U.S. patent application number 16/051725 was filed with the patent office on 2018-11-22 for novel vaccines against multiple subtypes of influenza virus.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to Ruxandra Draghia-Akli, Dominick Laddy, David B. Weiner, Jian Yan.
Application Number | 20180333480 16/051725 |
Document ID | / |
Family ID | 40718429 |
Filed Date | 2018-11-22 |
United States Patent
Application |
20180333480 |
Kind Code |
A1 |
Draghia-Akli; Ruxandra ; et
al. |
November 22, 2018 |
Novel Vaccines Against Multiple Subtypes of Influenza Virus
Abstract
An aspect of the present invention is directed towards DNA
plasmid vaccines capable of generating in a mammal an immune
response against a plurality of influenza virus subtypes,
comprising a DNA plasmid and a pharmaceutically acceptable
excipient. The DNA plasmid is capable of expressing a consensus
influenza antigen in a cell of the mammal in a quantity effective
to elicit an immune response in the mammal, wherein the consensus
influenza antigen comprises consensus hemagglutinin (HA),
neuraminidase (NA), matrix protein, nucleoprotein, M2
ectodomain-nucleo-protein (M2e-NP), or a combination thereof.
Preferably the consensus influenza antigen comprises HA, NA,
M2e-NP, or a combination thereof. The DNA plasmid comprises a
promoter operably linked to a coding sequence that encodes the
consensus influenza antigen. Additionally, an aspect of the present
invention includes methods of eliciting an immune response against
a plurality of influenza virus subtypes in a mammal using the DNA
plasmid vaccines provided.
Inventors: |
Draghia-Akli; Ruxandra;
(Brussels, BE) ; Weiner; David B.; (Merion,
PA) ; Yan; Jian; (Wallingford, PA) ; Laddy;
Dominick; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Family ID: |
40718429 |
Appl. No.: |
16/051725 |
Filed: |
August 1, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15416604 |
Jan 26, 2017 |
10076565 |
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16051725 |
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12269824 |
Nov 12, 2008 |
9592285 |
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15416604 |
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60987284 |
Nov 12, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/34 20130101;
C12N 2760/16034 20130101; A61K 2039/54 20130101; A61P 31/12
20180101; A61K 2039/58 20130101; A61K 39/145 20130101; A61K 38/208
20130101; A61P 31/16 20180101; A61K 2039/70 20130101; A61P 37/04
20180101; C12N 7/00 20130101; A61K 9/0019 20130101; A61K 38/2086
20130101; A61K 2300/00 20130101; C12N 2760/16122 20130101; A61K
38/2086 20130101; A61K 2300/00 20130101; A61K 39/12 20130101; C12N
2760/16134 20130101; C07K 14/005 20130101; A61K 2039/53 20130101;
A61K 38/208 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C12N 7/00 20060101 C12N007/00; A61K 9/00 20060101
A61K009/00 |
Claims
1-53. (canceled)
54. A composition for inducing an immune response against
influenza, comprising one or more nucleotide sequences selected
from the group consisting of: a) a nucleotide sequence encoding a
consensus neuraminidase (NA) antigen, and b) a nucleotide sequence
encoding a consensus M2 ectodomain-nucleoprotein (M2e-NP) antigen
.
55. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus NA antigen is selected from the group
consisting of: a nucleotide sequence encoding SEQ ID NO: 4, a
nucleotide sequence encoding an amino acid sequence having at least
90% homology to SEQ ID NO: 4, and a fragment of a nucleotide
sequence encoding SEQ ID NO: 4. 56 (New) The composition of claim
54, wherein the nucleotide sequence encoding the consensus NA
antigen encodes an amino acid sequence having at least 90% homology
to SEQ ID NO: 4.
57. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus NA antigen encodes an amino acid sequence
comprising SEQ ID NO: 4.
58. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus NA antigen is selected from the group
consisting of: a nucleotide sequence comprising SEQ ID NO: 3, a
nucleotide sequence having at least 90% homology to SEQ ID NO: 3,
and a fragment of a nucleotide sequence comprising SEQ ID NO:
3.
59. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus NA antigen comprises a nucleotide sequence
having at least 90% homology to SEQ ID NO: 3.
60. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus NA antigen comprises SEQ ID NO:3.
61. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus M2e-NP antigen is selected from the group
consisting of: a nucleotide sequence encoding SEQ ID NO: 8, a
nucleotide sequence encoding an amino acid sequence having at least
90% homology to SEQ ID NO: 8, and a fragment of a nucleotide
sequence encoding SEQ ID NO: 8.
62. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus M2e-NP antigen encodes an amino acid
sequence having at least 90% homology to SEQ ID NO: 8.
63. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus M2e-NP antigen encodes an amino acid
sequence comprising SEQ ID NO: 8.
64. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus M2e-NP antigen is selected from the group
consisting of: a nucleotide sequence comprising SEQ ID NO: 7, a
nucleotide sequence having at least 90% homology to SEQ ID NO: 7,
and a fragment of a nucleotide sequence comprising SEQ ID NO:
7.
65. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus M2e-NP antigen comprises a nucleotide
sequence having at least 90% homology to SEQ ID NO: 7.
66. The composition of claim 54, wherein the nucleotide sequence
encoding the consensus M2e-NP antigen comprises SEQ ID NO: 7.
67. The composition of claim 54, further comprising one or more
nucleotide sequences encoding one or more consensus influenza
antigens selected from the group consisting of: consensus H1,
consensus H3, and consensus H5.
68. The composition of claim 54, wherein the composition comprises
a DNA plasmid comprising the one or more nucleotide sequences.
69. The composition of claim 68, wherein the DNA plasmid comprises
a promoter operably linked to the one or more nucleotide
sequences.
70. The composition of claim 54, comprising a plurality of
different DNA plasmids; Wherein one of the plurality of DNA
plasmids comprises the nucleotide sequence that encodes the
consensus NA, one of the plurality of DNA plasmids comprises the
nucleotide sequence that encodes the consensus M2e-NP, and one of
the plurality of DNA plasmids comprises a nucleotide sequence that
encodes a consensus hemagglutinin (HA) antigen.
71. A method of eliciting an immune response against influenza
comprising administering to a subject a composition comprising one
or more nucleotide sequences selected from the group consisting of:
a) a nucleotide sequence encoding a consensus neuraminidase (NA)
antigen, and b) a nucleotide sequence encoding a consensus M2
ectodomain-nucleoprotein (M2e-NP) antigen.
72. The method of claim 71, further comprising electroporating
cells of the subject with a pulse of energy at a constant current
effective to permit entry of the composition in the cells.
73. The method of claim 71, wherein the administering step
comprises injecting the composition intradermally, subcutaneously,
or intramuscularly.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/987,284, filed Nov. 12, 1997, the contents of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to improved influenza
vaccines, improved methods for inducing immune responses, and for
prophylactically and/or therapeutically immunizing individuals
against influenza.
BACKGROUND
[0003] The use of nucleic acid sequences to vaccinate against
animal and human diseases has been studied. Studies have focused on
effective and efficient means of delivery in order to yield
necessary expression of the desired antigens, resulting immunogenic
response and ultimately the success of this technique. One method
for delivering nucleic acid sequences such as plasmid DNA is the
electroporation (EP) technique. The technique has been used in
human clinical trials to deliver anti-cancer drugs, such as
bleomycin, and in many preclinical studies on a large number of
animal species.
[0004] The influenza virus genome is contained on eight single
(non-paired) RNA strands that code for eleven proteins (HA, NA, NP,
M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The segmented nature of
the genome allows for the exchange of entire genes between
different viral strains during cellular cohabitation. The eight RNA
segments are: HA, which encodes hemagglutinin (about 500 molecules
of hemagglutinin are needed to make one virion); NA, which encodes
neuraminidase (about 100 molecules of neuraminidase are needed to
make one virion); NP, which encodes nucleoprotein; M, which encodes
two matrix proteins (the M1 and the M2) by using different reading
frames from the same RNA segment (about 3000 matrix protein
molecules are needed to make one virion); NS, which encodes two
distinct non-structural proteins (NS1 and NEP) by using different
reading frames from the same RNA segment; PA, which encodes an RNA
polymerase; PB1, which encodes an RNA polymerase and PB1-F2 protein
(induces apoptosis) by using different reading frames from the same
RNA segment; and PB2, which encodes an RNA polymerase.
[0005] Influenza hemagglutinin (HA) is expressed on the surface of
influenza viral particles and is responsible for initial contact
between the virus and its host cell. HA is a well-known immunogen.
Influenza A strain H5N1, an avian influenza strain, particularly
threatens the human population because of its HA protein (H5)
which, if slightly genetically reassorted by natural mutation, has
greatly increased infectivity of human cells as compared to other
strains of the virus. Infection of infants and older or
immunocompromised adult humans with the viral H5N1 strain is often
correlated to poor clinical outcome. Therefore, protection against
the H5N1 strain of influenza is a great need for the public.
[0006] There are two classes of anti-influenza agents available,
inhibitors of influenza A cell entry/uncoating (such as antivirals
amantadine and rimantadine) and neuraminidase inhibitors (such as
antivirals oseltamivir, zanamivir). These antiviral agents inhibit
the cellular release of both influenza A and B. Concerns over the
use of these agents have been reported due to findings of strains
of virus resistant to these agents.
[0007] Influenza vaccines are a popular seasonal vaccine and many
people have experienced such vaccinations. However, the
vaccinations are limited in their protective results because the
vaccines are specific for certain subtypes of virus. The Centers
for Disease Control and Prevention promote vaccination with a "flu
shot" that is a vaccine that contains three influenza viruses
(killed viruses): one A (H3N2) virus, one A (H1N1) virus, and one B
virus. They also report that the viruses in the vaccine change each
year based on international surveillance and scientists'
estimations about which types and strains of viruses will circulate
in a given year. Thus, it is apparent that vaccinations are limited
to predictions of subtypes, and the availability of a specific
vaccine to that subtype.
[0008] There still remains a need for effective influenza vaccines
that are economical and effective across numerous subtypes.
Further, there remains a need for an effective method of
administering DNA vaccines to a mammal in order to provide
immunization against influenza either prophylatically or
therapeutically.
SUMMARY OF THE INVENTION
[0009] One aspect of the present invention relates to a DNA plasmid
vaccine capable of generating in a mammal an immune response
against a plurality of influenza virus subtypes, comprising a DNA
plasmid and a pharmaceutically acceptable excipient. The DNA
plasmid is capable of expressing a consensus influenza antigen in a
cell of the mammal in a quantity effective to elicit an immune
response in the mammal, wherein the consensus influenza antigen
comprises consensus hemagglutinin (HA), neuraminidase (NA), matrix
protein, nucleoprotein, M2 ectodomain-nucleo-protein (M2e-NP), or a
combination thereof. Preferably the consensus influenza antigen
comprises HA, NA, M2e-NP, or a combination thereof. The DNA plasmid
comprises a promoter operably linked to a coding sequence that
encodes the consensus influenza antigen. Preferably, the DNA
plasmid vaccine is one having a concentration of total DNA plasmid
of 1 mg/ml or greater.
[0010] Another aspect of the present invention relates to DNA
plasmids capable of expressing a consensus influenza antigen in a
cell of the mammal, the consensus influenza antigen comprising
consensus hemagglutinin (HA), neuraminidase (NA), matrix protein,
nucleoprotein, M2 ectodomain-nucleo-protein (M2e-NP), or a
combination thereof. Preferably the consensus influenza antigen
comprises HA, NA, M2e-NP, or a combination thereof. The DNA plasmid
comprises a promoter operably linked to a coding sequence that
encodes the consensus influenza antigen.
[0011] Another aspect of the present invention relates to methods
of eliciting an immune response against a plurality of influenza
virus subtypes in a mammal. The methods include delivering a DNA
plasmid vaccine to tissue of the mammal, the DNA plasmid vaccine
comprising a DNA plasmid capable of expressing a consensus
influenza antigen in a cell of the mammal to elicit an immune
response in the mammal, the consensus influenza antigen comprising
consensus HA, NA, M2e-NP or a combination thereof, and
electroporating cells of the tissue with a pulse of energy at a
constant current effective to permit entry of the DNA plasmids in
the cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The numerous objects and advantages of the present invention
may be better understood by those skilled in the art by reference
to the accompanying figures, in which:
[0013] FIG. 1 displays a schematic representation (plasmid maps) of
the DNA plasmid constructs used in the studies described herein.
Consensus HA, NA and M2e-NP constructs were generated by analyzing
primary virus sequences from 16 H5 viruses that have proven fatal
to humans in recent years, and over 40 human N1 viruses (Los Alamos
National Laboratory's Influenza Sequence Database). After
generating the consensus sequences, the constructs were optimized
for mammalian expression, including the addition of a Kozak
sequence, codon optimization, and RNA optimization. These
constructs were then subcloned into the pVAX vector (Invitrogen,
Carlsbad, CA). Plasmids pGX2001 (consensus HA), pGX2002 (consensus
NA), pGX2003 (consensus M2e-NP) are shown. The plasmid pCMVSEAP,
displayed, encodes the reporter protein secreted embryonic alkaline
phosphatase (SEAP).
[0014] FIG. 2 displays a bar graph of the results of the HI titers
in pig serum at Day 35 post-injection. The highest titers were
found in the group administered 2 mg of HA-expressing plasmid at a
current setting of 0.5 A (120.+-.40; *P=0.11 versus 2 mg/0.3 A and
*P=0.02 versus 2 mg/0.1 A). The three groups administered
descending doses of plasmid and electroporated at 0.5 A also
demonstrated decreasing HI titers.
[0015] FIG. 3 displays a bar graph of the IFN-.gamma. ELISpot
counts. The counts were highest in pigs administered 2 mg of HA and
2 mg of NA plasmid vaccine (for a total of 4 mg plasmid) and
electroporated with 0.3 A of current (2000 spots) and in the group
administered 0.8 mg of HA and 0.8 mg of NA plasmid vaccine (for a
total of 1.6 mg plasmid) electroporated with 0.5 A of current (934
spots). For comparison purposes, the cellular immune responses of
an unimmunized control group are depicted.
[0016] FIGS. 4A and 4B display bar graphs showing results from
muscle biopsies from treated pigs at Day 14 and Day 35: FIG. 4A
displays a bar graph showing the mean pathology scores for all
groups. FIG. 4B displays a bar graph showing the muscle necrosis
and fibrosis scores. The group injected with 6 mg total plasmid and
electroporated at 0.5 A exhibited the highest mean pathology score
(*P<0.0002 as compared to controls). The pathology scores were
significantly reduced by Day 35 compared to Day 14 in all groups
(P<0.05) except for the 0.3 mg/0.3 A group (P=0.057) and 2.4 mg/
0.1 A group (P=1.0).
[0017] FIG. 5 displays the percent change in weight of ferrets
after challenge with H5N1 virus
(A/Viet/1203/2004(H5N1)/PR8-IBCDC-RG). Ferrets that were vaccinated
with HA, HA+M2e-NP or HA+M2e-NP+NA lost significantly less weight
than control animals (*P<0.005 versus controls) in the 9 days
post-challenge period. One animal in the HA vaccine group actually
gained weight post-challenge.
[0018] FIG. 6 displays a graph showing the body temperatures of
ferrets during the 9 days post-challenge. Control animals showed
higher body temperatures than the vaccinated animals. The body
temperature on day 5 is not depicted as it was measured at a
different time of day and all the temperatures regardless of group
were lower.
[0019] FIG. 7 displays a bar graph of results from HI titers in
ferrets after vaccination; the assay was performed using
reassortant viruses obtained from the Center for Disease Control:
A/Viet/1203/04 or Indo/05/2005 influenza strains.
[0020] FIG. 8 displays a bar graph of results from HI titers
measured three weeks after the second immunization. Macaques
immunized ID followed by EP showed significantly higher HI titers
than all other groups (P<0.03). Non-treated controls did not
exhibit any HI titers.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] The following abbreviated, or shortened, definitions are
given to help the understanding of the preferred embodiments of the
present invention. The abbreviated definitions given here are by no
means exhaustive nor are they contradictory to the definitions as
understood in the field or dictionary meaning. The abbreviated
definitions are given here to supplement or more clearly define the
definitions known in the art.
Definitions
[0022] Sequence homology for nucleotides and amino acids as used
herein may be determined using FASTA, BLAST and Gapped BLAST
(Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is
incorporated herein by reference in its entirety) and PAUP* 4.0b10
software (D. L. Swofford, Sinauer Associates, Massachusetts).
Briefly, the BLAST algorithm, which stands for Basic Local
Alignment Search Tool is suitable for determining sequence
similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410,
which is incorporated herein by reference in its entirety).
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov). One measure of similarity provided
by the BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match
between two nucleotide sequences would occur by chance. For
example, a nucleic acid is considered similar to another if the
smallest sum probability in comparison of the test nucleic acid to
the other nucleic acid is less than about 1, preferably less than
about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001. "Percentage of similarity" can be
calculated using PAUP*4.0b10 software (D. L. Swofford, Sinauer
Associates, Massachusetts). The average similarity of the consensus
sequence is calculated compared to all sequences in the phylogenic
tree.
[0023] As used herein, the term "genetic construct" or "nucleic
acid construct" is used interchangeably and refers to the DNA or
RNA molecules that comprise a nucleotide sequence which encodes
protein. The coding sequence, or "encoding nucleic acid sequence,"
includes initiation and termination signals operably linked to
regulatory elements including a promoter and polyadenylation signal
capable of directing expression in the cells of the individual to
whom the nucleic acid molecule is administered.
[0024] As used herein, the term "expressible form" refers to
nucleic acid constructs that contain the necessary regulatory
elements operable linked to a coding sequence that encodes a
protein such that when present in the cell of the individual, the
coding sequence will be expressed.
[0025] The term "constant current" is used herein to define a
current that is received or experienced by a tissue, or cells
defining said tissue, over the duration of an electrical pulse
delivered to same tissue. The electrical pulse is delivered from
the electroporation devices described herein. This current remains
at a constant amperage in said tissue over the life of an
electrical pulse because the electroporation device provided herein
has a feedback element, preferably having instantaneous feedback.
The feedback element can measure the resistance of the tissue (or
cells) throughout the duration of the pulse and cause the
electroporation device to alter its electrical energy output (e.g.,
increase voltage) so current in same tissue remains constant
throughout the electrical pulse (on the order of microseconds), and
from pulse to pulse. In some embodiments, the feedback element
comprises a controller.
[0026] The term "feedback" or "current feedback" is used
interchangeably and means the active response of the provided
electroporation devices, which comprises measuring the current in
tissue between electrodes and altering the energy output delivered
by the EP device accordingly in order to maintain the current at a
constant level. This constant level is preset by a user prior to
initiation of a pulse sequence or electrical treatment. Preferably,
the feedback is accomplished by the electroporation component,
e.g., controller, of the electroporation device, as the electrical
circuit therein is able to continuously monitor the current in
tissue between electrodes and compare that monitored current (or
current within tissue) to a preset current and continuously make
energy-output adjustments to maintain the monitored current at
preset levels. In some embodiments, the feedback loop is
instantaneous as it is an analog closed-loop feedback.
[0027] The terms "electroporation," "electro-permeabilization," or
"electro-kinetic enhancement" ("EP") as used interchangeably herein
refer to the use of a transmembrane electric field pulse to induce
microscopic pathways (pores) in a bio-membrane; their presence
allows biomolecules such as plasmids, oligonucleotides, siRNA,
drugs, ions, and water to pass from one side of the cellular
membrane to the other.
[0028] The term "decentralized current" is used herein to define
the pattern of electrical currents delivered from the various
needle electrode arrays of the electroporation devices described
herein, wherein the patterns minimize, or preferably eliminate, the
occurrence of electroporation related heat stress on any area of
tissue being electroporated.
[0029] The term "feedback mechanism" as used herein refers to a
process performed by either software or hardware (or firmware),
which process receives and compares the impedance of the desired
tissue (before, during, and/or after the delivery of pulse of
energy) with a present value, preferably current, and adjusts the
pulse of energy delivered to achieve the preset value. The term
"impedance" is used herein when discussing the feedback mechanism
and can be converted to a current value according to Ohm's law,
thus enabling comparisons with the preset current. In a preferred
embodiment, the "feedback mechanism" is performed by an analog
closed loop circuit.
[0030] The term "immune response" is used herein to mean the
activation of a host's immune system, e.g., that of a mammal, in
response to the introduction of influenza consensus antigen via the
provided DNA plasmid vaccines. The immune response can be in the
form of a cellular or humoral response, or both.
[0031] The term "consensus" or "consensus sequence" is used herein
to mean a synthetic nucleic acid sequence, or corresponding
polypeptide sequence, constructed based on analysis of an alignment
of multiple subtypes of a particular influenza antigen, that can be
used to induce broad immunity against multiple subtypes or
serotypes of a particular influenza antigen. Consensus influenza
antigens include HA, including consensus H1, H2, H3, or H5, NA, NP,
matrix protein, and nonstructural protein. Also, synthetic antigens
such as fusion proteins, e.g., M2e-NP, can be manipulated to
consensus sequences (or consensus antigens).
[0032] The term "adjuvant" is used herein to mean any molecule
added to the DNA plasmid vaccines described herein to enhance
antigenicity of the influenza antigen encoded by the DNA plasmids
and encoding nucleic acid sequences described hereinafter.
[0033] The term "subtype" or "serotype" is used herein
interchangeably and in reference to influenza viruses, and means
genetic variants of an influenza virus antigen such that one
subtype is recognized by an immune system apart from a different
subtype (or, in other words, each subtype is different in antigenic
character from a different subtype).
[0034] In some embodiments, there are DNA plasmids capable of
expressing a consensus influenza antigen in a cell of the mammal,
the consensus influenza antigen comprising consensus hemagglutinin
(HA), neuraminidase (NA), matrix protein, nucleoprotein, M2
ectodomain-nucleo-protein (M2e-NP), or a combination thereof.
Preferably the consensus influenza antigen comprises HA, NA,
M2e-NP, or a combination thereof. The DNA plasmid comprises a
promoter operably linked to a coding sequence that encodes the
consensus influenza antigen.
[0035] In some embodiments, the present invention provides DNA
plasmid vaccines that are capable of generating in a mammal an
immune response against a plurality of influenza virus subtypes,
the DNA plasmid vaccines comprising a DNA plasmid and a
pharmaceutically acceptable excipient. The DNA plasmid is capable
of expressing a consensus influenza antigen in a cell of the mammal
in a quantity effective to elicit an immune response in the mammal,
wherein the consensus influenza antigen comprises consensus
hemagglutinin (HA), neuraminidase (NA), matrix protein,
nucleoprotein, M2 ectodomain-nucleo-protein (M2e-NP), or a
combination thereof. Preferably the consensus influenza antigen
comprises HA, NA, M2e-NP, or a combination thereof. The DNA plasmid
comprises a promoter operably linked to a coding sequence that
encodes the consensus influenza antigen. In some embodiments, the
DNA plasmid vaccine is one having a concentration of total DNA
plasmid of 1 mg/ml or greater. The immune response can be a
cellular or humoral response, or both; preferably, the immune
response is both cellular and humoral.
[0036] In some embodiments, the DNA plasmid can further include an
IgG leader sequence attached to an N-terminal end of the coding
sequence and operably linked to the promoter. In addition, in some
embodiments, the DNA plasmid can further include a polyadenylation
sequence attached to the C-terminal end of the coding sequence. In
some embodiments, the DNA plasmid is codon optimized.
[0037] In some embodiments of the present invention, the DNA
plasmid vaccines can further include an adjuvant. In some
embodiments, the adjuvant is selected from the group consisting of:
alpha-interferon, gamma-interferon, platelet derived growth factor
(PDGF), TNF.alpha., TNF.beta., GM-CSF, epidermal growth factor
(EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial
thymus-expressed chemokine (TECK), mucosae-associated epithelial
chemokine (MEC), IL-12, IL-15, MHC, CD80,CD86 including IL-15
having the signal sequence deleted and optionally including the
signal peptide from IgE. Other genes which may be useful adjuvants
include those encoding: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES,
L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1,
LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2,
LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L,
vascular growth factor, fibroblast growth factor, IL-7, nerve
growth factor, vascular endothelial growth factor, Fas, TNF
receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD,
NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos,
c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB,
Inactive NIK, SAP K, SAP-1, .INK, interferon response genes, NFkB,
Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK
LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C,
NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof. In some
preferred embodiments, the adjuvant is selected from IL-12, IL-15,
CTACK, TECK, or MEC.
[0038] In some embodiments, the pharmaceutically acceptable
excipient is a transfection facilitating agent, which can include
the following: surface active agents, such as immune-stimulating
complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog
including monophosphoryl lipid A, muramyl peptides, quinone
analogs, vesicles such as squalene and squalene, hyaluronic acid,
lipids, liposomes, calcium ions, viral proteins, polyanions,
polycations, or nanoparticles, or other known transfection
facilitating agents. Preferably, the transfection facilitating
agent is a polyanion, polycation, including poly-L-glutamate (LGS),
or lipid. Preferably, the transfection facilitating agent is
poly-L-glutamate, and more preferably, the poly-L-glutamate is
present in the DNA plasmid vaccine at a concentration less than 6
mg/ml. In some embodiments, the concentration of poly-L-glutamate
in the DNA plasmid vaccine is less than 4 mg/ml, less than 2 mg/ml,
less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,
less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050
mg/ml, or less than 0.010 mg/ml.
[0039] In some embodiments, the DNA plasmid vaccine can include a
plurality of different DNA plasmids. In some examples, the
different DNA plasmids include a DNA plasmid comprising a nucleic
acid sequence that encodes a consensus HA, a DNA plasmid comprising
a sequence that encodes a consensus NA, and a DNA plasmid
comprising a sequence that encodes a consensus M2e-NP. In some
embodiments, the consensus HA is a consensus H1, consensus H2,
consensus H3, or consensus H5. Preferably, the consensus HA is
nucleotide sequence that is SEQ ID NO:1 (H5N1 HA consensus DNA),
SEQ ID NO:9 (consensus H1 DNA), SEQ ID NO: 11 (consensus H3 DNA),
or SEQ ID NO:13 (consensus H5). The consensus HA can also be a
nucleotide sequence encoding a polypeptide of the sequence SEQ ID
NO: 2, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:14. In some
embodiments, the consensus NA is a nucleotide sequence that is SEQ
ID NO: 3, or a nucleotide sequence encoding a polypeptide of the
sequence SEQ ID NO: 4. In some embodiments, the consensus M2e-NP is
a nucleotide sequence that is SEQ ID NO:7, or a nucleotide sequence
encoding a polypeptide of the sequence SEQ ID NO:8. In one
preferred embodiment, the DNA plasmid vaccine includes a DNA
plasmid comprising a sequence that encodes a consensus H1, a DNA
plasmid comprising a sequence that encodes a consensus H2, a DNA
plasmid comprising a sequence that encodes a consensus H3, a DNA
plasmid comprising a sequence that encodes a consensus H5, a DNA
plasmid comprising a sequence that encodes a consensus NA, and a
DNA plasmid comprising a sequence that encodes a consensus
M2e-NP.
[0040] In some embodiments, the DNA plasmid vaccine can include a
plurality of different DNA plasmids, including at least one DNA
plasmid that can express consensus influenza antigens and at least
one that can express one influenza subtype antigen. In some
examples, the different DNA plasmids that express consensus antigen
include a DNA plasmid comprising a nucleic acid sequence that
encodes a consensus HA, a DNA plasmid comprising a sequence that
encodes a consensus NA, and a DNA plasmid comprising a sequence
that encodes a consensus M2e-NP. In some embodiments, the DNA
plasmid vaccine comprises a DNA plasmid that can express a
consensus HA antigen, e.g., consensus H1, H3 or H5, and a DNA
plasmid that can express any one of the following influenza A
antigens: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,
H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8, N9, NP, M1, M2, NS1,
or NEP, or a combination thereof. In some embodiments, the DNA
plasmid vaccine comprises a DNA plasmid that can express a
consensus NA antigen and a DNA plasmid that can express any one of
the following influenza A antigens: H1, H2, H3, H4, H5, H6, H7, H8,
H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7,
N8, N9, NP, M1, M2, NS1, or NEP, or a combination thereof. In some
embodiments, the DNA plasmid vaccine comprises a DNA plasmid that
can express a consensus M2e-NP and a DNA plasmid that can express
any one of the following influenza A antigens: H1, H2, H3, H4, H5,
H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4,
N5, N6, N7, N8, N9, NP, M1, M2, NS1, or NEP, or a combination
thereof.
[0041] In some embodiments, the DNA plasmid vaccine can be
delivered to a mammal to elicit an immune response; preferably the
mammal is a primate, including human and nonhuman primate, a cow,
pig, chicken, dog, or ferret. More preferably, the mammal is a
human primate.
[0042] One aspect of the present invention relates to methods of
eliciting an immune response against a plurality of influenza virus
subtypes in a mammal. The methods include delivering a DNA plasmid
vaccine to tissue of the mammal, the DNA plasmid vaccine comprising
a DNA plasmid capable of expressing a consensus influenza antigen
in a cell of the mammal to elicit an immune response in the mammal,
the consensus influenza antigen comprising consensus HA, NA, M2e-NP
or a combination thereof, and electroporating cells of the tissue
with a pulse of energy at a constant current effective to permit
entry of the DNA plasmids in the cells.
[0043] In some embodiments, the methods of the present invention
include the delivering step, which comprises injecting the DNA
plasmid vaccine into intradermic, subcutaneous or muscle tissue.
Preferably, these methods include using an in vivo electroporation
device to preset a current that is desired to be delivered to the
tissue; and electroporating cells of the tissue with a pulse of
energy at a constant current that equals the preset current. In
some embodiments, the electroporating step further comprises:
measuring the impedance in the electroporated cells; adjusting
energy level of the pulse of energy relative to the measured
impedance to maintain a constant current in the electroporated
cells; wherein the measuring and adjusting steps occur within a
lifetime of the pulse of energy.
[0044] In some embodiments, the electroporating step comprises
delivering the pulse of energy to a plurality of electrodes
according to a pulse sequence pattern that delivers the pulse of
energy in a decentralized pattern.
[0045] In some embodiments, the DNA plasmid influenza vaccines of
the invention comprise nucleotide sequences that encode a consensus
HA, or a consensus HA and a nucleic acid sequence encoding
influenza proteins selected from the group consisting of: SEQ ID
NOS: 4, 6, and 8. SEQ ID NOS:1 and 13 comprise the nucleic acid
sequence that encodes consensus H5N1 HA and H5 of influenza virus,
respectively. SEQ ID NOS:2 and 14 comprise the amino acid sequence
for H5N1 HA and H5 of influenza virus, respectively. In some
embodiments of the invention, the vaccines of the invention
comprise SEQ ID NO:3 or SEQ ID NO:4. SEQ ID NO:3 comprises the
nucleic acid sequence that encodes influenza H1N1 and H5N1
(H1N1/H5N1) NA consensus sequences. SEQ ID NO:4 comprises the amino
acid sequence for influenza H1N1/H5N1 NA consensus sequences. In
some embodiments of the invention, the vaccines of the invention
comprise SEQ ID NO:5 or SEQ ID NO:6. SEQ ID NO:5 comprises the
nucleic acid sequence that encodes influenza H1N1/H5N1 M1 consensus
sequences. SEQ ID NO:6 comprises the amino acid sequence for
influenza H1N1/H5N1 M1 consensus sequences. In some embodiments of
the invention, the vaccines of the invention comprise SEQ ID NO:7
or SEQ ID NO:8. SEQ ID NO:7 comprises the nucleic acid sequence
that encodes influenza H5N1 M2E-NP consensus sequence. SEQ ID NO:8
comprises the amino acid sequence for influenza H5N1 M2E-NP
consensus sequence. In some embodiments of the invention, the
vaccines of the invention comprise SEQ ID NO:9 or SEQ ID NO:10. SEQ
ID NO:9 comprises the nucleic acid sequence that encodes influenza
H1N1 HA consensus sequences. SEQ ID NO:4 comprises the amino acid
sequence for influenza H1N1 HA consensus sequences. In some
embodiments of the invention, the vaccines of the invention
comprise SEQ ID NO:11 or SEQ ID NO:12. SEQ ID NO:11 comprises the
nucleic acid sequence that encodes influenza H3N1 HA consensus
sequences. SEQ ID NO:12 comprises the amino acid sequence for
influenza H3N1 HA consensus sequences. The consensus sequence for
influenza virus strain H5N1 HA includes the immunodominant epitope
set forth in SEQ ID NO:1 or SEQ ID NO:13. The influenza virus H5N1
HA amino acid sequence encoded by SEQ ID NO:1 is SEQ ID NO:2, and
that encoded by SEQ ID NO:13 is SEQ ID NO:14. The consensus
sequence for influenza virus H1N1/H5N1 NA includes the
immunodominant epitope set forth in SEQ ID NO:3. The influenza
virus strains H1N1/H5N1 NA amino acid sequence encoded by SEQ ID
NO:3 is SEQ ID NO:4. The consensus sequence for influenza virus
strains H1N1/H5N1 M1 includes the immunodominant epitope set forth
in SEQ ID NO:5. The influenza virus H1N1/H5N1 M1 amino acid
sequence encoded by SEQ ID NO:5 is SEQ ID NO:6. The consensus
sequence for influenza virus H5N1 M2E-NP includes the
immunodominant epitope set forth in SEQ ID NO:7. The influenza
virus H5N1 M2E-NP amino acid sequence encoded by SEQ ID NO:7 is SEQ
ID NO:8. Vaccines of the present invention may include protein
products encoded by the nucleic acid molecules defined above or any
fragments of proteins.
[0046] The present invention also comprises DNA fragments that
encode a polypeptide capable of eliciting an immune response in a
mammal substantially similar to that of the non-fragment for at
least one influenza subtype. The DNA fragments are fragments
selected from at least one of the various encoding nucleotide
sequences of the present invention, including SEQ ID NOS:1, 3, 5,
7, 9, 11, and 13, and can be any of the following described DNA
fragments, as it applies to the specific encoding nucleic acid
sequence provided herein. In some embodiments, DNA fragments can
comprise 30 or more, 45 or more, 60 or more, 75 or more, 90 or
more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or
more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or
more, 540 or more, 600 or more, 660 or more, 720 or more, 780 or
more, 840 or more, 900 or more, 960 or more, 1020 or more, 1080 or
more, 1140 or more, 1200 or more, 1260 or more, 1320 or more, 1380
or more, 1440 or more, 1500 or more, 1560 or more, 1620 or more,
1680 or more, or 1740 or more nucleotides. In some embodiments, DNA
fragments can comprise coding sequences for the immunoglobulin E
(IgE) leader sequences. In some embodiments, DNA fragments can
comprise fewer than 60, fewer than 75, fewer than 90, fewer than
120, fewer than 150, fewer than 180, fewer than 210, fewer than
240, fewer than 270, fewer than 300, fewer than 360, fewer than
420, fewer than 480, fewer than 540, fewer than 600, fewer than
660, fewer than 720, fewer than 780, fewer than 840, fewer than
900, fewer than 960, fewer than 1020, fewer than 1080, fewer than
1140, fewer than 1200, fewer than 1260, fewer than 1320, fewer than
1380, fewer than 1440, fewer than 1500, fewer than 1560, fewer than
1620, fewer than 1680, or fewer than 1740 nucleotides. Preferably,
the DNA fragments are fragments of SEQ ID NOS:1, 3, 7, 9, 11 or 13,
and more preferably fragments of SEQ ID NOS:1, 5, 9, 11, or 13, and
even more preferably fragments of SEQ ID NOS:1, 9, or 13.
[0047] The present invention also comprises polypeptide fragments
that are capable of eliciting an immune response in a mammal
substantially similar to that of the non-fragment for at least one
influenza subtype. The polypeptide fragments are selected from at
least one of the various polypeptide sequences of the present
invention, including SEQ ID NOS:2, 4, 6, 8, 10, 12, and 14, and can
be any of the following described polypeptide fragments, as it
applies to the specific polypeptide sequence provided herein. In
some embodiments, polypeptide fragments can comprise 15 or more, 30
or more, 45 or more, 60 or more, 75 or more, 90 or more, 105 or
more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or
more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or
more, 540 or more, or 565 or more amino acids. In some embodiments,
polypeptide fragments can comprise fewer than 30, fewer than 45,
fewer than 60, fewer than 75, fewer than 90, fewer than 120, fewer
than 150, fewer than 180, fewer than 210, fewer than 240, fewer
than 270, fewer than 300, fewer than 360, fewer than 420, fewer
than 480, fewer than 540, or fewer than 565 amino acids.
Preferably, the polypeptide fragments are fragments of SEQ ID
NOS:2, 4, 8, 10, 12, or 14, and more preferably fragments of SEQ ID
NOS:2, 6, 10, 12, or 14, and even more preferably fragments of SEQ
ID NOS:2, 10, or 14.
[0048] The determination of a fragment eliciting an immune response
in a mammal substantially similar to that of the non-fragment for
at least one influenza subtype can be readily determined by one of
ordinary skill. The fragment can be analyzed to contain at least
one, preferably more, antigenic epitopes as provided by a publicly
available database, such as the Los Alamos National Laboratory's
Influenza Sequence Database. In addition, immune response studies
can be routinely assessed using mice and HI titers and ELISpots
analysis, such as that shown in the Examples below.
[0049] According to some embodiments of the invention, methods of
inducing or eliciting an immune response in mammals against a
plurality of influenza viruses comprise administering to the
mammals: a) the influenza strain H5N1 consensus HA protein,
functional fragments thereof, or expressible coding sequences
thereof; and b) one or more isolated encoding nucleic acid
molecules provided herein, protein encoded by such nucleic acid
molecules, or fragments thereof.
[0050] According to some embodiments of the invention, methods of
inducing or eliciting an immune response in mammals against a
plurality of influenza viruses comprise administering to the
mammals: a) the influenza strain H1N1 and influenza strain H5N1
consensus NA protein, functional fragments thereof, or expressible
coding sequences thereof and b) one or more isolated encoding
nucleic acid molecules provided herein, protein encoded by such
nucleic acid molecules, or fragments thereof.
[0051] According to some embodiments of the invention, methods of
inducing or eliciting an immune response in mammals against a
plurality of influenza viruses comprise administering to the
mammals: a) the influenza strain H1N1 and influenza strain H5N1
consensus M1 protein, functional fragments thereof, or expressible
coding sequences thereof and b) one or more isolated encoding
nucleic acid molecules provided herein, protein encoded by such
nucleic acid molecules, or fragments thereof.
[0052] According to some embodiments of the invention, methods of
inducing or eliciting an immune response in mammals against a
plurality of influenza viruses comprise administering to the
mammals: a) the influenza strain H5N1 M2E-NP consensus protein,
functional fragments thereof, or expressible coding sequences
thereof and b) one or more isolated encoding nucleic acid molecules
provided herein, protein encoded by such nucleic acid molecules, or
fragments thereof.
[0053] According to some embodiments of the invention, methods of
inducing or eliciting an immune response in mammals against a
plurality of influenza viruses comprise administering to the
mammals: a) the influenza strain H1N1 HA consensus protein,
functional fragments thereof, or expressible coding sequences
thereof and b) one or more isolated encoding nucleic acid molecules
provided herein, protein encoded by such nucleic acid molecules, or
fragments thereof.
[0054] According to some embodiments of the invention, methods of
inducing or eliciting an immune response in mammals against a
plurality of influenza viruses comprise administering to the
mammals: a) the influenza strain H3N1 HA consensus protein,
functional fragments thereof, or expressible coding sequences
thereof; and b) one or more isolated encoding nucleic acid
molecules provided herein, protein encoded by such nucleic acid
molecules, or fragments thereof.
[0055] In some embodiments of the invention, the vaccines of the
invention include at least two of the following sequences: SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or any
combination of two or more sequences from the aforementioned
list.
Vaccines
[0056] In some embodiments, the invention provides improved
vaccines by providing proteins and genetic constructs that encode
proteins with epitopes that make them particularly effective as
immunogens against which immune responses can be induced.
Accordingly, vaccines can be provided to induce a therapeutic or
prophylactic immune response.
[0057] According to some embodiments of the invention, a vaccine
according to the invention is delivered to an individual to
modulate the activity of the individual's immune system and thereby
enhance the immune response. When a nucleic acid molecule that
encodes the protein is taken up by cells of the individual the
nucleotide sequence is expressed in the cells and the protein are
thereby delivered to the individual. Aspects of the invention
provide methods of delivering the coding sequences of the protein
on nucleic acid molecule such as plasmid.
[0058] According to some aspects of the present invention,
compositions and methods are provided which prophylactically and/or
therapeutically immunize an individual.
[0059] When taken up by a cell, the DNA plasmids can stay present
in the cell as separate genetic material. Alternatively, RNA may be
administered to the cell. It is also contemplated to provide the
genetic construct as a linear minichromosome including a
centromere, telomeres and an origin of replication. Genetic
constructs include regulatory elements necessary for gene
expression of a nucleic acid molecule. The elements include: a
promoter, an initiation codon, a stop codon, and a polyadenylation
signal. In addition, enhancers are often required for gene
expression of the sequence that encodes the target protein or the
immunomodulating protein. It is necessary that these elements be
operable linked to the sequence that encodes the desired proteins
and that the regulatory elements are operably in the individual to
whom they are administered.
[0060] Initiation codons and stop codon are generally considered to
be part of a nucleotide sequence that encodes the desired protein.
However, it is necessary that these elements are functional in the
mammals to whom the nucleic acid construct is administered. The
initiation and termination codons must be in frame with the coding
sequence.
[0061] Promoters and polyadenylation signals used must be
functional within the cells of the individual.
[0062] Examples of promoters useful to practice the present
invention, especially in the production of a genetic vaccine for
humans, include but are not limited to promoters from simian virus
40 (SV40), mouse mammary tumor virus (MMTV) promoter, human
immunodeficiency virus (HIV) such as the bovine immunodeficiency
virus (BIV) long terminal repeat (LTR) promoter, Moloney virus,
avian leukosis virus (ALV), cytomegalovirus (CMV) such as the CMV
immediate early promoter, Epstein Barr virus (EBV), Rous sarcoma
virus (RSV) as well as promoters from human genes such as human
actin, human myosin, human hemoglobin, human muscle creatine and
human metalothionein; in other embodiments, promoters can be tissue
specific promoters, such as muscle or skin specific promoters,
natural or synthetic. Examples of such promoters are described in
US patent application publication no. US20040175727, which is
incorporated hereby in its entirety.
[0063] Examples of polyadenylation signals useful to practice the
present invention, especially in the production of a genetic
vaccine for humans, include but are not limited to SV40
polyadenylation signals, LTR polyadenylation signals, bovine growth
hormone (bGH) polyadenylation signals, human growth hormone (hGH)
polyadenylation signals, and human .beta.-globin polyadenylation
signals. In particular, the SV40 polyadenylation signal that is in
pCEP4 plasmid (Invitrogen, San Diego, CA), referred to as the SV40
polyadenylation signal, can be used.
[0064] In addition to the regulatory elements required for DNA
expression, other elements may also be included in the DNA
molecule. Such additional elements include enhancers. The enhancer
may be selected from the group including but not limited to: human
actin, human myosin, human hemoglobin, human muscle creatine and
viral enhancers such as those from CMV, RSV and EBV.
[0065] Genetic constructs can be provided with mammalian origin of
replication in order to maintain the construct extrachromosomally
and produce multiple copies of the construct in the cell. Plasmids
pVAX1, pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain
the Epstein Barr virus origin of replication and nuclear antigen
EBNA-1 coding region which produces high copy episomal replication
without integration.
[0066] In order to maximize protein production, regulatory
sequences may be selected which are well suited for gene expression
in the cells the construct is administered into. Moreover, codons
that encode said protein may be selected which are most efficiently
transcribed in the host cell. One having ordinary skill in the art
can produce DNA constructs that are functional in the cells.
[0067] In some embodiments, nucleic acid constructs may be provided
in which the coding sequences for the proteins described herein are
linked to IgE signal peptide. In some embodiments, proteins
described herein are linked to IgE signal peptide.
[0068] In some embodiments for which protein is used, for example,
one having ordinary skill in the art can, using well known
techniques, can produce and isolate proteins of the invention using
well known techniques. In some embodiments for which protein is
used, for example, one having ordinary skill in the art can, using
well known techniques, inserts DNA molecules that encode a protein
of the invention into a commercially available expression vector
for use in well known expression systems. For example, the
commercially available plasmid pSE420 (Invitrogen, San Diego,
Calif.) may be used for production of protein in Escherichia coli
(E.coli). The commercially available plasmid pYES2 (Invitrogen, San
Diego, Calif.) may, for example, be used for production in
Saccharomyces cerevisiae strains of yeast. The commercially
available MAXBAC.TM. complete baculovirus expression system
(Invitrogen, San Diego, Calif.) may, for example, be used for
production in insect cells. The commercially available plasmid
pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) may, for example,
be used for production in mammalian cells such as Chinese hamster
ovary (CHO) cells. One having ordinary skill in the art can use
these commercial expression vectors and systems or others to
produce protein by routine techniques and readily available
starting materials. (See e.g., Sambrook et al., Molecular Cloning a
Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
Thus, the desired proteins can be prepared in both prokaryotic and
eukaryotic systems, resulting in a spectrum of processed forms of
the protein.
[0069] One having ordinary skill in the art may use other
commercially available expression vectors and systems or produce
vectors using well known methods and readily available starting
materials. Expression systems containing the requisite control
sequences, such as promoters and polyadenylation signals, and
preferably enhancers are readily available and known in the art for
a variety of hosts. See e.g., Sambrook et al., Molecular Cloning a
Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989).
Genetic constructs include the protein coding sequence operably
linked to a promoter that is functional in the cell line, or cells
of targeted tissue, into which the constructs are transfected.
Examples of constitutive promoters include promoters from
cytomegalovirus (CMV) or SV40. Examples of inducible promoters
include mouse mammary leukemia virus or metallothionein promoters.
Those having ordinary skill in the art can readily produce genetic
constructs useful for transfecting cells with DNA that encodes
protein of the invention from readily available starting materials.
The expression vector including the DNA that encodes the protein is
used to transform the compatible host which is then cultured and
maintained under conditions wherein expression of the foreign DNA
takes place.
[0070] The protein produced is recovered from the culture, either
by lysing the cells or from the culture medium as appropriate and
known to those in the art. One having ordinary skill in the art
can, using well known techniques, isolate protein that is produced
using such expression systems. The methods of purifying protein
from natural sources using antibodies which specifically bind to a
specific protein as described above may be equally applied to
purifying protein produced by recombinant DNA methodology.
[0071] In addition to producing proteins by recombinant techniques,
automated peptide synthesizers may also be employed to produce
isolated, essentially pure protein. Such techniques are well known
to those having ordinary skill in the art and are useful if
derivatives which have substitutions not provided for in
DNA-encoded protein production.
[0072] The nucleic acid molecules may be delivered using any of
several well known technologies including DNA injection (also
referred to as DNA vaccination) with and without in vivo
electroporation, liposome mediated, nanoparticle facilitated,
recombinant vectors such as recombinant adenovirus, recombinant
adenovirus associated virus and recombinant vaccinia. Preferably,
the nucleic acid molecules such as the DNA plasmids described
herein are delivered via DNA injection and along with in vivo
electroporation.
[0073] Routes of administration include, but are not limited to,
intramuscular, intransally, intraperitoneal, intradermal,
subcutaneous, intravenous, intraarterially, intraoccularly and oral
as well as topically, transdermally, by inhalation or suppository
or to mucosal tissue such as by lavage to vaginal, rectal,
urethral, buccal and sublingual tissue. Preferred routes of
administration include intramuscular, intraperitoneal, intradermal
and subcutaneous injection. Genetic constructs may be administered
by means including, but not limited to, traditional syringes,
needleless injection devices, "microprojectile bombardment gone
guns", or other physical methods such as electroporation ("EP"),
"hydrodynamic method", or ultrasound.
[0074] Examples of electroporation devices and electroporation
methods preferred for facilitating delivery of the DNA vaccines of
the present invention, include those described in U.S. Pat. No.
7,245,963 by Draghia-Akli, et al., U.S. Patent Pub. 2005/0052630
submitted by Smith, et al., the contents of which are hereby
incorporated by reference in their entirety. Also preferred, are
electroporation devices and electroporation methods for
facilitating delivery of the DNA vaccines provided in co-pending
and co-owned U.S. patent application, Ser. No. 11/874072, filed
Oct. 17, 2007, which claims the benefit under 35 USC 119(e) to U.S.
Provisional Applications Ser. Nos. 60/852,149, filed Oct. 17, 2006,
and 60/978,982, filed Oct. 10, 2007, all of which are hereby
incorporated in their entirety. Preferable, the electroporation
device is the CELLECTRA.TM. device (VGX Pharmaceuticals, Blue Bell,
Pa.), including the intramuscular (IM) and intradermal (ID)
models.
[0075] U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes
modular electrode systems and their use for facilitating the
introduction of a biomolecule into cells of a selected tissue in a
body or plant. The modular electrode systems comprise a plurality
of needle electrodes; a hypodermic needle; an electrical connector
that provides a conductive link from a programmable
constant-current pulse controller to the plurality of needle
electrodes; and a power source. An operator can grasp the plurality
of needle electrodes that are mounted on a support structure and
firmly insert them into the selected tissue in a body or plant. The
biomolecules are then delivered via the hypodermic needle into the
selected tissue. The programmable constant-current pulse controller
is activated and constant-current electrical pulse is applied to
the plurality of needle electrodes. The applied constant-current
electrical pulse facilitates the introduction of the biomolecule
into the cell between the plurality of electrodes. The entire
content of U.S. Pat. No. 7,245,963 is hereby incorporated by
reference.
[0076] U.S. Patent Pub. 2005/0052630 submitted by Smith, et al.
describes an electroporation device which may be used to
effectively facilitate the introduction of a biomolecule into cells
of a selected tissue in a body or plant. The electroporation device
comprises an electro-kinetic device ("EKD device") whose operation
is specified by software or firmware. The EKD device produces a
series of programmable constant-current pulse patterns between
electrodes in an array based on user control and input of the pulse
parameters, and allows the storage and acquisition of current
waveform data. The electroporation device also comprises a
replaceable electrode disk having an array of needle electrodes, a
central injection channel for an injection needle, and a removable
guide disk. The entire content of U.S. Patent Pub. 2005/0052630 is
hereby incorporated by reference.
[0077] The electrode arrays and methods described in U.S. Pat. No.
7,245,963 and U.S. Patent Pub. 2005/0052630 are adapted for deep
penetration into not only tissues such as muscle, but also other
tissues or organs. Because of the configuration of the electrode
array, the injection needle (to deliver the biomolecule of choice)
is also inserted completely into the target organ, and the
injection is administered perpendicular to the target issue, in the
area that is pre-delineated by the electrodes The electrodes
described in U.S. Pat. No. 7,245,963 and U.S. Patent Pub.
2005/005263 are preferably 20 mm long and 21 gauge.
[0078] The following is an example of methods of the present
invention, and is discussed in more detail in the patent references
discussed above: electroporation devices can be configured to
deliver to a desired tissue of a mammal a pulse of energy producing
a constant current similar to a preset current input by a user. The
electroporation device comprises an electroporation component and
an electrode assembly or handle assembly. The electroporation
component can include and incorporate one or more of the various
elements of the electroporation devices, including: controller,
current waveform generator, impedance tester, waveform logger,
input element, status reporting element, communication port, memory
component, power source, and power switch. The electroporation
component can function as one element of the electroporation
devices, and the other elements are separate elements (or
components) in communication with the electroporation component. In
some embodiments, the electroporation component can function as
more than one element of the electroporation devices, which can be
in communication with still other elements of the electroporation
devices separate from the electroporation component. The present
invention is not limited by the elements of the electroporation
devices existing as parts of one electromechanical or mechanical
device, as the elements can function as one device or as separate
elements in communication with one another. The electroporation
component is capable of delivering the pulse of energy that
produces the constant current in the desired tissue, and includes a
feedback mechanism. The electrode assembly includes an electrode
array having a plurality of electrodes in a spatial arrangement,
wherein the electrode assembly receives the pulse of energy from
the electroporation component and delivers same to the desired
tissue through the electrodes. At least one of the plurality of
electrodes is neutral during delivery of the pulse of energy and
measures impedance in the desired tissue and communicates the
impedance to the electroporation component. The feedback mechanism
can receive the measured impedance and can adjust the pulse of
energy delivered by the electroporation component to maintain the
constant current.
[0079] In some embodiments, the plurality of electrodes can deliver
the pulse of energy in a decentralized pattern. In some
embodiments, the plurality of electrodes can deliver the pulse of
energy in the decentralized pattern through the control of the
electrodes under a programmed sequence, and the programmed sequence
is input by a user to the electroporation component. In some
embodiments, the programmed sequence comprises a plurality of
pulses delivered in sequence, wherein each pulse of the plurality
of pulses is delivered by at least two active electrodes with one
neutral electrode that measures impedance, and wherein a subsequent
pulse of the plurality of pulses is delivered by a different one of
at least two active electrodes with one neutral electrode that
measures impedance.
[0080] In some embodiments, the feedback mechanism is performed by
either hardware or software. Preferably, the feedback mechanism is
performed by an analog closed-loop circuit. Preferably, this
feedback occurs every 50 .mu.s, 20 .mu.s, 10 .mu.s or 1 .mu.s, but
is preferably a real-time feedback or instantaneous (i.e.,
substantially instantaneous as determined by available techniques
for determining response time). In some embodiments, the neutral
electrode measures the impedance in the desired tissue and
communicates the impedance to the feedback mechanism, and the
feedback mechanism responds to the impedance and adjusts the pulse
of energy to maintain the constant current at a value similar to
the preset current. In some embodiments, the feedback mechanism
maintains the constant current continuously and instantaneously
during the delivery of the pulse of energy. A pharmaceutically
acceptable excipient can include such functional molecules as
vehicles, adjuvants, carriers or diluents, which are known and
readily available to the public. Preferably, the pharmaceutically
acceptable excipient is an adjuvant or transfection facilitating
agent. In some embodiments, the nucleic acid molecule, or DNA
plasmid, is delivered to the cells in conjunction with
administration of a polynucleotide function enhancer or a genetic
vaccine facilitator agent (or transfection facilitating agent).
[0081] Polynucleotide function enhancers are described in U.S. Pat.
Nos. 5,593,972, 5,962,428 and International Application Serial
Number PCT/US94/00899 filed Jan. 26, 1994, which are each
incorporated herein by reference. Genetic vaccine facilitator
agents are described in U.S. Ser. No. 021,579 filed Apr. 1, 1994,
which is incorporated herein by reference. The transfection
facilitating agent can be administered in conjunction with nucleic
acid molecules as a mixture with the nucleic acid molecule or
administered separately simultaneously, before or after
administration of nucleic acid molecules. Examples of transfection
facilitating agents includes surface active agents such as
immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant,
LPS analog including monophosphoryl lipid A, muramyl peptides,
quinone analogs and vesicles such as squalene and squalene, and
hyaluronic acid may also be used administered in conjunction with
the genetic construct. In some embodiments, the DNA plasmid
vaccines may also include a transfection facilitating agent such as
lipids, liposomes, including lecithin liposomes or other liposomes
known in the art, as a DNA-liposome mixture (see for example
W09324640), calcium ions, viral proteins, polyanions, polycations,
or nanoparticles, or other known transfection facilitating agents.
Preferably, the transfection facilitating agent is a polyanion,
polycation, including poly-L-glutamate (LGS), or lipid.
[0082] In some preferred embodiments, the DNA plasmids are
delivered with an adjuvant that are genes for proteins which
further enhance the immune response against such target proteins.
Examples of such genes are those which encode other cytokines and
lymphokines such as alpha-interferon, gamma-interferon, platelet
derived growth factor (PDGF), TNF.alpha., TNF.beta., GM-CSF,
epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10,
IL-12, IL-18, MHC, CD80,CD86 and IL-15 including IL-15 having the
signal sequence deleted and optionally including the signal peptide
from IgE. Other genes which may be useful include those encoding:
MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin,
E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95,
PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4,
mutant forms of IL-18, CD40, CD4OL, vascular growth factor,
fibroblast growth factor, IL-7, nerve growth factor, vascular
endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55,
WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER,
TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2,
p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,
.INK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,
TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40
LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1,
TAP2 and functional fragments thereof.
[0083] The pharmaceutical compositions according to the present
invention comprise DNA quantities of from about 1 nanogram to 100
milligrams; about 1 microgram to about 10 milligrams; or preferably
about 0.1 microgram to about 10 milligrams; or more preferably
about 1 milligram to about 2 milligram. In some preferred
embodiments, pharmaceutical compositions according to the present
invention comprise about 5 nanogram to about 1000 micrograms of
DNA. In some preferred embodiments, the pharmaceutical compositions
contain about 10 nanograms to about 800 micrograms of DNA. In some
preferred embodiments, the pharmaceutical compositions contain
about 0.1 to about 500 micrograms of DNA. In some preferred
embodiments, the pharmaceutical compositions contain about 1 to
about 350 micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 25 to about 250
micrograms of DNA. In some preferred embodiments, the
pharmaceutical compositions contain about 100 to about 200
microgram DNA.
[0084] The pharmaceutical compositions according to the present
invention are formulated according to the mode of administration to
be used. In cases where pharmaceutical compositions are injectable
pharmaceutical compositions, they are sterile, pyrogen free and
particulate free. An isotonic formulation is preferably used.
Generally, additives for isotonicity can include sodium chloride,
dextrose, mannitol, sorbitol and lactose. In some cases, isotonic
solutions such as phosphate buffered saline are preferred.
Stabilizers include gelatin and albumin. In some embodiments, a
vasoconstriction agent is added to the formulation. In some
embodiments, a stabilizing agent that allows the formulation to be
stable at room or ambient temperature for extended periods of time,
such as LGS or other polycations or polyanions is added to the
formulation.
[0085] In some embodiments, methods of eliciting an immune response
in mammals against a consensus influenza antigen include methods of
inducing mucosal immune responses. Such methods include
administering to the mammal one or more of CTACK protein, TECK
protein, MEC protein and functional fragments thereof or
expressible coding sequences thereof in combination with an DNA
plasmid including a consensus influenza antigen, described above.
The one or more of CTACK protein, TECK protein, MEC protein and
functional fragments thereof may be administered prior to,
simultaneously with or after administration of the DNA plasmid
influenza vaccines provided herein. In some embodiments, an
isolated nucleic acid molecule that encodes one or more proteins of
selected from the group consisting of: CTACK, TECK, MEC and
functional fragments thereof is administered to the mammal.
EXAMPLES
[0086] The present invention is further illustrated in the
following Examples. It should be understood that these Examples,
while indicating preferred embodiments of the invention, are given
by way of illustration only. From the above discussion and these
Examples, one skilled in the art can ascertain the essential
characteristics of this invention, and without departing from the
spirit and scope thereof, can make various changes and
modifications of the invention to adapt it to various usages and
conditions. Thus, various modifications of the invention in
addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0087] Preferably the DNA formulations for use with a muscle or
skin EP device described herein have high DNA concentrations,
preferably concentrations that include milligram to tens of
milligram quantities, and preferably tens of milligram quantities,
of DNA in small volumes that are optimal for delivery to the skin,
preferably small injection volume, ideally 25-200 microliters
(.mu.L). In some embodiments, the DNA formulations have high DNA
concentrations, such as 1 mg/mL or greater (mg DNA/volume of
formulation). More preferably, the DNA formulation has a DNA
concentration that provides for gram quantities of DNA in 200 .mu.L
of formula, and more preferably gram quantities of DNA in 100 .mu.L
of formula.
[0088] The DNA plasmids for use with the EP devices of the present
invention can be formulated or manufactured using a combination of
known devices and techniques, but preferably they are manufactured
using an optimized plasmid manufacturing technique that is
described in a commonly owned, co-pending U.S. provisional
application U.S. Ser. No. 60/939,792, which was filed on May 23,
2007. In some examples, the DNA plasmids used in these studies can
be formulated at concentrations greater than or equal to 10 mg/mL.
The manufacturing techniques also include or incorporate various
devices and protocols that are commonly known to those of ordinary
skill in the art, in addition to those described in U.S. Ser. No.
60/939792, including those described in a commonly owned patent,
U.S. Pat. No. 7,238,522, which issued on Jul. 3, 2007. The high
concentrations of plasmids used with the skin EP devices and
delivery techniques described herein allow for administration of
plasmids into the ID/SC space in a reasonably low volume and aids
in enhancing expression and immunization effects. The commonly
owned application and patent, U.S. Ser. No. 60/939,792 and U.S.
Pat. No. 7,238,522, respectively, are hereby incorporated in their
entirety.
Example 1
Plasmid Constructs
[0089] A ubiquitous cytomegalovirus (CMV) promoter drives the
expression of human secreted embryonic alkaline phosphatase (SEAP)
reporter transgene product in the pCMV-SEAP vector. Plasmids were
obtained using a commercially available kit (Qiagen Inc.,
Chatsworth, Calf.). Endotoxin levels were at less than 0.01
EU/.mu.g, as measured by Kinetic Chromagenic LAL (Endosafe,
Charleston, S.C.). Consensus HA and NA constructs were generated by
analyzing primary virus sequences from 16 H5 viruses that have
proven fatal to humans in recent years, and over 40 human N1
viruses. These sequences were downloaded from the Los Alamos
National Laboratory's Influenza Sequence Database. After generating
the consensus sequences, the constructs were optimized for
mammalian expression, including the addition of a Kozak sequence,
codon optimization, and RNA optimization. These constructs were
then subcloned into the pVAX vector (Invitrogen, Carlsbad, Calif.).
Unless indicated otherwise, plasmid preparations were diluted in
sterile water and formulated 1% weight/weight with poly-L-glutamate
sodium salt (LGS) (MW=10.5 kDa average)(Sigma, St. Louis, Mo.),
further HPLC purified at VGX Pharmaceuticals, Immune Therapeutics
Division (The Woodlands, Tex.).
Example 2
Treatment of Pigs
[0090] Pigs were divided into 10 groups.times.4 pigs per group for
a total of 40 pigs (Table 1). Pigs were acclimated for 4 days,
weighed and ear-tagged. On Study Day 0, pigs were weighed, bled and
anesthetized using a combination pre-anesthetic for
pigs--ketamine--(20 mg/kg), xylazine--(2.2 mg/kg) and atropine
(0.04 mg/kg), and then anesthetized using isoflurane (induction at
5%, maintenance at 2-3%). Pigs (n=4/group) were injected with 0.6
mL of CMV-HA (a pVAX based construct that expresses a consensus H5
antigen), CMV-NA (a pVAX based construct that expresses a consensus
N1 antigen), and CMV-SEAP (a construct expressing the reporter gene
secreated ambryonic alkaline phosphatase, SEAP) plasmid (the last
one added to increase plasmid concentration, and viscosity of the
solution for the "muscle damage" assessment)+1.0% wt/wt LGS at
varying plasmid concentrations and current intensities. The
plasmids were prepared according to the materials and methods
provided in Example 1. After 4 s, animals were electroporated using
the adaptive constant current CELLECTRA.TM. intramuscular (IM)
system (VGX Pharmaceuticals, Blue Bell, Pa.) equipped with 5 needle
electrodes and operated with the following pulse parameters: 52
millisecond pulses, 1 second between pulses, 3 pulses with varying
current (0.1, 0.3 and 0.5 A).
TABLE-US-00001 TABLE 1 Groups for the pig vaccine experiment Total
Conc Dose (mg/ Construct (mg/ Injection Group Plasmid mL) (mg)/pig
pig) Volume A n 1 HA, NA, 10 2 6 600 .mu.l 0.5 4 SEAP 2 HA, NA, 4
0.8 2.4 600 .mu.l 0.5 4 SEAP 3 HA, NA, 1.5 0.1 0.3 600 .mu.l 0.5 4
SEAP 4 HA, NA, 10 2 6 600 .mu.l 0.3 4 SEAP 5 HA, NA, 4 0.8 2.4 600
.mu.l 0.3 4 SEAP 6 HA, NA, 1.5 0.1 0.3 600 .mu.l 0.3 4 SEAP 7 HA,
NA, 10 2 6 600 .mu.l 0.1 4 SEAP 8 HA, NA, 4 0.8 2.4 600 .mu.l 0.1 4
SEAP 9 HA, NA, 1.5 0.1 0.3 600 .mu.l 0.1 4 SEAP 10 None N/A N/A N/A
N/A N/A 4
The area surrounding each injection site was tattooed for rapid
identification for biopsy at Days 14 and 35 post-injection.
[0091] Pigs were allowed to recover from anesthesia and were
closely monitored for 24 hours to ensure full recovery. Any pigs
that did not fully recover within 2 to 3 hours post-treatment were
noted. Pigs were weighed and bled on Day 10, Day 21 and Day 35. The
pigs were administered a second vaccination on Day 21. Blood was
collected in 2 purple top tubes, 1.0 mL for CBC and differentials
(Antech Diagnostics, Irvine, Calif.); 10 mL for IFN-.gamma.
ELISpots against HA and NA antigens, and separate falcon tubes
which were allowed to clot and centrifuged to isolate serum then
aliquoted into tubes on ice. On Day 35, all pigs were exsanguinated
under surgical plane of anesthesia and needle punch biopsies of the
injection sites were taken for histology.
Hemagglutination Inhibition (HI) Assay
[0092] Pig sera were treated with receptor destroying enzyme (RDE)
by diluting one part serum with three parts enzyme and incubated
overnight in 37.degree. C. water bath. The enzyme was inactivated
by 30 min incubation at 56.degree. C. followed by addition of six
parts PBS for a final dilution of 1/10. HI assays were performed in
V-bottom 96-well microtiter plates, using four HA units of virus
and 1% horse red blood cells as previously described (Stephenson,
I., et al., Virus Res., 103 (1-2): 91-5 (July 2004)).
[0093] The highest titers as demonstrated by the HI assay (FIG. 2)
were found in sera from the group administered 2 mg of
HA-expressing plasmid at a current setting of 0.5 A (120.+-.40;
P=0.11 versus 2 mg/0.3 A and P=0.02 versus 2 mg/0.1 A); the titers
decreased with the intensity of the electric field for the group
that received 2 mg of each plasmid; if either plasmid quantity of
current were decreased thereafter, the titers were more variable,
and non-different between groups.
[0094] The HI titers were highest in the group administered 2 mg of
HA-expressing plasmid and electroporated at 0.5 A. Furthermore, the
titers decline with descending plasmid doses in the group
electroporated at 0.5 A, and with the intensity of the electric
field. The lower plasmid quantities or lower current intensities
appeared to increase the intra-group variability.
HA and NA IFN-.gamma. ELISpots
[0095] ELISpot were performed as previously described using
IFN-.gamma. capture and detection antibodies (MabTech, Sweden)
(Boyer J D, et al., J Med Primatol, 34(5-6):262-70 (October 2005)).
Antigen-specific responses were determined by subtracting the
number of spots in the negative control wells from the wells
containing peptides. Results are shown as the mean value
(spots/million splenocytes) obtained for triplicate wells. The
group administered 2 mg of each plasmid (for a total of 4 mg) at a
current setting of 0.3 A attained the highest cellular immune
response as measured by the IFN-.gamma. ELISpot of 537.+-.322 SFU
per million cells. The average responses of all other groups were
within background levels of the assay. The individual ELISpot
responses of two animals attaining the highest cellular immune
response are highlighted in FIG. 3.
CBC Results
[0096] Lymphocytes reached the highest levels at Day 21 of the
study and in the groups administered the highest dose of vaccines,
regardless of current setting, although the groups with the highest
dose (4 mg of total plasmid, 2 mg each) and highest current setting
(0.5 A) demonstrates highest lymphocyte response, 40% higher than
controls (12670.+-.1412 vs. 7607.+-.1603 lymphocyte counts/100
blood, respectively; P<0.002).
Muscle Histopathology
[0097] The injection sites were identified and punch biopsies were
taken at Days 14 and 35 post-treatment after the pigs were
exsanguinated. The tissues were fixed in buffered formalin for 24
hours then washed 3.times. in PBS and stored in 70% alcohol. The
biopsy samples were submitted to Antech Diagnostics where they were
processed and sections stained with hematoxylin and eosin
(H&E). All the slides were evaluated by a single
board-certified pathologist who scored them 0 to 5 for pathological
criteria (Table 2) in various tissue layers (Table 3). The mean
score was calculated for each group at each time point.
TABLE-US-00002 TABLE 2 Biopsy pathology scoring parameters Score
Criteria 0 Not present, no inflammatory cells 1 Minimal, 1-20
inflammatory cells/100 .times. high-powered field (HPF) 2 Mild,
21-40 inflammatory cells/100 .times. HPF 3 Moderate, 41-75
inflammatory cells/100 .times. HPF 4 Moderate to Marked/Severe,
76-100 inflammatory cells/100 .times. HPF 5 Marked Severe, >100
inflammatory cells/100 .times. HPF
TABLE-US-00003 TABLE 3 Biopsy tissue layers and pathological
parameters Anatomy Location Pathology Parameter Dermal Superficial
neovascularization Dermal Pylogranulomatous inflammation Dermal
Overlying erosion & inflammatory crusting Dermal Focal fibrosis
Subcutaneous Pylogranulomatous inflammation with intralesional
collagen necrosis Subcutaneous Lymphacytic and plasmalytic
inflammation Skeletal muscle Lymphacytic and plasmalytic and
eosinophilic inflammation Skeletal muscle Myocyte
degeneration/necrosis Skeletal muscle Fibrosis
[0098] The histopathology was scored from the muscle biopsy (FIG.
4A) at 14 and 35 days after plasmid injection and EP based on a 0
to 5 scale criteria (Table 2). Overall pathology scores following
electroporation declined in the tissue layers (Table 3) from Day 14
to Day 35. The group that received 6 mg of total plasmid at 0.3 A
settings exhibited the highest total pathology scores at Day 14
(18.3.+-.6.4, P<0.0002 versus control), correlating with the
highest average lymphocyte responses. All pathology scores at Day
35 approached levels of non-treated control levels (range of 6.67
to 4.25). Nevertheless, when the muscle necrosis and fibrosis
(typically associated with the EP procedure) (Gronevik E, et al., J
Gene Med, 7(2):218-27 (2005 February)).were analyzed separately
(FIG. 4B), the scores ranged between 1 and 2, with no difference
between groups or between treated groups and controls, while the
higher scores were associated with lymphatic, plasmacytic or
eosinophilic inflammation due to immune responses. Significantly,
these scores also declined from day 14 to day 35
post-treatment.
Data Analysis
[0099] Data were analyzed using Microsoft Excel Statistics package.
Values shown in the figures are the mean.+-.SEM. Specific values
were obtained by comparison using one-way ANOVA and subsequent
t-test. A value of p <0.05 was set as the level of statistical
significance.
Example 3
Treatment of Ferrets
[0100] Twenty male ferrets (Triple F Farms, Sayre, Pa.), 4-6 months
of age or at least 1 kg body weight, were used in this study and
housed at BIOQUAL, Inc. (Rockville, Md.). The ferret study design
is in Table 4. Animals were allowed to acclimate for two weeks
prior to the study. Animals were immunized (under anesthesia) at
Week 0, 4, and 9. Blood was drawn every 2 weeks. After the third
immunization, animals were moved into a BSL-3 facility and
challenged at Week 13 with a very potent strain of avian influenza
(H5N1) and then followed for two more weeks post-challenge. For two
weeks after challenge, animals were monitored daily, and body
weights, temperature and clinical scores were recorded. Activity
level was monitored and recorded; death were documented.
[0101] This study tested the efficacy of HA, NA and M2e-NP DNA
vaccine delivered IM followed by electroporation using the
CELLECTRA.TM. adaptive constant current electroporation
intramuscular (IM) system (VGX Pharmaceuticals, Blue Bell, Pa.) in
an influenza challenge model in ferrets. The DNA plasmids were
prepared according to the materials and methods provided in Example
1. As outlined in Table 4, animals in Groups 2, 3 and 4 received
0.2 mg of the respective influenza plasmid vaccine. In order to
correct for dose, groups which received 1 plasmid vaccine (Groups 2
and 3) or no vaccine (control Group 1), the difference was made up
by pVAX empty vector such that all animals in every group received
a total dose of 0.6 mg of plasmid. The conditions of
electroporation were, using a 5 needle electrode array: 0.5 Amps,
52 msec pulse width, 1 sec between pulses, 4 sec delay between
injection and electroporation.
TABLE-US-00004 TABLE 4 Groups for the Influenza challenge
experiment in ferrets Total Vaccine vaccine in Dose (mg) Total
Group Plasmids/Antigens per Plasmid volume n 1 None (pVAX only) 0
mg 0.6 mg 4 in 0.6 mL 2 H5 + pVAX 0.2 mg 0.6 mg 4 in 0.6 mL 3 NA +
pVAX 0.2 mg 0.6 mg 4 in 0.6 mL 4 H5, NA, M2e-NP 0.2 mg 0.6 mg 4 in
0.6 mL
Hemagglutination Inhibition (HI) Assay
[0102] Sera were treated with receptor destroying enzyme (RDE) by
diluting one part serum with three parts enzyme and incubated
overnight in 37.degree. C. water bath. The enzyme was inactivated
by 30 min incubation at 56.degree. C. followed by addition of six
parts PBS for a final dilution of 1/10. HI assays were performed in
V-bottom 96-well microtiter plates, using four HA units of virus
and 1% horse red blood cells as previously described (Stephenson,
I., et al., Virus Res., 103(1-2):91-5 (July 2004)). The viruses
used for the HI assay are reassortant strains we obtained from the
Center for Disease Control: A/Viet/1203/2004(H5N1)/PR8-IBCDC-RG
(clade 1 virus) and A/Indo/05/2005 (H5N1)/PR8-IBCDC-RG2 (clade 2
virus). The ferret model of influenza infection is considered to be
more reflective of human disease and a more rigorous challenge
model. Ferrets exhibit similar symptoms to humans infected with
influenza and similar tissue tropism with regards to human and
avian influenza viruses. Serum collected at different time points
throughout the study was used to detect HI activity against H5N1
viruses. As shown in FIG. 7, both groups containing the consensus
H5 specific HA construct attained protective levels of antibody
(>1:40) after two immunizations and were also able to inhibit a
clade 2 H5N1 virus. In other words, the HI assay was positive
against both viral strains despite the consensus HA strain was
based on clade 1 viruses.
Data Analysis
[0103] Data were analyzed using Microsoft Excel Statistics package.
Values shown in the figures are the mean.+-.SEM. Specific values
were obtained by comparison using one-way ANOVA and subsequent
t-test. A value of p<0.05 was set as the level of statistical
significance.
Ferret Influenza Challenge
[0104] The results of the influenza challenge are depicted in FIGS.
5 and 6. Control animals lost 25% of their body weight on average
post-challenge (FIG. 5), while animals vaccinated with HA (Group 1)
or HA+M2e-NP+NA (Group 4) lost between 9 and 10% (*P<0.004
versus controls). Body temperatures were elevated in control
animals until all control animals were either found dead or
euthanized by Day 8 (FIG. 6). All animals vaccinated, regardless of
which vaccine regimen, survived the challenge and showed fewer
signs of infection as compared to the control animals as evidenced
by their clinical scores (Table 5). Control animals worsen as far
as clinical scores (nasal discharge, cough, lethargy), and died
between day 5 and day 7 post-challenge. As shown in Table 5, the
severity of the clinical scores in vaccinated animals was inversely
correlated with the antibody titers (higher antibody titers, lower
clinical scores, better clinical outcome).
TABLE-US-00005 TABLE 5 Results for Challenged Ferrets
Post-challenge Observations HI Titers 3 wks Vaccines Ferret Day 1
Day 2 Day 3 Day 5 Day 6 Day 7 Day 8 Day 9 Pre-challenge 891 0_1 1_1
1_1 0_1 0_1 1_1* <20 Control 890 0_1 0_1 1_1 0_1* <20 (pVAX)
877 0_1 0_1 1_1 0_2 2_3 FD <20 876 0_1 0_1 0_1 0_1 2_3 1_3*
<20 878 0_1 0_1 1_1 0_1 0_1 0_1 0_1 0_1 40 H5 879 0_1 0_1 0_1
0_1 0_1 0_1 0_1 0_1 320 888 0_1 0_1 0_1 0_1 0_1 0_1 0_1 0_1 160 889
0_1 0_1 0_1 0_1 0_1 0_1 0_1 0_1 320 881 0_1 0_1 1_0 0_1 0_1 1_1 0_1
0_1 <20 M2-NP 880 0_1 0_1 0_0 0_1 0_1 0_1 0_1 0_1 <20 883 0_1
1_1 1_1 0_1 0_1 1_1 0_1 0_1 <20 882 0_1 0_1 1_1 0_1 0_1 1_2 0_2
0_1 <20 885 0_1 0_1 0_1 0_1 0_1 0_1 0_1 0_1 1280 H5 + M2- 884
0_1 0_1 0_1 0_1 0_1 0_1 0_1 0_1 320 NP + NA 886 1_1 1_1 0_1 0_1 0_1
1_1 0_1 1_1 160 887 0_1 1_1 0_0 0_1 0_1 1_1 0_1 0_1 640 Table 5
Note: Clinical scores are depicted for the post-challenge
observation period. A "*" indicates the animal was euthanized; FD =
found dead. The first clinical score in each column is for nasal
symptoms: 0 = none; 1 = nasal discharge; 2 = breathing from mouth.
The second score is for activity: 0 = sleeping; 1 = bright and
alert; 2 = alert but non-responsive; 3 = lethargic. The HI titers
for each animal measured 3 weeks pre-challenge are depicted for
comparison purposes.
ExamplE 4
Intradermal Delivery Comparisons with Intramuscular Delivery in
Primates
[0105] Rhesus macaques were immunized in these studies. Animals
were acclimated for 2 months prior to the start of experiments. The
study progressed as follows: Week 0--performed 1st immunization
(plasmid dose administration) and baseline bleed; Week 2 performed
bleed; Week 3 performed 2nd immunization (plasmid dose
administration); Week 5 performed bleed; Week 6 performed 3rd
immunization (plasmid dose administration) and bleed; Week 8
performed bleed.
TABLE-US-00006 TABLE 6 Total Study DNA DNA Group Constructs Nr.
Route of Admin Dose (mg) A DNA 6 + 9 5 IM CELLECTRA .TM. EP 1 mg/ 2
Const B DNA 6 + 9 5 ID CELLECTRA .TM. EP 1 mg/ 2 Const C DNA 1 + 6
+ 5 IM Syringe 1 mg/ 4 9 + 10 Const D Negative Control 5 N/A 0
TABLE-US-00007 DNA Construct # Encoding Antigen 1 Non-influenza
antigen control plasmid 6 Influenza H5 consensus 9 Non-influenza
antigen control plasmid 10 Non-influenza antigen control
plasmid
[0106] All plasmids were formulated at 10 mg/mL in water for
injection+1% LGS, as described in previous examples, above, and
mixed into a single solution PER STUDY GROUP(S) (Groups C, D, G,
and H, in above table, Table 6). The correct injection volume for
each group designated IM CELLECTRA.TM. EP (VGX Pharmaceuticals), ID
CELLECTRA.TM. EP (VGX Pharmaceuticals), and IM Syringe was
calculated. For the ID administration, if the required injection
volume surpassed 100 .mu.L per site, the formulation was split into
a number of injection sites (2, 3, or 6 depending on how many total
mg of vaccine were administered). The animals that received IM
injection(s) were given the entire formulation in one single
site.
[0107] The CELLECTRA.TM. adaptive constant current device used in
the pigs experiments, ferret experiments and nonhuman experiments
described in the Examples. The electroporation conditions were as
following: for the IM injection and electroporation groups, the
conditions were: 0.5 Amps, 52 msec/pulse, three pulses, 4 sec delay
between plasmid injection and electroporation. For the ID injection
and electroporation groups, the conditions were: 0.2 Amps, 52
msec/pulse, three pulses, 4 sec delay between plasmid injection and
electroporation.
[0108] Hemagglutination Inhibition (HI) Assay--monkey sera were
treated with receptor destroying enzyme (RDE) by diluting one part
serum with three parts enzyme and incubated overnight in 37.degree.
C. water bath. The enzyme was inactivated by 30 min incubation at
56.degree. C. followed by addition of six parts PBS for a final
dilution of 1/10. HI assays were performed in V-bottom 96-well
microtiter plates, using four HA units of virus and 1% horse red
blood cells. The data presented herein are the results after the
second immunization (bleed collected before the third
immunization).
[0109] HI titers were measured three weeks after the second
immunization. The results can be seen displayed in the graph in
FIG. 8. Monkeys receiving the HA plasmid vaccine via ID injection
followed by electroporation demonstrated more than twice the
average titers of the IM +EP group and almost three times the
average titers of the IM group alone (*P<0.03). Non-treated
controls did not exhibit any HI titers.
Example 5
Cross Protection in Primates
[0110] Using Delivery method--ID injection followed by
electroporation (EP)
[0111] Studies in non-human primates with the influenza vaccine
(including H5, NA and M2e-NP consensus antigens, see above)
indicated that ID injection followed by electroporation elicited
higher antibody responses to the vaccine antigens than in IM
injections. In one of our non-human primate studies (NHP) animals
were vaccinated per Table 7.
TABLE-US-00008 TABLE 7 Study design and conditions. Rhesus macaques
were immunized at weeks 0, 4, and 8. Concentration Group n/group
Antigen Delivery (mg/plasmid) EP Conditions 1 5 pVax (sham) IM 1
mg/construct 0.5 Amps, 3 pulses, 52 msec, 1 sec between pulses 2 5
H5, NA, IM 1 mg/construct 0.5 Amps, 3 pulses, 52 msec, 1 sec M2e-NP
between pulses 3 5 M2e-NP IM 1 mg/construct 0.5 Amps, 3 pulses, 52
msec, 1 sec between pulses 4 5 H5, NA, ID 1 mg/construct 0.2 Amps,
2 .times. 2 pulses, 52 msec, 1 sec M2e-NP between pulses
Each animal received three vaccinations, and HAI titers and
microneutralization were performed for both the same clade and
cross-clades. As shown, the consensus vaccine offered broad
protection not only within the same clade, but also cross-clades.
Results are included in Table 8.
TABLE-US-00009 TABLE 8 Results of hemagglutination (HAI) and
microneutralization assays. Clade 1 Clade 2.1 Clade 2.2 Clade 2.3.4
A/Vietnam A/Indonesia A/Turkey A/Anhui HAI Assay 2nd immunization
VGX-3400IM 160 (80-320) 36 (20-80) 110 (0-320).sup.4/5 80 (40-160)
VGX-3400ID 664 (40-1280) 120 (20-320) 205 (0-320).sup.4/5 592
(40-1280) 3rd immunization VGX-3400IM 288 (160-640) 32
(0-80).sup.3/5 36 (20-80) 84 (20-160) VGX-3400ID 416 (160-640) 64
(0-160).sup.2/5 145 (20-320) 276 (20-640) Microneutralization 3rd
immunization VGX-3400IM 144 (40-360) 8 (0-40).sup.1/5 32
(0-80).sup.2/5 88 (0-160).sup.4/5 VGX-3400ID 740 (20-2560) 96
(0-320).sup.3/5 296 (0-1280).sup.3/5 1172 (20-2560) Values
presented indicate the mean titer, the range (in parenthesis) and
the number of responders if less than 5/5 (in superscript). Note:
HAI titers > 1:20 are generally considered seroprotective in the
NHP model.
[0112] The needles in the ID electroporation device are much
shorter (.about.5 mm), of a lower gauge, and do not elicit muscle
contractions or visible pain responses in the animals tested to
date. Furthermore, the required electric field for efficacious ID
EP is lower than that required for an optimum IM delivery. ID
injection has been shown to elicit better immune responses to
influenza vaccine antigens. (Holland D, et. al. (2008). J Inf Dis.
198:650-58.) Usually, a lower dose is needed in vaccines delivered
ID compared to IM delivery to achieve similar humoral responses.
Sequence CWU 1
1
1711707DNAArtificial SequenceInfluenza H5N1 HA consensus sequence
1atggaaaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc
60atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga aaaaaacgtg
120accgtgaccc acgcccagga catcctggaa aagacccaca acggcaagct
gtgcgacctg 180gacggcgtga agcccctgat cctgcgggac tgcagcgtgg
ccggctggct gctgggcaac 240cccatgtgcg acgagttcat caacgtgccc
gagtggagct acatcgtgga gaaggccaac 300cccgtgaacg acctgtgcta
ccccggcgac ttcaacgact acgaggaact gaagcacctg 360ctgtcccgga
tcaaccactt cgagaagatc cagatcatcc ccaagagcag ctggtccagc
420cacgaggcca gcctgggcgt gagcagcgcc tgcccatacc agggcaagtc
cagcttcttc 480cggaacgtgg tgtggctgat caagaagaac agcacctacc
ccaccatcaa gcggagctac 540aacaacacca accaggaaga tctgctggtc
ctgtggggca tccaccaccc caacgacgcc 600gccgagcaga ccaagctgta
ccagaacccc accacctaca tcagcgtggg caccagcacc 660ctgaaccagc
ggctggtgcc ccggatcgcc acccggtcca aggtgaacgg ccagagcggc
720cggatggaat tcttctggac catcctgaag cccaacgatg ccatcaactt
cgagagcaac 780ggcaacttca tcgcccccga gtacgcctac aagatcgtga
agaagggcga cagcaccatc 840atgaagagcg agctggaata cggcaactgc
aacaccaagt gccagacccc catgggcgcc 900atcaacagca gcatgccctt
ccacaacatc caccccctga ccatcggcga gtgccccaag 960tacgtgaaga
gcaacaggct ggtgctggcc accggcctgc ggaacagccc ccagcgggag
1020cggcgggccg ccgcccgggg cctgttcggc gccatcgccg gcttcatcga
gggcggctgg 1080cagggcatgg tggacgggtg gtacggctac caccacagca
atgagcaggg cagcggctac 1140gccgccgaca aagagagcac ccagaaggcc
atcgacggcg tcaccaacaa ggtgaacagc 1200atcatcgaca agatgaacac
ccagttcgag gccgtgggcc gggagttcaa caacctggaa 1260cggcggatcg
agaacctgaa caagaaaatg gaagatggct tcctggacgt gtggacctac
1320aacgccgagc tgctggtgct gatggaaaac gagcggaccc tggacttcca
cgacagcaac 1380gtgaagaacc tgtacgacaa agtgcggctg cagctgcggg
acaacgccaa agagctgggc 1440aacggctgct tcgagttcta ccacaagtgc
gacaacgagt gcatggaaag cgtgcggaac 1500ggcacctacg actaccccca
gtacagcgag gaagcccggc tgaagcggga ggaaatcagc 1560ggcgtgaaac
tggaaagcat cggcatctac cagatcctga gcatctacag caccgtggcc
1620agcagcctgg ccctggccat catggtggcc ggcctgagcc tgtggatgtg
cagcaacggc 1680agcctgcagt gccggatctg catctag 17072568PRTArtificial
SequenceInfluenza H5N1 HA consensus sequence 2Met 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 Val 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 Ser 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 Ser Thr 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 Lys 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 Arg Ile Ala Thr Arg Ser Lys
Val Asn Gly Gln Ser 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 Thr 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 Ala Ala Ala 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 Ile 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 31466DNAArtificial
SequenceInfluenza H1N1&H5N1 NA consensus sequence 3ggtaccgaat
tcgccaccat ggactggacc tggatcctgt tcctggtggc cgctgccacc 60cgggtgcaca
gcatgaaccc caaccagaag atcatcacca tcggcagcat ctgcatggtg
120atcggcatcg tgagcctgat gctgcagatc ggcaacatga tcagcatctg
ggtgtcccac 180agcatccaga ccggcaacca gcaccaggcc gagcccatca
gcaacaccaa ctttctgacc 240gagaaggccg tggccagcgt gaccctggcc
ggcaacagca gcctgtgccc catcagcggc 300tgggccgtgt acagcaagga
caacagcatc cggatcggca gcaagggcga cgtgttcgtg 360atccgggagc
ccttcatcag ctgcagccac ctggaatgcc ggaccttctt cctgacccag
420ggggccctgc tgaacgacaa gcacagcaac ggcaccgtga aggacagaag
cccctaccgg 480accctgatga gctgccccgt gggcgaggcc cccagcccct
acaacagccg gttcgagagc 540gtggcctggt ccgccagcgc ctgccacgac
ggcaccagct ggctgaccat cggcatcagc 600ggccctgaca acggcgccgt
ggccgtgctg aagtacaacg gcatcatcac cgacaccatc 660aagagctggc
ggaacaacat cctgcggacc caggaaagcg agtgcgcctg cgtgaacggc
720agctgcttca ccgtgatgac cgacggcccc agcaacggcc aggccagcta
caagatcttc 780aagatggaaa agggcaaggt ggtgaagagc gtggagctgg
acgcccccaa ctaccactac 840gaggaatgca gctgctaccc cgacgccggc
gagatcacct gcgtgtgccg ggacaactgg 900cacggcagca accggccctg
ggtgtccttc aaccagaacc tggaatacca gatcggctac 960atctgcagcg
gcgtgttcgg cgacaacccc aggcccaacg atggcaccgg cagctgcggc
1020cctgtgagcg ccaacggcgc ctacggcgtg aagggcttca gcttcaagta
cggcaacggc 1080gtgtggatcg gccggaccaa gagcaccaac agcagatccg
gcttcgagat gatctgggac 1140cccaacggct ggaccgagac cgacagcagc
ttcagcgtga agcaggacat cgtggccatc 1200accgactggt ccggctacag
cggcagcttc gtgcagcacc ccgagctgac cggcctggac 1260tgcatccggc
cctgcttttg ggtggagctg atcagaggca ggcccaaaga gagcaccatc
1320tggaccagcg gcagcagcat cagcttttgc ggcgtgaaca gcgacaccgt
gagctggtcc 1380tggcccgacg gcgccgagct gcccttcacc atcgacaagt
acccctacga cgtgcccgac 1440tacgcctgat gagcggccgc gagctc
14664476PRTArtificial SequenceInfluenza H1N1&H5N1 NA consensus
sequence 4Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr
Arg Val 1 5 10 15 His Ser Met Asn Pro Asn Gln Lys Ile Ile Thr Ile
Gly Ser Ile Cys 20 25 30 Met Val Ile Gly Ile Val Ser Leu Met Leu
Gln Ile Gly Asn Met Ile 35 40 45 Ser Ile Trp Val Ser His Ser Ile
Gln Thr Gly Asn Gln His Gln Ala 50 55 60 Glu Pro Ile Ser Asn Thr
Asn Phe Leu Thr Glu Lys Ala Val Ala Ser 65 70 75 80 Val Thr Leu Ala
Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala 85 90 95 Val Tyr
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val 100 105 110
Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg 115
120 125 Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser
Asn 130 135 140 Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met
Ser Cys Pro 145 150 155 160 Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser
Arg Phe Glu Ser Val Ala 165 170 175 Trp Ser Ala Ser Ala Cys His Asp
Gly Thr Ser Trp Leu Thr Ile Gly 180 185 190 Ile Ser Gly Pro Asp Asn
Gly Ala Val Ala Val Leu Lys Tyr Asn Gly 195 200 205 Ile Ile Thr Asp
Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr 210 215 220 Gln Glu
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met 225 230 235
240 Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe Lys Met
245 250 255 Glu Lys Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro
Asn Tyr 260 265 270 His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly
Glu Ile Thr Cys 275 280 285 Val Cys Arg Asp Asn Trp His Gly Ser Asn
Arg Pro Trp Val Ser Phe 290 295 300 Asn Gln Asn Leu Glu Tyr Gln Ile
Gly Tyr Ile Cys Ser Gly Val Phe 305 310 315 320 Gly Asp Asn Pro Arg
Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val 325 330 335 Ser Ala Asn
Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly 340 345 350 Asn
Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly 355 360
365 Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser
370 375 380 Phe Ser Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser
Gly Tyr 385 390 395 400 Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr
Gly Leu Asp Cys Ile 405 410 415 Arg Pro Cys Phe Trp Val Glu Leu Ile
Arg Gly Arg Pro Lys Glu Ser 420 425 430 Thr Ile Trp Thr Ser Gly Ser
Ser Ile Ser Phe Cys Gly Val Asn Ser 435 440 445 Asp Thr Val Ser Trp
Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr 450 455 460 Ile Asp Lys
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 465 470 475 5875DNAArtificial
SequenceInfluenza H1N1&H5N1 M1 consensus sequence 5ggtaccggat
ccgccaccat ggactggacc tggattctgt tcctggtggc cgctgccacc 60cgggtgcaca
gcatgagcct gctgaccgag gtggagacct acgtgctgtc catcatcccc
120agcggccctc tgaaggccga gatcgcccag cggctggaag atgtgttcgc
cggcaagaac 180accgacctgg aagccctgat ggaatggctg aaaacccggc
ccatcctgag ccccctgacc 240aagggcatcc tgggcttcgt gttcaccctg
accgtgccca gcgagcgggg cctgcagcgg 300cggagattcg tgcagaacgc
cctgaacggc aacggcgacc ccaacaacat ggaccgggcc 360gtgaagctgt
acaagaagct gaagcgggag atcaccttcc acggcgccaa agaggtggcc
420ctgagctaca gcacaggcgc cctggccagc tgcatgggcc tgatctacaa
ccggatgggc 480accgtgacca ccgaggtggc cttcggcctg gtgtgcgcca
cctgcgagca gatcgccgac 540agccagcaca gatcccaccg gcagatggcc
accaccacca accccctgat ccggcacgag 600aaccggatgg tcctggcctc
caccaccgcc aaggccatgg aacagatggc cggcagcagc 660gagcaggccg
ccgaagccat ggaagtggcc agccaggcca ggcagatggt gcaggccatg
720cggaccatcg gcacccaccc cagcagcagc gccggactgc gggacgacct
gctggaaaac 780ctgcaggcct accagaaacg gatgggcgtg cagatgcagc
ggttcaagta cccctacgac 840gtgcccgact acgcctgatg agcggccgcg agctc
8756279PRTArtificial SequenceInfluenza H1N1&H5N1 M1 consensus
sequence 6Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr
Arg Val 1 5 10 15 His Ser Met Ser Leu Leu Thr Glu Val Glu Thr Tyr
Val Leu Ser Ile 20 25 30 Ile Pro Ser Gly Pro Leu Lys Ala Glu Ile
Ala Gln Arg Leu Glu Asp 35 40 45 Val Phe Ala Gly Lys Asn Thr Asp
Leu Glu Ala Leu Met Glu Trp Leu 50 55 60 Lys Thr Arg Pro Ile Leu
Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe 65 70 75 80 Val Phe Thr Leu
Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg 85 90 95 Phe Val
Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp 100 105 110
Arg Ala Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile Thr Phe His 115
120 125 Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala
Ser 130 135 140 Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr
Thr Glu Val 145 150 155 160 Ala Phe Gly Leu Val Cys Ala Thr Cys Glu
Gln Ile Ala Asp Ser Gln 165 170 175 His Arg Ser His Arg Gln Met Ala
Thr Thr Thr Asn Pro Leu Ile Arg 180 185 190 His Glu Asn Arg Met Val
Leu Ala Ser Thr Thr Ala Lys Ala Met Glu 195 200 205 Gln Met Ala Gly
Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala 210 215 220 Ser Gln
Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His 225 230 235
240 Pro Ser Ser Ser Ala Gly Leu Arg Asp Asp Leu Leu Glu Asn Leu Gln
245 250 255 Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys
Tyr Pro 260 265 270 Tyr Asp Val Pro Asp Tyr Ala 275
71700DNAArtificial SequenceInfluenza H5N1 M2E-NP consensus sequence
7ggtaccgaat tcgccaccat ggactggacc tggatcctgt tcctggtcgc tgccgccacc
60agggtgcaca gcagcctgct gaccgaggtg gagaccccca cccggaacga gtggggctgc
120cggtgcagcg acagcagcga ccggggcagg aagcggagaa gcgccagcca
gggcaccaag 180cggagctacg agcagatgga aacaggcggc gagcggcaga
acgccaccga gatccgggcc 240agcgtgggca gaatggtcgg cggcatcggc
cggttctaca tccagatgtg caccgagctg 300aagctgtccg actacgaggg
ccggctgatc cagaacagca tcaccatcga gcggatggtg 360ctgtccgcct
tcgacgagcg gcggaacaga tacctggaag agcaccccag cgccggcaag
420gaccccaaga aaaccggcgg acccatctac cggcggaggg acggcaagtg
ggtgcgggag 480ctgatcctgt acgacaaaga ggaaatccgg cggatctggc
ggcaggccaa caacggcgag 540gacgccacag ccggcctgac ccacctgatg
atctggcaca gcaacctgaa cgacgccacc 600taccagcgga caagggctct
ggtccggacc ggcatggacc cccggatgtg cagcctgatg 660cagggcagca
cactgcccag aagaagcgga gccgctggcg cagccgtgaa gggcgtgggc
720accatggtga tggaactgat ccggatgatc aagcggggca tcaacgaccg
gaatttttgg 780aggggcgaga acggcaggcg gacccggatc gcctacgagc
ggatgtgcaa catcctgaag 840ggcaagttcc agacagccgc ccagcgggcc
atgatggacc aggtccggga gagccggaac 900cccggcaacg ccgagatcga
ggacctgatc ttcctggcca gaagcgccct gatcctgcgg 960ggcagcgtgg
cccacaagag ctgcctgccc gcctgcgtgt acggactggc cgtggccagc
1020ggctacgact tcgagcggga gggctacagc ctggtcggca tcgacccctt
ccggctgctg 1080cagaactccc aggtgttcag cctgatccgg cccaacgaga
accccgccca caagtcccag 1140ctggtctgga tggcctgcca cagcgccgcc
ttcgaggatc tgagagtgag cagcttcatc 1200cggggcacca gagtggtgcc
caggggccag ctgtccacca ggggcgtgca gatcgccagc 1260aacgagaaca
tggaagccat ggacagcaac accctggaac tgcggagccg gtactgggcc
1320atccggacca gaagcggcgg caacaccaac cagcagcggg ccagcgccgg
acagatcagc 1380gtgcagccca ccttctccgt gcagcggaac ctgcccttcg
agagggccac catcatggcc 1440gccttcaccg gcaacaccga gggccggacc
agcgacatgc ggaccgagat catcaggatg 1500atggaaagcg ccaggcccga
ggacgtgagc ttccagggca ggggcgtgtt cgagctgtcc 1560gatgagaagg
ccaccaaccc catcgtgccc agcttcgaca tgaacaacga gggcagctac
1620ttcttcggcg acaacgccga ggaatacgac aactacccct acgacgtgcc
cgactacgcc 1680tgatgagcgg ccgcgagctc 17008554PRTArtificial
SequenceInfluenza H5N1 M2E-NP consensus sequence 8Met Asp Trp Thr
Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5
10 15 His Ser Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Glu
Trp 20 25 30 Gly Cys Arg Cys Ser Asp Ser Ser Asp Arg Gly Arg Lys
Arg Arg Ser 35 40 45 Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln
Met Glu Thr Gly Gly 50 55 60 Glu Arg Gln Asn Ala Thr Glu Ile Arg
Ala Ser Val Gly Arg Met Val 65 70 75 80 Gly Gly Ile Gly Arg Phe Tyr
Ile Gln Met Cys Thr Glu Leu Lys Leu 85 90 95 Ser Asp Tyr Glu Gly
Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu Arg 100 105 110 Met Val Leu
Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu Glu 115 120 125 His
Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile Tyr 130 135
140 Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp Lys
145 150 155 160 Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly
Glu Asp Ala 165 170 175 Thr Ala Gly Leu Thr His Leu Met Ile Trp His
Ser Asn Leu Asn Asp 180 185 190 Ala Thr Tyr Gln Arg Thr Arg Ala Leu
Val Arg Thr Gly Met Asp Pro 195 200 205 Arg Met Cys Ser Leu Met Gln
Gly Ser Thr Leu Pro Arg Arg Ser Gly 210 215 220 Ala Ala Gly Ala Ala
Val Lys Gly Val Gly Thr Met Val Met Glu Leu 225 230 235 240 Ile Arg
Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg Gly 245 250 255
Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn Ile 260
265 270 Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp
Gln 275 280 285 Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu
Asp Leu Ile 290 295 300 Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly
Ser Val Ala His Lys 305 310 315 320 Ser Cys Leu Pro Ala Cys Val Tyr
Gly Leu Ala Val Ala Ser Gly Tyr 325 330 335 Asp Phe Glu Arg Glu Gly
Tyr Ser Leu Val Gly Ile Asp Pro Phe Arg 340 345 350 Leu Leu Gln Asn
Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu Asn 355 360 365 Pro Ala
His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala Ala 370 375 380
Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Val Val 385
390 395 400 Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser
Asn Glu 405 410 415 Asn Met Glu Ala Met Asp Ser Asn Thr Leu Glu Leu
Arg Ser Arg Tyr 420 425 430 Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn
Thr Asn Gln Gln Arg Ala 435 440 445 Ser Ala Gly Gln Ile Ser Val Gln
Pro Thr Phe Ser Val Gln Arg Asn 450 455 460 Leu Pro Phe Glu Arg Ala
Thr Ile Met Ala Ala Phe Thr Gly Asn Thr 465 470 475 480 Glu Gly Arg
Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met Glu 485 490 495 Ser
Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe Glu 500 505
510 Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp Met
515 520 525 Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu
Tyr Asp 530 535 540 Asn Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 545 550
91728DNAArtificial SequenceInfluenza H1 consensus sequence
9ggtaccaagc ttgccaccat gaaggtgaaa ctgctggtgc tgctgtgcac cttcaccgcc
60acctacgccg acaccatctg catcggctac cacgccaaca acagcaccga caccgtggat
120accgtgctgg aaaagaacgt gaccgtgacc cacagcgtga acctgctgga
agatagccac 180aacggcaagc tgtgcctgct gaaaggcatc gcccccctgc
agctgggcaa ctgcagcgtg 240gccggctgga tcctgggcaa ccccgagtgc
gagctgctga tttccaaaga aagctggtcc 300tacatcgtgg agacccccaa
ccccgagaac ggcacctgct accccggcta cttcgccgac 360tacgaggaac
tgcgggagca gctgtccagc gtgagcagct tcgagcggtt cgagatcttc
420cccaaagaga gcagctggcc caaccacacc gtgaccggcg tgagcgccag
ctgctcccac 480aatggcaaga gcagcttcta ccggaacctg ctgtggctga
ccggcaagaa cggcctgtac 540cccaacctga gcaagagcta cgccaataac
aaagaaaagg aagtgctggt gctgtggggc 600gtgcaccacc cccccaacat
cggcgaccag cgggccctgt accacaccga gaacgcctac 660gtgagcgtgg
tgtccagcca ctacagccgg cggttcaccc ccgagatcgc caagcggccc
720aaagtgcggg accaggaagg ccggatcaac tactactgga ccctgctgga
acccggcgac 780accatcatct tcgaggccaa cggcaacctg atcgccccca
gatacgcctt cgccctgagc 840cggggcttcg gcagcggcat catcaccagc
aacgccccca tggacgagtg cgacgccaag 900tgccagaccc ctcagggagc
tattaacagc agcctgccct tccagaacgt gcaccccgtg 960accatcggcg
agtgccccaa gtacgtgcgg agcgccaagc tgcggatggt gaccggcctg
1020cggaacatcc ccagcatcca gagcaggggc ctgttcggcg ccatcgccgg
cttcatcgag 1080ggcggctgga ccggcatggt ggacgggtgg tacggctacc
accaccagaa cgagcagggc 1140agcggctacg ccgccgacca gaagagcacc
cagaacgcca tcaacggcat caccaacaag 1200gtgaacagcg tgatcgagaa
gatgaacacc cagttcaccg ccgtgggcaa agagttcaac 1260aagctggaac
ggcggatgga aaacctgaac aagaaggtgg acgacggctt cctggacatc
1320tggacctaca acgccgagct gctggtgctg ctggaaaacg agcggaccct
ggacttccac 1380gacagcaacg tgaagaacct gtacgagaag gtgaaaagcc
agctgaagaa caacgccaaa 1440gagatcggca acggctgctt cgagttctac
cacaagtgca acgacgagtg catggaaagc 1500gtgaagaatg gcacctacga
ctaccccaag tacagcgagg aaagcaagct gaaccgggag 1560aagatcgacg
gcgtgaagct ggaaagcatg ggcgtgtacc agatcctggc catctacagc
1620accgtcgctt ccagcctcgt cctgctcgtg tccctgggcg ccatctcctt
ttggatgtgc 1680agcaacggca gcctgcagtg ccggatctgc atctgatgac tcgagctc
172810565PRTArtificial SequenceInfluenza H1 consensus sequence
10Met Lys Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Leu Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Asn Cys Ser
Val Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Leu Leu
Ile Ser Lys Glu Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 100 105 110 Ala Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 130 135
140 Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu
Tyr Pro Asn 165 170 175 Leu Ser Lys Ser Tyr Ala Asn Asn Lys Glu Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Val His His Pro Pro Asn Ile
Gly Asp Gln Arg Ala Leu Tyr 195 200 205 His Thr Glu Asn Ala Tyr Val
Ser Val Val Ser Ser His Tyr Ser Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 225 230 235 240 Gly Arg
Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Pro
Met 275 280 285 Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala
Ile Asn Ser 290 295 300 Ser Leu Pro Phe Gln Asn Val His Pro Val Thr
Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Ile Pro Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly
Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu 385
390 395 400 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn
Lys Leu 405 410 415 Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe
Tyr His Lys Cys Asn Asp Glu Cys Met Glu Ser Val Lys 485 490 495 Asn
Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505
510 Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gln
515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile Cys Ile 565
111731DNAArtificial SequenceInfluenza H3 consensus sequence
11ggtaccaagc ttgccaccat gaaaaccatc atcgccctga gctacatcct gtgcctggtg
60ttcgcccaga agctgcccgg caacgacaac agcaccgcca ccctgtgtct gggccaccac
120gccgtgccca acggcaccat cgtgaaaaca atcaccaacg accagatcga
ggtgaccaac 180gccaccgagc tggtgcagag cagcagcacc ggcggcatct
gcgacagccc ccaccagatc 240ctggacggcg agaactgcac cctgatcgac
gccctgctgg gcgaccctca gtgcgacggc 300ttccagaaca aaaagtggga
cctgttcgtg gagcggagca aggcctacag caactgctac 360ccctacgacg
tgcccgacta cgccagcctg cggagcctgg tggccagcag cggcaccctg
420gaattcaaca acgagagctt caactggacc ggcgtgaccc agaacggcac
cagcagcgcc 480tgcaagcggc ggagcaacaa cagcttcttt tccagactga
actggctgac ccacctgaag 540ttcaagtacc ccgccctgaa cgtgaccatg
cccaacaatg agaagttcga caagctgtac 600atctggggcg tgcaccaccc
cggcaccgac aatgaccaga tcagcctgta cgcccaggcc 660agcggccgga
tcaccgtgag caccaagcgg agccagcaga ccgtgatccc caacatcggc
720agccggccca gagtgagaga catccccagc cggatcagca tctactggac
aatcgtgaag 780cccggcgaca tcctgctgat caactccacc ggcaacctga
tcgcccccag gggctacttc 840aagatcagaa gcggcaagag cagcatcatg
cggagcgacg cccccatcgg caagtgcaac 900agcgagtgca tcacccccaa
tggcagcatc cccaacgaca agcccttcca gaacgtgaac 960cggatcacct
acggcgcctg ccccagatac gtgaagcaga acaccctgaa gctggccacc
1020ggcatgcgga acgtgcccga gaagcagacc cggggcatct tcggcgccat
cgccggcttc 1080atcgagaacg gctgggaggg catggtggac gggtggtacg
gcttccggca ccagaactcc 1140gagggcatcg gccaggccgc cgacctgaag
agcacccagg ccgccatcaa ccagatcaac 1200ggcaagctga accggctgat
cggcaagacc aacgagaagt tccaccagat cgaaaaagaa 1260ttcagcgagg
tggagggccg gatccaggac ctggaaaagt acgtggagga caccaagatc
1320gacctgtgga gctacaacgc cgagctgctg gtcgccctgg aaaaccagca
caccatcgac 1380ctgaccgaca gcgagatgaa caagctgttc gagcggacca
agaagcagct gcgggagaac 1440gccgaggaca tgggcaacgg ctgctttaag
atctaccaca agtgcgacaa cgcctgcatc 1500ggcagcatcc ggaacggcac
ctacgaccac gacgtgtacc gggacgaggc cctgaacaac 1560cggttccaga
tcaagggcgt ggagctgaag agcggctaca aggactggat cctgtggatc
1620agcttcgcca tcagctgctt tctgctgtgc gtggccctgc tgggattcat
catgtgggcc 1680tgccagaagg gcaacatccg ctgcaacatc tgcatctgat
gactcgagct c 173112566PRTArtificial SequenceInfluenza H3 consensus
sequence 12Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val
Phe Ala 1 5 10 15 Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr
Leu Cys Leu Gly 20 25 30 His His Ala Val Pro Asn Gly Thr Ile 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 Gly Ile Cys Asp Ser
Pro His Gln Ile Leu Asp Gly Glu Asn Cys 65 70 75 80 Thr Leu Ile Asp
Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe Gln 85 90 95 Asn Lys
Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn 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 Asn Glu Ser Phe Asn Trp
Thr 130 135 140 Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg
Arg Ser Asn 145 150 155 160 Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu
Thr His Leu Lys Phe 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 Gly Thr Asp Asn Asp Gln Ile 195 200 205 Ser Leu Tyr Ala
Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys Arg 210 215 220 Ser Gln
Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Arg Val Arg 225 230 235
240 Asp Ile Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly
245 250 255 Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro
Arg Gly 260 265 270 Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met
Arg Ser Asp Ala 275 280 285 Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile
Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro Phe Gln Asn
Val Asn Arg Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Arg Tyr Val
Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg Asn Val
Pro Glu Lys Gln Thr 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 Gln Asn Ser Glu Gly Ile 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 Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile
Glu Lys Glu Phe Ser 405 410 415 Glu 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
Lys 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 Asp Val Tyr Arg Asp Glu Ala
Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser
Gly Tyr Lys 515 520 525 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser
Cys Phe Leu Leu Cys 530 535 540 Val Ala 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 131791DNAArtificial SequenceInfluenza H5 consensus sequence
13atggactgga cctggatcct gttcctggtg gccgctgcca cccgggtgca cagcatggaa
60aagatcgtgc tgctgttcgc catcgtgagc ctggtgaaga gcgaccagat ctgcatcggc
120taccacgcca acaacagcac cgagcaggtg gacaccatca tggaaaaaaa
cgtgaccgtg 180acccacgccc aggacatcct ggaaaagacc cacaacggca
agctgtgcga cctggacggc 240gtgaagcccc tgatcctgcg ggactgcagc
gtggccggct ggctgctggg caaccccatg 300tgcgacgagt tcatcaacgt
gcccgagtgg agctacatcg tggagaaggc caaccccgtg 360aacgacctgt
gctaccccgg cgacttcaac gactacgagg aactgaagca cctgctgtcc
420cggatcaacc acttcgagaa gatccagatc atccccaaga gcagctggtc
cagccacgag 480gccagcctgg gcgtgagcag cgcctgccca taccagggca
agtccagctt cttccggaac 540gtggtgtggc tgatcaagaa gaacagcacc
taccccacca tcaagcggag ctacaacaac 600accaaccagg aagatctgct
ggtcctgtgg ggcatccacc accccaacga cgccgccgag 660cagaccaagc
tgtaccagaa ccccaccacc tacatcagcg tgggcaccag caccctgaac
720cagcggctgg tgccccggat cgccacccgg tccaaggtga acggccagag
cggccggatg 780gaattcttct ggaccatcct gaagcccaac gatgccatca
acttcgagag caacggcaac 840ttcatcgccc ccgagtacgc ctacaagatc
gtgaagaagg gcgacagcac catcatgaag 900agcgagctgg aatacggcaa
ctgcaacacc aagtgccaga cccccatggg cgccatcaac 960agcagcatgc
ccttccacaa catccacccc ctgaccatcg gcgagtgccc caagtacgtg
1020aagagcaaca ggctggtgct ggccaccggc ctgcggaaca gcccccagcg
ggagcggcgg 1080aggaagaagc ggggcctgtt cggcgccatc gccggcttca
tcgagggcgg ctggcagggc 1140atggtggacg ggtggtacgg ctaccaccac
agcaatgagc agggcagcgg ctacgccgcc 1200gacaaagaga gcacccagaa
ggccatcgac ggcgtcacca acaaggtgaa cagcatcatc 1260gacaagatga
acacccagtt cgaggccgtg ggccgggagt tcaacaacct ggaacggcgg
1320atcgagaacc tgaacaagaa aatggaagat ggcttcctgg acgtgtggac
ctacaacgcc 1380gagctgctgg tgctgatgga aaacgagcgg accctggact
tccacgacag caacgtgaag 1440aacctgtacg acaaagtgcg gctgcagctg
cgggacaacg ccaaagagct gggcaacggc 1500tgcttcgagt tctaccacaa
gtgcgacaac gagtgcatgg aaagcgtgcg gaacggcacc 1560tacgactacc
cccagtacag cgaggaagcc cggctgaagc gggaggaaat cagcggcgtg
1620aaactggaaa gcatcggcat ctaccagatc ctgagcatct acagcaccgt
ggccagcagc 1680ctggccctgg ccatcatggt ggccggcctg agcctgtgga
tgtgcagcaa cggcagcctg 1740cagtgccgga tctgcatcta cccctacgac
gtgcccgact acgcctgatg a 179114595PRTArtificial SequenceInfluenza H5
consensus sequence 14Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala
Ala Ala Thr Arg Val 1 5 10 15 His Ser Met Glu Lys Ile Val Leu Leu
Phe Ala Ile Val Ser Leu Val 20 25 30 Lys Ser Asp Gln Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Glu 35 40 45 Gln Val Asp Thr Ile
Met Glu Lys Asn Val Thr Val Thr His Ala Gln 50 55 60 Asp Ile Leu
Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly 65 70 75 80 Val
Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu 85 90
95 Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr
100 105 110 Ile Val Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro
Gly Asp 115 120 125 Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser
Arg Ile Asn His 130 135 140 Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser
Ser Trp Ser Ser His Glu 145 150 155 160 Ala Ser Leu Gly Val Ser Ser
Ala Cys Pro Tyr Gln Gly Lys Ser Ser 165 170 175 Phe Phe Arg Asn Val
Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro 180 185 190 Thr Ile Lys
Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val 195 200 205 Leu
Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu 210 215
220 Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
225 230 235 240 Gln Arg Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val
Asn Gly Gln 245 250 255 Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu
Lys Pro Asn Asp Ala 260 265 270 Ile Asn Phe Glu Ser Asn Gly Asn Phe
Ile Ala Pro Glu Tyr Ala Tyr 275 280 285 Lys Ile Val Lys Lys Gly Asp
Ser Thr Ile Met Lys Ser Glu Leu Glu 290 295 300 Tyr Gly Asn Cys Asn
Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn 305 310 315 320 Ser Ser
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys 325 330 335
Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg 340
345 350 Asn Ser Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe
Gly 355 360 365 Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met
Val Asp Gly 370 375 380 Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly
Ser Gly Tyr Ala Ala 385 390 395 400 Asp Lys Glu Ser Thr Gln Lys Ala
Ile Asp Gly Val Thr Asn Lys Val 405 410 415 Asn Ser Ile Ile Asp Lys
Met Asn Thr Gln Phe Glu Ala Val Gly Arg 420 425 430 Glu Phe Asn Asn
Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met 435 440 445 Glu Asp
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val 450 455 460
Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys 465
470 475 480 Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala
Lys Glu 485 490 495 Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn Glu Cys 500 505 510 Met Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr Ser Glu 515 520 525 Glu Ala Arg Leu Lys Arg Glu Glu
Ile Ser Gly Val Lys Leu Glu Ser 530 535 540 Ile Gly Ile Tyr Gln Ile
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser 545 550 555 560 Leu Ala Leu
Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser 565 570 575 Asn
Gly Ser Leu Gln Cys Arg Ile Cys Ile Tyr Pro Tyr Asp Val Pro 580 585
590 Asp Tyr Ala 595 154733DNAArtificial SequenceDNA plasmid having
encoding sequence for influenza consensus H5N1 HA 15gctgcttcgc
gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca
attacggggt cattagttca tagcccatat atggagttcc gcgttacata
120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc
cattgacgtc aatgggtgga 240gtatttacgg taaactgccc acttggcagt
acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg
gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt
cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac
caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac
gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc
tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg
actcactata gggagaccca agctggctag cgtttaaact taagcttggt
720accgccacca tggactggac ctggatcctg ttcctggtgg ccgctgccac
ccgggtgcac 780agcatggaaa agatcgtgct gctgttcgcc atcgtgagcc
tggtgaagag cgaccagatc 840tgcatcggct accacgccaa caacagcacc
gagcaggtgg acaccatcat ggaaaaaaac 900gtgaccgtga cccacgccca
ggacatcctg gaaaagaccc acaacggcaa gctgtgcgac 960ctggacggcg
tgaagcccct gatcctgcgg gactgcagcg tggccggctg gctgctgggc
1020aaccccatgt gcgacgagtt catcaacgtg cccgagtgga gctacatcgt
ggagaaggcc 1080aaccccgtga acgacctgtg ctaccccggc gacttcaacg
actacgagga actgaagcac 1140ctgctgtccc ggatcaacca cttcgagaag
atccagatca tccccaagag cagctggtcc 1200agccacgagg ccagcctggg
cgtgagcagc gcctgcccat accagggcaa gtccagcttc 1260ttccggaacg
tggtgtggct gatcaagaag aacagcacct accccaccat caagcggagc
1320tacaacaaca ccaaccagga agatctgctg gtcctgtggg gcatccacca
ccccaacgac 1380gccgccgagc agaccaagct gtaccagaac cccaccacct
acatcagcgt gggcaccagc 1440accctgaacc agcggctggt gccccggatc
gccacccggt ccaaggtgaa cggccagagc 1500ggccggatgg aattcttctg
gaccatcctg aagcccaacg atgccatcaa cttcgagagc 1560aacggcaact
tcatcgcccc cgagtacgcc tacaagatcg tgaagaaggg cgacagcacc
1620atcatgaaga gcgagctgga atacggcaac tgcaacacca agtgccagac
ccccatgggc 1680gccatcaaca gcagcatgcc cttccacaac atccaccccc
tgaccatcgg cgagtgcccc 1740aagtacgtga agagcaacag gctggtgctg
gccaccggcc tgcggaacag cccccagcgg 1800gagcggcgga ggaagaagcg
gggcctgttc ggcgccatcg ccggcttcat cgagggcggc 1860tggcagggca
tggtggacgg gtggtacggc taccaccaca gcaatgagca gggcagcggc
1920tacgccgccg acaaagagag cacccagaag gccatcgacg gcgtcaccaa
caaggtgaac 1980agcatcatcg acaagatgaa cacccagttc gaggccgtgg
gccgggagtt caacaacctg 2040gaacggcgga tcgagaacct gaacaagaaa
atggaagatg gcttcctgga cgtgtggacc 2100tacaacgccg agctgctggt
gctgatggaa aacgagcgga ccctggactt ccacgacagc 2160aacgtgaaga
acctgtacga caaagtgcgg ctgcagctgc gggacaacgc caaagagctg
2220ggcaacggct gcttcgagtt ctaccacaag tgcgacaacg agtgcatgga
aagcgtgcgg 2280aacggcacct acgactaccc ccagtacagc gaggaagccc
ggctgaagcg ggaggaaatc 2340agcggcgtga aactggaaag catcggcatc
taccagatcc tgagcatcta cagcaccgtg 2400gccagcagcc tggccctggc
catcatggtg gccggcctga gcctgtggat gtgcagcaac 2460ggcagcctgc
agtgccggat ctgcatctac ccctacgacg tgcccgacta cgcctgatga
2520ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
ttctagttgc 2580cagccatctg ttgtttgccc ctcccccgtg ccttccttga
ccctggaagg tgccactccc 2640actgtccttt cctaataaaa tgaggaaatt
gcatcgcatt gtctgagtag gtgtcattct 2700attctggggg gtggggtggg
gcaggacagc aagggggagg attgggaaga caatagcagg 2760catgctgggg
atgcggtggg ctctatggct tctactgggc ggttttatgg acagcaagcg
2820aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc
aaagtaaact 2880ggatggcttt cttgccgcca aggatctgat ggcgcagggg
atcaagctct gatcaagaga 2940caggatgagg atcgtttcgc atgattgaac
aagatggatt gcacgcaggt tctccggccg 3000cttgggtgga gaggctattc
ggctatgact gggcacaaca gacaatcggc tgctctgatg 3060ccgccgtgtt
ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
3120ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
gccacgacgg 3180gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
gggaagggac tggctgctat 3240tgggcgaagt gccggggcag gatctcctgt
catctcacct tgctcctgcc gagaaagtat 3300ccatcatggc tgatgcaatg
cggcggctgc atacgcttga tccggctacc tgcccattcg 3360accaccaagc
gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
3420atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
ttcgccaggc 3480tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
ccatggcgat gcctgcttgc 3540cgaatatcat ggtggaaaat ggccgctttt
ctggattcat cgactgtggc cggctgggtg 3600tggcggaccg ctatcaggac
atagcgttgg ctacccgtga tattgctgaa gagcttggcg 3660gcgaatgggc
tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
3720tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac
aatttcctga 3780tgcggtattt tctccttacg catctgtgcg gtatttcaca
ccgcatcagg tggcactttt 3840cggggaaatg tgcgcggaac ccctatttgt
ttatttttct aaatacattc aaatatgtat 3900ccgctcatga gacaataacc
ctgataaatg cttcaataat agcacgtgct aaaacttcat 3960ttttaattta
aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
4020taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
aggatcttct 4080tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
caaaaaaacc accgctacca 4140gcggtggttt gtttgccgga tcaagagcta
ccaactcttt ttccgaaggt aactggcttc 4200agcagagcgc agataccaaa
tactgttctt ctagtgtagc cgtagttagg ccaccacttc 4260aagaactctg
tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
4320gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
accggataag 4380gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
ccagcttgga gcgaacgacc 4440tacaccgaac tgagatacct acagcgtgag
ctatgagaaa gcgccacgct tcccgaaggg 4500agaaaggcgg acaggtatcc
ggtaagcggc agggtcggaa caggagagcg cacgagggag 4560cttccagggg
gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
4620gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
cgccagcaac 4680gcggcctttt tacggttcct ggccttttgc tggccttttg
ctcacatgtt ctt 4733164418DNAArtificial SequenceDNA plasmid having
encoding sequence for influenza consensus NA 16gctgcttcgc
gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca
attacggggt cattagttca tagcccatat atggagttcc gcgttacata
120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc
cattgacgtc aatgggtgga 240gtatttacgg taaactgccc acttggcagt
acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg
gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt
cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac
caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac
gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc
tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg
actcactata gggagaccca agctggctag cgtttaaact taagcttggt
720accgagctcg gatccactag tccagtgtgg tggaattcgc caccatggac
tggacctgga 780tcctgttcct ggtggccgct gccacccggg tgcacagcat
gaaccccaac cagaagatca 840tcaccatcgg cagcatctgc atggtgatcg
gcatcgtgag cctgatgctg cagatcggca 900acatgatcag catctgggtg
tcccacagca tccagaccgg caaccagcac caggccgagc 960ccatcagcaa
caccaacttt ctgaccgaga aggccgtggc cagcgtgacc ctggccggca
1020acagcagcct gtgccccatc agcggctggg ccgtgtacag caaggacaac
agcatccgga 1080tcggcagcaa gggcgacgtg ttcgtgatcc gggagccctt
catcagctgc agccacctgg 1140aatgccggac cttcttcctg acccaggggg
ccctgctgaa cgacaagcac agcaacggca 1200ccgtgaagga cagaagcccc
taccggaccc tgatgagctg ccccgtgggc gaggccccca 1260gcccctacaa
cagccggttc gagagcgtgg cctggtccgc cagcgcctgc cacgacggca
1320ccagctggct gaccatcggc atcagcggcc ctgacaacgg cgccgtggcc
gtgctgaagt 1380acaacggcat catcaccgac accatcaaga gctggcggaa
caacatcctg cggacccagg 1440aaagcgagtg cgcctgcgtg aacggcagct
gcttcaccgt gatgaccgac ggccccagca 1500acggccaggc cagctacaag
atcttcaaga tggaaaaggg caaggtggtg aagagcgtgg 1560agctggacgc
ccccaactac cactacgagg aatgcagctg ctaccccgac gccggcgaga
1620tcacctgcgt gtgccgggac aactggcacg gcagcaaccg gccctgggtg
tccttcaacc 1680agaacctgga ataccagatc ggctacatct gcagcggcgt
gttcggcgac aaccccaggc 1740ccaacgatgg caccggcagc tgcggccctg
tgagcgccaa cggcgcctac ggcgtgaagg 1800gcttcagctt caagtacggc
aacggcgtgt ggatcggccg gaccaagagc accaacagca 1860gatccggctt
cgagatgatc tgggacccca acggctggac cgagaccgac agcagcttca
1920gcgtgaagca ggacatcgtg gccatcaccg actggtccgg ctacagcggc
agcttcgtgc 1980agcaccccga gctgaccggc ctggactgca tccggccctg
cttttgggtg gagctgatca 2040gaggcaggcc caaagagagc accatctgga
ccagcggcag cagcatcagc ttttgcggcg 2100tgaacagcga caccgtgagc
tggtcctggc ccgacggcgc cgagctgccc ttcaccatcg 2160acaagtaccc
ctacgacgtg cccgactacg cctgatgagc ggccgctcga gtctagaggg
2220cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
atctgttgtt 2280tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
ctcccactgt cctttcctaa 2340taaaatgagg aaattgcatc gcattgtctg
agtaggtgtc attctattct ggggggtggg 2400gtggggcagg acagcaaggg
ggaggattgg gaagacaata gcaggcatgc tggggatgcg 2460gtgggctcta
tggcttctac tgggcggttt tatggacagc aagcgaaccg gaattgccag
2520ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg
gctttcttgc 2580cgccaaggat ctgatggcgc aggggatcaa gctctgatca
agagacagga tgaggatcgt 2640ttcgcatgat tgaacaagat ggattgcacg
caggttctcc ggccgcttgg gtggagaggc 2700tattcggcta tgactgggca
caacagacaa tcggctgctc tgatgccgcc gtgttccggc 2760tgtcagcgca
ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
2820aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
ccttgcgcag 2880ctgtgctcga cgttgtcact gaagcgggaa gggactggct
gctattgggc gaagtgccgg 2940ggcaggatct cctgtcatct caccttgctc
ctgccgagaa agtatccatc atggctgatg 3000caatgcggcg gctgcatacg
cttgatccgg ctacctgccc attcgaccac caagcgaaac 3060atcgcatcga
gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
3120acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
gcgagcatgc 3180ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
cttgccgaat atcatggtgg 3240aaaatggccg cttttctgga ttcatcgact
gtggccggct gggtgtggcg gaccgctatc 3300aggacatagc gttggctacc
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 3360gcttcctcgt
gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
3420ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg
tattttctcc 3480ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
cttttcgggg aaatgtgcgc 3540ggaaccccta tttgtttatt tttctaaata
cattcaaata tgtatccgct catgagacaa 3600taaccctgat aaatgcttca
ataatagcac gtgctaaaac ttcattttta atttaaaagg 3660atctaggtga
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
3720ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
tccttttttt 3780ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
taccagcggt ggtttgtttg 3840ccggatcaag agctaccaac tctttttccg
aaggtaactg gcttcagcag agcgcagata 3900ccaaatactg ttcttctagt
gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3960ccgcctacat
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
4020tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
gcggtcgggc 4080tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
cgacctacac cgaactgaga 4140tacctacagc gtgagctatg agaaagcgcc
acgcttcccg aagggagaaa ggcggacagg 4200tatccggtaa gcggcagggt
cggaacagga gagcgcacga gggagcttcc agggggaaac 4260gcctggtatc
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
4320tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
ctttttacgg 4380ttcctggcct tttgctggcc ttttgctcac atgttctt
4418174652DNAArtificial SequenceDNA plasmid having encoding
sequence for influenza consensus M2e-NP 17gctgcttcgc gatgtacggg
ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca attacggggt
cattagttca tagcccatat atggagttcc gcgttacata 120acttacggta
aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
180aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
aatgggtgga 240gtatttacgg taaactgccc acttggcagt acatcaagtg
tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg gtaaatggcc
cgcctggcat tatgcccagt acatgacctt 360atgggacttt cctacttggc
agtacatcta cgtattagtc atcgctatta ccatggtgat 420gcggttttgg
cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
480tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
gggactttcc 540aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
ggtaggcgtg tacggtggga 600ggtctatata agcagagctc tctggctaac
tagagaaccc actgcttact ggcttatcga 660aattaatacg actcactata
gggagaccca agctggctag cgtttaaact taagcttggt 720accgagctcg
gatccactag tccagtgtgg tggaattcgc caccatggac tggacctgga
780tcctgttcct ggtcgctgcc gccaccaggg tgcacagcag cctgctgacc
gaggtggaga 840cccccacccg gaacgagtgg ggctgccggt gcagcgacag
cagcgaccgg ggcaggaagc 900ggagaagcgc cagccagggc accaagcgga
gctacgagca gatggaaaca ggcggcgagc 960ggcagaacgc caccgagatc
cgggccagcg tgggcagaat ggtcggcggc atcggccggt 1020tctacatcca
gatgtgcacc gagctgaagc tgtccgacta cgagggccgg ctgatccaga
1080acagcatcac catcgagcgg atggtgctgt ccgccttcga cgagcggcgg
aacagatacc 1140tggaagagca ccccagcgcc ggcaaggacc ccaagaaaac
cggcggaccc atctaccggc 1200ggagggacgg caagtgggtg cgggagctga
tcctgtacga caaagaggaa atccggcgga 1260tctggcggca ggccaacaac
ggcgaggacg ccacagccgg cctgacccac ctgatgatct 1320ggcacagcaa
cctgaacgac gccacctacc agcggacaag ggctctggtc cggaccggca
1380tggacccccg gatgtgcagc ctgatgcagg gcagcacact gcccagaaga
agcggagccg 1440ctggcgcagc cgtgaagggc gtgggcacca tggtgatgga
actgatccgg atgatcaagc 1500ggggcatcaa cgaccggaat ttttggaggg
gcgagaacgg caggcggacc cggatcgcct 1560acgagcggat gtgcaacatc
ctgaagggca agttccagac agccgcccag cgggccatga 1620tggaccaggt
ccgggagagc cggaaccccg gcaacgccga gatcgaggac ctgatcttcc
1680tggccagaag cgccctgatc ctgcggggca gcgtggccca caagagctgc
ctgcccgcct 1740gcgtgtacgg actggccgtg gccagcggct acgacttcga
gcgggagggc tacagcctgg 1800tcggcatcga ccccttccgg ctgctgcaga
actcccaggt gttcagcctg atccggccca 1860acgagaaccc cgcccacaag
tcccagctgg tctggatggc ctgccacagc gccgccttcg 1920aggatctgag
agtgagcagc ttcatccggg gcaccagagt ggtgcccagg ggccagctgt
1980ccaccagggg cgtgcagatc gccagcaacg agaacatgga agccatggac
agcaacaccc 2040tggaactgcg gagccggtac tgggccatcc ggaccagaag
cggcggcaac accaaccagc 2100agcgggccag cgccggacag atcagcgtgc
agcccacctt ctccgtgcag cggaacctgc 2160ccttcgagag ggccaccatc
atggccgcct tcaccggcaa caccgagggc cggaccagcg 2220acatgcggac
cgagatcatc aggatgatgg aaagcgccag gcccgaggac gtgagcttcc
2280agggcagggg cgtgttcgag ctgtccgatg agaaggccac caaccccatc
gtgcccagct 2340tcgacatgaa caacgagggc agctacttct tcggcgacaa
cgccgaggaa tacgacaact 2400acccctacga cgtgcccgac tacgcctgat
gagcggccgc tcgagtctag agggcccgtt 2460taaacccgct gatcagcctc
gactgtgcct tctagttgcc agccatctgt tgtttgcccc 2520tcccccgtgc
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
2580gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
tggggtgggg 2640caggacagca agggggagga ttgggaagac aatagcaggc
atgctgggga tgcggtgggc 2700tctatggctt ctactgggcg gttttatgga
cagcaagcga accggaattg ccagctgggg 2760cgccctctgg taaggttggg
aagccctgca aagtaaactg gatggctttc ttgccgccaa 2820ggatctgatg
gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca
2880tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
aggctattcg 2940gctatgactg ggcacaacag acaatcggct gctctgatgc
cgccgtgttc cggctgtcag 3000cgcaggggcg cccggttctt tttgtcaaga
ccgacctgtc cggtgccctg aatgaactgc 3060aagacgaggc agcgcggcta
tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc 3120tcgacgttgt
cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
3180atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
gatgcaatgc 3240ggcggctgca tacgcttgat ccggctacct gcccattcga
ccaccaagcg aaacatcgca 3300tcgagcgagc acgtactcgg atggaagccg
gtcttgtcga tcaggatgat ctggacgaag 3360agcatcaggg gctcgcgcca
gccgaactgt tcgccaggct caaggcgagc atgcccgacg 3420gcgaggatct
cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
3480gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
tatcaggaca 3540tagcgttggc tacccgtgat attgctgaag agcttggcgg
cgaatgggct gaccgcttcc 3600tcgtgcttta cggtatcgcc gctcccgatt
cgcagcgcat cgccttctat cgccttcttg 3660acgagttctt ctgaattatt
aacgcttaca atttcctgat gcggtatttt ctccttacgc 3720atctgtgcgg
tatttcacac cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc
3780cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
acaataaccc 3840tgataaatgc ttcaataata gcacgtgcta aaacttcatt
tttaatttaa aaggatctag 3900gtgaagatcc tttttgataa tctcatgacc
aaaatccctt aacgtgagtt ttcgttccac 3960tgagcgtcag accccgtaga
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 4020gtaatctgct
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
4080caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
gataccaaat 4140actgttcttc tagtgtagcc gtagttaggc caccacttca
agaactctgt agcaccgcct 4200acatacctcg ctctgctaat cctgttacca
gtggctgctg ccagtggcga taagtcgtgt 4260cttaccgggt tggactcaag
acgatagtta ccggataagg cgcagcggtc gggctgaacg 4320gggggttcgt
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
4380cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
caggtatccg 4440gtaagcggca gggtcggaac aggagagcgc acgagggagc
ttccaggggg aaacgcctgg 4500tatctttata gtcctgtcgg gtttcgccac
ctctgacttg agcgtcgatt tttgtgatgc 4560tcgtcagggg ggcggagcct
atggaaaaac gccagcaacg cggccttttt acggttcctg 4620gccttttgct
ggccttttgc tcacatgttc tt 4652
* * * * *
References