U.S. patent application number 10/180593 was filed with the patent office on 2003-09-04 for stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens.
This patent application is currently assigned to Mount Sinai School of Medicine of New York University. Invention is credited to Srivastava, Pramod K..
Application Number | 20030165516 10/180593 |
Document ID | / |
Family ID | 22782837 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165516 |
Kind Code |
A1 |
Srivastava, Pramod K. |
September 4, 2003 |
Stress protein-peptide complexes as prophylactic and therapeutic
vaccines against intracellular pathogens
Abstract
Disclosed is a family of vaccines that contain stress
protein-peptide complexes which when administered to a mammal are
operative to initiate in the mammal a cytotoxic T cell response
against cells infected with a preselected intracellular pathogen.
Also disclosed are methodologies for preparing and administering
vaccines containing such stress protein-peptide complexes.
Inventors: |
Srivastava, Pramod K.;
(Riverdale, NY) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Mount Sinai School of Medicine of
New York University
|
Family ID: |
22782837 |
Appl. No.: |
10/180593 |
Filed: |
June 25, 2002 |
Related U.S. Patent Documents
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Application
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10180593 |
Jun 25, 2002 |
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09412420 |
Oct 5, 1999 |
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6455503 |
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09412420 |
Oct 5, 1999 |
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08704727 |
Jun 19, 1997 |
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6048530 |
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08704727 |
Jun 19, 1997 |
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PCT/US95/03311 |
Mar 16, 1995 |
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PCT/US95/03311 |
Mar 16, 1995 |
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08210421 |
Mar 16, 1994 |
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5961979 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
Y02A 50/466 20180101;
A61K 47/646 20170801; C12N 2760/16122 20130101; A61K 39/39
20130101; A61K 2039/55522 20130101; A61P 31/00 20180101; A61K
2039/57 20130101; A61K 2039/622 20130101; Y02A 50/41 20180101; Y10S
530/806 20130101; A61K 39/385 20130101; Y02A 50/403 20180101; A61K
2039/6043 20130101; A61P 37/04 20180101; C07K 14/47 20130101; Y02A
50/30 20180101; C07K 17/02 20130101; Y02A 50/478 20180101; A61K
39/12 20130101; Y10S 436/823 20130101; C12N 2760/16134
20130101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 039/00 |
Claims
What is claimed is:
1. A vaccine for administration to a mammal for inducing in the
mammal a cytotoxic T cell response against a preselected
intracellular pathogen, the vaccine comprising: (a) an immunogenic
stress protein-peptide complex operative to initiate in said mammal
a cytotoxic T cell response against said pathogen, said complex
comprising, a peptide that is present in a eukaryotic cell infected
with said pathogen but not present in said cell when said cell is
not infected with said pathogen, complexed with a stress protein;
and (b) a pharmaceutically acceptable carrier.
2. A vaccine for administration to a mammal for inducing in said
mammal resistance to infection by a preselected intracellular
pathogen, the vaccine comprising: (a) an immunogenic stress
protein-peptide complex operative to initiate in said mammal, by
means of a cytotoxic T cell response in said mammal, resistance to
infection by said pathogen, said complex comprising, a peptide that
is present in a eukaryotic cell infected with said pathogen but not
present in said cell when said cell is not infected with said
pathogen, complexed with a stress protein; and (b) a
pharmaceutically acceptable carrier.
3. The composition of claim 1 or 2, wherein said stress protein is
a member of the stress protein families selected from the group
consisting of Hsp60, Hsp70, and Hsp90.
4. The composition of claim 1 or 2, wherein said stress protein is
a gp96.
5. The composition of claim 1 or 2 further comprising a
cytokine.
6. The composition of claim 5, wherein said cytokine is selected
from the group consisting of IL-1.alpha., IL-1.beta., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IFN.alpha., IFN.beta., IFN.gamma., TNF.alpha., TNF.beta., G-CSF,
GM-CSF, GM-CSF and TGF-.beta..
7. The composition of claim 1 or 2, wherein said eukaryotic cell is
an immortalized eukaryotic cell.
8. The composition of claim 1 or 2, wherein said mammal is a
human.
9. The composition of claim 1 or 2, wherein said pathogen is a
virus.
10. The composition of claim 9, wherein said virus is selected from
the group consisting of hepatitis type A, hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex
type I, herpes simplex type II, rinderpest, rhinovirus, echovirus,
rotavirus, respiratory synctial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsachie virus, mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I, and human
immunodeficiency virus type II.
11. The composition of claim 1 or 2, wherein said pathogen is a
bacteria.
12. The composition of claim 11, wherein said bacteria is selected
from the group consisting of Mycobacteria, Rickettsia, Neisseria
and Legionella.
13. The composition of claim 1 or 2, wherein said pathogen is a
protozoa.
14. The composition of claim 13, wherein said protozoa is selected
from the group consisting of Leishmania, Trypanosoma and
Kokzidioa.
15. The composition of claim 1 or 2, wherein said pathogen is an
intracellular parasite.
16. The composition of claim 15, wherein said parasite is selected
from the group consisting of Chlamydia and Rickettsia.
17. The composition of claim 1 or 2, wherein said peptide is non
covalently complexed with said stress protein.
18. A method of inducing in a mammal a cytotoxic T cell response
against a preselected intracellular pathogen that causes disease in
said mammal, the method comprising: administering to said mammal a
vaccine comprising, (a) an immunogenic stress protein-peptide
complex operative to initiate in said mammal a cytotoxic T cell
response against said pathogen and comprising, a peptide that is
present in a eukaryotic cell infected with said pathogen but not
present in said cell when said cell is not infected with said
pathogen, complexed with a stress protein, and (b) a
pharmaceutically acceptable carrier, in an amount sufficient to
elicit in said mammal a cytotoxic T cell response against said
pathogen.
19. A method of inducing in a mammal resistance to infection by a
preselected intracellular pathogen that-causes disease in said
mammal, the method comprising: administering to said mammal a
vaccine comprising, (a) an immunogenic stress protein-peptide
complex operative to initiate in said mammal cytotoxic T cell
response against said pathogen and comprising, a peptide that is
present in a eukaryotic cell infected with said pathogen but not
present in said cell when said cell is not infected with said
pathogen, complexed with a stress protein, and (b) a
pharmaceutically acceptable carrier, in an amount sufficient to
induce in said mammal, by means of the cytotoxic T cell response in
said mammal, resistance to infection by said pathogen.
20. The method of claim 18 or 19, wherein said cytotoxic T cell
response is mediated by the class I major histocompatibility
complex.
21. The method of claim 18 or 19, wherein said stress protein is a
member of the stress protein families selected from the group
consisting of Hsp60, Hsp70 , and Hsp90.
22. The method of claim 18 or 19, wherein said stress protein is a
gp96.
23. The method of claim 18 or 19, wherein said composition further
comprises a cytokine.
24. The method of claim 23, wherein said cytokine is selected from
the group consisting of IL-1.alpha. IL-1.beta., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN.alpha.,
IFN.beta., IFN.gamma., TNF.alpha., TNF.beta., G-CSF, GM-CSF, GM-CSF
and TGF-.beta..
25. The method of claim 18 or 19, wherein said eukaryotic cell is
an immortalized eukaryotic cell.
26. The method of claim 18 or 19, wherein said mammal is a
human.
27. The method of claim 18 or 19, wherein said vaccine is
administered prophylactically to said mammal for stimulating in
said manual a cytotoxic T cell response for preventing subsequent
infection of said mammal by said pathogen.
28. The method of claim 18 or 19, wherein said vaccine is
administered therapeutically to said mammal for stimulating in said
mammal a cytotoxic T cell response against said pathogen presently
infecting said mammal.
29. The method of claim 18 or 19, wherein said vaccine is
administered to said mammal in an amount in the range of about 0.1
to about 1000 micrograms of complex/kg body weight of
mammal/immunization.
30. The method of claim 29, wherein said amount is in range of
about 0.5 to about 100 micrograms of complex/kg body weight of
mammal/immunization.
31. A method for preparing a vaccine for inducing in a mammal a
cytotoxic T cell response against a preselected intracellular
pathogen, the method comprising: (a) harvesting from a eukaryotic
cell infected with said pathogen an immunogenic stress
protein-peptide complex comprising, a peptide that is present in
said cell infected with said pathogen but not present in said cell
when said cell is not infected with said pathogen complexed with a
stress protein, said complex, when administered to said mammal,
being operative at initiating in said mammal a cytotoxic T cell
response against said pathogen; and (b) combining said complex with
a pharmaceutically acceptable carrier.
32. A method for preparing a vaccine for inducing in a mammal a
cytotoxic T cell response against a preselected intracellular
pathogen, the method comprising: (a) reconstituting in vitro, a
peptide that is present in a eukaryotic cell infected with said
pathogen but not present in said cell when said cell is not
infected with said pathogen and a stress protein, thereby to
generate a stress protein-peptide complex, which when administered
to said mammal is operative to initiate a cytotoxic T cell response
against said pathogen in said mammal; and (b) combining said
complex with a pharmaceutically acceptable carrier.
33. The method of claim 32, wherein said stress protein is
harvested in the presence of ATP prior to reconstitution.
34. The method of claim 32, wherein said stress protein is treated
with low pH prior to reconstitution.
35. The method of claim 31 or 32, wherein said stress protein is a
member of the stress protein families selected from the group
consisting of Hsp60, Hsp70 and Hsp90.
36. The method of claim 31 or 32, wherein said stress protein is a
gp96.
Description
FIELD OF THE INVENTION
[0001] The invention relates generally to the field of vaccine
development. More particularly, the invention relates to the
development of prophylactic and therapeutic vaccines effective
against intracellular pathogens.
BACKGROUND OF THE INVENTION
[0002] The development of vaccines directed against intracellular
pathogens, for example, viruses, bacteria, protozoa, fungi, and
intracellular parasites, is ongoing. The development and use of
vaccines has proved invaluable in preventing the spread of disease
in man. For example, in 1967, smallpox was endemic in 33 countries
with 10 to 15 million cases being reported annually. At that time,
the World Health Organization introduced a program to eradicate
smallpox. Approximately one decade later, smallpox was successfully
eradicated from the human population.
[0003] Theoretically, an ideal vaccine has a long shelf life, is
capable of inducing with a single dose long lasting immunity
against a preselected pathogen and all of its phenotypic variants,
is incapable of causing the disease to which the vaccine is
directed against, is effective therapeutically and
prophylactically, is prepared easily and economically using
standard methodologies, and can be administered easily in the
field.
[0004] Presently four major classes of vaccine have been developed
against mammalian diseases. These include: live-attenuated
vaccines; non living whole vaccines; vector vaccines; and subunit
vaccines. Several reviews discuss the preparation and utility of
these classes of vaccines. See for example, Subbarao et al. (1992)
in Genetically Engineered Vaccines, edited by Ciardi et al., Plenum
Press, New York; and Melnick (1985) in High Technology Route to
Virus Vaccines, edited by Dreesman et al., published by the
American Society for Microbiology, the disclosures of which are
incorporated herein by reference. A summary of the advantages and
disadvantages of each of the four classes of vaccines is set forth
below.
[0005] Live attenuated vaccines comprise live but attenuated
pathogens, i.e., non-virulent pathogens, that have been "crippled"
by means of genetic mutations. The mutations prevent the pathogens
from causing disease in the recipient or vaccinee. The primary
advantage of this type of vaccine is that the attenuated organism
stimulates the immune system of the recipient in the same manner as
the wild type pathogen by mimicking the natural infection.
Furthermore, the attenuated pathogens replicate in the vaccinee
thereby presenting a continuous supply of antigenic determinants to
the recipient's immune system. As a result, live vaccines can
induce strong, long lasting immune responses against the wild type
pathogen. In addition, live vaccines can stimulate the production
of antibodies which neutralize the pathogen. Also they can induce
resistance to the pathogen at its natural portal of entry into the
host. To date, live attenuated vaccines have been developed
against: smallpox; yellow fever; measles; mumps; rubella;
poliomyelitis; adenovirus; and tuberculosis.
[0006] Live attenuated vaccines, however, have several inherent
problems. First, there is always a risk that the attenuated
pathogen may revert back to a virulent phenotype. In the event of
phenotypic reversion, the vaccine may actually induce the disease
it was designed to provide immunity against. Second, it is
expensive and can be impractical to develop live vaccines directed
against pathogens that continuously change their antigenic
determinants. For example, researchers have been unable to develop
a practical live vaccine against the influenza virus because the
virus continually changes the antigenic determinants of its coat
proteins. Third, live attenuated vaccines may not be developed
against infections caused by retroviruses and transforming viruses.
The nucleic acids from these viruses may integrate into the
recipients genome with the potential risk of inducing cancer in the
recipient. Fourth, during the manufacture of live attenuated
vaccines adventitious agents present in the cells in which the
vaccine is manufactured may be copurified along with the attenuated
pathogen. Alien viruses that have been detected in vaccine
preparations to date include the avian leukosis virus, the simian
papovavirus SV40, and the simian cytomegalovirus. Fifth, live
vaccine preparations can be unstable therefore limiting their
storage and use in the field. Presently, attempts are being made to
develop stabilizing agents which enhance the longevity of the
active vaccines.
[0007] Non living whole vaccines comprise non viable whole
organisms. The pathogens are routinely inactivated either by
chemical treatment, i.e., formalin inactivation, or by treatment
with lethal doses of radiation. Non living whole vaccines have been
developed against: pertussis; typhus; typhoid fever; paratyphoid
fever; and particular strains of influenza.
[0008] In principle, non living vaccines usually are safe to
administer because it is unlikely that the organisms will cause
disease in the host. Furthermore, since the organism is dead the
vaccines tend to be stable and have long shelf lives. There are,
however, several disadvantages associated with non living whole
vaccines. First, considerable care is required in their manufacture
to ensure that no live pathogens remain in the vaccine. Second,
vaccines of this type generally are ineffective at stimulating
cellular responses and tend to be ineffective against intracellular
pathogens. Third, the immunity elicited by non viable vaccines is
usually short-lived and must be boosted at a later date. This
process repeatedly entails reaching the persons in need of
vaccination and also raises the concern about hypersensitizing the
vaccinee against the wild type pathogen.
[0009] Vector vaccines, also known as live recombinant vehicle
vaccines, may be prepared by incorporating a gene encoding a
specific antigenic determinant of interest into a living but
harmless virus or bacterium. The harmless vector organism is in
turn to be injected into the intended recipient. In theory, the
recombinant vector organism replicates in the host producing and
presenting the antigenic determinant to the host's immune system.
It is contemplated that this type of vaccine will be more effective
than the non-replicative type of vaccine. For such a vaccine to be
successful, the vector must be viable, and be either naturally
non-virulent or have an attenuated phenotype.
[0010] Currently preferred vectors include specific strains of:
vaccinia (cowpox) virus, adenovirus, adeno-associated virus,
salmonella and mycobacteria. Live strains of vaccinia virus and
mycobacteria have been administered safely to humans in the form of
smallpox and tuberculosis (BCG) vaccines, respectively. They have
been shown to express foreign proteins and exhibit little or no
conversion into virulent phenotypes. Several types of vector
vaccines using the BCG vector currently are being developed against
the human immunodeficiency virus (HIV). For example, the HIV
antigenic proteins: gag; env; HIV protease; reverse transcriptase;
gp120 and gp41 have been introduced, one at a time, into the BCG
vector and shown to induce T cell mediated immune responses against
the HIV proteins in animal models (Aldovini et al. (1991) Nature
351:479-482; Stover et al. (1991) Nature 351:456-460; Colston
(1991) Nature 351:442-443).
[0011] Vector vaccines are capable of carrying a plurality of
foreign genes thereby permitting simultaneous vaccination against a
variety of preselected antigenic determinants. For example,
researchers have engineered several HIV genes into the vaccinia
virus genome thereby creating multivalent vaccines which therefore
are, in theory, capable of simultaneously stimulating a response
against several HIV proteins.
[0012] There are several disadvantages associated with vector
vaccines. First, it is necessary to identify suitable strains of
viable but non-pathogenic organisms that may act as carriers for
the genes of interest. Second, vector vaccines can be prepared only
when a potentially protective antigenic determinants has been
identified and characterized. Accordingly, vector vaccines cannot
be prepared against pathogens whose antigenic determinant has not
yet been identified or are so variable that the prospect of
identifying the antigenic determinant for each variant is
impractical. Third, the genes encoding the preselected antigenic
determinant must be stably transfected and expressed in the
preferred carrier organism. Consequently, the methodologies
required for developing this type of vaccine are both labor
intensive and time consuming. Fourth, it has not yet been
established that recombinant vector vaccines effectively immunize a
recipient against a preselected pathogen.
[0013] Subunit vaccines usually comprise a subcellular component
purified from the pathogen of interest. Subunit vaccines usually
are safe to administer because it is unlikely that the subcellular
components will cause disease in the recipient. The purified
subcellular component may be either a defined subcellular fraction,
purified protein, nucleic acid or polysaccharide having an
antigenic determinant-capable of stimulating an immune response
against the pathogen. The antigenic components can be purified from
a preparation of disrupted pathogen. Alternatively, the antigenic
proteins, nucleic acids or polysaccharides may be synthesized using
procedures well known in the art. Diseases that have been treated
with subunit type vaccines include: cholera; diphtheria; hepatitis
type B; poliomyelitis; tetanus; and specific strains of
influenza.
[0014] There are, however, several disadvantages associated with
subunit vaccines. First, it is important to identify and
characterize the protective antigenic determinant. This can be a
labor intensive and time consuming process. As a result it may be
impractical to develop subunit vaccines against pathogens with
highly variable antigenic deterninants. Second, subunit vaccines
generally are ineffective at stimulating cytotoxic T cell responses
and so they may be ineffective at stimulating an immune response
against intracellular pathogens. Third, the immunity elicited by
subunit vaccines is usually short-lived, and like the non living
whole vaccines must be boosted at a later date therefore raising
the concern about hypersensitizing the vaccinee against the wild
type pathogen.
[0015] Heretofore, many of the inactivated whole and subunit
vaccines have not been sufficiently immunogenic by themselves to
induce strong, protective responses. As a result, immunostimulants
including, for example, aluminum hydroxide; intact mycobacteria;
and/or mycobacterial components have been co-administered with
these vaccines to enhance the immune response stimulated by the
vaccine. Recently, experiments have shown that mycobacterial heat
shock proteins may act as carriers for peptide vaccines thereby
enhancing the immunogenicity of the peptides in vivo (Lussow et al.
(1991) Eur. J. Immunol. 21:2297-2302). Further studies have shown
that administering a composition to mice comprising an antigenic
peptide chemically crosslinked to a purified mycobacterial stress
protein stimulates a humoral (antibody mediated) rather than a
temporal (cell mediated) response against the antigenic peptide
Barrios et al. (1992) Eur. J. Immunol. 22:1365-1372).
[0016] However, because it is generally believed that cellular
responses are required for immunizing against intracellular
pathogens (see for example, "Advanced Immunology," Male et al.
(1991) Gower Medical Publishing; Raychaudhuri et al. (1993)
Immunology Today 14: 344-348) it is contemplated that conventional
subunit and inactivated whole organism vaccines may be ineffective
at stimulating immune responses, specifically cytotoxic T cell
responses, against intracellular pathogens.
[0017] It is an object of the instant invention to provide a safe
subunit vaccine comprising a stress protein-peptide complex for
administration to a mammal that is capable of inducing, by means of
a cytotoxic T cell response, resistance to infection by a
preselected intracellular pathogen. The vaccines prepared in
accordance with the invention may be used to elicit an immune
response against an intracellular pathogens whose antigenic
determinants have been identified, have not yet been identified, or
where it is impractical to isolate and characterize each of the
antigenic determinants. The vaccines prepared in accordance with
the invention may be prophylactically and therapeutically effective
against preselected pathogens.
[0018] Another object of the invention is to provide a method for
inducing in a mammal resistance to infection by an intracellular
pathogen by administering to the mammal a stress protein-peptide
subunit vaccine. Another object is to provide a method for rapidly
and cost effectively producing commercially feasible quantities of
the stress protein-peptide vaccines from a cell or cell line
infected with the intracellular pathogen or alternatively from a
cell or cell line transfected with, and expressing a gene encoding
a specific antigenic determinant. Still another object is to
provide a method for preparing an immunogenic stress
protein-peptide subunit vaccine by reconstituting in vitro
immunologically unreactive stress proteins and peptides thereby to
produce immunoreactive complexes capable of stimulating an immune
response against a preselected intracellular pathogen.
[0019] These and other objects and features of the invention will
be apparent from the description, drawings, and claims which
follow.
SUMMARY OF THE INVENTION
[0020] It has now been discovered that a subunit vaccine containing
a stress protein-peptide complex when isolated from cells infected
with a preselected intracellular pathogen and then administered to
a mammal can effectively stimulate cellular immune responses
against cells infected with the same pathogen. Specifically, the
immune response is mediated through the cytotoxic T cell cascade
which targets and destroys cells containing intracellular
pathogens.
[0021] The vaccines prepared in accordance with the methodologies
described herein provide an alternative approach for stimulating
cellular immunity thereby obviating the use of live (attenuated or
otherwise) intracellular pathogens. In addition, the vaccines
described herein are ideal for inducing immune responses against
intracellular pathogens having either defined or as yet undefined
immunogenic determinants. Furthermore the vaccines may be used to
induce immune responses against intracellular pathogens whose
antigenic determinants are either diverse or constantly changing
thereby making the isolation and characterization of antigenic
determinants impractical.
[0022] In a preferred aspect, the invention comprises a vaccine
that can be administered to a mammal for inducing in the mammal a
cytotoxic T cell response against a preselected intracellular
pathogen. Also, it is contemplated that the vaccines may induce in
the mammal, by means of a cytotoxic T cell response, resistance to
infection by the preselected intracellular pathogen. The vaccines
manufactured in accordance with the principles described herein
contain an immunogenic stress protein-peptide complex that is
capable of stimulating in the recipient a cytotoxic T cell response
directed against cells infected with the pathogen of interest. The
complex when combined with a pharmaceutically acceptable carrier,
adjuvant, or excipient may be administered to a mammal using
techniques well known in the art.
[0023] The term "vaccine", as used herein, is understood to mean
any composition containing a stress protein-peptide complex having
at least one antigenic determinant which when administered to a
mammal stimulates in the mammal an immune response against the
antigenic determinant.
[0024] The term "stress protein" as used herein, is understood to
mean any cellular protein which satisfies the following criteria.
It is a protein whose intracellular concentration increases when a
cell is exposed to stressful stimuli, is capable of binding other
proteins or peptides, and is capable of releasing the bound
proteins or peptides in the presence of adenosine triphosphate
(ATP) or low pH. Stressful stimuli include, but are not limited to,
heat shock, nutrient deprivation, metabolic disruption, oxygen
radicals, and infection with intracellular pathogens.
[0025] It will be apparent to the artisan upon reading this
disclosure that other recombinant stress proteins, including non
native forms, truncated analogs, muteins, fusion proteins as well
as other proteins capable of mimicking the peptide binding and
immunogenic properties of a stress protein may be used in the
preparation of stress protein-peptide vaccines disclosed
herein.
[0026] The first stress proteins to be identified were the heat
shock proteins (Hsp). As their name suggests, Hsps are induced by a
cell in response to heat shock. Three major families of Hsp have
been identified and are called Hsp60, Hsp70 and Hsp90 because of
their respective molecular weights of about 60, 70, and 90 kD. Many
members of these families subsequently were found to be induced in
response to other stressful stimuli, such as those mentioned
above.
[0027] Stress proteins are found in all prokaryotes and eukaryotes
and exhibit a remarkable level of evolutionary conservation. For
example, DnaK, the Hsp70 from E. coli has about 50% amino acid
sequence identity with Hsp70 proteins from eukaryotes (Bardwell et
al. (1984) Proc. Natl. Acad. Sci. 81:848-852). The Hsp60 and Hsp90
families also exhibit similarly high levels of intrafamilial
conservation (Hickey et al. (1989) Mol. Cell Biol. 9:2615-2626;
Jindal (1989) Mol. Cell. Biol. 9:2279-2283). In addition, it has
been discovered that the Hsp-60, Hsp-70, and Hsp-90 families are
composed of proteins that are related to the stress proteins in
sequence, for example, having greater than 35% amino acid identity,
but whose expression levels typically remain unaltered under
conditions stressful to the host cell. An example of such a protein
includes the constitutively expressed cytosolic protein Hsc 70 to
which is related in amino acid sequence to the stress-induced
protein Hsp 70. Accordingly, it is contemplated the definition of
stress protein, as used herein, embraces other proteins, muteins,
analogs, and variants thereof having at least 35% to 55%,
preferably 55% to 75%, and most preferably 75% to 95% amino acid
identity with members of the three families whose expression levels
in a cell are stimulated in response to stressful stimuli.
[0028] The term "peptide", as used herein, is understood to mean
any amino acid sequence that is present in a eukaryotic cell
infected with an intracellular pathogen but which is not present in
a similar cell when the cell is not infected with the same
pathogen. The definition embraces peptides that not only originate
from the pathogen itself but also peptides which are synthesized by
the infected cell in response to infection by the intracellular
pathogen.
[0029] The term "immunogenic stress protein-peptide complex", as
used herein, is understood to mean any complex containing a stress
protein and a peptide that is capable of inducing an immune
response in a mammal. The peptides preferably are non covalently
associated with the stress protein. The complexes may include, but
are not limited to, Hsp60-peptide, Hsp70-peptide and Hsp90-peptide
complexes. In a preferred aspect of the invention a stress protein
belonging to the Hsp90 family, namely gp96 can be used to generate
an effective vaccine containing a gp96-peptide complex. Since the
peptides can be dissociated from the complex in the presence of ATP
or low pH potentially antigenic peptides can be isolated from cells
infected with a preselected intracellular pathogen. Consequently,
the antigenic determinants for potentially any intracellular
pathogen of interest can be identified readily using the
methodologies described herein.
[0030] The term "cytotoxic T cell", as used herein, is understood
to mean any T lymphocyte expressing the cell surface glycoprotein
marker CD8 that is capable of targeting and lysing a target cell
which bears a class I histocompatibility complex on its cell
surface and which is infected with an intracellular pathogen. The
term "cytotoxic T cell response" is understood to mean any
cytotoxic activity that is mediated by cytotoxic T cells.
[0031] As used herein, the term "intracellular pathogen" is
understood to mean any viable organism, including, but not limited
to, viruses, bacteria, fungi, protozoa and intracellular parasites,
capable of existing within a mammalian cell and causing a disease
in the mammal.
[0032] In a preferred aspect of the invention, the stress
protein-peptide vaccines have particular utility in treating human
diseases caused by intracellular pathogens. It is contemplated that
the vaccines developed using the principles described herein will
be useful in treating diseases of other mammals, for example, farm
animals including: cattle; horses; goats; sheep; and pigs, and
household pets including: cats; and dogs.
[0033] Vaccines may be prepared that stimulate cytotoxic T cell
responses against cells infected with viruses including, but not
limited to, hepatitis type A, hepatitis type B, hepatitis type C,
influenza, varicella, adenovirus, herpes simplex type I (HSV-I),
herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory synctial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsachie virus, mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I (HIV-I), and human
immunodeficiency virus type II (HIV-II). Vaccines also may be
prepared that stimulate cytotoxic T cell responses against cells
infected with intracellular bacteria, including, but not limited
to, Mycobacteria, Rickettsia, Mycoplasma, Neisseria and Legionella.
Vaccines also may be prepared that stimulate cytotoxic T cell
responses against cells infected with intracellular protozoa,
including, but not limited to, Leishmania, Kokzidioa, and
Trypanosoma. Vaccines may be prepared that stimulate cytotoxic T
cell responses against cells infected with intracellular parasites
including, but not limited to, Chlamydia and Rickettsia.
[0034] In another preferred embodiment of the invention, the stress
protein-peptide vaccine may also contain a therapeutically
effective amount of a cytokine. As used herein, the term "cytokine"
is meant to mean any secreted polypeptide that influences the
function of other cells mediating an immune response. Currently,
preferred cytokines include: interleukin-1.alpha. (IL-1.alpha.),
interleukin-1.beta. (L-1.beta.), interleukin-2 (IL-2),
interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5),
interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),
interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11
(IL-11), interleukin-12 (IL-12), interferon .alpha. (IFN.alpha.),
interferon .beta. (IFN.beta.), interferon .gamma., (IFN.gamma.),
tumor necrosis factor .alpha. (TNF.alpha.)), tumor necrosis factor
.beta. (TNF.beta.), granulocyte colony stimulating factor (G-CSF),
granulocyte/macrophage colony stimulating factor (GM-CSF), and
transforming growth factor .beta. (TGF-.beta.). It is contemplated
that other but as yet undiscovered cytokines may be effective in
the invention. In addition, conventional antibiotics may be
co-administered with the stress protein-peptide complex. The choice
of a suitable antibiotic or a combination thereof, however, will be
dependent upon the disease in question.
[0035] It has been discovered that the vaccine stimulates the
cytotoxic T cell response via the major histocompatibility complex
(MHC) class I cascade. Thus, it is contemplated that the cytotoxic
T cell response may be enhanced further by co-administering the
vaccine with a therapeutically effective amount of one or more of
cytokines that potentiate or modulate cytotoxic T cell
responses.
[0036] Another preferred embodiment, the invention provides a
method for stimulating in a mammal a cellular immune response,
specifically a cytotoxic T cell response, against cells infected
with a preselected intracellular pathogen. The method involves
administering to the mammal a vaccine made in accordance with the
principles disclosed herein in an amount sufficient to elicit in
the mammal a cytotoxic T cell response against the preselected
intracellular pathogen.
[0037] The vaccine may be administered prophylactically to a mammal
in order to stimulate in the mammal a cytotoxic T cell response
that prevents subsequent infection of the mammal by the
intracellular pathogen. Alternatively, the vaccine may be
administered therapeutically to a mammal having a disease caused by
an intracellular pathogen. It is contemplated that the vaccine may
stimulate a cytotoxic T cell response against cells presently
infected with the intracellular pathogen.
[0038] The dosage and means of administration of the family of
stress protein-peptide vaccines necessarily will depend upon the
nature of the complex, the intracellular pathogen and the nature of
the disease in question. The complex should be administered in an
amount sufficient to initiate a cytotoxic T cell response against
the intracellular pathogen. In general, the amount of stress
protein-peptide complex administered may range from about 0.1 to
about 1000 micrograms of complex/kg body weight of the
mammal/immunization, and preferably in the range of about 0.5 to
100 micrograms of complex/kg body weight of the
mammal/immunization. The recipient preferably should be vaccinated
four times at weekly intervals. If necessary, the responses may be
boosted at a later date by subsequent administration of the
vaccine. It is contemplated, however, that the optimal dosage and
vaccination schedule may be determined empirically for each stress
protein-peptide vaccine complex by an artisan using conventional
techniques well known in the art.
[0039] In another aspect, the invention provides a variety of
methodologies for preparing commercially available amounts of the
stress-protein peptide vaccines which when administered to a mammal
induce in the mammal a cytotoxic T cell response against cells
infected with a preselected antigen. In one approach, the stress
protein-peptide complex may be harvested using conventional protein
purification methodologies from a sample of tissue, an isolated
cell or immortalized cell line infected with the preselected
intracellular pathogen, or an isolated cell or immortalized cell
line transfected with, and expressing a gene encoding a preselected
antigenic determinant. The purified complex subsequently may be
stored or combined with a pharmaceutically acceptable carrier for
administration as a vaccine.
[0040] Alternatively, the stress protein-peptide complex may be
prepared by reconstituting a potentially antigenic peptide and a
stress protein in vitro. For example, the antigenic peptide may be
eluted from either a purified stress protein-peptide complex or a
MHC-peptide complex using methodologies well known in the art.
Specifically, the peptides may be eluted from the stress
protein-peptide complex by incubating the complex in the presence
of ATP or low pH. Alternatively, the peptides may be eluted from
the MHC-peptide complex by incubating the complex in the presence
of trifluoroacetic acid (TFA). The resulting peptides may be
purified by reverse phase HPLC and their amino acid sequences
determined by standard protein sequencing methodologies. Peptides
of defined sequence then may be synthesized using conventional
peptide synthesis methodologies. Stress proteins may be purified
directly from cells naturally expressing the stress proteins.
Alternatively, recombinant stress proteins, including non native
forms, truncated analogs, muteins, fusion proteins as well as other
constructs capable of mimicking the peptide binding and immunogenic
properties of stress proteins may be expressed using conventional
recombinant DNA methodologies. For example, a recombinant stress
protein may be expressed from recombinant DNA in either a
eukaryotic or prokaryotic expression system and purified from the
expression system. The two purified components then may be combined
in vitro to generate a synthetic and completely defined stress
protein-peptide complex. The immunogenicity and specificity of the
recombinant-complexes subsequently may be assayed in vitro and in
vivo to identify useful candidate complexes that stimulate
cytotoxic T cell responses against a preselected intracellular
pathogen. Once identified, the synthetic complexes may be prepared
on any scale, stored as is, or combined with pharmaceutically
acceptable carriers for administration to mammals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The foregoing and other objects and features of the
invention, as well as the invention itself, may be more fully
understood from the following description, when read together with
the accompanying drawings, in which:
[0042] FIG. 1 shows antigen specific cytotoxic T cell activity of
splenocytes derived from mice immunized with a gp96-peptide complex
harvested from BALB/c fibroblasts transfected with the
nucleoprotein (NP) gene from the PR8 influenza virus. The cytotoxic
activity was assayed by the release of .sup.51Cr from BALB/c
fibroblasts expressing the NP gene (filled circles), BALB/c
fibroblasts expressing the NP gene but treated with the anti-MHC
type I antisera K44 (empty circles) and from the syngeneic non-NP
transfected cell line 5117 (asterisks).
[0043] FIG. 2 shows antigen specific cytotoxic T cell activity of
splenocytes derived from mice immunized with gp96-peptide complex
harvested from SV40 transformed SVB6 cells. The cytotoxic activity
was assayed by the release of .sup.51Cr from SVB6 cells (filled
circles) and from a non-SV40 transformed syngeneic cell line, MCA
(empty circles).
[0044] FIGS. 3A-3D shows antigen specific cytotoxic T cell
activities of splenocytes derived from two mice immunized with a
reconstituted Hsp70-peptide complex where the peptide has the
sequence SLSDLRGYVYQGL (SEQ. ID. NO. 1). Prior to performing the
assay, the splenocytes derived from each mouse were stimulated
either once (3A and 3C) or twice (3B and 3D) in vitro with lethally
irradiated cells transfected with, and expressing the peptide
SLSDLRGYVYQGL (SEQ. ID. NO. 1). Cytotoxic activity was assayed by
the release of .sup.51Cr from EL4 cells expressing the peptide
(filled triangle) and from EL4 cells not expressing the peptide
(empty triangles).
DETAILED DESCRIPTION
[0045] The invention is based on the discovery that a stress
protein-peptide complex when isolated from a eukaryotic cell
infected with a preselected intracellular pathogen and then
administered to a mammal can stimulate a cytotoxic T cell response
directed against cells infected with the same pathogen. This
discovery provides a significant advance to the field of vaccine
development.
[0046] In accordance with the invention, the aforementioned
discovery is exploited to provide a family of vaccines which may be
used to immunize mammals against diseases caused by intracellular
pathogens. In principle, the vaccines can be prepared against any
intracellular pathogen of interest, for example: viruses; bacteria;
protozoa; fungi; or intracellular parasites. Generic methodologies
useful for preparing vaccines against all of these classes of
pathogens are discussed in detail hereinbelow.
[0047] As will be appreciated by those skilled in the art, the
stress protein-peptide vaccines described herein have several
advantages over the vaccines currently available. First, the stress
protein-peptide vaccines provide an alternative approach for
stimulating cellular immunity and obviate the use of intact
intracellular (attenuated or otherwise) pathogens. Second, since
the vaccines do not-contain intact organisms this reduces the risk
of causing the disease the vaccine was designed to induce immunity
against. Third, the vaccines described herein are ideal for
inducing immune responses against either defined antigenic
determinants isolated from an intracellular pathogen or as yet
undefined antigenic determinants. Furthermore, vaccines may be
prepared that are effective against pathogens that normally evade
the immune system by evolving new antigenic coat proteins, i.e.,
the influenza virus. Fourth, vaccines of this type may in principle
be prepared against any intracellular pathogen of interest. Fifth,
the vaccines may be prepared synthetically using the methodologies
described hereinafter thereby providing completely defined vaccines
that are suitable for administration to humans.
[0048] It is contemplated that the vaccines may be administered
either prophylactically or therapeutically. When administered
prophylactically the vaccine may stimulate in the mammal a
cytotoxic T cell response that permits the vaccinee to resist
subsequent infection by the intracellular pathogen. Alternatively,
when administered therapeutically the vaccine may stimulate in the
mammal a cytotoxic T cell response against a pathogen which is
presently infecting and causing disease in the mammal.
[0049] The specific component of the vaccine that induces in the
recipient a specific cytotoxic T cell response against the pathogen
is a stress protein-peptide complex. The peptide may be any amino
acid sequence that is present in a eukaryotic cell infected with an
intracellular pathogen but which is not present when such a cell is
not infected with the same pathogen. This includes peptides that
not only originate from the pathogen itself but also are
synthesized by the infected cell in response to infection by the
intracellular pathogen.
[0050] The immunogenic complexes may be purified from any
eukaryotic cell, including: whole tissues; isolated cells; and
immortalized eukaryotic cell lines infected with the intracellular
pathogen. The complexes may be purified by using conventional
protein purification techniques well known in the art. For example,
it is contemplated that an immunogenic complex capable of
stimulating a cytotoxic T cell response against the influenza virus
may be harvested from a eukaryotic cell line that is infected with
the influenza virus.
[0051] In addition, it has been found that the peptide can be
eluted from the stress protein-complex either in the presence of
ATP or low pH. Neither the peptide nor the stress protein
individually are effective at inducing a cytotoxic T cell response.
These experimental conditions, however, may be exploited to isolate
peptides from infected cells which may contain potentially useful
antigenic determinants. Once isolated, the amino acid sequence of
each antigenic peptide may be determined using conventional amino
acid sequencing methodologies. Consequently, the antigenic
determinants for potentially any intracellular pathogen of interest
can be identified readily using the methodologies described herein.
As discussed in detail hereinafter, this property may be exploited
in the preparation of completely synthetic vaccines.
[0052] Similarly, it has been found that potentially immunogenic
peptides may be eluted from MHC-peptide complexes using techniques
well known in the art. See for example, Falk et al. (1990) Nature
348:248-251; Rotzsche et al. (1990) Nature 348:252-254; Elliott et
al. (1990) Nature 348:195-197; Falk et al. (1991) Nature
351:290-296, Demotz et al. (1989) Nature 334:682-684; Rotzsche et
al. (1990) Science 249:283-287, the disclosures of which are
incorporated herein by reference. Although the peptides eluted from
the MHC complexes may define a potentially protective antigenic
determinant, it is appreciated that administration of the isolated
peptide in a conventional subunit vaccine may be ineffective at
stimulating a cytotoxic T cell response in the recipient.
Consequently, it is contemplated that the peptides eluted from
MHC-peptide complexes may be reconstituted with a stress protein,
using the methodologies described herein, thereby to generate a
stress protein-peptide complex effective at stimulating a cytotoxic
T cell response capable of targeting and lysing cells expressing
the antigenic peptide.
[0053] Stress proteins useful in the practice of the instant
invention may be defined as any cellular protein that satisfies the
following criteria. It is a protein whose intracellular
concentration increases when a cell is exposed to a stressful
stimuli, it is capable of binding other proteins or peptides, and
it is capable of releasing the bound proteins or peptides in the
presence of adenosine triphosphate (ATP) or low pH.
[0054] The first stress proteins to be identified were the heat
shock proteins (Hsp). As their name implies, Hsps are synthesized
by a cell in response to heat shock. To date, three major families
of Hsp have been identified based on molecular weight. The families
have been called Hsp60, Hsp70 and Hsp90 where the numbers reflect
the approximate molecular weight of the stress proteins in kD. Many
members of these families subsequently were found to be induced in
response to other stressful stimuli including, but not limited to,
nutrient deprivation, metabolic disruption, oxygen radicals, and
infection with intracellular pathogens. See for example: Welch (May
1993) Scientific American 56-64; Young (1990) Annu. Rev. Immunol.
8:401-420; Craig (1993) Science 260:1902-1903; Gething et al.
(1992) Nature 355:33-45; and Lindquist et al. (1988) Annu. Rev.
Genetics 22:631-677, the disclosures of which are incorporated
herein by reference. Accordingly, it is contemplated that stress
proteins belonging to all three families may be useful in the
practice of the instant invention.
[0055] The major stress proteins can accumulate to very high levels
in stressed cells, but they occur at low to moderate levels in
cells that have not been stressed. For example, the highly
inducible mammalian Hsp70 is hardly detectable at normal
temperatures but becomes one of the most actively synthesized
proteins in the cell upon heat shock (Welch et al. (1985), J. Cell.
Biol. 101:1198-1211). In contrast, Hsp90 and Hsp60 proteins are
abundant at normal temperatures in most, but not all, mammalian
cells and are further induced by heat (Lai et al. (1984), Mol.
Cell. Biol. 4:2802-10; van Bergen en Henegouwen et al. (1987),
Genes Dev. 1:525-531).
[0056] Stress proteins are among the most highly conserved proteins
in existence. For example, DnaK, the Hsp70 from E. coli has about
50% amino acid sequence identity with Hsp70 proteins from
eukaryotes (Bardwell et al. (1984) Proc. Natl. Acad. Sci.
81:848-852). The Hsp60 and Hsp90 families also show similarly high
levels of intrafamilial conservation (Hickey et al. (1989) Mol.
Cell Biol. 9:2615-2626; Jindal (1989) Mol. Cell. Biol.
9:2279-2283). In addition, it has been discovered that the Hsp60,
Hsp70 and Hsp90 families are composed of proteins that are related
to the stress proteins in sequence, for example, having greater
than 35% amino acid identity, but whose expression levels typically
remain unaltered under conditions stressful to the host cell. An
example of such a protein includes the constitutively expressed
cystolic protein Hsc 70 which is related in amino acid sequence to
the stress-induced protein Hsp 70. It is, therefore, contemplated
that the definition of stress protein, as used herein, embraces
other proteins, muteins, analogs, and variants thereof having at
least 35% to 55%, preferably 55% to 75%, and most preferably 75% to
95% amino acid identity with members of the three families whose
expression levels in a cell are enhanced in response to a stressful
stimulus. The purification of stress proteins belonging to these
three families is described below.
[0057] The immunogenic stress protein-peptide complexes of the
invention may include any complex containing a stress protein and a
peptide that is capable of inducing an immune response in a mammal.
The peptides preferably are non covalently associated with the
stress protein. Preferred complexes may include, but are not
limited to, Hsp60-peptide, Hsp70-peptide and Hsp90-peptide
complexes. For example, a stress protein called gp96 which is
present in the endoplasmic reticulum of eukaryotic cells and is
related to the cytoplasmic Hsp90s can be used to generate an
effective vaccine containing a gp96-peptide complex.
[0058] Another family of low molecular weight heat shock proteins
has now been identified and is called Hsp 25/Hsp 27. The
purification of these proteins is discussed below. It is
contemplated that these low molecular weight proteins may also have
utility in the instant invention.
[0059] It has been discovered also that the stress protein-peptide
complexes of the invention can be prepared from cells infected with
an intracellular pathogen as well as cells that have been
transformed by an intracellular pathogen. For example, immunogenic
stress protein peptide complexes may be isolated from eukaryotic
cells transformed with a transforming virus such as SV40, see
below.
[0060] In a preferred aspect of the invention, the purified stress
protein-peptide vaccines may have particular utility in the
treatment of human diseases caused by intracellular pathogens. It
is appreciated, however, that the vaccines developed using the
principles described herein will be useful in treating diseases of
other mammals, for example, farm animals including: cattle; horses;
sheep; goats; and pigs, and household pets including: cats; and
dogs, that similarly are caused by intracellular pathogens.
[0061] In accordance with the methods described herein, vaccines
may be prepared that stimulate cytotoxic T cell responses against
cells infected with viruses including, but not limited to,
hepatitis type A, hepatitis type B, hepatitis type C, influenza,
varicella, adenovirus, HSV-I, HSV-II, rinderpest rhinovirous,
echovirus, rotavirus, respiratory synctial virus, papilloma virus,
papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsachie virus, mumps virus, measles virus, rubella virus, polio
virus, HIV-I, and HIV-II. Similarly, vaccines may also be prepared
that stimulate cytotoxic T cell responses against cells infected
with intracellular bacteria, including, but not limited to,
Mycobacteria, Rickettsia, Mycoplasma, Neisseria and Legionella. In
addition, vaccines may also be prepared that stimulate cytotoxic T
cell responses against cells infected with intracellular protozoa,
including, but not limited to, Leishmania, Kokzidioa, and
Trypanosoma. Furthermore, vaccines may be prepared that stimulate
cytotoxic T cell responses against cells infected with
intracellular parasites including, but not limited to, Chlamydia
and Rickettsia.
[0062] I. Propagation of Infected Eukaryotic Cells
[0063] As will be appreciated by those skilled in the art, the
protocols described herein may be used to isolate stress
protein-peptide complexes from any eukaryotic cell, for example,
tissues, isolated cells or immortalized eukaryotic cell lines
infected with a preselected intracellular pathogen.
[0064] When immortalized animal cell lines are used as a source of
the stress protein-peptide complex it is of course important to use
cell lines that can be infected with the pathogen of interest. In
addition, it is preferable to use cells that are derived from the
same species as the intended recipient of the vaccine.
[0065] For example, in order to prepare a stress protein-peptide
complex for administration to humans that may be effective against
HIV-I, the virus may be propagated in human cells which include,
but are not limited to, human CD4+ T cells, HepG2 cells, and U937
promonocytic cells. In order to prepare a stress protein-peptide
complex for administration to humans that may be effective against
HIV-II, the virus may be propagated in, for example, human CD4+ T
cells. Similarly, influenza viruses may be propagated in, for
example, human fibroblast cell lines and MDCK cells, and
mycobacteria may be cultured in, for example, human Schwaan
cells.
[0066] If the intracellular pathogens do not lyse the infected
cells then the infected cells are cultured under the same
conditions as the normal uninfected cells. For example,
mycobacteria may be propagated in nerve cultures of the sensory
ganglia of newborn Swiss white mice. The nerve cells are cultured
in a growth medium containing 70% Dulbecco modified Eagle minimal
essential medium (DMEM) with 0.006% glucose, 20% fetal calf serum,
10% chicken embryo extract and cytosine arabinoside. After eight to
ten days, the cultures are inoculated with 5-8.times.10.sup.6
mycobacteria isolated from fresh nodules of untreated lepromatous
leprosy patients. The infected cells may be cultured at 37.degree.
C., for up to 6 weeks, after which the infected cells are harvested
and the stress protein-peptide complexes isolated. See for example,
Mukherjee et al. (1985) J. Clin. Micro. 21:808-814, the disclosure
of which is incorporated herein by reference.
[0067] If, on the other hand, the host cells are lysed by the
pathogen of interest (as in the case of influenza virus) the cells
may still be grown under standard conditions except the cells are
washed and harvested just prior to lysis of the host cell. For
example, during the purification of stress protein-peptide
complexes from influenza infected cells, fibroblasts (or other cell
types) are infected for 1 hour at 37.degree. C. with 5-10 plaque
forming units (PFU) of virus per cell. The infected cells may be
cultured in plain DMEM medium for 24 hours at 37.degree. C. After
24 hours the cells are washed and harvested prior to lysis. The
stress protein-peptide complexes may be isolated using the
procedures set forth below.
[0068] In addition, when the gene encoding a particular antigenic
determinant has been identified, the gene of interest may be
transfected and expressed in an immortalized human or other
mammalian cell line using techniques well known in the art. See for
example "Current Protocols in Molecular Biology" (1989), eds.
Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J
A and Struhl K, Wiley Interscience, the disclosure of which is
incorporated by reference herein. The transfected cells may be
grown under standard conditions and the complexes isolated
subsequently.
[0069] II. Preparation of Stress Proteins and Immunogenic Stress
Protein-peptide Complexes
[0070] Methods for preparing Hsp70-peptide complexes, Hsp90-peptide
complexes, gp96-peptide complexes, Hsp70, Hsp25/Hsp27, and Hsp60
are set forth below.
[0071] (a) Purification of Hsp70-peptide Complexes
[0072] A pellet of infected cells is resuspended in 3 volumes of
1.times. Lysis buffer consisting of 5 mM sodium phosphate buffer
(pH7), 150 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM
phenyl methyl sulfonyl fluoride (PMSF). The pellet is sonicated, on
ice, until >99% cells are lysed as judged by microscopic
examination. Alternatively, the cells may be lysed by mechanical
shearing. In this procedure, the cells are resuspended in 30 mM
sodium bicarbonate pH 7.5, 1 mM PMSF, incubated on ice for 20 min.
and then homogenized in a dounce homogenizer until >95% cells
are lysed.
[0073] The lysate is centrifuged at 1000 g for 10 minutes to remove
unbroken cells, nuclei and other debris. The supernatant from this
centrifugation step is then recentrifuged at 100,000 g for 90
minutes.
[0074] The supernatant is mixed for 2-3 hours at 4.degree. C. with
Con A Sepharose equilibrated with PBS containing 2 mM Ca.sup.2+ and
2 mM Mg.sup.2+. When the cells are lysed by mechanical shearing,
the supernatant is diluted with equal volume of 233 Lysis Buffer
before proceeding. Then the slurry is packed into a column and
washed with 1.times. lysis buffer. The material that does not bind
is dialyzed for 36 hours (three times, 100 volumes each time)
against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM
PMSF. The dialyzate is centrifuged for 20 min. at 17,000 rpm
(Sorvall SS34 rotor) and the resulting supernatant applied to a
Mono Q FPLC column (Pharmacia) equilibrated in 20 mM Tris-Acetate
pH 7.5, 20 mM NaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. Then
the proteins are eluted with a 20 mM to 500 mM NaCl gradient. The
fractions are characterized by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDPAGE) and
immunoblotting using an appropriate anti-Hsp70 antibody (such as
clone N27F3-4 from StressGen).
[0075] The fractions that are strongly immunoreactive with the
antibody are pooled and the Hsp70-peptide complexes precipitated
with ammonium sulfate. The complex is precipitated in the 50%-70%
ammonium sulfate cut. The protein pellet is harvested by
centrifugation at 17,000 rpm (SS34 Sorvall rotor) and washed with
70% ammonium sulfate. Then the pellet is solubilized and the
residual ammonium sulfate removed by gel filtration on a
Sephadex.RTM. G25 column (Pharmacia).
[0076] The Hsp70-peptide complex can be purified to apparent
homogeneity using this method. Up to 1 mg of Hsp70-peptide complex
can be purified from 1 g of cells/tissue.
[0077] (b) Purification of Hsp70
[0078] The Hsp70 polypeptide may be purified from the Hsp70-peptide
complex by ATP agarose chromatography. See for example, Welch et
al. (1985) Mol. Cell. Biol. 5:1229, the disclosure of which is
incorporated herein by reference. Briefly, MgCl.sub.2 is added to
the previously isolated complex to a final concentration of 3 mM.
Then, the complex is applied to an ATP agarose column (Sigma
Chemical Co.) equilibrated in 20 mM Tris-Acetate (pH 7.5), 20 mM
NaCl, 0.1 mM EDTA, 15 mM 2-mercaptoethanol, 3 mM MgCl.sub.2. The
column is washed extensively with the equilibration buffer
containing 0.5M NaCl, and then washed with buffer without the NaCl.
Then the Hsp70 is eluted from the column with equilibration buffer
containing 3 mM ATP (Sigma Chemical Co.).
[0079] (c) Purification of Hsp90-peptide Complexes
[0080] A pellet of infected cells is resuspended in 3 volumes of
1.times. Lysis buffer consisting of 5 mM sodium phosphate buffer
(pH7), 150 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM PMSF.
The cell pellet is sonicated, on ice, until >95% cells are lysed
as judged by microscopic examination. Alternatively, the cells may
be lysed by mechanical shearing, as before
[0081] The lysate is centrifuged at 1000 g for 10 minutes to remove
unbroken cells, nuclei and other debris. The supernatant from this
centrifugation step subsequently is recentrifuged at 100,000 g for
90 minutes.
[0082] Then, the supernatant is mixed for 2-3 hours at 4.degree. C.
with Con A Sepharose equilibrated with PBS containing 2 mM
Ca.sup.2+ and 2 mM Mg.sup.2+. When the cells are lysed by
mechanical shearing, the supernatant is diluted with equal volume
of 2.times. Lysis Buffer before proceeding. Then, the slurry is
packed into a column and washed with 1.times. lysis buffer. The
material that does not bind is dialyzed for 36 hours (three times,
100 volumes each time) against 20 mM sodium phosphate pH 7.4, 1 mM
EDTA, 250 mM NaCl, 1 mM PMSF. The dialyzate is centrifuged at
17,000 rpm (Sorvall SS34 rotor) for 20 min. The resulting
supernatant is applied to a Mono Q FPLC column (Pharmacia)
equilibrated with lysis buffer and the bound proteins eluted with a
salt gradient of 200 mM to 600 mM NaCl.
[0083] The eluted fractions are analyzed by SDS-PAGE and the Hsp90
complexes identified by immunoblotting using an anti-Hsp90 antibody
(for example, 3G3 from Affinity Bioreagents). Hsp90 can be purified
to apparent homogeneity using this procedure. Approximately 150-200
.mu.g of Hsp90 can be purified routinely from 1 g of
cells/tissue.
[0084] (d) Purification of gp96 Peptide Complexes
[0085] A pellet of infected cells is resuspended in 4 volumes of
buffer consisting of 30 mM sodium bicarbonate buffer (pH7.5) and 1
mM PMSF and the cells allowed to swell on ice for 20 min. The cell
pellet then is homogenized in a Dounce homogenizer (the appropriate
clearance of the homogenizer will vary according to each cells
type) on ice until >95% cells are lysed.
[0086] The lysate is centrifuged at 1000 g for 10 minutes to remove
unbroken cells, nuclei and other debris. The supernatant from this
centrifugation step then is recentrifuged at 100,000 g for 90
minutes. The gp96-peptide complex can be purified either from the
100,000 g pellet or from the supernatant.
[0087] When purified from the supernatant, the supernatant is
diluted with equal volume of 2.times. Lysis Buffer and the
supernatant mixed for 2-3 hours at 4.degree. C. with Con A
Sepharose equilibrated with PBS containing 2mM Ca.sup.2+ and 2 mM
Mg.sup.2+. Then, the slurry is packed into a column and washed with
1.times. lysis buffer until the OD.sub.280 drops to baseline. Then,
the column is washed with 1/2 column bed volume of 10%
.alpha.-methyl mannoside (.alpha.-MM) dissolved in PBS containing 2
mM Ca.sup.2+ and 2 mM Mg.sup.2+, the column sealed with a piece of
parafilm, and incubated at 37.degree. C. for 15 min. Then the
column is cooled to room temperature and the parafilm removed from
the bottom of the column. Five column volumes of the .alpha.-MM
buffer are applied to the column and the eluate analyzed by
SDS-PAGE. Typically the resulting material is about 60-95% pure,
however this depends upon the cell type and the tissue-to-lysis
buffer ratio used. Then the sample is applied to a Mono Q FPLC
column (Pharmacia) equilibrated with a buffer containing 5 mM
sodium phosphate, pH7. The proteins then are eluted from the column
with a 0-1 M NaCl gradient and the gp96 fraction elutes between 400
mM and 550 mM NaCl.
[0088] This procedure, however, may be modified by two additional
steps, used either alone or in combination, to consistently produce
apparently homogeneous gp96-peptide complexes. One optional step
involves an ammonium sulfate precipatation prior to the Con A
purification step and the other optional step involves
DEAE-Sepharose purification after the Con A purification step but
before the Mono Q FPLC step.
[0089] In the first optional step, the supernatant resulting from
the 100,000 g centrifugation step is brought to a final
concentration of 50% ammonium sulfate by the addition ammonium
sulfate. The ammonium sulfate is added slowly while gently stirring
the solution in a beaker placed in a tray of ice water. The
solution is stirred for about 2 to 12 h. at 4.degree. C. and the
resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor).
The supernatant resulting from this step is removed, brought to 70%
ammonium sulfate saturation by the addition of ammonium sulfate
solution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor). The
resulting pellet from this step is harvested and suspended in PBS
containing 70% ammonium sulfate in order to rinse the pellet. This
mixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the
pellet dissolved in PBS containing 2 mM Ca.sup.2 and Mg.sup.2+.
Undissolved material is removed by a brief centrifugation at 15,000
rpm (Sorvall SS34 rotor). Then, the solution is mixed with Con A
Sepharose and the procedure followed as before.
[0090] In the second optional step, the gp96 containing fractions
eluted from the Con A column are pooled and the buffer exchanged
for 5 mM sodium phosphate buffer, pH 7,300 mM NaCl by dialysis, or
preferably by buffer exchange on a Sephadex G25 column. After
buffer exchange, the solution is mixed with DEAE-Sepharose
previously equilibrated with 5 mM sodium phosphate buffer, pH 7,
300 mM NaCl. The protein solution and the beads are mixed gently
for 1 hour and poured into a column. Then, the column is washed
with 5 mM sodium phosphate buffer, pH 7, 300 mM NaCl, until the
absorbance at 280 nM drops to baseline. Then, the bound protein is
eluted from the column with five volumes of 5 mM sodium phosphate
buffer, pH 7, 700 mM NaCl. Protein containing fractions are pooled
and diluted with 5 mM sodium phosphate buffer, pH 7 in order to
lower the salt concentration to 175 mM. The resulting material then
is applied to the Mono Q FPLC column (Pharmacia) equilibrated with
5 mM sodium phosphate buffer, pH 7 and the protein that binds to
the Mono Q FPLC column (Pharmacia) is eluted as described
before.
[0091] It is appreciated, however, that one skilled in the art may
assess, by routine experimentation, the benefit of incorporating
the optional steps into the purification protocol. In addition, it
is appreciated also that the benefit of adding each of the optional
steps will depend upon the source of the starting material.
[0092] When the gp96 fraction is isolated from the 100,000 g
pellet, the pellet is suspended in 5 volumes of PBS containing
either 1% sodium deoxycholate or 1% octyl glucopyranoside (but
without the Mg.sup.2+ and Ca.sup.2+) and incubated on ice for 1 h.
The suspension is centrifuged at 20,000 g for 30 min and the
resulting supernatant dialyzed against several changes of PBS (also
without the Mg.sup.2+ and Ca.sup.2+) to remove the detergent. The
dialysate is centrifuged at 100,000 g for 90 min, the supernatant
harvested, and calcium and magnesium are added to the supernatant
to give final concentrations of 2 mM, respectively. Then the sample
is purified by either the unmodified or the modified method for
isolating gp96-peptide complex from the 100,000 g supernatant, see
above.
[0093] The gp96-peptide complexes can be purified to apparent
homogeneity using this procedure. About 10-20 .mu.g of gp96 can be
isolated from 1 g cells/tissue.
[0094] (e) Purification of HSP25 and HSP27
[0095] The purification of Hsp25 and Hsp27 polypeptides has been
disclosed previously and so is not discussed in detail herein. See
for example Jakob et al. (1993) J. Biol. Chem. 268:1517-1520, the
disclosure of which is incorporated herein by reference.
[0096] Briefly, the cell lysates are precipitated with 35% ammonium
sulfate. The pellet is harvested by centrifugation, solubilized in
buffer and fractionated by ion exchange chromatography using a DEAE
Sepharose CL-6B column (Pharmacia Biotechnology, Inc.). The
proteins are eluted with 50-200 mM NaCl gradient. The fractions
containing Hsp25 and Hsp27 are identified by immunoblotting using
suitable antibodies. The fractions are combined and fractionated by
size exclusion chromatography on a Superose 6 gel filtration column
(Pharmacia).
[0097] (f) Purification of Hsp60
[0098] The purification of Hsp60 has been discussed in detail
previously and so is not discussed in detail herein. See for
example, Vitanen et al. (1992) J. Biol. Chem. 267: 695-698, the
disclosure of which is incorporated herein by reference.
[0099] Briefly, a mitochondrial matrix lysate is applied to a Mono
Q FPLC column equilibrated with 50 mM sodium phosphate, 1 mM
MgCl.sub.2, 1 mM EGTA, pH 6.9. The proteins are eluted with a 0-1 M
NaCl gradient. The fractions containing Hsp65 are pooled and
fractionated by ATP agarose chromatography as discussed above.
[0100] III. Preparation of Recombinant Stress Proteins
[0101] It is contemplated that recombinant stress proteins and
amino acid sequence variants thereof may be prepared using
conventional recombinant DNA methodologies. For example,
recombinant DNAs encoding either a known stress protein or a
homologue can be inserted into a suitable host cell, the protein
expressed, harvested, renatured if necessary, and purified. Stress
proteins currently known in the art are summarized in Table I,
below.
[0102] The processes for manipulating, amplifying, and recombining
DNA which encode amino acid sequences of interest are generally
well known in the art, and therefore, not described in detail
herein. Methods of identifying and isolating genes encoding members
of the stress protein families also are well understood, and are
described in the patent and other literature.
[0103] Accordingly, the construction of DNAs encoding biosynthetic
constructs as disclosed herein can be performed using known
techniques involving the use of various restriction enzymes which
make sequence specific cuts in DNA to produce blunt ends or
cohesive ends, DNA ligases, techniques enabling enzymatic addition
of sticky ends to blunt-ended DNA, construction of synthetic DNAs
by assembly of short or medium length oligonucleotides, cDNA
synthesis techniques, and synthetic probes for isolating genes of
members of the stress protein families. Various promoter sequences
and other regulatory DNA sequences used in achieving expression,
and various types of host cells are also known and available.
Conventional transfection techniques, and equally conventional
techniques for cloning and subcloning DNA are useful in the
practice of this invention and known
1TABLE 1 Families of Stress Proteins from Gething, et al., Infra
Organism/ Organelle Hsp 60 Hsp 70 Hsp 90 E. coli GroEL DnaK HtpG
(C62.5) Yeast /cytosol Ssal-4p Hsp 83/Hsc83 /endoplasmic Karp2
(BiP) reticulum /mitochondria Hsp 60 (Mif4p) Ssclp Drosophila Hsp
68 Hsp 70 Hsc 1.2.4 Mammals /cytosol Hsp 70 (p73) Hsp 90 (Hsp83)
Hsc 70 (p72) Hsp 87 /endoplasmic BiP (Grp 78) Grp 94 (Erp99)
reticulum gp96 /mitochondria Hsp 60 (Hsp 8) Hsp 70 (Grp 75) Plants
/endoplasmic b70 (BiP) reticulum /chloroplasts RUSBP Alternative
names are shown in parentheses.
[0104] to those skilled in the art. Various types of vectors may be
used such as plasmids and viruses including animal viruses and
bacteriophages. The vectors may exploit various marker genes which
impart to a successfully transfected cell a detectable phenotypic
property that-can be used to identify which of a family of clones
has successfully incorporated the recombinant DNA of the
vector.
[0105] DNA molecules encoding potentially useful stress proteins
may be obtained by a variety of methods. Genes of interest may be
purified from standard cDNA libraries using colony or plaque
hybridization technologies or by using polymerase chain reaction
(PCR) methodologies, all of which are well known in the art. See
for example, "Molecular Cloning: A Laboratory Manual, 2nd Edition"
Sambrook et al. (1989), Cold Spring Harbor Press, the disclosure of
which is incorporated herein by reference. Alternatively, the
preferred genes can be generated by the assembly of synthetic
oligonucleotides produced in a conventional, automated,
polynucleotide synthesizer followed by ligation with appropriate
ligases. For example, overlapping, complementary DNA fragments
comprising 15 bases may be synthesized semi manually using
phosphoramidite chemistry, with end segments left unphosphorylated
to prevent polymerization during ligation. One end of the synthetic
DNA is left with a "sticky end" corresponding to the site of action
of a particular restriction endonuclease, and the other end is left
with an end corresponding to the site of action of another
restriction endonuclease. Alternatively, this approach can be fully
automated. The DNA encoding the biosynthetic constructs may be
created by synthesizing longer single strand fragments (e.g.,
50-100 nudeotides long) in, for example, an Applied Biosystems
oligonucleotide synthesizer, and then ligating the fragments.
[0106] The recombinant DNA constructs then may be integrated into
an expression vector and transfected into an appropriate host cell
for protein expression. Useful host cells include E. coli,
Saccharomyces the insect/baculovirus cell system, myeloma cells,
and various other mammalian cells. In E. coli and other microbial
hosts, the synthetic genes can be expressed as fusion proteins.
Expression in eukaryotes can be accomplished by the transfection of
DNA sequences encoding the biosynthetic protein of interest into
myeloma or other type of cell line.
[0107] The vector additionally may include various sequences to
promote correct expression of the recombinant protein, including
transcriptional promoter and termination sequences, enhancer
sequences, preferred ribosome binding site sequences, preferred
mRNA leader sequences, preferred protein processing sequences,
preferred signal sequences for protein secretion, and the like. The
DNA sequence encoding the gene of interest also may be manipulated
to remove potentially inhibiting sequences or to minimize unwanted
secondary structure formation. The recombinant protein also may be
expressed as a fusion protein. After being translated, the protein
may be purified from the cells themselves or recovered from the
culture medium.
[0108] For example, if the gene is to be expressed in E. coli, it
may first be cloned into an expression vector. This is accomplished
by positioning the engineered gene downstream of a promoter
sequence such as Trp or Tac, and a gene coding for a leader peptide
such as fragment B of protein A (FB). The resulting fusion proteins
accumulate in refractile bodies in the cytoplasm of the cells, and
may be harvested after disruption of the cells by French press or
sonication. The refractile bodies are solubilized, and the
expressed proteins refolded and cleaved by methods already
established for many other recombinant proteins.
[0109] Expression of the engineered genes in eukaryotic cells
requires the establishment of appropriate cells and cell lines that
are easy to transfect, are capable of stably maintaining foreign
DNA with an unrearranged sequence, and which have the necessary
cellular components for efficient transcription, translation,
post-translation modification, and secretion of the protein. In
addition, a suitable vector carrying the gene of interest also is
necessary. DNA vector design for transfection into mammalian cells
should include appropriate sequences to promote expression of the
gene of interest as described supra, including appropriate
transcription initiation, termination, and enhancer sequences, as
well as sequences that enhance translation efficiency, such as the
Kozak consensus sequence. Preferred DNA vectors also include a
marker gene and means for amplifying the copy number of the gene of
interest. A detailed review of the state of the art of the
production of foreign proteins in mammalian cells, including useful
cells, protein expression-promoting sequences, marker genes, and
gene amplification methods, is disclosed in Genetic Engineering
7:91-127 (1988).
[0110] The best-characterized transcription promoters useful for
expressing a foreign gene in a particular mammalian cell are the
SV40 early promoter, the adenovirus promoter (AdMLP), the mouse
metallothionein-1 promoter (mMT-1), the Rous sarcoma virus (RSV)
long terminal repeat (LTR), the mouse mammary tumor virus long
terminal repeat (MMTV-LTR), and the human cytomegalovirus major
intermediate-early promoter (hCMV). The DNA sequences for all of
these promoters are known in the art and are available
commercially.
[0111] The use of a selectable DHFR gene in a dhfr cell line is a
well characterized method useful in the amplification of genes in
mammalian cell systems. Briefly, the DHFR gene is provided on the
vector carrying the gene of interest, and addition of increasing
concentrations of the cytotoxic drug methotrexate leads to
amplification of the DHFR gene copy number, as well as that of the
associated gene of interest. DHFR as a selectable, amplifiable
marker gene in transfected Chinese hamster ovary cell lines (CHO
cells) is particularly well characterized in the art. Other useful
amplifiable marker genes include the adenosine deaminase (ADA) and
glutamine synthetase (GS) genes.
[0112] The choice of cells/cell lines is also important and depends
on the needs of the experimenter. Monkey kidney cells (COS) provide
high levels of transient gene expression, providing a useful means
for rapidly testing vector construction and the expression of
cloned genes. COS cells are transfected with a simian virus 40
(SV40) vector carrying the gene of interest. The transfected COS
cells eventually die, thus preventing the long term production of
the desired protein product. However, transient expression does not
require the time consuming process required for the development of
a stable cell line. Among established cell lines, CHO cells may be
the best-characterized to date. CHO cells are capable of expressing
proteins from a broad range of cell types. The general
applicability of CHO cells and its successful production for a wide
variety of human proteins in unrelated cell types emphasizes the
underlying similarity of all mammalian cells.
[0113] The various cells, cell lines and DNA sequences that can be
used for mammalian cell expression of the recombinant stress
protein constructs of the invention are well characterized in the
art and are readily available. Other promoters, selectable markers,
gene amplification methods and cells also may be used to express
the proteins of this invention. Particular details of the
transfection, expression, and purification of recombinant proteins
are well documented in the art and are understood by those having
ordinary skill in the art. Further details on the various technical
aspects of each of the steps used in recombinant production of
foreign genes in mammalian cell expression systems can be found in
a number of texts and laboratory manuals in the art, such as, for
example, Current Protocols in Molecular Biology, (1989) eds.
Ausubel et al., Wiley Interscience.
[0114] IV. Isolation of Potentially Immunogenic Peptides
[0115] As mentioned previously, potentially immunogenic peptides
may be isolated from either stress protein-peptide complexes or
MHC-peptide complexes. Protocols for isolating peptides from either
of these complexes are set forth below.
[0116] (a) Peptides from Stress Protein-peptide Complexes
[0117] Two methods may be used to elute the peptide from a stress
protein-peptide complex. One approach involves incubating the
stress protein-peptide complex in the presence of ATP, the other
involves incubating the complexes in a low pH buffer.
[0118] Briefly, the complex of interest is centrifuged through a
Centricon 10 assembly (Millipore) to remove any low molecular
weight material loosely associated with the complex. The large
molecular weight fraction may be removed and analyzed by SDSPAGE
while the low molecular weight may be analyzed by HPLC as described
below. In the ATP incubation protocol, the stress protein-peptide
complex in the large molecular weight fraction is incubated with 10
mM ATP for 30 minutes at room temperature. In the low pH protocol,
acetic acid is added to the stress protein-peptide complex to give
a final concentration of 10% (vol/vol) and the mixture incubated in
a boiling water bath for 10 minutes. See for example, Van Bleek et
al. (1990) Nature 348:213-216; and Li et al. (1993) EMBO Journal
12:3143-3151, the disclosures of which are incorporated herein by
reference.
[0119] The resulting samples are centrifuged through an Centricon
10 assembly as mentioned previously. The high and low molecular
weight fractions are recovered. The remaining large molecular
weight stress protein-peptide complexes can be reincubated with ATP
or low pH to remove any remaining peptides.
[0120] The resulting lower molecular weight fractions are pooled,
concentrated by evaporation and dissolved in 0.1% trifluoroacetic
acid (TFA). Then, the dissolved material is fractionated by reverse
phase high pressure liquid chromatography (HPLC), using for example
a VYDAC C18 reverse phase column equilibrated with 0.1% TFA. The
bound material subsequently is eluted by developing the column with
a linear gradient of 0 to 80% acetonitrile in 0.1% TFA at a flow
rate of about 0.8 ml/min. The elution of the peptides can be
monitored by OD.sub.210 and the fractions containing the peptides
collected.
[0121] (b) Peptides from MHC-peptide Complexes
[0122] The isolation of potentially immunogenic peptides from MHC
molecules is well known in the art and so is not described in
detail herein. See for example, Falk et al. (1990) Nature
348:248-251; Rotzsche et al. (1990) Nature 348:252-254; Elliott et
al. (1990) Nature 348:195-197; Falk et al. (1991) Nature
351:290-296, Demotz et al. (1989) Nature 343:682-684; Rotzsche et
al. (1990) Science 249:283-287.
[0123] Briefly, MHC-peptide complexes may be isolated by a
conventional immunoaffinity procedure. Then the peptides are eluted
from the MHC-peptide complex by incubating the complexes in the
presence of about 0.1% TFA in acetonitrile. The extracted peptides
may be fractionated and purified by reverse phase HPLC, as
before.
[0124] The amino acid sequences of the eluted peptides may be
determined either by manual or automated amino acid sequencing
techniques well known in the art. Once the amino acid sequence of a
potentially protective peptide has been determined the peptide may
be synthesized in any desired amount using conventional peptide
synthesis or other protocols well known in the art.
[0125] V. Synthesis of Potentially Useful Immunogenic Peptides
[0126] Peptides having the same amino acid sequence as those
isolated above may be synthesized by solid-phase peptide synthesis
using procedures similar to those described by Merrifield (1963) J.
Am. Chem. Soc., 85: 2149. During synthesis, N-.alpha.-protected
amino acids having protected side chains are added stepwise to a
growing polypeptide chain linked by its C-terminal end to an
insoluble polymeric support i.e., polystyrene beads. The peptides
are synthesized by linking an amino group of an
N-.alpha.-deprotected amino acid to an .alpha.-carboxy group of an
N-.alpha.-protected amino acid that has been activated by reacting
it with a reagent such as dicyclohexylcarbodiimide. The attachment
of a free amino group to the activated carboxyl leads to peptide
bond formation. The most commonly used N-.alpha.-protecting groups
include Boc which is acid labile and Fmoc which is base labile.
[0127] Briefly, the C-terminal N-a-protected amino acid is first
attached to the polystyrene beads. The N-.alpha.-protecting group
is then removed. The deprotected .alpha.-amino group is coupled to
the activated .alpha.-carboxylate group of the next
N-.alpha.-protected amino acid. The process is repeated until the
desired peptide is synthesized. The resulting peptides then are
cleaved from the insoluble polymer support and the amino acid side
chains deprotected. Longer peptides can be derived by condensation
of protected peptide fragments. Details of appropriate chemistries,
resins, protecting groups, protected amino acids and reagents are
well known in the art and so are not discussed in detail herein.
See for example, Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, (1989), and Bodanszky, Peptide
Chemistry, A Practical Textbook, 2nd Ed, Springer-Berlog (1993),
the disclosures of which are incorporated herein by reference.
[0128] Purification of the resulting peptides is accomplished using
conventional procedures, such as preparative HPLC using gel
permeation, partition and/or ion exchange chromatography. The
choice of appropriate matrices and buffers are well known in the
art and so are not described in detail herein.
[0129] VI. Reconstitution of Stress Protein-peptide Complexes
[0130] As will be appreciated by those skilled in the art, the
peptides, either isolated from the complexes using the
aforementioned procedures or chemically synthesized, may be
reconstituted with a variety of naturally purified or recombinant
stress proteins in vitro to generate immunogenic stress
protein-peptide complexes. A preferred protocol for reconstituting
a stress protein and a peptide in vitro is discussed below.
[0131] Prior to reconstitution the stress proteins are pretreated
with ATP or low pH to remove any peptides that may be associated
with the stress-protein of interest. When the ATP procedure is
used, excess ATP is removed from the preparation by the addition of
apyranase as discussed in Levy et al. (1991) Cell 67:265-274, the
disclosure of which is incorporated herein by reference. When the
low pH procedure is used the buffer is readjusted to neutral pH by
the addition of pH modifying reagents.
[0132] The peptide (1 mg) and the pretreated stress protein (9 mg)
are admixed to give an approximate molar ratio of 5 peptides:1
stress protein. Then, the mixture is incubated for 3 hours at room
temperature in a binding buffer containing 20 mM sodium phosphate,
pH 7.2, 350 mM NaCl, 3 mM MgCl.sub.2, 1 mM PMSF. The preparations
are centrifuged through Centricon 10 assembly (Millipore) to remove
any unbound peptide. The association of the peptides with the
stress proteins can be assayed by SDS-PAGE and radioautography when
radiolabelled peptides are used to reconstitute the complexes.
[0133] Following reconstitution, the candidate immunogenic stress
protein-peptide complexes can be tested in vitro using for example
the mixed lymphocyte target cell assay (MLTC) described below. Once
potential immunogenic constructs have been isolated they can be
characterized further in animal models using the preferred
administration protocols and excipients discussed below.
[0134] VII. Determination of Immunogenicity of Stress
Protein-Peptide Complexes
[0135] The purified and reconstituted stress protein-peptide
complexes can be assayed for immunogenicity using the mixed
lymphocyte target-culture assay (MLTC) well known in the art.
[0136] Briefly, mice are injected subcutaneously with the candidate
stress protein-peptide complexes. Other mice are injected with
either other stress protein-peptide complexes or whole infected
cells which act as positive controls for the assay. The mice are
injected twice, 7-10 days apart. Ten days after the last
immunization, the spleens are removed and lymphocytes released from
the excised spleens. The released lymphocytes may be restimulated
in vitro by the subsequent addition of dead cells which prior to
death had expressed the complex of interest.
[0137] For example, 8.times.10.sup.6 immune spleen cells may be
stimulated with either 4.times.10.sup.4 mitomycin C treated or
.gamma.-irradiated (5-10,000 rads) cells (the cells having been
infected with the intracellular pathogen or transfected with an
appropriate gene) in 3 ml RPMI medium containing 10% fetal calf
serum. In certain cases 33% secondary mixed lymphocyte culture
supernatant may be included in the culture medium as a source of T
cell growth factors. See for example, Glasebrook et al. (1980) J.
Exp. Med. 151; 876. In order to test the primary cytotoxic T cell
response after immunization, spleen cells may be cultured without
stimulation. In some experiments spleen cells of the immunized mice
also may be restimulated with antigenically distinct cells, to
determine the specificity of the cytotoxic T cell response.
[0138] Six days later the cultures are tested for cytotoxicity in a
4 hour .sup.51Cr-release assay. See for example, Palladino et al.
(1987) Cancer Res. 47:5074-5079 and Blachere et al. (1993) J.
Immunotherapy 14;352-356, the disclosures of which are incorporated
herein by reference. In this assay, the mixed lymphocyte culture is
added to a target cell suspension to give different effector:target
(E:T) ratios (usually 1:1 to 40:1). The target cells are
prelabelled by incubating 1.times.10.sup.6 target cells in culture
medium containing 200 mCi .sup.51Cr/ml for one hour at 37.degree.
C. The cells are washed three times following labeling. Each assay
point (E:T ratio) is performed in triplicate and the appropriate
controls incorporated to measure spontaneous .sup.51Cr release (no
lymphocytes added to assay) and 100% release (cells lysed with
detergent). After incubating the cell mixtures for 4 hours, the
cells are pelleted by centrifugation at 200 g for 5 minutes. The
amount of .sup.51Cr released into the supernatant is measured by a
gamma counter. The percent-cytotoxicity is measured as cpm in the
test sample minus spontaneously released cpm divided by the total
detergent released cpm minus spontaneously released cpm.
[0139] In order to block the MHC class I cascade a concentrated
hybridoma supernatant derived from K-44 hybridoma cells (an
anti-MHC class I hybridoma) is added to the test samples to a final
concentration of 12.5%.
[0140] VIII. Formulation and Vaccination Regimes
[0141] Once candidate stress protein-peptide complexes have been
identified they may be administered either to an animal model or to
the intended recipient to stimulate cytotoxic T cell responses
against the preselected intracellular pathogen. The stress
protein-peptide complexes of the invention may be either stored or
prepared for administration by mixing with physiologically
acceptable carriers, excipients, or stabilizers. These materials
should be non-toxic to the intended recipient at dosages and
concentrations employed.
[0142] If the complex is water soluble then it may be formulated in
an appropriate buffer, for example phosphate buffered saline (5 mM
sodium phosphate, 150 mM NaCl, pH7.1) or other physiologically
compatible solutions. Alternatively, if the resulting complex has
poor solubility in aqueous solvents then it may be formulated with
a non-ionic surfactant such as Tween, or polyethylene glycol.
[0143] Useful solutions for oral or parenteral administration may
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, (Gennaro, A., ed.), Mack Pub., 1990. Formulations may
include, for example, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like. Formulations for direct administration, in particular,
may include glycerol and other compositions of high viscosity.
Biocompatible, preferably bioresorbable polymers, including, for
example, hyaluronic acid, collagen, tricalcium phosphate,
polybutyrate, polylactide, polyglycolide and lactide/glycolide
copolymers, may be useful excipients to control the release of the
stress protein-peptide complexes in vivo.
[0144] Formulations for inhalation administration may contain as
excipients, for example, lactose. Aqueous solutions may contain,
for example, polyoxyethylene-9-lauryl ether, glycocholate and
deoxycholate. Oily solutions may be useful administration in the
form of nasal drops. Gels may be applied topically
intranasally.
[0145] The compounds provided herein can be formulated into
pharmaceutical compositions by admixture with pharmaceutically
acceptable non toxic excipients and carriers. In addition the
formulations may optionally contain one or more adjuvants.
Preferred adjuvants include, but are not limited to, pluronic
tri-block copolymers, muramyl dipeptide and its derivatives,
detoxified endotoxin, saponin and its derivatives such as QS-21 and
liposomes. The present invention further envisages sustained
release formulations in which the complex is released over an
extended period of time.
[0146] The dosage and means of administration of the family of
stress protein-peptide vaccines prepared in accordance with the
invention will necessarily depend upon the nature of the complex,
the intracellular pathogen and the nature of the disease in
question. The complex should be administered in an amount
sufficient to initiate a cytotoxic T cell response against the
intracellular pathogen. The preferred dosage of drug to be
administered also is likely to depend on such variables as the type
of disease, the age, sex and weight of the intended recipient, the
overall health status of the particular patient, the relative
biological efficacy of the compound selected, the formulation of
the compound, the presence and types of excipients in the
formulation, and the route of administration.
[0147] In general terms, the compounds of this invention may be
provided in an aqueous physiological buffer solution containing
about 0.001 to 10% w/v compound for parenteral administration.
Typical doses range from about 0.1 to about 1000 micrograms of
complex/kg body weight of recipient/immunization; and preferably
range from about 0.5 to about 100 micrograms of complex/kg body
weight of recipient/immunization. It is contemplated that between
about 10 to about 250 micrograms of complex will be administered
per dose to a human subject weighing about 75 kg. These quantities,
however, may vary according to the adjuvant-co-administered with
the complex.
[0148] The vaccines may be administered using standard protocols
which include, but are not limited to, intramuscular, subcutaneous,
intradermal, intraperitoneal, intravenous, intravaginal,
intrarectal, oral, sublingual, transcutaneous, and intranasal
administration Preferably the recipient should be vaccinated four
times at weekly intervals. If necessary, the responses may be
boosted at a later date by subsequent administration of the
vaccine. It is contemplated that the optimal dosage and vaccination
schedule may be determined empirically for each stress
protein-peptide vaccine using techniques well known in the art.
[0149] Various cytokines, antibiotics, and other bioactive agents
also may be administered with the stress protein complexes. For
example, various known cytokines, i.e., IL-1.alpha., IL-1.beta.,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IFN.alpha., IFN.beta., IFN.gamma., TNF.alpha., TNF.beta.,
G-CSF, GM-CSF, and TGF-.beta. may be co-administered with the
complexes in order to maximize the physiological response. It is
anticipated, however, that other but as yet undiscovered cytokines
may be effective in the invention. In addition, conventional
antibiotics may be co-administered with the stress protein-peptide
complex. The choice of suitable antibiotics will also depend upon
the disease in question.
EXAMPLE 1
Immunogenicity of Stress Protein-peptide Complexes Isolated From
Cells Transfected With a Gene Encoding an Antigenic Determinant
[0150] FIG. 1 shows the antigen specific cytotoxic T cell activity
of splenocytes derived from mice immunized with a gp96-peptide
complex harvested from BALB/c fibroblasts transfected with the
nucleoprotein (NP) gene from the PR8 influenza virus.
[0151] Briefly, gp96-peptide preparations were isolated from BALB/c
cells transfected with and expressing the NP gene of the PR8
influenza virus. The gp96-peptide complex was isolated from 100,000
g supernatant by the unmodifed gp60-peptide complex purification
protocol. Then, the preparations were used to immunize naive BALB/c
mice. The mice were injected twice subcutaneously with the
gp96-peptide complexes at ten day intervals. The mice were
sacrificed and the spleen cells obtained. The spleen cells were
stimulated twice in vitro by the additional lethally irradiated
BALB/c cells expressing the NP gene using the mixed target
lymphocyte culture (MLTC) assay described above. Six days later the
cultures were tested for cytotoxicity using the .sup.51Cr release
assay. In order to block the MHC type I cascade the spleen cells
were incubated with the supernatant derived from K-44 hybridoma
(containing anti-MHC type I immunoglobulins) culture.
[0152] The cytotoxic activity was assayed by the release of 51Cr
from BALB/c fibroblasts expressing the NP gene (filled circles),
BALB/c cells expressing the NP gene but treated with the anti-MHC
type I antisera (empty circles) and from the syngeneic non-NP
transfected cell line 5117 (asterisks). The spleens of the mice
immunized with the gp96 complex showed strong MHC class
I-restricted cytotoxic T cell activity against BALB/c cells
expressing the NP gene, but not against the syngeneic non-NP
transfected cell line 5117. Furthermore, the anti MHC type I
antisera blocked the response. Therefore, it is apparent that
immunization with a stress protein-peptide complex elicits a
specific cytotoxic T cell response against the peptide in the
complex and that the MHC class I cascade plays an integral role in
stimulating the cytotoxic T cell response against cells infected
with intracellular pathogens.
EXAMPLE 2
Immunogenicity of Stress Protein-peptide Complexes Isolated from
SV40 Transformed Cells
[0153] FIG. 2 shows the antigen specific cytotoxic T cell activity
of splenocytes derived from mice immunized with gp96-peptide
complex harvested from SV40 transformed SVB6 cells.
[0154] Briefly, gp96-peptide complexes were isolated from SV40
transformed SVB6 cells and used to immunize naive (57BL/6) mice.
The gp96-peptide complex was isolated from 100,000 g supernatant by
the unmodifed gp60-peptide complex purification protocol. The mice
were injected twice subcutaneously with the complex at ten day
intervals. The mice were sacrificed, the spleen cells isolated and
stimulated in vitro by the addition of lethally L-radiated SV40
transformed SVB6 cells by the MLTC procedure. Six days later the
cells were assayed for cytotoxicity using the .sup.51Cr release
assay. The cytotoxic activity was assayed by the release of
.sup.51Cr from SVB6 cells (filled triangles) and from a non SV40
transfected syngeneic cell line, MCA (empty triangles). MHC class I
mediated activity was assayed also by adding anti-MHC class I
immunoglobulins derived from the K-44 hybridoma cell line to the
spleen cells.
[0155] The spleen cells isolated from mice immunized with the
gp96-peptide complex showed strong MHC class I-restricted activity
against the SV40 transfected SVB6 cells but not against the non
transfected cells.
EXAMPLE 3
Reconstitution of Immunogenic Stress Protein-peptide Complexes In
Vitro
[0156] FIGS. 3A-3D show antigen specific cytotoxic T cell
activities of splenocytes derived from two mice immunized with
reconstituted Hsp70-peptide complex.
[0157] Briefly, uncomplexed Hsp70 was purified by the procedure
described above and the peptide (SLSDLRGYVYQCL, SEQ. ID. NO.: 1)
was synthesized by solid phase peptide synthesis. The peptide (1
mg) and ATP treated Hsp70 (9 mg) were admixed and incubated for 3
hours at room temperature in a binding buffer containing 20 mM
sodium phosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl.sub.2, 1 mM PMSF.
The resulting preparation was centrifuged through Centricon 10
assembly (Millipore) to remove unbound peptide.
[0158] The resulting complex was used to immunize two naive mice.
The spleen cells were isolated from the mice and stimulated twice
in vitro by the addition of lethally irradiated EL4 cells
transfected with, and expressing a minigene encoding the peptide
SLSDLRGYVYQGL (SEQ. ID. NO.: 1), using the MLTC procedure. The
cytotoxicities of spleen cells from both mice were assayed after
the first (3A and 3C) and second (3B and 3D) stimulations by the
.sup.51Cr release assay. The release of .sup.51Cr was measured from
EL4 cells (hollow triangles) and from EL4 cells transfected with,
and expressing the peptide SLSDLRGYVYQGL (SEQ. ID. NO.: 1) (filled
triangles). The results show that stress proteins and peptides can
be reconstituted successfully in vitro to give specific immunogenic
stress protein-peptide complexes.
[0159] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
Sequence CWU 1
1
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