lll lll D IID lll ll DIII DII DD lll DIII D ll Dl ll

Transcription

lll lll D IID lll ll DIII DII DD lll DIII D ll Dl ll
USOO8 133 723B2
(12)
(54)
United States Patent
(10)
Draghia-Akli et al.
(45)
VACCINES AGAINST MULTIPLE SUBTYPES
OF INFLUENZA VIRUS
(75) Inventors: Ruxandra Draghia-Akli, Brussels (BE);
David B Weiner, Merion, PA (US); Jian
Yan, Havertown, PA (US); Dominick J
Laddy, Germantown, MD (US)
(73) Assignee: The Trustees of The University of
Pennsylvania, Philadelphia, PA (US)
(*) Notice:
Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 0 days.
(21)
Appl. No.: 13/158,150
(22)
Filed:
Jun. 10, 2011
(65)
Prior Publication Data
US 2011/0305664 Al
Dec. 15, 2011
Related U.S. Application Data
(62) Division of application No. 12/269,824, filed on Nov.
12, 2008.
(60) Provisional application No. 60/987,284, filed on Nov.
12, 2007.
(51)
(52)
(58)
Int. Cl.
C12N 15/00
(2006.01)
C12P 21106
(2006.01)
A61K391145
(2006.01)
CO7H 21/02
(2006.01)
U.S. Cl. ..................... 435/320.1; 435/69.1; 435/7.1;
424/206.1; 536/23.1
Field of Classification Search ........................ None
See application file for complete search history.
(56)
References Cited
U.S. PATENT DOCUMENTS
5,593,972 A
5,908,780 A
5,962,428 A
7,238,522 B2
7,245,963 B2
7,262,045 B2
2004/0175727 Al
2005/0052630 Al
2006/0024670 Al
2006/0165684 Al
2008/0091135 Al
1/1997
6/1999
10/1999
7/2007
7/2007
8/2007
9/2004
3/2005
2/2006
7/2006
4/2008
Weiner et al.
Jones
Carrano et al.
Hebel et al.
Draghia-Akli et al.
Schwartz et al.
Draghia-Akli et al.
Smith et al.
Luke et al.
Utku
Draghia-Akli
FOREIGN PATENT DOCUMENTS
WO
WO
W09324640
W09416737
12/1993
8/1994
WO
WO
WO
Patent No.:
US 8,133,723 B2
Date of Patent:
Mar. 13, 2012
2007011904
2008091657
2008124331
1/2007
7/2008
10/2008
OTHER PUBLICATIONS
Stobie, L. et al., "The role of antigen and IL-12 in sustaining Thl
memory cells in vivo: IL- 12 is required to maintain memory/effector
Thl cells sufficient to mediate protection to an infectious parasite
challenge", PNAS, 2000, 97:8427-8432.
Soh J-W et al., "Novel roles of specific isoforms of protein kinase C
in activation of the c-fos serum response element", Molecular and
Cell Biology, 1999, 19:1313-1324.
Wong et al., "DNA vaccination against respiratory influenza virus
infection", Vaccine, 2001, 19:2461-2467.
Nicol, F. et al., "Poly-L-glutamate, an anionic polymer, enhances
transgene expression for plasmids delivered by intramuscular injection with an in vivo electroporation", Gene Therapy, 2002, 9:13511358.
Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs", Nuc. Acids Res., 1997,
25:3389-3402.
Altschul et al., "Basic local alignment search tool", J. Mol. Biol.,
1990, 215:403-410.
Holland, D. et al., "Intradermal influenza vaccine administered using
a new microinjection system produces superior immunogenicity in
elderly adults: a randomized controlled trial", J Inf Dis, 2008,
198:650-658.
Gronevik, E. et al., "Gene expression and immune response kinetics
using e!ectroporation-mediated DNA delivery to muscle', J Gene
Med, 2005, 7(2)218-227.
Primary Examiner Bo Peng
(74) Attorney, Agent, or Firm
(57)
Pepper Hamilton LLP
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-nucleoprotein (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.
52 Claims, 5 Drawing Sheets
Figure 1
Nru 1(10)
pUC OI'i
~,Nco I (412)
o I (412)
pUC i
J
pCMV
~
T7 Hindlll (713)
Hind III (713)
~--•
am HI (731)
co RI (754)
Nco 1(729)
Ram HI
pGX2002-NA
(744)
Pstl (998)
pGX2001
Nco 1(35-
PCMV
4733 bp
Nco 1(764)
4418 bp
Ncol (3207)
Bam HI (779)
st1(893)
t Psi I (1132)
Kan
Kan
......
NA
Pst I (1714)
Eco RI (1511)
bGHA
HA
Nco 1(1674)
Nat 1 (2201)
Xbal(2213)
bGHpA
Xba 1(2520
AmpR
Pstl (2196)
fin dill (891)
Pst I (2472)
T7 promoter
pUC ri
dill (713)
Nco I (344 1)L~.
pGX2003-M2e-NP
Kan
~
4652 bp
am HI (731)
RI (754)
I Nco I (764)
Barn HI (779)
[
8VMA nnlvA small
.,.,,
Pst I (1829)
Notl (2435)
SEAP
pCMVS
7582 by
A
Nco I(1469)
M2e-NP
bGHpA
CoIEI orig'
Nco 1(2024)
NEO(R)
Sp€ promoter
SV40 origin
Xba 1(3025)
SV40 early promoter
bGH polyA
Kpn 1 (3873)
florigin
180
160
140
120
Figure 2
100
so
6o
40
20
0
P
* (P < 0.05 vs. 2mgl4.3A
and 2mg/0.1A)
D•
2500
548
2000
mU
0
NA (1 +2+3)
U HA (1+2+3)
1500
Figure 3
E
w
1452
272
1000
.
U
500
662
0
0
43
63
0
Week 0
Week 5
4 mgfO.3A
Week 0
Week 5
1.6 mg10,5A
Week 0
Week 5
Unimmunized control
HA (1+2+3) and NA (1+2+3)
= HA and NA peptide pools
25
` 20
0
LI
U,
15
0
Figure 4A
0
10
2 5
0
8
* (P < 0.0002 as compared to controls)
3.0
® Day 14
2.0
■ Day 35
1.7
1.6
1.5
1.5
1.1
Q.6
1.Q
QD
.8
1-00.9 Q.8
~}
0.L
c'
O'
o
O'
d•
o'
,o'
0.8
Figure 4B
0.00
54019.'
w
to
-10.00
Figure 5
-15.00
2M
-20.00
a
m
-25.00
108
--------------------------------------------
---+— Control
105 ----------------- -- H5
—e—M2-NP
fl9
1 104
---H5+M2-NP+NA
103 -------d
E 102 ---- ---- ----~,
s
c
m
---
101
-----
100
----------- ---------- -----------------
99
Figure 6
- -- --------
----------- -----
-
--------------------------
98
0
1
2
3
4
6
Days Post-challenge
7
8
9
900
800
700
60D
Figure 7
500
a 400
Y 300
12
P 200
x
100
0
control
H5
M2-NP
H5 + NA + M2-NP
160
140
Figure 8
120
100
in
80
60
= 40
20
0
HA IM+EP
HA ID+EP
HA IM alone
non-treated
control
US 8,133,723 B2
1
VACCINES AGAINST MULTIPLE SUBTYPES
OF INFLUENZA VIRUS
ral 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.
Influenza vaccines are a popular seasonal vaccine and
CROSS REFERENCE TO RELATED
many
people have experienced such vaccinations. However,
APPLICATIONS
5
the vaccinations are limited in their protective results because
the vaccines are specific for certain subtypes of virus. The
This application is a divisional of U.S. patent application
Centers for Disease Control and Prevention promote vacciSer. No. 12/269,824, filed Nov. 12, 2008, pending, which
nation with a "flu shot" that is a vaccine that contains three
claims the benefit of U.S. Provisional Application No.
influenza viruses (killed viruses): one A (H3N2) virus, one A
60/987,284, filed Nov. 12, 2007, the contents of which are 10 (H1N1) virus, and one B virus. They also report that the
incorporated herein by reference.
viruses in the vaccine change each year based on international
surveillance and scientists' estimations about which types
FIELD OF THE INVENTION
and strains of viruses will circulate in a given year. Thus, it is
apparent that vaccinations are limited to predictions of subThe present invention relates to improved influenza vac- 15 types, and the availability of a specific vaccine to that subtype.
cines, improved methods for inducing immune responses,
There still remains a need for effective influenza vaccines
and for prophylactically and/or therapeutically immunizing
that are economical and effective across numerous subtypes.
individuals against influenza.
Further, there remains a need for an effective method of
administering DNA vaccines to a mammal in order to provide
BACKGROUND
20 immunization against influenza either prophylactically or
therapeutically.
The use of nucleic acid sequences to vaccinate against
SUMMARY OF THE INVENTION
animal and human diseases has been studied. Studies have
focused on effective and efficient means of delivery in order
One aspect of the present invention relates to a DNA plasto yield necessary expression of the desired antigens, result- 25
mid
vaccine capable of generating in a mammal an immune
ing immunogenic response and ultimately the success of this
response against a plurality of influenza virus subtypes, comtechnique. One method for delivering nucleic acid sequences
prising a DNA plasmid and a pharmaceutically acceptable
such as plasmid DNA is the electroporation (EP) technique.
excipient. The DNA plasmid is capable of expressing a conThe technique has been used in human clinical trials to deliver
sensus influenza antigen in a cell of the mammal in a quantity
anti-cancer drugs, such as bleomycin, and in many preclinical 30 effective to elicit an immune response in the mammal,
studies on a large number of animal species.
wherein the consensus influenza antigen comprises consenThe influenza virus genome is contained on eight single
sus hemagglutinin (HA), neuraminidase (NA), matrix pro(non-paired) RNA strands that code for eleven proteins (HA,
tein, nucleoprotein, M2 ectodomain-nucleo-protein (M2eNA, NP, Ml, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The
NP), or a combination thereof. Preferably the consensus
segmented nature of the genome allows for the exchange of 35 influenza antigen comprises HA, NA, M2e-NP, or a combientire genes between different viral strains during cellular
nation thereof. The DNA plasmid comprises a promoter opercohabitation. The eight RNA segments are: HA, which
ably linked to a coding sequence that encodes the consensus
encodes hemagglutinin (about 500 molecules of hemagglutiinfluenza antigen. Preferably, the DNA plasmid vaccine is
nin are needed to make one virion); NA, which encodes
one having a concentration of total DNA plasmid of 1 mg/ml
neuraminidase (about 100 molecules of neuraminidase are 40 or greater.
needed to make one virion); NP, which encodes nucleoproAnother aspect of the present invention relates to DNA
tein; M, which encodes two matrix proteins (the Ml and the
plasmids capable of expressing a consensus influenza antigen
M2) by using different reading frames from the same RNA
in a cell of the mammal, the consensus influenza antigen
segment (about 3000 matrix protein molecules are needed to
comprising consensus hemagglutinin (HA), neuraminidase
make one virion); NS, which encodes two distinct non-struc- 45 (NA), matrix protein, nucleoprotein, M2 ectodomain-nucleotural proteins (NS1 and NEP) by using different reading
protein (M2e-NP), or a combination thereof. Preferably the
frames from the same RNA segment; PA, which encodes an
consensus influenza antigen comprises HA, NA, M2e-NP, or
RNA polymerase; PB 1, which encodes an RNA polymerase
a combination thereof. The DNA plasmid comprises a proand PB1-F2 protein (induces apoptosis) by using different
moter operably linked to a coding sequence that encodes the
reading frames from the same RNA segment; and PB2, which 50 consensus influenza antigen.
encodes an RNA polymerase.
Another aspect of the present invention relates to methods
Influenza hemagglutinin (HA) is expressed on the surface
of eliciting an immune response against a plurality of influof influenza viral particles and is responsible for initial conenza virus subtypes in a mammal. The methods include delivtact between the virus and its host cell. HA is a well-known
ering a DNA plasmid vaccine to tissue of the mammal, the
immunogen. Influenza A strain H5N1, an avian influenza 55 DNA plasmid vaccine comprising a DNA plasmid capable of
strain, particularly threatens the human population because of
its HA protein (H5) which, if slightly genetically reassorted
mammal to elicit an immune response in the mammal, the
by natural mutation, has greatly increased infectivity of
consensus influenza antigen comprising consensus HA, NA,
human cells as compared to other strains of the virus. InfecM2e-NP or a combination thereof, and electroporating cells
tion of infants and older or immunocompromised adult 60 of the tissue with a pulse of energy at a constant current
humans with the viral H5N1 strain is often correlated to poor
effective to permit entry of the DNA plasmids in the cells.
clinical outcome. Therefore, protection against the H5N1
strain of influenza is a great need for the public.
BRIEF DESCRIPTION OF THE DRAWINGS
There are two classes of anti-influenza agents available,
inhibitors of influenza A cell entry/uncoating (such as antivi- 65
The numerous objects and advantages of the present invenrals amantadine and rimantadine) and neuraminidase inhibition may be better understood by those skilled in the art by
tors (such as antivirals oseltamivir, zanamivir). These antivireference to the accompanying figures, in which:
US 8,133,723 B2
3
4
FIG. 1 displays a schematic representation (plasmid maps)
of the present invention. The abbreviated definitions given
of the DNA plasmid constructs used in the studies described
here are by no means exhaustive nor are they contradictory to
herein. Consensus HA, NA and M2e-NP constructs were
the definitions as understood in the field or dictionary meangenerated by analyzing primary virus sequences from 16 H5
ing. The abbreviated definitions are given here to supplement
viruses that have proven fatal to humans in recent years, and 5 or more clearly define the definitions known in the art.
over 40 human Ni viruses (Los Alamos National Laboratory's Influenza Sequence Database). After generating the conDefinitions
sensus sequences, the constructs were optimized for mammalian expression, including the addition of a Kozak sequence,
Sequence homology for nucleotides and amino acids as
codon optimization, and RNA optimization. These constructs
io used herein may be determined using FASTA, BLAST and
were then subcloned into the pVAX vector (Invitrogen, CarlsGapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25,
bad, Calif.). Plasmids pGX2001 (consensus HA), pGX2002
3389,
which is incorporated herein by reference in its
(consensus NA), pGX2003 (consensus M2e-NP) are shown.
entirety) and PAUP* 4.0b10 software (D. L. Swofford,
The plasmid pCMVSEAP, displayed, encodes the reporter
Sinauer Associates, Massachusetts). Briefly, the BLAST
protein secreted embryonic alkaline phosphatase (SEAP).
FIG. 2 displays a bar graph of the results of the HI titers in 15 algorithm, which stands for Basic Local Alignment Search
Tool is suitable for determining sequence similarity (Altschul
pig serum at Day 35 post-injection. The highest titers were
et al., J. Mol. Biol., 1990,215,403-410, whichis incorporated
found in the group administered 2 mg of HA-expressing
herein by reference in its entirety). Software for performing
plasmid at a current setting of 0.5 A (120±40; *P-0.11 versus
BLAST analyses is publicly available through the National
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 20 Center for Biotechnology Information (http://www.ncbi.nat 0.5 A also demonstrated decreasing HI titers.
lm.nih.gov). One measure of similarity provided by the
FIG. 3 displays a bar graph of the IFN-y ELISpot counts.
BLAST algorithm is the smallest sum probability (P(N)),
The counts were highest in pigs administered 2 mg of HA and
which provides an indication of the probability by which a
2 mg of NA plasmid vaccine (for a total of 4 mg plasmid) and
match between two nucleotide sequences would occur by
electroporated with 0.3 A of current (2000 spots) and in the 25 chance. For example, a nucleic acid is considered similar to
group administered 0.8 mg of HA and 0.8 mg of NA plasmid
another if the smallest sum probability in comparison of the
vaccine (for a total of 1.6 mg plasmid) electroporated with 0.5
test nucleic acid to the other nucleic acid is less than about 1,
A of current (934 spots). For comparison purposes, the celpreferably less than about 0.1, more preferably less than
lular immune responses of an unimmunized control group are
about 0.01, and most preferably less than about 0.001. "Perdepicted.
30 centage of similarity" can be calculated using PAUP* 4.0b10
FIGS. 4A and 4B display bar graphs showing results from
software (D. L. Swofford, Sinauer Associates, Massachumuscle biopsies from treated pigs at Day 14 and Day 35: FIG.
setts). The average similarity of the consensus sequence is
4A displays a bar graph showing the mean pathology scores
calculated compared to all sequences in the phylogenic tree.
for all groups. FIG. 4B displays a bar graph showing the
As used herein, the term "genetic construct" or "nucleic
muscle necrosis and fibrosis scores. The group injected with
6 mg total plasmid and electroporated at 0.5 A exhibited the 35 acid construct" is used interchangeably and refers to the DNA
or RNA molecules that comprise a nucleotide sequence
highest mean pathology score (*P<0.0002 as compared to
which encodes protein. The coding sequence, or "encoding
controls). The pathology scores were significantly reduced by
nucleic acid sequence," includes initiation and termination
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
signals operably linked to regulatory elements including a
(P=1.0).
40 promoter and polyadenylation signal capable of directing
FIG. 5 displays the percent change in weight of ferrets after
expression in the cells of the individual to whom the nucleic
challenge with H5N1 virus (A/Viet/1203/2004(H5N1)/PR8acid molecule is administered.
IBCDC-RG). Ferrets that were vaccinated with HA,
As used herein, the term "expressible form" refers to
HA+M2e-NP or HA+M2e-NP+NA lost significantly less
nucleic acid constructs that contain the necessary regulatory
weight than control animals (*P<0.005 versus controls) in the 45 elements operable linked to a coding sequence that encodes a
9 days post-challenge period. One animal in the HA vaccine
protein such that when present in the cell of the individual, the
group actually gained weight post-challenge.
coding sequence will be expressed.
FIG. 6 displays a graph showing the body temperatures of
The term "constant current" is used herein to define a
ferrets during the 9 days post-challenge. Control animals
current that is received or experienced by a tissue, or cells
showed higher body temperatures than the vaccinated animals. The body temperature on day 5 is not depicted as it was 5o defining said tissue, over the duration of an electrical pulse
delivered to same tissue. The electrical pulse is delivered from
measured at a different time of day and all the temperatures
the electroporation devices described herein. This current
regardless of group were lower.
remains at a constant amperage in said tissue over the life of
FIG. 7 displays a bar graph of results from HI titers in
an electrical pulse because the electroporation device proferrets after vaccination; the assay was performed using reassortant viruses obtained from the Center for Disease Control: 55 vided herein has a feedback element, preferably having
instantaneous feedback. The feedback element can measure
A/Viet/1203/04 or Indo/05/2005 influenza strains.
the resistance of the tissue (or cells) throughout the duration
FIG. 8 displays a bar graph of results from HI titers meaof the pulse and cause the electroporation device to alter its
sured three weeks after the second immunization. Macaques
electrical energy output (e.g., increase voltage) so current in
immunized ID followed by EP showed significantly higher
HI titers than all other groups (P<0.03). Non-treated controls 60 same tissue remains constant throughout the electrical pulse
(on the order of microseconds), and from pulse to pulse. In
did not exhibit any HI titers.
some embodiments, the feedback element comprises a conDETAILED DESCRIPTION OF PREFERRED
troller.
EMBODIMENTS
The term "feedback" or "current feedback" is used inter6 5 changeably and means the active response of the provided
The following abbreviated, or shortened, definitions are
electroporation devices, which comprises measuring the curgiven to help the understanding of thepreferred embodiments
rent in tissue between electrodes and altering the energy
US 8,133,723 B2
output delivered by the EP device accordingly in order to
mammal, the consensus influenza antigen comprising conmaintain the current at a constant level. This constant level is
sensus hemagglutinin (HA), neuraminidase (NA), matrix
preset by a user prior to initiation of a pulse sequence or
protein, nucleoprotein, M2 ectodomain-nucleo-protein
electrical treatment. Preferably, the feedback is accomplished
(M2e-NP), or a combination thereof. Preferably the consenby the electroporation component, e.g., controller, of the elec- 5 sus influenza antigen comprises HA, NA, M2e-NP, or a comtroporation device, as the electrical circuit therein is able to
bination thereof. The DNA plasmid comprises a promoter
continuously monitor the current in tissue between electrodes
operably linked to a coding sequence that encodes the conand compare that monitored current (or current within tissue)
sensus influenza antigen.
to a preset current and continuously make energy-output
In some embodiments, the present invention provides
adjustments to maintain the monitored current at preset lev- io DNA plasmid vaccines that are capable of generating in a
els. In some embodiments, the feedback loop is instantaneous
mammal an immune response against a plurality of influenza
as it is an analog closed-loop feedback.
virus subtypes, the DNA plasmid vaccines comprising a DNA
The terms "electroporation," "electro-permeabilization,"
plasmid and a pharmaceutically acceptable excipient. The
or "electro-kinetic enhancement" ("EP") as used interDNA plasmid is capable of expressing a consensus influenza
changeably herein refer to the use of a transmembrane electric 15 antigen in a cell of the mammal in a quantity effective to elicit
field pulse to induce microscopic pathways (pores) in a bioan immune response in the mammal, wherein the consensus
membrane; their presence allows biomolecules such as plasinfluenza antigen comprises consensus hemagglutinin (HA),
mids, oligonucleotides, siRNA, drugs, ions, and water to pass
neuraminidase (NA), matrix protein, nucleoprotein, M2
from one side of the cellular membrane to the other.
ectodomain-nucleo-protein (M2e-NP), or a combination
The term "decentralized current" is used herein to define 20 thereof. Preferably the consensus influenza antigen comthe pattern of electrical currents delivered from the various
prises HA, NA, M2e-NP, or a combination thereof. The DNA
needle electrode arrays of the electroporation devices
plasmid comprises a promoter operably linked to a coding
described herein, wherein the patterns minimize, or prefersequence that encodes the consensus influenza antigen. In
ably eliminate, the occurrence of electroporation related heat
some embodiments, the DNA plasmid vaccine is one having
stress on any area of tissue being electroporated.
25 a concentration of total DNA plasmid of 1 mg/ml or greater.
The term "feedback mechanism" as used herein refers to a
The immune response can be a cellular or humoral response,
process performed by either software or hardware (or firmor both; preferably, the immune response is both cellular and
ware), which process receives and compares the impedance
humoral.
of the desired tissue (before, during, and/or after the delivery
In some embodiments, the DNA plasmid can further
of pulse of energy) with a present value, preferably current, 30 include an IgG leader sequence attached to an N-terminal end
and adjusts the pulse of energy delivered to achieve the preset
of the coding sequence and operably linked to the promoter.
value. The term "impedance" is used herein when discussing
In addition, in some embodiments, the DNA plasmid can
the feedback mechanism and can be converted to a current
further include a polyadenylation sequence attached to the
value according to Ohm's law, thus enabling comparisons
C-terminal end of the coding sequence. In some embodiwith the preset current. In a preferred embodiment, the "feed- 35 ments, the DNA plasmid is codon optimized.
back mechanism" is performed by an analog closed loop
In some embodiments of the present invention, the DNA
circuit.
plasmid vaccines can further include an adjuvant. In some
The term "immune response" is used herein to mean the
embodiments, the adjuvant is selected from the group conactivation of a host's immune system, e.g., that of a mammal,
sisting of: alpha-interferon, gamma-interferon, platelet
in response to the introduction of influenza consensus antigen 4o derived growth factor (PDGF), TNFa, TNF(3, GM-CSF, epivia the provided DNA plasmid vaccines. The immune
dermal growth factor (EGF), cutaneous T cell-attracting
response can be in the form of a cellular or humoral response,
chemokine (CTACK), epithelial thymus-expressed chemokor both.
ine (TECK), mucosae-associated epithelial chemokine
The term "consensus" or "consensus sequence" is used
(MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15
herein to mean a synthetic nucleic acid sequence, or corre- 45 having the signal sequence deleted and optionally including
sponding polypeptide sequence, constructed based on analythe signal peptide from IgE. Other genes which may be useful
sis of an alignment of multiple subtypes of a particular influadjuvants include those encoding: MCP-1, MIP-la, MIP-Ip,
enza antigen, that can be used to induce broad immunity
IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34,
against multiple subtypes or serotypes of a particular influG1yCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95,
enza antigen. Consensus influenza antigens include HA, 50 PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,
including consensus Hl, H2, H3, or H5, NA, NP, matrix
G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular
protein, and nonstructural protein. Also, synthetic antigens
growth factor, fibroblast growth factor, IL-7, nerve growth
such as fusion proteins, e.g., M2e-NP, can be manipulated to
factor, vascular endothelial growth factor, Fas, TNF receptor,
consensus sequences (or consensus antigens).
Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD,
The term "adjuvant" is used herein to mean any molecule 55 NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6,
added to the DNA plasmid vaccines described herein to
Caspase ICE, Fos, c jun, Sp-1, Ap-1, Ap-2, p38, p65Rel,
enhance antigenicity of the influenza antigen encoded by the
MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,
DNA plasmids and encoding nucleic acid sequences
JNK, interferon response genes, NFkB, Bax, TRAIL,
described hereinafter.
TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,
The term "subtype" or "serotype" is used herein inter- 6o RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D,
changeably and in reference to influenza viruses, and means
MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E,
genetic variants of an influenza virus antigen such that one
NKG2F, TAPi, TAP2 and functional fragments thereof. In
subtype is recognized by an immune system apart from a
some preferred embodiments, the adjuvant is selected from
different subtype (or, in other words, each subtype is different
IL-12, IL-15, CTACK, TECK, or MEC.
in antigenic character from a different subtype).
65
In some embodiments, the pharmaceutically acceptable
In some embodiments, there are DNA plasmids capable of
excipient is a transfection facilitating agent, which can
include the following: surface active agents, such as immune-
US 8,133,723 B2
7
8
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 ofpoly-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.
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 HI, 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 HI 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 HI, 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.
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 HI, H3 or
H5, and a DNA plasmid that can express any one of the
following influenza A antigens: HI, H2, H3, H4, H5, H6, H7,
H8, H9, H10, H11, H12, H13, H14, H15, H16, Ni, N2, N3,
N4, N5, N6, N7, N8, N9, NP, MI, 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: HI, H2, H3,
H4, H5, H6, H7, H8, H9, H10, Hi i, H12, H13, H14, H15,
HI6, 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: HI,
H2, H3, H4, H5, H6, H7, H8, H9, H10, HI1, H12, H13, H14,
H15, H16, NI, N2, N3, N4, N5, N6, N7, N8, N9, NP, Ml, M2,
NS 1, or NEP, or a combination thereof.
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.
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
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.
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.
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.
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/H5NI) 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
MI consensus sequences. SEQ ID NO:6 comprises the amino
acid sequence for influenza H1N1/H5N1 MI 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
5
10
15
20
25
30
35
40
45
50
55
6o
65
US 8,133,723 B2
10
ID NO:9 or SEQ ID NO:10. SEQ ID NO:9 comprises the
at least one influenza subtype. The polypeptide fragments are
nucleic acid sequence that encodes influenza H1N1 HA conselected from at least one of the various polypeptide
sensus sequences. SEQ ID NO:4 comprises the amino acid
sequences of the present invention, including SEQ ID NOS:2,
sequence for influenza H1N1 HA consensus sequences. In
4, 6, 8, 10, 12, and 14, and can be any of the following
some embodiments of the invention, the vaccines of the 5 described polypeptide fragments, as it applies to the specific
invention comprise SEQ ID NO:11 or SEQ ID NO:12. SEQ
polypeptide sequence provided herein. In some embodiID NO: 11 comprises the nucleic acid sequence that encodes
ments, polypeptide fragments can comprise 15 or more, 30 or
influenza H3N1 HA consensus sequences. SEQ ID NO: 12
more, 45 or more, 60 or more, 75 or more, 90 or more, 105 or
comprises the amino acid sequence for influenza H3N1 HA
more, 120 or more, 150 or more, 180 or more, 210 or more,
consensus sequences. The consensus sequence for influenza
10 240 or more, 270 or more, 300 or more, 360 or more, 420 or
virus strain H5N1 HA includes the immunodominant epitope
more, 480 or more, 540 or more, or 565 or more amino acids.
set forth in SEQ ID NO:1 or SEQ ID NO:13. The influenza
In some embodiments, polypeptide fragments can comprise
virus H5N1 HA amino acid sequence encoded by SEQ ID
fewer than 30, fewer than 45, fewer than 60, fewer than 75,
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 15 fewer than 90, fewer than 120, fewer than 150, fewer than
180, fewer than 210, fewer than 240, fewer than 270, fewer
H1N1/H5N1 NA includes the immunodominant epitope set
than 300, fewer than 360, fewer than 420, fewer than 480,
forth in SEQ ID NO:3. The influenza virus strains H1N1/
fewerthan
540, orfewerthan 565 amino acids. Preferably, the
H5N1 NA amino acid sequence encoded by SEQ ID NO:3 is
polypeptide fragments are fragments of SEQ ID NOS:2, 4, 8,
SEQ ID NO:4. The consensus sequence for influenza virus
strains H1N1/H5N1 Ml includes the immunodominant 20 10, 12, or 14, and more preferably fragments of SEQ ID
NOS:2, 6, 10, 12, or 14, and even more preferably fragments
epitope set forth in SEQ ID NO:5. The influenza virus H1N1/
H5N1 Ml amino acid sequence encoded by SEQ ID NO:5 is
of SEQ ID NOS:2, 10, or 14.
SEQ ID NO:6. The consensus sequence for influenza virus
The determination of a fragment eliciting an immune
H5N1 M2E-NP includes the immunodominant epitope set
response in a mammal substantially similar to that of the
forth in SEQ ID NO:7. The influenza virus H5N1 M2E-NP 25 non-fragment for at least one influenza subtype can be readily
amino acid sequence encoded by SEQ ID NO:7 is SEQ ID
determined by one of ordinary skill. The fragment can be
NO:8. Vaccines of the present invention may include protein
analyzed to contain at least one, preferably more, antigenic
products encoded by the nucleic acid molecules defined
epitopes as provided by a publicly available database, such as
above or any fragments of proteins.
the Los Alamos National Laboratory's Influenza Sequence
The present invention also comprises DNA fragments that 3o Database. In addition, immune response studies can be rouencode a polypeptide capable of eliciting an immune
tinely assessed using mice and HI titers and ELISpots analyresponse in a mammal substantially similar to that of the
sis, such as that shown in the Examples below.
non-fragment for at least one influenza subtype. The DNA
According to some embodiments ofthe invention, methods
fragments are fragments selected from at least one of the
of inducing or eliciting an immune response in mammals
various encoding nucleotide sequences of the present inven- 35 against a plurality of influenza viruses comprise administertion, including SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13, and can
ing to the mammals: a) the influenza strain H5N1 consensus
be any of the following described DNA fragments, as it
HA protein, functional fragments thereof, or expressible codapplies to the specific encoding nucleic acid sequence proing sequences thereof; and b) one or more isolated encoding
vided herein. In some embodiments, DNA fragments can
nucleic acid molecules provided herein, protein encoded by
comprise 30 or more, 45 or more, 60 or more, 75 or more, 90 40 such nucleic acid molecules, or fragments thereof.
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,
against a plurality of influenza viruses comprise administer720 or more, 780 or more, 840 or more, 900 or more, 960 or
ing to the mammals: a) the influenza strain H1N1 and influmore, 1020 or more, 1080 or more, 1140 or more, 1200 or 45 enza strain H5N1 consensus NA protein, functional fragmore, 1260 or more, 1320 or more, 1380 or more, 1440 or
ments thereof, or expressible coding sequences thereof and b)
more, 1500 or more, 1560 or more, 1620 or more, 1680 or
one or more isolated encoding nucleic acid molecules promore, or 1740 or more nucleotides. In some embodiments,
vided herein, protein encoded by such nucleic acid molDNA fragments can comprise coding sequences for the
ecules, or fragments thereof.
immunoglobulin E (IgE) leader sequences. In some embodi- 50
ments, DNA fragments can comprise fewer than 60, fewer
than 75, fewer than 90, fewer than 120, fewer than 150, fewer
against a plurality of influenza viruses comprise administerthan 180, fewer than 210, fewer than 240, fewer than 270,
ing to the mammals: a) the influenza strain H1N1 and influfewer than 300, fewer than 360, fewer than 420, fewer than
enza strain H5N1 consensus Ml protein, functional frag480, fewer than 540, fewer than 600, fewer than 660, fewer 55 ments thereof, or expressible coding sequences thereof and b)
than 720, fewer than 780, fewer than 840, fewer than 900,
one or more isolated encoding nucleic acid molecules profewer than 960, fewer than 1020, fewer than 1080, fewer than
vided herein, protein encoded by such nucleic acid mol1140, fewer than 1200, fewer than 1260, fewer than 1320,
ecules, or fragments thereof.
fewer than 1380, fewer than 1440, fewer than 1500, fewer
than 1560, fewer than 1620, fewer than 1680, or fewer than 60 of inducing or eliciting an immune response in mammals
1740 nucleotides. Preferably, the DNA fragments are fragagainst a plurality of influenza viruses comprise administerments of SEQ ID NOS: 1, 3, 7, 9, 11 or 13, and more prefering to the mammals: a) the influenza strain H5N1 M2E-NP
ably fragments of SEQ ID NOS:1, 5, 9, 11, or 13, and even
consensus protein, functional fragments thereof, or expressmore preferably fragments of SEQ ID NOS:1, 9, or 13.
ible coding sequences thereof and b) one or more isolated
The present invention also comprises polypeptide frag- 65 encoding nucleic acid molecules provided herein, protein
ments that are capable of eliciting an immune response in a
encoded by such nucleic acid molecules, or fragments
mammal substantially similar to that of the non-fragment for
thereof.
US 8,133,723 B2
11
12
Examples of promoters useful to practice the present
invention, especially in the production of a genetic vaccine
against a plurality of influenza viruses comprise administerfor humans, include but are not limited to promoters from
ing to the mammals: a) the influenza strain H1N1 HA consimian virus 40 (SV40), mouse mammary tumor virus
sensus protein, functional fragments thereof, or expressible 5 (MMTV) promoter, human immunodeficiency virus (HIV)
coding sequences thereof and b) one or more isolated encodsuch as the bovine immunodeficiency virus (BIV) long tering nucleic acid molecules provided herein, protein encoded
minal repeat (LTR) promoter, Moloney virus, avian leukosis
by such nucleic acid molecules, or fragments thereof.
virus (ALV), cytomegalovirus (CMV) such as the CMV
immediate early promoter, Epstein Barr virus (EBV), Rous
of inducing or eliciting an immune response in mammals io sarcoma virus (RSV) as well as promoters from human genes
against a plurality of influenza viruses comprise administersuch as human actin, human myosin, human hemoglobin,
ing to the mammals: a) the influenza strain H3N1 HA conhuman muscle creatine and human metalothionein; in other
sensus protein, functional fragments thereof, or expressible
embodiments, promoters can be tissue specific promoters,
coding sequences thereof; and b) one or more isolated encodsuch as muscle or skin specific promoters, natural or syning nucleic acid molecules provided herein, protein encoded 15 thetic. Examples of such promoters are described in US
by such nucleic acid molecules, or fragments thereof.
patent application publication no. US20040175727, which is
In some embodiments of the invention, the vaccines of the
incorporated hereby in its entirety.
invention include at least two of the following sequences:
Examples of polyadenylation signals useful to practice the
SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
present invention, especially in the production of a genetic
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, 20 vaccine for humans, include but are not limited to SV40
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
polyadenylation signals, LTR polyadenylation signals,
NO:12, SEQ ID NO:13, and SEQ ID NO:14, or any combibovine growth hormone (bGH) polyadenylation signals,
nation of two or more sequences from the aforementioned
human growth hormone (hGH) polyadenylation signals, and
list.
human (3-globin polyadenylation signals. In particular, the
Vaccines
25 SV40 polyadenylation signal that is in pCEP4 plasmid (InvitIn some embodiments, the invention provides improved
rogen, San Diego, Calif.), referred to as the SV40 polyadevaccines by providing proteins and genetic constructs that
nylation signal, can be used.
encode proteins with epitopes that make them particularly
In addition to the regulatory elements required for DNA
effective as immunogens against which immune responses
expression, other elements may also be included in the DNA
can be induced. Accordingly, vaccines can be provided to 30 molecule. Such additional elements include enhancers. The
induce a therapeutic or prophylactic immune response.
enhancer may be selected from the group including but not
According to some embodiments of the invention, a vaclimited to: human actin, human myosin, human hemoglobin,
cine according to the invention is delivered to an individual to
human muscle creatine and viral enhancers such as those
modulate the activity of the individual's immune system and
from CMV, RSV and EBV.
thereby enhance the immune response. When a nucleic acid 35
Genetic constructs can be provided with mammalian origin
molecule that encodes the protein is taken up by cells of the
of replication in order to maintain the construct extrachromoindividual the nucleotide sequence is expressed in the cells
somally and produce multiple copies of the construct in the
and the protein are thereby delivered to the individual.
cell. Plasmids pVAX1, pCEP4 and pREP4 from Invitrogen
Aspects of the invention provide methods of delivering the
(San Diego, Calif.) contain the Epstein Barr virus origin of
coding sequences of the protein on nucleic acid molecule 4o replication and nuclear antigen EBNA-1 coding region which
such as plasmid.
produces high copy episomal replication without integration.
According to some aspects of the present invention, comIn order to maximize protein production, regulatory
positions and methods are provided which prophylactically
sequences may be selected which are well suited for gene
and/or therapeutically immunize an individual.
expression in the cells the construct is administered into.
When taken up by a cell, the DNA plasmids can stay 45 Moreover, codons that encode said protein may be selected
present in the cell as separate genetic material. Alternatively,
which are most efficiently transcribed in the host cell. One
RNA may be administered to the cell. It is also contemplated
having ordinary skill in the art can produce DNA constructs
to provide the genetic construct as a linear minichromosome
that are functional in the cells.
including a centromere, telomeres and an origin of replicaIn some embodiments, nucleic acid constructs may be
tion. Genetic constructs include regulatory elements neces- 50 provided in which the coding sequences for the proteins
sary for gene expression of a nucleic acid molecule. The
described herein are linked to IgE signal peptide. In some
elements include: a promoter, an initiation codon, a stop
embodiments, proteins described herein are linked to IgE
codon, and a polyadenylation signal. In addition, enhancers
signal peptide.
are often required for gene expression of the sequence that
In some embodiments for which protein is used, for
encodes the target protein or the immunomodulating protein. 55 example, one having ordinary skill in the art can, using well
It is necessary that these elements be operable linked to the
known techniques, can produce and isolate proteins of the
sequence that encodes the desired proteins and that the reguinvention using well known techniques. In some embodilatory elements are operably in the individual to whom they
ments for which protein is used, for example, one having
are administered.
ordinary skill in the art can, using well known techniques,
Initiation codons and stop codon are generally considered 60 inserts DNA molecules that encode a protein of the invention
to be part of a nucleotide sequence that encodes the desired
into a commercially available expression vector for use in
protein. However, it is necessary that these elements are funcwell known expression systems. For example, the commertional in the mammals to whom the nucleic acid construct is
cially available plasmid pSE420 (Invitrogen, San Diego,
administered. The initiation and termination codons must be
Calif.) may be used for production of protein in Escherichia
in frame with the coding sequence.
65 coli (E. coli). The commercially available plasmid pYES2
Promoters and polyadenylation signals used must be func(Invitrogen, San Diego, Calif.) may, for example, be used for
tional within the cells of the individual.
production in Saccharomyces cerevisiae strains of yeast. The
US 8,133,723 B2
13
14
commercially available MAXBACTM complete baculovirus
intradermal and subcutaneous injection. Genetic constructs
expression system (Invitrogen, San Diego, Calif.) may, for
may be administered by means including, but not limited to,
example, be used for production in insect cells. The commertraditional syringes, needleless injection devices, "microcially available plasmid pcDNA I or pcDNA3 (Invitrogen,
projectile bombardment gone guns", or other physical methSan Diego, Calif.) may, for example, be used for production 5 ods such as electroporation ("EP"), "hydrodynamic method",
in mammalian cells such as Chinese hamster ovary (CHO)
or ultrasound.
cells. One having ordinary skill in the art can use these comExamples of electroporation devices and electroporation
mercial expression vectors and systems or others to produce
methods preferred for facilitating delivery of the DNA vacprotein by routine techniques and readily available starting
cines of the present invention, include those described in U.S.
materials. (See e.g., Sambrook et al., Molecular Cloning a io Pat. No. 7,245,963 by Draghia-Akli, et al., U.S. Patent Pub.
Laboratory Manual, Second Ed. Cold Spring Harbor Press
2005/0052630 submitted by Smith, et al., the contents of
(1989)). Thus, the desired proteins can be prepared in both
which are hereby incorporated by reference in their entirety.
prokaryotic and eukaryotic systems, resulting in a spectrum
Also preferred, are electroporation devices and electroporaof processed forms of the protein.
tion methods for facilitating delivery of the DNA vaccines
One having ordinary skill in the art may use other commer- 15 provided in co-pending and co-owned U.S. patent application
cially available expression vectors and systems or produce
Ser. No. 11/874,072, filed Oct. 17, 2007, which claims the
vectors using well known methods and readily available startbenefit under 35 USC 119(e) to U.S. Provisional Application
ing materials. Expression systems containing the requisite
Ser. Nos. 60/852,149, filed Oct. 17, 2006, and 60/978,982,
control sequences, such as promoters and polyadenylation
filed Oct. 10, 2007, all of which are hereby incorporated in
signals, and preferably enhancers are readily available and 20 their entirety. Preferable, the electroporation device is the
known in the art for a variety of hosts. See e.g., Sambrook et
CELLECTRATM device (VGX Pharmaceuticals, Blue Bell,
al., Molecular Cloning a Laboratory Manual, Second Ed.
Pa.), including the intramuscular (IM) and intradermal (ID)
Cold Spring Harbor Press (1989). Genetic constructs include
models.
the protein coding sequence operably linked to a promoter
U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes
that is functional in the cell line, or cells of targeted tissue, into 25 modular electrode systems and their use for facilitating the
which the constructs are transfected. Examples of constituintroduction of a biomolecule into cells of a selected tissue in
tive promoters include promoters from cytomegalovirus
a body or plant. The modular electrode systems comprise a
(CMV) or SV40. Examples of inducible promoters include
plurality of needle electrodes; a hypodermic needle; an elecmouse mammary leukemia virus or metallothionein promottrical connector that provides a conductive link from a proers. Those having ordinary skill in the art can readily produce 30 grammable constant-current pulse controller to the plurality
genetic constructs useful for transfecting cells with DNA that
of needle electrodes; and a power source. An operator can
encodes protein of the invention from readily available startgrasp the plurality of needle electrodes that are mounted on a
ing materials. The expression vector including the DNA that
support structure and firmly insert them into the selected
encodes the protein is used to transform the compatible host
tissue in a body or plant. The biomolecules are then delivered
which is then cultured and maintained under conditions 35 via the hypodermic needle into the selected tissue. The prowherein expression of the foreign DNA takes place.
grammable constant-current pulse controller is activated and
The protein produced is recovered from the culture, either
constant-current electrical pulse is applied to the plurality of
by lysing the cells or from the culture medium as appropriate
needle electrodes. The applied constant-current electrical
and known to those in the art. One having ordinary skill in the
pulse facilitates the introduction of the biomolecule into the
art can, using well known techniques, isolate protein that is 40 cell between the plurality of electrodes. The entire content of
produced using such expression systems. The methods of
U.S. Pat. No. 7,245,963 is hereby incorporated by reference.
purifying protein from natural sources using antibodies
U.S. Patent Pub. 2005/0052630 submitted by Smith, et al.
which specifically bind to a specific protein as described
describes an electroporation device which may be used to
above may be equally applied to purifying protein produced
effectively facilitate the introduction of a biomolecule into
by recombinant DNA methodology.
45 cells of a selected tissue in a body or plant. The electroporaIn addition to producing proteins by recombinant techtion device comprises an electro-kinetic device ("EKD
niques, automated peptide synthesizers may also be
device") whose operation is specified by software or firmemployed to produce isolated, essentially pure protein. Such
ware. The EKD device produces a series of programmable
techniques are well known to those having ordinary skill in
constant-current pulse patterns between electrodes in an array
the art and are useful if derivatives which have substitutions 5o based on user control and input of the pulse parameters, and
not provided for in DNA-encoded protein production.
allows the storage and acquisition of current waveform data.
The nucleic acid molecules may be delivered using any of
The electroporation device also comprises a replaceable elecseveral well known technologies including DNA injection
trode disk having an array of needle electrodes, a central
(also referred to as DNA vaccination) with and without in
injection channel for an injection needle, and a removable
vivo electroporation, liposome mediated, nanoparticle facili- 55 guide disk. The entire content of U.S. Patent Pub. 2005/
tated, recombinant vectors such as recombinant adenovirus,
0052630 is hereby incorporated by reference.
recombinant adenovirus associated virus and recombinant
The electrode arrays and methods described in U.S. Pat.
vaccinia. Preferably, the nucleic acid molecules such as the
No. 7,245,963 and U.S. Patent Pub. 2005/0052630 are
DNA plasmids described herein are delivered via DNA injecadapted for deep penetration into not only tissues such as
tion and along with in vivo electroporation.
60 muscle, but also other tissues or organs. Because of the conRoutes of administration include, but are not limited to,
figuration of the electrode array, the injection needle (to
intramuscular, intransally, intraperitoneal, intradermal, subdeliver the biomolecule of choice) is also inserted completely
cutaneous, intravenous, intraarterially, intraoccularly and
into the target organ, and the injection is administered peroral as well as topically, transdermally, by inhalation or suppendicular to the target issue, in the area that is pre-delineated
pository or to mucosal tissue such as by lavage to vaginal, 65 by the electrodes The electrodes described in U.S. Pat. No.
rectal, urethral, buccal and sublingual tissue. Preferred routes
7,245,963 and U.S. Patent Pub. 2005/005263 are preferably
of administration include intramuscular, intraperitoneal,
20 mm long and 21 gauge.
US 8,133,723 B2
15
16
The following is an example of methods of the present
constant current continuously and instantaneously during the
delivery of the pulse of energy.
invention, and is discussed in more detail in the patent referA pharmaceutically acceptable excipient can include such
ences discussed above: electroporation devices can be confunctional molecules as vehicles, adjuvants, carriers or dilufigured to deliver to a desired tissue of a mammal a pulse of
energy producing a constant current similar to a preset current 5 ents, which are known and readily available to the public.
Preferably, the pharmaceutically acceptable excipient is an
input by a user. The electroporation device comprises an
adjuvant or transfection facilitating agent. In some embodielectroporation component and an electrode assembly or
ments, the nucleic acid molecule, or DNA plasmid, is delivhandle assembly. The electroporation component can include
ered to the cells in conjunction with administration of a polyand incorporate one or more of the various elements of the
io nucleotide function enhancer or a genetic vaccine facilitator
electroporation devices, including: controller, current waveagent (or transfection facilitating agent). Polynucleotide
form generator, impedance tester, waveform logger, input
function enhancers are described in U.S. Pat. Nos. 5,593,972,
element, status reporting element, communication port,
5,962,428 and International Application Serial Number PCT/
memory component, power source, and power switch. The
US94/00899 filed Jan. 26, 1994, which are each incorporated
electroporation component can function as one element of the 15 herein by reference. Genetic vaccine facilitator agents are
electroporation devices, and the other elements are separate
described in U.S. Ser. No. 021,579 filedApr. 1, 1994, which
elements (or components) in communication with the elecis incorporated herein by reference. The transfection facilitroporation component. In some embodiments, the electropotating agent can be administered in conjunction with nucleic
ration component can function as more than one element of
acid molecules as a mixture with the nucleic acid molecule or
the electroporation devices, which can be in communication 20 administered separately simultaneously, before or after
with still other elements of the electroporation devices sepaadministration of nucleic acid molecules. Examples of transrate from the electroporation component. The present invenfection facilitating agents includes surface active agents such
tion is not limited by the elements of the electroporation
as immune-stimulating complexes (ISCOMS), Freunds
devices existing as parts of one electromechanical or
incomplete adjuvant, LPS analog including monophosphoryl
mechanical device, as the elements can function as one device 25 lipidA, muramyl peptides, quinone analogs and vesicles such
or as separate elements in communication with one another.
as squalene and squalene, and hyaluronic acid may also be
The electroporation component is capable of delivering the
used administered in conjunction with the genetic construct.
pulse of energy that produces the constant current in the
In some embodiments, the DNA plasmid vaccines may also
desired tissue, and includes a feedback mechanism. The elecinclude a transfection facilitating agent such as lipids, lipotrode assembly includes an electrode array having a plurality 30 somes, including lecithin liposomes or other liposomes
of electrodes in a spatial arrangement, wherein the electrode
known in the art, as a DNA-liposome mixture (see for
assembly receives the pulse of energy from the electroporaexample W09324640), calcium ions, viral proteins, polyantion component and delivers same to the desired tissue
ions, polycations, or nanoparticles, or other known transfecthrough the electrodes. At least one of the plurality of election facilitating agents. Preferably, the transfection facilitattrodes is neutral during delivery of the pulse of energy and 35 ing agent is a polyanion, polycation, including poly-Lmeasures impedance in the desired tissue and communicates
glutamate (LGS), or lipid.
the impedance to the electroporation component. The feedIn some preferred embodiments, the DNA plasmids are
back mechanism can receive the measured impedance and
delivered with an adjuvant that are genes for proteins which
can adjust the pulse of energy delivered by the electroporation
further enhance the immune response against such target
component to maintain the constant current.
40 proteins. Examples of such genes are those which encode
In some embodiments, the plurality of electrodes can
other cytokines and lymphokines such as alpha-interferon,
deliver the pulse of energy in a decentralized pattern. In some
gamma-interferon, platelet derived growth factor (PDGF),
embodiments, the plurality of electrodes can deliver the pulse
TNFa, TNF(3, GM-CSF, epidermal growth factor (EGF),
of energy in the decentralized pattern through the control of
IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC,
the electrodes under a programmed sequence, and the pro- 45 CD80, CD86 and IL-15 including IL-15 having the signal
grammed sequence is input by a user to the electroporation
sequence deleted and optionally including the signal peptide
component. In some embodiments, the programmed
from IgE. Other genes which may be useful include those
sequence comprises a plurality of pulses delivered in
encoding: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-sesequence, wherein each pulse of the plurality of pulses is
lectin, P-selectin, E-selectin, CD34, G1yCAM-1, MadCAMdelivered by at least two active electrodes with one neutral 50 1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,
electrode that measures impedance, and wherein a subseICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4,
quent pulse of the plurality of pulses is delivered by a different
mutant forms of IL-18, CD40, CD40L, vascular growth facone of at least two active electrodes with one neutral electrode
tor, fibroblast growth factor, IL-7, nerve growth factor, vasthat measures impedance.
cular endothelial growth factor, Fas, TNF receptor, Flt, ApoIn some embodiments, the feedback mechanism is per- 55 1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,
formed by either hardware or software. Preferably, the feedDR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase
back mechanism is performed by an analog closed-loop cirICE, Fos, c jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88,
cuit. Preferably, this feedback occurs every 50 µs, 20 µs, 10 µs
IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK,
or 1 µs, but is preferably a real-time feedback or instantaneous
interferon response genes, NFkB, Bax, TRAIL, TRAILrec,
(i.e., substantially instantaneous as determined by available 6o TRAILrecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK
techniques for determining response time). In some embodiLIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB,
ments, the neutral electrode measures the impedance in the
NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2
desired tissue and communicates the impedance to the feedand functional fragments thereof.
back mechanism, and the feedback mechanism responds to
The pharmaceutical compositions according to the present
the impedance and adjusts the pulse of energy to maintain the 65 invention comprise DNA quantities of from about 1 nanoconstant current at a value similar to the preset current. In
gram to 100 milligrams; about 1 microgram to about 10
some embodiments, the feedback mechanism maintains the
milligrams; or preferably about 0.1 microgram to about 10
US 8,133,723 B2
17
18
milligrams; or more preferably about 1 milligram to about 2
delivery to the skin, preferably small injection volume, idemilligram. In some preferred embodiments, pharmaceutical
ally 25-200 microliters (µL). In some embodiments, the DNA
compositions according to the present invention comprise
formulations have high DNA concentrations, such as 1
about 5 nanogram to about 1000 micrograms of DNA. In
mg/mL or greater (mg DNA/volume of formulation). More
some preferred embodiments, the pharmaceutical composi- 5 preferably, the DNA formulation has a DNA concentration
tions contain about 10 nanograms to about 800 micrograms of
that provides for gram quantities of DNA in 200 µL of forDNA. In some preferred embodiments, the pharmaceutical
mula, and more preferably gram quantities of DNA in 100 µL
compositions contain about 0.1 to about 500 micrograms of
of formula.
DNA. In some preferred embodiments, the pharmaceutical
The DNA plasmids for use with the EP devices of the
compositions contain about 1 to about 350 micrograms of 10 present invention can be formulated or manufactured using a
DNA. In some preferred embodiments, the pharmaceutical
combination of known devices and techniques, but preferably
compositions contain about 25 to about 250 micrograms of
they are manufactured using an optimized plasmid manufacDNA. In some preferred embodiments, the pharmaceutical
turing technique that is described in a commonly owned,
compositions contain about 100 to about 200 microgram
co-pending U.S. provisional application U.S. Ser. No.
DNA.
15 60/939,792, which was filed on May 23, 2007. In some
The pharmaceutical compositions according to the present
examples, the DNA plasmids used in these studies can be
invention are formulated according to the mode of adminisformulated at concentrations greater than or equal to 10
tration to be used. In cases where pharmaceutical composimg/mL. The manufacturing techniques also include or incortions are injectable pharmaceutical compositions, they are
porate various devices and protocols that are commonly
sterile, pyrogen free and particulate free. An isotonic formu- 20 known to those of ordinary skill in the art, in addition to those
lation is preferably used. Generally, additives for isotonicity
described in U.S. Ser. No. 60/939,792, including those
can include sodium chloride, dextrose, mannitol, sorbitol and
described in a commonly owned patent, U.S. Pat. No. 7,238,
lactose. In some cases, isotonic solutions such as phosphate
522, which issued on Jul. 3, 2007. The high concentrations of
buffered saline are preferred. Stabilizers include gelatin and
plasmids used with the skin EP devices and delivery techalbumin. In some embodiments, a vasoconstriction agent is 25 niques described herein allow for administration of plasmids
added to the formulation. In some embodiments, a stabilizing
into the ID/SC space in a reasonably low volume and aids in
agent that allows the formulation to be stable at room or
enhancing expression and immunization effects. The comambient temperature for extended periods of time, such as
monly owned application and patent, U.S. Ser. No. 60/939,
LGS or other polycations or polyanions is added to the for792 and U.S. Pat. No. 7,238,522, respectively, are hereby
mulation.
30 incorporated in their entirety.
In some embodiments, methods of eliciting an immune
response in mammals against a consensus influenza antigen
Example 1
include methods of inducing mucosal immune responses.
Such methods include administering to the mammal one or
Plasmid Constructs
more of CTACK protein, TECK protein, MEC protein and 35
functional fragments thereof or expressible coding sequences
A ubiquitous cytomegalovirus (CMV) promoter drives the
thereof in combination with an DNA plasmid including a
expression of human secreted embryonic alkaline phosconsensus influenza antigen, described above. The one or
phatase (SEAP) reporter transgene product in the pCMVmore of CTACK protein, TECK protein, MEC protein and
SEAP vector. Plasmids were obtained using a commercially
functional fragments thereof may be administered prior to, 40 available kit (Qiagen Inc., Chatsworth, Calif.). Endotoxin
simultaneously with or after administration of the DNA plaslevels were at less than 0.01 EU/µg, as measured by Kinetic
mid influenza vaccines provided herein. In some embodiChromagenic LAL (Endosafe, Charleston, S.C.). Consensus
ments, an isolated nucleic acid molecule that encodes one or
HA and NA constructs were generated by analyzing primary
more proteins of selected from the group consisting of:
virus sequences from 16 H5 viruses that have proven fatal to
CTACK, TECK, MEC and functional fragments thereof is 45 humans in recent years, and over 40 human Ni viruses. These
administered to the mammal.
sequences were downloaded from the Los Alamos National
Laboratory's Influenza Sequence Database. After generating
EXAMPLES
the consensus sequences, the constructs were optimized for
mammalian expression, including the addition of a Kozak
The present invention is further illustrated in the following 50 sequence, codon optimization, and RNA optimization. These
Examples. It should be understood that these Examples,
constructs were then subcloned into the pVAX vector (Invitwhile indicating preferred embodiments of the invention, are
rogen, Carlsbad, Calif.). Unless indicated otherwise, plasmid
given by way of illustration only. From the above discussion
preparations were diluted in sterile water and formulated 1%
and these Examples, one skilled in the art can ascertain the
weight/weight with poly-L-glutamate sodium salt (LGS)
essential characteristics of this invention, and without depart- 55 (MW=10.5 kDa average)(Sigma, St. Louis, Mo.), further
ing from the spirit and scope thereof, can make various
HPLC purified at VGX Pharmaceuticals, Immune Therapeuchanges and modifications of the invention to adapt it to
tics Division (The Woodlands, Tex.).
various usages and conditions. Thus, various modifications of
the invention in addition to those shown and described herein
Example 2
will be apparent to those skilled in the art from the foregoing 60
description. Such modifications are also intended to fall
Treatment of Pigs
within the scope of the appended claims.
Preferably the DNA formulations for use with a muscle or
Pigs were divided into 10 groupsx4 pigs per group for a
skin EP device described herein have high DNA concentratotal of 40 pigs (Table 1). Pigs were acclimated for 4 days,
tions, preferably concentrations that include milligram to tens 65 weighed and ear-tagged. On Study Day 0, pigs were weighed,
of milligram quantities, and preferably tens of milligram
bled and anesthetized using a combination pre-anesthetic for
quantities, of DNA in small volumes that are optimal for
pigs ketamine (20 mg/kg), xylazine (2.2 mg/kg) and
US 8,133,723 B2
19
20
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 Ni 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 CELLECTRATM 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 1
Groups for the pie vaccine experiment
Group Plasmid
1
2
3
4
5
6
7
8
9
10
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
HA, NA,
SEAP
None
Cone
(mg/
mL)
10
Construct
(mg)/pig
Total
Dose
(mg/pig)
Injection
Volume
A
n
2
6
600 iJ
0.5
4
4
0.8
2.4
600 µl
0.5
4
1.5
0.1
0.3
600 p1
0.5
4
2
6
600 p1
0.3
4
4
0.8
2.4
600
ld
0.3
4
1.5
0.1
0.3
600 p1
0.3
4
2
6
600 ld
0.1
4
4
0.8
2.4
600 p1
0.1
4
1.5
0.1
0.3
600 ld
0.1
4
N/A
N/A
N/A
N/A
N/A
4
10
10
The area surrounding each injection site was tattooed for
rapid identification for biopsy at Days 14 and 35 post-injection.
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 posttreatment were noted. Pigs were weighed andbled 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-y 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
Pig sera were treated with receptor destroying enzyme
(RDE) by diluting one part serum with three parts enzyme and
Incubated overnight in37°
37 C. water bath. The enzyme was
inactivated by 30 min incubation at 56° 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(12):91-5 (July 2004)).
The highest titers as demonstrated by the HI assay (FIG. 2)
5 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
10 quantity of current were decreased thereafter, the titers were
more variable, and non-different between groups.
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
15 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-y ELISpots
ELISpot were performed as previously described using
20 IFN-y capture and detection antibodies (MabTech, Sweden)
(Boyer J D, et al., JMedPrimatol, 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
25
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-y ELISpot of
30 537±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
35
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 lympho40
cyte response, 40% higher than controls (12670±1412 vs.
7607±1603 lymphocyte counts/100 blood, respectively;
P<0.002).
Muscle Histopathology
The injection sites were identified and punch biopsies were
45 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 3x in PBS and stored in 70%
alcohol. The biopsy samples were submitted to Antech Diagnostics where they were processed and sections stained with
50 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
55 each time point.
TABLE 2
Biopsy pathology scoring parameters
60
65
Score Criteria
0
1
2
3
4
5
Not present, no inflammatory cells
Minimal, 1-20 inflammatory cells/100x high-powered field (HPF)
Mild, 21-40 inflammatory cells/100x HPF
Moderate, 41-75 inflammatory cells/100x HPF
Moderate to Marked/Severe, 76-100 inflammatory cells/100x HPF
Marked Severe, >100 inflammatory cells/100x HPF
US 8,133,723 B2
21
22
TABLE 3
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.
Biopsy tissue layers and pathological parameters
Anatomy Location Pathology Parameter
5
Dermal
Dermal
Dermal
Dermal
Subcutaneous
Subcutaneous
Skeletal muscle
Skeletal muscle
Skeletal muscle
Superficial neovascularization
Pylogranulomatous inflammation
Overlying erosion & inflammatory crusting
Focal fibrosis
Pylogranulomatous inflammation with intralesional
collagen necrosis
Lymphacytic and plasmalytic inflammation
Lymphacytic and plasmalytic and eosinophilic
inflammation
Myocyte degeneration/necrosis
Fibrosis
10
TABLE 4
Groups for the Influenza challenge experiment in ferrets
15
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., JGene 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
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
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.
This study tested the efficacy of HA, NA and M2e-NP
DNA vaccine delivered IM followed by electroporation using
the CELLECTRATM 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
20
Vaccine
Total
Dose (mg)
vaccine in
per
Total
Group Plasmids/Antigens Plasmid volume
1
None (pVAX only)
0 mg
2
H5 + pVAX
0.2mg
3
NA+pVAX
0.2 mg
4
H5, NA, M2e-NP
0.2 mg
0.6 mg
in 0.6 mL
0.6mg
in 0.6 mL
0.6 mg
in 0.6 mL
0.6 mg
in 0.6 mL
4
4
4
4
25
Hemagglutination Inhibition (HI) Assay
Sera were treated with receptor destroying enzyme (RDE)
by diluting one part serum with three parts enzyme and incu30 bated overnight in 37° C. water bath. The enzyme was inactivated by 30 min incubation at 56°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
35 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
40
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
45 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
50
two immunizations and were also able to inhibit a Glade 2
H5N1 virus. In other words, the HI assay was positive against
both viral strains despite the consensus HA strain was based
on Glade 1 viruses.
55 Data Analysis
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 ofp<0.05 was set as the level of
60
statistical significance.
Ferret Influenza Challenge
The results of the influenza challenge are depicted in FIGS.
5 and 6. Control animals lost 25% of their body weight on
65 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 tem-
US 8,133,723 B2
24
23
peratures were elevated in control animals until all control
All plasmids were formulated at 10 mg/mL in water for
animals were either found dead or euthanized by Day 8 (FIG.
injection+1% LGS, as described in previous examples,
6). All animals vaccinated, regardless of which vaccine regiabove, and mixed into a single solution PER STUDY GROUP
men, survived the challenge and showed fewer signs of infec(S) (Groups C, D, G, and H, in above table, Table 6). The
tion as compared to the control animals as evidenced by their 5 correct injection volume for each group designated IM CELclinical scores (Table 5). Control animals worsen as far as
LECTRATM EP (VGX Pharmaceuticals), ID CELLECclinical scores (nasal discharge, cough, lethargy), and died
TRATM EP (VGX Pharmaceuticals), and IM Syringe was
between day 5 and day 7 post-challenge. As shown in Table 5,
calculated. For the ID administration, if the required injection
the severity of the clinical scores in vaccinated animals was 10 volume surpassed 100 tL per site, the formulation was split
inversely correlated with the antibody titers (higher antibody
into a number of injection sites (2, 3, or 6 depending on how
titers, lower clinical scores, better clinical outcome).
many total mg of vaccine were administered). The animals
TABLE 5
Results for Challenged Ferrets
Post-challenge Observations
Vaccines
Ferret Day 1
Control
(pVAX)
H5
M2-NP
H5 + M2-NP + NA
891
890
877
876
878
879
888
889
881
880
883
882
885
884
886
887
0l
01
01
01
01
01
01
01
01
0 1
01
01
01
0 1
11
0 1
HI Titers 3 wks
Day 2
Day 3
Day 5
Day 6
Day 7
1 1
01
01
01
01
01
01
01
01
0 1
11
01
01
0 1
11
11
1 1
11
11
01
11
01
01
01
10
00
11
11
01
0 1
01
00
0l
0 1*
02
01
01
01
01
01
01
0 1
01
01
01
0 1
01
01
0l
11*
23
23
01
01
01
01
01
0 1
01
01
01
0 1
01
01
FD
1 3*
01
01
01
01
11
0 1
11
12
01
0 1
11
11
Day 8
01
01
01
01
01
0 1
01
02
01
0 1
01
01
Day 9
Pre-challenge
01
01
01
01
01
0 1
01
01
01
0 1
11
01
<20
<20
<20
<20
40
320
160
320
<20
<20
<20
<20
1280
320
160
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
40
Intradermal Delivery Comparisons with
Intramuscular Delivery in Primates
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.
45
that received IM injection(s) were given the entire formulation in one single site.
The CELLECTRATM 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.
TABLE 6
Study
Group DNA Constructs
A
B
C
D
DNA 6+ 9
DNA 6+ 9
DNA 1+ 6+ 9+ 10
Negative Control
Nr. Route ofAdmin
5 IM CELLECTRA TM EP
5 ID CELLECTRA TM EP
5 IM Syringe
5 N/A
Dose
1 mg/Const
1 mg/Const
1 mg/Const
DNA Construct #
Encoding Antigen
1
6
9
10
Non-influenza antigen control plasmid
Influenza H5 consensus
Total
DNA (mg)
2
2
4
0
US 8,133,723 B2
25
26
Hemagglutination Inhibition (HI) Assay monkey sera
three times the average titers of the IM group alone
were treated with receptor destroying enzyme (RDE) by
(*P<0.03). Non-treated controls did not exhibit any HI titers.
diluting one part serum with three parts enzyme and incubated overnight in 37° C. water bath. The enzyme was inacExample 5
tivatedby 30 min incubation at 56°C. followed by addition of 5
six parts PBS for a final dilution of 1/10. HI assays were
performed in V-bottom 96-well microtiter plates, using four
Cross Protection in Primates
HA units of virus and 1% horse red blood cells. The data
presented herein are the results after the second immunization 10 Using Delivery Method ID Injection Followed by Elec(bleed collected before the third immunization).
troporation (EP)
Studies in non-human primates with the influenza vaccine
HI titers were measured three weeks after the second
(including H5, NA and M2e-NP consensus antigens, see
immunization. The results can be seen displayed in the graph
above) indicated that ID injection followed by electroporain FIG. 8. Monkeys receiving the HA plasmid vaccine via ID 15 tion elicited higher antibody responses to the vaccine antigens
injection followed by electroporation demonstrated more
than in IM injections. In one of our non-human primate studthan twice the average titers of the IM+EP group and almost
ies (NHP) animals were vaccinated per Table 7.
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
2
5
H5, NA,
IM
1 mg/construct
3
5
M2e-NP
IM
1 mg/construct
4
5
H5, NA,
ID
1 mg/construct
0.2 Amps, 2X2 pulses, 52 msec, 1 sec
between pulses
M2e-NP
between pulses
between pulses
M2e-NP
between pulses
35
Each animal received three vaccinations, and HAI titers and
microneutralization were performed for both the same Glade
and cross-clades. As shown, the consensus vaccine offered
broad protection not only within the same Glade, but also
cross-clades. Results are included in Table 8.
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
110 (0-320)415
205 (0-320)415
80 (40-160)
592 (40-1280)
36(20-80)
84(20-160)
276 (20-640)
VGX-3400 IM
VGX-3400 ID
3rd immunization
160 (80-320)
36 (20-80)
664 (40-1280) 120 (20-320)
VGX-3400 IM
VGX-3400 ID
Microneutralization
3rd immunization
288 (160-640)
416 (160-640)
32 (0-80)315
VGX-3400 IM
VGX-3400 ID
144 (40-360)
740 (20-2560)
8 (0-40)115
32 (0-80)2/5
88 (0-160)415
96 (0-320)315 296 (0-1280)315 1172 (20-2560)
64 (0-160) 2/5 145 (20-320)
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.
LOLT
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663222222 2663 33 23 66663 6633363 223 3363 333663
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US 8,133,723 B2
29
30
-continued
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 2
LENGTH: 568
TYPE: PRT
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Influenza H5N1 HA consensus sequence
<400> SEQUENCE: 2
Met 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
OZOT
D66D6jD62D 66DDD66jP BD DDD66P DDDOP2D26D 66DIJBIBDB 6D62D6jDjP
096
02~0660~26 2002~2266 002260022 000666 600066002 20606600
006
66022066 60066606 000~266 066006060 0002~0606 20622666
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08L
DD 6D PID6eDD662 0066022062 0000660260 026~26~600 OD D6ID6e
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US 8,133,723 B2
34
33
-continued
cctgtgagcg ccaacggcgc ctacggcgtg aagggcttca gcttcaagta cggcaacggc
1080
gtgtggatcg gccggaccaa gagcaccaac agcagatccg gcttcgagat gatctgggac
1140
cccaacggct ggaccgagac cgacagcagc ttcagcgtga agcaggacat cgtggccatc
1200
accgactggt ccggctacag cggcagcttc gtgcagcacc ccgagctgac cggcctggac
1260
tgcatccggc cctgcttttg ggtggagctg atcagaggca ggcccaaaga gagcaccatc
1320
tggaccagcg gcagcagcat cagcttttgc ggcgtgaaca gcgacaccgt gagctggtcc
1380
tggcccgacg gcgccgagct gcccttcacc atcgacaagt acccctacga cgtgcccgac
1440
tacgcctgat gagcggccgc gagctc
1466
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 4
LENGTH: 476
TYPE: PRT
FEATURE:
OTHER INFORMATION: Influenza H1N1&HSN1 NA consensus sequence
<400> SEQUENCE: 4
Met 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
US 8,133,723 B2
35
36
-continued
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
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 5
LENGTH: 875
TYPE: DNA
FEATURE:
OTHER INFORMATION: Influenza H1N1&HSN1 Ml consensus sequence
<400> SEQUENCE: 5
ggtaccggat ccgccaccat ggactggacc tggattctgt tcctggtggc cgctgccacc
60
cgggtgcaca gcatgagcct gctgaccgag gtggagacct acgtgctgtc catcatcccc
120
agcggccctc tgaaggccga gatcgcccag cggctggaag atgtgttcgc cggcaagaac
180
accgacctgg aagccctgat ggaatggctg aaaacccggc ccatcctgag ccccctgacc
240
aagggcatcc tgggcttcgt gttcaccctg accgtgccca gcgagcgggg cctgcagcgg
300
cggagattcg tgcagaacgc cctgaacggc aacggcgacc ccaacaacat ggaccgggcc
360
gtgaagctgt acaagaagct gaagcgggag atcaccttcc acggcgccaa agaggtggcc
420
ctgagctaca gcacaggcgc cctggccagc tgcatgggcc tgatctacaa ccggatgggc
480
accgtgacca ccgaggtggc cttcggcctg gtgtgcgcca cctgcgagca gatcgccgac
540
agccagcaca gatcccaccg gcagatggcc accaccacca accccctgat ccggcacgag
600
aaccggatgg tcctggcctc caccaccgcc aaggccatgg aacagatggc cggcagcagc
660
gagcaggccg ccgaagccat ggaagtggcc agccaggcca ggcagatggt gcaggccatg
720
cggaccatcg gcacccaccc cagcagcagc gccggactgc gggacgacct gctggaaaac
780
ctgcaggcct accagaaacg gatgggcgtg cagatgcagc ggttcaagta cccctacgac
840
gtgcccgact acgcctgatg agcggccgcg agctc
875
<210> SEQ ID NO 6
<211> LENGTH: 279
US 8,133,723 B2
37
38
-continued
<212>
<213>
<220>
<223>
TYPE: PRT
FEATURE:
OTHER INFORMATION: Influenza HlNl&HSN1 Ml consensus sequence
<400> SEQUENCE: 6
Met 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
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 7
LENGTH: 1700
TYPE: DNA
FEATURE:
OTHER INFORMATION: Influenza H5N1 M2E -NP consensus sequence
<400> SEQUENCE: 7
ggtaccgaat tcgccaccat ggactggacc tggatcctgt tcctggtcgc tgccgccacc
60
agggtgcaca gcagcctgct gaccgaggtg gagaccccca cccggaacga gtggggctgc
120
cggtgcagcg acagcagcga ccggggcagg aagcggagaa gcgccagcca gggcaccaag
180
cggagctacg agcagatgga aacaggcggc gagcggcaga acgccaccga gatccgggcc
240
agcgtgggca gaatggtcgg cggcatcggc cggttctaca tccagatgtg caccgagctg
300
OTT
SOT
OOT
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08
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09
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0
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OE
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ST
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6606006 00060060 66220000 66606066
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600~260
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US 8,133,723 B2
41
42
-continued
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
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US 8,133,723 B2
-continued
<400> SEQUENCE: 10
Met 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
OOZT
DPPD 6PDD PPD DD6DD 66PDDD D6e 6ee6jDD 6D D6DD66PDD6 6D D6666
OTT
DD D 6DDD PD66DD D6 6D66666 D 66j66jPD 666e666jD6 6D 66D
080T
D D66DD6D jPDD6D66D jD D6666D DD 6D66 e6DDD6j6D e66D6jPD66
OZOT
oDPDo66j06 ee6joDoPoe e6eo6Pe6j6 oliP0000 6jDD6D66D jDD D 66D
096
D2266D226 2DDDDD62 2D6D22DDD D~2D6D66
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D22D6622D 66DDDDDD 6D6D666D 6DD6D 6622D66D6 6D6
Ob8
000666
08L
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6226DDDD D6D66D
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DD6D6D6D DD66D2262 DDD66D66 DD66D22D
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6000066o 6o60066 6600666o 6006006o
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09E
DD6D22D 6D2~DD662 2D666D66 66D6DD 6666
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06606066 20000606 66060006 060~2600 00602262 60660660
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or
D22DD666 26D~26DD2 6D22DDD
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6I66IDD616 DDDD6 26DDD6D
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0
S£S
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TPA nel nel TPA nel .zaS zaS pW TPA zLLL .zaS .tAJ eTI pw nel eTI
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S6b
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US 8,133,723 B2
49
50
-continued
ggcaagctga accggctgat cggcaagacc aacgagaagt tccaccagat cgaaaaagaa
1260
ttcagcgagg tggagggccg gatccaggac ctggaaaagt acgtggagga caccaagatc
1320
gacctgtgga gctacaacgc cgagctgctg gtcgccctgg aaaaccagca caccatcgac
1380
ctgaccgaca gcgagatgaa caagctgttc gagcggacca agaagcagct gcgggagaac
1440
gccgaggaca tgggcaacgg ctgctttaag atctaccaca agtgcgacaa cgcctgcatc
1500
ggcagcatcc ggaacggcac ctacgaccac gacgtgtacc gggacgaggc cctgaacaac
1560
cggttccaga tcaagggcgt ggagctgaag agcggctaca aggactggat cctgtggatc
1620
agcttcgcca tcagctgctt tctgctgtgc gtggccctgc tgggattcat catgtgggcc
1680
tgccagaagg gcaacatccg ctgcaacatc tgcatctgat gactcgagct c
1731
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 12
LENGTH: 566
TYPE: PRT
FEATURE:
OTHER INFORMATION: Influenza H3 consensus sequence
<400> SEQUENCE: 12
Met 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
US 8,133,723 B2
52
51
-continued
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
<210>
<211>
<212>
<213>
<220>
<223>
SEQ ID NO 13
LENGTH: 1791
TYPE: DNA
FEATURE:
OTHER INFORMATION: Influenza H5 consensus sequence
<400> SEQUENCE: 13
atggactgga cctggatcct gttcctggtg gccgctgcca cccgggtgca cagcatggaa
60
aagatcgtgc tgctgttcgc catcgtgagc ctggtgaaga gcgaccagat ctgcatcggc
120
taccacgcca acaacagcac cgagcaggtg gacaccatca tggaaaaaaa cgtgaccgtg
180
acccacgccc aggacatcct ggaaaagacc cacaacggca agctgtgcga cctggacggc
240
gtgaagcccc tgatcctgcg ggactgcagc gtggccggct ggctgctggg caaccccatg
300
tgcgacgagt tcatcaacgt gcccgagtgg agctacatcg tggagaaggc caaccccgtg
360
aacgacctgt gctaccccgg cgacttcaac gactacgagg aactgaagca cctgctgtcc
420
SZT
OZT
STT
nT0 and sTH usy aTI 6r .zag na1 nel sTH sAZ naZ nT0 nT0 .tAy dsy
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SOT
OOT
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S6
06
S8
TPA aTI zAy aaS dzy nT0 o.zd TPA usV aTI aiid nT0 dsy sAD IGH ozd
08
SL
OL
S9
usy AID na1 naZ dzy ATE) 2Ty TPA .zag sAD dsy 6r naI aII n91 o.zd
09
SS
OS
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US 8,133,723 B2
55
56
-continued
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 Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile
340
345
350
Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
355
360
365
Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
370
375
380
Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser
385
390
395
400
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405
410
415
Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
420
425
430
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met
435
440
445
Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450
455
460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly
465
470
475
480
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
485
490
495
Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
500
505
510
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515
520
525
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
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-continued
caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
4140
actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
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acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
4260
cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 4320
gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
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tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
4560
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4652
20
What is claimed is:
1.
A DNA plasmid vaccine capable of generating in a
mammal an immune response against a plurality of influenza
virus subtypes, comprising:
a DNA plasmid 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, the
consensus influenza antigen comprising consensus
hemagglutinin(HA)subtype HI and
a pharmaceutically acceptable excipient;
the DNA plasmid comprising a promoter operably linked
to a coding sequence that encodes the consensus influenza antigen HA subtype HI, and the DNA plasmid
vaccine having a concentration of total DNA plasmid of
I mg/ml or greater,
wherein the coding sequence that encodes the consensus
HA subtype HI comprises SEQ ID NO:9.
2. The DNA plasmid vaccine of claim 1, wherein the coding sequence that encodes the consensus HA subtype HI
further comprises a coding sequence that encodes an IgG
leader sequence attached at the 5' end of the coding sequence
that encodes the consensus HA subtype HI and is operably
linked to the promoter.
3. The DNA plasmid vaccine of claim 1, wherein coding
sequence that encodes the consensus HA subtype HI further
comprises a coding sequence that encodes a polyadenylation
sequence attached at the 3' end of the coding sequence that
encodes the consensus HA subtype HI.
4. The DNA plasmid vaccine of claim 1 wherein the pharmaceutically acceptable excipient is an adjuvant.
5. The DNA plasmid vaccine of claim 4, wherein the adjuvant is selected from the group consisting of: IL-12 and IL-15.
6. The DNA plasmid vaccine of claim 1, wherein the pharmaceutically acceptable excipient is a transfection facilitating agent.
7. The DNA plasmid vaccine of claim 6, wherein the transfection facilitating agent is a polyanion, polycation, or lipid.
8. The DNA plasmid vaccine of claim 6, wherein the transfection facilitating agent is poly-L-glutamate.
9. The DNA plasmid vaccine of claim 6, wherein the transfection facilitating agent is poly-L-glutamate at a concentration less than 6 mg/ml.
10. The DNA plasmid vaccine of claim 1, wherein said
DNA plasmid vaccines comprises a plurality of different
DNA plasmids; said DNA plasmid vaccine further comprising
one ofthe plurality of DNAplasmids comprises a sequence
that encodes a consensus NA, and
one ofthe plurality of DNAplasmids comprises a sequence
that encodes a consensus M2e-NP.
25 11. The DNA plasmid vaccine of claim 10, wherein the
consensus NA is SEQ ID NO:4.
12. The DNA plasmid vaccine of claim 10, wherein the
sequence that encodes consensus NA is SEQ ID NO:3.
30
consensus M2e-NP is SEQ ID NO:8.
sequence that encodes consensus M2e-NP is SEQ ID NO:7.
15. The DNA plasmid vaccine of claim 1, further compris35 ing:
a DNA plasmid comprising a sequence that encodes a
consensus H3,
consensus H5,
40
consensus NA, and
consensus M2e-NP.
45
consensus H3 is SEQ ID NO:12.
sequence encoding consensus H3 is SEQ ID NO: 11.
consensus NA is SEQ ID NO:4.
50
sequence encoding consensus NA is SEQ ID NO:3.
20. The DNA plasmid vaccine of claim 15, wherein consensus M2e-NP is SEQ ID NO:8.
55
sequence encoding consensus M2e-NP is SEQ ID NO:7.
mammal is a primate.
60 mammal is a primate.
immune response is a humoral response.
immune response is a cellular response.
65
immune response is a combined humoral response and cellular response.
US 8,133,723 B2
71
72
27. A method of eliciting an immune response against a
38. The DNA plasmid of claim 32, wherein the consensus
plurality of influenza virus subtypes in a mammal, comprisM2e-NP is SEQ ID NO:8.
ing,
39. The DNA plasmid of claim 32, wherein the encoding
delivering a DNA plasmid vaccine of claim 1 to tissue of
sequence that encodes consensus M2e-NP is SEQ ID NO:7.
the mammal, and
5
40. The DNA plasmid of claim 32, wherein the DNA
electroporating cells of the tissue with a pulse of energy at
plasmid comprises SEQ ID NO: 15, SEQ ID NO:16 or SEQ
a constant current effective to permit entry of the DNA
ID NO:17.
plasmids in the cells.
41. A DNA molecule comprising a nucleotide sequence
28. The method of claim 27, wherein the delivering step
comprising SEQ ID NO:9 or a fragment thereof that is an at
comprises: injecting the DNA plasmid vaccine into intrader- io least 1380 or more nucleotide fragment of SEQ ID NO:9.
mic, subcutaneous or muscle tissue.
42. The DNA molecule of claim 41 comprising a nucle29. The method of claim 27, comprising:
otide sequence that is a fragment of SEQ ID NO:9 that is at
presetting a current that is desired to be delivered to the
least 1440 or more nucleotide fragment of SEQ ID NO:9.
tissue; and
43. The DNA molecule of claim 41 comprising a nucleelectroporating cells of the tissue with a pulse of energy at 15 otide sequence that is a fragment of SEQ ID NO:9 that is at
a constant current that equals the preset current.
30. The method of claim 29, wherein the electroporating
44. The DNA molecule of claim 41 comprising a nuclestep further comprises: measuring the impedance in the elecotide sequence that is a fragment of SEQ ID NO:9 that is at
troporated cells; adjusting energy level of the pulse of energy
relative to the measured impedance to maintain a constant 20
45. The DNA molecule of claim 41 comprising a nuclecurrent in the electroporated cells; wherein the measuring and
adjusting steps occur within a lifetime of the pulse of energy.
31. The method of claim 29, wherein the electroporating
46. The DNA molecule of claim 41 comprising a nuclestep comprises: delivering the pulse of energy to a plurality of
electrodes according to a pulse sequence pattern that delivers 25 least 1680 or more nucleotide fragment of SEQ ID NO:9.
the pulse of energy in a decentralized pattern.
47. The DNA molecule of claim 41 comprising a nucle32. A DNA plasmid comprising a coding sequence operotide sequence that is a fragment of SEQ ID NO:9 that is at
ably linked to a promoter which functions in a cell of a
mammal, wherein the coding sequence encodes consensus
48. The DNA molecule of claim 41 comprising SEQ ID
influenza antigen hemagglutinin (HA) subtype HI and com- so NO:9.
prises SEQ ID NO:9.
33. The DNA plasmid of claim 32, wherein coding
NO:9 operably linked to a promoter which functions in a cell
of a mammal.
comprises a coding sequence that encodes an IgG leader
sequence attached at the 5' end of the coding sequence that 35 NO:9 and further comprising a coding sequence that encodes
encodes the consensus HA subtype Hi and operably linked to
an IgE leader sequence.
the promoter.
34. The DNA plasmid of claim 32, wherein coding
NO:9 and further comprising a polyadenylation signal linked
thereto.
comprises a coding sequence that encodes a polyadenylation 40
52. The DNA molecule of claim 41 further comprising one
sequence attached at the 3' end of the coding sequence that
or more coding sequences that encode influenza antigens
encodes the consensus HA subtype HI.
selected from the group consisting of: a sequence that
encodes an influenza H3 antigen, a sequence that encodes an
sequence that encodes consensus HA is SEQ ID NO:9.
influenza H5 antigen, a sequence that encodes an influenza
36. The DNA plasmid of claim 32, wherein the consensus 45 NA antigen, and a sequence that encodes an influenza M2eNA is SEQ ID NO:4.
NP antigen.
sequence that encodes consensus NA is SEQ ID NO:3.
UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION
PATENT NO.
APPLICATION NO.
DATED
INVENTOR(S)
: 8,133,723 B2
: 13/158150
: March 13, 2012
: Draghia-Akli et al.
Page 1 of 1
It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
The Title page under item (73) Assignee please add: VGX Pharmaceuticals, Inc., Blue Bell, PA (US)
Signed and Sealed this
Fifteenth Day of May, 2012
David J. Kappos
Director of the United States Patent and Trademark Office

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Transcription

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