U.S. patent application number 15/423675 was filed with the patent office on 2017-10-26 for recombinant bacterium to decrease tumor growth.
The applicant listed for this patent is The Arizona Board of Regents for and on Behalf of Arizona State University. Invention is credited to Roy Curtiss, III, Wei Kong.
Application Number | 20170306338 15/423675 |
Document ID | / |
Family ID | 45004891 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170306338 |
Kind Code |
A1 |
Curtiss, III; Roy ; et
al. |
October 26, 2017 |
RECOMBINANT BACTERIUM TO DECREASE TUMOR GROWTH
Abstract
The present invention encompasses a recombinant bacterium
capable of reducing tumor growth.
Inventors: |
Curtiss, III; Roy; (Paradise
Valley, AZ) ; Kong; Wei; (Phoenix, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Arizona Board of Regents for and on Behalf of Arizona State
University |
Scottsdale |
AZ |
US |
|
|
Family ID: |
45004891 |
Appl. No.: |
15/423675 |
Filed: |
February 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13700591 |
Apr 15, 2013 |
9598697 |
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PCT/US11/38588 |
May 31, 2011 |
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15423675 |
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61349425 |
May 28, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/74 20130101;
C12R 1/42 20130101; C12N 1/36 20130101; C12N 15/74 20130101 |
International
Class: |
C12N 15/74 20060101
C12N015/74; A61K 35/74 20060101 A61K035/74; C12N 1/36 20060101
C12N001/36; C12R 1/42 20060101 C12R001/42 |
Goverment Interests
GOVERNMENTAL RIGHTS
[0002] This invention was made with government support under R01
AI065779, R01 A1056289, and R21 CA152456-01 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A recombinant bacterium, wherein the bacterium is capable of: a.
increased expression of a nucleic acid encoding a chemoreceptor
that directs chemotaxis towards tumors, b. accumulation in a
quiescent tumor, c. hyper-invasion of a tumor, d. reduced fitness
in normal tissue, e. enhanced stimulation of the host innate immune
responses, f. delivering a tumor specific DNA vaccine vector to a
tumor cell, and g. increased bacterium-induced host programmed cell
death.
2. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2,
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA, .DELTA.(wza-wcaM)-8,
.DELTA.relA198::araC P.sub.BAD lacI TT, .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD, .DELTA.pagP81::P.sub.lpp lpxE,
.DELTA.endA2311[[1123]].
3. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311[[1123]],
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar.
4. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311[[1123]]
.DELTA.P.sub.tar::P.sub.trc .DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr.
5. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311[[1123]],
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg.
6. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lac TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311[[1123]],
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA.
7. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311[[1123]],
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA, .DELTA.purA.
8. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2,
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA, .DELTA.(wza-wcaM)-8,
.DELTA.relA198::araC P.sub.BAD lacI TT, .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD, .DELTA.pagP81::P.sub.lpp lpxE,
.DELTA.endA2311[[1123]], .DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888
tar, .DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA, .DELTA.purA,
.DELTA.P.sub.sopE2::P.sub.trc sopE2.
9. A bacterium of claim 1, wherein the bacterium comprises the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2,
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA, .DELTA.(wza-wcaM)-8,
.DELTA.relA 198::araC P.sub.BAD lacI TT, .DELTA.(araC PBAD)-18::P22
P.sub.R araBAD, .DELTA.pagP81::P.sub.lpp lpxE,
.DELTA.endA2311[[1123]], .DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888
tar, .DELTA.P.sub.tsr.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA, .DELTA.purA,
.DELTA.P.sub.sopE2::P.sub.trc sopE2, .DELTA.P.sub.tlpA::P.sub.ansB
tlpA.
10. A recombinant bacterium, wherein the bacterium is capable of:
a. regulated attenuation, b. regulated lysis, c. increased
expression of a nucleic acid encoding a chemoreceptor that directs
chemotaxis towards tumors, d. accumulation in a quiescent tumor, e.
hyper-invasion of a tumor, f. reduced fitness in normal tissue, g.
enhanced stimulation of the host innate immune responses, h.
delivering a tumor specific DNA vaccine vector to a tumor cell, and
i. increased bacterium-induced host programmed cell death.
11. A method of inhibiting tumor growth, the method comprising
administering a recombinant bacterium of claim 1 to a tumor.
12. A method of treating cancer in a subject, the method comprising
administering a recombinant bacterium of claim 1 to the subject,
wherein the subject has cancer.
13. A recombinant bacterium, wherein the bacterium is capable of:
a. increased expression of Tar and Tsr, and b. decreased expression
of Trg.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/700,591, filed on Apr. 15, 2013, now
granted as U.S. Pat. No. 9,598,697 on Mar. 21, 2017, which, in
turn, is a 35 U.S.C. .sctn.371 national stage filing of
International Application No. PCT/US11/38588, filed on May 31,
2011, which in turn claims priority to U.S. Provisional Patent
Application No. 61/349,425, filed on May 28, 2010, the entire
contents of each of which are expressly incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The invention encompasses a recombinant bacterium capable of
reducing tumor size.
BACKGROUND OF THE INVENTION
[0004] Conventional therapies for cancer as radiotherapy and
chemotherapy are characterized by poor survival rates in many forms
of cancer. This is due to multiple factors including the
development of drug-resistant tumor cells and the presence of
undetectable micrometastases at the time of diagnosis and
treatment. The other substantial limitation of conventional cancer
chemotherapy and radiotherapy is the toxicity of these agents to
normal tissue. A major challenge in treating cancer is the
difficulty of bringing therapy to poorly perfused areas of solid
tumors, which are often most resistant to chemo- and radiotherapy.
This has prompted the development of many new approaches for the
treatment of cancer, including the delivery of anti-cancer genes to
the tumor site in various gene therapy protocols. These genetic
approaches include delivering genes encoding pro-drug activating
enzymes, cytotoxic, anti-angiogenic proteins or cell-targeted
toxins to the tumors. However, current gene therapy strategies
require local administration of vectors, which limits their
usefulness. Hence, there is a need in the art for an effective and
largely non-toxic therapy to fight tumor growth and metastasis.
REFERENCE TO COLOR FIGURES
[0005] The application file contains at least one photograph
executed in color. Copies of this patent application publication
with color photographs will be provided by the Office upon request
and payment of the necessary fee.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIGS. 1A and 1B. Invasion (FIG. 1A) and replication (FIG.
1B) of S. typhimurium strains in Int-407 cell line.
[0007] FIGS. 2A, 2B and 2C. Colonization of mice with S.
typhimurium strains at day 6 post-inoculation. (FIG. 2A) Peyer's
patches, (FIG. 2B) spleen, (FIG. 2C) liver.
[0008] FIGS. 3A and 3B. Improved DNA vaccine vector pYA4545 (FIG.
3B) and parent vector (FIG. 3A).
[0009] FIG. 4. Synthesis of EGFP from pYA4545 harboring EGFP gene
in INT-407 cell line and Vero cell line.
[0010] FIG. 5. Depicts an illustration of the suicide vector
pYA4946.
[0011] FIG. 6. Depicts an illustration of the suicide vector
pYA4947.
[0012] FIG. 7. Confirmation of the over-expression of Tar in strain
.chi.11371 by western blot analysis using mouse anti-Flag tag and
goat-anti-mouse IgG antisera.
[0013] FIG. 8. Confirmation of the over-expression of Tsr in strain
.chi.11372 by western blot analysis using mouse anti-c-Myc tag and
goat anti-mouse IgG antisera.
[0014] FIGS. 9A, 9B and 9C. Chemotaxis assay of strain harboring
tar deletion-insertion mutation versus its parent S. typhimurium
UK-1 wild-type strain. (FIG. 9A) depicts the OD600 versus time and
CFU/ml versus time, (FIG. 9B) illustrates the chemotaxis assay, and
(FIG. 9C) depicts the distance traveled by each strain.
[0015] FIGS. 10A, 10B and 10C. Chemotaxis assay of strain harboring
tsr deletion-insertion mutation versus its parent S. typhimurium
UK-1 wild-type strain. (FIG. 10A) depicts the OD600 versus time and
CFU/ml versus time, (FIG. 10B) illustrates the chemotaxis assay,
and (FIG. 10C) depicts the distance traveled by each strain.
[0016] FIG. 11. Depicts an illustration of the suicide vector
pYA5077.
[0017] FIGS. 12A and 12B. Chemotaxis assay of strain harboring trg
deletion mutation versus its parent S. typhimurium UK-1 wild-type
strain. (FIG. 12A) illustrates the chemotaxis assay, and (FIG. 12B)
depicts the distance traveled by each strain.
[0018] FIG. 13. Depicts an illustration of the suicide vector
pYA4948.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides a recombinant bacterium that
may be used to inhibit the growth of a tumor or tumor cell. In
addition, the invention encompasses methods of use thereof.
I. Recombinant Bacterium
[0020] A recombinant bacterium of the invention is typically an
anaerobic bacterium. An anaerobic bacterium may be an obligate
anaerobe (e.g. a bacterium from the genera Bacteroides,
Bifidobacteria, or Clostridium) an aerotolerant bacterium (e.g. a
bacterium from the genus Enterococci), or a facultative anaerobe
(e.g. a bacterium from the family Enterobacteriaceae, the genus
Streptococcus, the genus Lactobacillus, the genus Staphylococcus,
or the genus Corynebacterium). The Enterobacteriaceae family
comprises species from the following genera: Alterococcus,
Aquamonas, Aranicola, Arsenophonus, Brenneria, Budvicia,
Buttiauxella, Candidatus Phlomobacter, Cedeceae, Citrobacter,
Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella,
Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella,
Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus,
Plesiomonas, Pragia, Proteus, Providencia, Rahnella, Raoultella,
Salmonella, Samsonia, Serratia, Shigella, Sodalis, Tatumella,
Trabulsiella, Wigglesworthia, Xenorhbdus, Yersinia, Yokenella. In
certain embodiments, the recombinant bacterium is typically a
pathogenic species of the Enterobaceteriaceae. Due to their
clinical significance, Escherichia coli, Shigella, Edwardsiella,
Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia,
Proteus, Morganella, Providencia and Yersinia are considered to be
particularly useful. In other embodiments, the recombinant
bacterium may be a species or strain commonly used for a
vaccine.
[0021] Some embodiments of the instant invention comprise a species
or subspecies of the Salmonella genera. For instance, the
recombinant bacterium may be a Salmonella enterica serovar. In an
exemplary embodiment, a bacterium of the invention may be derived
from S. enterica serovar typhimurium, hereafter referred to as S.
typhimurium, and also from S. typhi, S. paratyphi, S. enteritidis,
S. choleraesius, S. arizona, or S. Dublin. In an exemplary
embodiment, the recombinant bacterium is derived from S.
typhimurium.
[0022] A bacterium of the invention may comprise one or more
mutations as detailed below. In particular, a bacterium may
comprise one or more mutations to increase invasiveness, to
maximize bacterium localization in tumor quiescence, and to reduce
bacterium normal tissue fitness (section (a) below), one or more
mutations to enhance the stimulation of host innate immune
responses (section (b) below), one or more mutations to increase
bacterium-induced host programmed cell death (section (c) below),
one or more mutations to induce lysis of the bacterium (section (d)
below), one or more vectors to express a nucleic acid encoding an
antigen or effector protein (section (e) below), one or more
mutations to attenuate the bacterium (section (f) below), and/or
one or more mutations to enhance the performance of the bacterium
as a tumor therapy (section (g) below).
(a) Hyper-Invasiveness and Maximized Localization in Tumors
[0023] A recombinant bacterium of the invention may also be
hyper-invasive. As used herein, "hyper-invasive" refers to a
bacterium that can invade a tumor more efficiently than a wild-type
bacterium of the same strain. Invasion may be determined by methods
known in the art, e.g. CFUs/g of tumor tissue.
[0024] In one embodiment, a recombinant bacterium of the invention
may comprise a mutation to increase the expression of a nucleic
acid encoding a chemoreceptor that directs chemotaxis towards
tumors or increases penetration of tumors. For instance, in one
embodiment, the expression of the nucleic acid encoding the
aspartate and maltose receptor, e.g. tar, may be increased. In
particular, the promoter of the nucleic acid encoding the receptor
may be replaced with a constitutive promoter. By way of
non-limiting example, a bacterium may comprise a
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar mutation. This allows
constitutive expression of tar, even when lacI is expressed. In
another embodiment, the expression of the nucleic acid encoding the
serine receptor, e.g. tsr, may be increased. In particular, the
promoter of the nucleic acid encoding the receptor may be replaced
with a constitutive promoter. By way of non-limiting example, a
bacterium may comprise a .DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888
tsr mutation. This allows constitutive expression of tsr, even when
lacI is expressed. Additionally, the expression of a nucleic acid
encoding a chemoreceptor may be modified by altering the codons of
the chemoreceptor nucleic acid to optimize expression in the
recombinant bacterium, and/or to alter the translational efficiency
of the mRNA and/or to increase the stability of the mRNA.
[0025] In certain embodiments, a recombinant bacterium may comprise
a mutation that decreases the expression of a nucleic acid encoding
a chemoreceptor that directs chemotaxis towards necrosis. This
allows bacterial accumulation in a quiescent tumor, as opposed to
necrotic cells. For instance, the expression of the nucleic acid
encoding the ribose/galactose receptor, e.g. trg, may be decreased.
In particular, the trg sequence may be deleted or mutated to
prevent or decrease expression of the nucleic acid or translation
of the nucleic acid into the corresponding protein. Non-limiting
examples of suitable mutations may include the .DELTA.trg and the
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg mutations, which will result
in cessation of Trg synthesis in vivo due to the lack of rhamnose.
In one embodiment, a bacterium of the invention may comprise both a
mutation that increases the expression of one or more nucleic acids
that encode a chemoreceptor and a mutation that decreases the
expression of one or more different nucleic acids that encode a
chemoreceptor.
[0026] In another embodiment, a recombinant bacterium may comprise
a mutation that decreases the fitness of the bacterium in a normal
cell (as opposed to a tumor cell). For instance, a bacterium may
comprise a mutation that eliminates the production of adenosine
monophosphate (AMP) from inosine monophosphate (IMP). For instance,
the S. typhimurium purA gene may be deleted resulting in a
purine-deficient auxotroph. Such a mutant could grow in tumor
associated necrotized tissue in vivo, but would have very
restricted growth in healthy tissues, which have a very limited
supply of purines.
[0027] In other embodiments, a recombinant bacterium may further
comprise a mutation that increases expression of hilA. For
instance, the promoter of hilA may be mutated to enable
constitutive expression of hilA. A non-limiting example may include
a P.sub.hilA::P.sub.trc.DELTA.lacO hilA mutation, such as
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA. Such a mutation
replaces the wild-type hilA promoter with the P.sub.trc promoter
that lacks the lacO operator sequence. This allows constitutive
expression of hilA, even when lacI is expressed. Alternatively,
deletion of the lrp nucleic acid sequence may be used to increase
hilA expression. In another alternative embodiment, a recombinant
bacterium may comprise a .DELTA.P.sub.hilA::P.sub.hilA256 hilA
mutation.
(b) Enhanced Stimulation of the Host Innate Immune Responses
[0028] The human immune system naturally grows stronger while
fighting bacteria, including Salmonella. It is widely believed that
one of the main triggers of host inflammation is the recognition of
microbial products by receptors of the innate immune system.
Consequently, in some embodiments, a recombinant bacterium of the
invention may be capable of stimulation of innate immune responses.
In an exemplary embodiment, the bacterium is capable of stimulating
enhanced host innate immune responses, compared to a wild-type
bacterium of the same strain.
[0029] In one embodiment, a recombinant bacterium of the invention
may overexpress a guanidyl nucleotide exchange factor (e.g. SopE2)
and/or an inositol polyphosphatase (e.g. SopB), that activate
Rho-family GTPases in a functionally redundant manner to mediate
the innate immune responses. In some embodiments, the native
promoter of such nucleic acid sequences may be replaced with
P.sub.trc to enable the regulated delayed synthesis of SopE2 and/or
SopB. In certain embodiments, the start codon of the sopE2 and/or
sopB genes may be modified to alter its expression level. For
instance, the start codon may be changed from GTG to ATG. In
addition, the second and third codons can be made more A rich to
further increase translation efficiency.
(c) Increased Bacterium-Induced Host Programmed Cell Death
[0030] Programmed cell death of a host cell invaded by a bacterium
of the invention is advantageous if the host cell is a tumor cell.
Consequently, in some embodiments, a recombinant bacterium of the
invention may be capable of increased bacterium-induced host
programmed cell death compared to a wild-type bacterium of the same
strain. Non-limiting examples of bacterium-induced host programmed
cell death may include apoptosis and pyroptosis. Methods of
detecting and measuring bacterium-induced host programmed cell
death are known in the art.
[0031] In one embodiment, a bacterium of the invention capable of
increasing bacterium-induced host programmed cell death may
comprise a mutation that causes over-synthesis of a bacterial
protein or effector, after the bacteria accumulate in tumor cells,
to affect a pathway inducing apoptosis/pyroptosis. Non-limiting
examples of such a mutation may include mutations causing in vivo
upregulation of a deubiquitinase-encoding nucleic acid sequence,
such as Salmonella sseL, and/or a ToII IL1 Receptor (TIR)-like
protein A (TIR-like protein A) nucleic acid sequence (e.g. the
Salmonella enteritidis tlpA), and/or a member of the YopJ/Avr
family (e.g. the Salmonella typhimurium avrA). In particular, a
recombinant bacterium of the invention may comprise a mutation that
increases the tumor specific expression of S. typhimurium tlpA. By
way of non-limiting example, the ansB promoter, which is
preferentially activated in tumor cells, may be operably linked to
tlpA. Also, the SD sequence of tlpA may be modified to facilitate
tumor-specific synthesis of TlpA. For instance, the sequence may be
modified to AGGA. In certain embodiments, a bacterium may be
capable of regulated lysis, such that the bacterium releases the
increased amounts of TlpA upon lysis, thereby increasing
bacterium-induced host programmed cell death.
[0032] In other embodiments described herein, a recombinant
bacterium of the invention may also be used to deliver a nucleic
acid vaccine vector, such that the vaccine vector encodes a nucleic
acid sequence that increases bacterium-induced host programmed cell
lysis. For instance, the vaccine vector may encode Fas ligand
(FasL) and/or the tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL). This is discussed in more detail
in section (e) below.
(d) Lysis
[0033] In another embodiment, a recombinant bacterium of the
invention is capable of regulated lysis. Lysis of the bacterium
within the host cell may release a bolus of antigen, or
alternatively, may release a nucleic acid vaccine vector for
transcription by the tumor cell. Lysis also provides a means of
biocontainment.
[0034] In some embodiments, a recombinant bacterium capable of
regulated lysis may comprise a mutation in a required constituent
of the peptidoglycan layer of the bacterial cell wall. For
instance, the bacterium may comprise a mutation in a nucleic acid
sequence encoding a protein involved in muramic acid synthesis,
such as murA. It is not possible to alter murA by deletion,
however, because a .DELTA.murA mutation is lethal and can not be
isolated. This is because the missing nutrient required for
viability is a phosphorylated muramic acid that cannot be
exogenously supplied since enteric bacteria cannot internalize it.
Consequently, the murA nucleic acid sequence may be altered to make
expression of murA dependent on a nutrient (e.g., arabinose) that
can be supplied during the growth of the bacterium. For example,
the alteration may comprise a .DELTA.P.sub.murA:: araC P.sub.BAD
murA deletion-insertion mutation. During in vitro growth of the
bacterium, this type of mutation makes synthesis of muramic acid
dependent on the presence of arabinose in the growth medium. During
growth of the bacterium in a host, however, arabinose is absent.
Consequently, the bacterium is non-viable and/or avirulent in a
host unless the bacterium further comprises at least one
extrachromosomal vector comprising a nucleic acid sequence, that
when expressed, substantially functions as murA. Recombinant
bacteria with a .DELTA.P.sub.murA::TT araC P.sub.BAD murA
deletion-insertion mutation grown in the presence of arabinose
exhibit effective colonization of effector lymphoid tissues after
oral administration prior to cell death due to cell wall-less
lysing.
[0035] Similarly, in various embodiments a recombinant bacterium
may comprise the araC P.sub.BAD c2 cassette inserted into the asdA
nucleic acid sequence that encodes aspartate semialdehyde
dehydrogenase, a necessary enzyme for DAP synthesis, a required
component of the peptidoglycan layer of the bacterial cell wall.
The chromosomal asdA nucleic acid sequence is typically inactivated
to enable use of plasmid vectors encoding the wild-type asdA
nucleic acid sequence in the balanced-lethal host-vector system.
This allows stable maintenance of plasmids in vivo in the absence
of any drug resistance attributes that are not permissible in live
bacterial vaccines.
[0036] In one embodiment, .DELTA.asdA27::TT araC P.sub.BAD c2 has
an improved SD sequence and a codon optimized c2 nucleic acid
sequence. The C2 repressor synthesized in the presence of arabinose
is used to repress nucleic acid sequence expression from P22
P.sub.R and P.sub.L promoters. In another embodiment,
.DELTA.asdA27::TT araC P.sub.BAD c2 has the 1104 base-pair asdA
nucleic acid sequence deleted (1 to 1104, but not including the TAG
stop codon) and the 1989 base-pair fragment containing T4 ipIII TT
araC P.sub.BAD c2 inserted. The c2 nucleic acid sequence in
.DELTA.asdA27::TT araC P.sub.BAD c2 has a SD sequence that was
optimized to TAAGGAGGT. It also has an improved P.sub.BAD promoter
such that the -10 sequence is improved from TACTGT to TATAAT.
Furthermore, it has a codon optimized c2 nucleic acid sequence, in
which the second codon was modified from AAT to AAA. In some
additional embodiments, the C2 repressor binding sites may be
modified so that as C2 decreases the P22 P.sub.R araBAD nucleic
acid sequences are expressed at a higher level than in wild-type
strains.
[0037] In exemplary embodiments, the bacterium may comprise a
mutation in the murA nucleic acid sequence encoding the first
enzyme in muramic acid synthesis and the asdA nucleic acid sequence
essential for DAP synthesis. By way of non-limiting example, these
embodiments may comprise the chromosomal deletion-insertion
mutations .DELTA.asdA19::TT araC P.sub.BAD c2 or .DELTA.asdA27::TT
araC P.sub.BAD c2 and .DELTA.P.sub.murA7::TT araC P.sub.BAD murA or
.DELTA.P.sub.murA12:TT araC P.sub.BAD murA or
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA. This host-vector grows
in LB broth with 0.1% L-arabinose, but is unable to grow in or on
media devoid of arabinose since it undergoes cell wall-less death
by lysis. In another embodiment, the onset of programmed lysis may
be delayed about one cell division by including a .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD mutation, which initially
prevents breakdown of accumulated arabinose at the time of
inoculation. Later, however, this mutation allows breakdown of
residual arabinose to reduce the likelihood of expressin any araC
P.sub.BAD regulated nucleic acid sequences.
[0038] Bacterium that comprise these mutations also comprise a
plasmid that contains a nucleic acid sequence that substitutes for
murA and asdA. This allows the bacterium to grow in permissive
environments, e.g. when arabinose is present. For instance plasmid
vector pYA3681 contains the murA nucleic acid sequence (with
altered start codon sequences from ATG to GTG to decrease
translation efficiency) under the control of an araC P.sub.BAD
promoter. The second nucleic acid sequence under the direction of
this promoter is the asdA nucleic acid sequence (with altered start
codon sequences from ATG to GTG to decrease translation
efficiency). The P22 P.sub.R promoter is in the anti-sense
direction of both the asdA nucleic acid sequence and the murA
nucleic acid sequence. The P22 P.sub.R is repressed by the C2
repressor made during growth of the strain in media with arabinose
(due to the .DELTA.asdA::TT araC P.sub.BAD c2 deletion-insertion).
However C2 concentration decreases due to cell division in vivo to
cause P.sub.R directed synthesis of anti-sense mRNA to further
block translation of asdA and murA mRNA. The araC P.sub.BAD
sequence is also not from E. coli B/r as originally described but
represents a sequence derived from E. coli K-12 strain .chi.289
with tighter control and less leakiness in the absence of
arabinose. In the preferred embodiment, transcription terminators
(TT) flank all of the domains for controlled lysis, replication,
and expression so that expression in one domain does not affect the
activities of another domain. As a safety feature, the plasmid asdA
nucleic acid sequence does not replace the chromosomal asdA
mutation since they have a deleted sequence in common.
Additionally, the E. coli murA nucleic acid sequence was used in
the plasmid instead of using the Salmonella murA nucleic acid
sequence. In addition to being fully attenuated, this construction
exhibits complete biological containment. This property enhances
safety and minimizes the potential for exposure of individuals not
intended for tumor treatment.
[0039] One of skill in the art will recognize that other nutrients
besides arabinose may be used in the above mutations. By way of
non-limiting example, xylose, mannose, and rhamnose regulatory
systems may also be used.
[0040] In some embodiments of the invention, the recombinant
bacterium may further comprise araBAD and araE mutations to
preclude breakdown and leakage of internalized arabinose such that
asdA and murA nucleic acid sequence expression continues for a cell
division or two after oral immunization into an environment that is
devoid of external arabinose. Additionally, a bacterium may
comprise a mutation in a protein involved in GDP-fucose synthesis
to preclude formation of colonic acid. Non-limiting examples of
such a mutation include .DELTA.(gmd-fcl)-26. A bacterium may also
comprise a mutation like .DELTA.relA (e.g., .DELTA.relA1123) that
uncouples cell wall-less death from dependence on protein
synthesis.
[0041] Lysis of the bacterium will typically release lipid A, an
endotoxin. So, a bacterium of the invention may comprise a mutation
that reduces the toxicity of lipid A. Non-limiting examples may
include a mutation that causes synthesis of the mono-phosphoryl
lipid A. This form of lipid A is non-toxic, but still serves as an
adjuvant agonist. For instance, in one embodiment, a recombinant
bacterium may comprise a .DELTA.pagP81::P.sub.lpp lpxE mutation. In
particular embodiments, the lpxE sequence may be codon optimized
for high-level expression in the recombinant bacterium.
[0042] A recombinant bacterium may also comprise a .DELTA.relA:TT
araC P.sub.BAD lacI TT deletion-insertion mutation so that growth
of the strain in the presence of arabinose causes synthesis of LacI
to initially repress synthesis of protein antigens encoded by
sequences under the control of P.sub.trc. As a consequence of cell
division in vivo during colonization of lymphoid tissues, LacI
becomes diluted and expression of P.sub.trc controlled genes
commences with synthesis of the protective antigen to stimulate
induction of immune responses. In all cases the regulated delayed
lysis phenotype is totally attenuating with no persistence of
bacteria cells in vivo and no survival of bacteria cells if
excreted. This regulated delayed lysis system has been described by
Kong et al. (2008. Regulated programmed lysis of recombinant
Salmonella in host tissues to release protective antigens and
confer biological containment. Proc. Natl. Acad. Sci. USA
105:9361-9366) and Curtiss and Kong (US Patent 2006/0140975), each
of which is hereby incorporated by reference in its entirety. In
certain embodiments, a recombinant bacterium of the invention may
further comprise mutations to increase the expression of lacI. For
instance, the SD sequence of lacI may be modified, the start codon
may be modified, and/or structural codons may be modified to
maximize transcription efficiency in the recombinant bacterium. In
a specific embodiment, the SD sequence of lad may be modified from
AGGG to AGGA and/or the start codon may be modified from GTG to
ATG.
(e) Expression of a Nucleic Acid Encoding an Antigen or Effector
Protein
[0043] A recombinant bacterium of the invention may express or
deliver one or more nucleic acids that encode one or more antigens
or effector proteins. For instance, in one embodiment, a
recombinant bacterium may be capable of the regulated expression of
a nucleic acid sequence encoding an antigen or effector protein. In
another embodiment, a recombinant bacterium may comprise a nucleic
acid vaccine vector. Each of the above embodiments is described in
more detail below. Other means of expressing or delivering one or
more nucleic acids that encode one or more antigens are known in
the art.
[0044] In one embodiment, the antigen is tumor specific antigen. In
another embodiment, the antigen is an effector protein designed to
illicit an innate immune response. For instance, in one embodiment,
the effector protein is FasL and/or TRAIL. Additional examples of
antigens may be found in sections i. and ii. below and in the
Examples.
[0045] In some embodiments, antigens of the invention may be
delivered via a type 2 or a type 3 secretion system.
i. Regulated Expression
[0046] The present invention encompasses a recombinant bacterium
capable of the regulated expression of at least one nucleic acid
sequence encoding an antigen or effector protein of interest.
Generally speaking, such a bacterium comprises a chromosomally
integrated nucleic acid sequence encoding a repressor and a vector.
Each is discussed in more detail below.
A. Chromosomally Integrated Nucleic Acid Sequence Encoding a
Repressor
[0047] A recombinant bacterium of the invention that is capable of
the regulated expression of at least one nucleic acid sequence
encoding an antigen or effector protein comprises, in part, at
least one chromosomally integrated nucleic acid sequence encoding a
repressor. Typically, the nucleic acid sequence encoding a
repressor is operably linked to a regulatable promoter. The nucleic
acid sequence encoding a repressor and/or the promoter may be
modified from the wild-type nucleic acid sequence so as to optimize
the expression level of the nucleic acid sequence encoding the
repressor.
[0048] Methods of chromosomally integrating a nucleic acid sequence
encoding a repressor operably-linked to a regulatable promoter are
known in the art and detailed in the examples. Generally speaking,
the nucleic acid sequence encoding a repressor should not be
integrated into a locus that disrupts colonization of the host by
the recombinant bacterium, or attenuates the bacterium. In one
embodiment, the nucleic acid sequence encoding a repressor may be
integrated into the relA nucleic acid sequence. In another
embodiment, the nucleic acid sequence encoding a repressor may be
integrated into the endA nucleic acid sequence.
[0049] In some embodiments, at least one nucleic acid sequence
encoding a repressor is chromosomally integrated. In other
embodiments, at least two, or at least three nucleic acid sequences
encoding repressors may be chromosomally integrated into the
recombinant bacterium. If there is more than one nucleic acid
sequence encoding a repressor, each nucleic acid sequence encoding
a repressor may be operably linked to a regulatable promoter, such
that each promoter is regulated by the same compound or condition.
Alternatively, each nucleic acid sequence encoding a repressor may
be operably linked to a regulatable promoter, each of which is
regulated by a different compound or condition.
1. Repressor
[0050] As used herein, "repressor" refers to a biomolecule that
represses transcription from one or more promoters. Generally
speaking, a suitable repressor of the invention is synthesized in
high enough quantities during the in vitro growth of the bacterial
strain to repress the transcription of the nucleic acid sequence
encoding an antigen or effector protein of interest on the vector,
as detailed below, and not impede the in vitro growth of the
strain. Additionally, a suitable repressor will generally be
substantially stable, i.e. not subject to proteolytic breakdown.
Furthermore, a suitable repressor will be diluted by about half at
every cell division after expression of the repressor ceases, such
as in a non-permissive environment (e.g. an animal or human
host).
[0051] The choice of a repressor depends, in part, on the species
of the recombinant bacterium used. For instance, the repressor is
usually not derived from the same species of bacteria as the
recombinant bacterium. For instance, the repressor may be derived
from E. coli if the recombinant bacterium is from the genus
Salmonella. Alternatively, the repressor may be from a
bacteriophage.
[0052] Suitable repressors are known in the art, and may include,
for instance, LacI of E. coli, C2 encoded by bacteriophage P22, or
C1 encoded by bacteriophage .lamda.. Other suitable repressors may
be repressors known to regulate the expression of a regulatable
nucleic acid sequence, such as nucleic acid sequences involved in
the uptake and utilization of sugars. In one embodiment, the
repressor is LacI. In another embodiment, the repressor is C2. In
yet another embodiment, the repressor is C1.
2. Regulatable Promoter
[0053] The chromosomally integrated nucleic acid sequence encoding
a repressor is operably linked to a regulatable promoter. The term
"promoter", as used herein, may mean a synthetic or
naturally-derived molecule that is capable of conferring,
activating or enhancing expression of a nucleic acid. A promoter
may comprise one or more specific transcriptional regulatory
sequences to further enhance expression and/or to alter the spatial
expression and/or temporal expression of a nucleic acid. The term
"operably linked," as used herein, means that expression of a
nucleic acid sequence is under the control of a promoter with which
it is spatially connected. A promoter may be positioned 5'
(upstream) of the nucleic acid sequence under its control. The
distance between the promoter and a nucleic acid sequence to be
expressed may be approximately the same as the distance between
that promoter and the native nucleic acid sequence it controls. As
is known in the art, variation in this distance may be accommodated
without loss of promoter function.
[0054] The regulated promoter used herein generally allows
transcription of the nucleic acid sequence encoding a repressor
while in a permissive environment (i.e. in vitro growth), but
ceases transcription of the nucleic acid sequence encoding a
repressor while in a non-permissive environment (i.e. during growth
of the bacterium in an animal or human host). For instance, the
promoter may be sensitive to a physical or chemical difference
between the permissive and non-permissive environment. Suitable
examples of such regulatable promoters are known in the art.
[0055] In some embodiments, the promoter may be responsive to the
level of arabinose in the environment. Generally speaking,
arabinose may be present during the in vitro growth of a bacterium,
while typically absent from host tissue. In one embodiment, the
promoter is derived from an araC-P.sub.BAD system. The
araC-P.sub.BAD system is a tightly regulated expression system,
which has been shown to work as a strong promoter induced by the
addition of low levels of arabinose. The araC-araBAD promoter is a
bidirectional promoter controlling expression of the araBAD nucleic
acid sequences in one direction, and the araC nucleic acid sequence
in the other direction. For convenience, the portion of the
araC-araBAD promoter that mediates expression of the araBAD nucleic
acid sequences, and which is controlled by the araC nucleic acid
sequence product, is referred to herein as P.sub.BAD. For use as
described herein, a cassette with the araC nucleic acid sequence
and the araC-araBAD promoter may be used. This cassette is referred
to herein as araC-P.sub.BAD. The AraC protein is both a positive
and negative regulator of P.sub.BAD. In the presence of arabinose,
the AraC protein is a positive regulatory element that allows
expression from P.sub.BAD. In the absence of arabinose, the AraC
protein represses expression from P.sub.BAD. This can lead to a
1,200-fold difference in the level of expression from
P.sub.BAD.
[0056] Other enteric bacteria contain arabinose regulatory systems
homologous to the araC-araBAD system from E. coli. For example,
there is homology at the amino acid sequence level between the E.
coli and the S. typhimurium AraC proteins, and less homology at the
DNA level. However, there is high specificity in the activity of
the AraC proteins. For example, the E. coli AraC protein activates
only E. coli P.sub.BAD (in the presence of arabinose) and not S.
typhimurium P.sub.BAD. Thus, an arabinose regulated promoter may be
used in a recombinant bacterium that possesses a similar arabinose
operon, without substantial interference between the two, if the
promoter and the operon are derived from two different species of
bacteria.
[0057] Generally speaking, the concentration of arabinose necessary
to induce expression is typically less than about 2%. In some
embodiments, the concentration is less than about 1.5%, 1%, 0.5%,
0.2%, 0.1%, or 0.05%. In other embodiments, the concentration is
0.05% or below, e.g. about 0.04%, 0.03%, 0.02%, or 0.01%. In an
exemplary embodiment, the concentration is about 0.05%.
[0058] In other embodiments, the promoter may be responsive to the
level of maltose in the environment. Generally speaking, maltose
may be present during the in vitro growth of a bacterium, while
typically absent from host tissue. The malT nucleic acid sequence
encodes MalT, a positive regulator of four maltose-responsive
promoters (P.sub.PQ, P.sub.EFG, P.sub.KBM, and P.sub.S). The
combination of malT and a mal promoter creates a tightly regulated
expression system that has been shown to work as a strong promoter
induced by the addition of maltose. Unlike the araC-P.sub.BAD
system, malT is expressed from a promoter (P.sub.T) functionally
unconnected to the other mal promoters. P.sub.T is not regulated by
MalT. The malEFG-malKBM promoter is a bidirectional promoter
controlling expression of the malKBM nucleic acid sequences in one
direction, and the malEFG nucleic acid sequences in the other
direction. For convenience, the portion of the malEFG-malKBM
promoter that mediates expression of the malKBM nucleic acid
sequence, and which is controlled by the malT nucleic acid sequence
product, is referred to herein as P.sub.KBM, and the portion of the
malEFG-malKBM promoter that mediates expression of the malEFG
nucleic acid sequence, and that is controlled by the malT nucleic
acid sequence product, is referred to herein as P.sub.EFG. Full
induction of P.sub.KBM requires the presence of the MalT binding
sites of P.sub.EFG. For use in the vectors and systems described
herein, a cassette with the malT nucleic acid sequence and one of
the mal promoters may be used. This cassette is referred to herein
as malT-P.sub.mal. In the presence of maltose, the MalT protein is
a positive regulatory element that allows expression from
P.sub.mal.
[0059] In still other embodiments, the promoter may be sensitive to
the level of rhamnose in the environment. Analogous to the
araC-P.sub.BAD system described above, the rhaRS-P.sub.rhaB
activator-promoter system is tightly regulated by rhamnose.
Expression from the rhamnose promoter (P.sub.rha) is induced to
high levels by the addition of rhamnose, which is common in
bacteria but rarely found in host tissues. The nucleic acid
sequences rhaBAD are organized in one operon that is controlled by
the P.sub.rhaBAD promoter. This promoter is regulated by two
activators, RhaS and RhaR, and the corresponding nucleic acid
sequences belong to one transcription unit that is located in the
opposite direction of the rhaBAD nucleic acid sequences. If
L-rhamnose is available, RhaR binds to the P.sub.rhaRS promoter and
activates the production of RhaR and RhaS. RhaS together with
L-rhamnose in turn binds to the P.sub.rhaBAD and the P.sub.rhaT
promoter and activates the transcription of the structural nucleic
acid sequences. Full induction of rhaBAD transcription also
requires binding of the Crp-cAMP complex, which is a key regulator
of catabolite repression.
[0060] Although both L-arabinose and L-rhamnose act directly as
inducers for expression of regulons for their catabolism, important
differences exist in regard to the regulatory mechanisms.
L-Arabinose acts as an inducer with the activator AraC in the
positive control of the arabinose regulon. However, the L-rhamnose
regulon is subject to a regulatory cascade; it is therefore subject
to even tighter control than the araC P.sub.BAD system. L-Rhamnose
acts as an inducer with the activator RhaR for synthesis of RhaS,
which in turn acts as an activator in the positive control of the
rhamnose regulon. In the present invention, rhamnose may be used to
interact with the RhaR protein and then the RhaS protein may
activate transcription of a nucleic acid sequence operably-linked
to the P.sub.rhaBAD promoter.
[0061] In still other embodiments, the promoter may be sensitive to
the level of xylose in the environment. The xylR-P.sub.xylA system
is another well-established inducible activator-promoter system.
Xylose induces xylose-specific operons (xylE, xylFGHR, and xylAB)
regulated by XylR and the cyclic AMP-Crp system. The XylR protein
serves as a positive regulator by binding to two distinct regions
of the xyl nucleic acid sequence promoters. As with the
araC-P.sub.BAD system described above, the xylR-P.sub.xylAB and/or
xylR-P.sub.xylFGH regulatory systems may be used in the present
invention. In these embodiments, xylR P.sub.xylAB xylose
interacting with the XylR protein activates transcription of
nucleic acid sequences operably-linked to either of the two
P.sub.xyl promoters.
[0062] The nucleic acid sequences of the promoters detailed herein
are known in the art, and methods of operably-linking them to a
chromosomally integrated nucleic acid sequence encoding a repressor
are known in the art and detailed in the examples.
3. Modification to Optimize Expression
[0063] A nucleic acid sequence encoding a repressor and regulatable
promoter detailed above, for use in the present invention, may be
modified so as to optimize the expression level of the nucleic acid
sequence encoding the repressor. The optimal level of expression of
the nucleic acid sequence encoding the repressor may be estimated,
or may be determined by experimentation. Such a determination
should take into consideration whether the repressor acts as a
monomer, dimer, trimer, tetramer, or higher multiple, and should
also take into consideration the copy number of the vector encoding
the antigen or effector protein of interest, as detailed below. In
an exemplary embodiment, the level of expression is optimized so
that the repressor is synthesized while in the permissive
environment (i.e. in vitro growth) at a level that substantially
inhibits the expression of the nucleic acid sequence encoding an
antigen or effector protein of interest, and is substantially not
synthesized in a non-permissive environment, thereby allowing
expression of the nucleic acid sequence encoding an antigen or
effector protein of interest.
[0064] As stated above, the level of expression may be optimized by
modifying the nucleic acid sequence encoding the repressor and/or
promoter. As used herein, "modify" refers to an alteration of the
nucleic acid sequence of the repressor and/or promoter that results
in a change in the level of transcription of the nucleic acid
sequence encoding the repressor, or that results in a change in the
level of synthesis of the repressor. For instance, in one
embodiment, modify may refer to altering the start codon of the
nucleic acid sequence encoding the repressor. Generally speaking, a
GTG or TTG start codon, as opposed to an ATG start codon, may
decrease translation efficiency ten-fold. In another embodiment,
modify may refer to altering the Shine-Dalgarno (SD) sequence of
the nucleic acid sequence encoding the repressor. The SD sequence
is a ribosomal binding site generally located 6-7 nucleotides
upstream of the start codon. The SD consensus sequence is AGGAGG,
and variations of the consensus sequence may alter translation
efficiency. In yet another embodiment, modify may refer to altering
the distance between the SD sequence and the start codon. In still
another embodiment, modify may refer to altering the -35 sequence
for RNA polymerase recognition. In a similar embodiment, modify may
refer to altering the -10 sequence for RNA polymerase binding. In
an additional embodiment, modify may refer to altering the number
of nucleotides between the -35 and -10 sequences. In an alternative
embodiment, modify may refer to optimizing the codons of the
nucleic acid sequence encoding the repressor to alter the level of
translation of the mRNA encoding the repressor. For instance, non-A
rich codons initially after the start codon of the nucleic acid
sequence encoding the repressor may not maximize translation of the
mRNA encoding the repressor. Similarly, the codons of the nucleic
acid sequence encoding the repressor may be altered so as to mimic
the codons from highly synthesized proteins of a particular
organism. In a further embodiment, modify may refer to altering the
GC content of the nucleic acid sequence encoding the repressor to
change the level of translation of the mRNA encoding the repressor.
Modify can also mean optimization of codons to increase the
stability of the mRNA to increase its half-life and thus the number
of times it can be translated.
[0065] In some embodiments, more than one modification or type of
modification may be performed to optimize the expression level of
the nucleic acid sequence encoding the repressor. For instance, at
least one, two, three, four, five, six, seven, eight, or nine
modifications, or types of modifications, may be performed to
optimize the expression level of the nucleic acid sequence encoding
the repressor.
[0066] By way of non-limiting example, when the repressor is LacI,
then the nucleic acid sequence of LacI and the promoter may be
altered so as to increase the level of LacI synthesis. In one
embodiment, the start codon of the LacI repressor may be altered
from GTG to ATG. In another embodiment, the SD sequence may be
altered from AGGG to AGGA. In yet another embodiment, the codons of
lacI may be optimized according to the codon usage for highly
synthesized proteins of the recombinant bacterium. In a further
embodiment, the start codon of lacI may be altered, the SD sequence
may be altered, and/or the codons of lacI may be optimized.
[0067] Methods of modifying the nucleic acid sequence encoding the
repressor and/or the regulatable promoter are known in the art and
detailed in the examples.
4. Transcription Termination Sequence
[0068] In some embodiments, the chromosomally integrated nucleic
acid sequence encoding the repressor further comprises a
transcription termination sequence. A transcription termination
sequence may be included to prevent inappropriate expression of
nucleic acid sequences adjacent to the chromosomally integrated
nucleic acid sequence encoding the repressor and regulatable
promoter.
B. Vector
[0069] A recombinant bacterium of the invention that is capable of
the regulated expression of at least one nucleic acid sequence
encoding an antigen or effector protein comprises, in part, a
vector. The vector comprises a nucleic acid sequence encoding at
least one antigen or effector protein of interest operably linked
to a promoter. The promoter is regulated by the chromosomally
encoded repressor, such that the expression of the nucleic acid
sequence encoding an antigen or effector protein of interest is
repressed during in vitro growth of the bacterium, but the
bacterium is capable of high level synthesis of the antigen or
effector protein in an animal or human host. In certain
embodiments, however, the promoter may also be regulated by a
plasmid encoded repressor.
[0070] As used herein, "vector" refers to an autonomously
replicating nucleic acid unit. The present invention can be
practiced with any known type of vector, including viral, cosmid,
phasmid, and plasmid vectors. The most preferred type of vector is
a plasmid vector.
[0071] As is well known in the art, plasmids and other vectors may
possess a wide array of promoters, multiple cloning sequences,
transcription terminators, etc., and vectors may be selected so as
to control the level of expression of the nucleic acid sequence
encoding an antigen by controlling the relative copy number of the
vector. In some instances in which the vector might encode a
surface localized adhesin as the antigen, or an antigen capable of
stimulating T-cell immunity, it may be preferable to use a vector
with a low copy number such as at least two, three, four, five,
six, seven, eight, nine, or ten copies per bacterial cell. A
non-limiting example of a low copy number vector may be a vector
comprising the pSC101 ori.
[0072] In other cases, an intermediate copy number vector might be
optimal for inducing desired immune responses. For instance, an
intermediate copy number vector may have at least 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 copies per bacterial cell. A non-limiting example of an
intermediate copy number vector may be a vector comprising the p15A
ori.
[0073] In still other cases, a high copy number vector might be
optimal for the induction of maximal antibody responses. A high
copy number vector may have at least 31, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, or 100 copies per bacterial cell. In
some embodiments, a high copy number vector may have at least 100,
125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400
copies per bacterial cell. Non-limiting examples of high copy
number vectors may include a vector comprising the pBR ori or the
pUC ori.
[0074] Additionally, vector copy number may be increased by
selecting for mutations that increase plasmid copy number. These
mutations may occur in the bacterial chromosome but are more likely
to occur in the plasmid vector.
[0075] Preferably, vectors used herein do not comprise antibiotic
resistance markers to select for maintenance of the vector.
1. Antigen or Effector Protein
[0076] As used herein, "antigen" refers to a biomolecule capable of
eliciting an immune response in a host. In some embodiments, an
antigen may be a protein, or fragment of a protein, or a nucleic
acid. In an exemplary embodiment, the antigen elicits a protective
immune response. As used herein, "protective" means that the immune
response decreases the size of a tumor, decreases metastases,
and/or contributes to the lessening of any symptoms associated with
a tumor. The use of the term "protective" in this invention does
not necessarily require that the host is completely protected from
the effects of the tumor. As used herein, "effector protein" refers
to a biomolecule capable of inhibiting tumor cell growth. In some
embodiments, an effector protein may induce programmed cell death
(e.g. apoptosis or pyrotosis) in tumor cells, or may otherwise
decrease the size of a tumor, decrease metastases, or contribute to
the lessening of any symptoms associated with a tumor
[0077] It is not necessary that the vector comprise the complete
nucleic acid sequence of the antigen or effector protein. It is
only necessary that the antigen sequence used be capable of
eliciting an immune response, or the effector protein be capable of
eliciting the desired effect. The antigen or effector protein may
be one that was not found in that exact form in the parent
organism. For example, a sequence coding for an antigen or effector
protein comprising 100 amino acid residues may be transferred in
part into a recombinant bacterium so that a peptide comprising only
75, 65, 55, 45, 35, 25, 15, or even 10, amino acid residues is
produced by the recombinant bacterium. Alternatively, if the amino
acid sequence of a particular antigen, effector protein, or
fragment thereof is known, it may be possible to chemically
synthesize the nucleic acid fragment or analog thereof by means of
automated nucleic acid sequence synthesizers, PCR, or the like and
introduce said nucleic acid sequence into the appropriate copy
number vector.
[0078] In another alternative, a vector may comprise a long
sequence of nucleic acid encoding several nucleic acid sequence
products, one or all of which may be antigenic or be effector
proteins. In some embodiments, a vector of the invention may
comprise a nucleic acid sequence encoding at least one antigen or
effector protein, at least two antigens or effector proteins, at
least three antigens or effector proteins, or more than three
antigens or effector proteins. These antigens or effector proteins
may be encoded by two or more open reading frames operably linked
to be expressed coordinately as an operon, wherein each antigen or
effector proteins is synthesized independently. Alternatively, the
two or more antigens or effector proteins may be encoded by a
single open reading frame such that the antigens or effector
proteins are synthesized as a fusion protein.
[0079] In certain embodiments, an antigen of the invention may
comprise a B cell epitope or a T cell epitope. Alternatively, an
antigen to which an immune response is desired may be expressed as
a fusion to a carrier protein that contains a strong promiscuous T
cell epitope and/or serves as an adjuvant and/or facilitates
presentation of the antigen to enhance, in all cases, the immune
response to the antigen or its component part. This can be
accomplished by methods known in the art. Fusion to tenus toxin
fragment C, CT-B, LT-B and hepatitis virus B core are particularly
useful for these purposes, although other epitope presentation
systems such as hepatitis B virus and woodchuck hepatitis virus
cores are well known in the art.
[0080] In further embodiments, a nucleic acid sequence encoding an
antigen or effector protein of the invention may comprise a
secretion signal. In other embodiments, an antigen or effector
protein of the invention may be toxic to the recombinant
bacterium.
[0081] In one embodiment, an effector protein may be SopE2. In some
embodiments, the native promoter may be replaced with P.sub.trc to
enable the regulated delayed synthesis of SopE2. In certain
embodiments, the start codon of sopE2 may be modified to alter its
expression level. For instance, the start codon may be changed from
GTG to ATG.
2. Promoter Regulated by Repressor
[0082] The vector comprises a nucleic acid sequence encoding at
least one antigen operably-linked to a promoter regulated by the
repressor, encoded by a chromosomally integrated nucleic acid
sequence. One of skill in the art would recognize, therefore, that
the selection of a repressor dictates, in part, the selection of
the promoter operably-linked to a nucleic acid sequence encoding an
antigen or effector protein of interest. For instance, if the
repressor is LacI, then the promoter may be selected from the group
consisting of LacI responsive promoters, such as P.sub.trc,
P.sub.lac, P.sub.T7lac and P.sub.tac. If the repressor is C2, then
the promoter may be selected from the group consisting of C2
responsive promoters, such as P22 promoters P.sub.L and P.sub.R. If
the repressor is C1, then the promoter may be selected from the
group consisting of C1 responsive promoters, such as .lamda.
promoters P.sub.L and P.sub.R.
[0083] In each embodiment herein, the promoter regulates expression
of a nucleic acid sequence encoding the antigen or effector
protein, such that expression of the nucleic acid sequence encoding
an antigen or effector protein is repressed when the repressor is
synthesized (i.e. during in vitro growth of the bacterium), but
expression of the nucleic acid sequence encoding an antigen or
effector protein is high when the repressor is not synthesized
(i.e. in an animal or human host). Generally speaking, the
concentration of the repressor will decrease with every cell
division after expression of the nucleic acid sequence encoding the
repressor ceases. In some embodiments, the concentration of the
repressor decreases enough to allow high level expression of the
nucleic acid sequence encoding an antigen or effector protein after
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 divisions of the
bacterium. In an exemplary embodiment, the concentration of the
repressor decreases enough to allow high level expression of the
nucleic acid sequence encoding an antigen or expression protein
after about 5 divisions of the bacterium in an animal or human
host.
[0084] In certain embodiments, the promoter may comprise other
regulatory elements. For instance, the promoter may comprise lacO
if the repressor is LacI. This is the case with the lipoprotein
promoter P.sub.lpp that is regulated by LacI since it possesses the
LacI binding domain lacO.
[0085] In one embodiment, the repressor is a LacI repressor and the
promoter is P.sub.trc.
3. Expression of the Nucleic Acid Sequence Encoding an Antigen or
Effector Protein
[0086] As detailed above, generally speaking the expression of the
nucleic acid sequence encoding the antigen or effector protein
should be repressed when the repressor is synthesized. For
instance, if the repressor is synthesized during in vitro growth of
the bacterium, expression of the nucleic acid sequence encoding the
antigen or effector protein should be repressed. Expression may be
"repressed" or "partially repressed" when it is about 50%, 45%,
40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or even less than 1% of
the expression under non-repressed conditions. Thus although the
level of expression under conditions of "complete repression" might
be exceeding low, it is likely to be detectable using very
sensitive methods since repression can never by absolute.
[0087] Conversely, the expression of the nucleic acid sequence
encoding the antigen or effector protein should be high when the
expression of the nucleic acid sequence encoding the repressor is
repressed. For instance, if the nucleic acid sequence encoding the
repressor is not expressed during growth of the recombinant
bacterium in the host, the expression of the nucleic acid sequence
encoding the antigen or effector protein should be high. As used
herein, "high level" expression refers to expression that is strong
enough to elicit an immune response to the antigen or to see the
effects of the effector protein on the tumor cell. Consequently,
the copy number correlating with high level expression can and will
vary depending on the antigen or effector protein and the type of
immune response desired. Methods of determining whether an antigen
elicits an immune response such as by measuring antibody levels or
antigen-dependant T cell populations or antigen-dependant cytokine
levels are known in the art, and methods of measuring levels of
expression of antigen or effector protein encoding sequences by
measuring levels of mRNA transcribed or by quantitating the level
of antigen or effector protein synthesis are also known in the
art.
ii. Nucleic Acid Vaccine Vector
[0088] A recombinant bacterium of the invention may encompass a
nucleic acid vaccine vector. Such a vector is typically designed to
be transcribed in the nucleus of the host cell to produce mRNA
encoding one or more antigens or effector proteins of interest. To
increase performance, a nucleic acid vaccine vector should be
targeted to the nucleus of a host cell, and should be resistant to
nuclease attack.
[0089] In one embodiment of the invention, a nucleic acid vaccine
vector may be targeted to the nucleus using a DNA nuclear targeting
sequence. Such a sequence allows transcription factors of the host
cell to bind to the vector in the cytoplasm and escort it to the
nucleus via the nuclear localization signal-mediated machinery. DNA
nuclear targeting sequences are known in the art. For instance, the
SV40 enhancer may be used. In particular, a single copy of a 72-bp
element of the SV40 enhancer may be used, or a variation thereof.
The SV40 enhancer may be used in combination with the CMV
immediate-early gene enhancer/promoter.
[0090] Additionally, DNA binding sites for eukaryotic transcription
factors may be included in the vaccine vector. These sites allow
transcription factors such as NF-.kappa.B and AP-2 to bind to the
vector, allowing the nuclear location signal to mediate import of
the vector to the nucleus.
[0091] A nucleic acid vaccine vector of the invention may also be
resistant to eukaryotic nuclease attack. In particular, the
polyadenalytion signal may be modified to increase resistance to
nuclease attack. Suitable polyadenylation signals that are
resistant to nuclease attack are known in the art. For instance,
the SV40 late poly A signal may be used. Alternatively, other poly
A adenylation signal sequences could be derived from other DNA
viruses known to be successful in infecting avian and/or mammalian
species.
[0092] A bacterium comprising a nucleic acid vaccine vector may
also comprise a mutation that eliminates the periplasmic
endonuclease I enzyme, such as a .DELTA.endA mutation, e.g.
.DELTA.endA2311. This type of mutation is designed to increase
vector survival upon the vector's release into the host cell.
[0093] In one embodiment, a nucleic acid vaccine vector may
comprise a promoter that is over-expressed in a tumor. For
instance, the hexokinase type II promoter may be used. In an
exemplary embodiment, the hexokinase type II promoter may be
operably linked to a nucleic acid encoding Fas ligand.
(f) Attenuation
[0094] In each of the above embodiments, a recombinant bacterium of
the invention may also be attenuated. "Attenuated" refers to the
state of the bacterium wherein the bacterium has been weakened from
its wild-type fitness by some form of recombinant or physical
manipulation. This includes altering the genotype of the bacterium
to reduce its ability to cause disease. However, the bacterium's
ability to colonize the tumor is, preferably, not substantially
compromised. For instance, in one embodiment, regulated attenuation
allows the recombinant bacterium to express one or more nucleic
acids encoding products important for the bacterium to withstand
stresses encountered in the host after immunization. This allows
efficient invasion and colonization of tumor tissues before the
recombinant bacterium is regulated to display the attenuated
phenotype.
[0095] In one embodiment, a recombinant bacterium may be attenuated
by regulating LPS O-antigen. In other embodiments, attenuation may
be accomplished by altering (e.g., deleting) native nucleic acid
sequences found in the wild type bacterium. For instance, if the
bacterium is Salmonella, non-limiting examples of nucleic acid
sequences which may be used for attenuation include: a pab nucleic
acid sequence, a pur nucleic acid sequence, an aro nucleic acid
sequence, asdA, a dap nucleic acid sequence, nadA, pncB, galE, pmi,
fur, rpsL, ompR, htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc,
poxA, galU, mviA, sodC, recA, ssrA, sirA, inv, hilA, rpoE, flgM,
tonB, slyA, and any combination thereof. Exemplary attenuating
mutations may be aroA, aroC, aroD, cdt, cya, crp, phoP, phoQ, ompR,
galE, and htrA.
[0096] In certain embodiments, the above nucleic acid sequences may
be placed under the control of a sugar regulated promoter wherein
the sugar is present during in vitro growth of the recombinant
bacterium, but substantially absent within an animal or human host.
The cessation in transcription of the nucleic acid sequences listed
above would then result in attenuation and the inability of the
recombinant bacterium to induce disease symptoms.
[0097] The bacterium may also be modified to create a
balanced-lethal host-vector system, although other types of systems
may also be used (e.g., creating complementation heterozygotes).
For the balanced-lethal host-vector system, the bacterium may be
modified by manipulating its ability to synthesize various
essential constituents needed for synthesis of the rigid
peptidoglycan layer of its cell wall. In one example, the
constituent is diaminopimelic acid (DAP). Various enzymes are
involved in the eventual synthesis of DAP. In one example, the
bacterium is modified by using a .DELTA.asdA mutation to eliminate
the bacterium's ability to produce .beta.-aspartate semialdehyde
dehydrogenase, an enzyme essential for the synthesis of DAP. One of
skill in the art can also use the teachings of U.S. Pat. No.
6,872,547 for other types of mutations of nucleic acid sequences
that result in the abolition of the synthesis of DAP. These nucleic
acid sequences may include, but are not limited to, dapA, dapB,
dapC, dapD, dapE, dapF, and asdA. Other modifications that may be
employed include modifications to a bacterium's ability to
synthesize D-alanine or to synthesize D-glutamic acid (e.g.,
.DELTA.murI mutations), which are both unique constituents of the
peptidoglycan layer of the bacterial cell wall
[0098] Yet another balanced-lethal host-vector system comprises
modifying the bacterium such that the synthesis of an essential
constituent of the rigid layer of the bacterial cell wall is
dependent on a nutrient (e.g., arabinose) that can be supplied
during the growth of the microorganism. For example, a bacterium
may comprise the .DELTA.P.sub.murA::TT araC P.sub.BAD murA
deletion-insertion mutation. This type of mutation makes synthesis
of muramic acid (another unique essential constituent of the
peptidoglycan layer of the bacterial cell wall) dependent on the
presence of arabinose that can be supplied during growth of the
bacterium in vitro.
[0099] Other means of attenuation are known in the art.
i. Regulated Attenuation
[0100] The present invention also encompasses a recombinant
bacterium capable of regulated attenuation. Generally speaking, the
bacterium comprises a chromosomally integrated regulatable
promoter. The promoter replaces the native promoter of, and is
operably linked to, at least one nucleic acid sequence encoding an
attenuation protein, such that the absence of the function of the
protein renders the bacterium attenuated. In some embodiments, the
promoter is modified to optimize the regulated attenuation
[0101] In each of the above embodiments described herein, more than
one method of attenuation may be used. For instance, a recombinant
bacterium of the invention may comprise a regulatable promoter
chromosomally integrated so as to replace the native promoter of,
and be operably linked to, at least one nucleic acid sequence
encoding an attenuation protein, such that the absence of the
function of the protein renders the bacterium attenuated, and the
bacterium may comprise another method of attenuation detailed in
section I above.
A. Attenuation Protein
[0102] Herein, "attenuation protein" is meant to be used in its
broadest sense to encompass any protein the absence of which
attenuates a bacterium. For instance, in some embodiments, an
attenuation protein may be a protein that helps protect a bacterium
from stresses encountered in the gastrointestinal tract or
respiratory tract. Non-limiting examples may be the RpoS, PhoPQ,
OmpR, Fur, and Crp proteins. In other embodiments, the protein may
be necessary to synthesize a component of the cell wall of the
bacterium, or may itself be a necessary component of the cell wall
such as the protein encoded by murA.
[0103] The native promoter of at least one, two, three, four, five,
or more than five attenuation proteins may be replaced by a
regulatable promoter as described herein. In one embodiment, the
promoter of one of the proteins selected from the group comprising
RpoS, PhoPQ, OmpR, Fur, and Crp may be replaced. In another
embodiment, the promoter of two, three, four or five of the
proteins selected from the group comprising RpoS, PhoPQ, OmpR, Fur,
and Crp may be replaced.
[0104] If the promoter of more than one attenuation protein is
replaced, each promoter may be replaced with a regulatable
promoter, such that the expression of each attenuation protein
encoding sequence is regulated by the same compound or condition.
Alternatively, each promoter may be replaced with a different
regulatable promoter, such that the expression of each attenuation
protein encoding sequence is regulated by a different compound or
condition such as by the sugars arabinose, maltose, rhamnose or
xylose.
B. Regulatable Promoter
[0105] The native promoter of a nucleic acid sequence encoding an
attenuation protein is replaced with a regulatable promoter
operably linked to the nucleic acid sequence encoding an
attenuation protein. The term "operably linked," is defined
above.
[0106] The regulatable promoter used herein generally allows
transcription of the nucleic acid sequence encoding the attenuation
protein while in a permissive environment (i.e. in vitro growth),
but cease transcription of the nucleic acid sequence encoding an
attenuation protein while in a non-permissive environment (i.e.
during growth of the bacterium in an animal or human host). For
instance, the promoter may be responsive to a physical or chemical
difference between the permissive and non-permissive environment.
Suitable examples of such regulatable promoters are known in the
art and detailed above.
[0107] In some embodiments, the promoter may be responsive to the
level of arabinose in the environment, as described above. In other
embodiments, the promoter may be responsive to the level of
maltose, rhamnose, or xylose in the environment, as described
above. The promoters detailed herein are known in the art, and
methods of operably linking them to a nucleic acid sequence
encoding an attenuation protein are known in the art.
[0108] In certain embodiments, a recombinant bacterium of the
invention may comprise any of the following: .DELTA.P.sub.fur::TT
araC P.sub.BAD fur, .DELTA.P.sub.crp::TT araC P.sub.BAD crp,
.DELTA.P.sub.phoPQ::TT araC P.sub.BAD phoPQ, or a combination
thereof. Growth of such strains in the presence of arabinose leads
to transcription of the fur, phoPQ, and/or crp nucleic acid
sequences, but nucleic acid sequence expression ceases in a host
because there is no free arabinose. Attenuation develops as the
products of the fur, phoPQ, and/or the crp nucleic acid sequences
are diluted at each cell division. Strains with the
.DELTA.P.sub.fur and/or the .DELTA.P.sub.phoPQ mutations are
attenuated at oral doses of 10.sup.9 CFU, even in three-week old
mice at weaning. Generally speaking, the concentration of arabinose
necessary to induce expression is typically less than about 2%. In
some embodiments, the concentration is less than about 1.5%, 1%,
0.5%, 0.2%, 0.1%, or 0.05%. In certain embodiments, the
concentration may be about 0.04%, 0.03%, 0.02%, or 0.01%. In an
exemplary embodiment, the concentration is about 0.05%. Higher
concentrations of arabinose or other sugars may lead to acid
production during growth that may inhibit desirable cell densities.
The inclusion of mutations such as .DELTA.araBAD or mutations that
block the uptake and/or breakdown of maltose, rhamnose, or xylose,
however, may prevent such acid production and enable use of higher
sugar concentrations with no ill effects.
[0109] When the regulatable promoter is responsive to arabinose,
the onset of attenuation may be delayed by including additional
mutations, such as .DELTA.araBAD23, which prevents use of arabinose
retained in the cell cytoplasm at the time of oral immunization,
and/or .DELTA.araE25 that enhances retention of arabinose. Thus,
inclusion of these mutations may be beneficial in at least two
ways: first, enabling higher culture densities, and second enabling
a further delay in the display of the attenuated phenotype that may
result in higher densities in effector lymphoid tissues to further
enhance immunogenicity.
C. Modifications
[0110] Attenuation of the recombinant bacterium may be optimized by
modifying the nucleic acid sequence encoding an attenuation protein
and/or promoter. Methods of modifying a promoter and/or a nucleic
acid sequence encoding an attenuation protein are the same as those
detailed above with respect to repressors in section (d).
[0111] In some embodiments, more than one modification may be
performed to optimize the attenuation of the bacterium. For
instance, at least one, two, three, four, five, six, seven, eight
or nine modifications may be performed to optimize the attenuation
of the bacterium. In various exemplary embodiments of the
invention, the SD sequences and/or the start codons for the fur
and/or the phoPQ virulence nucleic acid sequences may be altered so
that the production levels of these nucleic acid products are
optimal for regulated attenuation.
(g) Other Mutations
[0112] In some embodiments, a recombinant bacterium of the
invention may also comprise a .DELTA.P.sub.crp::TT araC P.sub.BAD
crp deletion-insertion mutation. Since the araC P.sub.BAD cassette
is dependent both on the presence of arabinose and the binding of
the catabolite repressor protein Crp, a .DELTA.P.sub.crp::TT araC
P.sub.BAD crp deletion-insertion mutation may be included as an
additional means to reduce expression of any nucleic acid sequence
under the control of the P.sub.BAD promoter. This means that when
the bacterium is grown in a non-permissive environment (i.e. no
arabinose) both the repressor itself and the Crp protein cease to
be synthesized, consequently eliminating both regulating signals
for the araC P.sub.BAD regulated nucleic acid sequence. This double
shut off of araC P.sub.BAD may constitute an additional safety
feature ensuring the genetic stability desired of phenotypes.
[0113] Generally speaking, the activity of the Crp protein requires
interaction with cAMP, but the addition of glucose, which may
inhibit synthesis of cAMP, decreases the ability of the Crp protein
to regulate transcription from the araC P.sub.BAD promoter.
Consequently, to avoid the effect of glucose on cAMP, glucose may
be substantially excluded from the growth media, or variants of crp
may be isolated or constructed that synthesize a Crp protein that
is not dependent on cAMP to regulate transcription from P.sub.BAD.
Two such alterations in the crp gene have been made with amino acid
substitution mutations T127I, Q170K and L195R to result in the
crp-70 gene modification and with amino acid substitutions I112L,
T127I and A144T to result in the crp-72 gene modification. Both
constructions have been made with araC P.sub.BAD to yield the
.DELTA.P.sub.crp70:: araC P.sub.BAD crp-70 and
.DELTA.P.sub.crp72::TT araC P.sub.BAD crp-72 deletion-insertion
mutations. In both cases, synthesis of the Crp protein induced by
arabinose is insensitive to the addition of glucose. This strategy
may also be used in other systems responsive to Crp, such as the
systems responsive to rhamnose and xylose described above.
(h) Exemplary Bacterium
[0114] In an exemplary embodiment, a bacterium may comprise one or
more mutations to increase invasiveness (section (a) above), one or
more mutations that enhance stimulation of host innate immune
responses (section (b) above), one or more mutations to increase
bacterium-induced host programmed cell death (section (c) above),
one or more mutations to induce lysis of the bacterium (section (d)
above), one or more vectors to express a nucleic acid encoding an
antigen or effector protein (section (e) above), one or more
mutations to attenuate the bacterium (section (f) above), and one
or more mutations to enhance the performance of the bacterium as a
vaccine (section (g) above).
[0115] In one embodiment, a bacterium of the invention may comprise
the following mutations: .DELTA.asdA27:TT araC P.sub.BAD c2
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA .DELTA.(wza-wcaM)-8
.DELTA.relA198::araC P.sub.BAD lacI TT .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD .DELTA.pagP81::P.sub.lpp lpxE
.DELTA.endA2311[[1123]] .DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888
hilA. In another embodiment, a bacterium of the invention may
comprise the following mutations: .DELTA.P.sub.murA25::TT araC
P.sub.BAD murA .DELTA.(wza-wcaM)-8 .DELTA.relA198::araC P.sub.BAD
lacI TT .DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.endA2311
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr .DELTA.trg or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA .DELTA.purA. In yet
another embodiment, a bacterium of the invention may comprise the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA .DELTA.(wza-wcaM)-8
.DELTA.relA198::araC P.sub.BAD lacI TT .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD .DELTA.pagP81::P.sub.lpp lpxE
.DELTA.endA2311.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr .DELTA.trg or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA .DELTA.purA
.DELTA.P.sub.sopE2::P.sub.trc sopE2. In still another embodiment, a
bacterium of the invention may comprise the following mutations:
.DELTA.asdA27::TT araC P.sub.BAD c2 .DELTA.P.sub.murA25::TT araC
P.sub.bad murA .DELTA.(wza-wcaM)-8 .DELTA.relA 198::araC P.sub.BAD
lacI TT .DELTA.(araC PBAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.endA2311
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.O888 tsr .DELTA.trg or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA .DELTA.purA
.DELTA.P.sub.sopE2::P.sub.trc sopE2 .DELTA.P.sub.tlpA::P.sub.ansB
tlpA.
[0116] In a certain embodiment, the bacterium comprises the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2,
.DELTA.P.sub.murA25:: TT araC P.sub.BAD murA, .DELTA.(wza-wcaM)-8,
.DELTA.relA198::araC P.sub.BAD lacI TT, .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD, .DELTA.pagP81::P.sub.lpp lpxE,
.DELTA.endA2311.
[0117] In another embodiment, the bacterium comprises the following
mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.((wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311,
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar.
[0118] In still another embodiment, the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311,
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr.
[0119] In yet another embodiment, the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311,
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg.
[0120] In a further embodiment, the bacterium comprises the
following mutations: .DELTA.P.sub.murA25::TT araC P.sub.BAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC P.sub.BAD lacI TT,
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD,
.DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311,
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA.
[0121] In still a further embodiment, the bacterium comprises the
following mutations: .DELTA.PmurA25::TT araC PBAD murA,
.DELTA.(wza-wcaM)-8, .DELTA.relA198::araC PBAD lacI TT,
.DELTA.(araC PBAD)-18::P22 PR araBAD, .DELTA.pagP81::Plpp lpxE,
.DELTA.endA2311, .DELTA.Ptar::Ptrc .DELTA.lacO888 tar,
.DELTA.Ptsr::Ptrc .DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.Ptrg::rhaRS-PrhaB trg, .DELTA.PhilA::Ptrc .DELTA.lacO888
hilA, .DELTA.purA.
[0122] In an alternative embodiment, the bacterium comprises the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2,
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA, .DELTA.(wza-wcaM)-8,
.DELTA.relA198::araC P.sub.BAD lacI TT, .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD, .DELTA.pagP81::P.sub.lpp lpxE,
.DELTA.endA2311, .DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA, .DELTA.purA,
.DELTA.P.sub.sopE2::P.sub.trc sopE2.
[0123] In yet another alternative, the bacterium comprises the
following mutations: .DELTA.asdA27::TT araC P.sub.BAD c2,
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA, .DELTA.(wza-wcaM)-8,
.DELTA.rel 198::araC P.sub.BAD lacI TT, .DELTA.(araC PBAD)-18::P22
P.sub.R araBAD, .DELTA.pagP81::P.sub.lpp lpxE, .DELTA.endA2311,
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar,
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr, .DELTA.trg, or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg,
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA, .DELTA.purA,
.DELTA.P.sub.sopE2::P.sub.trc sopE2, .DELTA.P.sub.tlpA::P.sub.ansB
tlpA.
II. Pharmaceutical Compositions and Administration
[0124] Pharmaceutical compositions of the present invention may be
administered to any host susceptible to tumors and the recombinant
bacterium. Such hosts may include all vertebrates, for example,
mammals, including domestic animals, agricultural animals,
laboratory animals, and humans, and various species of birds,
including domestic birds and birds of agricultural importance.
Preferably, the host is a warm-blooded animal.
[0125] In exemplary embodiments, the recombinant bacterium is alive
when administered to a host in a pharmaceutical composition of the
invention. Suitable pharmaceutical composition formulations and
methods of administration are detailed below.
(a) Pharmaceutical Composition
[0126] A pharmaceutical composition comprising a recombinant
bacterium of the invention may optionally comprise one or more
possible additives, such as carriers, preservatives, stabilizers,
and other substances.
[0127] In another embodiment, the composition may comprise a
pharmaceutical carrier (or excipient). Such a carrier may be any
solvent or solid material for encapsulation that is non-toxic to
the inoculated host and compatible with the recombinant bacterium.
A carrier may give form or consistency, or act as a diluent.
Suitable pharmaceutical carriers may include liquid carriers, such
as normal saline and other non-toxic salts at or near physiological
concentrations, and solid carriers not used for humans, such as
talc or sucrose, or animal feed. Carriers may also include
stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers. Carriers and excipients as well as
formulations for parenteral and nonparenteral drug delivery are set
forth in Remington's Pharmaceutical Sciences 19th Ed. Mack
Publishing (1995). When used for administering via the bronchial
tubes, the pharmaceutical composition is preferably presented in
the form of an aerosol.
[0128] Care should be taken when using additives so that the live
recombinant bacterium is not killed, or have its ability to
effectively colonize tumor tissues compromised by the use of
additives. Stabilizers, such as lactose or monosodium glutamate
(MSG), may be added to stabilize the pharmaceutical formulation
against a variety of conditions, such as temperature variations or
a freeze-drying process.
[0129] The dosages of a pharmaceutical composition of the invention
can and will vary depending on the recombinant bacterium, the
regulated antigen or effector protein, and the intended host, as
will be appreciated by one of skill in the art. Generally speaking,
the dosage need only be sufficient to elicit an anti-tumor response
in a majority of hosts. Routine experimentation may readily
establish the required dosage. Typical initial dosages of a
pharmaceutical composition for oral administration could be about
1.times.10.sup.7 to 1.times.10.sup.10 CFU depending upon the age of
the host to be immunized. Administering multiple dosages may also
be used as needed to provide the desired level of anti-tumor
activity. In some embodiments, parental administration is preferred
(e.g. for treatment of internal solid tumors). In such embodiments,
doses may range from about 1.times.10.sup.5 to 1.times.10.sup.8
CFU.
(b) Methods of Administration
[0130] A pharmaceutical composition may be administered orally
intravenously, intramuscularly, or by subcutaneous injection. In
some embodiments, these compositions are formulated for
administration by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). Accordingly,
these compositions are preferably combined with pharmaceutically
acceptable vehicles such as saline, Ringer's solution, dextrose
solution, and the like.
III. Methods of Use
[0131] A further aspect of the invention encompasses methods of
using a recombinant bacterium of the invention. For instance, in
one embodiment the invention provides a method for inhibiting tumor
growth. The method generally comprises administering a recombinant
bacterium of the invention to a subject. In another embodiment, the
invention provides a method for treating cancer. The method
generally comprises administering a recombinant bacterium of the
invention to a subject.
[0132] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. Those of skill in the art
should, however, in light of the present disclosure, appreciate
that many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention, therefore all
matter set forth or shown in the accompanying drawings is to be
interpreted as illustrative and not in a limiting sense.
EXAMPLES
[0133] The following examples illustrate various iterations of the
invention.
Introduction for Examples 1-8
[0134] Colorectal cancer is the second leading cause of
cancer-related deaths in the United States (after lung cancer).
According to the American Cancer Society, almost 150,000 new cases
of colorectal cancer were diagnosed and approximately 50,000 people
died from the disease last year. Multidrug resistance and the
presence of undetectable micrometastases, which are caused by at
least two mechanisms: i.e. limited drug penetration and poor cell
susceptibility (59, 80), significantly reduce the effectiveness of
most cancer therapeutics, mainly radiotherapy and chemotherapy.
Uneven perfusion in tumors creates populations of cells that are
physically distant from therapeutics in the bloodstream and are
quiescent due to nutrient deficiencies (105). The other substantial
limitation of conventional cancer chemotherapy and radiotherapy is
the toxicity of these agents to normal tissue (131). This has
prompted the development of many new approaches for the treatment
of cancer including the delivery of anti-cancer genes to the tumor
site in various gene therapy protocols (85, 119). However, current
gene therapy protocols require local administration of vectors,
which limits their usefulness. Also the nonselectivity of the
available gene delivery systems renders cancer gene therapy
strategies potentially toxic to normal cell populations. Although
there have been recent advances in adjuvant therapy, there are no
major breakthroughs in the treatment of colorectal cancers.
[0135] Motile facultative anaerobes have the potential to actively
penetrate into tumor tissue and overcome diffusion limitation,
where they could attack quiescent cancer cells that are impervious
to standard chemo- and radiotherapies (49, 67, 78, 113). The
preferential accumulation of bacteria in certain experimental
tumors was initially reported in the 1950s when spores of
Clostridium tetani were shown to germinate exclusively in the tumor
after intravenous administration into tumor-bearing mice (96). It
was assumed that the obligate anaerobic bacteria were replicating
in the necrotic/hypoxic centers of these tumors, leaving the well
oxygenated normal tissues unaffected. In an initial clinical trial
in humans using spores of Clostridia incapable of producing toxins,
most patients showed no objective regression (23). Thus, further
studies were abandoned due to the lack of clinical efficiency. More
recently, investigators have attempted to use the tumor-targeting
properties of Clostridia for the selective delivery of pro-drug
converting enzymes (106). Once germinated within the tumor, these
Clostridia destroy adjacent tumor cells through the secretion of
degradative enzymes, at the same time the host reacts to the
bacterial infection by producing cytokines that lead to the influx
of inflammatory cells. On the one hand, the inflammatory reaction
restricts bacterial growth, and on the other hand, it may also
contribute to the destruction of tumor cells (2). Anaerobic
Bifidobacteria have also been investigated and shown to colonize
tumors. In contrast to Clostridia, Bifidobacteria are
non-pathogenic bacteria found naturally in the digestive tract of
humans and other mammals and therefore may represent a safer
alternative compared to Clostridia (137). Strains of these
bacteria, which were modified to produce cytosine deaminase and the
antiangiogenic protein endostatin (103), resulted in inhibition of
angiogenesis and retardation of growth of the tumor after systemic
administration. In addition, oral administration of Bifidobacterium
longum carrying the endostatin gene was efficient in a liver tumor
model (52). However, the utility of anaerobes as anti-cancer agents
is limited by the absolute requirement for anoxic conditions, which
restrict their activity to large tumors.
[0136] However, these restrictions do not apply to facultative
anaerobes such as the gram-negative bacterium S. typhimurium. These
bacteria have the potential to colonize not only the anaerobic
necrotic parts of the tumor but also oxygenated proliferative and
quiescent tumor regions as well as metastatic lesions (49). This
promoted an extensive research on using attenuated strains of S.
typhimurium for tumor therapy. The capacity of Salmonella to
preferentially target and replicate in tumor tissue has been used
in tumor therapy. This was first demonstrated in 1997, in which
injected auxotrophic Salmonella were shown to specifically
accumulate in the malignant tissue of tumor-bearing mice (113). The
ratio of bacteria in the tumor to bacteria in normal tissues ranged
between 250:1 and 9000:1 and this specific accumulation was
accompanied by retarded tumor growth. Other studies have confirmed
these findings and further shown that attenuated Salmonella have
bacteriolytic activity (120). Although the precise molecular
mechanisms for this antitumor effect remain elusive, it has been
reported that infected tumor cells present antigens of bacterial
origin and become targets for Salmonella-specific T cells (7). It
is proposed that massive recruitment of both innate and adaptive
effector cells at the site of infection and Salmonella-induced
cross-presentation of tumor antigens contribute to the antitumor
activity (7). Furthermore, Salmonella strains have been constructed
in order to deliver therapeutic molecules such as the herpes
simplex thymidine kinase protein (113), endostatin (87) and
thrombospondin-1 (88). A safety barrier for the utilization
bacteria as systemically administered anti-cancer agents in humans
is that they often massively stimulate TNF-.alpha. induction, which
might lead to a cytokine cascade responsible for septic shock (28).
This effect is mediated by lipid A of gram-negative bacteria, which
is a component of the bacterial outer membrane. By disrupting the
msbB gene, which encodes a myristil transferase involved in the
synthesis of this lipid moiety, TNF-.alpha. induction could be
reduced without losing the tumor-targeting and tumor inhibiting
properties (91). A safe attenuated strain of S. typhimurium
VNP20009, was generated, in which the purI gene and msbB gene were
deleted and remained susceptible to antibiotics (28). In initial
clinical trials, this strain showed tumor colonization but no tumor
regression was observed (133), even when a pro-drug converting
enzyme was expressed (110). Both bacterial and tumor-related
factors have been implicated for the preferential accumulation of
Salmonella in tumors. It has been shown that chemoattractive
compounds produced by quiescent tumor cells contribute to the
preferential accumulation (78). However, the administration of
Salmonella to tumor-bearing animals also caused bacteria to
colonize normal tissues, albeit transiently and to a lesser extent.
In case of constitutive expression of therapeutic genes, this might
cause adverse side effects. The increased specificity of the growth
in tumor tissue could be recently achieved by creating
Leu/Arg-dependent auxotrophic Salmonella mutants (146, 147). These
mutants were cleared from normal tissue even in immunodeficient
mice, whereas the tumors were still colonized. Also, it would be
desirable to use regulated promoters that can be turned on either
specifically in tumors or at certain time points when the bacteria
have been cleared from normal tissues. In addition, several studies
have found that S. typhimurium purA auxotrophs are fully attenuated
and undetectable 21 days after inoculation in healthy tissue. This
is because there is an insufficient supply of unphosporylated
purines available in healthy tissues to enable growth. Therefore,
the means to improve the bacterial carrier for efficient tumor
therapy may be investigated.
[0137] Immune cells play an important role in the control of
spontaneous tumors such as melanoma that express endogenous tumor
antigens (126). Innate immune cells respond to "danger" signals,
which can be provided by growing tumors due to cell transformation
and disruption of the surrounding microenvironment. Ideally, these
signals induce inflammation, activation of innate effector cells
with anti-tumor activity and stimulation of dendritic cells (DC) to
present tumor-derived antigens and to trigger an adaptive immune
response (15). Tumors often exhibit strategies to escape this
immunosurveillance, such as exclusion of immune cells from tumor
sites, impairing antigen presentation by DCs and poor
immunogenicity due to reduced expression of MHC molecules and
co-stimulatory proteins (98). The strategies have been developed to
manipulate the innate immune responses by administration of
adjuvants, cytokines or ligands for co-stimulatory proteins
directly triggering innate immune cells (15). As the global
activation of the innate immune system often leads to toxicity, it
is desirable to combine this approach with specific targeting of
the tumor, e.g. providing effector molecules specifically at the
tumor site. In this respect, the use of bacteria as vaccine vectors
and delivery systems for therapeutic molecules represents a very
promising alternative. More than 100 years ago, William B. Coley
observed that, when patients with sarcomas developed acute
streptococcal infections, their tumors regressed due to the
stimulation of the innate immune system (29). The background of the
attenuated bacterial carrier strain and the type of mutation
selected to achieve attenuation critically affect the extent and
quality of elicited immune responses (42, 136).
[0138] The apoptosis-inducing Fas ligand (FasL) is a membrane
protein that belongs to the tumor necrosis factor family. After
binding to its receptor (Fas), it initiates an apoptotic signal in
the Fas-sensitive cells (132). This mechanism is of particular
importance for a variety of physiological and pathological
conditions, including the killing of transformed target cells by
cytotoxic T lymphocytes and natural killer cells (116). However, it
has been shown that systemic administration of recombinant FasL
induced lethal liver injury (115). TNF-related apoptosis inducing
ligand (TRAIL) is a protein that has been a focus of cancer
research since 1995, because of its ability to induce apoptosis in
cancer cells by stimulating death receptors on the cell surface,
while leaving normal cells relatively unaffected. It is believed
that TRAIL is part of the body's natural defense system against
cancer. The Examples below describe the use of attenuated S.
typhimurium with multiple improved features to deliver
tumor-specific synthesized FasL and/or TRAIL as cytotoxic and
immunostimulatory therapeutic proteins. Although initial studies
using S. typhimurium constructs provide preclinical data from
experiments with mice, the ultimate use of these delivery systems
in humans might more logically rely on human host-adapted S. typhi
and S. paratyphi A vaccine vector systems.
[0139] Attenuation of Salmonella vectors should decrease, if not
eliminate, induction of undesirable disease symptoms while
retaining immunogenicity. Such Salmonella vectors should be
sufficiently invasive and persistent and minimize unnecessary
tissue damage. However, it is difficult to achieve a balance
between these desirable safety features and immunogenicity. Many
means to attenuate Salmonella make them less able to tolerate
stresses encountered after administration. To address these
problems, strains were designed that display features of wild-type
virulent strains of Salmonella at the time of inoculation to enable
strains for effective colonization and then exhibit a regulated
delayed attenuation in vivo to preclude inducing disease symptoms
(30, 34, 82). Using live attenuated Salmonella as carriers of
homologous and heterologous antigens, the inventors and others have
developed a variety of attenuating mutations and antibiotic
resistance-free balanced-lethal plasmid stabilization systems (22,
56, 57, 109). However, biological containment systems are
recommended to address potential risks posed by the unintentional
release of these genetically modified organisms into the
environment as a subject of considerable concern (37, 84). Such
release can lead to unintentional infections and the possible
transfer of cloned genes that might represent virulence attributes
in some cases (31, 100). An approach has been to develop a
biological containment system that will allow Salmonella strains
enough time to colonize the host tissues, a requirement for
delivery of selected proteins and DNA vaccine vectors but
eventually leads to Salmonella cell death by programmed cell lysis,
thus preventing Salmonella strain persistence in vivo and spread
into the environment (82). Also, release of the synthesized
proteins or DNA vaccine vectors from the RAS strains with
programmed lysis phenotype would be much more efficient than a
non-lysis delivery system.
[0140] To significantly improve on these accomplishments,
innovative improvements in these processes may be investigated and
perfected. These efforts are described in the Examples below.
Example 1
Available Improved Means to Genetically Alter S. Typhimurium to
Display Regulated Delayed or Constitutive Synthesis of Selected
Proteins Aerobically and Anaerobically and to Diminish Toxicity of
Lipid A to Reduce the Possibility of Septic Shock
[0141] Overexpression of foreign protein by recombinant attenuated
S. Typhimurium (RAS) strains reduces colonizing ability and thus
immunogenicity. It was for this reason that Chatfield et al. (25)
proposed the use of the nirB promoter that is more active
anaerobically than aerobically in accord with a more likely in vivo
anaerobic environment. The promoter, P.sub.trc, that had been used
by the inventors (55, 74, 125) is constitutively active under most
environments but is more transcriptionally active in both anaerobic
and aerobic conditions than the nirB promoter (25). Therefore,
.DELTA.relA198::araC P.sub.BAD lacI TT (90) was generated, so that
Salmonella strains growing in the presence of arabinose should
synthesize the LacI repressor to inhibit transcription from
P.sub.trc in Salmonella until after administration when the
Salmonella strain is already colonizing internal targeted tissues.
This technology was incrementally improved to ultimately increase
expression of the lacI gene 40-fold by changing (i) the SD sequence
from AGGG to AGGA, (ii) the start codon from GTG to ATG, and (iii)
structural codons to maximize transcription efficiency in
Salmonella. In addition, an alternative P.sub.trc promoter that
lacks the operator lacO sequence was created to enable constitutive
synthesis of selected protein, if necessary, even when the lacI
gene in the host strain is expressed.
[0142] The regulated delayed lysis phenotype results in the release
of the lipid A endotoxin which is inflammatory via interaction with
TLR4 and MD2 (114), and also induces TNF-.alpha. mediated septic
shock (79). To preclude this, the .DELTA.pagP81::P.sub.lpp lpxE
deletion-insertion mutation was generated and fully evaluated. This
construction contains the lpxE gene from Francisella tularensis
that has been codon-optimized for high-level expression in
Salmonella, and the resulting strain produces the mono-phosphoryl
lipid A, which is totally non-toxic and yet is a safe adjuvant for
recruitment of innate immunity (5). Therefore, a strain with the
optimal lipid A form to enhance innate immunity and reduce septic
shock may now be constructed.
Example 2
Construction of Hyper-Invasive Strains to Enhance Delivery of
Selected Protein and DNA Vaccine Vector
[0143] One of the major mechanisms of S. typhimurium invasion of
animal hosts is to enter and traverse the epithelial monolayer
lining the intestine through microfold (M) cells (50, 69, 71, 128).
The expression of genes required for invasion of M cells is tightly
regulated by a variety of regulatory factors that are activated by
specific environmental conditions. The hilA (hyper-invasion locus)
regulator encodes an OmpR/ToxR family transcriptional regulator
that activates the expression of invasion genes in response to both
environmental and genetic regulatory factors (9, 10). The
regulation of hilA expression is a key point for controlling
expression of the invasive phenotype (8, 43, 92). To improve M cell
mediated Salmonella invasion for efficient oral administration, the
hilA promoter was replaced with an artificial
P.sub.trc.DELTA.lacO888 promoter that lacks the operator lacO
sequence to enable constitutive synthesis of HilA even when the
lacI gene in the host strain is expressed. The S. typhimurium
strain 9971 (.DELTA.P.sub.hilA::.sub.Ptrc.DELTA.lacO888 hilA) was
able to invade and replicate in human intestinal Int-407 cells (MOI
50:1) (FIGS. 1A and 1B) and colonize mouse tissues in significantly
greater numbers than the wild-type strain (FIGS. 2A, 2B and
2C).
Example 3
Construction of S. Typhimurium Vaccine Strains with Regulated
Expression of Genes for the Synthesis of Essential Components of
the Peptidoglycan Enabling Regulated Delayed Lysis after
Colonization to Release Selected Proteins and DNA Vaccine Vectors
In Vivo and Confer Attenuation and Complete Biological
Containment
[0144] To eliminate use of plasmid vectors with non-permitted drug
resistance genes and to stabilize plasmid vectors in recombinant
attenuated Salmonella strains in vivo, a balanced-lethal Salmonella
host-vector system with deletion of the asdA gene to impose an
obligate requirement for diaminopimelic acid (DAP) was developed
(13), and a plasmid vector with the wild-type asdA gene (32, 55).
The murA gene encodes the first enzyme in muramic acid synthesis
(18). DAP and muramic acid are essential unique constituents of
peptidoglycan. The asdA and murA systems were combined, providing
redundant mechanisms to ensure cell death. A regulated delayed
lysis system was devised for antigen delivery after colonization of
host lymphoid tissues that relies on using a more tightly regulated
araC P.sub.BAD activator-promoter (82) than the original sequence
from E. coli B/r (60) for the arabinose-dependant synthesis of the
Asd and MurA enzymes. This system is composed of two parts. The
first component is the S. typhimurium strain 8937
(.DELTA.asdA19::TT araC P.sub.BAD c2 TT .DELTA.P.sub.murA7::TT araC
P.sub.BAD murA .DELTA.(gmd-fcl)-26 .DELTA.relA1123 .DELTA.endA2311)
with deletion of the asdA gene, arabinose-regulated expression of
murA and additional mutations to enhance complete lysis and antigen
delivery. Unlike strains with asdA deletions, which can be grown by
addition of DAP to the growth medium, strains with murA deletions
are lethal due to an inability to supply the phosphorylated
substrate for the MurA enzyme. Therefore, a conditional-lethal
arabinose-dependant murA mutation was created by replacing the
chromosomal murA promoter with the araC P.sub.BAD
activator-promoter. Although arabinose is present in plant foods,
most is in a complex form unavailable to support growth of strains
with .DELTA.P.sub.muraA7::TT araC P.sub.BAD murA deletion-insertion
mutations. Thus a strain with the .DELTA.P.sub.murA7::TT araC
P.sub.BAD murA mutation undergoes about two cell divisions and then
commences to lyse in media without arabinose (82). The A
(gmd-fcl)-26 mutation deletes genes encoding enzymes for GDP-fucose
synthesis, thereby precluding the formation of colanic acid, a
polysaccharide made in response to stress associated with cell wall
damage (141). This mutation was included because it was observed
that under some conditions, asdA mutants can survive if they
produce copious amounts of colonic acid (33). Therefore, by
deleting the genes required for colanic acid synthesis, this
possibility was circumvented. The .DELTA.relA1123 mutation
uncouples cell wall-less death from dependence on protein synthesis
to further ensure that the bacteria do not survive in vivo or after
excretion and to allow for maximum antigen production in the face
of amino acid starvation resulting from a lack of aspartate
semi-aldehyde synthesis due to the asdA mutation (38, 75). The
second component is plasmid pYA3681, which encodes
arabinose-regulated murA and asdA expression and C2-regulated
synthesis of anti-sense asdA and murA mRNA transcribed from the P22
P.sub.R promoter with opposite polarity at the 3' end of the asdA
gene. The mRNA translation efficiency was reduced for both the murA
and asdA genes by changing their start codons from ATG to GTG. An
arabinose-regulated c2 gene is present in the chromosome due to the
.DELTA.asdA19::TT araC P.sub.BAD c2 TT deletion-insertion. The
cloning of a sequence encoding a protective antigen is under
P.sub.trc control. Transcription terminators (TT) flank all of the
domains for controlling lysis, replication and expression so that
expression of a function in one domain does not affect the
activities of another domain. As a safety feature, the plasmid asdA
and murA gene sequences cannot replace the chromosomal asdA and
murA mutations. .chi.8937(pYA3681) exhibits arabinose-dependent
growth. Upon invasion of host tissues, an arabinose-free
environment, transcription of asdA, murA and c2 ceases and
concentrations of their gene products decrease due to cell
division. The drop in C2 concentration results in activation of
P.sub.R, driving synthesis of anti-sense mRNA to block translation
of any residual asdA and murA mRNA (82). This host-vector grows in
LB broth with 0.2% L-arabinose as well as the wild-type strain
.chi.3761, but is unable to grow in or on media devoid of arabinose
since it undergoes cell wall-less death by lysis (82). Vaccine
strains with this regulated lysis system are totally avirulent at
oral doses in excess of 10.sup.9 CFU to BALB/c mice and, by release
of a bolus of protective antigen upon lysis, induce very good
immune responses (82). These Salmonella host-vector systems are
ideal for delivery of selected proteins that are difficult to
secrete due to structural attributes. In addition, they provide
complete biological containment with no persistence in vivo and no
survival if excreted (82).
[0145] The regulated lysis phenotype commences as the products of
arabinose-regulated genes are diluted at each cell division. Onset
of programmed lysis can be delayed about one cell division by
including the .DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
mutation, which initially prevents breakdown of accumulated
arabinose at `the time of inoculation but later allows breakdown of
residual arabinose to reduce likelihood of expressing any araC
P.sub.BAD regulated genes. This mutation also prevents acid
production by metabolism of arabinose that must be included in the
growth medium for Salmonella strains exhibiting the regulated lysis
phenotype. This is important to maximize generation of an invasive
phenotype during growth of Salmonella strains. It should be noted
that the -10, SD and C2 repressor binding sites have been altered
so that as C2 decreases the araBAD genes are expressed at a higher
level than in wild-type strains. We have also constructed the
much-improved .DELTA.asdA27::TT araC P.sub.BAD c2 and
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA deletion-insertion
mutations that share the tightly-regulated araC P.sub.BAD cassette
and a better spacing of the regulatory sequences was included in
.DELTA.P.sub.murA mutation.
Example 4
Construction of S. Typhimurium Strains with Regulated Lysis
Phenotype to Release Improved DNA Vector, with Enhanced Plasmid
Nuclear Import and Resistance to Attack from Mammalian
Nucleases
[0146] A second regulated delayed lysis host-vector system was
devised that harbors DNA vaccine vector pYA3650 with the same
regulatory domain that contributes to the lysis phenotype encoded
on pYA3681 but with a eukaryotic expression promoter. The S.
typhimurium host strain .chi.8888 (.DELTA.asdA19::TT araC P.sub.BAD
c2 TT .DELTA.P.sub.murA7:TT araC P.sub.BAD murA,
.DELTA.(gmd-fcl)-26, .DELTA.relA1123, .DELTA.endA2311,
.DELTA.araBAD1923 .DELTA.araE25) includes a .DELTA.endA mutation to
eliminate the periplasmic endonuclease I enzyme (41) to increase
plasmid survival upon its release into host cells. .DELTA.araBAD
and .DELTA.araE mutations were also included to block arabinose
catabolism with production of acid during vaccine growth and enable
efficient and rapid breakdown of arabinose encountered in vivo or
in intestinal contents. Other attributes of the regulated delayed
lysis system are described above. It should be emphasized, that all
DNA vaccine host-vector constructions derived from .chi.8888 with
pYA3650 are strictly arabinose-dependent for growth in liquid or on
solid media. Although use of non-viral DNA vaccine vectors offers
advantages, such as decreasing inflammatory responses, gene
expression in vivo remains much lower than observed with their
viral counterparts. One reason for such low expression is that
bacterial plasmids, unlike many viruses, have not evolved
mechanisms to target the nucleus in non-dividing cells and make use
of the cell's protein synthesis machinery to produce the antigen of
interest (19, 21, 107). Plasmid nuclear import is dependent on DNA
nuclear targeting sequences (DTS) (39, 40) several of which have
been identified (135). The DTS frequently contain transcription
factor binding sites, which allow transcription factors to bind to
the plasmid in the cytoplasm and escort it to the nucleus by the
nuclear localization signal-mediated machinery. The SV40 enhancer,
which is known to bind to over 10 distinct transcription factors,
is an excellent DTS (12). The minimum requirement for this function
is a single copy of a 72-bp element of the SV40 enhancer, in
combination with the CMV immediate-early gene enhancer/promoter
(CMV E/P) (4). Nuclease degradation of DNA vaccine vectors after
delivery and during trafficking to the nucleus is another barrier
that leads to inefficient DNA vaccination. Homopurine-rich tracts
in the bovine growth hormone polyadenylation signal (BGH poly A)
were identified as labile sequences, and replacement of BGH poly A
with SV40 late poly A has improved resistance to attack from
mammalian nucleases (24, 117). To increase the efficiency of the
DNA vaccine vector system, the 72 bp DTS (I) of the SV40 enhancer
was inserted into pYA3650 and also replaced the BGH poly A with the
SV40 late poly A resulting in pYA4050. The synthesis of eukaryotic
transcription factors, e.g., NF-.kappa.B and .DELTA.P-2, are
stimulated by Salmonella infection (25, 129, 139). Newly
synthesized transcription factors can bind to non-viral DNA vaccine
vectors in the cytoplasm, allowing the nuclear locating signal to
mediate import of plasmid DNA into the nucleus. Depending on the
position of these transcription factor binding sites relative to
the trans-gene, the binding sites could also act as transcriptional
enhancers that further increase gene expression levels. Therefore,
artificial DNA binding sites for NF-.kappa.B and AP-2 (SEQ ID
NO:1--GGGGACTTTCCGGGGACTTTCCTCCCCACGCGGGGGACT
TTCCGCCACGGGCGGGGACTTTCCGGGGACTTTCC) were designed and inserted
them upstream of CMV E/P in pYA4050 as a DNA nuclear targeting and
enhancer sequence to yield the improved DNA vaccine vector pYA4545
(FIG. 3B). The plasmid pYA4545 allows rapid nuclear import and
high-level synthesis of the enhanced green fluorescent proteins
(EGFP) in multiple tested mammalian cells (FIG. 4).
Example 5
The Roles of TIR-Like Protein A (TlpA), Deubiquitinase (SseL) and a
Member of the YopJ/Avr Family of Proteins (AvrA) in
Salmonella-Induced Host Cell Apoptosis
[0147] Invasive Salmonella induces pyroptosis/apoptosis in a
fraction of infected macrophages (46, 93). Macrophages infected by
Salmonella triggers caspase-1-dependent proinflammatory programmed
cell death, i.e., a recently recognized process termed pyroptosis,
which is distinguished from other forms of cellular demise by its
unique mechanism, features and inflammatory outcome (17, 45, 63,
102). Salmonella strains harboring mutations in the genes encoding
the SPI-1 T3SS, including invA, invG, invJ, prgH, sipB, sipC, sipD
and spaO, are not cytotoxic (27, 70, 97, 108). Salmonella
enteritidis gene tlpA (for (TIR)-like protein A) is predicted to
encode a protein with homology to the ToII/interleukin-1 receptor
(TIR) domain of the mammalian ToII like receptors (TLRs) (53, 83,
111). Like many important bacterial virulence factors, TlpA also
acts as mimics of mammalian proteins to subvert normal host cell
processes. As analogous to the previously characterized SipB
protein of S. typhimurium, TlpA promotes activation of the protease
caspase-1, resulting in caspase-dependent secretion of IL-1.beta.
and host cell apoptosis. Salmonella deubiquitinase (SseL) is
required for Salmonella-induced cytotoxicity of macrophages.
Salmonella sseL mutant strains did not show a replication defect or
induce altered levels of cytokine production upon infection of
macrophages, but were defective for the delayed cytotoxic effect.
Salmonella AvrA effector presumably involved in the metabolism of
ubiquitin or related molecules, have evolved to inhibit the
anti-apoptotic NF-.kappa.B pathway. Taken together, TlpA, SseL and
AvrA effectors are directly involved in inducing apoptosis of host
cells infected by Salmonella, it is hypothesized that release of
over-synthesized TlpA, SseL and AvrA effectors by a S. typhimurium
strain with the regulated lysis phenotype after the strain
accumulated in tumor tissue, could be a potential means to further
enhance inducing apoptosis in tumor cells.
Materials and Methods for Examples 6-8
Bacterial Strains, Media and Bacterial Growth.
[0148] All strains are derived from the S. typhimurium strain UK-1
(35). Defined deletion mutations with and without specific
insertions are described in the following sections. These genetic
constructions can be introduced into any strain using suicide
vectors, transduction and novel allele replacement methods
previously described (73, 127). LB broth and agar (94) are used as
complex media for propagation and plating of bacteria. MacConkey
agar with 0.5% lactose (Lac) and arabinose (Ara) will be used to
enumerate bacteria from mice. Bacterial growth is monitored
spectrophotometrically and/or by plating.
Molecular and Genetic Procedures.
[0149] Methods for DNA isolation, restriction enzyme digestion, DNA
cloning and use of PCR for construction and verification of vectors
are standard (121). E. coli K-12 strain 6212 was used for initial
cloning. DNA sequence analysis may be performed at nominal charge
in the DNA Sequence Laboratory in the School of Life Sciences. All
oligonucleotide and/or gene segment syntheses may be done
commercially. Phage P22HTint (123, 124) may be used to transduce
mutations of a selectable phenotype from one S. typhimurium strain
into other strains. Conjugational transfer of suicide vectors may
be performed by standard methods (104, 118) using the suicide
vector donor strain .chi.7213. Plasmid constructs may be evaluated
by DNA sequencing, ability to complement various S. typhimurium
mutant strains and for ability to specify synthesis of proteins
using gel electrophoresis and western blot analyses.
Strain Characterization.
[0150] Multiple gene modifications are routinely included in the
strains, and complete biochemical and genetic characterizations are
performed after every step in strain construction for stability of
plasmid maintenance, integrity and selected protein synthesis
ability when strains are grown in the presence of arabinose and/or
DAP over a 50 generation period. Moreover, an LPS gel is run to
make sure rough variants are not selected (64). Multiple mutant
strains therefore grow at almost the same rate and to the same
density as wild-type parental strains when grown under permissive
conditions. With many regulated functions, it is critical that
strains commence to synthesize selected protein and often deliver
them prior to cell lysis. Strains synthesizing GFP have been used
to monitor these events. So far, selected protein synthesis
commences several divisions before lysis commences. Engineered
Salmonella strain stability are also evaluated, due to possible
recombinational events, and to date have detected no problems.
Motility tests and use of specific antisera for given flagellar
antigens may be used to reveal presence of flagella. Presence of
fimbrial adhesins may be assayed using agglutination of yeast and
red blood cells. Metabolic attributes of candidate vaccine strains
may be evaluated using API-20E tests.
Cell Biology.
[0151] The ability of various constructed Salmonella strains to
attach to, invade into and survive in various murine and human
epithelial and/or macrophage cell lines may be quantitated by well
established methods (36, 54) that are routinely used.
Cell Culture and Cylindroid Formation.
[0152] Human colon cancer cells, LS174T may be obtained from the
American Type Culture Collection (Manassas, Va.) and cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum (FBS) and 26 mM HEPES buffer at 37 C and 5% CO2. Cell
aggregates may be grown in tissue culture flasks coated with 20
mg/ml poly (2-hydroxyethyl methacrylate) for 9 days to form
spheroids. Formation of tumor cylindroids may be done as described
by Kasinskas (78). Briefly, cylindroids may be formed by
constraining spheroids between the bottom surface of a 96-well
plate and the top surface of a set of polycarbonate cylindrical
plugs attached to a polycarbonate lid. The diameter of each
cylindroid is dependent on the initial size of the spheroid used in
its formation. Spheroids ranging from 150 to 1,000 .mu.m in
diameter will be selected based on their size, symmetry, and
overall integrity. After being constrained, cylindroids were
allowed to equilibrate for 22 h in 100 .mu.L DMEM to relieve
mechanical stress and establish oxygen and metabolic gradients
before subjection to further experimentation (78).
Establish LS174T Cell Line Stably over Expressing Red Fluorescence
Protein RFP.
[0153] RFP open reading frame may be inserted into plasmid
pSELECT-puro-MCS to result in plasmid pSELECT-puro-RFP. This
plasmid may be transfected into LS174T cells. After 2 days of
culture, cells may be selected by addition of puromycin to the
culture medium. Subsequently, puromycin-resistant cells may be
cloned into sublines expressing RFP, designated as LS174T/RFP
cells.
Establish a Real-Time Whole-Body Imaging of an Orthotopic Colon
Cancer Model Stably.
[0154] All animal experiments may be performed according to the
National Institutes of Health Guide for Care and Use of
Experimental Animals with approve by the Animal Care Committee of
Arizona State University. Male BALB/c six to eight-week old mice
may be used in the study. Although mouse subcutaneous tumor models
are easy to establish and monitor, it is clear that this model
cannot replicate the original anatomic site of colorectal cancer.
Due to the difference in microenvironment, colorectal cancer cells
growing under the skin have been shown to change their phenotype
and almost always fail to progress and metastasize (62, 81). In
fact, tumor response to therapy can vary dramatically depending on
whether cancer cells are implanted in an ectopic (subcutaneous)
versus orthotopic location (142). Orthotopic mouse models of
colorectal cancer, which feature cancer cells growing in their
natural location, replicate human disease with high fidelity (134).
To establish an orthotopic mouse model, subconfluent cultures of
LS174T/RFP cells may be harvested by treatment with 0.25% trypsin
and 1 mM EDTA-4Na in Hank's balanced salt solution (HBSS), washed
and suspended at a density of 2.times.10.sup.8 cells/ml in DMEM.
The single cell suspension of LS174T/RFP (1.times.10.sup.7
cells/100 .mu.l/mouse) may be injected into the mouse cecal wall
(134). Tumor-bearing mice (after tumors reach a size that is
clearly visible) may be used to monitor and measure the
accumulation and anti-tumor activity of the RAS strains in
vivo.
Statistical Analysis.
[0155] Experiments may be performed three times and the data may be
presented as mean.+-.SD. Student's t-test will be carried out to
assess the statistical difference. P<0.05 may be considered to
be significant.
Example 6
To Construct and Characterize Recombinant Attenuated S. Typhimurium
(RAS) Strains that are Hyper-Invasive, Allow Constitutive
Over-Synthesis of Serine and Aspartate Chemoreceptors to Maximize
Salmonella Localization in Tumor Quiescence, Display Regulated
Delayed Protein Synthesis Attributes to Facilitate Maximal
Colonization of Tumor Tissues and Exhibit Regulated Delayed Lysis
Phenotype
Introduction.
[0156] The starting S. typhimurium delivery strain has the genotype
.DELTA.asdA27::TT araC P.sub.BAD c2 .DELTA.P.sub.murA25::TT araC
P.sub.BAD murA (to enable regulated delayed lysis),
.DELTA.(wza-wcaM)-8 (to block synthesis of colanic acid that can
enable survival of bacteria undergoing cell wall-less death),
.DELTA.relA198::araC P.sub.BAD lacI TT (to enable regulated delayed
synthesis of protective antigens), .DELTA.(araC P.sub.BAD)-18::P22
P.sub.R araBAD (to block arabinose catabolism with production of
acid during vaccine growth and enable efficient and rapid breakdown
of arabinose encountered in vivo or in intestinal contents),
.DELTA.pagP81::P.sub.lpp lpxE (to diminish toxicity of lipid A) and
.DELTA.endA2311 (to reduce destruction of DNA vaccines upon their
release during lysis by eliminating endonuclease). Our core
genotype includes the mutations of .DELTA.asdA27::TT araC P.sub.BAD
c2 and .DELTA.P.sub.murA25::TT araC P.sub.BAD murA. The
much-improved .DELTA.P.sub.murA25::TT araC P.sub.BAD murA
deletion-insertion mutation has a tightly-regulated araC P.sub.BAD
cassette and a better spacing of the regulatory sequences. A strain
with this deletion-insertion mutation when grown in medium with
0.05 percent arabinose produces the MurA enzyme at the same level
as the wild-type S. typhimurium UK-1 strain.
[0157] The effectiveness of most chemotherapeutics is limited by
their inability to deeply penetrate into tumor tissue and their
ineffectiveness against quiescent cells (80, 105). Motile S.
typhimurium, which are specifically attracted to compounds produced
by quiescent cancer cells, could overcome this therapeutic barrier
(67, 113). S. typhimurium accumulate within the necrotic regions of
tumors formed both in vitro and in vivo, and chemotaxis is
essential to initiate bacterial accumulation (49, 78). There are
five chemotaxis-specific transmembrane receptors in S. typhimurium
(16), four of which bind specific chemical ligands including
aspartate/maltose, serine, citrate, and ribose/galactose (14, 66,
143). It has been shown that chemoreceptors direct bacterial
chemotaxis within cylindroids: the aspartate and maltose receptor
(Tar) initiates chemotaxis toward cylindroids, the serine receptor
(Tsr) initiates penetration, and the ribose/galactose receptor
(Trg) directs S. typhimurium toward necrosis (77). By deleting the
ribose/galactose receptor Trg, bacterial accumulation took place in
locations to tumor quiescence, and had a greater individual effect
on inducing apoptosis than a wild-type strain (77). A better means
of down regulating the trg gene in vivo is to make a
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg construction that will
result in cessation of Trg synthesis in vivo with its level
decreasing by half after every cell division. Furthermore,
overexpression of tar results in longer response to aspartate and
maltose (122). The role of chemoreceptors in enhancing the
accumulation of S. typhimurium within quiescent cells in tumor may
be explored by up-regulating expression of the Tar and Tsr
chemoreceptors.
Construction of the RAS Strain that Preferably Localize in Tumor
Tissue.
[0158] It is hypothesized that up-regulating the synthesis of the
Tar and Tsr chemoreceptors could enhance the accumulation of the
Salmonella in tumor quiescence. Therefore, the promoters of tar and
tsr genes of strain .chi.11409 were replaced, respectively, with
the P.sub.trc.DELTA.lacO888 promoter that lacks the operator lacO
sequence to enable constitutive synthesis of Tar and Tsr even when
the lacI gene in the host strain is expressed. We have constructed
suicide vectors pYA4946 (pRE112 based suicide vector for
construction of .DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
deletion-insertion mutation) and pYA4947 (pRE112 based suicide
vector for construction of
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr deletion-insertion
mutation) (FIG. 5 and FIG. 6). The
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar and
.DELTA.P.sub.trsr::P.sub.trc.DELTA.lacO888 tsr deletion-insertion
mutations were introduced into S. typhimurium UK-1 wild-type strain
.chi.3761, resulting in strains .chi.11371
(.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar) and .chi.11372
(.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr). The
over-expressions of Tar in strain .chi.11371 and Tsr in strain
.chi.11372 were confirmed by western blot analysis using mouse
anti-Flag tag and mouse anti-c-Myc tag, respectively.
Goat-anti-mouse IgG antisera served as secondary antibody (FIG. 7
and FIG. 8). The chemotaxis assays were carried out, and strain
.chi.11371 was significantly attracted by 50 .mu.m and 100 .mu.m
aspartate on swarm plate comparing with its parent S. typhimurium
UK-1 wild-type strain .chi.3761 (FIGS. 9A, 9B and 9C). Furthermore,
strain .chi.11372 was significantly attracted by 10 .mu.m serine on
swarm plate (FIGS. 10A, 10B and 10C). These two mutations were also
introduced into regulated delayed lysis strain .chi.11283,
resulting in strains .chi.11374 (.DELTA.asdA27::TT araC P.sub.BAD
c2 .DELTA.(araC P.sub.BAD)-5::P22 P.sub.R araBAD
.DELTA.(wza-wcaM)-8 .DELTA.pmi-2426 .DELTA.relA198::araC P.sub.BAD
lacI TT .DELTA.P.sub.MurA25::TT araC P.sub.BAD murA
.DELTA.pagP81::P.sub.lpp lpxE
.DELTA.P.sub.Tar::P.sub.trc.DELTA.lacO888 tar) and .chi.11375
(.DELTA.asdA27::TT araC P.sub.BAD c2 .DELTA.(araC P.sub.BAD)-5::P22
P.sub.R araBAD .DELTA.(wza-wcaM)-8 .DELTA.pmi-2426
.DELTA.relA198-::araC P.sub.BAD lacI TT .DELTA.P.sub.murA25::TT
araC P.sub.BAD murA .DELTA.pagP81::P.sub.lpp lpxE
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr). The
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar and
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr deletion-insertion
mutations were also introduced into strain .chi.11409. The
resulting strains were strain .chi.11410 (.DELTA.P.sub.murA25::TT
araC P.sub.BAD murA .DELTA.(wza-wcaM)-8 .DELTA.relA198::araC
P.sub.BAD lacI TT .DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.endA2311
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar) and strain
.chi.11514 (.DELTA.P.sub.murA25::TT araC P.sub.BAD murA
.DELTA.(wza-wcaM)-8 .DELTA.relA198::araC P.sub.BAD lacI TT
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE
.DELTA.endA2311.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr).
Construction of the RAS Strain that Preferably Localizes in Tumor
Quiescence.
[0159] By deleting the ribose/galactose receptor Trg, bacterial
accumulation took place in locations to tumor quiescence, and had a
greater individual effect on inducing apoptosis than a wild-type
strain. We have constructed suicide vector pYA5077 (pRE112 based
suicide vector for construction of .DELTA.trg mutation) (FIG. 11).
The .DELTA.trg mutation was introduced into S. typhimurium UK-1
wild-type strain .chi.3761, resulting in strain .chi.11525. The
chemotaxis assays was performed, and strain .chi.11525 was not
attracted by 10 .mu.M galactose on swarm plate (FIGS. 12A and 12B).
Therefore, the mutation .DELTA.trg was included into .chi.11514.
The resulting strain was .chi.11515 (.DELTA.P.sub.murA25::TT araC
P.sub.BAD murA A(wza-wcaM)-8 .DELTA.relA198::araC P.sub.BAD lacI TT
.DELTA.(araC P.sub.BAD)-18::P22 PR araBAD .DELTA.pagP81::P.sub.lpp
lpxE .DELTA.endA2311.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 trs .DELTA.trg).
Furthermore, a better means of down regulating the trg gene in vivo
is to make a construction that will result in cessation of Trg
synthesis in vivo with its level decreasing by half after every
cell division. In this regard, the mutation .DELTA.trg was replaced
with mutation-insertion .DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg. The
resulting strain was .chi.11516 (.DELTA.P.sub.murA25::TT araC
P.sub.BAD murA .DELTA.(wza-wcaM)-8 .DELTA.relA198::araC P.sub.BAD
lacI TT .DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.endA2311
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg).
Construction of the S. Typhimurium Strain that Displays
Super-Invasive Phenotype.
[0160] It is evident that an engineered S. typhimurium strain
whether delivering selected protein or a DNA vaccine vector will
likely be improved by increasing its invasiveness. Therefore, the
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA deletion-insertion
mutation was included into strain .chi.11515 or strain .chi.11516
to result in strain .chi.11517 (.DELTA.asdA27::TT araC P.sub.BAD c2
.DELTA.P.sub.murA25::TT araC P.sub.BAD murA .DELTA.(wza-wcaM)-8
.DELTA.relA198::araC P.sub.BAD lacI TT .DELTA.(araC
P.sub.BAD)-18::P22 P.sub.R araBAD .DELTA.pagP81::P.sub.lpp lpxE
.DELTA.endA2311 .DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr .DELTA.trg or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA).
Construction of the RAS .DELTA.purA Auxotrophs Strain to
Selectively Colonize in the Necrotized Tumor Tissue.
[0161] The possibility of utilizing S. typhimurium auxotrophs has
been considered for selective growth in tumors, since the bacteria
would be both attenuated for use in vivo and preferentially survive
in the necrotized tissue in and around tumors, utilizing the cell
lysate for its own needs. The .DELTA.purA mutation was introduced
into strain .chi.11517 to result in strain .chi.11518
(.DELTA.asdA27::TT araC P.sub.BAD c2 .DELTA.PmurA25::TT araC
P.sub.BAD murA .DELTA.(wza-wcaM)-8 .DELTA.relA198::araC P.sub.BAD
lacI TT .DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.endA2311
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr .DELTA.trg or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA .DELTA.purA). We
have also constructed balanced-lethal vector-host system strain
.chi.11203 (.DELTA.asdA .DELTA.purA) and regulated delayed lysis
system strain .chi.11204 (.DELTA.asdA::TT araC P.sub.BAD c2
.DELTA.murA::TT araC P.sub.BAD murA .DELTA.(gmd-fcl)
.DELTA.relA::araC P.sub.BAD lacI TT .DELTA.pmi .DELTA.araBAD
.DELTA.purA) to test the effects of .DELTA.purA mutation on
selective colonization in the necrotized tumor tissue. The plasmids
pYA4545 or pYA4545-GFP (DNA vaccine vector pYA4545 harboring
prokaryotic expressing green fluorescent protein (GFP)) may then be
transformed into the RAS strains .chi.11409, .chi.11410,
.chi.11514, .chi.11515, .chi.11516, .chi.11517, and .chi.11518,
respectively, to test the efforts of these modifications.
Capillary Assay to Quantify Bacterial Chemotaxis.
[0162] The ability of the RAS strains to migrate toward
chemoattractant molecules may be quantified using the
needle-syringe capillary assay (99). Briefly, the RAS strains
.chi.11409 (pYA4545), .chi.11410 (pYA4545), .chi.11514 (pYA4545),
.chi.11515 (pYA4545), .chi.11516 (pYA4545), .chi.11517 (pYA4545),
and .chi.11518 (pYA4545) may be grown to mid-logarithmic phase,
centrifuged, washed, and suspended in motility buffer to a final
concentration of 3.2.times.10.sup.7 CFU/ml bacterial (1, 102).
Hypodermic needles (25 gauge) attached to 1 ml syringes may be
filled with 0.1 ml of chemoattractant solution containing 0.1 mM
serine or 1 mM aspartate. The needle-syringe assemblies may be
inserted into 200 .mu.l pipette tips containing the bacterial
suspension and incubated at 35.degree. C. for 1 h. After
incubation, the content of the needles may be removed, diluted, and
plated to quantify the bacterial numbers (CFU). Chemotactic ability
may be reported as the ratio of the average number of bacteria that
accumulated in the chemoattractant capillaries to the average
number of bacteria that accumulated in the chemoattractant-free
controls.
Accumulation of Bacteria in Cylindroids, Image Acquisition and
Analysis.
[0163] Before inoculation into cylindroid cultures (See Materials
and Methods section), the RAS strains .chi.11409 (pYA4545-GFP),
.chi.11410 (pYA4545-GFP), .chi.11514 (pYA4545-GFP), .chi.11515
(pYA4545-GFP), .chi.11516 (pYA4545-GFP), .chi.11517 (pYA4545-GFP),
and .chi.11518 (pYA4545-GFP) may be grown at 37.degree. C. to
mid-logarithmic phase (OD.sub.600 0.3-0.5) from single colony
cultures. Individual colonies may be chosen from agar plates
following confirmation of GFP expression using fluorescence
microscopy. These bacterial cultures may be centrifuged at 4,000
rpm for 10 min and resuspended in DMEM with 10% FBS and 26 mM HEPES
buffer to a final concentration of 500 CFU/ml. Equilibrated
cylindroid cultures may be inoculated with 100 .mu.l of 500 CFU/ml
S. typhimurium. Time-lapse fluorescent images may be acquired at
10-min intervals up to 34 h after inoculation using time-lapse
microscopy (Nikon Eclipse TE300 Inverted Microscope). Excitation
light may be shuttered between acquisitions to prevent
photobleaching. To test the influence of aspartate and serine on
the accumulation of RAS strains .chi.11409 (pYA4545-GFP),
.chi.11410 (pYA4545-GFP), .chi.11514 (pYA4545-GFP), .chi.11515
(pYA4545-GFP), .chi.11516 (pYA4545-GFP), .chi.11517 (pYA4545-GFP),
and .chi.11518 (pYA4545-GFP), cylindroids may be prepared as
described above, except cylindroids may be equilibrated in the
medium containing 1 and/or 5 mM of added aspartate or serine.
Bacteria added to the cylindroids may be suspended in medium
containing corresponding concentrations (1 and/or 5 mM) of
aspartate or serine. The accumulation of bacteria and fluorescent
dyes in cylindroids may be quantified as described by Kasinskas
(78).
Analysis of Bacteria Fitness in Normal Tissue versus Tumor Tissue
in LS174T Tumor Orthotopic Mice.
[0164] The LD.sub.50 of the RAS strains .chi.1409 (pYA4545),
.chi.11410 (pYA4545), .chi.11514 (pYA4545), .chi.11515 (pYA4545),
.chi.11516 (pYA4545), .chi.11517 (pYA4545), and .chi.11518
(pYA4545) may be determined in BALB/c mice and their abilities of
colonizating mouse Peyer's patches, spleen and liver monitored.
Then the accumulation of each strain in tumor versus normal tissue
in LS174T tumor orthotopic mice will be monitored post i.v. or oral
inoculation. The strain with the best attributes will be named as
Strain H.
Discussion.
[0165] (i) .DELTA.P.sub.hilA::P.sub.hilA256 hilA deletion-insertion
mutation in S. typhimurium UK-1 strain was also created, in that
both upstream and downstream AT-track sequences of hilA promoter
region recognized by the nucleoid-associated protein H-NS to
silence hilA gene expression, were deleted to construct strain
.chi.9974. Strain .chi.9974 is more invasive than wild-type S.
typhimurium UK-1, but less than strain .chi.9971
(.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA). If Strain A,
that has .DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA mutation,
induces unexpected pro-inflammatory response, the
.DELTA.P.sub.hilA::P.sub.hilA256 hilA mutation may be considered
instead. (ii) the expression levels of the selected chemoreceptors
may always be modulated by changing the 2nd and 3rd codon (altering
translational efficiency of mRNA), and by altering codons in the
chemoreceptor genes (in order to optimize for a high-level
expression in Salmonella). These modifications may be included if
needed and these constructs may be evaluated to establish the final
RAS strain.
Example 7
To Construct and Characterize the Improved RAS Strains with
Regulated Delayed Synthesis of S. Typhimurium T3SS Effectors SopE2
and/or SopB that Stimulate Innate Immune Responses, and to Explore
the Means that Provoke the Human Immune System
Introduction.
[0166] The human immune system naturally grows stronger while
fighting bacteria, including Salmonella. The potential stimulation
of innate immune responses by the ideal bacterial vector to provoke
the human immune system may be investigated. It is widely believed
that one of the main triggers of host inflammation is the
recognition of microbial products by receptors of the innate immune
system (3, 11, 76). Intestinal epithelial cells, however, are a
special case in that they are exposed to massive amounts of
bacterial products potentially able to activate innate immune
receptors. Therefore, signaling through these receptors,
particularly surface TLRs, must be prevented from uncontrolled
inflammation, which would be detrimental to the host. How this
negative regulation of innate immune receptor activation is exerted
remains poorly understood. However, it recently has been shown that
S. typhimurium can stimulate innate immune responses in cultured
epithelial cells through the activity of bacterial effector
proteins, such as the guanidyl nucleotide exchange factor SopE2 and
an inositol polyphosphatase SopB, which are delivered by its T3SS
in a manner independent of innate immune receptors (20). SopE2 and
SopB are good candidates to mediate the innate immune responses
since they activate Rho-family GTPases in a functionally redundant
manner (51, 112, 148). Rho GTPases are important regulators of gene
transcription and cytokine expression in infection. One role of Rho
proteins in the signaling networks is to activate nuclear factor KB
(NF-.kappa.B), which is a central regulator of innate and adaptive
immunity. Activation of NF-.kappa.B results in expression of many
inflammatory and anti-apoptotic factors, and modulation of diverse
immune responses (47, 58).
Construction of the S. Typhimurium Strain Exhibiting Regulated
Delayed Synthesis of S. Typhimurium T3SS Effector SopE2.
[0167] To modulate production of the immune stimulants in RAS
strains, the promoter of the sopE2 gene in the RAS strain H or its
derivatives may be replaced with the P.sub.trc promoter to enable
the regulated delayed expression of the sopE2 gene facilitating a
delayed stimulation of the immune system. This may avoid an
unexpected level of pro-inflammatory responses before Salmonella
colonization since growth of the RAS strain in LB broth with 0.2%
arabinose causes synthesis of LacI due to the .DELTA.relA198::araC
P.sub.BAD lacI TT deletion-insertion mutation. We have constructed
suicide vector pYA4948 (pRE112 based suicide vector for
construction of .DELTA.P.sub.sopE2::P.sub.trc sopE2
deletion-insertion mutation) (FIG. 13). The
.DELTA.P.sub.sopE2::P.sub.trc sopE2 deletion-insertion mutation was
introduced into S. typhimurium UK-1 wild-type strain .chi.3761,
resulting strain .chi.11376 (.DELTA.P.sub.sopE2::P.sub.trc sopE2).
This mutation was also introduced into regulated delayed lysis
strain .chi.11283, resulting strains .chi.11376 (.DELTA.asdA27::TT
araC P.sub.BAD c2 .DELTA.(araC P.sub.BAD)-5::P22 P.sub.R araBAD
.DELTA.(wza-wcaM)-8 .DELTA.pmi-2426 .DELTA.relA198::araC P.sub.BAD
lacI TT .DELTA.P.sub.MurA25::TT araC P.sub.BAD murA
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.P.sub.sopE2::P.sub.trc sopE2).
The .DELTA.P.sub.sopE2::P.sub.trc sopE2 deletion-insertion mutation
will also be introduced into Strain H. This may result in Strain I.
the genotype of strain I will most likely be (.DELTA.asdA27::TT
araC P.sub.BAD c2 .DELTA.P.sub.murA25::TT araC P.sub.BAD murA
.DELTA.(wza-wcaM)-8 .DELTA.relA198::araC P.sub.BAD lacI TT
.DELTA.(araC P.sub.BAD)-18::P22 P.sub.R araBAD
.DELTA.pagP81::P.sub.lpp lpxE .DELTA.endA2311
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA .DELTA.purA
.DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr .DELTA.trg
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.O888 hilA .DELTA.pur A
.DELTA.P.sub.sopE2::P.sub.trc sopE2). The regulated delayed
synthesis of SopE2 will be confirmed in these strains by western
blot using anti-SopE2 antibody.
Evaluation of the Improved RAS Strain by Monitoring Innate Immune
Responses.
[0168] S. typhimurium induces innate immune responses in cultured
epithelial cells. The transcriptional program stimulated by
wild-type S. typhimurium infection that activates several genes
whose products are pro-inflammatory such as several chemokines
(Interleukin-8 (IL-8), IL1a, IL11, IL1 R1 and IL4R, etc). The
Salmonella effector SopE2 activates Rho-family GTPases, which can
lead to MAPK and NF-.kappa.B activation (26, 61, 140). Therefore,
the ability of the RAS strain over-expressing SopE2 to stimulate
innate immune responses may be investigated by examining its effect
on the expression of IL-8 and activation of p38 MAPK as described
below.
Detect the Stimulation of IL-8 Expression.
[0169] The RAS Strain H (pYA4545) and Strain I (pYA4545) may be
grown at 37.degree. C. over night in the medium with arabinose
starvation to release P.sub.trc promoter from LacI repressor. Human
intestinal Int-407 cell may be infected by the RAS Strain H
(pYA4545) or Strain I (pYA4545). Two days after infection, infected
cells may be lysed and the levels of IL-8 may be determined using
Human IL-8 ELISA Kit (BioVendor Laboratory Medicine, Inc., Brno,
CZ).
Detect the Activation of p38 MAPK by Measuring the Level of
Phospho-P38 MAPK.
[0170] The 48 h post-infected human intestinal Int-407 cells by RAS
Strain H (pYA4545) and Strain I (pYA4545) may be lysed with the
lysis buffer (10 mM Tris-HCl, pH 7.5, 40 mM Na pyrophosphate, 5 mM
EDTA, 150 mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.025% SDS, 1 mM
Na orthovanadate and complete protease inhibitor cocktail (Roche)).
Proteins from cell lysates may be separated by SDS-PAGE, and
phospho-P38 MAPK, total P38 MAPK may be examined by western
immunoblotting using mouse anti-phospho-P38 [Thr 180, Tyr 182]
(Cell Signaling Technology, Danvers, Mass.) and rabbit anti-P38,
while actin may be used as a control and detected using rabbit
anti-actin (Santa Cruz Biotechnology, Santa Cruz, Calif.).
Discussion.
[0171] If necessary, sopE2 expression level may be enhanced by
changing the sopE2 start codon from GTG to ATG. The best candidate
from these studies may be included in the RAS strain for
anti-cancer agent delivery.
Example 8
To Construct and Evaluate an Improved RAS Delivery System Allowing
Oversynthesis and Release of a Bacterial Virulence Factor
Controlled by a Salmonella Promoter Preferentially Activated inside
Tumors, and Simultaneous Release of a DNA Vaccine Vector Encoding a
Tumor-Specific Synthesized Fas Ligand and/or TRIAL to Trigger Tumor
Apoptosis
Introduction.
[0172] Invaded Salmonella can induce pyroptosis/apoptosis in a
fraction of infected macrophages. Salmonella enteritidis TIR-like
protein A (TlpA) is involved in induction of the host cell
apoptosis (Example 5). An improved RAS strain described above may
be designed and constructed to over-synthesize TlpA after they
accumulate in tumor cells. Over-synthesized TlpA may be released in
the tumor tissues by regulated cell lysis after Salmonella
colonizes to induce tumor cell apoptosis. Success of gene therapy
strategies for cancer by using genetic elements or toxic molecules
largely depends on the cancer-specific delivery and expression of
the therapeutic molecules at high level (65, 86, 138). It is known
that the promoter of S. typhimurium ansB gene (encoding periplasmic
L-asparaginase II) is preferentially activated inside tumors (6,
68). The potential of genetically engineered promoter of ansB for
tumor-specific expression of TlpA may be investigated to enhance
induction of the apoptosis in tumor cells. On the other hand, the
delayed regulated lysis system should ensure Salmonella to release
with adequate time a DNA vaccine vector in host tissues by
programmed cell lysis, thereby enhancing the probability of
efficient DNA delivery. Moreover, the improved DNA vaccine vector
pYA4545 allows a rapid nuclear import and high-level synthesis of
the encoded gene. Therefore, the potential of the improved RAS
strain that could release TlpA and simultaneously release a DNA
vaccine vector encoding death ligand Fas and/or TRAIL by the
programmed cell lysis may be explored.
Constructing the Improved RAS Strain with Overexpression of the
Salmonella Enteritidis tlpA Gene Directed by a Salmonella Promoter
Preferentially Activated Inside of Tumors.
[0173] The promoter and Shine-Dalgarno (SD) sequence of the tlpA
gene in Strain C may be replaced with P.sub.ansB promoter and a
strong SD sequence AGGA that should facilitate tumor-specific
synthesis of TlpA, and subsequently induce apoptosis in tumor
cells. The resulting strain may be Strain J (.DELTA.asdA27::TT araC
P.sub.BAD c2 .DELTA.P.sub.murA25::TT araC P.sub.BAD murA
.DELTA.(wza-wcaM)-8 .DELTA.relA 198::araC P.sub.BAD lacI TT
.DELTA.(araC PBAD)-18::P22 P.sub.R araBAD .DELTA.pagP81::P.sub.lpp
lpxE .DELTA.endA2311 .DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888
hilA .DELTA.purA .DELTA.P.sub.tar::P.sub.trc.DELTA.lacO888 tar
.DELTA.P.sub.tsr::P.sub.trc.DELTA.lacO888 tsr .DELTA.trg or
.DELTA.P.sub.trg::rhaRS-P.sub.rhaB trg
.DELTA.P.sub.hilA::P.sub.trc.DELTA.lacO888 hilA .DELTA.purA
.DELTA.P.sub.sopE2::P.sub.trc sopE2 .DELTA.P.sub.tlpA::P.sub.ansB
tlpA). It is well established that cell proliferation and death are
important in the regulation of development and homeostasis in
multicellular organisms (44, 72), and physiological cell death is
usually accomplished through apoptosis. However, uncontrolled
growth and proliferation, and blocked apoptosis are major
characteristics of cancer cells (144). The effects of RAS Strain H,
I, and J harboring a DNA vaccine vector on cell proliferation and
apoptosis in the tumor cell line, and the anti-tumor activity may
be evaluated using the colon cancer mouse model.
Constructing an Improved RAS Strain Harboring the Improved DNA
Vaccine Vector Encoding a Tumor-Specific Synthesized FasL to
Trigger Tumor Cell Death.
[0174] The anti-tumor activity of FasL is well known. However,
systemic administration of recombinant FasL appears to induce
lethal liver injury (115), making the untargeted systemic delivery
an unacceptable strategy. The use of tumor-selective promoters for
targeted gene therapy of cancer depends on their strong and
selective activities. Hexokinase type II (HK II) catalyzes the
first committed step of glycolysis, which is over-expressed in
tumors, and no longer responsive to normal physiological
inhibitors, e.g., glucagon (89). The P.sub.CMV promoter of the
improved DNA vaccine vector pYA4545 may be replaced with the HK II
gene promoter to control the synthesis of Fas ligand. The resulting
plasmid (pYA4545PHK IIFasL) may be transformed into RAS Strain H,
Strain I, and Strain J, respectively, to evaluate their anti-cancer
efficacy. The synthesis of TlpA and FasL in normal cells (human
Int-407 cells) and LS174T human colon cancer cell cultures may be
compared. These RAS strain FasL delivery systems may be fully
characterized as described in the Materials and Methods section.
The LD.sub.50 of each strain may be determined in BALB/c mice and
their abilities of colonizating mouse Peyer's patches, spleen and
liver monitored. The efficacy of these RAS strain FasL delivery
systems to reduce tumor mass may be measured in LS174T/RFP tumor
cells (human colon cancer cells, LS174T stably over expressing
RFP), and in a real-time whole-body imaging of an orthotopic colon
cancer model. The effectiveness of different Salmonella
administration routes (including intratumoral injection,
intravenous (i.v.) injection, intraperitoneal (I.P.) injection and
oral administration) on the efficacy of these RAS strains may be
specifically compared to reduce tumor mass. The most efficient
administration route may be applied to future clinical tests. We
have also constructed balanced-lethal vector harboring TRAIL
encoding gene pYA5078 and Lysis vector specifying TRAIL encoding
gene pYA5079. The vector pYA5079 was introduced into regulated
delayed lysis system strain .chi.11204 (.DELTA.asdA::TT araC
P.sub.BAD c2 .DELTA.murA::TT araC P.sub.BAD murA .DELTA.(gmd-fcl)
.DELTA.relA::araC P.sub.BAD lacI TT .DELTA.pmi .DELTA.araBAD
.DELTA.purA) to test the effects of .DELTA.purA mutation on
selective colonization in the necrotized tumor tissue. The vector
pYA5078 will also be introduced into balanced-lethal host strain to
test the effects of .DELTA.purA mutation.
Examination of Cancer Cell-Specific Expression of TlpA and FasL by
Immunostaining and Microscopic Imaging.
[0175] Human Int-407 cells and human colon cancer LS174T cells may
be infected with RAS Strain H (pYA4545), RAS strain H (pYA4545+PHK
IIFasL), RAS strain I (pYA4545+PHK IIFasL), and RAS strain J
(pYA4545+PHK IIFasL), respectively. After incubating for a proper
time, cells may be fixed with 2.0% paraformaldehyde and
permeabilized with 0.5% Triton-X in PBS. The rabbit anti-TlpA or
FasL antibody may be used as the primary antibodies, and detected
by an Alexa-488-conjugated secondary antibody (Molecular Probes,
Eugene, Oreg. USA). Infected cells may be counterstained by
4',6-diamino-2-phenylindole (DAPI), and mounted in Vectashield
mounting medium (Vector Laboratories, Burlingame, Calif., USA).
Microscopic images of cultured cells may be collected using an
inverted microscope Leica Microsystems Heidelberg Gmbh.
Anti-Tumor Efficacy Assay in Cell Culture.
[0176] i. Cell Proliferation Assay.
[0177] Incorporation of bromodeoxyuridine (Brd-U) may be examined
using a cell proliferation enzyme-linked immunosorbent assay
(ELISA) kit (Roche Diagnostics, Mannheim, Germany) by following the
manufacturer's instructions. Briefly, human Int-407 and LS174T
cells may be plated at a density of 10-104 cells per well into
96-well tissue culture plates and allowed to adhere overnight.
Cells may be infected with RAS Strain H (pYA4545), RAS Strain H
(pYA4545+PHK IIFasL), Strain I (pYA4545+PHK IIFasL), and Strain J
(pYA4545+PHK IIFasL), respectively, and may continue to be cultured
for a proper time. The infected cells may be labeled with Brd-U for
8 h at the end of culture. Each condition may be measured in
triplicate and the results may be analyzed by Student's t test.
ii. Apoptosis Assay.
[0178] Apoptosis may be characterized by a series of morphological
changes such as chromatin condensation, cell shrinkage, membrane
blebbing, packing of organelles, the formation of apoptotic bodies,
internucleosomal DNA fragmentation (48, 145). The cleavage of DNA
double strand can be visualized in a laddering pattern on agarose
gel indicates a late event and is a hallmark of apoptosis (95).
Therefore human Int-407 and LS174T cells may again be infected with
the same RAS strains as described above. DNA fragmentation assays
may be performed as described (130). The DNA solution may be
electrophoresed on 2% agarose gel. DNA fragments may be visualized
under UV light.
iii. Measure Apoptosis/Pyroptosis Induction by Cell Morphology.
[0179] The LS174T/RFP cells may be grown on glass cover slips to
about 60% confluency. Cells may be infected with the same RAS
strains as described above. Afterwards, cells may be fixed with
PBS-23.7% formaldehyde, and permeabilized with PBS-20.1% Triton
X-100. Cells may be visualized using microscope Leica Microsystems
Heidelberg Gmbh.
Anti-Tumor Efficacy Assay in Orthtopic Mouse Models.
[0180] i. Whole-Body Imaging of the Efficacy of the RAS Strains on
the Growth of a Human Colorectal Tumor.
[0181] Whole-body imaging of orthotopic colorectal tumor-bearing
mice (five mice/group) may be used for growth models and infection
studies. Uninfected healthy mice and tumor-bearing mice may be the
controls. For intratumoral injection, RAS Strain H (pYA4545), RAS
Strain H (pYA4545+PHK IIFasL), Strain I (pYA4545+PHK IIFasL), and
Strain J (pYA4545+PHK IIFasL), may be harvested at late logarithmic
phase, respectively, washed, diluted with PBS, and injected
directly into the central areas of the RFP-labeled tumors under
fluorescence guidance. A total of 100 .mu.l injection at two sites
(50 .mu.l each) and 10.sup.9 CFU per tumor may be used. For the
i.v. injection, the same strains described above may be injected
into the tail vein of RFP orthotopic tumor-bearing mice (10.sup.7
CFU per 100 .mu.l of PBS). For the I.P. injection, the same strains
listed above may be injected into the peritoneum (body cavity) of
RFP orthotopic tumor-bearing mice (10.sup.5 CFU per 100 .mu.l of
PBS). For oral administration, tumor-bearing mice may be deprived
of food and water 4 h before oral infection. The above strains may
be inoculated orally (10.sup.9 CFU per 20 .mu.l of PBS). Food and
water may be returned 30 min after infection. Whole-body
fluorescence imaging techniques May be used to track the effect on
the red fluorescent protein (RFP)-labeled target tumors using the
Lumina Imaging System IVIS-200 (Xenogen) by following instructions
of the manufacturer. Tumor size may be determined by fluorescence
imaging on days 11, 16, 21, 25, 30 and 35.
ii. Demonstrate Apoptotic Cell Death of Tumor Tissues.
[0182] Mice may be killed on Day 36 after inoculation of the RAS
strains. Specimens from the tumor tissues may be collected, fixed
in 10% neutral formaldehyde for 6 h and paraffin-embedded, and 5
.mu.m-thick consecutive sections may be sliced. To demonstrate
apoptotic cell death of tumor tissues on paraffin-embedded
sections, terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling (TUNEL) assay may be performed using In Situ Cell
Death Detection Kit (Roche Diagnostics, Basel, Switzerland)
according to the manufacturer's instructions. Positive index (PI)
may be counted from five randomly selected high-power fields under
light microscope, and expressed as a percentage of total cells
counted.
Discussion.
[0183] (i) If the activity of P.sub.ansB promoter is not high
enough to trigger expected high-level expression of the bacterial
virulence factor genes in tumor cells, P.sub.ansB promoter may be
engineered by modifying its -35 and -10 regions. The codons of the
tlpA gene may be further optimized for high-level expression in
Salmonella.
[0184] (ii) On the other hand, the plasmid stability of the DNA
vaccine vector in Salmonella during infection and the timing of
Salmonella cell lysis to release DNA vaccine vector are critical
for desired anti-tumor efficacy. If needed, the improved regulated
delayed lysis vector pYA4545 (pUC ori) derivatives may be
constructed. These vectors may have pSC101 ori, p15A ori and pBR
ori, respectively, such that plasmid stability during Salmonella
infection and the timing of lysis in vivo for release of DNA
vaccine vectors may be improved.
[0185] The success of using a multiple functional RAS host-vector
delivery system to overcome therapeutic resistance and increase
treatment efficiency may significantly reduce systemic toxicity,
limit the deleterious effects of metastatic disease, and increase
life expectancy. Future human trials using similarly genetically
modified S. typhi strains may demonstrate the ability of
administration of the multiple functional RAS host-vector
anti-cancer system to reduce local recurrence and metastatic
disease in stage-four colorectal cancer patients.
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Sequence CWU 1
1
1174DNAArtificial SequenceHOMO SAPIENS 1ggggactttc cggggacttt
cctccccacg cgggggactt tccgccacgg gcggggactt 60tccggggact ttcc
74
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