U.S. patent application number 09/997848 was filed with the patent office on 2003-02-06 for helper virus-free herpesvirus amplicon particles and uses thereof.
Invention is credited to Bowers, William J., Dewhurst, Stephen, Evans, Thomas D., Federoff, Howard J., Frelinger, John G., Rosenblatt, Joseph D., Tolba, Khaled A., Willis, Richard A..
Application Number | 20030027322 09/997848 |
Document ID | / |
Family ID | 26940582 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027322 |
Kind Code |
A1 |
Federoff, Howard J. ; et
al. |
February 6, 2003 |
Helper virus-free herpesvirus amplicon particles and uses
thereof
Abstract
The invention features new helper virus-free methods for making
herpesvirus amplicon particles that can be used in immunotherapies,
including those for treating any number of infectious diseases and
cancers (including chronic lymphocytic leukemia, other cancers in
which blood cells become malignant, lymphomas (e.g. Hodgkin's
lymphoma or non-Hodgkin's type lymphomas). Described herein are
methods of making helper virus-free HSV amplicon particles; cells
that contain those particles (e.g., packaging cell lines or
patients'cells, infected in vivo or ex vivo); particles produced
according to those methods; and methods of treating a patient with
an hf-HSV particle made according to those methods.
Inventors: |
Federoff, Howard J.;
(Rochester, NY) ; Bowers, William J.; (Webster,
NY) ; Frelinger, John G.; (Pittsford, NY) ;
Willis, Richard A.; (Denver, CO) ; Evans, Thomas
D.; (Davis, CA) ; Dewhurst, Stephen;
(Rochester, NY) ; Tolba, Khaled A.; (Rochester,
NY) ; Rosenblatt, Joseph D.; (Ft. Lauderdale,
FL) |
Correspondence
Address: |
LEE CREWS, PH.D.
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Family ID: |
26940582 |
Appl. No.: |
09/997848 |
Filed: |
November 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60253858 |
Nov 29, 2000 |
|
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60250079 |
Nov 30, 2000 |
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Current U.S.
Class: |
435/235.1 ;
435/325; 435/456 |
Current CPC
Class: |
A61K 2039/55522
20130101; A61K 2039/5258 20130101; A61P 31/00 20180101; A61K
2039/55561 20130101; A61K 2039/5156 20130101; C12N 2710/16622
20130101; A61K 39/0011 20130101; A61K 2039/54 20130101; C12N
2710/16643 20130101; A61K 2039/5256 20130101; A61K 2039/5154
20130101; A61K 2039/57 20130101; A61K 48/00 20130101; C12N 7/00
20130101; A61P 35/00 20180101; C07K 14/005 20130101; C07K 14/70575
20130101; C12N 15/86 20130101; C12N 2710/16662 20130101 |
Class at
Publication: |
435/235.1 ;
435/456; 435/325 |
International
Class: |
C12N 007/00; C12N
015/86 |
Goverment Interests
[0002] The work described herein was funded, in part, by grants
from the National Institutes of Health. The government may,
therefore, have certain rights in the invention.
Claims
What is claimed is:
1. A method of generating a herpesvirus amplicon particle, the
method comprising providing a cell that has been stably transfected
with a nucleic acid sequence that encodes an accessory protein; and
transfecting the cell with (a) one or more packaging vectors that,
individually or collectively, encode one or more HSV structural
proteins but do not encode a functional herpesvirus
cleavage/packaging site and (b) an amplicon plasmid comprising a
sequence that encodes a functional herpesvirus cleavage/packaging
site and a herpesvirus origin of DNA replication.
2. A method of generating a herpesvirus amplicon particle, the
method comprising transfecting a cell with (a) one or more
packaging vectors that, individually or collectively, encode one or
more HSV structural proteins but do not encode a functional
herpesvirus cleavage/packaging site; (b) an amplicon plasmid
comprising a sequence that encodes a functional herpesvirus
cleavage/packaging site, a herpesvirus origin of DNA replication,
and a sequence that encodes an immunomodulatory protein, a
tumor-specific antigen, or an antigen of an infectious agent; and
(c) a nucleic acid sequence that encodes an accessory protein.
3. The method of claim 1 or claim 2, wherein the herpesvirus is an
alpha herpesvirus or an Epstein-Barr virus.
4. The method of claim 3, wherein the alpha herpesvirus is a
Varicella-Zoster virus, a pseudorabies virus, or a herpes simplex
virus.
5. The method of claim 1 or claim 2, wherein the accessory protein
inhibits the expression of a gene in the cell.
6. The method of claim 5, wherein the accessory protein is a virion
host shutoff protein.
7. The method of claim 6, wherein the virion host shutoff protein
is an HSV-1 virion host shutoff protein, an HSV-2 virion host
shutoff protein, an HSV-3 virion host shutoff protein, bovine
herpesvirus 1 virion host shutoff protein, bovine herpesvirus 1.1
virion host shutoff protein, gallid herpesvirus 1 virion host
shutoff protein, gallid herpesvirus 2 virion host shutoff protein,
suid herpesvirus 1 virion host shutoff protein, baboon herpesvirus
2 virion host shutoff protein, pseudorabies virus virion host
shutoff protein, cercopithecine herpesvirus 7 virion host shutoff
protein, meleagrid herpesvirus 1 virion host shutoff protein,
equine herpesvirus 1 virion host shutoff protein, or equine
herpesvirus 4 virion host shutoff protein.
8. The method of claim 6, wherein the virion host shutoff protein
is operatively coupled to its native transcriptional control
elements.
9. The method of claim 1 or claim 2, wherein the cell is further
transfected with a sequence encoding a VP16 protein, wherein the
VP16 protein is transiently or stably expressed.
10. The method of claim 9, wherein the VP16 protein is HSV1 VP16,
HSV-2 VP16, bovine herpesvirus 1 VP16, bovine herpesvirus 1.1 VP16,
gallid herpesvirus 1 VP16, gallid herpesvirus 2 VP16, meleagrid
herpesvirus 1 VP16, or equine herpesvirus 4 VP16.
11. The method of claim 1 or claim 2, wherein the one or more
packaging vectors comprises a cosmid, a yeast artificial
chromosome, a bacterial artificial chromosome, a human artificial
chromosome, or an F element plasmid.
12. The method of claim 1 or claim 2, wherein the one or more
packaging vectors comprises a set of cosmids comprising cos
6.DELTA.a, cos 28, cos 14, cos 56, and cos 48.DELTA.a.
13. The method of claim 1 or claim 2, wherein the one or more
packaging vectors, individually or collectively, express the
structural herpesvirus proteins.
14. The method of claim 1 or claim 2, wherein the herpesvirus
origin of DNA replication is not present in the one or more
packaging vectors.
15. The method of claim 1, wherein the amplicon plasmid further
comprises a sequence encoding a therapeutic agent.
16. The method of claim 15, wherein the therapeutic agent is a
protein or an RNA molecule.
17. The method of claim 16, wherein the RNA molecule is an
antisense RNA molecule, RNAi, or a ribozyme.
18. The method of claim 16, wherein the protein is a receptor, a
signaling molecule, a transcription factor, a growth factor, an
apoptosis inhibitor, an apoptosis promoter, a DNA replication
factor, an enzyme, a structural protein, a neural protein, or a
histone.
19. The method of claim 16, wherein the protein is an
immunomodulatory protein, a tumor-specific antigen, or an antigen
of an infectious agent.
20. The method of claim 19, wherein the immunomodulatory protein is
a cytokine or a costimulatory molecule.
21. The method of claim 20, wherein the cytokine is an interleukin,
an interferon, or a chemokine.
22. The method of claim 20, wherein the costimulatory molecule is a
B7 molecule or CD40L.
23. The method of claim 19, wherein the tumor-specific antigen is a
prostate specific antigen.
24. The method of claim 19, wherein the infectious agent is a
virus.
25. The method of claim 24, wherein the virus is a human
immunodeficiency virus.
26. The method of claim 19, wherein the antigen of an infectious
agent is gp120.
27. The method of claim 19, wherein the infectious agent is a
bacterium or parasite.
28. The method of claim 2, wherein the immunomodulatory protein is
a cytokine or a costimulatory molecule.
29. The method of claim 28, wherein the cytokine is an interleukin,
an interferon, or a chemokine.
30. The method of claim 28, wherein the costimulatory molecule is a
B7 molecule or CD40L.
31. The method of claim 2, wherein the tumor-specific antigen is a
prostate specific antigen.
32. The method of claim 2, wherein the infectious agent is a
virus.
33. The method of claim 32, wherein the virus is a human
immunodeficiency virus.
34. The method of claim 2, wherein the antigen of an infectious
agent is gp120.
35. The method of claim 2, wherein the infectious agent is a
bacterium or parasite.
36. The method of claim 1 or claim 2, wherein the amplicon plasmid
further comprises a promoter.
37. A cell transfected by the method of claim 1 or transduced by a
herpesvirus amplicon particle made by the method of claim 1.
38. The cell of claim 37, wherein the cell is a neuron, a blood
cell, a hepatocyte, a keratinocyte, a melanocyte, a neuron, a glial
cell, an endocrine cell, an epithelial cell, a muscle cell, a
prostate cell, or a testicular cell.
39. A cell transfected by the method of claim 2 or transduced by a
herpesvirus amplicon particle made by the method of claim 2.
40. The cell of claim 39, wherein the cell is a neuron, a blood
cell, a hepatocyte, a keratinocyte, a melanocyte, a neuron, a glial
cell, an endocrine cell, an epithelial cell, a muscle cell, a
prostate cell, or a testicular cell.
41. The cell of claim 39, wherein the cell is a malignant cell.
42. The cell of claim 39, wherein the cell is infected with an
infectious agent.
43. The cell of claim 42, wherein the infectious agent is a virus,
a bacterium, or a parasite.
44. The cell of claim 43, wherein the virus is an immunodeficiency
virus.
45. A herpesvirus amplicon particle made by the method of claim
1.
46. The herpesvirus amplicon particle of claim 45, wherein the
herpesvirus is an alpha herpesvirus or an Epstein-Barr virus.
47. The herpesvirus amplicon particle of claim 46, wherein the
alpha herpesvirus is a Varicella-Zoster virus, a pseudorabies
virus, or a herpes simplex virus.
48. The herpesvirus amplicon particle of claim 47, wherein the
herpes simplex virus is a type 1 or a type 2 herpes simplex
virus.
49. A herpesvirus amplicon particle made by the method of claim
2.
50. The herpesvirus amplicon particle of claim 49, wherein the
herpesvirus is an alpha herpesvirus or an Epstein-Barr virus.
51. The herpesvirus amplicon particle of claim 50, wherein the
alpha herpesvirus is a Varicella-Zoster virus, a pseudorabies
virus, or a herpes simplex virus.
52. The herpesvirus amplicon particle of claim 51, wherein the
herpes simplex virus is a type 1 or a type 2 herpes simplex
virus.
53. A method of treating a patient who has cancer, or who may
develop cancer, the method comprising administering to the patient
an HSV amplicon particle of claim 19, wherein the protein is an
immunomodulatory protein or a tumor-specific antigen, or an HSV
amplicon particle made by the method of claim 2, wherein the
protein is an immunomodulatory protein or a tumor-specific
antigen.
54. A method of treating a patient who has cancer, or who may
develop cancer, the method comprising administering to the patient
the cell of claim 37, wherein the amplicon plasmid further encodes
an immunomodulatory protein or a tumor-specific antigen, or the
cell of claim tumor-specific antigen, or an HSV amplicon particle
made by the method of claim 39, wherein the protein is an
immunomodulatory protein or a tumor-specific antigen.
55. A method of treating a patient who has a disease caused by an
infectious agent, or who may contract a disease caused by an
infectious agent, the method comprising administering to the
patient the herpesvirus amplicon particle of claim 45, wherein the
amplicon plasmid further comprises a sequence that encodes an
antigen of the infectious agent, or the cell of claim 39, wherein
the amplicon plasmid comprises a sequence that encodes an antigen
of an infectious agent.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of two earlier-filed
provisional applications: U.S. Ser. No. 60/253,858, filed Nov. 29,
2000, and U.S. Ser. No. 60/250,079, filed Nov. 30, 2000. The
contents of these provisional applications are hereby incorporated
by reference in the present application in their entirety.
FIELD OF THE INVENTION
[0003] The present invention related to improved methods for making
helper virus-free preparations of herpesvirus amplicon particles;
the particles per se; and methods of using the particles to treat
patients, including patients who have cancer or an infectious
disease.
BACKGROUND
[0004] Herpes simplex virus (HSV) is a DNA virus capable of rapidly
and efficiently infecting a wide variety of cell types (Leib and
Olivo, BioEssays 15:547-554, 1993). Plasmid-based viral vectors
derived from HSV, termed amplicons, are easy to construct and
package into viral particles.
SUMMARY
[0005] The compositions and methods of the present invention are
based on a number of discoveries, including the discoveries that:
(1) cells transduced with HSV amplicon vectors can process proteins
encoded by the vectors for class I MHC presentation; (2) when used
to deliver a viral antigen, herpes virus-based amplicon vectors can
induce a cell-mediated immune response that is equivalent to that
induced by live herpesvirus vectors and that exceeds that induced
by a modified vaccinia Ankara vector; (3) animals immunized with
HSV amplicon-transduced dendritic cells respond by producing
antigen-specific cytotoxic T lymphocytes (e.g., animals immunized
with an HSV-gp120 amplicon display a cell-mediated immune
response); (4) animals infected with HSV-gp120 also exhibit a
humoral immune response; (5) the expression of virion host shutoff
(vhs) proteins in helper virus-free packaging systems improves
amplicon titer and vector stocks prepared in this way do not
exhibit the pseudotransduction phenomenon (to further enhance
packaging efficiency, an HSV transcriptional activator can be
introduced into packaging cells); and (6) helper virus-free
amplicon preparations are superior to helper virus-containing
amplicon preparations (see the studies below).
[0006] Accordingly, the invention features new helper virus-free
methods for making herpesvirus amplicon particles that can be used
in immunotherapies, including those for treating any number of
infectious diseases and cancers (including chronic lymphocytic
leukemia, other cancers in which blood cells become malignant,
lymphomas (e.g. Hodgkin's lymphoma or non-Hodgkin's type
lymphomas), melanoma, glioblastoma, astrocytoma, pancreatic cancer,
a cancer of the reproductive system, a cancer of the endocrine
system, neuroblastoma, breast cancer, colorectal cancer, stomach
cancer, cancer of the throat or mouth, lung cancer, or bladder
cancer). The invention features: methods of making helper
virus-free HSV amplicon particles; cells that contain those
particles (e.g., packaging cell lines or patients' cells, infected
in vivo or ex vivo); particles produced according to those methods
(such particles, regardless of the method by which they are
produced, may be abbreviated herein as "hf-HSV" particles); and
methods of treating a patient with an hf-HSV particle made
according to those methods. For example, hf-HSV particles
(including those made according to the methods described herein)
that contain one or more genes encoding one or more therapeutic
proteins, can be used to transduce cells. For example, one can
transduce cells that contain an infectious agent (such as a virus
or bacterium) or that have become malignant (e.g., malignant cells
of the prostate, skin, bladder, breast, endocrine system, or
gastrointestinal tract). The therapeutic protein (discussed further
below) can be an immunostimulatory protein and may be a neoantigen
(e.g., a tumor-specific antigen, such as prostate-specific antigen
(PSA))
[0007] In one embodiment, a cell that contains an infectious agent
or a cell that is malignant is transduced (in vivo or ex vivo) with
an hf-HSV amplicon particle that encodes an immunostimulatory
protein (i.e., any immunomodulatory protein or peptide that, when
expressed by a target cell, induces or enhances an immune response
to that cell). For example, a patient who has cancer can be treated
with an HSV amplicon particle (or a cell within which it is
contained) that expresses a protein that acts as a general
stimulator of the immune system or a specific protein, such as a
tumor-specific antigen (these particles and cells can be those made
by the methods described herein). Similarly, a patient who has an
infectious disease can be treated with an HSV amplicon particle (or
a cell within which it is contained) that expresses a protein that
acts as a general stimulator of the immune system or a specific
antigen associated with (i.e., expressed by) the infectious agent
(here again, the patients that are treated for an infectious
disease can be treated with particles or cells made by the methods
described herein).
[0008] Immunostimulatory proteins include cytokines, including
chemotactic cytokines (also known as chemokines) and interleukins,
adhesion molecules (e.g., I-CAM) and costimulatory factors
necessary for activation of B cells or T cells.
[0009] The hf-HSV particles can be made according to methods known
in the art (Applicants know of no suggestion that any previously
made particles or cells should be used for the treatment of cancer
or infectious disease) or according to the new methods described
below (the novel methods for producing herpesvirus amplicon
particles produce particles that are different from those produced
to date, even those produced by helper virus-free methods, and
these particles (and the cells that contain them) can be used to
treat not only cancer and infectious disease, but also any
condition that would benefit from the administration of a protein
(e.g., neurological conditions in which neurotransmitters are not
adequately available).
[0010] More specifically, the invention features a method of
generating a herpesvirus amplicon particle. In one embodiment, the
method comprises: (1) providing a cell that has been stably
transfected with a nucleic acid sequence that encodes an accessory
protein (alternatively, a transiently transfected cell can be
provided); and (2) transfecting the cell with (a) one or more
packaging vectors that, individually or collectively, encode one or
more HSV structural proteins but do not encode a functional
herpesvirus cleavage/packaging site and (b) an amplicon plasmid
comprising a sequence that encodes a functional herpesvirus
cleavage/packaging site and a herpesvirus origin of DNA
replication. In another embodiment, the method comprises
transfecting a cell with (a) one or more packaging vectors that,
individually or collectively, encode one or more HSV structural
proteins but do not encode a functional herpesvirus
cleavage/packaging site; (b) an amplicon plasmid comprising a
sequence that encodes a functional herpesvirus cleavage/packaging
site, a herpesvirus origin of DNA replication, and a sequence that
encodes an immunomodulatory protein, a tumor-specific antigen, or
an antigen of an infectious agent; and (c) a nucleic acid sequence
that encodes an accessory protein.
[0011] In either of these methods, one or more of the following
additional limitations may apply. For example, in either method,
the herpesvirus can be any of the more than 100 known species of
herpesvirus. For example, the herpesvirus can be an alpha
herpesvirus (e.g., a Varicella-Zoster virus, a pseudorabies virus,
or a herpes simplex virus (e.g., type 1 or type 2 HSV) or an
Epstein-Barr virus. Similarly, both methods require sequences that
encode an accessory protein and, in either method, the accessory
protein can be a protein that inhibits the expression of a gene in
the cell. For example, the accessory protein can be a virion host
shutoff (vhs) protein (e.g., an HSV-1 vhs protein, an HSV-2 vhs
protein, an HSV-3 vhs protein, bovine herpesvirus 1 vhs protein,
bovine herpesvirus 1.1 vhs protein, gallid herpesvirus 1 vhs
protein, gallid herpesvirus 2 virion hsp, suid herpesvirus 1 vhs
protein, baboon herpesvirus 2 vhs protein, pseudorabies vhs
protein, cercopithecine herpesvirus 7 vhs protein, meleagrid
herpesvirus 1 vhs protein, equine herpesvirus 1 vhs protein, or
equine herpesvirus 4 vhs protein). Any of these proteins can be
operatively coupled to its native transcriptional control
element(s) or to an artificial control element (i.e., a control
element that does not normally regulate its expression in
vivo).
[0012] The methods by which herpesvirus amplicon particles are
generated can also include a step in which the cell is transfected
with a sequence encoding a VP16 protein, which may be transiently
or stably expressed. Alternatively, or in addition, one can
engineer a transcriptional activator to mimic VP16 (e.g., a
pseudo-activator that recognizes cis elements but uses a different
transcriptional activation domain).
[0013] The VP16 protein can be HSV1 VP16, HSV-2 VP16, bovine
herpesvirus 1 VP16, bovine herpesvirus 1.1 VP16, gallid herpesvirus
1 VP16, gallid herpesvirus 2 VP16, meleagrid herpesvirus 1 VP16, or
equine herpesvirus 4 VP16.
[0014] The vhs and VP16 encoding sequences can be introduced into a
cell on the same vector or on two different vectors or on two
different types of vectors (e.g., both sequences can be introduced
in the same plasmid, in two different plasmids, or in a plasmid and
cosmid). Sequences encoding vhs and/or VP16 can be transiently or
stably introduced into cells (these methods are routine in the
art), and the invention features a cell that is transiently or
stably transfected with one or both of the sequences that encode
one or more of a vhs or VP16 protein.
[0015] As noted above, the herpesvirus (e.g., HSV) amplicon
particles are made by methods that employ one or more packaging
vectors, which may comprise a cosmid (and may include a set of
cosmids), a yeast artificial chromosome, a bacterial artificial
chromosome, a human artificial chromosome, or an F element plasmid.
A single packaging vector can encode the entire genome of a
herpesvirus, or the genome may be divided between two or more
vectors. For example, the packaging vectors can include a set of
cosmids (e.g., a set of cosmids comprising cos6.DELTA.a, cos28,
cos14, cos56, and cos48.DELTA.a). One or more packaging vectors,
individually or collectively, can express the structural
herpesvirus proteins. The herpesvirus origin of DNA replication is
not present in the one or more packaging vectors.
[0016] In the method first described above (the method that employs
a transiently or stably transfected cell), the amplicon plasmid can
also include a sequence encoding a therapeutic agent. The
therapeutic agent can be a protein or an RNA molecule (e.g., an
antisense RNA molecule, RNAi, or a ribozyme). In the event the
therapeutic agent is a protein, the protein can be a receptor
(e.g., a receptor for a growth factor or neurotransmitter), a
signaling molecule (e.g., a growth factor or neurotransmitter), a
transcription factor, a factor that promotes or inhibits apoptosis,
a DNA replication factor, an enzyme, a structural protein, a neural
protein, or a histone. The protein can also be an immunomodulatory
protein (e.g., a cytokine, such as an interleukin, an interferon,
or a chemokine, or a costimulatory molecule, such as a B7 molecule
or CD40L), a tumor-specific antigen (e.g., PSA), or an antigen of
an infectious agent (e.g., a virus such as a human immunodeficiency
virus, a herpesvirus, a papillomavirus, an influenza virus, or
Ebola virus, a bacterium (e.g., an Escherichia (e.g., E. coli)
Staphylococcus, Campylobacter (e.g., C. jejuni), Listeria (e.g., L.
monocytogenes), Salmonella, Shigella or Bacillus (e.g., Bacillus
anthracis)), or a parasite.
[0017] In the second method described above, the amplicon plasmid
encodes an immunomodulatory protein, a tumor-specific antigen, or
the antigen of an infectious agent (including those described
above). It will be apparent to one of ordinary skill in the art
which therapeutic agents can be expressed to generate particles and
cells useful for treating which conditions. For example, one would
select an antigen expressed by HIV (e.g., gp120) to treat a patient
who is infected, or who may become infected, with HIV.
[0018] The amplicon plasmid can include a promoter to increase the
efficiency of expression of the therapeutic agent.
[0019] In addition, the invention features kits containing one or
more of the herpesvirus amplicon particles described herein; one of
more of the cells containing them; or one or more of the components
useful in generating either the particles or the cells. For
example, a kit can include a packaging vector and an amplicon
plasmid. Optionally, the kit can also contain stably transfected
cells. Optionally, the kit can include instructions for use.
[0020] The particles generated by the methods of the invention, and
cells that contain those particles, are also within the scope of
the invention. The particles and cells that come within the scope
of the invention include any of those made using the methods
described herein. The cell can be virtually any differentiated
cell, including a neuron, a blood cell, a hepatocyte, a
keratinocyte, a melanocyte, a neuron, a glial cell, an endocrine
cell, an epithelial cell, a muscle cell, a prostate cell, or a
testicular cell. The cell can also be a malignant cell (including
any of those that arise from the differentiated cells just listed;
e.g., a neuroblastoma, a lymphoma or leukemia cell, a
hepatocarcinoma cell etc.). Alternatively, or in addition, the cell
can be any cell that is infected with an infectious agent
(including a virus, a bacterium, or a parasite, including, but not
limited to, those types described herein).
[0021] Gene therapy vectors based on the herpes simplex virus have
a number of features that make them advantageous in gene therapies.
They exhibit a broad cellular tropism, they have the capacity to
package large amounts of genetic material (and thus can be used to
express multiple genes or gene sequences), they have a high
transduction efficiency, and they are maintained episomally, which
makes them less prone to insertional mutagenesis. In addition to
infecting many different types of cells, HSV vectors can transduce
non-replicating or slowly replicating cells, which has therapeutic
advantages. For example, freshly isolated cells can be transduced
in tissue culture, where conditions may not be conducive to cell
replication. The ability of HSV vectors to infect non-replicating
or poorly replicating cells also means that cells (such as tumor
cells) that have been irradiated can still be successfully treated
with HSV vectors.
[0022] The transduction procedure can also be carried out fairly
quickly; freshly harvested human tumors have been successfully
transduced within about 20 minutes. As a result, cells (such a
tumor cells) can be removed from a patient, treated, and
readministered to the patient in the course of a single operative
procedure (one would readminister tumor cells following
transduction with, for example, an immunostimulatory agent (HSV
vectors encoding immunomodulatory proteins and cells transduced
with such vectors can confer specific antitumor immunity that
protects against tumor growth in vivo).
[0023] On the other hand, it is inherently difficult to manipulate
a large viral genome (150 kb) and HSV-encoded regulatory and
structural viral proteins may be toxic (Frenkel et al., Gene Ther.
1 Suppl. 1:S40-46, 1994).
[0024] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, useful methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflicting subject matter, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
[0025] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a panel of four photomicrographs. Murine dendrite
cells were photographed using phase contrast optics and fluorescent
light after infection with HSV-creGFP or HSV-OVA amplicons
(MOI=1).
[0027] FIG. 2 is a schematic representation of an infection
procedure and photographs of activated T cells following co-culture
with infected dendritic cells.
[0028] FIG. 3 is a schematic representation of an immunization and
line graphs of the resulting cytotoxic T lymphocyte (CTL)
response.
[0029] FIG. 4 is a bar graph representing the expression of IL-12
p70 (ng/ml) following treatment of dendritic cells (antigen
presenting cells (APCs)) with one of two HSV amplicons (one that
expresses PSA and one that expresses p35) followed by activation
with oligonucleotides that contain an immunostimulatory CpG
sequence or oligonucleotides in which the CpG sequence is altered
to GpC.
[0030] FIG. 5 is a photograph of a Western blot. Lysates were
prepared from HSVgp120-infected NIH 3T3 cells.
[0031] FIG. 6 is a series of four bar graphs illustrating the
cellular responses to class I-restricted peptides from gp120.
[0032] FIG. 7 is a bar graph made by analyzing the humoral response
in mice immunized with HSVgp120 (anti-env IgG responses in
serum).
[0033] FIG. 8 is a graph plotting the results of a cell lysis assay
(JAM). HSVgp120 mediated induction of CTL activity.
[0034] FIG. 9 is a series of four bar graphs illustrating the
effect of administering an HSV-gp120 amplicon by three common
routes of administration (intramuscular, subcutaneous, or
intraperitoneal).
[0035] FIG. 10 is a Table of essential HSV-1 genes.
[0036] FIG. 11 shows three Tables. The uppermost concerns IL-2
production following transduction of CLL cells with helper
virus-containing and helper virus-free amplicon stocks; the middle
table concerns the % of CLL cells expressing B7.1 and CD40L
following transduction with helper virus-containing and helper
virus-free amplicon stocks; the lower table concerns
gamma-interferon levels in supernatant derived from CTL assays
using CLL cells transduced with helper virus-free amplicon
stocks.
DETAILED DESCRIPTION
[0037] Helper virus-free systems for packaging herpesvirus
particles, including those described herein, include the use of at
least one vector (herein, the packaging vector) that, upon delivery
to a cell that supports herpesvirus replication, will form a DNA
segment (or segments) capable of expressing sufficient structural
herpesvirus proteins that they are capable of assembling into
herpesvirus particles. For example, sets of cosmids have been
isolated that contain overlapping clones that represent the entire
genomes of a variety of herpesviruses (see U.S. Pat. No.
5,998,208). The packaging vectors are prepared so that none of the
viruses used will contain a functional herpesvirus
cleavage-packaging site containing sequence. This sequence is
referred to as the "a" sequence (and is not encoded by the
packaging vector(s)). The "a" sequence can be deleted from the
packaging vector(s) by any of a variety of techniques practiced by
those of ordinary skill in the art. For example, one can simply
delete the entire sequence (by, for example, the techniques
described in U.S. Pat. No. 5,998,208). Alternatively, one can
delete a sufficient portion of the sequence to render it incapable
of packaging. Another alternative is to insert nucleotides into the
site that render it non-functional.
[0038] The core of the herpesvirus particle is formed from a
variety of structural genes that create the capsid matrix. It is
necessary to have those genes for matrix formation present in a
susceptible cell used to prepare particles. Preferably, the
necessary envelope proteins are also expressed. In addition, there
are a number of other proteins present on the surface of a
herpesvirus particle. Some of these proteins help mediate viral
entry into certain cells. Thus, the inclusion or exclusion of the
functional genes encoding these proteins will depend upon the
particular use of the particle.
[0039] The amplicon plasmid contains a herpesvirus
cleavage/packaging site containing sequence and an origin of DNA
replication (ori) that is recognized by the herpesvirus DNA
replication proteins and enzymes. This vector permits packaging of
desired nucleotide inserts in the absence of helper viruses. In
some embodiments, the amplicon plasmid contains at least one
heterologous DNA sequence that encodes a therapeutic agent,
optionally and operatively linked to a promoter sequence.
[0040] Herpesvirus (e.g., HSV)-based vectors have several features
that make them attractive for use in gene therapies. As noted
above, they transduce cells in a highly efficient manner, they can
infect post-mitotic cells, and they have the ability to package
large amounts of genetic material. The amplicon plasmid,
essentially a eukaryotic expression plasmid, can contain one or
more of the following elements: (i) an HSV-derived origin of DNA
replication (ori) and packaging sequence ("a" sequence); (ii) a
transcription unit driven typically the the HSV-1 immediate early
(IE) 4/5 promoter followed by an SV-40 polyadenylation site; and
(iii) a bacterial origin of replication and an antibiotic
resistance gene for propagation in E. coli (Frenkel, supra; Spaete
and Frenkel, Cell 30:295-304, 1982).
[0041] Amplicon plasmids are dependent upon helper virus function
to provide the replication machinery and structural proteins
necessary for packaging amplicon plasmid DNA into viral particles.
Helper packaging function is usually provided by a
replication-defective virus that lacks an essential viral
regulatory gene. The final product of helper virus-based packaging
contains a mixture of varying proportions of helper and amplicon
virions. Recently, helper virus-free amplicon packaging methods
were developed by providing a packaging-deficient helper virus
genome via a set of five overlapping cosmids (Fraefel et al., J.
Virol. 70:7190-7197, 1996) or by using a bacterial artificial
chromosome (BAC) that encodes for the entire HSV genome minus its
cognate cleavage/packaging signals (Stavropoulos and Strathdee, J.
Virol. 72:7137-7143, 1998; Saeki et al., Hum Gene Ther.
9:2787-2794, 1998).
[0042] Conditions Amenable to Treatment
[0043] The compositions of the present invention (including
herpesvirus particles and cells that contain them) can be used to
treat patients who have been, or who may become, infected with a
wide variety of agents (including viruses such as a human
immunodeficiency virus, human papilloma virus, herpes simplex
virus, influenza virus, pox viruses, bacteria, such as E. coli or a
Staphylococcus, or a parasite) and with a wide variety of cancers.
A patient can be treated after they have been diagnosed as having a
cancer or an infectious disease or, since the agents of the present
invention can be formulated as vaccines, patients can be treated
before they have developed cancer or contracted an infectious
disease. Thus, "treatment" encompasses prophylactic treatment.
[0044] Chronic lymphocytic leukemia (CLL) is a malignancy of mature
appearing small B lymphocytes that closely resemble those in the
mantle zone of secondary lymphoid follicles (Caligaris-Cappio and
Hamblin, J. Clin. Oncol. 17:399-408, 1999). CLL remains a largely
incurable disease of the elderly with an incidence of more than 20
per 100,000 above the age of 70, making it the most common leukemia
in the United States and Western Europe. CLL, which arises from an
antigen-presenting B cell that has undergone a non-random genetic
event (del13q14-23.1, trisomy 12, del 11q22-23 and del6q21-23
(Dohner et al., J. Mol. Med. 77:266-281, 1999) and clonal
expansion, exhibits a unique tumor-specific antigen in the form of
surface immunoglobulin. CLL cells possess the ability to
successfully process and present this tumor antigen, a
characteristic that makes the disease an attractive target for
immunotherapy (Bogen et al., Eur. J. Immunol. 16:1373-1378, 1986;
Bogen et al., Int. Rev. Immunol. 10:337-355, 1993; Kwak et al., N.
Engl. J. Med. 327:1209-1215, 1992; and Trojan et al., Nat. Med.
6:667-672, 2000). However, the lack of expression of co-stimulatory
molecules on CLL cells renders them inefficient effectors of T cell
activation, a prerequisite for generation of anti-tumor immune
responses (Hirano et al., Leukemia 10:1168-1176, 1996). This
failure to activate T cells has been implicated in the
establishment of tumor-specific tolerance (Cardoso et al., Blood
88:41-48, 1996). Reversal of preexisting tolerance can,
potentially, be achieved by up-regulating a panel of co-stimulatory
molecules (B7.1, B7.2 and ICAM-I) (Grewal and Flavell, Immunol.
Rev. 153:85-106, 1996) through the activation of CD40
receptor-mediated signaling and concomitant enhancement of antigen
presentation machinery (Khanna et al., J. Immunol. 159:5982-5785,
1997; Lanzavecchia, Nature 393:413-414, 1998; Diehl et al., Nat.
Med. 5:774-779, 1999; Sotomayor et al., Nat. Med. 5:780-787,
1999).
[0045] Applying the information above in effective gene therapies
for CLL has been hampered by the lack of a safe and reliable vector
that can be used to transduce primary leukemia cells. In contrast
to tumor cell lines, CLL cells are effectively post-mitotic; only a
small fraction of the population enters the cell cycle (Andreeff et
al., Blood 55:282-293, 1980). Although both retroviral and
adenoviral vectors have been employed in different clinical trials
for cancer gene therapy, both systems exhibit limitations (Uckert
and Walther, Pharmacol. Ther. 63:323-347, 1994; Vile et al., Mol
Biotechnol. 5:139-158, 1996; Collins, Ernst Schering Research
Foundation Workshop, 2000; Hitt et al., Adv. Pharmacol. 40:137-206,
1997; Kochanek, Hum. Gene Ther. 10:2451-2459, 1999). For example,
the low levels of integrin receptors for adenovirus on CLL cells
mandates the use of very high adenovirus titers, preactivation of
the CLL cell with IL-4 and/or anti-CD40/CD40L (Cantwell et al.,
Blood 88:4676-4683, 1996; Huang et al., Gene Ther. 4:1093-1099,
1997), or adenovirus modification with polycations to achieve
clinically meaningful levels of transgene expression (Howard et
al., Leukemia 13:1608-1616, 1999).
[0046] In some of the Examples below, HSV amplicon particles were
used to transduce primary human B-cell chronic lymphocytic leukemia
(CLL) cells. The vectors were constructed to encode
.beta.-galactosidase (by inclusion of the lacZ gene), B7.1 (also
known as CD80), or CD40L (also known as CD154), and they were
packaged using either a standard helper virus (HSVlac, HSVB7.1, and
HSVCD40L) or by a helper virus-free method (hf-HSVlac, hf-HSVB7.1,
and hf-HSVCD40L). CLL cells transduced with these vectors were
studied for their ability to stimulate allogeneic T cell
proliferation in a mixed lymphocyte tumor reaction (MLTR). A
vigorous T cell proliferative response was obtained using cells
transduced with hf-HSVB7.1 but not with HSVB7.1. CLL cells
transduced with either HSVCD40L or hf-HSVCD40L were also compared
for their ability to up-regulate resident B7.1 and function as T
cell stimulators. Significantly enhanced B7.1 expression was seen
in response to CD40L delivered by hf-HSVCD40L amplicon stock
(compared to HSVCD40L). CLL cells transduced with hf-HSVCD40L were
also more effective at stimulating T cell proliferation than those
transduced with HSVCD40L stocks. These studies support the
conclusion that HSV amplicons are efficient vectors for gene
therapy, particularly of hematologic malignancies, and that helper
virus-free amplicon preparations are better suited for use in
therapeutic compositions.
[0047] Therapeutic Agents
[0048] As noted, the hf-HSV amplicon particles described herein
(and the cells that contain them) can express a heterologous
protein (i.e., a full-length protein or a portion thereof (e.g., a
functional domain or antigenic peptide) that is not naturally
encoded by a herpesvirus). The heterologous protein can be any
protein that conveys a therapeutic benefit on the cells in which
it, by way of infection with an hf-HSV amplicon particle, is
expressed or a patient who is treated with those cells.
[0049] The therapeutic agents can be immunomodulatory (e.g.,
immunostimulatory) proteins (as described in U.S. Pat. No.
6,051,428). For example, the heterologous protein can be an
interleukin (e.g., IL-1, IL-2, IL-4, IL-10, or IL-15), an
interferon (e.g., IFN.gamma.), a granulocyte macrophage colony
stimulating factor (GM-CSF), a tumor necrosis factor (e.g.,
TNF.alpha.), a chemokine (e.g., RANTES, MCP-1, MCP-2, MCP-3,
DC-CK1, MIP-1.alpha., MIP-3.alpha., MIP-.beta., MIP-3.beta., an
.alpha. or C-X-C chemokine (e.g., IL-8, SDF-1.beta., 8DF-1.alpha.,
GRO, PF-4 and MIP-2). Other chemokines that can be usefully
expressed are in the C family of chemokines (e.g., lymphotactin and
CX3C family chemokines).
[0050] Intercellular adhesion molecules are transmembrane proteins
within the immunoglobulin superfamily that act as mediators of
adhesion of leukocytes to vascular endothelium and to one another.
The vectors described herein can be made to express ICAM-1 (also
known as CD54), and/or another cell adhesion molecule that binds to
T or B cells (e.g., ICAM-2 and ICAM-3).
[0051] Costimulatory factors that can be expressed by the vectors
described herein are cell surface molecules, other than an antigen
receptor and its ligand, that are required for an efficient
lymphocytic response to an antigen (e.g., B7 (also known as CD80)
and CD40L).
[0052] When used for gene therapy, the transgene encodes a
therapeutic transgene product, which can be either a protein or an
RNA molecule.
[0053] Therapeutic RNA molecules include, without limitation,
antisense RNA, inhibitory RNA (RNAi), and an RNA ribozyme. The RNA
ribozyme can be either cis or trans acting, either modifying the
RNA transcript of the transgene to afford a functional RNA molecule
or modifying another nucleic acid molecule. Exemplary RNA molecules
include, without limitation, antisense RNA, ribozymes, or RNAi to
nucleic acids for huntingtin, alpha synuclein, scatter factor,
amyloid precursor protein, p53, VEGF, etc.
[0054] Therapeutic proteins include, without limitation, receptors,
signaling molecules, transcription factors, growth factors,
apoptosis inhibitors, apoptosis promoters, DNA replication factors,
enzymes, structural proteins, neural proteins, and histone or
non-histone proteins. Exemplary protein receptors include, without
limitation, all steroid/thyroid family members, nerve growth factor
(NGF), brain derived neurotrophic factor (BDNF), neutotrophins 3
and 4/5, glial derived neurotrophic factor (GDNF), cilary
neurotrophic factor (CNTF), persephin, artemin, neurturin, bone
morphogenetic factors (B M1's), c-ret, gp 130, dopamine receptors
(D 1D5), muscarinic and nicotinic cholinergic receptors, epidermal
growth factor (EGF), insulin and insulin-like growth factors,
leptin, resistin, and orexin. Exemplary protein signaling molecules
include, without limitation, all of the above-listed receptors plus
MAPKs, ras, rac, ERKs, NFK.beta., GSK3.beta., AKT, and PI3K.
Exemplary protein transcription factors include, without
limitation, .about.300, CBP, HIF-1alpha, NPAS1 and 2, HIF-1.beta.,
p53, p73, nurr 1, nurr 77, MASHs, REST, and NCORs. Exemplary neural
proteins include, without limitation, neurofilaments, GAP-43,
SCG-10, etc. Exemplary enzymes include, without limitation, TH,
DBH, aromatic amino acid decarboxylase, parkin, unbiquitin E3
ligases, ubiquitin conjugating enzymes, cholineacetyltransferase,
neuropeptide processing enzymes, dopamine, VMAT and other
catecholamine transporters. Exemplary histones include, without
limitation, H1-5. Exemplary non-histones include, without
limitation, ND10 proteins, PML, and HMG proteins. Exemplary pro-and
anti-apoptotic proteins include, without limitation, bax, bid, bak,
bcl-xs, bcl-xl, bcl-2, caspases, SMACs, and IAPs.
[0055] The one or more vectors individually or collectively
encoding all essential HSV genes but excluding all
cleavage/packaging signals can either be in the form of a set of
vectors or a single bacterial-artificial chromosome ("BAC"), which
is formed, for example, by combining the set of vectors to create a
single, doublestranded vector. Preparation and use of a five cosmid
set is disclosed in (Fraefel et al., "Helper virus-free transfer of
herpes simplex virus type 1 plasmid vectors into neural cells, " J.
Virol., 70:7190-7197, 1996). Ligation of the cosmids together to
form a single BAC is disclosed in Stavropoulos and Strathdee (J.
Virol. 72:7137-43, 1998). The BAC described in Stavropoulos and
Strathdee includes a pac cassette inserted at a BamHI site located
within the UL41 coding sequence, thereby disrupting expression of
the HSV-1 virion host shutoff protein.
[0056] By "essential HSV genes", it is intended that the one or
more vectors include all genes that encode polypeptides that are
necessary for replication of the amplicon vector and structural
assembly of the amplicon particles. Thus, in the absence of such
genes, the amplicon vector is not properly replicated and packaged
within a capsid to form an amplicon particle capable of adsorption.
Such "essential HSV genes" have previously been reported in review
articles by Roizrnan (Proc. Natl. Acad. Sci. USA 11:307-1 13, 1996;
Acta Viroloeica 43:75-80, 1999. Another source for identifying such
essential genes is available at the Internet site operated by the
Los Alamos National Laboratory, Bioscience Division, which reports
the entire HSV-1 genome and includes a table identifying the
essential HSV-1 genes. The genes currently identified as essential
are listed in the Table provided as FIG. 10.
[0057] Formulation and Administration of hf-HSV Amplicon
Particles
[0058] The hf-HSV amplicon particles described herein can be
administered to patients directly or indirectly; alone or in
combination with other therapeutic agents; and by any route of
administration. For example, the hf-HSV amplicon particles can be
administered to a patient indirectly by administering cells
transduced with the vector to the patient. Alternatively, or in
addition, an hf-HSV amplicon particle could be administered
directly. For example, an hf-HSV amplicon particle that expresses
an immunostimulatory protein or a tumor-specific antigen can be
introduced into a tumor by, for example, injecting the vector into
the tumor or into the vicinity of the tumor (or, in the event the
cancer is a blood-bourne tumor, into the bloodstream).
[0059] Administration of HSV-immunomodulatory protein amplicons
encoding cytokines such as IL-2, GM-CSF and RANTES, intercellular
adhesion molecules such as ICAM-1 and costimulatory factors such as
B7.1 all provide therapeutic benefit in the form of reduction of
preexisting tumor size, a vaccine-effect protecting against tumor
growth after a subsequent challenge, or both (see U.S. Pat. No.
6,051,428; see also Kutubuddin et a., Blood 93:643-654, 1999). The
helper virus-free HSV vectors disclosed herein can be administered
in the same manner.
[0060] The herpesvirus amplicon particles described herein, and
cells that contain them, can be administered, directly or
indirectly, with other species of HSV-transduced cells (e.g.,
HSV-immunomodulatory transduced cells) or in combination with other
therapies, such as cytokine therapy. Such administrations may be
concurrent or they may be done sequentially. Thus, in one
embodiment, HSV amplicon particles, the vectors with which they are
made (i.e., packaging vectors, amplicon plasmids, and vectors that
express an accessory protein) can be injected into a living
organism or patient (e.g., a human patient) to treat, for example,
cancer or an infectious disease. In further embodiments, one or
more of these entities can be administered after administration of
a therapeutically effective amount of a cytokine.
EXAMPLES
Example 1
[0061] HSV Amplicon Vector-mediated Transduction of Murine
Dendritic Cells.
[0062] We have constructed amplicon particles that encode the model
tumor antigen ovalbumin (HSV-OVA) and human prostate-specific
antigen (HSV-PSA), a protein that is expressed specifically in
prostate epithelium and prostate carcinoma cells.
[0063] As shown in FIG. 1, dendritic cells can be transduced with
HSV amplicons. Murine dendritic cells were infected overnight with
HSV-creGFP or, as a negative control, a comparable vector that did
not include a fluorescent marker (HSV-OVA). The cells were viewed
under a microscope (without fixation) with phase contrast optics
and with fluorescent light appropriate for visualizing GFP. The
cells, as they appeared by phase contrast following transduction
with the HSV-creGFP amplicon and the HSV-OVA amplicion, are shown
in the upper and lower left-hand panels of FIG. 1, respectively.
When viewed with fluorescent light, the cells successfully
transduced with the HSV-creGFP amplicon fluoresce (upper right-hand
panel of FIG. 1), but none of the HSV-OVA-transduced cells do
(lower right-hand panel of FIG. 1).
Example 2
[0064] Dendritic Cells Transduced with HSV Amplicons Present
Antigen to T Cell Hybridomas.
[0065] As in Example 1, murine dendritic cells (obtained from a
C57Bl/6.times.BALB/cByJ)F1 mouse) were infected with an HSV-OVA
amplicon and, as a negative control, a comparable population of
dendritic cells were infected with an HSV-PSA amplicon. The
dendritic cells were then cultured overnight with CTL hybridoma B3Z
cells that (1) have been transfected with a construct in which the
lacZ gene, encoding .beta.-galactosidase, is placed under the
control of an IL-2 promoter (NFAT) and (2) become activated in the
presence of ovalbumin. (We have also developed class I-restricted
CTL hybridomas specific for PSA). The construct is illustrated at
the top of FIG. 2. Following T cell activation, the NFAT promoter
is bound, the lacZ gene is transcribed, and the cells in which
.beta.-galactosidase is produced turn blue upon staining with X-gal
(a standard assay). The hybridoma cells, as they appear following
X-gal staining, are shown in the lower half of FIG. 2. No T cells
co-cultured with HSV-PSA-transfected dendritic cells turned blue
(left-hand photograph), but many of those co-cultured with
HSV-OVA-transfected cells did (right-hand panel). The fact that T
cells were activated means that the dendritic cells were not only
successfully transduced, but also processed OVA for class I MHC
presentation.
[0066] Infection of DCs with HSV-PSA and co-culture with CTL
hybridomas specific for PSA can be used to evaluate presentation of
PSA. In fact, infection with an HSV-based amplicon that expresses
any antigen of interest can be similarly tested for
presentation.
Example 3
[0067] Mice Immunized with HSV Amplicon-transduced Dendritic Cells
Respond by Producing Antigen-specific Cytotoxic T Lymphocytes.
[0068] Dendritic cells were infected in cell culture with one of
two amplicons: an HSV-PSA amplicon or an HSV-OVA amplicon, each at
an MOI of 1. The transduced cells were used to immunize mice
(BALB/c mice were immunized with HSV-PSA-transduced dendritic cells
and C57Bl/6 mice were immunized with HSV-OVA-transduced dendritic
cells, as illustrated in FIG. 3). The cells were injected
subcutaneously on day 1 and day 7. Splenocytes were subsequently
obtained from the immunized animals and placed in cell culture
where they were re-stimulated for five days with irradiated,
lipopolysaccharide-treated B cells blasts with the immunodominant
peptide of PSA or OVA. CTL responses were measured using a standard
.sup.51Cr release assay. The results, which are presented in FIG. 3
as plots of % specific lysis vs. E:T ratio (the ratio of effector
cell to target cell), demonstrate that mice immunized with
dendritic cells infected with HSV-OVA or HSV-PSA generate specific
CTL responses that can be detected in vitro.
Example 4
[0069] Dendritic Cells Infected with HSV-p35 Amplicons and
Activated with CpG Oligonucleotides Produce Increased Levels of
IL-12 p70 Heterodimer.
[0070] We have also used amplicons to express IL-12 in activated
DCs to enhance Th1-mediated responses (FIG. 4). IL-12 is a product
of activated APCs and is an important activator of NK and T cell
responses. Dendritic cells were infected in cell culture with one
of two amplicons: an HSV-PSA amplicon (which served as a control)
or an HSV-p35 amplicon (p35 is a subunit of IL-12). Following
infection, the dendritic cells were activated with oligonucleotides
that contain an immunostimulatory sequence (CpG) or with control
oligonucleotides in which the CpG sequence is altered to GpC.
Supernatants were collected 48 hours later and tested in an IL-12
ELISA specific for IL-12 p70 heterodimer. As shown in FIG. 4, IL-12
p70 expression was almost nil in cells that were infected with
either HSV-PSA or HSV-p35 and stimulated with the control
oligonucleotides. There was a low level of IL-12 p70 expression
when HSV-PSA-infected cells were stimulated with CpG
oligonucleotides and robust expression from HSV-p35-infected cells
stimulated with CpG oligonucleotides. These experiments demonstrate
that, as shown above, dendritic cells can be successfully
transduced with HSV-based amplicons and that the antigen encoded by
the amplicon can be induced by appropriate stimuli.
[0071] Taken together, the studies described above support the use
of DCs infected with HSV-1 amplicon particles in investigations of
CTL activation and in immunotherapies to treat cancer and other
diseases. The studies described herein provide direct evidence that
these HSV-based amplicons can effectively infect cells that remain
functional in their ability to present antigen, which is crucial to
their use as therapeutic agents (e.g., when formulated as
vaccines).
Example 5
[0072] Fibroblasts Infected with an HSV-gp120 Amplicon Express
gp120.
[0073] Immunotherapeutic agents for the treatment of HIV infection
are likely to be more effective if they can induce or enhance
CD4.sup.+- and CD8.sup.+-T cell activity. To develop such agents,
we generated an amplicon vector that encodes the HIV envelope
glycoprotein (HSVgp120). The construct was packaged using a
modified BAC-based expression system, and gp120 expression was
initially monitored by Western blot analysis. As described further
below, NIH 3T3 cells infected with HSVgp120 produced high levels of
the HIV glycoprotein.
[0074] NIH 3T3 cells were cultured and infected with an HSV-gp120
amplicon. Lysates were then prepared and the proteins in them were
analyzed. More specifically, 20 .mu.g samples of cell lysates were
isolated from uninfected NIH 3T3 cells (this sample served as a
control) and HSV-gp120-infected NIH 3T3 cells, separated
electrophoretically on a 10% SDS-polyacrylamide gel, and
transferred to a nylon membrane that was incubated with an HIV
gp120-specific antibody (Clontech, Inc.). The gp120-specific bands
were visualized on film using chemiluminescent detection. As shown
in FIG. 5, uninfected cells expressed virtually no gp120, whereas
HSV-gp120-infected cells expressed substantial amounts of this
protein. The lanes designated 1 .mu.l and 10 .mu.l in FIG. 5
represent two different volumes of virus stock used to infect the
cells. This high level of expression demonstrates that fibroblasts
can be readily infected with an HSV amplicon.
Example 6
[0075] Animals Immunized with an HSV-gp120 Amplicon Display a
Cell-mediated Immune Response.
[0076] We next tested the ability of the HSV-gp120 vector to elicit
gp120-specific immune responses in BALB/c mice. We were able to
detect strong responses to a single intramuscular injection, at
both the humoral and cellular level. Anti-Env IgG antibodies were
generated (see below and FIG. 6). Cellular immune responses were
detected in an interferon-gamma Elispot assay using the class
I-restricted V3 peptide recognized by the mice (RGPGRAFVT (SEQ ID
NO:1); see Example 7 and FIG. 7)). In these experiments, HSV
amplicons expressing a modified MN gp120 induced interferon
gamma-producing T cells that were equivalent to those induced by
live herpesvirus vectors, and that far exceeded those induced by a
modified vaccinia Ankara vector.
[0077] To determine whether animals immunized with an HSV-gp120
amplicon could later mount a cell-mediated immune response to the
gp120 antigen, mice were immunized with either (1) an HSV-gp120
amplicon, (2) a sequence encoding the V3 peptide (MVA.H), or (3) an
HSV-lacZ amplicon. "Nave" mice constituted a fourth group.
Following immunization, the mice were sacrificed and their
splenocytes were placed in culture. The cellular responses to a
class I-restricted peptide from gp120 (RGPGRAFVTI (SEQ ID NO:1))
were measured by interferon gamma Elispot. Splenocytes incubated
without the gp120 peptide served as another control for this study.
The number of interferon-gamma-positive spots per well was plotted
for each animal, in triplicate, with three dilutions of input
splenocytes (100,000; 200,000; and 400,000 cells/well). The results
are shown in FIG. 6. The designations A1-A4 represent splenocytes
obtained from individual animals, and the (+) and (-) symbols
beneath those designations mark splenocytes incubated with or
without the specific gp120 peptide. As shown in FIG. 6, the number
of interferon gamma-positive spots (which is indicative of the
ability of the cells to mount a cell-mediated immune response) was
low and not significantly different in splenocytes obtained from
mice that were immunized with MVA or HSV-lacZ or that were not
immunized at all (nave). However, significantly more of the
splenocytes obtained from HSV-gp120-immunized mice produced
interferon following exposure to the gp120 peptide in culture.
Example 7
[0078] Animals Infected with HSV-gp120 Also Exhibit a Humoral
Immune Response.
[0079] Mice were immunized with either an HSV-gp120 amplicon or an
HSV-lacZ amplicon (which served as a negative control). Serum was
obtained either before the animals were infected or three weeks
afterward and analyzed for anti-env IgG antibodies. The results are
shown in FIG. 7. The numbers on the y-axis represent individual
animals (four were immunized with HSV-gp120 and two were immunized
with HSV-lacZ); the astericks above some bars of the graph
represent titers detected at the 1:160 final dilution; and the "+"
above other bars denotes titers determined at the 1:10 dilution.
The anti-env IgG response in serum obtained three weeks after
immunization with HSV-gp120 was substantially greater than in serum
obtained from the animals prior to immunization or in serum
obtained from animals immunized with HSV-lacZ. Thus, humoral as
well as cell-mediated immune responses result.
Example 8
[0080] HSV-gp120 Induces CTL Activity in Vivo.
[0081] BALB/c mice (n=3) were inoculated with an HSV-gp120 amplicon
(10.sup.6 pfu) by intramuscular injection. The mice were sacrificed
21 days later, and splenocytes were harvested and placed in
culture, where they were restimulated in the presence of LPS blasts
loaded with the HIVgp120 specific peptide RGPRAFVTI (SEQ ID NO:1).
After five days, these effector cells were mixed at various ratios
with radiolabeled P815 target cells, either pulsed with peptide (+)
or unpulsed (-). Cell killing was assessed using the JAM assay
method described by Matzinger et al. (J. Immunol. Methods
145:185-92, 1991). The data, shown in FIG. 8, were expressed in
terms of % cytotoxicity at each effector to target (E:T) ratio. A1,
A2, and A3 denote data obtained from individual animals. These data
demonstrate that a single intramuscular injection of an HSV-gp120
vector is sufficient to produce a strong, peptide-specific,
cytotoxic effector response in the treated animals.
Example 9
[0082] Subcutaneous Administration of an HSV-gp120 Amplicon Can
Produce a Greater Cellular Immune Response than Other Routes of
Administration.
[0083] To study the effect of the route of administration on the
strength of the immune response generated, BALB/c mice were
inoculated with the same vector, an HSV-gp120 amplicon (10.sup.6
pfu) administered either intramuscularly (into the thigh),
subcutaneously (at the base of the tail), or intraperiotoneally.
Control mice received 10.sup.6 pfu of the HSV-lacZ vector
intramuscularly. All animals were sacrificed 21 days later, and
their splenocytes were harvested and subjected to an
interferon-gamma Elispot assay using either an HIVgp120 specific
peptide (RGPRAFVTI (SEQ ID NO:1); designated "+" in FIG. 9) or no
peptide (designated "-" in FIG. 9). A1, A2, and A3 designate
splenocytes obtained from individual animals. As shown in FIG. 9,
while all routes of administration produced some number of
interferon-gamma-positive spots per well, the greatest number were
produced when the antigen had been administered subcutaneously.
Thus, subcutaneous inoculation with HSV-gp120 produced the best
cellular immune response (at least as defined in this assay system
under the parameters used).
[0084] The experiments described above show that amplicons can
infect DCs, which function in vitro and in vivo. Moreover, direct
injection of amplicons results in effective immunization in vivo.
Thus, these vectors provide a useful platform for a variety of
antigens, including HIV antigens, and the HSV amplicon-based vector
systems described herein can be used to treat HIV infection.
Example 10
[0085] Production of a Helper Virus-free Amplicon Particle
[0086] As noted above, HSV-based amplicon particles are attractive
gene delivery tools, and they are particularly well suited for
delivering gene products to neurons (e.g. neurons in the central
nervous system) because they are easy to manipulate, can carry
large transgenes, and are naturally neurotropic (Geller and
Breakefield, Science 241:1667-1669, 1988; Spaete and Frenkel, Cell
30:305-310, 1982; Federoff et al., Proc. Natl. Acad. Sci. USA
89:1636-1640, 1992; Federoff in Cells: A Laboratory Manual, Spector
et al., Eds., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,
1997; Frenkel et al., in Eucaryotic Viral Vectors, Gluzman, Ed.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1982). Efforts
to bring this vector system into the clinical arena to treat
neurodegenerative disease have been hampered by potential
cytotoxicites that are associated with traditional methods of virus
packaging. This problem involves the co-packaging of helper virus
that encodes cytotoxic and immunogenic viral proteins. Newer
methods of packaging have been developed that result in helper
virus-free amplicon stocks (Fraefel et al., J. Virol. 70:7190-7197,
1996; Stavropoulos and Strathdee, J. Virol. 72:7137-7143, 1998; see
also U.S. Pat. Nos. 5,851,826 and 5,998,208). Stocks prepared by
these methods, however, are typically low titer (<10.sup.5
expression units/ml), allowing for only modest scale
experimentation, primarily in vitro. Such low titers make large
animal studies difficult, if not impossible. Present helper
virus-free packaging strategies lead to not only lower amplicon
titers, but also to stocks that exhibit a high frequency of
pseudotransduction events when used to infect a variety of cell
types.
[0087] Optimal propagation of wild-type HSV virions requires
orderly progression of .alpha., .beta., and .gamma. gene
transcription following infection of a host cell. This is achieved
by delivery of co-packaged proteins, carried by the virion, that
help co-opt the cell's transcription machinery and transactivation
of viral .alpha. gene promoters. This information is fundamental to
the development of our helper virus-free system. Helper virus-based
packaging involves superinfection of an amplicon DNA-transfected
monolayer of packaging cells with a replication-defective helper
virus. The helper virus genome, as in the case of wild-type HSV, is
delivered to the cell in a complex with co-packaged proteins,
including VP16 and virion host shutoff (vhs). The HSV vhs protein
functions to inhibit the expression of genes in infected cells via
destabilization of both viral and host mRNAs. Because vhs plays
such a vital role in establishing the HSV replicative cycle and is
a potential structural protein, we hypothesized that its presence
during amplicon packaging accounted for the higher titers obtained
with helper virus-based packaging systems. VP16 is another
co-packaged protein that resides in the helper virus nucleocapsid
and is responsible for activating transcription of HSV
immediate-early genes to initiate the cascade of lytic
cycle-related viral protein expression.
[0088] In contrast to helper virus-based packaging systems, helper
virus-free systems involve co-transfection of naked DNA forms of
either an HSV genome-encoding cosmid set or BAC reagent with an
amplicon vector (e.g., a plasmid). Thus, the HSV genome gains
access to the cell without co-packaged vhs or VP16. The initiation
and temporal progression of HSV gene expression is, we speculated,
not optimal for production of packaged amplicon vectors due to the
absence of these important HSV proteins. To test our
hypothesis--that the efficiency of amplicon packaging would be
increased by introducing vhs and/or VP16 during the initial phase
of virus propagation--we included a vhs-encoding DNA segment in the
packaging protocol as a co-transfection reagent. In some instances,
packaging cells were "pre-loaded" with VP16 to mimic its presence
during helper virus-mediated amplicon packaging. As shown below,
these modifications led to a 30- to 50-fold enhancement of packaged
amplicon vector titers, nearly approximatig titers obtained using
helper virus-based traditional approaches. In addition, the viral
stocks failed to exhibit the pseudotransduction phenomenon. These
improvements make large-scale in vivo applications much more
likely. The methods used to make a helper virus-free amplicon
particles are described first, followed by a description of the
results obtained.
[0089] Cell culture: Baby hamster kidney (BHK) cells were
maintained as described by Lu et al. (Human Gene Ther. 6:421-430,
1995). NIH 3T3 cells were originally obtained from the American
Type Culture Collection and were maintained in Dulbecco's modified
Eagle medium (DMED) supplemented with 10% fetal bovine serum,
penicillin, and streptomycin.
[0090] Plasmid construction: The HSVPrPUC/CMVegfp amplicon plasmid
was constructed by cloning the 0.8-kb cytomegalovirus (CMV)
immediate early promoter and 0.7-kb enhanced gree fluorescent
protein cDNA (Clontech, Inc.) into the BamHI restriction enzyme
site of the pHSVPrPUC amplicon vector (Geller et al., Proc. Natl.
Acad. Sci. USA 87:8950-8954, 1990). A 3.5 kb HpaI/HindIII fragment
encompassing the UL41 (vhs) open reading frame and its 5' and 3'
transcriptional regulatory elements was removed from cos56
(Cunningham and Davison, Virol. 197:116-124, 1993) and cloned into
pBSKSII (Stratagene, Inc.) to create pBSKS(vhs). For construction
of pGRE.sub.5vp16, the VP16 coding sequence was amplified by PCR
from pBAC-V2 using gene-specific oligonucleotides that possess
EcoRI (5'-CGGAATTCCGCAGGTTTTGTAATGTATGTGCTCGT-3' (SEQ ID NO:2) and
HindIII (5'-CTCCGAAGCTTAAGCCCGATATCGTCTTTCCCGTATCA-3' (SEQ ID
NO:3)) restriction enzyme sequences that facilitate cloning into
the pGRE.sub.5-2 vector (Mader and White, Proc. Natl. Acad. Sci.
USA 90:5603-5607, 1993).
[0091] Helper virus-free Amplicon Packaging: On the day prior to
transfection, 2.times.10.sup.6 BHK cells were seeded on a 60-mm
culture dish and incubated overnight at 37.degree. C. The following
procedures were followed for cosmid-based packaging. The day of
transfection, 250 .mu.l Opti-MEM (Gibco-BRL, Bethesda, Md.), 0.4
.mu.g of each of five cosmid DNAs (kindly provided by Dr. A.
Geller, and 0.5 .mu.g amplicon vector DNA, with or without varying
amounts of pBSKS(vhs) plasmid DNA were combined in a sterile
polypropylene tube (Fraefel et al., J. Virol. 70:7190-7197, 1996).
The following procedures were followed for BAC-based packaging. 250
.mu.l Opti-MEM (Gibco-BRL, Bethesda, Md.), 3.5 .mu.g of pBAC-V2 DNA
(kindly provided by Dr. C. Strathdee, and 0.5 .mu.g amplicon vector
DNA, with or without varying amounts of pBSKS(vhs) plasmid DNA were
combined in a sterile polypropylene tube (Stavropoulos and
Strathdee, J. Virol. 72:7137-7143, 1998). The protocol for both
cosmid- and BAC-based packaging was identical from the following
step forward. Ten microliters of Lipofectamine Plus.TM. reagent
(Gibco-BRL) were added over a 30-second period to the DNA mix and
allowed to incubate at room temperature for 20 minutes. In a
separate tube, 15 .mu.l Lipofectamine (Gibco-BRL) were mixed with
250 .mu.l Opti-MEM. Follwing the 20 minute incubation, the contents
of the two tubes were combined over a one-minute period and then
incubated for an additional 20 minutes at room temperature. During
the second incubation, the medium in the seeded 60 mm dish was
removed and replaced with 2 ml Opti-MEM. The transfection mix was
added to the flask and allowed to incubate at 37.degree. C. for
five hours. The transfection mix was then diluted with an equal
volume of DMEM plus 20% FBS, 2% penicillin/streptomycin, and 2 mM
hexamethylene bis-acetamide (HMBA), and incubated overnight at
34.degree. C. The following day, medium was removed and replaced
with DMEM plus 10% FBS, 1% penicillin/streptomycin, and 2 mM HMBA.
The packaging flask was incubated an additional three days and
virus was harvested and stored at -80.degree. C. until
purification. Viral preparations were subsequently thawed,
sonicated, and clarified by centrifugation (3000.times.g for 20
minutes). Viral samples were stored at -80.degree. C. until
use.
[0092] For concentrated viral stocks, viral preparations were
subsequently thawed, sonicated, clarified by centriftigation, and
concentrated by ultracentrifugation through a 30% sucrose cushion
(Geschwind et al., Providing pharmacological access to the brain in
Methods in Neuroscience, Conn, Ed., Academic Press, Orlando, Fla.,
1994). Viral pellets were resuspended in 100 .mu.l PBS and stored
at -80.degree. C. until use. For packaging experiments examining
the effect of VP16 on amplicon titers, the cells plated for
packaging were first allowed to adhere to the 60 mm culture dish
for 5 hours and subsequently transfected with pGRE.sub.5vp 16 using
the Lipofectamine reagent as described above. Following a five-hour
incubation, the transfection mix was removed, complete medium (DMEM
plus 10% FBS, 1% penicillin/streptomycin) was added, and the
cultures were incubated at 37.degree. C. until the packaging
co-transfection step the next day.
[0093] Viral titering: Amplicon titers were determined by counting
the number of cells expressing enhanced green fluorescent protein
(HSVPrPUC/CMVegfp amplicon) or .beta.-galactosidase (HSVlac
amplicon). Briefly, 10 .mu.l of concentrated amplicon stock was
incubated with confluent monolayers (2.times.10.sup.5 expressing
particles) of NIH 3T3 cells plated on glass coverslips. Following a
48-hr incubation, cells were either fixed with 4% paraformaldehyde
for 15 min at RT and mounted in Mowiol for fluorescence microscopy
(eGFP visualization), or fixed with 1% glutaraldehyde and processed
for X-gal histochemistry to detect the lacZ transgene product.
Fluorescent or X-gal-stained cells were enumerated, expression
titer calculated, and represented as either green-forming units per
ml (gfu/ml) or blue-forming units per ml (bfu/ml),
respectively.
[0094] TaqMan Quantitative PCR System: To isolate total DNA for
quantitation of amplicon genomes in packaged stocks, virions were
lysed in 100-mM potassium phosphate pH 7.8 and 0.2% Triton X-100.
Two micrograms of genomic carrier DNA was added to each sample. An
equal volume of 2.times.Digestion Buffer (0.2 M NaCl, 20 mM Tris-Cl
pH 8.0, 50 mM EDTA, 0.5% SDS, 0.2 mg/ml proteinase K) was added to
the lysate and the sample was incubated at 56.degree. C. for 4 hrs.
Samples were processed further by one phenol:chloroform, one
chloroform extraction, and a final ethanol precipitation. Total DNA
was quantitated and 50 ng of DNA was analyzed in a PE7700
quantitative PCR reaction using a designed lacZ-specific
primer/probe combination multiplexed with an 18S rRNA-specific
primer/probe set. The lacZ probe sequence was 5'-6F
AM-ACCCCGTACGTCTTCCCGAGCG-TAMRA-3' (SEQ ID NO:4); the lacZ sense
primer sequence was 5'- GGGATCTGCCATTGTCAGACAT-3' (SEQ ID NO:5);
and the lacZ antisense primer sequence was 5'-
TGGTGTGGGCCATAATTCAA-3'(SEQ ID NO:______). The 18S rRNA probe
sequence was 5'-JOE-TGCTGGCACCAGACTTGCCCTC- -TAMRA-3' (SEQ ID
NO:6); the 18S sense primer sequence was 5'-CGGCTACCACATCCAAGGAA-3'
(SEQ ID NO:7); and the 18S antisense primer sequence was
5'-GCTGGAATTACCGCGGCT-3' (SEQ ID NO:8).
[0095] Each 25-.mu.l PCR sample contained 2.5 .mu.l (50 ng) of
purified DNA, 900 nM of each primer, 50 nM of each probe, and 12.5
.mu.l of 2.times.Perkin-Elmer Master Mix. Following a 2-min
50.degree. C. incubation and 2-min 95.degree. C. denaturation step,
the samples were subjected to 40 cycles of 95.degree. C. for 15
sec. and 60.degree. C. for 1 min. Fluorescent intensity of each
sample was detected automatically during the cycles by the
Perkin-Elmer Applied Biosystem Sequence Detector 7700 machine. Each
PCR run included the following: no-template control samples,
positive control samples consisting of either amplicon DNA (for
lacZ) or cellular genomic DNA (for 18S rRNA), and standard curve
dilution series (for lacZ and 18S). Following the PCR run,
"real-time" data were analyzed using Perkin-Elmer Sequence Detector
Software version 1.6.3 and the standard curves. Precise quantities
of starting template were determined for each titering sample and
results were expressed as numbers of vector genomes per ml of
original viral stock.
[0096] Western blot analysis: BHK cell monolayers (2.times.10.sup.6
cells) transfected with varying packaging components were lysed
with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.5% SDS, and 50
mM Tris-Cl, pH 8). Equal amounts of protein were
electrophoretically separated on a 10% SDS-PAGE gel and transferred
to a PVDF membrane. The resultant blot was incubated with an
anti-VP16 monoclonal antibody (Chemicon, Inc.), and specific VP16
immunoreactive band visualized using an alkaline phosphatase-based
chemiluminescent detection kit (ECL).
[0097] Cytotoxicity Assays: The effect of BAC-packaged HSVlac
stocks prepared in the presence or absence of VP16 and/or vhs on
cell viability was determined using a lactate dehydrogenase (LDH)
release-based assay (Promega Corp., Madison, Wis.). Equivalent
expression units of virus from each packaging sample were used to
transduce 5.times.10.sup.3 NIH 3T3 cells in 96-well flat-bottomed
culture dishes. Quantitation of LDH release was performed according
to manufacturer's instructions. Viability data were represented as
normalized cell viability index.
[0098] Stereotactic injections: Mice were anesthetized with Avertin
at a dose of 0.6 ml per 25 g body weight. After positioning in an
ASI murine stereotactic apparatus, the skull was exposed via a
midline incision, and burr holes were drilled over the following
coordinates (bregma, +0.5 mm; lateral -2.0 mm; and deep, -3.0 mm)
to target infections to the striatum. A 33 GA steel needle was
gradually advanced to the desired depth, and 3 .mu.l (equivalent in
vitro titer) HSVPrPUC/CMVegfp virus was infused via a
microprocessor-controlled pump over 10 minutes (UltraMicroPump,
World Precision Instruments, Sarasota Springs, Fla.). The injector
unit was mounted on a precision small animal stereotaxic frame (ASI
Instruments, Warren, Mich.) micromanipulator at a 90.degree. angle
using a mount for the injector. Viral injections were performed at
a constant rate of 300 nl/min. The needle was removed slowly over
an additional 10-minute period.
[0099] Tissue preparation and GFP visualization: Infected mice were
anesthetized four days later, a catheter was placed into the left
ventricle, and intracardiac perfusion was initiated with 10 ml of
heparinized saline (5,000 U/L saline) followed by 60 ml of chilled
4% PFA. Brains were extracted and postfixed for 1-2 hours in 4% PFA
at 4.degree. C. Subsequently, brains were cryoprotected in a series
of sucrose solutions with a final solution consisting of a 30%
sucrose concentration (w/v) in PBS. Forty micron serial sections
were cut on a sliding microtome (Micron/Zeiss, Thornwood, N.Y.) and
stored in a cryoprotective solution (30% sucrose (w/v), 30%
ethylene glycol in 0.1 M phosphate buffer (pH 7.2)) at -20.degree.
C. until processed for GFP visualization. Sections were placed into
Costar net wells (VWR, Springfield, N.J.) and incubated for 2 hrs
in 0.1 M Tris buffered saline (TBS) (pH 7.6). Upon removal of
cryoprotectant, two additional 10 min washes in 0.1 M TBS with
0.25% Triton X-100 (Sigma, St. Louis, Mo.) were performed. Sections
were mounted with a fine paint brush onto subbed slides, allowed to
air dry, and mounted with an aqueous mounting media, Mowiol.
GFP-positive cells were visualized with a fluorescent microscope
(Axioskop, Zeiss, Thornwood, N.Y.) utilizing a FITC cube (Chroma
Filters, Brattleboro, Vt.). All images used for morphological
analyses were digitally acquired with a 3-chip color CCD camera at
200.times. magnification (DXC-9000, Sony, Montvale, N.J.).
[0100] Morphological analyses: Cell counts were performed on
digital images acquired within 24 hrs of mounting. At the time of
tissue processing coronal slices were stored serially in three
separate compartments. All compartments were processed for cell
counting and GFP(+) cell numbers reflect cell counts throughout the
entire injection site. All spatial measurements were acquired using
an image analysis program (Image-Pro Plus, Silver Spring, Md.) at a
final magnification of 200.times.. Every section was analyzed using
identical parameters in three different planes of focus throughout
the section to prevent repeated scoring of GFP(+) cells. Each field
was analyzed by a computer macro to count cells based on the
following criteria: object area, image intensity (fluorescent
signal) and plane of focus. Only cells in which the cell body was
unequivocally GFP(+) and nucleus clearly defined were counted.
Every section that contained a GFP(+) cell was counted. In
addition, a watershed separation technique was applied to every
plane of focus in each field to delineate overlapping cell bodies.
The watershed method is an algorithm that is designed to erode
objects until they disappear, then dilates them again such that
they do not touch.
[0101] Statistical Analyses: Statistical analyses were carried out
using one-way analyses of variance (ANOVA) with plasmid construct
as the between-group variable. Two-way repeated measure analyses of
variance (RMANOVA) were carried out using plasmid construct as the
between-group variable and time interval as a within-group
variable.
[0102] Results: Prior to the methods described herein, widespread
use of helper virus-free HSV particles has been hampered by helper
virus-mediated cytotoxicity associated with traditionally packaged
amplicon stocks or by the low titers obtained from helper
virus-free production methods. Helper virus-free methods of
packaging hold the most promise as resultant stocks exhibit little
or no cytotoxicity. As shown here, modifications to such packaging
strategies could be made to increase viral titers.
[0103] We utilized both cosmid- and BAC-based methods of helper
virus-free packaging previously described (Fraefel et al., J. Virol
70:719-7197, 1996; Stavropoulos and Strathdee, J. Virol.
72:7137-7143, 1998; and Saeki et al., Hum. Gene Ther. 9:2787-2794,
1998). The low titers observed for helper virus-free methods may be
a result of the sub-optimal state of the HSV genome at the
beginning of amplicon production, as the genome is without
co-packaged viral regulators vhs and VP16. To determine if
introduction of vhs into the packaging scheme could increase
amplicon titers and quality, we cloned a genomic segment of the
UL41 gene into pBluescript and added this plasmid (pBSKS(vhs)) to
the co-transfection protocols to provide vhs in trans. The genomic
copy of UL41 contained the transcriptional regulatory region and
flanking cis elements believed to confer native UL41 gene
expression during packaging. When pBSKS(vhs) was added to the
packaging protocols for production of a .beta.-galactosidase
(lacZ)-expressing amplicon (HSVlac), a maximum of 10-fold enhanced
amplicon expression titers was observed for both cosmid- and
BAC-based strategies. As observed previously, the expression titers
for HSVlac virus produced by the BAC-based method were
approximately 500- to 1000-fold higher than stocks produced using
the modified cosmid set. Even though titers were disparate between
the differently prepared stocks, the effect of additionally
expressed vhs on amplicon titers was analogous.
[0104] The punctate appearance of reporter gene product
(pseudotransduction), a phenomenon associated with first-generation
helper virus-free stocks, was substantially diminished in vitro
when vhs was included in BAC-based packaging of a
.beta.-galactosidase-expressing (HSVlac) or an enhanced green
fluorescent (GFP)-expressing virus (HSVPrPUC/CMVegfp).
Pseudotransduction was not observed, as well, for cosmid-packaged
amplicon stocks prepared in the presence of vhs. To assess the
ability of the improved amplicon stocks to mediate gene delivery in
vivo, BAC-packaged HSVPrPUC/CMVegfp virus prepared in the absence
or presence of pBSKS(vhs) was injected stereotactically into the
striata of C57BL/6 mice (see above). Four days following infection,
animals were sacrificed and analyzed for GFP-positive cells present
in the striatum. The numbers of cells transduced by
HSVPrPUC/CMVegfp prepared in the presence of vhs were significantly
higher than in animals injected with stocks produced in the absence
of vhs. In fact, it was difficult to definitively identify
GFP-positive cells in animals transduced with vhs(-) amplicon
stocks.
[0105] The mechanism by which vhs expression resulted in higher
apparent amplicon titers in helper virus-free packaging could be
attributed to one or several properties of vls. The UL41 gene
product is a component of the viral tegument and could be
implicated in structural integrity, and its absence could account
for the appearance of punctate gene product material following
transduction. For example, the viral particles may be unstable as a
consequence of lacking vhs. Thus, physical conditions, such as
repeated freeze-thaw cycles or long-term storage, may have led to
inactivation or destruction of vhs-lacking virions at a faster rate
than those containing vhs.
[0106] The stability of HSVPrPUC/CMVegfp packaged via the BAC
method in the presence or absence of vhs was analyzed initially
with a series of incubations at typically used experimental
temperatures. Viral aliquots from prepared stocks of
HSVPrPUC/CMVegfp were incubated at 4, 22, or 37.degree. C. for
periods up to three hours. Virus recovered at time points 0, 30,
60, 120, and 180 minutes were analyzed for their respective
expression titer on NIH 3T3 cells. The rates of decline in viable
amplicon particles, as judged by their ability to infect and
express GFP, did not differ significantly between the vhs(+) and
vhs(-) stocks. Another condition that packaged amplicons encounter
during experimental manipulation is freeze-thaw cycling. Repetitive
freezing and thawing of virus stocks is known to diminish numbers
of viable particles, and potentially the absence of vhs in the
tegument of BAC-packaged amplicons leads to sensitivity to freeze
fracture. To test this possibility, viral aliquots were exposed to
a series of four freeze-thaw cycles. Following each cycle, samples
were removed and titered for GFP expression on NIH 3T3 cells as
described previously. At the conclusion of the fourth freeze-thaw
cycle, the vhs(-) HSVPrPUC/CMVegfp stock exhibited a 10-fold
diminution in expression titers as opposed to only a 2-fold
decrease for vhs(+) stocks. This observation suggests that not only
do vhs(+) stocks have increased expression titers, but the virions
are more stable when exposed to temperature extremes, as determined
by repetitive freeze-thaw cycling.
[0107] The native HSV genome enters the host cell with several
viral proteins besides vhs, including the strong transcriptional
activator VP16. Once within the cell, VP16 interacts with cellular
transcription factors and HSV genome to initiate immediate-early
gene transcription. Under helper virus-free conditions,
transcriptional initiation of immediate-early gene expression from
the HSV genome may not occur optimally, thus leading to lower than
expected titers. To address this issue, a VP16 expression construct
was introduced into packaging cells prior to cosmid/BAC, amplicon,
and pBSKS(vhs) DNAs, and resultant amplicon titers were measured.
To achieve regulated expression a glucocorticoid-controlled VP16
expression vector was used (pGRE.sub.5vp16).
[0108] The pGRE.sub.5vp16 vector was introduced into the packaging
cells 24 hours prior to transfection of the regular packaging DNAs.
HSVlac was packaged in the presence or absence of vhs and/or VP16
and resultant amplicon stocks were assessed for expression titer.
Some packaging cultures received 100-nM dexamethasone at the time
of pGREsvp16 transfection to strongly induce VP16 expression;
others received no dexamethasone. Introduction of pGRE.sub.5vp16 in
an uninduced (basal levels) or induced state (100 nM dexamethasone)
had no effect on HSVlac titers when vhs was absent from the cosmid-
or BAC-based protocol. In the presence of vhs, addition of
pGRE.sub.5vp16 led to either a two- or five-fold enhancement of
expression titers over those of stocks packaged with only vhs
(cosmid- and BAC-derived stocks). The effect of "uninduced"
pGRE.sub.5vp16 on expression titers suggested that VP16 expression
was occurring in the absence of dexamethasone. To examine this,
Western blot analysis with a VP16-specific monoclonal antibody was
performed using lysates prepared from BHK cells transfected with
the various packaging components. Cultures transfected with
pGRE.sub.5vp16/BAC/pBSKS(vhs) in the absence of dexamethasone did
show VP16 levels intermediate to cultures transfected either with
BAC alone (lowest) or those transfected with
pGRE.sub.5vp16/BAC/pBSKS(vhs) in the presence of 100 nM
dexamethasone (highest)(FIG. 4C). There was no difference in level
of pGRE.sub.5vp16-mediated expression in the presence or absence of
BAC, nor did dexamethasone treatment induce VP16 expression from
the BAC.
[0109] VP16-mediated enhancement of packaged amplicon expression
titers could be due to increased DNA replication and packaging of
amplicon genomes. Conversely, the additional VP16 that is expressed
via pGRE.sub.5vp16 could be incorporated into virions and act by
increasing vector-directed expression in transduced cells. To test
the possibility that VP16 is acting by increasing replication in
the packaging cells, concentrations of vector genomes in
BAC-derived vector stocks were determined. HSVlac stocks produced
in the presence or absence of vhs and/or VP16 were analyzed using a
"real-time" quantitative PCR method. The concentration of vector
genome was increased two-fold in stocks prepared in the presence of
VP16 and this increase was unaffected by the presence of vhs.
[0110] There is a possibility that addition of viral proteins, like
vhs and VP16, to the packaging process may lead to vector stocks
that are inherently more cytotoxic. The amplicon stocks described
above were examined for cytotoxicity using a lactate dehydrogenase
(LDH) release-based cell viability assay. Packaged amplicon stocks
were used to transduce NIH 3T3 cells and 48 hours following
infection, viability of the cell monolayers was assessed by the
LDH-release assay. Amplicon stocks produced in the presence of vhs
and VP16 displayed less cytotoxicity on a per virion basis than
stocks packaged using the previously published BAC-based protocol
(Stavropoulos and Strathdee, supra).
[0111] Significance: Wild-type HSV virions contain multiple
regulatory proteins that prepare an infected host cell for virus
propagation. These virally encoded regulators, which are localized
to the tegument and nucleocapsid, include vhs and VP16,
respectively. The UL41 gene-encoded vhs protein exhibits an
essential endoribonucleolytic cleavage activity during lytic growth
that destabilizes both cellular and viral mRNA species (Smibert et
al., J. Gen. Virol. 73:467-470, 1992). Vhs-mediated ribonucleolytic
activity appears to prefer the 5' ends of mRNAs over 3' termini,
and the activity is specific for mRNA, as vhs does not act upon
ribosomal RNAs (Karr and Read, Virology 264: 195-204, 1999). Vhs
also serves a structural role in virus particle maturation as a
component of the tegument. HSV isolates that possess disruptions in
UL41 demonstrate abnormal regulation of IE gene transcription and
significantly lower titers than wild-type HSV-1 (Read and frenkel,
J. Virol. 46:498-512, 1983), presumably due to the absence of vhs
activity. Therefore, because vhs is essential for efficient
production of viable wild-type HSV particles, it likely plays a
similarly important role in packaging of HSV-1-derived amplicon
vectors.
[0112] The term "pseudotransduction" refers to virion
expression-independent transfer of biologically active
vector-encoded gene product to target cells (Liu et al., J. Virol.
70:2497-2502, 1996; Alexander et al. Human Gene Ther. 8:1911-1920,
1997. This phenomenon was originally described with retrovirus and
adeno-associated virus vector stocks and was shown to result in an
overestimation of gene transfer efficiencies. .beta.-galactosidase
and alkaline phosphatase are two commonly expressed reporter
proteins that have been implicated in pseudotransduction,
presumably due to their relatively high enzymatic stability and
sensitivity of their respective detection assays (Alexander et al.,
supra). Stocks of .beta.galactosidase expressing HSVlac and
GFP-expressing HSVPrPUC/CMVegfp exhibited high levels of
pseudotransduction when packaged in the absence of vhs. Upon
addition of vhs to the previously described helper virus-free
packaging protocols, a 10-fold increase in expression titers and
concomitant decrease in pseudotransduction were observed in
vitro.
[0113] Vhs-mediated enhancement of HSV amplicon packaging was even
more evident when stocks were examined in vivo. GFP-expressing
cells in animals transduced with vhs(+) stocks were several
hundred-fold greater in number than in animals receiving vhs(-)
stocks. This could have been due to differences in virion
stability, where decreased particle stability could have led to
release of co-packaged reporter gene product observed in the case
of vhs(-) stocks. Additionally, the absence of vhs may have
resulted in packaging of reporter gene product into particles that
consist of only tegument and envelope (Rixon et al., J. Gen. Virol.
73:277-284, 1992). Release of co-packaged reporter gene product in
either case could potentially activate a vigorous immune response
in the CNS, resulting in much lower than expected numbers of
vector-expressing cells.
[0114] Pre-loading of packaging cells with low levels of the potent
HSV transcriptional activator VP16 led to a 2- to 5-fold additional
increase in amplicon expression titers only in the presence of vhs
for cosmid- and BAC-based packaging systems, respectively. This
observation indicates the transactivation and structural functions
of VP16 were not sufficient to increase viable viral particle
production when vhs was absent, and most likely led to generation
of incomplete virions containing amplicon genomes as detected by
quantitative PCR. When vhs was present for viral assembly, however,
VP16-mediated enhancement of genome replication led to higher
numbers of viable particles formed. Quantitative PCR analysis of
amplicon stocks produced in the presence of VP16 and vhs showed
that viral genomes were increased only 2-fold while expression
titers were increased 5-fold over stocks produced in the presence
of vhs only. This result suggests that a portion of the effect
related to VP16-mediated enhancement of genome replication while
the additional .about.2-fold enhancement in expression titers may
be attributed to the structural role of VP16. The effect of VP16 on
expression titers was not specific to amplicons possessing the
immediate-early 4/5 promoter of HSV, as amplicons with other
promoters were packaged to similar titers in the presence of VP16
and vhs.
[0115] VP16 is a strong transactivator protein and structural
component of the HSV virion (Post et al., Cell 24:555-565, 1981).
VP16-mediated transcriptional activation occurs via interaction of
VP16 and two cellular factors, Oct-1 (O'Hare and Goding, Cell
52:435-445, 1988; Preston et al., Cell 52:425-434, 1988; Stern et
al., Nature 341:624-630, 1989) and HCF (wilson et al, Cell
74:115-125, 1993; Xiao and Capone, Mol. Cell Biol. 10:4974-4977,
1990) and subsequent binding of the complex to TAATGARAT elements
found within HSV IE promoter regions (O'Hare, Semin. Virol.
4:145-155, 1993. This interaction results in robust up-regulation
of IE gene expression. Neuronal splice-variants of the related
Oct-2 transcription factor have been shown to block IE gene
activation via binding to TAATGARAT elements (Lillycrop et al.,
Neuron 7:381-390, 1991) suggesting that cellular transcription
factors may also play a role in limiting HSV lytic growth.
[0116] The levels of VP16 appear to be important in determining its
effect on expression titers. Low, basal levels of VP16 (via
uninduced pGRE.sub.5vp 16) present in the packaging cell prior to
introduction of the packaging components induced the largest effect
on amplicon expression titers. Conversely, higher expression of
VP16 (via dexamethasone-induced pGRE.sub.5vp16) did not enhance
virus production to the same degree and may have, in fact,
abrogated the process. The presence of glucocorticoids in the serum
components of growth medium is the most likely reason for this
low-level VP16 expression, as charcoal-stripped sera significantly
reduces basal expression from this construct. Perhaps only a low
level or short burst of VP16 is required to initiate IE gene
transcription, but excessive VP16 leads to disruption of the
temporal progression through the HSV lytic cycle, possibly via
inhibition of vhs activity. Moreover, evidence has arisen to
suggest vbs activity is downregulated by interaction with newly
synthesized VP16 during the HSV lytic cycle, thereby allowing for
accumulation of viral mRNAs after host transcripts have been
degraded (Schmelter et al., J. Virol 70:2124-2131, 1996; Smibert et
al., J. Virol. 68:2333-2346, 1994; Lam et al., EMBO J.
15:2575-2581, 1996). Therefore, a delicate regulatory protein
balance may be required to attain optimal infectious particle
propagation. Additionally, the 100-nM dexamethasone treatment used
to induce VP16 expression may have a deleterious effect on cellular
gene activity and/or interfere with replication of the
OriS-containing amplicon genome in packaging cells. High levels of
dexamethasone have been shown previously to repress HSV-1
OriS-dependent replication by an unknown mechanism Hardwicke and
Schaffer, J. Virol. 71:3580-3587, 1997). Inhibition of
OriS-dependent replication does not appear to be responsible for
our results, however, since quantitative PCR analysis of amplicon
stocks produced in the presence and absence of dexamethasone
indicated no change in genome content as a function of drug
concentration. It is interesting to note that amplicon stocks were
prepared in the presence of hexamethylene bisacetamide (HMBA). HMBA
has been shown to compensate for the absence of VP16, thus leading
to the transactivation of immediate early gene promoters (McFarlane
et al., J. Gen. Virol. 73:285-292, 1992. In the absence of HMBA
pre-loading a packaging cell with VP16 could impart an even more
dramatic effect on titers.
[0117] Ectopic expression of vhs and VP16 did not lead to amplicon
stocks that exhibited higher cytotoxicity than helper virus-free
stocks prepared in the traditional manner when examined by an
LDH-release assay. Stocks prepared by the various methods were
equilibrated to identical expression titers prior to exposure to
cells. The heightened cytotoxicity in stocks produced in the
absence of vhs and/or VP16 may reflect that larger volumes of these
stocks were required to obtain similar expression titers as the
vhs/VP16-containing samples or the levels of defective particles in
the former may be significantly higher. Contaminating cellular
proteins that co-purify with the amplicon particles are most likely
higher in concentration in the traditional stocks, and probably
impart the higher toxicity profiles observed.
Example 11
[0118] Herpesvirus Amplicon Particles in the Treatment of
Hematologic Malignancies
[0119] The experiments described below were designed to test
viral-based amplicons as therapeutic agents for hematologic (and
other types of) malignancies. We transduced tumor cells ex vivo
with various HSV-based amplicons that encode different
co-stimulatory molecules, such as B7.1 (also known as CD80) and
CD40L (also known as CD154). In addition, we tested two HSV
amplicon stocks: one packaged using a helper virus (manufactured
via a replication-defective helper virus deleted in HSV ICP4) and
one prepared, helper virus-free, using a bacterial artificial
chromosome (BAC). Stocks packaged in either way were prepared to
express either B7.1 or CD40L. The helper virus-containing and the
helper virus-free stock were tested for their ability to transduce
freshly isolated human B cell chronic lymphocytic leukemia (CLL)
cells, to function as antigen-presenting cells, to stimulate T cell
proliferative responses and cytokine release, and to affect MHC-I
expression in transduced target CLL cells.
[0120] Using CLL cells, we found that: (1) both helper
virus-containing and helper virus-free virus stocks are able to
transduce primary human leukemia cells at high efficiencies, and
(2) cells transduced with helper virus-containing amplicon were
less efficient as APCs, and thus not as desirable as helper
virus-free preparations for use in immunotherapies. The
disadvantages of using a helper virus-containing preparation arise
from the transcription of certain genes within the HSV genome,
which is delivered largely intact into the host cell with the
helper virus. More specifically, we found: (1) loss of MHC-I on
cells transduced with helper virus-containing HSV amplicon stocks
(this is likely to be mediated by the ICP-47 gene product that is
introduced with the helper virus) and (2) increased cytotoxicity in
cells transduced by the helper virus-containing amplicon stock.
With respect to (1), loss of MHC-I hampers CD8-mediated CTL
activity and results in a loss of the ability to kill target tumor
cells. With respect to (2), the increased cytotoxicity in CLL cells
is most likely related to the introduction of pro-apoptotic genes
mediated by the helper virus. Due to these issues (inherent
immunosuppression and cytotoxicity), helper virus-free amplicon
preparations emerge as a superior choice for developing
immunotherapies to treat any number of infectious diseases and
cancers (including chronic lymphocytic leukemia).
[0121] Cell culture: Samples of blood (10 ml each) were obtained
from eight patients with an established diagnosis of CLL.
Peripheral blood lymphocytes (PBL) were isolated by density
gradient centrifugation on Ficoll-Paque.TM. Plus (Amersham
Pharmacia Biotech AB, Uppsala, Sweden). More than 97% of purified
PBL stained positive for CD19 by flow-cytometry. Allogeneic T cells
were purified from healthy donor PBL through a T cell enrichment
column (R&D Systems, Minneapolis, Minn.). More than 97% of the
purified lymphocytes obtained from the T cell column were CD3
positive by flow cytometry. Both CLL cells and T cells were
maintained in RPMI supplemented with 10% human AB serum. Baby
hamster kidney (BHK) and RR1 cell lines were maintained as
described in Kutubuddin et al. (Blood 93:643-654, 1999). The NIH
3T3 mouse fibroblast cell line was originally obtained from the
American Type Culture Collection (Manassas, Va.) and maintained in
Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum
(FBS).
[0122] Amplicon Construction: Coding sequences for E. coli
.beta.-galactosidase and human B7.1 (CD80) were cloned into the
polylinker region of the pHSVPrPUC plasmid (Geller et al., Proc.
Natl. Acad. Sci. USA 87:8950-8954, 1990) as described by Kutubuddin
et al. (Blood 93643-654, 1999). Murine CD40L (CD154; kindly
provided by Dr. Mark Gilber, Immunex Corp.) was cloned into the
BamHJ and EcoRI sites of the pHSVPrPUC amplicon vector.
[0123] Helper virus-based amplicon packaging: Amplicon DNA was
packaged into HSV-1 particles by transfecting 5 .mu.g of plasmid
DNA into RR1 cells with Lipofectamine as recommended by the
manufacturer (GIBCO-BRL). Following incubation for 24 hours, the
transfected monolayer was superinfected with the HSV strain
17-derived IE3 deletion mutant virus D30EBA (Paterson and Everett,
J. Gen. Virol. 71:1775-1783, 1990) at a multiplicity of infection
(MOI) of 0.2. Once cytopathic changes were observed in the infected
monolayer, the cells were harvested, freeze-thawed, and sonicated
using a cup sonicator (Misonix, Inc.). Viral supernatants were
clarified by centrifugation at 5000.times.g for ten minutes prior
to repreat passage on RR1 cells. This second viral passage was
harvested as above and concentrated for two hours by
ultracentrifugation on a 30% sucrose cushion as described by
Federoff (In Cells: A Laboratory Manual, Spector and Leinwand,
Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1997). Viral pellets were resuspended in PBS (Ca.sup.2+ and
Mg.sup.2+ free) and stored at -80.degree. C. for future use.
[0124] Helper virus-free amplicon packaging (HF-HSV): Amplicon
stocks were also prepared using a modified helper virus-free
packaging method. The packaging system utilizes a bacterial
artificial chromosom (BAC; kindly provided by C. Strathdee) that
contains the HSV genome without its cognate pac signals as a
co-transfection reagent with amplicon DNA. Because the amplicon
vector possesses pac signals, only the amplicon genome is packaged.
Briefly, on the day prior to transfection, 2.times.10.sup.7 BHK
cells were seeded in a T-150 flask and incubated overnight at
37.degree. C. The day of transfection, 1.8 ml Opti-MEM (Gibco-BRL,
Bethesda, Md.), 25 .mu.g of pBAC-V2 DNA (Stavropoulos and
Strathdee, supra), 7 .mu.g of pBS(vhs), and 3.6 .mu.g amplicon
vector DNA were combined in a sterile polypropylene tube. Seventy
microliters of Lipofectamine Plus reagent (Gibco-BRL) were added
over a period of 30 seconds to the DNA mix and allowed to incubate
at 22.degree. C. for 20 minutes. In a separate tube, 100 .mu.l
Lipofectamine (Gibco-BRL) was mixed with 1.8 ml Optim-MEM and also
incubated at 22.degree. C. for 20 minutes. Following the
incubations, the contents of the two tubes were combined over a
period of 30 seconds, and incubated for an additional 20 minutes at
22.degree. C. During this second incubation, the media in the
seeded T-150 flask was removed and replaced with 14 ml Opti-MEM.
The transfection mix was added to the flask and allowed to incubate
at 37.degree. C. for five hours. The transfection mix was then
diluted with an equal volume of DMEM plus 20% FBS, 2%
penicillin/streptomycin, and 2 mM hexamethylene bis-acetamide
(HMBA), and incubated overnight at 34.degree. C. The following day,
media was removed and replaced with DMEM plus 10% FBS, 1%
penicillin/streptomycin, and 2 mM HMBA. The packaging flask was
incubated an additional three days before virus was harvested and
stored at -80.degree. C. until purification. Viral preparations
were subsequently thawed, sonicated, clarified by centrifugation,
and concentrated by ultracentrifugation through a 30% sucrose
cushion. Viral pellets were resuspended in 100 .mu.l PBS (Ca.sup.2+
and Mg.sup.2+ free) and stored at -80.degree. C. for future
use.
[0125] Virus Titering: Helper virus-containing stocks were titered
for helper virus by standard plaque assay methods (Geschwind et
al., Brain Res. Mol. Brain Res. 24:327-335, 1994). Amplicon titers
for both helper virus-based and helper-free stocks were determined
as follows. NIH 3T3 cells were plated in a 24-well plate at a
density of 1.times.10.sup.5 cells/well and infected with the virus.
Twenty-four hours after viral infection, the monolayers were washed
twice in PBS and either fixed with 4% paraformaldehyde and stained
by X-gal histochemistry (HSVlac; 5 mM potassium ferricyanide; 5 mM
potassium ferrocyanide; 0.02% NP-40; 0.01% sodium deoxycholic acid;
2 mM MgCl.sub.2; and 1 mg/ml X-gal dissolved in PBS) or harvested
for total DNA using lysis buffer (100 mM NaCl, 10 mM Tris, pH 8.0,
25 mM EDTA, 0.5% SDS) followed by phenol/chloroform extraction and
ethanol precipitation. Real-time quantitative PCR was performed on
duplicate samples using primers corresponding to the
.beta.-lactamase gene present in the amplicon plasmid, according to
Bowers et al. (Mol. Ther. 1:294-299, 2000). Total DNA was
quantitated and 50 ng of DNA was analyzed in a PE7700 quantitative
PCR reaction using a designed .beta.-lactamase-specific
primer/probe combinatino multiplexed with an 18S rRNA-specific
primer/probe set. The .beta.-lactamase probe sequence was
5'-CAGGACCACTTCTGCGCTCGGC-3' (SEQ ID NO:9); the .beta.-lactamase
sense primer sequence was 5'-CTGGATGGAGGCGGATAAAGT-3' (SEQ ID
NO:10); and the .beta.-lactamaseantisense primer sequence was
5'-TGCTGGCACCAGACTTGCCCTC-3' (SEQ ID NO:11). The 18S rRNA probe
sequence was 5'-TGCTGGCACCAGACTTGCCCTC-3' (SEQ ID NO:12); the 18S
sense primer sequence was 5'-CGGCTACCACATCCAAGGAA-3' (SEQ ID
NO:13); and the 18S antisense primer sequence was
5'-GCTGGAATTACCGCGGCT-3' (SEQ ID NO:14). Helper virus titers
(pfu/ml), amplicon expression titers (bfu/ml), and amplicon
transduction titers (TU/ml) obtained from these methods were used
to calculate amplicontiter and thus standardize experimental viral
delivery. Amplicon titers of the various virus preparations ranged
from 4-5.times.10.sup.8 bfu/ml while helper titers were in the
range of 5-15.times.10.sup.7 pfu/ml.
[0126] Mixed lymphocyte tumor reaction (MLTR) assay: CLL cells were
transduced with equal transduction units of helper virus-containing
or helper virus-free amplicon stocks, were irradiated (20 Gy), and
were used as stimulators (2.5 or 5.times.10.sup.4 cells/well) with
allogeneic normal donor T cells (2.times.10.sup.5 cells in a final
volume of 200 .mu.l) in 96-well flat-bottom plates. All cultures
were performed in triplicate. The cells were incubated 5 days at
37.degree. C. in 5% CO.sub.2. Cells were pulsed with 1 .mu.Ci
(.sup.3H)-thymidine for the last 18 hours of the culture period
before being transferred onto a glass fiber filter and radioactive
counts measured by liquid scintillation counting. To determine the
involvement of Signal One, CLL cells were infected with equivalent
transduction units of HSVlac, HSVB7.1, hf-HSVlac, or hf-HSVB7.1 and
were used as stimulators as described above with or without phorbol
12-myristate 13-acetate (PMA) added to a final concentration of 10
ng/ml.
[0127] ELISA for IL-2 and .gamma.-interferon: Culture supernatant
(50 .mu.l) from every well of the MLTR plate was collected on day 4
prior to adding (.sup.3H)-thymidine and used in a standard sandwich
ELISA (R&D Systems) according to manufacturer
recommendations.
[0128] Cytotoxic T lymphocyte (CTL) Assay: T cells purified from
normal donor peripheral blood mononuclear cells (PBMC) were
incubated with uninfected irradiated CLL cells, helper virus-free
HSVlac-, or helper virus-free HSVCD40L-infected CLL cells at a
ratio of 4:1 and incubated for six days. A cytotoxicity assay was
performed by incubating primed T cells with 1.times.10.sup.4
51Cr-labeled CLL cells in a V-shaped 96-well plate at varying
effector:target ratios. Spontaneous release was measured by
incubating .sup.51Cr-labeled CLL cells alone while maximum release
was calculated by lysing the cells with 2% Triton-X. After a
six-hour incubation, supernatant was collected and radioactivity
was measured using a .gamma.-counter (Packard Instrument). Mean
values were calculated for the triplicate wells and the results are
expressed as % specific lysis according to the formula:
experimental counts--spontaneous counts/total counts--spontaneous
counts X 100.
[0129] Results
[0130] HSV amplicon-mediated gene transfer into CLL cells. The
utility of HSV-based amplicon vectors for transduction of CLL cells
was examined according to the methods described above. HSV amplicon
vectors encoding .beta.-galactoside, CD80 (B7.1) or CD154 (CD40L)
were packaged using either a standard helper virus (designated
HSVlac, HSVB7.1 and HSVCD40L) or a helper virus-free method
(designated hf-HSVlac, hf-HSVB7.1 and hf-HSVCD40L).
[0131] CLL cells were isolated by density gradient centrifugation
and .ltoreq.97% of the cells stained for CD19, a cell surface
marker for B lymphocytes. The cells were transduced with either
HSVlac, HSVB7.1, hf-HSVlac, or hf-HSVB7.1. X-gal histochemistry was
performed to detect the .beta.-galactosidase (lacZ) transgene
product expressed by HSVlac and hf-HSVlac, while fluorescence
activated cell sorting (FACS) analyses were performed on CLL cells
transduced with equivalent transduction units of HSVB7.1 and
hf-HSVB7.1 (FIG. 10). More than 70% of the cells stained for either
lacZ or B7.1 expression at an MOI of 1.0. In agreement with
previous studies using HSVlac, expression levels of
.beta.-galactosidase peaked at 2-3 days and persisted for up to 7
days post-infection. Hence, both helper virus-containing and helper
virus-free amplicon preparations appear to be effective for gene
transfer into CLL cells.
[0132] Effect of helper virus on host cell MHC-I expression.
Although both vector preparations were able to drive high-level
expression of B7.1 in CLL cells, it was possible that helper
virus-containing amplicon preparations disrupted MHC I-mediated
antigen presentation. ICP-47, a gene present in the D30EBA helper
virus, encodes a protein that blocks TAP-1 mediated peptide loading
into MHC I. Expression of such an immunosuppressive activity would
reduce the utility of HSV amplicon vectors for immunotherapeutic
strategies. To examine this possibility, CLL cells were transduced
with HSVB7.1 or hf-HSVB7.1 and examined by flow-cytometry for
levels of B7.1 and MHC I expression.
[0133] Significant down-regulation of MHC I in CLL cells transduced
with HSVB7.1 was observed compared to MHC I expression in
uninfected cells (FIG. 11). In contrast, transduction with
hf-HSB7.1 resulted in high levels of B7.1 expression and
maintenance of MHC I surface expression on B7.1-transduced cells.
These data highlight the role of HSV-encoded factors in modulation
of host immunity and underscore a fundamental difference in the
immunotherapeutic potential between helper virus-based and helper
virus-free amplicon preparations.
[0134] Allogeneic T cell activation by HSV amplicon-transduced CLL
cells. To assess functional differences in antigen presentation
following transduction with helper virus-containing or helper
virus-free amplicon stocks, the effects of B71. transduction on the
ability of CLL cells to stimulate T cell proliferation in an
allogeneic mixed leukocyte tumor reaction (MLTR) were analyzed. CLL
cells were transduced with either HSVlac, HSVB7.1, hf-HSVlac, or
hf-HSVB7.1 and transduced cells served as stimulators in an
allogeneic MLTR using T cells from a normal donor.
hf-HSVB7.1-transduced CLL cells were able to directly stimulate T
cell proliferation (FIG. 12). In spite of amplicon-directed
expression of B7.1 on at least 70% of the CLL cells,
HSVB7.1-transduced CLL cells failed to elicit a T cell
proliferative response, suggesting that the antigen presenting
capacity of the infected CLL cells had been seriously impaired.
This could have occurred through the loss of MHC I expression (as
shown in FIG. 11) or through some other mechanism mediated by the
helper virus. Phorbol 12-myristate 13-acetate (PMA) was used to
provide an extrinsic "signal one" to potentially compensate for the
adverse effect elicited by the helper virus on CLL cells, thereby
allowing transduced B7.1 to elicit a co-stimulatory signal to T
cells. Provision of extrinsic Signal One by PMA resulted in
significant proliferation in HSVB7.1-infected CLL cells (relative
to non-transduced or HSVlac-transduced CLL cells). PM treatment
also augmented proliferation in hf-HSVB7.1-transduced CLL cells,
suggesting that the full potential of T cell activation by these
transduced cells was not fully achieved by helper virus-free vector
delivery alone.
[0135] Another correlate to T cell activation relates to induction
of IL-2 secretion. Supernatants collected from the MLTR samples
described above were analyzed using an IL-2 ELISA. IL-2 levels were
highest when hf-HSVB7.1-transduced CLL cells were utilized as T
cell stimulators (the uppermost Table in FIG. 11) as compared to
HSVB7.1 or HSVlac-transduced cells. In other MLTR assays using
HSVB7.1-transduced CLL cells, IL-2 secretion was dependent on
provision of Signal One via PMA, as was observed with PMA-mediated
rescue of T cell stimulators.
[0136] Up-regulation of co-stimulatory molecules on CLL cells
transduced by HSV amplicons. Engagement of the CD40 receptor on
APCs is a critical step in the initiation of an immune response.
Up-regulation of costimulatory molecules on CLL cells induced by
CD40 receptor signaling correlates with a cell's ability to
function as an APC (van Kooten et al., Curr. Opin. Immunol.
9:330-337, 1997; Gruss et al., Leuk. Lymphoma 24:393-422, 1997). We
selected endogenous B7.1 expression as a surrogate marker for the
morphologic changes induced by CD40 receptor engagement in CLL
cells. To test for paracrine and autocrine induction of B7.1, CLL
cells were transduced with either hf-HSVCD40L or hf-HSVlac,
incubated for six days and subsequently analyzed for expression of
endogenous B7.1. As shown in FIG. 13, transduction with hf-HSVCD40L
resulted in up-regulation of B7.1 on CLL cells as compared to
untransduced and hf-HSVlac transduced cells.
[0137] The percentage of CLL cells expressing B7.1, CD40L, or both,
was quantitated by two-color flow cytometry (the middle Table in
FIG. 11). Although infection of CLL cells with HSVCD40L resulted in
more than 70% of the cells expressing CD40L, the percentage of
cells expressing endogenous B7.1 did not increase over background
levels observed in cells transduced with control vector. CLL cells
infected with hf-HSVCD40L exhibited a marked enhancement of B7.1
expression. The discrepancy at the level of endogenous B7.1
expression between CLL cells transduced with HSVCD40L and
hf-HSVCD40L cannot be attributed to different efficiencies of
infectivity as both groupd expressed similar levels of CD40L.
Similar experiments using CD19 expression as an endogenous cell
marker confirmed an inverse relationship between surface CD19
expression and CD40L expression in cells transduced with helper
virus-containing HSVCD40L, but not in cells transduced with
hf-HSVCD40L. These data suggested that transduction with HSVCD40L
resulted in a decrease in expression level of endogenous B7.1
[0138] Subsequently, the ability of CLL cells transduced by CD40L
to serve as stimulators in an allogeneic MLTR was examined. CLL
cells were transduced with hf-HSVlac, hf-HSVCD40L, HSVlac, or
HSVCD40L and incubated for 4-6 days to allow for up-regulation of
co-stimulatory molecules and then used as stimulators in an
allogeneic MLTR. Although similar levels of CD40L expression were
observed following transduction with either HSVCD40L or
hf-HSVCD40L, cells transduced with hf-HSVCD40L were more potent T
cell stimulators than those transduced with HSVCD40L or control
vectors.
[0139] hf-HSV amplicon transduced CLL stimulate allogeneic CTL.
Since the goal of immune therapy is to generate tumor-specific CTL,
and in view of the data above showing superiority of helper
virus-free stock, we tested the capacity of allogeneic T cells to
elicit a cytotoxic response against CLL cells transduced with
hf-HSVCD40L. T cells purified from normal donor peripheral blood
mononuclear cells (PBMC) were incubated for six days with
non-transduced/irradiated CLL cells, hf-HSVlac-, or
hf-HSVCD40L-transduced CLL cells. A cytotoxicity assay was
performed by incubating primed T cells with .sup.51Cr-labeled CLL
cells at varying effector to target ratios. Significantly higher
CTL activity was generated by priming with hf-HSVCD40L-transduced
CLL cells compared to control or hf-HSVlac-transduced cells. As
another index oc cytolytic T cell activation, we measured levels of
gamma-interferon secretion. High levels of IFN-gamma were secreted
by hf-HSVCD40L-transduced CLL stimulated T cells as detected by
ELISA (the lower Table in FIG. 11), suggesting that helper
virus-free amplicon stocks can effectively transduce CLL cells to
serve as tumor vaccines.
[0140] DCs pulsed with CTL peptide epitopes derived from tumor
antigens or transduced with adenoviral vectors that direct
expression of tumor antigens have been shown to elicit antitumor
CTL activity. However, each of these methods has limitations. For
example, to use peptides for tumor immunotherapy, one would have to
recognize CTL epitopes for tumor antigens in multiple HLA types
and, with adenoviral vectors, the viral gene products expressed in
transduced cells can lead to anti-vector immunity, which would
preclude multiple immunizations.
Example 12
[0141] LIGHT, a TNF Family Member Enhances the Antigen Presenting
Capacity of Chronic Lymphocytic Leukemia and Stimulates Autologous
Cytolytic T Cells.
[0142] CLL B cells possess the ability to process and present tumor
antigens, but lack expression of costimulatory molecules, rendering
them inefficient effectors of T-cell activation. We previously
demonstrated that helper virus-free preparations of Herpes Simplex
Virus (HSV) amplicon vectors encoding CD40L efficiently transduce
CLL B cells and render them capable of eliciting specific
anti-tumor T-cell responses (Tolba et al., Blood 98:287-295, 2001).
LIGHT (TNFSF14), a member of the TNF superfamily, represents a
strong candidate molecule as it efficiently activates T cells as
well as antigen-presenting cells (APC). We employed an HSV amplicon
vector expressing human LIGHT (hf-HSVLIGHT) to transduce CLL B
cells and compared the immunomodulatory function and T-cell
activation by hf-HSV-LIGHT to that of the previously described
CD40L-expressing amplicon (hf-HSVCD40L). hf-HSVLIGHT transduction
induced expression of endogenous B7.1, B7.2 and ICAM.1, albeit to a
lesser degree than observed in response to CLL B cells transduced
with hf-HSV-CD40L. hf-HSVLIGHT enhanced antigen-presenting capacity
of CLL B cells and stimulated T cell proliferation in an allogeneic
mixed lymphocyte tumor reaction (MLTR) through a dual mechanuism:
a) indirectly through induction of native B7.1/B7.2 and b) directly
via stimulation of Hve-A receptor on T cells. Finally, hf-HSVLIGHT
transduced CLL B cells successfully stimulated outgrowth of
autologous cytotoxic T-lymphocytes in vitro. These data suggest
that hf-HSVLIGHT transduction may be useful for induction of immune
responses to CLL and other B-cell lymphoid malignancies.
1TABLE 1 Essential HSV-1 Genes Genbank Gene* Protein(Function) I.D.
No. Accession No.** UL1 virion glycoprotein L (gL) 136775 CAA32337
UL5 component of DNA helicase-primase complex 74000 CAA32341 UL6
minor capsid protein 136794 CAA32342 UL7 unknown 136798 CAA32343
UL8 DNA helicase/primase complex associated protein 136802 CAA32344
UL8.5 unknown*** -- -- UL9 ori-binding protein 136806 CAA32345 UL15
DNA cleavage/packaging protein 139646 CAA32330 UL17 tegument
protein 136835 CAA32329 UL18 capsid protein, VP23 139191 CAA32331
UL19 major capsid protein, VP5 137571 CAA32332 UL22 virion
glycoprotein H, gH 138315 CAA32335 UL25 DNA packaging virion
protein 136863 CAA32317 UL26 serine protease, self-cleaves to form
VP21 & VP24 139233 CAA32318 UL26.5 capsid scaffolding protein,
VP22a 1944539 CAA32319 UL27 virion glycoprotein B, gB 138194
CAA32320 UL28 DNA cleavage and packaging protein, ICP18.5 124088
CAA32321 UL29 single-stranded DNA binding protein, ICP8 118746
CAA32322 UL30 DNA polymerase 118878 CAA32323 UL31 UL34-associated
nuclear protein 136875 CAA32324 UL32 cleavage and packaging protein
136879 CAA32307 UL33 capsid packaging protein 136883 CAA32308 UL34
membrane-associated virion protein 136888 CAA32309 UL36 very large
tegument protein, ICP1/2 135576 CAA32311 UL37 tegument protein,
ICP32 136894 CAA32312 UL38 capsid protein, VP19C 418280 CAA32313
UL42 DNA polymerase accessory protein 136905 CAA32305 UL48 alpha
trans-inducing factor, VP16 114359 CAA32298 UL49 putative
microtubule-associated protein, VP22 136927 CAA32299 UL49.5
membrane-associated virion protein 1944541 CAA32300 UL52 component
of DNA helicase/primase complex 136939 CAA32288 UL54 regulation and
transportation of RNA, ICP27 124180 CAA32290 .alpha.4 (RS1)
positive and negative gene regulator, ICP4 124141 CAA32286 CAA32278
US6 virion glycoprotein D, gD 73741 CAA32283 *The complete genome
of HSV-1 is reported at Genbank Accession No. X14112, which is
hereby incorporated by reference in its entirety. **Each of the
listed Accession Nos. which report an amino acid sequence for the
encoded proteins is hereby incorporated by reference in its
entirety. ***UL8.5 maps to a transcript which overlaps and is in
frame with the carboxyl terminal of UL9 (Baradaran et al.,
"Transcriptional analysis of the region of the herpes simplex virus
type 1 genome containing the UL8, UL9, and UL10 genes and
identification of a novel delayed-early gene product, OBPC," J.
Virol. 68(7):4251-4261 (1994), which is hereby # incorporated by
reference in its entirety).
* * * * *