U.S. patent application number 17/200012 was filed with the patent office on 2021-09-02 for modified serotype 28 adenoviral vectors.
This patent application is currently assigned to GenVec, Inc.. The applicant listed for this patent is GenVec, Inc.. Invention is credited to Douglas E. Brough, C. Richter King, Lisa Wei.
Application Number | 20210269827 17/200012 |
Document ID | / |
Family ID | 1000005594718 |
Filed Date | 2021-09-02 |
United States Patent
Application |
20210269827 |
Kind Code |
A1 |
Wei; Lisa ; et al. |
September 2, 2021 |
MODIFIED SEROTYPE 28 ADENOVIRAL VECTORS
Abstract
The invention provides a replication-deficient serotype 28
adenoviral vector characterized by comprising a portion of a
serotype 45 adenoviral hexon protein and/or a portion of a serotype
45 fiber protein in place of the endogenous serotype 28 hexon
and/or fiber protein.
Inventors: |
Wei; Lisa; (Gaithersburg,
MD) ; Brough; Douglas E.; (Gaithersburg, MD) ;
King; C. Richter; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GenVec, Inc. |
Gaithersburg |
MD |
US |
|
|
Assignee: |
GenVec, Inc.
Gaithersburg
MD
|
Family ID: |
1000005594718 |
Appl. No.: |
17/200012 |
Filed: |
March 12, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15700279 |
Sep 11, 2017 |
10947560 |
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17200012 |
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14403651 |
Nov 25, 2014 |
9790519 |
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PCT/US2013/042824 |
May 28, 2013 |
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15700279 |
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61652407 |
May 29, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2710/10021 20130101; C12N 2810/6018 20130101; C12N 2710/10345
20130101; C12N 2710/10043 20130101; C12N 2710/10343 20130101; C12N
2710/10322 20130101; C12N 15/86 20130101; C12N 2710/16634 20130101;
A61K 48/0008 20130101 |
International
Class: |
C12N 15/86 20060101
C12N015/86; A61K 48/00 20060101 A61K048/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made with Government support under Grant
Number 5R43AI077147-02 awarded by the National Institutes of
Health. The Government has certain rights in this invention.
Claims
1. A replication-deficient serotype 28 adenoviral vector comprising
one or both of the following: (a) at least a portion of an
adenovirus serotype 45 hexon protein in place of at least a portion
of the endogenous serotype 28 hexon protein, and (b) at least a
portion of an adenovirus serotype 45 fiber protein in place of at
least a portion of the endogenous serotype 28 fiber protein.
2. The adenoviral vector of claim 1, which comprises at least a
portion of an adenovirus serotype 45 hexon protein in place of at
least a portion of the endogenous serotype 28 hexon protein, or at
least a portion of an adenovirus serotype 45 fiber protein in place
of at least a portion of the endogenous serotype 28 fiber
protein.
3. The adenoviral vector of claim 1, which comprises at least a
portion of an adenovirus serotype 45 hexon protein in place of at
least a portion of the endogenous serotype 28 hexon protein, and at
least a portion of an adenovirus serotype 45 fiber protein in place
of at least a portion of the endogenous serotype 28 fiber
protein.
4. The adenoviral vector of claim 1, wherein the portion of an
adenovirus serotype 45 hexon protein comprises at least one
hypervariable region (HVR).
5. The adenoviral vector of claim 4, wherein the portion of an
adenovirus serotype 45 hexon protein comprises nine HVRs.
6. The adenoviral vector of claim 1, wherein the portion of an
adenovirus serotype 45 hexon protein comprises the amino acid
sequence of SEQ ID NO: 1, or an amino acid sequence that is at
least 91.4% identical to SEQ ID NO: 1.
7. The adenoviral vector of claim 6, wherein the portion of an
adenovirus serotype 45 hexon protein is encoded by the nucleic acid
sequence of SEQ ID NO: 2.
8. The adenoviral vector of claim 1, wherein the portion of an
adenovirus serotype 45 fiber protein comprises the amino acid
sequence of SEQ ID NO: 3, or an amino acid sequence that is at
least 67% identical to SEQ ID NO: 3.
9. The adenoviral vector of claim 8, wherein the portion of an
adenovirus serotype 45 fiber protein is encoded by the nucleic acid
sequence of SEQ ID NO: 4.
10. The adenoviral vector of claim 1, which comprises: (a) an amino
acid sequence of a serotype 28 adenovirus penton protein, (b) an
amino acid sequence of a serotype 28 adenovirus pIX protein, (c) an
amino acid sequence of a serotype 28 adenovirus p100 protein, (d)
an amino acid sequence of a serotype 28 adenovirus L1 52/55K
protein, or (e) any combination of (a)-(d).
11. The adenoviral vector of claim 1, wherein the adenoviral vector
requires complementation of a deficiency in one or more early
regions of the adenoviral genome for propagation and does not
require complementation of any other deficiency of the adenoviral
genome for propagation.
12. The adenoviral vector of claim 1, wherein the one or more early
regions are selected from the group consisting of the E1 region,
the E2 region, and the E4 region of the adenovirus genome.
13. The adenoviral vector of claim 12, wherein the adenoviral
vector requires complementation of a deficiency in the E1 region of
the adenoviral genome for propagation and does not require
complementation of any other deficiency of the adenoviral genome
for propagation.
14. The adenoviral vector of claim 12, wherein the adenoviral
vector requires complementation of a deficiency in the E1A region
or the E1B region of the adenoviral genome for propagation and does
not require complementation of any other deficiency of the
adenoviral genome for propagation.
15. The adenoviral vector of claim 12, wherein the adenoviral
vector requires at most complementation of a deficiency in the E4
region of the adenoviral genome for propagation and does not
require complementation of any other deficiency of the adenoviral
genome for propagation.
16. The adenoviral vector of claim 12, wherein the adenoviral
vector requires complementation of a deficiency in the E1 region of
the adenoviral genome and a deficiency in the E4 region of the
adenoviral genome for propagation and does not require
complementation of any other deficiency of the adenoviral genome
for propagation.
17. The adenoviral vector of claim 1, which comprises the nucleic
acid sequence of SEQ ID NO: 10.
18. The adenoviral vector of claim 1 further comprising a
transgene.
19. A composition comprising the adenoviral vector of claim 1 and a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of co-pending U.S.
application Ser. No. 15/700,279, filed Sep. 11, 2017, which is a
continuation of U.S. application Ser. No. 14/403,651, filed Nov.
25, 2014, now U.S. Pat. No. 9,790,519, which is a 371 of
International Patent Application No. PCT/US2013/042824, filed May
28, 2013, which claims the benefit of U.S. Provisional Patent
Application No. 61/652,407, filed May 29, 2012.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0003] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 79,166 Byte
ASCII (Text) file named "753312_ST25.TXT," created on Mar. 12,
2021.
BACKGROUND OF THE INVENTION
[0004] In vivo delivery of proteins in biologically relevant forms
and amounts has been an obstacle to drug and vaccine development
for decades. One solution that has proven to be a successful
alternative to traditional protein delivery approaches is the use
of exogenous nucleic acid sequences for production of proteins in
vivo. Gene transfer vectors ideally enter a wide variety of cell
types, have the capacity to accept large nucleic acid sequences,
are safe, and can be produced in quantities required for treating
patients. Viral vectors are gene transfer vectors with these
advantageous properties (see, e.g., Thomas et al., Nature Review
Genetics, 4: 346-358 (2003)). Furthermore, while many viral vectors
are engineered to infect a broad range of cell types, viral vectors
also can be modified to target specific cell types, which can
enhance the therapeutic efficacy of the vector (see, e.g., Kay et
al., Nature Medicine, 7(1): 33-40 (2001)).
[0005] Viral vectors that have been used with some success to
deliver exogenous proteins to mammalian cells for therapeutic
purposes include, for example, Retrovirus (see, e.g.,
Cavazzana-Calvo et al., Science, 288 (5466): 669-672 (2000)),
Lentivirus (see, e.g., Cartier et al., Science, 326: 818-823
(2009)), Adeno-associated virus (AAV) (see, e.g., Mease et al.,
Journal of Rheumatology, 27(4): 692-703 (2010)), Herpes Simplex
Virus (HSV) (see, e.g., Goins et al., Gene Ther., 16(4): 558-569
(2009)), Vaccinia Virus (see, e.g., Mayrhofer et al., J. Virol.,
83(10): 5192-5203 (2009)), and Adenovirus (see, e.g., Lasaro and
Ertl, Molecular Therapy, 17(8): 1333-1339 (2009)).
[0006] Despite their advantageous properties, widespread use of
viral gene transfer vectors is hindered by several factors. In this
respect, certain cells are not readily amenable to gene delivery by
currently available viral vectors. For example, lymphocytes are
impaired in the uptake of adenoviruses (Silver et al., Virology,
165: 377-387 (1988), and Horvath et al., J. Virology, 62(1):
341-345 (1988)). In addition, viral vectors that integrate into the
host cell's genome (e.g., retroviral vectors) have the potential to
cause insertion mutations in oncogenes (see, e.g., Cavazzana-Calvo
et al., supra, and Hacein-Bey-Abina et al., N. Engl. J Med., 348:
255-256 (2003)).
[0007] The use of viral vectors for gene transfer also is impeded
by the immunogenicity of viral vectors. A majority of the U.S.
population has been exposed to wild-type forms of many of the
viruses currently under development as gene transfer vectors (e.g.,
adenovirus). As a result, much of the U.S. population has developed
pre-existing immunity to certain virus-based gene transfer vectors.
Such vectors are quickly cleared from the bloodstream, thereby
reducing the effectiveness of the vector in delivering biologically
relevant amounts of a gene product. Moreover, the immunogenicity of
certain viral vectors prevents efficient repeat dosing, which can
be advantageous for "boosting" the immune system against pathogens
when viral vectors are used in vaccine applications, thereby
resulting in only a small fraction of a dose of the viral vector
delivering its payload to host cells.
[0008] Thus, there remains a need for improved viral vectors that
can be used to efficiently deliver genes to mammalian cells in
vivo. The invention provides such viral vectors.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention provides a replication-deficient serotype 28
adenoviral vector comprising one or both of the following: (a) at
least a portion of an adenovirus serotype 45 hexon protein in place
of at least a portion of the endogenous serotype 28 hexon protein,
and (b) at least a portion of an adenovirus serotype 45 fiber
protein in place of at least a portion of the endogenous serotype
28 fiber protein.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0010] FIG. 1 is a flow diagram of the construction of plasmid
pAC28UL19.H (45) by homologous recombination. Steps 1 through 10
illustrate the homologous recombination between the Ad28 viral
vector genome plasmid and the shuttle plasmid containing the Ad45
hexon followed by subsequent recombination containing the
CMVTetO.UL19 expression cassette as described in Example 1.
[0011] FIG. 2 is a graph depicting experimental data of CD8+ T-cell
responses induced by serotype 5 and 28 adenoviral vectors encoding
an HSV2 UL19 protein (Ad5 UL19 and Ad28 UL19, respectively), as
well as the mutant serotype 28 adenoviral vector encoding an HSV2
UL19 protein (Ad28 H/F UL19) described in Example 2.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The invention is predicated, at least in part, on the
generation of an adenoviral vector based on a serotype 28
adenovirus (Ad28) containing one or more modified capsid proteins,
which can effectively be used to deliver and express nucleic acid
sequences encoding therapeutic proteins. It is believed that a
vector based on Ad28 will provide improved delivery to human cells
because Ad28 seroprevalence is low in human populations. It is also
believed that a serotype 28 adenoviral vector containing a modified
hexon protein and/or a modified fiber protein as described herein
can stimulate a more robust T-cell response than an Ad28 vector
comprising wild-type hexon and fiber proteins when used in vaccine
applications.
[0013] A serotype 28 adenovirus is a member of the group D
adenoviruses. In addition to Ad28, the group D adenoviruses include
the following serotypes: 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24,
25, 26, 27, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47,
48, 49, 51, 53, 54, and 56 (see, e.g., Gema et al., J. Infectious
Diseases, 145(5): 678-682 (1982), and Robinson et al., Virology,
409(2): 141-147 (2011)). The genome of human Ad28 has been
sequenced and is available via the GenBank database (Accession No.
FJ824826.1).
[0014] The term "adenoviral vector," as used herein, refers to an
adenovirus in which the adenoviral genome has been manipulated to
accommodate a nucleic acid sequence that is non-native with respect
to the adenoviral genome. Adenovirus is a medium-sized (90-100 nm),
nonenveloped icosohedral virus containing approximately 36 kb of
double-stranded DNA. There are 49 human adenoviral serotypes,
categorized into 6 subgenera (A through F) based on nucleic acid
comparisons, fiber protein characteristics, and biological
properties (Crawford-Miksza et al., J. Virol., 70: 1836-1844
(1996)). The group C viruses (e.g., serotypes 2 and 5, or Ad2 and
Ad5) are well characterized, and currently are employed for gene
transfer studies, including human gene therapy trials (see, e.g.,
Rosenfeld et al., Science, 252: 431-434 (1991); Rosenfeld et al.,
Cell, 68: 143-155 (1992); Zabner, Cell, 75: 207-216 (1993); Crystal
et al., Nat. Gen., 8: 42-51 (1994); Yei et al., Gene Therapy, 1:
192-200 (1994); Chen et al., Proc. Natl. Acad. Sci., 91: 3054-3057
(1994); Yang et al., Nat. Gen., 7: 362-369 (1994); Zabner et al.,
Nat. Gen., 6: 75-83 (1994)). Typically, an adenoviral vector is
generated by introducing one or more mutations (e.g., deletion,
insertion, or substitution) into the adenoviral genome of the
adenovirus so as to accommodate the insertion of a non-native
nucleic acid sequence, for example, for gene transfer, into the
adenovirus.
[0015] The adenovirus capsid mediates the key interactions of the
early stages of the infection of a cell by the virus, and is
required for packaging adenovirus genomes at the end of the
adenovirus life cycle. The capsid comprises 252 capsomeres, which
includes 240 hexons, 12 penton base proteins, and 12 fibers
(Ginsberg et al., Virology, 28: 782-83 (1966)). In one embodiment
of the invention, one or more capsid proteins (also referred to
herein as "coat" proteins) of the adenoviral vector can be
manipulated to alter the binding specificity or recognition of the
vector for a viral receptor on a potential host cell. Such
manipulations can include deletion of regions of the fiber or
penton, insertions of various native or non-native ligands into
portions of the capsid proteins, and the like. Manipulation of
capsid proteins can broaden the range of cells infected by the
adenoviral vector or enable targeting of the adenoviral vector to a
specific cell type.
[0016] The adenoviral vector of the invention can comprise a
modified hexon protein. The adenovirus hexon protein is the largest
and most abundant protein in the adenovirus capsid. The hexon
protein is essential for virus capsid assembly, determination of
the icosahedral symmetry of the capsid (which in turn defines the
limits on capsid volume and DNA packaging size), and integrity of
the capsid. In addition, the hexon protein is a primary target for
modification in order to reduce neutralization of adenoviral
vectors (see, e.g., Gall et al., J. Virol., 72: 10260-264 (1998),
and Rux et al., J. Virol., 77(17): 9553-9566 (2003)). The major
structural features of the hexon protein are shared by adenoviruses
across serotypes, but the hexon protein differs in size and
immunological properties between serotypes (Jornvall et al., J.
Biol. Chem., 256(12): 6181-6186 (1981)). A comparison of 15
adenovirus hexon proteins reveals that the predominant antigenic
and serotype-specific regions of the hexon protein appear to be in
loops 1 and 2 (i.e., LI or l1, and LII or l2, respectively), within
which are seven to nine discrete hypervariable regions (HVR1 to HVR
7 or HVR9) varying in length and sequence between adenoviral
serotypes (Crawford-Miksza et al., J. Virol., 70(3): 1836-1844
(1996), and Bruder et al., PLoS ONE, 7(4): e33920 (2012)).
[0017] The hexon protein is "modified" in that it comprises a
non-native amino acid sequence in addition to or in place of a
wild-type hexon amino acid sequence of the serotype 28 adenoviral
vector. In this respect, at least a portion of the wild-type hexon
protein (e.g., the entire hexon protein) of the inventive serotype
28 adenoviral vector desirably is removed and replaced with a
corresponding portion of a hexon protein from a non-group D
adenovirus (e.g., a group A, B, C, E, or F adenovirus), or an
adenovirus that does not normally infect humans (e.g., a simian or
gorilla adenovirus). Alternatively and preferably, at least a
portion of the wild-type hexon protein of the serotype 28
adenoviral vector can be removed and replaced with a corresponding
portion of a hexon protein from another group D adenovirus. For
example, a portion of the wild-type hexon protein of the serotype
28 adenoviral vector can be removed and replaced with a
corresponding portion of a hexon protein from any group D
adenovirus (such as those described herein). Preferably, the
inventive serotype 28 adenoviral vector comprises at least a
portion of an adenovirus serotype 45 hexon protein in place of at
least a portion of the endogenous serotype 28 hexon protein. Any
suitable amino acid residue(s) of a wild-type hexon protein of the
serotype 28 adenoviral vector can be modified or removed, so long
as viral capsid assembly is not impeded. Similarly, amino acids can
be added to the hexon protein as long as the structural integrity
of the capsid is maintained. In a preferred embodiment, at least
50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or 100%) of the endogenous Ad28 hexon protein is modified or
removed.
[0018] A "portion" of an amino acid sequence comprises at least
three amino acids (e.g., about 3 to about 800 amino acids).
Preferably, a "portion" comprises 10 or more (e.g., 10 or more, 15
or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or
more, or 100 or more) amino acid residues, but less than the entire
wild-type hexon protein (e.g., 900 or less, 800 or less, 700 or
less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or
less, or 100 or less amino acid residues). For example, a portion
can be about 10 to about 700 amino acids (e.g., about 10, 100, 300,
500, or 600 amino acids), about 10 to about 500 amino acids (e.g.,
about 20, 50, 200, or 400 amino acids), or about 10 to about 300
amino acids (e.g., about 15, 40, 60, 70, 90, 150, 250, or 290 amino
acids), or a range defined by any two of the foregoing values. More
preferably, a "portion" comprises no more than about 600 amino
acids (e.g., about 10 to about 550 amino acids, about 10 to about
500 amino acids, or about 50 to about 300 amino acids, or a range
defined by any two of the foregoing values).
[0019] A "portion" of a nucleic acid sequence comprises at least
ten nucleotides (e.g., about 10 to about 5000 nucleotides).
Preferably, a "portion" of a nucleic acid sequence comprises 10 or
more (e.g., 15 or more, 20 or more, 25 or more, 30 or more, 35 or
more, 40 or more, 45 or more, 50 or more, or 100 or more)
nucleotides, but less than 5,000 (e.g., 4900 or less, 4000 or less,
3000 or less, 2000 or less, 1000 or less, 800 or less, 500 or less,
300 or less, or 100 or less) nucleotides. Preferably, a portion of
a nucleic acid sequence is about 10 to about 3500 nucleotides
(e.g., about 10, 20, 30, 50, 100, 300, 500, 700, 1000, 1500, 2000,
2500, or 3000 nucleotides), about 10 to about 1000 nucleotides
(e.g., about 25, 55, 125, 325, 525, 725, or 925 nucleotides), or
about 10 to about 500 nucleotides (e.g., about 15, 30, 40, 50, 60,
70, 80, 90, 150, 175, 250, 275, 350, 375, 450, 475, 480, 490, 495,
or 499 nucleotides), or a range defined by any two of the foregoing
values. More preferably, a "portion" of a nucleic acid sequence
comprises no more than about 3200 nucleotides (e.g., about 10 to
about 3200 nucleotides, about 10 to about 3000 nucleotides, or
about 30 to about 500 nucleotides, or a range defined by any two of
the foregoing values).
[0020] Desirably, the portion of an adenovirus serotype 45 hexon
protein comprises at least one hypervariable region (HVR) in place
of an endogenous Ad28 HVR. Thus, at least one HVR of the hexon
protein of the inventive serotype 28 adenoviral vector is removed
and replaced with at least one HVR from a wild-type serotype 45
adenovirus. In one embodiment, the serotype 28 adenoviral vector
can comprise one or more of HVR1, HVR2, HVR3, HVR4, HVR5, HVR6,
HVR7, HVR8, or HVR9 of a wild-type serotype 45 adenovirus hexon
protein in place of the corresponding endogenous Ad28 HVR.
Preferably, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) HVRs of
the hexon protein of the serotype 28 adenoviral vector are removed
and replaced with corresponding HVRs from a serotype 45 adenovirus.
More preferably, the inventive serotype 28 adenoviral vector
comprises all nine HVRs of a serotype 45 adenovirus hexon protein
in place of the corresponding endogenous Ad28 HVRs. In one
embodiment, the entire wild-type hexon protein of the serotype 28
adenoviral vector is replaced with the entire hexon protein of a
serotype 45 adenovirus.
[0021] Nucleic acid sequences that encode all or a portion of a
serotype 28 or 45 adenovirus hexon protein are publicly available
(see, e.g., GenBank Accession Nos. DQ149626.1 and AB330126.1).
Amino acid sequences that comprise a full-length serotype 28 or 45
adenovirus hexon protein, or portions thereof, also are publicly
available (see, e.g., GenBank Accession Nos. ABA00010.1 and
BAG48822). In one embodiment, the portion of an adenovirus serotype
45 hexon protein comprises, for example, the amino acid sequence of
SEQ ID NO: 1, and a nucleic acid sequence that encodes a portion of
a serotype 45 adenovirus hexon protein comprises, for example, SEQ
ID NO: 2. In another embodiment, the portion of an adenovirus
serotype 45 hexon protein comprises an amino acid sequence that is
at least 91.4% identical (e.g., at least 91.5% identical, at least
92% identical, at least 92.5% identical, at least 93% identical, at
least 93.5% identical, at least 94% identical, at least 94.5%
identical, at least 95% identical, at least 95.5% identical, at
least 96% identical, at least 96.5% identical, at least 97%
identical, at least 97.5% identical, at least 98% identical, at
least 98.5% identical, at least 99% identical, or at least 99.5%
identical) to SEQ ID NO: 1.
[0022] Nucleic acid or amino acid sequence "identity," as described
herein, can be determined by comparing a nucleic acid or amino acid
sequence of interest to a reference nucleic acid or amino acid
sequence. The number of nucleotides or amino acid residues that
have been changed and/or modified (such as, e.g., by point
mutations, insertions, or deletions) in the reference sequence so
as to result in the sequence of interest are counted. The total
number of such changes is subtracted from the total length of the
sequence of interest, and the difference is divided by the length
of the sequence of interest and expressed as a percentage. A number
of mathematical algorithms for obtaining the optimal alignment and
calculating identity between two or more sequences are known and
incorporated into a number of available software programs. Examples
of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for
alignment of nucleic acid and amino acid sequences), BLAST programs
(e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA
programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence
alignment and sequence similarity searches). Sequence alignment
algorithms also are disclosed in, for example, Altschul et al., J.
Molecular Biol., 215(3): 403-410 (1990); Beigert et al., Proc. Nat.
Acad. Sci. USA, 106(10): 3770-3775 (2009); Durbin et al., eds.,
Biological Sequence Analysis: Probalistic Models of Proteins and
Nucleic Acids, Cambridge University Press, Cambridge, U.K. (2009),
Soding, Bioinformatics, 21(7): 951-960 (2005); Altschul et al.,
Nucleic Acids Res., 25(17): 3389-3402 (1997); and Gusfield,
Algorithms on Strings, Trees and Sequences, Cambridge University
Press, Cambridge, U.K. (1997)).
[0023] The adenoviral vector of the invention can comprise a
modified fiber protein. The adenovirus fiber protein is a
homotrimer of the adenoviral polypeptide IV that has three domains:
the tail, shaft, and knob (Devaux et al., J. Molec. Biol., 215:
567-88 (1990), and Yeh et al., Virus Res., 33: 179-98 (1991)). The
fiber protein mediates primary viral binding to receptors on the
cell surface via the knob and the shaft domains (Henry et al., J.
Virol., 68(8): 5239-46 (1994)). The amino acid sequences for
trimerization are located in the knob, which appears necessary for
the amino terminus of the fiber (the tail) to properly associate
with the penton base (Novelli et al., Virology, 185: 365-76
(1991)). In addition to recognizing cell receptors and binding the
penton base, the fiber contributes to serotype identity. Fiber
proteins from different adenoviral serotypes differ considerably
(see, e.g., Green et al., EMBO J., 2: 1357-65 (1983), Chroboczek et
al., Virology, 186: 280-85 (1992), and Signas et al., J. Virol.,
53: 672-78 (1985)). Thus, the fiber protein has multiple functions
that are key to the life cycle of adenovirus.
[0024] The fiber protein is "modified" in that it comprises a
non-native amino acid sequence, in addition to or in place of a
wild-type fiber amino acid sequence of the inventive serotype 28
adenoviral vector. In this respect, a portion of the wild-type
fiber protein (e.g., the fiber tail, the fiber shaft, the fiber
knob, or the entire fiber protein) of the inventive serotype 28
adenoviral vector can be removed and replaced with a corresponding
portion of a fiber protein from a non-group D adenovirus (e.g., a
group A, B, C, E, or F adenovirus), or an adenovirus that does not
infect humans (e.g., a simian or gorilla adenovirus). Alternatively
and preferably, at least a portion of the wild-type fiber protein
of the inventive serotype 28 adenoviral vector can be removed and
replaced with a corresponding portion of a fiber protein from
another group D adenovirus. For example, a portion of the wild-type
fiber protein of the inventive serotype 28 adenoviral vector can be
removed and replaced with a corresponding portion of a fiber
protein from any group D adenovirus (such as those described
herein). Preferably, the inventive serotype 28 adenoviral vector
comprises at least a portion of an adenovirus serotype 45 fiber
protein in place of at least a portion of the endogenous serotype
28 fiber protein. Any suitable amino acid residue(s) of a wild-type
fiber protein of the serotype 28 adenoviral vector that mediates or
assists in the interaction between the fiber knob and the native
cellular receptor can be modified or removed, so long as the fiber
protein is able to trimerize. Similarly, amino acids can be added
to the fiber knob as long as the fiber protein retains the ability
to trimerize. In a preferred embodiment, at least 75% (e.g., at
least 80%, at least 85%, at least 90%, at least 95%, or 100%) of
the endogenous Ad28 fiber protein is modified or removed.
[0025] Nucleic acid sequences that encode all or a portion of a
serotype 28 or 45 adenovirus fiber protein are publicly available
(see, e.g., GenBank Accession Nos. AB361404.1, Y14242.1,
FM210554.1, and AB361421.1). Amino acid sequences that comprise a
full-length serotype 28 or 45 adenovirus fiber protein, or portions
thereof, also are publicly available (see, e.g., GenBank Accession
Nos. ACQ91171, CAR66130.1, BAG71098.1, and CAH18767.1). In one
embodiment, the portion of an adenovirus serotype 45 fiber protein
comprises the amino acid sequence of SEQ ID NO: 3, and a nucleic
acid sequence that encodes a portion of a serotype 45 adenovirus
fiber protein comprises, for example, SEQ ID NO: 4. In another
embodiment, the portion of an adenovirus serotype 45 fiber protein
comprises an amino acid sequence that is at least 67% identical
(e.g., at least 68% identical, at least 69% identical, at least 70%
identical, at least 71% identical, at least 72% identical, at least
73% identical, at least 74% identical, at least 75% identical, at
least 76% identical, at least 77% identical, at least 78%
identical, at least 79% identical, at least 80% identical, at least
81% identical, at least 82% identical, at least 83% identical, at
least 84% identical, at least 85% identical, at least 86%
identical, at least 87% identical, at least 88% identical, at least
89% identical, at least 90% identical, at least 91% identical, at
least 92% identical, at least 93% identical, at least 94%
identical, at least 95% identical, at least 96% identical, at least
97% identical, at least 98% identical, or at least 99% identical)
to SEQ ID NO: 3.
[0026] The inventive serotype 28 adenoviral vector comprises the
aforementioned modified hexon protein, the aforementioned modified
fiber protein, or the modified hexon protein and the modified fiber
protein. For example, the inventive serotype 28 adenoviral vector
comprises at least a portion of an adenovirus serotype 45 hexon
protein in place of at least a portion of the endogenous serotype
28 hexon protein, or at least a portion of an adenovirus serotype
45 fiber protein in place of at least a portion of the endogenous
serotype 28 fiber protein. Alternatively, the inventive serotype 28
adenoviral vector comprises at least a portion of an adenovirus
serotype 45 hexon protein in place of at least a portion of the
endogenous serotype 28 hexon protein, and at least a portion of an
adenovirus serotype 45 fiber protein in place of at least a portion
of the endogenous serotype 28 fiber protein.
[0027] Modifications to adenovirus coat proteins, including methods
for generating chimeric hexon and fiber proteins, are described in,
for example, e.g., U.S. Pat. Nos. 5,543,328; 5,559,099; 5,712,136;
5,731,190; 5,756,086; 5,770,442; 5,846,782; 5,871,727; 5,885,808;
5,922,315; 5,962,311; 5,965,541; 6,057,155; 6,127,525; 6,153,435;
6,329,190; 6,455,314; 6,465,253; 6,576,456; 6,649,407; and
6,740,525; U.S. Patent Application Publications 2001/0047081 A1,
2002/0099024 A1, 2002/0151027 A1, 2003/0022355 A1, and 2003/0099619
A1, and International Patent Application Publications WO
1996/007734, WO 1996/026281, WO 1997/020051, WO 1998/007865, WO
1998/007877, WO 1998/040509, WO 1998/054346, WO 2000/015823, WO
2001/058940, and WO 2001/092549.
[0028] The adenoviral vector can be replication-competent,
conditionally replication-competent, or replication-deficient.
[0029] A replication-competent adenoviral vector can replicate in
typical host cells, i.e., cells typically capable of being infected
by an adenovirus. A replication-competent adenoviral vector can
have one or more mutations as compared to the wild-type adenovirus
(e.g., one or more deletions, insertions, and/or substitutions) in
the adenoviral genome that do not inhibit viral replication in host
cells. For example, the adenoviral vector can have a partial or
entire deletion of the adenoviral early region known as the E3
region, which is not essential for propagation of the adenoviral
genome.
[0030] A conditionally-replicating adenoviral vector is an
adenoviral vector that has been engineered to replicate under
pre-determined conditions. For example, replication-essential gene
functions, e.g., gene functions encoded by the adenoviral early
regions, can be operably linked to an inducible, repressible, or
tissue-specific transcription control sequence, e.g., promoter. In
such an embodiment, replication requires the presence or absence of
specific factors that interact with the transcription control
sequence. Conditionally-replicating adenoviral vectors are further
described in U.S. Pat. No. 5,998,205.
[0031] A replication-deficient adenoviral vector is an adenoviral
vector that requires complementation of one or more gene functions
or regions of the adenoviral genome that are required for
replication, as a result of, for example, a deficiency in the one
or more replication-essential gene function or regions, such that
the adenoviral vector does not replicate in typical host cells,
especially those in a human to be infected by the adenoviral
vector.
[0032] A deficiency in a gene function or genomic region, as used
herein, is defined as a disruption (e.g., deletion) of sufficient
genetic material of the adenoviral genome to obliterate or impair
the function of the gene (e.g., such that the function of the gene
product is reduced by at least about 2-fold, 5-fold, 10-fold,
20-fold, 30-fold, or 50-fold) whose nucleic acid sequence was
disrupted (e.g., deleted) in whole or in part. Deletion of an
entire gene region often is not required for disruption of a
replication-essential gene function. However, for the purpose of
providing sufficient space in the adenoviral genome for one or more
transgenes, removal of a majority of one or more gene regions may
be desirable. While deletion of genetic material is preferred,
mutation of genetic material by addition or substitution also is
appropriate for disrupting gene function. Replication-essential
gene functions are those gene functions that are required for
adenovirus replication (e.g., propagation) and are encoded by, for
example, the adenoviral early regions (e.g., the E1, E2, and E4
regions), late regions (e.g., the L1, L2, L3, L4, and L5 regions),
genes involved in viral packaging (e.g., the IVa2 gene), and
virus-associated RNAs (e.g., VA-RNA-1 and/or VA-RNA-2).
[0033] Whether the adenoviral vector is replication-competent or
replication-deficient, the adenoviral vector retains at least a
portion of a serotype 28 adenoviral genome. The adenoviral vector
can comprise any portion of a serotype 28 adenoviral genome,
including protein coding and non-protein coding regions. Desirably,
the adenoviral vector comprises at least one nucleic acid sequence
that encodes a serotype 28 adenovirus protein. The adenoviral
vector can comprise any suitable adenovirus protein, or a nucleic
acid sequence that encodes any suitable adenovirus protein, such
as, for example, a protein of any one of the early region genes
(i.e., E1A, E1B, E2A, E2B, E3, and/or E4 regions), or a protein
encoded by any one of the late region genes, which encode the virus
structural proteins (i.e., L1, L2, L3, L4, and L5 regions).
[0034] The adenoviral vector desirably comprises one or more amino
acid sequences of the pIX protein, the penton protein, the p100
protein, the L1 52/55K protein of a serotype 28 adenovirus, or any
combination of the foregoing. The adenoviral vector can comprise a
full-length amino acid sequence of a serotype 28 adenovirus
protein. Alternatively, the adenoviral vector can comprise a
portion of a full-length amino acid sequence of a serotype 28
adenovirus protein. An amino acid sequence of a serotype 28
adenovirus pIX protein comprises, for example, SEQ ID NO: 5. An
amino acid sequence of a serotype 28 adenovirus penton protein
comprises, for example, SEQ ID NO: 6. An amino acid sequence of a
serotype 28 adenovirus p100 protein comprises, for example, SEQ ID
NO: 7. An amino acid sequence of a serotype 28 adenovirus L1 52/55K
protein comprises, for example, SEQ ID NO: 8. The adenoviral vector
also desirably comprises a nucleic acid sequence encoding a DNA
polymerase protein of a serotype 28 adenovirus or a portion
thereof. A nucleic acid sequence encoding a DNA polymerase of a
serotype 28 adenovirus comprises, for example, SEQ ID NO: 9. The
adenoviral vector desirably comprises one or more of SEQ ID NOs:
5-9.
[0035] In another embodiment, the invention provides a virus-like
particle comprising one or more amino acid sequences of the pIX
protein, the penton protein, the p100 protein, the L1 52/55K
protein of a serotype 28 adenovirus, or any combination of the
foregoing, as well as the serotype 45 hexon protein and/or the
serotype 45 fiber protein described herein. A "virus-like particle"
consists of one or more viral coat proteins that assemble into
viral particles, but lacks any viral genetic material (see, e.g.,
Miyanohara et al., J. Virol., 59: 176-180 (1986), Gheysen et al.,
Cell, 59: 103-112 (1989), and Buonaguro et al., ASHI Quarterly, 29:
78-80 (2005)).
[0036] Preferably, the adenoviral vector is replication-deficient,
such that the replication-deficient adenoviral vector requires
complementation of at least one replication-essential gene function
of one or more regions of the adenoviral genome for propagation
(e.g., to form adenoviral vector particles).
[0037] The replication-deficient adenoviral vector can be modified
in any suitable manner to cause the deficiencies in the one or more
replication-essential gene functions in one or more regions of the
adenoviral genome for propagation. The complementation of the
deficiencies in the one or more replication-essential gene
functions of one or more regions of the adenoviral genome refers to
the use of exogenous means to provide the deficient
replication-essential gene functions. Such complementation can be
effected in any suitable manner, for example, by using
complementing cells and/or exogenous DNA (e.g., helper adenovirus)
encoding the disrupted replication-essential gene functions.
[0038] The adenoviral vector can be deficient in one or more
replication-essential gene functions of only the early regions
(i.e., E1-E4 regions) of the adenoviral genome, only the late
regions (i.e., L1-L5 regions) of the adenoviral genome, both the
early and late regions of the adenoviral genome, or all adenoviral
genes (i.e., a high capacity adenovector (HC-Ad)). See Morsy et
al., Proc. Natl. Acad. Sci. USA, 95: 965-976 (1998); Chen et al.,
Proc. Natl. Acad. Sci. USA, 94: 1645-1650 (1997); and Kochanek et
al., Hum. Gene Ther., 10: 2451-2459 (1999). Examples of
replication-deficient adenoviral vectors are disclosed in U.S. Pat.
Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and
7,195,896, and International Patent Application Publications WO
1994/028152, WO 1995/002697, WO 1995/016772, WO 1995/034671, WO
1996/022378, WO 1997/012986, WO 1997/021826, and WO
2003/022311.
[0039] The early regions of the adenoviral genome include the E1,
E2, E3, and E4 regions. The late regions of the adenoviral genome
include the L1, L2, L3, L4, and L5 regions. The adenoviral vector
also can have a mutation in the major late promoter (MLP), as
discussed in International Patent Application Publication WO
2000/000628, which can render the adenoviral vector
replication-deficient if desired.
[0040] The E1 region comprises the E1A and E1B subregions, and one
or more deficiencies in replication-essential gene functions in the
E1 region can include one or more deficiencies in
replication-essential gene functions in either or both of the E1A
and E1B subregions, thereby requiring complementation of the
deficiency in the E1A subregion and/or the E1B subregion of the
adenoviral genome for the adenoviral vector to propagate (e.g., to
form adenoviral vector particles).
[0041] The E2 region comprises the E2A and E2B subregions, and one
or more deficiencies in replication-essential gene functions in the
E2 region can include one or more deficiencies in
replication-essential gene functions in either or both of the E2A
and E2B subregions, thereby requiring complementation of the
deficiency in the E2A subregion and/or the E2B subregion of the
adenoviral genome for the adenoviral vector to propagate (e.g., to
form adenoviral vector particles).
[0042] The E3 region does not include any replication-essential
gene functions, such that a deletion of the E3 region in part or in
whole does not require complementation of any gene functions in the
E3 region for the adenoviral vector to propagate (e.g., to form
adenoviral vector particles). In the context of the invention, the
E3 region is defined as the region that initiates with the open
reading frame that encodes a protein with high homology to the
12.5K protein from the E3 region of human adenovirus 28 (NCBI
reference sequence FJ824826) and ends with the open reading frame
that encodes a protein with high homology to the 14.7K protein from
the E3 region of human adenovirus 28 (NCBI reference sequence
FJ824826). The E3 region may be deleted in whole or in part, or
retained in whole or in part. The size of the deletion may be
tailored so as to retain an adenoviral vector whose genome closely
matches the optimum genome packaging size. A larger deletion will
accommodate the insertion of larger heterologous nucleic acid
sequences in the adenoviral genome. In one embodiment of the
invention, the L4 polyadenylation signal sequences, which reside in
the E3 region, are retained.
[0043] The E4 region comprises multiple open reading frames (ORFs).
An adenoviral vector with a deletion of all of the open reading
frames of the E4 region except ORF6, and in some cases ORF3, does
not require complementation of any gene functions in the E4 region
for the adenoviral vector to propagate (e.g., to form adenoviral
vector particles). Conversely, an adenoviral vector with a
disruption or deletion of ORF6, and in some cases ORF3, of the E4
region (e.g., with a deficiency in a replication-essential gene
function based in ORF6 and/or ORF3 of the E4 region), with or
without a disruption or deletion of any of the other open reading
frames of the E4 region or the native E4 promoter, polyadenylation
sequence, and/or the right-side inverted terminal repeat (ITR),
requires complementation of the deficiency in the E4 region
(specifically, of ORF6 and/or ORF3 of the E4 region) for the
adenoviral vector to propagate (e.g., to form adenoviral vector
particles).
[0044] The one or more regions of the adenoviral genome that
contain one or more deficiencies in replication-essential gene
functions desirably are one or more early regions of the adenoviral
genome, i.e., the E1, E2, and/or E4 regions, optionally with the
deletion in part or in whole of the E3 region. In other words, the
adenoviral vector requires, at most, complementation of a
deficiency in one or more early regions of the adenoviral genome
for propagation.
[0045] The replication-deficient adenoviral vector also can have
one or more mutations as compared to the wild-type adenovirus
(e.g., one or more deletions, insertions, and/or substitutions) in
the adenoviral genome that do not inhibit viral replication in host
cells. Thus, in addition to one or more deficiencies in
replication-essential gene functions, the adenoviral vector can be
deficient in other respects that are not replication-essential. For
example, the adenoviral vector can have a partial or entire
deletion of the adenoviral early region known as the E3 region,
which is not essential for propagation of the adenoviral
genome.
[0046] In one embodiment, the adenoviral vector is
replication-deficient and requires, at most, complementation of the
E1 region of the adenoviral genome, for propagation (e.g., to form
adenoviral vector particles). Thus, the replication-deficient
adenoviral vector requires complementation of at least one
replication-essential gene function of the E1A subregion and/or the
E1B subregion of the adenoviral genome (denoted an E1-deficient
adenoviral vector) for propagation (e.g., to form adenoviral vector
particles). The adenoviral vector can be deficient in at least one
replication-essential gene function (desirably all
replication-essential gene functions) of the E1 region of the
adenoviral genome and at least one gene function of the
nonessential E3 region of the adenoviral genome (denoted an
E1/E3-deficient adenoviral vector). Such an adenoviral vector
requires, at most, complementation of a deficiency in the E1 region
of the adenoviral genome for propagation.
[0047] In one embodiment, the adenoviral vector is
replication-deficient and requires, at most, complementation of the
E2 region, preferably the E2A subregion, of the adenoviral genome,
for propagation (e.g., to form adenoviral vector particles). Thus,
the replication-deficient adenoviral vector requires
complementation of at least one replication-essential gene function
of the E2A subregion of the adenoviral genome (denoted an
E2A-deficient adenoviral vector) for propagation (e.g., to form
adenoviral vector particles). The adenoviral vector can be
deficient in at least one replication-essential gene function
(desirably all replication-essential gene functions) of the E2A
region of the adenoviral genome and at least one gene function of
the nonessential E3 region of the adenoviral genome (denoted an
E2A/E3-deficient adenoviral vector). Such an adenoviral vector
requires, at most, complementation of a deficiency in the E2 region
of the adenoviral genome for propagation.
[0048] In one embodiment, the adenoviral vector is
replication-deficient and requires, at most, complementation of the
E4 region of the adenoviral genome, for propagation (e.g., to form
adenoviral vector particles). Thus, the replication-deficient
adenoviral vector requires complementation of at least one
replication-essential gene function of the E4 region of the
adenoviral genome (denoted an E4-deficient adenoviral vector) for
propagation (e.g., to form adenoviral vector particles). The
adenoviral vector can be deficient in at least one
replication-essential gene function (desirably all
replication-essential gene functions) of the E4 region of the
adenoviral genome and at least one gene function of the
nonessential E3 region of the adenoviral genome (denoted an
E3/E4-deficient adenoviral vector). Such an adenoviral vector
requires, at most, complementation of a deficiency in the E4 region
of the adenoviral genome for propagation.
[0049] In one embodiment, the adenoviral vector requires
complementation of the E1 and E2 (e.g., E2A) regions of the
adenoviral genome for propagation (denoted an E1/E2-deficient
adenoviral vector), wherein the adenoviral vector also can be
deficient in at least one gene function of the E3 region (denoted
an E1/E2/E3-deficient adenoviral vector). Such an adenoviral vector
requires, at most, complementation of a deficiency in the E1 region
and a deficiency in the E2 region of the adenoviral genome for
propagation.
[0050] In one embodiment, the adenoviral vector is
replication-deficient and requires, at most, complementation of the
E1 and E4 regions of the adenoviral genome for propagation (e.g.,
to form adenoviral vector particles). Thus, the
replication-deficient adenoviral vector requires complementation of
at least one replication-essential gene function of both the E1 and
E4 regions of the adenoviral genome (denoted an E1/E4-deficient
adenoviral vector) for propagation (e.g., to form adenoviral vector
particles). The adenoviral vector can be deficient in at least one
replication-essential gene function (desirably all
replication-essential gene functions) of the E1 region of the
adenoviral genome, at least one replication-essential gene function
of the E4 region of the adenoviral genome, and at least one gene
function of the nonessential E3 region of the adenoviral genome
(denoted an E1/E3/E4-deficient adenoviral vector). Such an
adenoviral vector requires, at most, complementation of a
deficiency in the E1 region and a deficiency in the E4 region of
the adenoviral genome for propagation.
[0051] In a preferred embodiment, the adenoviral vector requires,
at most, complementation of a deficiency in the E1 region of the
adenoviral genome for propagation, and does not require
complementation of any other deficiency of the adenoviral genome
for propagation. In another preferred embodiment, the adenoviral
vector requires, at most, complementation of a deficiency in both
the E1 and E4 regions of the adenoviral genome for propagation, and
does not require complementation of any other deficiency of the
adenoviral genome for propagation.
[0052] The adenoviral vector, when deficient in multiple
replication-essential gene functions of the adenoviral genome
(e.g., an E1/E4-deficient adenoviral vector), can include a spacer
sequence to provide viral growth in a complementing cell line
similar to that achieved by adenoviruses or adenoviral vectors
deficient in a single replication-essential gene function (e.g., an
E1-deficient adenoviral vector). The spacer sequence can contain
any nucleotide sequence or sequences which are of a desired length,
such as sequences at least about 15 base pairs (e.g., between about
15 nucleotides and about 12,000 nucleotides), preferably about 100
nucleotides to about 10,000 nucleotides, more preferably about 500
nucleotides to about 8,000 nucleotides, even more preferably about
1,500 nucleotides to about 6,000 nucleotides, and most preferably
about 2,000 to about 3,000 nucleotides in length, or a range
defined by any two of the foregoing values. The spacer sequence can
be coding or non-coding and native or non-native with respect to
the adenoviral genome, but does not restore the
replication-essential function to the deficient region. The spacer
also can contain an expression cassette. More preferably, the
spacer comprises a polyadenylation sequence and/or a gene that is
non-native with respect to the adenovirus or adenoviral vector. The
use of a spacer in an adenoviral vector is further described in,
for example, U.S. Pat. No. 5,851,806 and International Patent
Application Publication WO 1997/021826.
[0053] The replication-deficient adenoviral vector of the invention
can be produced in complementing cell lines that provide gene
functions not present in the replication-deficient adenovirus or
adenoviral vector, but required for viral propagation, at
appropriate levels in order to generate high titers of viral vector
stock. Such complementing cell lines are known and include, but are
not limited to, 293 cells (described in, e.g., Graham et al., J.
Gen. Virol., 36: 59-72 (1977)), PER.C6 cells (described in, e.g.,
International Patent Application Publication WO 1997/000326, and
U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells
(described in, e.g., International Patent Application Publication
WO 1995/34671 and Brough et al., J. Virol., 71: 9206-9213 (1997)).
Other suitable complementing cell lines to produce the
replication-deficient adenoviral vector of the invention include
complementing cells that have been generated to propagate
adenoviral vectors encoding transgenes whose expression inhibits
viral growth in host cells (see, e.g., U.S. Patent Application
Publication 2008/0233650). Additional suitable complementing cells
are described in, for example, U.S. Pat. Nos. 6,677,156 and
6,682,929, and International Patent Application Publication WO
2003/020879. In some instances, the cellular genome need not
comprise nucleic acid sequences, the gene products of which
complement for all of the deficiencies of a replication-deficient
adenoviral vector. One or more replication-essential gene functions
lacking in a replication-deficient adenoviral vector can be
supplied by a helper virus, e.g., an adenoviral vector that
supplies in trans one or more essential gene functions required for
replication of the replication-deficient adenoviral vector.
Alternatively, the inventive adenoviral vector can comprise a
non-native replication-essential gene that complements for the one
or more replication-essential gene functions lacking in the
inventive replication-deficient adenoviral vector. For example, an
E1/E4-deficient adenoviral vector can be engineered to contain a
nucleic acid sequence encoding E4 ORF 6 that is obtained or derived
from a different adenovirus (e.g., an adenovirus of a different
serotype than the inventive adenoviral vector, or an adenovirus of
a different species than the inventive adenoviral vector).
[0054] An example of an E1/E3-deficient serotype 28 adenoviral
vector comprising a serotype 45 hexon protein and a serotype 45
fiber protein as described herein comprises the nucleic acid
sequence of SEQ ID NO: 10. Using the publicly available genome
information for Ad28, however, one of ordinary skill in the art
would be able generate other Ad28 vectors with similar deficiencies
and/or modifications using routine methods known in the art and/or
described herein.
[0055] The adenoviral vector can further comprise one or more
exogenous or non-native nucleic acids, which can be positioned at
any suitable place in the adenoviral vector. By removing all or
part of the adenoviral genome, for example, the E1, E3, and E4
regions of the adenoviral genome, the resulting adenoviral vector
is able to accept inserts of exogenous nucleic acid sequences while
retaining the ability to be packaged into adenoviral capsids. An
exogenous nucleic acid sequence can be inserted at any position in
the adenoviral genome so long as insertion in the position allows
for the formation of adenovirus or the adenoviral vector particle.
The exogenous nucleic acid sequence preferably is positioned in the
E1 region, the E3 region, or the E4 region of the adenoviral
genome. In embodiments where the adenoviral vector comprises
multiple exogenous nucleic acid sequences (e.g., 2, 3, 4 or more
exogenous nucleic acid sequences), at least one exogenous nucleic
acid sequence is positioned in the E1 region, and at least one
exogenous nucleic acid sequence is positioned in the E4 region. For
example, when the adenoviral vector comprises three exogenous
nucleic acid sequences, two exogenous nucleic acid sequences can be
positioned in the E1 region, and one exogenous nucleic acid
sequence can be positioned in the E4 region.
[0056] An "exogenous" or "non-native" nucleic acid sequence is any
nucleic acid sequence (e.g., DNA, RNA, or cDNA sequence) that is
not a naturally occurring nucleic acid sequence of an adenovirus in
a naturally occurring position. Thus, the non-native nucleic acid
sequence can be naturally found in an adenovirus, but located at a
non-native position within the adenoviral genome and/or operably
linked to a non-native promoter. The terms "non-native nucleic acid
sequence," "heterologous nucleic acid sequence," and "exogenous
nucleic acid sequence" are synonymous and can be used
interchangeably in the context of the invention. The non-native
nucleic acid sequence preferably is DNA and preferably encodes a
protein (i.e., one or more nucleic acid sequences encoding one or
more proteins).
[0057] The non-native nucleic acid can be in the form of a
transgene. The term "transgene" is defined herein as a non-native
nucleic acid sequence that is operably linked to appropriate
regulatory elements (e.g., a promoter), such that the non-native
nucleic acid sequence can be expressed to produce a protein (e.g.,
peptide or polypeptide). The regulatory elements (e.g., promoter)
can be native or non-native to the adenovirus or adenoviral
vector.
[0058] The non-native nucleic acid sequence can encode a
therapeutic protein that can be used to prophylactically or
therapeutically treat a mammal for a disease. Examples of suitable
therapeutic proteins include cytokines, toxins, tumor suppressor
proteins, growth factors, hormones, receptors, mitogens,
immunoglobulins, neuropeptides, neurotransmitters, and enzymes.
Alternatively, the non-native nucleic acid sequence can encode an
antigen of a pathogen (e.g., a bacterium or a virus), and the
adenoviral vector can be used as a vaccine. For example, the
non-native nucleic acid sequence can encode an antigen of a Herpes
Simplex Virus-2, including, but not limited to an HSV-2 UL47
protein (e.g., SEQ ID NO: 11 or SEQ ID NO: 13), and/or an HSV-2
UL19 protein (e.g., SEQ ID NO: 12).
[0059] The invention provides a composition comprising the
adenoviral vector described herein and a carrier therefor (e.g., a
pharmaceutically acceptable carrier). The composition desirably is
a physiologically acceptable (e.g., pharmaceutically acceptable)
composition, which comprises a carrier, preferably a
physiologically (e.g., pharmaceutically) acceptable carrier, and
the adenoviral vector. Any suitable carrier can be used within the
context of the invention, and such carriers are well known in the
art. The choice of carrier will be determined, in part, by the
particular use of the composition (e.g., administration to an
animal) and the particular method used to administer the
composition. Ideally, in the context of replication-deficient
adenoviral vectors, the pharmaceutical composition preferably is
free of replication-competent adenovirus. The pharmaceutical
composition optionally can be sterile.
[0060] Suitable compositions include aqueous and non-aqueous
isotonic sterile solutions, which can contain anti-oxidants,
buffers, and bacteriostats, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The composition
can be presented in unit-dose or multi-dose sealed containers, such
as ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, water, immediately prior to use.
Extemporaneous solutions and suspensions can be prepared from
sterile powders, granules, and tablets. Preferably, the carrier is
a buffered saline solution. More preferably, the adenovirus or
adenoviral vector is part of a composition formulated to protect
the adenovirus or adenoviral vector from damage prior to
administration. For example, the composition can be formulated to
reduce loss of the adenovirus or adenoviral vector on devices used
to prepare, store, or administer the adenovirus or adenoviral
vector, such as glassware, syringes, or needles. The composition
can be formulated to decrease the light sensitivity and/or
temperature sensitivity of the adenovirus or adenoviral vector. To
this end, the composition preferably comprises a pharmaceutically
acceptable liquid carrier, such as, for example, those described
above, and a stabilizing agent selected from the group consisting
of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and
combinations thereof. Use of such a composition will extend the
shelf life of the adenovirus or adenoviral vector, and facilitate
its administration. Formulations for adenovirus or adenoviral
vector-containing compositions are further described in, for
example, U.S. Pat. Nos. 6,225,289, 6,514,943, and International
Patent Application Publication WO 2000/034444.
[0061] The composition also can be formulated to enhance
transduction efficiency. In addition, one of ordinary skill in the
art will appreciate that the adenoviral vector can be present in a
composition with other therapeutic or biologically-active agents.
For example, factors that control inflammation, such as ibuprofen
or steroids, can be part of the composition to reduce swelling and
inflammation associated with in vivo administration of the
adenoviral vector. If the adenoviral vector is used to deliver an
antigen-encoding nucleic acid sequence to a host, immune system
stimulators or adjuvants, e.g., interleukins, lipopolysaccharide,
double-stranded RNA, and/or TNFSF14/LIGHT (see, e.g., Zhang et al.,
J. Virol. Methods, 153(2): 142-148 (2008)) can be administered to
enhance or modify any immune response to the antigen. Antibiotics,
i.e., microbicides and fungicides, can be utilized to treat
existing infection and/or reduce the risk of future infection, such
as infection associated with gene transfer procedures.
[0062] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0063] This example demonstrates the construction of a serotype 28
adenoviral vector comprising a serotype 45 adenovirus hexon protein
and a serotype 45 adenovirus fiber protein, which encodes a Herpes
Simplex Virus-2 (HSV) antigen.
[0064] A human serotype 28 adenoviral vector was prepared with a
deletion in the E1 region, a portion of the hexon protein replaced
with a portion of the hexon protein of a human serotype 45
adenovirus, and an insertion of a codon-optimized nucleic acid
sequence encoding a wild-type HSV-2 UL19 protein into the E1 region
(Ad28UL19 H(Ad45)). Ad28UL19 H(Ad45) is replication-deficient due
to the deletion of the essential replication function provided by
E1. In place of the E1 sequences, a CMVtetO UL19 expression
cassette was introduced to provide the HSV-2 UL19 nucleic acid
sequence. The expression cassette, located at the E1 region
deletion junction, was oriented right-to-left with respect to the
viral genome.
[0065] Ad28UL19 H(Ad45) has an E1 region deletion of nucleotides
462 through 3111. The deletion includes the E1A protein and part of
the E1B protein, which renders the vector replication-incompetent
in noncomplementing cell lines.
[0066] The construction of plasmid pAC28UL19.H(45) to provide
Ad28UL19 H(Ad45) is schematically depicted in FIG. 1. Specifically,
wild-type Ad28 virus DNA was rescued into a plasmid backbone by
homologous recombination by using pAd28RSQ plasmid to make an Ad28
genome-containing plasmid (pACE28) (see FIG. 1, Step 3). A CMV tetO
EGFP expression plasmid was introduced into the Ad28
genome-containing plasmid by homologous recombination to make an
E1-deleted Ad28 genome-containing plasmid (pAC28EGFP) (462-3111 t
ef) (see FIG. 1, Steps 4 and 5). PCR amplification and subcloning
of the Ad45 hexon sequence into a shuttle plasmid was carried out
by standard restriction enzyme subcloning. The Ad45 hexon nucleic
acid sequence was inserted into pAC28EGFP plasmid by homologous
recombination in E. coli to generate the Ad45 hexon-containing Ad28
adenoviral genome in plasmid form (pAC28EGFP.H(45)) (see FIG. 1,
Steps 7 and 8). The HSV-2 UL19 gene was inserted into an adenoviral
vector expression shuttle plasmid by standard restriction enzyme
subcloning. The CMVTetO.UL19 gene was inserted into the
pAC28EGFP.H(45) plasmid by homologous recombination in E. coli to
generate the UL19-expressing Ad28 adenovector genome containing the
Ad45 hexon sequence in plasmid form (pAC28UL19.H(45)) (see FIG. 1,
Steps 9 and 10).
[0067] Plasmid pAC28UL19.H(45) was linearized with the restriction
endonuclease PmeI. Adherent 293-ORF6 cells were transfected with
the linearized plasmid, thereby resulting in conversion of the
adenoviral genome (i.e., DNA) of the linearized plasmid into the
viral vector Ad28UL19H(45).
[0068] The Ad28UL19H(45) adenoviral vector was expanded by serial
passaging to generate high titer cell-virus lysate. The adenoviral
vector was purified from the lysates by density-equilibrium
centrifugation in cesium chloride gradients. The purified
adenoviral vector was dialyzed into Final Formulation Buffer (FFB).
The structural integrity of the genome of the adenoviral vector was
confirmed by PCR, and the expression of the HSV-2 UL19 nucleic acid
sequence was confirmed by Western blot.
[0069] A similar method was used to incorporate a portion of the
Ad45 fiber nucleic acid sequence in place of a portion of the Ad28
fiber nucleic acid sequence.
[0070] The results of this example confirm the production of a
serotype 28 adenoviral vector in accordance with the invention.
Example 2
[0071] This example demonstrates that the administration of the
inventive serotype 28 adenoviral vector encoding one or more HSV
antigens induces an immune response against HSV.
[0072] A serotype 28 adenoviral vector comprising portions of the
hexon and fiber proteins from a serotype 45 adenovirus vector (Ad28
H/F) was produced using the methods described in Example 1. A first
such adenoviral vector comprised a nucleic acid sequence encoding a
wild-type HSV-2 UL47 protein (SEQ ID NO: 11). A second such
adenoviral vector comprised a codon-optimized nucleic acid sequence
encoding a wild-type HSV-2 UL19 protein (SEQ ID NO: 12).
[0073] T-cell response following a single intramuscular
administration of each of the adenoviral vector-delivered antigens
(1.times.10.sup.9 PU) was assessed in a mouse model and compared to
natural infection (1.times.10.sup.6 PFU of HSV administered
intravaginally) and control (i.e., formulation buffer (FFB)). A
single administration of the adenoviral vector encoding either
HSV-2 antigen induced a T-cell response that was greater than the
T-cell response induced by a natural HSV infection.
[0074] The results of this example confirm that administration of
the inventive serotype 28 adenoviral vector comprising a nucleic
acid sequence encoding an HSV antigen induces an immune response
against HSV.
Example 3
[0075] This example demonstrates that the administration of the
inventive serotype 28 adenoviral vector encoding one or more HSV
antigens induces an immune response against HSV.
[0076] Mice were divided into groups of four, and each group
received a single intramuscular administration of one of the
following: (a) an E1-deleted serotype 5 adenoviral vector encoding
an HSV2 UL19 protein, (b) an E1-deleted serotype 28 adenoviral
vector encoding an HSV2 UL19 protein, (c) the Ad28 H/F vector of
the invention as described in Example 2, which encodes an HSV2 UL19
protein, and (d) formulation buffer (FFB; negative control). CD8+
T-cell responses following vaccination with the adenoviral vectors
(1.times.10.sup.9 PU) were assessed. The elicited T-cell responses
are plotted in the graph of FIG. 2.
[0077] As is apparent from the results depicted in FIG. 2, a single
administration of the Ad28H/F vector elicited a strong CD8+ T-cell
response that was comparable to the CD8+ T-cell response elicited
by the Ad5 vector.
[0078] The results of this example confirm that administration of
the inventive serotype 28 adenoviral vector can induce a strong
immune response against HSV.
[0079] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0080] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0081] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
131531PRTAdenovirus 1Arg Ser Gln Arg Leu Thr Leu Arg Phe Val Pro
Val Asp Arg Glu Asp1 5 10 15Thr Thr Tyr Ser Tyr Lys Ala Arg Phe Thr
Leu Ala Val Gly Asp Asn 20 25 30Arg Val Leu Asp Met Ala Ser Thr Tyr
Phe Asp Ile Arg Gly Val Leu 35 40 45Asp Arg Gly Pro Ser Phe Lys Pro
Tyr Ser Gly Thr Ala Tyr Asn Ser 50 55 60Leu Ala Pro Lys Ser Ala Pro
Asn Pro Ser Gln Trp Asp Ala Lys Glu65 70 75 80Lys Glu Gly Val Ala
Gln Thr Glu Lys Asn Val Leu Lys Thr Phe Gly 85 90 95Val Ala Ala Thr
Gly Gly Phe Asn Ile Thr Asp Gln Gly Leu Leu Leu 100 105 110Gly Thr
Glu Glu Thr Ala Glu Asn Val Lys Lys Asp Ile Tyr Ala Glu 115 120
125Lys Thr Phe Gln Pro Glu Pro Gln Val Gly Glu Glu Asn Trp Gln Glu
130 135 140Ser Glu Ala Phe Tyr Gly Gly Arg Ala Ile Lys Lys Asp Thr
Lys Met145 150 155 160Lys Pro Cys Tyr Gly Ser Phe Ala Arg Pro Thr
Asn Glu Lys Gly Gly 165 170 175Gln Ala Lys Phe Lys Thr Leu Asp Gly
Gln Val Thr Lys Asp Pro Asp 180 185 190Ile Asp Phe Ala Tyr Phe Asp
Val Pro Gly Gly Lys Ala Pro Thr Gly 195 200 205Ser Ser Leu Pro Glu
Glu Tyr Lys Ala Asp Ile Ile Leu Tyr Thr Glu 210 215 220Asn Val Asn
Leu Glu Thr Pro Asp Thr His Ile Val Tyr Lys Pro Gly225 230 235
240Lys Glu Asp Asp Asn Ser Glu Ile Asn Leu Thr Gln Gln Ser Met Pro
245 250 255Asn Arg Pro Asn Tyr Ile Gly Phe Arg Asp Asn Phe Val Gly
Leu Met 260 265 270Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala
Gly Gln Ala Ser 275 280 285Gln Leu Asn Ala Val Val Asp Leu Gln Asp
Arg Asn Thr Glu Leu Ser 290 295 300Tyr Gln Leu Leu Leu Asp Ser Leu
Gly Asp Arg Thr Arg Tyr Phe Ser305 310 315 320Met Trp Asn Ser Ala
Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile 325 330 335Glu Asn His
Gly Val Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu 340 345 350Asn
Gly Thr Gly Thr Asn Ser Thr Tyr Gln Gly Val Lys Ile Thr Gly 355 360
365Asn Asn Asp Gly Asp Leu Glu Thr Glu Trp Glu Arg Asp Glu Ala Ile
370 375 380Ser Arg Gln Asn Gln Ile Cys Lys Gly Asn Val Tyr Ala Met
Glu Ile385 390 395 400Asn Leu Gln Ala Asn Leu Trp Lys Ser Phe Leu
Tyr Ser Asn Val Ala 405 410 415Leu Tyr Leu Pro Asp Ser Tyr Lys Tyr
Thr Pro Ala Asn Val Thr Leu 420 425 430Pro Ala Asn Thr Asn Thr Tyr
Glu Tyr Met Asn Gly Arg Val Val Ala 435 440 445Pro Ser Leu Val Asp
Ala Tyr Ile Asn Ile Gly Ala Arg Trp Ser Leu 450 455 460Asp Pro Met
Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly465 470 475
480Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe
485 490 495His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu
Leu Leu 500 505 510Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg
Lys Asp Val Asn 515 520 525Met Ile Leu 53021596DNAAdenovirus
2ccggtcccag cgtctgacgc tgcgcttcgt gcccgtggat cgcgaggaca ccacgtactc
60gtacaaggcg cgcttcactc tggccgtggg agacaaccgg gtgctagaca tggccagcac
120ttactttgac atccgcggcg tcctggaccg cggtcccagc ttcaaaccct
actcgggcac 180ggcttacaac agcctggccc ccaagagcgc tcccaatccc
agccagtggg atgcaaagga 240aaaggaagga gttgcccaaa cagaaaaaaa
tgttttaaaa acatttggtg ttgccgctac 300aggtggtttt aatattacag
atcagggttt gttacttgga actgaggaaa cagctgaaaa 360cgttaaaaag
gatatctatg cagagaaaac tttccagcct gaacctcaag ttggtgaaga
420aaactggcag gaaagtgaag ccttttatgg aggaagggct attaagaaag
acaccaaaat 480gaagccatgc tatggttcat ttgccagacc cactaatgaa
aaaggaggac aggctaaatt 540taaaacacta gatgggcaag ttacaaaaga
tccagatatt gactttgctt actttgacgt 600ccctggcgga aaagctccaa
caggcagtag tctaccggaa gaatacaaag cagatataat 660tttgtacaca
gaaaatgtta atctggaaac accagatact cacatagtgt ataaacctgg
720caaagaagat gacaattctg aaattaactt aacacaacag tccatgccaa
acagacccaa 780ctacattggc tttagggaca actttgtagg tctcatgtac
tacaacagta ctggcaacat 840gggtgtgctg gctggtcagg cctctcagtt
gaatgctgtg gtggacttgc aagacagaaa 900caccgagctg tcttaccagc
tcttgctaga ttctctgggt gacagaacca gatactttag 960catgtggaac
tctgcggttg acagttatga tcccgatgtc aggatcattg agaatcacgg
1020tgtggaagat gaacttccaa actattgctt cccattgaat ggcactggta
ccaattccac 1080ctatcaaggt gtaaaaatta caggtaataa tgatggcgat
cttgaaaccg aatgggaaag 1140agatgaagca atctctagac aaaaccaaat
ctgcaagggc aacgtctatg ccatggagat 1200caacctccag gccaacctgt
ggaagagttt tctgtactcg aacgtagccc tgtacctgcc 1260tgactcatac
aagtacacgc cggccaacgt cacgctgccc gccaacacca acacctacga
1320gtacatgaac ggccgcgtgg tagccccctc gctggtggac gcttacatca
acatcggcgc 1380ccgctggtcg ctggatccca tggacaatgt aaacccattc
aaccaccacc gcaacgcggg 1440cctgcgctac cgttccatgt tgttgggcaa
cggtcgctac gtgcccttcc acatccaagt 1500gccccaaaag ttctttgcca
tcaagaacct gcttctgctc ccgggctcct acacctacga 1560gtggaacttc
cgcaaggacg tcaacatgat cctgca 15963324PRTAdenovirus 3Leu Ser Leu Lys
Leu Ala Asp Pro Ile Ala Ile Val Asn Gly Asp Val1 5 10 15Ser Leu Lys
Val Gly Gly Gly Leu Thr Leu Gln Glu Gly Asn Leu Thr 20 25 30Val Asp
Ala Lys Ala Pro Leu Gln Val Ala Asn Asp Asn Lys Leu Glu 35 40 45Leu
Ser Tyr Ala Asp Pro Phe Glu Val Lys Asp Thr Lys Leu Gln Leu 50 55
60Lys Val Gly His Gly Leu Lys Val Ile Asp Glu Lys Thr Ser Ser Gly65
70 75 80Leu Gln Ser Leu Ile Gly Asn Leu Val Val Leu Thr Gly Lys Gly
Ile 85 90 95Gly Thr Gln Glu Leu Lys Asp Lys Asp Asp Glu Thr Lys Asn
Ile Gly 100 105 110Val Gly Ile Asn Val Arg Ile Gly Lys Asn Glu Ser
Leu Ala Phe Asp 115 120 125Lys Asp Gly Asn Leu Val Ala Trp Asp Asn
Glu Asn Asp Arg Arg Thr 130 135 140Leu Trp Thr Thr Pro Asp Thr Ser
Pro Asn Cys Lys Ile Ser Thr Glu145 150 155 160Lys Asp Ser Lys Leu
Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile 165 170 175Leu Ala Ser
Val Ser Leu Leu Ala Val Ala Gly Ser Tyr Leu Asn Met 180 185 190Thr
Ala Ser Thr Gln Lys Ser Ile Lys Val Ser Leu Met Phe Asp Ser 195 200
205Lys Gly Leu Leu Met Thr Thr Ser Ser Ile Asp Lys Gly Tyr Trp Asn
210 215 220Tyr Arg Asn Lys Asn Ser Val Val Gly Thr Ala Tyr Glu Asn
Ala Ile225 230 235 240Pro Phe Met Pro Asn Leu Val Ala Tyr Pro Arg
Pro Asn Thr Pro Asp 245 250 255Ser Lys Ile Tyr Ala Arg Ser Lys Ile
Val Gly Asn Val Tyr Leu Ala 260 265 270Gly Leu Ala Tyr Gln Pro Ile
Val Ile Thr Val Ser Phe Asn Gln Glu 275 280 285Lys Asp Ala Ser Cys
Ala Tyr Ser Ile Thr Phe Glu Phe Ala Trp Asn 290 295 300Lys Asp Tyr
Val Gly Gln Phe Asp Thr Thr Ser Phe Thr Phe Ser Tyr305 310 315
320Ile Ala Gln Glu4975DNAAdenovirus 4ttgtcactca aactggctga
cccaatagcc atcgtcaatg gggatgtctc actcaaggtg 60ggaggtggac tcactttgca
agaaggaaac ctaactgttg atgcaaaggc tccattgcaa 120gttgcaaatg
acaacaaatt ggagctttct tatgcagacc catttgaggt taaagacact
180aagctacaat taaaagtagg tcatggttta aaagtaatag atgaaaaaac
ttcttcaggt 240cttcaaagtc taattggaaa tctcgtagtt ttaacaggaa
aaggaattgg cactcaagaa 300ttaaaagaca aagacgatga aactaaaaat
ataggagttg gaataaatgt gagaataggg 360aaaaacgaaa gtctggcgtt
tgacaaagat ggaaatttgg tagcatggga taatgaaaac 420gacaggcgca
ctctatggac aactccagac acatctccaa attgtaaaat aagtactgaa
480aaagactcca aacttacttt agtccttact aaatgcggat ctcaaattct
agcaagtgtg 540tctttgcttg ctgtcgctgg aagttatctt aatatgacag
ctagtactca aaagagtata 600aaggtatctt tgatgtttga ctcaaaaggg
cttctaatga ctacatcttc tattgataaa 660ggatattgga attatagaaa
taaaaacagc gttgttggaa ctgcttatga aaacgcaatt 720ccatttatgc
caaatttagt ggcttatcca agacctaaca cgccagactc taaaatttat
780gctagaagca aaattgttgg aaatgtttat ttagcaggtt tggcttacca
accaattgtc 840ataacagtta gttttaatca ggagaaggat gcaagttgtg
cttactcaat aacatttgaa 900tttgcctgga acaaagacta cgttggtcaa
tttgatacca cctcctttac cttctcttat 960attgcccaag aatga
9755134PRTAdenovirus 5Met Asn Gly Thr Gly Gly Pro Phe Glu Gly Gly
Leu Phe Ser Pro Tyr1 5 10 15Leu Thr Thr Arg Leu Pro Gly Trp Ala Gly
Val Arg Gln Asn Val Met 20 25 30Gly Ser Thr Val Asp Gly Arg Pro Val
Leu Pro Ala Asn Ser Ser Thr 35 40 45Met Thr Tyr Ala Thr Val Gly Ser
Ser Ser Leu Asp Ser Thr Ala Ala 50 55 60Ala Ala Ala Ala Ala Ala Ala
Met Thr Ala Thr Arg Leu Ala Ser Ser65 70 75 80Tyr Met Pro Ser Ser
Gly Ser Ser Pro Ser Val Pro Ser Ser Ile Ile 85 90 95Ala Glu Glu Lys
Leu Leu Ala Leu Leu Ala Glu Leu Glu Ala Leu Ser 100 105 110Arg Gln
Leu Ala Ala Leu Thr Gln Gln Val Ser Glu Leu Arg Glu Gln 115 120
125Gln Gln Gln Gln Asn Lys 1306519PRTAdenovirus 6Met Arg Arg Ala
Val Val Ser Ser Ser Pro Pro Pro Ser Tyr Glu Ser1 5 10 15Val Met Ala
Gln Ala Thr Leu Glu Val Pro Phe Val Pro Pro Arg Tyr 20 25 30Met Ala
Pro Thr Glu Gly Arg Asn Ser Ile Arg Tyr Ser Glu Leu Ala 35 40 45Pro
Gln Tyr Asp Thr Thr Arg Val Tyr Leu Val Asp Asn Lys Ser Ala 50 55
60Asp Ile Ala Ser Leu Asn Tyr Gln Asn Asp His Ser Asn Phe Leu Thr65
70 75 80Thr Val Val Gln Asn Asn Asp Phe Thr Pro Ala Glu Ala Ser Thr
Gln 85 90 95Thr Ile Asn Phe Asp Glu Arg Ser Arg Trp Gly Gly Asp Leu
Lys Thr 100 105 110Ile Leu His Thr Asn Met Pro Asn Val Asn Glu Tyr
Met Phe Thr Ser 115 120 125Lys Phe Lys Ala Arg Val Met Val Ala Arg
Lys His Pro Lys Asp Val 130 135 140Asp Ala Ser Asp Leu Ser Lys Asp
Ile Leu Glu Tyr Asp Trp Phe Glu145 150 155 160Phe Thr Leu Pro Glu
Gly Asn Phe Ser Glu Thr Met Thr Ile Asp Leu 165 170 175Met Asn Asn
Ala Ile Leu Glu Asn Tyr Leu Gln Val Gly Arg Gln Asn 180 185 190Gly
Val Leu Glu Ser Asp Ile Gly Val Lys Phe Asp Ser Arg Asn Phe 195 200
205Lys Leu Gly Trp Asp Pro Val Thr Lys Leu Val Met Pro Gly Val Tyr
210 215 220Thr Tyr Glu Ala Phe His Pro Asp Val Val Leu Leu Pro Gly
Cys Gly225 230 235 240Val Asp Phe Thr Glu Ser Arg Leu Ser Asn Leu
Leu Gly Ile Arg Lys 245 250 255Lys Gln Pro Phe Gln Glu Gly Phe Arg
Ile Met Tyr Glu Asp Leu Val 260 265 270Gly Gly Asn Ile Pro Ala Leu
Leu Asn Val Lys Glu Tyr Leu Lys Asp 275 280 285Lys Glu Glu Ala Gly
Thr Ala Asp Ala Asn Thr Ile Lys Ala Gln Asn 290 295 300Asp Ala Val
Pro Arg Gly Asp Asn Tyr Ala Ser Ala Ala Glu Ala Lys305 310 315
320Ala Ala Gly Lys Glu Ile Glu Leu Lys Ala Ile Leu Lys Asp Asp Ser
325 330 335Asn Arg Ser Tyr Asn Val Ile Glu Gly Thr Thr Asp Thr Leu
Tyr Arg 340 345 350Ser Trp Tyr Leu Ser Tyr Thr Tyr Gly Asp Pro Glu
Lys Gly Val Gln 355 360 365Ser Trp Thr Leu Leu Thr Thr Pro Asp Val
Thr Cys Gly Ala Glu Gln 370 375 380Val Tyr Trp Ser Leu Pro Asp Leu
Met Gln Asp Pro Val Thr Phe Arg385 390 395 400Ser Thr Gln Gln Val
Ser Asn Tyr Pro Val Val Gly Ala Glu Leu Met 405 410 415Pro Phe Arg
Ala Lys Ser Phe Tyr Asn Asp Leu Ala Val Tyr Ser Gln 420 425 430Leu
Ile Arg Ser Tyr Thr Ser Leu Thr His Val Phe Asn Arg Phe Pro 435 440
445Asp Asn Gln Ile Leu Cys Arg Pro Pro Ala Pro Thr Ile Thr Thr Val
450 455 460Ser Glu Asn Val Pro Ala Leu Thr Asp His Gly Thr Leu Pro
Leu Arg465 470 475 480Ser Ser Ile Arg Gly Val Gln Arg Val Thr Val
Thr Asp Ala Arg Arg 485 490 495Arg Thr Cys Pro Tyr Val Tyr Lys Ala
Leu Gly Ile Val Ala Pro Arg 500 505 510Val Leu Ser Ser Arg Thr Phe
5157732PRTAdenovirus 7Met Glu Glu Gln Pro Arg Lys Gln Glu Gln Glu
Glu Asp Leu Thr Thr1 5 10 15His Glu Gln Pro Lys Ile Glu Gln Asp Leu
Gly Phe Glu Glu Pro Ala 20 25 30Arg Leu Glu Pro Pro Gln Asp Glu Gln
Glu His Glu Gln Asp Ala Gly 35 40 45Gln Glu Glu Thr Asp Ala Gly Leu
Glu His Gly Tyr Leu Gly Gly Glu 50 55 60Glu Asp Val Leu Leu Lys His
Leu Gln Arg Gln Ser Leu Ile Leu Arg65 70 75 80Asp Ala Leu Ala Asp
Arg Ser Glu Thr Pro Leu Ser Val Glu Glu Leu 85 90 95Cys Arg Ala Tyr
Glu Leu Asn Leu Phe Ser Pro Arg Val Pro Pro Lys 100 105 110Arg Gln
Pro Asn Gly Thr Cys Glu Pro Asn Pro Arg Leu Asn Phe Tyr 115 120
125Pro Val Phe Ala Val Pro Glu Ala Leu Ala Thr Tyr His Ile Phe Phe
130 135 140Lys Asn Gln Lys Ile Pro Val Ser Cys Arg Ala Asn Arg Thr
Arg Ala145 150 155 160Asp Ala Leu Leu Ala Leu Gly Pro Gly Ala His
Ile Pro Asp Ile Ala 165 170 175Ser Leu Glu Glu Val Pro Lys Ile Phe
Glu Gly Leu Gly Arg Asp Glu 180 185 190Thr Arg Ala Ala Asn Ala Leu
Lys Glu Thr Ala Glu Glu Glu Gly His 195 200 205Thr Ser Ala Leu Val
Glu Leu Glu Gly Asp Asn Ala Arg Leu Ala Val 210 215 220Leu Lys Arg
Ser Val Glu Leu Thr His Phe Ala Tyr Pro Ala Val Asn225 230 235
240Leu Pro Pro Lys Val Met Arg Arg Ile Met Asp Gln Leu Ile Met Pro
245 250 255His Ile Glu Ala Leu Asp Glu Ser Gln Glu Gln Arg Pro Glu
Asp Val 260 265 270Arg Pro Val Val Ser Asp Glu Met Leu Ala Arg Trp
Leu Gly Thr Arg 275 280 285Asp Pro Gln Ala Leu Glu Gln Arg Arg Lys
Leu Met Leu Ala Val Val 290 295 300Leu Val Thr Leu Glu Leu Glu Cys
Met Arg Arg Phe Phe Ser Asp Pro305 310 315 320Glu Thr Leu Arg Lys
Val Glu Glu Thr Leu His Tyr Thr Phe Arg His 325 330 335Gly Phe Val
Arg Gln Ala Cys Lys Ile Ser Asn Val Glu Leu Thr Asn 340 345 350Leu
Val Ser Cys Leu Gly Ile Leu His Glu Asn Arg Leu Gly Gln Thr 355 360
365Val Leu His Ser Thr Leu Lys Gly Glu Ala Arg Arg Asp Tyr Val Arg
370 375 380Asp Cys Ile Phe Leu Phe Leu Cys His Thr Trp Gln Ala Ala
Met Gly385 390 395 400Val Trp Gln Gln Cys Leu Glu Asp Glu Asn Leu
Lys Glu Leu Asp Lys 405 410 415Val Leu Ala Arg Asn Leu Lys Lys Leu
Trp Thr Gly Phe Asp Glu Arg 420 425 430Thr Val Ala Ser Asp Leu Ala
Gln Ile Val Phe Pro Glu Arg Leu Arg 435 440 445Gln Thr Leu Lys Gly
Gly Leu Pro Asp Phe Met Ser Gln Ser Met Ile 450 455 460Gln Asn Tyr
Arg Thr Phe Ile Leu Glu Arg Ser Gly Met Leu Pro Ala465 470 475
480Thr Cys Asn Ala Phe Pro Ser Asp Phe Val Pro Leu Ser Tyr Arg Glu
485 490 495Cys Pro Pro Pro Leu Trp Ser His Cys Tyr Leu Leu Gln Leu
Ala Asn 500 505 510Tyr Ile Ala Tyr His Ser Asp Val Ile Glu Asp Val
Ser Gly Glu Gly 515
520 525Leu Leu Glu Cys His Cys Arg Cys Asn Leu Cys Ser Pro His Arg
Ser 530 535 540Leu Val Cys Asn Pro Gln Leu Leu Ser Glu Thr Gln Val
Ile Gly Thr545 550 555 560Phe Glu Leu Gln Gly Pro Gln Glu Ser Thr
Ala Pro Leu Lys Leu Thr 565 570 575Pro Gly Leu Trp Thr Ser Ala Tyr
Leu Arg Lys Phe Val Pro Glu Asp 580 585 590Tyr His Ala His Glu Ile
Lys Phe Phe Glu Asp Gln Ser Arg Pro Gln 595 600 605His Ala Asp Leu
Thr Ala Cys Val Ile Thr Gln Gly Ala Ile Leu Ala 610 615 620Gln Leu
His Ala Ile Gln Lys Ser Arg Gln Glu Phe Leu Leu Lys Lys625 630 635
640Gly Arg Gly Val Tyr Leu Asp Pro Gln Thr Gly Glu Val Leu Asn Pro
645 650 655Gly Leu Pro Gln His Ala Glu Glu Glu Ala Gly Ala Ala Ser
Gly Gly 660 665 670Asp Gly Arg Arg Met Gly Gln Pro Gly Arg Gly Gly
Arg Met Gly Gly 675 680 685Gly Asp Arg Gly Gly Arg Ile Gly Arg Gly
Gly Arg Gly Ala Gly Asn 690 695 700Arg Ala Ala Arg Arg Arg Thr Ile
Arg Ala Gly Ser Pro Gly Gly His705 710 715 720Gly Tyr Asn Leu Arg
Ser Ser Gly Gln Ala Ser Ser 725 7308373PRTAdenovirus 8Met His Pro
Val Leu Arg Gln Met Arg Pro Thr Pro Pro Ala Thr Thr1 5 10 15Ala Thr
Ala Ala Val Ala Gly Ala Gly Ala Val Ala Pro Pro Gln Thr 20 25 30Glu
Met Asp Leu Glu Glu Gly Glu Gly Leu Ala Arg Leu Gly Ala Pro 35 40
45Ser Pro Glu Arg His Pro Arg Val Gln Leu Gln Lys Asp Val Arg Pro
50 55 60Ala Tyr Val Pro Pro Gln Asn Leu Phe Arg Asp Arg Ser Gly Glu
Glu65 70 75 80Pro Glu Glu Met Arg Asp Cys Arg Phe Arg Ala Gly Arg
Glu Leu Arg 85 90 95Glu Gly Leu Asp Arg Gln Arg Val Leu Arg Asp Glu
Asp Phe Glu Pro 100 105 110Asn Glu Gln Thr Gly Ile Ser Pro Ala Arg
Ala His Val Ala Ala Ala 115 120 125Asn Leu Val Thr Ala Tyr Glu Gln
Thr Val Lys Gln Glu Arg Asn Phe 130 135 140Gln Lys Ser Phe Asn Asn
His Val Arg Thr Leu Ile Ala Arg Glu Glu145 150 155 160Val Ala Leu
Gly Leu Met His Leu Trp Asp Leu Ala Glu Ala Ile Val 165 170 175Gln
Asn Pro Asp Ser Lys Pro Leu Thr Ala Gln Leu Phe Leu Val Val 180 185
190Gln His Ser Arg Asp Asn Glu Ala Phe Arg Glu Ala Leu Leu Asn Ile
195 200 205Ala Glu Pro Glu Gly Arg Trp Leu Leu Glu Leu Ile Asn Ile
Leu Gln 210 215 220Ser Ile Val Val Gln Glu Arg Ser Leu Ser Leu Ala
Glu Lys Val Ala225 230 235 240Ala Ile Asn Tyr Ser Val Leu Ser Leu
Gly Lys Phe Tyr Ala Arg Lys 245 250 255Ile Tyr Lys Thr Pro Tyr Val
Pro Ile Asp Lys Glu Val Lys Ile Asp 260 265 270Ser Phe Tyr Met Arg
Met Ala Leu Lys Val Leu Thr Leu Ser Asp Asp 275 280 285Leu Gly Val
Tyr Arg Asn Asp Arg Ile His Lys Ala Val Ser Thr Ser 290 295 300Arg
Arg Arg Glu Leu Ser Asp Arg Glu Leu Met Leu Ser Leu Arg Arg305 310
315 320Ala Leu Val Gly Gly Ala Ala Gly Gly Glu Glu Ser Tyr Phe Asp
Met 325 330 335Gly Ala Asp Leu His Trp Gln Pro Ser Arg Arg Ala Leu
Glu Ala Ala 340 345 350Tyr Gly Pro Glu Asp Leu Glu Glu Asp Glu Glu
Glu Glu Glu Asp Ala 355 360 365Pro Ala Ala Gly Tyr
37093531DNAAdenovirus 9atggccttgg ttcaaagtca cggggccagt ggtcttcacg
cagaggcggc agatccagga 60tgtcaaccga cgcgtcgtcg cgcacgccag cgctctcagg
gcgcagcacc gggacctgcc 120cgagcgccac gccgacgtgc ctctgccgcc
cctgcccgcg gggccggaac cgccgctgcc 180gccgggagcg cgtccgcgac
accgcttcta aaagcgcacc gcggcacggt cgtggccccg 240cgcagctacg
ggctcatgca atgcgtggac acggccacca actcacccgt agaaatcaag
300taccatctgc atctcaagca cgccctcacc cgcctctacg aggtcaacct
cagaaccctg 360cccccggacc tggatctccg cgacaccatg gacagctccc
aactgcgcgc cctcgtcttc 420gctctccgcc cccgccgcgc cgagatctgg
acctggctcc cgcgcgggct cgtcagcctc 480tccgtcctcg aggagcccca
gggtgagtcc cacgcaggcg aacatgaaaa ccaccagcca 540gggccgccac
tcctgaagtt cctcctcaag ggacgcgctg tgtatctcgt ggatgaggta
600cagcccgtgc agcgctgcga gtactgcgga cgcttttaca agcatcagca
cgagtgctcg 660gttcgccggc gggatttcta ctttcatcac atcaacagcc
actcgtccaa ctggtggcag 720gaaatccagt tcttcccaat cggctctcat
cctcgcacgg agaggctctt tgtcacctac 780gatgtagaaa cctacacctg
gatggggtcc ttcggcaagc agctcgtccc cttcatgctg 840gtcatgaaat
tctccgggga ccccgagctg atcgccctgg ctcgcgatct cgccgtgcgc
900ttacgctggg atcgctggga gcgggacccc ctcaccttct actgcgtcac
accagaaaag 960atggccgtgg gccagcagtt ccgcctcttt cgcgacgagc
tccagaccct catggcccgc 1020gagctctggg cttccttcat gcaagccaac
ccacatctcc aggagtgggc gctcgagcag 1080cacggcctgc aatgccccga
ggacctcacc tacgaggagc tcaaaaagct gccgcacatc 1140aaaggccgcc
cgcgattcat ggaactctac atcgtcgggc acaacatcaa cggcttcgac
1200gagatcgtcc tcgccgccca ggtgatcaac aaccgagcct ccgtcccggg
ccctttccgc 1260atcacccgca atttcatgcc gcgggcaggc aagattctct
tcaatgacgt cactttcgct 1320ctgcctaacc ccctctcgaa gaagcgcacc
gatttcgagc tctgggagca cggcggctgc 1380gacgactcgg acttcaagta
ccagttcttg aaagtcatgg tcagggacac cttcgccctg 1440acgcacacct
cgctccgcaa ggccgctcaa gcttacgccc tccccgtgga gaagggctgc
1500tgtccctaca aggccgtgaa ccatttctac atgctgggct cttaccgtgc
ggacgatcga 1560ggattcccgc tccgggagta ctggaaggat gacgaggagt
acgccctcaa ccgcgagctg 1620tgggagaaga aaggagaagc gggttatgac
atcatccgcg aaacgctgga ctactgcgcc 1680atggacgtcc tcgtcaccgc
cgagctcgtc gccaagctgc aagactccta cgcgcacttc 1740atccgcgact
cggtccgcct gccccacgcc cactttaaca tcttccaacg gcccaccatc
1800tcctccaact cgcacgccat ctttcgccag atcgtcttcc gcgccgagca
gccccagcgc 1860accaatctcg gcccctcctt cttggccccc tcgcacgagt
tgtatgacta cgtgcgcgcc 1920agcatccgcg gggggcgctg ttatcccacc
tacatcggca tcctctcgga gcccatctat 1980gtctatgaca tctgcggcat
gtacgcctcc gccctcacgc atcccatgcc ctggggtccg 2040cccctcaacc
cctacgagcg agcgctggcc gcccgcgagt ggcagatggc cttggatgat
2100gcatcctcaa aaatcgatta ttttgacaag gaactctgtc cgggcatctt
caccatcgat 2160gcggaccccc ctgacgagca cctcctggat gtgctgcccc
cgttctgctc gcgcaagggt 2220ggcagactct gctggaccaa cgagcccctg
cgcggcgagg tggccaccag cgtggacctg 2280gtcaccctgc ataaccgcgg
ctggcgcgtc aggatcgtgc ccgacgagcg caccaccgtc 2340ttccccgaat
ggaagtgcgt cgcgcgcgag tatgtccagc tcaacatcgc ggccaaggag
2400cgcgccgacc gtgacaaaaa tcagaccatg agatccatcg ccaagcttct
ctccaacgcc 2460ctctatggct cctttgccac caagcttgac aataaaaaaa
tagtcttttc tgaccagatg 2520gatgaaagtc tcctaaaaag catcgcggca
gggcaagcca acatcaaatc ctcctcgttt 2580ctagaaactg acaacctgag
tgccgaggtc atgcccgctc tcgagaggga atacctaccc 2640caacagctgg
cgctcgtgga cagcgacgcg gaagagagtg aggacgagca cagacccgcc
2700cccttttata cccccccgtc ggggaccccc ggtcacgtgg cctacaccta
caagccaatc 2760accttcttgg atgcggagga gggggacatg tgcttgcaca
cggtggaaaa ggtggacccc 2820ctggtggaca acgaccgcta cccctcgcac
gtggcctcct ttgtcttggc gtggacgcgc 2880gccttcgtct cagagtggtc
cgaatttctc tacgaggagg accgcgggac gcccctgcag 2940gacaggccaa
tcaagtccat ctacggggac accgacagcc tgtttgtcac cgagcgcgga
3000cacagactca tggagacgcg aggtaagaag cgcatcaaaa agaacggggg
aaaactggtt 3060tttgaccccg aacaacccga gctcacctgg ctcgtcgagt
gcgagaccgt ctgcgcccac 3120tgcggagcgg acgcgttcgc ccccgagtcc
gtctttctcg cacccaagct atacgccctg 3180caatccctcc tctgtcccgc
ctgtgggcgc tcttccaagg gcaagctccg cgccaagggc 3240cacgccgccg
aggccctcaa ctacgagctc atggtcaact gctatctcgc cgacgcgcag
3300ggcgaagacc gtgcccgttt cagcaccagc aggatgagtc tcaagcgaac
ccttgcaagc 3360gcccagcccg gggcccaccc cttcaccgtg acggagacaa
ccctcacgcg gaccctgaga 3420ccctggaagg acatgacgct ggccgcgctg
gacgcccatc gtctcgtgcc ctacagtcgc 3480agtcgtccca acccccgaaa
cgaggaagtc tgctggatcg agatgccgta g 35311028607DNAArtificial
SequenceSynthetic 10catcatcaat aatatacccc acaaagtaaa caaaagttaa
tatgcaaatg agcttttgaa 60tttagggcgg ggccgtcgct gattggccgt tgcaagaacc
gttagtgacg tcacgacgca 120cggccgacgc tcgccgcgga ggcgtggcct
agcccggaag caagtcgcgg ggctgatgac 180gtataaaaaa gcggacttta
gacccggaaa cggccgattt tcccgcggcc acgcccggat 240atgaggtaat
tctgggcgga tgcaagtgaa attaggtcat tttggcgcga aaactgaatg
300aggaagtgaa aagcgaaaaa taccggtccc tcccagggcg gaatatttac
cgagggccga 360gagactttga ccgattacgt gggggtttcg attgcggtgt
ttttttcgcg aatttccgcg 420tccgtgtcaa agtccggtgt ttatgtcaca
gatcagctga tagctgctgt tggagaacga 480tgccttctcc agggtgaacc
tgaacggcat ctttgacatg gatgtctcgg tgtacaagat 540cctgagatac
gatgagacca agtccagggt gcgcgcttgc gagtgcgggg gcagacacac
600caggatgcag ccagtggccc tggatgtgac cgaggagctg agaccagacc
acctggtgat 660ggcctgtacc gggaccgagt tcagctccag tggggaggac
acagattaga ggtaggtttt 720tgagtagtgg gcgtggctaa tgtgagtata
aaggtgggtg tcttacgagg gtctttttgc 780ttttctgcag acatcatgaa
cgggaccggc gggcccttcg aaggggggct ttttagccct 840tatttgacaa
cccgcctgcc gggatgggcc ggagttcgtc agaatgtgat gggatcgacg
900gtggatgggc gcccagtgct tccagcaaat tcctcgacca tgacctacgc
gaccgtgggg 960agctcgtcgc tcgacagcac cgccgcagcc gcggcagccg
cagccgccat gacagcgacg 1020agactggcct cgagctacat gcccagcagc
ggcagcagcc cctctgtgcc cagttccatc 1080atcgccgagg agaaactgct
ggccctgctg gccgagctgg aagccctgag ccgccagctg 1140gccgccctga
cccagcaggt gtctgagctc cgcgagcagc agcagcagca aaataaatga
1200ttcaataaac acagattctg attcaaacag caaagcatct ttattattta
ttttttcgcg 1260cgcggtaggc cctggtccac ctctcccgat cattgagagt
gcggtggatt ttttccagga 1320cccggtagag gtgggattgg atgttgaggt
acatgggcat gagcccgtcc cgggggtgga 1380ggtagcacca ctgcatggcc
tcgtgctctg gggtcgtgtt gtagatgatc cagtcatagc 1440aggggcgctg
ggcgtggtgc tggatgatgt ctttaaggag gagactgatg gccacgggga
1500gccccttggt gtaggtgttg gcaaagcggt tgagctggga aggatgcatg
cggggggaga 1560tgatgtgcag tttggcctgg atcttgaggt tggcaatgtt
gccgcccaga tcccgcctgg 1620ggttcatgtt gtgcaggacc accaggacgg
tgtagcccgt gcacttgggg aacttatcat 1680gcaacttgga agggaatgcg
tggaagaatt tggagacgcc cttgtgcccg cccaggtttt 1740ccatgcactc
atccatgatg atggcgatgg gcccgtgggc tgcggctttg gcaaagacgt
1800ttctggggtc agagacatcg taattatgct cctgggtgag atcgtcataa
gacattttaa 1860tgaatttggg gcggagggtg ccagattggg ggacgatggt
tccctcgggc cccggggcga 1920agttcccctc gcagatctgc atctcccagg
ctttcatctc ggaggggggg atcatgtcca 1980cctgcggggc tatgaaaaaa
acggtttccg gggcgggggt gatgagctgc gaggagagca 2040ggtttcttaa
cagctgggac ttgccgcacc cggtcgggcc gtatatgacc ccgatgacgg
2100gttgcaggtg gtagttcaag gacatgcagc tgccgtcgtc ccggaggagg
ggggccacct 2160cgttgagcat gtctctgact tggaggtttt cccggacgag
ctcgccgagg aggcggtccc 2220cgcccagcga gagcagctct tgcagggaag
caaagttttt caggggcttg agcccgtcgg 2280ccatgggcat cttggcgagg
gtctgcgaga ggagctccag gcggtcccag agctcggtga 2340cgtgctctac
ggcatctcga tccagcagac ttcctcgttt cgggggttgg gacgactgcg
2400actgtagggc acgagacgat gggcgtccag cgcggccagc gtcatgtcct
tccagggtct 2460cagggtccgc gtgagggttg tctccgtcac ggtgaagggg
tgggccccgg gctgggcgct 2520tgcaagggtt cgcttgagac tcatcctgct
ggtgctgaaa cgggcacggt cttcgccctg 2580cgcgtcggcg agatagcagt
tgaccatgag ctcgtagttg agggcctcgg cggcgtggcc 2640cttggcgcgg
agcttgccct tggaagagcg cccacaggcg ggacagagga gggattgcag
2700ggcgtatagc ttgggtgcga gaaagacgga ctcgggggcg aacgcgtccg
ctccgcagtg 2760ggcgcagacg gtctcgcact cgacgagcca ggtgagctcg
ggttgttcgg ggtcaaaaac 2820cagttttccc ccgttctttt tgatgcgctt
cttacctcgc gtctccatga gtctgtgtcc 2880gcgctcggtg acaaacaggc
tgtcggtgtc cccgtagatg gacttgattg gcctgtcctg 2940caggggcgtc
ccgcggtcct cctcgtagag aaattcggac cactctgaga cgaaggcgcg
3000cgtccacgcc aagacaaagg aggccacgtg cgaggggtag cggtcgttgt
ccaccagggg 3060gtccaccttt tccaccgtgt gcaagcacat gtccccctcc
tccgcatcca agaaggtgat 3120tggcttgtag gtgtaggcca cgtgaccggg
ggtccccgac gggggggtat aaaagggggc 3180gggtctgtgc tcgtcctcac
tctcttccgc gtcgctgtcc acgagcgcca gctgttgggg 3240taggtattcc
ctctcgagag cgggcatgac ctcggcactc aggttgtcag tttctagaaa
3300cgaggaggat ttgatgttgg cttgccctgc cgcgatgctt tttaggagac
tttcatccat 3360ctggtcagaa aagactattt ttttattgtc aagcttggtg
gcaaaggagc catagagggc 3420gttggagaga agcttggcga tggatctcat
ggtctgattt ttgtcacggt cggcgcgctc 3480cttggccgcg atgttgagct
ggacatactc gcgcgcgacg cacttccatt cggggaagac 3540ggtggtgcgc
tcgtcgggca cgatcctgac gcgccagccg cggttatgca gggtgaccag
3600gtccacgctg gtggccacct cgccgcgcag gggctcgttg gtccagcaga
gtctgccacc 3660cttgcgcgag cagaacgggg gcagcacatc caggaggtgc
tcgtcagggg ggtccgcatc 3720gatggtgaag atgcccggac agagttcctt
gtcaaaataa tcgatttttg aggatgcatc 3780atccaaggcc atctgccact
cgcgggcggc cagcgctcgc tcgtaggggt tgaggggcgg 3840accccagggc
atgggatgcg tgagggcgga ggcgtacatg ccgcagatgt catagacata
3900gatgggctcc gagaggatgc cgatgtaggt gggataacag cgccccccgc
ggatgctggc 3960gcgcacgtag tcatacaact cgtgcgaggg ggccaagaag
gaggggccga gattggtgcg 4020ctggggctgc tcggcgcgga agacgatctg
gcgaaagatg gcgtgcgagt tggaggagat 4080ggtgggccgt tggaagatgt
taaagtgggc gtggggcagg cggaccgagt cgcggatgaa 4140gtgcgcgtag
gagtcttgca gcttggcgac gagctcggcg gtgacgagga cgtccatggc
4200gcagtagtcc agcgtttcgc ggatgatgtc ataacccgct tctcctttct
tctcccacag 4260ctcgcggttg agggcgtact cctcgtcatc cttccagtac
tcccggagcg ggaatcctcg 4320atcgtccgca cggtaagagc ccagcatgta
gaaatggttc acggccttgt agggacagca 4380gcccttctcc acggggaggg
cgtaagcttg agcggccttg cggagcgagg tgtgcgtcag 4440ggcgaaggtg
tccctgacca tgactttcaa gaactggtac ttgaagtccg agtcgtcgca
4500gccgccgtgc tcccagagct cgaaatcggt gcgcttcttc gagagggggt
taggcagagc 4560gaaagtgacg tcattgaaga gaatcttgcc tgcccgcggc
atgaaattgc gggtgatgcg 4620gaaagggccc gggacggagg ctcggttgtt
gatcacctgg gcggcgagga cgatctcgtc 4680gaagccgttg atgttgtgcc
cgacgatgta gagttccatg aatcgcgggc ggcctttgat 4740gtgcggcagc
tttttgagct cctcgtaggt gaggtcctcg gggcattgca ggccgtgctg
4800ctcgagcgcc cactcctgga gatgtgggtt ggcttgcatg aaggaagccc
agagctcgcg 4860ggccatgagg gtctggagct cgtcgcgaaa gaggcggaac
tgctggccca cggccatctt 4920ttctggtgtg acgcagtaga aggtgagggg
gtcccgctcc cagcgatccc agcgtaagcg 4980cacggcgaga tcgcgagcca
gggcgatcag ctcggggtcc ccggagaatt tcatgaccag 5040catgaagggg
acgagctgct tgccgaagga ccccatccag gtgtaggttt ctacatcgta
5100ggtgacaaag agcctctccg tgcgaggatg agagccgatt gggaagaact
ggatttcctg 5160ccaccagttg gacgagtggc tgttgatgtg atgaaagtag
aaatcccgcc ggcgaaccga 5220gcactcgtgc tgatgcttgt aaaagcgtcc
gcagtactcg cagcgctgca cgggctgtac 5280ctcatccacg agatacacag
cgcgtccctt gaggaggaac ttcaggagtg gcggccctgg 5340ctggtggttt
tcatgttcgc ctgcgtggga ctcaccctgg ggctcctcga ggacggagag
5400gctgacgagc ccgcgcggga gccaggtcca gatctcggcg cggcgggggc
ggagagcgaa 5460gacgagggcg cgcagttggg agctgtccat ggtgtcgcgg
agatccaggt ccgggggcag 5520ggttctgagg ttgacctcgt agaggcgggt
gagggcgtgc ttgagatgca gatggtactt 5580gatttctacg ggtgagttgg
tggccgtgtc cacgcattgc atgagcccgt agctgcgcgg 5640ggccacgacc
gtgccgcggt gcgcttttag aagcggtgtc gcggacgcgc tcccggcggc
5700agcggcggtt ccggccccgc gggcaggggc ggcagaggca cgtcggcgtg
gcgctcgggc 5760aggtcccggt gctgcgccct gagagcgctg gcgtgcgcga
cgacgcgtcg gttgacatcc 5820tggatctgcc gcctctgcgt gaagaccact
ggccccgtga ctttgaacct gaaagacagt 5880tcaacagaat caatctcggc
gtcattgacg gcggcctgac gcaggatctc ttgcacgtcg 5940cccgagttgt
cctggtaggc gatttcggac atgaactgct cgatctcctc ctcctggaga
6000tcgccgcggc cagcgcgctc gacggtggcg gcgaggtcat tcgagatgcg
acccatgagc 6060tgcgagaagg cgcccaggcc gctctcgttc cagacgcggc
tgtagaccac gtccccgtcg 6120gcgtcgcgcg cgcgcatgac cacctgcgcg
aggttgagct ccacgtgccg cgtgaagacg 6180gcgtagttgc gcaggcgctg
gaagaggtag ttgagggtgg tggcgatgtg ctcggtgacg 6240aagaagtaca
tgatccagcg gcgcaggggc atctcgctga tgtcgccgat ggcctccagc
6300ctttccatgg cctcgtagaa atccacggcg aagttgaaaa actgggcgtt
gcgggccgag 6360accgtgagct cgtcttccaa gagccggatg agctcggcga
tggtggcgcg cacctcgcgc 6420tcgaaacccc cgggggcctc ctcctcttcc
tcttcttcca tgacgacctc ttcttctatt 6480tcttcctctg ggggcggtgg
tggtggcggg gcccgacgac gacggcggcg caccgggaga 6540cggtcgacga
agcgctcgat catctccccg cggcggcgac gcatggtttc ggtgacggcg
6600cgaccccgtt cgcgaggacg cagcgtgaag acgccgccgg tcatctcccg
gtaatggggc 6660gggtccccgt tgggcagcga gagggcgctg acgatgcatc
ttatcaattg cggtgtaggg 6720gacgtgagcg cgtcgagatc gaccggatcg
gagaatcttt cgaggaaagc gtctagccaa 6780tcgcagtcgc aaggtaagct
caaacacgta gcagccctgt ggacgctgtt agaattgcgg 6840ttgctgatga
tgtaattgaa gtaggcgttt ttaaggcggc ggatggtggc gaggaggacc
6900aggtccttgg gtcccgcttg ctggatgcgg agccgctcgg ccatgcccca
ggcctggccc 6960tgacaccggc tcaggttctt gtagtagtca tgcatgagcc
tttcaatgtc atcactggcg 7020gaggcggagt cttccatgcg ggtgaccccg
acgcccctga gcggctgcac gagcgccagg 7080tcggcgacga cgcgctcggc
gaggatggcc tgttgcacgc gggtgagggt gtcctggaag 7140tcgtccatgt
cgacgaagcg gtggtaggcc cctgtgttga tggtgtaggt gcagttggcc
7200atgagcgacc agttgacggt ctgcaggccg ggttgcacga cctcggagta
cctgatccgc 7260gagaaggcgc gcgagtcgaa gacgtagtcg ttgcaggtgc
gcacgaggta ctggtagccg 7320actaggaagt gcggcggcgg ctggcggtag
agtggccagc gctgggtggc cggcgctccc 7380ggggccaggt cctcgagcat
gaggcggtgg tagccgtaga ggtagcggga catccaggtg 7440atgccggcgg
cagtggtgga ggcgcgcggg aactcgcgga cgcggttcca gatgttgcgc
7500agcggcagga aatagtccat ggtcggcacg gtctggccgg tgagacgcgc
gcagtcattg 7560acgctctaga ggcaaaaacg aaagcggttg agcgggctct
tcctccgtag cctggcggaa 7620cgcaaacggg ttaggccgcg tgtgtacccc
ggttcgagtc ccctcgaatc aggctggagc 7680cgcgactaac gtggtattgg
cactcccgtc tcgacccgag cccgatagcc gccaggatac 7740ggcggagagc
cctttttgcc ggccgagggg agtcgctaga cttgaaagcg gccgaaaacc
7800ctgccgggta gtggctcgcg cccgtagtct ggagaagcat cgccagggtt
gagtcgcggc 7860agaacccggt tcgcggacgg ccgcggcgag cgggacttgg
tcaccccgcc gatttaaaga
7920cccacagcca gccgacttct ccagttacgg gagcgagccc ccttttttct
ttttgccaga 7980tgcatcccgt cctgcgccaa atgcgtccca cccccccggc
gaccaccgcg accgcggccg 8040tagcaggcgc cggcgctgta gccccgccac
agacagagat ggacttggaa gagggcgaag 8100ggctggcgag actgggggcg
ccgtccccgg agcgacaccc ccgcgtgcag ctgcagaagg 8160acgtgcgccc
ggcgtacgtg cctccgcaga acctgttcag ggaccgcagc ggggaggagc
8220ccgaggagat gcgcgactgc cggtttcggg cgggcaggga gctgcgcgag
ggcctggacc 8280gccagcgcgt gctgcgcgac gaggatttcg agccgaacga
gcagacgggg atcagccccg 8340cgcgcgcgca cgtggcggcg gccaacctgg
tgacggccta cgagcagacg gtgaagcagg 8400agcgcaactt ccaaaagagt
ttcaacaacc acgtgcgcac cctgatcgcg cgcgaggagg 8460tggccctggg
cttgatgcac ctgtgggacc tggcggaggc catcgtgcag aacccggaca
8520gcaagcctct gacggcgcag ctgttcctgg tggtgcagca cagcagggac
aacgaggcgt 8580tcagggaggc gctgctgaac atcgccgagc ccgagggtcg
ctggctgctg gagctgatta 8640acatcttgca gagcatcgta gtgcaggagc
gcagcttgag cctggccgag aaggtggcgg 8700cgatcaacta ctcggtgctg
agcctgggca agttttacgc gcgcaagatt tacaagacgc 8760cgtatgtgcc
catagacaag gaggtgaaga tagacagctt ttacatgcgc atggcgctca
8820aggtgctgac gctgagcgac gacctgggcg tgtaccgcaa cgaccgcatc
cacaaggccg 8880tgagcacgag ccggcggcgc gagctgagcg accgcgagct
gatgctgagc ctgcgccggg 8940cgctggtagg gggcgccgcc ggcggcgagg
agtcctactt cgacatgggg gcggacctgc 9000attggcagcc gagccggcgc
gccttggagg ccgcctacgg tccagaggac ttggaagagg 9060atgaggaaga
ggaggaggat gcacccgctg cggggtactg acgcctccgt gatgtgtttt
9120tagatgtccc agcaagcccc ggaccccgcc ataagggcgg cgctgcaaag
ccagccgtcc 9180ggtctagcat cggacgactg ggaggccgcg atgcaacgca
tcatggccct gacgacccgc 9240aaccccgagt cctttagaca acagccgcag
gccaacagac tctcggccat tctggaggcg 9300gtggtcccct ctcggaccaa
ccccacgcac gagaaggtgc tggcgatcgt gaacgcgctg 9360gcggagaaca
aggccatccg tcccgacgag gccgggctgg tgtacaacgc cctgctggag
9420cgcgtgggcc gctacaacag cacgaacgtg cagtccaacc tggaccggct
ggtgacggac 9480gtgcgcgagg ccgtggcgca gcgcgagcgg ttcaagaacg
agggcctggg ctcgctggtg 9540gcgctgaacg ccttcctggc gacgcagccg
gcgaacgtgc cgcgcgggca ggacgattac 9600accaacttta tcagcgcgct
gcggctgatg gtgaccgagg tgccccagag cgaggtgtac 9660cagtcgggcc
ctgactactt tttccagacg agccggcagg gcttgcagac ggtgaacctg
9720agtcaggctt tcaagaacct gcgcgggctg tggggcgtgc aggcgcccgt
gggcgaccgg 9780tcgacggtga gcagcttgct gacgcccaac tcgcggctgc
tgctgttgct gatcgcgccc 9840tttactgaca gcggcagcgt aaaccgcaac
tcgtacctgg gccacctgct gacgctgtac 9900cgcgaggcca taggccaggc
acaggtggac gagcagacct tccaggaaat tacgagcgtg 9960agccgcgcgc
tggggcagaa cgacaccgac agtctgaggg ccaccctgaa ctttttgctg
10020accaatagac agcagaagat cccggcgcag tacgcactgt cggccgagga
ggaaaggatc 10080ctgagatatg tgcagcagag cgtagggctg ttcctgatgc
aggagggtgc cacccccagc 10140gccgcgctgg acatgaccgc gcgcaacatg
gaacctagca tgtacgccgc caaccggccg 10200ttcatcaata agctgatgga
ctacctgcac cgcgcggcgg ccatgaacac ggactacttt 10260accaacgcca
tcctgaaccc gcactggctc ccgccgccgg gtttctacac gggcgagtac
10320gacatgcccg accccaacga cgggttcctg tgggacgacg tggacagcgc
ggtgttctcg 10380ccggcctttc aaaagcgtca ggaggcgccg ccgagcgagg
gcgcggtggg gagaagcccc 10440tttcctagct tagggagttt gcatagcttg
ccgggctcgg tgaacagcgg cagggtgagc 10500cggccgcgct tgctgggcga
ggacgagtac ctgaacgact cgctactgca gccgccgcgg 10560gccaagaacg
ccatggccaa taacgggata gagagtctgg tggacaaact gaaccgctgg
10620aagacctacg ctcaggacca tagggacgcg cccgcgccgc ggcgacagcg
ccacgaccgg 10680cagcggggcc tggtgtggga cgacgaggac tcggccgacg
atagcagcgt gttggacttg 10740ggcgggagcg gtggggccaa cccgttcgca
catctgcagc ccaaactggg gcggcggatg 10800ttttgaaatg caaaataaaa
ctcaccaagg ccatagcgtg cgttctcttc cttgttagag 10860atgaggcgcg
cggtggtgtc ttcctctcct cctccctcgt acgagagcgt gatggcgcag
10920gcgaccctgg aggttccgtt tgtgcctccg cggtatatgg ctcctacgga
gggcagaaac 10980agcattcgtt actcggagct ggctccgcag tacgacacca
ctcgcgtgta cttggtggac 11040aacaagtcgg cggacatcgc ttctctgaac
taccaaaacg accacagcaa cttcctgacc 11100acggtggtgc agaacaacga
tttcaccccc gccgaggcca gcacgcagac gataaatttt 11160gacgagcggt
cgcggtgggg cggtgatctg aagaccattc tgcacaccaa catgcccaat
11220gtgaacgagt acatgttcac cagcaagttt aaggcgcggg tgatggtggc
tagaaagcat 11280cccaaagatg tagatgccag tgatttaagc aaggatatct
tagagtatga ttggtttgag 11340tttaccctgc ccgagggcaa cttttccgag
accatgacca tagacctgat gaacaacgcc 11400atcttggaaa actacttgca
agtggggcgg caaaatggcg tgctggagag cgatatcggt 11460gtcaagtttg
acagcaggaa tttcaagctg ggctgggacc cggtgaccaa gctggtgatg
11520cctggggtct acacctacga ggccttccac ccggacgtgg tgctgctgcc
gggctgcggg 11580gtggacttca ccgagagtcg tctgagcaac ctcctgggca
ttcgcaagaa gcaacctttc 11640caagagggct tcagaatcat gtatgaggat
ctagtagggg gcaacatccc cgccctcctg 11700aatgtcaagg agtatctgaa
ggataaggaa gaagctggca cagcagatgc aaataccatt 11760aaggctcaga
atgatgcagt cccaagagga gataactatg catcagcggc agaagccaaa
11820gcagcaggaa aagaaattga gttgaaggcc attttgaaag atgattcaaa
cagaagctac 11880aatgtgatcg agggaaccac agacaccctg taccgcagtt
ggtacctgtc ctatacctac 11940ggtgatcccg agaagggagt gcagtcgtgg
acactgctta ccaccccgga cgtcacctgc 12000ggcgcggagc aagtctactg
gtcgctgccg gacctcatgc aagaccccgt caccttccgc 12060tctacccagc
aagtcagcaa ctaccccgtg gtcggcgccg agctcatgcc tttccgcgcc
12120aagagctttt acaacgacct cgccgtctac tctcagctca tccgcagcta
cacctccctc 12180acccacgtct tcaaccgctt ccccgacaac cagatcctct
gccgcccgcc cgcgcccacc 12240atcaccaccg tcagtgaaaa cgtgcctgct
ctcacagatc acgggacgct accgctgcgc 12300agcagtatcc gcggagtcca
gcgagtgacc gtcactgacg cccgtcgccg cacctgtccc 12360tacgtctaca
aggccctggg catagtcgcg ccgcgcgtgc tttccagtcg caccttctaa
12420aaaatgtcta ttctcatctc gcccagcaat aacaccggct ggggtcttac
taggcccagc 12480accatgtacg gaggagccaa gaagcgctcc cagcagcacc
ccgtccgcgt ccgcggccac 12540ttccgcgctc cctggggcgc ttacaagcgc
gggcggactt ccaccgccgc cgtgcgcacc 12600accgtcgacg acgtcatcga
ctcggtggtc gccgacgcgc gcaactatac ccccgccccc 12660tccaccgtgg
acgcggtcat cgacagcgtg gtggccgatg cacgcgacta tgccagacgc
12720aagagccggc ggcgacggat cgccaggcgc caccggagca cgcccgccat
gcgcgccgcc 12780cgggctctgc tgcgccgcgc cagacgcacg ggccgccggg
ccatgatgcg agccgcgcgc 12840cgcgctgcca ctgcacccac ccccgcaggc
aggactcgca gacgagcggc cgccgccgcc 12900gccgcggcca tttctagcat
gaccagaccc aggcgcggaa acgtgtactg ggtgcgcgac 12960tccgtcacgg
gcgtgcgcgt gcccgtgcgc acccgtcctc ctcgtccctg atctaatgct
13020tgtgtcctcc cccgcaagcg acgatgtcaa agcgcaaaat caaggaggag
atgctccagg 13080tcgtcgcccc ggagatttac ggacccccgg accagaaacc
ccgcaaaatc aagcgggtta 13140aaaaaaagga tgaggtggac gagggggcag
tagagtttgt gcgcgagttc gctccgcggc 13200ggcgcgtaaa ttggaagggg
cgcagggtgc agcgcgtgtt gcggcccggc acggcggtgg 13260tgttcacgcc
cggcgagcgg tcctcggtca ggagcaagcg tagctatgac gaggtgtacg
13320gcgacgacga catcctggac caggcggcgg agcgggcggg cgagttcgcc
tacgggaagc 13380ggtcgcgcga agaggagctg atctcgctgc cgctggacga
aagcaacccc acgccgagcc 13440tgaagcccgt gaccctgcag caggtgctgc
cccaggcgat gctgctgccg agccgcgggg 13500tcaagcgcga gggcgagagc
atgtacccga ccatgcagat catggtgccc aagcgccggc 13560gcgtggagga
cgtgctggac accgtgaaaa tggatgtgga gcccgaggtc aaggtgcgcc
13620ccatcaagca ggtggcgccg ggcctgggcg tgcagaccgt ggacattcag
atccccaccg 13680acatggatgt cgacaaaaaa ccctcgacca gcatcgaggt
gcagactgac ccctggctcc 13740cagcctccac cgctaccgtc tctacttcta
ccgccgccac ggctaccgag cctacaagga 13800ggcgaagatg gggcgccgcc
agccggctga tgcccaacta cgtgttgcat ccttccatca 13860tcccgacgcc
gggctaccgc ggcacccggt actacgccag ccgcaggcgc ccagccagca
13920aacgccgccg ccgcaccgcc acccgccgcc gtctggcccc cgcccgcgtg
cgccgcgtaa 13980ccacgcgccg gggccgctcg ctcgttctgc ccaccgtgcg
ctaccacccc agcatccttt 14040aatccgtgtg ctgtgatact gttgcagaga
gatggctctc acttgccgcc tgcgcatccc 14100cgtcccgaat taccgaggaa
gatcccgccg caggagaggc atggcaggca gcggcctgaa 14160ccgccgccgg
cggcgggcca tgcgcaggcg cctgagtggc ggctttctgc ccgcgctcat
14220ccccataatc gccgcggcca ttggcacgat cccgggcata gcttccgttg
cgctgcaggc 14280gtcgcagcgc cgttgatgtg cgaataaagc ctctttagac
tctgacacac ctggtcctgt 14340atatttttag aatggaagac atcaattttg
cgtccctggc tccgcggcac ggcacgcggc 14400cgttcatggg cacctggaac
gagatcggca ccagccagct gaacgggggc gccttcaatt 14460ggagcagtgt
ctggagcggg cttaaaaatt tcggctcgac gctccggacc tatgggaaca
14520aggcctggaa tagtagcacg gggcagttgt taagggaaaa gctcaaagac
cagaacttcc 14580agcaaaaggt ggtggacggc ctggcctcgg gcattaacgg
ggtggtggac atcgcgaacc 14640aggccgtgca gcgcgagata aacagccgcc
tggacccgcg gccgcccacg gtggtggaga 14700tggaagatgc aactcttccg
ccgcccaaag gcgagaagcg cccgcggccc gacgcggagg 14760agacgatcct
gcaggtggac gagccgccct cgtacgagga ggccgtcaag gccggcatgc
14820ccaccacgcg catcatcgcg ccgctggcca cgggtgtaat gaaacccgcc
acccttgacc 14880tgcctccacc acccacgccc gctccaccga aggcagctcc
ggtcgtgcag gcccccccgg 14940tggcgaccgc cgtgcgccgc gtccccgccc
gccgccaggc ccagaactgg cagagcacgc 15000tgcacagtat cgtgggcctg
ggagtgaaaa gtctgaagcg ccgccgatgc tattgagaga 15060gaggaaagag
gacactaaag ggagagctta acttgtatgt gccttaccgc cagagaacgc
15120gcgaagatgg ccaccccctc gatgatgccg cagtgggcgt acatgcacat
cgccgggcag 15180gacgcctcgg agtacctgag cccgggtctg gtgcagtttg
cccgcgccac cgacacgtac 15240ttcagcctgg gcaacaagtt taggaacccc
acggtggctc ccacccacga tgtgaccacg 15300gaccggtccc agcgtctgac
gctgcgcttc gtgcccgtgg atcgcgagga caccacgtac 15360tcgtacaagg
cgcgcttcac tctggccgtg ggagacaacc gggtgctaga catggccagc
15420acttactttg acatccgcgg cgtcctggac cgcggtccca gcttcaaacc
ctactcgggc 15480acggcttaca acagcctggc ccccaagagc gctcccaatc
ccagccagtg ggatgcaaag 15540gaaaaggaag gagttgccca aacagaaaaa
aatgttttaa aaacatttgg tgttgccgct 15600acaggtggtt ttaatattac
agatcagggt ttgttacttg gaactgagga aacagctgaa 15660aacgttaaaa
aggatatcta tgcagagaaa actttccagc ctgaacctca agttggtgaa
15720gaaaactggc aggaaagtga agccttttat ggaggaaggg ctattaagaa
agacaccaaa 15780atgaagccat gctatggttc atttgccaga cccactaatg
aaaaaggagg acaggctaaa 15840tttaaaacac tagatgggca agttacaaaa
gatccagata ttgactttgc ttactttgac 15900gtccctggcg gaaaagctcc
aacaggcagt agtctaccgg aagaatacaa agcagatata 15960attttgtaca
cagaaaatgt taatctggaa acaccagata ctcacatagt gtataaacct
16020ggcaaagaag atgacaattc tgaaattaac ttaacacaac agtccatgcc
aaacagaccc 16080aactacattg gctttaggga caactttgta ggtctcatgt
actacaacag tactggcaac 16140atgggtgtgc tggctggtca ggcctctcag
ttgaatgctg tggtggactt gcaagacaga 16200aacaccgagc tgtcttacca
gctcttgcta gattctctgg gtgacagaac cagatacttt 16260agcatgtgga
actctgcggt tgacagttat gatcccgatg tcaggatcat tgagaatcac
16320ggtgtggaag atgaacttcc aaactattgc ttcccattga atggcactgg
taccaattcc 16380acctatcaag gtgtaaaaat tacaggtaat aatgatggcg
atcttgaaac cgaatgggaa 16440agagatgaag caatctctag acaaaaccaa
atctgcaagg gcaacgtcta tgccatggag 16500atcaacctcc aggccaacct
gtggaagagt tttctgtact cgaacgtagc cctgtacctg 16560cctgactcat
acaagtacac gccggccaac gtcacgctgc ccgccaacac caacacctac
16620gagtacatga acggccgcgt ggtagccccc tcgctggtgg acgcttacat
caacatcggc 16680gcccgctggt cgctggatcc catggacaat gtaaacccat
tcaaccacca ccgcaacgcg 16740ggcctgcgct accgttccat gttgttgggc
aacggtcgct acgtgccctt ccacatccaa 16800gtgccccaaa agttctttgc
catcaagaac ctgcttctgc tcccgggctc ctacacctac 16860gagtggaact
tccgcaagga cgtcaacatg atcctgcaga gttccctcgg aaacgatctg
16920cgcgtcgacg gcgcctccgt ccgcttcgac agcgtcaacc tctacgccac
cttcttcccc 16980atggcgcaca acaccgcctc caccctggaa gccatgctgc
gcaacgacac caacgaccag 17040tccttcaacg actacctctc ggccgccaac
atgctctacc ccatcccggc caaggccacc 17100aacgtgccca tctccatccc
ctcacgcaac tgggccgcct tccgcggctg gagtttcacc 17160aggctcaaga
ccaaggaaac tccctcgcta ggctcgggtt tcgacccata ctttgtctac
17220tcgggctcca tcccctatct cgacgggacc ttctacctca atcacacctt
caagaaggtc 17280tccatcatgt tcgactcctc ggtcagctgg cccggcaacg
accggctgct cacgccgaac 17340gagttcgaga tcaagcgcag cgtcgacggg
gagggctaca acgtggccca atgcaacatg 17400accaaggact ggttcctcgt
ccagatgctc tcccactaca acatcggcta ccagggcttc 17460cacgtgcccg
agggctacaa ggaccgcatg tactccttct tccgcaactt ccagcccatg
17520agcaggcagg tggtcgatga gatcaactac aaggactaca aggccgtcac
cctgcccttc 17580cagcacaaca actcgggctt caccggctac ctcgcaccca
ccatgcgtca ggggcagccc 17640taccccgcca acttccccta cccgctcatc
ggccagacag ccgtgccctc cgtcacccag 17700aaaaagttcc tctgcgacag
ggtcatgtgg cgcatcccct tctccagcaa cttcatgtcc 17760atgggcgccc
tcaccgacct gggtcagaac atgctctacg ccaactcggc ccatgcgctc
17820gacatgacct tcgaggtgga ccccatggat gagcccaccc tcctctatct
tctcttcgaa 17880gttttcgacg tggtcagagt gcaccagccg caccgcggcg
tcatcgaggc cgtctacctg 17940cgcacgccct tctccgccgg aaacgccacc
acataagcat gagcggctcc agcgaaagag 18000agctcgcggc catcgtgcgc
gacctgggct gcgggcccta ctttttgggc acccacgaca 18060agcgcttccc
tggcttcctc gccggcgaca agctggcctg cgccatcgtc aacacggccg
18120gccgcgagac cggaggcgtg cactggctcg ccttcggctg gaacccgcgc
tcgcgcacct 18180gctacatgtt cgacccattt gggttctcgg accgccggct
caagcagatt tacagcttcg 18240agtacgaggc tatgctgcgc cgaagcgccc
tggcttcctc gccagaccgc tgtctcagcc 18300tcgagcagtc cacccagacc
gtgcaggggc ccgactccgc cgcctgcgga cttttctgtt 18360gcatgttctt
gcatgccttc gtgcactggc ccgaccgacc catggacggg aaccccacca
18420tgaacttgct gacgggggtg cccaacggca tgctacaatc gccacaggtg
ctgcccaccc 18480tccggcgcaa ccaggaggag ctctaccgct tcctcgcgcg
ccactcccct tactttcgat 18540cccaccgcgc cgccatcgaa cacgccaccg
cttttgacaa aatgaaacaa ctgcgtgtat 18600ctcaataaac agcactttta
ttttacatgc actggagtat atgcaagtta tttaaaagtc 18660gaaggggttc
tcgcgctcgt cgttgtgcgc cgcgctgggg agggccacgt tgcggtactg
18720atacttgggc tgccacttga actcggggat caccagtttg ggcactgggg
tctcggggaa 18780ggtctcgctc cacatgcgcc ggctcatctg cagggcgccc
agtatgtccg gggcggagat 18840cttgaaatcg cagttggggc cggtgctctg
cgcgcgcgag ttgcggtaca cggggttgca 18900gcactggaac accatcagac
tggggtactt cacactggcc agcacgctct tgtcgctgat 18960ctgatccttg
tccaggtcct cggcgttgct caggccgaac ggggtcatct tgcacagctg
19020gcggcccagg aagggcacgc tctgaggctt gtggttacac tcgcagtgca
cgggcatcag 19080catcatcccc gcaccgcgct gcatattcgg gtagagggcc
ttgacaaagg ccgagatctg 19140cttgaaagct tgctgggcct tggccccctc
gctgaagaac agaccacagc tcttcccgct 19200gaactggtta ttcccgcacc
cggcatcatg cacgcagcag cgcgcgtcat ggctggtcag 19260ttgcaccacg
ctccgtcccc agcggttctg ggtcaccttg gccttgctgg gttgctcctt
19320caacgcgcgc tgcccgttct cgctggtcac atccatctcc accacgtggt
ccttgtggat 19380catcaccgtc ccatgcagac acttgagctg gccttccacc
tcggtgcagc cgtgatccca 19440cagggcgcat ccggtgcact cccaattctt
gtgtgcgatc ccgctgtggc tgaagatgta 19500accttgcaac atgcggccca
tgacggtgct aaatgctttc tgggtggtga aggtcagttg 19560catcccgcgg
acctcctcgt tcatccaggt ctggcacatc ttctggaaga tctcggtctg
19620ctcgggcatg agcttgtaag catcgcgcag gccgctgtcg acgcggtagc
gttccatcag 19680tacgttcatg gtatccatgc ccttctccca ggacgagacc
agaggcagac tcagggggtt 19740gcgcacgttc aggacacctg gggtcgcggg
ctcgacgatg cgttttccgt ccttgtcttc 19800cttcaacaga accggaggct
ggctgaatcc cactcccacg atcacggcat cttcctgggg 19860catctcttcg
tcggggtcta ccttggtcac atgcttggtc tttctggctt gcttcttttt
19920tggaggactg tccacgggga ccacgtcctc ctcggaagac ccggagccca
cccgctgata 19980ctttcggcgc ttggtgggca gaggaggtgg cggcgagggg
ctcctctcct gctccggcgg 20040atagcgcgcc gacccgtggc cccggggcgg
agtggcctct cggtccatga accggcgcac 20100gtcctgactg ccgccggcca
ttatttccta ggggaagatg gaggagcagc cgcgtaagca 20160ggagcaggag
gaggacttaa ccacccacga gcaacccaaa atcgagcagg acctgggctt
20220cgaagagccg gctcgtctag aacccccaca ggatgaacag gagcacgagc
aagacgcagg 20280ccaggaggag accgacgctg ggctcgagca tggctacctg
ggaggagagg aggatgtgct 20340gctgaaacac ctgcagcgcc agtccctcat
cctccgggat gccctggccg accggagcga 20400aacccccctc agcgtcgagg
agctgtgtcg ggcctacgag ctcaacctat tctcgccgcg 20460cgtgcccccc
aaacgccagc ccaacggcac atgcgagccc aacccgcgtc tcaacttcta
20520tcccgtcttt gcggtccccg aggcccttgc cacctatcac atctttttca
agaaccaaaa 20580gatccccgtc tcctgtcgcg ccaaccgcac ccgcgccgac
gcgctcctcg ctctggggcc 20640cggcgcacac atacctgata tcgcttccct
ggaagaggtg cccaagatct tcgaagggct 20700cggtcgggac gagacgcgcg
cggcgaacgc tctgaaagaa acagcagagg aagagggtca 20760cactagcgcc
ctggtagagt tggaaggcga caacgctagg ctggccgtgc tcaagcgcag
20820tgtcgagctc acccacttcg cctaccccgc cgtcaacctc ccgcccaagg
tcatgcgtcg 20880catcatggat cagctcatca tgccccacat cgaggccctc
gatgaaagtc aggagcagcg 20940ccccgaggac gtccggcccg tggtcagcga
cgagatgctc gcgcgctggc tcggaacccg 21000cgacccccag gctttggaac
agcggcgcaa actcatgctg gccgtggtcc tggtcaccct 21060cgagctcgaa
tgcatgcgcc gcttcttcag cgaccccgag accctacgca aagtcgagga
21120aaccctgcac tacactttca gacacggctt cgtcaggcag gcctgcaaga
tctccaacgt 21180ggagctgacc aacctggtct cctgcctggg tatccttcac
gagaaccgcc tggggcagac 21240cgtgctccac tctaccctga agggcgaggc
gcgtcgggac tatgtccgcg actgcatctt 21300tctctttctc tgccacacat
ggcaagcggc catgggcgtg tggcagcagt gtctcgagga 21360cgagaacctg
aaggagctgg acaaggttct tgctagaaac cttaaaaagc tgtggacggg
21420cttcgacgag cgcaccgtcg cctcggacct ggcccagatc gtcttccccg
agcgcctgag 21480gcagacgctg aaaggcgggc tgccagactt catgagccag
agcatgatac aaaactaccg 21540cactttcatt ctcgagcgat ctggaatgct
gcccgccacc tgcaacgcct tcccctccga 21600ctttgtcccg ctgagctacc
gcgagtgtcc cccgccgctg tggagccatt gctacctctt 21660gcagctggcc
aactacatcg cctaccactc ggacgtgatc gaggacgtga gcggcgaggg
21720gcttctcgag tgccactgcc gctgcaacct gtgctccccg caccgctccc
tggtctgcaa 21780cccccagctc ctgagcgaga cccaggtcat cggtaccttc
gagctgcaag gtccgcagga 21840gtccaccgct ccgctgaaac tcacgccggg
gttgtggact tccgcgtacc tgcgcaaatt 21900tgtacccgag gactaccacg
cccatgagat aaagttcttc gaggaccaat cgcgtccgca 21960gcacgcggat
ctcacggcct gcgtcatcac ccagggcgcg atcctcgccc aattgcacgc
22020catccaaaaa tcccgccaag agtttcttct gaaaaagggt agaggggtct
acctggaccc 22080ccagacgggc gaggtgctca acccgggtct cccccagcat
gccgaggaag aagcaggagc 22140cgctagtgga ggagatggaa gaagaatggg
acagccaggc agaggaggac gaatgggagg 22200aggagacaga ggaggaagaa
ttggaagagg tggaagagga gcaggcaaca gagcagcccg 22260tcgccgcacc
atccgcgccg gcagccccgg cggtcacgga tacaacctcc gcagctccgg
22320ccaagcctcc tcgtagatgg gatcgagtga agggtgacgg taagcacgag
cggcagggct 22380accgatcatg gagggcccac aaagccgcga tcatcgcctg
cttgcaagac tgcgggggga 22440acatcgcttt cgcccgccgc tacctgctct
tccaccgcgg ggtgaacatc ccccgcaacg 22500tgttgcatta ctaccgtcac
cttcacagct aagaaaaaat cagaagtaag aggagtcgcc 22560ggaggaggcc
tgaggatcgc ggcgaacgag cccttgacca ccagggagct gaggaaccgg
22620atcttcccca ctctttatgc catttttcag cagagtcgag gtcagcagca
agagctcaaa 22680gtaaaaaacc ggtctctgcg ctcgctcacc cgcagttgct
tgtaccacaa aaacgaagat 22740cagctgcagc gcactctcga agacgccgag
gctctgttcc acaagtactg cgcgctcact 22800cttaaagact aaggcgcgcc
cacccggaaa aaaggcggga attacctcat cgccaccatg 22860agcaaggaga
ttcccacccc ttacatgtgg agctatcagc cccagatggg cctggccgcg
22920ggcgcctccc aagactactc cacacgcatg aactggctca gtgccggccc
ctcgatgatc
22980tcacgggtca acggggtccg cagtcatcga aaccagatat tgttggagca
ggcggcggtc 23040acctccacgc ccagggcaaa gctcaacccg cgtaattggc
cctccaccct ggtgtatcag 23100gaaatccccg ggccgactac cgtactactt
ccgcgtgacg cactggccga agtccgcatg 23160actaactcag gtgtccagct
ggccggcggc gcttcccggt gcccgctccg cccacaatcg 23220ggtataaaaa
ccctggtgat ccgaggcaga ggcacacagc tcaacgacga gttggtgagc
23280tcttcgatcg gtctgcgacc ggacggagtg ttccaactag ccggagccgg
gagatcctcc 23340ttcactccca accaggccta cctgaccttg cagagcagct
cttcggagcc tcgctccgga 23400ggcatcggaa ccctccagtt cgtggaggag
tttgtgccct cggtctactt caaccccttc 23460tcgggatcgc caggcctcta
cccggacgag ttcataccga acttcgacgc agtgagagaa 23520gcggtggacg
gctacgactg aatgtcccat ggtgactcgg ctgagctcgc tcggttgagg
23580catctggacc actgccgccg cctgcgctgc ttcgcccggg agagctgcgg
actcatctac 23640tttgagtttc ccgaggagca ccccaacggc cctgcacacg
gagtgcgaat caacgtagag 23700ggcaccaccg agtctcacct ggtcaggttc
ttcacccagc aacccttcct ggtcgagcgg 23760gaccggggcg ccaccaccta
caccgtctac tgcatctgtc ctaacccgaa gttgcatgag 23820aatttttgct
gtactctttg tggtgagttt aataaaagct gaactaagaa cctactttgg
23880aatcccttgt cgtcatcctc gaaacaagac cgtcttcttt accaaccaga
ccaaggttcg 23940tctgaactgc acaaccaaca ggaagtacct tctctggact
ttccaaaaca cctcactcgc 24000tgttgtcaat acccgtgacg acgtcaaccc
catagtcatc acccagcagt cgggcgagac 24060caacggctgc atccactgct
cctgcgaaag ccccgagtgc atctactccc tcctcaagac 24120cctttgcgga
ctccgcgacc tcctccccat gaactgatgt tgattaaaag cccaaaaacc
24180aatcataccc ttcccccatt tccccatccc caattactca taagaataaa
tcattggaac 24240taatcattca ataaagatca cttacttgaa atctgaaagt
atgtctctgg tgtagttgtt 24300cagcagcacc tcggtaccct cctcccagct
ctggtactcc agtccccggc gggcggcgaa 24360cttcctccac accttgaaag
ggatgtcaaa ttcctggtcc acaattttca ttgtcttccc 24420tctcagatga
caaagaggct ccgggtggaa gatgacttca accccgtcta cccctatggc
24480tacgcgcgga atcagaatat ccccttcctc actcccccct ttgtttcttc
cgatggattc 24540caaaacttcc cacccggggt attgtcactc aaactggctg
acccaatagc catcgtcaat 24600ggggatgtct cactcaaggt gggaggtgga
ctcactttgc aagaaggaaa cctaactgtt 24660gatgcaaagg ctccattgca
agttgcaaat gacaacaaat tggagctttc ttatgcagac 24720ccatttgagg
ttaaagacac taagctacaa ttaaaagtag gtcatggttt aaaagtaata
24780gatgaaaaaa cttcttcagg tcttcaaagt ctaattggaa atctcgtagt
tttaacagga 24840aaaggaattg gcactcaaga attaaaagac aaagacgatg
aaactaaaaa tataggagtt 24900ggaataaatg tgagaatagg gaaaaacgaa
agtctggcgt ttgacaaaga tggaaatttg 24960gtagcatggg ataatgaaaa
cgacaggcgc actctatgga caactccaga cacatctcca 25020aattgtaaaa
taagtactga aaaagactcc aaacttactt tagtccttac taaatgcgga
25080tctcaaattc tagcaagtgt gtctttgctt gctgtcgctg gaagttatct
taatatgaca 25140gctagtactc aaaagagtat aaaggtatct ttgatgtttg
actcaaaagg gcttctaatg 25200actacatctt ctattgataa aggatattgg
aattatagaa ataaaaacag cgttgttgga 25260actgcttatg aaaacgcaat
tccatttatg ccaaatttag tggcttatcc aagacctaac 25320acgccagact
ctaaaattta tgctagaagc aaaattgttg gaaatgttta tttagcaggt
25380ttggcttacc aaccaattgt cataacagtt agttttaatc aggagaagga
tgcaagttgt 25440gcttactcaa taacatttga atttgcctgg aacaaagact
acgttggtca atttgatacc 25500acctccttta ccttctctta tattgcccaa
gaatgaaaga ccaataaacg tgtttttcat 25560ttgaaaattt tcatgtatct
ttattgattt ttacaccagc acgggtagtc agtctcccac 25620caccagccca
tttcacagtg taaacaattc tctcagcacg ggtggcctta aataggggaa
25680tgttctgatt agtgcgggaa ctgaacttgg ggtctataat ccacacagtt
tcctggcgag 25740ccaaacgggg gtcggtgatt gagatgaagc cgtcctctga
aaagtcatcc aagcgggcct 25800cgcagtccaa ggtcacagtc tggtggaatg
agaagaacgc acagattcat actcggaaaa 25860caggatgggt ctgtgcctct
ccatcagcgc cctcaacagt ctttgccgcc ggggctcggt 25920gcggctgctg
cagatgggat cgggatcgca agtctctctg actatgatcc ccacagcctt
25980cagcatcagt ctcctggtgc gtcgggcaca gcaccgcatc ctgatctcgc
tcatgttctc 26040acagtaagtg cagcacataa tcaccatgtt attcagcagc
ccataattca ggatgctcca 26100gccaaagctc atgttgggga tgatggaacc
cacgtgacca tcataccaga tgcggcagta 26160tatcaggtgc ctgcccctca
tgaacacact gcccatatac atgatctctt tgggcatgtt 26220tctgttcaca
atctgccggt accaggggaa tcgctggttg aacatgcacc cgtaaatgac
26280tctcctgaac cacacggcca gcagggtgcc tcccgcccga cactgcaggg
agcccgggga 26340tgaacagtgg caatgcagga tccagcgctc gtacccgctc
accatctgag ctctcaccaa 26400gtccagggta gcaggacaca ggcacactga
catacatctt tttaaaattt ttatttcctc 26460tggggacagg atcatatccc
aggggactgg aaactcttgg agcagggtaa agccagcagc 26520acatggcaat
ccacggacag aacttacatt atgataatct gcatgatcac aatcgggcaa
26580cagagggtgt tgttcagtca gagaggccct ggtctcctca tcagatcgtg
gtaaacgggc 26640cctgcgatat ggatgatggc ggagcaagct cgactgatcc
tcggtttgca ttgtagtgga 26700ttctcttgcg taccttgtcg tacttctgcc
agcagaaatg ggcccttgaa cagcagatac 26760ctctccttct cctgtctttc
cgctgctgac gctcagtcat ccaactgaag tacagccatt 26820cccgcaggtt
ctcgagcagc tcctcagcat ctgatgaaac aaaagttctg tccatgcgga
26880ttccccttaa cacatcagcc aggacattgt aggccatccc aatccagtta
atgcagcctg 26940gtctatcatt cagaggaggt gggggaagaa ctggaagaac
catttttatt ccaagcggtc 27000tcgaaggatg ataaagtgca agtcacgcag
gtgacagcgt tccccgccgc tgtgctggtg 27060gaaacagaca gccaggtcaa
aacctactct attttcaagg tgctcgactg tggcttcgag 27120cagtggctct
acgcgtacat ccagcataag aatcacatta aaggctggcc ctccatcgat
27180ttcatcaatc atcaggttac actcattcac cattcccagg taattctcat
ttttccagcc 27240ttgaattatt tctacaaatt gttggtgtaa gtccactccg
cacatgtgga aaagttccca 27300cagcgccccc tccactttca taatcaggca
gaccttcata atagcaacag atctggctgc 27360tccaccacct gcagcgtgtt
caaaacaaca agattcaatg agtttctgcc ctctgccctg 27420agctcgcgtc
tcagcgtcag ctgtaaaaag tcactcaagt cctcggccac tacagatgcc
27480aattcagagc cagggctaag cgtgggactg gcaagcgtga tggagtactt
taatgctcca 27540aagctagcac ccaaaaactg cacgctggaa taagctctct
ttgtgtcacc ggtgattcct 27600tccaaaaggt gagtgataaa gcgaggtagg
tgctctctaa tcatagcagt aatggaaaag 27660tcctctaaat aagtcactag
ggccccaggg accacaatgt ggtagctgac agcgcgtcgc 27720tcaagcatgg
ttagtagaga tgagagtctg aaaaacagaa agcatgcact aaaccagagt
27780ggcaagtctt actgaaggaa aaatcactct ctccagcagc aaagtgccca
ctgggtggcc 27840ctctcggaca tacaaaaatc gatccgtgtg gttaaagagc
agcacagtta gctactgtct 27900tctcccagca aagatcacat cggactgggt
tagtatgccc ctggaatggt agtcattcaa 27960ggccataaat ctgccttggt
agccattagg aatcagcacg ctcactctca agtgaaccaa 28020aaccacccca
tgcggaggaa tgtggaaaga ttcggggcaa aagaaattat atctattgct
28080agtcccttcc tggacgggag cgattcctcc agggctatct atgaaagcat
acagagattc 28140agccatagct cagcccgctt accagtagac agagagcaca
gcagtacaag cgccaacagc 28200agcgactgac tacccactaa cccagctccc
tatttaaagg caccttacac tgacgtaatg 28260accaaaggtc taaaaacccc
gccaaaaaaa aacacacacg ccctgggtgt ttttcgcgaa 28320aacacttccg
cgttctcact tcctcgtatc gatttcgtga ctcaacttcc gggttcccac
28380gttacgtcac ttctgccctt acatgtaact cagccgtagg gtgccatctt
gcccacgtcc 28440aagatggctt ccatgtccgg ccacgcctcc gcggcgaccg
tcagccgtgc gtcgtgacgt 28500cactaacggt tcttgcaacg gccaatcagc
gacggccccg ccctaaattc aaaagctcat 28560ttgcatatta acttttgttt
actttgtggg gtatattatt gatgatg 28607112094DNAHerpes Simplex Virus
11atgtctgtga gaggacacgc cgtgagaaga agaagagcca gcaccagatc tcatgcccca
60tctgcccaca gagctgatag ccctgtggag gatgaacctg aaggcggcgg agtgggactg
120atgggatacc tgcgggccgt gttcaacgtg gacgacgatt ctgaagtgga
agccgctggc 180gaaatggcct ctgaggaacc ccctcctcgg agaagaagag
aggctagagg ccaccctgga 240agcagaagag cctctgaagc cagagctgct
gcccctccta gaagagctag cttccccaga 300cctagaagcg tgacagccag
atcccagagc gtgagaggca gaagagatag cgccatcacc 360agagccccta
gaggcggata tctgggcccc atggacccta gagatgtgct gggaagagtg
420ggcggatcta gagtggtgcc tagccccctg tttctggacg agctgaacta
cgaagaggac 480gattatcctg ccgccgtggc ccatgatgat ggacctggcg
ccagaccttc tgccacagtg 540gagatcctgg ccggaagagt gtctggacct
gaactgcagg ccgccttccc tctggataga 600ctgacaccta gagtggccgc
ctgggatgag tctgtgagat ctgccctggc tctgggacat 660ccagccggct
tttacccttg tcccgatagc gcctttggcc tgtctagagt gggcgtgatg
720cactttgcta gccctgccga ccctaaggtg ttcttcaggc agacactgca
gcagggcgaa 780gctctggctt ggtacgtgac aggcgacgcc attctggatc
tgaccgacag aagagccaag 840accagccctt ctagagccat gggctttctg
gtggacgcca ttgtgagagt ggccatcaat 900ggatgggtgt gcggcacaag
actgcacaca gagggcagag gctctgagct ggatgataga 960gccgccgaac
tgagaaggca gtttgcctct ctgacagccc tgagacctgt gggagctgct
1020gctgtgcctc tgctgtctgc tggcggagct gctcctcctc atcctggacc
tgatgccgcc 1080gtgtttagaa gcagcctggg cagcctgctg tattggcctg
gcgtgagagc cctgctgggc 1140agagattgta gagtggctgc cagatacgca
ggcaggatga cctatattgc cacaggcgcc 1200ctgctggcta gattcaatcc
tggcgccgtg aaatgtgtgc tgcctaggga agccgctttt 1260gctggcagag
tgctggatgt gctggccgtg ctggctgaac agacagtgca gtggctgtct
1320gtggtggtgg gagctagact gcatcctcac tctgcccatc ctgcctttgc
cgatgtggaa 1380caggaagccc tgtttagagc cctgccactg ggatctccag
gcgtggtggc cgctgaacat 1440gaagctctgg gcgatacagc tgctagaagg
ctgctggcca catctggact gaatgccgtg 1500ctgggagccg ctgtgtatgc
tctgcatacc gccctggcta cagtgacact gaagtacgcc 1560ctggcctgtg
gagatgctag aagaaggcgg gacgacgctg ctgctgctag ggctgtgctg
1620gctacaggac tgatcctgca gagactgctg ggactggctg atacagtggt
ggcttgtgtg 1680gccctggctg cttttgatgg cggaagcaca gctcctgaag
tgggcaccta cacccctctg 1740agatacgcct gtgtgctgag agctacccag
cctctgtacg ccagaacaac ccctgccaag 1800ttctgggctg atgtgagagc
cgctgccgaa catgtggatc tgagacctgc ctcttctgct 1860cctagagccc
ctgtgtctgg aactgccgat ccagccttcc tgctggaaga tctggccgct
1920tttcctcctg cccctctgaa tagcgagtct gtgctgggcc ctagagtgag
agtggtggac 1980atcatggccc agttccggaa actgctgatg ggcgatgaag
aaacagccgc cctgagagcc 2040cacgtgtcag gaagaagagc tacaggcctg
ggaggacctc ctagaccttg atga 2094124125DNAHerpes Simplex Virus
12atggctgccc ctgccagaga tccccctggc tacagatacg ccgctgccat cctgcctacc
60ggcagcatcc tgagcaccat cgaggtggcc agccacagac ggctgttcga cttcttcgcc
120gccgtgcgga gcgatgagaa cagcctgtac gacgtggagt tcgacgccct
gctgggcagc 180tactgcaaca ccctgagcct ggtccggttc ctggaactgg
gcctgagcgt ggcctgtgtg 240tgcaccaagt tccccgagct ggcctacatg
aacgagggcc gggtgcagtt tgaggtgcac 300cagcccctga tcgccagaga
tggcccccac cctgtggagc agcccgtgca caactacatg 360accaaggtga
tcgacagacg ggccctgaat gccgccttta gcctggccac agaggccatc
420gccctgctga caggcgaagc cctggatggc accggcatca gcctgcacag
gcagctgagg 480gccattcagc agctggcccg gaatgtgcag gctgtgctgg
gcgcctttga gagaggcacc 540gccgaccaga tgctgcacgt gctgctggaa
aaggcccctc ctctggccct gctgctgccc 600atgcagagat acctggacaa
cggccggctg gccacaagag tggccagagc caccctggtg 660gccgagctga
agcggagctt ctgcgatacc agcttcttcc tgggcaaggc cggccacaga
720agggaagcca tcgaggcctg gctggtcgat ctgaccaccg ccacccagcc
atctgtggcc 780gtgcccagac tgacccacgc cgacaccaga ggcagacccg
tggatggcgt gctggtgacc 840acagccgcca tcaagcagcg gctgctgcag
agcttcctga aggtggagga caccgaggcc 900gatgtgcctg tgacctacgg
cgagatggtg ctgaacggcg ccaatctggt gaccgccctg 960gtgatgggca
aagccgtgag atccctggac gacgtgggca gacacctgct ggacatgcag
1020gaagagcagc tggaagccaa ccgggagacc ctggatgagc tggaaagcgc
ccctcagacc 1080accagagtgc gggccgatct ggtggccatc ggcgacaggc
tggtgttcct ggaagctctg 1140gaacggcgga tctacgccgc caccaacgtg
ccttaccctc tggtcggcgc catggacctg 1200accttcgtgc tgcccctggg
cctgttcaac cccgccatgg aaaggtttgc cgcccacgcc 1260ggcgatctgg
tgcctgcccc tggccaccct gaacccagag ccttcccccc cagacagctg
1320ttcttctggg gcaaggacca ccaggtgctg agactgagca tggaaaacgc
cgtgggcacc 1380gtgtgtcacc ccagcctgat gaacatcgac gccgccgtgg
gcggagtgaa ccacgatcct 1440gtggaggccg ccaatcccta cggcgcctac
gtggctgctc ctgctggacc aggcgccgac 1500atgcagcagc ggtttctgaa
cgcctggcgg cagagactgg cccacggcag agtgagatgg 1560gtggccgagt
gccagatgac cgccgagcag ttcatgcagc ccgacaacgc caacctggcc
1620ctggaactgc accccgcctt cgatttcttt gccggcgtgg ccgatgtgga
actgccaggc 1680ggcgaagtgc ctccagctgg ccctggcgcc atccaggcca
catggcgggt ggtgaacggc 1740aatctgccac tggccctgtg ccctgtggcc
ttcagagatg ccagggggct ggaactggga 1800gtgggcaggc atgccatggc
ccctgccaca attgccgccg tgaggggcgc cttcgaggac 1860agatcctacc
ccgccgtgtt ctacctgctg caggccgcca tccatggcaa cgagcacgtg
1920ttctgcgccc tggccagact ggtgacccag tgcatcacca gctactggaa
caacaccaga 1980tgcgccgcct tcgtgaacga ctacagcctg gtgtcctaca
tcgtgaccta cctgggcggc 2040gacctgcctg aggaatgcat ggccgtgtac
cgggacctgg tggcccatgt ggaggctctg 2100gcccagctgg tcgacgactt
caccctgcct ggccctgagc tgggaggaca ggcccaggcc 2160gaactgaacc
acctgatgcg ggaccctgct ctgctgcctc ccctggtctg ggattgcgac
2220ggcctgatga gacacgccgc cctggacagg caccgggact gcagaatcga
tgccggcgga 2280cacgagcctg tgtacgctgc cgcctgcaat gtggccaccg
ccgacttcaa ccggaacgac 2340ggcaggctgc tgcacaacac ccaggccaga
gccgccgatg ccgccgacga cagacctcac 2400agacccgccg actggaccgt
gcaccacaag atctactact acgtgctggt gcccgccttc 2460agcagaggca
ggtgctgtac agccggcgtg cggttcgaca gagtgtacgc caccctgcag
2520aacatggtgg tgcccgagat tgcccctggc gaggaatgcc ccagcgaccc
cgtgacagat 2580cctgcccacc ctctgcaccc tgccaacctg gtggctaaca
ccgtgaagcg gatgttccac 2640aacggcaggg tggtggtgga tggccctgcc
atgctgaccc tgcaggtgct ggcccacaac 2700atggccgaga ggaccacagc
cctgctgtgt tctgccgccc ctgacgctgg cgccaatacc 2760gccagcaccg
ccaacatgcg gatcttcgac ggcgccctgc atgctggcgt cctgctgatg
2820gccccccagc acctggatca caccatccag aacggcgagt acttttacgt
gctgcccgtg 2880cacgccctgt ttgccggcgc tgaccacgtg gccaacgccc
ccaatttccc ccctgccctg 2940agggatctgg ccagggacgt gcctctggtg
cctcctgccc tgggcgccaa ctacttcagc 3000agcatccggc agcctgtggt
gcagcatgcc agagaatctg ccgctggcga gaacgccctg 3060acctacgccc
tgatggccgg ctacttcaag atgagccccg tggccctgta tcaccagctg
3120aaaaccggcc tgcaccctgg cttcggcttt accgtggtgc ggcaggacag
attcgtgacc 3180gagaacgtgc tgttcagcga gagagccagc gaggcctact
tcctgggcca gctccaggtg 3240gccagacacg aaacaggcgg gggagtgaat
ttcaccctga cccagcccag aggcaacgtg 3300gatctgggcg tgggctatac
agccgtggcc gccaccggca ccgtgagaaa ccccgtgacc 3360gacatgggca
acctgcccca gaacttctac ctgggcaggg gagcccctcc cctgctggat
3420aatgccgccg ctgtgtacct gaggaacgcc gtggtggccg gcaatagact
cggacccgcc 3480cagcctctgc ctgtgttcgg ctgcgcccag gtgccaagac
gggccggaat ggaccacgga 3540caggacgccg tgtgcgagtt catcgccacc
cccgtggcca ccgacatcaa ctactttcgg 3600aggccctgca accctagagg
aagggccgct ggcggagtgt atgccggcga caaggaaggc 3660gacgtcatcg
ccctgatgta cgaccacggc cagagcgatc ccgccagacc ttttgccgcc
3720accgccaacc cttgggccag ccagaggttc agctacggcg atctgctgta
caatggcgcc 3780taccacctca atggcgccag ccctgtgctg tccccctgct
tcaagttctt cacagccgcc 3840gacatcaccg ccaagcaccg gtgcctggaa
aggctgatcg tggagaccgg cagcgccgtg 3900tctacagcca ccgccgccag
cgacgtgcag ttcaagaggc ccccaggctg cagagaactg 3960gtggaggacc
cttgcggcct gttccaggaa gcctacccca tcacctgcgc ctctgatcct
4020gccctgctgc ggagcgctag agatggcgag gcccacgcca gggaaaccca
cttcacccag 4080tacctgatct acgacgccag ccccctgaag ggcctgagcc tgtga
4125131686DNAHerpes Simplex Virus 13atggagactt cagtcagagg
gcacgcagta cggaggcgac gcgccagcac cagatcgcac 60gccccctcgg cacatagggc
ggactccccg gtggaggacg aacccgaagg agggggagtg 120ggactcatgg
aaaccgggta tctccgggca gtctttaacg tggatgatga cagcgaggta
180gaggcagcgg gtgaaatgga gacagcctcc gaagagccac cccctagacg
gcggagagaa 240gcgagaggac atcccggttc cagacgggca tcagaagcga
gggccgcagc tccgcctcga 300agggcgtcct tccctagacc acgctcagtg
acggcgaggt cgcagtccgt ccgcggcaga 360cgggactcgg ccatcactcg
agcgccgagg ggtgggtatc tgggaccgat ggagacagat 420ccgcgggacg
tgctggggcg agtcggaggc agccgggtgg tgccctcgcc cttgttcctc
480gatgagctta actacgagga ggatgactac cctgctgccg tcgcgcatga
cgacggaccc 540ggagcgaggc cgtccgcgac ggtcgagatt cttgcgggtc
gcgtgagcgg accagaattg 600caggcggcct tccctctcga tcgcttgacg
cccagggtag ccgcttggga tgaatccgta 660cgctcagcac tggcgctggg
gcacccagcc ggcttctacc catgcccgga ctccgccttt 720gggttgtcgc
gcgtaggggt catggagaca cactttgcat cgcctgctga tccgaaagtg
780ttctttcgac aaacactgca gcagggggag gctctggcat ggtatgtcac
tggggatgcg 840atcttggacc tcaccgatcg acgggccaaa acatcgccta
gccgggctat ggagacagga 900ttcctcctcg acgcggctgt acgggtcgcg
atcaacggtt gggtatgtgg aacgagattg 960cataccgaag gacgcggatc
ggaacttgac gatcgcgcag cagaacttcg gagacagttc 1020gcatcgctca
cggccctccg gccagtggga gcagccgcgg tgccattgct ctccgcggga
1080ggggctgcgc ctccccaccc gggtccggat gcggccgtgt tccgctcatc
gttggggtca 1140ttgctttact ggcctggcgt ccgggctctg cttggcaggg
actgcagggt cgccgccaga 1200tacgcgggta ggatggaaac gacctatatc
gcgacggggg cactgctcgc caggttcaat 1260cccggtgccg tgaagtgtgt
cctgccgcga gaagccgcgt tcgctgggag agtgctggac 1320gtcctggcgg
tgctggcaga gcaaacagta cagtggctct cggtggtagt cggtgcccgc
1380ttgcatcccc actcagcgca cccggcattt gccgacgtcg aacaggaggc
gctgtttcgc 1440gcccttcccc ttgggtcacc cggagtggtc gcagctgagc
acgaagccct tggggataca 1500gcggcacgcc ggttgctggc gacatcgggt
cttaatgcgg tgttgggtgc ggcggtgtac 1560gcgctccata ccgcgctggc
caccgtcact ttgaaatacg cactggcgtg cggggatgca 1620cgccgacgca
gggatgacgc ggcagccgcc agagctgtac ttgcgaccgg attgattctt 1680cagtag
1686
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