U.S. patent application number 11/861413 was filed with the patent office on 2008-07-10 for intracellular delivery vehicles.
Invention is credited to Darren E. Higgins, Daniel A. Portnoy.
Application Number | 20080166366 11/861413 |
Document ID | / |
Family ID | 22460889 |
Filed Date | 2008-07-10 |
United States Patent
Application |
20080166366 |
Kind Code |
A1 |
Portnoy; Daniel A. ; et
al. |
July 10, 2008 |
Intracellular Delivery Vehicles
Abstract
The invention provides methods and compositions relating to
intracellular delivering of agents to eukaryotic cells. The
compositions include microbial delivery vehicles such as
nonvirulent bacteria comprising a first gene encoding a nonsecreted
foreign cytolysin operably linked to a heterologous promoter and a
second gene encoding a different foreign agent. The foreign agent
may be a nucleic acid or protein, and is frequently bioactive in
and therapeutic to the target eukaryote. In addition, the invention
provides eukaryotic cells comprising the subject nonvirulent
bacteria and nonhuman eukaryotic host organisms comprising such
cells.
Inventors: |
Portnoy; Daniel A.;
(Berkeley, CA) ; Higgins; Darren E.; (Berkeley,
CA) |
Correspondence
Address: |
RICHARD ARON OSMAN
4070 CALLE ISABELLA
SAN CLEMENTE
CA
92672
US
|
Family ID: |
22460889 |
Appl. No.: |
11/861413 |
Filed: |
September 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10627452 |
Jul 25, 2003 |
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11861413 |
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09949109 |
Sep 7, 2001 |
6599502 |
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10627452 |
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09469197 |
Dec 21, 1999 |
6287556 |
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09949109 |
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09133914 |
Aug 13, 1998 |
6004815 |
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09469197 |
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Current U.S.
Class: |
424/184.1 ;
506/10 |
Current CPC
Class: |
C12N 15/87 20130101;
A61P 35/00 20180101; A61P 37/00 20180101; C12N 15/63 20130101; A61K
39/00 20130101 |
Class at
Publication: |
424/184.1 ;
506/10 |
International
Class: |
C40B 30/06 20060101
C40B030/06; A61K 39/00 20060101 A61K039/00 |
Goverment Interests
[0002] The disclosed inventions were made with Government support
under Grant (Contract) Nos. A127655-10 BD37 awarded by the National
Institutes of Health. The government may have rights in these
inventions.
Claims
1. A method of determining whether a library contains or encodes an
immunogenic polypeptide, the method comprising: (a) providing a
cell member library, which cell member library comprises a
plurality of cell members, each cell member comprising a first
polynucleotide encoding a polypeptide encoded by the genome of a
pathogenic organism, the first polynucleotide operably linked to a
promoter such that each cell member of the cell member library
produces its respective polypeptide; (b) individually contacting
each cell member of the cell member library with a second cell
capable of (i) endocytosing the contacted cell member and (ii)
displaying the polypeptide produced by the endocytosed cell member
on its surface through the MHC class I pathway; (c) individually
contacting each second cell of step (b) with a sample containing a
least one cytotoxic T-lymphocyte derived from a mammal previously
infected with the pathogenic organism; and (d) detecting whether a
cytotoxic T-lymphocyte within the sample is activated, wherein
activation of the cytotoxic T-lymphocyte indicates that the
polypeptide is immunogenic, thereby determining whether the cell
member library contains or encodes an immunogenic polypeptide.
2. The method of claim 1, wherein the step of providing a cell
member library comprises: providing a plurality of first
polynucleotides, which plurality represents a genomic library of
the pathogenic organism; and contacting the plurality of first
polynucleotides with a plurality of cell members so that first
polynucleotides are introduced into cell members and the provided
cell member library is thereby generated.
3. The method of claim 1 or 2, wherein the each cell member of the
cell member library further comprises a second polynucleotide
encoding a pore-forming protein.
4. The method of claim 3, wherein the pore-forming protein is
listeriolysin O.
5. The method of claim 1 or 2, wherein the second cell is a
macrophage.
6. The method of claim 1 or 2, wherein the each cell member of the
library is killed prior to the contacting step (b).
7. The method of claim 1 or 2, wherein prior to the contacting step
(c), the second cell is killed.
8. The method of claim 1 or 2, wherein, prior to contacting step
(b), a replica of the library is made.
9. The method of claim 8, further comprising a step of: (e)
recovering the first polynucleotide encoding the polypeptide
identified in step (d) from a replica copy of the cell member
library.
10. The method of claim 1 or 2, further comprising a step of: (e)
identifying an epitope sufficient for cytotoxic T-lymphocyte
activation within the polypeptide determined to be immunogenic in
step (d).
11. A vaccine comprising at least one epitope identified by the
method of claim 10 and a pharmaceutically acceptable carrier.
12. A vaccine comprising at least one immunogenic polypeptide
identified by the method of claim 1 or 2 and a pharmaceutically
acceptable carrier.
13. The method of claim 1 or 2, wherein the contacting step (c) is
performed using a sample containing a plurality of cytotoxic
T-lymphocytes.
14. The method of claim 1 or 2, further comprising performing the
method steps (c) and (d) at least one further time using the cell
member library.
15. The method of claim 1 or 2, wherein a different sample
containing a different cytotoxic T-lymphocyte is contacted in step
(c) each time the method is performed.
16. The method of claim 1 or 2, wherein the cell member library
comprises a plurality of bacterial cell members.
17. The method of claim 16, wherein the cell member library
comprises a plurality of E. coli cell members.
18. The method of claim 1 or 2, wherein the pathogenic organism is
a bacterium.
19. The method of claim 1 or 2, wherein the pathogenic organism is
a virus.
20. The method of claim 1 or 2, wherein the promoter is an
inducible promoter.
21. The method of claim 1 or 2, wherein each cell member of the
cell member library comprises at least one of a plurality of
different first polynucleotides.
22. The method of claim 21, wherein each cell member of the cell
member library comprises a different first polynucleotide.
23. The method of claim 21, wherein the plurality of first
polynucleotides comprises a genomic library of the pathogenic
organism.
24. The method of claim 23, wherein the genomic library comprises
polynucleotides encoding each polypeptide encoded by the genome of
the pathogenic organism.
25. The method of claim 24, wherein the step of detecting comprises
detecting specific antigens of the pathogenic organism.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of and claims priority
under 35 U.S.C..sctn. 120, of U.S. Ser. No. 10/627,452, filed Jul.
25, 2003, now abandoned, which is a continuation of U.S. Ser. No.
09/949,109, filed Sep. 7, 2001, now U.S. Pat. No. 6,599,502, which
is a continuation of Ser. No. 09/469,197, filed Dec. 21, 1999, now
U.S. Pat. No. 6,287,556, which is a continuation of U.S. Ser. No.
09/133,914, filed Aug. 13, 1998, now U.S. Pat. No 6,004,815, which
are incorporated herein by reference.
INTRODUCTION
[0003] 1. Field of the Invention
[0004] The field of this invention is microbial-based intracellular
delivery of agents to eukaryotic cells.
[0005] 2. Background
[0006] The efficient delivery of macromolecules to the cytosol of
mammalian cells is of fundamental importance in such processes as
the generation of transfected phenotypes and the study of protein
function and localization. Furthermore, delivery of macromolecules
to the cytosol is also important for the induction of cell-mediated
immunity and is a significant challenge facing the rational design
of vaccines to intracellular pathogens. Numerous methodologies
currently exist for delivering macromolecules to mammalian cells.
These include but are not limited to: mechanical techniques such as
electroporation (1) and microinjection (2); fusion methodologies
such as fusion with vesicles and liposomes (2); chemical treatments
employing the use of ATP or EDTA (3) or the external addition of
molecules mixed with pore-forming toxins such as .alpha.-toxin of
Staphylococcus aureus (4). Many of these methods have a
disadvantage in that the molecule to be delivered may require
laborious purification (i.e. protein) or the delivery method is
limited to use in vitro. In order to overcome these obstacles,
investigators have sought to use biological vectors that can enter
tissues, cells and specific cellular compartments for the delivery
of macromolecules. These vectors are often derived from either
retroviruses or bacteria that have evolved to invade and replicate
in specific hosts, organs, or cell types. While retroviral vectors
have been used for the delivery and subsequent expression of DNA in
host cells (5-7), bacterial vectors have been exploited primarily
for the delivery of antigenic proteins and more recently adapted
for the delivery of DNA to mammalian cells (8-12).
Relevant Literature
[0007] Lee et al. (1997) U.S. Pat. No. 5,643,599 and Lee et al.
(1996) J. Biol. Chem. 271, 7249-7252 describe hemolysin loaded
liposomes for intracellular delivery of macromolecules. Dietrich,
G., et al. (1998) Nature Biotech. 16, 138-139 and Ikonomidis, G.,
et al. (1997) Vaccine 15, 433-440 describe the use of Listeria
monocytogenes as a macrophage delivery vehicle. Sizemore, D. R.,
Branstrom, A. A., & Sadoff, J. C. (1995) Science 270, 299-302
and Courvalin, P., et al. (1995) C. R. Acad. Sci. III 318,
1207-1212 describe the use of attenuated Shigella and invasive
strains of Shigella flexneri and E. coli, respectively, as a DNA
delivery vehicle. Hess, J., et al. (1998) Proc. Natl. Acad. Sci.
U.S.A. 95, 5299-5304; and Darji, A., et al. (1995) J. Biotechnol.
43, 205-212 describe the expression of listeriolysin in several
heterologous systems: an invasive E. coli, Mycobacterium bovis and
Listeria innocua, respectively. Moriishi et al. (1996) FEMS
Immunol. Med. Microbiol. 16, 213-222, 217 describe the
transformation of an E. coli with a plasmid encoding listeriolysin.
Sanderson, S., Campbell, D. J., & Shastri, N. (1995) J. Exp.
Med. 182, 1751-1757, describe the cloning of a Listeria
monocytogenes genomic library in E. coli. Higgins and Portnoy
(1998) Nature Biotech. 16, 181-185 review bacterial delivery of
DNA.
SUMMARY OF THE INVENTION
[0008] The invention provides methods and compositions relating to
intracellular delivering of agents to eukaryotic cells. The
compositions include microbial delivery vehicles such as
nonvirulent bacteria comprising a first gene encoding a nonsecreted
foreign cytolysin operably linked to a heterologous promoter and a
second gene encoding a different foreign agent. In particular
embodiments, the bacteria may be variously invasive to the target
cell, autolysing within target cell endosomes and preferably, a
laboratory strain of E. coli. The cytolysin may lack a functional
signal sequence, and is preferably a listeriolysin. The foreign
agent may be a nucleic acid or protein, and is frequently bioactive
in and therapeutic to the target eukaryote. In addition, the
invention provides eukaryotic cells comprising the subject
nonvirulent bacteria and nonhuman eukaryotic host organisms
comprising such cells. The invention also provides methods for
introducing foreign agents into eukaryotic cells comprising the
step of contacting the cell in vivo or in vitro with the subject
bacteria under conditions whereby the agent enters the cell. In
particular embodiments, the bacterium is endocytosed into a vacuole
of the cell, undergoes lysis and the cytolysin mediates transfer of
the agent from the vacuole to the cytosol of the cell.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Presentation of SL8/K.sup.b complex to B3Z
T-cells.
[0010] FIGS. 2A and 2B. Time requirement for presentation of
SL8/K.sup.b complex.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0011] The following preferred embodiments and examples are offered
by way of illustration and not by way of limitation.
[0012] The subject bacteria comprise a first gene encoding a
nonsecreted foreign cytolysin operably linked to a heterologous
promoter. A wide variety of foreign (i.e. not native to the
microbial delivery vehicle) cytolysins may be used so long as the
cytolysin is not significantly secreted by the microbe and
facilitates cytosolic delivery of the foreign agent as determined
by the assays described below. Exemplary cytolysins include
phospholipases (see, e.g., Camilli, A., et al., J. Exp. Med.
173:751-754 (1991)), pore-forming toxins (e.g., an alpha-toxin),
natural cytolysins of gram-positive bacteria, such as listeriolysin
O (LLO, e.g. Mengaud, J., et al., Infect. Immun. 56:766-772 (1988)
and Portnoy, et al., Infect. Immun. 60:2710-2717 (1992)),
streptolysin O (SLO, e.g. Palmer M, et al., 1998, Biochemistry
37(8):2378-2383) and perfringolysin O (PFO, e.g. Rossjohn J. et
al., Cell 89(5):685-692). Where the target cell is phagosomal, acid
activated cytolysins may be advantageously used. For example,
listeriolysin O exhibits greater pore-forming ability at mildly
acidic pH (the pH conditions within the phagosome), thereby
facilitating delivery of the liposome contents to the cytoplasm
(see, e.g., Portnoy, et al., Infect. Immun. 60:2710-2717 (1992)).
Furthermore, natural cytolysins are readily modified to generate
mutants which are screened in the assays described below or
otherwise known in the art (e.g. Awad M M, et al., Microb Pathog.
1997, 22(5): 275-284) desired activity modifications. In general,
the screening assays measure the ability of a candidate cytolysin
to confer on a bacterium the ability to render a target cell
vacuole permeable to a label (e.g., a fluorescent or radioactive
label) that is contained in the vacuole. In a particular example,
the invention provides mutations in natural cytolysin wherein
highly conserved cysteine residues (e.g., cysteine 460 in PFO,
cysteine 486 in LLO) are replaced by conservative amino acid
substitutions which are not subject to reduction in order to
prepare oxidation/reduction-insensitive cytolysin mutants which
exhibit improved lytic activity. Alternatively, mutant cytolysins
are selected from naturally occurring mutants by, for example,
identifying bacteria which contain cytolysins that are capable of
lysing cells over a narrow pH range, preferably the pH range which
occurs in phagosomes (pH 5.0-6.0), or under other conditions (e.g.,
ionic strength) which occur in the targeted phagosomes. Nonsecreted
cytolysins may be provided by various mechanisms, e.g. absence of a
functional signal sequence, a secretion incompetent microbe, such
as microbes having genetic lesions (e.g. a functional signal
sequence mutation), or poisoned microbes, etc.
[0013] The bacteria also comprise a second gene encoding a foreign
agent different from the cytolysin, and the subject methods may be
used to deliver a wide variety of such foreign agents for a variety
of applications, including diagnosis, therapy including
prophylactics such as immunizations (see, e.g. HIV vaccine, Table
1) and treatments such as gene therapy (especially of single gene
disorders amenable to localized treatment, see Table 1, below),
biosynthesis, etc.; essentially any agent that the microbial host
can be engineered to produce. In a particular embodiment, the agent
is largely retained by the microbe until lysis within the target
cell vacuole. Note that the first and second genes may be the same,
i.e. the same nucleic acid encodes both the cytolysin and the
foreign agent. For example, in a particular embodiment, the foreign
agent is expressed in frame with the cytolysin as a fusion protein.
In other embodiments, the microbes are engineered to deliver
libraries of agents for screening, e.g. Tenson T. et al., J Biol
Chem 1997 Jul. 11; 272(28):17425-17430.
[0014] A wide variety of nucleic acid-based agents may be
delivered, including expression vectors, probes, primers, antisense
nucleic acids, knockout/in vectors, ribozymes, etc. For example,
the subject bacteria are used to deliver nucleic acids which
provide templates for transcription or translation or provide
modulators of transcription and/or translation by hybridizing to
selected endogenous templates, see, e.g. U.S. Pat. No. 5,399,346
for a non-limiting list of genes that can be administered using
gene therapy and diseases that can be treated by gene therapy. For
example, polynucleotide agents may provide a coding region operably
linked to a transcriptional regulatory region functional in a
target mammalian cell, e.g. a human cytomegalovirus (CMV) promoter.
In particular, the polynucleotide may encode a transcription
factor, whereby expression of the transcription factor in the
target cell provides activation or de-activation of targeted gene
expression in the target cell. In another example, RNA virus
infected cells are targeted by microbes delivering viral
RNA-specific ribozymes, e.g. HIV-infected T-cells, leukemia virus
infected leukocytes, hepatitis C infected liver cells. In yet
another embodiment, labeled probes are delivered which effect in
situ hybridization-based diagnostics.
[0015] A wide variety of polypeptide-based agents may also be
delivered, including antibiotics, insecticides, fungicides,
anti-viral agents, anti-protozoan agents, enzymes, anti-cancer
agents (e.g. cyclin dependent kinase (CDK) inhibitors such as P16,
P21 or P27), antibodies, anti-inflammatory peptides, transcription
factors, antigenic peptides, etc. Exemplary therapeutically active
polypeptides which can be delivered by the subject invention are
described in Nature Biotech 16(2), entire issue, etc. In a
particular embodiment, the invention provides for the delivery to
antigen-presenting cells of antigenic polypeptides which are
presented in association with MHC proteins. In another particular
embodiment, both nucleic acids and proteins are delivered together
contemporaneously, in the same administration or in the same
microbe. In some such applications, the nucleic acids and proteins
can act in concert, e.g. an integrating vector and an integrase,
and RNA and a reverse transcriptase, a transposon and a
transposase, etc.
[0016] The subject methods may also be used to deliver a wide
variety of other foreign agents that are synthesized by the host
microbe. For example, microbes may be selected for, or engineered
to contain, biosynthetic machinery to produce any microbiologically
producible agent compatible with the subject methods (e.g.
sufficiently microbe impermeant to provide effective delivery to
the target cell). Preferred such agents are those that are
contraindicated for convenient direct (e.g. oral) administration,
because of, for example, gut inactivation, toxicity, intolerance,
impermeability, etc. In fact, even agents providing significant
toxicity to the microbial host find use so long as an effective
amount of the agent may be loaded (by synthesis) or maintained in
the microbe (see, e.g. LLO toxicity, below).
[0017] A wide variety of nonvirulent, non-pathogenic bacteria may
be used; preferred microbes are relatively well characterized
strains, particularly laboratory strains of E. coli, such as
MC4100, MC1061, DH5.alpha., etc. Other bacteria that can be
engineered for the invention include well-characterized,
nonvirulent, non-pathogenic strains of Listeria monocytogenes,
Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis,
etc. In a particular embodiment, the bacteria are attenuated to be
nonreplicative, nonintegrative into the host cell genome, and/or
non-motile inter- or intra-cellularly. A wide variety of suitable
means for microbial attenuation are known in the art. In another
particular embodiment, the bacteria are dead or non-viable prior to
endocytosis by the target cell or administration to the target
organism, obviating any microbial growth or metabolism in the
target cell. A wide variety of suitable means for killing or
rendering the bacteria nonviable are known in the art, including
fixation with organic solvents such as methanol, UV irradiation,
heat, freeze-drying, etc. Preferred methods preserve the ability of
the microbial membrane and/or wall to retain the cytolysin and the
foreign agent. In this embodiment, the first and second genes are
sufficiently expressed to load the microbe with an effective amount
of the cytolysin and foreign agent prior to microbial cell death.
Generally the bacteria contain (i.e. are loaded by expression
within the bacteria with) with from about ten to one thousand,
preferably from about one hundred to one thousand cytolysin
molecules per bacterium.
[0018] The microbes of the invention can be used to deliver the
foreign agent to virtually any target cell capable of endocytosis
of the subject microbe, including phagocytic, non-phagocytic,
pathogenic or diseased cells. Exemplary target animal cells include
epithelial cells, endothelial cells, muscle cells, liver cells,
pancreatic cells, neural cells, fibroblasts, tumor cells,
leukocytes such as macrophages, neutrophils, B-cells, T-cells,
monocytes, etc., etc. The subject methods generally require
microbial uptake by the target cell and subsequent lysis within the
target cell vacuole (including phagosomes and endosomes). While
phagocytic target cells generally provide for microbial uptake and
lysis, for many cell types, it is necessary to provide the bacteria
with an invasin to facilitate or mediate uptake by the target cell
and an autolysin to facilitate or mediate autolysis of the bacteria
within the target cell vacuole. A wide variety of suitable invasins
and autolysins are known in the art. For example, both Sizemore et
al. (Science, 1995, 270:299-302) and Courvalin et al. (C.R. Acad.
Sci. Paris, 1995, 318:1207-12) teach expression of an invasin to
effect endocytosis of the bacterium by a target cell and suitable
microbial autolysins are described by Cao et al., Infect Immun
1998, 66(6): 2984-2986; Margot et al., J. Bacteriol 1998,
180(3):749-752; Buist et al., Appl Environ Microbiol, 1997,
63(7):2722-2728; Yamanaka et al., FEMS Microbiol Lett, 1997,
150(2): 269-275; Romero et al., FEMS Microbiol Lett, 1993,
108(1):87-92; Betzner and Keck, Mol Gen Genet, 1989, 219(3):
489-491; Lubitz et al., J. Bacteriol, 1984, 159(1):385-387; and
Tomasz et al., J. Bacteriol, 1988, 170(12): 5931-5934. Providing
the advantage of delayed lysis are temperature-sensitive
autolysins, time-sensitive autolysins (see, e.g. Chang et al.,
1995, J Bact 177, 3283-3294; Raab et al., 1985, J Mol Biol 19,
95-105; Gerds et al., 1995, Mol Microbiol 17, 205-210) and
addiction (poison/antidote) autolysins, (see e.g. Magnuson R. et
al., 1996, J Biol. Chem. 271(31), 18705-18710; Smith A S, et al.,
1997, Mol. Microbiol. 26(5), 961-970).
[0019] Administration of the microbe to target cells may be in
vitro or in vivo according to conventional methodologies. In either
case, the methods generally involve growing the microbes, inducing
the expression of the first and second genes, and contacting the
target cells with an effective amount of bacteria sufficient to
effect the desired activity of the foreign agent in the target
cell. Immunofluorescense may be used to image and track the
contents of the bacteria upon administration to the cells in vivo
or in vitro.
[0020] In vitro or ex vivo administration generally involves
contacting the target cell with an effective amount of the microbes
of the invention. Exemplary in vitro administrations are described
and/or cited by reference below. In vitro applications include
protein delivery (e.g. for functional determinations, toxin
delivery to targeted cells in culture, half-life, degradation and
localization determinations), nucleic acid delivery (e.g. DNA to
transfected cell lines, genomic libraries to screen and identify
specific antigens, i.e. expression cloning, etc.)
[0021] In vivo administration generally involves administering a
pharmaceutical composition containing a therapeutically effective
amount of the microbes of the invention. Generally, the
therapeutically effective amount is between about 1 .mu.g and 100
mg/kg, preferably between about 1 .mu.g and 1 mg/kg. The microbes
are formulated into a pharmaceutical composition by combination
with an appropriate pharmaceutically acceptable excipient in
accordance with routine procedures known to one of ordinary skill
in the art. The microbes may be used alone or in appropriate
association, as well as in combination with other pharmaceutically
active compounds. The microbes may be formulated into preparations
in solid, semisolid, or liquid form such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, and
injections, in usual ways for topical, nasal, oral, parenteral, or
surgical administration. Administration in vivo can be oral,
mucosal, nasal, bronchial, parenteral, subcutaneous, intravenous,
intra-arterial, intra-muscular, intra-organ, intra-tumoral,
surgical or in general by any means typical of a gene therapy
administration. Administration will be selected as is appropriate
for the targeted host cells. Target cells may also be removed from
the subject, treated ex vivo, and the cells then replaced into the
subject. Exemplary methods for in vivo administration are described
in Shen et al., Proc Natl Acad Sci USA 1995, 92(9):3987-3991;
Jensen et al, Immunol Rev 1997, 158: 147-157; Szalay et al., Proc
Natl Acad Sci USA 1995, 92(26):12389-12392; Belyi et al, FEMS
Immunol Med Microbiol 1996, 13(3): 211-213; Frankel et al., J.
Immunol. 1995, 155(10):4775-4782; Goossens et al., Int Immunol
1995, 7(5):797-805; Schafer et al., J. Immunol. 1992, 149(1):53-59;
and Linde et al., Vaccine 1991, 9(2):101-105.
[0022] The foregoing methods and compositions are demonstrated to
be effective in a wide variety of exemplary applications. In one
application, a K12 strain of E. coli is engineered with a signal
sequence deficient LLO gene operably linked to the constitutive tet
promoter for expressing the cytolysin in the bacterium and a second
gene encoding a truncated BRCA1 cancer antigen, under regulatory
control of a trc or tac promoter. The cytolysin and cancer antigen
are expressed to maximum levels, the bacteria are then fixed with
methanol, and the killed bacteria loaded with the cytolysin and
cancer antigen are then injected into solid breast tumors in three
weekly injections. At four weeks, a cancer antigen-specific
cytotoxic T-cell response (CTL response) and tumor size reduction
is detected. As shown in Table 1, analogous studies conducted in a
variety of animals and animal cell types, both in vivo and in
vitro, using a variety of agents, secretory deficient cytolysins,
bacterial types and methods demonstrate consistent delivery of the
agent to the target cell cytosol, as measured by agent activity,
immunoassay, or other delivery monitoring assays described
herein.
TABLE-US-00001 TABLE 1 Microbial-Based Delivery Target Cell
Indication Agent Lysin Bacteria Administration transformed human
acites tumor in nude mice human tumor antigen hTA1 LLO E. coli,
JM109 (DE3) intraperitoneal macrophage injection rat liver
hepatocellular carcinoma p51 tumor suppressor LLO.sup.M1 E. coli,
DP-E3619, in situ; intratumor invasin/autolysin injection rat
kidney genetic nephopathy angiotensin converting enzyme LLO.sup.M2
S. typhimurium, attenuated, ex vivo invasin/autolysin mouse brain
neurodegeneration cFos gene expression construct LLO.sup.M3 S.
typhimurium, attenuated, in situ; intracranial invasin/autolysin
implant mouse pancreas transformation .gamma.-interferon expression
construct LLO.sup.M4 E. coli, JM109 (DE3) in vitro pig muscle
muscular atrophy insulin-like growth factor I (IGF-I) PLO E. coli,
DP-E3618, in situ; i.m. invasin/autolysin injection human breast
transformation anti-estrogen receptor antibody SLO E. coli,
DP-E3617, in vitro expression construct invasin/autolysin human
prostate localized prostatic carcinoma ribozyme or antisense
against CDK LLO S. typhimurium, attenuated in situ; intratumor 2 or
CDK4 invasin/autolysin injection human lymphoid lymphoma tumor
necrosis factor (TNF) LLO E. coli, DP-E3616 ex vivo human bone
marrow mylomoid leukemia Hepatocyte growth factor/scatter SLO E.
coli, DP-E3615 ex vivo factor (HGF/SF) human lymphoid HIV infection
HIV RT gene-specific ribozyme LLO S. typhimurium, attenuated ex
vivo human hepatic cells Hepatitis C infection Hepatitis C
virus-specific ribozyme LLO S. typhimurium, attenuated, in vivo;
direct invasin/autolysin injection human hepatic cells diabetes
insulin receptor expression LLO S. typhimurium, attenuated, in
vivo; direct construct invasin/autolysin injection human beta islet
cells diabetes insulin expression construct LLO S. typhimurium,
attenuated, in vivo; direct invasin/autolysin injection murine
fibroblast fibroblastoma diptheria toxin LLO S. typhimurium,
attenuated, in vivo; direct invasin/autolysin injection feline
retinal cells retinal degenerative disease cGMP
phosphodiesterase-beta LLO E. coli, JM109 (DE3), intraocular
invasin/autolysin injection human cytotoxic T- melanoma melanosomal
proteins LLO E. coli, JM109 (DE3) in vivo; IV cells injection human
epithelium Herpes infection antisense RNAseP construct LLO S.
typhimurium, attenuated, in vivo; oral, opical invasin/autolysin
abrasion murine macrophages IL-2 production NFAT LLO S. typhimurium
in vitro LLO.sup.M1-M4 are LLO mutants M1(Cys486Ser),
M2(Cys486Met), M3(Trp492Ala) and M4(del491-493), respectively . .
.
EXAMPLES
I. Delivery of Protein to the Cytosol of Macrophages Using
Escherichia coli K-12 Expressing Listeriolysin O
[0023] Listeria monocytogenes is a bacterial pathogen that
replicates within the cytosol of mammalian cells. L. monocytogenes
has been used extensively as a model for the study of cell-mediated
immunity and as a model pathogen for understanding the basis of
intracellular pathogenesis (13, 14). Following internalization into
host cells, bacteria are initially contained within host vacuoles
then subsequently lyse these vacuoles to gain access to the
cytosol. The ability of L. monocytogenes to lyse the vacuole and
enter the cytosol is primarily mediated by listeriolysin O (LLO).
LLO is a member of a family of related pore-forming cytolysins
secreted by diverse species of gram positive bacteria (15). LLO
encapsulated into pH-sensitive liposomes has been used as a vehicle
to deliver co-encapsulated protein to the cytosol of macrophages
(16). Moreover, purified LLO when mixed with foreign proteins and
added to mammalian cells can mediate the delivery of protein to the
cytosol and has been exploited for delivery to host cells both in
vitro and in vivo (17-19). However, both of these methods require
the purification of LLO and the protein to be delivered. Here, we
show that Escherichia coli expressing cytoplasmic LLO can be used
to efficiently deliver co-expressed proteins to the cytosol of
macrophages. The utility of this system to deliver a large active
protein to the cytosol was demonstrated by the delivery of E. coli
.beta.-galactosidase (.beta.-gal). Using chicken ovalbumin (OVA) we
demonstrate the rapid delivery of protein to the cytosol of
macrophages and the ability of the E. coli/LLO system to
efficiently deliver OVA to the MHC class I pathway of antigen
processing and presentation. Moreover, the time required for
processing and presentation of an OVA-derived peptide to CD8.sup.+
T cells, when OVA was delivered using this system, was equivalent
to that previously reported when purified OVA was introduced into
the cytosol by alternative methods such as scrape-loading or
liposomes (16, 20, 21).
[0024] Bacterial Strains and Plasmids. All bacterial strains and
plasmids used in this report are listed in Table 2.
TABLE-US-00002 TABLE 2 E. coli strains and plasmids used in this
work Strain or Plasmid Description Reference or source pACYC184
cloning vector; Tc.sup.r Cm.sup.r (23) pET28a over-expression
vector; Kan.sup.r (Novagen, Inc.) pET29b over-expression vector;
Kan.sup.r (Novagen, Inc.) pTL61T lacZ transcriptional fusion
vector; Ap.sup.r (24) pBluescript SK- cloning vector; Ap.sup.r
(Stratagene, Inc.) pTrcHisC/Ova pTrcHisC::ova (D. Campbell, UCB)
pDP3615 pACYC184 tet::hly Herein pDP3616 pET28a pT7::ova Herein
pDH70 pBluescript SK- pT7::lacZ Herein MC4100(DE3) F.sup.- araD139
.DELTA.(argF-lac) U169 rpsL150 (Schifferli, U.Penn)
(Str.sup.r)relA1 flbB5301 deoC1 ptsF25 rbsR with DE3, a .lamda.
prophage carrying the T7 RNA polymerase gene JM109(DE3) endA1 recA1
gyrA96 thi hsdR17 relA1 supE44 (Promega, Co.) .DELTA.(lac-proAB)
[F' traD36 proAB lacI.sup.q Z.DELTA.M15] DE3 DP-E3615 MC4100(DE3)
harboring pDP3615 Herein DP-E3616 MC4100(DE3) harboring pDP3616
Herein DP-E3617 MC4100(DE3) harboring pDP3615 and pDP3616 Herein
DP-E3618 JM109(DE3) harboring pDH70 Herein DP-E3619 JM109(DE3)
harboring pDP3615 and pDH70 Herein Ap.sup.r, ampicillin resistant;
Tc.sup.r, tetracycline resistant; Kan.sup.r, kanamycin resistant;
Cm.sup.r, chloramphenicol resistant; Str.sup.r, streptomycin
resistant
[0025] Plasmid pDP3615 was generated by PCR amplification of the
hly gene encoding LLO lacking its secretion signal sequence (22).
DNA sequences encoding mature LLO were first PCR amplified and
cloned into pET26b (Novagen, Inc., Madison, Wis.) using
oligonucleotide primer 5'-GGAATTCCATATGAAGGATGCATCTGCATTCAAT-3'
(SEQ ID NO: 1) generating a NdeI restriction site at the gene
fragment and primer 5'-CGGGATCCTTATTATTCGATTGGATTATCTACT-3' (SEQ ID
NO:2) generating a BamHI restriction site at the 3' end of the gene
fragment. Following ligation into the pET29b vector, the DNA
sequences encoding mature LLO along with the upstream translation
initiation site found in pET29b were amplified using primer
5'-CGCGATATCCTCTAGAAATAATTTTG-3' (SEQ ID NO:3) generating an EcoRI
restriction site at the 5' end of the gene fragment and the same
primer used previously to generate a BamHI restriction site at the
3' end of the gene fragment. The amplified fragment was ligated
into pACYC184 (23) placing transcription of the mature hly gene
under control of the tet gene promoter. Plasmid pDP3616 was
generated by subcloning a NcoI-HindIII fragment containing DNA
sequences encoding truncated OVA from plasmid pTrcH is C/OVA. The
DNA fragment was ligated into the over-expression vector pET28a
(Novagen, Inc., Madison, Wis.). Plasmid pDH70 was generated by PCR
amplification of the promoterless lacZ gene in plasmid pTL61T (24)
using oligonucleotide primers
5'-AGGCGTCGACGGTTAATACGACCGGGATCGAG-3' (SEQ ID NO:4) and
5'-AGGCGTCGACAGGCCTTACGCGAAATACGGGCAGACATGG-3' (SEQ ID NO:5)
generating SalI restriction sites at both the 5' and 3' ends of the
fragment. The amplified fragment was ligated into pBluescript SK-
(Stratagene, Inc., La Jolla, Calif.) placing transcription of the
lacZ gene under control of a phage T7 promoter. Plasmid DNA was
transferred to E. coli strains by transformation, using standard
methods (25). E. coli strains were grown in Luria-Bertani (LB)
medium. The strains were stored at -70.degree. C. in LB medium plus
40% glycerol. Antibiotics were used at the following
concentrations: ampicillin, 100 .mu.g/ml; chloramphenicol, 40
.mu.g/ml; and kanamycin, 30 .mu.g/ml.
[0026] Expression of Target Proteins. E. coli strains were
inoculated from a LB agar plate into 2 mls of LB medium and grown
overnight to stationary phase at 37.degree. C. with aeration.
Cultures were diluted 1:100 in 10 mls of LB medium in 250 ml flasks
and grown 2 hours with aeration at 30.degree. C. Target protein
expression was induced by the addition of
isopropyl-.beta.-D-thiogalactopyranoside (IPTG) to 1 mM and growth
continued until cultures reached an OD.sub.600 of 0.5. Equivalent
numbers of bacteria were centrifuged (14,000.times.g) for 1 minute
and washed once with phosphate buffered saline (PBS). Washed
samples were suspended in Final Sample Buffer (0.0625M Tris pH 6.8,
2% SDS, 10% glycerol, 0.01% bromophenol blue) boiled for 5 minutes
and total cellular protein analyzed by polyacrylamide gel
electrophoresis followed by staining with Coomassie Brilliant
Blue.
[0027] Determination of Hemolytic Activity. Following bacterial
growth and induction of target proteins, 1 ml aliquots of bacteria
were centrifuged (14,000.times.g) for 1 minute and washed once with
PBS. Samples were resuspended in 1 ml of PBS and lysed by
sonication. Soluble extract fractions were obtained by centrifuging
lysed samples for 10 minutes (14,000.times.g) at 4.degree. C. and
saving the supernatant. Hemolytic activity in the soluble fractions
was determined as previously described (26) and is expressed as the
reciprocal of the dilution of extracts required to lyse 50% of
sheep erythrocytes.
[0028] Cell Culture. Cell lines were maintained in RPMI 1640 medium
or DMEM supplemented with 10% fetal bovine serum (Hyclone
Laboratories, Inc., Logan, Utah), 2 mM glutamine, 1 mM pyruvate, 50
.mu.M 2-mercaptoethanol, penicillin (200 units/ml), and
streptomycin (200 .mu.g/ml) at 37.degree. C. in a 5% CO.sub.2/air
atmosphere. The IC-21 and Raw 309 Cr. 1 mouse macrophage cell lines
were obtained from the American Type Culture Collection (ATCC,
Rockford, Md.). The B3Z T-cell hybrid is a LacZ-inducible CD8.sup.+
T-cell hybridoma specific for OVA residues 257-264, SIINFEKL (SL8)
(SEQ ID NO:6), presented on the murine K.sup.b MHC class I molecule
(27, 28).
[0029] Delivery of Protein to the Cytosol of Macrophages. IC-21
cells were seeded onto 18 mm glass coverslips in 35 mm dishes in
RPMI medium without antibiotics. One hour prior to addition of
bacteria, dishes were placed at 4.degree. C. Medium was removed
from the dishes and E. coli were added in cold RPMI medium without
antibiotics to obtain an infection ratio of one
bacterium/macrophage. Samples were incubated at 4.degree. C. for
one hour to allow association of bacteria and macrophages. Samples
were washed five times with 3 mls of cold PBS and 37.degree. C.
RPMI medium added. Samples were incubated at 37.degree. C. in a 5%
CO.sub.2/air atmosphere for up to one hour. At varying intervals
during incubation of macrophages and E. coli, coverslips were
removed and fixed for subsequent detection of protein.
[0030] Detection of Target Protein in Macrophages. Following
delivery of 1-gal to macrophages, coverslips were fixed in cold 2%
formaldehyde/0.2% glutaraldehyde for 5 minutes at 4.degree. C.
.beta.-gal activity was detected by staining with
5-bromo-4-chloro-3-indolyl .beta.-galactopyranoside (X-gal, Sigma
Immunochemicals, St. Louis, Mo.) as previously described (27). For
detection of OVA, coverslips were fixed in 3.2% electron microscopy
grade paraformaldehyde (Electron Microscopy Sciences, Ft.
Washington, Pa.) overnight at 4.degree. C. in aluminum foil wrapped
containers. OVA was detected by immunofluorescence as previously
described (29) with the exception that polyclonal rabbit anti-OVA
antibody (Calbiochem, San Diego, Calif.) and LRSC-conjugated donkey
anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, Pa.)
were used for detection.
[0031] Antigen Presentation Assays. Presentation of SL8/K.sup.b
complex to B3Z cells was determined as previously described (30).
Briefly, E. coli were added to 1.times.10.sup.5 antigen presenting
cells (APCs) in each well of a 96-well microtiter plate. Following
one hour of incubation at 37.degree. C. in a 5% CO.sub.2/air
atmosphere, extracellular bacteria were removed by washing three
times with PBS and 1.times.10.sup.5 B3Z T cells were added to each
well in medium containing 100 .mu.g/ml gentamicin. Following 15
hours of incubation at 37.degree. C. in a 5% CO.sub.2/air
atmosphere, cultures were washed once with PBS and lysed by
addition of 100 .mu.l PBS buffer containing 100 .mu.M
2-mercaptoethanol, 9 mM MgCl.sub.2, 0.125% NP40, and 0.15 mM
chlorophenolred-.beta.-galactoside (CPRG, Calbiochem, San Diego,
Calif.). After 4-6 hours at 37.degree. C., 50 .mu.l of stop buffer
(300 mM glycine and 15 mM EDTA in water) was added and the
absorbance at 570 nm of each well was determined using a 96-well
plate reader. Where appropriate, APCs were fixed with 1%
paraformaldehyde prior to the addition of B3Z cells as described
(31). Synthetic SL8 peptide was obtained from Research Genetics,
Inc. (Huntsville, Ala.) and was incubated with APCs and B3Z cells
at a saturating concentration of 90 nM in RPMI medium.
[0032] Expression of Listeriolysin O and Target Proteins. To
facilitate expression of mature cytoplasmic LLO in E. coli, the hly
gene encoding LLO lacking its N-terminal signal sequence (22) was
cloned into the plasmid vector pACYC184 to generate pDP3615 as
described in Materials and Methods. Transcription of the truncated
hly gene in pDP3615 is under the control of the constitutive tet
gene promoter. Proteins to be delivered to the cytosol of
macrophages were expressed from co-resident plasmids in E. coli. We
chose chicken ovalbumin (OVA) as one of the representative proteins
to deliver to the cytosol of macrophages. OVA is not toxic to E.
coli and can be readily expressed to high levels (32). A plasmid
encoding truncated (32 kD) OVA was transformed into E. coli along
with pDP3615. In order to determine if a large protein with a
measurable enzymatic activity could be delivered to the cytosol of
macrophages, we expressed .beta.-galactosidase (.beta.-gal) along
with LLO in E. coli. A plasmid containing the gene encoding
.beta.-gal, was transformed into E. coli along with plasmid
pDP3615. Expression of both OVA and .beta.-gal in these strains is
under the control of IPTG-inducible phage T7 RNA polymerase. We
next analyzed the hemolytic activity and protein expression
profiles of these strains. Following IPTG induction, OVA and
.beta.-gal were expressed to approximately 20% of the total E. coli
cellular protein as determined by SDS-PAGE. To verify expression of
active LLO protein within E. coli, hemolytic activity contained in
the soluble fraction of E. coli extracts was determined as
described above. All of the strains expressing LLO contained
approximately 500-600 hemolytic units of activity in the soluble
extracts. No measurable hemolytic activity was found in the culture
medium in which the E. coli were grown. These data indicate that
functional LLO protein was contained within the E. coli cells and
not secreted to the extracellular environment.
[0033] Delivery of Protein to the Cytosol of Macrophages. To
examine the ability of E. coli expressing LLO to deliver a
co-expressed target protein to the cytosol of macrophages, E. coli
expressing LLO and OVA were added to macrophages to obtain an
infection ratio of approximately one bacterium/macrophage. The
presence of OVA either in phagosomes or in the cytosol was
determined by immunofluorescence microscopy. When bacteria
expressing OVA in the absence of LLO were added to macrophages,
protein was contained within phagosomes and no OVA could be
detected in the cytosol of macrophages within one hour following
phagocytosis. In contrast, when bacteria expressing both LLO and
OVA were added to macrophages, OVA protein could be detected
throughout the entire cytosolic compartment within 30 minutes of
bacterial uptake. Moreover, at least 50% of macrophages that had
phagocytosed a single bacterium demonstrated release of OVA into
the cytosol within 30 minutes following bacterial uptake. In some
instances, OVA protein could be detected leaking from phagosomes
into the cytosol as early as 10 minutes post-phagocytosis of
bacteria.
[0034] Since release of OVA into the cytosol occurs subsequent to
degradation of the E. coli within macrophage phagosomes, it was
possible that proteins contained within the bacteria were also
partially degraded or inactivated before release into the cytosol.
To examine whether full-length .beta.-gal could be delivered to the
cytosol of macrophages and retain its biological activity, E. coli
expressing LLO and .beta.-gal were added to macrophages to obtain
an infection ratio of approximately one bacterium/macrophage.
.beta.-gal activity in the cytosol was then determined by staining
macrophages with X-gal. Our data indicate that .beta.-gal activity
could be detected throughout the cytosol within 30 minutes
following bacterial uptake. Following phagocytosis of E. coli
expressing .beta.-gal in the absence of LLO, .beta.-gal activity
was detected sequestered within phagosomes. .beta.-gal is a 116 kD
protein that functions as a 465 kD tetramer (33). Whether
.beta.-gal is associated as a tetramer prior to release into the
cytosol is unknown, but these data indicate that at a minimum a 116
kD protein can be delivered to the cytosol of macrophages using the
E. coli/LLO delivery system and still retain its enzymatic
activity.
[0035] Delivery of OVA to the MHC Class I Pathway for Antigen
Presentation. Immunity to intracellular bacterial pathogens and
viruses often requires the generation of cytotoxic T-lymphocytes
(CTLs) that recognize and kill infected cells (13, 34, 35).
Efficient processing and presentation of antigens to CTLs typically
requires the endogenous synthesis of the antigen within the cytosol
of the infected cell or introduction of the antigenic protein into
the cytosol of an antigen presenting cell (APC) (36, 37). Once in
the cytosol, proteases process the antigen to peptide epitopes
which are subsequently presented on the surface of the APC in
association with major histocompatibility (MHC) class I molecules
for recognition by CD8.sup.+ CTLs (37-39).
[0036] We wished to examine the ability of E. coli expressing LLO
and an antigenic protein to deliver the antigen to the cytosol of
macrophages for processing and presentation on MHC class I
molecules. E. coli expressing LLO and OVA were added to macrophages
and the processing and presentation of a peptide epitope derived
from OVA was accessed using the B3Z T-cell hybrid. B3Z is a
LacZ-inducible CD8.sup.+ T-cell hybrid specific for OVA residues
257-264 (SIINFEKL, SEQ ID NO:6) presented on the murine K.sup.b MHC
class I molecule (27, 28). The presentation of the SIINFEKL epitope
(SL8) to B3Z cells results in the induction of 1-gal synthesis by
B3Z. The amount of .beta.-gal produced can be measured by the
hydrolysis of the chromogenic substrate CPRG and is an indication
of the amount of SL8/K.sup.b complexes presented on the surface of
APCs (27, 30). Bacteria were added to macrophages and phagocytosis
allowed to proceed for one hour, followed by the removal of
extracellular bacteria and addition of B3Z T-cells. As shown in
FIG. 1, processing and presentation of SL8/K.sup.b to B3Z T-cells
occurred when E. coli expressing both LLO and OVA were added to
macrophages. In this figure, the indicated number of E. coli were
added to 1.times.10.sup.5 Raw 309 Cr.1 APCs in each well of a
96-well plate. Following one hour of incubation at 37.degree. C. in
a 5% CO.sub.2/air atmosphere, extracellular bacteria were removed
by washing with PBS and 1.times.10.sup.5 B3Z T-cells were added to
each well in medium containing 100 .mu.g/ml gentamicin. Following
15 hours of incubation at 37.degree. C. in a 5% CO.sub.2/air
atmosphere, presentation of SL8/K.sup.b to B3Z cells was assayed as
described (27, 30) and indicated as an increase in the absorbance
at 570 nm. E. coli strains added to APCs expressed LLO, DP-E3615
(.largecircle.); OVA, DP-E3616 ( ); or LLO and OVA, DP-E3617,
(.quadrature.). (.box-solid.) indicates the level of activation
obtained when APCs were incubated with 90 nM synthetic SL8 and B3Z
cells. Data presented is from triplicate groups of wells from one
of several repeated experiments with identical results. As shown,
antigen presentation could be detected with as few as 1 bacterium
added/10 macrophages. At a ratio of 10 bacteria added/macrophage,
which resulted in the phagocytosis of one to two
bacteria/macrophage, the level of presentation was equivalent to
the maximal activity achieved by incubating macrophages with a
saturating dose (90 nM) of synthetic SL8 peptide. No presentation
of SL8/K.sup.b complex could be detected when E. coli expressing
OVA in the absence of LLO were added to macrophages. Equivalent
results were obtained when primary bone marrow and peritoneal
derived macrophages were used in antigen presentation experiments.
The decrease in absorbance seen when 10.sup.7 and 10.sup.8 LLO
expressing bacteria were added was due to visible damage to the
macrophages.
[0037] Processing and Presentation of SL8/K.sup.b Complex is Rapid.
It has been previously demonstrated that OVA is efficiently
processed and peptide/MHC complexes presented on the surface of
APCs within two to four hours following delivery of OVA to the
cytosol using alternative methods such as encapsulation in
liposomes or scrape-loading (16, 20, 21). Data in FIG. 1 indicate
that considerable processing and presentation of antigenic peptides
can occur with as few as one bacterium added/macrophage. However,
processing and presentation of antigen was allowed to occur for
greater than 15 hours prior to measuring T-cell activation. It is
possible that the time required for efficient processing of antigen
has been altered by delivering protein to the cytosol using this
method. We wished to examine the time necessary for antigen
processing and presentation of peptide/MHC complexes when protein
is delivered using the E. coli/LLO system.
[0038] Paraformaldehyde fixation of macrophages prevents further
phagocytosis of bacteria and has been shown to crosslink surface
MHC class I molecules to associated .beta..sub.2-microglobulin
(31). Thus, fixation stabilizes peptide/MHC complexes present on
the cell surface and arrests any further processing and
presentation of peptides. We examined the time required for antigen
processing and presentation by fixing APCs with paraformaldehyde at
varying intervals after addition of bacteria and measuring
SL8/K.sup.b presentation to B3Z T-cells. First, the effect on
antigen presentation of fixing macrophages prior to the addition of
bacteria was addressed (FIG. 2A). In FIG. 2A, 1.times.10.sup.6 E.
coli strain DP-E3617 expressing LLO and OVA were added to
1.times.10.sup.5 IC-21 macrophages in each well of a 96-well plate.
Processing and presentation of SL8/K.sup.b was assayed as described
in FIG. 1. Immediately prior to the addition of bacteria, APCs were
either left untreated (Not Fixed) or fixed (Pre-Fixed) with 1%
paraformaldehyde as described (31). Fixing APCs prior to the
addition of bacteria completely abrogated the ability of
macrophages to process and present SL8/K.sup.b complex to B3Z
T-cells, as evident by an equivalent response as that seen when no
bacteria were added to the macrophages (FIG. 1).
[0039] In FIG. 2B, the time requirement for processing and
presentation of SL8/K.sup.b complex was addressed. Here,
1.times.10.sup.6E. coli strain DP-E3617 expressing LLO and OVA were
added to 1.times.10.sup.5 IC-21 APCs in each well of a 96-well
plate. Following one hour of incubation at 37.degree. C. in a 5%
CO.sub.2/air atmosphere, extracellular bacteria were removed by
washing with PBS and APCs were either immediately fixed with 1%
paraformaldehyde (1 hour) or incubated further in media containing
100 .mu.g/ml gentamicin. At one hour intervals, APCs were fixed
with 1% paraformaldehyde until all time points were completed.
Following completion of the four hour time interval, APCs were
washed with PBS and 1.times.10.sup.5 B3Z T cells added to each
well. Presentation of SL8/K.sup.b was assayed as described above.
Labels indicate the time elapsed post addition of bacteria prior to
fixation of APCs. Dark shaded bars indicate samples to which E.
coli strain DP-E3617 were added. Light shaded bar indicates samples
to which no bacteria but 90 nM synthetic SL8 was added with B3Z
cells. The (Not Fixed) samples received no fixation prior to the
addition of B3Z cells. Data presented is from triplicate groups of
wells from one of three experiments with identical results.
[0040] Fixing APCs at one hour following the addition of E. coli
expressing LLO and OVA resulted in sufficient antigen presentation
to yield activation of B3Z cells to a level slightly higher than
those seen in the absence of fixing (compare 1 hour fixed to Not
Fixed). The increased level of SL8/K.sup.b presentation following
fixation can be attributed to crosslinking of surface MHC class I
molecules resulting in increased stability of peptide-MHC complexes
(31). Consistent with previous studies of OVA delivery to APCs (16,
20, 21), the maximal presentation of SL8/K.sup.b complex occurred
when processing of OVA was allowed to continue for two hours prior
to fixation. This level of SL8/K.sup.b presentation was equivalent
to that seen with the addition of 90 nM synthetic SL8 in the
absence of fixation (FIG. 2B). Additional analysis indicated that
fixing APCs later than two hours after addition of bacteria,
resulted in a decreased level of SL8/K.sup.b presentation (FIG. 2B,
3 and 4 hour time points). This is consistent with dissociation of
surface peptide/MHC complexes prior to crosslinking MHC class I
molecules by fixation (31). These data indicate that no delay in
the processing and presentation of SL8/K.sup.b complex occurred
when OVA was delivered to the cytosol using the E. coli/LLO
delivery system.
[0041] The results of this example demonstrate that E. coli
expressing cytoplasmic LLO can be used to deliver a co-expressed
protein to the cytosol of macrophages. The delivery of protein to
macrophages was rapid and efficient with protein first appearing in
the cytosol within ten minutes following bacterial uptake.
Moreover, large enzymatically active proteins can be introduced
into the cytosol using this method as demonstrated by the delivery
of active .beta.-gal. The mechanism of delivery may be as follows.
Subsequent or concomitant to phagocytosis, the E. coli are killed
and degraded within phagosomes causing the release of LLO and the
target protein from the bacteria. LLO acts by forming large pores
in the phagosomal membrane thus releasing the target protein into
the cytosol. In any event, any protein that can be synthesized in
E. coli can be delivered to the macrophage cytosol.
[0042] LLO is an essential determinant of pathogenesis whose role
is to mediate release of L. monocytogenes from a phagosome. The
biological properties of LLO make it well suited for use in our
system. For example, LLO has an acidic pH optimum which facilitates
its action in a phagosome (40, 41). However, it was unclear whether
LLO released by degraded E. coli would retain its biological
activity. The data presented here demonstrate that the amount of
LLO expressed was sufficient to allow the rapid release of protein
into the cytosol. Based on SDS-PAGE analysis of known quantities of
purified LLO protein, we estimate approximately 1.times.10.sup.5
molecules of LLO per E. coli cell. Using our disclosure, one can
now determine how many molecules of LLO are actually needed to
introduce a pore into a phagosome as described (42), where it was
determined to take only approximately 50 molecules of streptolysin
O, a homologous pore-forming cytolysin, to form a pore in red cell
membranes. A second property of LLO is its relative lack of
toxicity thought to be due to its proteolysis in the cytosol of
host cells (43). Indeed, secretion by L. monocytogenes of a related
pore-forming hemolysin, perfringolysin O, resulted in death of the
infected cells (44). Other facultative intracellular pathogens,
Shigellae, Salmonellae, and Yersiniae all induce macrophage
apoptosis (14, 45), yet infection with L. monocytogenes is
relatively benign (46). In the current study, even though we
estimate each recombinant E. coli contained approximately
1.times.10.sup.5 molecules of LLO, there was no evidence of
toxicity until there were about 25 bacteria/macrophage.
[0043] There are a number of advantages and applications of the E.
coli/LLO delivery system. Many methodologies for delivering protein
to the cytosol of macrophages require the prior purification of the
protein to be delivered. With the E. coli/LLO mediated delivery of
protein, no protein purification is required, only expression of
the target protein in E. coli is necessary. Furthermore, with many
alternative methods, delivery is restricted to minute amounts of
protein or a limited number of cells and the in vivo delivery of
protein can not be achieved (2). Using the E. coli/LLO system, high
levels of protein can be delivered to the cytosol of virtually all
of the cells in culture. In addition, by expressing protein under
the control of inducible promoters, the level of protein produced
and ultimately delivered to the cytosol of macrophages can be
controlled. This system can be used in vivo and by expressing
invasive determinants from other bacterial species, the E. coli may
be modified to enter cells other than macrophages. Furthermore,
this system has applications for the delivery of pathogen-specific
protein antigens or DNA.
[0044] The results of this example show that the E. coli/LLO system
is particularly effective for the introduction of protein into the
MHC class I pathway of antigen processing and presentation. We were
able to detect antigen presentation with less than 1 bacterium/10
macrophages and observed a maximal response with as few as 1 to 2
bacteria/macrophage (FIG. 1). In addition, efficient processing and
surface presentation of peptide/MHC complexes occurred rapidly,
within 1-2 hours following addition of bacteria to macrophages
(FIG. 2B). Delivery to the MHC class I pathway was enhanced greater
than 4-logs compared to E. coli expressing OVA alone. This is a
similar level of enhancement to that reported when OVA linked to
beads was compared to soluble OVA for presentation with MHC class 1
molecules (47). It is clear from subsequent studies that the beads,
like LLO, mediated disruption of the phagosome (21, 48). However,
there was one report in which E. coli expressing OVA was able to
deliver OVA to the MHC class I pathway (49). Here, delivery was
proposed to occur by a non-conventional pathway involving
extracellular peptide regurgitation of phagosomal processed
antigens instead of transfer of protein from the phagosomal
compartment to the cytosol. Nonetheless, our data clearly show
undetectable levels of antigen presentation when E. coli lacking
LLO yet expressing OVA to 20% of the total cellular protein were
used to deliver OVA to macrophages. Perhaps the T-cells used in our
studies were unable to detect presentation of SL8/K.sup.b
complexes, when E. coli expressing OVA alone were used, because of
inefficient processing and presentation via the non-conventional
pathway. In the previous study, OVA was generated as fusions to Crl
or LamB proteins. The efficiency of processing and presentation of
epitopes from OVA has been shown to be dependent on the protein
context surrounding the epitope (50, 51). Therefore, it is possible
that the fusion proteins used in the previous study are processed
more efficiently than the truncated OVA used in this report.
[0045] Recently, an E. coli expression cloning strategy for the
identification of CD4.sup.+ T cell-stimulating antigens has been
reported (30). However, this method has not been successfully used
to identify CD8.sup.+CTL-stimulating antigens since proteins
expressed in E. coli do not gain efficient access to the MHC class
I pathway for antigen presentation. The results of this study
indicate that the E. coli/LLO delivery system provides an
expression cloning strategy for the identification of
pathogen-specific CD8.sup.+ CTL-stimulating antigens. The
identification of these pathogen-specific epitopes is an important
step in the rational design of vaccine strategies against these
infectious agents. These antigens are further characterized to
determine the peptide epitopes recognized by CTLs, as well as the
natural function the antigenic protein plays in the interaction of
the pathogen and host cells.
[0046] The efficiency of antigen delivery provides for the E.
coli/LLO system to be used for the induction of CTLs in vivo. The
efficient in vivo delivery of antigens to generate a protective
immune response is a significant challenge in vaccine development.
The use of bacterial vectors that have evolved to invade and
replicate in mammalian cells such as Shigella (11, 12), Salmonella
(8-10), and Listeria monocytogenes (52-55) are being explored as
methods for the delivery of both protein antigens and DNA. Although
these vehicles have had success in eliciting protective immune
responses, the in vivo use of pathogenic bacteria has inherent
risks. One strategy to overcome these obstacles has been to
engineer attenuated E. coli that can invade and enter the cytosol
of host cells for the delivery of macromolecules. E. coli deficient
in the production of diaminopimelate (DAP), an essential cell wall
component, undergo lysis during growth in the absence of DAP (56).
DAP-minus E. coli carrying the 200 kb virulence plasmid pWR100 from
Shigella flexneri have been engineered to deliver DNA to mammalian
cells (11). These E. coli have the ability to invade cultured cells
and enter the cytosol similar to S. flexneri, yet following brief
replication, spontaneously lyse in the cytosol and allow for the
delivery of DNA for subsequent expression in the host cell.
However, the presence of the pWR100 virulence plasmid poses
limitations on the suitability of this microbe for many
applications. The rational design of safe delivery vectors is
therefore of paramount importance when constructing new
methodologies for in vivo delivery. Since the E. coli/LLO system
does not contain any virulence associated determinants other than
LLO, it is uniquely situated to safely deliver antigens to
macrophages in vivo to generate a protective immune response.
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[0103] All publications and patent applications cited in this
specification are herein incorporated by reference as if individual
publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teaching of this invention that certain changes and
modifications may be made thereto without departing spirit or scope
of the appended claims.
Sequence CWU 1
1
6134DNAListeria monocytogenes 1ggaattccat atgaaggatg catctgcatt
caat 34233DNAListeria monocytogenes 2cgggatcctt attattcgat
tggattatct act 33326DNAListeria monocytogenes 3cgcgatatcc
tctagaaata attttg 26432DNAListeria monocytogenes 4aggcgtcgac
ggttaatacg accgggatcg ag 32540DNAListeria monocytogenes 5aggcgtcgac
aggccttacg cgaaatacgg gcagacatgg 4068PRTmurine 6Ser Ile Ile Asn Phe
Glu Lys Leu 1 5
* * * * *