U.S. patent application number 14/882749 was filed with the patent office on 2016-08-11 for combination therapy for use in cancer therapy.
The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Yvonne PATERSON.
Application Number | 20160228530 14/882749 |
Document ID | / |
Family ID | 55747250 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160228530 |
Kind Code |
A1 |
PATERSON; Yvonne |
August 11, 2016 |
COMBINATION THERAPY FOR USE IN CANCER THERAPY
Abstract
The present invention provides methods of treating anal or
vaginal tumors and cancers, comprising the step of administering to
a subject a combination therapy comprising a chemo-radiation
therapy and a recombinant Listeria strain.
Inventors: |
PATERSON; Yvonne;
(Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
|
Family ID: |
55747250 |
Appl. No.: |
14/882749 |
Filed: |
October 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62063828 |
Oct 14, 2014 |
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62065973 |
Oct 20, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/585 20130101;
A61K 39/0011 20130101; A61K 2039/54 20130101; A61K 39/12 20130101;
A61K 2039/545 20130101; A61K 2039/523 20130101; A61K 2039/6037
20130101; A61K 31/513 20130101; A61K 2039/572 20130101; A61P 35/00
20180101; A61N 5/10 20130101; A61K 2039/522 20130101; C12N 7/00
20130101; C12N 2710/20034 20130101; C07K 16/3046 20130101; A61K
31/407 20130101; A61K 39/0011 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61N 5/10 20060101 A61N005/10; C12N 7/00 20060101
C12N007/00; A61K 31/407 20060101 A61K031/407; A61K 31/513 20060101
A61K031/513 |
Claims
1. A method of treating an anal or vaginal tumor anal or vaginal
cancer, or an anal or vaginal neoplasia in a human subject, the
method comprising the step of administering to said subject a
combination therapy comprising a chemo-radiation therapy and a
recombinant Listeria strain, said Listeria strain comprising a
recombinant nucleic acid, said nucleic acid comprising a first open
reading frame encoding a recombinant polypeptide comprising an
N-terminal fragment of an LLO protein fused to a heterologous
antigen or fragment thereof, thereby treating said anal or vaginal
tumor anal or vaginal cancer, or an anal or vaginal neoplasia in
said human subject.
2. The method of claim 1, wherein said tumor, cancer, or neoplasia
is an intraepithelial cancer or neoplasia.
3. (canceled)
4. The method of claim 1, wherein said chemo-radiation therapy is
administered following a first administration of said recombinant
Listeria strain.
5. The method of claim 1, wherein said chemo-radiation therapy is
administered prior to the administration of said recombinant
Listeria strain.
6. The method of claim 1, wherein said chemo-radiation therapy is
administered following a first administration of said recombinant
Listeria strain and prior to one to three booster administrations
of said recombinant Listeria strain.
7. The method of claim 1, wherein said chemo-radiation therapy is
administered concurrently with said recombinant Listeria
strain.
8. The method of claim 1, wherein said method comprises
administering four doses of said recombinant Listeria.
9. The method of claim 8, wherein the first dose of said
recombinant Listeria is administered prior to chemo-radiation
therapy and the 2.sup.nd-4.sup.th doses are administered every 28
days after completion of radiation.
10. The method of claim 8, wherein the first dose of said
recombinant Listeria is administered before chemo-radiation
therapy, wherein the second dose of said recombinant Listeria is
administered during chemo-radiation therapy, and wherein the
3.sup.rd-4.sup.th doses are administered every 28 days following
the completion of chemo-radiation therapy.
11. The method of claim 1, wherein said chemo-radiation therapy
comprises mitomycin and fluorouracil (5-FU) and radiation
therapy.
12. The method of claim 11, wherein said chemo-radiation therapy
comprises administering 2 courses of mitomycin, 5-FU with
concurrent radiation (54 Gy in 30 fractions by intensity modulated
radiation therapy).
13. The method of claim 11, wherein said radiation therapy lasts
about 6 weeks.
14. The method of claim 1, wherein said Listeria comprises a
mutation or deletion in the endogenous prfA gene.
15. The method of claim 14, wherein said recombinant nucleic acid
further comprises a second open reading frame encoding a mutant
prfA gene, wherein said mutant prfA gene complements the prfA
genomic mutation or deletion, thereby inducing an immune response
against said anal or vaginal tumor or anal or vaginal cancer.
16. The method of claim 15, wherein said mutant prfA gene encodes a
PrfA protein comprising a D133V mutation.
17. (canceled)
18. The method of claim 1, wherein said administering is
intravenous or oral administering.
19. The method of claim 1, wherein said N-terminal fragment of an
LLO protein comprises SEQ ID NO: 2.
20. The method of claim 1, wherein said recombinant Listeria strain
is administered to said human subject at a dose of
1.times.10.sup.9-3.31.times.10.sup.10 organisms.
21. The method of claim 1, wherein said recombinant Listeria strain
is a recombinant Listeria monocytogenes strain.
22. The method of claim 1, wherein said recombinant Listeria strain
has been passaged through an animal host, prior to the step of
administering.
23. The method of claim 1, wherein said recombinant polypeptide is
expressed by said recombinant Listeria strain.
24. The method of claim 1, wherein said recombinant Listeria strain
comprises a plasmid that encodes said recombinant polypeptide.
25. (canceled)
26. The method of claim 1, further comprising the step of
inoculating said human subject with an immunogenic composition that
comprises or directs expression of an HPV16 or HPV18 E6 or E7
antigen.
27. The method of claim 1, wherein said recombinant Listeria strain
has been stored in a frozen or lyophilized cell bank.
28. A method for inducing an anti-tumor cytotoxic T cell response
in a human subject, comprising the step of administering to said
subject the combination therapy of claim 1.
29. A method of treating a human subject against a tumor or cancer,
comprising the step of administering to said subject the
recombinant Listeria strain of claim 1.
30. The method of claim 29, wherein said administering is
intravenous or oral administering.
31. The method of claim 28, wherein said immune response comprises
increasing a level of interferon-gamma producing cells, increasing
a level of TNF-alpha producing cells, or both.
32. (canceled)
33. The method of claim 28, wherein said immune response comprises
an increase of tumor infiltration by T effector cells.
34. The method of claim 33, wherein said T effector cells are CD8+T
cells or CD4+T cells.
35. The method of claim 28, wherein said immune response further
comprises epitope spreading, induction of broad-based response to
self-derived tumor antigens, or both.
36. (canceled)
37. The method of claim 28, wherein said immune response further
comprises improvement of the overall balance of suppressor and
effector immune cells in the tumor microenvironment or improvement
in the systemic balance of suppressor and effector immunocytes.
38-150. (canceled)
151. The method claim 12, wherein said radiation therapy lasts
about 6 weeks.
152. The method of claim 1, wherein said heterologous antigen is an
HPV16 or HPV18 E6 or E7 antigen.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/063,828, filed on Oct. 14, 2014 and U.S.
Provisional Application No. 62/065,973, filed on Oct. 20, 2014,
both of which are incorporated by reference herein in their
entirety.
FIELD OF INVENTION
[0002] The present invention provides methods of treating anal or
vaginal tumors and anal or vaginal cancers, comprising the step of
administering to a subject a combination therapy comprising a
chemo-radiation therapy and a recombinant Listeria strain.
BACKGROUND OF THE INVENTION
[0003] Listeria monocytogenes (Lm) is a food-borne gram-positive
bacterium that can occasionally cause disease in humans, in
particular elderly individuals, newborns, pregnant women and
immunocompromised individuals. In addition to strongly activating
innate immunity and inducing a cytokine response that enhances
antigen-presenting cell (APC) function, Lm has the ability to
replicate in the cytosol of APCs after escaping from the
phagolysosome, mainly through the action of the listeriolysin O
(LLO) protein. This unique intracellular life cycle allows antigens
secreted by Lm to be processed and presented in the context of both
MHC class I and II molecules, resulting in potent cytotoxic
CD8.sup.+ and Th1 CD4.sup.+ T-cell-mediated immune responses. Lm
has been extensively investigated as a vector for cancer
immunotherapy in pre-clinical models.
[0004] Persistent infection with high-oncogenic risk human
papillomavirus (HR-HPV) types is recognized as a necessary, but not
sufficient, cause of invasive carcinoma of the cervix (ICC). HPVs
16 and 18 are the most prevalent types in malignant lesions,
accounting for over 70% of ICC and over 50% of high-grade precursor
lesions.
[0005] Anal cancer is a rare malignancy that begins in the anus,
which is the opening at the end of the rectum. The American Cancer
Society estimates that 7,270 cases of anal cancer will be diagnosed
in 2015 (with the incidence still increasing) and about 1,010
deaths will occur that year from anal cancer. Approximately half of
all anal cancers are diagnosed before the malignancy has spread
beyond the primary site, whereas 13% to 25% are diagnosed after the
cancer has spread to the lymph nodes, and 10% are diagnosed after
the cancer has spread to distant organs, or has metastasized. When
it is found early, anal cancer is highly treatable, however, if the
cancer has spread to distant organs, about one in five patients
lives for five years or more.
[0006] Receptive anal intercourse is strongly related to the
development of anal cancer. Anal infection with human
papillomavirus (HPV) resulting in genital warts is a major risk
factor for the cancer, and immunocompromised patients, are prone to
get anal cancer. In this subgroup, the prognosis is worse, than for
non-immunocompromised patients.
[0007] Anal cancer is primarily treated with a combination of
chemotherapy and radiation. This reduces the need for a colostomy
and carries a 5-year survival rate of over 70%. Despite this, the
treatment of anal cancer, in this fashion, has not changed since
1974 and surgery is reserved only for patients failing the above
therapy. Hence, there exists a need for alternative approaches to
treating anal cancer. One such approach is an immunotherapeutic
approach using recombinant attenuated live vaccine vectors, such as
a Listeria monocytogenes vaccine vector. The present invention
provides an attenuated live Listeria vaccine vector for treating
anal cancer.
SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention relates to a method of
treating an anal or vaginal tumor or anal or vaginal cancer in a
human subject, the method comprising the step of administering to
said subject a combination therapy comprising a chemo-radiation
therapy and a recombinant Listeria strain, said Listeria strain
comprising a recombinant nucleic acid, said nucleic acid comprising
a first open reading frame encoding a recombinant polypeptide
comprising an N-terminal fragment of an LLO protein fused to a
heterologous antigen or fragment thereof, thereby treating said
anal or vaginal tumor or anal or vaginal cancer in said human
subject.
[0009] In another aspect, the present invention relates to a method
of treating an anal or vaginal neoplasia in a human subject, the
method comprising the step of administering to said subject a
combination therapy comprising a chemo-radiation therapy and a
recombinant Listeria strain, said Listeria strain comprising a
recombinant nucleic acid, said nucleic acid comprising a first open
reading frame encoding a recombinant polypeptide comprising an
N-terminal fragment of an LLO protein fused to a heterologous
antigen or fragment thereof, thereby treating said anal or vaginal
neoplasia in said human subject.
[0010] In a further aspect, the present invention relates to the
use of a composition comprising a recombinant Listeria strain, said
Listeria strain comprising a recombinant nucleic acid, said nucleic
acid comprising a first open reading frame encoding a recombinant
polypeptide comprising an N-terminal fragment of an LLO protein
fused to a heterologous antigen or fragment thereof for treating an
anal or vaginal tumor or anal or vaginal cancer in a human subject,
the treatment further comprising the step of administering to said
subject a chemo-radiation therapy, thereby treating said anal or
vaginal tumor or anal or vaginal cancer in said human subject.
[0011] In a yet further aspect, the present invention relates to
the use of a composition comprising a recombinant Listeria strain,
said Listeria strain comprising a recombinant nucleic acid, said
nucleic acid comprising a first open reading frame encoding a
recombinant polypeptide comprising an N-terminal fragment of an LLO
protein fused to a heterologous antigen or fragment thereof for
treating an anal or vaginal neoplasia in a human subject, the
treatment further comprising the step of administering to said
subject a chemo-radiation therapy, thereby treating said anal or
vaginal neoplasia in said human subject.
[0012] In a related aspect, the present invention relates to a
method of eliciting an anti-tumor cytotoxic T cell response in a
human subject comprising administering to said subject said
combination therapy.
[0013] Other features and advantages disclosed herein will become
apparent from the following detailed description examples and
figures. It should be understood, however, that the detailed
description and the specific examples while indicating preferred
embodiments of the invention are given by way of illustration only,
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The subject matter regarded as the invention is particularly
pointed out and distinctly claimed in the concluding portion of the
specification. The invention, however, both as to organization and
method of operation, together with objects, features, and
advantages thereof, may best be understood by reference to the
following detailed description when read with the accompanying
drawings in which:
[0015] FIG. 1. Lm-E7 and Lm-LLO-E7 use different expression systems
to express and secrete E7. Lm-E7 was generated by introducing a
gene cassette into the orfZ domain of the L. monocytogenes genome
(A). The hly promoter drives expression of the hly signal sequence
and the first five amino acids (AA) of LLO followed by HPV-16 E7.
B), Lm-LLO-E7 was generated by transforming the PrfA-strain XFL-7
with the plasmid pGG-55. pGG-55 has the hly promoter driving
expression of a nonhemolytic fusion of LLO-E7. pGG-55 also contains
the PrfA gene to select for retention of the plasmid by XFL-7 in
vivo.
[0016] FIG. 2. Lm-E7 and Lm-LLO-E7 secrete E7. Lm-Gag (lane 1),
Lm-E7 (lane 2), Lm-LLO-NP (lane 3), Lm-LLO-E7 (lane 4), XFL-7 (lane
5), and 10403S (lane 6) were grown overnight at 37.degree. C. in
Luria-Bertoni broth. Equivalent numbers of bacteria, as determined
by OD at 600 nm absorbance, were pelleted and 18 ml of each
supernatant was TCA precipitated. E7 expression was analyzed by
Western blot. The blot was probed with an anti-E7 mAb, followed by
HRP-conjugated anti-mouse (Amersham), then developed using ECL
detection reagents.
[0017] FIG. 3. Tumor immunotherapeutic efficacy of LLO-E7 fusions.
Tumor size in millimeters in mice is shown at 7, 14, 21, 28 and 56
days post tumor-inoculation. Naive mice: open-circles; Lm-LLO-E7:
filled circles; Lm-E7: squares; Lm-Gag: open diamonds; and
Lm-LLO-NP: filled triangles.
[0018] FIG. 4. Splenocytes from Lm-LLO-E7-immunized mice
proliferate when exposed to TC-1 cells. C57BL/6 mice were immunized
and boosted with Lm-LLO-E7, Lm-E7, or control rLm strains.
Splenocytes were harvested 6 days after the boost and plated with
irradiated TC-1 cells at the ratios shown. The cells were pulsed
with .sup.3H thymidine and harvested. Cpm is defined as
(experimental cpm)-(no-TC-1 control).
[0019] FIG. 5 A. Induction of E7-specific IFN-gamma-secreting
CD8.sup.+ T cells in the spleens and the numbers penetrating the
tumors, in mice administered TC-1 tumor cells and subsequently
administered Lm-E7, Lm-LLO-E7, Lm-ActA-E7, or no vaccine (naive).
FIG. 5 B. Induction and penetration of E7 specific CD8.sup.+ cells
in the spleens and tumors of the mice described for (A).
[0020] FIG. 6. Listeria constructs containing PEST regions induce a
higher percentage of E7-specific lymphocytes within the tumor. FIG.
6 A. representative data from 1 experiment. FIG. 6 B. average and
SE of data from all 3 experiments.
[0021] FIG. 7A. Effect of passaging on bacterial load (virulence)
of recombinant Listeria vaccine vectors. Top panel. Lm-Gag. Bottom
panel. Lm-LLO-E7. FIG. 7B. Effect of passaging on bacterial load of
recombinant Lm-E7 in the spleen. Average CFU of live bacteria per
milliliter of spleen homogenate from four mice is depicted.
[0022] FIG. 8 shows induction of antigen-specific CD8.sup.+ T-cells
for HIV-Gag and LLO after administration of passaged Lm-Gag versus
unpassaged Lm-Gag. Mice were immunized with 10.sup.3 (A, B, E, F)
or 10.sup.5 (C, D, G, H) CFU passaged Listeria vaccine vectors, and
antigen-specific T-cells were analyzed. B, D, F, H: unpassaged
Listeria vaccine vectors. A-D immune response to MHC class I
HIV-Gag peptide. E-H: immune response to an LLO peptide. I:
splenocytes from mice immunized with 10.sup.5 CFU passaged Lm-Gag
stimulated with a control peptide from HPV E7.
[0023] FIG. 9A shows plasmid isolation throughout LB stability
study. FIG. 9B shows plasmid isolation throughout TB stability
study. FIG. 9C shows quantitation of TB stability study.
[0024] FIG. 10 shows numbers of viable bacteria chloramphenicol
(CAP)-resistant and CAP-sensitive colony-forming units (CFU) from
bacteria grown in LB. Dark bars: CAP.sup.+; white bars: CAP.sup.-.
The two dark bars and two white bars for each time point represent
duplicate samples.
[0025] FIG. 11 shows numbers of viable bacteria CAP-resistant and
CAP-sensitive CFU from bacteria grown in TB. Dark bars: CAP.sup.+;
white bars: CAP. The two dark bars and two white bars for each time
point represent duplicate samples.
[0026] FIG. 12. Actual chromatograms showing the region of the
D133V mutation (arrows). The mixture ratio is shown in
parentheses.
[0027] FIG. 13. Representation of the location of the ADV451, 452
and 453 primers and the segment of the PrfA gene amplified in the
reaction.
[0028] FIG. 14. Specificity of the PCR reaction using primers
ADV451 and ADV453.
[0029] FIG. 15. Specificity of the PCR reaction using primers
ADV452 and ADV453.
[0030] FIG. 16. Sensitivity of the PCR reaction to detect the
wild-type PrfA sequence using the primer ADV452 and 1 .mu.g as the
initial amount of DNA.
[0031] FIG. 17. Sensitivity of the PCR reaction to detect the
wild-type PrfA sequence using the primer ADV452 and 5 .mu.g as the
initial amount of DNA.
[0032] FIG. 18. Average density of the bands from the PCR depicted
in FIG. 16.
[0033] FIG. 19. Average density of the bands from the PCR depicted
in FIG. 17.
[0034] FIG. 20. Validation of the PCR reaction to detect the
wild-type PrfA sequence using the primer ADV452.
[0035] FIG. 21. Average density of the bands from the PCR depicted
in FIG. 16.
[0036] FIG. 22. Analysis of the D133V PrfA mutation in the
Lm-LLO-E7. A, Original image used for densitometry; B, Image was
digitally enhanced to facilitate the visualization of the low
density bands.
[0037] FIG. 23. Shows treatment schedule #1 of the patient. The
schedule comprises administering a first dose of ADXS-HPV (ADX)
before chemo-radiation and the 2-4.sup.th doses are given every 28
days after completion of radiation.
[0038] FIG. 24. Shows treatment schedule #2 of the patient where
the second dose of ADXS-HPV is administered during
chemo-radiation.
[0039] It will be appreciated that for simplicity and clarity of
illustration, elements shown in the Figs. have not necessarily been
drawn to scale. For example, the dimensions of some of the elements
may be exaggerated relative to other elements for clarity. Further,
where considered appropriate, reference numerals may be repeated
among the Figs. to indicate corresponding or analogous
elements.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention disclose, in some embodiments, methods
of treating, protecting against, and inducing an immune response
against a disease, comprising the step of administering to a
subject a recombinant Listeria strain, expressing a fusion peptide
comprising a listeriolysin O (LLO) fragment and a heterologous
antigen expressed by said disease or fragment thereof. The present
invention also provides methods for inducing an anti-disease
cytotoxic T-cell (CTL) response in a human subject and treating
disorders, and symptoms associated with said disease comprising
administration of the recombinant Listeria strain. In one
embodiment, provided herein is a recombinant Listeria strain, said
recombinant Listeria strain comprising a recombinant nucleic acid,
said nucleic acid comprising a first open reading frame encoding a
recombinant polypeptide comprising a first an N-terminal fragment
of an LLO protein fused to a heterologous antigen or fragment
thereof, and wherein said recombinant nucleic acid further
comprises a second open reading frame encoding a mutant prfA gene.
In one embodiment, the mutant PrfA gene is one that encodes a point
mutation from amino acid D or Asp or Aspartate (or Aspartic acid)
to amino acid V or Val or Valine at the 133.sup.rd amino acid
position. In one embodiment, a recombinant Listeria strain
disclosed herein comprises a prfA mutation or deletion that is
complemented via a plasmid comprised by the same Listeria, wherein
the plasmid comprises a mutant prfA gene encoding a mutant PrfA
protein comprising a D133V amino acid substitution.
[0041] In another embodiment, the recombinant Listeria is an
attenuated Listeria. "Attenuation" and "attenuated" may encompass a
bacterium, virus, parasite, infectious organism, prion, tumor cell,
gene in the infectious organism, and the like, that is modified to
reduce toxicity to a host. The host can be a human or animal host,
or an organ, tissue, or cell. The bacterium, to give a non-limiting
example, can be attenuated to reduce binding to a host cell, to
reduce spread from one host cell to another host cell, to reduce
extracellular growth, or to reduce intracellular growth in a host
cell. Attenuation can be assessed by measuring, e.g., an indicum or
indicia of toxicity, the LD.sub.50, the rate of clearance from an
organ, or the competitive index (see, e.g., Auerbuch, et al. (2001)
Infect. Immunity 69:5953-5957). Generally, an attenuation results
an increase in the LD.sub.50 and/or an increase in the rate of
clearance by at least 25%; more generally by at least 50%; most
generally by at least 100% (2-fold); normally by at least 5-fold;
more normally by at least 10-fold; most normally by at least
50-fold; often by at least 100-fold; more often by at least
500-fold; and most often by at least 1000-fold; usually by at least
5000-fold; more usually by at least 10,000-fold; and most usually
by at least 50,000-fold; and most often by at least
100,000-fold.
[0042] It will be well appreciated by a skilled artisan that the
term "Attenuated gene" may encompass a gene that mediates toxicity,
pathology, or virulence, to a host, growth within the host, or
survival within the host, where the gene is mutated in a way that
mitigates, reduces, or eliminates the toxicity, pathology, or
virulence. The reduction or elimination can be assessed by
comparing the virulence or toxicity mediated by the mutated gene
with that mediated by the non-mutated (or parent) gene. "Mutated
gene" encompasses deletions, point mutations, and frameshift
mutations in regulatory regions of the gene, coding regions of the
gene, non-coding regions of the gene, or any combination thereof.
In one embodiment, provided herein is a method of treating an anal
or vaginal tumor or anal or vaginal cancer in a human subject, the
method comprising the step of administering to said subject a
combination therapy comprising a chemo-radiation therapy and a
recombinant Listeria strain, said Listeria strain comprising a
recombinant nucleic acid, said nucleic acid comprising a first open
reading frame encoding a recombinant polypeptide comprising an
N-terminal fragment of an LLO protein fused to a heterologous
antigen or fragment thereof, thereby treating said anal or vaginal
tumor or anal or vaginal cancer in said human subject.
[0043] In one embodiment, said chemo-radiation therapy is
administered following a first administration of said recombinant
Listeria strain. In another embodiment, said chemo-radiation
therapy is administered prior to the administration of said
recombinant Listeria strain. In another embodiment, said
chemo-radiation therapy is administered following a first
administration of said recombinant Listeria strain and prior to one
to three booster administrations of said recombinant Listeria
strain. In another embodiment, said chemo-radiation therapy is
administered concurrently with said recombinant Listeria
strain.
[0044] In one embodiment, the method disclosed herein comprises
administering four doses of a recombinant Listeria provided herein.
In one embodiment the recombinant Listeria expresses a fusion
protein of N-terminal LLO and a heterologous antigen. In another
embodiment, the heterologous antigen is human papilloma virus E7
antigen (HPV-E7). In another embodiment, the HPV antigen is
HPVE6.
[0045] In one embodiment, the first dose of said recombinant
Listeria in the combination therapy provided herein is administered
prior to chemo-radiation therapy and the 2.sup.nd-4.sup.th doses
are administered every 28 days after completion of radiation (see
Example 11 herein). In another embodiment, the first dose of said
recombinant Listeria is administered before chemo-radiation
therapy, the second dose of ADXS-HPV is administered during
chemo-radiation therapy, and the 3.sup.rd-4.sup.th doses are
administered every 28 days following the completion of
chemo-radiation therapy (see Example 11).
[0046] In one embodiment, provided herein is a chemo-radiation
regiment or chemo-radiation therapy for use in combination with the
recombinant Listeria provided herein. In another embodiment, the
chemo-radiation therapy provided herein comprises mitomycin and
fluorouracil (5-FU) and radiation therapy. In another embodiment,
the chemo-radiation therapy can comprise any other chemotherapeutic
agents known in the art, including but not limited to,
Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan,
Nitrosoureas, Temozolomide, Azacitidine, Azathioprine,
Capecitabine, Cytarabine, Doxifluridine, Gemcitabine, Hydroxyurea,
Mercaptopurine, Methotrexate, or Tioguanine (formerly
Thioguanine).
[0047] In one embodiment, the chemo-radiation regiment or
chemo-radiation therapy provided herein comprises administering 2
courses of mitomycin, 5-FU with concurrent radiation (54 Gy in 30
fractions by intensity modulated radiation therapy).
[0048] In one embodiment, the radiation provided herein lasts about
6 weeks. In another embodiment, the radiation lasts 3 weeks. In
another embodiment, the radiation lasts 4 weeks. In another
embodiment, the radiation lasts, 5 weeks. In another embodiment,
the radiation lasts 7 weeks. In another embodiment, the radiation
lasts 8 weeks. In another embodiment, the radiation lasts 6-8
weeks. In another embodiment, the radiation lasts 4-6 weeks. In
another embodiment, the radiation lasts 2-4 weeks. In another
embodiment, the radiation lasts 8-10 weeks.
[0049] In another embodiment, provided herein is a method of
eliciting an anti-tumor cytotoxic T cell response in a human
subject comprising administering to said subject said combination
therapy.
[0050] In one embodiment, provided herein is a method for inducing
an immune response against a tumor or a cancer in a human subject,
the method comprising the step of administering to said subject a
recombinant Listeria strain comprising a recombinant nucleic acid,
said nucleic acid comprising a first open reading frame encoding a
recombinant polypeptide comprising an N-terminal fragment of an LLO
protein fused to a heterologous antigen or fragment thereof, is,
wherein said recombinant nucleic acid further comprises a second
open reading frame encoding a mutant prfA gene, thereby inducing an
immune response against a tumor or a cancer. In one embodiment, the
present invention provides a method of treating a cancer in a human
subject, comprising the step of administering to the subject the
recombinant Listeria strain provided herein. In another embodiment,
the present invention provides a method of protecting a human
subject against a cervical cancer, comprising the step of
administering to the subject the recombinant Listeria strain
provided herein. In another embodiment, the recombinant Listeria
strain expresses the recombinant polypeptide. In another
embodiment, the recombinant Listeria strain comprises a plasmid
that encodes the recombinant polypeptide. In another embodiment,
the method further comprises the step of boosting the human subject
with a recombinant Listeria strain of the present invention. In
another embodiment, the method further comprises the step of
boosting the human subject with an immunogenic composition
comprising a heterologous antigen or fragment thereof provided
herein. In another embodiment, the method further comprises the
step of boosting the human subject with an immunogenic composition
that directs a cell of the subject to express the heterologous
antigen. In another embodiment, the cell is a tumor cell. In
another embodiment, the method further comprises the step of
boosting the human subject with the vaccine of the present
invention.
[0051] In one embodiment, the present invention provides a method
of inducing an anti-tumor or an anti-cancer immune response in a
human subject, the method comprising the step of administering to
said subject a composition comprising a recombinant Listeria strain
comprising a recombinant nucleic acid, said nucleic acid comprising
a first open reading frame encoding a recombinant polypeptide
comprising an N-terminal fragment of an LLO protein fused to a
heterologous antigen or fragment thereof, wherein said recombinant
nucleic acid further comprises a second open reading frame encoding
a metabolic enzyme, thereby inducing an immune response against a
tumor or a cancer. In another embodiment, said Listeria comprises a
mutation in the endogenous prfA gene. In another embodiment, the
Listeria comprises a mutation or deletion in the endogenous dal/dat
and actA genes.
[0052] In one embodiment, the nucleic acid molecule provided herein
comprises a first open reading frame encoding recombinant
polypeptide comprising a heterologous antigen or fragment thereof.
In another embodiment, the recombinant polypeptide further
comprises a N-terminal LLO fused to the heterologous antigen. In
another embodiment, the nucleic acid molecule provided herein
further comprises a second open reading frame encoding a metabolic
enzyme. In another embodiment, the metabolic enzyme complements an
endogenous gene that is lacking in the chromosome of the
recombinant Listeria strain. In another embodiment, the metabolic
enzyme encoded by the second open reading frame is an alanine
racemase enzyme (dal). In another embodiment, the metabolic enzyme
encoded by the second open reading frame is a D-amino acid
transferase enzyme (dat). In another embodiment, the Listeria
strains provided herein comprise a mutation, a deletion or
inactivation in the genomic dal, dat, or actA genes. In another
embodiment, the Listeria strains provided herein comprise a
mutation, a deletion or inactivation in the genomic dal, dat, and
actA genes. In another embodiment, the Listeria lack the genomic
dal, dat or actA genes. In another embodiment, the Listeria lack
the genomic dal, dat and actA genes.
[0053] In one embodiment, the fragment thereof in the context of
LLO proteins and ActA proteins disclosed herein refer to a peptide
or polypeptide comprising an amino acid sequence of at least 5
contiguous amino acid residues of the LLO or ActA proteins. In
another embodiment, the term refers to a peptide or polypeptide
comprising an amino acid sequence of at least of at least 10
contiguous amino acid residues, at least 15 contiguous amino acid
residues, at least 20 contiguous amino acid residues, at least 25
contiguous amino acid residues, at least 40 contiguous amino acid
residues, at least 50 contiguous amino acid residues, at least 60
contiguous amino residues, at least 70 contiguous amino acid
residues, at least 80 contiguous amino acid residues, at least 90
contiguous amino acid residues, at least 100 contiguous amino acid
residues, at least 125 contiguous amino acid residues, at least 150
contiguous amino acid residues, at least 175 contiguous amino acid
residues, at least 200 contiguous amino acid residues, at least 250
contiguous amino acid residues of the amino acid sequence, at least
300 contiguous amino acid residues, at least 350 contiguous amino
acid residues of, at least 400 contiguous amino acid residues, or
at least 450 contiguous amino acid residues of an LLO or ActA
protein or polypeptide.
[0054] In another embodiment, the fragment is a functional fragment
that elicits an immune response against a disease-associated
antigen when in the form of an N-terminal LLO/heterologous antigen
fusion protein or N-terminal ActA/heterologous antigen fusion
protein). In another embodiment, the fragment is an immunogenic
fragment. In another embodiment, the fragment is functional in a
non-fused form.
[0055] The present invention, in certain embodiments, provides
codon optimization of a nucleic acid heterologous to Listeria, or
of a nucleic acid endogenous to Listeria. The optimal codons
utilized by L. monocytogenes for each amino acid are shown US
Patent Publication 2007/0207170, which is hereby incorporated by
reference herein. A nucleic acid is codon-optimized if at least one
codon in the nucleic acid is replaced with a codon that is more
frequently used by L. monocytogenes for that amino acid than the
codon in the original sequence.
[0056] The N-terminal LLO protein fragment and heterologous antigen
are, in another embodiment, fused directly to one another. In
another embodiment, the genes encoding the N-terminal LLO protein
fragment and the heterologous antigen are fused directly to one
another. In another embodiment, the N-terminal LLO protein fragment
and the heterologous antigen are attached via a linker peptide. In
another embodiment, the N-terminal LLO protein fragment and the
heterologous antigen are attached via a heterologous peptide. In
another embodiment, the N-terminal LLO protein fragment is
N-terminal to the heterologous antigen. In another embodiment, the
N-terminal LLO protein fragment is the N-terminal-most portion of
the fusion protein. As disclosed herein, recombinant Listeria
strains expressing LLO-antigen fusions induce anti-tumor immunity
(Example 1), elicit antigen-specific T cell proliferation (Example
2), generate antigen-specific, and tumor-infiltrating T cells
(Example 3).
[0057] In another embodiment, the present invention provides a
method of treating a cervical cancer in a human subject, comprising
the step of administering to the subject a recombinant Listeria
strain, the recombinant Listeria strain comprising a recombinant
polypeptide comprising an N-terminal fragment of an LLO protein and
an HPV E7 antigen, whereby the recombinant Listeria strain induces
an immune response against the E7 antigen, thereby treating a
cervical cancer in a human subject. In another embodiment, the
recombinant Listeria strain expresses the recombinant polypeptide.
In another embodiment, the recombinant Listeria strain comprises a
plasmid that encodes the recombinant polypeptide.
[0058] In another embodiment, the present invention provides a
method of protecting a human subject against a cervical cancer,
comprising the step of administering to the subject a recombinant
Listeria strain, the recombinant Listeria strain comprising a
recombinant polypeptide comprising an N-terminal fragment of an LLO
protein and an HPV E7 antigen, whereby the recombinant Listeria
strain induces an immune response against the E7 antigen, thereby
protecting a human subject against a cervical cancer. In another
embodiment, the recombinant Listeria strain expresses the
recombinant polypeptide. In another embodiment, the recombinant
Listeria strain comprises a plasmid that encodes the recombinant
polypeptide.
[0059] In another embodiment, the present invention provides a
method for inducing an immune response against a cervical cancer in
a human subject, comprising the step of administering to the
subject a recombinant Listeria strain, the recombinant Listeria
strain comprising a recombinant polypeptide comprising an
N-terminal fragment of an LLO protein and an HPV E7 antigen,
thereby inducing an immune response against a cervical cancer in a
human subject. In another embodiment, the recombinant Listeria
strain expresses the recombinant polypeptide. In another
embodiment, the recombinant Listeria strain comprises a plasmid
that encodes the recombinant polypeptide.
[0060] In another embodiment, the present invention provides a
method of treating a cervical cancer in a human subject, comprising
the step of administering to the subject a recombinant Listeria
strain, the recombinant Listeria strain comprising a recombinant
polypeptide comprising an N-terminal fragment of an ActA protein
and heterologous antigen, whereby the recombinant Listeria strain
induces an immune response against the heterologous antigen,
thereby treating a cervical cancer in a human subject. In another
embodiment, the recombinant Listeria strain expresses the
recombinant polypeptide. In another embodiment, the recombinant
Listeria strain comprises a plasmid that encodes the recombinant
polypeptide.
[0061] In another embodiment, the present invention provides a
method of protecting a human subject against a cervical cancer,
comprising the step of administering to the subject a recombinant
Listeria strain, the recombinant Listeria strain comprising a
recombinant polypeptide comprising an N-terminal fragment of an
ActA protein and a heterologous antigen, whereby the recombinant
Listeria strain induces an immune response against the heterologous
antigen, thereby protecting a human subject against a cervical
cancer. In another embodiment, the recombinant Listeria strain
expresses the recombinant polypeptide. In another embodiment, the
recombinant Listeria strain comprises a plasmid that encodes the
recombinant polypeptide.
[0062] In another embodiment, the present invention provides a
method for inducing an immune response against a cervical cancer in
a human subject, comprising the step of administering to the
subject a recombinant Listeria strain, the recombinant Listeria
strain comprising a recombinant polypeptide comprising an
N-terminal fragment of an heterologous protein and a heterologous
antigen, thereby inducing an immune response against a cervical
cancer in a human subject. In another embodiment, the recombinant
Listeria strain expresses the recombinant polypeptide. In another
embodiment, the recombinant Listeria strain comprises a plasmid
that encodes the recombinant polypeptide.
[0063] The N-terminal ActA protein fragment and the heterologous
antigen are, in another embodiment, fused directly to one another.
In another embodiment, the genes encoding the N-terminal ActA
protein fragment and heterologous antigen are fused directly to one
another. In another embodiment, the N-terminal ActA protein
fragment and heterologous antigen are attached via a linker
peptide. In another embodiment, the N-terminal ActA protein
fragment and heterologous antigen are attached via a heterologous
peptide. In another embodiment, the N-terminal ActA protein
fragment is N-terminal to the heterologous antigen. In another
embodiment, the N-terminal ActA protein fragment is the
N-terminal-most portion of the fusion protein.
[0064] In another embodiment, the present invention provides a
method of inducing an immune response against a cervical cancer in
a human subject, comprising the step of administering to the
subject a recombinant Listeria strain, the recombinant Listeria
strain comprising a recombinant polypeptide comprising a PEST amino
acid sequence-containing peptide and a heterologous antigen,
whereby the recombinant Listeria strain induces an immune response
against the heterologous antigen, thereby treating a cervical
cancer in a human subject. In another embodiment, the recombinant
Listeria strain expresses the recombinant polypeptide. In another
embodiment, the recombinant Listeria strain comprises a plasmid
that encodes the recombinant polypeptide. In another embodiment,
the method protects a human subject against a cervical. In another
embodiment, the method treats a cervical cancer in said human
subject.
[0065] The PEST amino acid amino acid sequence-containing peptide
and heterologous antigen are, in another embodiment, fused directly
to one another. In another embodiment, the genes encoding the PEST
amino acid sequence-containing peptide and heterologous antigen are
fused directly to one another. In another embodiment, the PEST
amino acid sequence-containing peptide and heterologous antigen are
attached via a linker peptide. In another embodiment, the PEST
amino acid sequence-containing peptide and heterologous antigen are
attached via a heterologous peptide. In another embodiment, the
PEST amino acid sequence-containing peptide is N-terminal to the
heterologous antigen. In another embodiment, the PEST amino acid
sequence-containing peptide is the N-terminal-most portion of the
fusion protein.
[0066] In another embodiment, the present invention provides a
method for vaccinating a human subject against an HPV, comprising
the step of administering to the subject the recombinant Listeria
strain provided herein, wherein the Listeria expresses an HPV E7
antigen and wherein the Listeria expresses a mutant PrfA gene. In
another embodiment, the mutant PrfA gene is a D133V PrfA mutation.
In another embodiment, the mutant PrfA gene is in a plasmid in said
recombinant Listeria. In another embodiment, the recombinant
Listeria strain expresses the recombinant polypeptide. In another
embodiment, the recombinant Listeria strain comprises a plasmid
that encodes the recombinant polypeptide. In one embodiment, the
recombinant Listeria strain comprises a bivalent episomal
expression vector, the vector comprising a first, and a second
nucleic acid molecule encoding a heterologous antigenic polypeptide
or a functional fragment thereof, wherein the first and the second
nucleic acid molecules each encode the heterologous antigenic
polypeptide or functional fragment thereof in an open reading frame
with an N-terminal or truncated or detoxified Listeriolysin O
protein (LLO), or a truncated ActA protein, or a PEST amino acid
sequence.
[0067] In one embodiment, the heterologous antigens expressed by
bivalent expression vector are HPV16 E7 and HPV18 E7.
[0068] In one embodiment, the recombinant Listeria strain comprises
a trivalent episomal expression vector, the vector comprising a
first, second, and third nucleic acid molecule, each encoding a
heterologous antigenic polypeptide or a functional fragment
thereof, wherein the first through third nucleic acid molecules
each encode the heterologous antigenic polypeptide or functional
fragment thereof in an open reading frame with an N-terminal or
truncated or detoxified Listeriolysin O protein (LLO, a truncated
ActA protein, or a PEST amino acid sequence.
[0069] In one embodiment, the bivalent, trivalent or quadravalent
recombinant Listeria strains disclosed herein express at least one
heterologous antigen from an open reading frame in a
extrachromosomal plasmid or episome. In another embodiment, the
bivalent, or trivalent recombinant Listeria strains disclosed
herein express at least one heterologous antigen from an open
reading frame from at least one extrachromosomal plasmid or
episome. In another embodiment, the bivalent recombinant Listeria
strains disclosed herein express two heterologous antigens each
from an open reading frame of two extrachromosomal plasmids or
episomes. In another embodiment, the trivalent recombinant Listeria
strains disclosed herein express three heterologous antigens each
from an open reading frame of three extrachromosomal plasmids or
episomes. In another embodiment, the quadravalent recombinant
Listeria strains disclosed herein express four heterologous
antigens each from an open reading frame of four extrachromosomal
plasmids or episomes.
[0070] In another embodiment, the bivalent, trivalent, or
quadravalent recombinant Listeria strains disclosed herein express
at least one heterologous antigen from an open reading frame in the
genome of the Listeria. In another embodiment, the bivalent,
trivalent, or quadravalent recombinant Listeria strains provided
herein express at least one heterologous antigen from both, an
extrachromosomal plasmid or episome, and from the genome of a
Listeria provided herein. In another embodiment, each heterologous
antigen is expressed in a fusion protein with a PEST-containing
polypeptide or peptide provided herein.
[0071] In one embodiment, bivalent and multivalent recombinant
Listeria encompassed by the present invention include those
described in US Pub. No. 2011/0129499, and in US Pub No.
2012/0135033, both of which are incorporated by reference in their
entirety herein.
[0072] In another embodiment, the subject is at risk for developing
an HPV-mediated carcinogenesis (e.g. a cervical cancer). In another
embodiment, the subject is HPV-positive. In another embodiment, the
subject exhibits cervical intraepithelial neoplasia. In another
embodiment, the subject exhibits a squamous intraepithelial lesion.
In another embodiment, the subject exhibits a dysplasia in the
cervix.
[0073] In one embodiment, there heterologous antigen is any tumor
associated antigen known in the art and provided herein. In another
embodiment, the heterologous antigen is an autoimmune antigen. In
another embodiment, the heterologous antigen is an infectious
disease antigen. In another embodiment, the heterologous antigen is
an HPV-related antigen.
[0074] The HPV that is the target of methods disclosed herein is,
in another embodiment, an HPV 16. In another embodiment, the HPV is
an HPV-18. In another embodiment, the HPV is selected from HPV-16
and HPV-18. In another embodiment, the HPV is an HPV-31. In another
embodiment, the HPV is an HPV-35. In another embodiment, the HPV is
an HPV-39. In another embodiment, the HPV is an HPV-45. In another
embodiment, the HPV is an HPV-51. In another embodiment, the HPV is
an HPV-52. In another embodiment, the HPV is an HPV-58. In another
embodiment, the HPV is a high-risk HPV type. In another embodiment,
the HPV is a mucosal HPV type.
[0075] In another embodiment, the present invention provides a
method of vaccinating a human subject against an antigen of
interest, the method comprising the step of administering
intravenously to the human subject a recombinant Listeria strain
comprising or expressing the antigen of interest, wherein the first
peptide is selected from (a) an N-terminal fragment of an LLO
protein; (b) an ActA protein or N-terminal fragment thereof; and
(c) a PEST amino acid sequence-containing peptide, thereby
vaccinating a human subject against an antigen of interest.
[0076] In another embodiment, the present invention provides a
method of vaccinating a human subject against an antigen of
interest, the method comprising the step of administering
intravenously to the human subject an immunogenic composition,
comprising a fusion of a first peptide to the antigen of interest,
wherein the first peptide is selected from (a) an N-terminal
fragment of an LLO protein; (b) an ActA protein or N-terminal
fragment thereof; and (c) a PEST amino acid sequence-containing
peptide, thereby vaccinating a human subject against an antigen of
interest.
[0077] In another embodiment, the present invention provides a
method of vaccinating a human subject against an antigen of
interest, the method comprising the step of administering
intravenously to the human subject a recombinant Listeria strain
comprising a recombinant polypeptide, the recombinant polypeptide
comprising a first peptide fused to the antigen of interest,
wherein the first peptide is selected from (a) an N-terminal
fragment of an LLO protein; (b) an ActA protein or N-terminal
fragment thereof; and (c) a PEST amino acid sequence-containing
peptide, thereby vaccinating a human subject against an antigen of
interest.
[0078] In another embodiment, the present invention provides a
method of inducing a CTL response in a human subject against an
antigen of interest, the method comprising the step of
administering to the human subject a recombinant Listeria strain
comprising or expressing the antigen of interest, thereby inducing
a CTL response in a human subject against an antigen of interest.
In another embodiment, the step of administering is intravenous
administration.
[0079] As disclosed herein, recombinant Listeria strains expressing
LLO-antigen fusions induce anti-tumor immunity (Example 1), elicit
antigen-specific T cell proliferation (Example 2), generate
antigen-specific, and tumor-infiltrating T cells (Example 3). Thus,
vaccines disclosed herein are efficacious at inducing immune
responses against E7 and E6.
[0080] In another embodiment, the present invention provides a
method for inducing a regression of a cancer in a subject,
comprising the step of administering to the subject the recombinant
Listeria strain provided herein
[0081] In another embodiment, the present invention provides a
method for reducing an incidence of relapse of a cancer in a
subject, comprising the step of administering to the subject the
recombinant Listeria strain provided herein.
[0082] In another embodiment, the present invention provides a
method for suppressing a formation of a tumor in a subject,
comprising the step of administering to the subject the recombinant
Listeria strain provided herein.
[0083] In another embodiment, the present invention provides a
method for inducing a remission of a cancer in a subject,
comprising the step of administering to the subject the recombinant
Listeria strain provided herein.
[0084] In another embodiment, the present invention provides a
method for impeding a growth of a tumor in a human subject,
comprising the step of administering to the subject the recombinant
Listeria strain provided herein.
[0085] In another embodiment, the present invention provides a
method for reducing a size of a tumor in a subject, comprising the
step of administering to the subject the recombinant Listeria
strain provided herein.
[0086] In one embodiment, the disease is an infectious disease, an
autoimmune disease, a respiratory disease, a pre-cancerous
condition or a cancer.
[0087] It will be well appreciated by the skilled artisan that the
term "pre-cancerous condition" may encompass dysplasias,
preneoplastic nodules; macroregenerative nodules (MRN); low-grade
dysplastic nodules (LG-DN); high-grade dysplastic nodules (HG-DN);
biliary epithelial dysplasia; foci of altered hepatocytes (FAH);
nodules of altered hepatocytes (NAH); chromosomal imbalances;
aberrant activation of telomerase; re-expression of the catalytic
subunit of telomerase; expression of endothelial cell markers such
as CD31, CD34, and BNH9 (see, e.g., Terracciano and Tomillo (2003)
Pathologica 95:71-82; Su and Bannasch (2003) Toxicol. Pathol.
31:126-133; Rocken and Carl-McGrath (2001) Dig. Dis. 19:269-278;
Kotoula, et al. (2002) Liver 22:57-69; Frachon, et al. (2001) J.
Hepatol. 34:850-857; Shimonishi, et al. (2000) J. Hepatobiliary
Pancreat. Surg. 7:542-550; Nakanuma, et al. (2003) J. Hepatobiliary
Pancreat. Surg. 10:265-281). Methods for diagnosing cancer and
dysplasia are disclosed (see, e.g., Riegler (1996) Semin
Gastrointest. Dis. 7:74-87; Benvegnu, et al. (1992) Liver 12:80-83;
Giannini, et al. (1987) Hepatogastroenterol. 34:95-97; Anthony
(1976) Cancer Res. 36:2579-2583).
[0088] In one embodiment, an infectious disease is one caused by,
but not limited to, any one of the following pathogens:
BCG/Tuberculosis, Malaria, Plasmodium falciparum, plasmodium
malariae, plasmodium vivax, Rotavirus, Cholera, Diptheria-Tetanus,
Pertussis, Haemophilus influenzae, Hepatitis B, Human papilloma
virus, Influenza seasonal), Influenza A (H1N1) Pandemic, Measles
and Rubella, Mumps, Meningococcus A+C, Oral Polio Vaccines, mono,
bi and trivalent, Pneumococcal, Rabies, Tetanus Toxoid, Yellow
Fever, Bacillus anthracis (anthrax), Clostridium botulinum toxin
(botulism), Yersinia pestis (plague), Variola major (smallpox) and
other related pox viruses, Francisella tularensis (tularemia),
Viral hemorrhagic fevers, Arenaviruses (LCM, Junin virus, Machupo
virus, Guanarito virus, Lassa Fever), Bunyaviruses (Hantaviruses,
Rift Valley Fever), Flaviruses (Dengue), Filoviruses (Ebola,
Marburg), Burkholderia pseudomallei, Coxiella burnetii (Q fever),
Brucella species (brucellosis), Burkholderia mallei (glanders),
Chlamydia psittaci (Psittacosis), Ricin toxin (from Ricinus
communis), Epsilon toxin of Clostridium perfringens, Staphylococcus
enterotoxin B, Typhus fever (Rickettsia prowazekii), other
Rickettsias, Food- and Waterborne Pathogens, Bacteria
(Diarrheagenic E. coli, Pathogenic Vibrios, Shigella species,
Salmonella BCG/, Campylobacter jejuni, Yersinia enterocolitica),
Viruses (Caliciviruses, Hepatitis A, West Nile Virus, LaCrosse,
Calif. encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus,
Kyasanur Forest Virus, Nipah virus, hantaviruses, Tickborne
hemorrhagic fever viruses, Chikungunya virus, Crimean-Congo
Hemorrhagic fever virus, Tickborne encephalitis viruses, Hepatitis
B virus, Hepatitis C virus, Herpes Simplex virus (HSV), Human
immunodeficiency virus (HIV), Human papillomavirus (HPV)), Protozoa
(Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia,
Entamoeba histolytica, Toxoplasma), Fungi (Microsporidia), Yellow
fever, Tuberculosis, including drug-resistant TB, Rabies, Prions,
Severe acute respiratory syndrome associated coronavirus
(SARS-CoV), Coccidioides posadasii, Coccidioides immitis, Bacterial
vaginosis, Chlamydia trachomatis, Cytomegalovirus, Granuloma
inguinale, Hemophilus ducreyi, Neisseria gonorrhea, Treponema
pallidum, Trichomonas vaginalis, or any other infectious disease
known in the art that is not listed herein.
[0089] In another embodiment, the infectious disease is a livestock
infectious disease. In another embodiment, livestock diseases can
be transmitted to man and are called "zoonotic diseases." In
another embodiment, these diseases include, but are not limited to,
Foot and mouth disease, West Nile Virus, rabies, canine parvovirus,
feline leukemia virus, equine influenza virus, infectious bovine
rhinotracheitis (IBR), pseudorabies, classical swine fever (CSF),
IBR, caused by bovine herpesvirus type 1 (BHV-1) infection of
cattle, and pseudorabies (Aujeszky's disease) in pigs,
toxoplasmosis, anthrax, vesicular stomatitis virus, rhodococcus
equi, Tularemia, Plague (Yersinia pestis), trichomonas.
[0090] In another embodiment, the disease provided herein is a
respiratory or inflammatory disease. In another embodiment, the
respiratory or inflammatory disease is chronic obstructive
pulmonary disease (COPD). In another embodiment, the disease is
asthma.
[0091] In one embodiment, live attenuated Listeria strains are
capable of alleviating asthma symptoms without co-administration of
other therapeutic agents, such as anti-inflammatory agents or
bronchodilators. In another embodiment, the methods provided herein
further comprise the step of co-administering to a subject the live
attenuated Listeria strain and one or more therapeutic agents. In
another embodiment, the therapeutic agent is an anti-asthmatic
agent. In another embodiment, the agent is an anti-inflammatory
agent, a non-steroidal anti-inflammatory agent, an antibiotic, an
antichlolinerginc agent, a bronchodilator, a corticosteroid, a
short-acting beta-agonist, a long-acting beta-agonist, combination
inhalers, an antihistamine, or combinations thereof.
[0092] In one embodiment, the disease provided herein is a cancer
or a tumor. In one embodiment, the tumor is cancerous. In another
embodiment, the cancer is breast cancer. In another embodiment, the
cancer is a cervical cancer. In another embodiment, the cancer is a
Her2 containing cancer. In another embodiment, the cancer is a
melanoma. In another embodiment, the cancer is pancreatic cancer.
In another embodiment, the cancer is ovarian cancer. In another
embodiment, the cancer is gastric cancer. In another embodiment,
the cancer is a carcinomatous lesion of the pancreas. In another
embodiment, the cancer is pulmonary adenocarcinoma. In another
embodiment, it is a glioblastoma multiforme. In another embodiment,
the cancer is colorectal adenocarcinoma. In another embodiment, the
cancer is pulmonary squamous adenocarcinoma. In another embodiment,
the cancer is gastric adenocarcinoma. In another embodiment, the
cancer is an ovarian surface epithelial neoplasm (e.g. a benign,
proliferative or malignant variety thereof). In another embodiment,
the cancer is an oral squamous cell carcinoma. In another
embodiment, the cancer is non-small-cell lung carcinoma. In another
embodiment, the cancer is an endometrial carcinoma. In another
embodiment, the cancer is a bladder cancer. In another embodiment,
the cancer is a head and neck cancer. In another embodiment, the
cancer is a prostate carcinoma. In another embodiment, the cancer
is oropharyngeal cancer. In another embodiment, the cancer is lung
cancer. In another embodiment, the cancer is anal cancer. In
another embodiment, the cancer is colorectal cancer in another
embodiment, the cancer is vaginal cancer. In another embodiment,
the cancer is esophageal cancer. The cervical tumor targeted by
methods disclosed herein is, in another embodiment, a squamous cell
carcinoma. In another embodiment, the cervical tumor is an
adenocarcinoma. In another embodiment, the cervical tumor is an
adenosquamous carcinoma. In another embodiment, the cervical tumor
is a small cell carcinoma. In another embodiment, the cervical
tumor is any other type of cervical tumor known in the art.
[0093] In one embodiment, the disease provided herein is a
neoplasia. In another embodiment, the neoplasia is anal
intraepithelial neoplasia (AIN). In another embodiment, the
neoplasia is vaginal intraepithelial neoplasia (VIN).
[0094] The cervical tumor targeted by methods disclosed herein is,
in another embodiment, a squamous cell carcinoma. In another
embodiment, the cervical tumor is an adenocarcinoma. In another
embodiment, the cervical tumor is an adenosquamous carcinoma. In
another embodiment, the cervical tumor is a small cell carcinoma.
In another embodiment, the cervical tumor is any other type of
cervical tumor known in the art.
[0095] In one embodiment, the antigen provided herein is a
heterologous tumor antigen, which is also referred to herein as
"tumor antigen" "antigenic polypeptide," or "foreign antigen." In
another embodiment, the antigen is Human Papilloma Virus-E7
(HPV-E7) antigen, which in one embodiment, is from HPV16 (in one
embodiment, GenBank Accession No. AAD33253) and in another
embodiment, from HPV18 (in one embodiment, GenBank Accession No.
P06788). In another embodiment, the antigenic polypeptide is
HPV-E6, which in one embodiment, is from HPV16 (in one embodiment,
GenBank Accession No. AAD33252, AAM51854, AAM51853, or AAB67615)
and in another embodiment, from HPV18 (in one embodiment, GenBank
Accession No. P06463). In another embodiment, the antigenic
polypeptide is a Her/2-neu antigen. In another embodiment, the
antigenic polypeptide is Prostate Specific Antigen (PSA) (in one
embodiment, GenBank Accession No. CAD30844, CAD54617, AAA58802, or
NP_001639). In another embodiment, the antigenic polypeptide is
Stratum Corneum Chymotryptic Enzyme (SCCE) antigen (in one
embodiment, GenBank Accession No. AAK69652, AAK69624, AAG33360,
AAF01139, or AAC37551). In another embodiment, the antigenic
polypeptide is Wilms tumor antigen 1, which in another embodiment
is WT-1 Telomerase (GenBank Accession. No. P49952, P22561,
NP_659032, CAC39220.2, or EAW68222.1). In another embodiment, the
antigenic polypeptide is hTERT or Telomerase (GenBank Accession.
No. NM003219 (variant 1), NM198255 (variant 2), NM 198253 (variant
3), or NM 198254 (variant 4). In another embodiment, the antigenic
polypeptide is Proteinase 3 (in one embodiment, GenBank Accession
No. M29142, M75154, M96839, X55668, NM 00277, M96628 or X56606). In
another embodiment, the antigenic polypeptide is Tyrosinase Related
Protein 2 (TRP2) (in one embodiment, GenBank Accession No.
NP_001913, ABI73976, AAP33051, or Q95119). In another embodiment,
the antigenic polypeptide is High Molecular Weight Melanoma
Associated Antigen (HMW-MAA) (in one embodiment, GenBank Accession
No. NP_001888, AAI28111, or AAQ62842). In another embodiment, the
antigenic polypeptide is Testisin (in one embodiment, GenBank
Accession No. AAF79020, AAF79019, AAG02255, AAK29360, AAD41588, or
NP659206). In another embodiment, the antigenic polypeptide is
NY-ESO-1 antigen (in one embodiment, GenBank Accession No.
CAA05908, P78358, AAB49693, or NP_640343). In another embodiment,
the antigenic polypeptide is PSCA (in one embodiment, GenBank
Accession No. AAH65183, NP_005663, NP_082492, 043653, or CAB97347).
In another embodiment, the antigenic polypeptide is Interleukin
(IL) 13 Receptor alpha (in one embodiment, GenBank Accession No.
NP000631, NP001551, NP032382, NP598751, NP001003075, or NP_999506).
In another embodiment, the antigenic polypeptide is Carbonic
anhydrase IX (CAIX) (in one embodiment, GenBank Accession No.
CAI13455, CAI10985, EAW58359, NP_001207, NP_647466, or
NP_001101426). In another embodiment, the antigenic polypeptide is
carcinoembryonic antigen (CEA) (in one embodiment, GenBank
Accession No. AAA66186, CAA79884, CAA66955, AAA51966, AAD15250, or
AAA51970). In another embodiment, the antigenic polypeptide is
MAGE-A (in one embodiment, GenBank Accession No. NP786885,
NP786884, NP005352, NP004979, NP005358, or NP 005353). In another
embodiment, the antigenic polypeptide is survivin (in one
embodiment, GenBank Accession No. AAC51660, AAY15202, ABF60110,
NP001003019, or NP 001082350). In another embodiment, the antigenic
polypeptide is GP100 (in one embodiment, GenBank Accession No.
AAC60634, YP_655861, or AAB31176). In another embodiment, the
antigenic polypeptide is any other antigenic polypeptide known in
the art. In another embodiment, the antigenic peptide of the
compositions and methods disclosed herein comprise an immunogenic
portion of the antigenic polypeptide.
[0096] In another embodiment, the antigen is HPV-E6. In another
embodiment, the antigen is telomerase (TERT). In another
embodiment, the antigen is LMP-1. In another embodiment, the
antigen is p53. In another embodiment, the antigen is mesothelin.
In another embodiment, the antigen is EGFRVIII. In another
embodiment, the antigen is carboxic anhydrase IX (CAIX). In another
embodiment, the antigen is PSMA. In another embodiment, the antigen
is HMW-MAA. In another embodiment, the antigen is HIV-1 Gag. In
another embodiment, the antigen is Tyrosinase related protein 2. In
another embodiment, the antigen is selected from HPV-E7, HPV-E6,
Her-2, HIV-1 Gag, LMP-1, p53, PSMA, carcinoembryonic antigen (CEA),
LMP-1, kallikrein-related peptidase 3 (KLK3), KLK9, Muc, Tyrosinase
related protein 2, Muc1, FAP, IL-13R alpha 2, PSA
(prostate-specific antigen), gp-100, heat-shock protein 70
(HSP-70), beta-HCG, EGFR-III, Granulocyte colony-stimulating factor
(G-CSF), Angiogenin, Angiopoietin-1, Del-1, Fibroblast growth
factors: acidic (aFGF) or basic (bFGF), Follistatin, Granulocyte
colony-stimulating factor (G-CSF), Hepatocyte growth factor
(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,
Placental growth factor, Platelet-derived endothelial cell growth
factor (PD-ECGF), Platelet-derived growth factor-BB (PDGF-BB),
Pleiotrophin (PTN), Progranulin, Proliferin, Transforming growth
factor-alpha (TGF-alpha), Transforming growth factor-beta
(TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascular
endothelial growth factor (VEGF)/vascular permeability factor
(VPF), VEGFR, VEGFR2 (KDR/FLK-1) or a fragment thereof, FLK-1 or an
epitope thereof, FLK-E1, FLK-E2, FLK-I1, endoglin or a fragment
thereof, Neuropilin 1 (NRP-1), Angiopoietin 1 (Ang1), Tie2,
Platelet-derived growth factor (PDGF), Platelet-derived growth
factor receptor (PDGFR), Transforming growth factor-beta
(TGF-.beta.), endoglin, TGF-.beta. receptors, monocyte chemotactic
protein-1 (MCP-1), VE-cadherin, CD31, ephrin, ICAM-1, V-CAM-1,
VAP-1, E-selectin, plasminogen activators, plasminogen activator
inhibitor-1, Nitric oxide synthase (NOS), COX-2, AC133, or Id1/Id3,
Angiopoietin 3, Angiopoietin 4, Angiopoietin 6, CD105, EDG, HHT1,
ORW, ORW1 or a TGFbeta co-receptor, or a combination thereof. In
another embodiment, the antigen is a chimeric Her2/neu antigen as
disclosed in US Patent Application Publication No. 2011/0142791,
which is incorporated by reference herein in its entirety. The use
of fragments of antigens provided herein is also encompassed by the
present invention.
[0097] In another embodiment, the heterologous tumor antigen
provided herein is a tumor-associated antigen, which in one
embodiment, is one of the following tumor antigens: a MAGE
(Melanoma-Associated Antigen E) protein, e.g. MAGE 1, MAGE 2, MAGE
3, MAGE 4, a tyrosinase; a mutant ras protein; a mutant p53
protein; p97 melanoma antigen, a ras peptide or p53 peptide
associated with advanced cancers; the HPV 16/18 antigens associated
with cervical cancers, KLH antigen associated with breast
carcinoma, CEA (carcinoembryonic antigen) associated with
colorectal cancer, a MART1 antigen associated with melanoma, or the
PSA antigen associated with prostate cancer. In another embodiment,
the antigen for the compositions and methods provided herein are
melanoma-associated antigens, which in one embodiment are TRP-2,
MAGE-1, MAGE-3, gp-100, tyrosinase, HSP-70, beta-HCG, or a
combination thereof. It is to be understood that a skilled artisan
would be able to use any heterologous antigen not mentioned herein
but known in the art for use in the methods and compositions
provided herein. It is also to be understood that the present
invention provides, but is not limited by, an attenuated Listeria
comprising a nucleic acid that encodes at least one of the antigens
disclosed herein. The present invention encompasses nucleic acids
encoding mutants, muteins, splice variants, fragments, truncated
variants, soluble variants, extracellular domains, intracellular
domains, mature sequences, and the like, of the disclosed antigens.
Provided are nucleic acids encoding epitopes, oligo- and
polypeptides of these antigens. Also provided are codon optimized
embodiments, that is, optimized for expression in Listeria. The
cited references, GenBank Acc. Nos., and the nucleic acids,
peptides, and polypeptides disclosed herein, are all incorporated
herein by reference in their entirety. In another embodiment, the
selected nucleic acid sequence can encode a full length or a
truncated gene, a fusion or tagged gene, and can be a cDNA, a
genomic DNA, or a DNA fragment, preferably, a cDNA. It can be
mutated or otherwise modified as desired. These modifications
include codon optimizations to optimize codon usage in the selected
host cell or bacteria, i.e. Listeria. The selected sequence can
also encode a secreted, cytoplasmic, nuclear, membrane bound or
cell surface polypeptide.
[0098] In one embodiment, vascular endothelial growth factor (VEGF)
is an important signaling protein involved in both vasculogenesis
(the formation of the embryonic circulatory system) and
angiogenesis (the growth of blood vessels from pre-existing
vasculature). In one embodiment, VEGF activity is restricted mainly
to cells of the vascular endothelium, although it does have effects
on a limited number of other cell types (e.g. stimulation
monocyte/macrophage migration). In vitro, VEGF has been shown to
stimulate endothelial cell mitogenesis and cell migration. VEGF
also enhances microvascular permeability and is sometimes referred
to as vascular permeability factor.
[0099] In one embodiment, all of the members of the VEGF family
stimulate cellular responses by binding to tyrosine kinase
receptors (the VEGFRs) on the cell surface, causing them to
dimerize and become activated through transphosphorylation. The
VEGF receptors have an extracellular portion consisting of 7
immunoglobulin-like domains, a single transmembrane spanning region
and an intracellular portion containing a split tyrosine-kinase
domain.
[0100] In one embodiment, VEGF-A is a VEGFR-2 (KDR/Flk-1) ligand as
well as a VEGFR-1 (Ht-1) ligand. In one embodiment, VEGFR-mediates
almost all of the known cellular responses to VEGF. The function of
VEGFR-1 is less well defined, although it is thought to modulate
VEGFR-2 signaling, in one embodiment, via sequestration of VEGF
from VEGFR-2 binding, which in one embodiment, is particularly
important during vasculogenesis in the embryo. In one embodiment,
VEGF-C and VEGF-D are ligands of the VEGFR-3 receptor, which in one
embodiment, mediates lymphangiogenesis.
[0101] In one embodiment, the compositions disclosed herein
comprise a VEGF receptor or a fragment thereof, which in one
embodiment, is a VEGFR-2 and, in another embodiment, a VEGFR-1,
and, in another embodiment, VEGFR-3.
[0102] In one embodiment, vascular Endothelial Growth Factor
Receptor 2 (VEGFR2) is highly expressed on activated endothelial
cells (ECs) and participates in the formation of new blood vessels.
In one embodiment, VEGFR2 binds all 5 isoforms of VEGF. In one
embodiment, signaling of VEGF through VEGFR2 on ECs induces
proliferation, migration, and eventual differentiation. In one
embodiment, the mouse homologue of VEGFR2 is the fetal liver kinase
gene-1 (Elk-1), which is a strong therapeutic target, and has
important roles in tumor growth, invasion, and metastasis. In one
embodiment, VEGFR2 is also referred to as kinase insert domain
receptor (a type III receptor tyrosine kinase) (KDR), cluster of
differentiation 309 (CD309), FLK1, Ly73, Krd-1, VEGFR, VEGFR-2, or
6130401C07.
[0103] In other embodiments, the antigen is derived from a fungal
pathogen, bacteria, parasite, helminth, or viruses. In other
embodiments, the antigen is selected from tetanus toxoid,
hemagglutinin molecules from influenza virus, diphtheria toxoid,
HIV gp120, HIV gag protein, IgA protease, insulin peptide B,
Spongospora subterranea antigen, vibriose antigens, Salmonella
antigens, pneumococcus antigens, respiratory syncytial virus
antigens, Haemophilus influenza outer membrane proteins,
Helicobacter pylori urease, Neisseria meningitidis pilins, N.
gonorrhoeae pilins, the melanoma-associated antigens (TRP-2,
MAGE-1, MAGE-3, gp-100, tyrosinase, MART-1, HSP-70, beta-HCG),
human papilloma virus antigens E1 and E2 from type HPV-16, -18,
-31, -33, -35 or -45 human papilloma viruses, the tumor antigens
CEA, the ras protein, mutated or otherwise, the p53 protein,
mutated or otherwise, Muc1, or pSA.
[0104] In other embodiments, the antigen is associated with one of
the following diseases; cholera, diphtheria, Haemophilus, hepatitis
A, hepatitis B, influenza, measles, meningitis, mumps, pertussis,
small pox, pneumococcal pneumonia, polio, rabies, rubella, tetanus,
tuberculosis, typhoid, Varicella-zoster, whooping cough3 yellow
fever, the immunogens and antigens from Addison's disease,
allergies, anaphylaxis, Bruton's syndrome, cancer, including solid
and blood borne tumors, eczema, Hashimoto's thyroiditis,
polymyositis, dermatomyositis, type 1 diabetes mellitus, acquired
immune deficiency syndrome, transplant rejection, such as kidney,
heart, pancreas, lung, bone, and liver transplants, Graves'
disease, polyendocrine autoimmune disease, hepatitis, microscopic
polyarteritis, polyarteritis nodosa, pemphigus, primary biliary
cirrhosis, pernicious anemia, coeliac disease, antibody-mediated
nephritis, glomerulonephritis, rheumatic diseases, systemic lupus
erthematosus, rheumatoid arthritis, seronegative
spondylarthritides, rhinitis, sjogren's syndrome, systemic
sclerosis, sclerosing cholangitis, Wegener's granulomatosis,
dermatitis herpetiformis, psoriasis, vitiligo, multiple sclerosis,
encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis,
Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic
mucocutaneous candidiasis, urticaria, transient
hypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM
syndrome, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune hemolytic anemia, autoimmune thrombocytopenia,
autoimmune neutropenia, Waldenstrom's macroglobulinemia,
amyloidosis, chronic lymphocytic leukemia, non-Hodgkin's lymphoma,
malarial circumsporozite protein, microbial antigens, viral
antigens, autoantigens, and lesteriosis.
[0105] In another embodiment, an HPV E6 antigen is utilized instead
of or in addition to an E7 antigen in a method disclosed herein for
treating, protecting against, or inducing an immune response
against a cervical cancer.
[0106] In another embodiment, an ActA protein fragment is utilized
instead of or in addition to an LLO fragment in a method disclosed
herein for treating, protecting against, or inducing an immune
response against a cervical cancer.
[0107] In another embodiment, a PEST amino acid sequence-containing
protein fragment is utilized instead of or in addition to an LLO
fragment in a method disclosed herein for treating, protecting
against, or inducing an immune response against a cervical
cancer.
[0108] In another embodiment, disclosed herein is an immunogenic
composition comprising a recombinant Listeria of the present
invention. In another embodiment, the immunogenic composition of
methods and compositions disclosed herein comprises a recombinant
vaccine vector of the present invention. In another embodiment, the
immunogenic composition comprises a plasmid of the present
invention. In another embodiment, the immunogenic composition
comprises an adjuvant. In one embodiment, a vector disclosed herein
may be administered as part of a vaccine composition.]
[0109] In another embodiment, a vaccine disclosed herein is
delivered with an adjuvant. In one embodiment, the adjuvant favors
a predominantly Th1-mediated immune response. In another
embodiment, the adjuvant favors a Th1-type immune response. In
another embodiment, the adjuvant favors a Th1-mediated immune
response. In another embodiment, the adjuvant favors a
cell-mediated immune response over an antibody-mediated response.
In another embodiment, the adjuvant is any other type of adjuvant
known in the art. In another embodiment, the immunogenic
composition induces the formation of a T cell immune response
against a target protein present on a tumor cell.
[0110] In another embodiment, the present invention provides a
method for inducing an anti-E7 cytotoxic T cell (CTL) response in a
human subject, comprising the step of administering to the subject
a recombinant Listeria strain, the recombinant Listeria strain
comprising a recombinant polypeptide comprising an N-terminal
fragment of an LLO protein and an HPV E7 antigen, thereby inducing
an anti-E7 CTL response in a human subject. In another embodiment,
the recombinant Listeria strain comprises a plasmid that encodes
the recombinant polypeptide. In another embodiment, the method
further comprises the step of boosting the subject with a
recombinant Listeria strain of the present invention. In another
embodiment, the method further comprises the step of boosting the
subject with an immunogenic composition comprising an E7 antigen.
In another embodiment, the method further comprises the step of
boosting the subject with an immunogenic composition that directs a
cell of the subject to express an E7 antigen. In another
embodiment, the CTL response is capable of therapeutic efficacy
against an HPV-mediated disease, disorder, or symptom. In another
embodiment, the CTL response is capable of prophylactic efficacy
against an HPV-mediated disease, disorder, or symptom.
[0111] In another embodiment, the present invention provides a
method of treating or ameliorating an HPV-mediated disease,
disorder, or symptom in a subject, comprising the step of
administering to the subject a recombinant Listeria strain, the
recombinant Listeria strain comprising a recombinant polypeptide
comprising an N-terminal fragment of an LLO protein and an HPV E7
antigen, whereby the recombinant Listeria strain induces an immune
response against the E7 antigen, thereby treating or ameliorating
an HPV-mediated disease, disorder, or symptom in a subject. In
another embodiment, the subject is a human subject. In another
embodiment, the subject is a non-human mammal. In another
embodiment, the subject is any other type of subject known in the
art.
[0112] The HPV causing the disease, disorder, or symptom is, in
another embodiment, an HPV 16. In another embodiment, the HPV is an
HPV-18. In another embodiment, the HPV is an HPV-31. In another
embodiment, the HPV is an HPV-35. In another embodiment, the HPV is
an HPV-39. In another embodiment, the HPV is an HPV-45. In another
embodiment, the HPV is an HPV-51. In another embodiment, the HPV is
an HPV-52. In another embodiment, the HPV is an HPV-58. In another
embodiment, the HPV is a high-risk HPV type. In another embodiment,
the HPV is a mucosal HPV type.
[0113] In another embodiment, the HPV-mediated disease, disorder,
or symptom is genital warts. In another embodiment, the
HPV-mediated disease, disorder, or symptom is non-genital warts. In
another embodiment, the HPV-mediated disease, disorder, or symptom
is a respiratory papilloma. In another embodiment, the HPV-mediated
disease, disorder, or symptom is any other HPV-mediated disease,
disorder, or symptom known in the art.
[0114] In another embodiment, an HPV E6 antigen is utilized instead
of or in addition to an E7 antigen in a method disclosed herein for
treating or ameliorating an HPV-mediated disease, disorder, or
symptom.
[0115] In another embodiment, an ActA protein fragment is utilized
instead of or in addition to an LLO fragment in a method disclosed
herein for treating or ameliorating an HPV-mediated disease,
disorder, or symptom.
[0116] In another embodiment, a PEST amino acid sequence-containing
protein fragment is utilized instead of or in addition to an LLO
fragment in a method disclosed herein for treating or ameliorating
an HPV-mediated disease, disorder, or symptom.
[0117] In another embodiment, an HPV E6 antigen is utilized instead
of or in addition to an E7 antigen in a method disclosed herein for
treating or ameliorating an HPV-mediated disease, disorder, or
symptom.
[0118] The antigen of methods and compositions disclosed herein is,
in another embodiment, an HPV E7 protein. In another embodiment,
the antigen is an HPV E6 protein. In another embodiment, the
antigen is any other HPV protein known in the art.
[0119] "E7 antigen" refers, in another embodiment, to an E7
protein. In another embodiment, the term refers to an E7 fragment.
In another embodiment, the term refers to an E7 peptide. In another
embodiment, the term refers to any other type of E7 antigen known
in the art.
[0120] The E7 protein of methods and compositions disclosed herein
is, in another embodiment, an HPV 16 E7 protein. In another
embodiment, the E7 protein is an HPV-18 E7 protein. In another
embodiment, the E7 protein is an HPV-31 E7 protein. In another
embodiment, the E7 protein is an HPV-35 E7 protein. In another
embodiment, the E7 protein is an HPV-39 E7 protein. In another
embodiment, the E7 protein is an HPV-45 E7 protein. In another
embodiment, the E7 protein is an HPV-51 E7 protein. In another
embodiment, the E7 protein is an HPV-52 E7 protein. In another
embodiment, the E7 protein is an HPV-58 E7 protein. In another
embodiment, the E7 protein is an E7 protein of a high-risk HPV
type. In another embodiment, the E7 protein is an E7 protein of a
mucosal HPV type.
[0121] "E6 antigen" refers, in another embodiment, to an E6
protein. In another embodiment, the term refers to an E6 fragment.
In another embodiment, the term refers to an E6 peptide. In another
embodiment, the term refers to any other type of E6 antigen known
in the art.
[0122] The E6 protein of methods and compositions disclosed herein
is, in another embodiment, an HPV 16 E6 protein. In another
embodiment, the E6 protein is an HPV-18 E6 protein. In another
embodiment, the E6 protein is an HPV-31 E6 protein. In another
embodiment, the E6 protein is an HPV-35 E6 protein. In another
embodiment, the E6 protein is an HPV-39 E6 protein. In another
embodiment, the E6 protein is an HPV-45 E6 protein. In another
embodiment, the E6 protein is an HPV-51 E6 protein. In another
embodiment, the E6 protein is an HPV-52 E6 protein. In another
embodiment, the E6 protein is an HPV-58 E6 protein. In another
embodiment, the E6 protein is an E6 protein of a high-risk HPV
type. In another embodiment, the E6 protein is an E6 protein of a
mucosal HPV type.
[0123] The immune response induced by methods and compositions
disclosed herein is, in another embodiment, a T cell response. In
another embodiment, the immune response comprises a T cell
response. In another embodiment, the response is a CD8.sup.+ T cell
response. In another embodiment, the response comprises a CD8.sup.+
T cell response.
[0124] In one embodiment, compositions disclosed herein induce a
strong innate stimulation of interferon-gamma, which in one
embodiment, has anti-angiogenic properties. In one embodiment, a
Listeria disclosed herein induces a strong innate stimulation of
interferon-gamma, which in one embodiment, has anti-angiogenic
properties (Dominiecki et al., Cancer Immunol Immunother. 2005 May;
54(5):477-88. Epub 2004 Oct. 6, incorporated herein by reference in
its entirety; Beatty and Paterson, J Immunol. 2001 Feb. 15;
166(4):2276-82, incorporated herein by reference in its entirety).
In another embodiment, methods disclosed herein increase a level of
interferon-gamma producing cells. In one embodiment,
anti-angiogenic properties of Listeria are mediated by CD4.sup.+ T
cells (Beatty and Paterson, 2001). In another embodiment,
anti-angiogenic properties of Listeria are mediated by CD8.sup.+ T
cells. In another embodiment, IFN-gamma secretion as a result of
Listeria vaccination is mediated by NK cells, NKT cells, Th1
CD4.sup.+ T cells, TC1 CD8.sup.+ T cells, or a combination
thereof.
[0125] In another embodiment, compositions disclosed herein induce
production of one or more anti-angiogenic proteins or factors. In
one embodiment, the anti-angiogenic protein is IFN-gamma. In
another embodiment, the anti-angiogenic protein is pigment
epithelium-derived factor (PEDF); angiostatin; endostatin; fms-like
tyrosine kinase (sFlt)-1; or soluble endoglin (sEng). In one
embodiment, a Listeria disclosed herein is involved in the release
of anti-angiogenic factors, and, therefore, in one embodiment, has
a therapeutic role in addition to its role as a vector for
introducing an antigen to a subject.
[0126] In another embodiment, the administration of compositions
disclosed herein induces robust systemic antigen-specific immunity.
In another embodiment, the administration of compositions disclosed
herein induces epitope spreading. In another embodiment, the
administration of compositions disclosed herein induces broad-based
response to self-derived tumor or neoplasia antigens. In another
embodiment the immune response induced by methods and compositions
disclosed herein comprises an improvement of the overall balance of
suppressor and effector immune cells in the tumor or neoplasia
microenvironment (TME). In another embodiment the immune response
induced by methods and compositions disclosed herein comprises
improvement in the systemic balance of suppressor and effector
immunocytes.
[0127] In one embodiment, compositions and methods of use thereof
disclosed herein generate effector T cells that are able to
infiltrate the tumor or neoplasia, destroy tumor cells and
eradicate the disease. In another embodiment, methods of use of
this invention increase tumor infiltration by T effector cells. In
another embodiment, T effector cells comprise CD8+T cells. In
another embodiment, T effector cells comprise CD4+T cells.
[0128] In one embodiment, tumor infiltrating lymphocytes (TILs) are
associated with better prognosis in several tumors, such as colon,
ovarian and melanoma. In colon cancer, tumors without signs of
micrometastasis have an increased infiltration of immune cells and
a Th1 expression profile, which correlate with an improved survival
of patients. Moreover, the infiltration of the tumor by T cells has
been associated with success of immunotherapeutic approaches in
both pre-clinical and human trials. In one embodiment, the
infiltration of lymphocytes into the tumor site is dependent on the
up-regulation of adhesion molecules in the endothelial cells of the
tumor vasculature, generally by proinflammatory cytokines, such as
IFN-.gamma., TNF-.alpha. and IL-1. Several adhesion molecules have
been implicated in the process of lymphocyte infiltration into
tumors, including intercellular adhesion molecule 1 (ICAM-1),
vascular endothelial cell adhesion molecule 1 (V-CAM-1), vascular
adhesion protein 1 (VAP-1) and E-selectin. However, these
cell-adhesion molecules are commonly down-regulated in the tumor
vasculature. Thus, in one embodiment, cancer vaccines. As disclosed
herein increase TILs, up-regulate adhesion molecules (in one
embodiment, ICAM-1, V-CAM-1, VAP-1, E-selectin, or a combination
thereof), up-regulate pro-inflammatory cytokines (in one
embodiment, IFN-.gamma., TNF-.alpha., IL-1, or a combination
thereof), or a combination thereof.
[0129] The N-terminal LLO protein fragment of methods and
compositions disclosed herein comprises, in another embodiment, SEQ
ID No: 2. In another embodiment, the fragment comprises an LLO
signal peptide. In another embodiment, the fragment comprises SEQ
ID No: 2. In another embodiment, the fragment consists
approximately of SEQ ID No: 2. In another embodiment, the fragment
consists essentially of SEQ ID No: 2. In another embodiment, the
fragment corresponds to SEQ ID No: 2. In another embodiment, the
fragment is homologous to SEQ ID No: 2. In another embodiment, the
fragment is homologous to a fragment of SEQ ID No: 2. The
.DELTA.LLO used in some of the Examples was 416 AA long (exclusive
of the signal sequence), as 88 residues from the amino terminus
which is inclusive of the activation domain containing cysteine 484
were truncated. It will be clear to those skilled in the art that
any .DELTA.LLO without the activation domain, and in particular
without cysteine 484, are suitable for methods and compositions of
the present invention. In another embodiment, fusion of an E7 or E6
antigen to any .DELTA.LLO, including the PEST amino acid AA
sequence, SEQ ID NO: 1, enhances cell mediated and anti-tumor
immunity of the antigen.
[0130] The LLO protein utilized to construct vaccines disclosed
herein has, in another embodiment, the sequence:
[0131] MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEK
KHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSIN
QNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQ
DNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQL
IAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKA
VTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKS
VSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVP
IAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNY
DPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRT
VIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE (GenBank Accession No. P13128;
SEQ ID NO: 3; nucleic acid sequence is set forth in GenBank
Accession No. X15127). The first 25 AA of the proprotein
corresponding to this sequence are the signal sequence and are
cleaved from LLO when it is secreted by the bacterium. Thus, in
this embodiment, the full length active LLO protein is 504 residues
long. In another embodiment, the above LLO fragment is used as the
source of the LLO fragment incorporated in a vaccine of the present
invention.
[0132] In another embodiment, the N-terminal fragment of an LLO
protein utilized in compositions and methods disclosed herein has
the sequence:
TABLE-US-00001 (SEQ ID NO: 2)
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPK
TPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIV
VEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRD
SLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNV
SAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVIS
FKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGR
QVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGG
SAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVI
KNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD.
[0133] In another embodiment, the LLO fragment corresponds to about
AA 20-442 of an LLO protein utilized herein.
[0134] In another embodiment, the LLO fragment has the
sequence:
TABLE-US-00002 (SEQ ID NO: 4)
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPK
TPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIV
VEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRD
SLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYSNV
SAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVIS
FKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGR
QVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGG
SAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVI
KNNSEYIETTSKAYTD.
[0135] In another embodiment, "truncated LLO" or ".DELTA.LLO"
refers to a fragment of LLO that comprises the PEST amino acid
domain. In another embodiment, the terms refer to an LLO fragment
that comprises a PEST sequence.
[0136] In another embodiment, the terms refer to an LLO fragment
that does not contain the activation domain at the amino terminus
and does not include cysteine 484. In another embodiment, the terms
refer to an LLO fragment that is not hemolytic. In another
embodiment, the LLO fragment is rendered non-hemolytic by deletion
or mutation of the activation domain. In another embodiment, the
LLO fragment is rendered non-hemolytic by deletion or mutation of
cysteine 484. In another embodiment, the LLO fragment is rendered
non-hemolytic by deletion or mutation at another location.
[0137] In another embodiment, the LLO fragment consists of about
the first 441 AA of the LLO protein. In another embodiment, the LLO
fragment consists of about the first 420 AA of LLO. In another
embodiment, the LLO fragment is a non-hemolytic form of the LLO
protein.
[0138] In another embodiment, the LLO fragment contains residues of
a homologous LLO protein that correspond to one of the above AA
ranges. The residue numbers need not, in another embodiment,
correspond exactly with the residue numbers enumerated above; e.g.
if the homologous LLO protein has an insertion or deletion,
relative to an LLO protein utilized herein, then the residue
numbers can be adjusted accordingly.
[0139] In another embodiment, the LLO fragment is any other LLO
fragment known in the art.
[0140] In another embodiment, the recombinant Listeria strain is
administered to the human subject at a dose of
1.times.10.sup.9-3.31.times.10.sup.10 CFU. In another embodiment,
the dose is 5-500.times.10.sup.8 CFU. In another embodiment, the
dose is 7-500.times.10.sup.8 CFU. In another embodiment, the dose
is 10-500.times.10.sup.8 CFU. In another embodiment, the dose is
20-500.times.10.sup.8 CFU. In another embodiment, the dose is
30-500.times.10.sup.8 CFU. In another embodiment, the dose is
50-500.times.10.sup.8 CFU. In another embodiment, the dose is
70-500.times.10.sup.8 CFU. In another embodiment, the dose is
100-500.times.10.sup.8 CFU. In another embodiment, the dose is
150-500.times.10.sup.8 CFU. In another embodiment, the dose is
5-300.times.10.sup.8 CFU. In another embodiment, the dose is
5-200.times.10.sup.8 CFU. In another embodiment, the dose is
5-150.times.10.sup.8 CFU. In another embodiment, the dose is
5-100.times.10.sup.8 CFU. In another embodiment, the dose is
5-70.times.10.sup.8 CFU. In another embodiment, the dose is
5-50.times.10.sup.8 CFU. In another embodiment, the dose is
5-30.times.10.sup.8 CFU. In another embodiment, the dose is
5-20.times.10.sup.8 CFU. In another embodiment, the dose is
1-30.times.10.sup.9 CFU. In another embodiment, the dose is
1-20.times.10.sup.9 CFU. In another embodiment, the dose is
2-30.times.10.sup.9 CFU. In another embodiment, the dose is
1-10.times.10.sup.9 CFU. In another embodiment, the dose is
2-10.times.10.sup.9 CFU. In another embodiment, the dose is
3-10.times.10.sup.9 CFU. In another embodiment, the dose is
2-7.times.10.sup.9 CFU. In another embodiment, the dose is
2-5.times.10.sup.9 CFU. In another embodiment, the dose is
3-5.times.10.sup.9 CFU.
[0141] In another embodiment, the dose is 1.times.10.sup.9
organisms. In another embodiment, the dose is 1.5.times.10.sup.9
organisms. In another embodiment, the dose is 2.times.10.sup.9
organisms. In another embodiment, the dose is 3.times.10.sup.9
organisms. In another embodiment, the dose is 4.times.10.sup.9
organisms. In another embodiment, the dose is 5.times.10.sup.9
organisms. In another embodiment, the dose is 6.times.10.sup.9
organisms. In another embodiment, the dose is 7.times.10.sup.9
organisms. In another embodiment, the dose is 8.times.10.sup.9
organisms. In another embodiment, the dose is 10.times.10.sup.9
organisms. In another embodiment, the dose is 1.5.times.10.sup.10
organisms. In another embodiment, the dose is 2.times.10.sup.10
organisms. In another embodiment, the dose is 2.5.times.10.sup.10
organisms. In another embodiment, the dose is 3.times.10.sup.10
organisms. In another embodiment, the dose is 3.3.times.10.sup.10
organisms. In another embodiment, the dose is 4.times.10.sup.10
organisms. In another embodiment, the dose is 5.times.10.sup.10
organisms.
[0142] In another embodiment, the recombinant polypeptide of
methods disclosed herein is expressed by the recombinant Listeria
strain. In another embodiment, the expression is mediated by a
nucleotide molecule carried by the recombinant Listeria strain.
[0143] In another embodiment, the recombinant Listeria strain
expresses the recombinant polypeptide by means of a plasmid that
encodes the recombinant polypeptide. In another embodiment, the
plasmid comprises a gene encoding a bacterial transcription factor.
In another embodiment, the plasmid encodes a Listeria transcription
factor. In another embodiment, the transcription factor is PrfA. In
another embodiment, the PrfA is a mutant PrfA. In another
embodiment, the PrfA mutant protein contains a D133V amino acid
mutation.
[0144] In another embodiment, the plasmid comprises a gene encoding
a metabolic enzyme. In another embodiment, the metabolic enzyme is
a bacterial metabolic enzyme. In another embodiment, the metabolic
enzyme is a Listerial metabolic enzyme. In another embodiment, the
metabolic enzyme is an amino acid metabolism enzyme. In another
embodiment, the amino acid metabolism gene is involved in a cell
wall synthesis pathway. In another embodiment, the metabolic enzyme
is the product of a D-amino acid aminotransferase gene (dat). In
another embodiment, the metabolic enzyme is the product of an
alanine racemase gene (dal). In another embodiment, the metabolic
enzyme is any other metabolic enzyme known in the art.
[0145] In another embodiment, a method of present invention further
comprises the step of boosting the human subject with a recombinant
Listeria strain of the present invention. In another embodiment,
the recombinant Listeria strain used in the booster inoculation is
the same as the strain used in the initial "priming" inoculation.
In another embodiment, the booster strain is different from the
priming strain. In another embodiment, the same doses are used in
the priming and boosting inoculations. In another embodiment, a
larger dose is used in the booster. In another embodiment, a
smaller dose is used in the booster.
[0146] In another embodiment, a method of present invention further
comprises the step of inoculating the human subject with an
immunogenic composition comprising the E7 antigen. In another
embodiment, the immunogenic composition comprises a recombinant E7
protein or fragment thereof. In another embodiment, the immunogenic
composition comprises a nucleotide molecule expressing a
recombinant E7 protein or fragment thereof. In another embodiment,
the non-Listerial inoculation is administered after the Listerial
inoculation. In another embodiment, the non-Listerial inoculation
is administered before the Listerial inoculation.
[0147] "Boosting" refers, in another embodiment, to administration
of an additional vaccine dose to a subject. In another embodiment
of methods of the present invention, 2 boosts (or a total of 3
inoculations) are administered. In another embodiment, 3 boosts are
administered. In another embodiment, 4 boosts are administered. In
another embodiment, 5 boosts are administered. In another
embodiment, 6 boosts are administered. In another embodiment, more
than 6 boosts are administered.
[0148] The recombinant Listeria strain of methods and compositions
disclosed herein is, in another embodiment, a recombinant Listeria
monocytogenes strain. In another embodiment, the Listeria strain is
a recombinant Listeria seeligeri strain. In another embodiment, the
Listeria strain is a recombinant Listeria grayi strain. In another
embodiment, the Listeria strain is a recombinant Listeria ivanovii
strain. In another embodiment, the Listeria strain is a recombinant
Listeria murrayi strain. In another embodiment, the Listeria strain
is a recombinant Listeria welshimeri strain. In another embodiment,
the Listeria strain is a recombinant strain of any other Listeria
species known in the art.
[0149] The present invention provides a number of listerial species
and strains for making or engineering an attenuated Listeria of the
present invention. In one embodiment, the Listeria strain is L.
monocytogenes 10403S wild type (see Bishop and Hinrichs (1987) J.
Immunol. 139: 2005-2009; Lauer, et al. (2002) J. Bact. 184:
4177-4186.) In another embodiment, the Listeria strain is L.
monocytogenes DP-L4056 (phage cured) (see Lauer, et al. (2002) J.
Bact. 184: 4177-4186). In another embodiment, the Listeria strain
is L. monocytogenes DP-L4027, which is phage cured and deleted in
the hly gene (see Lauer, et al. (2002) J. Bact. 184: 4177-4186;
Jones and Portnoy (1994) Infect. Immunity 65: 5608-5613). In
another embodiment, the Listeria strain is L. monocytogenes
DP-L4029, which is phage cured, deleted in ActA (see Lauer, et al.
(2002) J. Bact. 184: 4177-4186; Skoble, et al. (2000) J. Cell Biol.
150: 527-538). In another embodiment, the Listeria strain is L.
monocytogenes DP-L4042 (delta PEST) (see Brockstedt, et al. (2004)
Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting
information). In another embodiment, the Listeria strain is L.
monocytogenes DP-L4097 (LLO-S44A) (see Brockstedt, et al. (2004)
Proc. Natl. Acad. Sci. USA 101: 13832-13837; supporting
information). In another embodiment, the Listeria strain is L.
monocytogenes DP-L4364 (delta 1p1A; lipoate protein ligase) (see
Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:
13832-13837; supporting information). In another embodiment, the
Listeria strain is L. monocytogenes DP-L4405 (delta in1A) (see
Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:
13832-13837; supporting information). In another embodiment, the
Listeria strain is L. monocytogenes DP-L4406 (delta in1B) (see
Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:
13832-13837; supporting information). In another embodiment, the
Listeria strain is L. monocytogenes CS-L0001 (delta ActA-delta
in1B) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA
101: 13832-13837; supporting information). In another embodiment,
the Listeria strain is L. monocytogenes CS-L0002 (delta ActA-delta
1p1A) (see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA
101: 13832-13837; supporting information). In another embodiment,
the Listeria strain is L. monocytogenes CS-L0003 (L461T-delta 1p1A)
(see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:
13832-13837; supporting information). In another embodiment, the
Listeria strain is L. monocytogenes DP-L4038 (delta ActA-LLO L461T)
(see Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:
13832-13837; supporting information). In another embodiment, the
Listeria strain is L. monocytogenes DP-L4384 (S44A-LLO L461T) (see
Brockstedt, et al. (2004) Proc. Natl. Acad. Sci. USA 101:
13832-13837; supporting information). In another embodiment, the
Listeria strain is L. monocytogenes. Mutation in lipoate protein
(see O'Riordan, et al. (2003) Science 302: 462-464). In another
embodiment, the Listeria strain is L. monocytogenes DP-L4017
(10403S hly (L461T), having a point mutation in hemolysin gene (see
U.S. Provisional Pat. Appl. Ser. No. 60/490,089 filed Jul. 24,
2003). In another embodiment, the Listeria strain is L.
monocytogenes EGD (see GenBank Acc. No. AL591824). In another
embodiment, the Listeria strain is L. monocytogenes EGD-e (see
GenBank Acc. No. NC_003210. ATCC Acc. No. BAA-679). In another
embodiment, the Listeria strain is L. monocytogenes DP-L4029
deleted in uvrAB (see U.S. Provisional Pat. Appl. Ser. No.
60/541,515 filed Feb. 2, 2004; U.S. Provisional Pat. Appl. Ser. No.
60/490,080 filed Jul. 24, 2003). In another embodiment, the
Listeria strain is L. monocytogenes ActA-/in1B-double mutant (see
ATCC Acc. No. PTA-5562). In another embodiment, the Listeria strain
is L. monocytogenes 1plA mutant or hly mutant (see U.S. Pat.
Applic. No. 20040013690 of Portnoy, et. al). In another embodiment,
the Listeria strain is L. monocytogenes DAL/DAT double mutant. (see
U.S. Pat. Applic. No. 20050048081 of Frankel and Portnoy. The
present invention encompasses reagents and methods that comprise
the above listerial strains, as well as these strains that are
modified, e.g., by a plasmid and/or by genomic integration, to
contain a nucleic acid encoding one of, or any combination of, the
following genes: hly (LLO; listeriolysin); iap (p60); in1A; in1B;
in1C; dal (alanine racemase); dat (D-amino acid aminotransferase);
plcA; plcB; actA; or any nucleic acid that mediates growth, spread,
breakdown of a single walled vesicle, breakdown of a double walled
vesicle, binding to a host cell, uptake by a host cell. The present
invention is not to be limited by the particular strains disclosed
above.
[0150] In another embodiment, a recombinant Listeria strain
disclosed herein has been passaged through an animal host. In
another embodiment, the passaging maximizes efficacy of the strain
as a vaccine vector. In another embodiment, the passaging
stabilizes the immunogenicity of the Listeria strain. In another
embodiment, the passaging stabilizes the virulence of the Listeria
strain. In another embodiment, the passaging increases the
immunogenicity of the Listeria strain. In another embodiment, the
passaging increases the virulence of the Listeria strain. In
another embodiment, the passaging removes unstable sub-strains of
the Listeria strain. In another embodiment, the passaging reduces
the prevalence of unstable sub-strains of the Listeria strain. In
another embodiment, the Listeria strain contains a genomic
insertion of the gene encoding the antigen-containing recombinant
peptide. In another embodiment, the Listeria strain carries a
plasmid comprising the gene encoding the antigen-containing
recombinant peptide. In another embodiment, the passaging is
performed as described herein (e.g. in Example 12). In another
embodiment, the passaging is performed by any other method known in
the art.
[0151] In another embodiment, the recombinant Listeria strain
utilized in methods disclosed herein has been stored in a frozen
cell bank. In another embodiment, the recombinant Listeria strain
has been stored in a lyophilized cell bank.
[0152] In another embodiment, the cell bank of methods and
compositions disclosed herein is a master cell bank. In another
embodiment, the cell bank is a working cell bank. In another
embodiment, the cell bank is Good Manufacturing Practice (GMP) cell
bank. In another embodiment, the cell bank is intended for
production of clinical-grade material. In another embodiment, the
cell bank conforms to regulatory practices for human use. In
another embodiment, the cell bank is any other type of cell bank
known in the art.
[0153] "Good Manufacturing Practices" are defined, in another
embodiment, by (21 CFR 210-211) of the United States Code of
Federal Regulations. In another embodiment, "Good Manufacturing
Practices" are defined by other standards for production of
clinical-grade material or for human consumption; e.g. standards of
a country other than the United States.
[0154] In another embodiment, a recombinant Listeria strain
utilized in methods disclosed herein is from a batch of vaccine
doses.
[0155] In another embodiment, a recombinant Listeria strain
utilized in methods disclosed herein is from a frozen or
lyophilized stock produced by methods provided in U.S. Pat. No.
8,114,414, which is incorporated by reference herein.
[0156] In another embodiment, a peptide disclosed herein is a
fusion peptide. In another embodiment, "fusion peptide" refers to a
peptide or polypeptide comprising 2 or more proteins linked
together by peptide bonds or other chemical bonds. In another
embodiment, the proteins are linked together directly by a peptide
or other chemical bond. In another embodiment, the proteins are
linked together with 1 or more AA (e.g. a "spacer") between the 2
or more proteins.
[0157] In another embodiment, a vaccine disclosed herein further
comprises an adjuvant. The adjuvant utilized in methods and
compositions disclosed herein is, in another embodiment, a
granulocyte/macrophage colony-stimulating factor (GM-CSF) protein.
In another embodiment, the adjuvant comprises a GM-CSF protein. In
another embodiment, the adjuvant is a nucleotide molecule encoding
GM-CSF. In another embodiment, the adjuvant comprises a nucleotide
molecule encoding GM-CSF. In another embodiment, the adjuvant is
saponin QS21. In another embodiment, the adjuvant comprises saponin
QS21. In another embodiment, the adjuvant is monophosphoryl lipid
A. In another embodiment, the adjuvant comprises monophosphoryl
lipid A. In another embodiment, the adjuvant is SBAS2. In another
embodiment, the adjuvant comprises SBAS2. In another embodiment,
the adjuvant is an unmethylated CpG-containing oligonucleotide. In
another embodiment, the adjuvant comprises an unmethylated
CpG-containing oligonucleotide. In another embodiment, the adjuvant
is an immune-stimulating cytokine. In another embodiment, the
adjuvant comprises an immune-stimulating cytokine. In another
embodiment, the adjuvant is a nucleotide molecule encoding an
immune-stimulating cytokine. In another embodiment, the adjuvant
comprises a nucleotide molecule encoding an immune-stimulating
cytokine. In another embodiment, the adjuvant is or comprises a
quill glycoside. In another embodiment, the adjuvant is or
comprises a bacterial mitogen. In another embodiment, the adjuvant
is or comprises a bacterial toxin. In another embodiment, the
adjuvant is or comprises any other adjuvant known in the art.
[0158] In another embodiment, a nucleotide disclosed herein is
operably linked to a promoter/regulatory sequence that drives
expression of the encoded peptide in the Listeria strain.
Promoter/regulatory sequences useful for driving constitutive
expression of a gene are well known in the art and include, but are
not limited to, for example, the P.sub.hlyA, P.sub.ActA, and p60
promoters of Listeria, the Streptococcus bac promoter, the
Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ
promoter. In another embodiment, inducible and tissue specific
expression of the nucleic acid encoding a peptide disclosed herein
is accomplished by placing the nucleic acid encoding the peptide
under the control of an inducible or tissue specific
promoter/regulatory sequence. Examples of tissue specific or
inducible promoter/regulatory sequences which are useful for his
purpose include, but are not limited to the MMTV LTR inducible
promoter, and the SV40 late enhancer/promoter. In another
embodiment, a promoter that is induced in response to inducing
agents such as metals, glucocorticoids, and the like, is utilized.
Thus, it will be appreciated that the invention includes the use of
any promoter/regulatory sequence, which is either known or unknown,
and which is capable of driving expression of the desired protein
operably linked thereto.
[0159] An N-terminal fragment of an ActA protein utilized in
methods and compositions disclosed herein has, in another
embodiment, the sequence set forth in SEQ ID NO: 5:
[0160] MRAMMVVFITANCITINPDHFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPR
YETAREVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAA
INEEASGADRPAIQVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVN
KKKVAKESVADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWV
RDKIDENPEVKKAIVDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETP
MLLGFNAPATSEPSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPT
EDELEIIRETASSLDSSFTRGDLASLRNAINRHSQNFSDFPPIPTEEELNGRGGRP. In
another embodiment, the ActA fragment comprises the sequence set
forth in SEQ ID NO: 5. In another embodiment, the ActA fragment is
any other ActA fragment known in the art.
[0161] In another embodiment, the recombinant nucleotide encoding a
fragment of an ActA protein comprises the sequence set forth in SEQ
ID NO: 6:
[0162]
Atgcgtgcgatgatggtggttttcattactgccaattgcattacgattaaccccgacataatatttg-
cagcgacagatagcgaa
gattctagtctaaacacagatgaatgggaagaagaaaaaacagaagagcaaccaagcgaggtaaatacgggac-
caagatacg
aaactgcacgtgaagtaagttcacgtgatattaaagaactagaaaaatcgaataaagtgagaaa-
tacgaacaaagcagacctaat
agcaatgttgaaagaaaaagcagaaaaaggtccaaatatcaataataacaacagtgaacaaactgagaatgcg-
gctataaatga
agaggcttcaggagccgaccgaccagctatacaagtggagcgtcgtcatccaggattgccatcggatagcgca-
gcggaaatta
aaaaaagaaggaaagccatagcatcatcggatagtgagcttgaaagccttacttatccggata-
aaccaacaaaagtaaataagaa
aaaagtggcgaaagagtcagttgcggatgcttctgaaagtgacttagattctagcatgcagtcagcagatgag-
tcttcaccacaac
ctttaaaagcaaaccaacaaccatttttccctaaagtatttaaaaaaataaaagatgcggggaaatgggtacg-
tgataaaatcgacg
aaaatcctgaagtaaagaaagcgattgttgataaaagtgcagggttaattgaccaattattaaccaaaaagaa-
aagtgaagaggta
aatgcttcggacttcccgccaccacctacggatgaagagttaagacttgctttgccagagacaccaatgcttc-
ttggttttaatgctc
ctgctacatcagaaccgagctcattcgaatttccaccaccacctacggatgaagagttaagacttgctttgcc-
agagacgccaatg
cttcttggttttaatgctcctgctacatcggaaccgagctcgttcgaatttccaccgcctccaacagaagatg-
aactagaaatcatcc
gggaaacagcatcctcgctagattctagttttacaagaggggatttagctagtttgagaaatgctattaatcg-
ccatagtcaaaatttc
tctgatttcccaccaatcccaacagaagaagagttgaacgggagaggcggtagacca. In
another embodiment, the recombinant nucleotide has the sequence set
forth in SEQ ID NO: 6. In another embodiment, the recombinant
nucleotide comprises any other sequence that encodes a fragment of
an ActA protein.
[0163] In another embodiment of the methods and compositions of the
present invention, a PEST amino acid AA sequence is fused to the E7
or E6 antigen. As disclosed herein, recombinant Listeria strains
expressing PEST amino acid sequence-antigen fusions induce
anti-tumor immunity (Example 3) and generate antigen-specific,
tumor-infiltrating T cells (Example 4). Further, enhanced cell
mediated immunity was demonstrated for fusion proteins comprising
an antigen and LLO containing the PEST amino acid AA sequence
KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 1).
[0164] Thus, fusion of an antigen to other LM PEST amino acid
sequences and PEST amino acid sequences derived from other
prokaryotic organisms will also enhance immunogenicity of the
antigen. The PEST amino acid AA sequence has, in another
embodiment, a sequence selected from SEQ ID NO: 7-12. In another
embodiment, the PEST amino acid sequence is a PEST amino acid
sequence from the LM ActA protein. In another embodiment, the PEST
amino acid sequence is KTEEQPSEVNTGPR (SEQ ID NO: 7),
KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 8), KNEEVNASDFPPPPTDEELR
(SEQ ID NO: 9), or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO:
10). In another embodiment, the PEST amino acid sequence is from
Streptolysin O protein of Streptococcus sp. In another embodiment,
the PEST amino acid sequence is from Streptococcus pyogenes
Streptolysin O, e.g. KQNTASTETTTTNEQPK (SEQ ID NO: 11) at AA 35-51.
In another embodiment, the PEST amino acid sequence is from
Streptococcus equisimilis Streptolysin O, e.g. KQNTANTETTTTNEQPK
(SEQ ID NO:12) at AA 38-54. In another embodiment, the PEST amino
acid sequence is another PEST amino acid AA sequence derived from a
prokaryotic organism. In another embodiment, the PEST amino acid
sequence is any other PEST amino acid sequence known in the
art.
[0165] PEST amino acid sequences of other prokaryotic organism can
be identified in accordance with methods such as described by, for
example Rechsteiner and Rogers (1996, Trends Biochem. Sci.
21:267-271) for LM. Alternatively, PEST amino acid AA sequences
from other prokaryotic organisms can also be identified based by
this method. Other prokaryotic organisms wherein PEST amino acid AA
sequences would be expected to include, but are not limited to,
other Listeria species. In another embodiment, the PEST amino acid
sequence is embedded within the antigenic protein. Thus, in another
embodiment, "fusion" refers to an antigenic protein comprising both
the antigen and the PEST amino acid amino acid sequence either
linked at one end of the antigen or embedded within the
antigen.
[0166] In another embodiment, the PEST amino acid sequence is
identified using any other method or algorithm known in the art,
e.g the CaSPredictor (Garay-Malpartida HM, Occhiucci JM, Alves J,
Belizario JE. Bioinformatics. 2005 June; 21 Suppl 1:i169-76). In
another embodiment, the following method is used:
[0167] A PEST index is calculated for each 30-35 AA stretch by
assigning a value of 1 to the amino acids Ser, Thr, Pro, Glu, Asp,
Asn, or Gln. The coefficient value (CV) for each of the PEST
residue is 1 and for each of the other AA (non-PEST) is 0.
[0168] In another embodiment, the LLO protein, ActA protein, or
fragment thereof disclosed herein need not be that which is set
forth exactly in the sequences set forth herein, but rather other
alterations, modifications, or changes can be made that retain the
functional characteristics of an LLO or ActA protein fused to an
antigen as set forth elsewhere herein. In another embodiment, the
present invention utilizes an analog of an LLO protein, ActA
protein, or fragment thereof. Analogs differ, in another
embodiment, from naturally occurring proteins or peptides by
conservative AA sequence differences or by modifications which do
not affect sequence, or by both.
[0169] In another embodiment, either a whole E7 protein or a
fragment thereof is fused to a LLO protein, ActA protein, or PEST
amino acid sequence-containing peptide to generate a recombinant
peptide of methods of the present invention. The E7 protein that is
utilized (either whole or as the source of the fragments) has, in
another embodiment, the sequence
[0170] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHY
NIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID No: 13). In
another embodiment, the E7 protein is a homologue of SEQ ID No: 13.
In another embodiment, the E7 protein is a variant of SEQ ID No:
13. In another embodiment, the E7 protein is an isomer of SEQ ID
No: 13. In another embodiment, the E7 protein is a fragment of SEQ
ID No: 13. In another embodiment, the E7 protein is a fragment of a
homologue of SEQ ID No: 13. In another embodiment, the E7 protein
is a fragment of a variant of SEQ ID No: 13. In another embodiment,
the E7 protein is a fragment of an isomer of SEQ ID No: 13.
[0171] In another embodiment, the sequence of the E7 protein
is:
[0172] MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARR
AEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ (SEQ ID No:
14). In another embodiment, the E6 protein is a homologue of SEQ ID
No: 14. In another embodiment, the E6 protein is a variant of SEQ
ID No: 14. In another embodiment, the E6 protein is an isomer of
SEQ ID No: 14. In another embodiment, the E6 protein is a fragment
of SEQ ID No: 14. In another embodiment, the E6 protein is a
fragment of a homologue of SEQ ID No: 14. In another embodiment,
the E6 protein is a fragment of a variant of SEQ ID No: 14. In
another embodiment, the E6 protein is a fragment of an isomer of
SEQ ID No: 14.
[0173] In another embodiment, the E7 protein has a sequence set
forth in one of the following GenBank entries: M24215, NC_004500,
V01116, X62843, or M14119. In another embodiment, the E7 protein is
a homologue of a sequence from one of the above GenBank entries. In
another embodiment, the E7 protein is a variant of a sequence from
one of the above GenBank entries. In another embodiment, the E7
protein is an isomer of a sequence from one of the above GenBank
entries. In another embodiment, the E7 protein is a fragment of a
sequence from one of the above GenBank entries. In another
embodiment, the E7 protein is a fragment of a homologue of a
sequence from one of the above GenBank entries. In another
embodiment, the E7 protein is a fragment of a variant of a sequence
from one of the above GenBank entries. In another embodiment, the
E7 protein is a fragment of an isomer of a sequence from one of the
above GenBank entries.
[0174] In another embodiment, either a whole E6 protein or a
fragment thereof is fused to a LLO protein, ActA protein, or PEST
amino acid sequence-containing peptide to generate a recombinant
peptide of methods of the present invention. The E6 protein that is
utilized (either whole or as the source of the fragments) has, in
another embodiment, the sequence
[0175] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFA
FRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCI
NCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID No: 15). In
another embodiment, the E6 protein is a homologue of SEQ ID No: 15.
In another embodiment, the E6 protein is a variant of SEQ ID No:
15. In another embodiment, the E6 protein is an isomer of SEQ ID
No: 15. In another embodiment, the E6 protein is a fragment of SEQ
ID No: 15. In another embodiment, the E6 protein is a fragment of a
homologue of SEQ ID No: 15. In another embodiment, the E6 protein
is a fragment of a variant of SEQ ID No: 15. In another embodiment,
the E6 protein is a fragment of an isomer of SEQ ID No: 15.
[0176] In another embodiment, the sequence of the E6 protein
is:
[0177] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFV
VYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPL
NPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV (SEQ ID No: 16). In
another embodiment, the E6 protein is a homologue of SEQ ID No: 16.
In another embodiment, the E6 protein is a variant of SEQ ID No:
16. In another embodiment, the E6 protein is an isomer of SEQ ID
No: 16. In another embodiment, the E6 protein is a fragment of SEQ
ID No: 16. In another embodiment, the E6 protein is a fragment of a
homologue of SEQ ID No: 16. In another embodiment, the E6 protein
is a fragment of a variant of SEQ ID No: 16. In another embodiment,
the E6 protein is a fragment of an isomer of SEQ ID No: 16.
[0178] In another embodiment, the E6 protein has a sequence set
forth in one of the following GenBank entries: M24215, M14119,
NC_004500, V01116, X62843, or M14119. In another embodiment, the E6
protein is a homologue of a sequence from one of the above GenBank
entries. In another embodiment, the E6 protein is a variant of a
sequence from one of the above GenBank entries. In another
embodiment, the E6 protein is an isomer of a sequence from one of
the above GenBank entries. In another embodiment, the E6 protein is
a fragment of a sequence from one of the above GenBank entries. In
another embodiment, the E6 protein is a fragment of a homologue of
a sequence from one of the above GenBank entries. In another
embodiment, the E6 protein is a fragment of a variant of a sequence
from one of the above GenBank entries. In another embodiment, the
E6 protein is a fragment of an isomer of a sequence from one of the
above GenBank entries.
[0179] In another embodiment, "homology" refers to identity to an
LLO sequence (e.g. to one of SEQ ID No: 2-4) of greater than 70%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 2-4 of greater than 64%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 68%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 72%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 75%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 78%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 80%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 82%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 83%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 85%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 87%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 88%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 90%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 92%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 93%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 95%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 96%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 97%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of greater than 98%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 2-4 of greater than 99%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 2-4 of 100%.
[0180] In another embodiment, "homology" refers to identity to an
E7 sequence (e.g. to one of SEQ ID No: 13-14) of greater than 70%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 62%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 64%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 68%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 72%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 75%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 78%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 80%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 82%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 83%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 85%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 87%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 88%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 90%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 92%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 93%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 95%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 96%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 97%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of greater than 98%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 13-14 of greater than 99%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 13-14 of 100%.
[0181] In another embodiment, "homology" refers to identity to an
E6 sequence (e.g. to one of SEQ ID No: 15-16) of greater than 70%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 64%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 68%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 72%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 75%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 78%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 80%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 82%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 83%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 85%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 87%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 88%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 90%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 92%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 93%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 95%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 96%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 97%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of greater than 98%.
In another embodiment, "homology" refers to identity to one of SEQ
ID No: 15-16 of greater than 99%. In another embodiment, "homology"
refers to identity to one of SEQ ID No: 15-16 of 100%.
[0182] In another embodiment, "homology" refers to identity to a
PEST amino acid sequence (e.g. to one of SEQ ID No: 1, and 7-12) or
to an ActA sequence (e.g. to one of SEQ ID No: 5-6) of greater than
70%. In another embodiment, "homology" refers to identity to one of
SEQ ID No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 60%. In
another embodiment, "homology" refers to identity to one of SEQ ID
No: 1, and 7-12 or SEQ ID No: 5-6 of greater than 64%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 68%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 72%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 75%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 78%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 80%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 82%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 83%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 85%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 87%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 88%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 90%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 92%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 93%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 95%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 96%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 97%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 98%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of greater than 99%. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 1,
and 7-12 or SEQ ID No: 5-6 of 100%.
[0183] Protein and/or peptide homology for any AA sequence listed
herein is determined, in one embodiment, by methods well described
in the art, including immunoblot analysis, or via computer
algorithm analysis of AA sequences, utilizing any of a number of
software packages available, via established methods. Some of these
packages include the FASTA, BLAST, MPsrch or Scanps packages, and
employ, in other embodiments, the use of the Smith and Waterman
algorithms, and/or global/local or BLOCKS alignments for analysis,
for example.
[0184] In another embodiment, the LLO protein, ActA protein, or
fragment thereof is attached to the antigen by chemical
conjugation. In another embodiment, glutaraldehyde is used for the
conjugation. In another embodiment, the conjugation is performed
using any suitable method known in the art.
[0185] In another embodiment, fusion proteins disclosed herein are
prepared by any suitable method, including, for example, cloning
and restriction of appropriate sequences or direct chemical
synthesis by methods discussed below. In another embodiment,
subsequences are cloned and the appropriate subsequences cleaved
using appropriate restriction enzymes. The fragments are then
ligated, in another embodiment, to produce the desired DNA
sequence. In another embodiment, DNA encoding the fusion protein is
produced using DNA amplification methods, for example polymerase
chain reaction (PCR). First, the segments of the native DNA on
either side of the new terminus are amplified separately. The 5'
end of the one amplified sequence encodes the peptide linker, while
the 3' end of the other amplified sequence also encodes the peptide
linker. Since the 5' end of the first fragment is complementary to
the 3' end of the second fragment, the two fragments (after partial
purification, e.g. on LMP agarose) can be used as an overlapping
template in a third PCR reaction. The amplified sequence will
contain codons, the segment on the carboxy side of the opening site
(now forming the amino sequence), the linker, and the sequence on
the amino side of the opening site (now forming the carboxyl
sequence). The insert is then ligated into a plasmid.
[0186] In another embodiment, the LLO protein, ActA protein, or
fragment thereof and the antigen, or fragment thereof are
conjugated by a means known to those of skill in the art. In
another embodiment, the antigen, or fragment thereof is conjugated,
either directly or through a linker (spacer), to the ActA protein
or LLO protein. In another embodiment, the chimeric molecule is
recombinantly expressed as a single-chain fusion protein.
[0187] In another embodiment, a fusion peptide disclosed herein is
synthesized using standard chemical peptide synthesis techniques.
In another embodiment, the chimeric molecule is synthesized as a
single contiguous polypeptide. In another embodiment, the LLO
protein, ActA protein, or fragment thereof; and the antigen, or
fragment thereof are synthesized separately, then fused by
condensation of the amino terminus of one molecule with the
carboxyl terminus of the other molecule, thereby forming a peptide
bond. In another embodiment, the ActA protein or LLO protein and
antigen are each condensed with one end of a peptide spacer
molecule, thereby forming a contiguous fusion protein.
[0188] In another embodiment, the peptides and proteins disclosed
herein are prepared by solid-phase peptide synthesis (SPPS) as
described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd
Edition, 1984, Pierce Chemical Company, Rockford, Ill.; or as
described by Bodanszky and Bodanszky (The Practice of Peptide
Synthesis, 1984, Springer-Verlag, New York). In another embodiment,
a suitably protected AA residue is attached through its carboxyl
group to a derivatized, insoluble polymeric support, such as
cross-linked polystyrene or polyamide resin. "Suitably protected"
refers to the presence of protecting groups on both the alpha-amino
group of the amino acid, and on any side chain functional groups.
Side chain protecting groups are generally stable to the solvents,
reagents and reaction conditions used throughout the synthesis, and
are removable under conditions which will not affect the final
peptide product. Stepwise synthesis of the oligopeptide is carried
out by the removal of the N-protecting group from the initial AA,
and couple thereto of the carboxyl end of the next AA in the
sequence of the desired peptide. This AA is also suitably
protected. The carboxyl of the incoming AA can be activated to
react with the N-terminus of the support-bound AA by formation into
a reactive group such as formation into a carbodiimide, a symmetric
acid anhydride or an "active ester" group such as
hydroxybenzotriazole or pentafluorophenly esters.
[0189] In another embodiment, the present invention provides a kit
comprising vaccine of the present invention, an applicator, and
instructional material that describes use of the methods of the
invention. Although model kits are described below, the contents of
other useful kits will be apparent to the skilled artisan in light
of the present disclosure.
EXPERIMENTAL DETAILS SECTION
Example 1
LLO-Antigen Fusions Induce Anti-Tumor Immunity
Materials and Experimental Methods (Examples 1-2)
Cell Lines
[0190] The C57BL/6 syngeneic TC-1 tumor was immortalized with
HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene. TC-1,
provided by T. C. Wu (Johns Hopkins University School of Medicine,
Baltimore, Md.) is a highly tumorigenic lung epithelial cell
expressing low levels of with HPV-16 E6 and E7 and transformed with
the c-Ha-ras oncogene. TC-1 was grown in RPMI 1640, 10% FCS, 2 mM
L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml streptomycin, 100
.mu.M nonessential amino acids, 1 mM sodium pyruvate, 50 micromolar
(mcM) 2-ME, 400 microgram (mcg)/ml G418, and 10% National
Collection Type Culture-109 medium at 37.degree. with 10% CO.sub.2.
C3 is a mouse embryo cell from C57BL/6 mice immortalized with the
complete genome of HPV 16 and transformed with pEJ-ras. EL-4/E7 is
the thymoma EL-4 retrovirally transduced with E7.
[0191] L. monocytogenes Strains and Propagation
[0192] Listeria strains used were Lm-LLO-E7 (hly-E7 fusion gene in
an episomal expression system; FIG. 1A), Lm-E7 (single-copy E7 gene
cassette integrated into Listeria genome), Lm-LLO-NP ("DP-L2028";
hly-NP fusion gene in an episomal expression system), and Lm-Gag
("ZY-18"; single-copy HIV-1 Gag gene cassette integrated into the
chromosome). E7 was amplified by PCR using the primers
5'-GGCTCGAGCATGGAGATACACC-3' (SEQ ID No: 17; XhoI site is
underlined) and 5'-GGGGACTAGTTTATGGTTTCTGAGAACA-3' (SEQ ID No: 18;
SpeI site is underlined) and ligated into pCR2.1 (Invitrogen, San
Diego, Calif.). E7 was excised from pCR2.1 by XhoI/SpeI digestion
and ligated into pGG-55. The hly-E7 fusion gene and the
pluripotential transcription factor PrfA were cloned into pAM401, a
multicopy shuttle plasmid (Wirth R et al, J Bacteriol, 165: 831,
1986), generating pGG-55. The hly promoter drives the expression of
the first 441 AA of the hly gene product, (lacking the hemolytic
C-terminus, referred to below as ".DELTA.LLO," and having the
sequence set forth in SEQ ID No: 25), which is joined by the XhoI
site to the E7 gene, yielding a hly-E7 fusion gene that is
transcribed and secreted as LLO-E7. Transformation of a PrfA
negative strain of Listeria, XFL-7 (provided by Dr. Hao Shen,
University of Pennsylvania), with pGG-55 selected for the retention
of the plasmid in vivo (FIGS. 1A-B). The hly promoter and gene
fragment were generated using primers
5'-GGGGGCTAGCCCTCCTTTGATTAGTATATTC-3' (SEQ ID No: 19; NheI site is
underlined) and 5'-CTCCCTCGAGATCATAATTTACTTCATC-3' (SEQ ID No: 20;
XhoI site is underlined). The PrfA gene was PCR amplified using
primers 5'-GACTACAAGGACGATGACCGACAAGTGATAACCCGGGATCTAAATAAATCCGTT
T-3' (SEQ ID No: 27; XbaI site is underlined) and
5'-CCCGTCGACCAGCTCTTCTTGGTGAAG-3' (SEQ ID No: 21; SalI site is
underlined). Lm-E7 was generated by introducing an expression
cassette containing the hly promoter and signal sequence driving
the expression and secretion of E7 into the orfZ domain of the LM
genome. E7 was amplified by PCR using the primers
5'-GCGGATCCCATGGAGATACACCTAC-3' (SEQ ID No: 22; BamHI site is
underlined) and 5'-GCTCTAGATTATGGTTTCTGAG-3' (SEQ ID No: 23; XbaI
site is underlined). E7 was then ligated into the pZY-21 shuttle
vector. LM strain 104035 was transformed with the resulting
plasmid, pZY-21-E7, which includes an expression cassette inserted
in the middle of a 1.6-kb sequence that corresponds to the orfX, Y,
Z domain of the LM genome. The homology domain allows for insertion
of the E7 gene cassette into the orfZ domain by homologous
recombination. Clones were screened for integration of the E7 gene
cassette into the orfZ domain. Bacteria were grown in brain heart
infusion medium with (Lm-LLO-E7 and Lm-LLO-NP) or without (Lm-E7
and ZY-18) chloramphenicol (20 .mu.g/ml). Bacteria were frozen in
aliquots at -80.degree. C. Expression was verified by Western
blotting (FIG. 2).
[0193] Western Blotting
[0194] Listeria strains were grown in Luria-Bertoni medium at
37.degree. C. and were harvested at the same optical density
measured at 600 nm. The supernatants were TCA precipitated and
resuspended in 1.times. sample buffer supplemented with 0.1 N NaOH.
Identical amounts of each cell pellet or each TCA-precipitated
supernatant were loaded on 4-20% Tris-glycine SDS-PAGE gels (NOVEX,
San Diego, Calif.). The gels were transferred to polyvinylidene
difluoride and probed with an anti-E7 monoclonal antibody (mAb)
(Zymed Laboratories, South San Francisco, Calif.), then incubated
with HRP-conjugated anti-mouse secondary Ab (Amersham Pharmacia
Biotech, Little Chalfont, U.K.), developed with Amersham ECL
detection reagents, and exposed to Hyperfilm (Amersham Pharmacia
Biotech).
[0195] Measurement of Tumor Growth
[0196] Tumors were measured every other day with calipers spanning
the shortest and longest surface diameters. The mean of these two
measurements was plotted as the mean tumor diameter in millimeters
against various time points. Mice were sacrificed when the tumor
diameter reached 20 mm Tumor measurements for each time point are
shown only for surviving mice.
[0197] Effects of Listeria Recombinants on Established Tumor
Growth
[0198] Six- to 8-wk-old C57BL/6 mice (Charles River) received
2.times.10.sup.5 TC-1 cells s.c. on the left flank. One week
following tumor inoculation, the tumors had reached a palpable size
of 4-5 mm in diameter. Groups of eight mice were then treated with
0.1 LD.sub.50 i.p. Lm-LLO-E7 (10.sup.7 CFU), Lm-E7 (10.sup.6 CFU),
Lm-LLO-NP (10.sup.7 CFU), or Lm-Gag (5.times.10.sup.5 CFU) on days
7 and 14.
[0199] .sup.51Cr Release Assay
[0200] C57BL/6 mice, 6-8 wk old, were immunized i.p. with
0.1LD.sub.50 Lm-LLO-E7, Lm-E7, Lm-LLO-NP, or Lm-Gag. Ten days
post-immunization, spleens were harvested. Splenocytes were
established in culture with irradiated TC-1 cells (100:1,
splenocytes:TC-1) as feeder cells; stimulated in vitro for 5 days,
then used in a standard .sup.51Cr release assay, using the
following targets: EL-4, EL-4/E7, or EL-4 pulsed with E7 H-2b
peptide (RAHYNIVTF). E:T cell ratios, performed in triplicate, were
80:1, 40:1, 20:1, 10:1, 5:1, and 2.5:1. Following a 4-h incubation
at 37.degree. C., cells were pelleted, and 50 .mu.l supernatant was
removed from each well. Samples were assayed with a Wallac 1450
scintillation counter (Gaithersburg, Md.). The percent specific
lysis was determined as [(experimental counts per minute
(cpm)-spontaneous cpm)/(total cpm-spontaneous cpm)].times.100.
[0201] TC-1-Specific Proliferation
[0202] C57BL/6 mice were immunized with 0.1 LD.sub.50 and boosted
by i.p. injection 20 days later with 1 LD.sub.50 Lm-LLO-E7, Lm-E7,
Lm-LLO-NP, or Lm-Gag. Six days after boosting, spleens were
harvested from immunized and naive mice. Splenocytes were
established in culture at 5.times.10.sup.5/well in flat-bottom
96-well plates with 2.5.times.10.sup.4, 1.25.times.10.sup.4,
6.times.10.sup.3, or 3.times.10.sup.3 irradiated TC-1 cells/well as
a source of E7 Ag, or without TC-1 cells or with 10 .mu.g/ml Con A.
Cells were pulsed 45 h later with 0.5 .mu.Ci
[.sup.3H]thymidine/well. Plates were harvested 18 h later using a
Tomtec harvester 96 (Orange, Conn.), and proliferation was assessed
with a Wallac 1450 scintillation counter. The change in cpm was
calculated as experimental cpm -no Ag cpm.
[0203] Flow Cytometric Analysis
[0204] C57BL/6 mice were immunized intravenously (i.v.) with 0.1
LD.sub.50 Lm-LLO-E7 or Lm-E7 and boosted 30 days later. Three-color
flow cytometry for CD8 (53-6.7, PE conjugated), CD62 ligand (CD62L;
MEL-14, APC conjugated), and E7 H-2Db tetramer was performed using
a FACSCalibur.RTM. flow cytometer with CellQuest.RTM. software
(Becton Dickinson, Mountain View, Calif.). Splenocytes harvested 5
days after the boost were stained at room temperature (rt) with
H-2Db tetramers loaded with the E7 peptide (RAHYNIVTF) or a control
(HIV-Gag) peptide. Tetramers were used at a 1/200 dilution and were
provided by Dr. Larry R. Pease (Mayo Clinic, Rochester, Minn.) and
by the NIAID Tetramer Core Facility and the NIH AIDS Research and
Reference Reagent Program. Tetramer.sup.+, CD8.sup.+, CD62L.sup.low
cells were analyzed.
B16F0-Ova Experiment
[0205] 24 C57BL/6 mice were inoculated with 5.times.10.sup.5
B16F0-Ova cells. On days 3, 10 and 17, groups of 8 mice were
immunized with 0.1 LD.sub.50 Lm-OVA (10.sup.6 cfu), Lm-LLO-OVA (108
cfu) and eight animals were left untreated.
[0206] Statistics
[0207] For comparisons of tumor diameters, mean and SD of tumor
size for each group were determined, and statistical significance
was determined by Student's t test. p<0.05 was considered
significant.
Results
[0208] Lm-E7 and Lm-LLO-E7 were compared for their abilities to
impact on TC-1 growth. Subcutaneous tumors were established on the
left flank of C57BL/6 mice. Seven days later tumors had reached a
palpable size (4-5 mm) Mice were vaccinated on days 7 and 14 with
0.1 LD.sub.50 Lm-E7, Lm-LLO-E7, or, as controls, Lm-Gag and
Lm-LLO-NP. Lm-LLO-E7 induced complete regression of 75% of
established TC-1 tumors, while tumor growth was controlled in the
other 2 mice in the group (FIG. 3). By contrast, immunization with
Lm-E7 and Lm-Gag did not induce tumor regression. This experiment
was repeated multiple times, always with very similar results. In
addition, similar results were achieved for Lm-LLO-E7 under
different immunization protocols. In another experiment, a single
immunization was able to cure mice of established 5 mm TC-1
tumors.
[0209] In other experiments, similar results were obtained with 2
other E7-expressing tumor cell lines: C3 and EL-4/E7. To confirm
the efficacy of vaccination with Lm-LLO-E7, animals that had
eliminated their tumors were re-challenged with TC-1 or EL-4/E7
tumor cells on day 60 or day 40, respectively Animals immunized
with Lm-LLO-E7 remained tumor free until termination of the
experiment (day 124 in the case of TC-1 and day 54 for
EL-4/E7).
[0210] Thus, expression of an antigen as a fusion protein with
.DELTA.LLO enhances the immunogenicity of the antigen.
Example 2
LM-LLO-E7 Treatment Elicits TC-1 Specific Splenocyte
Proliferation
[0211] To measure induction of T cells by Lm-E7 with Lm-LLO-E7,
TC-1-specific proliferative responses, a measure of
antigen-specific immunocompetence, were measured in immunized mice.
Splenocytes from Lm-LLO-E7-immunized mice proliferated when exposed
to irradiated TC-1 cells as a source of E7, at splenocyte: TC-1
ratios of 20:1, 40:1, 80:1, and 160:1 (FIG. 4). Conversely,
splenocytes from Lm-E7 and rLm control-immunized mice exhibited
only background levels of proliferation.
Example 3
Fusion of E7 to LLO, actA, or a Pest Amino Acid Sequence Enhances
E7-Specific Immunity and Generates Tumor-Infiltrating E7-Specific
CD8.sup.+ Cells
Materials and Experimental Methods
[0212] 500 mcl (microliter) of MATRIGEL.RTM., comprising 100 mcl of
2.times.10.sup.5 TC-1 tumor cells in phosphate buffered saline
(PBS) plus 400 mcl of MATRIGEL.RTM. (BD Biosciences, Franklin
Lakes, N.J.) were implanted subcutaneously on the left flank of 12
C57BL/6 mice (n=3). Mice were immunized intraperitoneally on day 7,
14 and 21, and spleens and tumors were harvested on day 28. Tumor
MATRIGELs were removed from the mice and incubated at 4.degree. C.
overnight in tubes containing 2 milliliters (ml) of RP 10 medium on
ice. Tumors were minced with forceps, cut into 2 mm blocks, and
incubated at 37.degree. C. for 1 hour with 3 ml of enzyme mixture
(0.2 mg/ml collagenase-P, 1 mg/ml DNAse-1 in PBS). The tissue
suspension was filtered through nylon mesh and washed with 5% fetal
bovine serum+0.05% of NaN.sub.3 in PBS for tetramer and IFN-gamma
staining.
[0213] Splenocytes and tumor cells were incubated with 1 micromole
(mcm) E7 peptide for 5 hours in the presence of brefeldin A at
10.sup.7 cells/ml. Cells were washed twice and incubated in 50 mcl
of anti-mouse Fc receptor supernatant (2.4 G2) for 1 hour or
overnight at 4.degree. C. Cells were stained for surface molecules
CD8 and CD62L, permeabilized, fixed using the permeabilization kit
Golgi-Stop.RTM. or Golgi-Plug.RTM. (Pharmingen, San Diego, Calif.),
and stained for IFN-gamma. 500,000 events were acquired using
two-laser flow cytometer FACSCalibur and analyzed using Cellquest
Software (Becton Dickinson, Franklin Lakes, N.J.). Percentages of
IFN-gamma secreting cells within the activated (CD62L.sup.low)
CD8.sup.+ T cells were calculated.
[0214] For tetramer staining, H-2D.sup.b tetramer was loaded with
phycoerythrin (PE)-conjugated E7 peptide (RAHYNIVTF, SEQ ID NO:
24), stained at rt for 1 hour, and stained with
anti-allophycocyanin (APC) conjugated MEL-14 (CD62L) and
FITC-conjugated CD8 at 4.degree. C. for 30 min Cells were analyzed
comparing tetramer.sup.+ CD8.sup.+ CD62L.sup.low cells in the
spleen and in the tumor.
Results
[0215] To analyze the ability of Lm-ActA-E7 to enhance antigen
specific immunity, mice were implanted with TC-1 tumor cells and
immunized with either Lm-LLO-E7 (1.times.10.sup.7 CFU), Lm-E7
(1.times.10.sup.6 CFU), or Lm-ActA-E7 (2.times.10.sup.8 CFU), or
were untreated (naive). Tumors of mice from the Lm-LLO-E7 and
Lm-ActA-E7 groups contained a higher percentage of
IFN-gamma-secreting CD8.sup.+ T cells (FIG. 5A) and
tetramer-specific CD8.sup.+ cells (FIG. 5B) than in Lm-E7 or naive
mice.
[0216] In another experiment, tumor-bearing mice were administered
Lm-LLO-E7, Lm-PEST-E7, Lm-.DELTA.PEST-E7, or Lm-E7epi, and levels
of E7-specific lymphocytes within the tumor were measured. Mice
were treated on days 7 and 14 with 0.1 LD.sub.50 of the 4 vaccines.
Tumors were harvested on day 21 and stained with antibodies to
CD62L, CD8, and with the E7/Db tetramer. An increased percentage of
tetramer-positive lymphocytes within the tumor were seen in mice
vaccinated with Lm-LLO-E7 and Lm-PEST-E7 (FIG. 6A). This result was
reproducible over three experiments (FIG. 6B).
[0217] Thus, Lm-LLO-E7, Lm-ActA-E7, and Lm-PEST-E7 are each
efficacious at induction of tumor-infiltrating CD8.sup.+ T cells
and tumor regression.
Example 4
Passaging of Listeria Vaccine Vectors Through Mice Elicits
Increased Immune Responses to Heterologous and Endogenous
Antigens
Materials and Experimental Methods
Bacterial Strains
[0218] L. monocytogenes strain 10403S, serotype 1 (ATCC, Manassas,
Va.) was the wild type organism used in these studies and the
parental strain of the constructs described below. Strain 10403S
has an LD.sub.50 of approximately 5.times.10.sup.4 CFU when
injected intraperitoneally into BALB/c mice. "Lm-Gag" is a
recombinant LM strain containing a copy of the HIV-1 strain HXB
(subtype B laboratory strain with a syncytia-forming phenotype) gag
gene stably integrated into the listerial chromosome using a
modified shuttle vector pKSV7. Gag protein was expressed and
secreted by the strain, as determined by Western blot. All strains
were grown in brain-heart infusion (BHI) broth or agar plates
(Difco Labs, Detroit, Mich.).
[0219] Bacterial Culture
[0220] Bacteria from a single clone expressing the passenger
antigen and/or fusion protein were selected and cultured in BHI
broth overnight. Aliquots of this culture were frozen at
-70.degree. C. with no additives. From this stock, cultures were
grown to 0.1-0.2 O.D. at 600 nm, and aliquots were again frozen at
-70.degree. C. with no additives. To prepare cloned bacterial
pools, the above procedure was used, but after each passage a
number of bacterial clones were selected and checked for expression
of the target antigen, as described herein. Clones in which
expression of the foreign antigen was confirmed were used for the
next passage.
[0221] Passage of Bacteria in Mice
[0222] 6-8 week old female BALB/c (H-2d) mice were purchased from
Jackson Laboratories (Bar Harbor, Me.) and were maintained in a
pathogen-free microisolator environment. The titer of viable
bacteria in an aliquot of stock culture, stored frozen at
-70.degree. C., was determined by plating on BHI agar plates on
thawing and prior to use. In all, 5.times.10.sup.5 bacteria were
injected intravenously into BALB/c mice. After 3 days, spleens were
harvested, homogenized, and serial dilutions of the spleen
homogenate were incubated in BHI broth overnight and plated on BHI
agar plates. For further passage, aliquots were again grown to
0.1-0.2 O.D., frozen at -70.degree. C., and bacterial titer was
again determined by serial dilution. After the initial passage
(passage 0), this sequence was repeated for a total of 4 times.
[0223] Intracellular Cytokine Stain for IFN-Gamma
[0224] Lymphocytes were cultured for 5 hours in complete RPMI-10
medium supplemented with 50 U/ml human recombinant IL-2 and 1
microliter/ml Brefeldin A (Golgistop.TM.; PharMingen, San Diego,
Calif.) in the presence or absence of either the cytotoxic T-cell
(CTL) epitope for HIV-GAG (AMQMLKETI; SEQ ID No: 25), Listeria LLO
(GYKDGNEYI; SEQ ID No: 26) or the HPV virus gene E7 (RAHYNIVTF (SEQ
ID No: 24), at a concentration of 1 micromole. Cells were first
surface-stained, then washed and subjected to intracellular
cytokine stain using the Cytofix/Cytoperm kit in accordance with
the manufacturer's recommendations (PharMingen, San Diego, Calif.).
For intracellular IFN-gamma stain, FITC-conjugated rat anti-mouse
IFN-gamma monoclonal antibody (clone XMG 1.2) and its isotype
control Ab (rat IgG1; both from PharMingen) was used. In all,
10.sup.6 cells were stained in PBS containing 1% Bovine Serum
Albumin and 0.02% sodium azide (FACS Buffer) for 30 minutes at
4.degree. C. followed by 3 washes in FACS buffer. Sample data were
acquired on either a FACScan.TM. flowcytometer or FACSCalibur.TM.
instrument (Becton Dickinson, San Jose, Calif.). Three-color flow
cytometry for CD8 (PERCP conjugated, rat anti-mouse, clone 53-6.7
Pharmingen, San Diego, Calif.), CD62L (APC conjugated, rat
anti-mouse, clone MEL-14), and intracellular IFN-gamma was
performed using a FACSCalibur.TM. flow cytometer, and data were
further analyzed with CELLQuest software (Becton Dickinson,
Mountain View, Calif.). Cells were gated on CD8 high and
CD62L.sup.low before they were analyzed for CD8.sup.+ and
intracellular IFN-gamma staining.
Results
Passaging in Mice Increases the Virulence of Recombinant Listeria
Monocytogenes
[0225] Three different constructs were used to determine the impact
of passaging on recombinant Listeria vaccine vectors. Two of these
constructs carry a genomic insertion of the passenger antigen: the
first comprises the HIV gag gene (Lm-Gag), and the second comprises
the HPV E7 gene (Lm-E7). The third (Lm-LLO-E7) comprises a plasmid
with the fusion gene for the passenger antigen (HPV E7) fused with
a truncated version of LLO and a gene encoding PrfA, the positive
regulatory factor that controls Listeria virulence factors. This
plasmid was used to complement a PrfA negative mutant so that in a
live host, selection pressures would favor conservation of the
plasmid, because without it the bacterium is avirulent. All 3
constructs had been propagated extensively in vitro for many
bacterial generations.
[0226] Passaging the bacteria resulted in an increase in bacterial
virulence, as measured by numbers of surviving bacteria in the
spleen, with each of the first 2 passages. For Lm-Gag and
Lm-LLO-E7, virulence increased with each passage up to passage 2
(FIG. 7A). The plasmid-containing construct, Lm-LLO-E7,
demonstrated the most dramatic increase in virulence. Prior to
passage, the initial immunizing dose of Lm-LLO-E7 had to be
increased to 10.sup.7 bacteria and the spleen had to be harvested
on day 2 in order to recover bacteria (whereas an initial dose of
10.sup.5 bacteria for Lm-Gag was harvested on day 3). After the
initial passage, the standard dosage of Lm-LLO-E7 was sufficient to
allow harvesting on day 3. For Lm-E7, virulence increased by 1.5
orders of magnitude over unpassaged bacteria (FIG. 7B).
[0227] Thus, passage through mice increases the virulence of
Listeria vaccine strains.
[0228] Passaging Increases the Ability of L. monocytogenes to
Induce CD8.sup.+ T Cells
[0229] Next, the effect of passaging on induction of
antigen-specific CD8.sup.+ T cells was determined by intracellular
cytokine staining with immunodominant peptides specific for
MHC-class I using HIV-Gag peptide AMQMLKETI (SEQ ID No: 25) and LLO
91-99 (GYKDGNEYI; SEQ ID No: 26). Injection of 10.sup.3 CFU
passaged bacteria (Lm-Gag) into mice elicited significant numbers
of HIV-Gag-specific CD8.sup.+ T cells, while the same dose of
non-passaged Lm-Gag induced no detectable Gag-specific CD8.sup.+ T
cells. Even increasing the dose of unpassaged bacteria 100-fold did
not compensate for their relative avirulence; in fact, no
detectable Gag-specific CD8.sup.+ T cells were elicited even at the
higher dose. The same dose increase with passaged bacteria
increased Gag-specific T cell induction by 50% (FIG. 8). The same
pattern of induction of antigen-specific CD8.sup.+ T cells was
observed with LLO-specific CD8.sup.+ T cells, showing that these
results were not caused by the properties of the passenger antigen,
since they were observed with LLO, an endogenous Listeria
antigen.
[0230] Thus, passage through mice increases the immunogenicity of
Listeria vaccine strains.
Example 5
A prfA-Containing Plasmid is Stable in an LM Strain with a prfA
Deletion in the Absence of Antibiotics
Materials and Experimental Methods
Bacteria
[0231] L. monocytogenes strain XFL7 contains a 300 base pair
deletion in the PrfA gene XFL7 carries pGG55 which partially
restores virulence and confers CAP resistance, and is described in
United States Patent Application Publication No. 200500118184.
[0232] Development of Protocol for Plasmid Extraction from
Listeria
[0233] 1 mL of Listeria monocytogenes Lm-LLO-E7 research working
cell bank vial was inoculated into 27 mL BH1 medium containing 34
.mu.g/mL CAP and grown for 24 hours at 37.degree. C. and 200
rpm.
[0234] Seven 2.5 mL samples of the culture were pelleted (15000 rpm
for 5 minutes), and pellets were incubated at 37.degree. C. with 50
.mu.l lysozyme solution for varying amounts of time, from 0-60
minutes.
[0235] Lysozyme solution: [0236] 29 .mu.l 1 M dibasic Potassium
Phosphate [0237] 21 .mu.l 1 M monobasic Potassium Phosphate [0238]
500 .mu.l 40% Sucrose (filter sterilized through 0.45/.mu.m filter)
[0239] 450 .mu.l water [0240] 60 .mu.l lysozyme (50 mg/mL)
[0241] After incubation with the lysozyme, the suspensions were
centrifuged as before and the supernatants discarded. Each pellet
was then subjected to plasmid extraction by a modified version of
the QIAprep Spin Miniprep Kit.RTM. (Qiagen, Germantown, Md.)
protocol. The changes to the protocol were as follows: [0242] 1.
The volumes of buffers PI, P2 and N3 were all increased threefold
to allow complete lysis of the increased biomass. [0243] 2. 2 mg/mL
of lysozyme was added to the resuspended cells before the addition
of P2. The lysis solution was then incubated at 37.degree. C. for
15 minutes before neutralization. [0244] 3. The plasmid DNA was
resuspended in 30 .mu.L rather than 50 .mu.L to increase the
concentration.
[0245] In other experiments, the cells were incubated for 15 min in
P1 buffer+Lysozyme, then incubated with P2 (lysis buffer) and P3
(neutraliztion buffer) at room temperature.
[0246] Equal volumes of the isolated plasmid DNA from each
subculture were run on an 0.8% agarose gel stained with ethidium
bromide and visualized for any signs of structural or segregation
instability.
[0247] The results showed that plasmid extraction from L.
monocytogenes Lm-LLO-E7 increases in efficiency with increasing
incubation time with lysozyme, up to an optimum level at
approximately 50 minutes incubation. 11002251 These results provide
an effective method for plasmid extraction from Listeria vaccine
strains.
[0248] Replica Plating
[0249] Dilutions of the original culture were plated onto plates
containing LB or TB agar in the absence or presence of 34 .mu.g/mL
CAP. The differences between the counts on selective and
non-selective agar were used to determine whether there was any
gross segregational instability of the plasmid.
Results
[0250] The genetic stability (i.e. the extent to which the plasmid
is retained by or remains stably associated with the bacteria in
the absence of selection pressure; e.g. antibiotic selection
pressure) of the pGG55 plasmid in L. monocytogenes strain XFL7 in
the absence of antibiotic was assessed by serial sub-culture in
both Luria-Bertani media (LB: 5 g/L NaCl, 10 g/ml soy peptone, 5
g/L yeast extract) and Terrific Broth media (TB: 10 g/L glucose,
11.8 g/L soy peptone, 23.6 g/L yeast extract, 2.2 g/L
KH.sub.2PO.sub.4, 9.4 g/L K.sub.2HPO.sub.4), in duplicate cultures.
50 mL of fresh media in a 250 mL baffled shake flask was inoculated
with a fixed number of cells (1 ODmL), which was then subcultured
at 24 hour intervals. Cultures were incubated in an orbital shaker
at 37.degree. C. and 200 rpm. At each subculture the OD.sub.600 was
measured and used to calculate the cell doubling time (or
generation) elapsed, until 30 generations were reached in LB and 42
in TB. A known number of cells (15 ODmL) at each subculture stage
(approximately every 4 generations) were pelleted by
centrifugation, and the plasmid DNA was extracted using the Qiagen
QIAprep Spin Miniprep.RTM. protocol described above. After
purification, plasmid DNA was subjected to agarose gel
electrophoresis, followed by ethidium bromide staining. While the
amount of plasmid in the preps varied slightly between samples, the
overall trend was a constant amount of plasmid with respect to the
generational number of the bacteria (FIGS. 9A-B). Thus, pGG55
exhibited stability in strain XFL7, even in the absence of
antibiotic.
[0251] Plasmid stability was also monitored during the stability
study by replica plating on agar plates at each stage of the
subculture. Consistent with the results from the agarose gel
electrophoresis, there was no overall change in the number of
plasmid-containing cells throughout the study in either LB or TB
liquid culture (FIGS. 10 and 11, respectively).
[0252] These findings demonstrate that PrfA-encoding plasmids
exhibit stability in the absence of antibiotic in Listeria strains
containing mutations in rfA gene.
Materials and Methods (Examples 6-10)
PCR Reagents
[0253] The primers used for amplification of the PrfA gene and
discrimination of the D133V mutation are shown in Table 1. Stock
solutions of the primers ADV451, 452 and 453 were prepared by
diluting the primers in TE buffer to 400 .mu.M. An aliquot of the
stock solution was further diluted to 20 .mu.M in water (PCR grade)
to prepare a working solution. Primers were stored at -20.degree.
C. The reagents used in the PCR are shown in Table 2.
TABLE-US-00003 TABLE 1 Primers ADV451, 452 and 453. Primer
Orientation Sequence (5'.fwdarw.3') Specificity ADV451 Forward
CCTAGCTAAATTTAATGT D133V (SEQ ID NO: 28) mutation ADV452 Forward
CCTAGCTAAATTTAATGA Wild-type (SEQ ID NO: 29) sequence ADV453
Reverse TAATTTTCCCCAAGTAGC Shared AGG sequence (SEQ ID NO: 30)
TABLE-US-00004 TABLE 2 PCR reagents. Catalog Description Provider
number 1 0.2 ml thin-walled PCR tubes: Applied N801-0612 GeneAmp
autoclaved reaction Biosystems tube with cap 2 Water (PCR reagent)
Sigma W1754 3 Taq DNA Polymerase with 10x reaction Sigma D1806
buffer containing 15 mM MgCl.sub.2 4 Set of deoxynucleotides
(dNTPs), Sigma D7295 10 mM each 5 Primers ADV451, ADV452 Invitrogen
and ADV453 6 Template DNA, midipreparations of pGG55 plasmids 7
Thermal cycler PTC200 MJ Research (48 wells block)
Plasmid DNA Preparation
[0254] pGG55 plasmids with (pGG55 D133V) and without (pGG55 WT) the
prfA mutation were extracted and purified by midipreparations
either from E. coli or Listeria monocytogenes using the
PureLink.TM. HiPure Plasmid Midiprep Kit (Invitrogen, K2100-05),
according to the manufacturer's instructions. For plasmid
purification from Listeria, bacterial strains carrying the pGG55
D133V or WT plasmids were streak plated from frozen stocks in BHI
agar plates supplemented with chloramphenicol (25 .mu.g/ml). A
single colony from each strain was grown in 5 ml of selective
medium (BHI broth with 25 .mu.g/ml of chloramphenicol) for 6 hours
with vigorous shaking at 37.degree. C. and subinoculated 1:500 in
100 ml of selective medium for overnight growth under similar
conditions. Bacteria from the overnight culture were harvested by
centrifugation at 4,000.times.g for 10 minutes and resuspended
buffer R3 (resuspension buffer) containing 2 mg/ml of lysozyme
(Sigma, L7001). The bacteria suspension was incubated for at least
1 hour at 37.degree. C. before proceeding to the regular protocol.
Concentration and purity of the eluted plasmids were measured in a
spectrophotometer at 260 nm and 280 nm. To prepare the template
DNAs, the pGG55 D133V and WT plasmids were resuspended in water to
a final concentration of 1 .mu.g/.mu.l from the midiprep stock
solution. For the pGG55 WT plasmid, serial 10-fold dilutions from
the 1 .mu.g/.mu.l solution were prepared, corresponding to
dilutions from 10.sup.-1 to 10.sup.-7.
[0255] prfA Specific PCR Protocol to Test Clinical Grade
Material
[0256] The reaction mixture contained 1.times.PCR buffer, 1.5 mM
MgCl.sub.2, 0.8 mM dNTPs, 0.4 .mu.M of each primer, 0.05 U/.mu.l of
Taq DNA polymerase and 0.04 .mu.g/.mu.l of the pGG55 D133V template
plasmid. For each test, 10 tubes were required and the key
components in each tube in a 25 .mu.l reaction are shown in the
Table 3. For the PCR reaction, a master mix was prepared with
enough reagents for 11 reactions as shown in Table 4, and 24 .mu.l
of this PCR mix was added to each tube. Subsequently, a total of 1
.mu.l of the serially diluted pGG55 WT plasmid was added to the
corresponding tubes: 1 .mu.g in tube 3; 100 pg in tube 4; 10 pg in
tube 5; 1 pg in tube 6; 100 fg in tube 7; 10 fg in tube 8; 1 fg in
tube 9; 0.1 fg in tube 10. This serial dilution was used to
calibrate a standard curve to determine the method sensitivity.
Additionally, 0.5 .mu.l of water and 0.5 .mu.l of primer ADV451 (20
.mu.M stock) were added in tube 1, and 1 .mu.l of water added in
tube 2, completing 25 .mu.l of final volume. The quantities of each
reagent per tube for a 25 .mu.l reaction are shown in Table 5. The
PCR cycling conditions used in the reaction are shown in Table
6.
[0257] After conclusion of the PCR reaction, 5 .mu.l of gel-loading
buffer (6.times., with bromophenol blue) was added to each sample
and 10 .mu.l were analyzed by electrophoresis in 1.2% agarose gel
in TBE buffer. The gel dimensions were 7 cm.times.7 cm.times.1 cm
with a 15 sample wells (1 mm.times.2 mm) comb. The gel was run at
100 V for .about.30 minutes, until the bromophenol blue dye reached
the middle of the gel. The gel was stained in ethidium bromide (0.5
.mu.g/ml) for 20 minutes, destaining in water for 10 minutes. The
gel is visualized by illumination with UV light and photographed.
The image was analyzed using a band densitometry software (Quantity
One version 4.5.1, BioRad).
TABLE-US-00005 TABLE 3 Set of individual PCR reactions to validate
the method to detect the presence of wild-type prfA sequence in
Lm-LLO-E7 samples. Expected Tube Primer A Primer B Template DNA
Function result 1 ADV451 ADV453 1 ng of pGG55 Positive control for
Positive (D133V) the ADV451 reaction 2 ADV452 ADV453 1 ng of pGG55
Negative control for Negative (D133V) the ADV452 reaction
(specificity) 3 ADV452 ADV453 1 ng of pGG55 Positive control for
Positive (wild-type) + 1 ng the ADV452 reaction of pGG55 (D133V) 4
ADV452 ADV453 100 pg of pGG55 Test the sensitivity of Positive
(wild-type) + 1 ng the reaction of pGG55 (D133V) 5 ADV452 ADV453 10
pg of pGG55 Test the sensitivity of Positive (wild-type) + 1 ng the
reaction of pGG55 (D133V) 6 ADV452 ADV453 1 pg of pGG55 Test the
sensitivity of Positive (wild-type) + 1 ng the reaction of pGG55
(D133V) 7 ADV452 ADV453 100 fg of pGG55 Test the sensitivity of
Positive (wild-type) + 1 ng the reaction pGG55 (D133V) 8 ADV452
ADV453 10 fg of pGG55 Test the sensitivity of Positive (wild-type)
+ the reaction pGG55 (D133V) 9 ADV452 ADV453 1 fg of pGG55 Test the
sensitivity of Weakly (wild-type) + the reaction positive pGG55
(D133V) 10 ADV452 ADV453 0.1 fg of pGG55 Test the sensitivity of To
be (wild-type) + the reaction determined pGG55 (D133V)
TABLE-US-00006 TABLE 4 Master PCR mix preparation. Reagent Quantity
(.mu.l) Water 206.25 Taq DNA Polymerase 10x reaction buffer 27.5
containing 15 mM MgCl.sub.2 Deoxynucleotides (dNTPs) 10 mM each 5.5
Primers ADV452 (20 .mu.M in water) 5.5 Primers ADV453 (20 .mu.M in
water) 5.5 pGG55 D133V (Lm-LLO-E7) plasmid (1 ng/.mu.l) 11 Taq DNA
Polymerase (5 U/.mu.l) 2.75 Total 264
TABLE-US-00007 TABLE 5 PCR protocol for validation of the method to
detect the presence of wild-type prfA sequence using primers
ADV451, 452 and 453. Reagent PCR Water 18.75 .mu.l PCR Buffer 10x +
MgCl.sub.2 15 mM 2.5 .mu.l Deoxynucleotides mix (dATP, dCTP, dGTP
and dTTP) 0.5 .mu.l 10 mM each Primer ADV452 (20 .mu.M) 0.5 .mu.l
Primer ADV453 (20 .mu.M) 0.5 .mu.l Taq DNA polymerase (5 U/.mu.l)
0.25 .mu.l Template DNA (1 ng/.mu.l) pGG55 D133V .sup. 1 .mu.l
Template DNA pGG55 WT (tubes 3 to 10).sup.a .sup. 1 .mu.l Final
volume per tube.sup.b 25 .mu.l .sup.apGG55 WT (1 ng in tube 3; 100
pg in tube 4; 10 pg in tube 5; 1 pg in tube 6; 100 fg in tube 7; 10
fg in tube 8; 1 fg in tube 9; 0.1 fg in tube 10). .sup.bIn tube 1,
add 0.5 .mu.l of water and 0.5 .mu.l of primer ADV451 (20 .mu.M
stock); in tube 2 add 1 .mu.l of water.
TABLE-US-00008 TABLE 6 PCR cycling conditions to detect the
presence of wild- type prfA sequence using primers ADV451, 452 and
453. Step Temperature Time Number of cycles 1. 94.degree. C. 2
minutes and 30 seconds 1 2. 94.degree. C. 30 seconds 1 3.
53.degree. C. 30 seconds 1 4. 72.degree. C. 30 seconds 1 5. Repeat
steps 2 to 4 12 6. 94.degree. C. 30 seconds 1 7. 50.degree. C. 30
seconds 1 8. 72.degree. C. 30 seconds 1 9. Repeat steps 6 to 8 23
10. 72.degree. C. 10 minutes 1
Sequencing:
[0258] Sequencing of the plasmids was done using the dideoxy
sequencing method. The plasmids pGG55 D133V and pGG55 WT were mixed
at different ratios (1:1, 1:10, 1; 100, 1:1,000 and 1:10,000). The
total amount of plasmid in the mixture was kept constant (500
.mu.g) and the plasmid containing the wild-type sequence was
10-fold serially diluted in relation to the D133V plasmid to
determine the sensitivity of the method.
Results
Example 6
Sequencing is not a Sensitive Method to Detect the Reversion of the
D133V Mutation
[0259] To estimate the sensitivity of sequencing in detecting the
wild-type prfA sequence, the pGG55 D133V and WT plasmids were mixed
at the different ratios and sequenced. The results are shown in
FIG. 12 and reveal that sequencing has a high specificity in
discriminating the prfA D133V mutation (FIG. 12). On the other
hand, the sensitivity is low and the maximum dilution of wild-type
prfA pGG55 plasmid with a detectable peak in the sequence was 1 in
10 (FIG. 12). In conclusion, although sequencing is very specific,
the sensitivity of the method is low and not appropriate to screen
for the presence of rare events such as revertants of the prfA
D133V mutation in Lm-LLO-E7 samples.
Example 7
Development of a Highly Specific and Sensitive PCR Method to Detect
Reversion of the D133V Mutation
[0260] Given the low sensitivity of sequencing to detect rare
events, it became imperative to develop a more sensitive method
with similar specificity to detect reversion of the D133V mutation
to wild-type. To achieve this goal, we designed a PCR-based method
that specifically amplifies the wild-type sequence and is sensitive
enough to detect at least 1 wild-type copy of prfA in 10,000,000
copies of the D133V mutated sequence. We designed 3 primers for
this method: ADV451, ADV452 and ADV453 (Table 1). Both ADV451 and
ADV452 are forward primers and differ in the last nucleotide at the
3' position to discriminate the A.fwdarw.T (D133V) mutation at
position 398 of the prfA gene. The ADV453 primer is the reverse
primer located approximately 300 by downstream the annealing site
of the ADV451 and ADV452 primers (FIG. 13). The expected PCR band
obtained with the primers ADV451 or ADV452 and ADV453 is 326 bp.
Under stringent conditions, the ADV451 primer should only amplify
the pGG55 D133V plasmid, whereas the ADV452 would be specific to
the wild-type prfA sequence.
Example 8
Specificity of the PCR Method
[0261] The reaction using the primer ADV451 was very specific and
amplified the mutated D133V prfA sequence (lanes 1 to 3), but not
the wild-type sequence (lanes 4 to 6). However, a very faint band
can be detected in lane 4, when 5 .mu.g of template DNA was used,
but not with 1 .mu.g (FIG. 14).
[0262] As shown in FIG. 15, the reaction with the ADV452 primer
only amplified the wild-type prfA sequence (lanes 4, 5 and 6), and
no bands were detected when the pGG55 carrying the D133V prfA
mutation was used as a template (lanes 1, 2 and 3), even when using
5 .mu.g of plasmid in the reaction (FIG. 16). In conclusion, the
PCR reactions with primers ADV451 and ADV452 are very specific and
able to discriminate the AT (D133V) mutation at position 398 of the
prfA gene in the pGG55 plasmid. Based on these results, we selected
the amount of 1 .mu.g as the standard amount of template DNA to be
used in the reaction.
Example 9
Sensitivity of the PCR Method
[0263] The sensitivity of the reaction was tested using 1 .mu.g of
template DNA. For the plasmid carrying the wild-type prfA sequence,
decreasing amounts of DNA (corresponding to 10-fold dilutions from
10.sup.-1 to 10.sup.-7), were also included in the reaction to
estimate the sensitivity. In these reactions only the primers
ADV452 and ADV453 were used. In a PCR reaction with 30 cycles (10
cycles with annealing temperature of 53.degree. C. and an
additional 20 cycles with annealing temperature of 50.degree. C.),
the sensitivity of the method was 1 in 100,000 (data not shown). As
shown in FIG. 5, increasing the number of PCR cycles to 37 improved
the visual sensitivity of the method to 10.sup.-6 for the detection
of D133V revertants, without significantly compromising the
specificity. A clear band was visible at the 10.sup.-6 dilution,
corresponding to a detection level of 1 copy of the wild-type
sequence in a million of the D133V mutant, when 1 .mu.g of plasmid
was used as the initial amount of DNA. Only a very weak band can be
visualized in lanes 1 and 9 after longer exposure, reassuring the
robust specificity of the method. On the other hand, when starting
with 5 .mu.g of DNA, a band could be easily detected at the
10.sup.-7 dilution, increasing the sensitivity of the PCR. However,
a similar band in intensity could also be detected with the pGG55
D133V plasmid, indicating the specificity limit of the method (FIG.
17). This band observed with the pGG55 D133V plasmid is likely due
to non-specific amplification of the D133V mutation with primer
ADV452 that can significantly accumulate with the increased number
of cycles. These results indicate that the sensitivity limit for
this method, without significantly compromising the specificity, is
situated between 1 to 1,000,000 and 1 to 10,000,000.
Example 10
Recombinant Listeria Expressing a Fusion Protein of LLO to
E7(Lm-LLO-E7)
[0264] This strain is approx. 4-5 logs more attenuated than the
wild-type parent strain 10403S and secretes the fusion protein
tLLO-E7. This immunotherapy is based on the backbone XFL7, which is
derived from 10403S by the irreversible deletion in the virulence
gene transcription activator prfA. PrfA regulates the transcription
of several virulence genes such as Listeriolysin O (LLO), ActA,
PlcA (phospholipase A), PlcB (phospholipase B) etc that are
required for in vivo intracellular growth and survival of L.
monocytogenes. The plasmid pGG55 is retained by the Lm-LLO-E7 in
vitro by means of selection with `chloramphenicol`. However for in
vivo retention of the plasmid by Lm-LLO-E7, it carries a copy of
mutated prfA (D133V), which has been demonstrated to be less active
than wild-type prfA in DNA binding and activating the transcription
of virulence genes. We have observed that complementation with
mutated prfA resulted in approx. 40 fold reduction in the amount of
secreted LLO from Lm-LLO-E7 when compared to wild-type strain
10403S. This implicates that possibly the strain Lm-LLO-E7 exhibits
a reduced expression of the virulence genes that are regulated by
prfA such as actA, inlA, inlB, inlC, plcB etc. In Lm-LLO-E7, the
complementation with mutated copy of prfA possibly causes a
reduction in the expression of different virulence genes that are
regulated by prfA resulting in overall attenuation of approx. 4-5
logs.
Example 11
Immunotherapy with Chemoradiation for Anal Cancer
[0265] Phase I-II Study
[0266] Eligibility Criteria [0267] Newly diagnosed locally advanced
anal cancer: [0268] Stages: T.gtoreq.4 cm or node +. [0269] PS 0-1
[0270] Staging by CT/MRI or PET scan. [0271] No significant cardiac
or pulmonary disease. Adequate bone marrow and renal function.
[0272] ADXS-HPV Treatment [0273] ADXS-HPV administered at
1.times.10.sup.9cfu [0274] IV Infusion of 500 ml over 30 minutes
[0275] Premedications for ADXS-HPV treatment: [0276] Naprosyn 500
mg BID, Day -1, 0 [0277] Promethazine 25 mg PO, BID (pre-dose, 8
hours) [0278] Post infusion antibiotics [0279] Ampicillin 500 mg
QID, Days 3-9
[0280] Treatment Schedule (See FIG. 23-24).
[0281] Patients with newly diagnosed anal cancer with a primary
tumor >4 cm or lymph node involvement, without distant
metastases, are eligible. All patients receive 2 courses of
mitomycin, 5-FU with concurrent radiation (54 Gy in 30 fractions by
intensity modulated radiation therapy [IMRT]). Patients receive 4
treatments of ADXS-HPV, 1.times.10.sup.9 colony forming units
intravenously once approximately every 28 days. In treatment
schedule #1 (FIG. 23), the first dose is given before
chemoradiation and the 2-4.sup.th doses are given every 28 days
after completion of radiation. In treatment schedule #2 (FIG. 24),
the second dose of ADXS-HPV is administered during
chemoradiation.
Results
[0282] 8 patients have been treated on the locally advanced anal
cancer trial. Seven have had completed treatment and have no
evidence of disease (NED) (Table 1). (Only the patient with the
grade 5 unrelated cardiac event is not alive and with NED. --see
Table 1)
TABLE-US-00009 Completed Treatment Patient Age Stage Standard
Treatment Outcome 1 59 IIIB (T3N3) Yes CR, NED 2 50 IIIB (T2N2) Yes
CR, NED 3 70 IIIA (T4N0) Yes CR, NED 4 55 IIIB (T3N3) Yes CR, NED 5
39 IIIA (T4N0) Yes CR, NED 6 71 II (4.5 cm, N0) Non tx related Gr5
Pending Cardiovascular 7 70 III T3NB Yes Pending 8 55 II (T3NO) Yes
Pending
[0283] Toxicities [0284] Rigors lasting for about 24 hours, often
requiring meperidine, are most common toxicity. [0285] No evidence
of bacterial infection. [0286] No overlapping toxicities with
chemoradiation (1 grade 3 neutropenia).
[0287] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended claims are intended to cover all such modifications
and changes as fall within the true spirit of the invention.
Sequence CWU 1
1
30132PRTListeria monocytogenes 1Lys Glu Asn Ser Ile Ser Ser Met Ala
Pro Pro Ala Ser Pro Pro Ala 1 5 10 15 Ser Pro Lys Thr Pro Ile Glu
Lys Lys His Ala Asp Glu Ile Asp Lys 20 25 30 2441PRTListeria
monocytogenes 2Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu
Val Ser Leu 1 5 10 15 Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala
Ser Ala Phe Asn Lys 20 25 30 Glu Asn Ser Ile Ser Ser Val Ala Pro
Pro Ala Ser Pro Pro Ala Ser 35 40 45 Pro Lys Thr Pro Ile Glu Lys
Lys His Ala Asp Glu Ile Asp Lys Tyr 50 55 60 Ile Gln Gly Leu Asp
Tyr Asn Lys Asn Asn Val Leu Val Tyr His Gly 65 70 75 80 Asp Ala Val
Thr Asn Val Pro Pro Arg Lys Gly Tyr Lys Asp Gly Asn 85 90 95 Glu
Tyr Ile Val Val Glu Lys Lys Lys Lys Ser Ile Asn Gln Asn Asn 100 105
110 Ala Asp Ile Gln Val Val Asn Ala Ile Ser Ser Leu Thr Tyr Pro Gly
115 120 125 Ala Leu Val Lys Ala Asn Ser Glu Leu Val Glu Asn Gln Pro
Asp Val 130 135 140 Leu Pro Val Lys Arg Asp Ser Leu Thr Leu Ser Ile
Asp Leu Pro Gly 145 150 155 160 Met Thr Asn Gln Asp Asn Lys Ile Val
Val Lys Asn Ala Thr Lys Ser 165 170 175 Asn Val Asn Asn Ala Val Asn
Thr Leu Val Glu Arg Trp Asn Glu Lys 180 185 190 Tyr Ala Gln Ala Tyr
Ser Asn Val Ser Ala Lys Ile Asp Tyr Asp Asp 195 200 205 Glu Met Ala
Tyr Ser Glu Ser Gln Leu Ile Ala Lys Phe Gly Thr Ala 210 215 220 Phe
Lys Ala Val Asn Asn Ser Leu Asn Val Asn Phe Gly Ala Ile Ser 225 230
235 240 Glu Gly Lys Met Gln Glu Glu Val Ile Ser Phe Lys Gln Ile Tyr
Tyr 245 250 255 Asn Val Asn Val Asn Glu Pro Thr Arg Pro Ser Arg Phe
Phe Gly Lys 260 265 270 Ala Val Thr Lys Glu Gln Leu Gln Ala Leu Gly
Val Asn Ala Glu Asn 275 280 285 Pro Pro Ala Tyr Ile Ser Ser Val Ala
Tyr Gly Arg Gln Val Tyr Leu 290 295 300 Lys Leu Ser Thr Asn Ser His
Ser Thr Lys Val Lys Ala Ala Phe Asp 305 310 315 320 Ala Ala Val Ser
Gly Lys Ser Val Ser Gly Asp Val Glu Leu Thr Asn 325 330 335 Ile Ile
Lys Asn Ser Ser Phe Lys Ala Val Ile Tyr Gly Gly Ser Ala 340 345 350
Lys Asp Glu Val Gln Ile Ile Asp Gly Asn Leu Gly Asp Leu Arg Asp 355
360 365 Ile Leu Lys Lys Gly Ala Thr Phe Asn Arg Glu Thr Pro Gly Val
Pro 370 375 380 Ile Ala Tyr Thr Thr Asn Phe Leu Lys Asp Asn Glu Leu
Ala Val Ile 385 390 395 400 Lys Asn Asn Ser Glu Tyr Ile Glu Thr Thr
Ser Lys Ala Tyr Thr Asp 405 410 415 Gly Lys Ile Asn Ile Asp His Ser
Gly Gly Tyr Val Ala Gln Phe Asn 420 425 430 Ile Ser Trp Asp Glu Val
Asn Tyr Asp 435 440 3529PRTListeria monocytogenes 3Met Lys Lys Ile
Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu 1 5 10 15 Pro Ile
Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30
Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35
40 45 Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Glu Ile Asp Lys
Tyr 50 55 60 Ile Gln Gly Leu Asp Tyr Asn Lys Asn Asn Val Leu Val
Tyr His Gly 65 70 75 80 Asp Ala Val Thr Asn Val Pro Pro Arg Lys Gly
Tyr Lys Asp Gly Asn 85 90 95 Glu Tyr Ile Val Val Glu Lys Lys Lys
Lys Ser Ile Asn Gln Asn Asn 100 105 110 Ala Asp Ile Gln Val Val Asn
Ala Ile Ser Ser Leu Thr Tyr Pro Gly 115 120 125 Ala Leu Val Lys Ala
Asn Ser Glu Leu Val Glu Asn Gln Pro Asp Val 130 135 140 Leu Pro Val
Lys Arg Asp Ser Leu Thr Leu Ser Ile Asp Leu Pro Gly 145 150 155 160
Met Thr Asn Gln Asp Asn Lys Ile Val Val Lys Asn Ala Thr Lys Ser 165
170 175 Asn Val Asn Asn Ala Val Asn Thr Leu Val Glu Arg Trp Asn Glu
Lys 180 185 190 Tyr Ala Gln Ala Tyr Pro Asn Val Ser Ala Lys Ile Asp
Tyr Asp Asp 195 200 205 Glu Met Ala Tyr Ser Glu Ser Gln Leu Ile Ala
Lys Phe Gly Thr Ala 210 215 220 Phe Lys Ala Val Asn Asn Ser Leu Asn
Val Asn Phe Gly Ala Ile Ser 225 230 235 240 Glu Gly Lys Met Gln Glu
Glu Val Ile Ser Phe Lys Gln Ile Tyr Tyr 245 250 255 Asn Val Asn Val
Asn Glu Pro Thr Arg Pro Ser Arg Phe Phe Gly Lys 260 265 270 Ala Val
Thr Lys Glu Gln Leu Gln Ala Leu Gly Val Asn Ala Glu Asn 275 280 285
Pro Pro Ala Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu 290
295 300 Lys Leu Ser Thr Asn Ser His Ser Thr Lys Val Lys Ala Ala Phe
Asp 305 310 315 320 Ala Ala Val Ser Gly Lys Ser Val Ser Gly Asp Val
Glu Leu Thr Asn 325 330 335 Ile Ile Lys Asn Ser Ser Phe Lys Ala Val
Ile Tyr Gly Gly Ser Ala 340 345 350 Lys Asp Glu Val Gln Ile Ile Asp
Gly Asn Leu Gly Asp Leu Arg Asp 355 360 365 Ile Leu Lys Lys Gly Ala
Thr Phe Asn Arg Glu Thr Pro Gly Val Pro 370 375 380 Ile Ala Tyr Thr
Thr Asn Phe Leu Lys Asp Asn Glu Leu Ala Val Ile 385 390 395 400 Lys
Asn Asn Ser Glu Tyr Ile Glu Thr Thr Ser Lys Ala Tyr Thr Asp 405 410
415 Gly Lys Ile Asn Ile Asp His Ser Gly Gly Tyr Val Ala Gln Phe Asn
420 425 430 Ile Ser Trp Asp Glu Val Asn Tyr Asp Pro Glu Gly Asn Glu
Ile Val 435 440 445 Gln His Lys Asn Trp Ser Glu Asn Asn Lys Ser Lys
Leu Ala His Phe 450 455 460 Thr Ser Ser Ile Tyr Leu Pro Gly Asn Ala
Arg Asn Ile Asn Val Tyr 465 470 475 480 Ala Lys Glu Cys Thr Gly Leu
Ala Trp Glu Trp Trp Arg Thr Val Ile 485 490 495 Asp Asp Arg Asn Leu
Pro Leu Val Lys Asn Arg Asn Ile Ser Ile Trp 500 505 510 Gly Thr Thr
Leu Tyr Pro Lys Tyr Ser Asn Lys Val Asp Asn Pro Ile 515 520 525 Glu
4416PRTListeria monocytogenes 4Met Lys Lys Ile Met Leu Val Phe Ile
Thr Leu Ile Leu Val Ser Leu 1 5 10 15 Pro Ile Ala Gln Gln Thr Glu
Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30 Glu Asn Ser Ile Ser
Ser Val Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45 Pro Lys Thr
Pro Ile Glu Lys Lys His Ala Asp Glu Ile Asp Lys Tyr 50 55 60 Ile
Gln Gly Leu Asp Tyr Asn Lys Asn Asn Val Leu Val Tyr His Gly 65 70
75 80 Asp Ala Val Thr Asn Val Pro Pro Arg Lys Gly Tyr Lys Asp Gly
Asn 85 90 95 Glu Tyr Ile Val Val Glu Lys Lys Lys Lys Ser Ile Asn
Gln Asn Asn 100 105 110 Ala Asp Ile Gln Val Val Asn Ala Ile Ser Ser
Leu Thr Tyr Pro Gly 115 120 125 Ala Leu Val Lys Ala Asn Ser Glu Leu
Val Glu Asn Gln Pro Asp Val 130 135 140 Leu Pro Val Lys Arg Asp Ser
Leu Thr Leu Ser Ile Asp Leu Pro Gly 145 150 155 160 Met Thr Asn Gln
Asp Asn Lys Ile Val Val Lys Asn Ala Thr Lys Ser 165 170 175 Asn Val
Asn Asn Ala Val Asn Thr Leu Val Glu Arg Trp Asn Glu Lys 180 185 190
Tyr Ala Gln Ala Tyr Ser Asn Val Ser Ala Lys Ile Asp Tyr Asp Asp 195
200 205 Glu Met Ala Tyr Ser Glu Ser Gln Leu Ile Ala Lys Phe Gly Thr
Ala 210 215 220 Phe Lys Ala Val Asn Asn Ser Leu Asn Val Asn Phe Gly
Ala Ile Ser 225 230 235 240 Glu Gly Lys Met Gln Glu Glu Val Ile Ser
Phe Lys Gln Ile Tyr Tyr 245 250 255 Asn Val Asn Val Asn Glu Pro Thr
Arg Pro Ser Arg Phe Phe Gly Lys 260 265 270 Ala Val Thr Lys Glu Gln
Leu Gln Ala Leu Gly Val Asn Ala Glu Asn 275 280 285 Pro Pro Ala Tyr
Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu 290 295 300 Lys Leu
Ser Thr Asn Ser His Ser Thr Lys Val Lys Ala Ala Phe Asp 305 310 315
320 Ala Ala Val Ser Gly Lys Ser Val Ser Gly Asp Val Glu Leu Thr Asn
325 330 335 Ile Ile Lys Asn Ser Ser Phe Lys Ala Val Ile Tyr Gly Gly
Ser Ala 340 345 350 Lys Asp Glu Val Gln Ile Ile Asp Gly Asn Leu Gly
Asp Leu Arg Asp 355 360 365 Ile Leu Lys Lys Gly Ala Thr Phe Asn Arg
Glu Thr Pro Gly Val Pro 370 375 380 Ile Ala Tyr Thr Thr Asn Phe Leu
Lys Asp Asn Glu Leu Ala Val Ile 385 390 395 400 Lys Asn Asn Ser Glu
Tyr Ile Glu Thr Thr Ser Lys Ala Tyr Thr Asp 405 410 415
5390PRTListeria monocytogenes 5Met Arg Ala Met Met Val Val Phe Ile
Thr Ala Asn Cys Ile Thr Ile 1 5 10 15 Asn Pro Asp Ile Ile Phe Ala
Ala Thr Asp Ser Glu Asp Ser Ser Leu 20 25 30 Asn Thr Asp Glu Trp
Glu Glu Glu Lys Thr Glu Glu Gln Pro Ser Glu 35 40 45 Val Asn Thr
Gly Pro Arg Tyr Glu Thr Ala Arg Glu Val Ser Ser Arg 50 55 60 Asp
Ile Lys Glu Leu Glu Lys Ser Asn Lys Val Arg Asn Thr Asn Lys 65 70
75 80 Ala Asp Leu Ile Ala Met Leu Lys Glu Lys Ala Glu Lys Gly Pro
Asn 85 90 95 Ile Asn Asn Asn Asn Ser Glu Gln Thr Glu Asn Ala Ala
Ile Asn Glu 100 105 110 Glu Ala Ser Gly Ala Asp Arg Pro Ala Ile Gln
Val Glu Arg Arg His 115 120 125 Pro Gly Leu Pro Ser Asp Ser Ala Ala
Glu Ile Lys Lys Arg Arg Lys 130 135 140 Ala Ile Ala Ser Ser Asp Ser
Glu Leu Glu Ser Leu Thr Tyr Pro Asp 145 150 155 160 Lys Pro Thr Lys
Val Asn Lys Lys Lys Val Ala Lys Glu Ser Val Ala 165 170 175 Asp Ala
Ser Glu Ser Asp Leu Asp Ser Ser Met Gln Ser Ala Asp Glu 180 185 190
Ser Ser Pro Gln Pro Leu Lys Ala Asn Gln Gln Pro Phe Phe Pro Lys 195
200 205 Val Phe Lys Lys Ile Lys Asp Ala Gly Lys Trp Val Arg Asp Lys
Ile 210 215 220 Asp Glu Asn Pro Glu Val Lys Lys Ala Ile Val Asp Lys
Ser Ala Gly 225 230 235 240 Leu Ile Asp Gln Leu Leu Thr Lys Lys Lys
Ser Glu Glu Val Asn Ala 245 250 255 Ser Asp Phe Pro Pro Pro Pro Thr
Asp Glu Glu Leu Arg Leu Ala Leu 260 265 270 Pro Glu Thr Pro Met Leu
Leu Gly Phe Asn Ala Pro Ala Thr Ser Glu 275 280 285 Pro Ser Ser Phe
Glu Phe Pro Pro Pro Pro Thr Asp Glu Glu Leu Arg 290 295 300 Leu Ala
Leu Pro Glu Thr Pro Met Leu Leu Gly Phe Asn Ala Pro Ala 305 310 315
320 Thr Ser Glu Pro Ser Ser Phe Glu Phe Pro Pro Pro Pro Thr Glu Asp
325 330 335 Glu Leu Glu Ile Ile Arg Glu Thr Ala Ser Ser Leu Asp Ser
Ser Phe 340 345 350 Thr Arg Gly Asp Leu Ala Ser Leu Arg Asn Ala Ile
Asn Arg His Ser 355 360 365 Gln Asn Phe Ser Asp Phe Pro Pro Ile Pro
Thr Glu Glu Glu Leu Asn 370 375 380 Gly Arg Gly Gly Arg Pro 385 390
61170DNAListeria monocytogenes 6atgcgtgcga tgatggtggt tttcattact
gccaattgca ttacgattaa ccccgacata 60atatttgcag cgacagatag cgaagattct
agtctaaaca cagatgaatg ggaagaagaa 120aaaacagaag agcaaccaag
cgaggtaaat acgggaccaa gatacgaaac tgcacgtgaa 180gtaagttcac
gtgatattaa agaactagaa aaatcgaata aagtgagaaa tacgaacaaa
240gcagacctaa tagcaatgtt gaaagaaaaa gcagaaaaag gtccaaatat
caataataac 300aacagtgaac aaactgagaa tgcggctata aatgaagagg
cttcaggagc cgaccgacca 360gctatacaag tggagcgtcg tcatccagga
ttgccatcgg atagcgcagc ggaaattaaa 420aaaagaagga aagccatagc
atcatcggat agtgagcttg aaagccttac ttatccggat 480aaaccaacaa
aagtaaataa gaaaaaagtg gcgaaagagt cagttgcgga tgcttctgaa
540agtgacttag attctagcat gcagtcagca gatgagtctt caccacaacc
tttaaaagca 600aaccaacaac catttttccc taaagtattt aaaaaaataa
aagatgcggg gaaatgggta 660cgtgataaaa tcgacgaaaa tcctgaagta
aagaaagcga ttgttgataa aagtgcaggg 720ttaattgacc aattattaac
caaaaagaaa agtgaagagg taaatgcttc ggacttcccg 780ccaccaccta
cggatgaaga gttaagactt gctttgccag agacaccaat gcttcttggt
840tttaatgctc ctgctacatc agaaccgagc tcattcgaat ttccaccacc
acctacggat 900gaagagttaa gacttgcttt gccagagacg ccaatgcttc
ttggttttaa tgctcctgct 960acatcggaac cgagctcgtt cgaatttcca
ccgcctccaa cagaagatga actagaaatc 1020atccgggaaa cagcatcctc
gctagattct agttttacaa gaggggattt agctagtttg 1080agaaatgcta
ttaatcgcca tagtcaaaat ttctctgatt tcccaccaat cccaacagaa
1140gaagagttga acgggagagg cggtagacca 1170714PRTListeria
monocytogenes 7Lys Thr Glu Glu Gln Pro Ser Glu Val Asn Thr Gly Pro
Arg 1 5 10 828PRTListeria monocytogenes 8Lys Ala Ser Val Thr Asp
Thr Ser Glu Gly Asp Leu Asp Ser Ser Met 1 5 10 15 Gln Ser Ala Asp
Glu Ser Thr Pro Gln Pro Leu Lys 20 25 920PRTListeria monocytogenes
9Lys Asn Glu Glu Val Asn Ala Ser Asp Phe Pro Pro Pro Pro Thr Asp 1
5 10 15 Glu Glu Leu Arg 20 1033PRTListeria monocytogenes 10Arg Gly
Gly Ile Pro Thr Ser Glu Glu Phe Ser Ser Leu Asn Ser Gly 1 5 10 15
Asp Phe Thr Asp Asp Glu Asn Ser Glu Thr Thr Glu Glu Glu Ile Asp 20
25 30 Arg 1117PRTStreptococcus sp. G148 11Lys Gln Asn Thr Ala Ser
Thr Glu Thr Thr Thr Thr Asn Glu Gln Pro 1 5 10 15 Lys
1217PRTStreptococcus equisimilis 12Lys Gln Asn Thr Ala Asn Thr Glu
Thr Thr Thr Thr Asn Glu Gln Pro 1 5 10 15 Lys 1398PRTHuman
papillomavirus type 16 13Met His Gly Asp Thr Pro Thr Leu His Glu
Tyr Met Leu Asp Leu Gln 1 5 10 15 Pro Glu Thr Thr Asp Leu Tyr Cys
Tyr Glu Gln Leu Asn Asp Ser Ser 20 25 30 Glu Glu Glu Asp Glu Ile
Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp 35 40 45 Arg Ala His Tyr
Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr 50 55 60 Leu Arg
Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu 65 70 75 80
Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln 85
90 95 Lys Pro 14105PRTHuman papillomavirus type 16 14Met His Gly
Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu 1 5 10 15 Pro
Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser 20
25
30 Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His
35 40 45 Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu
Cys Met 50 55 60 Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val
Glu Ser Ser Ala 65 70 75 80 Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe
Leu Asn Thr Leu Ser Phe 85 90 95 Val Cys Pro Trp Cys Ala Ser Gln
Gln 100 105 15158PRTHuman papillomavirus type 16 15Met His Gln Lys
Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro 1 5 10 15 Arg Lys
Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp 20 25 30
Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu 35
40 45 Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp
Gly 50 55 60 Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr
Ser Lys Ile 65 70 75 80 Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr
Gly Thr Thr Leu Glu 85 90 95 Gln Gln Tyr Asn Lys Pro Leu Cys Asp
Leu Leu Ile Arg Cys Ile Asn 100 105 110 Cys Gln Lys Pro Leu Cys Pro
Glu Glu Lys Gln Arg His Leu Asp Lys 115 120 125 Lys Gln Arg Phe His
Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met 130 135 140 Ser Cys Cys
Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 145 150 155
16158PRTHuman papillomavirus type 16 16Met Ala Arg Phe Glu Asp Pro
Thr Arg Arg Pro Tyr Lys Leu Pro Asp 1 5 10 15 Leu Cys Thr Glu Leu
Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys 20 25 30 Val Tyr Cys
Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala 35 40 45 Phe
Lys Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala 50 55
60 Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His
65 70 75 80 Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr
Asn Thr 85 90 95 Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys
Gln Lys Pro Leu 100 105 110 Asn Pro Ala Glu Lys Leu Arg His Leu Asn
Glu Lys Arg Arg Phe His 115 120 125 Asn Ile Ala Gly His Tyr Arg Gly
Gln Cys His Ser Cys Cys Asn Arg 130 135 140 Ala Arg Gln Glu Arg Leu
Gln Arg Arg Arg Glu Thr Gln Val 145 150 155 1722DNAArtificial
SequencePrimer 17ggctcgagca tggagataca cc 221828DNAArtificial
SequencePrimer 18ggggactagt ttatggtttc tgagaaca 281931DNAArtificial
SequencePrimer 19gggggctagc cctcctttga ttagtatatt c
312028DNAArtificial SequencePrimer 20ctccctcgag atcataattt acttcatc
282127DNAArtificial SequencePrimer 21cccgtcgacc agctcttctt ggtgaag
272225DNAArtificial SequencePrimer 22gcggatccca tggagataca cctac
252322DNAArtificial SequencePrimer 23gctctagatt atggtttctg ag
22249PRTHuman papillomavirus type 16 24Arg Ala His Tyr Asn Ile Val
Thr Phe 1 5 259PRTHuman immunodeficiency virus 25Ala Met Gln Met
Leu Lys Glu Thr Ile 1 5 269PRTListeria monocytogenes 26Gly Tyr Lys
Asp Gly Asn Glu Tyr Ile 1 5 2755PRTArtificial SequencePrimer 27Gly
Ala Cys Thr Ala Cys Ala Ala Gly Gly Ala Cys Gly Ala Thr Gly 1 5 10
15 Ala Cys Cys Gly Ala Cys Ala Ala Gly Thr Gly Ala Thr Ala Ala Cys
20 25 30 Cys Cys Gly Gly Gly Ala Thr Cys Thr Ala Ala Ala Thr Ala
Ala Ala 35 40 45 Thr Cys Cys Gly Thr Thr Thr 50 55
2818DNAArtificial SequenceADV451 forward primer 28cctagctaaa
tttaatgt 182918DNAArtificial SequenceADV452 forward primer
29cctagctaaa tttaatga 183021DNAArtificial SequenceADV453 reverse
primer 30taattttccc caagtagcag g 21
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