 600 605 cgc gag gac ctg aag 1839 Arg Glu Asp Leu Lys 610 2
613 PRT Pseudomonas aeruginosa 2 Ala Glu Glu Ala Phe Asp Leu Trp
Asn Glu Cys Ala Lys Ala Cys Val 1 5 10 15 Leu Asp Leu Lys Asp Gly
Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30 Ala Ile Ala Asp
Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45 Leu Glu
Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60
Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65
70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg
Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu
Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe Ile His Glu Leu Asn Ala
Gly Asn Gln Leu Ser His 115 120 125 Met Ser Pro Ile Tyr Thr Ile Glu
Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 Leu Ala Arg Asp Ala Thr
Phe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 Met Gln Pro
Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 Ala
Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185
190 Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr
195 200 205 Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly
Lys Ile 210 215 220 Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp
Leu Asp Ile Lys 225 230 235 240 Pro Thr Val Ile Ser His Arg Leu His
Phe Pro Glu Gly Gly Ser Leu 245 250 255 Ala Ala Leu Thr Ala His Gln
Ala Cys His Leu Pro Leu Glu Thr Phe 260 265 270 Thr Arg His Arg Gln
Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly 275 280 285 Tyr Pro Val
Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser 290 295 300 Trp
Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly 305 310
315 320 Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln
Ala 325 330 335 Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg
Phe Val Arg 340 345 350 Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala
Asn Ala Asp Val Val 355 360 365 Ser Leu Thr Cys Pro Val Ala Ala Gly
Glu Cys Ala Gly Pro Ala Asp 370 375 380 Ser Gly Asp Ala Leu Leu Glu
Arg Asn Tyr Pro Thr Gly Ala Glu Phe 385 390 395 400 Leu Gly Asp Gly
Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn 405 410 415 Trp Thr
Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg 420 425 430
Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln 435
440 445 Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp
Ala 450 455 460 Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu
Ala Tyr Gly 465 470 475 480 Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg
Gly Arg Ile Arg Asn Gly 485 490 495 Ala Leu Leu Arg Val Tyr Val Pro
Arg Ser Ser Leu Pro Gly Phe Tyr 500 505 510 Arg Thr Ser Leu Thr Leu
Ala Ala Pro Glu Ala Ala Gly Glu Val Glu 515 520 525 Arg Leu Ile Gly
His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly 530 535 540 Pro Glu
Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu 545 550 555
560 Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg
565 570 575 Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys
Glu Gln 580 585 590 Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro
Gly Lys Pro Pro 595 600 605 Arg Glu Asp Leu Lys 610 3 14 PRT
Artificial Sequence Description of Artificial SequencePseudomonas
aeruginosa (PAK strain) Type IV short pilin loop 3 Cys Thr Ser Asp
Gln Asp Glu Gln Phe Ile Pro Lys Gly Cys 1 5 10 4 14 PRT Artificial
Sequence Description of Artificial SequencePseudomonas aeruginosa
(T2A strain) Type IV short pilin loop 4 Cys Thr Ser Thr Gln Asp Glu
Met Phe Ile Pro Lys Gly Cys 1 5 10 5 14 PRT Artificial Sequence
Description of Artificial SequencePseudomonas aeruginosa (PAO and
90063 strains) Type IV short pilin loop 5 Cys Lys Ser Thr Gln Asp
Pro Met Phe Thr Pro Lys Gly Cys 1 5 10 6 14 PRT Artificial Sequence
Description of Artificial SequencePseudomonas aeruginosa (CD and
PA103 strains) Type IV short pilin loop 6 Cys Thr Ser Thr Gln Glu
Glu Met Phe Ile Pro Lys Gly Cys 1 5 10 7 14 PRT Artificial Sequence
Description of Artificial SequencePseudomonas aeruginosa (K122-4
strain) Type IV short pilin loop 7 Cys Thr Ser Asn Ala Asp Asn Lys
Tyr Leu Pro Lys Thr Cys 1 5 10 8 14 PRT Artificial Sequence
Description of Artificial SequencePseudomonas aeruginosa (KB7,
82932 and 82935 strains) Type IV short pilin loop 8 Cys Ala Thr Thr
Val Asp Ala Lys Phe Arg Pro Asn Gly Cys 1 5 10 9 14 PRT Artificial
Sequence Description of Artificial SequencePseudomonas aeruginosa
(1071 strain) Type IV short pilin loop 9 Cys Glu Ser Thr Gln Asp
Pro Met Phe Thr Pro Lys Gly Cys 1 5 10 10 19 PRT Artificial
Sequence Description of Artificial SequencePseudomonas aeruginosa
(577B strain) Type IV long pilin loop 10 Cys Asn Ile Thr Lys Thr
Pro Thr Ala Trp Lys Pro Asn Tyr Ala Pro 1 5 10 15 Ala Asn Cys 11 19
PRT Artificial Sequence Description of Artificial
SequencePseudomonas aeruginosa (1244, 9D2 and P1 strains) Type IV
long pilin loop 11 Cys Lys Ile Thr Lys Thr Pro Thr Ala Trp Lys Pro
Asn Tyr Ala Pro 1 5 10 15 Ala Asn Cys 12 19 PRT Artificial Sequence
Description of Artificial SequencePseudomonas aeruginosa (SBI-N
strain) Type IV long pilin loop 12 Cys Gly Ile Thr Gly Ser Pro Thr
Asn Trp Lys Ala Asn Tyr Ala Pro 1 5 10 15 Ala Asn Cys 13 33 PRT
Artificial Sequence Description of Artificial SequenceNeisseria
meningitidis (Z49820 strain) Type IV pilin loop 13 Cys Gly Leu Pro
Val Ala Arg Asp Asp Thr Asp Ser Ala Thr Asp Val 1 5 10 15 Lys Ala
Asp Thr Thr Asp Asn Ile Asn Thr Lys His Leu Pro Ser Thr 20 25 30
Cys 14 37 PRT Artificial Sequence Description of Artificial
SequenceNeisseria meningitidis (Z69262 strain) Type IV pilin loop
14 Cys Gly Gln Pro Val Thr Arg Gly Ala Gly Asn Ala Gly Lys Ala Asp
1 5 10 15 Asp Val Thr Lys Ala Gly Asn Asp Asn Glu Lys Ile Asn Thr
Lys His 20 25 30 Leu Pro Ser Thr Cys 35 15 31 PRT Artificial
Sequence Description of Artificial SequenceNeisseria meningitidis
(Z69261 strain) Type IV pilin loop 15 Cys Gly Gln Pro Val Thr Arg
Ala Lys Ala Asp Ala Asp Ala Ala Gly 1 5 10 15 Lys Asp Thr Thr Asn
Ile Asp Thr Lys His Leu Pro Ser Thr Cys 20 25 30 16 33 PRT
Artificial Sequence Description of Artificial SequenceNeisseria
gonorrhoeae (pilE; X66144 strain) Type IV pilin loop 16 Cys Gly Gln
Pro Val Thr Arg Thr Gly Asp Asn Asp Asp Thr Val Ala 1 5 10 15 Asp
Ala Lys Asp Gly Lys Glu Ile Asp Thr Lys His Leu Pro Ser Thr 20 25
30 Cys 17 35 PRT Artificial Sequence Description of Artificial
SequenceNeisseria gonorrhoeae (pilE; AF043648 strain) Type IV pilin
loop 17 Cys Gly Gln Pro Val Lys Arg Asp Ala Gly Ala Lys Thr Gly Ala
Asp 1 5 10 15 Asp Val Lys Ala Asp Gly Asn Asn Gly Ile Asn Thr Lys
His Leu Pro 20 25 30 Ser Thr Cys 35 18 67 PRT Artificial Sequence
Description of Artificial SequenceVibrio cholera (U09807 strain)
Type IV pilin loop 18 Cys Lys Thr Leu Val Thr Ser Val Gly Asp Met
Phe Pro Phe Ile Asn 1 5 10 15 Val Lys Glu Gly Ala Phe Ala Ala Val
Ala Asp Leu Gly Asp Phe Glu 20 25 30 Thr Ser Val Ala Asp Ala Ala
Thr Gly Ala Gly Val Ile Lys Ser Ile 35 40 45 Ala Pro Gly Ser Ala
Asn Leu Asn Leu Thr Asn Ile Thr His Val Glu 50 55 60 Lys Leu Cys 65
19 67 PRT Artificial Sequence Description of Artificial
SequenceVibrio cholera (X64098 strain) Type IV pilin loop 19 Cys
Lys Thr Leu Ile Thr Ser Val Gly Asp Met Phe Pro Tyr Ile Ala 1 5 10
15 Ile Lys Ala Gly Gly Ala Val Ala Leu Ala Asp Leu Gly Asp Phe Glu
20 25 30 Asn Ser Ala Ala Ala Ala Glu Thr Gly Val Gly Val Ile Lys
Ser Ile 35 40 45 Ala Pro Ala Ser Lys Asn Leu Asp Leu Thr Asn Ile
Thr His Val Glu 50 55 60 Lys Leu Cys 65 20 13 PRT Artificial
Sequence Description of Artificial SequencePasteurella multocida
(AF154834 strain) Type IV pilin loop 20 Cys Asn Gly Gly Ser Glu Val
Phe Pro Ala Gly Phe Cys 1 5 10 21 5 PRT Artificial Sequence
Description of Artificial Sequenceendoplasmic reticulum (ER)
retention domain in native Pseudomonas exotoxin A 21 Arg Glu Asp
Leu Lys 1 5 22 4
PRT Artificial Sequence Description of Artificial
Sequenceendoplasmic reticulum (ER) retention domain 22 Arg Glu Asp
Leu 1 23 4 PRT Artificial Sequence Description of Artificial
Sequenceendoplasmic reticulum (ER) retention domain 23 Lys Asp Glu
Leu 1 24 25 DNA Artificial Sequence Description of Artificial
SequenceForward primer 24 ggcccatatg cacctgatac cccat 25 25 24 DNA
Artificial Sequence Description of Artificial SequenceReverse
primer 25 gaattcagtt acttcaggtc ctcg 24 26 28 DNA Artificial
Sequence Description of Artificial SequenceForward primer 26
ggcccatatg gagggcggca gcctggcc 28 27 24 DNA Artificial Sequence
Description of Artificial SequenceReverse primer 27 gaattcagtt
acttcaggtc ctcg 24 28 26 DNA Artificial Sequence Description of
Artificial SequencePCR primer pilATG (26 nc) 28 gagatattca
tgaaagctca aaaagg 26 29 20 DNA Artificial Sequence Description of
Artificial SequencePCR primer nadB4 (20 nc) 29 atctccatcg
gcaccctgac 20 30 21 DNA Artificial Sequence Description of
Artificial SequencePCR primer nadB1 (21 nc) 30 tggaagtgga
agtggagaac c 21 31 90 DNA Artificial Sequence Description of
Artificial Sequencecoding strand of duplex 31 tggccctgac cctggccgcc
gccgagagcg agcgcttcgt ccggcagggc accggcaacg 60 acgaggccgg
cgcggcaaac ctgcagggcc 90 32 54 DNA Artificial Sequence Description
of Artificial SequenceSense oligonucleotide 32 ttgtactagt
gatcaggatg aacagtttat tccgaaaggt tgttcacgta tgca 54 33 54 DNA
Artificial Sequence Description of Artificial SequenceAntisense
oligonucleotide 33 tacgtgaaca acctttcgga ataaactgtt catcctgatc
actagtacaa tgca 54 34 17 PRT Artificial Sequence Description of
Artificial Sequencelong C-terminal peptide, peptide 1, amino acids
128-142 of PAK strain MOD_RES (1) Xaa = acetyl-lysine MOD_RES (17)
Xaa = lysinamide 34 Xaa Cys Thr Ser Asp Gln Asp Glu Gln Phe Ile Pro
Lys Gly Cys Ser 1 5 10 15 Xaa 35 7 PRT Artificial Sequence
Description of Artificial Sequencecore peptide, peptide 2, amino
acids 134-140 of PAK strain MOD_RES (1) Xaa = acetyl-aspartic acid
MOD_RES (7) Xaa = lysinamide 35 Xaa Glu Gln Phe Ile Pro Xaa 1 5 36
7 PRT Artificial Sequence Description of Artificial
Sequencescrambled peptide MOD_RES (1) Xaa = acetyl-glutamine
MOD_RES (7) Xaa = lysinamide 36 Xaa Ile Asp Pro Glu Phe Xaa 1 5
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References