U.S. patent application number 10/046649 was filed with the patent office on 2003-04-17 for stress proteins and uses therefor.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. Invention is credited to Young, Douglas, Young, Richard A..
Application Number | 20030073094 10/046649 |
Document ID | / |
Family ID | 22113364 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030073094 |
Kind Code |
A1 |
Young, Richard A. ; et
al. |
April 17, 2003 |
Stress proteins and uses therefor
Abstract
The present invention relates to stress proteins and methods of
modulating an individual's immune response. In particular, it
relates to the use of such stress proteins in immune therapy and
prophylaxis, which results in an induction or enhancement of an
individual's immune response and as an immunotherapeutic agent
which results in a decrease of an individual's immune response to
his or her own cells. The present invention also relates to
compositions comprising a stress protein joined to another
component, such as a fusion protein in which a stress protein is
fused to an antigen. Further, the present invention relates to a
method of generating antibodies to a substance using a conjugate
comprised of a stress protein joined to the substance.
Inventors: |
Young, Richard A.; (Weston,
MA) ; Young, Douglas; (Ruislip, GB) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
|
Family ID: |
22113364 |
Appl. No.: |
10/046649 |
Filed: |
January 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10046649 |
Jan 14, 2002 |
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08336251 |
Nov 3, 1994 |
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6338952 |
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08336251 |
Nov 3, 1994 |
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PCT/US94/06362 |
Jun 6, 1994 |
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08336251 |
Nov 3, 1994 |
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08073381 |
Jun 4, 1993 |
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08073381 |
Jun 4, 1993 |
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07804632 |
Dec 9, 1991 |
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07804632 |
Dec 9, 1991 |
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07366581 |
Jun 15, 1989 |
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07366581 |
Jun 15, 1989 |
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07207298 |
Jun 15, 1988 |
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07366581 |
Jun 15, 1989 |
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PCT/US89/02619 |
Jun 15, 1989 |
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Current U.S.
Class: |
435/6.15 ;
424/192.1; 435/69.3; 435/91.1 |
Current CPC
Class: |
A61K 39/39 20130101;
C07K 2319/735 20130101; A61K 2039/545 20130101; A61K 39/12
20130101; C07K 2319/35 20130101; A61K 39/04 20130101; C07K 2319/75
20130101; C07K 14/005 20130101; C12N 2740/16234 20130101; A61K
39/385 20130101; A61K 39/0008 20130101; A61K 2039/6043 20130101;
C07K 2319/21 20130101; C12N 2740/16222 20130101; C07K 2319/03
20130101; C07K 2319/20 20130101; C07K 14/35 20130101; A61K 39/00
20130101; C07K 2319/40 20130101; A61P 37/00 20180101; A61K 2039/70
20130101; C12N 15/62 20130101; A61K 39/21 20130101; C07K 2319/50
20130101; A61K 2039/55516 20130101 |
Class at
Publication: |
435/6 ; 435/91.1;
435/69.3; 424/192.1 |
International
Class: |
C12Q 001/68; C12N
015/09; C12P 019/34; A61K 039/00 |
Goverment Interests
[0002] Work described herein was funded by grants from the National
Institutes of Health (AI23545), the World Health Organization
Program for Vaccine Development, and the World Health
Organization/World Bank/United Nations Development Program Special
Program for Research and Training in Tropical Diseases. The United
States government has certain rights in the invention.
Claims
I claim:
1. A vaccine comprising all or a portion of a stress protein which
induces an immune response in an individual to whom it is
administered or all or a portion of a protein having an amino acid
sequence sufficiently homologous to the amino acid sequence of the
stress protein to be capable of inducing an immune response in an
individual to whom it is administered.
2. A vaccine of claim 1 in which the stress protein is a
mycobacterial stress protein or a protein having an amino acid
sequence sufficiently homologous to the amino acid sequence of the
mycobacterial stress protein to induce an immune response in the
individual to whom it is administered.
3. A vaccine for use in enhancing in an individual the immune
response to a pathogen, comprising all or a portion of a stress
protein of the pathogen against which the enhanced response is
desired.
4. A vaccine of claim 3 in which the stress protein is selected
from the group consisting of: mycobacterial stress proteins,
bacterial stress proteins, fungal stress proteins, viral stress
proteins and parasitic stress proteins.
5. A composition comprising all or a portion of a selected stress
protein, for use in producing or enhancing an immune response in an
individual, wherein the stress protein is in sufficient quantity to
elicit the desired immune response.
6. A composition comprising a stress protein for use in immunizing
an individual against a subsequent infection by a pathogen, wherein
the stress protein is in sufficient quantity to produce an immune
response to the stress protein.
7. The composition of claim 6 wherein the stress protein is a
stress protein of the pathogen.
8. A composition comprising all or a portion of a stress protein or
all or a portion of a protein having an amino acid sequence
sufficiently homologous to the amino acid sequence of the stress
protein for use in inducing in an individual immune tolerance
against a protein, under conditions appropriate for induction of
the desired tolerance.
9. A composition of claim 8, wherein the protein is a protein
associated with rheumatoid arthritis.
10. A vaccine for use in inducing an immune response in an
individual comprising all or a portion of a stress protein or all
or a portion of a protein having an amino acid sequence
sufficiently homologous to the amino acid sequence of the stress
protein conjugated to a substance to which an immune response is
desired or to a portion of the substance sufficient to induce an
immune response in an individual.
11. A vaccine of claim 10 in which the stress protein is a
mycobacterial stress protein or a protein having an amino acid
sequence sufficiently homologous to the amino acid sequence of the
mycobacterial stress protein to induce an immune response in an
individual to whom it is administered.
12. A vaccine of claim 10 in which the substance against which an
immune response is desired is selected from the group consisting
of: proteins, peptides, oligosaccharides, lipids, carbohydrates,
organic molecules and a combination thereof.
13. A vaccine for use in inducing an immune response in an
individual comprising a recombinant fusion protein which includes
all or a portion of a stress protein or all or a portion of a
protein having an amino acid sequence sufficiently homologous to
the amino acid sequence of the stress protein fused to a substance
against which an immune response is desired or to a portion of the
substance sufficient to induce an immune response in an
individual.
14. A vaccine of claim 13 in which the stress protein is a
mycobacterial stress protein or a protein having an amino acid
sequence sufficiently homologous to the amino acid sequence of the
mycobacterial stress protein to induce an immune response in an
individual to whom it is administered.
15. A vaccine of claim 14 in which the protein is the HIV gag or
pol protein.
16. A composition for use as an agent to induce immune tolerance,
comprising a stress protein conjugated to a substance to which an
immune response is desired.
17. A vaccine for use in enhancing in an individual an immune
response, comprising all or a portion of a stress protein
conjugated to a substance to which an immune response is desired or
to a portion of the substance sufficient to enhance an immune
response in the individual.
18. A vaccine of claim 17 in which the stress protein is selected
from the group consisting of: mycobacterial stress proteins,
bacterial stress proteins, fungal stress proteins, viral stress
proteins and parasitic stress proteins.
19. A composition comprising a stress protein for use in producing
or enhancing an immune response in an individual, wherein the
stress protein is in sufficient quantity to elicit the desired
immune response, and the stress protein is conjugated to a
substance against which an immune response is desired or to a
portion of the substance sufficient to produce or enhance an immune
response in the individual.
20. A composition comprising a stress protein for use in immunizing
an individual against a subsequent infection by a pathogen, wherein
the stress protein is in sufficient quantity to produce an immune
response sufficient to protect the individual against subsequent
infection by the pathogen, and the stress protein is conjugated to
a substance against which an immune response is desired or to a
portion of the substance sufficient to produce an immune response
in the individual.
21. A vaccine for use in inducing an immune response in an
individual comprising all or a portion of a stress protein or all
or a portion of a protein having an amino acid sequence
sufficiently homologous to the amino acid sequence of the stress
protein and a substance against which an immune response is desired
or to a portion of the substance sufficient to induce an immune
response in an individual.
22. A vaccine of claim 21 in which the stress protein is a
mycobacterial stress protein or a protein having an amino acid
sequence sufficiently homologous to the amino acid sequence of the
mycobacterial stress protein to induce an immune response in an
individual to whom it is administered.
23. A vaccine of claim 21 in which the substance against which an
immune response is desired is selected from the group consisting
of: proteins, peptides, oligosaccharides, lipids, carbohydrates,
organic molecules and any combination thereof.
24. A composition for use as an agent to induce immune tolerance,
comprising a stress protein and a substance to which an immune
response is desired.
25. A vaccine for use in enhancing in an individual an immune
response, comprising all or a portion of a stress protein and
either a substance to which an immune response is desired or a
portion of the substance sufficient to enhance an immune response
in the individual.
26. A vaccine of claim 25 in which the stress protein is selected
from the group consisting of: mycobacterial stress proteins,
bacterial stress proteins, fungal stress proteins, viral stress
proteins and parasitic stress proteins.
27. A composition comprising a stress protein and a substance
against which an immune response is desired or a portion of the
substance sufficient to produce or enhance an immune response in an
individual for use in producing or enhancing an immune response in
an individual, wherein the stress protein is in sufficient quantity
to elicit the desired immune response .
28. A composition comprising a stress protein and a substance
against which an immune response is desired or to a portion of the
substance sufficient to produce or enhance an immune response in
the individual for use in immunizing an individual against a
subsequent infection by a pathogen, wherein the stress protein is
in sufficient quantity to produce an immune response sufficient to
protect the individual against subsequent infection by the
pathogen.
29. A composition for use as an agent to induce an immune response
in an individual to whom it is administered, comprising all or a
portion of a stress protein or all or a portion of a protein having
an amino acid sequence sufficiently homologous to the amino acid
sequence of the stress protein to be capable of inducing an immune
response in an individual to whom it is administered.
30. A composition for use as an agent to induce an immune response
in an individual to whom it is administered, comprising all or a
portion of a stress protein or all or a portion of a protein having
an amino acid sequence sufficiently homologous to the amino acid
sequence of the stress protein conjugated to a substance against
which an immune response is desired or to a portion of the
substance sufficient to induce an immune response in the
individual.
31. A composition for use as an agent to induce an immune response
in an individual to whom it is administered, comprising a
recombinant fusion protein which includes a) all or a portion of a
stress protein or all or a portion of a protein having an amino
acid sequence sufficiently homologous to the amino acid sequence of
the stress protein and b) a substance against which an immune
response is desired or a portion of the substance sufficient to
induce an immune response in the individual.
32. A composition for use as an agent to induce immune tolerance,
comprising a stress protein.
33. A composition for use in treating an autoimmune disease,
comprising all or a portion of a stress protein or all or a portion
of a protein having an amino acid sequence sufficiently homologous
to the amino acid sequence of the stress protein to induce immune
tolerance in an individual to whom it is administered.
34. A composition of claim 36 for treating rheumatoid
arthritis.
35. A conjugate comprising a stress protein joined with a substance
against which an immune response is desired.
36. The conjugate of claim 35 wherein the stress protein is the
hsp70 or the hsp60 protein.
37. The conjugate of claim 35 wherein the substance is selected
from the group consisting of: proteins, peptides, oligosaccharides,
lipids, carbohydrates, organic molecules and combinations
thereof.
38. The conjugate of claim 37 wherein the protein is selected from
the group consisting of: ovalbumin, influenza virus hemagglutinin
protein, human immunodeficiency virus gag protein and human
immunodeficiency virus pol protein.
39. A fusion protein comprising a stress protein fused to a protein
against which an immune response is desired.
40. The fusion protein of claim 39 wherein the stress protein is a
heat shock protein and the protein is a human immunodeficiency
viral protein.
41. The fusion protein of claim 40 wherein the heat shock protein
is hsp70 and the human immunodeficiency viral protein is p24
protein.
42. A method of generating antibodies to a substance comprising the
steps of: a) introducing an effective amount of a conjugate
comprised of a stress protein joined to the substance into a
mammalian host; b) removing the antibodies produced by the host to
the substance from the host; and c) purifying the antibodies
thereby generating antibodies to the substance.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of the
corresponding International Application PCT/US94/06362, filed Jun.
6, 1994 and U.S. Ser. No. 08/073,381, filed Jun. 4, 1993, which is
a Continuation-in-Part of U.S. Ser. No. 07/804,632 filed Dec. 9,
1991, which is a File-Wrapper-Continuation of U.S. application Ser.
No. 07/366,581 filed Jun. 15, 1989, now abandoned, which is a
Continuation-in-Part of U. S. application Ser. No. 07/207,298 filed
Jun. 15, 1988, now abandoned, and the corresponding International
Application PCT/US89/02619 filed Jun. 15, 1989. The teachings of
PCT/US94/06362, U.S. Ser. No. 08/073,381, U.S. Ser. No. 07/804,632,
U.S. Ser. No. 07/366,581, U.S. Ser. No. 07/207,298 and
PCT/US89/02619 are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Although the function of stress proteins is not entirely
clear, it appears that some participate in assembly and structural
stabilization of certain cellular and viral proteins, and their
presence at high concentrations may have an additional stabilizing
effect during exposure to adverse conditions. Neidhardt, F. C. and
R. A. Van Bogelen, In: Escherichia coli and Salmonella typhimurium,
Cellular and Molecular Biology, (eds. Neidhardt, F. C., Ingraham,
J. L., Low, K. B., Magasanik, B. Schaechter, M. and Umbarger, H. E.
(Am. Soc. Microbiol., Washington, D.C.), pp. 1334-1345 (1987);
Pelham, H. R. B. Cell, 46:959-961 (1986); Takano, T. and T.
Kakefuda, Nature, 239:34-37 (1972); Georgopoulos, C. et al., New
Biology, 239:38-41 (1972). Phagocytic host cells produce a hostile
environment of foreign organisms, and the ability to produce stress
proteins has been implicated in the survival of bacterial pathogens
within macrophages Christman, M. F. et al., Cell, 41:753-762
(1985).
[0004] Mycobacterium (M.) tuberculosis and Mycobacterium (M.)
leprae are the etiologic agents of tuberculosis and leprosy,
respectively. These diseases afflict 20-30 million people and
continue to present a significant global health problem. Joint
International Union Against Tuberculosis and World Health
Organization Study Group, Tubercle, 63:157-169 (1982); Bloom, B.
and T. Godal, Rev. Infect Dis. 5:765-780 (1983). To develop more
effective tools for the diagnosis and prevention of these diseases,
it is important to understand the immune response to infection by
mycobacterial pathogens.
[0005] The antibody and T-cell responses to infection or
inoculation with killed mycobacteria have been studied in humans
and in animals. Human patients with tuberculosis or leprosy produce
serum antibodies directed against at least 12 mycobacterial
proteins. Some of these proteins are also recognized by
well-characterized murine monoclonal antibodies. Mice immunized
with mycobacterial lysates produce antibodies that are directed
predominantly to six M. tuberculosis and six M. leprae protein
antigens. Engers, H. D. Infect. Immun., 48:603-605 (1985); Engers,
H. D., Infect. Immun., 51:718-720 (1986). Genes encoding these 12
mycobacterial antigens have been cloned, and recombinant proteins
produced from these clones have been used to investigate the human
T-lymphocyte response to mycobacterial infection. Husson, R. N. and
R. A. Young, Proc. Natl. Acad. Sci., USA, 84:1679-1683 (1987);
Young, R.A. et al., Nature, 316:450-452 (1985); Britton, W. J. et
al., LePr. Rev., 57, Suppl. 2, 67-75 (1986).
[0006] Protection against mycobacterial disease involves
cell-mediated immunity. Joint International Union Against
Tuberculosis and World Health Organization Study Group, Tubercle,
63:157-169 (1982); Hahn, H. and S. H. E. Kaufman, Rev. Infect.
Dis., 3:1221-1250 (1981). T-lymphocytes cloned from patients or
from volunteers immunized with killed mycobacteria have been tested
for their ability to recognize the recombinant mycobacterial
proteins. Lymphocyte-proliferation assays demonstrate that most of
the antigens identified with monoclonal antibodies are involved in
the T-cell response to mycobacterial infection or vaccination in
mice and in humans. Limiting dilution analysis indicates that 20%
of the mycobacterial-reactive CD4.sup.+ T-lymphocytes in mice
immunized with M. tuberculosis recognize a single protein, the
65-kDa antigen. Kaufman, S. H. E. et al., Eur J. Immunol.,
17:351-357 (1987).
SUMMARY OF THE INVENTION
[0007] The present invention relates to stress proteins and methods
of modulating an individual's (such as a human, other mammal or
other vertebrate) immune response. In particular, it relates to the
use of such stress proteins in immune therapy or prophylaxis, which
results in an induction or enhancement of an individual's immune
response and as an immunotherapeutic agent which results in a
decrease of an individual's response to his or her own cells. In
the embodiment in which an individual's immune response is induced
or enhanced, the induced or enhanced response can be a response to
antigens, such as those derived from a pathogen or cancer cell, or
can be upregulation of the individual's immune status, such as in
an immune compromised individual. In immune prophylaxis, stress
proteins are administered to prevent or reduce the effects in an
individual of a pathogen, which can be any virus, microorganism,
parasite or other organism or substance (e.g., a toxin or toxoid)
which causes disease or to prevent or reduce the effects in an
individual of cancer cells. In preventing or reducing adverse
effects of pathogens which contain stress proteins (e.g., bacteria,
parasite, fungus) according to the method of the present invention,
an individual's immune response to the pathogen's stress protein(s)
is induced or enhanced through the administration of a vaccine
which includes the pathogen's stress protein(s) or other stress
proteins. The stress protein can be administered alone, as a member
or component of a conjugate (e.g., joined to another antigen by
chemical or recombinant means such as joined to a fusion partner
resulting in a fusion protein), or as an adjuvant or carrier
molecule to enhance or obtain a desired immune response to an
antigen.
[0008] The present invention also relates to compositions which are
conjugates comprised of a stress protein joined to another
substance or component. For example, the present invention relates
to a conjugate in which a stress protein is chemically linked to an
antigen, or in which a stress protein is fused to an antigen (e.g.,
a fusion protein).
[0009] The present invention also relates to a method of generating
monoclonal or polyclonal antibodies to a substance using a
conjugate comprised of a stress protein joined to the substance. In
this embodiment, an effective amount of the conjugate (i.e., an
amount which results in an immune response in the host) is
introduced into a mammalian host which results in production of
antibodies to the substance in the host. The antibodies are removed
from the host and purified using known techniques (e.g.,
chromatography).
[0010] Preventing or reducing adverse effects of viral pathogens
which do or do not contain stress proteins, as well as preventing
or reducing the adverse effects of cancer cells according to the
present method, is effected by enhancing an individual's immune
surveillance system. Enhancement of immune response can be effected
by modulating the immune cells by stimulation with a stress protein
(e.g., a bacterial stress protein).
[0011] In the embodiment in which an individual's immune response
is decreased, such as is used in treating autoimmune diseases,
stress proteins known to be involved in the autoimmune response are
administered to turn down an individual's immune response by
tolerizing the individual to the stress proteins. Alternatively,
the immune response to stress protein, which is known to occur in
autoimmune disease, is reduced by interfering with the ability of
immune cells which respond to stress proteins to do so.
[0012] A selected stress protein of the present invention can be
administered to an individual, according to the method of the
present invention, and result in an immune response which provides
protection against subsequent infection by a pathogen (e.g.,
bacteria, other infectious agents which produce stress proteins) or
reduction or prevention of adverse effects of cancer cells.
Alternatively, a selected stress protein can be administered to an
individual, generally over time, to induce immune tolerance against
the selected stress protein. For example, a selected stress protein
can be administered in multiple doses over time in order to induce
immune tolerance against an autoimmune disease such as rheumatoid
arthritis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A is a graph illustrating the sequence similarity
between portions of the M. tuberculosis 71-kDa antigen (residues
1-204; TB 71 kDa) and the E. coli DnaK protein (residues
430-639).
[0014] FIG. 1B is a graph illustrating the sequence similarity
between portions of the M. tuberculosis 65-kDa antigen (residues
1-540; TB 65 kDa) and the E. coli GroEL protein (residues
1-547).
[0015] FIG. 2 is a comparison of the amino acid sequence of the
human P1 protein (573 residues) (SEQ ID NO: 1) and the amino acid
sequence of the groEL protein (547 residues) (SEQ ID NO: 2).
[0016] FIG. 3 is a comparison of the amino acid sequence of the
human P1 protein (573 residues) (SEQ ID NO: 1), which is a homolog
of groEL protein, and the amino acid sequence of the 65 kDa M.
leprae protein (540 residues) (SEQ ID NO: 3).
[0017] FIG. 4 is a comparison of the amino acid sequence of the
human P1 protein (573 residues) (SEQ ID NO: 1), which is a homolog
of the groEL protein, and the amino acid sequence of the 65 kDa M.
tuberculosis protein (540 residues) (SEQ ID NO: 4).
[0018] FIG. 5 is a schematic representation of selected stress
protein fusion vectors which contain a polylinker with multiple
cloning sites permitting incorporation of a gene of interest.
[0019] FIG. 6 is a schematic representation of the stress protein
fusion vector, pKS70 containing the T7 RNA polymerase promoter, a
polylinker and the mycobacterial tuberculosis hsp70 gene, and the
stress protein fusion vector pKS72 containing the HIV p24 gag gene
subcloned into the pKS70 vector.
[0020] FIG. 7 is a graph illustrating the anti-p24 antibody titer
in mice injected with the p24-hsp70 fusion protein, p24 alone and
hsp70 alone.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Cells respond to a variety of stressful stimuli by
increasing the synthesis of specific stress proteins. The most
extensively studied cellular response to stressful stimuli is the
synthesis of heat shock proteins (hsp) by a cell, induced by a
sudden increase in temperature. Because many of the heat shock
proteins are also induced by other stresses, they are frequently
called stress proteins. Stress proteins and their relatives appear
to help assemble and disassemble protein complexes. In bacteria,
the major stress proteins, hsp70 and hsp60, occur at moderate
levels in cells that have not been stressed but accumulate to very
high levels in stressed cells. For example, hsp70 and hsp60
normally account for 1-3% of total E. coli protein, but can
accumulate to about 25% under stressful conditions. Eukaryotic
hsp70 and hsp60 proteins do not accumulate to these extreme levels.
Their levels range from undetectable to moderately abundant,
depending on the organism and cell type.
[0022] The present invention is based on the observation that
stress proteins are among the major antigens available for
presentation to T lymphocytes and may be common immune targets in a
broad spectrum of infectious diseases. Immune responses to stress
proteins are involved in immune surveillance by the body and a
variety of different T cell types has been shown to recognize
highly conserved stress protein determinants. Several observations,
described below, suggest a model of immune surveillance in which
self-reactive T cells provide a first line of defense against
infection or other invasion by pathogens, which include, but are
not limited to, viruses, microorganisms, other organisms,
substances such as toxins and toxoids, and agents which cause cell
transformation, by recognizing and helping to eliminate stressed
autologous cells, as well as cells infected with intracellular
pathogens. Without wishing to be bound by this model, it is
presented as one means by which it is possible to explain why
prokaryotic and eukaryotic cells respond to a variety of
potentially damaging stimuli, such as elevated temperature, by
increasing the synthesis of a family of proteins, referred to as
stress proteins, which are among the most highly conserved and
abundant proteins found in nature.
[0023] Investigation of antigens involved in the immune response to
the tuberculosis and leprosy bacilli (M. tuberculosis and M.
leprae) initially led to the observation that a variety of stress
proteins are among the major targets of the immune response, as is
described at greater length below.
[0024] Further assessment has demonstrated that stress proteins may
be common immune targets in a broad spectrum of infectious
diseases. Sequence analysis has revealed 70-kDa heat shock protein
homologues among major antigens of the protozoan parasites
Plasmodium falciparum (Bianco, A. E. et al., Proc. Natl. Acad.
Sci., USA, 83:8713-8717 (1986)) and Schistosoma mansoni (Hedstrom,
R. et al., J. Exp. Med., 165:1430-1435 (1987)) and the malarial
parasite Brugia malayi (Selkirk, M. E. et al., J. Cell Biochem.,
12D:290 (1988)). Similarly, homologues of GroEL have been found
among antigens involved in the immune response to Salmonella
typhimurium and Coxiella (Vodkin, M. H. and J. C. Williams, J.
Bacteriol, 170:1227 (1988)), as well as Bordetella pertussis (Del
Giudice, G., et al., J. of Imm., 150:2025-2032 (1993)). The
presence of stress proteins among major immune targets in a variety
of human pathogens is support for the idea that the stress response
may be a general component of infection and that stress proteins
should be considered among candidates for subunit vaccines. All
organisms respond to heat by inducing synthesis of heat shock
proteins (hsp), which are a group of proteins. This response is the
most highly conserved genetic system known and has been shown to
occur in every organism, including microorganisms, plants and
animals, investigated to date. Many of the characteristics of the
response are common to all organisms and the hsp are among the most
highly conserved proteins known. For example, hsp90 family and
hsp70 family proteins are present in widely diverse organisms. The
proteins in each family--even in such diverse organisms--show
approximately 50% identity at the amino acid level and at the
nonidentical residues, exhibit many similarities. Several of the
proteins induced by heat are also induced by a variety of other
stresses. The hsps or a closely related/similar protein are present
in all organisms at normal temperatures and have been shown to have
key functions in normal cell metabolism. Lindquist, S. and E. A.
Craig, Ann. Rev. Genet., 22:631-677 (1988). Because the stress
response is common to prokaryotes and eukaryotes and stress
proteins are among the most highly conserved in sequence, it is
reasonable to expect that an antigen from one pathogen could
immunize against another pathogen. Exposure to foreign stress
proteins early in life might, in fact, induce a degree a immunity
to a variety of infectious agents. If so, this could provide an
explanation for the observation that, for many pathogens, only a
fraction of infected individuals actually acquire clinical
disease.
[0025] The following is a description of the relationship which has
been observed between stress proteins and the immune response to
mycobacterial infection; of the observation and supporting
information that stress proteins are immune targets in many
infections by pathogens; of the role of stress proteins as immune
targets in transformed cells; of recognition of the fact that the
immune response to conserved stress protein determinants may play
an important role in autoimmune pathology in rheumatoid arthritis,
as well as in adjuvant arthritis; and of the role of stress
proteins in immune surveillance, as well as a model proposed for
immune surveillance in which self-reactive T cells provide a first
line of defense against infection and cell transformation.
[0026] Mycobacterial Stress Proteins are Taraets of the Immune
Response
[0027] An intriguing relationship between stress proteins and the
immune response to mycobacterial infection has been observed. A
more detailed examination of stress protein determinants and immune
response mechanisms is essential to understanding the relationship
among stress proteins, infection, and immunity.
[0028] In view of the involvement of proteins of M. tuberculosis
and M. leprae in humoral and cell-mediated immune responses and to
establish the functions of these proteins in the mycobacterial
cell, the DNA encoding several of the M. tuberculosis and M. leprae
antigens have been sequenced. The results, discussed in Example 1,
demonstrate that many of these mycobacterial protein antigens
exhibit striking sequence similarity to known stress-induced
proteins. Three of the M. leprae and two of the M. tuberculosis
protein antigens studied have been shown to exhibit striking
sequence similarity to known stress proteins. For reasons discussed
in Example 1, it is concluded that two of the M. leprae and two of
the M. tuberculosis antigens are homologues of the E. coli DnaK and
GroEL proteins.
[0029] In mice, immunization with mycobacterial lysates elicits
antibody responses to at least six M. tuberculosis protein antigens
and a similar number of M. leprae protein antigens. Monoclonal
antibodies specific for these proteins have been used to isolate
clones from .lambda.gtll DNA expression libraries of M.
tuberculosis and M. leprae. The sequence of the DNA clones revealed
that mycobacterial hsp70 (alias 70 kDa antigen) and hsp60 (alias 65
kDa antigen, GroEL) were the major targets of the murine antibody
response to both M. tuberculosis and M. leprae. Two additional hsp,
an 18 kDa member of the small hsp family and a 12 kDa homologue of
groES, were found among the M. leprae and M. tuberculosis antigens.
Young, D. B., et al., Proc. Natl. Acad. Sci., USA, 85:4267-4270
(1988); Shinnick, T. M., et al., Nuc. Acids Res., 17:1254
(1989).
[0030] The mycobacterial stress proteins are among the
immunodominant targets of both murine antibody and T cell
responses. In one study which summarized results obtained from 10
laboratories, a collection of 24 murine monoclonal antibodies
recognized 6 M. leprae proteins; 7 of these antibodies are directed
against 6 different determinants in the M. leprae hsp60. Engers, H.
D., et al., Infect. Immun., 48:603-605 (1985); Mehra, V., et al.,
Proc. Natl. Acad. Sci., USA, 83:7013-7017 (1986). In a similar
study, 3 of 33 monoclonal antibodies raised against M. tuberculosis
recognized the M. tuberculosis hsp60 protein. Engers, H. D., et
al., Infect. Immun., 51:718-720 (1986). Finally, limiting dilution
analysis indicates that 20% of the mycobacterial-reactive CD4+ T
lymphocytes in mice immunized with M. tuberculosis recognize this
antigen. Kaufmann, S. H., et al., Eur. J. Immunol., 17:351-357
(1987).
[0031] Although a rigorous quantitative analysis of the human
immune response to mycobacterial stress proteins has not yet been
reported, mycobacterial stress proteins are recognized by human
antibodies and T lymphocytes and the evidence suggests that these
proteins are among the major targets of the human cell mediated
immune response. Emmrich. F., et al., J. Exp. Med., 163:1024-1029
(1985); Mustafa, A. S., et al., Nature (London). 319:63-66 (1986);
Oftung, F., et al., J. Immunol., 138:927-931 (1987); Lamb, J. R.,
et al., EMBO J., 6:1245-1249 (1987). T lymphocytes from patients
with mycobacterial infection or from volunteers immunized with
mycobacteria have been cloned and tested for their ability to
recognize the mycobacterial stress proteins. In each of these
studies, some fraction of the human T cell clones were shown to
recognize one or more of the mycobacterial stress proteins.
[0032] Stress Proteins are Immune Targets in Infections by
Pathogens
[0033] The observation that stress proteins are important targets
of the immune response to mycobacterial infection and the knowledge
that the major stress proteins are conserved and abundant in other
organisms suggested that stress proteins are likely to be immune
targets in many infections by pathogens. Indeed, that is now
clearly the case. Antigens from a wide variety of infectious agents
have been identified as members of stress protein families. The
major stress protein antigen recognized by antibodies in bacterial
infections is hsp60. "Common antigen", an immunodominant protein
antigen long known to be shared by most bacterial species, turns
out to be hsp60. Shinnick, T. M., et al., Infect. Immun., 56:446
(1988); Thole, J. E. R., et al., Microbial Pathogenesis, 4:71-83
(1988). Stress proteins have also been identified as immune targets
in most major human parasite infections. Bianco, A. E., et al.,
Proc. Natl. Acad. Sci. USA, 83:8713 (1986); Nene, V., et al., Mol.
Biochem. Parasitol., 21:179 (1986); Ardeshir, F., et al., EMBO J.,
6:493 (1987); Hedstrom, R., et al., J. Exp. Med., 165:1430 (1987);
Selkirk, M. E., et al., J. Cell Biochem., 12D:290 (1988), Engman,
D. M., et al., J. Cell Biochem., 12D: Supplement, 290 (1988);
Smith, D. F., et al., J. Cell Biochem., 12D:296 (1988). Antibodies
to hsp70 have been identified in the sera of patients suffering
from malaria, trypanosomiasis, leishmaniasis, schistosomiasis and
filariasis. Hsp90 is also a target of antibodies in trypanosomiasis
and a member of the small hsp family is recognized in some patients
with schistosomiasis.
[0034] Proteins homologous to stress proteins have also been
identified in viruses. Recently, a protein encoded by the RNA
genome of the Beet Yellows Closterovirus, a plant virus, has been
shown to be homologous to hsp70. Agranovsky, A. A., et al., J. Mol.
Biol., 217: 603-610 (1991). In addition, stress protein induction
occurs in eukaryotic cells following infection by diverse viruses
in vitro. Collins, P. L., and Hightower, L. E., J. Virol.,
44:703-707 (1982); Nevins, J. R., Cell, 29:913-939 (982); Garry, R.
F. et al., Virology, 129:391-332 (1988); Khandjian, E. W. and
Turler, H., Mol. Cell Biol., 3:1-8 (1983); LaThangue, N. B., et
al., EMBO J., 3:267-277 (1984); Jindal, S. and Young, R., J. Viral,
66:5357-5362 (1992). CTL that recognize these neo-antigens could
limit the spread of virus by killing infected cells, possibly
before substantial amounts of mature virus are assembled, and by
secreting the lymphokine .gamma.-interferon. Pestka, S., in:
Methods Enzymol., Interferons, Part A., Vol. 79 Academic Press, New
York, pp. 667 (1981). Evidence consistent with this idea is
emerging. Koga et al., (1989) have shown that infection of primary
murine macrophages with CMV rendered them susceptible as targets
for MHC-I restricted CD8.sup.+ CTL specific for linear epitopes of
M. tuberculosis hsp60. Koga, T., et al. (1989). Although the
epitope recognized by these CTL on infected macrophages was not
defined, it is tempting to speculate that a cross-reactivity with
self hsp60 epitopes is being observed. Indeed, the same groups
showed that a homologous hsp60 is constitutively present in
macrophages and is upregulated by .gamma.-interferon
stimulation.
[0035] Stress Proteins as Immune Targets in Transformed Cells
[0036] Stress proteins appear to be produced at high levels in at
least some transformed cells. Bensaude, O. and Morange, M., EMBO
J., 2: 173-177 (1983). An 86 kDA murine tumor antigen has been
found to be homologous to representatives of the hsp90 family in
yeast and Drosophila. Ullrich, S. J., Proc. Natl. Acad. Sci., USA,
83: 3121-3125 (1986). Immunization of mice with the purified
protein led to inhibition of tumor growth in 95% of experimental
animals that had been seeded with cultured tumor cells. All of the
protected mice had high titers of anti-hsp90 serum antibody which
was able to precipitate murine hsp90 from lysates of heat shocked
mouse embryo cells. Again, a role for autoreactive lymphocytes is
implied, since T cells capable of recognizing autologous cells
stressed by transformation could help eliminate nascent tumor
cells.
[0037] Stress Proteins and Autoimmune Processes
[0038] Rheumatoid arthritis is characterized by a chronic
proliferative and inflammatory reaction in synovial membranes which
is thought to involve autoimmune processes. Rat adjuvant arthritis
resembles human rheumatoid arthritis in many respects, and has been
used as an experimental animal model for human disease. Pearson, C.
M., Arthritis Rheum., 7:80-86 (1964). Adjuvant arthritis can be
induced in rats with a single intradermal injection of killed M.
tuberculosis in complete Freund's adjuvant. An autoimmune process
involving T lymphocytes appears to be responsible for the
generation of the disease. Holoshitz, J., et al., Science,
219:56-58 (1983). T cell lines isolated from the draining lymph
nodes of arthritic rats and propagated in vitro by stimulation with
M. tuberculosis-pulsed syngeneic antigen presenting cells can cause
a transient form of the disease when transferred to irradiated
rats. Since care was taken in these experiments to exclude the
transfer of contaminating M. tuberculosis, this result strongly
suggests that the clinical effects of the disease are a consequence
of an autoimmune reaction in which the autoantigen is shared with
M. tuberculosis.
[0039] The rat and M. tuberculosis antigens recognized by the
arthritogenic T cells have been sought for a number of years. A
number of different proteins present in synovial membranes have
been proposed to be the cross-reactive rat antigen, but were later
discounted as procedures for the purification of these proteins
improved. van Eden, W., et al., Proc. Natl. Acad. Sci., USA,
82:5117-5120 (1985); Holoshitz, J., et al., Science, 219:56-58
(1983). The M. tuberculosis antigen recognized by the arthritogenic
T cells was recently shown to be a 65 kDa protein (van Eden, W., et
al., Nature, 331:171 (1988), which has now been shown to be hsp60
(see the Example 1). Using a combination of truncated recombinant
65 kDa proteins and peptides, a nine amino acid epitope of hsp60
has been identified as the minimum stimulatory sequence for
arthritogenic T cell clones in proliferation assays. Now that it is
clear that some arthritogenic T cells recognize the mycobacterial
hsp60, it is quite possible that the rat autoantigen is also
hsp60.
[0040] The results obtained in the adjuvant arthritis model led
investigators to determine whether T lymphocytes from human
rheumatoid arthritis patients also recognize mycobacterial
antigens. These investigators have found not only that patients
with rheumatoid arthritis have T cells that recognize M.
tuberculosis antigens, but that these T cells have diverse
phenotypes. Substantial proliferative responses to mycobacterial
extracts are observed with uncloned T cells (predominantly
CD4.sup.+) from both synovial infiltrates and peripheral blood,
although responses are generally greater in synovial infiltrates.
Abrahamson, T. G., et al., Scand. J. Immunol., 7:81-90 (1978);
Holoshitz, J., et al., Lancet ii, 305-306 (1986). Holoshitz et al.
found that 4 of 5 T cell clones isolated from human rheumatoid
synovia which respond to M. tuberculosis antigens were CD4.sup.-
CD8.sup.- cells with .gamma./.delta. T cell receptors. Holoshitz,
J., et al., Nature, 339:226-229 (1989). This observation is
interesting because .gamma./.delta. T cells have yet to be assigned
a role in immunity. One of the .gamma./.delta. clones was tested
for its ability to respond to purified mycobacterial hsp60 and was
found to be positive in proliferation assays. Due to the conserved
nature of stress proteins, these T cells have the potential for
autoreactivity. Lamb and coworkers have shown that polyclonal T
cells from synovial infiltrates recognize both mycobacterial hsp60
and hsp70. Lamb, J. R., et al., Intl. Immunol., in press (1989).
The population of T cells that recognize the mycobacterial stress
proteins were shown to respond to E. coli hsp60 and hsp70 and, most
interestingly, human hsp70 purified from heat shocked macrophages.
Thus, immune responses to conserved stress protein determinants,
perhaps initiated by bacterial infection (not necessarily by
mycobacteria), may play an important role in autoimmune pathology
in rheumatoid arthritis, as well as in adjuvant arthritis.
[0041] Stress Proteins and Immune Surveillance
[0042] A variety of different T cell types has now been shown to
recognize highly conserved stress protein determinants. The ability
of cells to respond to stress by increasing the levels of the
highly conserved stress proteins; the presence of T cells of
diverse phenotypes in healthy individuals that are capable of
recognizing self stress protein determinants; and observations that
stress responses are induced by pathogenic infections and by cell
transformation, all suggest a model of immune surveillance in which
self-reactive T cells provide a first line of defense against
infection and transformation by recognizing and helping to
eliminate stressed autologous cells, as well as cells infected with
intracellular pathogens. The pool of lymphocytes that recognize
conserved stress protein determinants might be induced during
establishment of natural microbial flora on the skin and in the
gut, and maintained by frequent stimulation by pathogens, such as
bacteria and viruses, as well as other stressful stimuli
encountered during a normal lifetime. This model is attractive
because it provides a way in which the immune system could exploit
the existence of conserved epitopes in stress proteins to respond
immediately to antigenically diverse pathogens and cellular
changes, producing an initial defense that need not await the
development of immunity to novel antigens.
[0043] The lymphocytes which recognize conserved stress protein
determinants must be capable of discriminating between normal and
stressed cells. Since many stress proteins are constitutively
expressed in normal cells, although at lower levels than in
stressed cells, the potential for autoreactivity is ever-present.
Normal cells may escape destruction by expressing only
substimulatory levels of stress protein determinants on their
surfaces. In addition, stress proteins may only be processed and
presented during stress, and it may be relevant that many stress
proteins have altered intracellular locations during stress.
Finally, immune regulatory networks may prevent activation of
autoreactive T cells under normal conditions. The regulatory
constraints required by this system might occasionally break down,
perhaps during stress caused by bacterial or viral infections,
leading to autoimmune disease. Rheumatoid arthritis may be such a
disease.
[0044] Modulation of Immune Response
[0045] The precise relationship between stress proteins and the
host immune response to infection is as yet undefined. When cells
are subjected to a variety of stresses, they respond by selectively
increasing the synthesis of a limited set of stress proteins. Some
stress proteins, including the products of DnaK and GroEL, are
major constituents of the cell under normal growth conditions and
are induced to even higher levels during stress. Lindquist, S.,
Annu. Rev. Biochem. 55:1151-1191 (1986); Neidhardt, F. C. and R. A.
VanBogelen, In Escherlchia coli and Salmonella Typhimurium,
Cellular and Molecular Biology, (eds. Neidhardt, F. C., Ingraham,
J. L. Low, K. B. Magasanik, B. Schaechter, M. and Umbarger, H. E.)
Am. Soc. Microbiol., Washington, D.C., pp. 1134-1345 (1987). It has
now been demonstrated that stress-related proteins are targets of
the immune response. Young, D. et al., Proc. Natl. Acad. Sci. USA,
85:4267-4270 (1988). It is reasonable to expect that immunodominant
antigens would be found among such abundant proteins, as has now
been shown to be the case.
[0046] According to the method of the present invention, it is
possible to modulate the immune response in an individual, such as
a human, other mammal or other vertebrate, by altering the
individual's response to stress proteins. In particular, it is
possible to enhance or induce an individual's response to a
pathogen (e.g., bacteria, virus, parasites, or other organism or
agent, such as toxins, toxoids) or to cancer cells or enhance or
induce an upregulation of an individual's immune status (such as in
an immune compromised individual or HIV-infected individual); and
to decrease an individual's autoimmune response, such as occurs in
some forms of arthritis. In addition, administration of a stress
protein using the method of the present invention provides
protection against subsequent infection by a pathogen. As
demonstrated herein, stress proteins contain regions of highly
conserved amino acid sequences and have been shown to be major
immunodominant antigens in bacterial and other infections.
Therefore, it is reasonable to expect stress proteins can be used
to elicit strong immune responses against a variety of pathogens.
The stress protein administered to induce or enhance an immune
response to pathogens can be the stress protein of the pathogen
against which an immune response is desired or other stress
protein, a portion of that protein of sufficient size to stimulate
the desired immune response or a protein or amino acid sequence
which is the functional equivalent of the stress protein in that it
is sufficiently homologous in amino acid sequence to that of the
stress protein to be capable of eliciting the desired response (an
immune response substantially similar to that which occurs in
response to the stress protein) in the individual to whom it is
administered. The term "sufficiently homologous in amino acid
sequence to that of the stress protein" means that the amino acid
sequence of the protein or polypeptide will generally show at least
40% identity with the stress protein amino acid sequence; in some
cases, the amino acid sequence of a functional equivalent exhibits
approximately 50% identity with the amino acid sequence of the
stress protein.
[0047] Any stress-induced proteins or their functional equivalents
can be used by the present invention to enhance or induce an immune
response in an individual (e.g. a human, other mammal or
vertebrate), against an infection by a pathogen, for immunotherapy
against cancer cells, for generally upregulating an individual's
immune status and for use in inducing immune tolerance in an
individual or animal.
[0048] The stress proteins of the present invention can be
administered in a variety of ways to modulate the immune response
of an individual (e.g., a human, other mammal or other vertebrate).
In one embodiment, the stress protein is administered as a vaccine
which is comprised of the stress protein or a portion of the stress
protein which is of sufficient size to stimulate the desired immune
response. In this embodiment, the vaccine can be a "specific
vaccine" which contains a specific stress protein of a particular
pathogen against which an immune response is desired, such as a
bacterial stress protein. In this case, since the pathogen's stress
proteins are distinguishable from those of the host, it is possible
to induce an immunoprophylactic response specific to the pathogen's
stress proteins. Blander, S. J., et al. J. Clin. Invest.,
91:717-723 (1993). This can be carried out by administering a
vaccine which includes all or a portion (e.g., sufficient amino
acid sequence to have the desired stimulatory effect on immune
response) of the pathogen's stress protein or of another protein
having an amino acid sequence sufficiently similar to that of the
stress protein sequence to stimulate the immune response to the
pathogen's stress protein. Alternatively, in the case of a pathogen
which does not contain stress proteins, (e.g. some viruses) or in
the condition of neoplasia, stress proteins or highly conserved
stress protein determinants, such as those shown to be recognized
by a variety of T cells, can be administered as a type of "general"
vaccine to achieve an upregulation of the immune response.
Administration of such a vaccine will enhance the existing immune
surveillance system. For instance, a vaccine which includes a
bacterial, or other stress protein can be administered to enhance
the immune system which will result in an immune response against a
pathogen which does not contain stress proteins. Alternatively,
this type of "general" vaccine can be used to enhance an
individual's immune response against cancer or to generally
upregulate an individual's immune status, such as in an immune
compromised individual (e.g., an individual undergoing chemotherapy
or an HIV-infected individual). In either case of this embodiment
(specific or general vaccine), the immune response to the stress
protein sequence will be increased and effects of the pathogen,
disease condition or immune impairment will be reduced (decreased,
prevented or eliminated).
[0049] In another embodiment, stress proteins can be used to
enhance immune surveillance by applying local heat or any other
substances or changes in condition which induce the stress response
in the individual being treated. (This can also be employed in
conjunction with the specific vaccine, described previously,
administered to enhance an immune response to a stress
protein-containing pathogen or in conjunction with the general
vaccine, described above, administered to enhance the immune
response against a pathogen which does not contain its own stress
proteins, cancer, or to upregulate the immune status of an
individual). For example, it is known that increased levels of
stress proteins are produced in many types of cancer cells.
Therefore, enhancement of the immune surveillance system, using
this embodiment of the present invention as described, can be used
to facilitate destruction and/or to prevent progression or
establishment of cancer cells.
[0050] The method of the present invention can also be used to
modify or modulate an individual's response to his or her own cells
(e.g., as in autoimmune diseases). There are at least two ways in
which the present invention can be used immunotherapeutically.
First, stress proteins, such as heat shock proteins (e.g., hsp 70
and hsp60), are known to be involved in autoimmune disease. It is,
thus, possible to turn down an individual's immune response,
resulting in the individual becoming more tolerant of the protein.
Second, because it is known that under some circumstances, one
component of the immune response in certain autoimmune diseases can
be to stress proteins, it is possible to selectively inhibit or
interfere with the ability of immune cells which normally interact
with such proteins to do so. This can be done, for example, by
administering monoclonal antibodies that bind to specific T cell
receptors and delete or disable such cells. Alternatively, rather
than knocking out immune cells, the stress response in cells can be
turned down by administering a drug capable of reducing a cell's
ability to undergo the stress response. For example, a drug
targeted to or specific for heat shock transcription factor, which
is needed to stimulate heat shock genes, can be administered. The
transcription factor is rendered nonfunctional or subfunctional
and, as a result, cells' ability to undergo the stress response is
also lessened.
[0051] In another embodiment of the present invention, the stress
protein is administered as a vaccine which is comprised of two
moieties: a stress protein and another substance (referred to as an
antigen, e.g. protein, peptide, carbohydrate, lipid, organic
molecule) against which an immune response is desired. The two
moieties are conjugated or joined to form a single unit.
conjugation can be achieved by chemical means known to those
skilled in the art (e.g. through a covalent bond between the stress
protein and the second moiety; reductive amination) or, as
demonstrated in Example 2, by recombinant techniques. If
recombinant techniques are used to produce the conjugate, the
result is a recombinant fusion protein which includes the stress
protein and the antigen in a single molecule. This makes it
possible to produce and purifiy a single recombinant molecule in
the vaccine production process. In this embodiment, the stress
protein can be seen to act as an adjuvant-free carrier, and it
stimulates strong humoral and T cell responses to the substance to
which the stress protein is fused. The stress protein can be
conjugated to any substance against which an immune response is
desired or to a portion of the substance sufficient to induce an
immune response in an individual to whom it is administered. The
substance includes but is not limited to proteins (e.g.,
ovalalbumin, Influenza virus Hemagglutinin, Human Immunodeficiency
Virus p24), peptides (e.g., Human Immunodeficiency Virus peptides,
melanoma antigen peptides), oligosaccharides (e.g., Neiserria
meningitidis group B, Streptococcus pneumoniae type 14, Hemophilis
influenzae type b), lipids, carbohydrates (e.g., glycolipid
antigens in human cancers such as GD3, GM2, Gb3, Forssman antigen,
Sialosyl-Le.sup.a antigen and glycoprotein antigens in human
cancers such as CEA, AFP, PSA, Tn antigen), organic molecules or a
combination thereof. Recent evidence demonstrating the
effectiveness of such a vaccine indicates that mycobacterial hsp70
proteins when conjugated to other proteins act as adjuvant-free
carriers. The humoral immune response to some peptides conjugated
to mycobacterial hsp70 administered without any adjuvant was very
similar to the antibody response to the same peptides administered
in Freund's complete adjuvant. Lussow, A. R., et al., Eur. J.
Immun., 21:2297-2302 (1991). Barrios, C. et al., Eur. J. Immun.,
22:1365-1372 (1992).
[0052] The present invention also relates to compositions which are
conjugates comprised a stress protein joined to another substance
or component. For example, the present invention relates to a
conjugate in which a stress protein is chemically linked to an
antigen, or in which a stress protein is fused to an antigen (e.g.,
a fusion protein).
[0053] As demonstrated in Example 3, the HIV p24 gag gene was
subcloned into the stress protein fusion vector pKS70 (FIG. 6),
containing the T7 RNA polymerase promoter, a polylinker and the
mycobacterial tuberculosis hsp70 gene. The resulting vector pKS72
(FIG. 6) was used to produce the p24-hsp70 fusion protein in E.
coli. Adjuvant-free, purified p24-hsp70 fusion protein was injected
into Balb/c mice and as shown in FIG. 7, the anti-p24 antibody
titer was 2.7 orders of magnitude higher in mice injected with the
p24-hsp70 fusion protein than in mice injected with p24 alone or
hsp70 alone. Mice injected with p24 and the adjuvant, alum, also
produced an antibody response to p24. Finally, a demonstrable T
cell response was seen in mice injected with the p24-hsp70 fusion
protein and in mice injected with p24 alone.
[0054] In another embodiment of the present invention, the stress
protein or a portion of the stress protein which is of sufficient
size to stimulate an immune response or an equivalent, is
administered as an adjuvant, with another substance (referred to as
an antigen) against which an immune response is desired. The stress
protein can be used as an adjuvant with any substance or antigen
against which an immune response is desired or to a portion of the
substance sufficient to induce an immune response in an individual
to whom it is administered. The substance includes proteins,
peptides, oligosaccharides, lipids, carbohydrates, organic
molecules or a combination thereof. Via linkage to a stress
protein, strong and specific B and T cell mediated immunity can be
generated in a mammalian host (e.g., mice, rabbits, humans) to
virtually any organic molecule. This is particularly useful 1) with
substances (e.g., antigens) which alone are non-immunogenic; 2)
when adjuvants cannot be used or do not work well in combination
with a particular antigen; 3) when the availability of purified
antigen is limited, particularly with fusion proteins where the
antigen is made using recombinant DNA technology; 4) where other
carrier molecules, such as KLH, BSA, OVA or thyrogloulin, which
additionally require adjuvants, are not effective or desirable; 5)
there is a genetic restriction in the immune response to the
antigen; 6) there is a pre-existing immunosuppression or
non-responsiveness to an antigen (e.g., pediatric vaccines where
infants and children under 2 years of age do not generate
protective immunity to carbohydrate antigens well); and 7) the type
of immune response achieved by other carriers or adjuvants is
undesirable or ineffectual (i.e., stress protein conjugates could
be used to bias toward either B or T cell immunity via proper dose,
route and inoculation regimen).
[0055] The present invention also relates to a method of generating
monoclonal or polyclonal antibodies to a substance using a
conjugate comprised of a stress protein joined to the substance. In
this embodiment, an effective amount of the conjugate (i.e., an
amount which results in an immune response in the host) is
introduced into a mammalian host which results in production of
antibodies to the substance in the host. The antibodies are remvoed
from the host and purified using known techniques (e.g.,
chromatography), thereby resulting in production of polyclonal
antibodies. Alternatively, the antibodies produced using the method
of the present invetion can be used to generate hybridoma cells
which produce monoclonal antibodies using known techniques (Kohler,
G., et al., Nature, 256:495(1975) Milstein et al., Nature,
266:550-552(1977); Koprowski et al., Proc. Natl. Acad. Sci,
74:2985-2988 (1977); Welsh, Nature, 266:495(1977); Maniatis, T. et
al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Lab., Cold Spring Harbor, N.Y. (1982)).
[0056] The stress protein, stress protein portion, stress protein
functional equivalent and the substance to which the stress protein
is fused or conjugated present in the vaccine can be produced or
obtained using known techniques. For example, the stress protein or
stress protein portion can be obtained (isolated) from a source in
which it occurs in nature, can be produced by cloning and
expressing a gene encoding the desired stress protein or stress
protein portion or can be synthesized chemically or
mechanically.
[0057] An effective dosage of the stress proteins of the present
invention as vaccines or adjuvants, to elicit specific cellular and
humoral immunity to stress proteins, or to substances conjugated to
the stress proteins, such as proteins or oligosaccharides, is in
the range of 0.1 to 1000 ug hsp per injection, depending on the
individual to whom the stress protein is being administered.
Lussow, A. R., et al., Eur. J. Immun., 21:2297-2302 (1991).
Barrios, C. et al., Eur. J. Immun., 22:1365-1372 (1992). The
appropriate dosage of the stress protein for each individual will
be determined by taking into consideration, for example, the
particular stress protein being administered, the type of
individual to whom the stress protein is being administered, the
age and size of the individual, the condition being treated or
prevented and the severity of the condition. Those skilled in the
art will be able to determine using no more than routine
experimentation, the appropriate dosage to administer to an
individual.
[0058] Various delivery systems can be used to administer an
effective dose of the vaccine of the present invention. Methods of
introduction include, for example, intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural
and oral routes. Any other convenient route of administration can
be used (infusion of a bolus injection, infusion of multiple
injections over time, absorption through epithelial or
mucocutaneous linings such as, oral mucosa, rectal and intestinal
mucosa) or a series of injections over time.
[0059] The present invention is further illustrated by the
following exemplification, which is not intended to be limiting in
any way.
EXEMPLIFICATION
EXAMPLE 1
Isolation and Characterization of Mycobacterial Stress Protein
Antigens
[0060] Recombinant DNA Clones
[0061] The isolation and characterization of M. tuberculosis and M.
leprae .lambda.gtll genomic DNA clones with murine monoclonal
antibodies have been described. Husson, R. N. and Young, R. A.,
Proc. Natl. Acad. Sci., USA 84:1679-1683 (1987); Young, R. A., et
al., Nature (London) 316:450-452 (1985). DNA was isolated from
these clones and was manipulated by standard procedures. Davis, R.
W., Advanced Bacterial Genetics: A Manual for Genetic Engineering
(Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.), (1980).
[0062] DNA Sequence Analysis
[0063] DNA was subcloned into vector M13mp18 or M13mp19 (New
England Biolabs), as suggested by the supplier. Dideoxynucleotide
chain-termination reactions and gel electrophoresis of the
sequenced reduced were as described. Davis, R. W., Advanced
Bacterial Genetics: A Manual for Genetic Engineering (Cold Spring
Harbor Lab., Cold Spring Harbor, N.Y.), (1980). DNA sequences were
determined for both strands of DNA. Computer analysis of sequences
with UWGCG programs was as described by Devereux, J., et al.,
Nucleic Acids Res., 12:387-395 (1984).
[0064] Immunoblot Analysis
[0065] Escherichia coil strain TG1 was transformed with the
following plasmids by standard procedures (Maniatis, T., et al.,
Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Lab.,
Cold Spring Harbor, N.Y.) (1982), with selection for ampicillin
resistance: pND5, a derivative of pBR325 containing the E. coli
GroEL genes (Jenkins, A. J., et al., Mol. Gen. Genet., 202:446-454
(1986); pUC8 (Vic, J., Gene, 19:259-268 (1982); pUC8 with insert
DNA for .lambda.gtll clone Y3178 (M. leprae 65-kDa antigen, Young,
R. A., et al., Nature, (London) 316:450-452 (1985)) ligated in the
EcoRI site.
[0066] Overnight cultures of E. coli strains in Luria-Bertani (LB)
medium were centrifuged and resuspended in isotonic
phosphate-buffered saline at a cell density corresponding to an
absorbance of 2 at 600 nm. An equal volume of sample buffer
containing 2% (wt/vol) NaDodSo.sub.4 was added, and, after heating
on a boiling water bath for 2 min, samples were electrophoresed on
12% (wt/vol) polyacrylamide gels in the presence of NaDodSO.sub.4.
Blots were prepared by electrophoretic transfer of the proteins to
a nitrocellulose membrane, and binding of monoclonal antibodies was
assayed with a peroxidase-conjugated secondary antibody as
described. Young, D. B., et al., Infect. Immun., 55:1421-1425
(1987).
[0067] Six M. tuberculosis and six M. leprae proteins have been
implicated in the immune response to the mycobacterial pathogens
(Table 1). To obtain clues to the normal cellular function of
several of these mycobacterial antigens, DNA clones encoding these
proteins, isolated by using monoclonal antibodies to probe lambda
gtll libraries (Husson, R. N. and Young, R. A., Proc. Natl. Acad.
Sci. USA, 84:1679-1683 (1987); Young, R. A., et al., Nature,
(London) 316:450-452 (1985)) were subjected to sequence analysis.
The sequences elucidated have been submitted to the GenBank
sequence database.
[0068] The Mycobacterial 71-k Da Antigen. The 71-k Da antigen of M.
tuberculosis is recognized by human T cells during infection (Table
1).
1TABLE 1 MYCOBACTERIAL PROTEIN ANTIGENS Subjected to Homology
Recognized by sequence with known Protein, kDA Human T Cells
analysis proteins M. tuberculosis 71 + + DnaK 65* + + GroEL 38 + -
- 19 + + None 14 + - - 12 ND - - M. leprae 70 ND - DnaK 65 + +
GroEL 36 + - - 28 + - - 18 + + Plant Hsp 12 ND - - Mycobacterial
protein antigens, their recognition by human T cells, and homology
of the deduced mycobacterial protein sequences to known # proteins
are summarized. ND, not determined; +, yes; -, no *Includes data
derived from study of the 65-kDA antigens of M. bovis BCG (Bacillus
Calmette-Gurein), which is identical to the M. tuberculosis 65-kDA
antigen. + A. S. Mustafa, J. R. Lamb, D. Young and R. A. Young,
unpublished data.
[0069] Mycrobacterial protein antigens, their recognition by human
T cells, and homology of the deduced mycobacterial protein
sequences to known proteins are summarized. ND, not determined; +,
yes; -, no
[0070] The insert DNA of lambdagtll clone Y3271 (Husson, R. N., et
al., Proc. Natl. Acad. Sci, USA, 84:1679-1683 (1987), was sequenced
to obtain amino acid sequence information for the 71-kDa antigen of
M. tuberculosis. This clone produces a beta-galactosidase fusion
protein containing the carboxyl-terminal one-third of the 71-kDa
antigen exhibiting 40% amino acid sequence identity with the
comparable segment of the dnaK gene product from E. coli (Bardwell,
J. C., et al., Proc. Natl. Sci., USA, 81:848-852 (1984)), (FIG. 1).
FIG. 1A shows the extent of sequence similarity between portions of
the mycobacterial and the E. coli 70-k Da polypeptides. Sequences
transcriptionally downstream from the mycobacterial 71-k Da gene
predict a 356-amino acid protein homologous to the is E. coli dnaJ
gene product (unpublished data), indicating that the E. coli
dnaK-dnaJ operon structure is conserved in M. tuberculosis and
consistent with the conclusion that the mycobacterial 71-kDa
antigen is a homologue of the E. coli dnaK gene product. The
product of the dnaK gene is a member of the 70-kDa heat shock
protein family that is highly conserved among prokaryotes and
eukaryotes (Bardwell, J. C., et al., Proc. Natl. Acad. Sci., USA,
81:848-852 (1984); Lindquist, S., Annu. Rev. Biochem., 55:1151-1191
(1986).
[0071] The M. leprae 70-k Da antigen cross-reacts with monoclonal
antibodies directed to the M. tuberculosis 70-kDa antigen. M.
tuberculosis and M. leprae are both members of the 70-k Da heat
shock protein family of stress proteins.
[0072] The mycobacterial 65-kDa antigen. The 65-kDa antigens of M.
tuberculosis and M. leprae are involved in the human T-cell
response to mycobacterial infection (Table 1). Genes encoding these
proteins have been isolated (Husson, R. N., and Young, R. A., Proc.
Natl. Acad. 35 Sci., USA, 84:1679-1683 (1987); Young, R. A., et
al., Nature, (London) 316:450-452 (1985)) and sequenced (Shinnick,
T. M., J. Bacteriol., 169:1080-1088 (1987); Mehram, V., et al.,
Proc. Natl. Acad. Sci., USA 83: 7013-7017 (1986)), revealing that
the amino acid sequences of the 65-kDa antigens of M. tuberculosis
(SEQ ID NO: 4) and M. leprae (SEQ ID NO: 3) are 95% identical.
These proteins sequences exhibited no significant sequence
similarity to proteins in the GenBank database.
[0073] Identification of these proteins was based on the
observation that some monoclonal antibodies directed against the
mycobacterial 65-kDa antigens cross-react with an E. coli protein
of 60 kDa. E. coli cells transformed with the plasmid pND5 (Sanger,
F., et al., Proc. Natl. Acad. Sci., USA 74: 5463-5467 (1977), which
contains the E. coli gro E genes, had been shown to accumulate
large amounts of the 60-kDa protein. A comparison of the
mycobacterial 65-kDa protein sequences with those determined for E.
coli groEl (C. Woolford, K. Tilly, C. Georgopoulous, and R. H.,
unpublished data) revealed the extent of the sequence similarity as
shown in FIG. 1B.
[0074] The 60-kDa Gro EL protein is a major stress protein in E.
coli. Lindquist, S., Annual. Rev. Biochem., 55:1151-1191 (1986);
Nature, 333: 330-334 (1988). There is some evidence that the
mycobacterial 65-kDa proteins accumulate in response to stress:
Mycobacterium bovis BCG (bacillus Calmette-Guerin) cultures grown
in zinc-deficient medium are substantially enriched in this protein
(De Bruyn, J., et al., Infect. Immun. 55: 245-252 (1987)). This
infers that the 65-kDa proteins of M. tuberculosis and M. leprae
are homologues of the E. coli Gro EL protein.
[0075] Other Mycobacterial Antigens
[0076] T lymphocytes that respond to the M. tuberculosis 19-kDa
antigen and the M. leprae 18-kDa antigen have been observed in
humans with tuberculosis and leprosy, respectively (Table 1). DNA
encoding these antigens was sequenced from the .lambda.gtll clones
Y3148 (Husson, R. N. and Young, R. A., Proc. Natl. Acad. Sci. USA
84: 1679-1683 (1987); and Y3179 (Young, R. A., et al., Nature,
(London) 316: 450-452 (1985)), respectively. The M. tuberculosis
19-kDa protein sequence predicted from the DNA exhibited no
significant sequence similarity to proteins in the GenBank
database.
[0077] However, the M. leprae 18-kDa protein sequence was similar
to the soybean 17-kDa protein heat shock protein, a protein
representation of a major class of plant heat shock proteins
(Schoffl, F. and Van Bogelen, R. A., In: Escherichia coli and
Salmonella typhimurium, Cellular and Molecular Biology, Am. Soc.
Microbiol., Washington, D.C. (1987).
EXAMPLE 2
Construction of Stress Protein-Fusion Vaccines for Use as
Adjuvant-Free Carriers in Immunizations
[0078] Recombinant Fusion Vectors
[0079] A series of stress protein fusion vectors for use in E. coli
were constructed and are shown in FIG. 5. These vectors contain the
T7 RNA polymerase promoter fused to the M. bovis BCG hsp70 gene or
the M. bovis BCG hsp60 gene. The vectors also contain a polylinker
with multiple cloning sites, permitting incorporation of a gene of
interest so that the antigen encoded by that gene is expressed as a
fusion protein with the stress protein. A subset of these vectors
permit incorporation of the foreign gene with a coding sequence for
a C-terminal 6-Histidine "tag" for ease of fusion protein
purification. Thus far, recombinant clones have been generated that
produce hsp70 proteins fused to HIV gag and HIV pol proteins.
[0080] Purification of Stress Protein Fusions
[0081] Two strategies have been developed to purify the recombinant
fusion proteins. The T7 system usually produces such large amounts
of protein that it forms inclusion bodies, permitting purification
by centrifugation. The preliminary results indicate that an
hsp70-HIV gag fusion protein accounts for about 20% of total E.
coli protein in the T7 system. If necessary, other fusion proteins
can be purified via the 6-Histidine "tag".
EXAMPLE 3
Adjuvant-Free Carrier Effect of HSP70 in Vitro
[0082] The stress protein fusion vector pKS70 (FIG. 6), containing
the T7 RNA polymerase promoter, a polylinker and the mycobacterial
tuberculosis hsp70 gene, was constructed. The HIV p24 gag gene was
subcloned into pKS70 using the Ndel and BamHI sites and the
resulting pKS72 vector (FIG. 6) was used to produce the p24-hsp70
fusion protein in E. coli. The fusion protein was purified as
inclusion bodies and further purified using ATP-agarose
chromatography and MonoQ ion exchange chromatography.
[0083] The p24-hsp70 protein in phosphate buffered saline (PBS), in
the absence of an adjuvant, was injected intraperitoneally into
Balb/c mice. As controls, the p24 protein alone in PBS or the hsp70
protein alone in PBS was injected into different groups of mice.
Three weeks later, the mice were boosted and finally, three weeks
after the boost, the mice were bled. The anti-p24 antibody titer
was then determined by ELISA. Mice injected with 25 pmoles of
p24-hsp70 had antibody levels 2.7 orders of magnitude higher than
mice injected with p24 alone or hsp70 alone (FIG. 7). Results of
experiments in which mice were injected with p24 and the adjuvant,
alum, also showed that there was an antibody response to p24. In
addition, mice injected with the p24-hsp70 fusion protein and mice
injected with p24 alone produced a demonstrable T cell
response.
[0084] Equivalents
[0085] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
Sequence CWU 1
1
* * * * *