U.S. patent application number 12/409351 was filed with the patent office on 2009-07-30 for dna vaccines and methods for the prevention of transplantation rejection.
Invention is credited to Alan P. Escher, Fengchun Li.
Application Number | 20090191218 12/409351 |
Document ID | / |
Family ID | 40899471 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191218 |
Kind Code |
A1 |
Li; Fengchun ; et
al. |
July 30, 2009 |
DNA Vaccines And Methods For The Prevention Of Transplantation
Rejection
Abstract
Methods for preventing, delaying the onset of, or treating
rejection of an allograft using a DNA vaccine, where the vaccine
can comprise a polynucleotide encoding a pro-apoptotic protein,
like BAX and/or a polynucleotide encoding an autoantigen or donor
antigen, like secreted glutamic acid decarboxylase 55.
Administering one of the DNA vaccines to a transplant recipient, as
described herein, can induce a donor specific tolerogenic
response.
Inventors: |
Li; Fengchun; (Loma Linda,
CA) ; Escher; Alan P.; (Redlands, CA) |
Correspondence
Address: |
SHELDON MAK ROSE & ANDERSON PC
100 Corson Street, Third Floor
PASADENA
CA
91103-3842
US
|
Family ID: |
40899471 |
Appl. No.: |
12/409351 |
Filed: |
March 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11913567 |
Nov 5, 2007 |
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PCT/US06/17763 |
May 5, 2006 |
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12409351 |
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60680249 |
May 11, 2005 |
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Current U.S.
Class: |
424/158.1 ;
424/184.1 |
Current CPC
Class: |
A61K 2039/577 20130101;
A61K 2039/545 20130101; A61K 45/06 20130101; A61K 39/001 20130101;
A61K 2039/53 20130101; A61P 37/06 20180101; A61K 9/0019 20130101;
A61K 2039/55561 20130101; A61K 2039/55516 20130101 |
Class at
Publication: |
424/158.1 ;
424/184.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00 |
Claims
1. A method for preventing, delaying the onset of, or treating
rejection of an allograft or an allogeneic transplant, comprising:
(A) selecting a recipient in need of a graft or transplant and an
allograft or allogeneic transplant donor; (B) grafting tissue or
transplanting a solid organ from the donor to the recipient; (C)
administering to the recipient one or more than one dose of a DNA
vaccine selected from the group consisting of: (1) a first plasmid
comprising (a) a polynucleotide encoding a pro-apoptotic protein;
(b) a promoter controlling the expression of the polynucleotide
encoding the pro-apoptotic protein; (2) a second plasmid
comprising: (a) a polynucleotide sequence encoding an autoantigen
or donor antigen; (b) a promoter controlling expression of the
polynucleotide sequence encoding the autoantigen or donor antigen;
and (c) a plurality of CpG motifs, where the CpG motifs are
methylated sufficiently to diminish the recipient's immune response
to unmethylated CpG motifs; (3) a third plasmid comprising: (a) a
polynucleotide sequence encoding an autoantigen or donor antigen;
(b) a promoter controlling expression of the polynucleotide
sequence encoding the autoantigen or donor antigen; (c) a
polynucleotide encoding a pro-apoptotic protein; (d) a promoter
controlling expression of the polynucleotide encoding the
pro-apoptotic protein; and (e) a plurality of CpG motifs, where the
CpG motifs are methylated sufficiently to diminish the recipient's
immune response to unmethylated CpG motifs; and (4) a combination
of the first plasmid and the second plasmid.
2. The method of claim 1, wherein engrafted tissues or transplanted
organs are selected from the group consisting of skin grafts, islet
cell transplants, and partial or whole organ transplants.
3. The method of claim 2, wherein the partial or whole organ
transplants are selected from the group consisting of hearts,
lungs, kidneys and livers.
4. The method of claim 1, where the DNA vaccine is one or more than
one plasmid comprising a plurality of methylated CpG motifs, where
the one or more than one plasmid is resistant to digestion by the
restriction enzyme HpaII.
5. The method of claim 1, where the DNA vaccine is one or more than
one plasmid comprising a plurality of CpG motifs, where the CpG
motifs of one or more than one plasmid are methylated by SssI
methylase.
6. The method of claim 1, where the promoter capable of expressing
the polynucleotide encoding the autoantigen or the donor antigen,
or the promoter capable of expressing the polynucleotide encoding
the pro-apoptotic protein, or both the promoter capable of
expressing the polynucleotide encoding the autoantigen or the donor
antigen, and the promoter capable of expressing the polynucleotide
encoding the pro-apoptotic protein maintain their promoter function
after methylation.
7. The method of claim 1, where the third plasmid further comprises
an internal ribosome entry site (IRES) sequence to permit
translation of the polynucleotide encoding the autoantigen or the
donor antigen, and the polynucleotide encoding the pro-apoptotic
protein from the same transcript.
8. The method of claim 1, where the DNA vaccine comprises the first
plasmid and the second plasmid in a ratio of between 1/1000 to
1000/1.
9. The method of claim 1, where DNA vaccine comprises the first
plasmid and the second plasmid in a ratio of between 1/100 to
100/1.
10. The method of claim 1, where DNA vaccine comprises the first
plasmid and the second plasmid in a ratio of between 1/10 to
10/1.
11. The method of claim 1, where the autoantigen or donor antigen
is selected from the group consisting of carbonic anhydrase II,
collagen, CYP2D6 (cytochrome P450, family 2, subfamily Device 400,
polypeptide 6), glutamic acid decarboxylase, secreted glutamic acid
decarboxylase 55, SEQ ID NO:1, insulin, myelin basic protein and
SOX-10 (SRY-box containing gene 10).
12. The method of claim 1, where the pro-apoptotic protein is
selected from the group consisting of BAX, SEQ ID NO:2, a modified
caspase, Tumor Necrosis Factor Receptor, Death Receptor 3 (DR3),
Death Receptor 4 (DR4), Death Receptor 5 (DR5) and a FAS
receptor.
13. The method of claim 7, where the internal ribosome entry site
(IRES) sequence is SEQ ID NO:3 from the EMC virus.
14. The method of claim 1, where the DNA vaccine is administered in
an effective dose, wherein an effective dose is an amount
sufficient to prevent, delay the onset or treat rejection of an
allograft or an allogeneic transplant by the recipient.
15. The method of claim 1, where the DNA vaccine is administered in
an effective dose, wherein an effective dose is an amount
sufficient to induce a donor-specific tolerogenic response.
16. The method of claim 1, where the DNA vaccine is administered in
a plurality of doses.
17. The method of claim 1, where the dose of the DNA vaccine is
about 0.001 mg/Kg of body weight of the recipient to about 100
mg/Kg of body weight of the recipient.
18. The method of claim 1, where the dose is administered weekly
between two times and 100 times.
19. The method of claim 1, where the DNA vaccine is administered by
an epidermal, intradermal, intramuscular, intranasal, intravenous,
intraperitoneal or oral route.
20. The method of claim 1, where the DNA vaccine is administered by
an injection proximal to the site of the allograft or allogeneic
transplant.
21. The method of claim 1, further comprising administering a dose
of one or more than one immunosuppressant agent before, on the day
of, and/or after engraftment or transplantation.
22. The method of claim 21, wherein the dose of one or more than
one immunosuppressant agent is administered simultaneously,
separately or sequentially.
23. The method of claim 21, wherein the one or more than one
immunosuppressant agent is selected from the group consisting of
corticosteroids, glucocorticoids. cyclophosphamide,
6-mercaptopurine, azathioprine, methotrexate cyclosporine,
mycophenolate mofetil, mycophenolic acid, tacrolimus, sirolimus,
everolimus, mizoribine, leflunomide, deoxyspergualin, brequinar,
azodicarbonamide, vitamin D analogs, antilymphocyte globulin,
antithymocyte globulin, anti-CD3 monoclonal antibodies,
anti-interleukin-2 receptor (anti-CD25) antibodies, anti-CD52
antibodies, anti-CD20 antibodies, anti-tumor necrosis factor
reagents and LFA-1 inhibitors.
24. The method of claim 21, comprising administering a single dose
of antilymphocyte globulin, at a dosage of about 1.6 mg/20 g of
body weight, on the day of engraftment or transplantation.
25. The method of claim 21, comprising administering rapamycin at a
dosage of from 0.05 to 15 mg/day.
26. A method for preventing, delaying the onset of, or treating
rejection of an allograft or an allogeneic transplant, comprising:
(A) selecting a graft or transplant recipient and an allograft or
allogeneic transplant donor; (B) grafting tissue or transplanting a
solid organ from the donor to the recipient; (C) inducing a
donor-specific immune response that elevates regulatory T cell
activity by administering to the recipient one or more than one
dose of a DNA vaccine comprising a first plasmid and a second
plasmid; (1) the first plasmid comprising: (a) a polynucleotide
encoding a pro-apoptotic protein; (b) a promoter controlling
expression of the polynucleotide encoding the pro-apoptotic
protein; and (2) a second plasmid comprising: (a) a polynucleotide
sequence encoding an autoantigen or donor antigen; (b) a promoter
controlling expression of the polynucleotide sequence encoding the
autoantigen or donor antigen; and (c) a plurality of CpG motifs,
where the CpG motifs are methylated sufficiently to diminish the
recipient's immune response to unmethylated CpG motifs.
27. The method of claim 26, wherein administration of the plasmids
elevates expression of Il-4 (interleukin 4).
28. The method of claim 26, wherein administration of the plasmids
further induces a donor-specific tolerogenic response.
29. The method of claim 26, wherein administration of the plasmids
elevates expression of inhibitory Fc receptor, Fc.gamma.IIb, and
Il-1 antagonist Il-1RA cytokine.
30. The method of claim 26, wherein administration of the plasmids
reduces an autoimmune response.
31. The method of claim 26, wherein administration of the plasmids
reduces expression of Tnf.alpha. (tumor necrosis factor) and
Ifn.gamma. (gamma interferon).
32. The method of claim 26, wherein the first plasmid and the
second plasmid are administered simultaneously or sequentially.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present Application claims the benefit of International
Patent Application No. PCT/US09/37027, titled "DNA Vaccines and
Methods for the Prevention of Transplantation Rejection," filed
Mar. 12, 2009, which claims the benefit of U.S. provisional patent
application 61/036,004, titled "DNA Construct for the Prevention of
Transplantation Rejection," filed Mar. 12, 2008, the contents of
which are all incorporated in this disclosure by reference in their
entirety. The present Application is also a continuation in part of
U.S. patent application Ser. No. 11/913,567, titled "Substances
Compositions and Methods for Preventing and Treating
Immune-Mediated Inflammatory Disorders," filed Nov. 5, 2007, which
is a 371 of International Patent Application PCT/US2006/17763
titled "Substances. Compositions and Methods for Preventing and
Treating Immune-Mediated Inflammatory Disorders," filed May 5,
2006, which claims the benefit of U.S. provisional patent
application 60/680,249, titled "Substances and Method for
Preventing and Treating Autoimmune Diseases," filed May 11, 2005,
the contents of which are all incorporated in this disclosure by
reference in their entirety.
BACKGROUND
[0002] Prevention of organ rejection in the clinic presently relies
on administration of cocktails of immunosuppressants (Tacrolimus,
Rapamycin, MMF, AZA, corticosteroids). The drugs are efficient for
prevention of acute rejection, but do not prevent chronic
rejection. In addition, because these drugs interfere with immune
responses non-specifically, their chronic use exposes patients to
high risks of cancer and infection. Other approaches that are being
tested are co-stimulatory blockade and bone-marrow chimerism.
However, "late onset chronic rejection, as well as the toxicity of
some of these regimens, remain as significant limitations that
hamper clinical application" (Ochiai et al., Front. Biosci. 2007,
12:4248-53).
SUMMARY
[0003] One embodiment of the present invention comprises a method
for preventing, delaying the onset of, or treating rejection of an
allograft or an allogeneic transplant. The method comprises: (A)
selecting a recipient in need of a graft or transplant and an
allograft or allogeneic transplant donor; (B) grafting tissue or
transplanting a solid organ from the donor to the recipient; and
(C) administering to the recipient one or more than one dose of a
DNA vaccine. The DNA vaccine is selected from the group consisting
of: three individual plasmids and a two plasmid vaccine. The first
plasmid comprises (a) a polynucleotide encoding a pro-apoptotic
protein; and (b) a promoter controlling the expression of the
polynucleotide encoding the pro-apoptotic protein. The second
plasmid comprises: (a) a polynucleotide sequence encoding an
autoantigen or donor antigen; (b) a promoter controlling expression
of the polynucleotide sequence encoding the autoantigen or donor
antigen; and (c) a plurality of CpG motifs, where the CpG motifs
are methylated sufficiently to diminish the recipient's immune
response to unmethylated CpG motifs. The third plasmid comprises:
(a) a polynucleotide sequence encoding an autoantigen or donor
antigen; (b) a promoter controlling expression of the
polynucleotide sequence encoding the autoantigen or donor antigen;
(c) a polynucleotide encoding a pro-apoptotic protein; (d) a
promoter controlling expression of the polynucleotide encoding the
pro-apoptotic protein; and (e) a plurality of CpG motifs, where the
CpG motifs are methylated sufficiently to diminish the recipient's
immune response to unmethylated CpG motifs. The two plasmid vaccine
comprises a combination of the first plasmid and the second
plasmid.
[0004] In some embodiments of the present invention, the engrafted
tissues or transplanted organs are selected from the group
consisting of skin grafts, islet cell transplants, and partial or
whole organ transplants. In additional embodiments of the method,
the partial or whole organ transplants are selected from the group
consisting of hearts, lungs, kidneys and livers.
[0005] In one embodiment of the method, the DNA vaccine is one or
more than one plasmid comprising a plurality of methylated CpG
motifs, where the one or more than one plasmid is resistant to
digestion by the restriction enzyme HpaII. In a preferred
embodiment, the DNA vaccine is one or more than one plasmid
comprising a plurality of CpG motifs, where the CpG motifs of one
or more than one plasmid are methylated by SssI methylase.
[0006] In one embodiment of the present invention, the promoter
capable of expressing the polynucleotide encoding the autoantigen
or the donor antigen, or the promoter capable of expressing the
polynucleotide encoding the pro-apoptotic protein, or both the
promoter capable of expressing the polynucleotide encoding the
autoantigen or the donor antigen, and the promoter capable of
expressing the polynucleotide encoding the pro-apoptotic protein
maintain their promoter function after methylation.
[0007] In another embodiment of the present method, the third
plasmid further comprises an internal ribosome entry site (IRES)
sequence, where the IRES sequence is SEQ ID NO:3 from the EMC
virus, to permit translation of the polynucleotide encoding the
autoantigen or the donor antigen, and the polynucleotide encoding
the pro-apoptotic protein from the same transcript.
[0008] In one embodiment of the present invention, the DNA vaccine
comprises the first plasmid and the second plasmid in a ratio of
between 1/1000 to 1000/1. In a preferred embodiment, the DNA
vaccine comprises the first plasmid and the second plasmid in a
ratio of between 1/100 to 100/1. In a particularly preferred
embodiment, the DNA vaccine comprises the first plasmid and the
second plasmid in a ratio of between 1/10 to 10/1.
[0009] In specific embodiments of the present invention, the
autoantigen or donor antigen is selected from the group consisting
of carbonic anhydrase II, collagen, CYP2D6 (cytochrome P450, family
2, subfamily Device 400, polypeptide 6), glutamic acid
decarboxylase, secreted glutamic acid decarboxylase 55, SEQ ID
NO:1, insulin, myelin basic protein and SOX-10 (SRY-box containing
gene 10) or any relevant autoantigen that is present in both the
transplant recipient and the donor allograft.
[0010] In additional specific embodiments, the pro-apoptotic
protein is selected from the group consisting of BAX, SEQ ID NO:2,
a modified caspase, Tumor Necrosis Factor Receptor, Death Receptor
3 (DR3), Death Receptor 4 (DR4), Death Receptor 5 (DR5) and a FAS
receptor.
[0011] In one embodiment of the method, the DNA vaccine is
administered in an effective dose, wherein an effective dose is an
amount sufficient to prevent, delay the onset, or treat rejection
of an allograft or an allogeneic transplant by the recipient.
[0012] In another embodiment of the method, the DNA vaccine is
administered in an effective dose, wherein an effective dose is an
amount sufficient to induce a donor-specific tolerogenic
response.
[0013] In preferred embodiments, the DNA vaccine is (a)
administered in a plurality of doses; (b) the dose of the DNA
vaccine is about 0.001 mg/Kg of body weight of the recipient to
about 100 mg/Kg of body weight of the recipient; and/or (c) the
dose is administered weekly between two times and 100 times.
[0014] In some embodiments the DNA vaccine is administered by an
epidermal, intradermal, intramuscular, intranasal, intravenous,
intraperitoneal or oral route. In a preferred embodiment, the DNA
vaccine is administered by an injection proximal to the site of the
allograft or allogeneic transplant.
[0015] In one embodiment, the method further comprises the step of
administering a dose of one or more than one immunosuppressant
agent before, on the day of and/or after engraftment or
transplantation. As will be appreciated by one of skill in the art,
with reference to the present disclosure, the dose of one or more
than one immunosuppressant agent can be administered
simultaneously, separately or sequentially.
[0016] In specific embodiments, the one or more than one
immunosuppressant agent is selected from the group consisting of
corticosteroids, glucocorticoids. cyclophosphamide,
6-mercaptopurine, azathioprine, methotrexate cyclosporine,
mycophenolate mofetil, mycophenolic acid, tacrolimus, sirolimus,
everolimus, mizoribine, leflunomide, deoxyspergualin, brequinar,
azodicarbonamide, vitamin D analogs, antilymphocyte globulin,
antithymocyte globulin, anti-CD3 monoclonal antibodies,
anti-interleukin-2 receptor (anti-CD25) antibodies, anti-CD52
antibodies, anti-CD20 antibodies, anti-tumor necrosis factor
reagents and LFA-1 inhibitors.
[0017] In a preferred embodiment, the method includes the step of
administering a single dose of antilymphocyte globulin, at a dosage
of about 1.6 mg/20 g of body weight, on the day of engraftment or
transplantation.
[0018] In another preferred embodiment, the method includes the
step of administering rapamycin at a dosage of from 0.05 to 15
mg/day.
[0019] One embodiment of the present invention provides a method
for preventing, delaying the onset of or treating rejection of an
allograft or an allogeneic transplant, comprising the steps of: (A)
selecting a graft or transplant recipient and an allograft or
allogeneic transplant donor; (B) grafting tissue or transplanting a
solid organ from the donor to the recipient; and (C) inducing a
donor-specific immune response that elevates regulatory T cell
activity by administering to the recipient one or more than one
dose of a DNA vaccine comprising a first plasmid and a second
plasmid. The first plasmid comprises: (a) a polynucleotide encoding
a pro-apoptotic protein; and (b) a promoter controlling the
expression of the polynucleotide encoding the pro-apoptotic
protein. The second plasmid comprises: (a) a polynucleotide
sequence encoding an autoantigen or donor antigen; (b) a promoter
controlling expression of the polynucleotide sequence encoding the
autoantigen or donor antigen; and (c) a plurality of CpG motifs,
where the CpG motifs are methylated sufficiently to diminish the
recipient's immune response to unmethylated CpG motifs.
[0020] In one version of this embodiment, administration of the
plasmids elevates expression of Il-4 (interleukin 4). In another
version of this embodiment, administration of the plasmids further
induces a donor-specific tolerogenic response. In yet another
version of this embodiment, administration of the plasmids elevates
expression of inhibitory Fc receptor, Fc.gamma.IIb, and Il-1ra. In
still another version of this embodiment, administration of the
plasmids reduces an autoimmune response. In an alternative version
of this embodiment, administration of the plasmids reduces
expression of Tnf.alpha. (tumor necrosis factor) and Ifn.gamma.
(gamma interferon). As will be appreciated by one of skill in the
art with reference to the present disclosure, alternative versions
of the method can include alternative steps where the first plasmid
and the second plasmid are administered simultaneously or
sequentially.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] These features, aspects and advantages of the present
invention will become better understood with regard to the
following description, appended claims and accompanying drawings
where:
[0022] FIG. 1 shows schematic depictions of the plasmids disclosed
herein, including plasmid vectors pSG5 and pND2, a plasmid
containing a polynucleotide encoding secreted glutamic acid
decarboxylase 55 (SGAD55) operably linked to a SV40 promoter
(pSG5-sgad55), a plasmid containing a polynucleotide encoding a
pro-apoptotic protein (hBAX) operably linked to an HCMV promoter
(pND2-hBAX), and a plasmid containing a polynucleotide encoding
SGAD55 and hBAX operably linked to an SV40 promoter
(pSG5-sga55-bax).
[0023] FIG. 2 shows the effects of DNA vaccination on skin
allograft survival. 7-week-old, age matched C57BL/6 recipients
(N=8-14) received skin grafts from BALB/c donor under minimum
immune suppression regimen (IS) that was ended on day 28, and
received a weekly i.d. injection of 50 .mu.g of the indicated
vaccine. The BAX and sGAD vaccines are non-CpG-methylated plasmid
DNA coding for BAX and sGAD alone, respectively. The MsGAD DNA
vaccine consists of CpG-methylated plasmid DNA coding for sGAD
alone. The MsGAD-BAX vaccine consists of a 4:1 ratio of MsGAD:BAX
plasmid DNA. A and B show allograft survival after immunization
with non-methylated and methylated vaccines, respectively. #,
P<0.003 compared to vector and IS alone (Mann-Whitney), @,
P<0.005 compared to methylated vector (Mvector) and IS alone,
.diamond-solid., P<0.04 compared to sGAD in A, .star-solid.,
P<0.04 compared to methylated, non-methylated vector controls
and IS alone (Kaplan-Meier).
[0024] FIG. 3 shows the results of quantitative gene expression
analysis in skin and LNs of recipient mice. C57BL/6 mice under
minimum immunosuppression received BALB/c skin grafts and were
immunized with the BAX or MsGAD-BAX DNA vaccine. Fresh skin
allografts (A) and fresh lymph nodes (LNs) (B) were taken 2 weeks
after transplant for qPCR analysis. In addition, LNs were
stimulated with self, donor, or third-party (C3H) antigens for
analysis (C). Results are shown as fold of gene expression relative
to non-vaccinated C57BL/6 mice under minimum immunosuppression
(control). In A and B, @, P<0.05 compared to control. In C, #,
@, P<0.05 compared to cells stimulated with self and third-party
antigens, respectively.
[0025] FIG. 4 shows the results of the adoptive transfer of immune
cells from pooled LNs and spleen. Adoptive transfer donor (C57BL/6)
received BALB/c skin graft and minimum immunosuppression.
Splenocytes and draining lymph node cells were then isolated on day
14, and injected i.p. on day -2 into adoptive transfer recipients
(C57BL/6, N=4-5) receiving donor (BALB/c, D) or third party (C3H,
T) skin grafts at day 0. The recipients also received 3 Gy
irradiation at day -3, and daily rapamycin i.p. @, P<0.05
compared to third party.
[0026] FIG. 5 shows the results of mixed lymphocyte reactions where
LN cells were isolated from recipients receiving donor skin grafts
and immune regimen for 2 weeks, and 4.times.10.sup.5 LN cells were
mixed with 4.times.10.sup.5 mitomycin treated splenocytes from
BALB/c donor or C3H third-party mice in presence of 2 or 5 .mu.g/ml
IL-2. @, P<0.05 compared to third party antigens.
DETAILED DESCRIPTION
[0027] As used in this disclosure, except where the context
requires otherwise, the term "comprise" and variations of the term,
such as "comprising," "comprises" and "comprised" are not intended
to exclude other additives, components, integers or steps.
[0028] As used in this disclosure, the terms "graft" and
"transplant" are used interchangeably and refer to an organ or
tissue taken from the body and grafted into another area of the
same individual or another individual.
[0029] As used in this disclosure, the term "allograft" comprises a
graft of tissue between individuals of the same species but of
disparate genotype; types of donors include cadaveric, living
related, and living unrelated.
[0030] As used in this disclosure, the term "allogeneic" denotes
individuals of the same species but of different genetic
constitution.
[0031] As used in this disclosure, the term "allogeneic
transplantation" denotes transplantation of an allograft.
[0032] As used in this disclosure, the term "DNA vaccine" comprises
DNA sequences that code for immunogenic proteins located in
appropriately constructed plasmids, which include strong promoters,
which when injected into an animal are taken up by cells and the
immunogenic proteins are expressed and elicit an immune
response.
[0033] As used in this disclosure, the term "autoantigen" comprises
an endogenous antigen that stimulates the production of
autoantibodies, as in an autoimmune reaction, as well as part of
such endogenous antigens, or modified endogenous antigens that
elicit the same response as the full endogenous antigen, as will be
understood by those with skill in the art with reference to this
disclosure. For example, in the context of this disclosure secreted
glutamic acid decarboxylase 55 and humanized BAX are both
autoantigens.
[0034] As used in this disclosure, the term "donor antigen"
comprises an antigen from an allograft that was transplanted into
the recipient to take the place of defective or absent cells or
tissues, such as for example skin grafts and islet cell
transplants, and partial or whole organ transplants, including
transplanted hearts, lungs, kidneys and livers, and that stimulates
the production of antibodies that produce an immune reaction, as
well as part of such donor antigens, or modified donor antigens
that elicit the same response as the full donor antigen, as will be
understood by those with skill in the art with reference to this
disclosure. For example, in the context of this disclosure,
secreted glutamic acid decarboxylase 55 is a donor antigen for skin
grafts and islet cell transplants.
[0035] Examples of a pro-apoptotic protein include BAX (SEQ ID
NO:2), a modified caspase, Tumor Necrosis Factor Receptor, Death
Receptor 3 (DR3), Death Receptor 4 (DR4), Death Receptor 5 (DR5)
and a FAS receptor. As used in this disclosure, the term "hBAX" and
"BAX" are interchangeable.
[0036] As will be understood by those with skill in the art with
reference to this disclosure, when reference is made to a protein
encoded by a polynucleotide sequence, the protein includes
"conservative substitutions" in which an amino acid is substituted
for another amino acid that has similar properties, such that one
skilled in the art of peptide chemistry would expect the secondary
structure and hydropathic nature of the polypeptide to be
substantially unchanged. A conservative substitution occurs when
one amino acid residue is replaced with another that has a similar
side chain. Amino acid residues having similar side chains are
known in the art and include families with basic side chains (e.g.,
lysine (Lys/K), arginine (Arg/R), histidine (His/H)), acidic side
chains (e.g., aspartic acid (Asp/Device 400), glutamic acid
(Glu/E)), uncharged polar side chains (e.g., glycine (Gly/G),
asparagine (Asn/N), glutamine (Gln/Q), serine (Ser/S), threonine
(Thr/T), tyrosine (Tyr/Y), cysteine (Cys/C)), nonpolar side chains
(e.g., alanine (Ala/A), valine (Val/V), leucine (Leu/L), isoleucine
(Ile/I), proline (Pro/P), phenylalanine (Phe/F), methionine
(Met/M), tryptophan (Trp/W)), .beta.-branched side chains (e.g.,
threonine (Thr/T), valine (Val/V), isoleucine (Ile/I)) and aromatic
side chains (e.g., tyrosine (Tyr/Y), phenylalanine (Phe/F),
tryptophan (Trp/W), histidine (His/H)).
[0037] As used in this disclosure, a CpG motif is a polynucleotide
region characterized by dinucleotides containing cytosine residues
in the sequence CG. As will be understood by those with skill in
the art with reference to this disclosure, bacterial DNAs
containing unmethylated CpG motifs, stimulate the immune system in
mammals to start a sequence of reactions leading to an immune
reaction and inflammation. However, methylated CpG motifs may be
applied to alleviate or inhibit the unwanted immune stimulation and
inflammation by bacterial DNA.
[0038] Apoptotic cells are routinely processed by antigen
presenting cells (APCs) like dendritic cells (DCs) to establish and
maintain antigen-specific tolerance. DNA vaccines can induce
apoptotic cells directly in vivo, and permit the engineering of the
induced apoptotic cells. Unlike the use of immunosuppressants and
co-stimulatory blockade, this is a "top down" approach that has the
potential to induce specific immunoregulation via physiological
modulation of APC function. In some embodiments, the present
invention provides for the use of three different plasmid DNA
constructs, and combinations thereof, as DNA vaccines to increase
survival of allografts. Specific embodiments of each of the
constructs causes increased survival of engrafted skin when
injected into mice receiving skin allografts.
[0039] According to one embodiment of the present invention, there
is provided a method of preventing, delaying the onset of, or
treating the rejection of an allograft or an allogeneic transplant.
In one embodiment, the method comprises, first, selecting a
recipient in need of a graft or transplant and an allograft or
allogeneic transplant donor. The selection can be made using
standard methods as will be understood by those with skill in the
art with reference to this disclosure.
[0040] According to one embodiment, the method further comprises
engrafting tissue or transplanting a solid organ from the donor to
the recipient to take the place of defective or absent cells or
tissues. Engrafted tissues or transplanted organs can include a
skin grafts, islet cell transplants, and partial or whole organ
transplants including transplanted hearts, lungs, kidneys and
livers.
[0041] According to one embodiment, the method further comprises
administering to the recipient a DNA vaccine comprising one or more
polynucleotides encoding (1) a pro-apoptotic protein, (2) an
autoantigen or donor antigen, or (3) a pro-apoptotic protein and an
autoantigen or donor antigen.
[0042] In one embodiment, a DNA vaccine for use in the present
invention comprises a plasmid, the plasmid comprising a
polynucleotide encoding a pro-apoptotic protein under the control
of a promoter capable of expressing the polynucleotide encoding the
pro-apoptotic protein.
[0043] In another embodiment, a DNA vaccine for use in the present
invention comprises a plasmid, the plasmid comprising a
polynucleotide encoding an autoantigen or a donor antigen operably
linked to a promoter capable of controlling the expression of the
polynucleotide encoding the autoantigen or the donor antigen, where
the plasmid comprises a plurality of CpG motifs, and where at least
some of the plurality of CpG motifs are methylated. In a preferred
embodiment, the CpG motifs are methylated sufficiently to inhibit
the recipient's immune response to unmethylated plasmid DNA. In a
particularly preferred embodiment, the plasmid is resistant to
digestion by the restriction endonuclease HpaII, which digests
unmethylated but not methylated DNA. In another embodiment, the CpG
motifs are methylated by SssI methylase.
[0044] In another embodiment, the plasmid comprising a
polynucleotide encoding an autoantigen or a donor antigen can
further comprise a polynucleotide encoding a pro-apoptotic protein
under the control of a promoter capable of expressing the
polynucleotide encoding the pro-apoptotic protein.
[0045] In a preferred embodiment, the promoter capable of
expressing the polynucleotide encoding the autoantigen or the donor
antigen, and the promoter capable of expressing the polynucleotide
encoding the pro-apoptotic protein, are a single promoter.
[0046] In a preferred embodiment, the promoter capable of
expressing the polynucleotide encoding the autoantigen or the donor
antigen, or the promoter capable of expressing the polynucleotide
encoding the pro-apoptotic protein, or both the promoter capable of
expressing the polynucleotide encoding the autoantigen or the donor
antigen, and the promoter capable of expressing the polynucleotide
encoding the pro-apoptotic protein, maintain their promoter
function after methylation.
[0047] In another embodiment, the plasmid comprises an internal
ribosome entry site (IRES) sequence, to permit translation of the
polynucleotide encoding the autoantigen or the donor antigen and
the polynucleotide encoding the pro-apoptotic protein from the same
transcript.
[0048] In another embodiment, the DNA vaccine of the present
invention comprises a first plasmid and a second plasmid, or
composition comprising a first plasmid and a second plasmid. The
first plasmid comprises a polynucleotide encoding an autoantigen or
a donor antigen under the control of a promoter capable of
expressing the polynucleotide encoding the autoantigen or the donor
antigen. The second plasmid comprises a polynucleotide encoding a
pro-apoptotic protein under the control of a promoter capable of
expressing the polynucleotide encoding the pro-apoptotic protein.
The first plasmid comprises a plurality of CpG motifs, and at least
some of the plurality of CpG motifs are methylated.
[0049] In a preferred embodiment, the promoter capable of
expressing the polynucleotide encoding the autoantigen or the donor
antigen maintains its promoter function after methylation.
[0050] In another embodiment, the second plasmid comprises a
plurality of CpG motifs, and at least some of the plurality of CpG
motifs are methylated.
[0051] In a preferred embodiment, the promoter capable of
expressing the polynucleotide encoding a pro-apoptotic protein
maintains its promoter function after methylation.
[0052] In one embodiment, the DNA vaccine comprises the first
plasmid and second plasmid in a ratio of between 1/1000 to 1000/1.
In another embodiment, the composition comprises the first plasmid
and second plasmid in a ratio of between 1/100 to 100/1. In another
embodiment, the composition comprises the first plasmid and second
plasmid in a ratio of between 1/10 to 10/1.
[0053] In one embodiment of the present invention, the recipient is
a mammal. In another embodiment, the recipient is a human.
[0054] In another embodiment, the autoantigen is selected from the
group consisting of carbonic anhydrase II, collagen, CYP2D6
(cytochrome P450, family 2, subfamily Device 400, polypeptide 6),
glutamic acid decarboxylase, secreted glutamic acid decarboxylase
55, SEQ ID NO:1, insulin, myelin basic protein and SOX-10 (SRY-box
containing gene 10).
[0055] In another embodiment, the pro-apoptotic protein is selected
from the group consisting of BAX, SEQ ID NO:2, a modified caspase,
Tumor Necrosis Factor Receptor, Death Receptor 3 (DR3), Death
Receptor 4 (DR4), Death Receptor 5 (DR5) and a FAS receptor.
[0056] In a preferred embodiment, the internal ribosome entry site
sequence is an internal ribosome binding site from the EMC virus,
SEQ ID NO:3.
[0057] The method comprises administering to the recipient one or
more than one dose of a DNA vaccine according to the present
invention. In a preferred embodiment, the DNA vaccine is
administered in a plurality of doses. In another preferred
embodiment, the dose is between about 0.001 mg/Kg of body weight of
the recipient and about 100 mg/Kg of body weight of the recipient.
In another preferred embodiment, the dose is between about 0.01
mg/Kg of body weight of the recipient and about 10 mg/Kg of body
weight of the recipient. In another preferred embodiment, the dose
is between about 0.1 mg/Kg of body weight of the recipient and
about 1 mg/Kg of body weight of the recipient. In another preferred
embodiment, the dose is about 0.05 mg/Kg of body weight of the
recipient. In a preferred embodiment, the recipient is a human and
the dose is between about 0.5 mg and 5 mg. In another preferred
embodiment, the recipient is a human and the dose is between about
1 mg and 4 mg. In another preferred embodiment, the recipient is a
human and the dose is between about 2.5 mg and 3 mg. In another
preferred embodiment, the dose is administered weekly between 2
times and about 100 times. In another preferred embodiment, the
dose is administered weekly between 2 times and about 20 times. In
another preferred embodiment, the dose is administered weekly
between 2 times and about 10 times. In another preferred
embodiment, the dose is administered weekly 4 times. In another
preferred embodiment, the dose is administered only once.
[0058] Administering the one or more than one dose of a DNA vaccine
to the recipient can be accomplished by any suitable route, as will
be understood by those with skill in the art with reference to this
disclosure. In one embodiment, administering to the recipient one
or more than one dose of a substance or a composition is performed
by a route selected from the group consisting of epidermal,
intradermal, intramuscular, intranasal, intravenous and oral. In a
preferred embodiment the DNA vaccine is administered proximal to
the site of the allograft or allogeneic transplant. For example,
the plasmid DNA does not have to be injected into a skin graft, but
can be injected directly intradermally into the recipient.
[0059] When the method comprises administering a first plasmid and
a second plasmid, the first plasmid and the second plasmid can be
administered either sequentially or simultaneously, as will be
understood by those with skill in the art with reference to this
disclosure.
[0060] When the method comprises administering a first plasmid and
a second plasmid, the method can further comprise inducing a
donor-specific immune response that elevates Th2-like activity,
which can include inducing expression of Il-4 in the allograft.
[0061] When the method comprises administering a first plasmid and
a second plasmid, the method can further comprise inducing
expression of Fc.alpha.IIb in the allograft.
[0062] When the method comprises administering a first plasmid and
a second plasmid, the method can further comprise decreasing
expression of pro-inflammatory Tnf-.alpha. and Ifn-.gamma.
genes.
[0063] In one embodiment, the method further comprises
administering a dose of one or more than one immunosuppressant
agent before, on the day of, and/or after engraftment or
transplantation.
[0064] When the method comprises administering one or more than one
immunosuppressant agent, the one or more than one immunosuppressant
agent can be administered simultaneously, separately or
sequentially.
[0065] In one embodiment, the one or more than one
immunosuppressant agent is selected from the group consisting of
corticosteroids, glucocorticoids. cyclophosphamide,
6-mercaptopurine (6-MP), azathioprine (AZA), methotrexate
cyclosporine, mycophenolate mofetil (MMF), mycophenolic acid (MPA),
tacrolimus (FK506), sirolimus ([SRL] rapamycin), everolimus
(Certican), mizoribine, leflunomide, deoxyspergualin, brequinar,
azodicarbonamide, vitamin D analogs, such as MC1288 and
bisindolylmaleimide VIII, antilymphocyte globulin, antithymocyte
globulin (ATG), anti-CD3 monoclonal antibodies, (Muromonab-CD3,
Orthoclone OKT3), anti-interleukin (IL)-2 receptor (anti-CD25)
antibodies, (Daclizumab, Zenapax, basiliximab, Simulect), anti-CD52
antibodies, (Alemtuzumab, Campath-1H), anti-CD20 antibodies
(Rituximab, Rituxan), anti-tumor necrosis factor (TNF) reagents
(Infliximab, Remicade, Adalimumab, Humira) and LFA-1 inhibitors
(Efalizumab, Raptiva).
[0066] The dosages of the immunosuppressant agents will vary
depending on the individual to be treated, the route of
administration, and the nature and severity of the condition to be
treated. For example, an initial dose of about 2 to 3 times the
maintenance dose may suitably be administered about 4 to 12 hours
before transplantation, followed by a daily dosage of 2 to 3 times
the maintenance dose for one to two weeks, before gradually
tapering down at a rate of about 5% a week to reach the maintenance
dose.
[0067] The skilled person may determine those dosages that provide
a therapeutic amount of an immunosuppressant agent at a level that
is tolerated. In a preferred embodiment, the method further
comprises administering a single dose of antilymphocyte globulin,
of about 1.6 mg/20 g of body weight on the day of engraftment or
transplantation. In another preferred embodiment, rapamycin may be
applied at a dosage range of from about 0.05 to about 15 mg/kg/day,
more preferably from about 0.25 to about 5 mg/kg/day and most
preferably from about 0.5 to about 1.5 mg/kg/day. Ideally, the
administration of doses of one or more than one immunosuppressant
agent can be curtailed after effective treatment with the DNA
vaccine.
[0068] In one embodiment, the method further comprises, after
administering the DNA vaccine, monitoring the recipient for
rejection of the allograft of transplant. In a preferred
embodiment, the recipient is monitored for rejection of the
allograft or transplant after tapering off or discontinuing the
administration of immunosuppressant agents.
Example 1
Plasmid DNA Constructs, Methylation, and Amplification
[0069] The following examples were designed to test whether our
pro-apoptotic DNA vaccination strategy is applicable to prevention
of solid allograft rejection. Our model suggests that injection of
plasmid DNA coding for BAX near the allograft would cause
recruitment of APCs after induction of apoptotic cells, the APCs
would process donor antigens under tolerogenic conditions, and a
protective, immunoregulatory response would be induced.
[0070] In order to compare the efficacy of different DNA vaccines
for preventing, delaying the onset of or treating the rejection of
an allograft or organ transplant according to the present
invention, several plasmids were prepared. Referring now to FIG. 1,
there are shown, respectively, a schematic depiction of pSG5, pND2,
pSG5-SGAD55, and pND2-hBAX.
[0071] Plasmid pSG5 was purchased from Stratagene (San Diego,
Calif. US). The remaining plasmids were produced using standard
techniques. Plasmid pND2-BAX carries a BAX cDNA under
transcriptional control of the CMV promoter, and plasmid
pSG5-SGAD55 carries a cDNA construct encoding a secreted form of
GAD65 under transcriptional control of the SV-40 promoter.
[0072] With reference to FIG. 1, the abbreviations shown are
standard, as will be understood by those with skill in the art with
reference to this disclosure, including: AMP (ampicillin resistance
gene for selection in E. coli); BGH pA (bovine growth hormone
polyadenylation sequence); ColE1 origin (origin of replication in
E. coli); f1 origin (origin of replication for filamentous phage f1
to generate single stranded DNA); hBAX (human bax cDNA), SEQ ID NO:
2; HCMV promoter (promoter from cytomegalovirus); HCMV intron
(intron from cytomegalovirus); MCS (multiple cloning site); pUC
origin (origin of replication for E. coli from pUC plasmid); sgad55
(secreted GAD cDNA construct), SEQ ID NO:1; SV40 promoter (simian
virus 40 promoter); SV40 pA (simian virus 40 polyadenylation
sequence); and T7 (T7 promoter).
[0073] Plasmids pSG5 and pSG-SGAD55 were methylated to produce
methylated pSG5, and methylated pSG5-SGAD55, by amplification in E.
coli strain ER1821 carrying a plasmid encoding the SssI methylase
(New England Biolabs, Beverly, Mass. US). SssI methylates the
dinucleotide motif CpG in DNA in a manner corresponding to
mammalian methylases by covalently adding a single methyl group to
the dinucleotide motif CpG.
[0074] Plasmid DNA was isolated after amplification in Escherichia
coli strain DH5.alpha. or ER1821 using the Endofree Plasmid DNA
Purification Kit (Qiagen, Valencia, Calif.). Successful methylation
was confirmed by digesting the isolated plasmid DNA with the
restriction enzyme HpaII which digests unmethylated but not
methylated DNA, where resistance to HpaII digestion indicates
successful methylation.
Example 2
Skin Allograft Survival
[0075] An allograft skin transplant model was used as proof of
principle because it is one of the most difficult models for
prevention of organ rejection. A combination of two of the plasmid
constructs (2-plasmid vaccine coding for secreted GAD, i.e.,
SGAD55, and pro-apoptotic BAX, with one plasmid methylated with
SssI methylase) has been used successfully as a DNA vaccine for
therapy of type 1 diabetes in NOD mice. However, each of the two
DNA constructs (plasmid DNA coding for BAX alone, or
SssI-methylated plasmid DNA coding for SGAD55 alone) were found to
be ineffective for therapy of diabetes on their own. Unexpectedly,
we now find that all three alternatives can prevent skin allograft
rejection.
[0076] The effects of i.d. injection of different DNA vaccines were
investigated in C57BL/6 mice receiving minimum immunosuppression
and skin allografts from BALB/c mice. 0.7.times.0.7 cm
full-thickness back skin grafts from BALB/c donors or C3H third
party were transplanted onto the back of C57BL/6 recipients. With
the exception of the untreated group, anti-lymphocyte
immunoglobulin ALG (1.6 mg/20 g BW) was given i.p. once on day 0.
Fifty .mu.g of the following plasmid DNAs: 1) vector alone, 2) DNA
coding for BAX alone, 3) SssI-methylated DNA coding for SGAD55
alone (Msgad55), or 4) DNA coding for BAX together with
SssI-methylated DNA coding for SGAD55 (Msgad55+bax), were injected
i.d. near the skin graft on day 0, 3, 7, and then weekly. Rapamycin
(1 mg/kg) was injected i.p. daily from days 0-27. Bandages were
removed on day 10, and skin graft rejection was defined as 85% loss
of the graft area, and confirmed by pathological analysis.
[0077] FIG. 2A shows that non-methylated plasmid DNA coding for BAX
alone could significantly delay skin rejection compared to mice
receiving vector DNA alone, but that non-methylated plasmid DNA
coding for sGAD alone did not. Injection of non-methylated plasmid
vector alone had no significant effect on allograft survival
compared to non-vaccinated, immunosuppressed mice.
[0078] In addition, we investigated the effects of CpG-methylation
of vaccine DNA and of combined delivery of plasmid DNA coding for
sGAD and BAX on skin allograft survival. CpG-methylation of the
vaccine coding for sGAD alone (MsGAD) resulted in increased
allograft survival (FIG. 2B), which was significant compared to
mice immunized with the non-methylated vaccine coding for sGAD
alone and CpG-methylated vector control. However, combined delivery
of CpG-methylated plasmid DNA coding for secreted GAD and plasmid
DNA coding for BAX resulted in increased survival only when
compared with mice vaccinated with vector controls and
non-immunized mice, which indicated an antagonistic effect of
BAX.
[0079] Without being held to any particular underlying mechanisms
behind the observed effects, we suspect that it may involve donor
antigen-specific immunoregulation for the constructs coding for
BAX, based on our previous model for a pro-apoptotic DNA
vaccination strategy. We do not know what the mechanism of action
is for the third construct (SssI-methylated DNA coding for
SGAD55).
Example 3
RNA Isolation and qPCR
[0080] We investigated the effects of the MsGAD-BAX vaccine, which
showed efficacy in both prevention of skin allograft rejection and
amelioration of new-onset diabetes in NOD mice in previous work,
and of the BAX vaccine on the expression of chosen genes in
transplanted BALB/c skin and freshly isolated LNs of recipient
C57BL/6 mice.
[0081] For immune analysis, draining lymph nodes of mice receiving
ALG and rapamycin alone, and from mice treated with the vaccine
coding for BAX and SGAD55, were taken 2 weeks after
transplantation. Lymph nodes were cultured in the presence of
inactivated splenocytes from C57/BL6 (recipient), BALB/c (donor),
or DBA (third party) as sources of antigens, and CD4+CD25+ and
CD4+CD25- cells were isolated for proliferation assays.
[0082] Quantitative PCR analysis of expression of selected genes
was performed with skin allografts, cultured lymph nodes, and
CD4+CD25+/CD25- cells isolated from proliferation assays. In
addition to the Il-4, Il-10, Tgf-.beta.1, Tnf-.alpha., and
Ifn-.gamma. genes, we quantified the expression of genes coding for
the co-stimulatory molecules CD80 and CD86 found on APCs, the
transcriptional factor FOXP3 synthesized by regulatory T cells
(Tregs), and the inhibitory receptor Fc.gamma.RIIB and
IL-1-antagonist IL-1RA cytokine, which are both up-regulated in
murine tolerogenic DCs.
[0083] Total RNA was isolated using Trizol LS reagent (Invitrogen,
Cartsbad, Calif.) from freshly taken skin allograft and draining
LNs 2 weeks after transplantation. RNA was also isolated from LNs
cultured for 3 days in as described above. qPCR was performed using
the iCycler system and SYBR green (Bio Rad, Hercules, Calif.) with
200 ng of total RNA as template and primers specific for the chosen
cDNAs, and for the GAPDH cDNA as a housekeeping gene.
[0084] In the transplanted skin, the most striking differences
between the two vaccines were increased expression of the Il-4 and
Fc.gamma.rIIb genes and decreased expression of the Tnf-.alpha. and
Ifn-.gamma. genes in mice immunized with MsGAD-BAX (FIG. 3A). In
LNs, the most apparent differences were increased expression of the
Foxp3, Il-10, Tgf-.beta.1, and Cd86 genes in mice immunized with
BAX, and decreased expression of the Tnf-.alpha. and Ifn-.gamma.
genes in mice immunized with MsGAD-BAX (FIG. 3B). Increased
expression of Cd86 was also observed in mice immunized with
MsGAD-BAX, but to an extent that was proportional to the amount of
delivered Bax cDNA. No significant change in the expression of the
Cd80 gene was observed (data not shown). These data indicated that
the two vaccines induced clearly distinct immune responses.
[0085] Several pieces of evidence indicated that BAX and MsGAD-BAX
induced donor-specific immune responses. First, gene expression
analysis of LNs from C57BL/6 mice receiving BALB/c skin allografts
and cultured for 3 days revealed several significant differences
when cells were stimulated with self, donor, or third party
antigens (FIG. 3C). Compared to stimulation with third party
antigens, cells from mice immunized with BAX and MsGAD-BAX and
stimulated with donor antigens showed a significant change in
expression in 8 and 5 of the 9 chosen genes, respectively. In
contrast, compared to stimulation with self antigens, cells from
mice immunized with BAX and MsGAD-BAX and stimulated with donor
antigens showed change in expression in 4 and 2 of the 9 genes,
respectively. These results indicated that the gene expression
profile after donor antigen stimulation was markedly different from
the third party antigen profile and more similar to the self
antigen profile. In addition, the finding that genes like
Fc.gamma.rIIb and Il-1ra, which are up-regulated in tolerogenic
DCs, were induced only when stimulated with donor antigens
corroborated the notion of a donor-specific tolerogenic
response.
Example 4
Adoptive Cell Transfer
[0086] Cells from spleen and draining axillary and cervical lymph
nodes (LNs) were isolated 2 weeks post donor skin graft transplant
and suspended in PBS; 5.times.10.sup.6 total cells per recipient
were injected i.p. at day -2, and 3 Gy TBI irradiation was given on
day -3. Donor or third party back skin grafts were transplanted on
day 0, and rapamycin (1 mg/kg) was given i.p. daily.
[0087] Results from adoptive transfer experiments indicated that
splenocytes and LN cells from C57BL/6 mice receiving BALB/c skin
graft and immunized with MsGAD-BAX could transfer survival of
donor, but not third party graft (FIG. 4). In contrast, cells from
mice immunized with BAX did not significantly prevent rejection of
either donor or third party graft.
Example 5
MLR
[0088] LNs were dispersed and stimulated with mitomycin C-treated
splenocytes from C57BL/6, BALB/c, and C3H mice for 5 days with 2 or
5 .mu.g/ml IL-2 for MLR using 2 .mu.M CFSE-labeled LN cells.
Results from MLR using CSFE-labeled LN cells cultured with
inactivated splenocytes from donor and third party antigens also
indicated a donor-specific immune response for each vaccine. In the
presence of 2 .mu.g/ml IL-2, LN cells from non-vaccinated,
immunosuppressed mice did not show a significant difference in
suppression when stimulated with donor or third party antigens
(FIG. 5A). In contrast, LN cells from mice immunized with BAX or
MsGAD-BAX showed a significant difference in their response to
third party antigen compared to donor antigen. Culture with 5
.mu.g/ml IL-2 did not restore proliferation and accentuated these
differences (FIG. 5B).
CONCLUSION
[0089] Specific embodiments of the present invention described
herein address the problems of preventing rejection of allografts
without having to know the identity of the donor antigens, and of
reducing the need for immunosuppressants that are known to have
serious side-effects over the long term. Our results indicate that
"pro-apoptotic" DNA vaccination can be applied successfully to
solid organ transplantation. Injection of BAX DNA appears to induce
a donor-specific immunoregulatory response that contributes to
increased graft survival.
[0090] One explanation for the observed effects is that vaccination
with plasmid DNA coding for BAX induces apoptotic cells that
recruit antigen presenting cells. The antigen presenting cells
process the tolerogenic apoptotic cells together with donor
antigens and protects the allograft, most likely via multiple
immune mechanisms. However, considering that expression of Foxp3,
Il-10 and Tgf-.beta.1 was consistently higher in non-vaccinated,
immunosuppressed animals, other mechanisms of tolerance induced by
our DNA vaccination strategy are likely to play a role in
expressing graft survival. Unexpectedly, injection of
SssI-methylated coding for SGAD55 alone could prolong graft
survival. The same vaccine was ineffective for treatment of type
diabetes in our previous studies. Consequently, the underlying
mechanism for graft survival using this approach remains to be
determined.
[0091] Our data indicates that both the BAX and MsGAD-BAX vaccines
induced a donor-specific immune response, albeit via two different
mechanisms. The BAX vaccine caused changes in gene expression
mainly in fresh LNs, most likely because of the recruitment of APCs
to LNs after plasmid DNA-mediated induction of apoptosis. Increased
expression of Cd86 associated with delivery of the bax cDNA was not
linked with concomitant Cd80 expression, which indicated induction
of Cd86. The CD86 molecule is the main ligand for CD28 and promotes
inflammation. Significantly, LN cells of BAX-immunized mice showed
highly increased expression of the Tnf-.alpha. and Ifn-.gamma.
genes when stimulated with self antigens, which was not observed
with cells stimulated with donor antigens or with cells from mice
immunized with MsGAD-BAX and stimulated with self antigens. These
results suggest that induction of recipient apoptotic cells near
the allograft could have amplified the autoimmune response that is
known to be induced by a skin allograft via an indirect
alloresponse.
[0092] In addition, fresh LNs and LN cells from BAX-immunized mice
stimulated with third party antigen showed increased expression of
Foxp3, Il-10, and Tgf-.beta.1, which is associated with
CD4.sup.+CD25.sup.+ regulatory T cell activity. However, it is
unlikely that such cells were responsible alone for increased
allograft survival, because adoptive transfer of spleen and LN
cells from BAX-immunized mice did not prevent rejection of either
donor or third party allografts. Rather, these results suggested
that the BAX vaccine promoted graft survival via a different
mechanism, like clonal deletion. The lesser efficacy of the BAX
vaccine could have been the result of the autoimmune response it
induced. Nevertheless, it the efficacy of the vaccine may be
improved using CpG-methylation of plasmid DNA to lower inflammation
resulting from interaction between bacterial plasmid DNA and TLR-9,
and by injecting the DNA outside of the inflammatory milieu induced
by the allograft.
[0093] In contrast with BAX, the MsGAD-BAX vaccine induced
expression in the allograft of Il-4, which indicated Th2-like
activity, and of Fc.gamma.IIb, which is up-regulated in tolerogenic
DCs. Moreover, the MsGAD-BAX vaccine caused decreased expression of
the pro-inflammatory Tnf-.alpha. and Ifn-.gamma. genes in both
transplanted skin and fresh LNs, did not appear to promote
autoimmunity, and induced cells that transferred graft survival in
a donor-specific manner. Notably, GAD is found in skin, which we
confirmed in skin allografts using qPCR (data not shown), and is
up-regulated in inflamed tissues. Therefore, the sGAD polypeptide
may act as a regulatory autoantigen that prevents allograft
rejection. Indeed, the strongest evidence that autoimmunity plays a
role in allograft rejection comes from experiments where
administration of autoantigens present in the donor graft can
prevent rejection of lung or heart allografts, or accelerate
rejection when injected with Freund's adjuvant into recipient. Our
finding that Il-4 expression was increased in skin allograft
corroborates the notion that this cytokine can be a key element in
the process.
[0094] In conclusion, plasmid DNA-mediated induction of recipient
apoptotic cells and delivery of an allograft-associated autoantigen
form the basis of a new DNA vaccination strategy that targets
allograft-induced autoimmunity to prevent transplant rejection.
This approach, which is readily amenable to manipulation at the
molecular and cellular levels for further improvement, provides a
promising means to control chronic rejection in which
allograft-induced autoimmunity is thought to play a significant
role.
[0095] Although the present invention has been discussed in
considerable detail with reference to certain preferred
embodiments, other embodiments are possible. Therefore, the scope
of the appended claims should not be limited to the description of
preferred embodiments contained in this disclosure.
Sequence CWU 1
1
311638DNAHomo sapiens 1atgtacagga tgcaactcct gtcttgcatt gcactaagtc
ttgcacttgt cacaaacagt 60gcacctactt acgcgtttct ccatgcaaca gacctgctgc
cggcgtgtga tggagaaagg 120cccactttgg cgtttctgca agatgttatg
aacattttac ttcagtatgt ggtgaaaagt 180ttcgatagat caaccaaagt
gattgatttc cattatccta atgagcttct ccaagaatat 240aattgggaat
tggcagacca accacaaaat ttggaggaaa ttttgatgca ttgccaaaca
300actctaaaat atgcaattaa aacagggcat cctagatact tcaatcaact
ttctactggt 360ttggatatgg ttggattagc agcagactgg ctgacatcaa
cagcaaatac taacatgttc 420acctatgaaa ttgctccagt atttgtgctt
ttggaatatg tcacactaaa gaaaatgaga 480gaaatcattg gctggccagg
gggctctggc gatgggatat tttctcccgg tggcgccata 540tctaacatgt
atgccatgat gatcgcacgc tttaagatgt tcccagaagt caaggagaaa
600ggaatggctg ctcttcccag gctcattgcc ttcacgtctg aacatagtca
tttttctctc 660aagaagggag ctgcagcctt agggattgga agagacagcg
tgattctgat taaatgtgat 720gagagaggga aaatgattcc atctgatctt
gaaagaagga ttcttgaagc caaacagaaa 780gggtttgttc ctttcctcgt
gagtgccaca gctggaacca ccgtgtacgg agcatttgac 840cccctcttag
ctgtcgctga catttgcaaa aagtataaga tctggatgca tgtggatgca
900gcttggggtg ggggattact gatgtcccga aaacacaagt ggaaactgag
tggcgtggag 960agggccaact ctgtgacgtg gaatccacac aagatgatgg
gagtcccttt gcagtggtct 1020gctctcctgg ttagagaaga gggattgatg
cagaattgca accaaatgca tgcctcctac 1080ctctttcagc aagataaaca
ttatgacctg tcctatgaca ctggagacaa ggccttacag 1140tgcggacgcc
acgttgatgt ttttaaacta tggctgatgt ggagggcaaa ggggactacc
1200gggtttgaag cgcatgttga taaatgtttg gagttggcag agtatttata
caacatcata 1260aaaaaccgag aaggatatga gatggtgttt gatgggaagc
ctgaggacac aaatgtctgc 1320ttctggtaca ttcctccaag cttgcgtact
ctggaagaca atgaagagag aatgagtcgc 1380ctctcgaagg tggctccagt
gattaaagcc agaatgatgg agtatggaac cacaatggtc 1440agctaccaac
ccttgggaga caaggtcaat ttcttccgca tggtcatctc aaacccagcg
1500gcaactcacc aagacattga cttcctgatt gaagaaatag aacgccttgg
acaagattta 1560taataacctt gctcaccaag ctgttccact tctctaggta
gcgacctcga gcggccgctc 1620gagggggggc ccggtacc 16382579DNAHomo
sapiens 2atggacgggt ccggggagca gcccagaggc ggggggccca ccagctctga
gcagatcatg 60aagacagggg cccttttgct tcagggtttc atccaggatc gagcagggcg
aatggggggg 120gaggcacccg agctggccct ggacccggtg cctcaggatg
cgtccaccaa gaagctgagc 180gagtgtctca agcgcatcgg ggacgaactg
gacagtaaca tggagctgca gaggatgatt 240gccgccgtgg acacagactc
cccccgagag gtctttttcc gagtggcagc tgacatgttt 300tctgacggca
acttcaactg gggccgggtt gtcgcccttt tctactttgc cagcaaactg
360gtgctcaagg ccctgtgcac caaggtgccg gaactgatca gaaccatcat
gggctggaca 420ttggacttcc tccgggagcg gctgttgggc tggatccaag
accagggtgg ttgggacggc 480ctcctctcct actttgggac gcccacgtgg
cagaccgtga ccatctttgt ggcgggagtg 540ctcaccgcct cgctcaccat
ctggaagaag atgggctga 5793619DNAEncephalomyocarditis virus
3tctagataat acgactcact atagggcgaa ttccccctct ccctcccccc cccctaacgt
60tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
120catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
tcttgacgag 180cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
ggtctgttga atgtcgtgaa 240ggaagcagtt cctctggaag cttcttgaag
acaaacaacg tctgtagcga ccctttgcag 300gcagcggaac cccccacctg
gcgacaggtg cctctgcggc caaaagccag gtgtataaga 360tacacctgca
aaggcggcac aaccccagtg ccacgttgtg agttggaata gttgtggaaa
420gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
cagaaggtac 480cccattgtat gggatctgat ctggggcctc ggtgcacatg
ctttacatgt gtttagtcga 540ggttaaaaaa cgtctaggcc ccccaaccac
ggggacgtgg ttttcctttg aaaaacacga 600ttattatatt gcctctaga 619
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