U.S. patent application number 15/408201 was filed with the patent office on 2017-08-03 for expression system for modulating an immune response.
The applicant listed for this patent is Admedus Vaccines Pty Ltd.. Invention is credited to Julia Louise Dutton, Ian Hector Frazer.
Application Number | 20170218393 15/408201 |
Document ID | / |
Family ID | 40566909 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170218393 |
Kind Code |
A1 |
Frazer; Ian Hector ; et
al. |
August 3, 2017 |
EXPRESSION SYSTEM FOR MODULATING AN IMMUNE RESPONSE
Abstract
The present invention discloses methods and compositions for
modulating the quality of an immune response to a target antigen in
a mammal, which response results from the expression of a
polynucleotide that encodes at least a portion of the target
antigen, wherein the quality is modulated by replacing at least one
codon of the polynucleotide with a synonymous codon that has a
higher or lower preference of usage by the mammal to confer the
immune response than the codon it replaces.
Inventors: |
Frazer; Ian Hector; (St.
Lucia, AU) ; Dutton; Julia Louise; (Yeronga,
AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Admedus Vaccines Pty Ltd. |
Wooloongabba |
|
AU |
|
|
Family ID: |
40566909 |
Appl. No.: |
15/408201 |
Filed: |
January 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12738284 |
Oct 14, 2010 |
9593340 |
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PCT/AU2008/001463 |
Oct 2, 2008 |
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15408201 |
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60980145 |
Oct 15, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/16122
20130101; A61K 2039/575 20130101; A61P 31/10 20180101; Y02A 50/469
20180101; A61K 39/245 20130101; C12N 2760/16134 20130101; C40B
40/08 20130101; A61P 31/18 20180101; A61P 35/02 20180101; Y02A
50/39 20180101; A61P 33/02 20180101; A61P 31/20 20180101; A61P
33/06 20180101; C12N 2710/16234 20130101; A61P 33/00 20180101; A61K
2039/55516 20130101; A61P 33/12 20180101; A61K 48/0075 20130101;
C12N 2710/16222 20130101; A61P 31/22 20180101; Y02A 50/30 20180101;
A61P 35/00 20180101; C12N 15/79 20130101; C12N 2800/22 20130101;
C40B 50/04 20130101; A61P 31/12 20180101; A61K 48/0066 20130101;
A61P 37/04 20180101; A61K 39/145 20130101; C12N 2710/20022
20130101; C12N 2770/24234 20130101; A61P 31/16 20180101; A61K
2039/53 20130101; C12N 15/67 20130101; C12N 2710/16634 20130101;
C12N 2710/20071 20130101; C12N 2710/16622 20130101; A61K 39/12
20130101; A61K 2039/54 20130101; A61P 31/04 20180101; C12N
2710/20034 20130101; A61K 2039/585 20130101; A61P 31/14 20180101;
C12N 15/85 20130101; A61P 31/06 20180101; A61P 33/04 20180101; C12N
2770/24222 20130101; A61K 39/29 20130101; C07K 14/005 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; A61K 48/00 20060101 A61K048/00; A61K 39/12 20060101
A61K039/12; A61K 39/245 20060101 A61K039/245; A61K 39/29 20060101
A61K039/29; C07K 14/005 20060101 C07K014/005; A61K 39/145 20060101
A61K039/145 |
Claims
1-41. (canceled)
42. A chimeric construct comprising a synthetic polynucleotide that
is operably connected to a regulatory polynucleotide, wherein the
synthetic polynucleotide is distinguished from a parent
polynucleotide that encodes a polypeptide that corresponds to at
least a portion of a target antigen by the replacement of a first
codon in the parent polynucleotide with a synonymous codon that has
a higher immune response preference than the first codon, wherein
the first and synonymous codons are selected according to TABLE 3:
TABLE-US-00013 TABLE 3 Synonymous First Codon Codon Ala.sup.GCG
Ala.sup.GCT Ala.sup.GCA Ala.sup.GCT Ala.sup.GCC Ala.sup.GCT
Arg.sup.CGG Arg.sup.CGA Arg.sup.CGG Arg.sup.CGT Arg.sup.CGG
Arg.sup.AGA Arg.sup.AGG Arg.sup.CGA Arg.sup.AGG Arg.sup.CGT
Arg.sup.AGG Arg.sup.AGA Glu.sup.GAG Glu.sup.GAA Gly.sup.GGC
Gly.sup.GGA Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG Gly.sup.GGA
Leu.sup.TTA Leu.sup.CTA Leu.sup.TTA Leu.sup.CTT Leu.sup.TTA
Leu.sup.TTG Leu.sup.TTG Leu.sup.CTA Leu.sup.TTG Leu.sup.CTT
phe.sup.TTC phe.sup.TTT pro.sup.CCG pro.sup.CCT pro.sup.CCA
pro.sup.CCT Ser.sup.AGT Ser.sup.TCG Ser.sup.AGT Ser.sup.TCT
Ser.sup.AGT Ser.sup.TCA Ser.sup.AGC Ser.sup.TCG Ser.sup.AGC
Ser.sup.TCT Ser.sup.AGC Ser.sup.TCA Ser.sup.AGC Ser.sup.TCC
Ser.sup.TCC Ser.sup.TCG Ser.sup.TCA Ser.sup.TCG Ser.sup.TCT
Ser.sup.TCG Thr.sup.ACT Thr.sup.ACG Thr.sup.ACT Thr.sup.ACA
Thr.sup.ACA Thr.sup.ACG Thr.sup.ACC Thr.sup.ACG Val.sup.GTA
Val.sup.GTT
43. The chimeric construct of claim 42, wherein at least about 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
96%, 97%, 98%, or 99% of the first codons of the parent
polynucleotide are replaced with synonymous codons in accordance
with TABLE 3.
44. The chimeric construct of claim 42, wherein the target antigen
is from a pathogenic organism.
45. The chimeric construct of claim 44, wherein the pathogenic
organism is selected from a virus, bacteria, fungi, parasite,
algae, protozoa and amoebae.
46. The chimeric construct of claim 42, wherein the target antigen
is a herpes simplex virus antigen.
47. The chimeric construct of claim 42, wherein the target antigen
is a herpes simplex virus antigen glycoprotein D.
48. The chimeric construct of claim 47, wherein the glycoprotein D
is gD2.
49. The chimeric construct of claim 42, wherein the target antigen
is an HIV antigen (e.g., the gene products of the HIV gag, pol, or
env genes, the Nef protein, and reverse transcriptase), hepatitis
viral antigens (e.g., the S, M, and L proteins of hepatitis B
virus, the pre-S antigen of hepatitis B virus, and other hepatitis
viral components), influenza viral antigens (e.g., hemagglutinin,
neuraminidase and other influenza viral components), a measles
viral antigen (e.g., the measles virus fusion protein and other
measles virus components), a rubella viral antigen (e.g., E1 and E2
proteins and other rubella virus components), a rotaviral antigen
(e.g., VP7sc and other rotaviral components), a cytomegaloviral
antigen (e.g., envelope glycoprotein B and other cytomegaloviral
antigen components), a respiratory syncytial viral antigen (e.g.,
the RSV fusion protein, the M2 protein and other respiratory
syncytial viral antigen components), a varicella zoster viral
antigen (e.g., 9PI, gpII, and other varicella zoster viral antigen
components), a Japanese encephalitis viral antigen (e.g., proteins
E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, and other Japanese encephalitis
viral antigen components), a rabies viral antigen (e.g., rabies
glycoprotein, rabies nucleoprotein and other rabies viral antigen
components), a papillomavirus antigen (e.g., the L1 and L2 capsid
proteins and the E6/E7 proteins).
50. The chimeric construct of claim 42, wherein the target antigen
is cancer or tumour antigen.
51. The chimeric construct of claim 42, further comprising a coding
sequence for an adjuvant.
52. The chimeric construct of claim 51, wherein the adjuvant is a
protein destabilizing element, which increased processing and
presentation of the polypeptide that corresponds to at least a
portion of the target antigen through the class I MHC pathway.
53. The chimeric contrast of claim 52, wherein the
protein-destabilizing element is an ubiquitin.
54. A pharmaceutical composition that is useful for modulating an
immune response to a target antigen in a mammal, which response is
conferred by the expression of a parent polynucleotide that encodes
a polypeptide corresponding to at least a portion of the target
antigen, the composition comprising a chimeric construct and a
pharmaceutically acceptable excipient and/or carrier, wherein the
chimeric construct comprises a synthetic polynucleotide that is
operably connected to a regulatory polynucleotide and that is
distinguished from the parent polynucleotide by the replacement of
a first codon in the parent polynucleotide with a synonymous codon
that has a different immune response preference than the first
codon and wherein the first and synonymous codons are selected
according to any one of TABLE 3.
55. The composition according to claim 49, further comprising an
adjuvant that enhances the effectiveness of the immune
response.
56. The composition according to claim 49, which is formulated for
transcutaneous administration.
57. The composition according to claim 49, which is formulated for
epidermal administration.
58. The composition according to claim 49, which is formulated for
dermal administration.
59. The composition according to claim 49, which is formulated for
intradermal administration.
60. The composition according to claim 49, which is formulated for
biolistic delivery.
61. The composition according to claim 49, which is formulated for
microneedle delivery.
62. The composition according to claim 49, which is formulated for
intradermal injection.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to gene expression.
More particularly, the present invention relates to methods for
modulating the quality of an immune response to a target antigen in
a mammal, which response results from the expression of a
polynucleotide that encodes at least a portion of the target
antigen, wherein the quality is modulated by replacing at least one
codon of the polynucleotide with a synonymous codon that has a
higher or lower preference of usage by the mammal to confer the
immune response than the codon it replaces. Even more particularly,
the present invention relates to the use of a protein-encoding
polynucleotide whose codon composition has been modified for
modulating the quality of an immune response to an antigen in a
mammal.
BACKGROUND OF THE INVENTION
[0002] The expression of foreign heterologous genes in transformed
cells is now commonplace. A large number of mammalian genes,
including, for example, murine and human genes, have been
successfully expressed in various host cells, including bacterial,
yeast, insect, plant and mammalian host cells. Nevertheless,
despite the burgeoning knowledge of expression systems and
recombinant DNA technology, significant obstacles remain when one
attempts to express a foreign or synthetic gene in a selected host
cell. For example, translation of a synthetic gene, even when
coupled with a strong promoter, often proceeds much more slowly
than would be expected. The same is frequently true of exogenous
genes that are foreign to the host cell. This lower than expected
translation efficiency is often due to the protein coding regions
of the gene having a codon usage pattern that does not resemble
those of highly expressed genes in the host cell. It is known in
this regard that codon utilization is highly biased and varies
considerably in different organisms and that biases in codon usage
can alter peptide elongation rates. It is also known that codon
usage patterns are related to the relative abundance of tRNA
isoacceptors, and that genes encoding proteins of high versus low
abundance show differences in their codon preferences.
[0003] The implications of codon preference phenomena on gene
expression are manifest in that these phenomena can affect the
translational efficiency of messenger RNA (mRNA). It is widely
known in this regard that translation of "rare codons", for which
the corresponding iso-tRNA is in low abundance relative to other
iso-tRNAs, may cause a ribosome to pause during translation which
can lead to a failure to complete a nascent polypeptide chain and
an uncoupling of transcription and translation. Thus, the
expression of an exogenous gene may be impeded severely if a
particular host cell of an organism or the organism itself has a
low abundance of iso-tRNAs corresponding to one or more codons of
the exogenous gene. Accordingly, a major aim of investigators in
this field is to first ascertain the codon preference for
particular cells in which an exogenous gene is to be expressed, and
to subsequently alter the codon composition of that gene for
optimized expression in those cells.
[0004] Codon-optimization techniques are known for improving the
translational kinetics of translationally inefficient protein
coding regions. Traditionally, these techniques have been based on
the replacement of codons that are rarely or infrequently used in
the host cell with those that are host-preferred. Codon frequencies
can be derived from literature sources for the highly expressed
genes of many organisms (see, for example, Nakamura et al., 1996,
Nucleic Acids Res 24: 214-215). These frequencies are generally
expressed on an `organism-wide average basis` as the percentage of
occasions that a synonymous codon is used to encode a corresponding
amino acid across a collection of protein-encoding genes of that
organism, which are preferably highly expressed.
[0005] Typically, codons are classified as: (a) "common" codons (or
"preferred" codons) if their frequency of usage is above about
4/3.times.the frequency of usage that would be expected in the
absence of any bias in codon usage; (b) "rare" codons (or
"non-preferred" codons) if their frequency of usage is below about
2/3.times.the frequency of usage that would be expected in the
absence of any bias in codon usage; and (c) "intermediate" codons
(or "less preferred" codons) if their frequency of usage is
in-between the frequency of usage of "common" codons and of "rare"
codons. Since an amino acid can be encoded by 2, 3, 4 or 6 codons,
the frequency of usage of any selected codon, which would be
expected in the absence of any bias in codon usage, will be
dependent upon the number of synonymous codons which code for the
same amino acid as the selected codon. Accordingly, for a
particular amino acid, the frequency thresholds for classifying
codons in the "common", "intermediate" and "rare" categories will
be dependent upon the number of synonymous codons for that amino
acid. Consequently, for amino acids having 6 choices of synonymous
codon, the frequency of codon usage that would be expected in the
absence of any bias in codon usage is 16% and thus the "common",
"intermediate" and "rare" codons are defined as those codons that
have a frequency of usage above 20%, between 10 and 20% and below
10%, respectively. For amino acids having 4 choices of synonymous
codon, the frequency of codon usage that would be expected in the
absence of codon usage bias is 25% and thus the "common",
"intermediate" and "rare" codons are defined as those codons that
have a frequency of usage above 33%, between 16 and 33% and below
16%, respectively. For isoleucine, which is the only amino acid
having 3 choices of synonymous codon, the frequency of codon usage
that would be expected in the absence of any bias in codon usage is
33% and thus the "common", "intermediate" and "rare" codons for
isoleucine are defined as those codons that have a frequency of
usage above 45%, between 20 and 45% and below 20%, respectively.
For amino acids having 2 choices of synonymous codon, the frequency
of codon usage that would be expected in the absence of codon usage
bias is 50% and thus the "common", "intermediate" and "rare" codons
are defined as those codons that have a frequency of usage above
60%, between 30 and 60% and below 30%, respectively. Thus, the
categorization of codons into the "common", "intermediate" and
"rare" classes (or "preferred", "less preferred" or "non
preferred", respectively) has been based conventionally on a
compilation of codon usage for an organism in general (e.g.,
`human-wide`) or for a class of organisms in general (e.g.,
`mammal-wide`). For example, reference may be made to Seed (see
U.S. Pat. Nos. 5,786,464 and 5,795,737) who discloses preferred,
less preferred and non-preferred codons for mammalian cells in
general. However, the present inventor revealed in WO 99/02694 and
in WO 00/42190 that there are substantial differences in the
relative abundance of particular iso-tRNAs in different cells or
tissues of a single multicellular organism (e.g., a mammal or a
plant) and that this plays a pivotal role in protein translation
from a coding sequence with a given codon usage or composition.
[0006] Thus, in contrast to the art-recognized presumption that
different cells of a multicellular organism have the same bias in
codon usage, it was revealed for the first time that one cell type
of a multicellular organism uses codons in a manner distinct from
another cell type of the same organism. In other words, it was
discovered that different cells of an organism can exhibit
different translational efficiencies for the same codon and that it
was not possible to predict which codons would be preferred, less
preferred or non preferred in a selected cell type. Accordingly, it
was proposed that differences in codon translational efficiency
between cell types could be exploited, together with codon
composition of a gene, to regulate the production of a protein in,
or to direct that production to, a chosen cell type.
[0007] Therefore, in order to optimize the expression of a
protein-encoding polynucleotide in a particular cell type, WO
99/02694 and in WO 00/42190 teach that it is necessary to first
determine the translational efficiency for each codon in that cell
type, rather than to rely on codon frequencies calculated on an
organism-wide average basis, and then to codon modify the
polynucleotide based on that determination.
[0008] The present inventor further disclosed in WO 2004/042059 a
strategy for enhancing or reducing the quality of a selected
phenotype that is displayed, or proposed to be displayed, by an
organism of interest. The strategy involves codon modification of a
polynucleotide that encodes a phenotype-associated polypeptide that
either by itself, or in association with other molecules, in the
organism of interest imparts or confers the selected phenotype upon
the organism. Unlike previous methods, however, this strategy does
not rely on data that provide a ranking of synonymous codons
according to their preference of usage in an organism or class of
organisms. Nor does it rely on data that provide a ranking of
synonymous codons according to their translational efficiencies in
one or more cells of the organism or class of organisms. Instead,
it relies on ranking individual synonymous codons that code for an
amino acid in the phenotype-associated polypeptide according to
their preference of usage by the organism or class of organisms, or
by a part thereof, for producing the selected phenotype.
SUMMARY OF THE INVENTION
[0009] The present invention is predicated in part on the
experimental determination of a ranking of individual synonymous
codons according to their preference for producing an immune
response, including a humoral immune response, to an antigen in a
mammal. Significantly, this ranking is not coterminous with a
ranking of codon frequency values derivable from an analysis of the
frequency with which codons are used to encode their corresponding
amino acids across a collection of highly expressed mammalian
protein-encoding genes, as for example disclosed by Seed (supra).
Nor is it coterminous with a ranking of translational efficiency
values obtained from an analysis of the translational efficiencies
of codons in specific cell types, as disclosed for example in WO
99/02694 for COS-1 cells and epithelial cells and in WO 2004/024915
for CHO cells. Indeed, the present inventors have determined that
codon modification of wild-type antigen-encoding polynucleotides to
replace codons found in the wild-type sequence with codons having a
higher preference for producing an immune response than the codons
they replaced significantly enhances the immune response to the
encoded antigen, as compared to the immune response obtained with
the wild-type sequence. As a result, the present invention enables
for the first time the construction of antigen-encoding
polynucleotides, which are codon-optimized for efficient production
of immune responses in a mammal.
[0010] Thus, in one aspect of the present invention, methods are
provided for constructing a synthetic polynucleotide from which a
polypeptide is producible to confer an immune response to a target
antigen in a mammal in a different quality than that conferred by a
parent polynucleotide that encodes the same polypeptide, wherein
the polypeptide corresponds to at least a portion of the target
antigen. These methods generally comprise: (a) selecting a first
codon of the parent polynucleotide for replacement with a
synonymous codon, wherein the synonymous codon is selected on the
basis that it exhibits a different preference for conferring an
immune response ("an immune response preference") than the first
codon in a comparison of immune response preferences; and (b)
replacing the first codon with the synonymous codon to construct
the synthetic polynucleotide, wherein the comparison of immune
response preferences of the codons is represented by TABLE 1:
TABLE-US-00001 TABLE 1 Amino Ranking of Immune Response Preferences
for Synonymous Acid Codons Ala Ala.sup.GCT > Ala.sup.GCC >
(Ala.sup.GCA, Ala.sup.GCG) Arg (Arg.sup.CGA, Arg.sup.CGC,
Arg.sup.CGT, Arg.sup.AGA) > (Arg.sup.AGG, Arg.sup.CGG) Asn
Asn.sup.AAC > Asn.sup.AAT Asp Asp.sup.GAC > Asp.sup.GAT Cys
Cys.sup.TGC > Cys.sup.TGT Glu Glu.sup.GAA > Glu.sup.GAG Gln
Gln.sup.CAA = Gln.sup.CAG Gly Gly.sup.GGA > (Gly.sup.GGG,
Gly.sup.GGT, Gly.sup.GGC) His His.sup.CAC = His.sup.CAT Ile
Ile.sup.ATC >> Ile.sup.ATT > Ile.sup.ATA Leu (Leu.sup.CTG,
Leu.sup.CTC) > (Leu.sup.CTA, Leu.sup.CTT) >> Leu.sup.TTG
> Leu.sup.TTA Lys Lys.sup.AAG = Lys.sup.AAA Phe Phe.sup.TTT >
Phe.sup.TTC Pro Pro.sup.CCC > Pro.sup.CCT >> (Pro.sup.CCA,
Pro.sup.CCG) Ser Ser.sup.TCG >> (Ser.sup.TCT, Ser.sup.TCA,
Ser.sup.TCC) >> (Ser.sup.AGC, Ser.sup.AGT) Thr Thr.sup.ACG
> Thr.sup.ACC >> Thr.sup.ACA > Thr.sup.ACT Tyr
Tyr.sup.TAC > Tyr.sup.TAT Val (Val.sup.GTG, Val.sup.GTC) >
Val.sup.GTT > Val.sup.GTA
[0011] Thus, a stronger or enhanced immune response to the target
antigen (e.g., an immune response that is at least about 110%,
150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% and all
integer percentages in between, of that produced from the parent
polynucleotide under identical conditions) can be achieved by
selecting a synonymous codon that has a higher immune response
preference than the first codon it replaces. In specific
embodiments, the synonymous codon is selected such that it has a
higher immune response preference that is at least about 10% (and
at least about 11% to at least about 1000% and all integer
percentages in between) higher than the immune response preference
of the codon it replaces. In illustrative examples of this type,
the first and synonymous codons are selected from TABLE 2:
TABLE-US-00002 TABLE 2 First Codon Synonymous Codon Ala.sup.GCG
Ala.sup.GCT Ala.sup.GCG Ala.sup.GCC Ala.sup.GCA Ala.sup.GCT
Ala.sup.GCA Ala.sup.GCC Ala.sup.GCC Ala.sup.GCT Arg.sup.CGG
Arg.sup.CGA Arg.sup.CGG Arg.sup.CGC Arg.sup.CGG Arg.sup.CGT
Arg.sup.CGG Arg.sup.AGA Arg.sup.AGG Arg.sup.CGA Arg.sup.AGG
Arg.sup.CGC Arg.sup.AGG Arg.sup.CGT Arg.sup.AGG Arg.sup.AGA
Asn.sup.AAT Asn.sup.AAC Asp.sup.GAT Asp.sup.GAC Cys.sup.TGT
Cys.sup.TGC Glu.sup.GAG Glu.sup.GAA Gly.sup.GGC Gly.sup.GGA
Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG Gly.sup.GGA Ile.sup.ATA
Ile.sup.ATC Ile.sup.ATA Ile.sup.ATT Ile.sup.ATT Ile.sup.ATC
Leu.sup.TTA Leu.sup.CTG Leu.sup.TTA Leu.sup.CTC Leu.sup.TTA
Leu.sup.CTA Leu.sup.TTA Leu.sup.CTT Leu.sup.TTA Leu.sup.TTG
Leu.sup.TTG Leu.sup.CTG Leu.sup.TTG Leu.sup.CTC Leu.sup.TTG
Leu.sup.CTA Leu.sup.TTG Leu.sup.CTT Leu.sup.CTT Leu.sup.CTG
Leu.sup.CTT Leu.sup.CTC Leu.sup.CTA Leu.sup.CTG Leu.sup.CTA
Leu.sup.CTC Phe.sup.TTC Phe.sup.TTT Pro.sup.CCG Pro.sup.CCC
Pro.sup.CCG Pro.sup.CCT Pro.sup.CCA Pro.sup.CCC Pro.sup.CCA
Pro.sup.CCT Pro.sup.CCT Pro.sup.CCC Ser.sup.AGT Ser.sup.TCG
Ser.sup.AGT Ser.sup.TCT Ser.sup.AGT Ser.sup.TCA Ser.sup.AGT
Ser.sup.TCC Ser.sup.AGC Ser.sup.TCG Ser.sup.AGC Ser.sup.TCT
Ser.sup.AGC Ser.sup.TCA Ser.sup.AGC Ser.sup.TCC Ser.sup.TCC
Ser.sup.TCG Ser.sup.TCA Ser.sup.TCG Ser.sup.TCT Ser.sup.TCG
Thr.sup.ACT Thr.sup.ACG Thr.sup.ACT Thr.sup.ACC Thr.sup.ACT
Thr.sup.ACA Thr.sup.ACA Thr.sup.ACG Thr.sup.ACA Thr.sup.ACC
Thr.sup.ACC Thr.sup.ACG Tyr.sup.TAT Tyr.sup.TAC Val.sup.GTA
Val.sup.GTG Val.sup.GTA Val.sup.GTC Val.sup.GTA Val.sup.GTT
Val.sup.GTT Val.sup.GTG Val.sup.GTT Val.sup.GTC
[0012] In other illustrative examples of this type, the first and
synonymous codons are selected from TABLE 3:
TABLE-US-00003 TABLE 3 First Codon Synonymous Codon Ala.sup.GCG
Ala.sup.GCT Ala.sup.GCA Ala.sup.GCT Ala.sup.GCC Ala.sup.GCT
Arg.sup.CGG Arg.sup.CGA Arg.sup.CGG Arg.sup.CGT Arg.sup.CGG
Arg.sup.AGA Arg.sup.AGG Arg.sup.CGA Arg.sup.AGG Arg.sup.CGT
Arg.sup.AGG Arg.sup.AGA Glu.sup.GAG Glu.sup.GAA Gly.sup.GGC
Gly.sup.GGA Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG Gly.sup.GGA
Leu.sup.TTA Leu.sup.CTA Leu.sup.TTA Leu.sup.CTT Leu.sup.TTA
Leu.sup.TTG Leu.sup.TTG Leu.sup.CTA Leu.sup.TTG Leu.sup.CTT
Phe.sup.TTC Phe.sup.TTT Pro.sup.CCG Pro.sup.CCT Pro.sup.CCA
Pro.sup.CCT Ser.sup.AGT Ser.sup.TCG Ser.sup.AGT Ser.sup.TCT
Ser.sup.AGT Ser.sup.TCA Ser.sup.AGC Ser.sup.TCG Ser.sup.AGC
Ser.sup.TCT Ser.sup.AGC Ser.sup.TCA Ser.sup.AGC Ser.sup.TCC
Ser.sup.TCC Ser.sup.TCG Ser.sup.TCA Ser.sup.TCG Ser.sup.TCT
Ser.sup.TCG Thr.sup.ACT Thr.sup.ACG Thr.sup.ACT Thr.sup.ACA
Thr.sup.ACA Thr.sup.ACG Thr.sup.ACC Thr.sup.ACG Val.sup.GTA
Val.sup.GTT
[0013] Suitably, in some of the illustrative examples noted above,
the method further comprises selecting a second codon of the parent
polynucleotide for replacement with a synonymous codon, wherein the
synonymous codon is selected on the basis that it exhibits a higher
immune response preference than the second codon in a comparison of
immune response preferences; and (b) replacing the second codon
with the synonymous codon, wherein the comparison of immune
response preferences of the codons is represented by TABLE 4:
TABLE-US-00004 TABLE 4 Second Codon Synonymous Codon Ala.sup.GCG
Ala.sup.GCT Ala.sup.GCG Ala.sup.GCC Ala.sup.GCA Ala.sup.GCT
Ala.sup.GCA Ala.sup.GCC Ala.sup.GCC Ala.sup.GCT Arg.sup.CGG
Arg.sup.CGA Arg.sup.CGG Arg.sup.CGC Arg.sup.CGG Arg.sup.CGT
Arg.sup.CGG Arg.sup.AGA Arg.sup.AGG Arg.sup.CGA Arg.sup.AGG
Arg.sup.CGC Arg.sup.AGG Arg.sup.CGT Arg.sup.AGG Arg.sup.AGA
Asn.sup.AAT Asn.sup.AAC Asp.sup.GAT Asp.sup.GAC Cys.sup.TGT
Cys.sup.TGC Glu.sup.GAG Glu.sup.GAA Gly.sup.GGC Gly.sup.GGA
Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG Gly.sup.GGA Ile.sup.ATA
Ile.sup.ATC Ile.sup.ATA Ile.sup.ATT Ile.sup.ATT Ile.sup.ATC
Leu.sup.TTA Leu.sup.CTG Leu.sup.TTA Leu.sup.CTC Leu.sup.TTA
Leu.sup.CTA Leu.sup.TTA Leu.sup.CTT Leu.sup.TTA Leu.sup.TTG
Leu.sup.TTG Leu.sup.CTG Leu.sup.TTG Leu.sup.CTC Leu.sup.TTG
Leu.sup.CTA Leu.sup.TTG Leu.sup.CTT Leu.sup.CTT Leu.sup.CTG
Leu.sup.CTT Leu.sup.CTC Leu.sup.CTA Leu.sup.CTG Leu.sup.CTA
Leu.sup.CTC Phe.sup.TTC Phe.sup.TTT Pro.sup.CCG Pro.sup.CCC
Pro.sup.CCG Pro.sup.CCT Pro.sup.CCA Pro.sup.CCC Pro.sup.CCA
Pro.sup.CCT Pro.sup.CCT Pro.sup.CCC Ser.sup.AGT Ser.sup.TCG
Ser.sup.AGT Ser.sup.TCT Ser.sup.AGT Ser.sup.TCA Ser.sup.AGT
Ser.sup.TCC Ser.sup.AGC Ser.sup.TCG Ser.sup.AGC Ser.sup.TCT
Ser.sup.AGC Ser.sup.TCA Ser.sup.AGC Ser.sup.TCC Ser.sup.TCC
Ser.sup.TCG Ser.sup.TCA Ser.sup.TCG Ser.sup.TCT Ser.sup.TCG
Thr.sup.ACT Thr.sup.ACG Thr.sup.ACT Thr.sup.ACC Thr.sup.ACT
Thr.sup.ACA Thr.sup.ACA Thr.sup.ACG Thr.sup.ACA Thr.sup.ACC
Thr.sup.ACC Thr.sup.ACG Tyr.sup.TAT Tyr.sup.TAC Val.sup.GTA
Val.sup.GTG Val.sup.GTA Val.sup.GTC Val.sup.GTA Val.sup.GTT
Val.sup.GTT Val.sup.GTG Val.sup.GTT Val.sup.GTC
[0014] Conversely, a weaker or reduced immune response to the
target antigen (e.g., an immune response that is at less than about
90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1% and all integer
percentages in between, of that produced from the parent
polynucleotide under identical conditions) can be achieved by
selecting a synonymous codon that has a lower immune response
preference than the first codon it replaces. In specific
embodiments of this type, the synonymous codon is selected such
that it has an immune response preference that is less than about
90% of the immune response preference of the codon it replaces. In
illustrative examples, the first and synonymous codons are selected
from the TABLE 5:
TABLE-US-00005 TABLE 5 Synonymous First Codon Codon Ala.sup.GCT
Ala.sup.GCG Ala.sup.GCT Ala.sup.GCA Ala.sup.GCT Ala.sup.GCC
Ala.sup.GCC Ala.sup.GCG Ala.sup.GCC Ala.sup.GCA Arg.sup.CGA
Arg.sup.AGG Arg.sup.CGA Arg.sup.CGG Arg.sup.CGC Arg.sup.AGG
Arg.sup.CGC Arg.sup.CGG Arg.sup.CGT Arg.sup.AGG Arg.sup.CGT
Arg.sup.CGG Arg.sup.AGA Arg.sup.AGG Arg.sup.AGA Arg.sup.CGG
Asn.sup.AAC Asn.sup.AAT Asp.sup.GAC Asp.sup.GAT Cys.sup.TGC
Cys.sup.TGT Glu.sup.GAA Glu.sup.GAG Gly.sup.GGA Gly.sup.GGC
Gly.sup.GGA Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG Ile.sup.ATC
Ile.sup.ATA Ile.sup.ATC Ile.sup.ATT Ile.sup.ATT Ile.sup.ATA
Leu.sup.CTG Leu.sup.CTA Leu.sup.CTG Leu.sup.CTT Leu.sup.CTG
Leu.sup.TTG Leu.sup.CTG Leu.sup.TTA Leu.sup.CTC Leu.sup.CTA
Leu.sup.CTC Leu.sup.CTT Leu.sup.CTC Leu.sup.TTG Leu.sup.CTC
Leu.sup.TTA Leu.sup.CTA Leu.sup.TTG Leu.sup.CTA Leu.sup.TTA
Leu.sup.CTT Leu.sup.TTG Leu.sup.CTT Leu.sup.TTA Leu.sup.TTG
Leu.sup.TTA Phe.sup.TTT Phe.sup.TTC Pro.sup.CCC Pro.sup.CCT
Pro.sup.CCC Pro.sup.CCA Pro.sup.CCC Pro.sup.CCG Pro.sup.CCT
Pro.sup.CCA Pro.sup.CCT Pro.sup.CCG Ser.sup.TCG Ser.sup.TCT
Ser.sup.TCG Ser.sup.TCA Ser.sup.TCG Ser.sup.TCC Ser.sup.TCG
Ser.sup.AGC Ser.sup.TCG Ser.sup.AGT Ser.sup.TCT Ser.sup.AGC
Ser.sup.TCT Ser.sup.AGT Ser.sup.TCA Ser.sup.AGC Ser.sup.TCA
Ser.sup.AGT Ser.sup.TCC Ser.sup.AGC Ser.sup.TCC Ser.sup.AGT
Thr.sup.ACG Thr.sup.ACC Thr.sup.ACG Thr.sup.ACA Thr.sup.ACG
Thr.sup.ACT Thr.sup.ACC Thr.sup.ACA Thr.sup.ACC Thr.sup.ACT
Thr.sup.ACA Thr.sup.ACT Tyr.sup.TAC Tyr.sup.TAT Val.sup.GTG
Val.sup.GTT Val.sup.GTG Val.sup.GTA Val.sup.GTC Val.sup.GTT
Val.sup.GTC Val.sup.GTA Val.sup.GTT Val.sup.GTA
[0015] In other illustrative examples, the first and synonymous
codons are selected from TABLE 6:
TABLE-US-00006 TABLE 6 Synonymous First Codon Codon Ala.sup.GCT
Ala.sup.GCG Ala.sup.GCT Ala.sup.GCA Ala.sup.GCT Ala.sup.GCC
Arg.sup.CGA Arg.sup.AGG Arg.sup.CGA Arg.sup.CGG Arg.sup.CGT
Arg.sup.AGG Arg.sup.CGT Arg.sup.CGG Arg.sup.AGA Arg.sup.AGG
Arg.sup.AGA Arg.sup.CGG Glu.sup.GAA Glu.sup.GAG Gly.sup.GGA
Gly.sup.GGC Gly.sup.GGA Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG
Leu.sup.CTA Leu.sup.TTG Leu.sup.CTA Leu.sup.TTA Leu.sup.CTT
Leu.sup.TTG Leu.sup.CTT Leu.sup.TTA Leu.sup.TTG Leu.sup.TTA
Phe.sup.TTT Phe.sup.TTC Pro.sup.CCT Pro.sup.CCA Pro.sup.CCT
Pro.sup.CCG Ser.sup.TCG Ser.sup.TCT Ser.sup.TCG Ser.sup.TCA
Ser.sup.TCG Ser.sup.TCC Ser.sup.TCG Ser.sup.AGC Ser.sup.TCG
Ser.sup.AGT Ser.sup.TCT Ser.sup.AGC Ser.sup.TCT Ser.sup.AGT
Ser.sup.TCA Ser.sup.AGC Ser.sup.TCA Ser.sup.AGT Ser.sup.TCC
Ser.sup.AGC Thr.sup.ACG Thr.sup.ACC Thr.sup.ACG Thr.sup.ACA
Thr.sup.ACG Thr.sup.ACT Thr.sup.ACA Thr.sup.ACT Val.sup.GTT
Val.sup.GTA
[0016] Suitably, in some of the illustrative examples noted above,
the method further comprises selecting a second codon of the parent
polynucleotide for replacement with a synonymous codon, wherein the
synonymous codon is selected on the basis that it exhibits a lower
immune response preference than the second codon in a comparison of
immune response preferences; and; (b) replacing the second codon
with the synonymous codon, wherein the comparison of immune
response preferences of the codons is represented by TABLE 7:
TABLE-US-00007 TABLE 7 Second Synonymous Codon Codon Ala.sup.GCT
Ala.sup.GCG Ala.sup.GCT Ala.sup.GCA Ala.sup.GCT Ala.sup.GCC
Ala.sup.GCC Ala.sup.GCG Ala.sup.GCC Ala.sup.GCA Arg.sup.CGA
Arg.sup.AGG Arg.sup.CGA Arg.sup.CGG Arg.sup.CGC Arg.sup.AGG
Arg.sup.CGC Arg.sup.CGG Arg.sup.CGT Arg.sup.AGG Arg.sup.CGT
Arg.sup.CGG Arg.sup.AGA Arg.sup.AGG Arg.sup.AGA Arg.sup.CGG
Asn.sup.AAC Asn.sup.AAT Asp.sup.GAC Asp.sup.GAT Cys.sup.TGC
Cys.sup.TGT Glu.sup.GAA Glu.sup.GAG Gly.sup.GGA Gly.sup.GGC
Gly.sup.GGA Gly.sup.GGT Gly.sup.GGA Gly.sup.GGG Ile.sup.ATC
Ile.sup.ATA Ile.sup.ATC Ile.sup.ATT Ile.sup.ATT Ile.sup.ATA
Leu.sup.CTG Leu.sup.CTA Leu.sup.CTG Leu.sup.CTT Leu.sup.CTG
Leu.sup.TTG Leu.sup.CTG Leu.sup.TTA Leu.sup.CTC Leu.sup.CTA
Leu.sup.CTC Leu.sup.CTT Leu.sup.CTC Leu.sup.TTG Leu.sup.CTC
Leu.sup.TTA Leu.sup.CTA Leu.sup.TTG Leu.sup.CTA Leu.sup.TTA
Leu.sup.CTT Leu.sup.TTG Leu.sup.CTT Leu.sup.TTA Leu.sup.TTG
Leu.sup.TTA Phe.sup.TTT Phe.sup.TTC Pro.sup.CCC Pro.sup.CCT
Pro.sup.CCC Pro.sup.CCA Pro.sup.CCC Pro.sup.CCG Pro.sup.CCT
Pro.sup.CCA Pro.sup.CCT Pro.sup.CCG Ser.sup.TCG Ser.sup.TCT
Ser.sup.TCG Ser.sup.TCA Ser.sup.TCG Ser.sup.TCC Ser.sup.TCG
Ser.sup.AGC Ser.sup.TCG Ser.sup.AGT Ser.sup.TCT Ser.sup.AGC
Ser.sup.TCT Ser.sup.AGT Ser.sup.TCA Ser.sup.AGC Ser.sup.TCA
Ser.sup.AGT Ser.sup.TCC Ser.sup.AGC Ser.sup.TCC Ser.sup.AGT
Thr.sup.ACG Thr.sup.ACC Thr.sup.ACG Thr.sup.ACA Thr.sup.ACG
Thr.sup.ACT Thr.sup.ACC Thr.sup.ACA Thr.sup.ACC Thr.sup.ACT
Thr.sup.ACA Thr.sup.ACT Tyr.sup.TAC Tyr.sup.TAT Val.sup.GTG
Val.sup.GTT Val.sup.GTG Val.sup.GTA Val.sup.GTC Val.sup.GTT
Val.sup.GTC Val.sup.GTA Val.sup.GTT Val.sup.GTA
[0017] In another aspect, the invention provides a synthetic
polynucleotide constructed according to any one of the above
methods.
[0018] In accordance with the present invention, synthetic
polynucleotides that are constructed by methods described herein
are useful for expression in a mammal to elicit an immune response
to a target antigen. Accordingly, in yet another aspect, the
present invention provides chimeric constructs that comprise a
synthetic polynucleotide of the invention, which is operably
connected to a regulatory polynucleotide.
[0019] In some embodiments, the chimeric construct is in the form
of a pharmaceutical composition that optionally comprises a
pharmaceutically acceptable excipient and/or carrier. Accordingly,
in another aspect, the invention provides pharmaceutical
compositions that are useful for modulating an immune response to a
target antigen in a mammal, which response is conferred by the
expression of a parent polynucleotide that encodes a polypeptide
corresponding to at least a portion of the target antigen. These
compositions generally comprise a chimeric construct and a
pharmaceutically acceptable excipient and/or carrier, wherein the
chimeric construct comprises a synthetic polynucleotide that is
operably connected to a regulatory polynucleotide and that is
distinguished from the parent polynucleotide by the replacement of
a first codon in the parent polynucleotide with a synonymous codon
that has a different immune response preference than the first
codon and wherein the first and synonymous codons are selected
according to any one of TABLES 2, 3, 5 and 6. In some embodiments,
the compositions further comprise an adjuvant that enhances the
effectiveness of the immune response. In some embodiments, the
composition is formulated for transcutaneous or dermal
administration, e.g., by biolistic or microneedle delivery or by
intradermal injection. Suitably, in embodiments in which a stronger
or enhanced immune response to the target antigen is desired, the
first and synonymous codons are selected according to TABLES 2 or
3. Conversely, in embodiments in which a weaker or reduced immune
response to the target antigen is desired, the first and synonymous
codons are selected according to TABLES 5 or 6.
[0020] In yet another aspect, the invention embraces methods of
modulating the quality of an immune response to a target antigen in
a mammal, which response is conferred by the expression of a parent
polynucleotide that encodes a polypeptide corresponding to at least
a portion of the target antigen. These methods generally comprise:
introducing into the mammal a synthetic polynucleotide that is
operably connected to a regulatory polynucleotide and that is
distinguished from the parent polynucleotide by the replacement of
a first codon in the parent polynucleotide with a synonymous codon
that has a different immune response preference than the first
codon and wherein the first and synonymous codons are selected
according to any one of TABLES 2, 3, 5 and 6. In these methods,
expression of the synthetic polynucleotide results in a different
quality (e.g., stronger or weaker) of immune response than the one
obtained through expression of the parent polynucleotide under the
same conditions. Suitably, the chimeric construct is introduced
into the mammal by delivering the construct to antigen-presenting
cells (e.g., dendritic cells, macrophages, Langerhans cells or
their precursors) of the mammal. In some embodiments, the chimeric
construct is introduced into the dermis and/or epidermis of the
mammal (e.g., by transcutaneous or intradermal administration) and
in this regard any suitable administration site is envisaged
including the abdomen. Generally, the immune response is selected
from a cell-mediated response and a humoral immune response. In
some embodiments, the immune response is a humoral immune response.
In other embodiments, the immune response is a cellular immune
response. In still other embodiments, the immune response is a
humoral immune response and a cellular immune response.
[0021] In a related aspect, the invention encompasses methods of
enhancing the quality of an immune response to a target antigen in
a mammal, which response is conferred by the expression of a parent
polynucleotide that encodes a polypeptide corresponding to at least
a portion of the target antigen. These methods generally comprise:
introducing into the mammal a chimeric construct comprising a
synthetic polynucleotide that is operably connected to a regulatory
polynucleotide and that is distinguished from the parent
polynucleotide by the replacement of a first codon in the parent
polynucleotide with a synonymous codon that has a higher immune
response preference than the first codon, wherein the first and
synonymous codons are selected according to TABLES 2 or 3. In these
methods, expression of the synthetic polynucleotide typically
results in a stronger or enhanced immune response than the one
obtained through expression of the parent polynucleotide under the
same conditions.
[0022] In another related aspect, the invention extends to methods
of reducing the quality of an immune response to a target antigen
in a mammal, which response is conferred by the expression of a
parent polynucleotide that encodes a polypeptide corresponding to
at least a portion of the target antigen. These methods generally
comprise: introducing into the mammal a chimeric construct
comprising a synthetic polynucleotide that is operably connected to
a regulatory polynucleotide and that is distinguished from the
parent polynucleotide by the replacement of a first codon in the
parent polynucleotide with a synonymous codon that has a lower
immune response preference than the first codon, wherein the first
and synonymous codons are selected according to TABLES 5 or 6. In
these methods, expression of the synthetic polynucleotide typically
results in a weaker or reduced immune response than the one
obtained through expression of the parent polynucleotide under the
same conditions.
[0023] Yet a further aspect of the present invention embraces
methods of enhancing the quality of an immune response to a target
antigen in a mammal, which response is conferred by the expression
of a first polynucleotide that encodes a polypeptide corresponding
to at least a portion of the target antigen. These methods
generally comprise: co-introducing into the mammal a first nucleic
acid construct comprising the first polynucleotide in operable
connection with a regulatory polynucleotide; and a second nucleic
acid construct comprising a second polynucleotide that is operably
connected to a regulatory polynucleotide and that encodes an
iso-tRNA corresponding to a codon of the first polynucleotide,
wherein the codon has a low or intermediate immune response
preference and is selected from the group consisting of
Ala.sup.GCA, Ala.sup.GCG, Ala.sup.GCC, Arg.sup.AGG, Arg.sup.CGG,
Asn.sup.AAT, Asp.sup.GAT, Cys.sup.TGT, Glu.sup.GAG, Gly.sup.GGG,
Gly.sup.GGT, Gly.sup.GGC, Ile.sup.ATA, Ile.sup.ATT, Leu.sup.TTG,
Leu.sup.TTA, Leu.sup.CTA, Leu.sup.CTT, Phe.sup.TTC, Pro.sup.CCA,
Pro.sup.CCG, Pro.sup.CCT, Ser.sup.AGC, ser.sup.AGT, Ser.sup.TCT,
Ser.sup.TCA, Ser.sup.TCC, Thr.sup.ACA, Thr.sup.ACT, Tyr.sup.TAT,
Val.sup.GTA and Val.sup.GTT. In specific embodiments, the codon has
a `low` immune response preference, and is selected from the group
consisting of Ala.sup.GCA, Ala.sup.GCG, Arg.sup.AGG, Arg.sup.CGG,
Asn.sup.AAT, Asp.sup.GAT, Cys.sup.TGT, Glu.sup.GAG, Gly.sup.GGG,
Gly.sup.GGT, Gly.sup.GGC, Ile.sup.ATA, Leu.sup.TTG, Leu.sup.TTA,
Phe.sup.TTC, Pro.sup.CCA, Pro.sup.CCG, Ser.sup.AGC, Ser.sup.AGT,
Thr.sup.ACT, Tyr.sup.TAT and Val.sup.GTA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted ALA E7 constructs and
controls (IgkC1, IgkS1-1, IgkS1-2, IgkS1-3, IgkS1-4 and IgkC2) as
further defined in Example 1 and Table 12. The sequences are
ligated into the KpnI and EcoRI sites of pCDNA3.
[0025] FIG. 2 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted ARG E7 constructs and
controls (IgkS1-5, IgkS1-6, IgkS1-7, IgkS1-8, IgkS1-9, IgkS1-10,
IgkC1 and IgkC2) as further defined in Example 1 and Table 12. The
sequences are ligated into the KpnI and EcoRI sites of pCDNA3.
[0026] FIG. 3 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted ASN and LYS E7 constructs
and controls (IgkC1, IgkS1-12, IgkS1-31 and IgkC2) as further
defined in Example 1 and Table 12. The sequences are ligated into
the KpnI and EcoRI sites of pCDNA3.
[0027] FIG. 4 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted ASP E7 constructs and
controls (IgkC1, IgkS1-13, IgkS1-14 and IgkC2) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3.
[0028] FIG. 5 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted CYS E7 constructs and
controls (IgkC1, IgkS1-15, IgkS1-16 and IgkC2) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3.
[0029] FIG. 6 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted GLU E7 constructs and
controls (IgkS1-17, IgkS1-18, IgkC2 and IgkC1) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3.
[0030] FIG. 7 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted GLN E7 constructs and
controls (IgkC1, IgkS1-19, IgkS1-20 and IgkC2) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3.
[0031] FIG. 8 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted GLY E7 constructs and
controls (IgkC1, IgkS1-21, IgkS1-22, IgkS1-23, IgkS1-24 and IgkC2)
as further defined in Example 1 and Table 12. The sequences are
ligated into the KpnI and EcoRI sites of pCDNA3.
[0032] FIG. 9 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted HIS E7 constructs and
controls (IgkC1, IgkS1-25, IgkS1-26 and IgkC2) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3.
[0033] FIG. 10 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted ILE E7 constructs and
controls (IgkC1, IgkS1-27, IgkS1-28, IgkS1-29 and IgkC2) as further
defined in Example 1 and Table 12. The sequences are ligated into
the KpnI and EcoRI sites of pCDNA3.
[0034] FIG. 11 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted LEU E7 constructs and
controls (IgkS1-50, IgkS1-51, IgkS1-52, IgkS1-53, IgkS1-54,
IgkS1-55, IgkC3 and IgkC4) as further defined in Example 1 and
Table 12. The sequences are ligated into the KpnI and EcoRI sites
of pCDNA3. The LEU E7 constructs are oncogenic (i.e., encode
wild-type E7 protein).
[0035] FIG. 12 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted PHE E7 constructs and
controls (IgkS1-32, IgkS1-33, IgkC1 and IgkC2) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3. Two LEU residues were mutated to PHE in
this sequence so that there are three instead of one PHE
residue.
[0036] FIG. 13 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted PRO E7 constructs and
controls (IgkS1-56, IgkS1-57, IgkS1-58, IgkS1-59, IgkC3 and IgkC4)
as further defined in Example 1 and Table 12. The sequences are
ligated into the KpnI and EcoRI sites of pCDNA3. The PRO E7
constructs are oncogenic (i.e., encode wild-type E7 protein).
[0037] FIG. 14 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted SER E7 constructs and
controls (IgkS1-34, IgkS1-35, IgkS1-36, IgkS1-37, IgkS1-38,
IgkS1-39, IgkC1 and IgkC2) as further defined in Example 1 and
Table 12. The sequences are ligated into the KpnI and EcoRI sites
of pCDNA3.
[0038] FIG. 15 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted THR E7 constructs and
controls (IgkC1, IgkS1-40, IgkS1-41, IgkS1-42, IgkS1-43 and IgkC2)
as further defined in Example 1 and Table 12. The sequences are
ligated into the KpnI and EcoRI sites of pCDNA3.
[0039] FIG. 16 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted TYR E7 constructs and
controls (IgkC1, IgkS1-44, IgkS1-45 and IgkC2) as further defined
in Example 1 and Table 12. The sequences are ligated into the KpnI
and EcoRI sites of pCDNA3.
[0040] FIG. 17 is a diagrammatic representation depicting a
nucleotide sequence alignment of secreted VAL E7 constructs and
controls (IgkC1, IgkS1-46, IgkS1-47, IgkS1-48, IgkS1-49 and IgkC2)
as further defined in Example 1 and Table 12. The sequences are
ligated into the KpnI and EcoRI sites of pCDNA3.
[0041] FIG. 18 is a graphical representation showing the response
to gene gun immunization with optimized and de-optimized E7
constructs measured by (a) ELISA, (b) Memory B cell ELISPOT, and
(c) IFN-.gamma. ELISPOT. For part (a) eight mice were immunized per
group (4 immunizations, 3 weeks apart) and the sera taken three
weeks after the final immunization; (left) E7 protein ELISA,
(right) E7 peptide 101 ELISA. Wells were done in duplicate. For
parts (b) and (c) mice were immunized twice, three weeks apart and
the spleens collected three weeks after the second immunization.
The spleens were pooled prior to analysis. The Memory B cell and
IFN-.gamma. ELISPOTs were conducted twice and three times,
respectively, and the wells done in triplicate. Three mice were
used per group per repeat. The results shown in parts (b) and (c)
are from individual experiments and are representative of the
complete data sets. The particular ELISPOT experimental data
included here were gathered together with the corresponding data in
FIG. 20 and therefore may be directly compared. Unpaired two-tailed
t-tests were used to compare the modified constructs to wild-type.
***P<0.001, **0.001.ltoreq.P.ltoreq.0.01,
*0.01.ltoreq.P.ltoreq.0.05, ns=not significant (P>0.05). In (a)
O1-O3 were not significantly different from MC as measured by
unpaired two-tailed t-tests. wt=wild-type codon usage E7;
O1-O3=codon-optimized E7 constructs 1 to 3; W-codon de-optimized
E7; MC=mammalian consensus codon usage E7.
[0042] FIG. 19 is a graphical representation showing the response
to immunization by intradermal injection with optimized and
de-optimized constructs measured by (a) ELISA, (b) Memory B cell
ELISPOT, and (c) IFN-.gamma. ELISPOT. For part (a) eight mice were
immunized per group (4 immunizations, 3 weeks apart) and the sera
taken three weeks after the final immunization; (left) E7 protein
ELISA, (right) E7 peptide 101 ELISA. Wells were done in duplicate.
For parts (b) and (c) mice were immunized twice, three weeks apart
and the spleens collected three weeks after the second
immunization. The spleens were pooled prior to analysis. The Memory
B cell and IFN-.gamma. ELISPOTs were conducted twice and three
times, respectively, and the wells done in triplicate. Three mice
were used per group per repeat. The results shown in parts (b) and
(c) are from individual experiments and are representative of the
complete data sets. The particular ELISPOT experimental data
included here were gathered together with the corresponding data in
FIG. 20 and therefore may be directly compared. Unpaired two-tailed
t-tests were used to compare the modified constructs to wild-type.
*** P<0.001, **0.001.ltoreq.P<0.01,
*0.01.ltoreq.P.ltoreq.0.05, ns=not significant (P>0.05). In (a)
O1-O3 were not significantly different from MC as measured by
unpaired two-tailed t-tests. wt=wild-type codon usage E7;
01-03=codon-optimized E7 constructs 1 to 3; W=codon de-optimized
E7; MC=mammalian consensus codon usage E7.
[0043] FIG. 20 is a graphical representation showing the results of
an ELISA that measures binding of serum from mice immunized with
various gD2 constructs by intradermal injection (white bars) or
gene gun immunization (black bars), to C-terminally His-tagged
gD2tr. Note that the His-tagged gD2tr protein was used in an
unpurified state (in CHO cell supernatant) and that background
readings of non-specific binding to control supernatant have been
subtracted from the results.
TABLE-US-00008 TABLE 8 BRIEF DESCRIPTION OF THE SEQUENCES SEQUENCE
ID NUMBER SEQUENCE LENGTH SEQ ID NO: 1 IgkS2-13 Asp GAT construct
nucleotide sequence 387 nts SEQ ID NO: 2 IgkS2-14 Asp GAC construct
nucleotide sequence 387 nts SEQ ID NO: 3 IgkS2-15 Cys TGT construct
nucleotide sequence 387 nts SEQ ID NO: 4 IgkS2-16 Cys TGC construct
nucleotide sequence 387 nts SEQ ID NO: 5 IgkS2-17 Glu GAG construct
nucleotide sequence 387 nts SEQ ID NO: 6 IgkS2-18 Glu GAA construct
nucleotide sequence 387 nts SEQ ID NO: 7 IgkS2-19 Gln CAG construct
nucleotide sequence 387 nts SEQ ID NO: 8 IgkS2-20 Gln CAA construct
nucleotide sequence 387 nts SEQ ID NO: 9 IgkS2-21 Gly GGG construct
nucleotide sequence 387 nts SEQ ID NO: 10 IgkS2-22 Gly GGA
construct nucleotide sequence 387 nts SEQ ID NO: 11 IgkS2-23 Gly
GGT construct nucleotide sequence 387 nts SEQ ID NO: 12 IgkS2-24
Gly GGC construct nucleotide sequence 387 nts SEQ ID NO: 13
IgkS2-27 Ile ATA construct nucleotide sequence 387 nts SEQ ID NO:
14 IgkS2-28 Ile ATT construct nucleotide sequence 387 nts SEQ ID
NO: 15 IgkS2-29 Ile ATC construct nucleotide sequence 387 nts SEQ
ID NO: 16 IgkS2-34 Ser AGT construct nucleotide sequence 387 nts
SEQ ID NO: 17 IgkS2-35 Ser AGC construct nucleotide sequence 387
nts SEQ ID NO: 18 IgkS2-36 Ser TCG construct nucleotide sequence
387 nts SEQ ID NO: 19 IgkS2-37 Ser TCA construct nucleotide
sequence 387 nts SEQ ID NO: 20 IgkS2-38 Ser TCT construct
nucleotide sequence 387 nts SEQ ID NO: 21 IgkS2-39 Ser TCC
construct nucleotide sequence 387 nts SEQ ID NO: 22 IgkS2-40 Thr
ACG construct nucleotide sequence 387 nts SEQ ID NO: 23 IgkS2-41
Thr ACA construct nucleotide sequence 387 nts SEQ ID NO: 24
IgkS2-42 Thr ACT construct nucleotide sequence 387 nts SEQ ID NO:
25 IgkS2-43 Thr ACC construct nucleotide sequence 387 nts SEQ ID
NO: 26 IgkS2-46 Val GTG construct nucleotide sequence 387 nts SEQ
ID NO: 27 IgkS2-47 Val GTA construct nucleotide sequence 387 nts
SEQ ID NO: 28 IgkS2-48 Val GTT construct nucleotide sequence 387
nts SEQ ID NO: 29 IgkS2-49 Val GTG construct nucleotide sequence
387 nts SEQ ID NO: 30 IgkS2-1 Ala GCG Linker nucleotide sequence
408 nts SEQ ID NO: 31 IgkS2-2 Ala GCA Linker nucleotide sequence
408 nts SEQ ID NO: 32 IgkS2-3 Ala GCT Linker nucleotide sequence
408 nts SEQ ID NO: 33 IgkS2-4 Ala GCC Linker nucleotide sequence
408 nts SEQ ID NO: 34 IgkS2-5 Arg AGG Linker nucleotide sequence
408 nts SEQ ID NO: 35 IgkS2-6 Arg AGA Linker nucleotide sequence
408 nts SEQ ID NO: 36 IgkS2-7 Arg CGG Linker nucleotide sequence
408 nts SEQ ID NO: 37 IgkS2-8 Arg CGA Linker nucleotide sequence
408 nts SEQ ID NO: 38 IgkS2-9 Arg CGT Linker nucleotide sequence
408 nts SEQ ID NO: 39 IgkS2-10 Arg CGC Linker nucleotide sequence
408 nts SEQ ID NO: 40 IgkS2-11 Asn AAT Linker nucleotide sequence
408 nts SEQ ID NO: 41 IgkS2-12 Asn AAC Linker nucleotide sequence
408 nts SEQ ID NO: 42 IgkS2-25 His CAT Linker nucleotide sequence
408 nts SEQ ID NO: 43 IgkS2-26 His CAC Linker nucleotide sequence
408 nts SEQ ID NO: 44 IgkS2-30 Lys AAG Linker nucleotide sequence
408 nts SEQ ID NO: 45 IgkS2-31 Lys AAA Linker nucleotide sequence
408 nts SEQ ID NO: 46 IgkS2-32 Phe TTT Linker nucleotide sequence
408 nts SEQ ID NO: 47 IgkS2-33 Phe TTC Linker nucleotide sequence
408 nts SEQ ID NO: 48 IgkS2-44 Tyr TAT Linker nucleotide sequence
408 nts SEQ ID NO: 49 IgkS2-45 Tyr TAC Linker nucleotide sequence
408 nts SEQ ID NO: 50 Influenza A Virus HA hemagglutinin (A/Hong
1707 nts Kong/213/03(H5N1)) BAE07201 wild-type SEQ ID NO: 51
Influenza A Virus HA hemagglutinin (A/Hong 568 aa
Kong/213/03(H5N1)) BAE07201 wild-type SEQ ID NO: 52 Influenza A
Virus HA hemagglutinin (A/Hong 1707 nts Kong/213/03(H5N1)) Codon
modified SEQ ID NO: 53 Influenza A Virus HA hemagglutinin 1701 nts
(A/swine/Korea/PZ72-1/2006 (H3N1)) DQ923506 wild-type SEQ ID NO: 54
Influenza A Virus HA hemagglutinin 566 aa
(A/swine/Korea/PZ72-1/2006 (H3N1)) DQ923506 wild-type SEQ ID NO: 55
Influenza A Virus HA hemagglutinin 1701 nts
(A/swine/Korea/PZ72-1/2006 (H3N1)) Codon modified SEQ ID NO: 56
Influenza A Virus NA neuraminidase (A/Hong 1410 nts
Kong/213/03(H5N1)) AB212056 wild-type SEQ ID NO: 57 Influenza A
Virus NA neuraminidase (A/Hong 469 aa Kong/213/03(H5N1)) AB212056
wild-type SEQ ID NO: 58 Influenza A Virus NA neuraminidase (A/Hong
1410 nts Kong/213/03(H5N1)) Codon modified SEQ ID NO: 59 Influenza
A Virus NA neuraminidase 1410 nts (A/swine/MI/PU243/04 (H3N1))
DQ150427 wild-type SEQ ID NO: 60 Influenza A Virus NA neuraminidase
469 aa (A/swine/MI/PU243/04 (H3N1)) DQ150427 wild-type SEQ ID NO:
61 Influenza A Virus NA neuraminidase 1410 nts (A/swine/MI/PU243/04
(H3N1)) Codon modified SEQ ID NO: 62 Hepatitis C Virus E1 (Serotype
1A, isolate H77) 576 nts AF009606 wild-type SEQ ID NO: 63 Hepatitis
C Virus E1 (Serotype 1A, isolate H77) NP 192 aa 751920 wild-type
SEQ ID NO: 64 Hepatitis C Virus E1 (Serotype 1A, isolate H77) Codon
576 nts modified SEQ ID NO: 65 Hepatitis C Virus E2 (Serotype 1A,
isolate H77) 1089 nts AF009606 wild-type SEQ ID NO: 66 Hepatitis C
Virus E2 (Serotype 1A, isolate H77) NP 363 aa 751921 wild-type SEQ
ID NO: 67 Hepatitis C Virus E2 (Serotype 1A, isolate H77) Codon
1089 nts modified SEQ ID NO: 68 Epstein Barr Virus (Type 1, gp350
B95-8) NC 007605 2724 nts wild-type SEQ ID NO: 69 Epstein Barr
Virus (Type 1, gp350 B95-8) CAD53417 907 aa wild-type SEQ ID NO: 70
Epstein Barr Virus (Type 1, gp350 B95-8) Codon 2724 nts modified
SEQ ID NO: 71 Epstein Barr Virus (Type 2, gp350 AG876) NC 009334
2661 nts wild-type SEQ ID NO: 72 Epstein Barr Virus (Type 2, gp350
AG876) YP 886 aa 001129462 wild-type SEQ ID NO: 73 Epstein Barr
Virus (Type 2, gp350 AG876) Codon 2661 nts Modified SEQ ID NO: 74
Herpes Simplex Virus 2 (Glycoprotein B strain HG52) 2715 nts NC
001798 wild-type SEQ ID NO: 75 Herpes Simplex Virus 2 (Glycoprotein
B strain HG52) 904 aa CAB06752 wild-type SEQ ID NO: 76 Herpes
Simplex Virus 2 (Glycoprotein B strain HG52) 2715 nts Codon
modified SEQ ID NO: 77 Herpes Simplex Virus (Glycoprotein D strain
HG52) 1182 nts NC 001798 wild-type SEQ ID NO: 78 Herpes Simplex
Virus (Glycoprotein D strain HG52) 393 aa NP 0044536 wild-type SEQ
ID NO: 79 Herpes Simplex Virus (Glycoprotein D strain HG52) 1182
nts Codon modified SEQ ID NO: 80 HPV-16 E7 wild-type 387 nts SEQ ID
NO: 81 HPV-16 E7 O1 387 nts SEQ ID NO: 82 HPV-16 E7 O2 387 nts SEQ
ID NO: 83 HPV-16 E7 O3 417 nts SEQ ID NO: 84 HPV-16 E7 W 387 nts
SEQ ID NO: 85 HSV-2 gD2 wild-type 1182 nts SEQ ID NO: 86 HSV-2 gD2
O1 1182 nts SEQ ID NO: 87 HSV-2 gD2 O2 1182 nts SEQ ID NO: 88 HSV-2
gD2 O3 1182 nts SEQ ID NO: 89 HSV-2 gD2 W 1182 nts SEQ ID NO: 90
Common forward primer 41 nts SEQ ID NO: 91 ODN-7909 24 nts
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0045] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0046] By "about" is meant a quantity, level, value, frequency,
percentage, dimension, size, or amount that varies by no more than
15%, and preferably by no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2%, 1% to a reference quantity, level, value, frequency,
percentage, dimension, size, or amount.
[0047] The terms "administration concurrently" or "administering
concurrently" or "co-administering" and the like refer to the
administration of a single composition containing two or more
actives, or the administration of each active as separate
compositions and/or delivered by separate routes either
contemporaneously or simultaneously or sequentially within a short
enough period of time that the effective result is equivalent to
that obtained when all such actives are administered as a single
composition. By "simultaneously" is meant that the active agents
are administered at substantially the same time, and desirably
together in the same formulation. By "contemporaneously" it is
meant that the active agents are administered closely in time,
e.g., one agent is administered within from about one minute to
within about one day before or after another. Any contemporaneous
time is useful. However, it will often be the case that when not
administered simultaneously, the agents will be administered within
about one minute to within about eight hours and preferably within
less than about one to about four hours. When administered
contemporaneously, the agents are suitably administered at the same
site on the subject. The term "same site" includes the exact
location, but can be within about 0.5 to about 15 centimeters,
preferably from within about 0.5 to about 5 centimeters. The term
"separately" as used herein means that the agents are administered
at an interval, for example at an interval of about a day to
several weeks or months. The active agents may be administered in
either order. The term "sequentially" as used herein means that the
agents are administered in sequence, for example at an interval or
intervals of minutes, hours, days or weeks. If appropriate the
active agents may be administered in a regular repeating cycle.
[0048] As used herein, the term "cis-acting sequence" or
"cis-regulatory region" or similar term shall be taken to mean any
sequence of nucleotides which is derived from an expressible
genetic sequence wherein the expression of the genetic sequence is
regulated, at least in part, by the sequence of nucleotides. Those
skilled in the art will be aware that a cis-regulatory region may
be capable of activating, silencing, enhancing, repressing or
otherwise altering the level of expression and/or
cell-type-specificity and/or developmental specificity of any
structural gene sequence.
[0049] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0050] As used herein, a "chimeric construct" refers to a
polynucleotide having heterologous nucleic acid elements. Chimeric
constructs include "expression cassettes" or "expression
constructs," which refer to an assembly that is capable of
directing the expression of the sequence(s) or gene(s) of interest.
An expression cassette generally includes control elements such as
a promoter that is operably linked to (so as to direct
transcription of) a synthetic polynucleotide of the invention, and
often includes a polyadenylation sequence as well. Within certain
embodiments of the invention, the chimeric construct may be
contained within a vector. In addition to the components of the
chimeric construct, the vector may include, one or more selectable
markers, a signal which allows the vector to exist as
single-stranded DNA (e.g., a M13 origin of replication), at least
one multiple cloning site, and a "mammalian" origin of replication
(e.g., a SV40 or adenovirus origin of replication).
[0051] As used herein, "conferred immune response," "immune
response that is conferred" and the like refer to a temporary or
permanent change in immune response to a target antigen, which
occurs or would occur after the introduction of a polynucleotide to
the mammal, and which would not occur in the absence of that
introduction. Typically, such a temporary or permanent change
occurs as a result of the transcription and/or translation of
genetic information contained within that polynucleotide in a cell,
or in at least one cell or cell type or class of cell within a
mammal or within a class of mammals, and can be used to distinguish
the mammal, or class of mammals to which the polynucleotide has
been provided from a similar mammal, or class of mammals, to which
the polynucleotide has not been provided.
[0052] By "corresponds to" or "corresponding to" is meant an
antigen which encodes an amino acid sequence that displays
substantial similarity to an amino acid sequence in a target
antigen. In general the antigen will display at least about 30, 40,
50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% similarity or identity to at least a portion of the target
antigen (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or 95% of the amino acid sequence of the target antigen).
[0053] By "effective amount," in the context of modulating an
immune response or treating or preventing a disease or condition,
is meant the administration of that amount of composition to an
individual in need thereof, either in a single dose or as part of a
series, that is effective for achieving that modulation, treatment
or prevention. The effective amount will vary depending upon the
health and physical condition of the individual to be treated, the
taxonomic group of individual to be treated, the formulation of the
composition, the assessment of the medical situation, and other
relevant factors. It is expected that the amount will fall in a
relatively broad range that can be determined through routine
trials.
[0054] The terms "enhancing an immune response," "producing a
stronger immune response" and the like refer to increasing an
animal's capacity to respond to a target antigen (e.g., a foreign
or disease-specific antigen or a self antigen), which can be
determined for example by detecting an increase in the number,
activity, and ability of the animal's cells that are primed to
attack such antigens or an increase in the titer or activity of
antibodies in the animal, which are immuno-interactive with the
target antigen. Strength of immune response can be measured by
standard immunoassays including: direct measurement of antibody
titers or peripheral blood lymphocytes; cytolytic T lymphocyte
assays; assays of natural killer cell cytotoxicity; cell
proliferation assays including lymphoproliferation (lymphocyte
activation) assays; immunoassays of immune cell subsets; assays of
T-lymphocytes specific for the antigen in a sensitized subject;
skin tests for cell-mediated immunity; etc. Such assays are well
known in the art. See, e.g., Erickson et al., 1993, J. Immunol.
151:4189-4199; Doe et al., 1994, Eur. J. Immunol. 24:2369-2376.
Recent methods of measuring cell-mediated immune response include
measurement of intracellular cytokines or cytokine secretion by
T-cell populations, or by measurement of epitope specific T-cells
(e.g., by the tetramer technique) (reviewed by McMichael, A. J.,
and O'Callaghan, C. A., 1998, J. Exp. Med. 187(9)1367-1371;
Mcheyzer-Williams, M. G., et al., 1996, Immunol. Rev. 150:5-21;
Lalvani, A., et al., 1997, J. Exp. Med. 186:859-865). Any
statistically significant increase in strength of immune response
as measured for example by immunoassay is considered an "enhanced
immune response" or "immunoenhancement" as used herein. Enhanced
immune response is also indicated by physical manifestations such
as fever and inflammation, as well as healing of systemic and local
infections, and reduction of symptoms in disease, i.e., decrease in
tumor size, alleviation of symptoms of a disease or condition
including, but not restricted to, leprosy, tuberculosis, malaria,
naphthous ulcers, herpetic and papillomatous warts, gingivitis,
arthrosclerosis, the concomitants of AIDS such as Kaposi's sarcoma,
bronchial infections, and the like. Such physical manifestations
also encompass "enhanced immune response" or "immunoenhancement" as
used herein. By contrast, "reducing an immune response," "producing
a weaker immune response" and the like refer to decreasing an
animal's capacity to respond to a target antigen, which can be
determined for example by conducting immunoassays or assessing
physical manifestations, as described for example above.
[0055] The terms "expression" or "gene expression" refer to
production of RNA message and/or translation of RNA message into
proteins or polypeptides.
[0056] By "expression vector" is meant any autonomous genetic
element capable of directing the synthesis of a protein encoded by
the vector. Such expression vectors are known by practitioners in
the art.
[0057] The term "gene" is used in its broadest context to include
both a genomic DNA region corresponding to the gene as well as a
cDNA sequence corresponding to exons or a recombinant molecule
engineered to encode a functional form of a product.
[0058] As used herein the term "heterologous" refers to a
combination of elements that are not naturally occurring or that
are obtained from different sources.
[0059] "Immune response" or "immunological response" refers to the
concerted action of lymphocytes, antigen-presenting cells,
phagocytic cells, granulocytes, and soluble macromolecules produced
by the above cells or the liver (including antibodies, cytokines,
and complement) that results in selective damage to, destruction of
or elimination from the body of cancerous cells, metastatic tumor
cells, metastatic breast cancer cells, invading pathogens, cells or
tissues infected with pathogens, or, in cases of autoimmunity or
pathological inflammation, normal human cells or tissues. In some
embodiments, an "immune response` encompasses the development in an
individual of a humoral and/or a cellular immune response to a
polypeptide that is encoded by an introduced synthetic
polynucleotide of the invention. As known in the art, the terms
"humoral immune response" includes and encompasses an immune
response mediated by antibody molecules, while a "cellular immune
response" includes and encompasses an immune response mediated by
T-lymphocytes and/or other white blood cells. Thus, an immune
response that is stimulated by a synthetic polynucleotide of the
invention may be one that stimulates the production of antibodies
(e.g., neutralizing antibodies that block bacterial toxins and
pathogens such as viruses entering cells and replicating by binding
to toxins and pathogens, typically protecting cells from infection
and destruction). The synthetic polynucleotide may also elicit
production of cytolytic T lymphocytes (CTLs). Hence, an
immunological response may include one or more of the following
effects: the production of antibodies by B-cells; and/or the
activation of suppressor T-cells and/or memory/effector T-cells
directed specifically to an antigen or antigens present in the
composition or vaccine of interest. In some embodiments, these
responses may serve to neutralize infectivity, and/or mediate
antibody-complement, or antibody dependent cell cytotoxicity (ADCC)
to provide protection to an immunized host. Such responses can be
determined using standard immunoassays and neutralization assays,
well known in the art. (See, e.g., Montefiori et al., 1988, J Clin
Microbiol. 26:231-235; Dreyer et al., 1999, AIDS Res Hum
Retroviruses 15(17):1563-1571). The innate immune system of mammals
also recognizes and responds to molecular features of pathogenic
organisms and cancer cells via activation of Toll-like receptors
and similar receptor molecules on immune cells. Upon activation of
the innate immune system, various non-adaptive immune response
cells are activated to, e.g., produce various cytokines,
lymphokines and chemokines. Cells activated by an innate immune
response include immature and mature dendritic cells of, for
example, the monocyte and plamsacytoid lineage (MDC, PDC), as well
as gamma, delta, alpha and beta T cells and B cells and the like.
Thus, the present invention also contemplates an immune response
wherein the immune response involves both an innate and adaptive
response.
[0060] A composition is "immunogenic" if it is capable of either:
a) generating an immune response against a target antigen (e.g., a
viral or tumor antigen) in an individual; or b) reconstituting,
boosting, or maintaining an immune response in an individual beyond
what would occur if the agent or composition was not administered.
An agent or composition is immunogenic if it is capable of
attaining either of these criteria when administered in single or
multiple doses.
[0061] "Immunomodulation," modulating an immune response" and the
like refer to the modulation of the immune system in response to a
stimulus and includes increasing or decreasing an immune response
to a target antigen or changing an immune response from one that is
predominantly a humoral immune response to one that is a more
cell-mediated immune response and vice versa. For example, it is
known in the art that decreasing the amount of antigen for
immunization can change the bias of the immune system from a
predominantly humoral immune response to a predominantly cellular
immune response.
[0062] By "isoaccepting transfer RNA" or "iso-tRNA" is meant one or
more transfer RNA molecules that differ in their anticodon
nucleotide sequence but are specific for the same amino acid.
[0063] As used herein, the term "mammal" refers to any mammal
including, without limitation, humans and other primates, including
non-human primates such as chimpanzees and other apes and monkey
species; farm animals such as cattle, sheep, pigs, goats and
horses; domestic mammals such as dogs and cats; and laboratory
animals including rodents such as mice, rats and guinea pigs. The
term does not denote a particular age. Thus, both adult and newborn
individuals are intended to be covered.
[0064] By "modulating," "modulate" and the like is meant increasing
or decreasing, either directly or indirectly, the quality of a
selected phenotype (e.g., an immune response). In certain
embodiments, "modulation" or "modulating" means that a
desired/selected immune response is more efficient (e.g., at least
10%, 20%, 30%, 40%, 50%, 60% or more), more rapid (e.g., at least
10%, 20%, 30%, 40%, 50%, 60% or more), greater in magnitude (e.g.,
at least 10%, 20%, 30%, 40%, 50%, 60% or more), and/or more easily
induced (e.g., at least 10%, 20%, 30%, 40%, 50%, 60% or more) than
if the parent polynucleotide had been used under the same
conditions as the synthetic polynucleotide. In other embodiments,
"modulation" or "modulating" means changing an immune response from
a predominantly antibody-mediated immune response as conferred by
the parent polynucleotide, to a predominantly cellular immune
response as conferred by the synthetic polynucleotide under the
same conditions. In still other embodiments, "modulation" or
"modulating" means changing an immune response from a predominantly
cellular immune response as conferred by the parent polynucleotide,
to a predominantly antibody-mediated immune response as conferred
by the synthetic polynucleotide under the same conditions.
[0065] By "natural gene" is meant a gene that naturally encodes the
protein. However, it is possible that the parent polynucleotide
encodes a protein that is not naturally-occurring but has been
engineered using recombinant techniques.
[0066] The term "5' non-coding region" is used herein in its
broadest context to include all nucleotide sequences which are
derived from the upstream region of an expressible gene, other than
those sequences which encode amino acid residues which comprise the
polypeptide product of the gene, wherein 5' non-coding region
confers or activates or otherwise facilitates, at least in part,
expression of the gene.
[0067] The term "oligonucleotide" as used herein refers to a
polymer composed of a multiplicity of nucleotide units
(deoxyribonucleotides or ribonucleotides, or related structural
variants or synthetic analogues thereof) linked via phosphodiester
bonds (or related structural variants or synthetic analogues
thereof). Thus, while the term "oligonucleotide" typically refers
to a nucleotide polymer in which the nucleotides and linkages
between them are naturally occurring, it will be understood that
the term also includes within its scope various analogues
including, but not restricted to, peptide nucleic acids (PNAs),
phosphoramidates, phosphorothioates, methyl phosphonates,
2-O-methyl ribonucleic acids, and the like. The exact size of the
molecule may vary depending on the particular application. An
oligonucleotide is typically rather short in length, generally from
about 10 to 30 nucleotides, but the term can refer to molecules of
any length, although the term "polynucleotide" or "nucleic acid" is
typically used for large oligonucleotides.
[0068] The terms "operably connected," "operably linked" and the
like as used herein refer to an arrangement of elements wherein the
components so described are configured so as to perform their usual
function. Thus, a given promoter operably linked to a coding
sequence is capable of effecting the expression of the coding
sequence when the proper enzymes are present. The promoter need not
be contiguous with the coding sequence, so long as it functions to
direct the expression thereof. Thus, for example, intervening
untranslated yet transcribed sequences can be present between the
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding sequence.
Terms such as "operably connected," therefore, include placing a
structural gene under the regulatory control of a promoter, which
then controls the transcription and optionally translation of the
gene. In the construction of heterologous promoter/structural gene
combinations, it is generally preferred to position the genetic
sequence or promoter at a distance from the gene transcription
start site that is approximately the same as the distance between
that genetic sequence or promoter and the gene it controls in its
natural setting; L e. the gene from which the genetic sequence or
promoter is derived. As is known in the art, some variation in this
distance can be accommodated without loss of function. Similarly,
the preferred positioning of a regulatory sequence element with
respect to a heterologous gene to be placed under its control is
defined by the positioning of the element in its natural setting;
i.e., the genes from which it is derived.
[0069] By "pharmaceutically-acceptable carrier" is meant a solid or
liquid filler, diluent or encapsulating substance that may be
safely used in topical or systemic administration.
[0070] The term "phenotype" means any one or more detectable
physical or functional characteristics, properties, attributes or
traits of an organism, tissue, or cell, or class of organisms,
tissues or cells, which generally result from the interaction
between the genetic makeup (i.e., genotype) of the organism,
tissue, or cell, or the class of organisms, tissues or cells and
the environment.
[0071] By "phenotypic preference" is meant the preference with
which an organism uses a codon to produce a selected phenotype.
This preference can be evidenced, for example, by the quality of a
selected phenotype that is producible by a polynucleotide that
comprises the codon in an open reading frame which codes for a
polypeptide that produces the selected phenotype. In certain
embodiment, the preference of usage is independent of the route by
which the polynucleotide is introduced into the organism. However,
in other embodiments, the preference of usage is dependent on the
route of introduction of the polynucleotide into the organism.
[0072] The term "polynucleotide" or "nucleic acid" as used herein
designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers
to oligonucleotides greater than 30 nucleotides in length.
[0073] "Polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
and to variants and synthetic analogues of the same. Thus, these
terms apply to amino acid polymers in which one or more amino acid
residues is a synthetic non-naturally occurring amino acid, such as
a chemical analogue of a corresponding naturally occurring amino
acid, as well as to naturally-occurring amino acid polymers. As
used herein, the terms "polypeptide," "peptide" and "protein" are
not limited to a minimum length of the product. Thus, peptides,
oligopeptides, dimers, multimers, and the like, are included within
the definition. Both full-length proteins and fragments thereof are
encompassed by the definition. The terms also include post
expression modifications of a polypeptide, for example,
glycosylation, acetylation, phosphorylation and the like. In some
embodiments, a "polypeptide" refers to a protein which includes
modifications, such as deletions, additions and substitutions
(generally conservative in nature), to the native sequence, so long
as the protein maintains the desired activity. These modifications
may be deliberate, as through site-directed mutagenesis, or may be
accidental, such as through mutations of hosts which produce the
proteins or errors due to PCR amplification.
[0074] The terms "polypeptide variant," and "variant" refer to
polypeptides that vary from a reference polypeptide by the
addition, deletion or substitution (generally conservative in
nature) of at least one amino acid residue. Typically, variants
retain a desired activity of the reference polypeptide, such as
antigenic activity in inducing an immune response against a target
antigen. In general, variant polypeptides are "substantially
similar" or substantially identical" to the reference polypeptide,
e.g., amino acid sequence identity or similarity of more than 50%,
generally more than 60%-70%, even more particularly 80%-85% or
more, such as at least 90%-95% or more, when the two sequences are
aligned. Often, the variants will include the same number of amino
acids but will include substitutions, as explained herein.
[0075] By "primer" is meant an oligonucleotide which, when paired
with a strand of DNA, is capable of initiating the synthesis of a
primer extension product in the presence of a suitable polymerizing
agent. The primer is preferably single-stranded for maximum
efficiency in amplification but may alternatively be
double-stranded. A primer must be sufficiently long to prime the
synthesis of extension products in the presence of the
polymerization agent. The length of the primer depends on many
factors, including application, temperature to be employed,
template reaction conditions, other reagents, and source of
primers. For example, depending on the complexity of the target
sequence, the oligonucleotide primer typically contains 15 to 35 or
more nucleotides, although it may contain fewer nucleotides.
Primers can be large polynucleotides, such as from about 200
nucleotides to several kilobases or more. Primers may be selected
to be "substantially complementary" to the sequence on the template
to which it is designed to hybridize and serve as a site for the
initiation of synthesis. By "substantially complementary", it is
meant that the primer is sufficiently complementary to hybridize
with a target nucleotide sequence. Preferably, the primer contains
no mismatches with the template to which it is designed to
hybridize but this is not essential. For example, non-complementary
nucleotides may be attached to the 5' end of the primer, with the
remainder of the primer sequence being complementary to the
template. Alternatively, non-complementary nucleotides or a stretch
of non-complementary nucleotides can be interspersed into a primer,
provided that the primer sequence has sufficient complementarity
with the sequence of the template to hybridize therewith and
thereby form a template for synthesis of the extension product of
the primer.
[0076] Reference herein to a "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a classical genomic gene, including the TATA box which
is required for accurate transcription initiation, with or without
a CCAAT box sequence and additional regulatory elements (i.e.
upstream activating sequences, enhancers and silencers) which alter
gene expression in response to developmental and/or environmental
stimuli, or in a tissue-specific or cell-type-specific manner. A
promoter is usually, but not necessarily, positioned upstream or
5', of a structural gene, the expression of which it regulates.
Furthermore, the regulatory elements comprising a promoter are
usually positioned within 2 kb of the start site of transcription
of the gene. Preferred promoters according to the invention may
contain additional copies of one or more specific regulatory
elements to further enhance expression in a cell, and/or to alter
the timing of expression of a structural gene to which it is
operably connected.
[0077] The term "quality" is used herein in its broadest sense and
includes a measure, strength, intensity, degree or grade of a
phenotype, e.g., a superior or inferior immune response.
[0078] The term "sequence identity" as used herein refers to the
extent that sequences are identical on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity" is calculated
by comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, I) or the identical
amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile,
Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met)
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. For the purposes of the present invention, "sequence
identity" will be understood to mean the "match percentage"
calculated by the DNASIS computer program (Version 2.5 for windows;
available from Hitachi Software engineering Co., Ltd., South San
Francisco, Calif., USA) using standard defaults as used in the
reference manual accompanying the software.
[0079] "Similarity" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions as
defined in Table 10. Similarity may be determined using sequence
comparison programs such as GAP (Deveraux et al. 1984, Nucleic
Acids Research 12, 387-395). In this way, sequences of a similar or
substantially different length to those cited herein might be
compared by insertion of gaps into the alignment, such gaps being
determined, for example, by the comparison algorithm used by
GAP.
[0080] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence",
"comparison window", "sequence identity", "percentage of sequence
identity" and "substantial identity". A "reference sequence" is at
least 12 but frequently 15 to 18 and often at least 25 monomer
units, inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.,
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
at least 6 contiguous positions, usually about 50 to about 100,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerized implementations of
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Drive Madison, Wis., USA) or by inspection and the best
alignment (i.e., resulting in the highest percentage homology over
the comparison window) generated by any of the various methods
selected. Reference also may be made to the BLAST family of
programs as for example disclosed by Altschul et al., 1997, Nucl.
Acids Res. 25:3389. A detailed discussion of sequence analysis can
be found in Unit 19.3 of Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter
15.
[0081] The term "synthetic polynucleotide" as used herein refers to
a polynucleotide that is formed by recombinant or synthetic
techniques and typically includes polynucleotides that are not
normally found in nature.
[0082] The term "synonymous codon" as used herein refers to a codon
having a different nucleotide sequence than another codon but
encoding the same amino acid as that other codon.
[0083] By "treatment," "treat," "treated" and the like is meant to
include both therapeutic and prophylactic treatment.
[0084] By "vector" is meant a nucleic acid molecule, preferably a
DNA molecule derived, for example, from a plasmid, bacteriophage,
or plant virus, into which a nucleic acid sequence may be inserted
or cloned. A vector preferably contains one or more unique
restriction sites and may be capable of autonomous replication in a
defined host cell including a target cell or tissue or a progenitor
cell or tissue thereof, or be integrable with the genome of the
defined host such that the cloned sequence is reproducible.
Accordingly, the vector may be an autonomously replicating vector,
i.e., a vector that exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a linear or closed circular plasmid, an extrachromosomal
element, a minichromosome, or an artificial chromosome. The vector
may contain any means for assuring self-replication. Alternatively,
the vector may be one which, when introduced into the host cell, is
integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. A vector system
may comprise a single vector or plasmid, two or more vectors or
plasmids, which together contain the total DNA to be introduced
into the genome of the host cell, or a transposon. The choice of
the vector will typically depend on the compatibility of the vector
with the host cell into which the vector is to be introduced. The
vector may also include a selection marker such as an antibiotic
resistance gene that can be used for selection of suitable
transformants. Examples of such resistance genes are well known to
those of skill in the art.
2. Abbreviations
[0085] The following abbreviations are used throughout the
application: [0086] nt=nucleotide [0087] nts=nucleotides [0088]
aa=amino acid(s) [0089] kb=kilobase(s) or kilobase pair(s) [0090]
kDa=kilodalton(s) [0091] d=day [0092] h=hour [0093] s=seconds
3. Immune Response Preference Ranking of Codons in Mammals
[0094] The present invention provides for the first time an immune
response preference ranking of individual synonymous codons in
mammals. This ranking was determined using a construct system that
comprises a series of reporter constructs each comprising a
different coding sequence for an antigenic polypeptide (e.g., a
papillomavirus E7 polypeptide), wherein the coding sequence of
individual constructs is distinguished from a parent coding
sequence that encodes the antigenic polypeptide by the substitution
of a single species of iso-accepting codon for each other species
of iso-accepting codon that is present in the parent coding
sequence. Accordingly, the coding sequence of individual synthetic
constructs uses the same iso-accepting codon to encode most
instances and preferably every instance of a particular amino acid
residue (e.g., Ala.sup.or for all alanines) in the antigenic
polypeptide and individual synthetic constructs differ in the
species of iso-accepting codon used to encode a particular amino
acid residue across the polypeptide sequence. As used herein, the
species of iso-accepting codon that is used to encode a particular
amino acid residue in the antigenic polypeptide is referred to as a
"standardized codon". An illustrative synthetic construct system is
described in Example 1, which covers the entire set of synonymous
codons that code for amino acids.
[0095] Test mammals (e.g., mice) were immunized with the synthetic
construct system in which individual mammals were immunized with a
different synthetic construct and the host immune response (e.g., a
humoral immune response or a cellular immune response) to the
antigenic polypeptide was determined for each construct. In
accordance with the present invention, the strength of immune
response obtained from individual synthetic constructs provides a
direct correlation to the immune preference of a corresponding
standardized codon in a test mammal. Accordingly, the stronger the
immune response produced from a given construct in a test mammal,
the higher the immune preference will be of the corresponding
standardized codon.
[0096] Comparison of the immune response preferences so determined
with the translational efficiencies derived from codon usage
frequency values for mammalian cells in general as determined by
Seed (see U.S. Pat. Nos. 5,786,464 and 5,795,737) reveals several
differences in the ranking of codons. For convenience, these
differences are highlighted in TABLE 9, in which Seed `preferred`
codons are highlighted with a blue background, Seed `less
preferred` codons are highlighted with a green background, and Seed
`non preferred` codons are highlighted with a grey background.
TABLE-US-00009 TABLE 9 Preferential codon usage as predicted
Experimentally determined codon by Seed for mammalian immune
response preferences in aa cells in general test mammals Ala GCC
>> (GCG, GCT, GCA) GCT > GCC > (GCA GCG) Arg CGC
>> (CGA, CGT, AGA, (CGA, CGC, CGT, AGA) > AGG, CGG) (AGG,
CGG) Asn AAC >> AAT AAC > AAT Asp GAC >> GAT GAC
> GAT Cys TGC >> TGT TGC > TGT Glu (GAA, GAG) GAA >
GAG Gln CAG >> CAA CAA = CAG Gly GGC > GGG > (GGT, GGA)
GGA > (GGG, GGT, GGC) His CAC >> CAT CAC = CAT Ile ATC
> ATT > ATA ATC >> ATT > ATA Leu CTG > CTC >
(TTA, CTA, (CTG, CTC) > (CTA, CTT) >> CTT, TTG) TTG >
TTA Lys AAG >> AAA AAG = AAA Phe TTC >> TTT TTT >
TTC Pro CCC >> (CCG, CCA, CCT) CCC > CCT >> (CCA,
CCG) Ser AGC > TCC > (TCG, AGT, TCG >> (TCT, TCA, TCC)
>> TCA, TCT) (AGC, AGT) Thr ACC >> (ACG, ACA, ACT) ACG
> ACC >> ACA > ACT Tyr TAC >> TAT TAC > TAT
Val GTG > GTC > (GTA, GTT) (GTG, GTC) > GTT > GTA
[0097] As will be apparent from the above table:
[0098] (i) several codons deemed by Seed to have a higher codon
usage ranking in mammalian cells than at least one other synonymous
codon have in fact a lower immune response preference ranking than
the or each other synonymous codon (e.g., Ala.sup.GCC has a higher
codon usage ranking but lower immune response preference ranking
than Ala.sup.GCT; Gly.sup.GGC has a higher codon usage ranking but
lower immune response preference ranking than Gly.sup.GGA;
Phe.sup.TTC has a higher codon usage ranking but lower immune
response preference ranking than Phe.sup.TTT; Ser.sup.AGC has a
higher codon usage ranking but lower immune response preference
ranking than any one of Ser.sup.TCG, Ser.sup.TCT, Ser.sup.TCG,
Ser.sup.TCA and Ser.sup.TCC; and Thr.sup.ACC has a higher codon
usage ranking but lower immune response preference ranking than
Thr.sup.ACG);
[0099] (ii) several codons deemed by Seed to have a lower codon
usage ranking in mammalian cells than at least one other synonymous
codon have in fact a higher immune response preference ranking than
the or each other synonymous codon (e.g., Ala.sup.GCT has a lower
codon usage ranking but higher immune response preference ranking
than Ala.sup.GCC; Gly.sup.GGA has a lower codon usage ranking but
higher immune response preference ranking than Gly.sup.GGC or
Gly.sup.GGG; Phe.sup.TTT has a lower codon usage ranking but higher
immune response preference ranking than Phe.sup.TTC; Ser.sup.TCG
has a lower codon usage ranking but higher immune response
preference ranking than Ser.sup.AGC or Ser.sup.TCC; Ser.sup.TCT and
Ser.sup.TCA have a lower codon usage ranking but higher immune
response preference ranking than Ser.sup.AGC; and Thr.sup.ACG has a
lower codon usage ranking but higher immune response preference
ranking than Thr.sup.ACC);
[0100] (iii) several codons deemed by Seed to have a higher codon
usage ranking in mammalian cells than another synonymous codon have
in fact the same immune response preference ranking as the other
synonymous codon (e.g., Gln.sup.CAG has a higher codon usage
ranking than, but the same immune response preference ranking as,
Gln.sup.CAA; His.sup.CAC has a higher codon usage ranking than, but
the same immune response preference ranking as, His.sup.CAT;
Leu.sup.CTG has a higher codon usage ranking than, but the same
immune response preference ranking as Leu.sup.CTC; Lys.sup.AAG has
a higher codon usage ranking than, but the same immune response
preference ranking as, Lys.sup.AAA; Val.sup.GTG has a higher codon
usage ranking than, but the same immune response preference ranking
as, Val.sup.GTC); and
[0101] (iv) several codons deemed by Seed to have the same codon
usage ranking in mammalian cells as at least one other synonymous
codon have in fact a different immune response preference ranking
than the or each other synonymous codon (e.g., Ala.sup.GCT has the
same codon usage ranking as, but a higher immune response
preference ranking than, Ala.sup.GCA and Ala.sup.GCG; Arg.sup.CGA,
Arg.sup.CGT and Arg.sup.AGA have the same codon usage ranking as,
but a higher immune response preference ranking than, Arg.sup.AGG
and Arg.sup.CGG; Glu.sup.GAA has the same codon usage ranking as,
but a higher immune response preference ranking than, Glu.sup.GAG;
Gly.sup.GGA ha the same codon usage ranking as, but a higher immune
response preference ranking than, Gly.sup.GGT; Leu.sup.CTA and
Leu.sup.CTT have the same codon usage ranking as, but a higher
immune response preference ranking than, Leu.sup.TTG and
Leu.sup.TTA; Pro.sup.CCT has the same codon usage ranking as, but a
higher immune response preference ranking than, Pro.sup.CCA or
Pro.sup.CCG; Ser.sup.TCG has the same codon usage ranking as, but a
higher immune response preference ranking than, any one of
Ser.sup.TCT, Ser.sup.TCA and Ser.sup.AGT; Ser.sup.TCT and
Ser.sup.TCA have the same codon usage ranking as, but a higher
immune response preference ranking than, Ser.sup.AGT; Thr.sup.ACG
has the same codon usage ranking as, but a higher immune response
preference ranking than, any one of Thr.sup.ACA and Thr.sup.ACT;
Thr.sup.ACG has the same codon usage ranking as, but a higher
immune response preference ranking than, Thr.sup.ACT; Val.sup.GTT
has the same codon usage ranking as, but a higher immune response
preference ranking than, Val.sup.GTA).
[0102] Accordingly, the present invention enables for the first
time the modulation of an immune response to a target antigen in a
mammal from a polynucleotide that encodes a polypeptide that
corresponds to at least a portion of the target antigen by
replacing at least one codon of the polynucleotide with a
synonymous codon that has a higher or lower preference for
producing an immune response than the codon it replaces. In some
embodiments, therefore, the present invention embraces methods of
constructing a synthetic polynucleotide from which a polypeptide is
producible to confer an enhanced or stronger immune response than
one conferred by a parent polynucleotide that encodes the same
polypeptide. These methods generally comprise selecting from TABLE
1 a codon (often referred to herein arbitrarily as a "first codon")
of the parent polynucleotide for replacement with a synonymous
codon, wherein the synonymous codon is selected on the basis that
it exhibits a higher immune response preference than the first
codon and replacing the first codon with the synonymous codon to
construct the synthetic polynucleotide. Illustrative selections of
the first and synonymous codons are made according to TABLE 2.
[0103] In some embodiments, the selection of the first and
synonymous codons is made according to TABLE 3, which is the same
as TABLE 2 with the exception that it excludes selections based on
codon usage rankings as disclosed by Seed. In illustrative examples
of this type, the selection of a second codon (and subsequent
codons if desired) for replacement with a synonymous codon is made
according to TABLE 4.
[0104] Where synonymous codons are classified into three ranks
(`high`, `intermediate` and `low` ranks) based on their immune
response preference ranking (e.g., the synonymous codons for Ala,
Ile, Leu, Pro, Ser, Thr and Val), it is preferred that the
synonymous codon that is selected is a high rank codon when the
first codon is a low rank codon. However, this is not essential and
the synonymous codon can be selected from intermediate rank codons.
In the case of two or more synonymous codons having similar immune
response preferences, it will be appreciated that any one of these
codons can be used to replace the first codon.
[0105] In other embodiments, the invention provides methods of
constructing a synthetic polynucleotide from which a polypeptide is
producible to confer a reduced or weaker immune response than one
conferred by a parent polynucleotide that encodes the same
polypeptide. These methods generally comprise selecting from TABLE
1 a first codon of the parent polynucleotide for replacement with a
synonymous codon, wherein the synonymous codon is selected on the
basis that it exhibits a lower immune response preference than the
first codon and replacing the first codon with the synonymous codon
to construct the synthetic polynucleotide. Illustrative selections
of the first and synonymous codons are made according to TABLE
5.
[0106] In some embodiments, the selection of the first and
synonymous codons is made according to TABLE 6, which is the same
as TABLE 5 with the exception that it excludes selections based on
codon usage rankings as disclosed by Seed. In illustrative examples
of this type, the selection of a second codon (and subsequent
codons if desired) for replacement with a synonymous codon is made
according to TABLE 7.
[0107] Where synonymous codons are classified into the three ranks
noted above, it is preferred that the synonymous codon that is
selected is a low rank codon when the first codon is a high rank
codon but this is not essential and thus the synonymous codon can
be selected from intermediate rank codons if desired.
[0108] Generally, the difference in strength of the immune response
produced in the mammal from the synthetic polynucleotide as
compared to that produced from the parent polynucleotide depends on
the number of first/second codons that are replaced by synonymous
codons, and on the difference in immune response preference ranking
between the first/second codons and the synonymous codons. Put
another way, the fewer such replacements, and/or the smaller the
difference in immune response preference ranking between the
synonymous and first/codons codons, the smaller the difference will
be in the immune response produced by the synthetic polynucleotide
and the one produced by the parent polynucleotide. Conversely, the
more such replacements, and/or the greater the difference in immune
response preference ranking between the synonymous and first/second
codons, the greater the difference will be in the immune response
produced by the synthetic polynucleotide and the one produced by
the parent polynucleotide.
[0109] It is preferable but not necessary to replace all the codons
of the parent polynucleotide with synonymous codons having
different (e.g., higher or lower) immune response preference
rankings than the first/second codons. Changes in the conferred
immune response can be accomplished even with partial replacement.
Generally, the replacement step affects at least about 5%, 10%,
15%, 20%, 25%, 30%, usually at least about 35%, 40%, 50%, and
typically at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99% or more of the first/second codons of the parent
polynucleotide. In embodiments in which a stronger or enhanced
immune response is required, it is generally desirable to replace
some, preferably most and more preferably all, low rank codons in a
parent polynucleotide with synonymous codons that are intermediate,
or preferably high rank codons. Typically, replacement of low with
intermediate or high rank codons will result in an increase in the
strength of immune response from the synthetic polynucleotide so
constructed, as compared to the one produced from the parent
polynucleotide under the same conditions. However, it is often
desirable to replace some, preferably most and more preferably all,
intermediate rank codons in the parent polynucleotide with high
rank codons, if stronger or more enhanced immune responses are
desired.
[0110] By contrast, in some embodiments in which a weaker or
reduced immune response is required, it is generally desirable to
replace some, preferably most and more preferably all, high rank
codons in a parent polynucleotide with synonymous codons that are
intermediate, or preferably low rank codons. Typically, replacement
of high with intermediate or low rank codons will result in a
substantial decrease in the strength of immune response from the
synthetic polynucleotide so constructed, as compared to the one
produced from the parent polynucleotide under the same condition.
In specific embodiments in which it is desired to confer a weaker
or more reduced immune response, it is generally desirable to
replace some, preferably most and more preferably all, intermediate
rank codons in the parent polynucleotide with low rank codons.
[0111] In illustrative examples requiring a stronger or enhanced
immune response, the number of; and difference in immune response
preference ranking between, the first/second codons and the
synonymous codons are selected such that the immune response
conferred by the synthetic polynucleotide is at least about 110%,
150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%,
900%, 1000%, or more, of the immune response conferred by the
parent polynucleotide under the same conditions. Conversely, in
some embodiments requiring a lower or weaker immune response, the
number of, and difference in phenotypic preference ranking between,
the first/second codons and the synonymous codons are selected such
that the immune response conferred by the synthetic polynucleotide
is no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%,
5%, or less of the immune response conferred by the parent
polynucleotide under the same conditions.
4. Modulating Immune Responses in Mammals by Expression of
Isoaccepting Transfer RNA-Encoding Polynucleotides
[0112] It is possible to take advantage of the immune response
preference rankings of codons discussed in Section 3 to modulate an
immune response to a target antigen by changing the level of
iso-tRNAs in the cell population which is the target of the
immunization. Accordingly, the invention also features methods of
enhancing the quality of an immune response to a target antigen in
a mammal, wherein the response is conferred by the expression of a
first polynucleotide that encodes a polypeptide corresponding to at
least a portion of the target antigen. These methods generally
comprise: introducing into the mammal a first nucleic acid
construct comprising the first polynucleotide in operable
connection with a regulatory polynucleotide. A second nucleic acid
construct is then introduced into the mammal, which comprises a
second polynucleotide that is operably connected to a regulatory
polynucleotide and that encodes an iso-tRNA corresponding to a low
immune preference codon of the first polynucleotide.
[0113] In practice, therefore, an iso-tRNA is introduced into the
mammal by the second nucleic acid construct when the iso-tRNA
corresponds to a low immune response preference codon in the first
polynucleotide, which are suitably selected from the group
consisting of Ala.sup.GCA, Ala.sup.GCG, Ala.sup.GCC, Arg.sup.AGG,
Arg.sup.CGG, Asn.sup.AAT, Asp.sup.GAT, Cys.sup.TGT, Glu.sup.GAG,
Gly.sup.GGG, Gly.sup.GGT, Gly.sup.GGC, Ile.sup.ATA, Ile.sup.ATT,
Leu.sup.TTG, Leu.sup.TTA, Leu.sup.CTA, Leu.sup.CTT, Phe.sup.TTC,
Pro.sup.CCA, Pro.sup.CCG, Pro.sup.CCT, Ser.sup.AGC, Ser.sup.AGT,
Ser.sup.TCT, Ser.sup.TCA, Ser.sup.TCC, Thr.sup.ACA, Thr.sup.ACT,
Tyr.sup.TAT, Val.sup.GTA and Val.sup.GTT. In specific embodiments,
the supplied iso-tRNAs are specific for codons that have `low`
immune response preference codons, which may be selected from the
group consisting of Ala.sup.GCA, Ala.sup.GCG, Arg.sup.AGG,
Arg.sup.CGG, Asn.sup.AAT, Asp.sup.GAT, Cys.sup.TGT, Glu.sup.GAG,
Gly.sup.GGG, Gly.sup.GGT, Gly.sup.GGC, Ile.sup.ATA, Leu.sup.TTG,
Leu.sup.TTA, Phe.sup.TTC, Pro.sup.CCA, Pro.sup.CCG, Ser.sup.AGC,
Ser.sup.AGT, Thr.sup.ACT, Tyr.sup.TAT and Val.sup.GTA. The first
construct (i.e., antigen-expressing construct) and the second
construct (i.e., the iso-tRNA-expressing construct) may be
introduced simultaneously or sequentially (in either order) and may
be introduced at the same or different sites. In some embodiments,
the first and second constructs are contained in separate vectors.
In other embodiments, they are contained in a single vector. If
desired, two or more second constructs may be introduced each
expressing a different iso-tRNA corresponding to a low preference
codon of the first polynucleotide. The first and second nucleic
acid constructs may be constructed and administered concurrently or
contemporaneously to a mammal according to any suitable method,
illustrative examples of which are discussed below for the chimeric
constructs of the invention.
[0114] In some embodiments, a plurality of different
iso-tRNA-expressing constructs (e.g., 2, 3, 4, 5, 6, 7, 8 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) are administered
concurrently or contemporaneously with the antigen-expressing
construct, wherein individual iso-tRNA-expressing constructs
express a different iso-tRNA than other iso-tRNA-expressing
constructs.
5. Antigens
[0115] Target antigens useful in the present invention are
typically proteinaceous molecules, representative examples of which
include polypeptides and peptides. Target antigens may be selected
from endogenous antigens produced by a host or exogenous antigens
that are foreign to the host. Suitable endogenous antigens include,
but are not restricted to, cancer or tumor antigens. Non-limiting
examples of cancer or tumor antigens include antigens from a cancer
or tumor selected from ABL1 proto-oncogene, AIDS related cancers,
acoustic neuroma, acute lymphocytic leukemia, acute myeloid
leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic
myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal
cancer, angiosarcoma, aplastic anemia, astrocytoma,
ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer,
bone cancers, bowel cancer, brain stem glioma, brain and CNS
tumors, breast cancer, CNS tumors, carcinoid tumors, cervical
cancer, childhood brain tumors, childhood cancer, childhood
leukemia, childhood soft tissue sarcoma, chondrosarcoma,
choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid
leukemia, colorectal cancers, cutaneous T-cell lymphoma,
dermatofibrosarcoma protuberans, desmoplastic small round cell
tumor, ductal carcinoma, endocrine cancers, endometrial cancer,
ependymoma, oesophageal cancer, Ewing's Sarcoma, Extra-Hepatic Bile
Duct Cancer, Eye Cancer, Eye: Melanoma, Retinoblastoma, Fallopian
Tube cancer, Fanconi anemia, fibrosarcoma, gall bladder cancer,
gastric cancer, gastrointestinal cancers,
gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell
tumors, gestational-trophoblastic-disease, glioma, gynecological
cancers, haematological malignancies, hairy cell leukemia, head and
neck cancer, hepatocellular cancer, hereditary breast cancer,
histiocytosis, Hodgkin's disease, human papillomavirus,
hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular
melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer,
Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma,
leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver
cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma,
non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid
tumor of kidney, medulloblastoma, melanoma, Merkel cell cancer,
mesothelioma, metastatic cancer, mouth cancer, multiple endocrine
neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma,
myeloproliferative disorders, nasal cancer, nasopharyngeal cancer,
nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage
syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer
(NSCLC), ocular cancers, esophageal cancer, oral cavity cancer,
oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas
cancer, paranasal cancer, parathyroid cancer, parotid gland cancer,
penile cancer, peripheral-neuroectodermal tumours, pituitary
cancer, polycythemia vera, prostate cancer, rare cancers and
associated disorders, renal cell carcinoma, retinoblastoma,
rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer,
sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung
cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal
cord tumors, squamous-cell-carcinoma-(skin), stomach cancer,
synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer,
transitional-cell-cancer-(bladder),
transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic
cancer, urethral cancer, urinary system cancer, uroplakins, uterine
sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstroms
macroglobulinemia, Wilms' tumor. In certain embodiments, the cancer
or tumor relates to melanoma. Illustrative examples of
melanoma-related antigens include melanocyte differentiation
antigen (e.g., gp100, MART, Melan-A/MART-1, TRP-1, Tyros, TRP2,
MC1R, MUC1F, MUC1R or a combination thereof) and melanoma-specific
antigens (e.g., BAGE, GAGE-1, gp100In4, MAGE-1 (e.g., GenBank
Accession No. X54156 and AA494311), MAGE-3, MAGE4, PRAME, TRP2IN2,
NYNSO1a, NYNSO1b, LAGE1, p97 melanoma antigen (e.g., GenBank
Accession No. M12154) p5 protein, gp75, oncofetal antigen, GM2 and
GD2 gangliosides, cdc27, p21ras, gp100.sup.Pmel117 or a combination
thereof. Other tumour-specific antigens include, but are not
limited to: etv6, aml1, cyclophilin b (acute lymphoblastic
leukemia); Ig-idiotype (B cell lymphoma); E-cadherin,
.alpha.-catenin, .beta.-catenin, .gamma.-catenin, p120ctn (glioma);
p21ras (bladder cancer); p21ras (biliary cancer); MUC family,
HER2/neu, c-erbB-2 (breast cancer); p53, p21ras (cervical
carcinoma); p21ras, HER2/neu, c-erbB-2, MUC family,
Cripto-1protein, Pim-1 protein (colon carcinoma); Colorectal
associated antigen (CRC)-CO17-1A/GA733, APC (colorectal cancer);
carcinoembryonic antigen (CEA) (colorectal cancer,
choriocarcinoma); cyclophilin b (epithelial cell cancer); HER2/neu,
c-erbB-2, ga733 glycoprotein (gastric cancer); .alpha.-fetoprotein
(hepatocellular cancer); Imp-1, EBNA-1 (Hodgkin's lymphoma); CEA,
MAGE-3, NY-ESO-1 (lung cancer); cyclophilin b (lymphoid
cell-derived leukemia); MUC family, p21ras (myeloma); HER2/neu,
c-erbB-2 (non-small cell lung carcinoma); Imp-1, EBNA-1
(nasopharyngeal cancer); MUC family, HER2/neu, c-erbB-2, MAGE-A4,
NY-ESO-1 (ovarian cancer); Prostate Specific Antigen (PSA) and its
antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu,
c-erbB-2, ga733 glycoprotein (prostate cancer); HER2/neu, c-erbB-2
(renal cancer); viral products such as human papillomavirus
proteins (squamous cell cancers of the cervix and esophagus);
NY-ESO-1 (testicular cancer); and HTLV-1 epitopes (T cell
leukemia).
[0116] Foreign or exogenous antigens are suitably selected from
antigens of pathogenic organisms. Exemplary pathogenic organisms
include, but are not limited to, viruses, bacteria, fungi
parasites, algae and protozoa and amoebae. Illustrative viruses
include viruses responsible for diseases including, but not limited
to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g.,
GenBank Accession No. E02707), and C (e.g., GenBank Accession No.
E06890), as well as other hepatitis viruses, influenza, adenovirus
(e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678),
yellow fever, Epstein-Barr virus and other herpesviruses such as
papillomavirus, Ebola virus, influenza virus, Japanese encephalitis
(e.g., GenBank Accession No. E07883), dengue (e.g., GenBank
Accession No. M24444), hantavirus, Sendai virus, respiratory
syncytial virus, orthomyxoviruses, vesicular stomatitis virus,
visna virus, cytomegalovirus and human immunodeficiency virus (HIV)
(e.g., GenBank Accession No. U18552). Any suitable antigen derived
from such viruses are useful in the practice of the present
invention. For example, illustrative retroviral antigens derived
from HIV include, but are not limited to, antigens such as gene
products of the gag, pol, and env genes, the Nef protein, reverse
transcriptase, and other HIV components. Illustrative examples of
hepatitis viral antigens include, but are not limited to, antigens
such as the S, M, and L proteins of hepatitis B virus, the pre-S
antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis
A, B, and C, viral components such as hepatitis C viral RNA.
Illustrative examples of influenza viral antigens include; but are
not limited to, antigens such as hemagglutinin and neuraminidase
and other influenza viral components. Illustrative examples of
measles viral antigens include, but are not limited to, antigens
such as the measles virus fusion protein and other measles virus
components. Illustrative examples of rubella viral antigens
include, but are not limited to, antigens such as proteins E1 and
E2 and other rubella virus components; rotaviral antigens such as
VP7sc and other rotaviral components. Illustrative examples of
cytomegaloviral antigens include, but are not limited to, antigens
such as envelope glycoprotein B and other cytomegaloviral antigen
components. Non-limiting examples of respiratory syncytial viral
antigens include antigens such as the RSV fusion protein, the M2
protein and other respiratory syncytial viral antigen components.
Illustrative examples of herpes simplex viral antigens include, but
are not limited to, antigens such as immediate early proteins,
glycoprotein D, and other herpes simplex viral antigen components.
Non-limiting examples of varicella zoster viral antigens include
antigens such as 9PI, gpII, and other varicella zoster viral
antigen components. Non-limiting examples of Japanese encephalitis
viral antigens include antigens such as proteins E, M-E, M-E-NS 1,
NS 1, NS 1-NS2A, 80% E, and other Japanese encephalitis viral
antigen components. Representative examples of rabies viral
antigens include, but are not limited to, antigens such as rabies
glycoprotein, rabies nucleoprotein and other rabies viral antigen
components. Illustrative examples of papillomavirus antigens
include, but are not limited to, the L1 and L2 capsid proteins as
well as the E6/E7 antigens associated with cervical cancers, See
Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe,
D. M., 1991, Raven Press, New York, for additional examples of
viral antigens.
[0117] Illustrative examples of fungi include Acremonium spp.,
Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces
dermatidis, Candida spp., Cladophialophora carrioni, Coccidioides
immitis, Conidiobolus spp., Cryptococcus spp., Curvularia spp.,
Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp.,
Fonsecaea compacta, Fonsecaea pedrosoi, Fusarium oxysporum,
Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var.
capsulatum, Histoplasma capsulatum var. duboisii, Hortaea
werneckit, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria
senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia
furfir, Microsporum spp., Neotestudina rosatti, Onychocola
canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa,
Piedraia hortae, Piedra iahortae, Pityriasis versicolor,
Pseudallescheria boydii, Pyrenochaeta romerot, Rhizopus arrhizus,
Scopulariopsis brevicaulis, Scytalidiwnum dimidatum, Sporothrix
schenckii, Trichophyton spp., Trichosporon spp., Zygomycete fungi,
Absidia corymblfera, Rhizomucor pusillus and Rhizopus arrhizus.
Thus, representative fungal antigens that can be used in the
compositions and methods of the present invention include, but are
not limited to, candida fungal antigen components; histoplasma
fungal antigens such as heat shock protein 60 (HSP60) and other
histoplasma fungal antigen components; cryptococcal fungal antigens
such as capsular polysaccharides and other cryptococcal fungal
antigen components; coccidioides fungal antigens such as spherule
antigens and other coccidioides fungal antigen components; and
tinea fungal antigens such as trichophytin and other coccidioides
fungal antigen components.
[0118] Illustrative examples of bacteria include bacteria that are
responsible for diseases including, but not restricted to,
diphtheria (e.g., Corynebacterium diphtheria), pertussis (e.g.,
Bordetella pertussis, GenBank Accession No. M35274), tetanus (e.g.,
Clostridium tetani, GenBank Accession No. M64353), tuberculosis
(e.g., Mycobacterium tuberculosis), bacterial pneumonias (e.g.,
Haemophilus influenzae.), cholera (e.g., Vibrio cholerae), anthrax
(e.g., Bacillus anthracis), typhoid, plague, shigellosis (e.g.,
Shigella dysenteriae), botulism (e.g., Clostridium botulinwnum),
salmonellosis (e.g., GenBank Accession No. L03833), peptic ulcers
(e.g., Helicobacter pylori), Legionnaire's Disease, Lyme disease
(e.g., GenBank Accession No. U59487), Other pathogenic bacteria
include Escherichia coli, Clostridium perfringens, Pseudomonas
aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. Thus,
bacterial antigens which can be used in the compositions and
methods of the invention include, but are not limited to: pertussis
bacterial antigens such as pertussis toxin, filamentous
hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other
pertussis bacterial antigen components; diphtheria bacterial
antigens such as diphtheria toxin or toxoid and other diphtheria
bacterial antigen components; tetanus bacterial antigens such as
tetanus toxin or toxoid and other tetanus bacterial antigen
components, streptococcal bacterial antigens such as M proteins and
other streptococcal bacterial antigen components; gram-negative
bacilli bacterial antigens such as lipopolysaccharides and other
gram-negative bacterial antigen components; Mycobacterium
tuberculosis bacterial antigens such as mycolic acid, heat shock
protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A
and other mycobacterial antigen components; Helicobacter pylori
bacterial antigen components, pneumococcal bacterial antigens such
as pneumolysin, pneumococcal capsular polysaccharides and other
pneumococcal bacterial antigen components; Haemophilus influenza
bacterial antigens such as capsular polysaccharides and other
Haemophilus influenza bacterial antigen components; anthrax
bacterial antigens such as anthrax protective antigen and other
anthrax bacterial antigen components; rickettsiae bacterial
antigens such as rompA and other rickettsiae bacterial antigen
component. Also included with the bacterial antigens described
herein are any other bacterial, mycobacterial, mycoplasmal,
rickettsial, or chlamydial antigens.
[0119] Illustrative examples of protozoa include protozoa that are
responsible for diseases including, but not limited to, malaria
(e.g., GenBank Accession No. X53832), hookworm, onchocerciasis
(e.g., GenBank Accession No. M27807), schistosomiasis (e.g.,
GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis,
leishmaniasis, giardiasis (GenBank Accession No. M33641),
amoebiasis, filariasis (e.g., GenBank Accession No. J03266),
borreliosis, and trichinosis. Thus, protozoal antigens which can be
used in the compositions and methods of the invention include, but
are not limited to: plasmodium falciparum antigens such as
merozoite surface antigens, sporozoite surface antigens,
circumsporozoite antigens, gametocyte/gamete surface antigens,
blood-stage antigen pf 155/RESA and other plasmodial antigen
components; toxoplasma antigens such as SAG-1, p30 and other
toxoplasma antigen components; schistosoma antigens such as
glutathione-S-transferase, paramyosin, and other schistosomal
antigen components; leishmania major and other leishmaniae antigens
such as gp63, lipophosphoglycan and its associated protein and
other leishmanial antigen components; and trypanosoma cruzi
antigens such as the 75-77 kDa antigen, the 56 kDa antigen and
other trypanosomal antigen components.
[0120] The present invention also contemplates toxin components as
antigens, illustrative examples of which include staphylococcal
enterotoxins, toxic shock syndrome toxin; retroviral antigens
(e.g., antigens derived from HIV), streptococcal antigens,
staphylococcal enterotoxin-A (SEA), staphylococcal enterotoxin-B
(SEB), staphylococcal enterotoxini-3 (SE.sub.1-3), staphylococcal
enterotoxin-D (SED), staphylococcal enterotoxin-E (SEE) as well as
toxins derived from mycoplasma, mycobacterium, and herpes
viruses.
6. Construction of Synthetic Polynucleotides
[0121] Replacement of one codon for another can be achieved using
standard methods known in the art. For example codon modification
of a parent polynucleotide can be effected using several known
mutagenesis techniques including, for example,
oligonucleotide-directed mutagenesis, mutagenesis with degenerate
oligonucleotides, and region-specific mutagenesis. Exemplary in
vitro mutagenesis techniques are described for example in U.S. Pat.
Nos. 4,184,917, 4,321,365 and 4,351,901 or in the relevant sections
of Ausubel, et al. (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley & Sons, Inc. 1997) and of Sambrook, et al., (MOLECULAR
CLONING. A LABORATORY MANUAL, Cold Spring Harbor Press, 1989).
Instead of in vitro mutagenesis, the synthetic polynucleotide can
be synthesized de novo using readily available machinery as
described, for example, in U.S. Pat. No. 4,293,652. However, it
should be noted that the present invention is not dependent on, and
not directed to, any one particular technique for constructing the
synthetic polynucleotide.
[0122] The parent polynucleotide is suitably a natural gene.
However, it is possible that the parent polynucleotide is not
naturally-occurring but has been engineered using recombinant
techniques. Parent polynucleotides can be obtained from any
suitable source, such as from eukaryotic or prokaryotic organisms,
including but not limited to mammals or other animals, and
pathogenic organisms such as yeasts, bacteria, protozoa and
viruses.
[0123] The invention also contemplates synthetic polynucleotides
encoding one or more desired portions of a target antigen. In some
embodiments, the synthetic polynucleotide encodes at least about 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 400, 500,
600, 700, 800, 900 or 1000, or even at least about 2000, 3000, 4000
or 5000 contiguous amino acid residues, or almost up to the total
number of amino acids present in a full-length target antigen. In
some embodiments, the synthetic polynucleotide encodes a plurality
of portions of the target antigen, wherein the portions are the
same or different. In illustrative examples of this type, the
synthetic polynucleotide encodes a multi-epitope fusion protein. A
number of factors can influence the choice of portion size. For
example, the size of individual portions encoded by the synthetic
polynucleotide can be chosen such that it includes, or corresponds
to the size of, T cell epitopes and/or B cell epitopes, and their
processing requirements. Practitioners in the art will recognize
that class I-restricted T cell epitopes are typically between 8 and
10 amino acid residues in length and if placed next to unnatural
flanking residues, such epitopes can generally require 2 to 3
natural flanking amino acid residues to ensure that they are
efficiently processed and presented. Class II-restricted T cell
epitopes usually range between 12 and 25 amino acid residues in
length and may not require natural flanking residues for efficient
proteolytic processing although it is believed that natural
flanking residues may play a role. Another important feature of
class II-restricted epitopes is that they generally contain a core
of 9-10 amino acid residues in the middle which bind specifically
to class II MHC molecules with flanking sequences either side of
this core stabilizing binding by associating with conserved
structures on either side of class II MHC antigens in a sequence
independent manner. Thus the functional region of class
II-restricted epitopes is typically less than about 15 amino acid
residues long. The size of linear B cell epitopes and the factors
effecting their processing, like class II-restricted epitopes, are
quite variable although such epitopes are frequently smaller in
size than 15 amino acid residues. From the foregoing, it is
advantageous, but not essential, that the size of individual
portions of the target antigen is at least 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 20, 25, 30 amino acid residues. Suitably, the size of
individual portions is no more than about 500, 200, 100, 80, 60,
50, 40 amino acid residues. In certain advantageous embodiments,
the size of individual portions is sufficient for presentation by
an antigen-presenting cell of a T cell and/or a B cell epitope
contained within the peptide.
[0124] As will be appreciated by those of skill in the art, it is
generally not necessary to immunize with a polypeptide that shares
exactly the same amino acid sequence with the target antigen to
produce an immune response to that antigen. In some embodiments,
therefore, the polypeptide encoded by the synthetic polynucleotide
is desirably a variant of at least a portion of the target antigen.
"Variant" polypeptides include proteins derived from the target
antigen by deletion (so-called truncation) or addition of one or
more amino acids to the N-terminal and/or C-terminal end of the
target antigen; deletion or addition of one or more amino acids at
one or more sites in the target antigen; or substitution of one or
more amino acids at one or more sites in the target antigen.
Variant polypeptides encompassed by the present invention will have
at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%,
typically at least about 90% to 95% or more, and more typically at
least about 96%, 97%, 98%, 99% or more sequence similarity or
identity with the amino acid sequence of the target antigen or
portion thereof as determined by sequence alignment programs
described elsewhere herein using default parameters. A variant of a
target antigen may differ from that antigen generally by as much
1000, 500, 400, 300, 200, 100, 50 or 20 amino acid residues or
suitably by as few as 1-15 amino acid residues, as few as 1-10,
such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue.
[0125] Variant polypeptides corresponding to at least a portion of
a target antigen may contain conservative amino acid substitutions
at various locations along their sequence, as compared to the
target antigen amino acid sequence. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art, which can be generally sub-classified as follows:
[0126] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having an acidic side chain
include glutamic acid and aspartic acid.
[0127] Basic: The residue has a positive charge due to association
with H ion at physiological pH or within one or two pH units
thereof (e.g., histidine) and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having a basic side chain
include arginine, lysine and histidine.
[0128] Charged: The residues are charged at physiological pH and,
therefore, include amino acids having acidic or basic side chains
(i.e., glutamic acid, aspartic acid, arginine, lysine and
histidine).
[0129] Hydrophobic: The residues are not charged at physiological
pH and the residue is repelled by aqueous solution so as to seek
the inner positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium. Amino acids having
a hydrophobic side chain include tyrosine, valine, isoleucine,
leucine, methionine, phenylalanine and tryptophan.
[0130] Neutral/polar: The residues are not charged at physiological
pH, but the residue is not sufficiently repelled by aqueous
solutions so that it would seek inner positions in the conformation
of a peptide in which it is contained when the peptide is in
aqueous medium. Amino acids having a neutral/polar side chain
include asparagine, glutamine, cysteine, histidine, serine and
threonine.
[0131] This description also characterizes certain amino acids as
"small" since their side chains are not sufficiently large, even if
polar groups are lacking, to confer hydrophobicity. With the
exception of proline, "small" amino acids are those with four
carbons or less when at least one polar group is on the side chain
and three carbons or less when not. Amino acids having a small side
chain include glycine, serine, alanine and threonine. The
gene-encoded secondary amino acid proline is a special case due to
its known effects on the secondary conformation of peptide chains.
The structure of proline differs from all the other
naturally-occurring amino acids in that its side chain is bonded to
the nitrogen of the .alpha.-amino group, as well as the a-carbon.
Several amino acid similarity matrices (e.g., PAM120 matrix and
PAM250 matrix as disclosed for example by Dayhoff et al. (1978) A
model of evolutionary change in proteins. Matrices for determining
distance relationships In M. O. Dayhoff (ed.), Atlas of protein
sequence and structure, Vol. 5, pp. 345-358, National Biomedical
Research Foundation, Washington D.C.; and by Gonnet et al., 1992,
Science 256(5062): 144301445), however, include proline in the same
group as glycine, serine, alanine and threonine. Accordingly, for
the purposes of the present invention, proline is classified as a
"small" amino acid.
[0132] The degree of attraction or repulsion required for
classification as polar or nonpolar is arbitrary and, therefore,
amino acids specifically contemplated by the invention have been
classified as one or the other. Most amino acids not specifically
named can be classified on the basis of known behavior.
[0133] Amino acid residues can be further sub-classified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side-chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of four carbon atoms or less,
inclusive of the carboxyl carbon, provided an additional polar
substituent is present; three or less if not. Small residues are,
of course, always nonaromatic. Dependent on their structural
properties, amino acid residues may fall in two or more classes.
For the naturally-occurring protein amino acids, sub-classification
according to the this scheme is presented in the Table 10.
TABLE-US-00010 TABLE 10 Original Residue Exemplary Substitutions
Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly
Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met
Leu, Ile, Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe
Val Ile, Leu
[0134] Conservative amino acid substitution also includes groupings
based on side chains. For example, a group of amino acids having
aliphatic side chains is glycine, alanine, valine, leucine, and
isoleucine; a group of amino acids having aliphatic-hydroxyl side
chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. For example, it is reasonable to expect that
replacement of a leucine with an isoleucine or valine, an aspartate
with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
will not have a major effect on the properties of the resulting
variant polypeptide. Conservative substitutions are shown in Table
11 below under the heading of exemplary substitutions. More
preferred substitutions are shown under the heading of preferred
substitutions. Amino acid substitutions falling within the scope of
the invention, are, in general, accomplished by selecting
substitutions that do not differ significantly in their effect on
maintaining (a) the structure of the peptide backbone in the area
of the substitution, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
After the substitutions are introduced, the variants are screened
for biological activity.
TABLE-US-00011 TABLE 11 EXEMPLARY AND PREFERRED AMINO ACID
SUBSTITUTIONS Preferred Original Residue Exemplary Substitutions
Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln,
His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn, His, Lys, Asn
Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu,
Val, Met, Ala, Phe, Leu Norleu Leu Norleu, Ile, Val, Met, Ala, Phe
Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile,
Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp,
Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu
[0135] Alternatively, similar amino acids for making conservative
substitutions can be grouped into three categories based on the
identity of the side chains. The first group includes glutamic
acid, aspartic acid, arginine, lysine, histidine, which all have
charged side chains; the second group includes glycine, serine,
threonine, cysteine, tyrosine, glutamine, asparagine; and the third
group includes leucine, isoleucine, valine, alanine, proline,
phenylalanine, tryptophan, methionine, as described in Zubay, G.,
Biochemistry, third edition, Wm.C. Brown Publishers (1993).
[0136] The invention further contemplates a chimeric construct
comprising a synthetic polynucleotide of the invention, which is
operably linked to a regulatory polynucleotide. The regulatory
polynucleotide suitably comprises transcriptional and/or
translational control sequences, which will be compatible for
expression in the organism of interest or in cells of that
organism. Typically, the transcriptional and translational
regulatory control sequences include, but are not limited to, a
promoter sequence, a 5' non-coding region, a cis-regulatory region
such as a functional binding site for transcriptional regulatory
protein or translational regulatory protein, an upstream open
reading frame, ribosomal-binding sequences, transcriptional start
site, translational start site, and/or nucleotide sequence which
encodes a leader sequence, termination codon, translational stop
site and a 3' non-translated region. Constitutive or inducible
promoters as known in the art are contemplated by the invention.
The promoters may be either naturally occurring promoters, or
hybrid promoters that combine elements of more than one promoter.
Promoter sequences contemplated by the present invention may be
native to the organism of interest or may be derived from an
alternative source, where the region is functional in the chosen
organism. The choice of promoter will differ depending on the
intended host or cell or tissue type. For example, promoters which
could be used for expression in mammals include the metallothionein
promoter, which can be induced in response to heavy metals such as
cadmium, the .beta.-actin promoter as well as viral promoters such
as the SV40 large T antigen promoter, human cytomegalovirus (CMV)
immediate early (IE) promoter, Rous sarcoma virus LTR promoter, the
mouse mammary tumor virus LTR promoter, the adenovirus major late
promoter (Ad MLP), the herpes simplex virus promoter, and a HPV
promoter, particularly the HPV upstream regulatory region (URR),
among others. All these promoters are well described and readily
available in the art.
[0137] Enhancer elements may also be used herein to increase
expression levels of the mammalian constructs. Examples include the
SV40 early gene enhancer, as described for example in Dijkema et
al. (1985, EMBO J. 4:761), the enhancer/promoter derived from the
long terminal repeat (LTR) of the Rous Sarcoma Virus, as described
for example in Gorman et al., (1982, Proc. Natl. Acad. Sci. USA
79:6777) and elements derived from human CMV, as described for
example in Boshart et al. (1985, Cell 41:521), such as elements
included in the CMV intron A sequence.
[0138] The chimeric construct may also comprise a 3' non-translated
sequence. A 3' non-translated sequence refers to that portion of a
gene comprising a DNA segment that contains a polyadenylation
signal and any other regulatory signals capable of effecting mRNA
processing or gene expression. The polyadenylation signal is
characterized by effecting the addition of polyadenylic acid tracts
to the 3' end of the mRNA precursor. Polyadenylation signals are
commonly recognized by the presence of homology to the canonical
form 5' AATAAA-3' although variations are not uncommon. The 3'
non-translated regulatory DNA sequence preferably includes from
about 50 to 1,000 nts and may contain transcriptional and
translational termination sequences in addition to a
polyadenylation signal and any other regulatory signals capable of
effecting mRNA processing or gene expression.
[0139] In some embodiments, the chimeric construct further contains
a selectable marker gene to permit selection of cells containing
the construct. Selection genes are well known in the art and will
be compatible for expression in the cell of interest.
[0140] It will be understood, however, that expression of
protein-encoding polynucleotides in heterologous systems is now
well known, and the present invention is not directed to or
dependent on any particular vector, transcriptional control
sequence or technique for expression of the polynucleotides.
Rather, synthetic polynucleotides prepared according to the methods
set forth herein may be introduced into a mammal in any suitable
manner in the form of any suitable construct or vector, and the
synthetic polynucleotides may be expressed with known transcription
regulatory elements in any conventional manner.
[0141] In addition, chimeric constructs can be constructed that
include sequences coding for adjuvants. Particularly suitable are
detoxified mutants of bacterial ADP-ribosylating toxins, for
example, diphtheria toxin, pertussis toxin (PT), cholera toxin
(CT), Escherichia coli heat-labile toxins (LT1 and LT2),
Pseudomonas endotoxin A, Clostridium botulinum C2 and C3 toxins, as
well as toxins from C. perfringens, C. spiriforma and C. difficile.
In some embodiments, the chimeric constructs include coding
sequences for detoxified mutants of E. coli heat-labile toxins,
such as the LT-K63 and LT-R72 detoxified mutants, described in U.S.
Pat. No. 6,818,222. In some embodiments, the adjuvant is a
protein-destabilising element, which increases processing and
presentation of the polypeptide that corresponds to at least a
portion of the target antigen through the class I MHC pathway,
thereby leading to enhanced cell-mediated immunity against the
polypeptide. Illustrative protein-destabilising elements include
intracellular protein degradation signals or degrons which may be
selected without limitation from a destabilising amino acid at the
amino-terminus of a polypeptide of interest, a PEST region or a
ubiquitin. For example, the coding sequence for the polypeptide can
be modified to include a destabilising amino acid at its
amino-terminus so that the protein so modified is subject to the
N-end rule pathway as disclosed, for example, by Bachmair et al. in
U.S. Pat. No. 5,093,242 and by Varshavsky et al. in U.S. Pat. No.
5,122,463. In some embodiments, the destabilising amino acid is
selected from isoleucine and glutamic acid, especially from
histidine tyrosine and glutamine, and more especially from aspartic
acid, asparagine, phenylalanine, leucine, tryptophan and lysine. In
certain embodiments, the destabilising amino acid is arginine. In
some proteins, the amino-terminal end is obscured as a result of
the protein's conformation (i.e., its tertiary or quaternary
structure). In these cases, more extensive alteration of the
amino-terminus may be necessary to make the protein subject to the
N-end rule pathway. For example, where simple addition or
replacement of the single amino-terminal residue is insufficient
because of an inaccessible amino-terminus, several amino acids
(including lysine, the site of ubiquitin joining to substrate
proteins) may be added to the original amino-terminus to increase
the accessibility and/or segmental mobility of the engineered amino
terminus. In some embodiments, a nucleic acid sequence encoding the
amino-terminal region of the polypeptide can be modified to
introduce a lysine residue in an appropriate context. This can be
achieved most conveniently by employing DNA constructs encoding
"universal destabilising segments". A universal destabilising
segment comprises a nucleic acid construct which encodes a
polypeptide structure, preferably segmentally mobile, containing
one or more lysine residues, the codons for lysine residues being
positioned within the construct such that when the construct is
inserted into the coding sequence of the protein-encoding synthetic
polynucleotide, the lysine residues are sufficiently spatially
proximate to the amino-terminus of the encoded protein to serve as
the second determinant of the complete amino-terminal degradation
signal. The insertion of such constructs into the 5' portion of a
polypeptide-encoding synthetic polynucleotide would provide the
encoded polypeptide with a lysine residue (or residues) in an
appropriate context for destabilization. In other embodiments, the
polypeptide is modified to contain a PEST region, which is rich in
an amino acid selected from proline, glutamic acid, serine and
threonine, which region is optionally flanked by amino acids
comprising electropositive side chains. In this regard, it is known
that amino acid sequences of proteins with intracellular half-lives
less than about 2 hours contain one or more regions rich in proline
(P), glutamic acid (E), serine (S), and threonine (T) as for
example shown by Rogers et al. (1986, Science 234 (4774): 364-368).
In still other embodiments, the polypeptide is conjugated to a
ubiquitin or a biologically active fragment thereof; to produce a
modified polypeptide whose rate of intracellular proteolytic
degradation is increased, enhanced or otherwise elevated relative
to the unmodified polypeptide.
[0142] One or more adjuvant polypeptides may be co-expressed with
an `antigenic` polypeptide that corresponds to at least a portion
of the target antigen. In certain embodiments, adjuvant and
antigenic polypeptides may be co-expressed in the form of a fusion
protein comprising one or more adjuvant polypeptides and one or
more antigenic polypeptides. Alternatively, adjuvant and antigenic
polypeptides may be co-expressed as separate proteins.
[0143] Furthermore, chimeric constructs can be constructed that
include chimeric antigen-coding gene sequences, encoding, e.g.,
multiple antigens/epitopes of interest, for example derived from a
single or from more than one target antigen. In certain
embodiments, multi-cistronic cassettes (e.g., bi-cistronic
cassettes) can be constructed allowing expression of multiple
adjuvants and/or antigenic polypeptides from a single mRNA using,
for example, the EMCV IRES, or the like. In other embodiments,
adjuvants and/or antigenic polypeptides can be encoded on separate
coding sequences that are operably connected to independent
transcription regulatory elements.
[0144] In some embodiments, the chimeric constructs of the
invention are in the form of expression vectors which are suitably
selected from self-replicating extrachromosomal vectors (e.g.,
plasmids) and vectors that integrate into a host genome. In
illustrative examples of this type, the expression vectors are
viral vectors, such as simian virus 40 (SV40) or bovine papilloma
virus (BPV), which has the ability to replicate as extrachromosomal
elements (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,
Gluzman ed., 1982; Sarver et al., 1981, Mol. Cell. Biol. 1:486).
Viral vectors include retroviral (lentivirus), adeno-associated
virus (see, e.g., Okada, 1996, Gene Ther. 3:957-964; Muzyczka,
1994, J. Clin. Invst. 94:1351; U.S. Pat. Nos. 6,156,303; 6,143,548
5,952,221, describing AAV vectors; see also U.S. Pat. Nos.
6,004,799; 5,833,993), adenovirus (see, e.g., U.S. Pat. Nos.
6,140,087; 6,136,594; 6,133,028; 6,120,764), reovirus, herpesvirus,
rotavirus genomes etc., modified for introducing and directing
expression of a polynucleotide or transgene in cells. Retroviral
vectors can include those based upon murine leukemia virus (see,
e.g., U.S. Pat. No. 6,132,731), gibbon ape leukemia virus (see,
e.g., U.S. Pat. No. 6,033,905), simian immuno-deficiency virus,
human immuno-deficiency virus (see, e.g., U.S. Pat. No. 5,985,641),
and combinations thereof.
[0145] Vectors also include those that efficiently deliver genes to
animal cells in vivo (e.g., stem cells) (see, e.g., U.S. Pat. Nos.
5,821,235 and 5,786,340; Croyle et al., 1998, Gene Ther. 5:645;
Croyle et al., 1998, Pharm. Res. 15:1348; Croyle et al., 1998, Hum.
Gene Ther. 9:561; Foreman et al., 1998, Hum. Gene Ther. 9:1313;
Wirtz et al., 1999, Gut 44:800). Adenoviral and adeno-associated
viral vectors suitable for in vivo delivery are described, for
example, in U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,604,090.
Additional vectors suitable for in vivo delivery include herpes
simplex virus vectors (see, e.g., U.S. Pat. No. 5,501,979),
retroviral vectors (see, e.g., U.S. Pat. Nos. 5,624,820, 5,693,508
and 5,674,703; and WO92/05266 and WO92/14829), bovine papilloma
virus (BPV) vectors (see, e.g., U.S. Pat. No. 5,719,054), CMV-based
vectors (see, e.g., U.S. Pat. No. 5,561,063) and parvovirus,
rotavirus and Norwalk virus vectors. Lentiviral vectors are useful
for infecting dividing as well as non-dividing cells (see, e.g.,
U.S. Pat. No. 6,013,516).
[0146] Additional viral vectors which will find use for delivering
the nucleic acid molecules encoding the antigens of interest
include those derived from the pox family of viruses, including
vaccinia virus and avian poxvirus. By way of example, vaccinia
virus recombinants expressing the chimeric constructs can be
constructed as follows. The antigen coding sequence is first
inserted into an appropriate vector so that it is adjacent to a
vaccinia promoter and flanking vaccinia DNA sequences, such as the
sequence encoding thymidine kinase (TK). This vector is then used
to transfect cells that are simultaneously infected with vaccinia.
Homologous recombination serves to insert the vaccinia promoter
plus the gene encoding the coding sequences of interest into the
viral genome. The resulting TK-recombinant can be selected by
culturing the cells in the presence of 5-bromodeoxyuridine and
picking viral plaques resistant thereto.
[0147] Alternatively, avipoxviruses, such as the fowlpox and
canarypox viruses, can also be used to deliver the genes.
Recombinant avipox viruses, expressing immunogens from mammalian
pathogens, are known to confer protective immunity when
administered to non-avian species. The use of an avipox vector is
particularly desirable in human and other mammalian species since
members of the avipox genus can only productively replicate in
susceptible avian species and therefore are not infective in
mammalian cells. Methods for producing recombinant avipoxviruses
are known in the art and employ genetic recombination, as described
above with. respect to the production of vaccinia viruses. See,
e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
[0148] Molecular conjugate vectors, such as the adenovirus chimeric
vectors described in Michael et al., J. Biol. Chem. (1993)
268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992)
89:6099-6103, can also be used for gene delivery.
[0149] Members of the Alphavirus genus, such as, but not limited
to, vectors derived from the Sindbis virus (SIN), Semliki Forest
virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will
also find use as viral vectors for delivering the chimeric
constructs of the present invention. For a description of
Sindbis-virus derived vectors useful for the practice of the
instant methods, see, Dubensky et al. (1996, J. Virol. 70:508-519;
and International Publication Nos. WO 95/07995, WO 96/17072); as
well as, Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, and
Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245. Exemplary vectors of
this type are chimeric alphavirus vectors comprised of sequences
derived from Sindbis virus and Venezuelan equine encephalitis
virus. See, e.g., Perri et al. (2003, J. Virol. 77: 10394-10403)
and International Publication Nos. WO 02/099035, WO 02/080982, WO
01/81609, and WO 00/61772.
[0150] In other illustrative embodiments, lentiviral vectors are
employed to deliver a chimeric construct of the invention into
selected cells or tissues. Typically, these vectors comprise a 5'
lentiviral LTR, a tRNA binding site, a packaging signal, a promoter
operably linked to one or more genes of interest, an origin of
second strand DNA synthesis and a 3' lentiviral LTR, wherein the
lentiviral vector contains a nuclear transport element. The nuclear
transport element may be located either upstream (5') or downstream
(3') of a coding sequence of interest (for example, a synthetic Gag
or Env expression cassette of the present invention). A wide
variety of lentiviruses may be utilized within the context of the
present invention, including for example, lentiviruses selected
from the group consisting of HIV, HIV-1, HIV-2, FIV, BIV, EIAV,
MVV, CAEV, and SIV. Illustrative examples of lentiviral vectors are
described in PCT Publication Nos. WO 00/66759, WO 00/00600, WO
99/24465, WO 98/51810, WO 99/51754, WO 99/31251, WO 99/30742, and
WO 99/15641. Desirably, a third generation SIN lentivirus is used.
Commercial suppliers of third generation SIN (self-inactivating)
lentiviruses include Invitrogen (ViraPower Lentiviral Expression
System). Detailed methods for construction, transfection,
harvesting, and use of lentiviral vectors are given, for example,
in the Invitrogen technical manual "ViraPower Lentiviral Expression
System version B 050102 25-0501", available at
http://www.invitrogen.com/Content/Tech-Online/molecular_biology/manuals_p-
-ps/virapower_lentiviral_system_man.pdf. Lentiviral vectors have
emerged as an efficient method for gene transfer. Improvements in
biosafety characteristics have made these vectors suitable for use
at biosafety level 2 (BL2). A number of safety features are
incorporated into third generation SIN (self-inactivating) vectors.
Deletion of the viral 3' LTR U3 region results in a provirus that
is unable to transcribe a full length viral RNA. In addition, a
number of essential genes are provided in trans, yielding a viral
stock that is capable of but a single round of infection and
integration. Lentiviral vectors have several advantages, including:
1) pseudotyping of the vector using amphotropic envelope proteins
allows them to infect virtually any cell type; 2) gene delivery to
quiescent, post mitotic, differentiated cells, including neurons,
has been demonstrated; 3) their low cellular toxicity is unique
among transgene delivery systems; 4) viral integration into the
genome permits long term transgene expression; 5) their packaging
capacity (6-14 kb) is much larger than other retroviral, or
adeno-associated viral vectors. In a recent demonstration of the
capabilities of this system, lentiviral vectors expressing GFP were
used to infect murine stem cells resulting in live progeny,
germline transmission, and promoter-, and tissue-specific
expression of the reporter (Ailles, L. E. and Naldini, L.,
HIV-1-Derived Lentiviral Vectors. In: Trono, D. (Ed.), Lentiviral
Vectors, Springer-Verlag, Berlin, Heidelberg, N.Y., 2002, pp.
31-52). An example of the current generation vectors is outlined in
FIG. 2 of a review by Lois et al. (2002, Science, 295 868-872).
[0151] The chimeric construct can also be delivered without a
vector. For example, the chimeric construct can be packaged as DNA
or RNA in liposomes prior to delivery to the subject or to cells
derived therefrom. Lipid encapsulation is generally accomplished
using liposomes which are able to stably bind or entrap and retain
nucleic acid. The ratio of condensed DNA to lipid preparation can
vary but will generally be around 1:1 (mg DNA:micromoles lipid), or
more of lipid. For a review of the use of liposomes as carriers for
delivery of nucleic acids, see, Hug and Sleight, (1991, Biochim.
Biophys. Acta. 1097:1-17); and Straubinger et al., in Methods of
Enzymology (1983), Vol. 101, pp. 512-527.
[0152] Liposomal preparations for use in the present invention
include cationic (positively charged), anionic (negatively charged)
and neutral preparations, with cationic liposomes particularly
preferred. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al., 1987, Proc.
Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone et al., 1989,
Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified
transcription factors (Debs et al., 1990, J. Biol. Chem.
265:10189-10192), in functional form.
[0153] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the trademark Lipofectin, from GIBCO BRL, Grand
Island, N.Y. (See, also, Feigner et al., 1987, Proc. Natl. Acad.
Sci. USA 84:7413-7416). Other commercially available lipids include
(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Alternative cationic
liposomes can be prepared from readily available materials using
techniques well known in the art. See, e.g., Szoka et al., 1978,
Proc. Natl. Acad. Sci. USA 75:4194-4198; PCT Publication No. WO
90/11092 for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
[0154] Similarly, anionic and neutral liposomes are readily
available, such as, from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0155] The liposomes can comprise multilammelar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs). The various liposome-nucleic acid complexes are prepared
using methods known in the art. See, e.g., Straubinger et al., in
METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al.,
1978, Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos et
al., 1975, Biochim. Biophys. Acta 394:483; Wilson et al., 1979,
Cell 17:77); Deamer and Bangham, 1976, Biochim. Biophys. Acta
443:629; Ostro et al., 1977, Biochem. Biophys. Res. Commun. 76:836;
Fraley et al., 1979, Proc. Natl. Acad. Sci. USA 76:3348); Enoch and
Strittmatter, 1979, Proc. Natl. Acad. Sci. USA 76:145); Fraley et
al., 1980, J. Biol. Chem. 255:10431; Szoka and Papahadjopoulos,
1978, Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder et
al., 1982, Science 215:166.
[0156] The chimeric construct can also be delivered in cochleate
lipid compositions similar to those described by Papahadjopoulos et
al., 1975, Biochem. Biophys. Acta. 394:483-491. See, also, U.S.
Pat. Nos. 4,663,161 and 4,871,488.
[0157] The chimeric construct may also be encapsulated, adsorbed
to, or associated with, particulate carriers. Such carriers present
multiple copies-of a selected chimeric construct to the immune
system. The particles can be taken up by professional antigen
presenting cells such as macrophages and dendritic cells, and/or
can enhance antigen presentation through other mechanisms such as
stimulation of cytokine release. Examples of particulate carriers
include those derived from polymethyl methacrylate polymers, as
well as microparticles derived from poly(lactides) and
poly(lactide-co-glycolides), known as PLO. See, e.g., Jeffery et
al., 1993, Pharm. Res. 10:362-368; McGee J. P., et al., 1997, J
Microencapsul. 14(2):197-210; O'Hagan D. T., et al., 1993, Vaccine
11(2):149-54.
[0158] Furthermore, other particulate systems and polymers can be
used for the in vivo delivery of the chimeric construct. For
example, polymers such as polylysine, polyarginine, polyornithine,
spermine, spermidine, as well as conjugates of these molecules, are
useful for transferring a nucleic acid of interest. Similarly, DEAE
dextran-mediated transfection, calcium phosphate precipitation or
precipitation using other insoluble inorganic salts, such as
strontium phosphate, aluminum silicates including bentonite and
kaolin, chromic oxide, magnesium silicate, talc, and the like, will
find use with the present methods. See, e.g., Felgner, P. L.,
Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of
delivery systems useful for gene transfer. Peptoids (Zuckerman, R.
N., et al., U.S. Pat. No. 5,831,005, issued Nov. 3, 1998) may also
be used for delivery of a construct of the present invention.
[0159] Additionally, biolistic delivery systems employing
particulate carriers such as gold and tungsten, are especially
useful for delivering chimeric constructs of the present invention.
The particles are coated with the synthetic expression cassette(s)
to be delivered and accelerated to high velocity, generally under a
reduced atmosphere, using a gun powder discharge from a "gene gun."
For a description of such techniques, and apparatuses useful
therefor, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,478,744. In illustrative
examples, gas-driven particle acceleration can be achieved with
devices such as those manufactured by PowderMed Pharmaceuticals PLC
(Oxford, UK) and PowderMed Vaccines Inc. (Madison, Wis.), some
examples of which are described in U.S. Pat. Nos. 5,846,796;
6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. This
approach offers a needle-free delivery approach wherein a dry
powder formulation of microscopic particles, such as polynucleotide
or polypeptide particles, are accelerated to high speed within a
helium gas jet generated by a hand held device, propelling the
particles into a target tissue of interest. Other devices and
methods that may be useful for gas-driven needle-less injection of
compositions of the present invention include those provided by
Bioject, Inc. (Portland, Oreg.), some examples of which are
described in U.S. Pat. Nos. 4,790,824; 5,064,413; 5,312,335;
5,383,851; 5,399,163; 5,520,639 and 5,993,412.
[0160] Alternatively, micro-cannula- and microneedle-based devices
(such as those being developed by Becton Dickinson and others) can
be used to administer the chimeric constructs of the invention.
Illustrative devices of this type are described in EP 1 092 444 A1,
and U.S. application Ser. No. 606,909, filed Jun. 29, 2000.
Standard steel cannula can also be used for intra-dermal delivery
using devices and methods as described in U.S. Ser. No. 417,671,
filed Oct. 14, 1999. These methods and devices include the delivery
of substances through narrow gauge (about 30 G) "micro-cannula"
with limited depth of penetration, as defined by the total length
of the cannula or the total length of the cannula that is exposed
beyond a depth-limiting feature. It is within the scope of the
present invention that targeted delivery of substances including
chimeric constructs can be achieved either through a single
microcannula or an array of microcannula (or "microneedles"), for
example 3-6 microneedles mounted on an injection device that may
include or be attached to a reservoir in which the substance to be
administered is contained.
7. Compositions
[0161] The invention also provides compositions, particularly
immunomodulating compositions, comprising one or more of the
chimeric constructs described herein. The immunomodulating
compositions may comprise a mixture of chimeric constructs, which
in turn may be delivered, for example, using the same or different
vectors or vehicles. Antigens may be administered individually or
in combination, in e.g., prophylactic (i.e., to prevent infection
or disease) or therapeutic (to treat infection or disease)
immunomodulating compositions. The immunomodulating compositions
may be given more than once (e.g., a "prime" administration
followed by one or more "boosts") to achieve the desired effects.
The same composition can be administered in one or more priming and
one or more boosting steps. Alternatively, different compositions
can be used for priming and boosting.
[0162] The immunomodulating compositions will generally include one
or more "pharmaceutically acceptable excipients or vehicles" such
as water, saline, glycerol, ethanol, etc. Additionally, auxiliary
substances, such as wetting or emulsifying agents, pH buffering
substances, and the like, may be present in such vehicles.
[0163] Immunomodulating compositions will typically, in addition to
the components mentioned above, comprise one or more
"pharmaceutically acceptable carriers." These include any carrier
which does not itself induce the production of antibodies harmful
to the individual receiving the composition. Suitable carriers
typically are large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, and lipid aggregates
(such as oil droplets or liposomes). Such carriers are well known
to those of ordinary skill in the art. A composition may also
contain a diluent, such as water, saline, glycerol, etc.
Additionally, an auxiliary substance, such as a wetting or
emulsifying agent, pH buffering substance, and the like, may be
present. A thorough discussion of pharmaceutically acceptable
components is available in Gennaro (2000) Remington: The Science
and Practice of Pharmacy. 20th ed., ISBN: 0683306472.
[0164] Pharmaceutically compatible salts can also be used in
compositions of the invention, for example, mineral salts such as
hydrochlorides, hydrobromides, phosphates, or sulfates, as well as
salts of organic acids such as acetates, proprionate, malonates, or
benzoates. Especially useful protein substrates are serum albumins,
keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,
ovalbumin, tetanus toxoid, and other proteins well known to those
of skill in the art.
[0165] The chimeric constructs of the invention can also be
adsorbed to, entrapped within or otherwise associated with
liposomes and particulate carriers such as PLG.
[0166] The chimeric constructs of the present invention are
formulated into compositions for delivery to a mammal. These
compositions may either be prophylactic (to prevent infection) or
therapeutic (to treat disease after infection). The compositions
will comprise a "therapeutically effective amount" of the gene of
interest such that an amount of the antigen can be produced in vivo
so that an immune response is generated in the individual to which
it is administered. The exact amount necessary will vary depending
on the subject being treated; the age and general condition of the
subject to be treated; the capacity of the subject's immune system
to synthesize antibodies; the degree of protection desired; the
severity of the condition being treated; the particular antigen
selected and its mode of administration, among other factors. An
appropriate effective amount can be readily determined by one of
skill in the art. Thus, a "therapeutically effective amount" will
fall in a relatively broad range that can be determined through
routine trials.
[0167] Once formulated, the compositions of the invention can be
administered directly to the subject (e.g., as described above).
Direct delivery of chimeric construct-containing compositions in
vivo will generally be accomplished with or without vectors, as
described above, by injection using either a conventional syringe,
needless devices such as Bioject.TM. or a gene gun, such as the
Accell.TM. gene delivery system (PowderMed Ltd, Oxford, England) or
microneedle device. The constructs can be delivered (e.g.,
injected) either subcutaneously, epidermally, intradermally,
intramuscularly, intravenous, intramucosally (such as nasally,
rectally and vaginally), intraperitoneally or orally. Delivery of
nucleic acid into cells of the epidermis is particularly preferred
as this mode of administration provides access to skin-associated
lymphoid cells and provides for a transient presence of nucleic
acid (e.g., DNA) in the recipient. Other modes of administration
include oral ingestion and pulmonary administration, suppositories,
needle-less injection, transcutaneous, topical, and transdermal
applications. Dosage treatment may be a single dose schedule or a
multiple dose schedule.
[0168] In order that the invention may be readily understood and
put into practical effect, particular preferred embodiments will
now be described by way of the following non-limiting examples.
EXAMPLES
Example 1
Synthetic Construct System for Determining the Immune Response
Preference of Codons in Mammals
Material and Methods
Primer Design/Synthesis and Sequence Manipulation
[0169] Oligonucleotides for site-directed mutagenesis were designed
according to the guidelines included in the mutagenesis kit manuals
(Quikchange II Site-directed Mutagenesis kit or Quikchange Multi
Site-directed Mutagenesis Kit; Stratagene, La Jolla Calif.). These
primers were synthesized and PAGE purified by Sigma (formerly
Proligo).
[0170] Oligonucleotides for whole gene synthesis were designed by
eye and synthesized by Sigma (formerly Proligo). The primers were
supplied as standard desalted oligos. No additional purification of
the oligonucleotides was carried out.
[0171] Sequence manipulation and analysis was carried out using the
suite of programs on Biomanager (ANGIS) and various other web-based
programs including BLAST at NCBI
(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cqi), NEBcutter
V2.0 from New England Biolabs
(http://tools.neb.com/NEBcutter2/index.php), the Translate Tool on
ExPASy (http://au.expasy.org/tools/dna.html), and the SignalP 3.0
server (http:/www.cb.dtu.dk/services/SignalP/).
[0172] Standard Cloning Techniques
[0173] Restriction enzyme digests, alkaline phosphatase treatments
and ligations were carried out according to the enzyme
manufacturers' instructions (various manufacturers including New
England Biolabs, Roche and Fermentas).
[0174] Purification of DNA from agarose gels and preparation of
mini-prep DNA were carried out using commercial kits (Qiagen,
Bio-Rad, Macherey-Nagel).
[0175] Agarose gel electrophoresis, phenol/chloroform extraction of
contaminant protein from DNA, ethanol precipitation of DNA and
other basic molecular biological procedures were carried out using
standard protocols, similar to those described in Current Protocols
in Molecular Biology (Ebook available via Wiley InterScience;
edited by Ausubel et al.).
[0176] Sequencing was carried out by the Australian Genome Research
Facility (AGRF, Brisbane).
[0177] Whole Gene Synthesis
[0178] Overlapping .about.35-50mer oligonucleotides (Sigma-Proligo)
were used to synthesize longer DNA sequences. Restriction enzyme
sites were incorporated to facilitate cloning. The method used to
synthesize the fragments is based on that given in Smith et al.
(2003). First, oligonucleotides for the top or bottom strand were
mixed and then phosphorylated using T4 polynucleotide kinase (PNK;
New England Biolabs). The oligonucleotide mixes were then purified
from the PNK by a standard phenol/chloroform extraction and sodium
acetate/ethanol (NaAc/EtOH) precipitation. Equal volumes of
oligonucleotide mixes for the top and bottom strands were then
mixed and the oligonucleotides denatured by heating at 95'C for 2
mins. The oligonucleotides were annealed by slowly cooling the
sample to 55'C and the annealed oligonucleotides ligated using Taq
ligase (New England Biolabs). The resulting fragment was purified
by phenol/CHCl.sub.3 extraction and NaAc/EtOH precipitation.
[0179] The ends of the fragments were filled in and the fragments
then amplified, using the outermost forward and reverse primers,
with the Clontech Advantage HF 2 PCR kit (Clontech) according to
the manufacturer's instructions. To fill in the ends the following
PCR was used: 35 cycles of a denaturation step of 94.degree. C. for
15 s, a slow annealing step where the temperature was ramped down
to 55.degree. C. over 7 minutes and then kept at 55.degree. C. for
2 min, and an elongation step of 72.degree. C. for 6 minutes. A
final elongation step for 7 min at 72.degree. C. was then carried
out. The second PCR to amplify the fragment involved: an initial
denaturation step at 94.degree. C. for 30 s, followed by 25 cycles
of 94.degree. C. for 15 s, 55.degree. C. 30 s and 68.degree. C. for
1 min, and a final elongation step of 68.degree. C. for 3 mins.
[0180] The fragments were then purified by gel electrophoresis,
digested and ligated into the relevant vector. Following
transformation of E. coli with the ligation mixture, mini-preps
were made for multiple colonies and the inserts sequenced.
Sometimes it was not possible to isolate clones with entirely
correct sequence. In those cases the errors were fixed by single or
multi site-directed mutagenesis.
[0181] Site-Directed Mutagenesis
[0182] Mutagenesis was carried out using the Quikchange II
Site-directed Mutagenesis kit or Quikchange Multi Site-directed
Mutagenesis Kit (Stratagene, La Jolla Calif.), with appropriate
PAGE (polyacrylamide gel electrophoresis)-purified primers (Sigma),
according to the manufacturer's instructions.
[0183] Preparation of Constructs
[0184] The details of the constructs used to generate the codon
preference table are summarized in TABLE 12. All constructs were
made using pCDNA3 from Invitrogen and were verified by sequencing
prior to use.
TABLE-US-00012 TABLE 12 SUMMARY OF SECRETORY E7 CONSTRUCT SERIES 1
AND 2 Construct AA & Codon CU of Sec Seq CU of E7 E7 Protein
Control Constructs IgkC1 N/A wt wt non-onc IgkC2 N/A mc mc non-onc
IgkC3 N/A wt wt onc IgkC4 N/A mc mc onc Secretory E7 construct
series 1 IgkS1-1 Ala GCG wt wt with all Ala non-onc gcg IgkS1-2 Ala
GCA wt wt with all Ala non-onc gca IgkS1-3 Ala GCT wt wt with all
Ala non-onc gct IgkS1-4 Ala GCC wt wt with all Ala non-onc gcc
IgkS1-5 Arg AGG wt wt with all Arg non-onc agg IgkS1-6 Arg AGA wt
wt with all Arg non-onc aga IgkS1-7 Arg CGG wt wt with all Arg
non-onc cgg IgkS1-8 Arg CGA wt wt with all Arg non-onc cga IgkS1-9
Arg CGT wt wt with all Arg non-onc cgt IgkS1-10 Arg CGC wt wt with
all Arg non-onc cgc IgkS1-11 Asn AAT wt wt with all Asn non-onc aat
IgkS1-12 Asn AAC wt wt with all Asn non-onc aac IgkS1-13 Asp GAT wt
with all Asp wt with all Asp non-onc gat gat IgkS1-14 Asp GAC wt
with all Asp wt with all Asp non-onc gac gac IgkS1-15 Cys TGT wt wt
with all Cys non-onc tgt IgkS1-16 Cys TGC wt wt with all Cys
non-onc tgc IgkS1-17 Glu GAG wt with all Glu wt with all Glu
non-onc gag gag IgkS1-18 Glu GAA wt with all Glu wt with all Glu
non-onc gaa gaa IgkS1-19 Gln CAG wt wt with all Gln non-onc cag
IgkS1-20 Gln CAA wt wt with all Gln non-onc caa IgkS1-21 Gly GGG wt
with all Gly wt with all Gly non-onc ggg ggg IgkS1-22 Gly GGA wt
with all Gly wt with all Gly non-onc gga gga IgkS1-23 Gly GGT wt
with all Gly wt with all Gly non-onc ggt ggt IgkS1-24 Gly GGC wt
with all Gly wt with all Gly non-onc ggc ggc IgkS1-25 His CAT wt wt
with all His non-onc cat IgkS1-26 His CAC wt wt with all His
non-onc cac IgkS1-27 Ile ATA wt wt with all Ile non-onc ata
IgkS1-28 Ile ATT wt wt with all Ile non-onc att IgkS1-29 Ile ATC wt
wt with all Ile non-onc atc IgkS1-30 Lys AAG wt wt with all Lys
non-onc aag IgkS1-31 Lys AAA wt wt with all Lys non-onc aaa
IgkS1-32 Phe TTT wt wt with all Phe non-onc L15F, ttt L22F IgkS1-33
Phe TTC wt wt with all Phe non-onc L15F, ttc L22F IgkS1-34 Ser AGT
wt with all Ser wt with all Ser non-onc agt agt IgkS1-35 Ser AGC wt
with all Ser wt with all Ser non-onc agc agc IgkS1-36 Ser TCG wt
with all Ser wt with all Ser non-onc tcg tcg IgkS1-37 Ser TCA wt
with all Ser wt with all Ser non-onc tca tca IgkS1-38 Ser TCT wt
with all Ser wt with all Ser non-onc tct tct IgkS1-39 Ser TCC wt wt
with all Ser non-onc tcc IgkS1-40 Thr ACG wt with all Thr wt with
all Thr non-onc acg acg IgkS1-41 Thr ACA wt with all Thr wt with
all Thr non-onc aca aca IgkS1-42 Thr ACT wt with all Thr wt with
all Thr non-onc act act IgkS1-43 Thr ACC wt with all Thr wt with
all Thr non-onc acc acc IgkS1-44 Tyr TAT wt wt with all Tyr non-onc
tat IgkS1-45 Tyr TAC wt wt with all Tyr non-onc tac IgkS1-46 Val
GTG wt with all Val wt with all Val non-onc gtg gtg IgkS1-47 Val
GTA wt with all Val wt with all Val non-onc gta gta IgkS1-48 Val
GTT wt with all Val wt with all Val non-onc gtt gtt IgkS1-49 Val
GTC wt with all Val wt with all Val non-onc gtc gtc IgkS1-50 Leu
CTG altered with Leu altered with Leu onc ctg ctg IgkS1-51 Leu CTA
altered with Leu altered with Leu onc cta cta IgkS1-52 Leu CTT
altered with Leu altered with Leu onc ctt ctt IgkS1-53 Leu CTC
altered with Leu altered with Leu onc ctc ctc IgkS1-54 Leu TTG
altered with Leu altered with Leu onc ttg ttg IgkS1-55 Leu TTA
altered with Leu altered with Leu onc tta tta IgkS1-56 Pro CCG
altered with Pro altered with Pro onc ccg ccg IgkS1-57 Pro CCA
altered with Pro altered with Pro onc cca cca IgkS1-58 Pro CCT
altered with Pro altered with Pro onc cct cct IgkS1-59 Pro CCC
altered with Pro altered with Pro onc ccc ccc Secretory E7
construct series 2 IgkS2-1 Ala GCG mc mc linkerA-onc IgkS2-2 Ala
GCA mc mc linkerA-onc IgkS2-3 Ala GCT mc mc linkerA-onc IgkS2-4 Ala
GCC mc mc linkerA-onc IgkS2-5 Arg AGG mc mc linkerR-onc IgkS2-6 Arg
AGA mc mc linkerR-onc IgkS2-7 Arg CGG mc mc linkerR-onc IgkS2-8 Arg
CGA mc mc linkerR-onc IgkS2-9 Arg CGT mc mc linkerR-onc IgkS2-10
Arg CGC mc mc linkerR-onc IgkS2-11 Asn AAT mc mc linkerN-onc
IgkS2-12 Asn AAC mc mc linkerN-onc IgkS2-13 Asp GAT wt with all Asp
wt with all Asp onc gat gat IgkS2-14 Asp GAC wt with all Asp wt
with all Asp onc gac gac IgkS2-15 Cys TGT wt wt with all Cys onc
tgt IgkS2-16 Cys TGC wt wt with all Cys onc tgc IgkS2-17 Glu GAG wt
with all Glu wt with all Glu onc gag gag IgkS2-18 Glu GAA wt with
all Glu wt with all Glu onc gaa gaa IgkS2-19 Gln CAG wt wt with all
Gln onc cag IgkS2-20 Gln CAA wt wt with all Gln onc caa IgkS2-21
Gly GGG wt with all Gly wt with all Gly onc ggg ggg IgkS2-22 Gly
GGA wt with all Gly wt with all Gly onc gga gga IgkS2-23 Gly GGT wt
with all Gly wt with all Gly onc ggt ggt IgkS2-24 Gly GGC wt with
all Gly wt with all Gly onc ggc ggc IgkS2-25 His CAT mc mc
linkerH-onc IgkS2-26 His CAC mc mc linkerH-onc IgkS2-27 Ile ATA wt
wt with all Ile onc ata IgkS2-28 Ile ATT wt wt with all Ile onc att
IgkS2-29 Ile ATC wt wt with all Ile onc atc IgkS2-30 Lys AAG mc mc
linkerK-onc IgkS2-31 Lys AAA mc mc linkerK-onc IgkS2-32 Phe TTT mc
mc linkerF-onc IgkS2-33 Phe TTC mc mc linkerF-onc IgkS2-34 Ser AGT
wt with all Ser wt with all Ser onc agt agt IgkS2-35 Ser AGC wt
with all Ser wt with all Ser onc agc agc IgkS2-36 Ser TCG wt with
all Ser wt with all Ser onc tcg tcg IgkS2-37 Ser TCA wt with all
Ser wt with all Ser onc tca tca IgkS2-38 Ser TCT wt with all Ser wt
with all Ser onc tct tct IgkS2-39 Ser TCC wt wt with all Ser onc
tcc IgkS2-40 Thr ACG wt with all Thr wt with all Thr onc acg acg
IgkS2-41 Thr ACA wt with all Thr wt with all Thr onc aca aca
IgkS2-42 Thr ACT wt with all Thr wt with all Thr onc act act
IgkS2-43 Thr ACC wt with all Thr wt with all Thr onc acc acc
IgkS2-44 Tyr TAT mc mc linkerY-onc IgkS2-45 Tyr TAC mc mc
linkerY-onc IgkS2-46 Val GTG wt with all Val wt with all Val onc
gtg gtg IgkS2-47 Val GTA wt with all Val wt with all Val onc gta
gta IgkS2-48 Val GTT wt with all Val wt with all Val onc gtt gtt
IgkS2-49 Val GTC wt with all Val wt with all Val onc gtc gtc IgkS2-
Asn AAT wt wt with all Asn linkerN-non-onc 11b aat IgkS2- Asn AAC
wt wt with all Asn linkerN-non-onc 12b aac AA = amino acid, CU =
codon usage, mc = mammalian consensus, wt = wild-type, onc =
oncogenic, non-onc = non-oncogenic, Sec seq = secretory sequence,
N/A = not applicable
[0185] Control Constructs
[0186] Control E7 constructs were based on those from Liu et al.
(2002). Both oncogenic (i.e. wild-type) and non-oncogenic E7
control constructs were made with wild-type or mammalian consensus
codon usage. "Non-oncogenic" E7 is E7 with D21 G, C24G, E26G
mutations, i.e. with mutations that have been reported to render E7
non-transforming (Edmonds and Vousden, 1989; Heck et al, 1992).
[0187] The secretory sequence was derived from Mus musculus IgK RNA
for the anti-HLA-DR antibody light chain (GenBank accession number
D84070). For some constructs the codon usage of this sequence was
modified.
[0188] Wild-Type Codon Usage Control Constructs:
[0189] The wild-type (wt) codon usage E7 construct from Liu et al.
was used as the template in a site-directed mutagenesis PCR to make
the wt codon usage non-oncogenic E7 construct.
[0190] The non-oncogenic and oncogenic wild-type codon usage E7
sequences were amplified to incorporate a 5' BamHI site and a 3'
EcoRI site. The resulting fragments were cloned into BamHI and
EcoRI cut pCDNA3 and sequenced. The secretory fragment was made by
whole gene synthesis using wild-type codon usage with flanking KpnI
and BamHI sites. The Kozak-secretory fragments were then ligated
into KpnI/BamHI cut pCDNA3-wtE7 (non-oncogenic or oncogenic) to
make pCDNA3-Igk-nE7 and pCDNA3-Igk-E7 (named IgkC1 and IgkC3
respectively; see TABLE 12). The identity of the constructs was
confirmed by sequencing.
[0191] Mammalian Consensus (Mc) Codon Usage Control Constructs:
[0192] As there were errors in the original mammalian consensus
(mc) E7 construct (L28F, Q70R and an E35 deletion; Liu et al.,
2002) it was not used. A me non-oncogenic E7 control construct was
synthesized by whole gene synthesis. A me oncogenic E7 (i.e.,
wild-type E7) control construct was subsequently made from the me
non-oncogenic E7 construct by single site-directed mutagenesis.
[0193] Secretory me oncogenic and non-oncogenic constructs were
made by amplifying the me E7 sequence with a forward primer that
introduced a BamHI site and a reverse primer that incorporated an
EcoRI site. The resulting E7 fragment was cloned into the
respective sites in pCDNA3 and sequenced. A me secretory sequence
flanked by KpnI and BamHI sites, 5' and 3' respectively, was
synthesised and ligated into the KpnI and BamHI sites of
pCDNA3-mcE7 (oncogenic or non-oncogenic) to make pCDNA3-mcIgk-mcnE7
and pCDNA3-mcIgk-mcE7 (named IgkC2 and IgkC4 respectively; see
TABLE 12). The identity of the constructs was confirmed by
sequencing.
[0194] Secreted Non-Oncogenic E7 Constructs with Predominantly
Wild-Type Codon Usage, Modified for Individual Codons
[0195] Plasmids encoding a non-oncogenic form of E7 were made for
all of the codons, with the exception of the Pro and Leu codons,
stop codons and codons for non-degenerate amino acids. As Phe
occurs just once in the E7 sequence, the codons for two Leu
residues, L15 and L22, were mutated to Phe codons. A combination of
techniques was used to make these constructs. When few mutations
were required single or multi site-directed mutagenesis of a
control construct encoding non-oncogenic E7 was performed (details
of the control construct are given above under "control
constructs"). When more extensive modifications were required whole
gene synthesis was employed. Regardless of the methods used these
constructs all include an E7 encoding sequence with identical
upstream and downstream sequence cloned into the KpnI and EcoRI
sites of pCDNA3. These constructs were then modified to include a
secretory sequence, as described below.
[0196] First, using the whole gene synthesis method, DNA fragments
that included a secretory sequence flanked by KpnI and BamHI sites
were synthesized. For some constructs the amino acid of interest
occurred in the secretory sequence so individual modified secretory
sequence fragments were made. For constructs for amino acids that
did not occur in the secretory sequence, wild-type secretory
sequence was used. These fragments were digested with KpnI and
BamHI. Then, using the relevant nE7 construct as a template and a
standard PCR protocol, a BamHI site was introduced at the 5' end of
the E7 sequence. The 3' EcoRI site was retained. The resulting E7
fragments were cut with BamHI and EcoRI, purified, and ligated into
pCDNA3. Following sequencing, the plasmids were cut with KpnI and
BamHI and ligated with the relevant KpnI/BamHI secretory sequences.
The sequences of the constructs were then confirmed. Constructs
IgkS1-1 to IgkS1-49 were made in this way (see TABLE 12 and FIGS. 1
to 11, 13 and 15 to 17 for sequence comparisons).
[0197] Secreted E7 Constructs with Individual Pro or Leu Codons
Modified
[0198] E7 DNA sequences in which the Pro or Leu codons were
individually modified were designed. The rest of the codon usage
for these E7 DNAs was the same for all of the Pro and Leu
constructs but differed from the wild-type or mammalian consensus
codon usage. [Note that this codon usage was based on our
preliminary data from immunizing mice with the GFP constructs.]
[0199] The Pro/LeuE7 DNA fragments, flanked by HindIII and BamHI
sites, were made by whole gene synthesis and cloned into the
HindIII and BamHI sites of pCDNA3. Using these constructs as
templates, a KpnI site was incorporated upstream and an EcoRI site
downstream, of the Pro/Leu E7 sequences by standard PCR methods.
The resulting fragments were cut with KpnI and EcoRI and cloned
into pCDNA3. These constructs were then used to make the secreted
E7 constructs with Pro or Lou codon modifications.
[0200] Firstly, using the whole gene synthesis method, DNA
fragments that included a secretory sequence flanked by KpnI and
BamHI sites were synthesized. As Pro and Leu occur in the secretory
sequence, individually modified secretory sequence fragments were
made for the different constructs. These fragments were digested
with KpnI and BamHI. Then, using the relevant Pro or Leu E7
construct as a template and a standard PCR protocol, a BamHI site
was introduced at the 5' end of the E7 sequence. The 3' EcoRI site
was retained. The resulting fragments were cut with BamHI and
EcoRI, purified, and ligated into pCDNA3. Following sequencing, the
plasmids were cut with KpnI and BamHI and ligated with the relevant
KpnI/BamHI secretory sequences. The resulting constructs were
sequenced and are denoted IgkS1-50 to IgkS1-59 (see TABLE 12 and
FIGS. 12 and 14 for sequence comparisons).
[0201] Secreted E7 Constructs with Predominantly Wild-Type Codon
Usage. Modified for Individual Codons
[0202] Constructs encoding a secreted form of oncogenic E7 (i.e.
wild-type E7 protein) were made by site-directed mutagenesis of the
plasmids encoding a secreted form of non-oncogenic E7. This was
done for constructs for codons for the following amino acids: Asp,
Cys, Glu, Gln, Gly, Ile, Ser, Thr and Val.
[0203] Site-directed mutagenesis was carried out using the
Quikchange II Site-directed Mutagenesis kit (Stratagene, La Jolla
Calif.) and appropriate PAGE (polyacrylamide gel
electrophoresis)-purified primers (Sigma) according to the
manufacturer's instructions. The pCDNA-kIgkX-nE7X series of
constructs were used as templates for the mutagenesis (i.e.
constructs IgkS1-13 to 24, IgkS1-27 to 29, IgkS1-34 to 43 and
IgkS1-46 to 49). The primers introduced the desired G21D, G24C,
G26E mutations.
[0204] The resulting constructs, IgkS2-13 to 24, IgkS2-27 to 29,
IgkS2-34 to 43 and IgkS2-46 to 49 (see Table 8, SEQ ID NOs: 1 to
29), have wild-type codon usage for the Igk secretory sequence and
E7 sequence with the exception that the codons for the relevant
amino acid were changed, and they encode oncogenic E7.
[0205] Linker Constructs
[0206] Constructs encoding the N-terminal Igk secretory sequence
followed by a linker sequence (XXGXGXX, where X is the relevant
amino acid for a particular construct and G is glycine) and the E7
protein were made for each of the following amino acids: Asn, Ala,
Lys, Arg, Phe, His and Tyr.
[0207] Fragments consisting of the Igk secretory sequence (with
mammalian consensus codon usage) and the linker sequences were made
by PCR using Taq polymerase and standard cycling conditions, as
recommended by the manufacturer.
[0208] The fragments were amplified from pCDNA3-kmcIgk-mcE7 using a
common forward primer
(5'TTGAATAGGTACCGCCGCCACCATGGAGACCGACACCCTCC3'; SEQ ID NO: 90) that
annealed to the KpnI site, the Kozak sequence and the beginning of
the Igk secretory sequence. The reverse primers were different for
each linker construct and annealed to the end of the Igk secretory
sequence (with mammalian consensus codon usage), introduced new
sequence that encoded the relevant linker sequence and a 3' BamHI
site.
[0209] The fragments were digested with KpnI/BamHI and were ligated
into KpnI/BamHI-cut pCDNA3-mcIgk-mcE7 (i.e. the Kozak sequence and
secretory sequence had been removed from the plasmid by digestion)
to make pCDNA3-mcIgk-linkerX-mcE7 (i.e., IgkS2-1 to 12, IgkS2-25
and 26, IgkS2-30 to 33 and IgkS2-44 and 45 as illustrated in Table
8, SEQ ID NOs: 30 to 49).
[0210] For Asn the fragments were also ligated into KpnI/BamHI-cut
pCDNA3-Igk-nE7Asn1/2 (i.e. IgkS1-11 and 12) to make
pCDNA3-mcIgk-linkerN1/2-nE7Asn1/2 (i.e., IgkS2-11b and IgkS2-12b,
see Table 12).
E7 Protein Expression
[0211] Cell Culture
[0212] CHO cells were cultured in DMEM (GIBCO from Invitrogen)
containing 10% foetal bovine serum (FBS) (DKSH), penicillin,
streptomycin and glutamine (GIBCO from Invitrogen) at 37.degree. C.
and 5% CO.sub.2. Cells were plated into 6-well plates at
3.times.10.sup.5/well, 24 hours prior to transfection. For each
transfection, 2 g of DNA was mixed with 50 .mu.L OptiMEM (GIBCO
from Invitrogen) and 4 .mu.L Plus reagent (Invitrogen) and
incubated at room temperature (RT) for 30 min. Lipofectamine
(Invitrogen; 5 .mu.L in 50 .mu.L OptiMEM) was added and the
complexes incubated at RT for 30 min. The cells were rinsed with
OptiMEM, 2 mL OptiMEM were added to each well, and the complexes
then added. The cells were incubated overnight at 37 C and 5%
CO.sub.2. The following morning the complexes were removed and 2 ml
of fresh DMEM containing 2% FBS added to each well.
[0213] Cell pellets and supernatants were collected about 40 h
after transfection. The cell pellets were resuspended in lysis
buffer (0.1% NP-40, 2 .mu.g/mL Aprotinin, 1 .mu.g/mL Leupeptin and
2 mM PMSF in PBS). Transfections were carried out in duplicate and
repeated. Control transfections, with empty vector (pCDNA3), were
also carried out.
[0214] Western Blotting
[0215] Western blots of the CHO cell supernatants or lysates were
carried out according to standard protocols. Briefly, this involved
firstly separating the samples by polyacrylamide gel
electrophoresis (PAGE). For cell lysates, 30 .mu.g of total protein
were loaded for each sample. For supernatants, 30 .mu.L of each was
loaded. The protein samples were boiled with SDS-PAGE loading
buffer for 10 mins before loading onto 12% SDS-PAGE gels and the
gels were run at 150-200V for approximately 1 h.
[0216] The separated proteins were then transferred from the gels
to PVDF membrane (100V for 1 h). The membranes were blocked with 5%
skim milk (in PBS/0.05% Tween 20 (PBS-T)) for 1 h at room
temperature and were then incubated with the primary antibody,
HPV-16 E7 Mouse Monoclonal Antibody (Zymed Laboratories) at a
concentration of 1:1000 in 5% skim milk (in PBS-T) overnight at
4.degree. C. Following washing of the membrane in PBS-T (3.times.10
min), secondary antibody, anti-mouse IgG (Sigma) in 5% skim milk,
was added and the membrane incubated at room temperature for 4 h.
The membranes were washed as before, incubated in a mixture
containing equal volumes of solution A (4.425 mL water, 50 .mu.L
luminol, 22 .mu.L p-coumaric and 500 .mu.L 1M Tris pH 8.5) and
solution B (4.5 mL water, 3 .mu.L 30% H.sub.2O.sub.2 and 500 .mu.L
1M Tris pH8.5) for 1 min, and then dried and wrapped in plastic
wrap. Film was exposed to the blots for various times (1 min, 3 min
or 10 min) and the film then developed.
Gene Gun Immunization Protocols
[0217] Plasmid Purification
[0218] All plasmids used for vaccination were grown in the
Escherichia coli strain DH5c and purified using the Nucleobond Maxi
Kit (Machery-Nagal). DNA concentration was quantitated
spectrophotometrically at 260 nm.
[0219] Preparation of DNA/Gold Cartridges
[0220] Coating of gold particles with plasmid DNA was performed as
described in the Biorad Helios Gene Gun System instruction manual
using a microcarrier loading quantity (MLQ) of 0.5 mg
gold/cartridge and a DNA loading ratio of 2 .mu.g DNA/mg gold. This
resulted in 1 .mu.g of DNA per prepared cartridge. In brief 50
.mu.L of 0.05M spermidine (Sigma) was added to 25 mg of 1.0 .mu.m
gold particles (Bio-Rad) and the spermidine/gold was sonicated for
3 seconds.. 50 .mu.g of plasmid DNA was then added, followed by the
dropwise addition of 100 .mu.L 1M CaCl.sub.2 while vortexing. The
mixture was allowed to precipitate at room temperature for 10 min,
then centrifuged to pellet the DNA/gold. The pellet was washed
three times with HPLC grade ethanol (Scharlau), before resuspension
in HPLC grade ethanol containing 0.5 mg/mL of polyvinylpyrrolidone
(PVP) (Bio-Rad). The gold/plasmid suspension was then coated onto
Tefzel tubing and 0.5 inch cartridges prepared.
[0221] Gene Gun Immunization of Mice
[0222] Groups of 8 female C57BL6/J (6-8 weeks old) (ARC, WA or
Monash Animal Services, VIC) were immunized on Day 0, Day21, Day 42
and Day 63 with the relevant DNA. The day before each immunization
the abdomen of each mouse was shaved and depilatory cream (Nair)
applied for 1 minute. DNA was delivered with the Helios gene gun
(Biorad) using a pressure of 400 psi. Mice were given 2 shots on
either side of the abdomen, with 1 .mu.g of DNA delivered per shot.
Serum was collected via intra-ocular bleed 2 days prior to initial
immunization and 2 weeks after each subsequent immunization (Day 2,
Day 35, Day 56 and Day 77).
[0223] ELISA to Measure E7 Immune Response
[0224] Nine peptides spanning the full-length of HPV16E7 (Frazer et
al., 1995) were used to measure the E7 antibody response. The
peptides were synthesised and purified to >70% purity by Auspep
(Melbourne). Peptides GF101 to 106 and GF108 to 109 described in
Frazer et al. were made. Note that instead of GF107, GF107a was
used: HYNIVTFCCKCDSTLRL.
[0225] GF102 D130, GF103 D5G/CSG/E10G and GF104E2G peptides, named
GF102n, GF103n and GF104n respectively, were also synthesised.
These peptides were used for the ELISA when measuring antibodies to
non-oncogenic E7 i.e. these peptides incorporate the mutations that
were made to make the E7 protein non-oncogenic.
[0226] Microtiter plates were coated overnight with 50 .mu.L of 10
.mu.g/mL E7 peptide per well. After coating, microtiter plates
(Maxisorp, Nunc) were washed two times with PBS/0.05% Tween 20
(PBS-T) and then blocked for two hours at 37.degree. C. with 100
.mu.L of 5% skim milk powder in PBS-T. After blocking, plates were
washed three times with PBS-T and 50 .mu.L of mouse sera at a
dilution of 1 in 100 was added for 2 hours at 37.degree. C. All
serum was assayed in duplicate wells. Plates were then washed three
times with PBS-T and 50 .mu.L of sheep anti-mouse IgG horseradish
peroxidise conjugate (Sigma) was added at a 1 in 1000 dilution.
After 1 hour plates were washed and 50 .mu.L of OPD substrate was
added. Absorbance was measured after 30 min and the addition of 25
.mu.L of 2.5 M HCl at 490 nm in a Multiskan EX plate reader
(Pathtech). Note controls were included: control primary antibody
for a positive control, secondary antibody only, and day 0
serum/serum from unimmunized mice as negative controls.
[0227] The immune response preferences of codons determined from
these experiments are tabulated in TABLE 1.
Example 2
Construction of Codon Modified Influenza a Virus (H5N1) Ha DNA for
Conferring an Enhanced Immune Response to H5N1 Ha
[0228] The wild-type nucleotide sequence of the influenza A virus,
HA gene for hemagglutinin (A/Hong Kong/213/03(H5N1), MDCK isolate,
embryonated chicken egg isolate) is shown in SEQ ID NO: 50 and
encodes the amino acid sequence shown in SEQ ID NO: 51. Several
codons within that sequence were mutated using the method described
in Example 1. Specifically, the method involved replacing codons of
the wild type nucleotide sequence with corresponding synonymous
codons having higher immune response preferences than the codons
they replaced, as represented in Table 1. An illustrative codon
modified nucleotide sequence comprising high immune response
preference codons is shown in SEQ ID NO: 52.
Example 3
Construction of Codon Modified Influenza a Virus (H3N1) DNA for
Conferring an Enhanced Immune Response to H3N1 Ha
[0229] The wild-type nucleotide sequence of the influenza A virus,
HA gene for hemagglutinin (A/swine/Korea/PZ72-1/2006(H3N1)) is
shown in SEQ ID NO: 53 and encodes the amino acid sequence shown in
SEQ ID NO: 54. Specifically, the method involved replacing codons
of the wild type nucleotide sequence with corresponding synonymous
codons having higher immune response preferences than the codons
they replaced, as represented in Table 1. An illustrative codon
modified nucleotide sequence comprising high immune response
preference codons is shown in SEQ ID NO: 55.
Example 4
Construction of Codon Modified Influenza a Virus (H5N1) Na DNA for
Conferring an Enhanced Immune Response to H5N1 Na
[0230] The wild-type nucleotide sequence of the influenza A virus,
NA gene for neuraminidase (A/Hong Kong/213/03(H5N1), NA gene
neuraminidase, MDCK isolate, embryonated chicken egg isolate) is
shown in SEQ ID NO: 56 and encodes the amino acid sequence shown in
SEQ ID NO: 57. Several codons within that sequence were mutated
using the method described in Example 1. Specifically, the method
involved replacing codons of the wild type nucleotide sequence with
corresponding synonymous codons having higher immune response
preferences than the codons they replaced, as represented in Table
1. An illustrative codon modified nucleotide sequence comprising
high immune response preference codons is shown in SEQ ID NO:
58.
Example 5
Construction of Codon Modified Influenza a Virus (H3N1) Na DNA for
Conferring an Enhanced Immune Response to H3N1 Na
[0231] The wild-type nucleotide sequence of the influenza A virus,
NA gene for neuraminidase (A/swine/MI/PU243/04(H3N1)) is shown in
SEQ ID NO: 59 and encodes the amino acid sequence shown in SEQ ID
NO: 60. Several codons within that sequence were mutated using the
method described in Example 1. Specifically, the method involved
replacing codons of the wild type nucleotide sequence with
corresponding synonymous codons having higher immune response
preferences than the codons they replaced, as represented in Table
1. An illustrative codon modified nucleotide sequence comprising
high immune response preference codons is shown in SEQ ID NO:
61.
Example 6
Construction of Codon Modified Hepatitis C Virus E1 (1AH77) DNA for
Conferring an Enhanced Immune Response to HCV E1 (1AH77)
[0232] The wild-type nucleotide sequence of the hepatitis C Virus
E1, (serotype 1A, isolate H77, from polyprotein nucleotide sequence
AF009606) is shown in SEQ ID NO: 62 and encodes the amino acid
sequence (NP 751920) shown in SEQ ID NO: 63. Several codons within
that sequence were mutated using the method described in Example 1.
Specifically, the method involved replacing codons of the wild type
nucleotide sequence with corresponding synonymous codons having
higher immune response preferences than the codons they replaced,
as represented in Table 1. An illustrative codon modified
nucleotide sequence comprising high immune response preference
codons is shown in SEQ ID NO: 64.
Example 7
Construction of Codon Modified Hepatitis C Virus E2 (1AH77) DNA for
Conferring an Enhanced Immune Response to HCV E2 (1AH77)
[0233] The wild-type nucleotide sequence of the hepatitis C Virus
E2, (serotype 1A, isolate H77, from polyprotein nucleotide sequence
AF009606) is shown in SEQ ID NO: 65 and encodes the amino acid
sequence (NP 751921) shown in SEQ ID NO: 66. Several codons within
that sequence were mutated using the method described in Example 1.
Specifically, the method involved replacing codons of the wild type
nucleotide sequence with corresponding synonymous codons having
higher immune response preferences than the codons they replaced,
as represented in Table 1. An illustrative codon modified
nucleotide sequence comprising high immune response preference
codons is shown in in SEQ ID NO: 67.
Example 8
Construction of Codon Modified Epstein--Barr Virus Type 1 Gp350 DNA
for Conferring an Enhanced Immune Response to EBV Type 1 Gp350
[0234] The wild-type nucleotide sequence of the Epstein--Barr
virus, EBV type 1 gp350 (Gene BLLF1, strand 77142-79865) is shown
in SEQ ID NO: 68 and encodes amino acid sequence (CAD53417) shown
in SEQ ID NO: 69. Several codons within that sequence were mutated
using the method described in Example 1. Specifically, the method
involved replacing codons of the wild type nucleotide sequence with
corresponding synonymous codons having higher immune response
preferences than the codons they replaced, as represented in Table
1. An illustrative codon modified nucleotide sequence comprising
high immune response preference codons is shown in SEQ ID NO:
70.
Example 9
Construction of Codon Modified Epstein--Barr Virus Type 2 Gp350 DNA
for Conferring an Enhanced Immune Response to EBV Type 2 Gp350
[0235] The wild-type nucleotide sequence of the Epstein--Barr
virus, EBV type 2 gp350 (Gene BLLF1, strand 77267-29936) is shown
in SEQ ID NO: 71 and encodes the amino acid sequence (YP 001129462)
shown in SEQ ID NO: 72. Several codons within that sequence were
mutated using the method described in Example 1. Specifically, the
method involved replacing codons of the wild type nucleotide
sequence with corresponding synonymous codons having higher immune
response preferences than the codons they replaced, as represented
in Table 1. An illustrative codon modified nucleotide sequence
comprising high immune response preference codons is shown in SEQ
ID NO: 73.
Example 10
Construction of Codon Modified Herpes Simplex Virus 2 Glycoprotein
B DNA for Conferring an Enhanced Immune Response to HSV-2
Glycoprotein B
[0236] The wild-type nucleotide sequence of the Herpes Simplex
virus 2, glycoprotein B strain H052 (genome strain NC 001798) is
shown in SEQ ID NO: 74 and encodes the amino acid sequence
(CAB06752) shown in SEQ ID NO: 75. Several codons within that
sequence were mutated using the method described in Example 1.
Specifically, the method involved replacing codons of the wild type
nucleotide sequence with corresponding synonymous codons having
higher immune response preferences than the codons they replaced,
as represented in Table 1. An illustrative codon modified
nucleotide sequence comprising high immune response preference
codons is shown in SEQ ID NO: 76.
Example 11
Construction of Codon Modified Herpes Simplex Virus 2 Glycoprotein
D DNA for Conferring an Enhanced Immune Response to HSV-2
Glycoprotein D
[0237] The wild-type nucleotide sequence of the Herpes Simplex
virus 2, glycoprotein D strain HG52 (genome strain NC 001798) is
shown in SEQ ID NO: 77 and encodes the amino acid sequence (NP
044536) shown in SEQ ID NO: 78. Several codons within that sequence
were mutated using the method described in Example 1. Specifically,
the method involved replacing codons of the wild type nucleotide
sequence with corresponding synonymous codons having higher immune
response preferences than the codons they replaced, as represented
in Table 1. An illustrative codon modified nucleotide sequence
comprising high immune response preference codons is shown in SEQ
ID NO: 79.
Example 12
Optimised E7 and HSV-2 Constructs Design and Synthesis of Optimal
and Least Optimal E7 Construct
[0238] One de-optimized (W) and three optimized (01-03) E7
constructs were designed and made using the codon preferences
summarized in Table 1 ("the Immune Coricode table"). The least
favourable codons were used for construct W. For the first
optimized construct, 01, whose sequence is shown in SEQ ID NO: 81,
all of the codons were modified to those codons determined most
optimal. 02, whose sequence is shown in SEQ ID NO: 82, is an
alternative optimized construct which involved changing all Ala to
GCT; Arg CGG and AGO to CGA and AGA, respectively; Glu to GAA; Gly
to GGA; Ile to ATC; all Leu to CTG; Phe to TTT, Pro to CCT or CCC,
Ser to TCG, Thr to ACG; and all Val except GTG to GTC. The O2
modifications avoided, with the exception of Leu and Ile, changing
codons to mammalian consensus-preferred codons. For O3, whose
sequence is shown in SEQ ID NO: 83, only certain amino acids for
which particularly distinct differences were observed between
codons, and for which the optimal codon(s) was not also a mammalian
consensus preferred codon, were modified. In particular, in O3 all
non-preferred Gly, Leu, Pro, Ser and Thr codons were changed to
GGA, CTC, CCT, TCG and ACG, respectively, and where a preferred
codon was already used it was not altered. Codons for other amino
acids in O3 were not modified.
Humoral and Cellular Responses to Biolistic Immunization with the
Optimal and Least Optimal E7 Constructs
[0239] As may be seen in FIG. 18 (a) all three optimized constructs
(O1 to 03) gave rise to significantly larger antibody responses
than the wild-type construct as measured by both the peptide ELISA
and a GST-E7 protein ELISA. The amplitudes of the response were not
statistically different between the three optimized constructs. The
de-optimized construct, W, whose sequence is shown in SEQ ID NO:
84, gave a very low antibody response, appearing slightly lower but
not statistically different from the wild-type (wt) codon usage
(CU) construct, whose sequence is shown in SEQ ID NO: 80. From the
IFN-.gamma. ELISPOT experiments, a representative example of which
is shown in FIG. 18, it appears that the codon preferences for
maximizing the antibody response are similar to those required for
maximising the T cell response: the de-optimized construct W failed
to give a measurable response in the IFN-.gamma. ELISPOT assay and
two of the optimized constructs (O2 and O3) gave statistically
significantly larger responses than the wild-type CU construct.
Over the three repeats the responses to O2 and O3 were not
statistically different from each other. Unexpectedly, and in
contrast to the antibody trend, in two of the three repeat
experiments O1 gave a similar cellular response to the wt CU
construct, which was less than that achieved by the O2 or O3
constructs.
Humoral and Cellular Responses to Immunization by Intradermal
Injection with the Optimal and Least Optimal E7 Constructs
[0240] The humoral and cellular responses of mice to the optimized,
wild-type CU and de-optimized constructs delivered by intradermal
injection were also measured and the results are summarized in FIG.
19. In general, similar trends were observed for intradermal
injection as for biolistic delivery.
[0241] From the E7 protein ELISA, it is apparent that the three
optimized constructs, O1-O3, were all significantly better at
generating antibodies than the wild-type construct and that the
de-optimized construct gave a very low antibody response similar to
wild-type. The optimized constructs all gave rise to significantly
more spots in the IFN-.gamma. ELISPOT than the wild-type construct
and the de-optimized construct failed to give rise to a measurable
response.
[0242] The amplitudes of the antibody responses to gene gun
immunization were larger than that for the intradermally (ID)
delivered vaccines, despite the ID immunization delivering more
than five times the dose.
Design and Synthesis of Optimal and Least Optimal HSV-2
Constructs
[0243] Three optimized (O1-O3; whose sequences are shown in SEQ ID
NO: 86-88, respectively) and a de-optimized construct (W; whose
sequence is shown in SEQ ID NO: 88) encoding full-length
glycoprotein D from Herpes Simplex Virus 2 (gD2) were prepared. A
control construct pCDNA3-gD2 with wt CU was also made. Wild-type
CU, whose sequence is shown in SEQ ID NO: 85, is close to MC
CU.
Humoral Responses to Biolistic and Intradermal Immunization with
the Optimal and Least Optimal gD2 Constructs
[0244] C57Bl/6 mice were immunized in two groups (8 mice/construct;
used intradermal injection (ID) and gene gun delivery) using the
same immunization protocol as for the E7 constructs.
[0245] Group 1 included pCDNA3-gD2 and pCDNA3-gD2 O1. Group 2
included pCDNA3-gD2, pCDNA3-gD2 O2, pCDNA3-gD2 O3, and pCDNA3-gD2
W.
[0246] Antibody responses were measured by an ELISA using plates
coated with CHO cell supernatant containing C-terminally His tagged
and truncated gD2. The truncation is at amino acid residue 331 and
removes the transmembrane region resulting in the protein being
secreted into the medium. Control ELISA plates coated with
supernatant from CHO cells transfected with empty vector were used
as a control.
[0247] For both biolistic and intradermal injection delivery routes
it was found that the three optimized constructs generated similar
levels of antibodies as the wt CU gD2 construct (FIG. 20). The
de-optimized construct, W gD2, was very poor at generating
antibodies, particularly when delivered by intradermal injection.
The two delivery methods resulted in similar levels of
antibodies.
[0248] To date, there are no DNA vaccines on the market for the
treatment or prevention of disease in humans. There is a need to
maximize the immune responses generated by DNA vaccines and the
present invention discloses ways of enhancing efficacy of DNA
vaccines by using codons that have a higher preference for
producing an immune response.
[0249] The study described in this Example has validated the Immune
Coricode table by applying it to optimization or de-optimization of
the HPV16 E7 and HSV-2 glycoprotein D (gD2) genes and demonstrating
that this does enhance or reduce, respectively, the antibody or
cellular response to biolistic delivery of these genes to mammals
such as mice.
Material and Methods
[0250] ELISPOT Assay
[0251] For the IFN-.gamma. ELISPOTs, mice were immunized twice, at
days 0 and 21, and the spleens were collected 3 weeks after the
second immunization.
[0252] Intradermal Injection Protocol
[0253] The timing and frequency of the immunizations by intradermal
injection were the same as for gene gun immunization. At each
immunization 5 .mu.g of DNA was injected per ear i.e. a total of 10
.mu.g was administered per immunization per mouse. Hair removal
prior to immunization was not necessary. The timing of bleeds and
spleen collection was the same as for the gene gun immunized
mice.
[0254] GST-E7 ELISA
[0255] The GST-E7 ELISA was carried out in the same way as the
peptide ELISA with the exception that the plates were coated
overnight with 50 .mu.L of 10 .mu.g/mL GST-tagged E7 protein
(kindly provided by the Frazer group from the Diamantina Institute,
The University of Queensland, Brisbane).
[0256] HSV-2 gD ELISA
[0257] This ELISA was carried out in the same way as the E7 ELISAs
with the exception that the plates were coated with supernatant
from CHO cells transfected with a vector encoding C-terminally
His-tagged and truncated gD2 protein. Control plates coated with
supernatant from CHO cells transfected with empty vector were also
used.
[0258] Detection of HPV-Specific Responses
[0259] For the detection of HPV-specific responses, 96-well filter
ELISPOT plates (Millipore) were coated overnight with 10 .mu.g/mL
HPV GST-tagged E7 protein in 0.1 M NaHCO.sub.3. For the detection
of total IgG secreting cells, 96-well filter ELISPOT plates were
coated overnight with 2 .mu.g/mL goat anti-mouse Ig (Sigma) in PBS
without MgCl.sub.2 and CaCl.sub.2. After coating, plates were
washed once with complete DMEM without FCS and then blocked with
complete DMEM supplemented with 10% FCS for one hour at 37.degree.
C. Cultured mouse spleen cells were washed and added to ELISPOT
plates at 10.sup.6 cells/100 .mu.L. For the detection of
HPV-specific memory B cells, plates were incubated overnight at
37.degree. C. and for measuring total IgG cells, plates were
incubated for 1 hour at 37.degree. C. For detection, we used
biotinylated goat anti-mouse IgG (Sigma) in PBS-T/1% FCS, followed
by 5 .mu.g/mL HRP-conjugated avidin (Pierce) and developed using
3-amino-9-ethylcarbozole (Sigma). Developed plates were counted
using an automated ELISPOT plate counter.
[0260] E7 IFN-.gamma. ELISPOT
[0261] 96-well filter plates (Millipore) were coated overnight with
4 .mu.g/mL of monoclonal antibody (AN18; Mabtech). After coating,
plates were washed once with complete RPMI and blocked for 2 hours
with complete RPMI with 10% foetal calf serum (FCS; CSL Ltd). Mouse
spleens were made into single cell suspensions and treated with ACK
lysis buffer, washed and resuspended at a concentration of 10'
cells/mL. Spleen cells (10.sup.6/well) were added to each well
followed by the addition of complete RPMI supplemented with
recombinant hIL-2 (ProSpec-Tany TechnoGene Ltd) and peptide to a
final concentration of 10 IU/well and 1 .mu.g/mL, respectively.
Medium containing hIL-2 without peptide was added to control wells.
Plates were incubated for approximately 18 hours at 37.degree. C.
in 5-8% CO.sub.2.
[0262] After overnight incubation, cells were lysed by rinsing the
plates in tap water and then washed six times in PBS/0.05% Tween 20
(PBS-T). For detection, biotinylated detection mAb (R4-6A2;
Mabtech) in PBS-T/2% FCS was added, followed by horse radish
peroxidase (HRP)-conjugated strepavidin and DAB (Sigma). Developed
plates were counted using an automated ELISPOT plate counter.
[0263] The disclosure of every patent, patent application, and
publication cited herein is hereby incorporated herein by reference
in its entirety.
[0264] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
[0265] Throughout the specification the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
Those of skill in the art will therefore appreciate that, in light
of the instant disclosure, various modifications and changes can be
made in the particular embodiments exemplified without departing
from the scope of the present invention. All such modifications and
changes are intended to be included within the scope of the
appended claims.
BIBLIOGRAPHY
[0266] Ausubel, F. M. (Ed.) 2007. Current Protocols in Molecular
Biology. Ebook (http://www.mrw.interscience.wiley.com/emrw/978
0471142720/cp/cpmb/toc). [0267] Edmonds, C., and Vousden, K. H.
(1989). A point mutational analysis of human papillomavirus type 16
E7 protein. Journal of Virology. 63: 2650-2656. [0268] Frazer, I.
H., Leippe, D. M., Dunn, L. A., Leim, A., Tindle, R. W., Fernando,
G. J., Phelps, W. C., and Lambert, P. F. (1995). Immunological
responses in human papillomavirus 16 E6/E7 transgenic mice to E7
protein correlate with the presence of skin disease. Cancer
Research. 55: 2635-2639. [0269] Heck, D. V., Yee, C. L., Howley, P.
M., and Munger, K. (1992). Efficiency of binding the retinoblastoma
protein correlates with the transforming capacity of the E7
oncoproteins of the human papillomaviruses. PNAS 89: 4442-4446.
[0270] Liu, W. J., Gao, F., Zhao, K N., Zhao, W., Fernando, G. J,
Thomas, R. And Frazer, I. H. (2002). Codon modified human
papillomavirus type 16 E7 DNA vaccine enhances cytotoxic
T-lymphocyte induction and anti-tumour activity. Virology 301:
43-52. [0271] Smith, H. O., Hutchison III, C. A., Pfannkoch, C. and
Venter, J. C. (2003). Generating a synthetic genome by whole genome
assembly: 4.times.174 bacteriophage from synthetic
oligonucleotides. PNAS. 100 (26): 15440-15445.
Sequence CWU 1
1
1541387DNAArtificial sequencePlasmid sequence 1ggtaccgccg
ccaccatgga gacagataca ctcctgctat gggtactgct gctctgggtt 60ccaggttcca
ctggtgatgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgatagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggatag agcccattac 240aatattgtaa ccttttgttg caagtgtgat
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagata ttcgtacttt
ggaagatctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 3872387DNAArtificial sequencePlasmid sequence
2ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagacacac ctacattgca tgaatatatg
120ttagacttgc aaccagagac aactgacctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg acgaaataga cggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 3873387DNAArtificial sequencePlasmid sequence
3ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg taagtgtgac
tctacgcttc ggttgtgtgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgtccc 360atctgttctc
agaagcccta agaattc 3874387DNAArtificial sequencePlasmid sequence
4ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgctatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgctg caagtgcgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 3875387DNAArtificial sequencePlasmid sequence
5ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgagtatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgagataga tggtccagct ggacaagcag
agccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaggacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 3876387DNAArtificial sequencePlasmid sequence
6ggtaccgccg ccaccatgga aacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagaaac aactgatctc tactgttatg aacaattaaa
tgacagctca 180gaagaagaag atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 3877387DNAArtificial sequencePlasmid sequence
7ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc agccagagac aactgatctc tactgttatg agcagttaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaggcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acagagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 3878387DNAArtificial sequencePlasmid sequence
8ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
aaaagcccta agaattc 3879387DNAArtificial sequencePlasmid sequence
9ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccagggtcca ctggggacgg atccatgcat ggggatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tgggccagct gggcaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggga cactagggat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38710387DNAArtificial sequencePlasmid sequence
10ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggatcca ctggagacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggaccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggaa cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38711387DNAArtificial sequencePlasmid sequence
11ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggtgatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggtcaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggta cactaggtat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38712387DNAArtificial sequencePlasmid sequence
12ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggctcca ctggcgacgg atccatgcat ggcgatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggcccagct ggccaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggcat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38713387DNAArtificial sequencePlasmid sequence
13ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatatagtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca tacgtacttt
ggaagacctg ttaatgggca cactaggaat agtgtgcccc 360atatgctctc
agaagcccta agaattc 38714387DNAArtificial sequencePlasmid sequence
14ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaattga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atttgctctc
agaagcccta agaattc 38715387DNAArtificial sequencePlasmid sequence
15ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaatcga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatatcgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca tccgtacttt
ggaagacctg ttaatgggca cactaggaat cgtgtgcccc 360atctgctctc
agaagcccta agaattc 38716387DNAArtificial sequencePlasmid sequence
16ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggtagta ctggtgacgg aagtatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagtagt 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
agtacgcttc ggttgtgcgt acaaagtaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgcagtc
agaagcccta agaattc 38717387DNAArtificial sequencePlasmid sequence
17ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggtagca ctggtgacgg aagcatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagcagc 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
agcacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgcagcc
agaagcccta agaattc 38718387DNAArtificial sequencePlasmid sequence
18ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcga ctggtgacgg atcgatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgactcgtcg 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tcgacgcttc ggttgtgcgt acaatcgaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctcgc
agaagcccta agaattc 38719387DNAArtificial sequencePlasmid sequence
19ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcaa ctggtgacgg atcaatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgactcatca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tcaacgcttc ggttgtgcgt acaatcaaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctcac
agaagcccta agaattc 38720387DNAArtificial sequencePlasmid sequence
20ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcta ctggtgacgg atctatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgactcttct 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaatctaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38721387DNAArtificial sequencePlasmid sequence
21ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgactcctcc 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tccacgcttc ggttgtgcgt acaatccaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctccc
agaagcccta agaattc 38722387DNAArtificial sequencePlasmid sequence
22ggtaccgccg ccaccatgga gacggacacg ctcctgctat gggtactgct gctctgggtt
60ccaggttcca cgggtgacgg atccatgcat ggagatacgc ctacgttgca tgaatatatg
120ttagatttgc aaccagagac gacggatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa cgttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcacg 300cacgtagaca ttcgtacgtt
ggaagacctg ttaatgggca cgctaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38723387DNAArtificial sequencePlasmid sequence
23ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca caggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aacagatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa cattttgttg caagtgtgac
tctacacttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacatt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38724387DNAArtificial sequencePlasmid sequence
24ggtaccgccg ccaccatgga gactgacact ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatactc ctactttgca tgaatatatg
120ttagatttgc aaccagagac tactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ctttttgttg caagtgtgac
tctactcttc ggttgtgcgt acaaagcact 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca ctctaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38725387DNAArtificial sequencePlasmid sequence
25ggtaccgccg ccaccatgga gaccgacacc ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ccggtgacgg atccatgcat ggagataccc ctaccttgca tgaatatatg
120ttagatttgc aaccagagac caccgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacccttc ggttgtgcgt acaaagcacc 300cacgtagaca ttcgtacctt
ggaagacctg ttaatgggca ccctaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38726387DNAArtificial sequencePlasmid sequence
26ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtgctgct gctctgggtg
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtga ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt gcaaagcaca 300cacgtggaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38727387DNAArtificial sequencePlasmid sequence
27ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggta
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtatgcccc 360atctgctctc
agaagcccta agaattc 38728387DNAArtificial sequencePlasmid sequence
28ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggttctgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtta ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt tcaaagcaca 300cacgttgaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtttgcccc 360atctgctctc
agaagcccta agaattc 38729387DNAArtificial sequencePlasmid sequence
29ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtcctgct gctctgggtc
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtca ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt ccaaagcaca 300cacgtcgaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtctgcccc 360atctgctctc
agaagcccta agaattc 38730408DNAArtificial sequencePlasmid linker
sequence 30ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgacgc ggcgggcgcg ggcgcggcgg gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc gcgcccacta
caacatcgtg accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg
tgcagagcac ccacgtggac atccgcaccc tggaggacct gctgatgggc
360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40831408DNAArtificial sequencePlasmid linker sequence 31ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgacgc agcaggcgca ggcgcagcag gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 40832408DNAArtificial
sequencePlasmid linker sequence 32ggtaccgccg ccaccatgga gaccgacacc
ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca ccggcgacgc tgctggcgct
ggcgctgctg gatccatgca cggcgacacc 120cccaccctgc acgagtacat
gctggacctg cagcccgaga ccaccgacct gtactgctac 180gagcagctca
acgacagcag cgaggaggag gacgagatcg acggccccgc cggccaggcc
240gagcccgacc gcgcccacta caacatcgtg accttctgct gcaagtgcga
cagcaccctg 300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc
tggaggacct gctgatgggc 360accctgggca tcgtgtgccc catctgctcc
cagaagccct aagaattc 40833408DNAArtificial sequencePlasmid linker
sequence 33ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgacgc cgccggcgcc ggcgccgccg gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc
gcgcccacta caacatcgtg accttctgct gcaagtgcga cagcaccctg
300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc tggaggacct
gctgatgggc 360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40834408DNAArtificial sequencePlasmid linker sequence 34ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgacag gaggggcagg ggcaggaggg gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 40835408DNAArtificial
sequencePlasmid linker sequence 35ggtaccgccg ccaccatgga gaccgacacc
ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca ccggcgacag aagaggcaga
ggcagaagag gatccatgca cggcgacacc 120cccaccctgc acgagtacat
gctggacctg cagcccgaga ccaccgacct gtactgctac 180gagcagctca
acgacagcag cgaggaggag gacgagatcg acggccccgc cggccaggcc
240gagcccgacc gcgcccacta caacatcgtg accttctgct gcaagtgcga
cagcaccctg 300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc
tggaggacct gctgatgggc 360accctgggca tcgtgtgccc catctgctcc
cagaagccct aagaattc 40836408DNAArtificial sequencePlasmid linker
sequence 36ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgaccg gcggggccgg ggccggcggg gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc gcgcccacta
caacatcgtg accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg
tgcagagcac ccacgtggac atccgcaccc tggaggacct gctgatgggc
360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40837408DNAArtificial sequencePlasmid linker sequence 37ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgaccg acgaggccga ggccgacgag gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 40838408DNAArtificial
sequencePlasmid linker sequence 38ggtaccgccg ccaccatgga gaccgacacc
ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca ccggcgaccg tcgtggccgt
ggccgtcgtg gatccatgca cggcgacacc 120cccaccctgc acgagtacat
gctggacctg cagcccgaga ccaccgacct gtactgctac 180gagcagctca
acgacagcag cgaggaggag gacgagatcg acggccccgc cggccaggcc
240gagcccgacc gcgcccacta caacatcgtg accttctgct gcaagtgcga
cagcaccctg 300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc
tggaggacct gctgatgggc 360accctgggca tcgtgtgccc catctgctcc
cagaagccct aagaattc 40839408DNAArtificial sequencePlasmid linker
sequence 39ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgaccg ccgcggccgc ggccgccgcg gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc gcgcccacta
caacatcgtg accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg
tgcagagcac ccacgtggac atccgcaccc tggaggacct gctgatgggc
360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40840408DNAArtificial sequencePlasmid linker sequence 40ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgacaa taatggcaat ggcaataatg gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 40841408DNAArtificial
sequencePlasmid linker sequence 41ggtaccgccg ccaccatgga gaccgacacc
ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca ccggcgacaa caacggcaac
ggcaacaacg gatccatgca cggcgacacc 120cccaccctgc acgagtacat
gctggacctg cagcccgaga ccaccgacct gtactgctac 180gagcagctca
acgacagcag cgaggaggag gacgagatcg acggccccgc cggccaggcc
240gagcccgacc gcgcccacta caacatcgtg accttctgct gcaagtgcga
cagcaccctg 300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc
tggaggacct gctgatgggc 360accctgggca tcgtgtgccc catctgctcc
cagaagccct aagaattc 40842408DNAArtificial sequencePlasmid linker
sequence 42ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgacca tcatggccat ggccatcatg gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc gcgcccacta
caacatcgtg accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg
tgcagagcac ccacgtggac atccgcaccc tggaggacct gctgatgggc
360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40843408DNAArtificial sequencePlasmid linker sequence 43ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgacca ccacggccac ggccaccacg gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 40844408DNAArtificial
sequencePlasmid linker sequence 44ggtaccgccg ccaccatgga gaccgacacc
ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca ccggcgacaa gaagggcaag
ggcaagaagg gatccatgca cggcgacacc 120cccaccctgc acgagtacat
gctggacctg cagcccgaga ccaccgacct gtactgctac 180gagcagctca
acgacagcag cgaggaggag gacgagatcg acggccccgc cggccaggcc
240gagcccgacc gcgcccacta caacatcgtg accttctgct gcaagtgcga
cagcaccctg 300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc
tggaggacct gctgatgggc 360accctgggca tcgtgtgccc catctgctcc
cagaagccct aagaattc 40845408DNAArtificial sequencePlasmid linker
sequence 45ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgacaa aaaaggcaaa ggcaaaaaag gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc gcgcccacta
caacatcgtg accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg
tgcagagcac ccacgtggac atccgcaccc tggaggacct gctgatgggc
360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40846408DNAArtificial sequencePlasmid linker sequence 46ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgactt ttttggcttt ggcttttttg gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 40847408DNAArtificial
sequencePlasmid linker sequence 47ggtaccgccg ccaccatgga gaccgacacc
ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca ccggcgactt cttcggcttc
ggcttcttcg gatccatgca cggcgacacc 120cccaccctgc acgagtacat
gctggacctg cagcccgaga ccaccgacct gtactgctac 180gagcagctca
acgacagcag cgaggaggag gacgagatcg acggccccgc cggccaggcc
240gagcccgacc gcgcccacta caacatcgtg accttctgct gcaagtgcga
cagcaccctg 300cgcctctgcg tgcagagcac ccacgtggac atccgcaccc
tggaggacct gctgatgggc 360accctgggca tcgtgtgccc catctgctcc
cagaagccct aagaattc 40848408DNAArtificial sequencePlasmid linker
sequence 48ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt gggtgctgct
gctctgggtg 60cccggctcca ccggcgacta ttatggctat ggctattatg gatccatgca
cggcgacacc 120cccaccctgc acgagtacat gctggacctg cagcccgaga
ccaccgacct gtactgctac 180gagcagctca acgacagcag cgaggaggag
gacgagatcg acggccccgc cggccaggcc 240gagcccgacc gcgcccacta
caacatcgtg accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg
tgcagagcac ccacgtggac atccgcaccc tggaggacct gctgatgggc
360accctgggca tcgtgtgccc catctgctcc cagaagccct aagaattc
40849408DNAArtificial sequencePlasmid linker sequence 49ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgacta ctacggctac ggctactacg gatccatgca cggcgacacc
120cccaccctgc acgagtacat gctggacctg cagcccgaga ccaccgacct
gtactgctac 180gagcagctca acgacagcag cgaggaggag gacgagatcg
acggccccgc cggccaggcc 240gagcccgacc gcgcccacta caacatcgtg
accttctgct gcaagtgcga cagcaccctg 300cgcctctgcg tgcagagcac
ccacgtggac atccgcaccc tggaggacct gctgatgggc 360accctgggca
tcgtgtgccc catctgctcc cagaagccct aagaattc 408501707DNAInfluenza A
Virus 50atggagaaaa tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga
tcagatttgc 60attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga
aaagaacgtt 120actgttacac atgcccaaga catactggaa aagacacaca
acgggaagct ctgcgatcta 180gatggagtga agcctctaat tttgagagat
tgtagtgtag ctggatggct cctcggaaac 240ccaatgtgtg acgaattcat
caatgtgccg gaatggtctt acatagtgga gaaggccaat 300ccagccaatg
acctctgtta cccaggggat ttcaacgact atgaagaatt gaaacaccta
360ttgagcagaa taaaccattt tgagaaaatt cagatcatcc ccaaaaattc
ttggtccagt 420catgaagcct cattaggggt gagctcagca tgtccatacc
aaggaaagtc ctcctttttc 480aggaatgtgg tatggcttat caaaaagaac
aatgcatacc caacaataaa gaggagctac 540aataatacca accaagaaga
tcttttggta ttgtggggga ttcaccatcc taatgatgcg 600gcagagcaga
ctaggctcta tcaaaaccca accacctaca tttccgttgg gacatcaaca
660ctaaaccaga gattggtacc aaaaatagct actagatcca aagtaaacgg
gcaaaatgga 720aggatggagt tcttctggac aattttaaaa ccgaatgatg
caatcaactt cgagagcaat 780ggaaatttca ttgctccaga atatgcatac
aaaattgtca agaaagggga ctcagcaatt 840atgaaaagtg aattggaata
tggtaactgc aacaccaagt gtcaaactcc aatgggggcg 900ataaactcta
gtatgccatt ccacaatata caccctctca ccatcgggga atgccccaaa
960tatgtgaaat caaacagatt agtccttgcg actgggctca gaaatagccc
tcaaagagag 1020agaagaagaa aaaagagagg attatttgga gctatagcag
gttttataga gggaggatgg 1080cagggaatgg tagatggttg gtatgggtac
caccatagca atgagcaggg gagtgggtac 1140gctgcagaca aagaatccac
tcaaaaggca atagatggag tcaccaataa ggtcaactcg 1200atcattgaca
aaatgaacac tcagtttgag gccgttggaa gggaatttaa taacttagaa
1260aggagaatag agaatttaaa caagaagatg gaagacggat tcctagatgt
ctggacttat 1320aatgctgaac ttctggttct catggaaaat gagagaactc
tagactttca tgactcaaat 1380gtcaagaacc tttacgacaa ggtccgacta
cagcttaggg ataatgcaaa ggagctgggt 1440aacggttgtt tcgagttcta
tcacaaatgt gataatgaat gtatggaaag tgtaagaaac 1500ggaacgtatg
actacccgca gtattcagaa gaagcaagac taaaaagaga ggaaataagt
1560ggagtaaaat tggagtcaat aggaacttac caaatactgt caatttattc
tacagtggcg 1620agttccctag cactggcaat catggtagct ggtctatctt
tatggatgtg ctccaatggg 1680tcgttacaat gcagaatttg catttaa
170751568PRTInfluenza A Virus 51Met Glu Lys Ile Val Leu Leu Phe Ala
Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr
His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu
Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys
Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro
Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile
Val 85 90 95 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly
Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg
Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Asn Ser
Trp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala Cys
Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val
Trp Leu Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile 165 170 175 Lys Arg
Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190
Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln 195
200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln
Arg 210 215 220 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly
Gln Asn Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys
Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile
Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser
Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr
Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro
Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315
320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly
Ala Ile 340 345 350 Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val
Asp Gly Trp Tyr 355 360 365 Gly Tyr His His Ser Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Lys 370 375 380 Glu Ser Thr Gln Lys Ala Ile Asp
Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 Ile Ile Asp Lys Met
Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe 405 410 415 Asn Asn Leu
Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430 Gly
Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 435 440
445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450 455 460 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu
Leu Gly 465 470 475 480 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu 485 490 495 Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Gln Tyr Ser Glu Glu Ala 500 505 510 Arg Leu Lys Arg Glu Glu Ile
Ser Gly Val Lys Leu Glu Ser Ile Gly 515 520 525 Thr Tyr Gln Ile Leu
Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu Ala Ile
Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555 560
Ser Leu Gln Cys Arg Ile Cys Ile 565 521707DNAArtificial
sequenceCodon modified Influenza A Virus sequence 52atggaaaaaa
tcgtgctgct gttcgctatc gtctcgctgg tcaaatcgga tcagatctgc 60atcggatacc
atgctaacaa ctcgacggaa caggtcgaca cgatcatgga aaagaacgtc
120acggtcacgc atgctcaaga catcctggaa aagacgcaca acggaaagct
gtgcgatctg 180gatggagtga agcctctgat cctgagagat tgttcggtcg
ctggatggct gctgggaaac 240cccatgtgtg acgaatttat caatgtgccc
gaatggtcgt acatcgtgga aaaggctaat 300cccgctaatg acctgtgtta
ccccggagat tttaacgact atgaagaact gaaacacctg 360ctgtcgagaa
tcaaccattt cgaaaaaatc cagatcatcc ccaaaaattc gtggtcgtcg
420catgaagctt cgctgggagt gtcgtcggct tgtccctacc aaggaaagtc
gtcgttcttt 480agaaatgtgg tctggctgat caaaaagaac aatgcttacc
ccacgatcaa gagatcgtac 540aataatacga accaagaaga tctgctggtc
ctgtggggaa tccaccatcc taatgatgct 600gctgaacaga cgagactgta
tcaaaacccc acgacgtaca tctcggtcgg aacgtcgacg 660ctgaaccaga
gactggtccc caaaatcgct acgagatcga aagtcaacgg acaaaatgga
720agaatggaat ttttttggac gatcctgaaa cccaatgatg ctatcaactt
tgaatcgaat 780ggaaatttta tcgctcccga atatgcttac aaaatcgtca
agaaaggaga ctcggctatc 840atgaaatcgg aactggaata tggaaactgc
aacacgaagt gtcaaacgcc catgggagct 900atcaactcgt cgatgccctt
tcacaatatc caccctctga cgatcggaga atgccccaaa 960tatgtgaaat
cgaacagact ggtcctggct acgggactga gaaattcgcc tcaaagagaa
1020agaagaagaa aaaagagagg actgttcgga gctatcgctg gattcatcga
aggaggatgg 1080cagggaatgg tcgatggatg gtatggatac caccattcga
atgaacaggg atcgggatac 1140gctgctgaca aagaatcgac gcaaaaggct
atcgatggag tcacgaataa ggtcaactcg 1200atcatcgaca aaatgaacac
gcagttcgaa gctgtcggaa gagaattcaa taacctggaa 1260agaagaatcg
aaaatctgaa caagaagatg gaagacggat ttctggatgt ctggacgtat
1320aatgctgaac tgctggtcct gatggaaaat gaaagaacgc tggacttcca
tgactcgaat 1380gtcaagaacc tgtacgacaa ggtccgactg cagctgagag
ataatgctaa ggaactggga 1440aacggatgtt ttgaatttta tcacaaatgt
gataatgaat gtatggaatc ggtcagaaac 1500ggaacgtatg actaccccca
gtattcggaa gaagctagac tgaaaagaga agaaatctcg 1560ggagtcaaac
tggaatcgat cggaacgtac caaatcctgt cgatctattc gacggtggct
1620tcgtcgctgg ctctggctat
catggtcgct ggactgtcgc tgtggatgtg ctcgaatgga 1680tcgctgcaat
gcagaatctg catctaa 1707531701DNAInfluenza A Virus 53atgaagacta
tcattgctct gagctacatt ttatgtctgg tcttcgctca aaaacttccc 60cgaaatgaca
acagcacggc aacgctgtgc ttgggacacc atgcagtgtc aaacggaaca
120ctagtgaaaa caatcacgaa tgaccaaatt gaagtgacta atgctactga
attggttcag 180agttcctcaa caggtagaat atgtgaccga cctcatcgaa
tccttgatgg ggaaaactgc 240acactgatag atgctctctt gggagaccct
cattgtgata gtttccaaaa caaggaatgg 300gacctttttg tagaacgcag
cacagcttac agcgactgtt acccttatga tgtgccggat 360tatgcctccc
ttaggtcact agttgcctca tccggcaccc tggagtttaa cgatgaaagt
420ttcgattgga ctggagtctc tcaggatgga acaagcaatg cttgcaaaag
gagatctgtt 480aaaagttttt ttagtagatt aaattggttg tacaaattag
aatacaaata tccagcactg 540aacgtgacta tgccaaacaa tgaaaaattt
gacaaattgt acatttgggg ggtgcaccac 600ccgagcacgg acagtgacca
aaccagtcta tatgttcaag catcagggag agtcacaatc 660tctaccaaaa
gaagccaaca aactgtaatc ccgaatatcg gatctagacc ctgggtaagg
720ggtatctcca gcagaataag catctattgg acaatagtaa aacctggaga
catacttatg 780attaacagca cagggaatct aatcgcccct cggggttact
tcaagatacg aagtggagaa 840agctcaataa tgaggtcaga tgcacccatt
gatagctgca attctgaatg catcactcca 900aatggaagca ttcccaataa
caaaccattt caaaatgtaa acaggatcac atatggggcc 960tgtcctagat
atgttaaaca aaaaactcta aaattggcaa cagggatgcg gaatgtacca
1020gagaaacaag ctaggggcat attcggcgcc atcgcaggtt tcatagaaaa
tggttgggag 1080ggaatggtag acggttggta cggttttagg catctaaatt
ctgagggctc aggacaagca 1140gcagacctca aaagcactca ggcagcaatt
aaccaaatca acgggaaact gaataggttg 1200gtcgaaaaaa caaacgagaa
attccatcaa attgaaaaag aattctcaga cgtggaaggg 1260agaattcagg
atctcgagaa atatgttgaa gacaccaaaa tagatctctg gtcatacaat
1320gcggagcttc ttgttgccct ggagaaccaa cacacaattg atctaactga
ctcagaaatg 1380aacaaactgt tcgaaagaac aaggaaacaa ctgagggaaa
atgctgagga catgggcaat 1440ggttgcttca aaatatacca caaatgtgac
aatgcctgca tagggtcgat cagaaatgga 1500acttatgacc ataatgtata
cagagacgaa gcattaaaca accgactcca tatcaaaggg 1560gttgagctga
agtcaggata caaagattgg atcttatgga tctcattttc catatcatgc
1620tttttgtttt gtgttgtttt gctggggttc atcatgtggg cctgccaaaa
aggcaacatt 1680aggtgcaaca tttgcatttg a 170154566PRTInfluenza A
Virus 54Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe
Ala 1 5 10 15 Gln Lys Leu Pro Arg Asn Asp Asn Ser Thr Ala Thr Leu
Cys Leu Gly 20 25 30 His His Ala Val Ser Asn Gly Thr Leu Val Lys
Thr Ile Thr Asn Asp 35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu
Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly Arg Ile Cys Asp Arg Pro
His Arg Ile Leu Asp Gly Glu Asn Cys 65 70 75 80 Thr Leu Ile Asp Ala
Leu Leu Gly Asp Pro His Cys Asp Ser Phe Gln 85 90 95 Asn Lys Glu
Trp Asp Leu Phe Val Glu Arg Ser Thr Ala Tyr Ser Asp 100 105 110 Cys
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120
125 Ala Ser Ser Gly Thr Leu Glu Phe Asn Asp Glu Ser Phe Asp Trp Thr
130 135 140 Gly Val Ser Gln Asp Gly Thr Ser Asn Ala Cys Lys Arg Arg
Ser Val 145 150 155 160 Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu Tyr
Lys Leu Glu Tyr Lys 165 170 175 Tyr Pro Ala Leu Asn Val Thr Met Pro
Asn Asn Glu Lys Phe Asp Lys 180 185 190 Leu Tyr Ile Trp Gly Val His
His Pro Ser Thr Asp Ser Asp Gln Thr 195 200 205 Ser Leu Tyr Val Gln
Ala Ser Gly Arg Val Thr Ile Ser Thr Lys Arg 210 215 220 Ser Gln Gln
Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg 225 230 235 240
Gly Ile Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245
250 255 Asp Ile Leu Met Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg
Gly 260 265 270 Tyr Phe Lys Ile Arg Ser Gly Glu Ser Ser Ile Met Arg
Ser Asp Ala 275 280 285 Pro Ile Asp Ser Cys Asn Ser Glu Cys Ile Thr
Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asn Lys Pro Phe Gln Asn Val
Asn Arg Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Arg Tyr Val Lys
Gln Lys Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg Asn Val Pro
Glu Lys Gln Ala Arg Gly Ile Phe Gly Ala Ile Ala 340 345 350 Gly Phe
Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly 355 360 365
Phe Arg His Leu Asn Ser Glu Gly Ser Gly Gln Ala Ala Asp Leu Lys 370
375 380 Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg
Leu 385 390 395 400 Val Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu
Lys Glu Phe Ser 405 410 415 Asp Val Glu Gly Arg Ile Gln Asp Leu Glu
Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn
Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His Thr Ile Asp
Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu Arg Thr Arg
Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470 475 480 Gly
Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser 485 490
495 Ile Arg Asn Gly Thr Tyr Asp His Asn Val Tyr Arg Asp Glu Ala Leu
500 505 510 Asn Asn Arg Leu His Ile Lys Gly Val Glu Leu Lys Ser Gly
Tyr Lys 515 520 525 Asp Trp Ile Leu Trp Ile Ser Phe Ser Ile Ser Cys
Phe Leu Phe Cys 530 535 540 Val Val Leu Leu Gly Phe Ile Met Trp Ala
Cys Gln Lys Gly Asn Ile 545 550 555 560 Arg Cys Asn Ile Cys Ile 565
551701DNAArtificial sequenceCodon modified Influenza A Viral
sequence 55atgaagacga tcatcgctct gtcgtacatc ctgtgtctgg tctttgctca
aaaactgccc 60cgaaatgaca actcgacggc tacgctgtgc ctgggacacc atgctgtgtc
gaacggaacg 120ctggtgaaaa cgatcacgaa tgaccaaatc gaagtgacga
atgctacgga actggtccag 180tcgtcgtcga cgggaagaat ctgtgaccga
cctcatcgaa tcctggatgg agaaaactgc 240acgctgatcg atgctctgct
gggagaccct cattgtgatt cgtttcaaaa caaggaatgg 300gacctgttcg
tcgaacgctc gacggcttac tcggactgtt acccttatga tgtgcccgat
360tatgcttcgc tgagatcgct ggtcgcttcg tcgggaacgc tggaattcaa
cgatgaatcg 420tttgattgga cgggagtctc gcaggatgga acgtcgaatg
cttgcaaaag aagatcggtc 480aaatcgttct tctcgagact gaattggctg
tacaaactgg aatacaaata tcccgctctg 540aacgtgacga tgcccaacaa
tgaaaaattc gacaaactgt acatctgggg agtgcaccac 600ccctcgacgg
actcggacca aacgtcgctg tatgtccaag cttcgggaag agtcacgatc
660tcgacgaaaa gatcgcaaca aacggtcatc cccaatatcg gatcgagacc
ctgggtcaga 720ggaatctcgt cgagaatctc gatctattgg acgatcgtca
aacctggaga catcctgatg 780atcaactcga cgggaaatct gatcgctcct
cgaggatact ttaagatccg atcgggagaa 840tcgtcgatca tgagatcgga
tgctcccatc gattcgtgca attcggaatg catcacgccc 900aatggatcga
tccccaataa caaacccttc caaaatgtca acagaatcac gtatggagct
960tgtcctagat atgtcaaaca aaaaacgctg aaactggcta cgggaatgcg
aaatgtcccc 1020gaaaaacaag ctagaggaat ctttggagct atcgctggat
ttatcgaaaa tggatgggaa 1080ggaatggtcg acggatggta cggattcaga
catctgaatt cggaaggatc gggacaagct 1140gctgacctga aatcgacgca
ggctgctatc aaccaaatca acggaaaact gaatagactg 1200gtcgaaaaaa
cgaacgaaaa atttcatcaa atcgaaaaag aattttcgga cgtggaagga
1260agaatccagg atctggaaaa atatgtcgaa gacacgaaaa tcgatctgtg
gtcgtacaat 1320gctgaactgc tggtcgctct ggaaaaccaa cacacgatcg
atctgacgga ctcggaaatg 1380aacaaactgt ttgaaagaac gagaaaacaa
ctgagagaaa atgctgaaga catgggaaat 1440ggatgcttta aaatctacca
caaatgtgac aatgcttgca tcggatcgat cagaaatgga 1500acgtatgacc
ataatgtcta cagagacgaa gctctgaaca accgactgca tatcaaagga
1560gtcgaactga agtcgggata caaagattgg atcctgtgga tctcgttctc
gatctcgtgc 1620ttcctgttct gtgtcgtcct gctgggattt atcatgtggg
cttgccaaaa aggaaacatc 1680agatgcaaca tctgcatctg a
1701561410DNAInfluenza A Virus 56atgaatccaa atcagaagat aacaaccatt
ggatcaatct gtatggtaat tggaatagtt 60agcttgatgt tacaaattgg gaacataatc
tcaatatggg ttagtcattc aattcaaaca 120gggaatcaac accaggctga
accatgcaat caaagcatta ttacttatga aaacaacacc 180tgggtaaacc
agacatatgt caacatcagc aataccaatt ttcttactga gaaagctgtg
240gcttcagtaa cattagcggg caattcatct ctttgcccca ttagtggatg
ggctgtatac 300agtaaggaca acggtataag aatcggttcc aagggggatg
tgtttgttat aagagagccg 360ttcatctcat gctcccactt ggaatgcaga
actttctttt tgactcaggg agccttgctg 420aatgacaagc attctaatgg
gaccgtcaaa gacagaagcc ctcacagaac attaatgagt 480tgtcccgtgg
gtgaggctcc ttccccatac aactcgaggt ttgagtctgt tgcttggtcg
540gcaagtgctt gtcatgatgg cactagttgg ttgacaattg gaatttctgg
cccagacaat 600ggggctgtgg ctgtattgaa atacaatggc ataataacag
acactatcaa gagttggagg 660aacaacataa tgagaactca agagtctgaa
tgtgcatgtg taaatggctc ttgctttact 720gttatgactg atggaccaag
taatgggcag gcttcataca aaatcttcag aatagaaaaa 780gggaaagtag
ttaaatcagc cgaattaaat gcccctaatt atcactatga ggagtgctcc
840tgttatcctg atgctggaga aatcacatgt gtgtgcaggg ataactggca
tggctcaaat 900cggccatggg tatctttcaa tcaaaatttg gagtatcgaa
taggatatat atgcagtgga 960gttttcggag acaatccacg ccccaatgat
gggacaggca gttgtggtcc ggtgtcccct 1020aaaggggcat atggaataaa
agggttctca tttaaatacg gcaatggtgt ttggatcggg 1080agaaccaaaa
gcactaattc caggagcggc tttgaaatga tttgggatcc aaatggatgg
1140actggtacgg acagtaattt ttcagtaaag caagatattg tagctataac
cgattggtca 1200ggatatagcg ggagttttgt ccagcatcca gaactgacag
gattagattg cataagacct 1260tgtttctggg ttgagctaat cagagggcgg
cccaaagaga gcacaatttg gactagtggg 1320agcagcatat ccttttgtgg
tgtaaatagt gacactgtgg gttggtcttg gccagacggt 1380gctgagttgc
cattcaccat tgacaagtag 141057469PRTInfluenza A Virus 57Met Asn Pro
Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Met Val 1 5 10 15 Ile
Gly Ile Val Ser Leu Met Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25
30 Trp Val Ser His Ser Ile Gln Thr Gly Asn Gln His Gln Ala Glu Pro
35 40 45 Cys Asn Gln Ser Ile Ile Thr Tyr Glu Asn Asn Thr Trp Val
Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Leu Thr
Glu Lys Ala Val 65 70 75 80 Ala Ser Val Thr Leu Ala Gly Asn Ser Ser
Leu Cys Pro Ile Ser Gly 85 90 95 Trp Ala Val Tyr Ser Lys Asp Asn
Gly Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg
Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn
Gly Thr Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser 145 150 155
160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser
165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Thr Ser Trp
Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala
Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser
Trp Arg Asn Asn Ile Met 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala
Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly
Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Arg Ile Glu
Lys Gly Lys Val Val Lys Ser Ala Glu Leu Asn Ala Pro 260 265 270 Asn
Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile 275 280
285 Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val
290 295 300 Ser Phe Asn Gln Asn Leu Glu Tyr Arg Ile Gly Tyr Ile Cys
Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly
Thr Gly Ser Cys Gly 325 330 335 Pro Val Ser Pro Lys Gly Ala Tyr Gly
Ile Lys Gly Phe Ser Phe Lys 340 345 350 Tyr Gly Asn Gly Val Trp Ile
Gly Arg Thr Lys Ser Thr Asn Ser Arg 355 360 365 Ser Gly Phe Glu Met
Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp 370 375 380 Ser Asn Phe
Ser Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser 385 390 395 400
Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405
410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro
Lys 420 425 430 Glu Ser Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe
Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp
Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465
581410DNAArtificial sequenceCodon modified Influenza A Virus
sequence 58atgaatccca atcagaagat cacgacgatc ggatcgatct gtatggtcat
cggaatcgtc 60tcgctgatgc tgcaaatcgg aaacatcatc tcgatctggg tctcgcattc
gatccaaacg 120ggaaatcaac accaggctga accctgcaat caatcgatca
tcacgtatga aaacaacacg 180tgggtcaacc agacgtatgt caacatctcg
aatacgaatt tcctgacgga aaaagctgtg 240gcttcggtca cgctggctgg
aaattcgtcg ctgtgcccca tctcgggatg ggctgtctac 300tcgaaggaca
acggaatcag aatcggatcg aagggagatg tgttcgtcat cagagaaccc
360tttatctcgt gctcgcacct ggaatgcaga acgtttttcc tgacgcaggg
agctctgctg 420aatgacaagc attcgaatgg aacggtcaaa gacagatcgc
ctcacagaac gctgatgtcg 480tgtcccgtgg gagaagctcc ttcgccctac
aactcgagat tcgaatcggt cgcttggtcg 540gcttcggctt gtcatgatgg
aacgtcgtgg ctgacgatcg gaatctcggg acccgacaat 600ggagctgtgg
ctgtcctgaa atacaatgga atcatcacgg acacgatcaa gtcgtggaga
660aacaacatca tgagaacgca agaatcggaa tgtgcttgtg tcaatggatc
gtgcttcacg 720gtcatgacgg atggaccctc gaatggacag gcttcgtaca
aaatctttag aatcgaaaaa 780ggaaaagtcg tcaaatcggc tgaactgaat
gctcctaatt atcactatga agaatgctcg 840tgttatcctg atgctggaga
aatcacgtgt gtgtgcagag ataactggca tggatcgaat 900cgaccctggg
tctcgtttaa tcaaaatctg gaatatcgaa tcggatatat ctgctcggga
960gtctttggag acaatccccg ccccaatgat ggaacgggat cgtgtggacc
cgtgtcgcct 1020aaaggagctt atggaatcaa aggattttcg ttcaaatacg
gaaatggagt ctggatcgga 1080agaacgaaat cgacgaattc gagatcggga
ttcgaaatga tctgggatcc caatggatgg 1140acgggaacgg actcgaattt
ctcggtcaag caagatatcg tcgctatcac ggattggtcg 1200ggatattcgg
gatcgttcgt ccagcatccc gaactgacgg gactggattg catcagacct
1260tgtttttggg tcgaactgat cagaggacga cccaaagaat cgacgatctg
gacgtcggga 1320tcgtcgatct cgttctgtgg agtcaattcg gacacggtgg
gatggtcgtg gcccgacgga 1380gctgaactgc cctttacgat cgacaagtag
1410591410DNAInfluenza A Virus 59atgaatacaa atcaaaaaat aataaccatt
ggaacagcct gtctgatagt cggaataatt 60agtctattat tgcagatagg agatatagtc
tcgttatgga taagccattc aattcagact 120ggagagaaaa accactctca
gatatgcagt caaagtgtca ttacatatga aaacaacaca 180tgggtgaacc
aaacttatgt aaacattggc aataccaata ttgctgatgg acagggagta
240aattcaataa tactagcggg caattcctct ctttgcccag taagtggatg
ggccatatac 300agcaaagaca atagcataag gatcggttcc aaaggagaca
tttttgtcat aagagaacta 360tttatctcat gctctcattt ggagtgcaga
actttttatc tgacccaagg tgctttgctg 420aatgacaagc attctaatgg
aaccgtcaaa gacaggagtc cttatagaac cttaatgagc 480tgcccgattg
gtgaagctcc ttctccgtac aattcaaggt tcgaatcagt tgcttggtca
540gcaagtgcat gccatgacgg aatgggatgg ctgacaatcg gaatttccgg
cccagataat 600ggagcagtgg ctgttttgaa atacaatggg ataataacag
atacaataaa aagttggagg 660aacaaaatac taagaacaca agaatcagaa
tgtgtctgta taaacggttc gtgtttcact 720ataatgactg atggcccaag
caatgggcag gcctcataca aaatattcaa aatgaagaaa 780gggaaaatta
ttaaatcagt ggagatgaat gcacctaatt accactatga ggaatgctcc
840tgttaccctg atacaggcaa agtggtgtgc gtgtgcagag acaattggca
tgcttcgaat 900agaccgtggg tctctttcga tcagaacctt aattatcaga
tagggtacat atgtagtggg 960gttttcggtg ataacccgcg ttctaatgat
gggagaggcg attgtgggcc agtactttct 1020aatggagcta atggagtgaa
aggattctca tttaggtatg gcaatggcgt ttggatagga 1080agaactaaaa
gcatcagctc tagaagtgga tttgagatga tttgggatcc gaatggatgg
1140acggaaaccg atagtagttt ctcgataaag caggatgtta tagcattaac
tgattggtca 1200ggatacagtg ggaactttgt ccaacatccc gaattaacag
gaatgaactg cataaagcct 1260tgtttctggg tagagttaat cagaggacag
cccaaggaga gaacaatctg gactagtgga 1320agcagcattt ctttctgtgg
tgtagacagt gaaaccgcaa gctggtcatg gccagacgga 1380gctgatctgc
cattcactat tgacaagtag 141060469PRTInfluenza A Virus 60Met Asn Thr
Asn Gln Lys Ile Ile Thr Ile Gly Thr Ala Cys Leu Ile 1 5 10 15 Val
Gly Ile Ile Ser Leu Leu
Leu Gln Ile Gly Asp Ile Val Ser Leu 20 25 30 Trp Ile Ser His Ser
Ile Gln Thr Gly Glu Lys Asn His Ser Gln Ile 35 40 45 Cys Ser Gln
Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln 50 55 60 Thr
Tyr Val Asn Ile Gly Asn Thr Asn Ile Ala Asp Gly Gln Gly Val 65 70
75 80 Asn Ser Ile Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser
Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly
Ser Lys Gly 100 105 110 Asp Ile Phe Val Ile Arg Glu Leu Phe Ile Ser
Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Tyr Leu Thr Gln Gly
Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp
Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Ile Gly
Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala
Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190
Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195
200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Lys Ile
Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Ser
Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asn Gly Gln
Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Met Lys Lys Gly Lys Ile Ile
Lys Ser Val Glu Met Asn Ala Pro 260 265 270 Asn Tyr His Tyr Glu Glu
Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Val Cys Val Cys
Arg Asp Asn Trp His Ala Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe
Asp Gln Asn Leu Asn Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315
320 Val Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Arg Gly Asp Cys Gly
325 330 335 Pro Val Leu Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser
Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser
Ile Ser Ser Arg 355 360 365 Ser Gly Phe Glu Met Ile Trp Asp Pro Asn
Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Ile Lys Gln Asp
Val Ile Ala Leu Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Asn
Phe Val Gln His Pro Glu Leu Thr Gly Met Asn 405 410 415 Cys Ile Lys
Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Gln Pro Lys 420 425 430 Glu
Arg Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val 435 440
445 Asp Ser Glu Thr Ala Ser Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro
450 455 460 Phe Thr Ile Asp Lys 465 611410DNAArtificial
sequenceCodon modified Influenza A Virus sequence 61atgaatacga
atcaaaaaat catcacgatc ggaacggctt gtctgatcgt cggaatcatc 60tcgctgctgc
tgcagatcgg agatatcgtc tcgctgtgga tctcgcattc gatccagacg
120ggagaaaaaa accactcgca gatctgctcg caatcggtca tcacgtatga
aaacaacacg 180tgggtgaacc aaacgtatgt caacatcgga aatacgaata
tcgctgatgg acagggagtc 240aattcgatca tcctggctgg aaattcgtcg
ctgtgccccg tctcgggatg ggctatctac 300tcgaaagaca attcgatcag
aatcggatcg aaaggagaca tcttcgtcat cagagaactg 360ttcatctcgt
gctcgcatct ggaatgcaga acgttctatc tgacgcaagg agctctgctg
420aatgacaagc attcgaatgg aacggtcaaa gacagatcgc cttatagaac
gctgatgtcg 480tgccccatcg gagaagctcc ttcgccctac aattcgagat
ttgaatcggt cgcttggtcg 540gcttcggctt gccatgacgg aatgggatgg
ctgacgatcg gaatctcggg acccgataat 600ggagctgtgg ctgtcctgaa
atacaatgga atcatcacgg atacgatcaa atcgtggaga 660aacaaaatcc
tgagaacgca agaatcggaa tgtgtctgta tcaacggatc gtgttttacg
720atcatgacgg atggaccctc gaatggacag gcttcgtaca aaatctttaa
aatgaagaaa 780ggaaaaatca tcaaatcggt ggaaatgaat gctcctaatt
accactatga agaatgctcg 840tgttaccctg atacgggaaa agtggtgtgc
gtgtgcagag acaattggca tgcttcgaat 900agaccctggg tctcgtttga
tcagaacctg aattatcaga tcggatacat ctgttcggga 960gtctttggag
ataacccccg ttcgaatgat ggaagaggag attgtggacc cgtcctgtcg
1020aatggagcta atggagtgaa aggattttcg ttcagatatg gaaatggagt
ctggatcgga 1080agaacgaaat cgatctcgtc gagatcggga ttcgaaatga
tctgggatcc caatggatgg 1140acggaaacgg attcgtcgtt ttcgatcaag
caggatgtca tcgctctgac ggattggtcg 1200ggatactcgg gaaacttcgt
ccaacatccc gaactgacgg gaatgaactg catcaagcct 1260tgtttttggg
tcgaactgat cagaggacag cccaaggaaa gaacgatctg gacgtcggga
1320tcgtcgatct cgttttgtgg agtcgactcg gaaacggctt cgtggtcgtg
gcccgacgga 1380gctgatctgc cctttacgat cgacaagtag
141062576DNAHepatitis C Virus E1 62taccaagtgc gcaattcctc ggggctttac
catgtcacca atgattgccc taactcgagt 60attgtgtacg aggcggccga tgccatcctg
cacactccgg ggtgtgtccc ttgcgttcgc 120gagggtaacg cctcgaggtg
ttgggtggcg gtgaccccca cggtggccac cagggacggc 180aaactcccca
caacgcagct tcgacgtcat atcgatctgc ttgtcgggag cgccaccctc
240tgctcggccc tctacgtggg ggacctgtgc gggtctgtct ttcttgttgg
tcaactgttt 300accttctctc ccaggcgcca ctggacgacg caagactgca
attgttctat ctatcccggc 360catataacgg gtcatcgcat ggcatgggat
atgatgatga actggtcccc tacggcagcg 420ttggtggtag ctcagctgct
ccggatccca caagccatca tggacatgat cgctggtgct 480cactggggag
tcctggcggg catagcgtat ttctccatgg tggggaactg ggcgaaggtc
540ctggtagtgc tgctgctatt tgccggcgtc gacgcg 57663192PRTHepatitis C
Virus E1 63Tyr Gln Val Arg Asn Ser Ser Gly Leu Tyr His Val Thr Asn
Asp Cys 1 5 10 15 Pro Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala
Ile Leu His Thr 20 25 30 Pro Gly Cys Val Pro Cys Val Arg Glu Gly
Asn Ala Ser Arg Cys Trp 35 40 45 Val Ala Val Thr Pro Thr Val Ala
Thr Arg Asp Gly Lys Leu Pro Thr 50 55 60 Thr Gln Leu Arg Arg His
Ile Asp Leu Leu Val Gly Ser Ala Thr Leu 65 70 75 80 Cys Ser Ala Leu
Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val 85 90 95 Gly Gln
Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Asp 100 105 110
Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala 115
120 125 Trp Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Leu Val Val
Ala 130 135 140 Gln Leu Leu Arg Ile Pro Gln Ala Ile Met Asp Met Ile
Ala Gly Ala 145 150 155 160 His Trp Gly Val Leu Ala Gly Ile Ala Tyr
Phe Ser Met Val Gly Asn 165 170 175 Trp Ala Lys Val Leu Val Val Leu
Leu Leu Phe Ala Gly Val Asp Ala 180 185 190 64576DNAArtificial
sequenceCodon modified Hepatitis C Virus E1 sequence 64taccaagtgc
gcaattcgtc gggactgtac catgtcacga atgattgccc taactcgtcg 60atcgtgtacg
aagctgctga tgctatcctg cacacgcccg gatgtgtccc ttgcgtccgc
120gaaggaaacg cttcgagatg ttgggtggct gtgacgccca cggtggctac
gagagacgga 180aaactgccca cgacgcagct gcgacgtcat atcgatctgc
tggtcggatc ggctacgctg 240tgctcggctc tgtacgtggg agacctgtgc
ggatcggtct tcctggtcgg acaactgttc 300acgttttcgc ccagacgcca
ctggacgacg caagactgca attgttcgat ctatcccgga 360catatcacgg
gacatcgcat ggcttgggat atgatgatga actggtcgcc tacggctgct
420ctggtggtcg ctcagctgct gcgaatcccc caagctatca tggacatgat
cgctggagct 480cactggggag tcctggctgg aatcgcttat ttttcgatgg
tgggaaactg ggctaaggtc 540ctggtcgtgc tgctgctgtt cgctggagtc gacgct
576651089DNAHepatitis C Virus E2 65gaaacccacg tcaccggggg aagtgccggc
cgcaccacgg ctgggcttgt tggtctcctt 60acaccaggcg ccaagcagaa catccaactg
atcaacacca acggcagttg gcacatcaat 120agcacggcct tgaactgcaa
tgaaagcctt aacaccggct ggttagcagg gctcttctat 180cagcacaaat
tcaactcttc aggctgtcct gagaggttgg ccagctgccg acgccttacc
240gattttgccc agggctgggg tcctatcagt tatgccaacg gaagcggcct
cgacgaacgc 300ccctactgct ggcactaccc tccaagacct tgtggcattg
tgcccgcaaa gagcgtgtgt 360ggcccggtat attgcttcac tcccagcccc
gtggtggtgg gaacgaccga caggtcgggc 420gcgcctacct acagctgggg
tgcaaatgat acggatgtct tcgtccttaa caacaccagg 480ccaccgctgg
gcaattggtt cggttgtacc tggatgaact caactggatt caccaaagtg
540tgcggagcgc ccccttgtgt catcggaggg gtgggcaaca acaccttgct
ctgccccact 600gattgtttcc gcaagcatcc ggaagccaca tactctcggt
gcggctccgg tccctggatt 660acacccaggt gcatggtcga ctacccgtat
aggctttggc actatccttg taccatcaat 720tacaccatat tcaaagtcag
gatgtacgtg ggaggggtcg agcacaggct ggaagcggcc 780tgcaactgga
cgcggggcga acgctgtgat ctggaagaca gggacaggtc cgagctcagc
840ccattgctgc tgtccaccac acagtggcag gtccttccgt gttctttcac
gaccctgcca 900gccttgtcca ccggcctcat ccacctccac cagaacattg
tggacgtgca gtacttgtac 960ggggtagggt caagcatcgc gtcctgggcc
attaagtggg agtacgtcgt tctcctgttc 1020ctcctgcttg cagacgcgcg
cgtctgctcc tgcttgtgga tgatgttact catatcccaa 1080gcggaggcg
108966363PRTHepatitis C Virus E2 66Glu Thr His Val Thr Gly Gly Ser
Ala Gly Arg Thr Thr Ala Gly Leu 1 5 10 15 Val Gly Leu Leu Thr Pro
Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn 20 25 30 Thr Asn Gly Ser
Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu 35 40 45 Ser Leu
Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr Gln His Lys Phe 50 55 60
Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg Leu Thr 65
70 75 80 Asp Phe Ala Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly
Ser Gly 85 90 95 Leu Asp Glu Arg Pro Tyr Cys Trp His Tyr Pro Pro
Arg Pro Cys Gly 100 105 110 Ile Val Pro Ala Lys Ser Val Cys Gly Pro
Val Tyr Cys Phe Thr Pro 115 120 125 Ser Pro Val Val Val Gly Thr Thr
Asp Arg Ser Gly Ala Pro Thr Tyr 130 135 140 Ser Trp Gly Ala Asn Asp
Thr Asp Val Phe Val Leu Asn Asn Thr Arg 145 150 155 160 Pro Pro Leu
Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly 165 170 175 Phe
Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile Gly Gly Val Gly 180 185
190 Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu
195 200 205 Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro
Arg Cys 210 215 220 Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro
Cys Thr Ile Asn 225 230 235 240 Tyr Thr Ile Phe Lys Val Arg Met Tyr
Val Gly Gly Val Glu His Arg 245 250 255 Leu Glu Ala Ala Cys Asn Trp
Thr Arg Gly Glu Arg Cys Asp Leu Glu 260 265 270 Asp Arg Asp Arg Ser
Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gln 275 280 285 Trp Gln Val
Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 290 295 300 Gly
Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr 305 310
315 320 Gly Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp Glu Tyr
Val 325 330 335 Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys
Ser Cys Leu 340 345 350 Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala
355 360 671089DNAArtificial sequenceCodon modified Hepatitis C
Virus E2 sequence 67gaaacgcacg tcacgggagg atcggctgga cgcacgacgg
ctggactggt cggactgctg 60acgcccggag ctaagcagaa catccaactg atcaacacga
acggatcgtg gcacatcaat 120tcgacggctc tgaactgcaa tgaatcgctg
aacacgggat ggctggctgg actgttttat 180cagcacaaat ttaactcgtc
gggatgtcct gaaagactgg cttcgtgccg acgcctgacg 240gatttcgctc
agggatgggg acctatctcg tatgctaacg gatcgggact ggacgaacgc
300ccctactgct ggcactaccc tcccagacct tgtggaatcg tgcccgctaa
gtcggtgtgt 360ggacccgtct attgctttac gccctcgccc gtggtggtgg
gaacgacgga cagatcggga 420gctcctacgt actcgtgggg agctaatgat
acggatgtct ttgtcctgaa caacacgaga 480ccccccctgg gaaattggtt
tggatgtacg tggatgaact cgacgggatt tacgaaagtg 540tgcggagctc
ccccttgtgt catcggagga gtgggaaaca acacgctgct gtgccccacg
600gattgttttc gcaagcatcc cgaagctacg tactcgcgat gcggatcggg
accctggatc 660acgcccagat gcatggtcga ctacccctat agactgtggc
actatccttg tacgatcaat 720tacacgatct ttaaagtcag aatgtacgtg
ggaggagtcg aacacagact ggaagctgct 780tgcaactgga cgcgaggaga
acgctgtgat ctggaagaca gagacagatc ggaactgtcg 840cccctgctgc
tgtcgacgac gcagtggcag gtcctgccct gttcgtttac gacgctgccc
900gctctgtcga cgggactgat ccacctgcac cagaacatcg tggacgtgca
gtacctgtac 960ggagtcggat cgtcgatcgc ttcgtgggct atcaagtggg
aatacgtcgt cctgctgttt 1020ctgctgctgg ctgacgctcg cgtctgctcg
tgcctgtgga tgatgctgct gatctcgcaa 1080gctgaagct 1089682724DNAEpstein
Barr Virus 68atggaggcag ccttgcttgt gtgtcagtac accatccaga gcctgatcca
tctcacgggt 60gaagatcctg gttttttcaa tgttgagatt ccggaattcc cattttaccc
cacatgcaat 120gtttgcacgg cagatgtcaa tgtaactatc aatttcgatg
tcgggggcaa aaagcatcaa 180cttgatcttg actttggcca gctgacaccc
catacgaagg ctgtctacca acctcgaggt 240gcatttggtg gctcagaaaa
tgccaccaat ctctttctac tggagctcct tggtgcagga 300gaattggctc
taactatgcg gtctaagaag cttccaatta acgtcaccac cggagaggag
360caacaagtaa gcctggaatc tgtagatgtc tactttcaag atgtgtttgg
aaccatgtgg 420tgccaccatg cagaaatgca aaaccccgtg tacctgatac
cagaaacagt gccatacata 480aagtgggata actgtaattc taccaatata
acggcagtag tgagggcaca ggggctggat 540gtcacgctac ccttaagttt
gccaacgtca gctcaagact cgaatttcag cgtaaaaaca 600gaaatgctcg
gtaatgagat agatattgag tgtattatgg aggatggcga aatttcacaa
660gttctgcccg gagacaacaa atttaacatc acctgcagtg gatacgagag
ccatgttccc 720agcggcggaa ttctcacatc aacgagtccc gtggccaccc
caatacctgg tacagggtat 780gcatacagcc tgcgtctgac accacgtcca
gtgtcacgat ttcttggcaa taacagtatc 840ctgtacgtgt tttactctgg
gaatggaccg aaggcgagcg ggggagatta ctgcattcag 900tccaacattg
tgttctctga tgagattcca gcttcacagg acatgccgac aaacaccaca
960gacatcacat atgtgggtga caatgctacc tattcagtgc caatggtcac
ttctgaggac 1020gcaaactcgc caaatgttac agtgactgcc ttttgggcct
ggccaaacaa cactgaaact 1080gactttaagt gcaaatggac tctcacctcg
gggacacctt cgggttgtga aaatatttct 1140ggtgcatttg cgagcaatcg
gacatttgac attactgtct cgggtcttgg cacggccccc 1200aagacactca
ttatcacacg aacggctacc aatgccacca caacaaccca caaggttata
1260ttctccaagg cacccgagag caccaccacc tcccctacct tgaatacaac
tggatttgct 1320gatcccaata caacgacagg tctacccagc tctactcacg
tgcctaccaa cctcaccgca 1380cctgcaagca caggccccac tgtatccacc
gcggatgtca ccagcccaac accagccggc 1440acaacgtcag gcgcatcacc
ggtgacacca agtccatctc catgggacaa cggcacagaa 1500agtaaggccc
ccgacatgac cagctccacc tcaccagtga ctaccccaac cccaaatgcc
1560accagcccca ccccagcagt gactacccca accccaaatg ccaccagccc
caccccagca 1620gtgactaccc caaccccaaa tgccaccagc cccaccttgg
gaaaaacaag tcctacctca 1680gcagtgacta ccccaacccc aaatgccacc
agccccacct tgggaaaaac aagccccacc 1740tcagcagtga ctaccccaac
cccaaatgcc accagcccca ccttgggaaa aacaagcccc 1800acctcagcag
tgactacccc aaccccaaat gccaccggcc ctactgtggg agaaacaagt
1860ccacaggcaa atgccaccaa ccacacctta ggaggaacaa gtcccacccc
agtagttacc 1920agccaaccaa aaaatgcaac cagtgctgtt accacaggcc
aacataacat aacttcaagt 1980tcaacctctt ccatgtcact gagacccagt
tcaaacccag agacactcag cccctccacc 2040agtgacaatt caacgtcaca
tatgccttta ctaacctccg ctcacccaac aggtggtgaa 2100aatataacac
aggtgacacc agcctctatc agcacacatc atgtgtccac cagttcgcca
2160gcaccccgcc caggcaccac cagccaagcg tcaggccctg gaaacagttc
cacatccaca 2220aaaccggggg aggttaatgt caccaaaggc acgccccccc
aaaatgcaac gtcgccccag 2280gcccccagtg gccaaaagac ggcggttccc
acggtcacct caacaggtgg aaaggccaat 2340tctaccaccg gtggaaagca
caccacagga catggagccc ggacaagtac agagcccacc 2400acagattacg
gcggtgattc aactacgcca agaccgagat acaatgcgac cacctatcta
2460cctcccagca cttctagcaa actgcggccc cgctggactt ttacgagccc
accggttacc 2520acagcccaag ccaccgtgcc agtcccgcca acgtcccagc
ccagattctc aaacctctcc 2580atgctagtac tgcagtgggc ctctctggct
gtgctgaccc ttctgctgct gctggtcatg 2640gcggactgcg cctttaggcg
taacttgtct acatcccata cctacaccac cccaccatat 2700gatgacgccg
agacctatgt ataa 272469907PRTEpstein Barr Virus 69Met Glu Ala Ala
Leu Leu Val Cys Gln Tyr Thr Ile Gln Ser Leu Ile 1 5 10 15 His Leu
Thr Gly Glu Asp Pro Gly Phe Phe Asn Val Glu Ile Pro Glu 20 25 30
Phe Pro Phe Tyr Pro Thr Cys Asn Val Cys Thr Ala Asp Val Asn Val 35
40 45 Thr Ile Asn Phe Asp Val Gly Gly Lys Lys His Gln Leu Asp Leu
Asp 50 55 60 Phe Gly Gln Leu Thr Pro His Thr Lys Ala Val
Tyr Gln Pro Arg Gly 65 70 75 80 Ala Phe Gly Gly Ser Glu Asn Ala Thr
Asn Leu Phe Leu Leu Glu Leu 85 90 95 Leu Gly Ala Gly Glu Leu Ala
Leu Thr Met Arg Ser Lys Lys Leu Pro 100 105 110 Ile Asn Val Thr Thr
Gly Glu Glu Gln Gln Val Ser Leu Glu Ser Val 115 120 125 Asp Val Tyr
Phe Gln Asp Val Phe Gly Thr Met Trp Cys His His Ala 130 135 140 Glu
Met Gln Asn Pro Val Tyr Leu Ile Pro Glu Thr Val Pro Tyr Ile 145 150
155 160 Lys Trp Asp Asn Cys Asn Ser Thr Asn Ile Thr Ala Val Val Arg
Ala 165 170 175 Gln Gly Leu Asp Val Thr Leu Pro Leu Ser Leu Pro Thr
Ser Ala Gln 180 185 190 Asp Ser Asn Phe Ser Val Lys Thr Glu Met Leu
Gly Asn Glu Ile Asp 195 200 205 Ile Glu Cys Ile Met Glu Asp Gly Glu
Ile Ser Gln Val Leu Pro Gly 210 215 220 Asp Asn Lys Phe Asn Ile Thr
Cys Ser Gly Tyr Glu Ser His Val Pro 225 230 235 240 Ser Gly Gly Ile
Leu Thr Ser Thr Ser Pro Val Ala Thr Pro Ile Pro 245 250 255 Gly Thr
Gly Tyr Ala Tyr Ser Leu Arg Leu Thr Pro Arg Pro Val Ser 260 265 270
Arg Phe Leu Gly Asn Asn Ser Ile Leu Tyr Val Phe Tyr Ser Gly Asn 275
280 285 Gly Pro Lys Ala Ser Gly Gly Asp Tyr Cys Ile Gln Ser Asn Ile
Val 290 295 300 Phe Ser Asp Glu Ile Pro Ala Ser Gln Asp Met Pro Thr
Asn Thr Thr 305 310 315 320 Asp Ile Thr Tyr Val Gly Asp Asn Ala Thr
Tyr Ser Val Pro Met Val 325 330 335 Thr Ser Glu Asp Ala Asn Ser Pro
Asn Val Thr Val Thr Ala Phe Trp 340 345 350 Ala Trp Pro Asn Asn Thr
Glu Thr Asp Phe Lys Cys Lys Trp Thr Leu 355 360 365 Thr Ser Gly Thr
Pro Ser Gly Cys Glu Asn Ile Ser Gly Ala Phe Ala 370 375 380 Ser Asn
Arg Thr Phe Asp Ile Thr Val Ser Gly Leu Gly Thr Ala Pro 385 390 395
400 Lys Thr Leu Ile Ile Thr Arg Thr Ala Thr Asn Ala Thr Thr Thr Thr
405 410 415 His Lys Val Ile Phe Ser Lys Ala Pro Glu Ser Thr Thr Thr
Ser Pro 420 425 430 Thr Leu Asn Thr Thr Gly Phe Ala Asp Pro Asn Thr
Thr Thr Gly Leu 435 440 445 Pro Ser Ser Thr His Val Pro Thr Asn Leu
Thr Ala Pro Ala Ser Thr 450 455 460 Gly Pro Thr Val Ser Thr Ala Asp
Val Thr Ser Pro Thr Pro Ala Gly 465 470 475 480 Thr Thr Ser Gly Ala
Ser Pro Val Thr Pro Ser Pro Ser Pro Trp Asp 485 490 495 Asn Gly Thr
Glu Ser Lys Ala Pro Asp Met Thr Ser Ser Thr Ser Pro 500 505 510 Val
Thr Thr Pro Thr Pro Asn Ala Thr Ser Pro Thr Pro Ala Val Thr 515 520
525 Thr Pro Thr Pro Asn Ala Thr Ser Pro Thr Pro Ala Val Thr Thr Pro
530 535 540 Thr Pro Asn Ala Thr Ser Pro Thr Leu Gly Lys Thr Ser Pro
Thr Ser 545 550 555 560 Ala Val Thr Thr Pro Thr Pro Asn Ala Thr Ser
Pro Thr Leu Gly Lys 565 570 575 Thr Ser Pro Thr Ser Ala Val Thr Thr
Pro Thr Pro Asn Ala Thr Ser 580 585 590 Pro Thr Leu Gly Lys Thr Ser
Pro Thr Ser Ala Val Thr Thr Pro Thr 595 600 605 Pro Asn Ala Thr Gly
Pro Thr Val Gly Glu Thr Ser Pro Gln Ala Asn 610 615 620 Ala Thr Asn
His Thr Leu Gly Gly Thr Ser Pro Thr Pro Val Val Thr 625 630 635 640
Ser Gln Pro Lys Asn Ala Thr Ser Ala Val Thr Thr Gly Gln His Asn 645
650 655 Ile Thr Ser Ser Ser Thr Ser Ser Met Ser Leu Arg Pro Ser Ser
Asn 660 665 670 Pro Glu Thr Leu Ser Pro Ser Thr Ser Asp Asn Ser Thr
Ser His Met 675 680 685 Pro Leu Leu Thr Ser Ala His Pro Thr Gly Gly
Glu Asn Ile Thr Gln 690 695 700 Val Thr Pro Ala Ser Ile Ser Thr His
His Val Ser Thr Ser Ser Pro 705 710 715 720 Ala Pro Arg Pro Gly Thr
Thr Ser Gln Ala Ser Gly Pro Gly Asn Ser 725 730 735 Ser Thr Ser Thr
Lys Pro Gly Glu Val Asn Val Thr Lys Gly Thr Pro 740 745 750 Pro Gln
Asn Ala Thr Ser Pro Gln Ala Pro Ser Gly Gln Lys Thr Ala 755 760 765
Val Pro Thr Val Thr Ser Thr Gly Gly Lys Ala Asn Ser Thr Thr Gly 770
775 780 Gly Lys His Thr Thr Gly His Gly Ala Arg Thr Ser Thr Glu Pro
Thr 785 790 795 800 Thr Asp Tyr Gly Gly Asp Ser Thr Thr Pro Arg Pro
Arg Tyr Asn Ala 805 810 815 Thr Thr Tyr Leu Pro Pro Ser Thr Ser Ser
Lys Leu Arg Pro Arg Trp 820 825 830 Thr Phe Thr Ser Pro Pro Val Thr
Thr Ala Gln Ala Thr Val Pro Val 835 840 845 Pro Pro Thr Ser Gln Pro
Arg Phe Ser Asn Leu Ser Met Leu Val Leu 850 855 860 Gln Trp Ala Ser
Leu Ala Val Leu Thr Leu Leu Leu Leu Leu Val Met 865 870 875 880 Ala
Asp Cys Ala Phe Arg Arg Asn Leu Ser Thr Ser His Thr Tyr Thr 885 890
895 Thr Pro Pro Tyr Asp Asp Ala Glu Thr Tyr Val 900 905
702724DNAArtificial sequenceCodon modified Epstein Barr Virus
sequence 70atggaagctg ctctgctggt gtgtcagtac acgatccagt cgctgatcca
tctgacggga 60gaagatcctg gattctttaa tgtcgaaatc cccgaatttc ccttctaccc
cacgtgcaat 120gtctgcacgg ctgatgtcaa tgtcacgatc aattttgatg
tcggaggaaa aaagcatcaa 180ctggatctgg acttcggaca gctgacgccc
catacgaagg ctgtctacca acctcgagga 240gctttcggag gatcggaaaa
tgctacgaat ctgttcctgc tggaactgct gggagctgga 300gaactggctc
tgacgatgcg atcgaagaag ctgcccatca acgtcacgac gggagaagaa
360caacaagtct cgctggaatc ggtcgatgtc tacttccaag atgtgttcgg
aacgatgtgg 420tgccaccatg ctgaaatgca aaaccccgtg tacctgatcc
ccgaaacggt gccctacatc 480aagtgggata actgtaattc gacgaatatc
acggctgtcg tgagagctca gggactggat 540gtcacgctgc ccctgtcgct
gcccacgtcg gctcaagact cgaatttttc ggtcaaaacg 600gaaatgctgg
gaaatgaaat cgatatcgaa tgtatcatgg aagatggaga aatctcgcaa
660gtcctgcccg gagacaacaa attcaacatc acgtgctcgg gatacgaatc
gcatgtcccc 720tcgggaggaa tcctgacgtc gacgtcgccc gtggctacgc
ccatccctgg aacgggatat 780gcttactcgc tgcgtctgac gccccgtccc
gtgtcgcgat tcctgggaaa taactcgatc 840ctgtacgtgt tctactcggg
aaatggaccc aaggcttcgg gaggagatta ctgcatccag 900tcgaacatcg
tgttttcgga tgaaatcccc gcttcgcagg acatgcccac gaacacgacg
960gacatcacgt atgtgggaga caatgctacg tattcggtgc ccatggtcac
gtcggaagac 1020gctaactcgc ccaatgtcac ggtgacggct ttctgggctt
ggcccaacaa cacggaaacg 1080gacttcaagt gcaaatggac gctgacgtcg
ggaacgcctt cgggatgtga aaatatctcg 1140ggagctttcg cttcgaatcg
aacgttcgac atcacggtct cgggactggg aacggctccc 1200aagacgctga
tcatcacgcg aacggctacg aatgctacga cgacgacgca caaggtcatc
1260ttttcgaagg ctcccgaatc gacgacgacg tcgcctacgc tgaatacgac
gggattcgct 1320gatcccaata cgacgacggg actgccctcg tcgacgcacg
tgcctacgaa cctgacggct 1380cctgcttcga cgggacccac ggtctcgacg
gctgatgtca cgtcgcccac gcccgctgga 1440acgacgtcgg gagcttcgcc
cgtgacgccc tcgccctcgc cctgggacaa cggaacggaa 1500tcgaaggctc
ccgacatgac gtcgtcgacg tcgcccgtga cgacgcccac gcccaatgct
1560acgtcgccca cgcccgctgt gacgacgccc acgcccaatg ctacgtcgcc
cacgcccgct 1620gtgacgacgc ccacgcccaa tgctacgtcg cccacgctgg
gaaaaacgtc gcctacgtcg 1680gctgtgacga cgcccacgcc caatgctacg
tcgcccacgc tgggaaaaac gtcgcccacg 1740tcggctgtga cgacgcccac
gcccaatgct acgtcgccca cgctgggaaa aacgtcgccc 1800acgtcggctg
tgacgacgcc cacgcccaat gctacgggac ctacggtggg agaaacgtcg
1860ccccaggcta atgctacgaa ccacacgctg ggaggaacgt cgcccacgcc
cgtcgtcacg 1920tcgcaaccca aaaatgctac gtcggctgtc acgacgggac
aacataacat cacgtcgtcg 1980tcgacgtcgt cgatgtcgct gagaccctcg
tcgaaccccg aaacgctgtc gccctcgacg 2040tcggacaatt cgacgtcgca
tatgcctctg ctgacgtcgg ctcaccccac gggaggagaa 2100aatatcacgc
aggtgacgcc cgcttcgatc tcgacgcatc atgtgtcgac gtcgtcgccc
2160gctccccgcc ccggaacgac gtcgcaagct tcgggacctg gaaactcgtc
gacgtcgacg 2220aaacccggag aagtcaatgt cacgaaagga acgccccccc
aaaatgctac gtcgccccag 2280gctccctcgg gacaaaagac ggctgtcccc
acggtcacgt cgacgggagg aaaggctaat 2340tcgacgacgg gaggaaagca
cacgacggga catggagctc gaacgtcgac ggaacccacg 2400acggattacg
gaggagattc gacgacgccc agacccagat acaatgctac gacgtatctg
2460cctccctcga cgtcgtcgaa actgcgaccc cgctggacgt tcacgtcgcc
ccccgtcacg 2520acggctcaag ctacggtgcc cgtccccccc acgtcgcagc
ccagattttc gaacctgtcg 2580atgctggtcc tgcagtgggc ttcgctggct
gtgctgacgc tgctgctgct gctggtcatg 2640gctgactgcg ctttcagacg
taacctgtcg acgtcgcata cgtacacgac gcccccctat 2700gatgacgctg
aaacgtatgt ctaa 2724712661DNAEpstein Barr Virus 71atggaggcag
ccttgcttgt gtgtcagtac accatccaga gccttatcca actcacgcgt 60gatgatcctg
gttttttcaa tgttgagatt ctggaattcc cattttaccc agcgtgcaat
120gtttgcacgg cagatgtcaa tgcaactatc aatttcgatg tcgggggcaa
aaagcataaa 180cttaatcttg actttggcct gctgacaccc catacaaagg
ctgtctacca acctcgaggt 240gcatttggtg gctcagaaaa tgccaccaat
ctctttctac tggagctcct tggtgcagga 300gaattggctc taactatgcg
gtctaagaag cttccaatta acatcaccac cggagaggag 360caacaagtaa
gcctggaatc tgtagatgtc tactttcaag atgtgtttgg caccatgtgg
420tgccaccatg cagaaatgca aaacccagta tacctaatac cagaaacagt
gccatacata 480aagtgggata actgtaattc taccaatata acggcagtag
taagggcaca ggggctggat 540gtcacgctac ccttaagttt gccaacatca
gctcaagact cgaatttcag cgtaaaaaca 600gaaatgctcg gtaatgagat
agatattgag tgtattatgg aggatggcga aatttcacaa 660gttctgcccg
gagacaacaa atttaacatc acctgcagtg gatacgagag ccatgttccc
720agcggcggaa ttctcacatc aacgagtccc gtggccaccc caatacctgg
tacagggtat 780gcatacagcc tgcgtctgac accacgtcca gtgtcacgat
ttcttggcaa taacagtata 840ctgtacgtgt tttactctgg gaatggaccg
aaggcgagcg ggggagatta ctgcattcag 900tccaacattg tgttctctga
tgagattcca gcttcacagg acatgccgac aaacaccaca 960gacatcacat
atgtgggtga caatgctacc tattcagtgc caatggtcac ttctgaggac
1020gcaaactcgc caaatgttac agtgactgcc ttttgggcct ggccaaacaa
cactgaaact 1080gactttaagt gcaaatggac tctcacctcg gggacacctt
cgggttgtga aaatatttct 1140ggtgcatttg cgagcaatcg gacatttgac
attactgtct cgggtcttgg cacggccccc 1200aagacactca ttatcacacg
aacggctacc aatgccacca caacaaccca caaggttata 1260ttctccaagg
cacccgagag caccaccacc tcccctacct tgaatacaac tggatttgct
1320gctcccaata caacgacagg tctacccagc tctactcacg tgcctaccaa
cctcaccgca 1380cctgcaagca caggccccac tgtatccacc gcggatgtca
ccagcccaac accagccggc 1440acaacgtcag gcgcatcacc ggtgacacca
agtccatctc cacgggacaa cggcacagaa 1500agtaaggccc ccgacatgac
cagccccacc tcagcagtga ctaccccaac cccaaatgcc 1560accagcccca
ccccagcagt gactacccca accccaaatg ccaccagccc caccttggga
1620aaaacaagtc ccacctcagc agtgactacc ccaaccccaa atgccaccag
ccccacccca 1680gcagtgacta ccccaacccc aaatgccacc atccccacct
tgggaaaaac aagtcccacc 1740tcagcagtga ctaccccaac cccaaatgcc
accagcccta ccgtgggaga aacaagtcca 1800caggcaaata ccaccaacca
cacattagga ggaacaagtt ccaccccagt agttaccagc 1860ccaccaaaaa
atgcaaccag tgctgttacc acaggccaac ataacataac ttcaagttca
1920acctcttcca tgtcactgag acccagttca atctcagaga cactcagccc
ctccaccagt 1980gacaattcaa cgtcacatat gcctttacta acctccgctc
acccaacagg tggtgaaaat 2040ataacacagg tgacaccagc ctctaccagc
acacatcatg tgtccaccag ttcgccagcg 2100ccccgcccag gcaccaccag
ccaagcgtca ggccctggaa acagttccac atccacaaaa 2160ccgggggagg
ttaatgtcac caaaggcacg ccccccaaaa atgcaacgtc gccccaggcc
2220cccagtggcc aaaagacggc ggttcccacg gtcacctcaa caggtggaaa
ggccaattct 2280accaccggtg gaaagcacac cacaggacat ggagcccgga
caagtacaga gcccaccaca 2340gattacggcg gtgattcaac tacgccaaga
acgagataca atgcgaccac ctatctacct 2400cccagcactt ctagcaaact
gcggccccgc tggactttta cgagcccacc ggttaccaca 2460gcccaagcca
ccgtgcctgt cccgccaacg tcccagccca gattctcaaa cctctccatg
2520ctagtactgc agtgggcctc tctggctgtg ctgacccttc tgctgctgct
ggtcatggcg 2580gactgcgcct tcaggcgtaa cttgtcgaca tcccatacct
acaccacccc accatatgat 2640gacgccgaga cctatgtata a
266172886PRTEpstein Barr Virus 72Met Glu Ala Ala Leu Leu Val Cys
Gln Tyr Thr Ile Gln Ser Leu Ile 1 5 10 15 Gln Leu Thr Arg Asp Asp
Pro Gly Phe Phe Asn Val Glu Ile Leu Glu 20 25 30 Phe Pro Phe Tyr
Pro Ala Cys Asn Val Cys Thr Ala Asp Val Asn Ala 35 40 45 Thr Ile
Asn Phe Asp Val Gly Gly Lys Lys His Lys Leu Asn Leu Asp 50 55 60
Phe Gly Leu Leu Thr Pro His Thr Lys Ala Val Tyr Gln Pro Arg Gly 65
70 75 80 Ala Phe Gly Gly Ser Glu Asn Ala Thr Asn Leu Phe Leu Leu
Glu Leu 85 90 95 Leu Gly Ala Gly Glu Leu Ala Leu Thr Met Arg Ser
Lys Lys Leu Pro 100 105 110 Ile Asn Ile Thr Thr Gly Glu Glu Gln Gln
Val Ser Leu Glu Ser Val 115 120 125 Asp Val Tyr Phe Gln Asp Val Phe
Gly Thr Met Trp Cys His His Ala 130 135 140 Glu Met Gln Asn Pro Val
Tyr Leu Ile Pro Glu Thr Val Pro Tyr Ile 145 150 155 160 Lys Trp Asp
Asn Cys Asn Ser Thr Asn Ile Thr Ala Val Val Arg Ala 165 170 175 Gln
Gly Leu Asp Val Thr Leu Pro Leu Ser Leu Pro Thr Ser Ala Gln 180 185
190 Asp Ser Asn Phe Ser Val Lys Thr Glu Met Leu Gly Asn Glu Ile Asp
195 200 205 Ile Glu Cys Ile Met Glu Asp Gly Glu Ile Ser Gln Val Leu
Pro Gly 210 215 220 Asp Asn Lys Phe Asn Ile Thr Cys Ser Gly Tyr Glu
Ser His Val Pro 225 230 235 240 Ser Gly Gly Ile Leu Thr Ser Thr Ser
Pro Val Ala Thr Pro Ile Pro 245 250 255 Gly Thr Gly Tyr Ala Tyr Ser
Leu Arg Leu Thr Pro Arg Pro Val Ser 260 265 270 Arg Phe Leu Gly Asn
Asn Ser Ile Leu Tyr Val Phe Tyr Ser Gly Asn 275 280 285 Gly Pro Lys
Ala Ser Gly Gly Asp Tyr Cys Ile Gln Ser Asn Ile Val 290 295 300 Phe
Ser Asp Glu Ile Pro Ala Ser Gln Asp Met Pro Thr Asn Thr Thr 305 310
315 320 Asp Ile Thr Tyr Val Gly Asp Asn Ala Thr Tyr Ser Val Pro Met
Val 325 330 335 Thr Ser Glu Asp Ala Asn Ser Pro Asn Val Thr Val Thr
Ala Phe Trp 340 345 350 Ala Trp Pro Asn Asn Thr Glu Thr Asp Phe Lys
Cys Lys Trp Thr Leu 355 360 365 Thr Ser Gly Thr Pro Ser Gly Cys Glu
Asn Ile Ser Gly Ala Phe Ala 370 375 380 Ser Asn Arg Thr Phe Asp Ile
Thr Val Ser Gly Leu Gly Thr Ala Pro 385 390 395 400 Lys Thr Leu Ile
Ile Thr Arg Thr Ala Thr Asn Ala Thr Thr Thr Thr 405 410 415 His Lys
Val Ile Phe Ser Lys Ala Pro Glu Ser Thr Thr Thr Ser Pro 420 425 430
Thr Leu Asn Thr Thr Gly Phe Ala Ala Pro Asn Thr Thr Thr Gly Leu 435
440 445 Pro Ser Ser Thr His Val Pro Thr Asn Leu Thr Ala Pro Ala Ser
Thr 450 455 460 Gly Pro Thr Val Ser Thr Ala Asp Val Thr Ser Pro Thr
Pro Ala Gly 465 470 475 480 Thr Thr Ser Gly Ala Ser Pro Val Thr Pro
Ser Pro Ser Pro Arg Asp 485 490 495 Asn Gly Thr Glu Ser Lys Ala Pro
Asp Met Thr Ser Pro Thr Ser Ala 500 505 510 Val Thr Thr Pro Thr Pro
Asn Ala Thr Ser Pro Thr Pro Ala Val Thr 515 520 525 Thr Pro Thr Pro
Asn Ala Thr Ser Pro Thr Leu Gly Lys Thr Ser Pro 530 535 540 Thr Ser
Ala Val Thr Thr Pro Thr Pro Asn Ala Thr Ser Pro Thr Pro 545 550 555
560 Ala Val Thr Thr Pro Thr Pro Asn Ala Thr Ile Pro Thr Leu Gly Lys
565 570 575 Thr Ser Pro Thr Ser Ala Val Thr Thr Pro Thr Pro Asn Ala
Thr Ser 580 585 590 Pro Thr Val Gly Glu Thr Ser Pro Gln Ala Asn Thr
Thr Asn His Thr 595 600 605 Leu Gly Gly Thr Ser Ser Thr Pro Val Val
Thr Ser Pro Pro
Lys Asn 610 615 620 Ala Thr Ser Ala Val Thr Thr Gly Gln His Asn Ile
Thr Ser Ser Ser 625 630 635 640 Thr Ser Ser Met Ser Leu Arg Pro Ser
Ser Ile Ser Glu Thr Leu Ser 645 650 655 Pro Ser Thr Ser Asp Asn Ser
Thr Ser His Met Pro Leu Leu Thr Ser 660 665 670 Ala His Pro Thr Gly
Gly Glu Asn Ile Thr Gln Val Thr Pro Ala Ser 675 680 685 Thr Ser Thr
His His Val Ser Thr Ser Ser Pro Ala Pro Arg Pro Gly 690 695 700 Thr
Thr Ser Gln Ala Ser Gly Pro Gly Asn Ser Ser Thr Ser Thr Lys 705 710
715 720 Pro Gly Glu Val Asn Val Thr Lys Gly Thr Pro Pro Lys Asn Ala
Thr 725 730 735 Ser Pro Gln Ala Pro Ser Gly Gln Lys Thr Ala Val Pro
Thr Val Thr 740 745 750 Ser Thr Gly Gly Lys Ala Asn Ser Thr Thr Gly
Gly Lys His Thr Thr 755 760 765 Gly His Gly Ala Arg Thr Ser Thr Glu
Pro Thr Thr Asp Tyr Gly Gly 770 775 780 Asp Ser Thr Thr Pro Arg Thr
Arg Tyr Asn Ala Thr Thr Tyr Leu Pro 785 790 795 800 Pro Ser Thr Ser
Ser Lys Leu Arg Pro Arg Trp Thr Phe Thr Ser Pro 805 810 815 Pro Val
Thr Thr Ala Gln Ala Thr Val Pro Val Pro Pro Thr Ser Gln 820 825 830
Pro Arg Phe Ser Asn Leu Ser Met Leu Val Leu Gln Trp Ala Ser Leu 835
840 845 Ala Val Leu Thr Leu Leu Leu Leu Leu Val Met Ala Asp Cys Ala
Phe 850 855 860 Arg Arg Asn Leu Ser Thr Ser His Thr Tyr Thr Thr Pro
Pro Tyr Asp 865 870 875 880 Asp Ala Glu Thr Tyr Val 885
732661DNAArtificial sequenceCodon modified Epstein Barr Virus
sequence 73atggaagctg ctctgctggt gtgtcagtac acgatccagt cgctgatcca
actgacgcgt 60gatgatcctg gattctttaa tgtcgaaatc ctggaatttc ccttctaccc
cgcttgcaat 120gtctgcacgg ctgatgtcaa tgctacgatc aattttgatg
tcggaggaaa aaagcataaa 180ctgaatctgg acttcggact gctgacgccc
catacgaagg ctgtctacca acctcgagga 240gctttcggag gatcggaaaa
tgctacgaat ctgttcctgc tggaactgct gggagctgga 300gaactggctc
tgacgatgcg atcgaagaag ctgcccatca acatcacgac gggagaagaa
360caacaagtct cgctggaatc ggtcgatgtc tacttccaag atgtgttcgg
aacgatgtgg 420tgccaccatg ctgaaatgca aaaccccgtc tacctgatcc
ccgaaacggt gccctacatc 480aagtgggata actgtaattc gacgaatatc
acggctgtcg tcagagctca gggactggat 540gtcacgctgc ccctgtcgct
gcccacgtcg gctcaagact cgaatttttc ggtcaaaacg 600gaaatgctgg
gaaatgaaat cgatatcgaa tgtatcatgg aagatggaga aatctcgcaa
660gtcctgcccg gagacaacaa attcaacatc acgtgctcgg gatacgaatc
gcatgtcccc 720tcgggaggaa tcctgacgtc gacgtcgccc gtggctacgc
ccatccctgg aacgggatat 780gcttactcgc tgcgtctgac gccccgtccc
gtgtcgcgat tcctgggaaa taactcgatc 840ctgtacgtgt tctactcggg
aaatggaccc aaggcttcgg gaggagatta ctgcatccag 900tcgaacatcg
tgttttcgga tgaaatcccc gcttcgcagg acatgcccac gaacacgacg
960gacatcacgt atgtgggaga caatgctacg tattcggtgc ccatggtcac
gtcggaagac 1020gctaactcgc ccaatgtcac ggtgacggct ttctgggctt
ggcccaacaa cacggaaacg 1080gacttcaagt gcaaatggac gctgacgtcg
ggaacgcctt cgggatgtga aaatatctcg 1140ggagctttcg cttcgaatcg
aacgttcgac atcacggtct cgggactggg aacggctccc 1200aagacgctga
tcatcacgcg aacggctacg aatgctacga cgacgacgca caaggtcatc
1260ttttcgaagg ctcccgaatc gacgacgacg tcgcctacgc tgaatacgac
gggattcgct 1320gctcccaata cgacgacggg actgccctcg tcgacgcacg
tgcctacgaa cctgacggct 1380cctgcttcga cgggacccac ggtctcgacg
gctgatgtca cgtcgcccac gcccgctgga 1440acgacgtcgg gagcttcgcc
cgtgacgccc tcgccctcgc cccgagacaa cggaacggaa 1500tcgaaggctc
ccgacatgac gtcgcccacg tcggctgtga cgacgcccac gcccaatgct
1560acgtcgccca cgcccgctgt gacgacgccc acgcccaatg ctacgtcgcc
cacgctggga 1620aaaacgtcgc ccacgtcggc tgtgacgacg cccacgccca
atgctacgtc gcccacgccc 1680gctgtgacga cgcccacgcc caatgctacg
atccccacgc tgggaaaaac gtcgcccacg 1740tcggctgtga cgacgcccac
gcccaatgct acgtcgccta cggtgggaga aacgtcgccc 1800caggctaata
cgacgaacca cacgctggga ggaacgtcgt cgacgcccgt cgtcacgtcg
1860ccccccaaaa atgctacgtc ggctgtcacg acgggacaac ataacatcac
gtcgtcgtcg 1920acgtcgtcga tgtcgctgag accctcgtcg atctcggaaa
cgctgtcgcc ctcgacgtcg 1980gacaattcga cgtcgcatat gcctctgctg
acgtcggctc accccacggg aggagaaaat 2040atcacgcagg tgacgcccgc
ttcgacgtcg acgcatcatg tgtcgacgtc gtcgcccgct 2100ccccgccccg
gaacgacgtc gcaagcttcg ggacctggaa actcgtcgac gtcgacgaaa
2160cccggagaag tcaatgtcac gaaaggaacg ccccccaaaa atgctacgtc
gccccaggct 2220ccctcgggac aaaagacggc tgtccccacg gtcacgtcga
cgggaggaaa ggctaattcg 2280acgacgggag gaaagcacac gacgggacat
ggagctcgaa cgtcgacgga acccacgacg 2340gattacggag gagattcgac
gacgcccaga acgagataca atgctacgac gtatctgcct 2400ccctcgacgt
cgtcgaaact gcgaccccgc tggacgttca cgtcgccccc cgtcacgacg
2460gctcaagcta cggtgcctgt cccccccacg tcgcagccca gattttcgaa
cctgtcgatg 2520ctggtcctgc agtgggcttc gctggctgtg ctgacgctgc
tgctgctgct ggtcatggct 2580gactgcgctt ttagacgtaa cctgtcgacg
tcgcatacgt acacgacgcc cccctatgat 2640gacgctgaaa cgtatgtcta a
2661742715DNAHerpes Simplex Virus 2 74atgcgcgggg ggggcttgat
ttgcgcgctg gtcgtggggg cgctggtggc cgcggtggcg 60tcggcggccc cggcggcccc
ggcggccccc cgcgcctcgg gcggcgtggc cgcgaccgtc 120gcggcgaacg
ggggtcccgc ctcccggccg ccccccgtcc cgagccccgc gaccaccaag
180gcccggaagc ggaaaaccaa aaagccgccc aagcggcccg aggcgacccc
gccccccgac 240gccaacgcga ccgtcgccgc cggccacgcc acgctgcgcg
cgcacctgcg ggaaatcaag 300gtcgagaacg ccgatgccca gttttacgtg
tgcccgcccc cgacgggcgc cacggtggtg 360cagtttgagc agccgcgccg
ctgcccgacg cgcccggagg ggcagaacta cacggagggc 420atcgcggtgg
tcttcaagga gaacatcgcc ccgtacaaat tcaaggccac catgtactac
480aaagacgtga ccgtgtcgca ggtgtggttc ggccaccgct actcccagtt
tatggggata 540ttcgaggacc gcgcccccgt tcccttcgag gaggtgatcg
acaagattaa caccaagggg 600gtctgccgct ccacggccaa gtacgtgcgg
aacaacatgg agaccaccgc gtttcaccgg 660gacgaccacg agaccgacat
ggagctcaag ccggcgaagg tcgccacgcg cacgagccgg 720gggtggcaca
ccaccgacct caagtacaac ccctcgcggg tggaggcgtt ccatcggtac
780ggcacgacgg tcaactgcat cgtcgaggag gtggacgcgc ggtcggtgta
cccgtacgat 840gagtttgtgc tggcgacggg cgactttgtg tacatgtccc
cgttttacgg ctaccgggag 900gggtcgcaca ccgagcacac cagctacgcc
gccgaccgct tcaagcaggt cgacggcttc 960tacgcgcgcg acctcaccac
gaaggcccgg gccacgtcgc cgacgacccg caacttgctg 1020acgaccccca
agtttaccgt ggcctgggac tgggtgccga agcgaccggc ggtctgcacc
1080atgaccaagt ggcaggaggt ggacgagatg ctccgcgccg agtacggcgg
ctccttccgc 1140ttctcctccg acgccatctc gaccaccttc accaccaacc
tgaccgagta ctcgctctcg 1200cgcgtcgacc tgggcgactg catcggccgg
gatgcccgcg aggccatcga ccgcatgttt 1260gcgcgcaagt acaacgccac
gcacatcaag gtgggccagc cgcagtacta cctggccacg 1320gggggcttcc
tcatcgcgta ccagcccctc ctcagcaaca cgctcgccga gctgtacgtg
1380cgggagtaca tgcgggagca ggaccgcaag ccccggaatg ccacgcccgc
gccactgcgg 1440gaggcgccca gcgccaacgc gtccgtggag cgcatcaaga
ccacctcctc gatcgagttc 1500gcccggctgc agtttacgta taaccacata
cagcgccacg tgaatgacat gctggggcgc 1560atcgccgtcg cgtggtgcga
gctgcagaac cacgagctga ctctctggaa cgaggcccgc 1620aagctcaacc
ccaacgccat cgcctccgcc accgtcggcc ggcgggtgag cgcgcgcatg
1680ctcggagacg tcatggccgt ctccacgtgc gtgcccgtcg ccccggacaa
cgtgatcgtg 1740cagaactcga tgcgcgtcag ctcgcggccg gggacgtgct
acagccgccc cctggtcagc 1800tttcggtacg aagaccaggg cccgctgatc
gaggggcagc tgggcgagaa caacgagctg 1860cgcctcaccc gcgacgcgct
cgagccgtgc accgtgggcc accggcgcta cttcatcttc 1920ggcgggggct
acgtgtactt cgaggagtac gcgtactctc accagctgag tcgcgccgac
1980gtcaccaccg tcagcacctt catcgacctg aacatcacca tgctggagga
ccacgagttt 2040gtgcccctgg aggtctacac gcgccacgag atcaaggaca
gcggcctgct ggactacacg 2100gaggtccagc gccgcaacca gctgcacgac
ctgcgctttg ccgacatcga cacggtcatc 2160cgcgccgacg ccaacgccgc
catgttcgcg gggctgtgcg cgttcttcga ggggatgggg 2220gacttggggc
gcgcggtcgg caaggtagtc atgggagtag tggggggcgt ggtgtcggcc
2280gtctcgggcg tgtcctcctt tatgtccaac cccttcgggg cgcttgccgt
ggggctgctg 2340gtcctggccg gcctggtcgc ggccttcttc gccttccgct
acgtcctgca actgcaacgc 2400aatcccatga aggccctgta tccgctcacc
accaaggaac tcaagacttc cgaccccggg 2460ggcgtgggcg gggaggggga
ggaaggcgcg gaggggggcg ggtttgacga ggccaagttg 2520gccgaggccc
gagaaatgat ccgatatatg gctttggtgt cggccatgga gcgcacggaa
2580cacaaggcca gaaagaaggg cacgagcgcc ctgctcagct ccaaggtcac
caacatggtt 2640ctgcgcaagc gcaacaaagc caggtactct ccgctccaca
acgaggacga ggccggagac 2700gaagacgagc tctaa 271575904PRTHerpes
Simplex Virus 2 75Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val
Gly Ala Leu Val 1 5 10 15 Ala Ala Val Ala Ser Ala Ala Pro Ala Ala
Pro Ala Ala Pro Arg Ala 20 25 30 Ser Gly Gly Val Ala Ala Thr Val
Ala Ala Asn Gly Gly Pro Ala Ser 35 40 45 Arg Pro Pro Pro Val Pro
Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg 50 55 60 Lys Thr Lys Lys
Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp 65 70 75 80 Ala Asn
Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His Leu 85 90 95
Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro 100
105 110 Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg
Cys 115 120 125 Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile
Ala Val Val 130 135 140 Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys
Ala Thr Met Tyr Tyr 145 150 155 160 Lys Asp Val Thr Val Ser Gln Val
Trp Phe Gly His Arg Tyr Ser Gln 165 170 175 Phe Met Gly Ile Phe Glu
Asp Arg Ala Pro Val Pro Phe Glu Glu Val 180 185 190 Ile Asp Lys Ile
Asn Thr Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr 195 200 205 Val Arg
Asn Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His Glu 210 215 220
Thr Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr Ser Arg 225
230 235 240 Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val
Glu Ala 245 250 255 Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile Val
Glu Glu Val Asp 260 265 270 Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe
Val Leu Ala Thr Gly Asp 275 280 285 Phe Val Tyr Met Ser Pro Phe Tyr
Gly Tyr Arg Glu Gly Ser His Thr 290 295 300 Glu His Thr Ser Tyr Ala
Ala Asp Arg Phe Lys Gln Val Asp Gly Phe 305 310 315 320 Tyr Ala Arg
Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr 325 330 335 Arg
Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val 340 345
350 Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp
355 360 365 Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser
Ser Asp 370 375 380 Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu
Tyr Ser Leu Ser 385 390 395 400 Arg Val Asp Leu Gly Asp Cys Ile Gly
Arg Asp Ala Arg Glu Ala Ile 405 410 415 Asp Arg Met Phe Ala Arg Lys
Tyr Asn Ala Thr His Ile Lys Val Gly 420 425 430 Gln Pro Gln Tyr Tyr
Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln 435 440 445 Pro Leu Leu
Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met 450 455 460 Arg
Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg 465 470
475 480 Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr
Ser 485 490 495 Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His
Ile Gln Arg 500 505 510 His Val Asn Asp Met Leu Gly Arg Ile Ala Val
Ala Trp Cys Glu Leu 515 520 525 Gln Asn His Glu Leu Thr Leu Trp Asn
Glu Ala Arg Lys Leu Asn Pro 530 535 540 Asn Ala Ile Ala Ser Ala Thr
Val Gly Arg Arg Val Ser Ala Arg Met 545 550 555 560 Leu Gly Asp Val
Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp 565 570 575 Asn Val
Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr 580 585 590
Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro 595
600 605 Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr
Arg 610 615 620 Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg Tyr
Phe Ile Phe 625 630 635 640 Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr
Ala Tyr Ser His Gln Leu 645 650 655 Ser Arg Ala Asp Val Thr Thr Val
Ser Thr Phe Ile Asp Leu Asn Ile 660 665 670 Thr Met Leu Glu Asp His
Glu Phe Val Pro Leu Glu Val Tyr Thr Arg 675 680 685 His Glu Ile Lys
Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg 690 695 700 Arg Asn
Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val Ile 705 710 715
720 Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Cys Ala Phe Phe
725 730 735 Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val
Met Gly 740 745 750 Val Val Gly Gly Val Val Ser Ala Val Ser Gly Val
Ser Ser Phe Met 755 760 765 Ser Asn Pro Phe Gly Ala Leu Ala Val Gly
Leu Leu Val Leu Ala Gly 770 775 780 Leu Val Ala Ala Phe Phe Ala Phe
Arg Tyr Val Leu Gln Leu Gln Arg 785 790 795 800 Asn Pro Met Lys Ala
Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr 805 810 815 Ser Asp Pro
Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly 820 825 830 Gly
Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840
845 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Arg
850 855 860 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn
Met Val 865 870 875 880 Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro
Leu His Asn Glu Asp 885 890 895 Glu Ala Gly Asp Glu Asp Glu Leu 900
762715DNAArtificial sequenceCodon modified Herpes Simplex Virus 2
sequence 76atgcgcggag gaggactgat ctgcgctctg gtcgtgggag ctctggtggc
tgctgtggct 60tcggctgctc ccgctgctcc cgctgctccc cgcgcttcgg gaggagtggc
tgctacggtc 120gctgctaacg gaggacccgc ttcgcgaccc ccccccgtcc
cctcgcccgc tacgacgaag 180gctcgaaagc gaaaaacgaa aaagcccccc
aagcgacccg aagctacgcc cccccccgac 240gctaacgcta cggtcgctgc
tggacacgct acgctgcgcg ctcacctgcg agaaatcaag 300gtcgaaaacg
ctgatgctca gttctacgtg tgcccccccc ccacgggagc tacggtggtg
360cagttcgaac agccccgccg ctgccccacg cgccccgaag gacagaacta
cacggaagga 420atcgctgtgg tctttaagga aaacatcgct ccctacaaat
ttaaggctac gatgtactac 480aaagacgtga cggtgtcgca ggtgtggttt
ggacaccgct actcgcagtt catgggaatc 540tttgaagacc gcgctcccgt
cccctttgaa gaagtgatcg acaagatcaa cacgaaggga 600gtctgccgct
cgacggctaa gtacgtgcga aacaacatgg aaacgacggc tttccaccga
660gacgaccacg aaacggacat ggaactgaag cccgctaagg tcgctacgcg
cacgtcgcga 720ggatggcaca cgacggacct gaagtacaac ccctcgcgag
tggaagcttt tcatcgatac 780ggaacgacgg tcaactgcat cgtcgaagaa
gtggacgctc gatcggtgta cccctacgat 840gaattcgtgc tggctacggg
agacttcgtg tacatgtcgc ccttctacgg ataccgagaa 900ggatcgcaca
cggaacacac gtcgtacgct gctgaccgct ttaagcaggt cgacggattt
960tacgctcgcg acctgacgac gaaggctcga gctacgtcgc ccacgacgcg
caacctgctg 1020acgacgccca agttcacggt ggcttgggac tgggtgccca
agcgacccgc tgtctgcacg 1080atgacgaagt ggcaggaagt ggacgaaatg
ctgcgcgctg aatacggagg atcgtttcgc 1140ttttcgtcgg acgctatctc
gacgacgttt acgacgaacc tgacggaata ctcgctgtcg 1200cgcgtcgacc
tgggagactg catcggacga gatgctcgcg aagctatcga ccgcatgttc
1260gctcgcaagt acaacgctac gcacatcaag gtgggacagc cccagtacta
cctggctacg 1320ggaggatttc tgatcgctta ccagcccctg ctgtcgaaca
cgctggctga actgtacgtg 1380cgagaataca tgcgagaaca ggaccgcaag
ccccgaaatg ctacgcccgc tcccctgcga 1440gaagctccct cggctaacgc
ttcggtggaa cgcatcaaga cgacgtcgtc gatcgaattt 1500gctcgactgc
agttcacgta taaccacatc cagcgccacg tgaatgacat gctgggacgc
1560atcgctgtcg cttggtgcga actgcagaac cacgaactga cgctgtggaa
cgaagctcgc 1620aagctgaacc ccaacgctat cgcttcggct acggtcggac
gacgagtgtc ggctcgcatg 1680ctgggagacg tcatggctgt ctcgacgtgc
gtgcccgtcg ctcccgacaa
cgtgatcgtg 1740cagaactcga tgcgcgtctc gtcgcgaccc ggaacgtgct
actcgcgccc cctggtctcg 1800ttccgatacg aagaccaggg acccctgatc
gaaggacagc tgggagaaaa caacgaactg 1860cgcctgacgc gcgacgctct
ggaaccctgc acggtgggac accgacgcta ctttatcttt 1920ggaggaggat
acgtgtactt tgaagaatac gcttactcgc accagctgtc gcgcgctgac
1980gtcacgacgg tctcgacgtt tatcgacctg aacatcacga tgctggaaga
ccacgaattc 2040gtgcccctgg aagtctacac gcgccacgaa atcaaggact
cgggactgct ggactacacg 2100gaagtccagc gccgcaacca gctgcacgac
ctgcgcttcg ctgacatcga cacggtcatc 2160cgcgctgacg ctaacgctgc
tatgtttgct ggactgtgcg ctttttttga aggaatggga 2220gacctgggac
gcgctgtcgg aaaggtcgtc atgggagtcg tgggaggagt ggtgtcggct
2280gtctcgggag tgtcgtcgtt catgtcgaac ccctttggag ctctggctgt
gggactgctg 2340gtcctggctg gactggtcgc tgcttttttt gcttttcgct
acgtcctgca actgcaacgc 2400aatcccatga aggctctgta tcccctgacg
acgaaggaac tgaagacgtc ggaccccgga 2460ggagtgggag gagaaggaga
agaaggagct gaaggaggag gattcgacga agctaagctg 2520gctgaagctc
gagaaatgat ccgatatatg gctctggtgt cggctatgga acgcacggaa
2580cacaaggcta gaaagaaggg aacgtcggct ctgctgtcgt cgaaggtcac
gaacatggtc 2640ctgcgcaagc gcaacaaagc tagatactcg cccctgcaca
acgaagacga agctggagac 2700gaagacgaac tgtaa 2715771182DNAHerpes
Simplex Virus 77atggggcgtt tgacctccgg cgtcgggacg gcggccctgc
tagttgtcgc ggtgggactc 60cgcgtcgtct gcgccaaata cgccttagca gacccctcgc
ttaagatggc cgatcccaat 120cgatttcgcg ggaagaacct tccggttttg
gaccagctga ccgacccccc cggggtgaag 180cgtgtttacc acattcagcc
gagcctggag gacccgttcc agccccccag catcccgatc 240actgtgtact
acgcagtgct ggaacgtgcc tgccgcagcg tgctcctaca tgccccatcg
300gaggcccccc agatcgtgcg cggggcttcg gacgaggccc gaaagcacac
gtacaacctg 360accatcgcct ggtatcgcat gggagacaat tgcgctatcc
ccatcacggt tatggaatac 420accgagtgcc cctacaacaa gtcgttgggg
gtctgcccca tccgaacgca gccccgctgg 480agctactatg acagctttag
cgccgtcagc gaggataacc tgggattcct gatgcacgcc 540cccgccttcg
agaccgcggg tacgtacctg cggctagtga agataaacga ctggacggag
600atcacacaat ttatcctgga gcaccgggcc cgcgcctcct gcaagtacgc
tctccccctg 660cgcatccccc cggcagcgtg cctcacctcg aaggcctacc
aacagggcgt gacggtcgac 720agcatcggga tgctaccccg ctttatcccc
gaaaaccagc gcaccgtcgc cctatacagc 780ttaaaaatcg ccgggtggca
cggccccaag cccccgtaca ccagcaccct gctgccgccg 840gagctgtccg
acaccaccaa cgccacgcaa cccgaactcg ttccggaaga ccccgaggac
900tcggccctct tagaggatcc cgccgggacg gtgtcttcgc agatcccccc
aaactggcac 960atcccgtcga tccaggacgt cgcgccgcac cacgcccccg
ccgcccccag caacccgggc 1020ctgatcatcg gcgcgctggc cggcagtacc
ctggcggtgc tggtcatcgg cggtattgcg 1080ttttgggtac gccgccgcgc
tcagatggcc cccaagcgcc tacgtctccc ccacatccgg 1140gatgacgacg
cgcccccctc gcaccagcca ttgttttact ag 118278393PRTHerpes Simplex
Virus 78Met Gly Arg Leu Thr Ser Gly Val Gly Thr Ala Ala Leu Leu Val
Val 1 5 10 15 Ala Val Gly Leu Arg Val Val Cys Ala Lys Tyr Ala Leu
Ala Asp Pro 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg
Gly Lys Asn Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro
Gly Val Lys Arg Val Tyr His 50 55 60 Ile Gln Pro Ser Leu Glu Asp
Pro Phe Gln Pro Pro Ser Ile Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala
Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 His Ala Pro
Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Asp Glu 100 105 110 Ala
Arg Lys His Thr Tyr Asn Leu Thr Ile Ala Trp Tyr Arg Met Gly 115 120
125 Asp Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Pro
130 135 140 Tyr Asn Lys Ser Leu Gly Val Cys Pro Ile Arg Thr Gln Pro
Arg Trp 145 150 155 160 Ser Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu
Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr
Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr
Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Arg Ala Ser
Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ala Ala Cys
Leu Thr Ser Lys Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240
Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245
250 255 Ala Leu Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Lys Pro
Pro 260 265 270 Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser Asp Thr
Thr Asn Ala 275 280 285 Thr Gln Pro Glu Leu Val Pro Glu Asp Pro Glu
Asp Ser Ala Leu Leu 290 295 300 Glu Asp Pro Ala Gly Thr Val Ser Ser
Gln Ile Pro Pro Asn Trp His 305 310 315 320 Ile Pro Ser Ile Gln Asp
Val Ala Pro His His Ala Pro Ala Ala Pro 325 330 335 Ser Asn Pro Gly
Leu Ile Ile Gly Ala Leu Ala Gly Ser Thr Leu Ala 340 345 350 Val Leu
Val Ile Gly Gly Ile Ala Phe Trp Val Arg Arg Arg Ala Gln 355 360 365
Met Ala Pro Lys Arg Leu Arg Leu Pro His Ile Arg Asp Asp Asp Ala 370
375 380 Pro Pro Ser His Gln Pro Leu Phe Tyr 385 390
791182DNAArtificial sequenceCodon modified Herpes Simplex Virus
sequence 79atgggacgtc tgacgtcggg agtcggaacg gctgctctgc tggtcgtcgc
tgtgggactg 60cgcgtcgtct gcgctaaata cgctctggct gacccctcgc tgaagatggc
tgatcccaat 120cgattccgcg gaaagaacct gcccgtcctg gaccagctga
cggacccccc cggagtgaag 180cgtgtctacc acatccagcc ctcgctggaa
gacccctttc agcccccctc gatccccatc 240acggtgtact acgctgtgct
ggaacgtgct tgccgctcgg tgctgctgca tgctccctcg 300gaagctcccc
agatcgtgcg cggagcttcg gacgaagctc gaaagcacac gtacaacctg
360acgatcgctt ggtatcgcat gggagacaat tgcgctatcc ccatcacggt
catggaatac 420acggaatgcc cctacaacaa gtcgctggga gtctgcccca
tccgaacgca gccccgctgg 480tcgtactatg actcgttctc ggctgtctcg
gaagataacc tgggatttct gatgcacgct 540cccgcttttg aaacggctgg
aacgtacctg cgactggtga agatcaacga ctggacggaa 600atcacgcaat
tcatcctgga acaccgagct cgcgcttcgt gcaagtacgc tctgcccctg
660cgcatccccc ccgctgcttg cctgacgtcg aaggcttacc aacagggagt
gacggtcgac 720tcgatcggaa tgctgccccg cttcatcccc gaaaaccagc
gcacggtcgc tctgtactcg 780ctgaaaatcg ctggatggca cggacccaag
cccccctaca cgtcgacgct gctgcccccc 840gaactgtcgg acacgacgaa
cgctacgcaa cccgaactgg tccccgaaga ccccgaagac 900tcggctctgc
tggaagatcc cgctggaacg gtgtcgtcgc agatcccccc caactggcac
960atcccctcga tccaggacgt cgctccccac cacgctcccg ctgctccctc
gaaccccgga 1020ctgatcatcg gagctctggc tggatcgacg ctggctgtgc
tggtcatcgg aggaatcgct 1080ttctgggtcc gccgccgcgc tcagatggct
cccaagcgcc tgcgtctgcc ccacatccga 1140gatgacgacg ctcccccctc
gcaccagccc ctgttctact ag 118280387DNAHuman papillomavirus type 16
80ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactgatctc tactgttatg agcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38781387DNAArtificial SequenceHPV-16 E7 O1
81ggtaccgccg ccaccatgga aacggacacg ctgctgctgt gggtcctgct gctgtgggtc
60cccggatcga cgggagacgg atcgatgcat ggagacacgc ccacgctgca tgaatacatg
120ctggacctgc aacccgaaac gacggacctg tactgctacg aacaactgaa
cgactcgtcg 180gaagaagaag acgaaatcga cggacccgct ggacaagctg
aacccgacag agctcattac 240aacatcgtca cgttctgctg caagtgcgac
tcgacgctgc gactgtgcgt ccaatcgacg 300cacgtcgaca tccgtacgct
ggaagacctg ctgatgggaa cgctgggaat cgtgtgcccc 360atctgctcgc
agaagcccta agaattc 38782387DNAArtificial SequenceHPV16 E7 O2
82ggtaccgccg ccaccatgga aacggacacg ctgctgctgt gggtcctgct gctgtgggtc
60cccggatcga cgggagacgg atcgatgcat ggagatacgc ctacgctgca tgaatatatg
120ctggatctgc aacccgaaac gacggatctg tactgttatg aacaactgaa
tgactcgtcg 180gaagaagaag atgaaatcga tggacccgct ggacaagctg
aacccgacag agctcattac 240aatatcgtca cgttttgttg caagtgtgac
tcgacgctgc gactgtgcgt ccaatcgacg 300cacgtcgaca tccgtacgct
ggaagacctg ctgatgggaa cgctgggaat cgtgtgcccc 360atctgctcgc
agaagcccta agaattc 38783417DNAArtificial SequenceHPV-16 E7 O3
83ggtaccgccg ccaccatgga gacggacacg ctcctgctct gggtactgct gctctgggtt
60cctggatcga cgggattgtg gacggatcga tgcatggaga tacgcctacg ctccatgaat
120atatgctcga tctccaacct ggttgagacg acggatctct actgttatga
gcaactcaat 180gactcgtcgg aggaggagga tgaattcata gatggacctg
ctggacaagc agaacctgac 240agagcccatt acaatattgt aacgtttgag
aattgttgca agtgtgactc gacgctccgg 300ctctgcgtac aatcgacgca
cgtagacatt cgtccctcta cgctcgaaga cctgctcatg 360ggaacgctcg
gaattgtgtg ccccatctgc tcgcagaagt gtgcccccta agaattc
41784387DNAArtificial SequenceHPV-16 E7 W 84ggtaccgccg ccaccatgga
gactgatact ttattattat gggtattatt attatgggtt 60ccaggtagta ctggtgatgg
cagtatgcat ggcgatactc caactttaca tgagtatatg 120ttagatttac
aaccagagac tactgattta tattgttatg agcaattaaa tgatagcagt
180gaggaggagg atgagataga tggtccagcg ggccaagcag agccggatcg
ggcgcattat 240aatatagtaa ctttctgttg taagtgtgat agtactttac
ggttatgtgt acaaagcact 300cacgtagata tacggacttt agaggattta
ttaatgggca ctttaggcat agtatgtcca 360atatgtagtc agaagccata agaattc
387851182DNAHerpes simplex virus type 2 85atggggcgtt tgacctccgg
cgtcgggacg gcggccctgc tagttgtcgc ggtgggactc 60cgcgtcgtct gcgccaaata
cgccttagca gacccctcgc ttaagatggc cgatcccaat 120cgatttcgcg
ggaagaacct tccggttttg gaccagctga ccgacccccc cggggtgaag
180cgtgtttacc acattcagcc gagcctggag gacccgttcc agccccccag
catcccgatc 240actgtgtact acgcagtgct ggaacgtgcc tgccgcagcg
tgctcctaca tgccccatcg 300gaggcccccc agatcgtgcg cggggcttcg
gacgaggccc gaaagcacac gtacaacctg 360accatcgcct ggtatcgcat
gggagacaat tgcgctatcc ccatcacggt tatggaatac 420accgagtgcc
cctacaacaa gtcgttgggg gtctgcccca tccgaacgca gccccgctgg
480agctactatg acagctttag cgccgtcagc gaggataacc tgggattcct
gatgcacgcc 540cccgccttcg agaccgcggg tacgtacctg cggctagtga
agataaacga ctggacggag 600atcacacaat ttatcctgga gcaccgggcc
cgcgcctcct gcaagtacgc tctccccctg 660cgcatccccc cggcagcgtg
cctcacctcg aaggcctacc aacagggcgt gacggtcgac 720agcatcggga
tgctaccccg ctttatcccc gaaaaccagc gcaccgtcgc cctatacagc
780ttaaaaatcg ccgggtggca cggccccaag cccccgtaca ccagcaccct
gctgccgccg 840gagctgtccg acaccaccaa cgccacgcaa cccgaactcg
ttccggaaga ccccgaggac 900tcggccctct tagaggatcc cgccgggacg
gtgtcttcgc agatcccccc aaactggcac 960atcccgtcga tccaggacgt
cgcgccgcac cacgcccccg ccgcccccag caacccgggc 1020ctgatcatcg
gcgcgctggc cggcagtacc ctggcggtgc tggtcatcgg cggtattgcg
1080ttttgggtac gccgccgcgc tcagatggcc cccaagcgcc tacgtctccc
ccacatccgg 1140gatgacgacg cgcccccctc gcaccagcca ttgttttact ag
1182861182DNAArtificial SequenceHSV-2 gD2 O1 86atgggacgtc
tgacgtcggg agtcggaacg gctgctctgc tggtcgtcgc tgtgggactc 60cgcgtcgtct
gcgctaaata cgctctggct gacccctcgc tgaagatggc tgaccccaac
120cgatttcgcg gaaagaacct gcccgtcctg gaccagctga cggacccccc
cggagtgaag 180cgtgtctacc acatccagcc ctcgctggaa gacccctttc
agcccccctc gatccccatc 240acggtgtact acgctgtgct ggaacgtgct
tgccgctcgg tgctcctcca tgctccctcg 300gaagctcccc agatcgtgcg
cggagcttcg gacgaagctc gaaagcacac gtacaacctg 360acgatcgctt
ggtaccgcat gggagacaac tgcgctatcc ccatcacggt catggaatac
420acggaatgcc cctacaacaa gtcgctcgga gtctgcccca tccgaacgca
gccccgctgg 480tcgtactacg actcgttttc ggctgtctcg gaagacaacc
tgggatttct gatgcacgct 540cccgcttttg aaacggctgg aacgtacctg
cgactcgtga agatcaacga ctggacggaa 600atcacgcaat ttatcctgga
acaccgagct cgcgcttcgt gcaagtacgc tctccccctg 660cgcatccccc
ccgctgcttg cctcacgtcg aaggcttacc aacagggagt gacggtcgac
720tcgatcggaa tgctcccccg ctttatcccc gaaaaccagc gcacggtcgc
tctctactcg 780ctcaaaatcg ctggatggca cggacccaag cccccctaca
cgtcgacgct gctgcccccc 840gaactgtcgg acacgacgaa cgctacgcaa
cccgaactcg tccccgaaga ccccgaagac 900tcggctctcc tcgaagaccc
cgctggaacg gtgtcgtcgc agatcccccc caactggcac 960atcccctcga
tccaggacgt cgctccccac cacgctcccg ctgctccctc gaaccccgga
1020ctgatcatcg gagctctggc tggatcgacg ctggctgtgc tggtcatcgg
aggaatcgct 1080ttttgggtcc gccgccgcgc tcagatggct cccaagcgcc
tccgtctccc ccacatccga 1140gacgacgacg ctcccccctc gcaccagccc
ctcttttact ag 1182871182DNAArtificial SequenceHSV-2 gD2 O2
87atgggacgtc tgacgtcggg agtcggaacg gctgctctgc tggtcgtcgc tgtgggactg
60cgcgtcgtct gcgctaaata cgctctggct gacccctcgc tgaagatggc tgatcccaat
120cgatttcgcg gaaagaacct gcccgtcctg gaccagctga cggacccccc
cggagtgaag 180cgtgtctacc acatccagcc ctcgctggaa gacccctttc
agcccccctc gatccccatc 240acggtgtact acgctgtgct ggaacgtgct
tgccgctcgg tgctgctgca tgctccctcg 300gaagctcccc agatcgtgcg
cggagcttcg gacgaagctc gaaagcacac gtacaacctg 360acgatcgctt
ggtatcgcat gggagacaat tgcgctatcc ccatcacggt catggaatac
420acggaatgcc cctacaacaa gtcgctggga gtctgcccca tccgaacgca
gccccgctgg 480tcgtactatg actcgttttc ggctgtctcg gaagataacc
tgggatttct gatgcacgct 540cccgcttttg aaacggctgg aacgtacctg
cgactggtga agatcaacga ctggacggaa 600atcacgcaat ttatcctgga
acaccgagct cgcgcttcgt gcaagtacgc tctgcccctg 660cgcatccccc
ccgctgcttg cctgacgtcg aaggcttacc aacagggagt gacggtcgac
720tcgatcggaa tgctgccccg ctttatcccc gaaaaccagc gcacggtcgc
tctgtactcg 780ctgaaaatcg ctggatggca cggacccaag cccccctaca
cgtcgacgct gctgcccccc 840gaactgtcgg acacgacgaa cgctacgcaa
cccgaactgg tccccgaaga ccccgaagac 900tcggctctgc tggaagatcc
cgctggaacg gtgtcgtcgc agatcccccc caactggcac 960atcccctcga
tccaggacgt cgctccccac cacgctcccg ctgctccctc gaaccccgga
1020ctgatcatcg gagctctggc tggatcgacg ctggctgtgc tggtcatcgg
aggaatcgct 1080ttttgggtcc gccgccgcgc tcagatggct cccaagcgcc
tgcgtctgcc ccacatccga 1140gatgacgacg ctcccccctc gcaccagccc
ctgttttact ag 1182881182DNAArtificial SequenceHSV-2 gD2 O3
88atgggacgtc tcacgtcggg agtcggaacg gcggccctgc tcgttgtcgc ggtgggactc
60cgcgtcgtct gcgccaaata cgccctcgca gacccctcgc tcaagatggc cgatcccaat
120cgatttcgcg gaaagaacct ccctgttctc gaccagctga cggacccccc
cggagtgaag 180cgtgtttacc acattcagcc ttcgctggag gaccctttcc
agcccccctc gatccctatc 240acggtgtact acgcagtgct ggaacgtgcc
tgccgctcgg tgctcctcca tgccccttcg 300gaggcccccc agatcgtgcg
cggagcttcg gacgaggccc gaaagcacac gtacaacctg 360acgatcgcct
ggtatcgcat gggagacaat tgcgctatcc ccatcacggt tatggaatac
420acggagtgcc cctacaacaa gtcgctcgga gtctgcccca tccgaacgca
gccccgctgg 480tcgtactatg actcgttttc ggccgtctcg gaggataacc
tgggattcct gatgcacgcc 540cccgccttcg agacggcggg aacgtacctg
cggctcgtga agataaacga ctggacggag 600atcacgcaat ttatcctgga
gcaccgggcc cgcgcctcgt gcaagtacgc tctccccctg 660cgcatccccc
ctgcagcgtg cctcacgtcg aaggcctacc aacagggagt gacggtcgac
720tcgatcggaa tgctcccccg ctttatcccc gaaaaccagc gcacggtcgc
cctctactcg 780ctcaaaatcg ccggatggca cggacccaag cccccttaca
cgtcgacgct gctgcctcct 840gagctgtcgg acacgacgaa cgccacgcaa
cccgaactcg ttcctgaaga ccccgaggac 900tcggccctcc tagaggatcc
cgccggaacg gtgtcgtcgc agatcccccc taactggcac 960atcccttcga
tccaggacgt cgcgcctcac cacgcccccg ccgccccctc gaaccctgga
1020ctgatcatcg gagcgctggc cggatcgacg ctggcggtgc tggtcatcgg
aggaattgcg 1080ttttgggtac gccgccgcgc tcagatggcc cccaagcgcc
tccgtctccc ccacatccgg 1140gatgacgacg cgcccccctc gcaccagcct
ctcttttact ag 1182891182DNAArtificial SequenceHSV-2 gD2 W
89atggggcggt tgactagtgg cgtagggact gcggcgttat tagtagtagc ggtaggctta
60cgggtagtat gtgcaaaata tgcgttagca gatccaagtt taaagatggc ggatccaaat
120cggttccggg ggaagaattt accggtattg gatcagttaa ctgatccacc
aggggtaaag 180cgggtatatc acatacagcc gagcttagag gatccgttcc
agccaccaag cataccgata 240actgtatatt atgcagtatt agagcgggcg
tgtcggagcg tattattaca tgcaccaagt 300gaggcgccac agatagtacg
gggggcaagt gatgaggcgc ggaagcacac ttataattta 360actatagcat
ggtatcggat gggcgataat tgtgcgatac caataactgt aatggagtat
420actgagtgtc catataataa gagtttgggg gtatgtccaa tacggactca
gccacggtgg 480agctattatg atagcttcag cgcagtaagc gaggataatt
taggcttctt aatgcacgcg 540ccagcattcg agactgcggg tacttattta
cggttagtaa agataaatga ttggactgag 600ataactcaat tcatattaga
gcaccgggca cgggcgagtt gtaagtatgc attaccatta 660cggataccac
cggcagcgtg tttaactagt aaggcatatc aacagggcgt aactgtagat
720agcataggga tgttaccacg gttcatacca gagaatcagc ggactgtagc
gttatatagc 780ttaaaaatag cagggtggca cggcccaaag ccaccgtata
ctagcacttt attaccgccg 840gagttaagtg atactactaa tgcgactcaa
ccagagttag taccggagga tccagaggat 900agtgcattat tagaggatcc
agcggggact gtaagtagtc agataccacc aaattggcac 960ataccgagta
tacaggatgt agcgccgcac cacgcaccag cggcaccaag caatccgggc
1020ttaataatag gcgcgttagc aggcagtact ttagcggtat tagtaatagg
cggtatagcg 1080ttctgggtac ggcggcgggc gcagatggcg ccaaagcggt
tacggttacc acacatacgg 1140gatgatgatg cgccaccaag tcaccagcca
ttgttctatt ag 11829041DNAArtificial sequenceCommon forward primer
90ttgaataggt accgccgcca ccatggagac cgacaccctc c 419124DNAArtificial
SequenceODN-7909 91tcgtcgtttt gtcgttttgt cgtt 24927PRTArtificial
SequenceSecretory sequence 92Xaa Xaa Gly Xaa Gly Xaa Xaa 1 5
93387DNAArtificial SequenceIgkC1 93ggtaccgccg ccaccatgga gacagacaca
ctcctgctat gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat
ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactggtctc tacggttatg ggcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38794387DNAArtificial SequenceIgkS1-1
94ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactggtctc tacggttatg ggcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagcg ggacaagcgg
aaccggacag agcgcattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38795387DNAArtificial SequenceIgKS1-2
95ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactggtctc tacggttatg ggcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagca ggacaagcag
aaccggacag agcacattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38796387DNAArtificial SequenceIgkS1-3
96ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactggtctc tacggttatg ggcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagctg
aaccggacag agctcattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38797387DNAArtificial SequenceIgkS1-4
97ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactggtctc tacggttatg ggcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagcc ggacaagccg
aaccggacag agcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgcttc ggttgtgcgt acaaagcaca 300cacgtagaca ttcgtacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 38798387DNAArtificial SequenceIgkC2 98ggtaccgccg
ccaccatgga gaccgacacc ctcctgctgt gggtgctgct gctctgggtg 60cccggctcca
ccggcgacgg atccatgcac ggcgacaccc ccaccctgca cgagtacatg
120ctggacctgc agcccgagac caccggcctg tacggctacg gccagctcaa
cgacagcagc 180gaggaggagg acgagatcga cggccccgcc ggccaggccg
agcccgaccg cgcccactac 240aacatcgtga ccttctgctg caagtgcgac
agcaccctgc gcctctgcgt gcagagcacc 300cacgtggaca tccgcaccct
ggaggacctg ctgatgggca ccctgggcat cgtgtgcccc 360atctgctccc
agaagcccta agaattc 38799387DNAArtificial SequenceIgkS1-5
99ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt
60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca tgaatatatg
120ttagatttgc aaccagagac aactggtctc tacggttatg ggcaattaaa
tgacagctca 180gaggaggagg atgaaataga tggtccagct ggacaagcag
aaccggacag ggcccattac 240aatattgtaa ccttttgttg caagtgtgac
tctacgctta ggttgtgcgt acaaagcaca 300cacgtagaca ttaggacttt
ggaagacctg ttaatgggca cactaggaat tgtgtgcccc 360atctgctctc
agaagcccta agaattc 387100387DNAArtificial SequenceIgkS1-6
100ggtaccgccg ccaccatgga gacagacaca ctcctgctat gggtactgct
gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac ctacattgca
tgaatatatg 120ttagatttgc aaccagagac aactggtctc tacggttatg
ggcaattaaa tgacagctca 180gaggaggagg atgaaataga tggtccagct
ggacaagcag aaccggacag agcccattac 240aatattgtaa ccttttgttg
caagtgtgac tctacgctta gattgtgcgt acaaagcaca 300cacgtagaca
ttagaacttt ggaagacctg ttaatgggca cactaggaat tgtgtgcccc
360atctgctctc agaagcccta agaattc 387101387DNAArtificial
SequenceIgkS1-7 101ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggaccg ggcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcggacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387102387DNAArtificial
SequenceIgkS1-8 102ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggaccg agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc gattgtgcgt acaaagcaca
300cacgtagaca ttcgaacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387103387DNAArtificial
SequenceIgkS1-9 103ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggaccg tgcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc gtttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387104387DNAArtificial
SequenceIgkS1-10 104ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggaccg cgcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc gcttgtgcgt acaaagcaca
300cacgtagaca ttcgcacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387105387DNAArtificial
SequenceIgkS1-12 105ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa cgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aacattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387106387DNAArtificial
SequenceIgkS1-31 106ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caaatgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaaacccta agaattc 387107387DNAArtificial
SequenceIgkS1-13 107ggtaccgccg ccaccatgga gacagataca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgatgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgatagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggatag agcccattac 240aatattgtaa
ccttttgttg caagtgtgat tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagata ttcgtacttt ggaagatctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387108387DNAArtificial
SequenceIgkS1-14 108ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagacacac
ctacattgca tgaatatatg 120ttagacttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg acgaaataga
cggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387109387DNAArtificial
SequenceIgkS1-15 109ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg taagtgtgac tctacgcttc ggttgtgtgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgtccc 360atctgttctc agaagcccta agaattc 387110387DNAArtificial
SequenceIgkS1-16 110ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgctg caagtgcgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387111387DNAArtificial
SequenceIgkS1-17 111ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgagtatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgagataga
tggtccagct ggacaagcag agccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaggacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387112387DNAArtificial
SequenceIgkS1-18 112ggtaccgccg ccaccatgga aacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagaaac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaagaagaag atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387113387DNAArtificial
SequenceIgkS1-19 113ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc agccagagac aactggtctc
tacggttatg ggcagttaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaggcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acagagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387114387DNAArtificial
SequenceIgkS1-20 114ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc aaaagcccta agaattc 387115387DNAArtificial
SequenceIgkS1-21 115ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccagggtcca ctggggacgg atccatgcat ggggatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactgggctc
tacgggtatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tgggccagct gggcaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggga cactagggat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387116387DNAArtificial
SequenceIgkS1-22 116ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggatcca ctggagacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggactc
tacggatatg gacaattaaa tgacagctca 180gaggaggagg atgaaataga
tggaccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggaa cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387117387DNAArtificial
SequenceIgkS1-23 117ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggtgatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg gtcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggtcaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggta cactaggtat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387118387DNAArtificial
SequenceIgkS1-24 118ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggctcca ctggcgacgg atccatgcat ggcgatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggcctc
tacggctatg gccaattaaa tgacagctca 180gaggaggagg atgaaataga
tggcccagct ggccaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggcat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387119387DNAArtificial
SequenceIgkS1-25 119ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300catgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387120387DNAArtificial
SequenceIgkS1-26 120ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcac ggagatacac
ctacattgca cgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccactac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387121387DNAArtificial
SequenceIgkS1-27 121ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatatagtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca tacgtacttt ggaagacctg ttaatgggca cactaggaat
agtgtgcccc 360atatgctctc agaagcccta agaattc 387122387DNAArtificial
SequenceIgkS1-28 122ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaattga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atttgctctc agaagcccta agaattc 387123387DNAArtificial
SequenceIgkS1-29 123ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaatcga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatatcgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca tccgtacttt ggaagacctg ttaatgggca cactaggaat
cgtgtgcccc 360atctgctctc agaagcccta agaattc 387124387DNAArtificial
SequenceIgkS1-50 124ggtaccgccg ccaccatgga aactgacact ctgctgctgt
gggtactgct gctgtgggtt 60ccaggatcga ctggagacgg atccatgcat ggagacactc
caactctgca tgaatatatg 120ctggacctgc aaccggaaac tactgacctg
tactgctatg aacaactgaa tgacagctcg 180gaagaagaag acgaaataga
cggacctgca ggacaagcag aaccagaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactctgc gcctgtgcgt acaaagcact
300catgtagaca ttcgcactct ggaagacctg ctgatgggaa ctctgggaat
tgtttgcccg 360atctgctcgc aaaagcctta agaattc 387125387DNAArtificial
SequenceIgkS1-51 125ggtaccgccg ccaccatgga aactgacact ctactactat
gggtactact actatgggtt 60ccaggatcga ctggagacgg atccatgcat ggagacactc
caactctaca tgaatatatg 120ctagacctac aaccggaaac tactgaccta
tactgctatg aacaactaaa tgacagctcg 180gaagaagaag acgaaataga
cggacctgca ggacaagcag aaccagaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactctac gcctatgcgt acaaagcact
300catgtagaca ttcgcactct agaagaccta ctaatgggaa ctctaggaat
tgtttgcccg 360atctgctcgc aaaagcctta agaattc 387126387DNAArtificial
SequenceIgkS1-52 126ggtaccgccg ccaccatgga aactgacact cttcttcttt
gggtacttct tctttgggtt
60ccaggatcga ctggagacgg atccatgcat ggagacactc caactcttca tgaatatatg
120cttgaccttc aaccggaaac tactgacctt tactgctatg aacaacttaa
tgacagctcg 180gaagaagaag acgaaataga cggacctgca ggacaagcag
aaccagaccg cgcacattac 240aatattgtaa ctttttgctg caagtgcgac
agtactcttc gcctttgcgt acaaagcact 300catgtagaca ttcgcactct
tgaagacctt cttatgggaa ctcttggaat tgtttgcccg 360atctgctcgc
aaaagcctta agaattc 387127387DNAArtificial SequenceIgkS1-53
127ggtaccgccg ccaccatgga aactgacact ctcctcctct gggtactcct
cctctgggtt 60ccaggatcga ctggagacgg atccatgcat ggagacactc caactctcca
tgaatatatg 120ctcgacctcc aaccggaaac tactgacctc tactgctatg
aacaactcaa tgacagctcg 180gaagaagaag acgaaataga cggacctgca
ggacaagcag aaccagaccg cgcacattac 240aatattgtaa ctttttgctg
caagtgcgac agtactctcc gcctctgcgt acaaagcact 300catgtagaca
ttcgcactct cgaagacctc ctcatgggaa ctctcggaat tgtttgcccg
360atctgctcgc aaaagcctta agaattc 387128387DNAArtificial
SequenceIgkS1-54 128ggtaccgccg ccaccatgga aactgacact ttgttgttgt
gggtattgtt gttgtgggtt 60ccaggatcga ctggagacgg atccatgcat ggagacactc
caactttgca tgaatatatg 120ttggacttgc aaccggaaac tactgacttg
tactgctatg aacaattgaa tgacagctcg 180gaagaagaag acgaaataga
cggacctgca ggacaagcag aaccagaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactttgc gcttgtgcgt acaaagcact
300catgtagaca ttcgcacttt ggaagacttg ttgatgggaa ctttgggaat
tgtttgcccg 360atctgctcgc aaaagcctta agaattc 387129387DNAArtificial
SequenceIgkS1-55 129ggtaccgccg ccaccatgga aactgacact ttattattat
gggtattatt attatgggtt 60ccaggatcga ctggagacgg atccatgcat ggagacactc
caactttaca tgaatatatg 120ttagacttac aaccggaaac tactgactta
tactgctatg aacaattaaa tgacagctcg 180gaagaagaag acgaaataga
cggacctgca ggacaagcag aaccagaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactttac gcttatgcgt acaaagcact
300catgtagaca ttcgcacttt agaagactta ttaatgggaa ctttaggaat
tgtttgcccg 360atctgctcgc aaaagcctta agaattc 387130387DNAArtificial
SequenceIgkC3 130ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactgatctc
tactgttatg agcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387131387DNAArtificial
SequenceIgkC4 131ggtaccgccg ccaccatgga gaccgacacc ctcctgctgt
gggtgctgct gctctgggtg 60cccggctcca ccggcgacgg atccatgcac ggcgacaccc
ccaccctgca cgagtacatg 120ctggacctgc agcccgagac caccgacctg
tactgctacg agcagctcaa cgacagcagc 180gaggaggagg acgagatcga
cggccccgcc ggccaggccg agcccgaccg cgcccactac 240aacatcgtga
ccttctgctg caagtgcgac agcaccctgc gcctctgcgt gcagagcacc
300cacgtggaca tccgcaccct ggaggacctg ctgatgggca ccctgggcat
cgtgtgcccc 360atctgctccc agaagcccta agaattc 387132387DNAArtificial
SequenceIgKS1-32 132ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagattttc aaccagagac aactggtttt
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387133387DNAArtificial
SequenceIgkS1-33 133ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttcc aaccagagac aactggtttc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttctgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387134387DNAArtificial
SequenceIgkS1-56 134ggtaccgccg ccaccatgga aactgacact ctcctgctat
gggtactgct gctctgggtt 60ccgggatcga ctggagacgg atccatgcat ggagacactc
cgactttgca tgaatatatg 120ctcgacttgc aaccggaaac tactgacctc
tactgctatg aacaattgaa tgacagctcg 180gaagaagaag acgaaataga
cggaccggca ggacaagcag aaccggaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactctcc gcttgtgcgt acaaagcact
300catgtagaca ttcgcacttt ggaagacctc ctcatgggaa ctttgggaat
tgtttgcccg 360atctgctcgc aaaagccgta agaattc 387135387DNAArtificial
SequenceIgkS1-57 135ggtaccgccg ccaccatgga aactgacact ctcctgctat
gggtactgct gctctgggtt 60ccaggatcga ctggagacgg atccatgcat ggagacactc
caactttgca tgaatatatg 120ctcgacttgc aaccagaaac tactgacctc
tactgctatg aacaattgaa tgacagctcg 180gaagaagaag acgaaataga
cggaccagca ggacaagcag aaccagaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactctcc gcttgtgcgt acaaagcact
300catgtagaca ttcgcacttt ggaagacctc ctcatgggaa ctttgggaat
tgtttgccca 360atctgctcgc aaaagccata agaattc 387136387DNAArtificial
SequenceIgkS1-58 136ggtaccgccg ccaccatgga aactgacact ctcctgctat
gggtactgct gctctgggtt 60cctggatcga ctggagacgg atccatgcat ggagacactc
ctactttgca tgaatatatg 120ctcgacttgc aacctgaaac tactgacctc
tactgctatg aacaattgaa tgacagctcg 180gaagaagaag acgaaataga
cggacctgca ggacaagcag aacctgaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactctcc gcttgtgcgt acaaagcact
300catgtagaca ttcgcacttt ggaagacctc ctcatgggaa ctttgggaat
tgtttgccct 360atctgctcgc aaaagcctta agaattc 387137387DNAArtificial
SequenceIgkS1-59 137ggtaccgccg ccaccatgga aactgacact ctcctgctat
gggtactgct gctctgggtt 60cccggatcga ctggagacgg atccatgcat ggagacactc
ccactttgca tgaatatatg 120ctcgacttgc aacccgaaac tactgacctc
tactgctatg aacaattgaa tgacagctcg 180gaagaagaag acgaaataga
cggacccgca ggacaagcag aacccgaccg cgcacattac 240aatattgtaa
ctttttgctg caagtgcgac agtactctcc gcttgtgcgt acaaagcact
300catgtagaca ttcgcacttt ggaagacctc ctcatgggaa ctttgggaat
tgtttgcccc 360atctgctcgc aaaagcccta agaattc 387138387DNAArtificial
SequenceIgkS1-34 138ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggtagta ctggtgacgg aagtatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagtagt 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac agtacgcttc ggttgtgcgt acaaagtaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgcagtc agaagcccta agaattc 387139387DNAArtificial
SequenceIgkS1-35 139ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggtagca ctggtgacgg aagcatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagcagc 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac agcacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgcagcc agaagcccta agaattc 387140387DNAArtificial
SequenceIgkS1-36 140ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcga ctggtgacgg atcgatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgactcgtcg 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tcgacgcttc ggttgtgcgt acaatcgaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctcgc agaagcccta agaattc 387141387DNAArtificial
SequenceIgkS1-37 141ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcaa ctggtgacgg atcaatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgactcatca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tcaacgcttc ggttgtgcgt acaatcaaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctcac agaagcccta agaattc 387142387DNAArtificial
SequenceIgkS1-38 142ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcta ctggtgacgg atctatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgactcttct 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaatctaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387143387DNAArtificial
SequenceIgkS1-39 143ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgactcctcc 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tccacgcttc ggttgtgcgt acaatccaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctccc agaagcccta agaattc 387144387DNAArtificial
SequenceIgkS1-40 144ggtaccgccg ccaccatgga gacggacacg ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca cgggtgacgg atccatgcat ggagatacgc
ctacgttgca tgaatatatg 120ttagatttgc aaccagagac gacgggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
cgttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcacg
300cacgtagaca ttcgtacgtt ggaagacctg ttaatgggca cgctaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387145387DNAArtificial
SequenceIgkS1-41 145ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca caggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aacaggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
cattttgttg caagtgtgac tctacacttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacatt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387146387DNAArtificial
SequenceIgkS1-42 146ggtaccgccg ccaccatgga gactgacact ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatactc
ctactttgca tgaatatatg 120ttagatttgc aaccagagac tactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ctttttgttg caagtgtgac tctactcttc ggttgtgcgt acaaagcact
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca ctctaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387147387DNAArtificial
SequenceIgkS1-43 147ggtaccgccg ccaccatgga gaccgacacc ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ccggtgacgg atccatgcat ggagataccc
ctaccttgca tgaatatatg 120ttagatttgc aaccagagac caccggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacccttc ggttgtgcgt acaaagcacc
300cacgtagaca ttcgtacctt ggaagacctg ttaatgggca ccctaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387148387DNAArtificial
SequenceIgkS1-44 148ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tatggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattat 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387149387DNAArtificial
SequenceIgkS1-45 149ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatacatg 120ttagatttgc aaccagagac aactggtctc
tacggttacg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387150387DNAArtificial
SequenceIgkS1-46 150ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtgctgct gctctgggtg 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtga
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt gcaaagcaca
300cacgtggaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtgtgcccc 360atctgctctc agaagcccta agaattc 387151387DNAArtificial
SequenceIgkS1-47 151ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtactgct gctctgggta 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtaa
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt acaaagcaca
300cacgtagaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtatgcccc 360atctgctctc agaagcccta agaattc 387152387DNAArtificial
SequenceIgkS1-48 152ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggttctgct gctctgggtt 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtta
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt tcaaagcaca
300cacgttgaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtttgcccc 360atctgctctc agaagcccta agaattc 387153387DNAArtificial
SequenceIgkS1-49 153ggtaccgccg ccaccatgga gacagacaca ctcctgctat
gggtcctgct gctctgggtc 60ccaggttcca ctggtgacgg atccatgcat ggagatacac
ctacattgca tgaatatatg 120ttagatttgc aaccagagac aactggtctc
tacggttatg ggcaattaaa tgacagctca 180gaggaggagg atgaaataga
tggtccagct ggacaagcag aaccggacag agcccattac 240aatattgtca
ccttttgttg caagtgtgac tctacgcttc ggttgtgcgt ccaaagcaca
300cacgtcgaca ttcgtacttt ggaagacctg ttaatgggca cactaggaat
tgtctgcccc 360atctgctctc agaagcccta agaattc 38715417PRTArtificial
SequenceSynthetic peptide used to measure the E7 antibody response
154His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg1
5 10 15Leu
* * * * *
References