U.S. patent application number 10/838659 was filed with the patent office on 2005-02-10 for vectors and methods for immunization or therapeutic protocols.
Invention is credited to Davis, Heather L., Krieg, Arthur M., Schorr, Joachim, Wu, Tong.
Application Number | 20050032734 10/838659 |
Document ID | / |
Family ID | 26724749 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032734 |
Kind Code |
A1 |
Krieg, Arthur M. ; et
al. |
February 10, 2005 |
Vectors and methods for immunization or therapeutic protocols
Abstract
The present invention shows that DNA vaccine vectors can be
improved by removal of CpG-N motifs and optional addition of CpG-S
motifs. In addition, for high and long-lasting levels of
expression, the optimized vector should include a promoter/enhancer
that is hot down-regulated by the cytokines induced by the
immunostimulatory CpG motifs. Vectors and methods of use for
immunostimulation are provided herein. The invention also provides
improved gene therapy vectors by determining the CpG-N and CpG-S
motifs present in the construct, removing stimulatory CpG (CpG-S)
motifs and/or inserting neutralizing CpG (CpG-N) motifs, thereby
producing a nucleic acid construct providing enhanced expression of
the therapeutic polypeptide. Methods of use for such vectors are
also included herein.
Inventors: |
Krieg, Arthur M.;
(Wellesley, MA) ; Davis, Heather L.; (Ottawa,
CA) ; Wu, Tong; (Hull, CA) ; Schorr,
Joachim; (Hilden, DE) |
Correspondence
Address: |
Patrick R.H. Waller, Ph.D.
Wolf, Greenfield & Sacks, P.C.
600 Atlantic Avenue
Boston
MA
02210
US
|
Family ID: |
26724749 |
Appl. No.: |
10/838659 |
Filed: |
May 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10838659 |
May 3, 2004 |
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09965101 |
Sep 26, 2001 |
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6821957 |
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09965101 |
Sep 26, 2001 |
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09082649 |
May 20, 1998 |
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6339068 |
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60047209 |
May 20, 1997 |
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60047233 |
May 20, 1997 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 39/292 20130101; A61K 39/12 20130101; A61K 39/39 20130101;
C12N 15/86 20130101; A61K 2039/53 20130101; C12N 2730/10134
20130101; A61K 2039/57 20130101; A61K 2039/55561 20130101; C12N
2710/10343 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 048/00 |
Claims
1-58. (Canceled)
59. A method for enhancing the expression of a polynucleotide in
vivo wherein the polynucleotide is contained in a nucleic acid
construct, the method comprising, determining the CpG-N and CpG-S
motifs present in the construct, removing stimulatory CpG (CpG-S)
motifs and/or inserting neutralizing CpG (CpG-N) motifs, thereby
producing a nucleic acid construct providing enhanced expression of
the polynucleotide.
60-82. (Canceled)
83. The method of claim 59, wherein the polynucleotide is an
antisense polynucleotide or a ribozyme.
84. A method for enhancing the expression of a polynucleotide in
vivo, the method comprising administering to a subject a nucleic
acid construct, wherein the construct is produced by determining
the CpG-N and CpG-S motifs present in the construct and removing
stimulatory CpG (CpG-S) motifs and/or inserting neutralizing CpG
(CpG-N) motifs, thereby enhancing expression of a polynucleotide
expressed by the nucleic acid construct in the subject.
85-107. (Canceled)
108. The method of claim 84, wherein the polynucleotide is an
antisense polynucleotide or a ribozyme.
Description
TECHNICAL FIELD
[0001] This invention relates generally to immune responses and
more particularly to vectors containing immunostimulatory CpG
motifs and/or a reduced number of neutralizing motifs and methods
of use for immunization purposes as well as vectors containing
neutralizing motifs and/or a reduced number of immunostimulatory
CpG motifs and methods of use for gene therapy protocols.
BACKGROUND
[0002] Bacterial DNA, but not vertebrate DNA, has direct
immunostimulatory effects on peripheral blood mononuclear cells
(PBMC) in vitro (Messina et al., J. Immunol. 147: 1759-1764, 1991;
Tokanuga et al., JNCI. 72: 955, 1994). These effects include
proliferation of almost all (>95%) B cells and increased
immunoglobulin (Ig) secretion (Krieg et al., Nature. 374: 546-549,
1995). In addition to its direct effects on B cells, CpG DNA also
directly activates monocytes, macrophages, and dendritic cells to
secrete predominantly Th 1 cytokines, including high levels of
IL-12 (Klinman, D., et al. Proc. Natl. Acad. Sci. USA. 93:
2879-2883 (1996); Halpern et al, 1996; Cowdery et al., J. Immunol.
156: 4570-4575 (1996). These cytokines stimulate natural killer
(NK) cells to secrete .gamma.-interferon (IFN-.gamma.) and to have
increased lytic activity (Klinman et al., 1996, supra; Cowdery et
al., 1996, supra; Yamamoto et al., J. Immunol. 148: 4072-4076
(1992); Ballas et al., J. Immunol. 157: 1840-1845 (1996)). These
stimulatory effects have been found to be due to the presence of
unmethylated CpG dinucleotides in a particular sequence context
(CpG-S motifs) (Krieg et al., 1995, supra). Activation may also be
triggered by addition of synthetic oligodeoxynucleotides (ODN) that
contain CpG-S motifs (Tokunaga et al., Jpn. J. Cancer Res. 79:
682-686 1988; Yi et al., J. Immunol. 156: 558-564, 1996; Davis et
al., J. Immunol. 160: 870-876, 1998).
[0003] Unmethylated CpG dinucleotides are present at the expected
frequency in bacterial DNA but are under-represented and methylated
in vertebrate DNA (Bird, Trends in Genetics. 3: 342-347, 1987).
Thus, vertebrate DNA essentially does not contain CpG stimulatory
(CpG-S) motifs and it appears likely that the rapid immune
activation in response to CpG-S DNA may have evolved as one
component of the innate immune defense mechanisms that recognize
structural patterns specific to microbial molecules.
[0004] Viruses have evolved a broad range of sophisticated
strategies for avoiding host immune defenses. For example, nearly
all DNA viruses and retroviruses appear to have escaped the defense
mechanism of the mammalian immune system to respond to
immunostimulatory CpG motifs. In most cases this has been
accomplished through reducing their genomic content of CpG
dinucleotides by 50-94% from that expected based on random base
usage (Karlin et al., J. Virol. 68: 2889-2897, 1994). CpG
suppression is absent from bacteriophage, indicating that it is not
an inevitable result of having a small genome. Statistical analysis
indicates that the CpG suppression in lentiviruses is an
evolutionary adaptation to replication in a eukaryotic host (Shaper
et al., Nucl. Acids Res. 18: 5793-5797, 1990).
[0005] Nearly all DNA viruses and retroviruses appear to have
evolved to avoid this defense mechanism through reducing their
genomic content of CpG dinucleotides by 50-94% from that expected
based on random base usage. CpG suppression is absent from
bacteriophage, indicating that it is not an inevitable result of
having a small genome. Statistical analysis indicates that the CpG
suppression in lentiviruses is an evolutionary adaptation to
replication in a eukaryotic host. Adenoviruses, however, are an
exception to this rule as they have the expected level of genomic
CpG dinucleotides. Different groups of adenovirae can have quite
different clinical characteristics. Serotype 2 and 5 adenoviruses
(Subgenus C) are endemic causes of upper respiratory infections and
are notable for their ability to establish persistent infections in
lymphocytes. These adenoviral serotypes are frequently modified by
deletion of early genes for use in gene therapy applications, where
a major clinical problem has been the frequent inflammatory immune
responses to the viral particles. Serotype 12 adenovirus (subgenus
A) does not establish latency, but can be oncogenic.
[0006] Despite high levels of unmethylated CpG dinucleotides,
serotype 2 adenoviral DNA surprisingly is nonstimulatory and can
actually inhibit activation by bacterial DNA. The arrangement and
flanking bases of the CpG dinucleotides are responsible for this
difference. Even though type 2 adenoviral DNA contains six times
the expected frequency of CpG dinucleotides, it has CpG-S motifs at
only one quarter of the frequency predicted by chance. Instead,
most CpG motifs are found in clusters of direct repeats or with a C
on the 5' side or a G on the 3' side. It appears that such CpG
motifs are immune-neutralizing (CpG-N) in that they block the
Th1-type immune activation by CpG-S motifs in vitro. Likewise, when
CpG-N ODN and CpG-S are administered with antigen, the
antigen-specific immune response is blunted compared to that with
CpG-S alone. When CpG-N ODN alone is administered in vivo with an
antigen, Th2-like antigen-specific immune responses are
induced.
[0007] B cell activation by CpG-S DNA is T cell independent and
antigen non-specific. However, B cell activation by low
concentrations of CpG DNA has strong synergy with signals delivered
through the B cell antigen receptor for both B cell proliferation
and Ig secretion (Krieg et al., 1995, sipra). This strong synergy
between the B cell signaling pathways triggered through the B cell
antigen receptor and by CpG-S DNA promotes antigen specific immune
responses. The strong direct effects (T cell independent) of CpG-S
DNA on B cells, as well as the induction of cytokines which could
have indirect effects on B-cells via T-help pathways, suggests
utility of CpG-S DNA as a vaccine adjuvant. This could be applied
either to classical antigen-based vaccines or to DNA vaccines.
CpG-S ODN have potent Th-1 like adjuvant effects with protein
antigens (Chu et al., J. Exp. Med 186: 1623-1631 1997; Lipford et
al., Eur. J. Immunol. 27: 2340-2344, 1997; Roman et al., Nature
Med. 3: 849-854, 1997; Weiner et al., Proc. Natl. Acad. Sci. USA.
94: 10833, 1997; Davis et al., 1998, supra, Moldoveanu et al., A
Novel Adjuvant for Systemic and Mucosal Immunization with Influenza
Virus. Vaccine (in press) 1998).
SUMMARY OF THE INVENTION
[0008] The present invention is based on the discovery that removal
of neutralizing motifs (e.g., CpG-N or poly G) from a vector used
for immunization purposes, results in an antigen-specific
immunostimulatory effect greater than with the starting vector.
Further, when neutralizing motifs (e.g., CpG-N or poly G) are
removed from the vector and stimulatory CpG-S motifs are inserted
into the vector, the vector has even more enhanced
immunostimulatory efficacy.
[0009] In a first embodiment, the invention provides a method for
enhancing the immunostimulatory effect of an antigen encoded by
nucleic acid contained in a nucleic acid construct including
determining the CpG-N and CpG-S motifs present in the construct and
removing neutralizing CpG (CpG-N) motifs and optionally inserting
stimulatory CpG (CpG-S) motifs in the construct, thereby producing
a nucleic acid construct having enhanced immunostimulatory
efficacy. Preferably, the CpG-S motifs in the construct include a
motif having the formula 5' X.sub.1CGX.sub.2 3' wherein at least
one nucleotide separates consecutive CpGs, X.sub.1 is adenine,
guanine, or thymine and X.sub.2 is cytosine, thymine, or
adenine.
[0010] In another embodiment, the invention provides a method for
stimulating a protective or therapeutic immune response in a
subject. The method includes administering to the subject an
effective amount of a nucleic acid construct produced by
determining the CpG-N and CpG-S motifs present in the construct and
removing neutralizing CpG (CpG-N) motifs and optionally inserting
stimulatory CpG (CpG-S) motifs in the construct, thereby producing
a nucleic acid construct having enhanced immunostimulatory efficacy
and stimulating a protective or therapeutic immune response in the
subject. Preferably, the nucleic acid construct contains a promoter
that functions in eukaryotic cells and a nucleic acid sequence that
encodes an antigen to which the immune response is direct toward.
Alternatively, an antigen can be admininstered simulataneously
(e.g., admixture) with the nucleic acid construct.
[0011] In another embodiment, the invention provides a method for
enhancing the expression of a therapeutic polypeptide in vivo
wherein the polypeptide is encoded by a nucleic acid contained in a
nucleic acid construct. The method includes determining the CpG-N
and CpG-S motifs present in the construct, optionally removing
stimulatory CpG (CpG-S) motifs and/or inserting neutralizing CpG
(CpG-N) motifs, thereby producing a nucleic acid construct
providing enhanced expression of the therapeutic polypeptide.
[0012] In yet another embodiment, the invention provides a method
for enhancing the expression of a therapeutic polypeptide in vivo.
The method includes administering to a subject a nucleic acid
construct, wherein the construct is produced by determining the
CpG-N and CpG-S motifs present in the construct and optionally
removing stimulatory CpG (CpG-S) motifs and/or inserting
neutralizing CpG (CpG-N) motifs, thereby enhancing expression of
the therapeutic polypeptide in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram of the construction of
pUK21-Al.
[0014] FIG. 2 is a schematic diagram of the construction of
pUK21-A2.
[0015] FIG. 3 is a schematic diagram of the construction of
pUK21-A.
[0016] FIG. 4 is a schematic diagram of the construction of
pMAS.
[0017] FIG. 5 is a diagram of DNA vector pMAS. The following
features are contained within pMAS. CMV promoter which drives
expression of inserted genes in eukaryotic cells. BGH polyA for
polyadenylation of transcribed mRNAs. ColE1 origin of replication
for high copy number growth in E. coli. Kanamycin resistance gene
for selection in E. coli. Polylinker for gene cloning. Unique
restriction enzyme sites DraI-BstRI-ScaI-AvaII-HpaII for inserting
immune stimulatory sequences.
[0018] FIG. 6 shows the effect of adding S-ODN to plasmid DNA
expressing reporter gene or antigen. ODN 1826 (10 or 100 .mu.g) was
added to DNA constructs (10 .mu.g) encoding hepatitis B surface
antigen (HBsAg) (pCMV-S, FIG. 6A) or luciferase (pCMV-luc, FIG. 6B)
DNA prior to intramuscular (IM) injection into mice. There was an
ODN dose-dependent reduction in the induction of antibodies against
HBsAg (anti-HBs, end-point dilution titers at 4 wk) by the pCMV-S
DNA (FIG. 6A) and in the amount of luciferase expressed in relative
light units per sec per mg protein (RLU/sec/mg protein at 3 days)
from the pCMV-luc DNA (FIG. 6B). This suggests that the lower
humoral response with DNA vaccine plus ODN was due to decreased
antigen expression. Each bar represents the mean of values derived
from 10 animals (FIG. 6A) or 10 muscles (FIG. 6B) ands vertical
lines represent the SEM. Numbers superimposed on the bars indicate
proportion of animals responding to the DNA vaccine (FIG. 6A); all
muscles injected with pCMV-luc expressed luciferase (FIG. 6B).
[0019] FIG. 7 shows the interference of ODN with plasmid DNA
depends on backbone and sequence. Luciferase activity (RLU/sec/mg
protein) in mouse muscles 3 days after they were injected with 10
.mu.g pCMV-luc DNA to which had been added no ODN (none=white bar)
or 100 .mu.g of an ODN, which had one of three backbones:
phosphorothioate (S=black bars: 1628, 1826, 1911, 1982, 2001 and
2017), phosphodiester (O=pale grey bar: 2061), or a
phosphorothioate-phosphodiester chimera (SOS=dark grey bars: 1585,
1844, 1972, 1980, 1981, 2018, 2021, 2022, 2023 and 2042). Three
S-ODN (1911, 1982 and 2017) and two SOS-ODN (1972 and 2042) did not
contain any immunostimulatory CpG motifs. One S-ODN (1628) and
three SOS-ODN (1585, 1972, 1981) had poly-G ends and one SOS-ODN
(2042) had a poly-G center. The (*) indicates ODN of identical
sequence but different backbone: 1826 (S-ODN), 1980 (SOS-ODN) and
2061 (O-ODN). All S-ODN (both CpG and non-CpG) resulted in
decreased luciferase activity whereas SOS-ODN did not unless they
had poly-G sequences.
[0020] FIG. 8 shows the effect of temporal or spatial separation of
plasmid DNA and S-ODN on gene expression. Luciferase activity
(RLU/sec/mg protein) in mouse muscles 3 or 14 days after they were
injected with 10 .mu.g pCMV-luc DNA. Some animals also received 10
.mu.g CpG-S ODN which was mixed with the DNA vaccine or was given
at the same time but at a different site, or was given 4 days prior
to or 7 days after the DNA vaccine. Only when the ODN was mixed
directly with the DNA vaccine did it interfere with gene
expression.
[0021] FIG. 9 shows the enhancement of in-vivo immune effects with
optimized DNA vaccines. Mice were injected with 10 .mu.g of pUK-S
(black bars), pMAS-S (white bars), pMCG16-S (pale grey bars) or
pMCG50-S (dark grey bars) plasmid DNA bilaterally (50 .mu.l at 0.1
mg/ml in saline) into the TA muscle. FIG. 9A shows the anti-HBs
antibody response at 6 weeks (detected as described in methods).
Bars represent the group means (n=5) for ELISA end-point dilution
titers (performed in triplicate), and vertical lines represent the
standard errors of the mean. The numbers on the bars indicate the
ratio of IgG2a:IgG1 antibodies at 4 weeks, as determined in
separate assays (also in triplicate) using pooled plasma. FIG. 9B
shows the cytotoxic T lymphocyte activity in specifically
restimulated (5 d) splenocytes taken from mice 8 wk after DNA
immunization. Bars represent the group means (n=3) for % specific
lysis (performed in triplicate) at an effector:target (E:T) ratio
of 10:1, dots represent the individual values. Non-specific lytic
activity determined with non-antigen-presenting target cells, which
never exceeds 10%, has been subtracted from values with
HBsAg-expressing target cells to obtain % specific lysis
values.
[0022] FIG. 10 shows induction of a Th2-like response by a CpG-N
motif and inhibition of the Th1-like response induced by a CpG-S
motif. Anti-HBs antibody titers (IgG1 and IgG-2a subclasses) in
BALB/c mice 12 weeks after IM immunization with recombinant HBsAg,
which was given alone (none) or with 10 .mu.g stimulatory ODN
(1826), 10 ug of neutralizing ODN (1631, CCGCGCGCGCGCGCGCGCG; 1984,
TCCATGCCGTTCCTGCCGTT; or 2010 GCGGCGGGCGGCGCGCGCCC; CpG
dinucleotides are underlined for clarity) or with 10 .mu.g
stimulatory ODN+10 .mu.g neutralizing ODN. To improve nuclease
resistance for these in vivo experiments, all ODN were
phosphorothioate-modified. Each bar represents the group mean (n=10
for none; n=15 for #1826 and n=5 for all other groups) for anti-HBs
antibody titers as determined by end-point dilution ELISA assay.
Black portions of bars indicate antibodies of IgG1 subclass
(Th2-like) and grey portions indicate IgG2a subclass (Th1-like).
The numbers above each bar indicate the IgG2a/IgG1 ratio where a
ratio>1 than indicates a predominantly Th1-like response and a
ratio<1 indicates a predominantly Th2-like response (a value of
0 indicates a complete absence of IgG2a antibodies).
[0023] FIG. 11 shows enhancement of in vivo-immune effects with
optimized DNA vaccines. Mice were injected with 10 .mu.g of pUK-S
(black bars), pMAS-S (white bars), pMCG16-S (pale grey bars) or
pMCG50-S (dark grey bars) plasmid DNA bilaterally (50 .mu.l at 0.1
mg/ml in saline) into the TA muscle. Panel A: The anti-HBs antibody
response at 6 weeks (detected as described in methods). Bars
represent the group means (n=5) for ELISA end-point dilution titers
(performed in triplicate), and vertical lines represent the
standard errors of the mean. The numbers on the bars indicate the
ratio of IgG2a:IgG1 antibodies at 4 weeks, as determined in
separate assays (also in triplicate) using pooled plasma. Panel B:
Cytotoxic T lymphocyte activity in specifically restimulated (5 d)
splenocytes taken from mice 8 wk after DNA immunization. Bars
represent the group means (n=3) for % specific lysis (performed in
triplicate) at an effector:target (E:T) ratio of 10:1, dots
represent the individual values. Non-specific lytic activity
determined with non-antigen-presenting target cells, which never
exceeds 10%, has been subtracted from values with HBsAg-expressing
target cells to obtain % specific lysis values.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides vectors for immunization or
therapeutic purposes based on the presence or absence of CpG
dinucleotide immunomodulating motifs. For immunization purposes,
immunostimulatory motifs (CpG-S) are desirable while
immunoinhibitory CpG motifs (CpG-N) are undesirable, whereas for
gene therapy purposes, CpG-N are desirable and CpG-S are
undesirable. Plasmid DNA expression cassettes were designed using
CpG-S and CpG-N motifs. In the case of DNA vaccines, removal of
CpG-N motifs and addition of CpG-S motifs should allow induction of
a more potent and appropriately directed immune response. The
opposite approach with gene therapy vectors, namely the removal of
CpG-S motifs and addition of CpG-N motifs, allows longer lasting
therapeutic effects by abrogating immune responses against the
expressed protein.
[0025] DNA Vaccines
[0026] DNA vaccines have been found to induce potent humoral and
cell-mediated immune responses. These are frequently Th1-like,
especially when the DNA is administered by intramuscular injection
(Davis, H. L. (1998) Gene-based Vaccines. In: Advanced Gene
Delivery: From Concepts to Pharmaceutical Products (Ed. A.
Rolland), Harwood Academic Publishers (in press); Donnelly et al.,
Life Sciences 60:163, 1997; Donnelly et al., Ann Rev. Immunol.
15:617, 1997; Sato et al., Science 273:352, 1996). Most DNA
vaccines comprise antigen-expressing plasmid DNA vectors. Since
such plasmids are produced in bacteria and then purified, they
usually contain several unmethylated immunostimulatory CpG-S
motifs. There is now convincing evidence that the presence of such
motifs is essential for the induction of immune responses with DNA
vaccines (see Krieg et al., Trends Microbiology. 6: 23-27, 1998).
For example, it has been shown that removal or methylation of
potent CpG-S sequences from plasmid DNA vectors reduced or
abolished the in vitro production of Th1 cytokines (e.g., IL-12,
IFN-.alpha., IFN-.gamma.) from monocytes and the in vivo antibody
and CTL response against an encoded antigen (.beta.-galactosidase)
(Sato et al., 1996, supra; Klinman et al., J. Immunol. 158:
3635-3639 (1997). Potent responses could be restored by cloning
CpG-S motifs back into the vectors (Sato et al., 1996, supra) or by
coadministering CpG-S ODN (Klinman et al., 1997, supra). The
humoral response in monkeys to a DNA vaccine can also be augmented
by the addition of E. coli DNA (Gramzinski et al., Molec. Med. 4:
109-119, 1998). It has also been shown that the strong Th1 cytokine
pattern induced by DNA vaccines can be obtained with a protein
vaccine by the coadministration of empty plasmid vectors (Leclerc
et al., Cell Immunology. 170: 97-106, 1997).
[0027] The present invention shows that DNA vaccine vectors can be
improved by removal of CpG-N motifs and further improved by the
addition of CpG-S motifs. In addition, for high and long-lasting
levels of expression, the optimized vector should preferably
include a promoter/enhancer, which is not down-regulated by the
cytokines induced by the immunostimulatory CpG motifs.
[0028] It has been shown that the presence of unmethylated CpG
motifs in the DNA vaccines is essential for the induction of immune
responses against the antigen, which is expressed only in very
small quantities (Sato et al., 1996, Klinman et al., 1997, supra).
As such, the DNA vaccine provides its own adjuvant in the form of
CpG DNA. Since single-stranded but not double-stranded DNA can
induce immunostimulation in vitro, the CpG adjuvant effect of DNA
vaccines in vivo is likely due to oligonucleotides resulting from
plasmid degradation by nucleases. Only a small portion of the
plasmid DNA injected into a muscle actually enters a cell and is
expressed; the majority of the plasmid is degraded in the
extracellular space.
[0029] The present invention provides DNA vaccine vectors further
improved by removal of undesirable immunoinhibitory CpG motifs and
addition of appropriate CpG immunostimulatory sequences in the
appropriate number and spacing. The correct choice of
immunostimulatory CpG motifs could allow one to preferentially
augment humoral or CTL responses, or to preferentially induce
certain cytokines.
[0030] The optimized plasmid cassettes of the invention are ready
to receive genes encoding any particular antigen or group of
antigens or antigenic epitopes. One of skill in the art can create
cassettes to preferentially induce certain types of immunity, and
the choice of which cassette to use would depend on the disease to
be immunized against.
[0031] The exact immunostimulatory CpG motif(s) to be added will
depend on the ultimate purpose of the vector. If it is to be used
for prophylactic vaccination, preferable motifs stimulate humoral
and/or cell-mediated immunity, depending on what would be most
protective for the disease in question. It the DNA vaccine is for
therapeutic purposes, such as for the treatment of a chronic viral
infection, then motifs which preferentially induce cell-mediated
immunity and/or a particular cytokine profile is added to the
cassette.
[0032] The choice of motifs also depends on the species to be
immunized as different motifs are optimal in different species.
Thus, there would be one set of cassettes for humans as well as
cassettes for different companion and food-source animals which
receive veterinary vaccination. There is a very strong correlation
between certain in vitro immunostimulatory effects and in vivo
adjuvant effect of specific CpG motifs. For example, the strength
of the humoral response correlates very well (r>0.9) with the in
vitro induction of TNF-.alpha., IL-6, IL-12 and B-cell
proliferation. On the other hand, the strength of the cytotoxic
T-cell response correlates well with in vitro induction of
IFN-.gamma..
[0033] Since the entire purpose of DNA vaccines is to enhance
immune responses, which necessarily includes cytokines, the
preferred promoter is not down-regulated by cytokines. For example,
the CMV immediate-early promoter/enhancer, which is used in almost
all DNA vaccines today, is turned off by IFN-.alpha. and
IFN-.gamma. (Gribaudo et al., Virology. 197: 303-311, 1993; Harms
& Splitter, Human Gene Ther. 6: 1291-1297, 1995; Xiang et al.,
Vaccine, 15: 896-898, 1997). Another example is the down-regulation
of a hepatitis B viral promoter in the liver of HBsAg-expressing
transgenic mice by IFN-.gamma. and TNF-.alpha. (Guidotti et al.,
Proc. Natl. Acad. Sci. USA. 91: 3764-3768, 1994).
[0034] Nevertheless, such viral promoters may still be used for DNA
vaccines as they are very strong, they work in several cell types,
and despite the possibility of promoter turn-off, the duration of
expression with these promoters has been shown to be sufficient for
use in DNA vaccines (Davis et al., Human Molec. Genetics. 2:
1847-1851, 1993). The use of CpG-optimized DNA vaccine vectors
could improve immune responses to antigen expressed for a limited
duration, as with these viral promoters. When a strong viral
promoter is desired, down-regulation of expression may be avoidable
by choosing CpG-S motfis that do not induce the cytokine(s) that
affect the promoter (Harms and Splitter, 1995 supra).
[0035] Other preferable promoters for use as described herein are
eukaryotic promoters. Such promoters can be cell- or
tissue-specific. Preferred cells/tissues for high antigen
expression are those which can act as professional antigen
presenting cells (APC) (e.g., macrophages, dendritic cells), since
these have been shown to be the only cell types that can induce
immune responses following DNA-based immunization (Ulmer et al.,
1996; Corr et al., J. Exp. Med., 184,1555-1560, 1996; Doe et al.,
Proc. Natl. Acad. Sci. USA, 93, 8578-8583, 1996; Iwasaki et al., J.
Immunol., 159: 11-141998). Examples of such a promoter are the
mammalian MHC I or MHC II promoters.
[0036] The invention also includes the use of a promoter whose
expression is up-regulated by cytokines. An example of this is the
mammalian MHC I promoter that has the additional advantage of
expressing in APC, which as discussed above is highly desirable.
This promoter has also been shown to have enhanced expression with
IFN-.gamma. (Harms & Splitter, 1995, supra).
[0037] After intramuscular injection of DNA vaccines, muscle fibers
may be efficiently transfected and produce a relatively large
amount of antigen that may be secreted or otherwise released (e.g.,
by cytolytic attack on the antigen-expressing muscle fibers) (Davis
et al., Current Opinions Biotech. 8: 635-640, 1997). Even though
antigen-expressing muscle fibers do not appear to induce immune
responses from the point of view of antigen presentation, B-cells
must meet circulating antigen to be activated, it is possible that
antibody responses are augmented by antigen secreted or otherwise
released from other cell types (e.g., myofibers, keratinocytes).
This may be particularly true for conformational B-cell epitopes,
which would not be conserved by peptides presented on APC. For this
purpose, expression in muscle tissue is particularly desirable
since myofibers are post-mitotic and the vector will not be lost
through cell-division, thus antigen expression can continue until
the antigen-expressing cell is destroyed by an immune repsonse
against it. Thus, when strong humoral responses are desired, other
preferred promoters are strong muscle-specific promoters such as
the human muscle-specific creatine kinase promoter (Bartlett et
al., 1996) and the rabbit .beta.-cardiac myosin heavy chain
(full-length or truncated to 781 bp) plus the rat myosin light
chain 1/3 enhancer.
[0038] In the case of DNA vaccines with muscle- or other non-APC
tissue-specific promoters, it may be preferable to administer it in
conjunction with a DNA vaccine encoding the same antigen but under
the control of a promoter that will work strongly in APC (e.g.,
viral promoter or tissue specific for APC). In this way, optimal
immune responses can be obtained by having good antigen
presentation as well as sufficient antigen load to stimulate
B-cells. A hybrid construct, such as the .beta.-actin promoter with
the CMV enhancer (Niwa et al, Gene. 108: 193-199, 1991) is also
desirable to circumvent some of the problems of strictly viral
promoters.
[0039] While DNA vaccine vectors may include a signal sequence to
direct secretion, humoral and cell-mediated responses are possible
even when the antigen is not secreted. For example, it has been
found in mice immunized with hepatitis B surface antigen
(HBsAg)-expressing DNA that the appearance of anti-HBs antibodies
is delayed for a few weeks if the HBsAg is not secreted (Michel et
al., 1995). As well, antibodies are induced in rabbits following IM
immunization with DNA containing the gene for cottontail rabbit
papilloma virus major capsid protein (L1), which has a nuclear
localization signal (Donnelly et al., 1996). In these cases, the
B-cells may not be fully activated until the expressed antigen is
released from transfected muscle (or other) cells upon lysis by
antigen-specific CTL.
[0040] Preferably, the CpG-S motifs in the construct include a
motif having the formula:
5' X.sub.1CGX.sub.2 3'
[0041] wherein at least one nucleotide separates consecutive CpGs,
X.sub.1 is adenine, guanine, or thymine and X.sub.2 is cytosine,
thymine, or adenine. Exemplary CpG-S oligonucleotide motifs include
GACGTT, AGCGTT, AACGC.sub.1T, GTCGTT and AACGAT. Another
oligonucleotide useful in the construct contains TCAACGTT. Further
exemplary oligonucleotides of the invention contain GTCG(T/C)T,
TGACGTT, TGTCG(T/C)T, TCCATGTCGTTCCTGTCGTT (SEQ ID NO:1),
TCCTGACGTTCCTGACGTT (SEQ ID NO:2) and TCGTCGTTTTGTCGTTTTGTCGTT (SEQ
ID NO:3).
[0042] Preferably CpG-N motifs contain direct repeats of CpG
dinucleotides, CCG trinucleotides, CGG trinucleotides, CCGG
tetranucleotides, CGCG tetranucleotides or a combination of any of
these motifs. In addition, the neutralizing motifs of the invention
may include oligos that contain a sequence motif that is a poly-G
motif, which may contain at least about four Gs in a row or two G
trimers, for example (Yaswen et al., Antisense Research and
Development 3:67, 1993; Burgess et al., PNAS 92:4051, 1995).
[0043] In the present invention, the nucleic acid construct is
preferably an expression vector. The term "expression vector"
refers to a plasmid, virus or other vehicle known in the art that
has been manipulated by insertion or incorporation of genetic
coding sequences. Polynucleotide sequence which encode polypeptides
can be operatively linked to expression control sequences.
[0044] "Operatively linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. An expression control sequence
operatively linked to a coding sequence is ligated such that
expression of the coding sequence is achieved under conditions
compatible with the expression control sequences. As used herein,
the term "expression control sequences" refers to nucleic acid
sequences that regulate the expression of a nucleic acid sequence
to which it is operatively linked. Expression control sequences are
operatively linked to a nucleic acid sequence when the expression
control sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signal for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons.
[0045] The term "control sequences" is intended to include, at a
minimum, components whose presence can influence expression, and
can also include additional components whose presence is
advantageous, for example, leader sequences and fusion partner
sequences. Expression control sequences can include a promoter.
[0046] The nucleic acid construct of the invention may include any
of a number of suitable transcription and translation elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, etc. may be used in
the expression vector (see e.g., Bitter et al., 1987, Methods in
Enzymology 153:516-544). When cloning in mammalian cell systems,
promoters derived from the genome of mammalian cells (e.g.
metallothionein promoter) or from mammalian viruses (e.g., the
retrovirus long terminal repeat; the adenoviral late promoter; the
vaccinia virus 7.5K promoter) may be used. Promoters produced by
recombinant DNA or synthetic techniques may also be used to provide
for transcription of the inserted polypeptide coding sequence.
[0047] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the polypeptide coding
sequence may be ligated to an adenovirus transcription/translation
control complex, e.g., the late promoter and tripartite leader
sequence. Alternatively, the vaccinia virus 7.5K promoter may be
used. (e.g., see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA
79: 7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864;
Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931).
Of particular interest are vectors based on bovine papilloma virus
which have the ability to replicate as extrachromosomal elements
(Sarver, et al., 1981, Mol. Cell. Biol. 1: 486). Shortly after
entry of this DNA into mouse cells, the plasmid replicates to about
100 to 200 copies per cell. Transcription of the inserted cDNA does
not require integration of the plasmid into the host's chromosome,
thereby yielding a high level of expression. These vectors can be
used for stable expression by including a selectable marker in the
plasmid, such as, for example, the neo gene. Alternatively, the
retroviral genome can be modified for use as a vector capable of
introducing and directing the expression of the gene of interest in
host cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci. USA
81:6349-6353). High level expression may also be achieved using
inducible promoters, including, but not limited to, the
metallothionine IIA promoter and heat shock promoters.
[0048] The polypeptide that acts as an antigen in the methods
described herein refers to an immunogenic polypeptide antigen,
group of antigens or peptides encoding particular epitopes.
[0049] A polynucleotide encoding such antigen(s) is inserted into
the nucleic acid construct as described herein. For example, a
nucleic acid sequence encoding an antigenic polypeptide derived
from a virus, such as Hepatitis B virus (HBV) (e.g., HBV surface
antigen), an antigen derived from a parasite, from a tumor, or a
bacterial antigen, is cloned into the nucleic acid construct
described herein. Virtually any antigen, groups of antigens, or
antigenic epitopes, can be used in the construct. Other antigens,
such as peptides that mimic nonpeptide antigens, such as
polysaccharides, are included in the invention.
[0050] Gene transfer into eukaryotic cells can be carried out by
direct (in vivo) or indirect (in vitro or ex vivo) means (Miller et
al., A. D. Nature. 357: 455-460, 1992). The DNA vector can also be
transferred in various forms and formulations. For example, pure
plasmid DNA in an aqueous solution (also called "naked" DNA) can be
delivered by direct gene transfer. Plasmid DNA can also be
formulated with cationic and neutral lipids (liposomes)
(Gregoriadis et al, 1996), microencapsulated (Mathiowitz et al.,
1997), or encochleated (Mannino and Gould Fogerite, 1995) for
either direct or indirect delivery. The DNA sequences can also be
contained within a viral (e.g., adenoviral, retroviral, herpesvius,
pox virus) vector, which can be used for either direct or indirect
delivery.
[0051] DNA vaccines will preferably be administered by direct (in
vivo) gene transfer. Naked DNA can be give by intramuscular (Davis
et al., 1993), intradermal (Raz et al., 1994; Condon et al., 1996;
Gramzinski et al., 1998), subcutaneous, intravenous (Yokoyama et
al., 1996; Liu et al., 1997), intraarterial (Nabel et al., 1993) or
buccal injection (Etchart et al., 1997; Hinkula et al., 1997).
Plasmid DNA may be coated onto gold particles and introduced
biolistically with a "gene-gun" into the epidermis if the skin or
the oral or vaginal mucosae (Fynan et al. Proc. Natl. Acad. Sci.
USA 90:11478, 1993; Tang et al, Nature 356:152, 1992; Fuller, et
al., J. Med. Primatol. 25:236, 1996; Keller et al., Cancer Gene
Ther., 3:186, 1996). DNA vaccine vectors may also be used in
conjunction with various delivery systems. Liposomes have been used
to deliver DNA vaccines by intramuscular injection (Gregoriadis et
al., FEBS Lett. 402:107, 1997) or into the respiratory system by
non-invasive means such as intranasal inhalation (Fynan et al.,
supra). Other potential delivery systems include microencapsulation
(Jones et al., 1998; Mathiowitz et al., 0.1997) or cochleates
(Mannino et al., 1995, Lipid matrix-based vaccines for mucosal and
systemic immunization. Vaccine Designs: The Subunit and Adjuvant
Approach, M. F. Powell and M. J. Newman, eds., Pleum Press, New
York, 363-387), which can be used for parenteral, intranasal (e.g.,
nasal spray) or oral (e.g. liquid, gelatin capsule, solid in food)
delivery. DNA vaccines can also be injected directly into tumors or
directly into lymphoid tissues (e.g., Peyer's patches in the gut
wall). It is also possible to formulate the vector to target
delivery to certain cell types, for example to APC. Targeting to
APC such as dendritic cells is possible through atachment of a
mannose moiety (dendritic cells have a high density of mannose
receptors) or a ligand for one of the other receptors found
preferentially on APC. There is no limitation as to the route that
the DNA vaccine is delivered, nor the manner in which it is
formulated as long as the cells that are transfected can express
antigen in such a way that an immune response is induced.
[0052] It some cases it may be desirable to carry out ex-vivo gene
transfer, in which case a number a methods are possible including
physical methods such as microinjection, electroportion or calcium
phosphate precipitation, or facilitated transfer methods such as
liposomes or dendrimers, or through the use of viral vectors. In
this case, the transfected cells would be subsequently administered
to the subject so that the antigen they expressed could induce an
immune response.
[0053] Nucleotide sequences in the nucleic acid construct can be
intentionally manipulated to produce CpG-S sequences or to reduce
the number of CpG-N sequences for immunization vectors. For
example, site-directed mutagenesis can be utilized to produce a
desired CpG motif. Alternatively, a particular CpG motif can be
synthesized and inserted into the nucleic acid construct. Further,
one of skill in the art can produce double-stranded CpG oligos that
have self-complementary ends that can be ligated together to form
long chains or concatemers that can be ligated into a plasmid, for
example. It will be apparent that the number of CpG motifs or
CpG-containing oligos that can be concatenated will depend on the
length of the individual oligos and can be readily determined by
those of skill in the art without undue experimentation. After
formation of concatemers, multiple oligos can be cloned into a
vector for use in the methods of the invention.
[0054] In one embodiment, the invention provides a method for
stimulating a protective immune response in a subject. The method
includes administering to the subject an immunomostimulatory
effective amount of a nucleic acid construct produced by removing
neutralizing CpG (CpG-N) motifs and optionally inserting
stimulatory CpG (CpG-S) motifs, thereby producing a nucleic acid
construct having enhanced immunostimulatory efficacy and
stimulating a protective immune response in the subject. The
construct typically further includes regulatory sequences for
expression of DNA in eukaryotic cells and nucleic acid sequences
encoding at least one polypeptide.
[0055] It is envisioned that methods of the present invention can
be used to prevent or treat bacterial, viral, parasitic or other
disease states, including tumors, in a subject. The subject can be
a human or may be a non-human such as a pig, cow, sheep, horse,
dog, cat, fish, chicken, for example. Generally, the terms
"treating," "treatment," and the like are used herein to mean
obtaining a desired pharmacologic and/or physiologic effect. The
effect may be prophylactic in terms of completely or partially
preventing a particular infection or disease (e.g., bacterial,
viral or parasitic disease or cancer) or sign or symptom thereof,
and/or may be therapeutic in terms of a partial or complete cure
for an infection or disease and/or adverse effect attributable to
the infection or disease. "Treating" as used herein covers any
treatment of (e.g., complete or partial), or prevention of, an
infection or disease in a non-human, such as a mammal, or more
particularly a human, and includes:
[0056] (a) preventing the disease from occurring in a subject that
may be at risk of becoming infected by a pathogen or that may be
predisposed to a disease (e.g., cancer) but has not yet been
diagnosed as having it;
[0057] (b) inhibiting the infection or disease, i.e., arresting its
development; or
[0058] (c) relieving or ameliorating the infection or disease,
i.e., cause regression of the infection or disease.
[0059] Delivery of polynucleotides can be achieved using a plasmid
vector as described herein, that can be administered as "naked DNA"
(i.e., in an aqueous solution), formulated with a delivery system
(e.g. liposome, cochelates, microencapsulated), or coated onto gold
particles. Delivery of polynucleotides can also be achieved using
recombinant expression vectors such as a chimeric virus. Thus the
invention includes a nucleic acid construct as described herein as
a pharmaceutical composition useful for allowing transfection of
some cells with the DNA vector such that antigen will be expressed
and induce a protective (to prevent infection) or a therapeutic (to
ameliorate symptoms attributable to infection or disease) immune
response. The pharmaceutical compositions according to the
invention are prepared by bringing the construct according to the
present invention into a form suitable for administration to a
subject using solvents, carriers, delivery systems, excipients, and
additives or auxiliaries. Frequently used solvents include sterile
water and saline (buffered or not). A frequently used carrier
includes gold particles, which are delivered biolistically (i.e.,
under gas pressure). Other frequently used carriers or delivery
systems include cationic liposomes, cochleates and microcapsules,
which may be given as a liquid, solution, enclosed within a
delivery capsule or incorporated into food.
[0060] The pharmaceutical compositions are preferably prepared and
administered in dose units. Liquid dose units would be injectable
solutions or nasal sprays or liquids to be instilled (e.g., into
the vagina) or swallowed or applied onto the skin (e.g., with
allergy tines, with tattoo needles or with a dermal patch). Solid
dose units would be DNA-coated gold particles, creams applied to
the skin or formulations incorporated into food or capsules or
embedded under the skin or mucosae or pressed into the skin (e.g.,
with allergy tines). Different doses will be required depending on
the activity of the compound, form and formulation, manner of
administration, and age or size of patient (i.e., pediatric versus
adult), purpose (prophylactic vs therapeutic). Doses will be given
at appropriate intervals, separated by weeks or months, depending
on the application. Under certain circumstances higher or lower, or
more frequent or less frequent doses may be appropriate. The
administration of a dose at a single time point may be carried out
as a single administration or a multiple administration (e.g.,
several sites with gene-gun or for intradermal injection or
different routes).
[0061] Whether the pharmaceutical composition is delivered locally
or systemically, it will induce systemic immune responses. By
"therapeutically effective dose" is meant the quantity of a vector
or construct according to the invention necessary to induce an
immune response that can prevent, cure, or at least partially
arrest the symptoms of the disease and its complications. Amounts
effective for this will of course depend on the mode of
administration, the age of the patient (pediatric versus adult) and
the disease state of the patient. Animal models may be used to
determine effective doses for the induction of particular immune
responses and in some cases for the prevention or treatment of
particular diseases.
[0062] The term "effective amount" of a nucleic acid molecule
refers to the amount necessary or sufficient to realize a desired
biologic effect. For example, an effective amount of a nucleic acid
construct containing at least one unmethylated CpG for treating a
disorder could be that amount necessary to induce an immune
response of sufficient magnitude to eliminate a tumor, cancer, or
bacterial, parasitic, viral or fungal infection. An effective
amount for use as a vaccine could be that amount useful for priming
and boosting a protective immune response in a subject. The
effective amount for any particular application can vary depending
on such factors as the disease or condition being treated, the
particular nucleic acid being administered (e.g. the number of
unmethylated CpG motifs (--S or --N) or their location in the
nucleic acid), the size of the subject, or the severity of the
disease or condition. One of ordinary skill in the art can
empirically determine the effective amount of a particular
oligonucleotide without necessitating undue experimentation. An
effective amount for use as a prophylactic vaccine is that amount
useful for priming and boosting a protective immune response in a
subject.
[0063] In one embodiment, the invention provides a nucleic acid
construct containing CpG motifs as described herein as a
pharmaceutical composition useful for inducing an immune response
to a bacterial, parasitic, fungal, viral infection, or the like, or
to a tumor in a subject, comprising an immunologically effective
amount of nucleic acid construct of the invention in a
pharmaceutically acceptable carrier. "Administering" the
pharmaceutical composition of the present invention may be
accomplished by any means known to the skilled artisan. By
"subject" is meant any animal, preferably a mammal, most preferably
a human. The term "immunogenically effective amount," as used in
describing the invention, is meant to denote that amount of nucleic
acid construct which is necessary to induce, in an animal, the
production of a protective immune response to the bacteria, fungus,
virus, tumor, or antigen in general.
[0064] In addition to the diluent or carrier, such compositions can
include adjuvants or additional nucleic acid constructs that
express adjuvants such as cytokines or co-stimulatory molecules.
Adjuvants include CpG motifs such as those described in co-pending
application Ser. No. 09/030,701.
[0065] The method of the invention also includes slow release
nucleic acid delivery systems such as microencapsulation of the
nucleic acid constructs or incorporation of the nucleic acid
constructs into liposomes. Such particulate delivery systems may be
taken up by the liver and spleen and are easily phagocytosed by
macrophages. These delivery systems also allow co-entrapment of
other immunomodulatory molecules, or nucleic acid constructs
encoding other immunomodulatory molecules, along with the
antigen-encoding nucleic acid construct, so that modulating
molecules may be delivered to the site of antigen synthesis and
antigen processing, allowing modulation of the immune system
towards protective responses.
[0066] Many different techniques exist for the timing of the
immunizations when a multiple immunization regimen is utilized. It
is possible to use the antigenic preparation of the invention more
than once to increase the levels and diversity of expression of the
immune response of the immunized animal. Typically, if multiple
immunizations are given, they will be spaced about four or more
weeks apart. As discussed, subjects in which an immune response to
a pathogen or cancer is desirable include humans, dogs, cattle,
horses, deer, mice, goats, pigs, chickens, fish, and sheep.
[0067] Examples of infectious virus to which stimulation of a
protective immune response is desirable include: Retroviridae
(e.g., human immunodeficiency viruses, such as HIV-1 (also referred
to as HTLV-III, LAV or HTLV-III/LAV, or HIV-II; and other isolates,
such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A
virus; enteroviruses, human coxsackie viruses, rhinoviruses,
echoviruses); Calciviridae (e.g., strains that cause
gastroenteritis); Togaviridae (e.g., equine encephalitis viruses,
rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis
viruses, yellow fever viruses); Coronaviridae (e.g.,
coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae
(e.g., parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g., influenza
viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (e.g., reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes viruses'); Poxviridae (variola
viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.,
African swine fever virus); and unclassified viruses (e.g., the
etiological agents of Spongiform encephalopathies, the agent of
delta hepatities (thought to be a defective satellite of hepatitis
B virus), the agents of non-A, non-B hepatitis (class 1=internally
transmitted; class 2=parenterally transmitted (i.e., Hepatitis C);
Norwalk and related viruses, and astroviruses).
[0068] Examples of infectious bacteria to which stimulation of a
protective immune response is desirable include: Helicobacter
pyloris, Borellia burgdorferi, Legionella pneumophilia,
Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.
intracellulare, M. kanzsaii, M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
influenzae, Bacillus antracis, corynebacterium diphtheriae,
corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pastutrella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidium,
Treponema pertenue, Leptospira, and Actinomyces israelli.
[0069] Examples of infectious fungi to which stimulation of a
protective immune response is desirable include: Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
Other infectious organisms (i.e., protists) include: Plasmodium
falcipanim and Toxoplasma gondii.
[0070] An "immunostimulatory nucleic acid molecule" or
oligonucleotide as used herein refers to a nucleic acid molecule,
which contains an unmethylated cytosine, guanine dinucleotide
sequence (i.e. "CpG DNA" or DNA containing a cytosine followed by
guanosine and linked by a phosphate bond) and stimulates (e.g. has
a mitogenic effect on, or induces or increases cytokine expression
by) a vertebrate lymphocyte. An immunostimulatory nucleic acid
molecule can be double-stranded or single-stranded. Generally,
double-stranded molecules are more stable in vivo, while
single-stranded molecules may have increased immune activity.
[0071] Unmethylated immunostimulatory CpG motifs, either within a
nucleic acid construct or an oligonucleotide, directly activate
lymphocytes and co-stimulate antigen-specific responses. As such,
they are fundamentally different form aluminum precipitates (alum),
currently the only adjuvant licensed for human use, which is
thought to act largely through adsorbing the antigen thereby
maintaining it available to immune cells for a longer period.
Further, alum cannot be added to all types of antigens (e.g., live
attentuated pathogens, some multivalent vaccines), and it induces
primarily Th2 type immune responses, namely humoral immunity but
rarely CTL. For many pathogens, a humoral response alone is
insufficient for protection, and for some pathogens can even be
detrimental.
[0072] In addition, an immunostimulatory oligonucleotide in the
nucleic acid construct of the invention can be administered prior
to, along with or after administration of a chemotherapy or other
immunotherapy to increase the responsiveness of malignant cells to
subsequent chemotherapy or immunotherapy or to speed the recovery
of the bone marrow through induction of restorative cytokines such
as GM-CSF. CpG nucleic acids also increase natural killer cell
lytic activity and antibody dependent cellular cytotoxicity (ADCC).
Induction of NK activity and ADCC may likewise be beneficial in
cancer immunotherapy, alone or in conjunction with other
treatments.
[0073] Gene Therapy
[0074] Plasmid or vector DNA may also be useful for certain gene
therapy applications. In most such cases, an immune response
against the encoded gene product would not be desirable. Thus, the
optimal plasmid DNA cassette for gene therapy purposes will have
all possible immunostimulatory (CpG-S) motifs removed and several
immunoinhibitory (CpG-N) motifs added in. An exemplary vector for
gene therapy purposes is described in the Examples.
[0075] Despite comparable levels of unmethylated CpG dinucleotides,
DNA from serotype 12 adenovirus is immune stimulatory, but serotype
2 is nonstimulatory and can even inhibit activation by bacterial
DNA. In type 12 genomes, the distribution of CpG-flanking bases is
similar to that predicted by chance. However, in type 2 adenoviral
DNA the immune stimulatory CpG-S motifs are outnumbered by a 15 to
30 fold excess of CpG dinucleotides in clusters of direct repeats
or with a C on the 5' side or a G on the 3' side. Synthetic
oligodeoxynucleotides containing these putative neutralizing
(CpG-N) motifs block immune activation by CpG-S motifs in vitro and
in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA
vaccine markedly enhanced its Th1-like function in vivo, which was
further increased by addition of CpG-S motifs. Thus, depending on
the CpG motif, prokaryotic DNA can be either immune-stimulatory or
neutralizing. These results have important implications for
understanding microbial pathogenesis and molecular evolution, and
for the clinical development of DNA vaccines and gene therapy
vectors.
[0076] Gene therapy, like DNA-based immunization, involves
introduction of new genes into cells of the body, where they will
be expressed to make a desired protein. However, in contrast to DNA
vaccines, an immune response against the expressed gene product is
not desired for gene therapy purposes. Rather, prolonged expression
of the gene product is desired to augment or replace the function
of a defective gene, and thus immune responses against the gene
product are definitely undesirable.
[0077] Plasmid DNA expression vectors are also used for gene
therapy approaches. They may be preferable to viral vectors (i.e.,
recombinant adenovirus or retrovirus), which themselves are
immunogenic (Newman, K. D., et al., J. Clin. Invest., 96:2955-2965,
1995; Zabner, J., et al., J. Clin. Invest., 97:1504-1511, 1996).
Immune responses directed against such vectors may interfere with
successful gene transfer if the same vector is used more than once.
Double-stranded DNA is poorly immunogenic (Pisetsky, D. S.
Antisense Res. Devel. 5: 219-225, 1995; Pisetsky, D. S. J. Immunol.
156: 421-423, 1996), and thus from this perspective, repeated use
is not a problem with plasmid DNA.
[0078] Nevertheless, even when gene transfer is carried out with
plasmid DNA vectors, expression of the introduced gene is often
short-lived and this appears to be due to immune responses against
the expressed protein (Miller, A. D. Nature. 357: 455-460, 1992;
Lasic, D. D., and Templeton, N. S. Advanced Drug Delivery Review.
20: 221-266, 1996). It is not a surprise that expression of a
foreign protein, as is the case with gene replacement strategies,
induces immune responses. Nevertheless, it is likely that the
presence of CpG-S motifs aggravates this situation. The finding
that removal of CpG-S motifs from DNA vaccines can abolish their
efficacy suggests that such a strategy may prove useful for
creating gene therapy vectors where immune responses against the
encoded protein are undesirable. Furthermore, the more recent
discovery of CpG-N motifs opens up the possibility of actually
abrogating unwanted immune responses through incorporating such
motifs into gene delivery vectors. In particular, the Th-2 bias of
CpG-N motifs may prevent induction of cytotoxic T-cells, which are
likely the primary mechanism for destruction of transfected
cells.
[0079] In another embodiment, the invention provides a method for
enhancing the expression of a therapeutic polypeptide in vivo
wherein the polypeptide is contained in a nucleic acid construct.
The construct is produced by removing stimulatory CpG (CpG-S)
motifs and optionally inserting neutralizing CpG (CpG-N) motifs,
thereby producing a nucleic acid construct providing enhanced
expression of the therapeutic polypeptide. Alternatively, the
invention envisions using the construct for delivery of antisense
polynucleotides or ribozymes.
[0080] Typical CpG-S motifs that are removed from the construct
include a motif having the formula:
5' X.sub.1CGX.sub.2 3'
[0081] wherein at least one nucleotide separates consecutive CpGs,
X.sub.1 is adenine, guanine, or thymine and X.sub.2 is cytosine,
thymine, or adenine. Exemplary CpG-S oligonucleotide motifs include
GACGTT, AGCGTT, AACGCT, GTCGTT and AACGAT. Another oligonucleotide
useful in the construct contains TCAACGTT. Further exemplary
oligonucleotides of the invention contain GTCG(T/C)T, TGACGTT,
TGTCG(T/C)T, TCCATGTCGTTCCTGTCGTT (SEQ ID NO:1),
TCCTGACGTTCCTGACGTT (SEQ ID NO:2) and TCGTCGTTTTGTCGTTTTGTCGTT (SEQ
ID NO:3). These motifs can be removed by site-directed mutagenesis,
for example.
[0082] Preferably CpG-N motifs contain direct repeats of CpG
dinucleotides, CCG trinucleotides, CGG trinucleotides, CCGG
tetranucleotides, CGCG tetranucleotides or a combination of any of
these motifs. In addition, the neutralizing motifs of the invention
may include oligos that contain a sequence motif that is a poly-G
motif, which may contain at least about four Gs in a row or two G
trimers, for example (Yaswen et al., Antisense Research and
Development 3:67, 1993; Burgess et al., PNAS 92:4051, 1995).
[0083] The present invention provides gene therapy vectors and
methods of use. Such therapy would achieve its therapeutic effect
by introduction of a specific sense or antisense polynucleotide
into cells or tissues affected by a genetic or other disease. It is
also possible to introduce genetic sequences into a different cell
or tissue than that affected by the disease, with the aim that the
gene product will have direct or indirect impact on the diseases
cells or tissues. Delivery of polynucleotides can be achieved using
a plasmid vector as described herein (in "naked" or formulated
form) or a recombinant expression vector (e.g., a chimeric
vector).
[0084] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with a heterologous cDNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. The selectable marker in a recombinant
plasmid or vector confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. For example, following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. A number of
selection systems may be used, including but not limited to the
herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell
11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska
& Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and
adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:
817) genes can be employed in tk-, hgprt- or aprt- cells
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77: 3567;
O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78: 2072; neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Recently,
additional selectable genes have been described, namely trpB, which
allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hartman
& Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85: 8047); and ODC
(ornithine decarboxylase) which confers resistance to the omithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine, DFMO
(McConlogue L., 1987, In: Current Communications in Molecular
Biology, Cold Spring Harbor Laboratory ed.).
[0085] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
When the subject is a human, a vector such as the gibbon ape
leukemia virus (GaLV) can be utilized. A number of additional
retroviral vectors can incorporate multiple genes. All of these
vectors can transfer or incorporate a gene for a selectable marker
so that transduced cells can be identified and generated.
[0086] Therapeutic peptides or polypeptides are typically included
in the vector for gene therapy. For example, immunomodulatory
agents and other biological response modifiers can be administered
for incorporation by a cell. The term "biological response
modifiers" is meant to encompass substances which are involved in
modifying the immune response. Examples of immune response
modifiers include such compounds as lymphokines. Lymphokines
include tumor necrosis factor, interleukins (e.g., IL-2, -4, -6,
-10 and -12), lymphotoxin, macrophage activating factor, migration
inhibition factor, colony stimulating factor, and alpha-interferon,
beta-interferon, and gamma-interferon and their subtypes. Also
included are polynucleotides which encode metabolic enzymes and
proteins, including Factor VIII or Factor IX. Other therapeutic
polypeptides include the cystic fibrosis transmembrane conductance
regulator (e.g., to treat cystic fibrosis); structural or soluble
muscle proteins such as dystrophin (e.g., to treat muscular
dystrophies); or hormones. In addition, suicide or tumor repressor
genes can be utilized in a gene therapy vector described
herein.
[0087] In addition, antisense polynucleotides can be incorporated
into the nucleic acid construct of the invention. Antisense
polynucleotides in context of the present invention includes both
short sequences of DNA known as oligonucleotides of usually 10-50
bases in length as well as longer sequences of DNA that may exceed
the length of the target gene sequence itself. Antisense
polynucleotides useful for the present invention are complementary
to specific regions of a corresponding target mRNA. Hybridization
of antisense polynucleotides to their target transcripts can be
highly specific as a result of complementary base pairing.
[0088] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1: 273
(1982)); the TK promoter of Herpes virus (McKnight, Cell 31: 355
(1982); the SV40 early promoter (Benoist et al., Nature 290: 304
(1981); the Rous sarcoma virus promoter (Gorman et al., Proc. Nat'l
Acad. Sci. USA 79: 6777 (1982); and the cytomegalovirus promoter
(Foecking et al., Gene 45: 101 (1980)) (See also discussion above
for suitable promoters).
[0089] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
fusion gene expression if the prokaryotic promoter is regulated by
a eukaryotic promoter. Zhou et al., Mol. Cell. Biol. 10: 4529
(1990); Kaufman et al., Nucl. Acids Res. 19: 4485 (1991).
[0090] It is desirable to avoid promoters that work well in APC
since that could induce an immune response. Thus, ubiquitous viral
promoters, such as CMV, should be avoided. Promoters specific for
the cell type requiring the gene therapy are desirable in many
instances. For example, with cystic fibrosis, it would be best to
have a promoter specific for the lung epithelium. In a situation
where a particular cell type is used as a platform to produce
therapeutic proteins destined for another site (for either direct
or indirect action), then the chosen promoter should work well in
the "factory" site. Muscle is a good example for this, as it is
post-mitotic, it could produce therapeutic proteins for years on
end as long as there is no immune response against the
protein-expressing muscle fibers. Therefore, use of strong muscle
promoters as described in the previous section are particularly
applicable here. Except for treating a muscle disease per se, use
of muscle is typically only suitable where there is a secreted
protein so that it can circulate and function elsewhere (e.g.,
hormones, growth factors, clotting factors).
[0091] Administration of gene therapy vectors to a subject, either
as a plasmid or as part of a viral vector can be affected by many
different routes. Plasmid DNA can be "naked" or formulated with
cationic and neutral lipids (liposomes), microencapsulated, or
encochleated for either direct or indirect delivery. The DNA
sequences can also be contained within a viral (e.g., adenoviral,
retroviral, herpesvius, pox virus) vector, which can be used for
either direct or indirect delivery. Delivery routes include but are
not limited to intramuscular, intradermal (Sato, Y. et al., Science
273: 352-354, 1996), intravenous, intra-arterial, intrathecal,
intrahepatic, inhalation, intravaginal instillation (Bagarazzi et
al., J. Med. Primatol. 26:27, 1997), intrarectal, intratumor or
intraperitoneal.
[0092] As much as 4.4 mg/kg/d of antisense polynucleotide has been
administered intravenously to a patient over a course of time
without signs of toxicity. Martin, 1998, "Early clinical trials
with GDM91, a systemic oligodeoxynucleotide", In: Applied
Oligonucleotide Technology, CA. Stein and A. M. Krieg, (Eds.), John
Wiley and Sons, Inc., New York, N.Y.). Also see Sterling, "Systemic
Antisense Treatment Reported," Genetic Engineering News 12: 1, 28
(1992).
[0093] Delivery of polynucleotides can be achieved using a plasmid
vector as described herein, that can be administered as "naked DNA"
(i.e., in an aqueous solution), formulated with a delivery system
(e.g., liposome, cochelates, microencapsulated). Delivery of
polynucleotides can also be achieved using recombinant expression
vectors such as a chimeric virus. Thus the invention includes a
nucleic acid construct as described herein as a pharmaceutical
composition useful for allowing transfection of some cells with the
DNA vector such that a therapeutic polypeptide will be expressed
and have a therapeutic effect (to ameliorate symptoms attributable
to infection or disease). The pharmaceutical compositions according
to the invention are prepared by bringing the construct according
to the present invention into a form suitable for administration to
a subject using solvents, carriers, delivery systems, excipients,
and additives or auxiliaries. Frequently used solvents include
sterile water and saline (buffered or not). One carrier includes
gold particles, which are delivered biolistically (i.e., under gas
pressure). Other frequently used carriers or delivery systems
include cationic liposomes, cochleates and microcapsules, which may
be given as a liquid solution, enclosed within a delivery capsule
or incorporated into food.
[0094] An alternative formulation for the administration of gene
therapy vectors involves liposomes. Liposome encapsulation provides
an alternative formulation for the administration of
polynucleotides and expression vectors. Liposomes are microscopic
vesicles that consist of one or more lipid bilayers surrounding
aqueous compartments. See, generally, Bakker-Woudenberg et al.,
Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993),
and Kim, Drugs 46: 618 (1993). Liposomes are similar in composition
to cellular membranes and as a result, liposomes can be
administered safely and are biodegradable. Depending on the method
of preparation, liposomes may be unilamellar or multilamellar, and
liposomes can vary in size with diameters ranging from 0.02 .mu.m
to greater than 10 .mu.m. See, for example, Machy et al., LIPOSOMES
IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and Ostro et
al., American J Hosp. Pharm. 46: 1576 (1989).
[0095] After intravenous administration, conventional liposomes are
preferentially phagocytosed into the reticuloendothelial system.
However, the reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means. Claassen et al., Biochim. Biophys. Acta 802: 428 (1984). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatised phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system. Allen et al., Biochim. Biophys. Acta
1068: 133 (1991); Allen et al., Biochim. Biohys. Acta 1150: 9
(1993). These Stealth.RTM. liposomes have an increased circulation
time and an improved targeting to tumors in animals. (Woodle et
al., Proc. Amer. Assoc. Cancer Res. 33: 2672 1992). Human clinical
trials are in progress, including Phase III clinical trials against
Kaposi's sarcoma. (Gregoriadis et al., Drugs 45: 15, 1993).
[0096] Expression vectors can be encapsulated within liposomes
using standard techniques. A variety of different liposome
compositions and methods for synthesis are known to those of skill
in the art. See, for example, U.S. Pat. No. 4,844,904, U.S. Pat.
No. 5,000,959, U.S. Pat. No. 4,863,740, U.S. Pat. No. 5,589,466,
U.S. Pat. No. 5,580,859, and U.S. Pat. No. 4,975,282, all of which
are hereby incorporated by reference.
[0097] Liposomes can be prepared for targeting to particular cells
or organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For instance, antibodies
specific to tumor associated antigens may be incorporated into
liposomes, together with gene therapy vectors, to target the
liposome more effectively to the tumor cells. See, for example,
Zelphati et al., Antisense Research and Development 3: 323-338
(1993), describing the use "immunoliposomes" containing vectors for
human therapy.
[0098] In general, the dosage of administered liposome-encapsulated
vectors will vary depending upon such factors as the patient's age,
weight, height, sex, general medical condition and previous medical
history. Dose ranges for particular formulations can be determined
by using a suitable animal model.
[0099] In addition to antisense, ribozymes can be utilized with the
gene therapy vectors described herein. Ribozymes are RNA molecules
possessing the ability to specifically cleave other single-stranded
RNA in a manner analogous to DNA restriction endonucleases. Through
the modification of nucleotide sequences which encode these RNAs,
it is possible to engineer molecules that recognize specific
nucleotide sequences in an RNA molecule and cleave it (Cech, J.
Amer. Med. Assn., 260:3030, 1988). A major advantage of this
approach is that, because they are sequence-specific, only mRNAs
with particular sequences are inactivated.
[0100] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0101] All references cited herein are hereby incorporated by
reference in their entirety. The following examples are intended to
illustrate but not limit the invention. While they are typical of
those that might be used, other procedures known to those skilled
in the art may alternatively be used.
EXAMPLE 1
Cloning of CpG Optimized Plasmid DNA Vectors
[0102] Plasmids and Other Reagents
[0103] The cloning vector pUK21, which contains one ColE1
replication region, kanamycin resistance gene and poly linker, was
provided by Martin Schleef of Qiagen Inc. (Qiagen, Hilden,
Germany). The expression vector pcDNA3 was purchased from
Invitrogen Corp. (Carlsbad, USA). E. coli strain DH5.alpha. was
used as the bacterial host.
[0104] Pwo DNA polymerase, T4 DNA ligase, dNTP and ATP were
purchased from Boerhinger Mannheim (Mannheim, Germany). T4 DNA
polymerase, large fragment of DNA polymerase I (klenow), T4 DNA
polynucleotide kinase, CIP (calf intestinal alkaline phosphatase)
and restriction enzymes were purchased from New England BioLabs
(Beverly, USA) and GIBCO BRL (Gaithersburg, USA). General
laboratory chemicals were from Sigma Chemical Corp. (St. Louis,
USA).
[0105] Recombinant DNA Techniques
[0106] Unless specified otherwise, all recombinant DNA methods were
as described by Sambrook et al. (1989). Plasmid DNA was prepared
with Qiagen Plasmid Kits (Qiagen Inc). DNA purification was carried
out by separating DNA fragments on an agarose gel and extracting
with QIAquick Gel Extraction Kit (Qiagen Inc). Double-stranded DNA
sequencing was performed with ABI PRISM automatic sequencing system
(Perkin Elmer Corp., Norwalk, USA). Oligonucleotides for primers
were synthesized with a DNA synthesizer, model Oligo 1000,
manufactured by Beckman Instrument Inc. (Palo Alto, USA). PCR was
performed with the Perkin Elmer PCR system 2400.
[0107] PCR Conditions
[0108] Cycling conditions for each PCR began with a 2-min
denaturation at 94.degree. C., followed by 25 cycles of
denaturation at 94.degree. C. for 15 sec, annealing at 55.degree.
C. for 30 sec, elongation at 72.degree. C. for 45 sec (adjusted
according to the size of DNA fragment), and completed with a 7-min
incubation at 72.degree. C. High-fidelity Pwo polymerase was used
when fragments were created for cloning and site-directed
mutagenesis.
[0109] Construction of Basic Expression Vector
[0110] The pUK21 vector was used as the starting material to
construct a basic expression vector, which was subsequently used
for construction of either a CpG-optimized DNA vaccine vectors or a
CpG-optimized gene therapy vectors. DNA sequences required for gene
expression in eukaryotic cells were obtained by PCR using the
expression vector pcDNA3 as a template.
[0111] (i) Insertion of the CMV (Human Cytomegalovirus) Major
Intermediate Early Promoter/Enhancer Region
[0112] The CMV promoter (from pcDNA3 position 209 to 863) was
amplified by PCR using 30 ng pcDNA3 as a template. The forward PCR
primer 5' CGT GGA TAT CCG ATG TAC GGG CCA GAT AT 3' (SEQ ID NO:4)
introduced an EcoRV site, and the reverse PCR primer 5' AGT CGC GGC
CGC AAT TTC GAT AAG CCA GTA AG 3' (SEQ ID NO:5)introduced a NotI
site. After digestion with EcoRV and NotI, a 0.7 kb PCR fragment
containing the CMV promoter was purified and inserted into the
pUK21 polylinker between XbaI and NotI sites. The XbaI sticky end
of pUK21 was filled in with the large fragment of T4 DNA polymerase
after digestion to create a blunt end. The inserted CMV promoter
was confirmed by sequencing. The resulting plasmid was pUK21-A1
(FIG. 1).
[0113] (ii) Insertion of the BGH polyA (Bovine Growth Hormone
Polyadenylation Signal)
[0114] BGH polyA (from pcDNA3 position 1018 to 1249) was amplified
by PCR using pcDNA3 as template. The forward PCR primer 5' ATT CTC
GAG TCT AGA CTA GAG CTC GCT GAT CAG CC 3' (SEQ ID NO:6) introduced
XhoI and XbaI sites, and the reverse PCR primer 5' ATT AGG CCT TCC
CCA GCA TGC CTG CTA TT 3' (SEQ ID NO:7) introduced a StiI site.
After digestion with XhoI and StuI, the 0.2 kb PCR fragment
containing the BGH polyA was purified, and ligated with the 3.7 kb
XhoI-StuI fragment of pUK21-A1. The inserted BGH polyA was
confirmed by sequencing. The resulting plasmid was pUK21-A2 (FIG.
2).
[0115] Note: Ligation of the EcoRV and XbaI-fill-in blunt ends in
the pUK21-Al construct recreated an XbaI site, but this site is
resistant to cleavage due to Dam methylation present in most
laboratory strains of E. coli, such as DH5.alpha., so the extra
XbaI site introduced by the forward PCR primer in the pUK21-A2
construct is available as a cloning site.
[0116] CpG Optimized DNA Vaccine Vector
[0117] The CpG-optimized DNA vaccine vectors were made from the
basic expression vector (pUK21-A2) in several steps:
[0118] Site-directed mutagenesis for removal of CpG-N motifs, with
care being taken to maintain the integrity of the open reading
frame. Where necessary, the mutated sequence was chosen to encode
the same amino acids as the original sequence.
[0119] Removal of unnecessary sequences (e.g., fl ori).
[0120] Addition of suitable polylinker sequence to allow easy
incorporation of CpG-S motifs.
[0121] Addition of CpG-S motifs which would be chosen to enhance a
particular immune response (humoral, cell-mediated, high levels of
a particular cytokine etc.).
[0122] The pUK21-A2 vector was used as the starting material for
construction of an optimized DNA vaccine vector. Site-directed
mutagenesis was carried out to mutate those CpG-N sequences that
were easy to mutate. As described below, 22 point-mutations were
made to change a total of 15 CpG-N motifs to alternative non-CpG
sequences. For 16 of these point mutations that were in coding
regions, the new sequences encoded the same amino acids as before
through alternative codon usage. The mutated sequences were all in
the kanamycin resistance gene or immediately adjacent regions. At
present, we did not mutate any CpG-N motifs in regions with
indispensable functions such as the ColE1, BGH poly A or polylinker
regions, or the promoter region (in this case CMV), however this
should be possible.
[0123] (i) Insertion of the f1 Origin of Replication Region
[0124] The f1 origin and two unique restriction enzyme sites (DraI
and ApaI) were introduced into pUK21-A2 for later vector
construction. f1 origin (from pcDNA3 position 1313 to 1729) was
amplified by PCR using pcDNA3 as template. The forward PCR primer
5' TAT AGG CCC TAT TTT AAA CGC GCC CTG TAG CGG CGC A 3' (SEQ ID
NO:8) introduced EcoO109I and DraI sites, and the reverse PCR
primer 5' CTA TGG CGC CTT GGG CCC AAT TTT TGT TAA ATC AGC TC 3'
(SEQ ID NO:9) introduced NarI and ApaI site. After digestion with
NarI and EcoO109I, the 0.4 kb PCR fragment containing the fl origin
was purified and ligated with the 3.3 kb NarI-EcoO109I fragment of
pUK21-A2, resulting in pUK21-A (FIG. 3).
[0125] (ii) Site-Directed Mutagenesis to Remove Immunoinhibitory
Sequences
[0126] Sixteen silent-mutations within the kanamycin resistance
gene and another six point-mutations within a non-essential DNA
region were designed in order to eliminate immunoinhibitory CpG-N
sequences. At this time, mutations were not made to CpG-N motifs
contained in regions of pUK21-A that had essential functions.
[0127] Site-directed mutagenesis was performed by overlap extension
PCR as described by Ge et al. (1997). The 1.3 kb AlwNI-EcoO109I
fragment of pUK21-A contained all 22 nucleotides to be mutated and
was regenerated by overlap extension PCR using mutagenic primers.
All the primers used for mutagenesis are listed in Table 1, and the
nucleotide sequence of this AlwNI-EcoO109I fragment is listed in
Table 2 (Note: the nucleotide numbering scheme is the same as the
backbone vector pUK21).
[0128] The mutagenesis was carried out as follows: In the first
round of overlap extension PCR, the pairs of primers:
Mu-0F/Mu-(4+5)R Mu-(4+5)F/Mu-9R, Mu-9F/Mu-13R and Mu-13F/Mu-0R were
used to introduce four point-mutations at positions 1351, 1363,
1717 and 1882. The PCR-generated EcoRI/AlwNI-EcoO109I/XbaI fragment
was inserted into the pcDNA3 polylinker between the EcoR I and XbaI
sites. The mutated MspI at position 1717 was used to identify the
pcDNA3-insert containing the appropriate mutant DNA fragment.
[0129] In the second round of overlap extension PCR, the
pcDNA3-insert from the first-round was used as a PCR template, the
pairs of primers: Mu-0F/Mu-2R, Mu-2F/Mu-7R, Mu-7F/Mu-10R and
Mu-10F/Mu-OR were used to introduce three point-mutations at
positions 1285, 1549 and 1759. The PCR-generated
EcoRI/AlwN-EcoO109I/XbaI fragment was inserted into the pcDNA3
polylinker between the EcoRI and XbaI sites. The SnaBI site created
by mutation at position 1759 was used to identify the pcDNA3-insert
containing the appropriate mutant DNA fragment.
[0130] In the third round of overlap extension PCR, the
pcDNA3-insert from the second-round was used as a template, the
pairs of primers: Mu-OF/Mu-3R, Mu-3F/Mu-8R, Mu-SF/Mu-14R and
Mu-14F/Mu-OR were used to introduce five point-mutations at
positions 1315, 1633, 1636, 1638 and 1924. The PCR-generated
EcoRI/AlwNI-EcoO109I/XbaI fragment was inserted into the pcDNA3
polylinker between the EcoRI and XbaI sites. The mutated MspI site
at position 1636 was used to identify the pcDNA3-insert containing
the appropriate DNA mutant fragment.
[0131] In the last round of overlap extension PCR, the
pcDNA3-insert from the third-round was used as a template, the
pairs of primers: Mu-OF/Mu-1R, Mu-1F/Mu-6R, Mu-6F/Mu-(11+12)R,
Mu-(11+12)F/Mu-15R and Mu-1 SF/Mu-0R were used to introduce
10-point mutations at positions 1144, 1145, 1148, 1149, 1152, 1153,
1453, 1777, 1795 and 1984. After digestion with the EcoO109I and
AlwNI, the PCR-generated 1.3 kb fragment was inserted into pUK21-A
to replace the corresponding part, resulting in pUK21-B. All the 22
point-mutations were confirmed by sequencing, and the PCR-generated
AlwNI-EcoO109I fragment was free from PCR errors.
[0132] (iii) Replacement of the f1 Origin with Unique Restriction
Enzyme Sites
[0133] Oligonucleotides 5' AAA TTC GAA AGT ACT GGA CCT GTT AAC A 3'
(SEQ ID NO:10) and its complementary strand 5'CGT GTT AAC AGG TCC
AGT ACT TTC GAA TTT 3' (SEQ ID NO:11) were synthesized, and
5'-phosphorylated. Annealing of these two phosphorylated oligos
resulted in 28 base pair double-stranded DNA containing three
unique restriction enzyme sites (ScaI, AvaII, HpaI), one sticky end
and one blunt end. Replacing the 0.4 kb NarI-DraI fragment of
pUK21-B with this double-stranded DNA fragment resulted in the
universal vector pMAS for DNA vaccine development (FIGS. 4 and
5).
[0134] (iv) Insertion of Immunostimulatory Motifs Into the Vector
pMAS
[0135] The vector is now ready for cloning CpG-S motifs. The exact
motif which would be added to the vector would depend on its
ultimate application, including the species it is to be used in and
whether a strong humoral and/or a cell-mediated response was
preferred. The following description gives an example of how a
varying number of a given motif could be added to the vector.
[0136] Insertion of murine-specific CpG-S motifs was carried out by
first synthesizing the oligonucleotide 5' GAC TCC ATG ACG TTC CTG
ACG TTT CCA TGA CGT TCC TGA CGT TG 3' (SEQ ID NO:12) which contains
four CpG-S motifs (underlined), and its complementary sequence 5'
GTC CAA CGT CAG GAA CGT CAT GGA AAC GTC AGG AAC GTC ATG GA 3' (SEQ
ID NO:13). This sequence is based on the CpG-S motifs contained in
oligo #1826, which has potent stimulatory effects on murine cells
in vitro and is a potent adjuvant for protein vaccines in vivo.
After 5'-phosphorylation, annealing was performed to create a 44 bp
double-stranded DNA fragment with AvaII-cut sticky ends.
Self-ligation of this 44 bp DNA fragment resulted in a mixture of
larger DNA fragments containing different copy numbers of the
stimulatory motif. These DNA fragments with different numbers of
mouse CpG-S motifs were inserted into the AvaII site of pMAS, which
was first dephosphorylated with CIP to prevent self-ligation. The
resulting recombinant plasmids maintained one AvaII site due to the
design of the synthetic oligonucleotide sequence allowing the
cloning process to be repeated until the desired number of CpG-S
motifs were inserted. Sixteen and 50 mouse CpG-S motifs were
inserted into the AvaII site of pMAS, creating pMCG-16 and pMCG-50
respectively. The DNA fragment containing 50 CpG-S motifs was
excised from pMCG-50, and inserted into HpaI-AvaII-ScaI-DraI linker
of pMCG-50, creating pMCG-100. The same procedure was followed to
create pMCG-200 (Table 3).
[0137] Two different sequences containing human-specific CpG-S
motifs were cloned in different numbers into pMAS to create two
series of vectors, pHCG and pHIS, following the same strategies as
described above.
[0138] The pHCG series of vectors contain multiple copies of the
following sequence 5' GAC TTC GTG TCG TTC TTC TGT CGT CTT TAG CGC
TTC TCC TGC GTG CGT CCC TTG 3' (SEQ ID NO:14) (CpG-S motifs are
underlined). This sequence incorporates various CpG-S motifs that
had previously been found to have potent stimulatory effects on
human cells in vitro. The vector pHCG-30, pHCG-50, pHCG-100 and
pHCG-200 contain 30, 50, 100 and 200 human CpG-S motifs
respectively (Table 3).
[0139] The pHIS series of vectors contain multiple copies of the
following sequence: 5' GAC TCG TCG TTT TGT CGT TTT GTC GTT TCG TCG
TTT TGT CGT TTT GTC GTT G 3' (SEQ ID NO:15) (CpG-S motifs are
underlined). This sequence is based on the CpG-S motifs in oligo
#2006, which has potent stimulatory effects on human cells in vitro
The vector pHIS-40, pHIS-64, pHIS-128 and pHIS-192 contain 40, 64,
128 and 192 human CpG motifs respectively (Table 3).
[0140] (v) Cloning of the Hepatitis B Surface Antigen Gene
[0141] To create a DNA vaccine, the S gene (subtype ayw) encoding
the hepatitis B surface antigen (HBsAg) was amplified by PCR and
cloned into the polylinker of pUK2'-A2 using the EcoRV and Pst I
restriction enzyme sites. The S gene was analyzed by sequencing,
and then subcloned into the same restriction enzyme sites of the
pMCG and pHCG series of vectors (Table 4).
[0142] The S gene (subtype adw2) encoding the hepatitis B surface
antigen (HBsAg) was cloned into the pHIS series of vectors
following the same strategy as described above (Table 4).
[0143] CpG Optimized Gene Therapy Vector
[0144] The optimized gene therapy vectors were constructed from the
basic expression vector (pUK21-A2) in several steps.
[0145] (i) Site-Directed Mutagenesis for Removal of CpG
Immunostimulatory Sequences Within pUK21-A2
[0146] Only point-mutations, which would not interfere with the
replication and function of the expression vector, pUK21-A2, were
designed. Seventy-five point-mutations, including 55 nucleotides
within non-essential regions and 20 silent-mutations within the
kanamycin resistance gene, were carried out following the same
strategy as described previously in (ii) Site-directed mutagenesis
to remove immunoinhibitory sequences. The point mutations
eliminated 64 CpG stimulatory motifs resulting in the vector pGT
(Table 5).
[0147] ii) Insertion of Unique Restriction Enzyme Sites Into
pGT
[0148] Oligonucleotides 5' GCC CTA GTA CTG TTA ACT TTA AAG GGC CC
3' (SEQ ID NO:16) and its complementary strand 5' GGC GGG CCC TTT
AAA GTT AAC AGT ACT AG 3' (SEQ ID. NO:17) were synthesized, and
5'-phosphorylated. Annealing of these two phosphorylated oligos
resulted in a 26 bp double-stranded DNA fragment containing four
unique restriction enzyme sites (ScaI, HpaI, DraI and ApaI) and two
EcoO109I-cut sticky ends. Insertion of this 26 bp DNA fragment into
pGT created the vector pGTU.
[0149] iii) Insertion of Immunoinhibitory Motifs Into the Vector
pGTU
[0150] Human CpG-N motifs were cloned into the pGTU following the
same strategies as described previously in (iv) Insertion of
immunostimulatory motifs into the vector pMAS. The oligonucleotide
5' GCC CTG GCG GGG ATA AGG CGG GGA TTT GGC GGG GGA TAA GGC GGG GAA
3' (SEQ ID NO:18) and its complementary strand 5' GGC CCC CGC CTT
ATC CCC GCC AAA TCC CCG CCT TAT CCC CGC CAG 3' (SEQ ID NO:19) (four
CpG motifs are underlined) were synthesized and phosphorylated.
Annealing of these two oligonucleotides created a double-stranded
DNA fragment, which was self-ligated first and then cloned into the
EcoO109I site of the vector pGTU. The recombinant plasmids will be
screened by restriction enzyme digestion and the vectors with the
desired number of CpG inhibitory motifs will be sequenced and
tested.
[0151] Immunization of Mice and Assay of Immune Responses
[0152] Female BALB/c mice aged 6-8 weeks (Charles River, Montreal)
were immunized with DNA vaccines of HBsAg-encoding DNA (see vectors
described above) by intramuscular injection into the tibialis
anterior (TA) muscle. The plasmid DNA was produced in E. coli and
purified using Qiagen endotoxin-free DNA purification mega columns
(Qiagen GmbH, Chatsworth, Calif.). DNA was precipitated and
redissolved in endotoxin-free PBS (Sigma, St. Louis Mo.) at a
concentration of 0.01, 0.1 or 1 mg/ml. Total doses of 1, 10 or 100
.mu.g were delivered by injection of 50 .mu.l bilaterally into the
TA muscles, as previously described (Davis et al., 1993b).
[0153] In some cases, 10 or 100 .mu.g of CpG ODN was added to the
DNA vaccine (pCMV-S, Davis et al., 1993b). The sequences and
backbones of the ODN used are outlined in Table 6.
[0154] Mice were bled via the retro-orbital plexus at various times
after immunization and recovered plasma was assayed for presence of
anti-HBs antibodies (total IgG or IgG1 and IgG2a isotypes) by
end-point dilution ELISA assay, as previously described (Davis et
al., 1993a).
[0155] For assay of CTL activity, mice were killed and their
spleens removed. Splenocytes were restimulated in vitro with
HBsAg-expressing cells and CTL activity was evaluated by chromium
release assay as previously described (Davis et al., 1998).
EXAMPLE 2
[0156] 1. In vitro Effects of CpG-N Motifs
[0157] Nearly all DNA viruses and retroviruses have 50-94% fewer
CpG dinucleotides than would be expected based on random base
usage. This would appear to be an evolutionary adaptation to avoid
the vertebrate defense mechanisms related to recognition of CpG-S
motifs. CpG suppression is absent from bacteriophage, indicating
that it is not an inevitable result of having a small genome.
Statistical analysis indicates that the CpG suppression in
lentiviruses is an evolutionary adaptation to replication in a
eukaryotic host. Adenoviruses, however, are an exception to this
rule as they have the expected level of genomic CpG dinucleotides.
Different groups of adenovirae can have quite different clinical
characteristics.
[0158] Unlike the genome of almost all DNA viruses and
retroviruses, some adenoviral genomes do not show suppression of
CpG dinucleotides (Karlin et al., 1994; Sun et al., 1997). Analysis
of different adenoviral genomes (types 2, 5, 12, and 40) reveals
surprising variability among each other and compared to human and
E. coli in the flanking bases around CpG dinucleotides (Table
7).
[0159] Adenoviral strains 2 and 5 belong to the same family but
strain 12 is quite distinct from them. Purified type 12 adenoviral
DNA induced cytokine secretion from human PBMC to a degree similar
to that seen with bacterial DNA (EC DNA) (Table 8). In contrast,
DNA from types 2 and 5 adenoviruses induced little or no production
of cytokines (Tables 3, 4). Remarkably, not only did type 2 and 5
adenoviral DNA fail to induce TNF-.alpha. or IFN-.gamma. secretion,
it actively inhibited the induction of this secretion by EC DNA
(Table 9). In contrast, type 12 adenoviral DNA had no discernible
inhibitory effects. These data suggested that type 2 and 5
adenoviral DNA contains sequence motifs that inhibit the cytokine
responses to the stimulatory motifs present.
[0160] The bases flanking CpG motifs determine whether a CpG
dinucleotide will cause immune stimulation, and may also determine
the type of cytokines secreted. The fact that type 2 and 5
adenoviral DNA was not only nonstimulatory but actually inhibitory
of CpG DNA, suggested that certain nonstimulatory CpG motifs may
even be able to block the stimulatory motifs and that the
inhibitory motifs should be over-represented in the genomes of
adenovirus type 2 and 5 compared to type 12 (or to human DNA). By
analysis of these genomes, it was possible to identify sequences
that could block the effects of known CpG-S sequences on in vitro B
cell proliferation (Table 10) and cytokine secretion (Table
11).
[0161] Sequences which were found to be immunoinhibitory by in
vitro assay were chosen to be mutated (wherever easily possible)
from the backbone of the DNA vaccine vector.
[0162] 2. CpG-S ODN Cannot be used as an Adjuvant for DNA
Vaccines
[0163] It has previously been shown that CpG-S ODN is a potent
vaccine adjuvant when given with HBsAg protein (Davis et al.,
1998). Antibodies against HBsAg (anti-HBs) were augmented many
times over those obtained with HBsAg alone or even HBsAg with alum
as adjuvant. In addition, the humoral response was more strongly
Th1, as indicated by a greater proportion of IgG2a than IgG1
isotypes of antibodies in immunized BALB/c mice. The strong Th1
effect of the CpG-S motifs was further demonstrated by the greatly
enhanced cytotoxic T-cell activity. One of the most potent CpG-S
ODN in mice was 1826, a 20-mer with 2 CpG-dinucleotides and made
with a synthetic phosphorothioate backbone (see Table 6 for
sequence).
[0164] In contrast to the success with protein antigens, attempts
to augment immune responses induced by a HBsAg-expressing DNA
vaccine by the addition of CpG-S ODN 1826 failed. Surprisingly, the
immune responses decreased with the addition of CpG-S ODN in a
dose-dependent manner (FIG. 6a). Addition of ODN #1826 to a
luciferase reporter gene construct (pCMV-luc, Davis et al., 1993b)
resulted in a dose-dependent decrease in luciferase expression
(FIG. 6b). This indicates that the negative effects of the CpG-S
ODN on the DNA vaccine were due to reduced gene expression rather
than an effect on the immune response against the gene product.
[0165] ODN #1826 used in the above studies is an ODN with a
phosphorothioate backbone (S-ODN) and it is possible that the
synthetic sulfur-containing backbone interfered with the ability of
the plasmid DNA to transfect target cells. Zhao et al. (1994)
investigated the effect of the backbone on binding, uptake and
degradation of ODN by mouse splenocytes and found that S-ODN had
the highest affinity for ODN-binding sites on the cell membrane and
could competitively inhibit binding of ODN made with a natural
phosphodiester backbone (O-ODN). A similar blocking of binding
might be taking place when S-ODN is mixed with plasmid DNA, which
contains a natural phosphodiester backbone like O-ODN. Furthermore,
it was shown that the affinity of ODN made with a
phophorothioate-phospho- diester chimeric backbone (SOS-ODN) for
ODN-binding sites was lower than that of S-ODN (Zhao et al., 1994).
Thus, we evaluated the effect of adding SOS-ODN 1980, which has the
identical sequence to S-ODN 1826, to pCMV-luc DNA and found that
even at a 100 .mu.g dose, this did not alter the expression of the
luciferase reporter gene (FIG. 7). While ODN with a chimeric
backbone (SOS-ODN) do not adversely affect the level of gene
expression (except when certain sequences such as a poly G are
present) (FIG. 7), this is not useful since SOS-ODN are apparently
also not sufficiently nuclease-resistant to exert a strong CpG
adjuvant effect (Table 12). Administering the CpG S-ODN at a
different time or site than the plasmid DNA does not interfere with
gene expression either (FIG. 8), however nor do these approaches
augment responses to DNA vaccines by administering the CpG S-ODN at
a different time or site than the plasmid DNA (Table 12). Thus it
appears that the immune system must see the antigen and the CpG-S
motif at the same time and the same place to augment
antigen-specific responses. Thus, at least for the present, it
appears necessary to clone CpG motifs into DNA vaccine vectors in
order to take advantage of their adjuvant effect.
EXAMPLE 3
CpG-Optimized DNA Vaccines
[0166] Eliminating 52 of 134 CpG-N motifs from a DNA vaccine
markedly enhanced its Th1-like function in vivo and immune
responses were further augmented by the addition of CpG-S motifs to
the DNA vaccine vectors (FIG. 9).
[0167] Titers of antibodies were increased by the removal of CpG-N
motifs. With the addition of 16 or 50 CpG-S motifs, humoral
responses became increasingly more Th1, with an ever greater
proportion of IgG2a antibodies. The anti-HBs titer was higher with
16 than 50 CpG-S motifs, perhaps because the strong cytokine
response with the greater number of motifs inhibited antigen
expression that was driven by the CMV promoter. Viral promoters
such as that from CMV are known to be down-regulated by cytokines
such as the IFNs (Gribaudo et al., 1993; Harms & Splitter,
1995; Xiang et al., 1997).
[0168] CTL responses were likewise improved by removal of CpG-N
motifs, and then more so by the addition of CpG-S motifs to the DNA
vaccines.
EXAMPLE 4
CpG-Optimized Gene Therapy Vectors
[0169] Oligodeoxynucleotides (ODN) and DNA Phosphodiester ODN were
purchased from Operon Technologies (Alameda, Calif.) and nuclease
resistant phosphorothioate ODN were purchased from Oligos Etc.
(Wilsonville, Oreg.) or Hybridon Specialty Products (Milford,
Mass.). All ODN had undetectable endotoxin levels (less than 1
ng/mg) by Limulus assay (Whittaker Bioproducts, Walkersville, Md.).
E. coli (strain B) DNA was purchased from Sigma (St. Louis, Mo.),
purified by repeated extraction with phenol:chloroform:isoamyl
alcohol (25:24:1) and/or Triton Xi 14 extraction and ethanol
precipitation and made single stranded by boiling for 10 min
followed by cooling on ice for 5 min. Highly purified type 2, 5,
and 12 adenoviral DNA was prepared from viral preparations using
standard techniques and processed in the same manner as the E. coli
DNA. Plasmids for DNA vaccination were purified using two rounds of
passage over Endo-free columns (Qiagen, Hilden, Germany).
[0170] Cell Cultures and ELISA assays for cytokines. ELISA assays
were performed using standard techniques and commercially available
reagents as previously described (Klinman, D., et al., Proc. Natl.
Acad. Sci. USA, 93, 2879-2883 (1996); Yi et al., J. Immunol., 157,
5394-5402 (1996)). Standard deviations of the triplicate wells were
<10%.
[0171] Construction of optimized DNA vectors. The starting material
was pUK21-A2, an expression vector containing the immediate early
promoter of human cytomegalovirus (CMV IE), the bovine growth
hormone (BGH) polyadenylation signal, and the kanamycin resistance
gene (Wu and Davis, unpublished). To avoid disrupting the plasmid
origin of replication, mutagenesis designed to eliminate CpG-N
motifs was restricted to the kanamycin resistance gene and
non-essential DNA sequences following the gene. A total of 22 point
mutations were introduced to alter 15 CpG-N motifs (a "motif"
refers to a hexamer containing one or more CpG dinucleotides)
containing 19 CpG dinucleotides, 12 of which were eliminated and 7
of which were transformed into CpG-S motifs. Site-directed
mutagenesis was performed by overlap extension PCR as described by
Ge et al. (Prosch, S., et al., Biol. Chem., 377, 195-201 (1996)).
The 1.3 kb AlwN I-EcoO109 I fragment of pUK21-A2, which contained
all 22 nucleotides to be mutated, was used as the template for PCR.
The 1.3 kb fragment was regenerated by four rounds of overlap
extension PCR using appropriate mutagenic primers, and substituted
for the original AlwN I-EcoO 109 I fragment, resulting in pUK21-B2.
All the mutations were confirmed by sequencing.
[0172] Another 37 CpG-N motifs were removed by replacing the fl
origin with a multiple cloning site. Oligonucleotides 5'
GCCCTATTTTAAATTCGAAAGTA- CTGGACCTGTTAACA 3' (SEQ ID NO:20) and its
complementary strand 5' CGTGTTAACAGGTCCAGTACTTTCGAATTTAAAATAG 3'
(SEQ ID NO:21) were synthesized, and 5'-phosphorylated. Annealing
of these two phosphorylated-oligos resulted in a 35 bp
double-stranded DNA fragment containing four unique restriction
enzyme sites (Dra I, Sca I, Ava II, Hpa I) and two sticky ends.
Replacing the 0.6 kb Nar I-EcoO109 I fragment of pUK21-B2, which
contained the entire f1 ori, with this double-stranded DNA fragment
resulted in the master vector pMAS.
[0173] Next, different numbers of CpG-S motifs were inserted into
the vector by allowing self-ligation of a 20 bp DNA fragment with
the sequence 5' GACTCCATGACGTTCCTGACGTTTCCATGACGTTCCTGACGTTG 3'
(SEQ ID NO:22) with a complementary strand and inserting different
numbers of copies into the Ava II site of pMAS. Recombinant clones
were screened and the two vectors were chosen for further testing
with 16 and 50 CpG-S motifs, and named pMCG16 and pMCG50
respectively.
[0174] To create a DNA vaccine, the S gene encoding ay subtype of
hepatitis B surface antigen (HBsAg) was amplified by PCR and cloned
into the EcoRV--PstI sites of the vectors, resulting in pUK-S,
pMAS-S, pMCG16-S, and pMCG50-S respectively. Vector sequences were
confirmed by sequencing and have been deposited in GenBank under
accession numbers AFO53406 (pUK-S), AFO53407 (pMAS-S), AFO53408
(pMCG16-S), and AFO53409 (pMCG50-S).
[0175] Immunization of mice against HBsAg: Immunization of 6-8 wk
old female BALB/c mice (Charles River, Montreal, QC) was by
injection into the tibialis anterior muscle (TA) of 1 .mu.g
recombinant HBsAg or 10 .mu.g HBsAg-expressing DNA vaccine (Chace,
J. H., et al., Immunopath, In press (1997)). Assay for antibodies
against HBsAg (anti-HBs) was by end point dilution and for
cytotoxic T lymphocytes (CTL) was by chromium release assay as
described previously.sup.19. Both the protein (.+-.ODN) and DNA
vaccines were resuspended in saline for injection.
EXAMPLE 5
[0176] Type 12 adenoviral DNA is immune stimulatory, but types 2
and 5 adenoviral DNA are immune neutralizing. To investigate
possible functional differences in the immune effects of various
prokaryotic DNAs, we determined their ability to induce cytokine
secretion from human PBMC. In contrast to bacterial DNA and genomic
DNA from type 12 adenovirus, DNA from types 2 and 5 adenovirus
failed to induce cytokine production (Table 8). In fact, despite
their similar frequency of CpG dinucleotides, type 2 or 5
adenoviral DNA severely reduced the cytokine expression induced by
co-administered immunostimulatory E. coli genomic DNA (Table 9).
This indicates that type 2 and 5 adenoviral DNA does not simply
lack CpG-S motifs, but contains sequences that actively suppress
those in E. coli DNA.
[0177] Identification of putative immune neutralizing CpG-N motifs
in type 2 and 5 adenoviral genomes. To identify possible non-random
skewing of the bases flanking the CpG dinucleotides in the various
adenoviral genomes, we examined their frequency of all 4096
hexamers. The six most common hexamers in the type 2 adenoviral
genome are shown in Table 7, along with their frequency in the Type
12 and E. coli genomes. Remarkably, all of these over-represented
hexamers contain either direct repeats of CpG dinucleotides, or
CpGs that are preceded by a C and/or followed by a G. These CpG-N
motifs are approximately three to six fold more common in the
immune inhibitory type 2 and 5 adenoviral genomes than in those of
immune-stimulatory type 12 adenoviral, E. coli or non-stimulatory
human genomic DNAs (Table 7). This hexamer analysis further
revealed that the frequency of hexamers containing CpG-S motifs
(e.g., GACGTT or AACGTT) in the type 2 adenoviral genome is as low
as that in the human genome: only 1/3 to 1/6 of that in E. coli and
type 12 adenoviral DNA (Table 7).
[0178] Effect of CpG-N motifs on the immune stimulatory effects of
CpG-S motifs. To determine whether these over-represented CpG-N
motifs could explain the neutralizing properties of type 2 and 5
adenoviral DNA, we tested the in vitro immune effects of synthetic
oligodeoxynucleotides bearing a CpG-S motif, one or more CpG-N
motifs, or combinations of both. An ODN containing a single CpG-S
motif induces spleen cell production of IL-6, IL-12, and
IFN-.gamma. (ODN 1619, Table 13). However, when the 3' end of this
ODN was modified by substituting either repeating CpG dinucleotides
or a CpG dinucleotide preceded by a C, the level of cytokine
production was reduced by approximately 50% (ODN 1952 and 1953,
Table 13). ODN consisting exclusively of these neutralizing CpG
(CpG-N) motifs induced little or no cytokine production (Table 14).
Indeed, addition of ODN containing one or more CpG-N motifs to
spleen cells along with the CpG-S ODN 1619 caused a substantial
decrease in the induction of IL-12 expression indicating that the
neutralizing effects can be exerted in trans (Table 14).
[0179] To determine whether the in vivo immune activation by ODN
containing CpG-S motifs would be reversed by CpG-N motifs, we
immunized mice with recombinant hepatitis B surface antigen
(HBsAg), with or without nuclease resistant
phosphorothioate-modified ODN containing various types of CpG
motifs. As expected, a CpG-S ODN promoted a high titer of
antibodies against HBsAg (anti-HBs antibodies) which were
predominantly of the IgG2a subclass, indicating a Th1-type immune
response (FIG. 10; ODN 1826). The various CpG-N ODN induced either
little or no production of anti-HBs antibodies (ODN 1631, 1984, and
2010) (FIG. 10). Mice immunized with combinations of CpG-S and
CpG-N ODN had a reduced level of anti-HBs antibodies compared to
mice immunized with CpG-S ODN alone, but these were still
predominantly IgG2a (FIG. 10).
[0180] Enhanced DNA vaccination by deletion of plasmid CpG-N
motifs. DNA vaccines can be highly effective inducers of Th1-like
immune responses (Raz, E., et al., Proc. Natl. Sci. Acad. USA, 93,
5141-5145 (1996); Donnelly, J. J., et al., Ann. Rev. Immunol., 15,
617-648 (1997)). Based on the in vivo and in vitro effects of CpG-N
motifs, we hypothesized that their presence within a DNA vaccine
would decrease its immunostimulatory effects. The starting vector,
pUK21-A2, contained 254 CpG dinucleotides, of which 134 were within
CpG-N motifs. In order to test the hypothesis that these CpG-N
motifs adversely affected the efficacy of this vector for DNA-based
vaccination, the number of CpG-N motifs was reduced, either by
mutation or deletion. Since mutations in the plasmid origin of
replication interfere with replication of the plasmid, we
restricted our initial mutations to the kanamycin resistance gene
and a nonessential flanking region. We were able to eliminate 19
CpG dinucleotides contained within 15 of the 20 CpG-N motifs in
these regions without changing the protein sequence. The F1 origin
of replication containing 37 CpG-N motifs and only 17 other CpG
dinucleotides was then deleted, creating the vector pMAS. This
vector was further modified by the introduction of 16 or 50 CpG-S
motifs, yielding vectors pMCG16 and pMCG50 respectively. The S gene
for HBsAg was then cloned into these vectors downstream from the
CMV promoter, to make pUK-S, pMAS-S, pMCG16-S, and pMCG50-S
respectively.
[0181] When tested for their ability to induce cytokine (IL-6 and
IL-12) secretion from cultured spleen cells, we found that the
pMAS-S, pMCG16-S and pMCG50-S vectors had significantly enhanced
immune stimulatory activity compared to pUK-S. When used as a DNA
vaccine, the anti-HBs response at 4 and 6 weeks was substantially
stronger with DNA vaccines from which CpG-N motifs had been
deleted, and even more so when 16 CpG-S motifs had been inserted.
The vector with 50 CpG-S motifs, however, was less effective at
inducing antibody production than that with 16 motifs. (FIG. 11A).
Removal of CpG-N motifs and addition of CpG-S motifs resulted in a
more than three-fold increase in the proportion of IgG2a relative
to IgG1 anti-HBs antibodies, indicating an enhanced Th-1 response.
This accentuated Th1 response also was demonstrated by the striking
progressive increases in CTL responses induced by vectors from
which CpG-N motifs were deleted and/or CpG-S motifs added (FIG.
11B).
[0182] The discovery of immune activating CpG-S motifs in bacterial
DNA has led to the realization that aside from encoding genetic
information, DNA can also function as a signal transducing
molecule. Our present results demonstrate that genomic DNA from
type 12 adenovirus is immune stimulatory, compatible with its
relatively high content of CpG-S motifs. In contrast, genomic DNA
from type 2 and 5 adenoviruses is not stimulatory, but rather is
immune neutralizing and blocks the cytokine induction of bacterial
DNA (Tables 8 and 9). To identify possible differences in the CpG
motifs present in these different adenoviral genomes, analyzed the
genomic frequency of all hexamer sequences was analyzed. This
analysis demonstrated that only the type 2 and 5 adenoviral genomes
had a dramatic overrepresentation of CpG motifs containing direct
repeats of CpG dinucleotides and/or CpGs preceded by a C and/or
followed by a G (Table 7). Synthetic ODN containing such putative
immune neutralizing (CpG-N) motifs not only did not induce cytokine
production in vitro, but also inhibited the ability of an immune
stimulatory CpG-S motif to induce cytokine expression (Tables 13,
14). These studies reveal that there are immune neutralizing CpG-N
as well as stimulatory CpG-S motifs and that there is a
surprisingly complex role for the bases flanking CpG dinucleotides
in determining these immune effects. In general, CpG-N motifs
oppose CpG-S motifs in cis or trans. The mechanism through which
CpG-N motifs work is not yet clear, but does not appear to involve
competition for cell uptake or binding to a CpG-S-specific binding
protein. Further studies are underway to determine the molecular
mechanisms through which CpG-N and CpG-S motifs exert their
respective immune effects.
[0183] The hexamers that contain CpG-N motifs are from 15 to 30
times more common in type 2 and 5 adenoviral genomes than those
that contain immune stimulatory CpG-S motifs. However, in type 12
adenoviral genomes the frequencies of hexamers containing CpG-N and
CpG-S motifs do not differ substantially from chance. These data
suggest that the immune neutralizing effects of types 2 and 5
adenoviral DNA are not merely a result of their propagation in
eukaryotic cells, but rather are due to the overall excess of CpG-N
compared to CpG-S motifs. It is tempting to speculate that the
marked over-representation of CpG-N motifs in the genomes of types
2 and 5 adenovirus may contribute to the biologic properties, such
as persistent infection of lymphocytes, which distinguish them from
type 12 adenovirus. The presence of large numbers of CpG-N motifs
within these adenoviral genomes may have played an important role
in the evolution of this virus by enabling it to avoid triggering
CpG-induced immune defenses. It will be interesting to determine
the general distribution of CpG-N and CpG-S motifs in different
families of microbial and viral genomes, and to explore their
possible roles in disease pathogenesis.
[0184] CpG-N motifs are also over-represented in the human genome,
where their hexamers are approximately two to five-fold more common
than CpG-S motifs. While this skewing is far less marked than that
in adenoviral DNA, it would still be expected to reduce or
eliminate any immune stimulatory effect from the unmethylated CpGs
present in CpG islands within vertebrate DNA. We and others have
found that even when predominantly or completely unmethylated,
vertebrate DNA is still not immune stimulatory (A. Krieg and P.
Jones, unpublished data) (Sun, S., et al., J. Immunol.,
159:3119-3125 (1997)) which is in keeping with its predominance of
CpG-N motifs (Table 7). Given the overall level of CpG suppression
in the human genome, the molecular mechanisms responsible for the
skewing of the frequency of CpG-N to CpG-S motifs are unclear. Such
a distortion from the expected random patterns would seem to
require the existence of pathways that preferentially mutate the
flanking bases of CpG-S motifs in vertebrate genomes, but do not
affect CpG-N motifs. Indeed, statistical analyses of vertebrate
genomes have provided evidence that CpGs flanked by A or T (as in
CpG-S motifs) mutate at a faster rate than CpGs flanked by C or G
(Bains, W., et al., Mutation Res., 267:43-54 (1992)).
[0185] Based on our in vitro experiments we hypothesized that the
presence of CpG-N motifs in DNA vaccines interferes with the
induction of the desired immune response. Indeed, the present study
demonstrates that elimination of CpG-N motifs from a DNA vaccine
leads to improved induction of antibodies. By removing 52 of the
CpG-N motifs from a DNA vaccine (45 were deleted and 7 turned into
CpG-S motifs) the serologic response was more than doubled; by then
adding an additional 16 CpG-S motifs, the response was enhanced
nearly 10 fold (FIG. 11A). Likewise, CTL responses were improved by
removing CpG-N motifs and even more so by adding 16 or 50 CpG-S
motifs (FIG. 11B). These increased responses are especially notable
in view of the fact that the total number of CpG dinucleotides in
the mutated vaccines is considerably below the original number.
[0186] The finding that the vector with 50 CpG-S motifs was
inferior to that with 16 motifs for induction of humoral immunity
was unexpected, and may be secondary to CpG-induced production of
type I interferons, and subsequent reduction in the amount of
antigen expressed. The decreased antibody response induced by
pMCG50-S seems unlikely to be explained by vector instability since
this vector gave the best CTL responses (FIG. 11B). Although the
pMCG50-S vector was slightly larger than pMCG16-S, the 10 .mu.g
dose still contained 93% as many plasmid copies as it did pMCG16-S,
so lower copy number is unlikely to account for the reduced
antibody levels. The current generation of DNA vaccines are quite
effective in mice, but much less effective in primates (Davis, H.
L., et al., Proc. Natl. Acad. Sci. USA, 93:7213-7218 (1996);
Letvin, N. L., et al., Proc. Natl. Acad. Sci. USA, 94:9378-9383
(1997); Fuller, D. H., et al., J. Med. Primatol., 25:236-241
(1996); Lu, S., et al., J. Virol., 70:3978-3991 (1996); Liu, M. A.,
et al., Vaccine, 15:909-919 (1997); Prince, A. M., et al., Vaccine,
15:9196-919 (1997); Gramzinski, R. A., et al., Molec. Med.,
4:109-119 (1998)). Our present results indicate that attaining the
full clinical potential of DNA vaccines will require using
engineered vectors in which CpG-N motifs have been deleted, and
CpG-S motifs added.
[0187] On the other hand, the field of gene therapy may benefit
from the discovery of CpG-N motifs through their insertion into
gene transfer vectors to prevent or reduce the induction of host
immune responses. Most of the CpG-N motifs in the adenoviral genome
are in the left hand (5') side, which is generally partially or
totally deleted for the preparation of gene therapy vectors,
especially with the "gutless" vectors (Kochanek, S., et al., Proc.
Natl. Acad. Sci. USA, 93:5731-5736 (1996)). This could lead to an
enhanced CpG-S effect. Since nucleic acids produced in viral
vectors are unmethylated, they may produce inflammatory effects if
they contain a relative excess of CpG-S over CpG-N motifs and are
delivered at an effective concentration (about 1 .mu.g/ml). Gene
therapy studies with adenoviral vectors have used doses Up to 10
infectious units (IU)/ml (which contains 0.4 .mu.g of DNA/ml based
on the genome size of 36 kb). Given that approximately 99% of
adenoviral particles are noninfectious, this corresponds to a DNA
dose of approximately 40 .mu.g/ml, which is well within the range
at which CpG DNA causes in vivo immune stimulatory effects; just 10
.mu.g/mouse induces IFN-.gamma. production acts as an adjuvant for
immunization (Davis, H. L., et al., J. Immunol., 160:870-876
(1998); Chu, R. S., et al., J. Exp.Med., 186:1623-1631 (1997);
Lipford, G. B., et al., Eur. J. Immunol., 27:2340-2344 (1997);
Weiner, G. J., et al., Proc. Natl. Acad. Sci. USA, 94:10833 (1997);
Moldoveanu, Z., et al., Vaccine, In press (1998)), and causes acute
pulmonary inflammation when delivered into mouse airways (Schwartz,
D., et al., J. Clin. Invest., 100:68-73 (1997)). Multiple
mechanisms besides the presence of CpG-S DNA are doubtless
responsible for the inflammatory responses that have limited the
therapeutic development of adenoviral vectors (Newman, K. D., et
al., J. Clin. Invest., 96:2955-2965 (1995); Zabner, J., et al., J.
Clin. Invest., 97:1504-1511 (1996)). Nonetheless, our present
results suggest that consideration be given to the maintenance or
insertion of CpG-N motifs in adenoviral vectors, and to the
engineering of backbones and inserts so that CpG-S motifs are
mutated in order to reduce immune activation.
[0188] In recent years, it has become clear that effective gene
expression need not require a viral delivery system. The use of
plasmids for gene delivery (with or without lipids or other
formulations) avoids some of the problems of viral vectors. On the
other hand, much larger doses of DNA are typically required, since
delivery is far less efficient than with a targeted system such as
a virus. For example, effective gene expression in mice typically
may require 500-1000 .mu.g DNA/mouse (Philip, R., et al., J. Biol.
Chem., 268:16087-16090 (1993); Wang, C., et al., J. Clin. Invest.,
95:1710-1715 (1995)). A recent human clinical trial using lipid/DNA
complexes and naked DNA for delivery of CFTR to the nasal
epithelium of patients with cystic fibrosis used doses of 1.25 mg
of plasmid/nostril (Zabner, J., et al., J. Clin. Invest.,
100:1529-1537 (1997)). The successful application of naked DNA
expression vectors for gene therapy will depend on the safety of
repeatedly delivering high doses of DNA. Since the plasmids used
for gene therapy typically contain several hundred unmethylated CpG
dinucleotides, many of which are in CpG-S motifs, some immune
activation may be expected to occur. Indeed, mice given repeated
doses of just 10 .mu.g of plasmid DNA daily develop elevated
lymphocyte levels and several humans who received intranasal
plasmid DNA had elevated serum IL-6 levels (Philip, R., et al., J.
Biol. Chem., 268:16087-16090 (1993)). Furthermore, delivery of 4 mg
of a gene therapy plasmid to cystic fibrosis patients in a recent
clinical trial caused acute onset of symptoms compatible with
immune activation, including fever, chills, and pulmonary
congestion. Another reason to avoid the presence of CpG-S motifs in
gene therapy vectors is that the cytokines that are produced-due to
the immune stimulation may reduce plasmid Vector expression,
especially when this is driven by viral promoters (Raz, E., et al.,
Proc. Natl. Acad. Sci. USA, 93:5141-5145 (1996)).
[0189] It is, therefore, highly desirable to develop improved gene
delivery systems with reduced immune activation. It is not possible
to simply methylate the CpG-S dinucleotides in gene therapy
plasmids, since methylation of promoters abolishes or severely
reduces their activity. The only promoter resistant to
methylation-induced silencing is the MMTV promoter, which contains
no essential CpGs, but is fairly weak. In any case, even when the
promoter is unmethylated, expression is still greatly reduced if
the coding sequences are methylated. In fact, even the strong CMV
IE promoter is completely inactivated by CpG methylation. Deletion
of all CpGs from an expression plasmid is not feasible since many
of these are located in the origin of replication (approximately
1.2 Kb long) where even single base changes can dramatically reduce
plasmid replication. For these reasons, we propose that addition of
CpG-N motifs, and/or mutation or conversion of CpG-S to CpG-N
motifs may lead to the generation of less immune stimulatory
vectors for gene therapy. Studies to investigate this possibility
are under way.
1TABLE 1 Primers used for site-directed mutagenesis. Mutated
nucleotides are underlined. Restriction enzyme sites for cloning
are indicated in bold. Forward primers: Mu-0F 5'
GTCTCTAGACAGCCACTGGTAACAGGATT 3' (845) (SEQ ID NO: 23-50,
respectively) Mu-1F (1144) 5' GTCGTTGTGTCGTCAAGTCAGCGTAATGC 3'
(1172) Mu-2F (1285) 5' TCGTTTCTGTAATGAAGGAG 3' (1304) Mu-3F (1315)
5' AAGGCAGTTCCATAGGATGG 3' (1334) Mu-(4 + 5)F (1348) 5'
TCGATCTGCGATTCCAACTCGTCCAACATCAATAC 3' (1382) Mu-6F (1453) 5'
TGGTGAGAATGGCAAAAGTT 3' (1472) Mu-7F (1548) 5'
CATTATTCATTCGTGATTGCG 3' (1568) Mu-8F (1633) 5'
ACGTCTCAGGAACACTGCCAGCGC 3' (1656) Mu-9F (1717) 5'
AGGGATCGCAGTGGTGAGTA 3' (1736) Mu-10F (1759) 5'
TATAAAATGCTTGATGGTCGG 3' (1779) Mu-(11 + 12)F (1777) 5'
GGGAAGAGGCATAAATTCTGTCAGCCAGTTTAGTC 3' (1811) Mu-13F (1882) 5'
TGGCTTCCCATACAAGCGAT 3' (1901) Mu-14F (1924) 5'
TACATTATCGCGAGCCCATT 3' (1943) Mu-15F (1984) 5' TGGCCTCGACGTTTCCCGT
3' (2002) Reverse primers: Mu-0R 5' ATCGAATTCAGGGCCTCGTGATACGCCTA
3' (2160) Mu-1R (1163) 5' TGACTTGACGACACAACGACAGCTCATGACCAAAATCCC
3' (1125) Mu-2R (1304) 5' CTCCTTCATTACAGAAACGACTTTTTAAAAATATGGTA 3'
(1266) Mu-3R (1334) 5' CCATCCTATGGAACTGCCTTGGTGAGTTTTCTCCTTC 3'
(1298) Mu-(4 + 5)R (1367) 5' GAGTTGGAATCGCAGATCGATACCAGGATCTTGC 3'
(1334) Mu-6R (1472) 5' AACTTTTGCCATTCTCACCAGATTCAGTCGT- CACTCA 3'
(1436) Mu-7R (1568) 5' CGCAATCACGAATGAATAATGGTTT- GGTTGATGCGAGTG 3'
(1530) Mu-8R (1652) 5' TGGCAGTGTTCCTGAGACGTTTGCATTCGATTCCTGTT 3'
(1615) Mu-9R (1736) 5' TACTCACCACTGCGATCCCTGGAAAAACAGCATTCCAG 3'
(1736) Mu-10R (1779) 5' CCGACCATCAAGCATTTTATACGTACTCCTGATGATGCA 3'
(1741) Mu-(11 + 12) (1796) 5'
CAGAATTTATGCCTCTTCCCACCATCAAGCATTTTATAC 3' (1758) Mu-13R (1901) 5'
ATCGCTTGTATGGGAAGCCAGATGCGCCAG- AGTTGTTT 3' (1882) Mu-14R (1943) 5'
AATGGGCTCGCGATAATGTAGGGCAATCAGGTGCGAC 3' (1907) Mu-15R (2002) 5'
ACGGGAAACGTCGAGGCCACGATTAAATTCCAACATGG 5' (1965)
[0190]
2TABLE 2 Nucleotide and amino acid sequences of the AlwNI-EcoO109I
fragment (SEQ ID NO: 80) kan (wt) 2180 AAGGGCCTCG TGATACGCCT
ATTTTTATAG GTTAATGTCA TGGGGGGGGG GGGGAAAGCC kan (wt) 2120
ACGTTGTGTC TCAAAATCTC TGATGTTACA TTGCACAAGA TAAAAATATA TCATCATGAA
kan (wt) 2060 CAATAAAACT GTCTGCTTAC ATAAACAGTA ATACAAGGGG
TGTTATGAGC CATATTCAAC kan (mu) ORF M S H I Q kan (wt) 2000
GGGAAACGTC GAGGCCGCGA TTAAATTCCA ACATGGATGC TGATTTATAT GGGTATAAAT
kan (mu) A ORF R E T S R P R L N S N M D A D L Y G Y K kan (wt)
1940 GGGCTCGCGA TAATGTCGGG CAATCAGGTG CGACAATCTA TCGCTTGTAT
GGGAAGCCCG kan (mu) A A ORF W A R D N V G Q S G A T I Y R L Y G K P
kan (wt) 1880 ATGCGCCAGA GTTGTTTCTG AAACATGGCA AAGGTAGCGT
TGCCAATGAT GTTACAGATG kan (mu) ORF D A P E L F L K H G K G S V A N
D V T D kan (wt) 1820 AGATGGTCAG ACTAAACTGG CTGACGGAAT TTATGCCTCT
TCCGACCATC AAGCATTTTA kan (mu) A C ORF E M V R L N W L T E F M P L
P T I K H F kan (wt) 1760 TCCGTACTCC TGATGATGCA TGGTTACTCA
CCACTGCGAT CCCCGGAAAA ACAGCATTCC kan (mu) A T ORF I R T P D D A W L
L T T A I P G K T A F kan (wt) 1700 AGGTATTAGA AGAATATCCT
GATTCAGGTG AAAATATTGT TGATGCGCTG GCAGTGTTCC kan (mu) ORF Q V L E E
Y P D S G E N I V D A L A V F kan (wt) 1640 TGCGCCGGTT GCATTCGATT
CCTGTTTGTA ATTGTCCTTT TAACAGCGAT CGCGTATTTC kan (mu) A A A ORF L R
R L H S I P V C N C P F N S D R V F kan (wt) 1580 GTCTCGCTCA
GGCGCAATCA CGAATGAATA ACGGTTTGGT TGATGCGAGT GATTTTGATG kan (mu) T
ORF R L A Q A Q S R M N N G L V D A S D F D kan (wt) 1520
ACGAGCGTAA TGGCTGGCCT GTTGAACAAG TCTGGAAAGA AATGCATAAA CTTTTGCCAT
kan (mu) ORF D E R N G W P V E Q V W K E M H K L L P kan (wt) 1460
TCTCACCGGA TTCAGTCGTC ACTCATGGTG ATTTCTCACT TGATAACCTT ATTTTTGACG
kan (mu) A ORF F S P D S V V T H G D F S L D N L I F D kan (wt)
1400 AGGGGAAATT AATAGGTTGT ATTGATGTTG GACGAGTCGG AATCGCAGAC
CGATACCAGG kan (mu) T T ORF E G K L I G C I D V G R V G I A D R Y Q
kan (wt) 1340 ATCTTGCCAT CCTATGGAAC TGCCTCGGTG AGTTTTCTCC
TTCATTACAG AAACGGCTTT kan (mu) T T ORF D L A I L W N C L G E F S P
S L Q K R L kan (wt) 1280 TTCAAAAATA TGGTATTGAT AATCCTGATA
TGAATAAATT GCAGTTTCAT TTGATGCTCG kan (mu) ORF F Q K Y G I D N P D M
N K L Q F H L M L kan (wt) 1220 ATGAGTTTTT CTAATCAGAA TTGGTTAATT
GGTTGTAACA CTGGCAGAGC ATTACGCTGA kan (mu) ORF D E F F kan (wt) 1160
CTTGACGGGA CGGCGCAAGC TCATGACCAA AATCCCTTAA CGTGAGTTTT CGTTCCACTG
kan(mu) AC AA AC kan (wt) 1100 AGCGTCAGAC CCCGTAGAAA AGATCAAAGG
ATCTTCTTGA GATCCTTTTT TTCTGCGCGT kan (wt) 1040 AATCTGCTGC
TTGCAAACAA AAAAACCACC GCTACCAGCG GTGGTTTGTT TGCCGGATCA kan (wt) 980
AGAGCTACCA ACTCTTTTTC CGAAGGTAAC TGGCTTCAGC AGAGCGCAGA TACCAAATAC
kan (wt) 920 TGTTCTTCTA GTGTAGCCGT AGTTAGGCCA CCACTTCAAG AACTCTGTAG
CACCGCCTAC kan (wt) 860 ATACCTCGCT CTGCTAATCC TGTTACCAGT GGCTGCTGCC
Note: Mutated nucleotides are underlined. The AlwNI and EcoO109I
sites are indicated in bold type. The nucleotide numbering scheme
is the same as the backbone vector pUK21.
[0191]
3TABLE 3 Plasmids containing immunostimulatory CpG motifs No CpG
Species-specificity and ODN Plasmid Backbone motifs equivalence of
CpG-S Insert pMCG-16 pMAS 16 mouse-specific CpG motif pMCG-50 pMAS
50 #1826.sup.1 pMCG-100 pMAS 100 pMCG-200 pMAS 200 pHCG-30 pMAS 30
human-specific CpG motif - pHCG-50 pMAS 50 no ODN equivalent.sup.2
pHCG-100 pMAS 100 pHCG-200 pMAS 200 pHIS-40 pMAS 40 human-specific
CpG motif pHIS-64 pMAS 64 #2006.sup.3 pHIS-128 pMAS 128 pHIS-192
pMAS 192+TZ,1/32 .sup.1sequence of 1826 is TCCATGACGTTCCTGACGTT
.sup.2sequence used as source of CpG motifs is
GACTTCGTGTCGTTCTTCTGTCGTCTTTAGCGCTTCTCCTG- CGTGCGTCCCTTG
.sup.3sequence of 2006 is TCGTCGTTTTGTCGTTTTGTCGTT
[0192]
4TABLE 4 Plasmids encoding hepatitis B surface antigen (derived
from ayw or adw subtypes of HBV) Plasmid Backbone Insert pUK-S
pUK21-A2 HBV-S (ayw) pUKAX-S pUK21-AX* HBV-S (ayw) pMAS-S pMAS
HBV-S (ayw) pMCG16-S pMCG-16 HBV-S (ayw) pMCG50-S pMCG-50 HBV-S
(ayw) pMCG100-S pMCG-100 HBV-S (ayw) pMCG200-S pMCG-200 HBV-S (ayw)
pHCG30-S pHCG-30 HBV-S (ayw) pHCG50-S pHCG-50 HBV-S (ayw) pHCG100-S
pHCG-100 HBV-S (ayw) pHCG200-S pHCG-200 HBV-S (ayw) pHIS20-S(ad)
pHIS-20 HBV-S(adw2) pHIS36-S(ad) pHIS-36 HBV-S(adw2) pHIS72-S(ad)
pHIS-72 HBV-S(adw2) pHIS108-S(ad) PHIS-108 HBV-S(adw2) *pUK21-AX
was created by deleting f1 origin from pUK21-A
[0193]
5TABLE 5 Sequence comparison of pUK21-A2 (SEQ ID NO: 83) and pGT
(SEQ ID NO: 84). 75 point-mutations (indicated with *) in pUK21-A2
results in the gene therapy vector (pGT) pUK21-A2 (1) GAATTCGAGC
TCCCGGGTAC CATGGCATGC ATCGATAGAT CTCGAGTCTA GACTAGAGCT pGT
GAATTCGAGC TCCCGGGTAC CATGGCATGC ATCGATAGAT CTCGAGTCTA GACTAGAGCT
--------- ----------- ---------- --------- ----------- ----------
pUK21-A2 (61) CGCTGATCAG CCTCGACTGT GCCTTCTAGT TGCCAGCCAT
CTGTTGTTTG CCCCTCCCCC pGT CGCTGATCAG CCTCGACTGT GCCTTCTAGT
TGCCAGCCAT CTGTTGTTTG CCCCTCCCCC --------- ----------- ----------
--------- ----------- ---------- pUK21-A2 (121) GTGCCTTCCT
TGACCCTGGA AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA pGT
GTGCCTTCCT TGACCCTGGA AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (181) ATTGCATCGC ATTGTCTGAG TAGGTGTCAT TCTATTCTGG
GGGGTGGGGT GGGGCAGGAC pGT ATTGCATCGC ATTGTCTGAG TAGGTGTCAT
TCTATTCTGG GGGGTGGGGT GGGGCAGGAC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (241) AGCAAGGGGG
AGGATTGGGA AGACAATAGC AGGCATGCTG GGGAAGGCCT CGGACTAGTG pGT
AGCAAGGGGG AGGATTGGGA AGACAATAGC AGGCATGCTG GGGAAGGCCT CGGACTAGTG
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (301) GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT
ATCCGCTCAC AATTCCACAC pGT CCGGAATCAT GGTCATAGCT GTTTCCTGTG
TGAAATTGTT ATCCGCTCAC AATTCCACAC *--*------ ---------- ----------
---------- ---------- ---------- pUK21-A2 (361) AACATACGAG
CCGCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC pGT
AACATCCGGG CCGCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC
-----*--*- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (421) TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC
GGGAAACCTG TCGTGCCAGC pGT TCACATTAAT TCCGTTCCGC TCACTGCCCG
CTTTCCAGTC GGGAAACCTG CCGTGCCAGC ---------- -*----*--- ----------
---------- ---------- *--------- pUK21-A2 (481) TGCATTAATG
AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG pGT
TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGCCGGTTT CCGTATTGGC CGCTCTTCCG
---------- ---------- ---------- ---*------ *--------* ----------
pUK21-A2 (541) CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT
GCGGCGAGCG GTATCAGCTC pGT CTTCCTCGCT CACTGACTCG CTGCGCTCGG
TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (601) ACTCAAAGGC
GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT pGT
ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (661) GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC
CGCGTTGCTG GCGTTTTTCC pGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC
GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (721) ATAGGCTCCG
CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA pGT
ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (781) ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG
AAGCTCCCTC GTGCGCTCTC pGT ACCCGACAGG ACTATAAAGA TACCAGGCGT
TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (841) CTGTTCCGAC
CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG pGT
CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (901) CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT
GTAGGTCGTT CGCTCCAAGC pGT CGCTTTCTCA TAGCTCACGC TGTAGGTATC
TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (961) TGGGCTGTGT
GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC pGT
TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (1021) GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT
GGCAGCAGCC ACTGGTAACA pGT TGGGCTGTGT GCACGAACCC CCCGTTCAGC
CCGACCGCTG CGCCTTATCC GGTAACTATC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (1081) GGATTAGCAG
AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT pGT
GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (1141) ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT
GCTGAAGCCA GTTACCTTCG pGT ACGGCTACAC TAGAAGAACA GTATTTGGTA
TCTGCGCTCT GCTGAAGCCA GTTACCTTCG ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (1201) GAAAAAGAGT
TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT pGT
GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (1261) GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC
CGCTGGTAGC GGTGGTTTTT pGT GAAAAAGAGT TGGTAGCTCT TGATCCGGCA
AACAAACCAC CGCTGGTAGC GGTGGTTTTT ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (1321) TTTCTACGGG
GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA pGT
TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (1381) GCTTGCGCCG TCCCGTCAAG TCAGCGTAAT GCTCTGCCAG
TGTTACAACC AATTAACCAA pGT GCTTGCGCCG TCCCGTCAAG TCACCGGAAT
GCTCTGCCAG TGTTACAACC AATTAACCAA ---------- ---------- ---*--*---
---------- ---------- ---------- pUK21-A2 (1441) TTCTGATTAG
AAAAACTCAT CGAGCATCAA ATGAAACTGC AATTTATTCA TATCAGGATT pGT
TTCTGATTAG AAAAACTCAT CCAGCATCAA ATGAAACTGC AATTTATTCA TATCAGGATT
---------- ---------- -*-------- ---------- ---------- ----------
pUK21-A2 (1501) ATCAATACCA TATTTTTGAA AAAGCCGTTT CTGTAATGAA
GGAGAAAACT CACCGAGGCA pGT ATCAATACCA TATTTTTGAA AAAGCCGTTT
CTGTAATGAA GGAGAAAACT CACCGAGGCA ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (1561) GTTCCATAGG
ATGGCAAGAT CCTGGTATCG GTCTGCGATT CCGACTCGTC CAACATCAAT * pGT
GTTCCATAGG ATGGCAAGAT CCTGGTATCG GTCTGCAATT CCGACTCGGC CAACATCAAT
---------- ---------- ---------- ------*--- --------*- ----------
pUK21-A2 (1621) ACAACCTATT AATTTCCCCT CGTCAAAAAT AAGGTTATCA
AGTGAGAAAT CACCATGAGT pGT ACAACCTATT AATTTCCCCT CATCAAAAAT
AAGGTTATCA AGTGAGAAAT CACCATGAGT ---------- ---------- -*--------
---------- ---------- ---------- pUK21-A2 (1681) GACGACTGAA
TCCGGTGAGA ATGGCAAAAG TTTATGCATT TCTTTCCAGA CTTGTTCAAC pGT
AACTACTGAA TCCGGTGAGA ATGGCAAAAG TTTATGCATT TCTTTCCAGA CTTGTTCAAC
*--*------ ---------- ---------- ---------- ---------- ----------
pUK21-A2 (1741) AGGCCAGCCA TTACGCTCGT CATCAAAATC ACTCGCATCA
ACCAAACCGT TATTCATTCG pGT AGGCCAGCCA TTACGCTCAT CATCAAAATC
GGAAGCATCA ACCAAACCGT TATTCATTCG ---------- --------*- ----------
***------- ---------- ---------- pUK21-A2 (1801) TGATTGCGCC
TGAGCGAGAC GAAATACGCG ATCGCTGTTA AAAGGACAAT TACAAACAGG pGT
GGATTGAGCC TGAGCCAGAC GGAATACGCG GTCGCTGTTA AAAGGACAAT TACAAACAGG
*-----*--- -----*---- -*-------- *--------- ---------- ----------
pUK21-A2 (1861) AATCGAATGC AACCGGCGCA GGAACACTGC CAGCGCATCA
ACAATATTTT CACCTGAATC pGT AATGGAATGC AACCGGCGGA GGAACACTGC
CAGAGCATCA ACAATATTTT CACCTGAATC ---*------ --------*- ----------
---*------ ---------- ---------- pUK21-A2 (1921) AGGATATTCT
TCTAATACCT GGAATGCTGT TTTTCCGGGG ATCGCAGTGG TGAGTAACCA pGT
AGGATATTCT TCTAATACCT GGAATGCTGT TTTTCCGGGG ATAGCAGTGG TGAGTAACCA
---------- ---------- ---------- ---------- --*------- ----------
pUK21-A2 (1981) TGCATCATCA GGAGTACGGA TAAAATGCTT GATGGTCGGA
AGAGGCATAA ATTCCGTCAG pGT TGCATCATCA GGAGTACGGA TAAAATGCTT
GATGGTCGGA AGAGGCATAA ATTCCGTCAG ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (2041) CCAGTTTAGT
CTGACCATCT CATCTGTAAC ATCATTGGCA ACGCTACCTT TGCCATGTTT pGT
CCAGTTTAGT CTGACCATCT CATCTGTAAC ATCATTGGCA ACGCTACCTT TGCCATGTTT
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (2101) CAGAAACAAC TCTGGCGCAT CGGGCTTCCC ATACAAGCGA
TAGATTGTCG CACCTGATTG pGT CAGAAACAAC TCCGGCGCGT CGGGCTTCCC
ATACAAGCGG TAGATTGTAG CACCTGATTG ---------- --*-----*- ----------
---------* --------*- ---------- pUK21-A2 (2161) CCCGACATTA
TCGCGAGCCC ATTTATACCC ATATAAATCA GCATCCATGT TGGAATTTAA pGT
CCCGACATTA TCGCGAGCCC ATTTATACCC ATATAAATCA GCATCCATGT TGGAATTTAA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (2221) TCGCGGCCTC GACGTTTCCC GTTGAATATG GCTCATAACA
CCCCTTGTAT TACTGTTTAT pGT TCGCGGCCTG GAGGTTTCCC GTTGAATATG
GCTCATAACA CCCCTTGTAT TACTGTTTAT ---------* --*------- ----------
---------- ---------- ---------- pUK21-A2 (2281) GTAAGCAGAC
AGTTTTATTG TTCATGATGA TATATTTTTA TCTTGTGCAA TGTAACATCA pGT
GTAAGCAGAC AGTTTTATTG TTCATGATGA TATATTTTTA TCTTGTGCAA TGTAACATCA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (2341) GAGATTTTGA GACACAACGT GGCTTTCCCC CCCCCCCCCA
TGACATTAAC CTATAAAAAT pGT GAGATTTTGA GACACACCGG GGCTTTCCCC
CCCCCCCCCA TGACATTAAC CTATAAAAAT ---------- ------*--* ----------
---------- ---------- ---------- pUK21-A2 (2401) AGGCGTATCA
CGAGGCCCTT TCGTCTCGCG CGTTTCGGTG ATGACGGTGA AAACCTCTGA pGT
AGCCGTATCC CGAGGCCCTT CCGTCTCGCG CGTTCCGGTG ATGCCGGTGA AAACCTCTGA
--*------* ---------- *--------- ----*----- ---*------ ----------
pUK21-A2 (2461) CACATGCAGC TCCCGGAGAC GGTCACAGCT TGTCTGTAAG
CGGATGCCGG GAGCAGACAA pGT CACATGCAGC TCCCGGAGAC GGTCACAGCT
TGTCTGTAAG CGGATGCCGG GAGCAGACAA ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (2521) GCCCGTCAGG
GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA pGT
GCCCGTCAGG GCGCGTCAGC GGGTGTTGGC GGGTGTCGGG GCTGGCTTAA CTATGCGGCA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (2581) TCAGAGCAGA TTGTACTGAG AGTGCACCAT AAAATTGTAA
ACGTTAATAT TTTGTTAAAA pGT TCAGAGCAGA TTGTACTGAG AGTGCACCAT
AAAATTGTAA CCGTTAATAT TTTGTTAAAA ---------- ---------- ----------
---------- *--------- ---------- pUK21-A2 (2641) TTCGCGTTAA
ATTTTTGTTA AATCAGCTCA TTTTTTAACC AATAGACCGA AATCGGCAAA pGT
TTCGCGTTAA ATTTTTGTTA AATCAGCTCA TTTTTTAACC AATAGACCGA AATCGGCAAA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (2701) ATCCCTTATA AATCAAAAGA ATAGCCCGAG ATAGAGTTGA
GTGTTGTTCC AGTTTGGAAC pGT ATCCCTTATA AATCAAAAGA ATAGCCCGAG
ATAGAGTTGA GTGTTGTTCC AGTTTGGAAC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (2761) AAGAGTCCAC
TATTAAACAA CGTGGACTCC AACGTCAAAG CGCGAAAAAC CGTCTATCAG pGT
AAGAGTCCAC TATTAAAGAC CGTGGACTCC ACCGTCAAAG GCCGAAAAAC CGTCTATCAG
---------- ---------* ---------- -*-------- -*-------- ----------
pUK21-A2 (2821) GGCGATGGCC CACCCCGATT TAGAGCTTGA CGGGGAAAGC
CGGCGAACGT GGCGAGAAAG pGT GCCGATGGCC CACCCCGATT TAGAGCTTGA
CGGGGAAAGC CGGCGCGCGT GCCGAGAAAG -*-------- ---------- ----------
---------- -----**--- -*-------- pUK21-A2 (2881) GAAGGGAAGA
AAGCGAAAGG AGCGGGCGCT AAGGCGCTGG CAAGTGTAGC GGTCACGCTG pGT
GAAGGGAAGA AACCGAAAGG AGCGGCCGCT AAGCCGCTGG CAAGTGTAGC GGTCCCGCTG
---------- --*------- -----*---- ---*------ ---------- ----*-----
pUK21-A2 (2941) CGCGTAACCA CCACACCCGC CGCGCTTAAT GCGCCGCTAC
AGGGCGCGTA CTATGGTTGC pGT CGCGTAACCA CCACACCCGC CGCGCTTAAT
CCGCCGCTAC AGGGCGCGTA CTATGGTTGC ---------- ---------- ----------
*--------- ---------- ---------- pUK21-A2 (3001) TTTGACGTAT
GCGGTGTGAA ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGC pGT
TTTGCCGTAT GCGGTGTGAA ATACCGCACA GATCCGTAAG GAGAAAATAC CGCATCAGCC
----*----- ---------- ---------- ---*------ ---------- --------*-
pUK21-A2 (3061) GCCATTCGCC ATTCAGGCTG CGCAACTGTT GGGAAGGGCG
ATCGGTGCGG GCCTCTTCGC pGT GCCATCCGCC ATTCAGGCTC CGCAACTGTT
GGGAAGGCCG ATCGGTGCGG GCCTCTCCGC -----*---- ---------* ----------
-------*-- ---------- ------*--- pUK21-A2 (3121) TATTACGCCA
GCTGGCGAAA GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG pGT
TATTCCGCCA GCTGCCGAAA GGGGGATGTG CTGCAAGCCG ATTAAGTTGG GTACCGCCAG
----*----- ----*----- ---------- -------*-- ---------- ---*------
pUK21-A2 (3181) GGTTTTCCCA GTCACGACGT TGTAAAACGA CGGCCAGTGA
ATTGTAATAC GACTCACTAT pGT GGTTTTCCCA GTCACGGCGG TGTAAACCGA
CGGCCAGTGA ATTGTAATCC GACTCACTAT ---------- ------*--* ------*---
---------- --------*- ---------- pUK21-A2 (3241) AGGGCGAATT
GGGGATCGAT CCACTAGTTC TAGATCCGAT GTACGGGCCA GATATACGCG pGT
AGGCCGAATT GGGGACCGAT CCACTAGTTC TAGATCCGAT GTACGGGCCA GATATACGCG
---*------ -----*---- ---------- ---------- ---------- ----------
pUK21-A2 (3301) TTGACATTGA TTATTGACTA GTTATTAATA GTAATCAATT
ACGGGGTCAT TAGTTCATAG pGT TTGACATTGA TTATTGACTA GTTATTAATA
GTAATCAATT ACGGGGTCAT TAGTTCATAG ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (3361) TTGACATTGA
TTATTGACTA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG pGT
TTGACATTGA TTATTGACTA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (3421) CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT
CCCATAGTAA CGCCAATAGG pGT CAACGACCCC CGCCCATTGA CGTCAATAAT
GACGTATGTT CCCATAGTAA CGCCAATAGG ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (3481) GACTTTCCAT
TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA pGT
GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (3541) TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC
AATGACGGTA AATGGCCCGC pGT TCAAGTGTAT CATATGCCAA GTACGCCCCC
TATTGACGTC AATGACGGTA AATGGCCCGC ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (3601) CTGGCATTAT
GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT pGT
CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT
---------- ---------- ---------- ---------- ---------- ----------
pUK21-A2 (3661) ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG
TACATCAATG GGCGTGGATA pGT ATTAGTCATC GCTATTACCA TGGTGATGCG
GTTTTGGCAG TACATCAATG GGCGTGGATA ---------- ---------- ----------
---------- ---------- ---------- pUK21-A2 (3721) GCGGTTTGAC
TCACGGGGAT
TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT pGT GCGGTTTGAC
TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT ----------
---------- ---------- ---------- ---------- ---------- pUK21-A2
(3781) TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC
CATTGACGCA pGT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC
AACTCCGCCC CATTGACGCA ---------- ---------- ---------- ----------
---------- ---------- pUK21-A2 (3841) AATGGGCGGT AGGCGTGTAC
GGTGGGAGGT CTATATAAGC AGAGCTCTCT GGCTAACTAG pGT AATGGGCGGT
AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTCTCT GGCTAACTAG ----------
---------- ---------- ---------- ---------- ---------- pUK21-A2
(3901) AGAACCCACT GCTTACTGGC TTATCGAAAT TGCGGCCGCC ACGGCGATAT
CGGATCCATA pGT AGAACCCACT GCTTACTGGC TTATCGAAAT TGCGGCCGCC
ACGGCGATAT CGGATCCATA ---------- ---------- ---------- ----------
---------- ---------- pUK21-A2 (3961) TGACGTCGAC GCGTCTGCAG AAGCTTC
pGT TGACGTCGAC GCGTCTGCAG AAGCTTC ---------- ----------
----------
[0194]
6TABLE 6 ODN used with plasmid DNA ODN code Backbone number
Sequence S-ODN 1826 TCCATGACGTTCCTGACGTT 1628 GGGGTCAACGTTGAGGGGGG
1911 TCCAGGACTTTCCTCAGGTT 1982 TCCAGGACTTCTCTCAGGTT 2017
CCCCCCCCCCCCCCCCCCCC O-ODN 2061 TCCATGACGTTCCTGACGTT 2001
GGCGGCGGCGGCGGCGGCGG SOS-ODN 1980 TCCATGACGTTCCTGACGTT 1585
GGGGTCAACGTTGAGGGGGG 1844 TCTCCCAGCGTGCGCCATAT 1972
GGGGTCTGTGCTTTTGGGGGG 2042 TCAGGGGTGGGGGGAACCTT 1981
GGGGTTGACGTTTTGGGGGG 2018 TCTAGCGTTTTTAGCGTTCC 2021
TCGTCGTTGTCGTTGTCGTT 2022 TCGTCGTTTTGTCGTTTTGTCGTT 2023
TCGTCGTTGTCGTTTTGTCGTT Note: (SEQ ID NO: 51-67, respectively)
[0195] SOS-ODN had two S-linkages at the 5' end, five S-linkages at
the 3' end, and O-linkages in between.
[0196] Three ODN used in this study were of the same
murine-specific immunostimulatory sequence in three different
backbones (1826, 2061 and 1980).
[0197] All ODN were synthesized by Hybridon (Milford, Mass.) or
Operon (Alameda, Calif.). ODN were ethanol precipitated and
resuspended in saline prior to use alone or as an additive to the
plasmid DNA solution.
7TABLE 7 Genomic frequencies of selected hexamers Genomic frequency
(.times.10.sup.-3) Adenovirus Adenovirus hexamer: Type 2 Type 12 E.
coli Human GCGCGC 1.614 0.498 0.462 0.153 GCGGCG 1.530 0.469 0.745
0.285 GGCGGC 1.419 0.440 0.674 0.388 CGCGCG 1.336 0.322 0.379 0.106
GCCGCC 1.280 0.410 0.466 0.377 CGCCGC 1.252 0.410 0.623 0.274
GACGTT 0.083 0.234 0.263 0.068 AACGTT 0.056 0.205 0.347 0.056
(CpG-S) The frequencies of hexamers in adenoviral and E. coli
genomes were kindly provided by J. Han (University of Alabama,
Birmingham), who also determined those for the human genome.sup.52.
The hexamer frequencies in type 5 adenovirus are essentially
identical to those in type 2, and are therefore not shown. The last
two hexamers are CpG-S motifs shown for comparison and are the most
stimulatory of all tested CpG-S motifs. Note that the expected
frequency of a randomly selected hexamer is 1/4096 = 0.244 .times.
10.sup.-3.
[0198]
8TABLE 8 Genomic DNA from type 12 but not type 2 adenovirus
stimulates cytokine secretion from human PBMC Experiment 1.sup.1
Experiment 2.sup.1 TNF-.alpha. IL-6 TNF-.alpha. IL-6 Cells 27 800
30 800 EC 3 .mu.g/ml 235 26,500 563 34,000 CT 10 .mu.g/ml 0 1,400 0
2,800 Adv 2; 3 .mu.g/ml 15.6 900 30 1,900 Adv 12; 3 .mu.g/ml 86
11,300 120 11,250 .sup.1PBMC were obtained from normal human donors
and cultured at 1 .times. 10.sup.5 cells/200 .mu.l in RPMI with 10%
autologous serum for 4 hr (TNF-.alpha. assay) or 24 hr (IL-6
assay). The level of cytokine present in culture supernatants was
determined by ELISA (pg/ml). Adv = adenovirus serotype
[0199]
9TABLE 9 Adenoviral type 5 DNA suppresses the cytokine response to
EC DNA by human PBMC IL-6 DNA Source (pg/ml).sup.1
IFN-(pg/ml).sup.1 TNF-(pg/ml).sup.1 EC DNA (50 .mu.g/ml) >3000
700 700 EC DNA (5 .mu.g/ml) >3000 400 675 EC DNA (0.5 .mu.g/ml)
>3000 200 350 EC DNA (0.05 .mu.g/ml) 3000 ND 100 Adenoviral DNA
(50 .mu.g/ml) 2500 0 0 Adenoviral DNA (5 .mu.g/ml) 1500 0 0 EC:
Adeno DNA (50:50 .mu.g/ml) 2000 35 675 EC: Adeno DNA (5:5 .mu.g/ml)
1500 40 ND .sup.1Represents the level of cytokine production above
that in wells cultured with cells alone without any DNA. Levels of
cytokines were determined by ELISA using Quantikine kits from
R&D Systems. ND = not done
[0200]
10TABLE 10 Inhibitory CpG motifs can block B cell pro- liferation
induced by a stimulatory CpG motif Oligonucleotide added cpm medium
194 1668 (TCCATGACGTTCCTGATGCT) 34,669 1668 + 1735
(GCGTTTTTTTTTGCG) 24,452 1720 (TCCATGAGCTTCCTGATGCT) 601 1720 +
1735 1109
[0201] Splenic B cells from a DBA/2 mouse were cultured at
5.times.10.sup.4 cells/100 .mu.l well in 96 well microtiter plates
in RPMI as previously described (Krieg, et al., 1995) with or
without the indicated phosphorothioate modified oligonucleotides at
a concentration of 60 ng/ml for 48 hr. The cells were then pulsed
with .sup.3H thymidine, harvested, and the cpm determined by
scintillation counting. The stimulatory CpG oligo 1668 was slightly
but significantly inhibited by the inhibitory motifs in oligo 1735.
The non CpG oligo 1720 is included as a negative control. (SEQ ID
NO:68-70, respectively).
11TABLE 11 Inhibitory effects of "bad" CpG motifs on the "good"
Oligo 1619 Note: The sequence of oligo 1619 is TCCATGTCGTTCCTGATGCT
1949 has only 1 GCG at the 3' end, which has essentially no
inhibitory activity Oligonucleotide added IL-12 in pg/ml medium 0
1619 alone 6 1619 + 1949 (TCCATGTCGTTCCTGATGCG) 16 1619 + 1952
(TCCATGTCGTTCCGCGCGCG) 0 1619 + 1953 (TCCATGTCGTTCCTGCCGCT) 0 1619
+ 1955 (GCGGCGGGCGGCGCGCGCCC) 0 Human PBMC were cultured in 96 well
microtiter plates at 10.sup.5/200 .mu.l for 24 hr in RPMI
containing 10% autologous serum. Supernatants were collected at the
end of the culture and tested for IL-12 by ELISA. All wells except
the control (medium) contained 60 .mu.g/ml of the stimulatory CpG
oligodeoxynucleotide 1619; stimulatory (1949) and inhibitory (all
other sequences have a strong inhibitory motif) oligos were added
to the #indicated wells at the same concentration at the beginning
of culture. All oligos have unmodified backbones.
[0202]
12TABLE 12 Effect of CpG-S ODN adjuvant on anti-HBs response in
mice immunized with HBsAg-expressing DNA vaccine (pCMV-S):
comparison of mixed formulation with temporal or spatial separation
of plasmid DNA and ODN CpG ODN Site and Time Relative (100 .mu.g)
to DNA vaccine Anti-HBs Titer Sequence Backbone (pCMV-S, 10 .mu.g)
at 12 wk None -- -- 6 379 .+-. 2 126 1826O O-ODN Mixed together
(same time, 4 395 .+-. 1 390 same muscle)
[0203]
13TABLE 13 Identification of neutralizing CpG motifs which reduce
the induction of cytokine secretion by a CpG-S motif in the same
ODN (cis-neutrali- zation) ODN-induced cytokine expression.sup.2
ODN sequence 5'-3'.sup.1 IL-6.sup.2 IL-12 IFN-.gamma. None <5
206 898 1619 TCCATGTCGTTCCTGATGCT 1405 3130 4628 1952
.............GCGCGCG 559 1615 2135 1953 ...............CC... 577
1854 2000 .sup.1Dots in the sequence of ODN 1952 and 1953 indicate
identity to ODN 1619; CpG dinucleotides are underlined for clarity.
ODN without CpG-N or CpG-S motifs had little or no effect on
cytokine production. The data shown are representative of 4
experiments. .sup.2All cytokines are given in pg/ml; measured by
ELISA on supernatants from DBA/2 spleen cells cultured in 96 well
plates at 2 .times. 10.sup.7 cells/ml for 24 hr with the indicated
ODN at 30 .mu.g/ml. Std. dev. of the triplicate wells was <7%.
None of the ODN induced significant amounts of IL-5.
[0204]
14TABLE 14 Inhibition of CpG-induced cytokine secretion by ODN
containing CpG-N motifs CpG-S-induced IL-12 IL-12 ODN sequence
5'-3' secretion.sup.1 secretion.sup.2 none 268 5453 1895
GCGCGCGCGCGCGCGCGCGC 123 2719 1896 CCGGCCGGCCGGCCGGCCGG 292 2740
1955 GCGGCGGGCGGCGCGCGCCC 270 2539 2037 TCCATGCCGTTCCTGCCGTT 423
2847 .sup.1BALB/c spleen cells were cultured in 96 well plates at 2
.times. 10.sup.7 cells/ml with the indicated ODN for 24 hr and then
the supernatants were assayed for IL-12 by ELISA (pg/ml).
.sup.2Cells were set up the same as in .sup.1 except that IL-12
secretion was induced by the addition of the CpG ODN 1619
(TCCATGACGTTCCTGATGCT) at 30 .mu.g/ml. The data shown are
representative of 5 experiments.
[0205] All references cited herein are hereby incorporated by
reference in their entirety. DNA vaccines given intramuscular:
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[0310] A number of embodiments of the present invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
84 1 20 DNA Artificial Sequence synthetic oligonucleotide 1
tccatgtcgt tcctgtcgtt 20 2 19 DNA Artificial Sequence synthetic
oligonucleotide 2 tcctgacgtt cctgacgtt 19 3 24 DNA Artificial
Sequence synthetic oligonucleotide 3 tcgtcgtttt gtcgttttgt cgtt 24
4 30 DNA Artificial Sequence synthetic oligonucleotide 4 ccgtggatat
ccgatgtacg ggccagatat 30 5 32 DNA Artificial Sequence synthetic
oligonucleotide 5 agtcgcggcc gcaatttcga taagccagta ag 32 6 35 DNA
Artificial Sequence synthetic oligonucleotide 6 attctcgagt
ctagactaga gctcgctgat cagcc 35 7 29 DNA Artificial Sequence
synthetic oligonucleotide 7 attaggcctt ccccagcatg cctgctatt 29 8 37
DNA Artificial Sequence synthetic oligonucleotide 8 tataggccct
attttaaacg cgccctgtag cggcgca 37 9 38 DNA Artificial Sequence
synthetic oligonucleotide 9 ctatggcgcc ttgggcccaa tttttgttaa
atcagctc 38 10 28 DNA Artificial Sequence synthetic oligonucleotide
10 aaattcgaaa gtactggacc tgttaaca 28 11 30 DNA Artificial Sequence
synthetic oligonucleotide 11 cgtgttaaca ggtccagtac tttcgaattt 30 12
44 DNA Artificial Sequence synthetic oligonucleotide 12 gactccatga
cgttcctgac gtttccatga cgttcctgac gttg 44 13 44 DNA Artificial
Sequence synthetic oligonucleotide 13 gtccaacgtc aggaacgtca
tggaaacgtc aggaacgtca tgga 44 14 54 DNA Artificial Sequence
synthetic oligonucleotide 14 gacttcgtgt cgttcttctg tcgtctttag
cgcttctcct gcgtgcgtcc cttg 54 15 52 DNA Artificial Sequence
synthetic oligonucleotide 15 gactcgtcgt tttgtcgttt tgtcgtttcg
tcgttttgtc gttttgtcgt tg 52 16 29 DNA Artificial Sequence synthetic
oligonucleotide 16 gccctagtac tgttaacttt aaagggccc 29 17 29 DNA
Artificial Sequence synthetic oligonucleotide 17 ggcgggccct
ttaaagttaa cagtactag 29 18 48 DNA Artificial Sequence synthetic
oligonucleotide 18 gccctggcgg ggataaggcg gggatttggc gggggataag
gcggggaa 48 19 45 DNA Artificial Sequence synthetic oligonucleotide
19 ggcccccgcc ttatccccgc caaatccccg ccttatcccc gccag 45 20 38 DNA
Artificial Sequence synthetic oligonucleotide 20 gccctatttt
aaattcgaaa gtactggacc tgttaaca 38 21 37 DNA Artificial Sequence
synthetic oligonucleotide 21 cgtgttaaca ggtccagtac tttcgaattt
aaaatag 37 22 20 DNA Artificial Sequence synthetic oligonucleotide
22 cgcgcgcgcg cgcgcgcgcg 20 23 29 DNA Artificial Sequence synthetic
oligonucleotide 23 gtctctagac agccactggt aacaggatt 29 24 29 DNA
Artificial Sequence synthetic oligonucleotide 24 gtcgttgtgt
cgtcaagtca gcgtaatgc 29 25 20 DNA Artificial Sequence synthetic
oligonucleotide 25 tcgtttctgt aatgaaggag 20 26 20 DNA Artificial
Sequence synthetic oligonucleotide 26 aaggcagttc cataggatgg 20 27
35 DNA Artificial Sequence synthetic oligonucleotide 27 tcgatctgcg
attccaactc gtccaacatc aatac 35 28 20 DNA Artificial Sequence
synthetic oligonucleotide 28 tggtgagaat ggcaaaagtt 20 29 21 DNA
Artificial Sequence synthetic oligonucleotide 29 cattattcat
tcgtgattgc g 21 30 24 DNA Artificial Sequence synthetic
oligonucleotide 30 acgtctcagg aacactgcca gcgc 24 31 20 DNA
Artificial Sequence synthetic oligonucleotide 31 agggatcgca
gtggtgagta 20 32 21 DNA Artificial Sequence synthetic
oligonucleotide 32 tataaaatgc ttgatggtcg g 21 33 35 DNA Artificial
Sequence synthetic oligonucleotide 33 gggaagaggc ataaattctg
tcagccagtt tagtc 35 34 20 DNA Artificial Sequence synthetic
oligonucleotide 34 tggcttccca tacaagcgat 20 35 20 DNA Artificial
Sequence synthetic oligonucleotide 35 tacattatcg cgagcccatt 20 36
19 DNA Artificial Sequence synthetic oligonucleotide 36 tggcctcgac
gtttcccgt 19 37 29 DNA Artificial Sequence synthetic
oligonucleotide 37 atcgaattca gggcctcgtg atacgccta 29 38 39 DNA
Artificial Sequence synthetic oligonucleotide 38 tgacttgacg
acacaacgac agctcatgac caaaatccc 39 39 39 DNA Artificial Sequence
synthetic oligonucleotide 39 ctccttcatt acagaaacga ctttttcaaa
aatatggta 39 40 37 DNA Artificial Sequence synthetic
oligonucleotide 40 ccatcctatg gaactgcctt ggtgagtttt ctccttc 37 41
34 DNA Artificial Sequence synthetic oligonucleotide 41 gagttggaat
cgcagatcga taccaggatc ttgc 34 42 37 DNA Artificial Sequence
synthetic oligonucleotide 42 aacttttgcc attctcacca gattcagtcg
tcactca 37 43 39 DNA Artificial Sequence synthetic oligonucleotide
43 cgcaatcacg aatgaataat ggtttggttg atgcgagtg 39 44 38 DNA
Artificial Sequence synthetic oligonucleotide 44 tggcagtgtt
cctgagacgt ttgcattcga ttcctgtt 38 45 38 DNA Artificial Sequence
synthetic oligonucleotide 45 tactcaccac tgcgatccct ggaaaaacag
cattccag 38 46 39 DNA Artificial Sequence synthetic oligonucleotide
46 ccgaccatca agcattttat acgtactcct gatgatgca 39 47 39 DNA
Artificial Sequence synthetic oligonucleotide 47 cagaatttat
gcctcttccc accatcaagc attttatac 39 48 38 DNA Artificial Sequence
synthetic oligonucleotide 48 atcgcttgta tgggaagcca gatgcgccag
agttgttt 38 49 37 DNA Artificial Sequence synthetic oligonucleotide
49 aatgggctcg cgataatgta gggcaatcag gtgcgac 37 50 38 DNA Artificial
Sequence synthetic oligonucleotide 50 acgggaaacg tcgaggccac
gattaaattc caacatgg 38 51 20 DNA Artificial Sequence synthetic
oligonucleotide 51 tccatgacgt tcctgacgtt 20 52 20 DNA Artificial
Sequence synthetic oligonucleotide 52 ggggtcaacg ttgagggggg 20 53
20 DNA Artificial Sequence synthetic oligonucleotide 53 tccaggactt
tcctcaggtt 20 54 20 DNA Artificial Sequence synthetic
oligonucleotide 54 tccaggactt ctctcaggtt 20 55 20 DNA Artificial
Sequence synthetic oligonucleotide 55 cccccccccc cccccccccc 20 56
20 DNA Artificial Sequence synthetic oligonucleotide 56 tccatgacgt
tcctgacgtt 20 57 20 DNA Artificial Sequence synthetic
oligonucleotide 57 ggcggcggcg gcggcggcgg 20 58 20 DNA Artificial
Sequence synthetic oligonucleotide 58 tccatgacgt tcctgacgtt 20 59
20 DNA Artificial Sequence synthetic oligonucleotide 59 ggggtcaacg
ttgagggggg 20 60 20 DNA Artificial Sequence synthetic
oligonucleotide 60 tctcccagcg tgcgccatat 20 61 21 DNA Artificial
Sequence synthetic oligonucleotide 61 ggggtctgtg cttttggggg g 21 62
20 DNA Artificial Sequence synthetic oligonucleotide 62 tcaggggtgg
ggggaacctt 20 63 20 DNA Artificial Sequence synthetic
oligonucleotide 63 ggggttgacg ttttgggggg 20 64 20 DNA Artificial
Sequence synthetic oligonucleotide 64 tctagcgttt ttagcgttcc 20 65
20 DNA Artificial Sequence synthetic oligonucleotide 65 tcgtcgttgt
cgttgtcgtt 20 66 24 DNA Artificial Sequence synthetic
oligonucleotide 66 tcgtcgtttt gtcgttttgt cgtt 24 67 22 DNA
Artificial Sequence synthetic oligonucleotide 67 tcgtcgttgt
cgttttgtcg tt 22 68 20 DNA Artificial Sequence synthetic
oligonucleotide 68 tccatgacgt tcctgatgct 20 69 15 DNA Artificial
Sequence synthetic oligonucleotide 69 gcgttttttt ttgcg 15 70 20 DNA
Artificial Sequence synthetic oligonucleotide 70 tccatgagct
tcctgatgct 20 71 20 DNA Artificial Sequence synthetic
oligonucleotide 71 tccatgtcgt tcctgatgct 20 72 20 DNA Artificial
Sequence synthetic oligonucleotide 72 tccatgtcgt tcctgatgcg 20 73
20 DNA Artificial Sequence synthetic oligonucleotide 73 tccatgtcgt
tccgcgcgcg 20 74 20 DNA Artificial Sequence synthetic
oligonucleotide 74 tccatgtcgt tcctgccgct 20 75 20 DNA Artificial
Sequence synthetic oligonucleotide 75 gcggcgggcg gcgcgcgccc 20 76
20 DNA Artificial Sequence synthetic oligonucleotide 76 gcgcgcgcgc
gcgcgcgcgc 20 77 20 DNA Artificial Sequence synthetic
oligonucleotide 77 ccggccggcc ggccggccgg 20 78 20 DNA Artificial
Sequence synthetic oligonucleotide 78 tccatgccgt tcctgccgtt 20 79
20 DNA Artificial Sequence synthetic oligonucleotide 79 tccatgacgt
tcctgatgct 20 80 1360 DNA Artificial Sequence plasmid DNA wild-type
Kanamycin resistance gene 80 aagggcctcg tgatacgcct atttttatag
gttaatgtca tggggggggg ggggaaagcc 60 acgttgtgtc tcaaaatctc
tgatgttaca ttgcacaaga taaaaatata tcatcatgaa 120 caataaaact
gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac 180
gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
240 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
gggaagcccg 300 atgcgccaga gttgtttctg aaacatggca aaggtagcgt
tgccaatgat gttacagatg 360 agatggtcag actaaactgg ctgacggaat
ttatgcctct tccgaccatc aagcatttta 420 tccgtactcc tgatgatgca
tggttactca ccactgcgat ccccggaaaa acagcattcc 480 aggtattaga
agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc 540
tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
600 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
gattttgatg 660 acgagcgtaa tggctggcct gttgaacaag tctggaaaga
aatgcataaa cttttgccat 720 tctcaccgga ttcagtcgtc actcatggtg
atttctcact tgataacctt atttttgacg 780 aggggaaatt aataggttgt
attgatgttg gacgagtcgg aatcgcagac cgataccagg 840 atcttgccat
cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt 900
ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
960 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
attacgctga 1020 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
cgtgagtttt cgttccactg 1080 agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt ttctgcgcgt 1140 aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 1200 agagctacca
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 1260
tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
1320 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1360 81 1360 DNA
Artificial Sequence plasmid DNA mutant Kanamycin resistance gene 81
aagggcctcg tgatacgcct atttttatag gttaatgtca tggggggggg ggggaaagcc
60 acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
tcatcatgaa 120 caataaaact gtctgcttac ataaacagta atacaagggg
tgttatgagc catattcaac 180 gggaaacgtc gaggccacga ttaaattcca
acatggatgc tgatttatat gggtataaat 240 gggctcgcga taatgtaggg
caatcaggtg cgacaatcta tcgcttgtat gggaagccag 300 atgcgccaga
gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg 360
agatggtcag actaaactgg ctgacagaat ttatgcctct tcccaccatc aagcatttta
420 tacgtactcc tgatgatgca tggttactca ccactgcgat ccctggaaaa
acagcattcc 480 aggtattaga agaatatcct gattcaggtg aaaatattgt
tgatgcgctg gcagtgttcc 540 tgagacgttt gcattcgatt cctgtttgta
attgtccttt taacagcgat cgcgtatttc 600 gtctcgctca ggcgcaatca
cgaatgaata atggtttggt tgatgcgagt gattttgatg 660 acgagcgtaa
tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat 720
tctcaccaga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
780 aggggaaatt aataggttgt attgatgttg gacgagttgg aatcgcagat
cgataccagg 840 atcttgccat cctatggaac tgccttggtg agttttctcc
ttcattacag aaacgacttt 900 ttcaaaaata tggtattgat aatcctgata
tgaataaatt gcagtttcat ttgatgctcg 960 atgagttttt ctaatcagaa
ttggttaatt ggttgtaaca ctggcagagc attacgctga 1020 cttgacgaca
caacgacagc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 1080
agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
1140 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
tgccggatca 1200 agagctacca actctttttc cgaaggtaac tggcttcagc
agagcgcaga taccaaatac 1260 tgttcttcta gtgtagccgt agttaggcca
ccacttcaag aactctgtag caccgcctac 1320 atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc 1360 82 269 PRT Artificial Sequence mutant
Kanamycin resistance gene 82 Met Ser His Ile Gln Arg Glu Thr Ser
Arg Pro Arg Leu Asn Ser Asn 1 5 10 15 Met Asp Ala Asp Leu Tyr Gly
Tyr Lys Trp Ala Arg Asp Asn Val Gly 20 25 30 Gln Ser Gly Ala Thr
Ile Tyr Arg Leu Tyr Gly Lys Pro Asp Ala Pro 35 40 45 Glu Leu Phe
Leu Lys His Gly Lys Gly Ser Val Ala Asn Asp Val Thr 50 55 60 Asp
Glu Met Val Arg Leu Asn Trp Leu Thr Glu Phe Met Pro Leu Pro 65 70
75 80 Thr Ile Lys His Phe Ile Arg Thr Pro Asp Asp Ala Trp Leu Leu
Thr 85 90 95 Thr Ala Ile Pro Gly Lys Thr Ala Phe Gln Val Leu Glu
Glu Tyr Pro 100 105 110 Asp Ser Gly Glu Asn Ile Val Asp Ala Leu Ala
Val Phe Leu Arg Arg 115 120 125 Leu His Ser Ile Pro Val Cys Asn Cys
Pro Phe Asn Ser Asp Arg Val 130 135 140 Phe Arg Leu Ala Gln Ala Gln
Ser Arg Met Asn Asn Gly Leu Val Asp 145 150 155 160 Ala Ser Asp Phe
Asp Asp Glu Arg Asn Gly Trp Pro Val Glu Gln Val 165 170 175 Trp Lys
Glu Met His Lys Leu Leu Pro Phe Ser Pro Asp Ser Val Val 180 185 190
Thr His Gly Asp Phe Ser Leu Asp Asn Leu Ile Phe Asp Glu Gly Lys 195
200 205 Leu Ile Gly Cys Ile Asp Val Gly Arg Val Gly Ile Ala Asp Arg
Tyr 210 215 220 Gln Asp Leu Ala Ile Leu Trp Asn Cys Leu Gly Glu Phe
Ser Pro Ser 225 230 235 240 Leu Gln Lys Arg Leu Phe Gln Lys Tyr Gly
Ile Asp Asn Pro Asp Met 245 250 255 Asn Lys Leu Gln Phe His Leu Met
Leu Asp Glu Phe Phe 260 265 83 3987 DNA Artificial Sequence plasmid
pUK21-A2 83 gaattcgagc tcccgggtac catggcatgc atcgatagat ctcgagtcta
gactagagct 60 cgctgatcag cctcgactgt gccttctagt tgccagccat
ctgttgtttg cccctccccc 120 gtgccttcct tgaccctgga aggtgccact
cccactgtcc tttcctaata aaatgaggaa 180 attgcatcgc attgtctgag
taggtgtcat tctattctgg ggggtggggt ggggcaggac 240 agcaaggggg
aggattggga agacaatagc aggcatgctg gggaaggcct cggactagtg 300
gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
360 aacatacgag ccgcggaagc ataaagtgta aagcctgggg tgcctaatga
gtgagctaac 420 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
gggaaacctg tcgtgccagc 480 tgcattaatg aatcggccaa cgcgcgggga
gaggcggttt gcgtattggg cgctcttccg 540 cttcctcgct cactgactcg
ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 600 actcaaaggc
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 660
gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
720 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
aggtggcgaa 780 acccgacagg actataaaga taccaggcgt ttccccctgg
aagctccctc gtgcgctctc 840 ctgttccgac cctgccgctt accggatacc
tgtccgcctt tctcccttcg ggaagcgtgg 900 cgctttctca tagctcacgc
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 960 tgggctgtgt
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 1020
gtcttgagtc
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 1080
ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
1140 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
gttaccttcg 1200 gaaaaagagt tggtagctct tgatccggca aacaaaccac
cgctggtagc ggtggttttt 1260 ttgtttgcaa gcagcagatt acgcgcagaa
aaaaaggatc tcaagaagat cctttgatct 1320 tttctacggg gtctgacgct
cagtggaacg aaaactcacg ttaagggatt ttggtcatga 1380 gcttgcgccg
tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa 1440
ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
1500 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
caccgaggca 1560 gttccatagg atggcaagat cctggtatcg gtctgcgatt
ccgactcgtc caacatcaat 1620 acaacctatt aatttcccct cgtcaaaaat
aaggttatca agtgagaaat caccatgagt 1680 gacgactgaa tccggtgaga
atggcaaaag tttatgcatt tctttccaga cttgttcaac 1740 aggccagcca
ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg 1800
tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
1860 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
cacctgaatc 1920 aggatattct tctaatacct ggaatgctgt ttttccgggg
atcgcagtgg tgagtaacca 1980 tgcatcatca ggagtacgga taaaatgctt
gatggtcgga agaggcataa attccgtcag 2040 ccagtttagt ctgaccatct
catctgtaac atcattggca acgctacctt tgccatgttt 2100 cagaaacaac
tctggcgcat cgggcttccc atacaagcga tagattgtcg cacctgattg 2160
cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
2220 tcgcggcctc gacgtttccc gttgaatatg gctcataaca ccccttgtat
tactgtttat 2280 gtaagcagac agttttattg ttcatgatga tatattttta
tcttgtgcaa tgtaacatca 2340 gagattttga gacacaacgt ggctttcccc
ccccccccca tgacattaac ctataaaaat 2400 aggcgtatca cgaggccctt
tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 2460 cacatgcagc
tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2520
gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
2580 tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat
tttgttaaaa 2640 ttcgcgttaa atttttgtta aatcagctca ttttttaacc
aatagaccga aatcggcaaa 2700 atcccttata aatcaaaaga atagcccgag
atagagttga gtgttgttcc agtttggaac 2760 aagagtccac tattaaagaa
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 2820 ggcgatggcc
caccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2880
gaagggaaga aagcgaaagg agcgggcgct aaggcgctgg caagtgtagc ggtcacgctg
2940 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta
ctatggttgc 3000 tttgacgtat gcggtgtgaa ataccgcaca gatgcgtaag
gagaaaatac cgcatcaggc 3060 gccattcgcc attcaggctg cgcaactgtt
gggaagggcg atcggtgcgg gcctcttcgc 3120 tattacgcca gctggcgaaa
gggggatgtg ctgcaaggcg attaagttgg gtaacgccag 3180 ggttttccca
gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat 3240
agggcgaatt ggggatcgat ccactagttc tagatccgat gtacgggcca gatatacgcg
3300 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
tagttcatag 3360 cccatatatg gagttccgcg ttacataact tacggtaaat
ggcccgcctg gctgaccgcc 3420 caacgacccc cgcccattga cgtcaataat
gacgtatgtt cccatagtaa cgccaatagg 3480 gactttccat tgacgtcaat
gggtggagta tttacggtaa actgcccact tggcagtaca 3540 tcaagtgtat
catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 3600
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
3660 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
ggcgtggata 3720 gcggtttgac tcacggggat ttccaagtct ccaccccatt
gacgtcaatg ggagtttgtt 3780 ttggcaccaa aatcaacggg actttccaaa
atgtcgtaac aactccgccc cattgacgca 3840 aatgggcggt aggcgtgtac
ggtgggaggt ctatataagc agagctctct ggctaactag 3900 agaacccact
gcttactggc ttatcgaaat tgcggccgcc acggcgatat cggatccata 3960
tgacgtcgac gcgtctgcag aagcttc 3987 84 3987 DNA Artificial Sequence
plasmid pGT 84 gaattcgagc tcccgggtac catggcatgc atcgatagat
ctcgagtcta gactagagct 60 cgctgatcag cctcgactgt gccttctagt
tgccagccat ctgttgtttg cccctccccc 120 gtgccttcct tgaccctgga
aggtgccact cccactgtcc tttcctaata aaatgaggaa 180 attgcatcgc
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 240
agcaaggggg aggattggga agacaatagc aggcatgctg gggaaggcct cggactagtg
300 ccggaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
aattccacac 360 aacatccggg ccgcggaagc ataaagtgta aagcctgggg
tgcctaatga gtgagctaac 420 tcacattaat tccgttccgc tcactgcccg
ctttccagtc gggaaacctg ccgtgccagc 480 tgcattaatg aatcggccaa
cgcgcgggga gagccggttt ccgtattggc cgctcttccg 540 cttcctcgct
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 600
actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
660 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
gcgtttttcc 720 ataggctccg cccccctgac gagcatcaca aaaatcgacg
ctcaagtcag aggtggcgaa 780 acccgacagg actataaaga taccaggcgt
ttccccctgg aagctccctc gtgcgctctc 840 ctgttccgac cctgccgctt
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 900 cgctttctca
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 960
tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
1020 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
actggtaaca 1080 ggattagcag agcgaggtat gtaggcggtg ctacagagtt
cttgaagtgg tggcctaact 1140 acggctacac tagaagaaca gtatttggta
tctgcgctct gctgaagcca gttaccttcg 1200 gaaaaagagt tggtagctct
tgatccggca aacaaaccac cgctggtagc ggtggttttt 1260 ttgtttgcaa
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 1320
tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
1380 gcttgcgccg tcccgtcaag tcaccggaat gctctgccag tgttacaacc
aattaaccaa 1440 ttctgattag aaaaactcat ccagcatcaa atgaaactgc
aatttattca tatcaggatt 1500 atcaatacca tatttttgaa aaagccgttt
ctgtaatgaa ggagaaaact caccgaggca 1560 gttccatagg atggcaagat
cctggtatcg gtctgcaatt ccgactcggc caacatcaat 1620 acaacctatt
aatttcccct catcaaaaat aaggttatca agtgagaaat caccatgagt 1680
aactactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
1740 aggccagcca ttacgctcat catcaaaatc ggaagcatca accaaaccgt
tattcattcg 1800 ggattgagcc tgagccagac ggaatacgcg gtcgctgtta
aaaggacaat tacaaacagg 1860 aatggaatgc aaccggcgga ggaacactgc
cagagcatca acaatatttt cacctgaatc 1920 aggatattct tctaatacct
ggaatgctgt ttttccgggg atagcagtgg tgagtaacca 1980 tgcatcatca
ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag 2040
ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
2100 cagaaacaac tccggcgcgt cgggcttccc atacaagcgg tagattgtag
cacctgattg 2160 cccgacatta tcgcgagccc atttataccc atataaatca
gcatccatgt tggaatttaa 2220 tcgcggcctg gaggtttccc gttgaatatg
gctcataaca ccccttgtat tactgtttat 2280 gtaagcagac agttttattg
ttcatgatga tatattttta tcttgtgcaa tgtaacatca 2340 gagattttga
gacacaccgg ggctttcccc ccccccccca tgacattaac ctataaaaat 2400
agccgtatcc cgaggccctt ccgtctcgcg cgttccggtg atgccggtga aaacctctga
2460 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
gagcagacaa 2520 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
gctggcttaa ctatgcggca 2580 tcagagcaga ttgtactgag agtgcaccat
aaaattgtaa ccgttaatat tttgttaaaa 2640 ttcgcgttaa atttttgtta
aatcagctca ttttttaacc aatagaccga aatcggcaaa 2700 atcccttata
aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac 2760
aagagtccac tattaaagac cgtggactcc accgtcaaag gccgaaaaac cgtctatcag
2820 gccgatggcc caccccgatt tagagcttga cggggaaagc cggcgcgcgt
gccgagaaag 2880 gaagggaaga aaccgaaagg agcggccgct aagccgctgg
caagtgtagc ggtcccgctg 2940 cgcgtaacca ccacacccgc cgcgcttaat
ccgccgctac agggcgcgta ctatggttgc 3000 tttgccgtat gcggtgtgaa
ataccgcaca gatccgtaag gagaaaatac cgcatcagcc 3060 gccatccgcc
attcaggctc cgcaactgtt gggaaggccg atcggtgcgg gcctctccgc 3120
tattccgcca gctgccgaaa gggggatgtg ctgcaagccg attaagttgg gtaccgccag
3180 ggttttccca gtcacggcgg tgtaaaccga cggccagtga attgtaatcc
gactcactat 3240 aggccgaatt ggggaccgat ccactagttc tagatccgat
gtacgggcca gatatacgcg 3300 ttgacattga ttattgacta gttattaata
gtaatcaatt acggggtcat tagttcatag 3360 cccatatatg gagttccgcg
ttacataact tacggtaaat ggcccgcctg gctgaccgcc 3420 caacgacccc
cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 3480
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
3540 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
aatggcccgc 3600 ctggcattat gcccagtaca tgaccttatg ggactttcct
acttggcagt acatctacgt 3660 attagtcatc gctattacca tggtgatgcg
gttttggcag tacatcaatg ggcgtggata 3720 gcggtttgac tcacggggat
ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3780 ttggcaccaa
aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 3840
aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
3900 agaacccact gcttactggc ttatcgaaat tgcggccgcc acggcgatat
cggatccata 3960 tgacgtcgac gcgtctgcag aagcttc 3987
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