Novel vaccines against multiple subtypes of influenza virus.

(54) Title: NOVEL VACCINES AGAINST MULTIPLE SUB-TYPES OF INFLUENZA VIRUSES.

(54) Title: NOVEL VACCINES AGAINST MULTIPLE SUBTYPES OF INFLUENZA VIRUS.

(57) Summary

The present invention relates to plasmid DNA vaccines capable of generating an immune response in a mammal against a plurality of influenza virus subtypes, comprising a plasmid DNA and a pharmaceutically acceptable excipient. The plasmid DNA is capable of expressing a consensual influenza antigen in a mammalian cell in an amount effective to elicit an immune response in the mammal, wherein the consensual influenza antigen comprises consensual hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, ectodomain-nucleo-protein M2 (M2e-NP), or a combination thereof. Preferably, the consensual influenza antigen comprises HA, MA, M2e-NP, or a combination thereof. Plasmid DNA comprises a promoter operably linked to a coding sequence encoding the consensual influenza antigen. Additionally, one aspect of the present invention includes methods of eliciting an immune response against a plurality of influenza virus subtypes in a mammal using the provided plasmid DNA vaccines.

(57) Abstract

An aspect of the present invention is directed towards DNA plasmid vaccines capable of generating in a mammal an immune response against a plurality of influenza virus subtypes, comprising a DNA plasmid and a pharmaceutically acceptable excipient. The DNA plasmid is capable of expressing a consensus influenza antigen in a cell of the mammal in a quantity effective to elicit an immune response in the mammal, wherein the consensus influenza antigen comprises consensus hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, M2 ectodomain-nucleo-protein (M2eNP), 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.

Institute

Mexican Property

Industrial

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PATENT TITLE NO. 339241

<td>Headlines):</td><td>THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA; VGX PHARMACEUTICALS, LLC.</td>

<td>Home:</td><td>3160 Chestnut Street, Suite 200, Philadelphia, Pennsylvania, 19104-6283, USA; 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, Pennsylvania, 19422, USA</td>

Denomination: NOVEL VACCINES AGAINST MULTIPLE SUB-TYPES OF INFLUENZA VIRUSES.

<td>Classification:</td><td>Int.CI.8: A61K39 / 145; C12N15 / 00</td>

Inventor (s): RUXANDRA DRAGHIA-AKLI; DAVID B. WEINER; JIAN YAN; DOMINICK LADDY

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MX / a / 2013/003382 'I

II vjgggi

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to presentation

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November 2008

Divisional Patent Number: 308182

PRIORITY

Country:

US *

Date:

Number:

November 2007

60/987,284

Validity: Twenty years

Expiration Date: November 12, 2028

The reference patent is granted confusing articles 1, 2, section V, 6, section III, and 58 of the Industrial Property Law.

In accordance with article 23 of I9 Industrial Law, this patent has a validity of twenty years, which cannot be extended, from the filing date of fa 80/10 * 1X1 Vpageuonal and will be subject to the payment of the tattoo to keep Agents rights. .................

"In this title the Industrial property subscribe (Official Journal of 01/2004, 06/16/2005, 01/25/2006, a), subsection iii) 4^orand 12th fra on 07/01/2002, 1 * 07/2004, of the Organic Statute flnel Institu ³° ^{and 5}mKor, Dlrectores

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based on Jo deposed by articles 8 ° Fracdonee M and 7 ° bis 2 of the Law of the Federation '....... ..........

/ 05 / 2008,08 / 01/2 s I and III of the Reg

07/2004 and 7/09/2007); to

Mexican Property In

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formed on 08/02/1994, 1/25 / 1W1W6, 12/26/1897, 05/17/1999, / 2010, 01/27/2012 and 04/09/2012); Articles 1, f fraction V ad Industrial (DOF14 / 12/1999, a), subsection iii), 16 sections I and III and 999, amended 10/10/2002, Í9 / 07/2004, wwswwcs: Departmental other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

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Issue Date: May 18, 2016

DIVISIONAL DEPUTY DIRECTOR OF PATENT FUND EXAMINATION, AREAS

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INST1T1 FROM&gt;

NOVEL VACCINES AGAINST MULTIPLE SUB-TYPES OF VlKÚS BE

INFLUENZA

FIELD OF THE INVENTION

The present invention relates to improved influenza vaccines, improved methods for inducing immune responses, and for prophylactically and / or therapeutically immunizing individuals against influenza.

BACKGROUND OF THE INVENTION

The use of nucleic acid sequences for vaccines against animal and human diseases has been studied. Studies have focused on effective and efficient means of delivery in order to produce a necessary expression of the desired antigens, resulting in an immunogenic response and ultimately the success of this technique. One method for delivery of nucleic acid sequences such as plasmid DNA is the electroporation (EP) technique. The technique has been used in human clinical trials to deliver anti-cancer drugs, such as bleomycin, and in many pre-clinical studies in large numbers of animal species.

The influenza virus genome is contained in eight simple (unpaired) RNA strands that code for eleven proteins (HA, NA, NP, MI, M2, NS1, NEP, PA, PB1, ir.

PB1-F2, PB2). The segmented nature of the genome Tija allows the exchange of entire genes between different viral strains during cell co-habitation. The eight RNA segments are: HA, which encodes hemagglutinin (approximately 500 hemagglutinin molecules are needed to compose a virion); NA, which encodes neuraminidase (approximately 100 neuraminidase molecules are needed to make a virion); NP, which encodes nucleoprotein; M, which codes for two matrix proteins (MI and M2) by using different reading tables of the same RNA segment (approximately 300 matrix protein molecules are needed to make a virion); NS, which encodes two different nonstructural proteins (NS1 and NEP) using different reading tables from the same RNA segment; PA, which encodes an RNA polymerase; PB1, which encodes an RNA polymerase and PB1-F2 protein (induces apoptosis) by using different reading tables of the same RNA segment; and PB2, which encodes an RNA polymerase.

Hemagglutinin influenza (HA) is expressed on the surface of viral influenza particles and is responsible for the initial contact between the virus and its host cell. HA is a well known immunogen. The H5N1 strain of influenza A, a strain of avian influenza, particularly threatens the human population due to its HA (H5) protein which, if lightly reclassified genetically via natural mutation, has increased ineffectiveness of mostly human cells in Comparison to infection of infants and immunodepressed people with the other strains

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older or adult humans

Viral H5N1 is sometimes correlated to poor clinical outcome

Therefore, protection against the H5N1 influenza strain is a major need for the public.

There are two classes of anti-influenza agents available, influenza A cell entry / non-coating inhibitors (such as amantadine and rimantadine antivirals) and neuraminidase inhibitors (such as antivirals oseltamivir, zanamivir). These antiviral agents inhibit the cellular release of influenza A and B. Interest in the use of these agents has been reported due to the findings of virus strains resistant to these agents.

Influenza vaccines are a popular seasonal vaccine, and many people have experienced such vaccines. However, vaccines are limited in their protective performance because the vaccines are specific for certain virus subtypes. The Centers for Disease Control and Prevention promotes vaccination with a flu shot that is a vaccine that contains three influenza viruses (killed viruses): one virus A (H3N2), one virus A (H1N1), and one virus B. They also report that the viruses in the vaccine change each year based on continuing international observations and estimates from scientists about. ')

INDUSTRIAL virus strains will circulate in a given year. Thus, it is obvious that vaccines are limited to subtype predictions, and the availability of a specific vaccine for that subtype.

There remains a need for effective influenza vaccines that are inexpensive and effective across numerous subtypes. Furthermore, there remains a need for an effective method of administering vaccines. DNA to a mammal to provide influenza immunization either prophylactically or therapeutically.

BRIEF DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a plasmid DNA vaccine capable of generating an immune response in a mammal against a plurality of influenza virus sub-types, comprising a plasmid DNA and a pharmaceutically acceptable excipient. The plasmid DNA is capable of expressing a consensual influenza antigen in a mammalian cell in an amount effective to produce an immune response in the mammal, wherein the consensual influenza antigen comprises consensual hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, ectodomain-nucleo-protein M2 (M2e-NP), or a combination thereof. Preferably, the consensual influenza antigen comprises HA, NA, M2e-NP, or a combination thereof.

Plasmid DNA comprises a promoter

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linked to a coding sequence that encodes the consensual influenza antigen.

Preferably, the plasmid DNA vaccine is one that has a total plasmid DNA concentration of 1 mg / ml or greater.

Another aspect of the present invention relates to plasmid DNA capable of expressing a consensual influenza antigen in a mammalian cell, the consensus influenza antigen comprising consensual hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, ectodomain-nucleo-protein M2 (M2e-NP), or a combination of these. Preferably, the consensus influenza antigen comprises HA, NA, N2e-NP, or a combination thereof. Plasmid DNA comprises a promoter operably linked to a coding sequence encoding the consensual influenza antigen.

<td>Other</td><td>aspect of</td><td>the</td><td>present invention relates to</td>

<td>methods for</td><td>produce</td><td>a</td><td>immune response against a</td>

<td>plurality of</td><td>subtypes</td><td>of</td><td>influenza virus in a</td>

mammal. The methods include delivering a plasmid DNA vaccine to mammalian tissue, the plasmid DNA vaccine comprising a plasmid DNA capable of expressing a consensual influenza antigen in a mammalian cell to elicit an immune response in the mammal, the consensual influenza antigen comprising HA, NA, M2e-NP

<img file="MX339241B_D0015.tif" />

consensual or a combination thereof, and the -e INS

OF THE?. ;

of tissue cells with a pulse of energy in a constant current effective to allow the entry of ADÑ '^orplasmid in cells.

BRIEF DESCRIPTION OF THE FIGURES

The numerous objectives and advantages of the present invention can be much better understood by those skilled in the art by referring to the accompanying figures, where:

Figure 1 displays a schematic representation (plasmid maps) of the plasmid DNA constructs used in the studies described here. Consensual HA, NA, and M2e-NP constructs are generated by analyzing primary virus sequences from 16 H5 viruses that are fatal to humans in recent years, and about 40 human NI viruses (Los Alamos National Laboratory's Influenza Sequence Database). After generating the consensual sequences, the constructs are optimized for mammalian expression, including the addition of a Kozak sequence, codon optimization, and RNA optimization. These constructs are then subcloned into the pVAX vector (Invitrogen, Carlsbad, CA). Plasmids pGX2001 (HA consensual), pGX2002 (NA consensual), pGX2003 (M2e-NP consensual) are shown. The unfolded plasmid pCMVSEAP encodes the Reporter Protein Secreted Embryonic Alkaline Phosphatase (SEAP).

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INSTI'Í '.

DE L Figure 2 displays a bar graph of

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The results of serum HI titrations of ee'Lilu in the * “dra- 35 after injection. The highest titers are found in the group administered with 2 mg of plasmid expressing HA at a current setting of 0.5A (20 + 40; * P = 0.11 versus 2 mg / 0.3A and * P = 0.02 versus 2 mg / Ο.ΙΑ). Three groups administered with descending doses of plasmid and electroporated at 0.5A also demonstrate descending HI titers.

Figure 3 displays a bar graph of the IFN-γ ELISpot counts. Counts are highest in pigs administered with 2 mg of HA and 2 mg of NA plasmid vaccine (for a total of 4 mg of plasmid) and electroporated with 0.3A current (2000 spots) and in the group administered with 0.8 mg of HA and 0.8 mg of plasmid DNA vaccine (for a total of 1.6 mg of plasmid) electroporated with 0.5A current (934 spots). For comparison purposes, the cellular immune responses of an immunized control group are plotted.

Figures 4A and 4B display bar graphs showing muscle biopsy results from pigs treated on day and day 35: Figure 4A displays a bar graph showing the average pathology scores for all groups. Figure 4B displays a graph of

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electroporated at 0.5A exhibit the highest paTUlugl · score (* P &lt;0.0002 compared to controls). Pathology scores are significantly reduced on day 35 compared to day 14 in all groups (P&gt; 0.05)

<td>except for</td><td>the group</td><td>0.3 mg / 0.3</td><td>group</td><td>A (P = 0.057) and</td><td> 2.4</td>

<td>mg / 0.1 group</td><td>A (P = 1.0).</td><td></td><td></td><td></td><td></td>

<td>The</td><td colspan="2">figure 5 displays the</td><td>change</td><td>percentage in</td><td>weight</td>

<td>ferrets</td><td>after</td><td colspan="2">of the challenge</td><td>with virus</td><td>H5N1</td>

(A / Viet / 1203/2004 (H5N1) / PR8-IBCDC-RG). Ferrets vaccinated with HA, HA + M2e-NP or HA + M2eNP + NA lose significantly less weight than control animals (* P &lt;0.005 versus controls) on day 9 after the challenge period. An animal in the HA vaccine group actually gains weight after the challenge.

Figure 6 displays a graph showing ferrets' body temperatures for 9 days after challenge. Control animals show higher body temperatures than vaccinated animals. Body temperature on day 5 is not represented as measured at a different time of day and all temperatures regardless of group are lower.

Figure 7 displays a bar graph of HI assessment results in ferrets after

<td>vaccination; he</td><td>test</td><td>I know</td><td>makes</td><td>using</td><td>mess¹</td><td>[reg</td>

<td>classifiers</td><td>obtained</td><td>of the</td><td>Center</td><td>for him</td><td>Otrol</td><td>of</td>

<td>Diseases:</td><td>strains</td><td>of</td><td>influenza</td><td colspan="2">A / Viet / 1203/04</td><td>or</td>

Indo / 05/2005.

Figure 8 displays a bar graph of HI titration results measured three weeks after the second immunization. Macaques immunized with ID followed by EP show significantly higher HI scores than all other groups (P &lt;0.03). Untreated controls do not exhibit some of the HI ratings.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviated, or shortened, definitions are provided to aid in understanding the preferred embodiments of the present invention. The abbreviated definitions given here are neither exhaustive nor contradictory to the definitions as understood in the field or the meaning found in the dictionaries. Abbreviated definitions are given herein to supplement or more clearly define definitions known in the art.

Definitions

Sequence homology for nucleotides and amino acids as used herein can be determined using FASTA, BLAST, and BLAST Gapped (Altschul et al., Nuc.

Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP * software (D; L.

DLL /. . ·

Swofford, Sinauer Associates, Massachusetts). Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, which is incorporated herein by reference in its whole). BLAST analysis software is available to the public through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). A measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide sequences can occur by chance. For example, one nucleic acid is considered similar to another if the probability of smallest addition compared to the test nucleic acid with the other nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and more preferably less than about 0.001. Percent similarity can be calculated using PAUP * 4.0bl0 software (DL Swofford, Snauer Associates, Massachusetts). The average similarity of the consensual sequence is calculated compared to all the sequences in the phylogenetic tree.

As used herein, the term genetic construct or nucleic acid construct is used interchangeably and refers to DNA or xf nucleic acid molecules of nucleotide sequence that encode the encoding sequence, or encoding sequence, includes initiation signals and termination operably linked to regulatory elements including a promoter and capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.

express contain linked

As used refers to such a protein herein, the term to constructs of elements sequence so that of the individual, the sequence can form nucleic acid that encoding operable necessary regulators that encode a when present in the cell encoding will express itself.

The term constant current is used herein to define a current that is received or experienced by a tissue, or cells that define the tissue, over the duration of an electrical pulse delivered to the same tissue. The electrical pulse is supplied from the electroporation devices described here. This current remains at a constant amperage in the tissue for the life of an electrical pulse because the electroporation device provided here has a feedback element, preferably having instantaneous feedback. The feedback element can measure the resistance of the fabric (or

-Μ. χνΐν.: 3. i ·!. ·· cells) throughout the duration of the pulse and p8? Gygear that the electroporation device alters its electrical energy output (e.g., increased voltage) so that the current in the same tissue remains constant through the electrical pulse (in the order of microseconds), and from pulse to pulse. In some embodiments, the feedback element comprises a controller.

The term feedback or current feedback is used interchangeably and means the active response of the provided electroporation devices, which comprises measuring the current in the tissue between electrodes and altering the power output supplied by the EP device accordingly, in order to keep the current at a constant level. This constant level is pre-set by a user prior to the initiation of a pulse sequence or electrical treatment. Preferably, the feedback is performed by the electroporation component, e.g. controller, of the electroporation device, since the electrical circuit in this is capable of continuously monitoring the current in tissue between electrodes and compares the monitored current (or current within tissue) to a pre-set current and continually makes energy output adjustments to keep the monitored current at pre-set levels. In some embodiments, the feedback loop is instantaneous as is analog closed-loop feedback. ¹

The terms electroporation, electropermeabilization, or electro-kinetic enhancement (EP) cuino is used interchangeably herein to refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; Their presence allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water to pass from one side of the cell membrane to another.

The term decentralized current is used herein to define the pattern of electrical currents delivered from the various needle electrode arrangements of the electroporation devices described herein, where the patterns minimize, or preferably eliminate, the occurrence of thermal charge related to electroporation in any area of ​​tissue that is electroporated.

The term feedback mechanism as used herein refers to a process performed by software or hardware (or firmware), the process of which receives and compares the desired tissue impedance (before, during, and / or after the supply of power pulse ) with a present value, preferably current, and adjusts the pulse of power supplied to achieve the preset value. The term impedance is used here when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus allowing '\ • ¿e ia;^:λ · 'λ ·. '· •' i / i; ¡M.us' ..-. I / λ comparisons with the preset current. In a preferred embodiment, the feedback mechanism is performed by an analogous closed loop circuit.

The term "immune response" is used herein to mean the activation of a host immune system, eg, of a mammal, in response to the introduction of consensual influenza antigen via the provided plasmid DNA vaccines. The immune response may be in the form of a cellular or humoral response, or both.

The term consensual or consensual sequence is used herein to mean a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of a multiple subtype alignment of a particular influenza antigen, which can be used to induce broad immunity against multiple subtypes or serotypes of a particular influenza antigen. Consensual influenza antigens include HA, including consensual H1, H2, H3, or H5, NA, NP, matrix protein, and nonstructural protein. Also, synthetic antigens such as fusion proteins, eg, M2e-NP, can be engineered to consensual sequences (or consensual antigens).

The term "adjuvant" is used herein to mean any molecule added to the plasmid DNA vaccines described herein to increase diΓ-antigen antigenicity.

Dt LA 7 '; . · Influenza encoded by plasmid DNAs and 'encoding the nucleic acid sequences described hereinafter.

The term subtype or serotype is used interchangeably herein and in reference to influenza viruses, and means genetic variants of an influenza virus antigen such that a subtype is recognized by an immune system other than a different subtype ( or, in other words, each subtype is antigenically different from a different subtype.)

In some embodiments, there is plasmid DNA capable of expressing a consensual influenza antigen in a mammalian cell, the consensus influenza antigen comprising consensual hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, ectodomain-nucleo- M2 protein (M2eNP), or their combination. Preferably the consensual influenza antigen comprises HA, NA, M2e-NP, or a combination thereof. Plasmid DNA comprises a promoter operably linked to a coding sequence encoding the consensual influenza antigen.

In some embodiments, the present invention provides plasmid DNA vaccines that are capable of eliciting in a mammal an immune response against a plurality of influenza virus subtypes, the plasmid DNA vaccines comprising a plasmid DNA and uA. .excipient

ÍKX INSTITUTE

FROM THE PHARMACEUTICALLY ACCEPTABLE. Plasmid DNA is^IN&amp; á? to de-express a consensual influenza antigen eif'UIlá Cell¹ of the··⁰¹¹·¹mammal in an amount effective to produce an immune response in the mammal, wherein the consensual influenza antigen comprises consensual hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, ectodomain-nucleo-protein M2 (M2e-NP) , or their combination. Preferably the consensual influenza antigen comprises HA, NA, M2e-NP, or a combination thereof. The plasmid DNA comprises a promoter operably linked to a coding sequence that encodes the consensual influenza antigen. In some embodiments, the plasmid DNA vaccine is one that has a total plasmid DNA concentration of 1 mg / ml or greater. The immune response can be a cellular or humoral response, or both; preferably, the immune response is the cellular or humoral response.

In some embodiments, the plasmid DNA may further include an IgG leader sequence attached to an N-terminal end of the coding sequence and operably linked to the promoter. Furthermore, in some embodiments, the plasmid DNA may further include a polyadenylation sequence attached to the C-terminus of the coding sequence. In some embodiments, the plasmid DNA is codon optimized.

In some modalities of the present ¡.in ^ eñciórt, las

Γ DE L /) r T ·; :. - /. 1 7&gt;.

plasmid DNA vaccines may additionally include an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of: alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFa, ΤΝΡβ, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell attractant chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the suppressed signal sequence and optionally including the IgE signal peptide. Other genes that may be useful adjuvants include those that encode: MCP-1, MIP-la, MIP-lp, IL8, RANTES, L-selectin, Pselectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA1, Mac-1, pl50.95, PECAM, ISAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF , DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspasa ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, NIK INACTIVE, SAP K, SAP-1, JNK, response genes Interferon, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB,

NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, ΤΑΡ1, ΤΆΡ2 and more fragments! NST, 7 functional. In some preferred embodiments, the adjuvant - · is selected from IL-12, IL-15, CTACK, TECK, or ΜΕΘτ —------- In some embodiments, the pharmaceutically acceptable excipient is a transfection facilitating agent, which may include the following: surface active agents, such as immune stimulating complexes (ISCOMS), incomplete Freund's adjuvant, LPS analog including monophosphoryl lipid A, muramil peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, ions of calcium, 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, poly-L-glutamate is present in the plasmid DNA vaccine in a concentration less than 6 mg / ml. In some embodiments, the concentration of poly-L-glutamate in the plasmid DNA 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 plasmid DNA vaccine can include a plurality of different En DNAs.

In some examples, the different plasmid DNAs include a plasmid DNA comprising a nucleic acid sequence encoding a consensual HA, a plasmid DNA comprising a sequence encoding a consensual NA, and a plasmid DNA comprising a sequence encoding a consensual M2e-NP. In some modalities, consensual HA is consensual HI, consensual H2, consensual H3, or consensual H5. Preferably, the consensual HA is a nucleotide sequence that is SEQ ID NO: 1 (consensual H5N1 HA DNA), SEQ ID NO: 9 (consensual HI DNA), SEQ ID NO: 11 (consensual H3 DNA), or SEQ ID NO: 13 (H5 consensual). The consensual 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 consensual 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, M2e-NP consensual 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 a preferred embodiment, the plasmid DNA vaccine includes a plasmid DNA comprising a sequence encoding a consensual HI, a plasmid DNA comprising a sequence encoding a consensual H2, a plasmid DNA comprising a sequence encoding a consensual H3, a plasmid DNA qje ^^ oijipréndé

INSTITUTE MFXiC / .MÚ a sequence encoding a consensual H5, a plasmid comprising a sequence that nnriifin a consensual NA, and a plasmid DNA comprising a sequence encoding a consensual M2e-NP.

In some embodiments, the plasmid DNA vaccine can include a plurality of different plasmid DNAs, including at least one plasmid DNA that can express

<td>antigens</td><td colspan="5">of consensual influenza and at least one that can</td>

<td>express</td><td>a</td><td>antigen</td><td>subtype</td><td>influenza</td><td>In some</td>

<td>examples,</td><td>the</td><td>different</td><td colspan="3">Plasmid DNA expressing antigen</td>

<td>consensual</td><td colspan="2">include a</td><td>DNA from</td><td>plasmid that</td><td>comprises a</td>

nucleic acid sequence encoding a consensual HA, a plasmid DNA comprising a sequence encoding a consensual NA, and a plasmid DNA comprising a sequence encoding a consensual M2e-NP. In some embodiments, the plasmid DNA vaccine comprises a plasmid DNA that can express a consensual HA antigen, eg, consensual H1, H3, or H5, and a plasmid DNA that can express any of the following influenza A antigens: Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hll, 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 plasmid DNA vaccine comprises a plasmid DNA that can express a consensus NA antigen and a DNA from

<img file="MX339241B_D0019.tif" />

OT INSTITUTE

H6, HV _Λ-Η8, L Η9 ·,: ...._....

plasmid that can express any of influenza A antigens: Hl, H2, H3, H4, H5,

H10, Hll, H12, H13, H14, H15, H16, NI, N2, N3,

N8, N9, NP, MI, M2, NS1, or NEP, or a combination thereof. In some embodiments, the plasmid DNA vaccine comprises a plasmid DNA that can express a consensual M2e-NP and a plasmid DNA that can express any of the following influenza A antigens: Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hll, 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 plasmid DNA vaccine can be delivered to a mammal to elicit an immune response; preferably the mammal is a primate, including a human and a non-human primate, a cow, a pig, a chicken, a dog, or a ferret. Most 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 subtypes of influenza viruses in a mammal. Methods include delivering a plasmid DNA vaccine to mammalian tissue, the plasmid DNA vaccine comprising a plasmid DNA capable of expressing a consensual influenza antigen in a mammalian cell to elicit an immune response in the mammal. , the influenza antigen with a pulse of energy in a constant erect current one to allow entry of the plasmid DNA into the cells.

In some embodiments, the methods of the present invention include the delivery step, which comprises the injection of the plasmid DNA vaccine into intradermal, subcutaneous, or muscle tissue. Preferably these methods include the use of an in vivo electroporation device to pre-adjust a current to be delivered to the tissue; and tissue electroporation cells with a pulse of energy in a constant current that equals the preset current. In some embodiments, the electroporation step further comprises: measuring the impedance in the electroporated cells; adjusting the energy level of the energy pulse relative to the measured impedance to maintain a constant current in the electroporated cells; where the measurement and adjustment stages occur within the lifetime of the energy pulse.

In some embodiments, the electroporation step comprises supplying the energy pulse to a plurality of electrodes according to a pulse sequence pattern that supplies the energy pulse in a decentralized pattern.

In some modalities,

<img file="MX339241B_D0020.tif" />

vaccinations

INDUSTRIAL

<img file="MX339241B_D0021.tif" />

Plasmid DNA of the invention comprises the nucleotide sequences that encode a consensual HA, or a consensual HA and a nucleic acid sequence that encodes 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 encoding HA and H5 H5N1 consensual influenza virus, respectively. SEQ ID NOS: 2 and 14 comprise the amino acid sequence for HA and H5 H5N1 of the influenza virus, respectively. In some embodiments of the invention, the vaccines of the invention comprise SEQ ID NO: 3 or SEQ ID

NO:

Four. SEQ ID NO: 3 comprises the nucleic acid sequence that

H1N1 and

H5N1 (H1N1 / H5N1)

NA.

SEQ ID NO: 4 comprises the amino acid sequence for consensual influenza sequences

H1N1 / H5N1

NA.

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 encoding influenza consensus sequences

H1N1 / H5N1

ME. SEQ ID

NO: 6 comprises the amino acid sequence for consensual influenza sequences

H1N1 / H5N1. 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 encoding the influenza M2E-NP H5N1 consensual sequence. SEQ ID NO: 8 comprises the consensual amino acid sequence for influenza M2E-NP H5N1. In some

<img file="MX339241B_D0022.tif" />

In embodiments of the invention, the vaccines of the invention comprise SEQ ID NO:

or SEQ ID NO: 10. SEQ ID NO: 9 comprises the nucleic acid sequence encoding the consensual HA H1N1 influenza sequences. SEQ ID NO: 4 comprises the amino acid sequence for consensual HA H1N1 influenza sequences. In some embodiments of the invention, the vaccines of the invention comprise SEQ ID NO: 11 or SEQ ID NO: 12. SEQ ID NO: 11 comprises the nucleic acid sequence encoding the consensus sequences of influenza HA H3N1. SEQ ID NO: 12 comprises the amino acid sequence for consensual H3N1 HA influenza sequences. Consensual sequence for H5N1 HA influenza virus strain includes epitope

<td colspan="2">immunodominant</td><td>exposed</td><td>at SEQ</td><td>ID</td><td>NO:</td><td>1 or SEQ</td><td>ID NO: 13.</td><td>The</td>

<td>sequence</td><td>of</td><td>amino acid</td><td>of the</td><td colspan="2">virus</td><td colspan="2">H1N1 influenza</td><td>HE HAS</td>

<td>encoded</td><td>by</td><td colspan="2">SEQ ID NO: 1</td><td>it is</td><td>I KNOW THAT</td><td>ID NO:</td><td>2, and that</td><td>it is</td>

encoded by SEQ ID NO: 13 is SEQ ID NO: 14. The consensual sequence for the H1N1 / H5N1 NA influenza virus includes the immunodominant epitope set forth in SEQ ID NO: 3. The amino acid sequence of strains of the H1N1 / influenza virus H5N1 NA encoded by SEQ ID NO: 3 is SEQ ID NO: 4. The consensual sequence for the H1N1 / H5N1 influenza virus strains includes the immunodominant epitope set forth in SEQ ID NO: 5.

The amino acid sequence of the H1N1 / H5N1 MI influenza virus encoded by SEQ ID NO: 5 is SEQ ID NO: 6. JL ^^ SEJu'lí'ntí'ia · consensual for the H5N1 142E-KF¿eÍ ^ influenza virus .includes the immunodominant epitope set forth in SEQ ID NO: 7. The amino acid sequence of the influenza virus H5N1 M2E-NP encoded by SEQ ID NO: 7 is SEQ ID NO: 8. The vaccines of the present invention can include protein products encoded by the nucleic acid molecules defined above or any of the protein fragments.

The present invention also comprises DNA fragments encoding a polypeptide capable of eliciting an immune response in a mammal substantially similar to that of the non-fragment for at least one influenza subtype. DNA fragments are fragments selected from at least one of the various coding nucleotide sequences of the present invention, include SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 13 and can be any of the following DNA fragments described, as applicable to the specific encoding nucleic acid sequence provided here. In some embodiments, DNA fragments can comprise 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or more, 540 or more, 600 or more, 660 or more, 720 or

<td>plus,</td><td colspan="2">780 or more,</td><td> 840</td><td colspan="2">or more, 900</td><td>or more</td><td> , 960</td><td>or</td><td>plus,</td><td> 1020</td><td>or</td><td>plus,</td>

<td> 1080</td><td>or</td><td>more, 1140</td><td>or</td><td>plus,</td><td>1200 or</td><td>plus,</td><td> 1260</td><td>or</td><td>plus,</td><td> 1320</td><td>or</td><td>plus,</td>

<td> 1380</td><td>or</td><td>more, 1440</td><td>or</td><td>plus,</td><td>1500 or</td><td>plus,</td><td> 1560</td><td>or</td><td>plus,</td><td> 1620</td><td>or</td><td>plus,</td>

1680 or more, or 1740 or more nucleotides. In some idadeS.,

INSTtTUTC DNA fragments can comprise coding sequences for the immunAjinhniin and leader sequences (IgE). In some embodiments, the DNA fragments may comprise less than 60, less than 75, less than 90, less than 12 0, less than 150, less than 180, less than 210, less than 24 0, less than 270, less than 300, less than 360, less than 420, less than 480, less than 540, less than 600, less than 660, less than 720, less than 780, less than 840, less than 900, less than 960, less than 1020, less than 1080, less than 1140, less than 1200, less than 1260, less than 1320, less than 1380, less than 1440, less than 1500, less than 1560, less than 1620, less than 1680, or less than 1740 nucleotides. Preferably, the DNA fragments are fragments of SEQ ID NOS: 1, 3, 7, 9, 11 or 13 and more preferably fragments of SEQ ID NOS: 1, 5, 9, 11 or 13, and even more preferably fragments of SEQ US ID: 1, 9 or 13.

The present invention also encompasses polypeptide fragments that are capable of eliciting an immune response in a mammal substantially similar to that of the no fragment for at least one influenza subtype. Polypeptide fragments are selected from at least one of the several polypeptide sequences of the present invention,

<td>including</td><td>I KNOW THAT</td><td>ID NOS: 2, 4,</td><td> 6,</td><td> 8, 10, 12,</td><td> 14,</td><td>and can be</td>

<td>anyone</td><td>of</td><td colspan="2">the following</td><td>fragments</td><td>of</td><td>polypeptide</td>

<td>described,</td><td colspan="2">as it applies to</td><td>the</td><td>sequence</td><td>of</td><td>polypeptide</td>

<img file="MX339241B_D0023.tif" />

specific provided here. In some modjlj / diaids, the

INSTITUTE _υ i; -DELA .J polypeptide fragments can comprise 15 or iriáhs, ·· - 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, ΙΌ? or ——— more, 150 or more, 180 or more, 210 or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or more, 540 or more, or 565 or more amino acids. In some embodiments the polypeptide fragments can comprise less than 30, less than 45, less than 60, less than 75, less than 90, less than 120, less than 150, less than 180, less than 210, less than 24 0, less than 2 70, less than 300, less than 360, less than 420, less than 480, less than 540, or less than 565 amino acids. Preferably, the polypeptide fragments are fragments of SEQ ID NOS: 2, 4, 8, 10, 12, or 14 and more preferably fragments of SEQ ID

NOS: 2, 6, 10, 12 or 14, and even more preferably fragments of SEQ ID NOS: 2, 10 or 14.

The determination of a fragment that elicits an immune response in a mammal substantially similar to that of the non-fragment for at least one subtype of influenza can be readily determined by one of ordinary skill in the art. The fragment can be analyzed to contain at least one, preferably more, antigenic epitopes as provided by a publicly available database, such as Los Alamos National Laboratory's Influenza Sequence Database. In addition, immune response studies can be routinely assessed using mice and HI assays and ELISpots analysis, the examples below.

<img file="MX339241B_D0024.tif" />

F just like ^AND instít;

: · DE L &lt;IND'U

According to some modalities of the lnVéílClSHT methods of induction or production of an immune response in mammals against a plurality of influenza viruses include administration to mammals: a) the consensual HA protein of the H5N1 influenza strain, its functional fragments, or their encodable sequences that can be expressed; and b) one or more isolated encoding nucleic acid molecules provided herein, protein encoded by said nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing or producing an immune response in mammals against a plurality of influenza viruses comprise administering to mammals: a) the H1N1 influenza strain and influenza strain consensual NA protein H5N1, its functional fragments, or its expressible coding sequences; and b) one or more isolated encoding nucleic acid molecules provided herein, protein encoded by said nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing or producing an immune response in mammals against a plurality of influenza viruses comprise the administration to mammals: a) the influenza strain H1N1 and the consensual MI protein of strain Jl &lt; ^ \ Jfhflufen-za _ _ institute

H5N1, its functional fragments, or their expressible coding and sequences; and b) one or more isolated encoding nucleic acid molecules provided herein, protein encoded by said nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing or producing an immune response in mammals against a plurality of influenza viruses comprise the administration to mammals: a) the consensus protein of influenza strain H5N1 M2E-NP, its functional fragments, or their encodable sequences that can be expressed; and b) one or more isolated encoding nucleic acid molecules provided herein, protein encoded by said nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing or producing an immune response in mammals against a plurality of influenza viruses comprise the administration to mammals: a) the consensus protein of the influenza strain H1N1 HA, its functional fragments , or their encodable sequences that can be expressed; and b) one or more isolated encoding nucleic acid molecules provided herein, protein encoded by said nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing or producing a response; irimune in

IN5TI 'J · &lt;. · · Of the V;

Mammals against a plurality of Inflüéhza viruses comprise administration to mammals: a) the consensual influenza strain H3N1 HA protein, its functional fragments, or its expressible coding sequences; and b) one or more isolated encoding nucleic acid molecules provided herein, protein encoded by said nucleic acid molecules, or fragments thereof.

In some embodiments of the invention, the vaccines of the invention include at least two of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, or any of the combinations of two or more sequences from the aforementioned list.

Vaccinations

In some embodiments, the invention provides improved vaccines by providing proteins and genetic constructs that encode proteins with epitopes that make them particularly effective as immunogens against inducible immune responses. Accordingly, vaccines can be provided to induce a therapeutic or prophylactic immune response.

According to some embodiments of the invention, a vaccine according to the invention is delivered to an individual to modulate the activity of the individual's immune system and thereby increasing rjsj ^ r ^ sta irimune.

INSTITL '·' ·

When a nucleic acid molecule that encodespripin ^ - ·²is taken up by the individual's cells, ^{one to} Nucleotide gpniisnf! ia _ .. is expressed in cells and protein is delivered in this way to the individual. Aspects of the invention provide methods of delivering the protein coding sequences to a nucleic acid molecule such as a plasmid.

In accordance with some aspects of the present invention, compositions and methods are provided which prophylactically and / or therapeutically immunize an individual.

When taken up by a cell, plasmid DNAs can be present in the cell as genetic material

<td>separated.</td><td>Alternatively, RNA can be administered to the</td>

cell, It is also contemplated to provide the genetic construct

<td>like a</td><td>linear mini chromosome including a centromere,</td>

<td>telomeres</td><td>and an origin of replication. Constructs</td>

<td>genetic</td><td>include regulatory elements necessary for the</td>

<td>expression</td><td>gene of a nucleic acid molecule. The</td>

<td>elements</td><td>include: a promoter, an initiation codon, a</td>

stop codon, and a polyadenylation signal. Furthermore, enhancers are sometimes required for gene expression of the sequence encoding the target protein or the immunomodulating protein. If it is necessary that these elements be operably linked to the coding sequence, they should be the desired proteins and that the .reg | a elements must be added if. '· INSTlTuT DE L / ii operable in the individual to whom they are administered. one·--¹

Start codons and stop codons oe are generally considered part of a nucleotide sequence that encodes the desired protein. However, these elements need to be functional in the mammals to which the nucleic acid construct is administered. The start and stop codons must be tabulated with the coding sequence.

Promoters and polyadenylation signals used must be functional within the cells of the individual.

Examples of promoters useful for the practice of the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from simian virus 40 (SV40), promoter from mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) such as the long terminal repeat promoter (LTR) of bovine immunodeficiency virus (BIV), Moloney virus, avian leukosis virus (ALV), cytomegalovirus (CMV) such as CMV immediate initial promoter, Epstein Barr virus (EBV), Rous sarcoma virus (RSV) as well as human gene promoters such as human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein; in other embodiments, promoters can be tissue-specific promoters, such as promoters specific for

USA No. US20040175727, which is incorporated by muscle or medium patent promoters in its entirety.

Examples of polyadenylation signals useful in practicing the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to LTR polyadenylation signals, bovine growth hormone (bGH) polyadenylation signals, human growth hormone (hGH) polyadenylation, and human β-globin polyadenylation signals. In particular, the polyadenylation signal

SV4 0 which is in plasmid pCEP4 (Invitrogen,

San Diego, CA)

SV40, can be used.

In addition to the regulatory elements required for DNA expression, other elements can also be included in the molecule of

DNA. Additional items include enhancers. The enhancer can be selected from the group that includes but is not limited to: human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those of

CMV, RSV and

EBV.

Genetic constructs can be provided of mammalian origin for replication in order to maintain the construct extrachromosomally and produce copies of the construct in the cell. Plasmids pVAXl, pCEP4 and pREP4 from Invitrogen (San Diego, CA) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region that produces high copy episomal replication without integration.

In order to maximize protein production, regulatory sequences can be selected which are suitable for gene expression in cells where the construct is administered. Furthermore, the codons encoding said protein can be selected which are more efficiently transcribed in the host cell. A person skilled in the art can produce DNA constructs that are functional in cells.

In some embodiments, nucleic acid constructs can be provided where the coding sequences for the proteins described herein are linked to IgE signal peptide. In some embodiments, the proteins described here bind to the IgE signal peptide.

In some embodiments for which the protein is used, for example, a person skilled in the art can, using well known techniques, can produce and isolate proteins of the invention using well known techniques. In some embodiments for which the protein is used, for example, one of skill in the art, using well-known techniques, molecule inserts encoding a protein of the invention, a commercially available expression vector for use in well known expression systems. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, California) can be used for protein production in Escherichia coli (E.coli). The commercially available plasmid pYES2 (Invitrogen, San Diego, CA) can, for example, be used for production in Saccharomyces cerevisiae strains of yeast. The commercially available MAXBAC ™ Complete Baculovirus Expression System (Invitrogen, San Diego, CA) can, for example, be used for production in insect cells. The commercially available plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego, CA) can, for example, be used for production in mammalian cells such as Chinese hamster ovary (CHO) cells. One of skill in the art can use these commercial or other vectors and expression systems to produce protein by means of routine techniques and readily available starting materials. (See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, second edition Coid Spring Harbor Press (1989)). In this way, the desired proteins can be prepared in prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein.

A person skilled in the art can use.:. &lt;»·« ¿Fc .'._- i,. · Φ. ·, · -.

other vectors and systems commercially or produce vectors

<img file="MX339241B_D0025.tif" />

Known and easily available starting materials. Expression systems containing the requisite control sequences, such as polyadenylation promoters and signals, and preferably enhancers are readily available and known in the art for a variety of hosts. See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, Second Edition Coid Spring Harbor Press (1989). Genetic constructs include the protein coding sequence operably linked to a promoter that is functional in the cell line, or target tissue cells, into which the constructs are transfected. Examples of constitutive promoters include cytomegalovirus (CMV) or SV40 promoters. Examples of inducible promoters include mouse mammary leukemia virus or metallothionein promoters. Those skilled in the art can readily produce genetic constructs useful for transfecting cells with DNA encoding protein of the invention from readily available starting materials. The expression vector including the DNA encoding the protein is used to transform the compatible host which is then cultured and maintained under conditions where expression of the foreign DNA takes place.

The protein produced is recovered from the culture, either by lysing the cells or from the culture medium.&gt; As appropriate and known to those of skill in the art. A person with experience in t-prnira can, using well known techniques, isolate protein that is produced using expression systems. Natural source purification methods using antibodies that specifically bind to a specific protein as described above can equally be applied to purification protein produced by recombinant DNA methodology.

In addition to producing proteins by means of recombinant techniques, automated peptide synthesizers can also be used to produce essentially pure, isolated protein. Such techniques are well known to those skilled in the art and are useful if derivatives having substitutions are not provided in DNA encoded protein production.

Nucleic acid molecules can be delivered using any of the well known technologies including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, facilitated nanoparticle, recombinant vectors such as recombinant adenovirus, virus associated with recombinant adenovirus and recombinant vaccinia. Preferably, nucleic acid molecules such as the DNA plasmids described herein are supplied via DNA injection and together with electroporation in vivo.

Administration routes inclüyB ¥ f^IED.WéIfó: -. river &lt;se. ΓΝ DVinChL - limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intra-arterial, intraocular and oral as well as topical, transdermal, inhalation or suppository or mucosal tissue such as by washing vaginal, rectal, urethral tissue, buccal and sublingual. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. Genetic constructs can be administered by means including, but not limited to, traditional syringes, needleless injection devices, microproject bombardment guns, and other methods such as electroporation (EP), hydrodynamic method, or ultrasound.

Examples of preferred electroporation devices and electroporation methods to facilitate delivery of the DNA vaccines of the present invention, include those described in US Patent No. 7,245,963 by Draghia-Akli, et al., US Patent Publication 2005 / 0052630 filed by Smith, et al., The contents of which are incorporated by reference in their entirety. Also preferred are electroporation devices and electroporation methods to facilitate the delivery of the DNA vaccines provided in co-pending and co-owned US Patent Application Serial No. 11/874072 filed October 17, 2007, that claims the 2006 and 60 / 978,982,

119 (e) a Provisional Requests for

60 / 852,149, filed October 17, filed October 10, 2007, all of which are hereby in their entirety.

Preferably, electroporation is the device

CELLECTRA ™ (VGX Pharmaceuticals, Blue Bell, intramuscular (IM) and intradermal models

US Patent No. 7,245,963

PA), including (ID).

by Draghia-Akli facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. Modular electrode systems comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a programmable constant current pulse conductive link to the plurality of needle electrodes; and a source of energy. An operator can grasp a plurality of needle electrodes that are mounted on a support structure and firmly insert them into selected tissue on a body or plant. The biomolecules are then delivered via the hypodermic needle into selected tissue. The programmable constant current pulse controller is activated and the constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cell among the plurality of electrodds. J content, full 'INSii:; · -', ί · '} of US Patent No. 7,245 ^ 963 hereby for reference. ~

US Patent Publication 2005/0052630 filed by Smith, et al. describes an electroporation device that can be used to effectively facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device (EKD device) whose operation is specified by software or firmware. The EKD device produces a series of programmable constant current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and enables the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disc having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disc. The full content of US Patent Publication 2005/0052630 is hereby incorporated for reference.

The electrode arrangements and methods described in

US Patent No. 7,245,963 and Patent Publication of

USA 2005/0052630 are adapted for deep penetration into not only tissues such as muscle, but also other tissues or organs. Due to the configuration of the electrode arrangement, the injection needle (to supply the biomolecule of choice) is also inserted ^ pioi-amAnt-A into the target organ, and the injection is administered perpendicular to the target tissue, in the area that is pre-delineated by the electrodes. The electrodes described in US Patent No. 7,245,963 and US Patent Publication 2005/005263 are preferably 20mm in length and 21 gauge.

The following is an example of methods of the present invention, and is discussed in greater detail in the patent references discussed above: Electroporation devices can be configured to supply a desired tissue of a mammal with a pulse of energy that produces a constant current. similar to a user preset current input. The electroporation device comprises an electroporation component and an electrode mount or handle mount. The electroporation component may include and incorporate one or more of the various elements of the electroporation devices, including: controller, current waveform generator, impedance analyzer, waveform recorder, input element, reporting element status, communication port, memory component, power source, and power switch. The electroporation component can function as an element of the electroporation devices, and the other elements are the — usomporenf e da, (or components) in communication with electroporation.

In some ways, electroporation can function as more than one element of electroporation devices, which can be in devices with other elements than electroporation that are still separate from the electroporation component. The present invention is not limited by the elements of existing electroporation devices as parts of an electromechanical or mechanical device, as the elements can function as one device or as separate elements in communication with each other. The electroporation component is capable of supplying the pulse of energy that produces the constant current in the tissue desired, and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives the pulse of energy from the electroporation component and supplies the same to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during the delivery of the energy pulse and measures the impedance in the desired tissue and communicates the impedance to the electroporation component. The feedback mechanism can receive the measured impedance and can adjust the pulse of energy supplied by the electroporation component for constant.

In some modalities, it can be supplied by the pulse of

<img file="MX339241B_D0026.tif" />

Plura 1 i give energy in a decentralized pattern. In some embodiments, the plurality of electrodes can supply the energy pulse in the decentralized pattern through control of the electrodes under a programmed sequence, and the programmed sequence is entered by a user to the electroporation component. In some embodiments, the programmed sequences comprise a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with a neutral electrode measuring impedance, and wherein a subsequent pulse of the A plurality of pulses are supplied by a different one from at least two active electrodes with a neutral electrode that measures impedance.

In some modalities, the feedback mechanism is performed by hardware or software.

Preferably, the feedback mechanism is performed by an analogous closed loop circuit. Preferably, this feedback occurs every 50 µl, 20 gs, 10 µβ, or 1 gs, but is preferably a real-time or instantaneous feedback (ie, substantially instantaneous as determined by available techniques for determining response time). In some modalities, the impedance in the desired tissue and * ·.

[The electrodialysis' iNST'TTOO to- tou · &lt;communicates the impedance feedback mechanism, and the .israo.-.— gives feedback responds to the impedance and adjusts the energy pulse to keep the current constant at a similar value to the preset current.

In some embodiments, the feedback mechanism maintains the constant current continuously and instantaneously during delivery of the power pulse.

A pharmaceutically acceptable carrier may include such functional molecules as vehicles, adjuvants, carriers, or diluents, which are known and readily available to the public. Preferably, the pharmaceutically acceptable excipient is an adjuvant or agent that facilitates transfection. In some embodiments, the nucleic acid molecule, or plasmid DNA, is delivered to cells in conjunction with the administration of a polynucleotide function enhancer or a genetic vaccine facilitating agent (or agent that facilitates transfection). Polynucleotide function enhancers are described in US Serial Number 5,593,972, 5,962,428 and International Application Serial Number PCT / US 94/00899 filed on January 26, 1994, each incorporated herein for reference. Genetic vaccine facilitating agents are described in US Serial Number 021,579 filed on AllriJ 1994 1 ', which_; í Instituí o »:: f is incorporated here for reference. The transfection agent ¥ á'cil.itiaciónili »** can be administered together with the nucleic acid molecules as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after the administration of acid molecules nucleic. Examples of transfection facilitating agents include surface active agents such as immune stimulating complexes (ISCOMS), incomplete Freunds adjuvant, LPS analog including monophosphoryl lipid A, muramil peptides, quinone analogs and vesicles such as squalene and squalene, and acid. Hyaluronic can also be used administered in conjunction with the genetic construct. In some embodiments, DNA plasmid vaccines can also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes, or other liposomes known in the art, such as a DNA-liposome mixture (see, eg, WO9324640 ), 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.

In some preferred embodiments, the plasmid DNA is supplied with an adjuvant which are genes for proteins that further increase the. r ^^ yiesta. immune against such target proteins. Examples of^1NS3S &amp; 7w &amp; éñés _v they are: INDuS ί'ΚΪΑΙ.

those encoding other cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNFa, ΤΝΡβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2 , IL-4, IL-5, IL-6,

IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL-15 including IL-15 having the signal sequence suppressed and optionally including the IgE signal peptide. Other genes that may be useful include those that encode: MCP-1, ΜΙΡ-Ια, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac- 1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, factor Growth Factor, Fibroblast Growth Factor, IL-7, Nerve Growth Factor, Vascular Endothelial Growth Factor, Fas, TNF Receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3 , AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, genes Interferon Response, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C,

NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

Pharmaceutical compositions according to

INSTITUTE .

FROM THE r microgram to approximately 10 mi i í ^ í'STflüS; "" Ts · preferably approximately micrograms approximately milligrams; or more pre fer ib1ement and approximately milligram to approximately milligrams. In some preferred embodiments, pharmaceutical compositions according to the present invention comprise from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 100 to about 200 micrograms of DNA.

The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particulate-free. A ''

Ñ ​​INSTñ. . OF THE_ÍL.

Isotonic formulation is preferably used. Generally, ·²Isotonicity additives may include soiHoy ClotUlU · - · dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizing agent that enables the formulation to be stable at room or local temperature for extended periods of time, such as LGS or other polycations or polyanions, is added to the formulation.

In some embodiments, methods of producing a mammalian immune response against a consensual influenza antigen include methods of inducing mucosal immune responses. The methods include administering to the mammal one or more of the CTACK protein, TECK protein, MEC protein and their functional fragments or expressible coding sequences thereof in combination with a DNA plasmid that includes a consensual influenza antigen, described above. The one or more CTACK protein, TECK protein, MEC protein and their functional fragments can be administered prior to, concurrently with or after administration of the plasmid DNA influenza vaccines provided herein. In some embodiments, an isolated nucleic acid molecule that encodes an or. maé / --proteins 'de'laÓ, selected from the group consisting of: CTACK, TECK,' MEC and their functional fragments are administered to the mammal.

EXAMPLES

The present invention is further illustrated in the following examples. It can be understood that these examples, while indicating preferred embodiments of the invention, are provided by way of illustration only. From the above discussion and these examples, a person skilled in the art can guess the essential characteristics of this invention, and without deviating from the essence and scope thereof, can make various changes and modifications of the invention to adapt them to various uses. and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art of the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Preferably the DNA formulations for use with a muscle or skin EP device described herein have high concentrations of DNA, preferably concentrations that include amounts from milligrams to tenths of a milligram, and preferably amounts of tenths of a milligram, of DNA in small volumes that they are optimal for supplying small injection to the skin, preferably |: d / volurrieñ 'de l · INST17Ü7C L' WW? '' -.W microliters¹¹ ΉμΕύ. 'Έή ideally 25-200 some modalities, DNA formulations have at the bottom¹ concentrations of

DNA, such as 1 mg / ml or more (mg of

DNA / formulation volume). More preferably, the DNA formulation has a DNA concentration that is provided for gram amounts of DNA in 200 µΐ of formula, and more preferably gram amounts of DNA in 100 µΐ of formula.

Plasmid DNA for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using an optimized plasmid manufacturing technique described in a provisional US application. co-pending, commonly owned U.S. serial No. 60 / 939,792, filed May 23, 2007. In some examples, the plasmid DNA used in these studies can be formulated in concentrations greater than or equal to 10 mg / ml. Manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in US Series No. 60/939792, which includes those described in a commonly owned patent. , US Patent No. 60/939792, which includes those described in a common property patent, files US 3 No. 7.2j3 8 'what \ ¿é; ·

2007. High concentrations of

July patent for plasmids used with the EP skin devices and delivery techniques described herein are allowed for administration of plasmids into the ID / SC space in a reasonable low volume and aids in increased expression and immunization effects. Common Property and Patent Application, US Serial No. 60 / 939,792 and US Patent No. 7,238,522, respectively, and thereby incorporated herein in its entirety.

EXAMPLE 1

Plasmid Constructs

A massive cytomegalovirus (CMV) promoter handles the expression of reporter transgene product (SEAP) embryonic alkaline phosphatase secreted in the pCMV-SEAP vector. Plasmids are obtained using a commercially available kit (Qiagen Inc., Chatsworth, CA). Endotoxin levels are less than 0.01 EU / gg, as measured by Kinetic Chromagenic LAL

<td>(Endorsement,</td><td>Charleston, SC). Consensus HA and NA Constructs</td>

<td>they generate</td><td>when analyzing primary virus sequences of viruses</td>

<td>16 H5 what</td><td>have been proven fatal to humans in years</td>

<td>recent,</td><td>or about 40 human NI viruses. These</td>

<td>sequences</td><td>are downloaded from Los Alamos National Laboratory's</td>

Influenza Sequence Database. Post-generation mammalian expression, including Kozak sequence, codon optimization, and

These constructs are then sterile and m

preparations formulate 1%

Carlsbad, CA)

<td>I know</td><td>subcloned</td><td>in</td><td colspan="2">the vector</td><td>pVAX</td>

<td>. TO</td><td>less than</td><td>I know</td><td>indicate</td><td>of</td><td>other</td>

<td>of</td><td>plasmid</td><td>I know</td><td>they dilute</td><td>in</td><td>Water</td>

<td>in</td><td>weight / weight</td><td>with</td><td>Get ouf of</td><td colspan="2">sodium of</td>

poly-L-glutamate (LGS) (MW = 10.5 kDa average) (Sigma, St.

Louis,

I immune

MO), plus purified HPLC at VGX Pharmaceuticals,

Therapeutics Divison (The Woodlands, TX).

EXAMPLE 2 Treatment of pigs

The pigs are divided into 10 groups x 4 pigs per group for a total of 40 pigs (table 1). Pigs are acclimatized for 4 days, weighed and tagged in the ear. In the day 0 study, pigs are weighed, mixed and anesthetized using a pig pre-anesthetic combination ketamine - (20 mg / kg), xylazine - (2.2 mg / kg) and atropine (0.04 mg / kg) , and then anesthetized using isoflurane (induction at 5% maintenance at 2-3%). Pigs (n = 4 / group) are injected with 0.6 ml of CMV-HA (a pVAX-based construct that expresses a consensual H5 antigen), CMV-NA (a pVAX-based construct that expresses a consensual NI antigen), and CMVSEAP (a construct expressing reporter gene secreted embryonic IK alkaline phosphatase, SE ^ .P) n &lt;sTg&gt; licked (at least one is added to increase plasmid concentration, and the viscosity of the solution for the assessment of muscle damage) + 1.0% w / w of LGS in the variation of plasmid concentrations and current intensities. Plasmids are prepared according to the materials and methods provided in Example 1. After 4s, the animals are electroporated using the CET J FCTRA intramuscular (IM) system^Mconstant current (VGX Pharmaceuticals, Blue Bell, PA) equipped with 5 needle electrodes and operated with the following pulse parameters: 52 millisecond pulses, 1 second between pulses, pulses with current variation (0.1, 0.3 and 0.5Ά ).

TABLE 1

Groups for the pig vaccine experiment

<td>Group</td><td>Plasmid</td><td></td><td>Conc. (mg / ml)</td><td>Construct (mg) / pig</td><td>Total dose (mg / pig)</td><td>Injection volume</td><td>Α</td><td>η</td>

<td> 1</td><td>HA, SEAP</td><td>NA,</td><td> 10</td><td> 2</td><td> 6</td><td>600 μΐ</td><td> 0.5</td><td> 4</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 2</td><td>HA, SEAP</td><td>NA,</td><td> 4</td><td> 0.8</td><td> 2.4</td><td>600 μΐ</td><td> 0.5</td><td> 4</td>

<td> 3</td><td>HA, SEAP</td><td>NA,</td><td> 1.5</td><td> 0.1</td><td> 0.3</td><td>600 μΐ</td><td> 0.5</td><td> 4</td>

<td> 4</td><td>HA, SEAP</td><td>NA,</td><td> 10</td><td> 2</td><td> 6</td><td>600 μΐ</td><td> 0.3</td><td> 4</td>

<td> 5</td><td>HA, SEAP</td><td>NA,</td><td> 4</td><td> 0.8</td><td> 2.4</td><td>600 μΐ</td><td> 0.3</td><td> 4</td>

<td> 6</td><td>HA, SEAP</td><td>NA,</td><td> 1.5</td><td> 0.1</td><td> 0.3</td><td>600 μΐ</td><td> 0.3</td><td> 4</td>

<td> 7</td><td>HA, SEAP</td><td>NA,</td><td> 10</td><td> 2</td><td> 6</td><td>600 μΐ</td><td> 0.1</td><td> 4</td>

<td> 8</td><td>HA, SEAP</td><td>NA,</td><td> 4</td><td> 0.8</td><td> 2.4</td><td>600 μΐ</td><td> 0.1</td><td> 4</td>

<td> 9</td><td>HA, SEAP</td><td>NA,</td><td> 1.5</td><td> 0.1</td><td> 0.3</td><td>600 μΐ</td><td> 0.1</td><td> 4</td>

<td> 10</td><td>None</td><td></td><td>N / A</td><td>N / A</td><td>N / A</td><td>Ν / Α</td><td>Ν / Α</td><td> 4</td>

The surrounding area of ​​each site is tattooed for rapid biopsy identification

<img file="MX339241B_D0027.tif" />

post injection.

Pigs are allowed to recover from anesthesia and are closely monitored for 24 hours to ensure full recovery. Any pig that does not fully recover within 2-3 hours post-treatment is scored. Pigs are weighed and purged on day 10, day 21, and day 35. Pigs are given a second vaccine on day 21. Blood is collected in 2 purple top tubes, 1.0 ml for CBC and differentials (Antech Diagnostics, Irvine, CA); 10 ml for IFN-γ ELISpots against HA and NA antigens, and separate Falcon tubes that allow the isolated serum to be coagulated and centrifuged after aliquoting in tubes on ice. On day 35, all pigs are bled to death under anesthesia surgery and needle punch biopsies from injection sites are taken for histology.

Hemagglutination Inhibition Assay (HI)

Pig serum is treated with a receptor-destroying enzyme (RDE) by diluting one part serum with three parts enzyme and incubated overnight in a 37 ° C water bath. The enzyme is inactivated by incubation for 30 minutes at 56 ° C followed by the addition of six parts of PBS for a final dilution of 1/10. HI assays are performed on V bottom well microtiter plates, using four units of horse red blood cell cells as described (Stephenson, I., et al., Virus Res. 103 (1-2); 91-5

IΜ P í ¿HA ¿'.ly-í ríU,. ^ \ Previously described

<img file="MX339241B_D0028.tif" />

(July 2004))

Highest ratings as demonstrated by trial

HI (figure 2) are found in serum of the group administered with 2 mg of plasmid that expresses HA in a current setting of 0.5A (120 + 40; P = 0.11 versus 2 mg / 0.3A and P = 0.02 versus 2 mg / Wave); titrations decrease with the intensity of the electric field for the group receiving 2 mg of each plasmid; if the amount of plasmid in the stream is decreased later, the titers are more variable; and not different between groups.

HI scores are highest in the group administered with 2 mg of HA expressing plasmid and electroporated at 0.5A. Furthermore, titrations decrease with decreased plasmid doses in the electroporated group by 0.5A, and with the intensity of the electric field. Lower amounts of plasmid or lower current intensities appear to increase intra-group variability.

IFN-γ ELISpots of HA and NA

ELISpot is performed as previously described using IFN-γ capture and detection antibodies (MabTech,

Sweden) (Boyer JD, et al., J Med Primatol, 34 (5-6): 262-70 (Oct. 2005). Antigen-specific responses are determined by subtraction of the number of spots in the negative control wells from wells containing peptides. The results are

Τ shown as the mean value (spots / million splenocates)! dtot for triplicate wells. '

The group administered with 2 mg of each plasmid 7 for "'" a total of 4 mg) in a current setting of 0.3A of 0.3A coupled with the highest cellular immune response as measured by IFN-γ ELISpot of 537+ 322 SFU per million cells. The average responses of all other groups are within the trial background levels. The individual ELISpot responses of two animals that bind the highest cellular immune response are highlighted in Figure 3.

<img file="MX339241B_D0029.tif" />

CBC results

Lymphocytes reach the highest levels on study day 21 and in the groups administered with the highest doses of vaccines, relative to current adjustment, although the groups with the highest doses (4 mg total plasmid, 2 mg each) and higher current setting (0.5A) demonstrates the highest lymphocyte response, 40% higher than controls (12670 ± 1412 vs. 7607 ± 1603 lymphocyte count / 100 blood, respectively; P &lt;0.002).

Muscular histopathology

Injection sites are identified and needle biopsies are taken on days 14 and 35 post-treatment after the pigs are bled out. The tissues are fixed in pH regulated formalin for 24 hours then washed 3X in PBS and stored in 70% alcohol. Biopsy samples are subjected to Diagnósti ^^ Tj ^ télhf dolóle ί ΠΚώτ; -, V '^: are processed and sections are stained with hematoxyliha and eosin (Ή &amp; Ε / τ'—

All slices are evaluated by a ceitiliuadu pathologist for the simple advice that registers them from 0 to 5 for pathological criteria (table 2) in several layers of tissue (table 3). The mean score is calculated for each group at each time point.

TABLE 2

Biopsy pathology marker parameters

<td>Marker</td><td>Criterion</td>

<td> 0</td><td>Not present, non-inflammatory cells</td>

<td> 1</td><td>Minimum, 1-20 inflammatory cells / 100x high energized field (HPF)</td>

<td> 2</td><td>Medium, 21-40 inflammatory cells / 100xHPF</td>

<td> 3</td><td>Moderate, 41-75 inflammatory cells / lOOxHPF</td>

<td> 4</td><td>Moderate marked / severe ,. 76-100 cells inflammatory / lOOxHPF</td>

<td> 5</td><td>Severe marking,&gt; 100 inflammatory cells / 100xHPF</td>

TABLE 3

Biopsy tissue layers and pathological parameters

<td>Anatomy location</td><td>Pathology parameter</td>

<td>Dermal</td><td>Superficial neovascularization</td>

<td>Dermal</td><td>Pilogranulomatous inflammation</td>

<td>Dermal</td><td>Overlying erosion &amp; inflammatory crusts</td>

<td>Dermal</td><td>Focal fibrosis</td>

<td>Subcutaneous</td><td>Pilogranulomatous inflammation with necrosis of intralesional collagen</td>

<td>Subcutaneous</td><td>Lymphatic and Plasma Inflammation</td>

<td>Skeletal muscle</td><td>Lymphatic and plasmalytic and eosinophilic inflammation</td>

<td>Skeletal muscle</td><td>Myocyte degeneration / necrosis</td>

<td>Skeletal muscle</td><td>Fibrosis</td>

Histopathology is recorded from the muscle biopsy (Figure 4A) on days 14 and 35 after - 'cié; la. · instít'ji or cy ··: ν ', --ΐ injection of plasmid and EP based on a scale criterion 0-a5 (table 2). Compleotopathological markers ..... ooguido'0— by electroporation decrease in tissue layers (table

3) from day 14 to day 35. The group that receives 6 mg of plasmid

<td>total in</td><td>settings 0.3Ά</td><td colspan="2">exhibit</td><td>the</td><td>markers</td><td>pathology</td>

<td>total more</td><td>high in the</td><td>day</td><td> 14</td><td> (18.3</td><td>± 6.4, P &lt;</td><td>0.0002 versus</td>

<td>control),</td><td colspan="2">correlating</td><td>with</td><td>the</td><td>answers</td><td>lymphocyte</td>

higher average. All pathology markers at day 35 improve levels from untreated control levels (range 6.67 to 4.25). However, when muscle necrosis and fibrosis (usually associated with the EP procedure) (Gronevik E, et al., J Gene Med, 7 (2): 218-27 (2005 Feb)), they are analyzed separately (Figure 4B), the markers range between 1 and 2, with no difference between groups or between treated groups and controls, while the highest markers are associated with lymphatic, plasmacytic or eosinophilic inflammation due to immune responses. Significantly, these markers also decrease from day 14 to day 35 post-treatment.

Analysis of data

The data is analyzed using the Microsoft Excel Statistics package. The values ​​shown in the figures are the mean ± SEM. Specific values ​​are obtained by comparison using

ANOVA in one way and consecutive t-test.

0.05 adjusts as the significance level

A Jr ^ lór '' dé 'p. &lt;

'·' Institute 'í; x; c' £ st adduce ·.? ·. , _

EXAMPLE 3

Ferret treatments

Twenty male ferrets (Triple F Farms, Sayre, PA), 46 months of age of at least 1 kg of body weight, are used in this study and are housed at BIOQUAL, Inc. (Rockville, MD).

The designed ferret study is in Table 4. Animals are allowed to acclimatize for two weeks prior to the study. Animals are immunized (under anesthesia) at weeks 0, 4 and 9.

The blood is drained every 2 weeks. After the third immunization, animals move a BSL-3 facility and are challenged at week 13 with each potent strain of avian influenza (H5N1), and then followed for an additional two weeks post challenge. For two weeks after the challenge, animals are monitored daily, and body weights, temperature, and clinical markers are recorded.

The level of activity is monitored and recorded;

death is documented.

This study tests the efficacy of the

HE HAS,

NA and M2e-NP DNA are supplied IM followed by electroporation using the adapted constant current electroporation intramuscular (IM) system

CELLECTRA ™ (VGX Pharmaceuticals, Blue Bell, PA) in a model of influenza in ferrets. The plasmid DNA is challenge prepared

ΙΑ / J τ 'nlwL j ^ j.®níplo

MEXICAN INSTITUTE V '-'-- .: Y. EE LA Ο'ΟΡ-Γ AO

one. As outlined in Table 4, animals in groups 2t ~ '3 and 4 receive 0.2 mg of the influenza plasmid vaccine.^{1 1}respective. In order to correct the dose, the groups receiving plasmid vaccine (groups 2 and 3) or no vaccine (control group 1), the difference is made by vacuum vector pVAX such that all animals in each group receive a dose 0.6 mg total plasmid. Electroporation conditions are, using a 5-needle electrode array; 0.5 Amps, pulse width of 52 msec, 1 sec between pulses, 4 second delay between injection and electroporation.

TABLE 4

Groups for influenza challenge experiment in ferrets

<td>Group</td><td>Plasmids / antigens</td><td>Dose of vaccine (mg) per plasmid</td><td>Total vaccine Total volume</td><td>In</td><td>n</td>

<td> 1 2 3 4</td><td>None (pVAX only) H5 + pVAX NA + pVAX H5, NA, M2e-NP</td><td>0 mg 0.2 mg 0.2 mg 0.2 mg</td><td>0.6 mg to 0.6 mi 0.6 mg to 0.6 mi 0.6 mg to 0.6 mi 0.6 mg to 0.6 mi</td><td></td><td> 4 4 4 4</td>

Hemagglutination Inhibition (HI) Assay

Serum is treated with receptor-destroying enzyme (RDE) by diluting one part serum with three parts enzyme and incubated overnight in a 37 ° C water bath. The enzyme is

IMPIAS inactivates for 30 minutes of incubation at 56 ° C according to R&gt;, by adding six parts of PBS for a final dilution of 1/10. HI assays are performed in V-bottom 96-well microtiter plates, using four HA units of virus and 1% horse red blood cell cells as previously described (Stephenson, I., et al., Virus Res, 103 ( 1-2): 915 (July 2004)). The viruses used for the HI assay are assorted strains obtained from the Center for Disease Control: A / Viet / 1203/2004 (H5N1) / PR8-IBCDC-RG (virus subclass 1) and A / lndo / 05/2005 (H5N1) / PR8-IBCDC-RG2 (subclass 2 virus). The ferret model of influenza infection is considered to be more reflective of human disease and a more rigorous challenge model. Ferrets exhibiting symptoms similar to influenza-infected humans and similar tissue tropism for avian and human influenza viruses. Serum collected at different time points throughout the study is used to detect HI activity against H5N1 virus. As shown in Figure 7, both groups containing the consensual H5-specific HA construct bound to protective antibody levels (&gt; 1:40) after two immunizations and are also capable of inhibiting a subtype 2 H5N1 virus. In other words , essay

HI that is positive against both viral strains represented in the consensual HA strain is based on subtype 1 viruses.

Analysis of data

The data is analyzed using

<img file="MX339241B_D0030.tif" />

Microsoft Excel package

Statistics. The values ​​shown in -the figures are the mean ± SEM. Specific values ​​are obtained by comparison using one-way ANOVA and consecutive t-test. A value of p &lt;0.05 is adjusted as the level of statistical significance.

Ferret Flu Challenge

The results of the influenza challenge are represented in figure 5 and 6. The control animals lose 25% of their body weight on average post-challenge (Figure 5), while the animals vaccinated with HA (Group 1) or HA + M2e NP + NA (Group 4) lose between 9 and 10% (* P &lt;0.004 versus controls). Body temperatures are raised in control animals until all control animals are dead or painlessly killed by day 8 (Figure 6). All vaccinated animals, with respect to which the vaccine regimen, survive the challenge and show some signs of infection as compared to control animals as evidenced by their clinical markers (Table 5). Control animals worsen as clinical markers (nasal discharge, cough, lethargy), and die on day 5 and day 7 post-challenge. As shown in Table 5, the severity of clinical markers in vaccinated animals inversely correlates with antibody titers (higher, lower antibody titers, better clinical outcomes).

<img file="MX339241B_D0031.tif" />

TABLE 5

Results for challenged ferrets

<td></td><td></td><td colspan="8">Post-challenge observations</td><td></td><td>HI titrators 3 weeks</td>

<td>Vaccinations</td><td>Ferret</td><td>Day 1</td><td>Day 2</td><td>Day 3</td><td>Day 5</td><td>Day 6</td><td>Day 7</td><td>Day 8</td><td>Day 9</td><td></td><td>Pre-challenge</td>

<td></td><td> 891</td><td> 0 1</td><td> 1 1</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td> 1 1*</td><td></td><td></td><td></td><td> &lt;20</td>

<td>Control</td><td> 890</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td>0 l *</td><td></td><td></td><td></td><td></td><td></td><td> &lt;20</td>

<td>(pVAX)</td><td> 877</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td> 02</td><td> 2 3</td><td>FD</td><td></td><td></td><td></td><td> &lt;20</td>

<td></td><td> 876</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 2 3</td><td> 1 3*</td><td></td><td></td><td></td><td> &lt;20</td>

<td></td><td> 878</td><td>0 l</td><td> 0 1</td><td> 1 1</td><td> 01</td><td> 0.1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 40</td>

<td>H5</td><td> 879</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 320</td>

<td></td><td> 888</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 01</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 160</td>

<td></td><td> 889</td><td> 0 1</td><td> 0 1</td><td> 0. 1</td><td>0 l</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 320</td>

<td></td><td> 881</td><td> 0 1</td><td> 0 1</td><td> 1 0</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td></td><td> &lt;20</td>

<td>M2-NP</td><td> 880</td><td> 0 1</td><td> 0 1</td><td> 0 0</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> &lt;20</td>

<td></td><td> 883</td><td> 0 1</td><td> 1 1</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td></td><td> &lt;20</td>

<td></td><td> 882</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td> 1 2</td><td> 0 2</td><td> 0 1</td><td></td><td> &lt;20</td>

<td></td><td> 885</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 1280</td>

<td>H5 + M2- NP + NA</td><td> 884</td><td> 01</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 320</td>

<td></td><td> 886</td><td>1 i</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td> 0 1</td><td> 1 1</td><td></td><td> 160</td>

<td></td><td> 887</td><td> 0 1</td><td> 1 1</td><td> 0 0</td><td> 0 1</td><td> 0 1</td><td> 1 1</td><td> 0 1</td><td> 0 1</td><td></td><td> 640</td>

Table 5: note: clinical markers are represented for post-challenge observation period. A * indicates that the animal has been slaughtered without pain; FD = found death. The first clinical marker in each column is for nasal symptoms; 0 = none; 1 = nasal discharge; 2 = mouth breathing. The second marker is for activity: 0 = sleep; 1 = breathing and alert; 2 = alert but not responsible; 3 = lethargic. HI ratings for each animal measured every 3 weeks pre-challenge comparison.

are represented for

<img file="MX339241B_D0032.tif" />

EXAMPLE 4

Comparisons of intradermal supply with intramuscular supply in primates

Rhesus macaques are immunized in these studies. The animals are acclimatized for 2 months before the start of experiments. The study progresses as follows: week 0 represents I^to immunization (administration of plasmid doses) and baseline blood; week 2 represents bleeding; week 3 represents 2^to immunization (administration of plasmid doses); week 5 represents blood; week 6 represents 3^to immunization (administration of plasmid doses) and bleeding; Week 8 represents bleeding.

TABLE 6

<td>Study group</td><td>DNA Constructs</td><td>Nr.</td><td>Administration route</td><td>Dose</td><td>Total DNA (mg)</td>

<td>TO</td><td>DNA 6 + 9</td><td> 5</td><td>IM CELLECTRA ™ EP</td><td>1 mg / Const</td><td> 2</td>

<td>B</td><td>DNA 6 + 9</td><td> 5</td><td>ID CELLECTRA ™ EP</td><td>1 mg / Const</td><td> 2</td>

<td>C</td><td>DNA 1 + 6 + 9 + 10</td><td> 5</td><td>IM syringe</td><td>1 mg / Const</td><td> 4</td>

<td>D</td><td>Negative control</td><td> 5</td><td>N / A</td><td></td><td> 0</td>

# of DNA construct coding antigen rr »antigen control plasmid

H5 consensual influenza

<img file="MX339241B_D0033.tif" />

antigen control plasmid 'S'lñ IrtflUéíTaá ”*¹'influenza-free antigen control plasmid

All plasmids are formulated in 10 mg / ml in water for injection + 1% LGS, as described in previous, previous examples, and mixed in a single solution PER

STUDY GROUP (S) (Groups C, D, G and H, in the previous table, table 6). The correct injection volume for each group designated IM CELLECTRA ™ EP (VGX Pharmaceuticals), ID CELLECTRA ™ EP (VGX Pharmaceuticals), and IM syringe is calculated. For ID administration, if the required injection volume exceeds 100 µΐ per site, the formulation is divided into a number of injection sites (2, 3, or 6 depending on how many total mg of vaccine are administered). Animals receiving IM injections are provided in the entire formulation in a single site.

The CELLECTRA ™ matched constant current device used in pig experiments, ferret experiments, and non-human experiments described in the examples. The electroporation conditions are as follows: for IM injection and electroporation groups, the conditions are: 0.5 Amps, 52 msec / pulse, three times, 4 sec delay between plasmid injection and electroporation.

For ID injection and electroporation groups, the

<td>conditions are:</td><td>0.2 Amps, 52 msec / pulse, three5 ^ ¿lac ^_and -4 'seuf OF</td>

INU'CS L i \ L '; L of delay between plasmid injection and electroporation.

Hemagglutination Inhibition (HI) Assay

Monkey serum is treated with receptor destroying enzyme (RDE) by diluting one part serum with three parts enzyme and incubating overnight in a 37 ° C water bath. The enzyme is inactivated by incubation for 30 minutes at 56 ° C followed by the addition of six parts of PBS for a final dilution of 1/10. HI assays are performed on V bottom well microtiter plates using four HA virus units and 1% horse red blood cell cells.

The data presented here i are the results after the second immunization (blood collected before the third immunization).

HI ratings are measured three times after the second immunization.

The results can be seen displayed on the graph in figure 8.

Monkeys receiving the HA plasmid vaccine via ID injection followed by electroporation demonstrate more than twice the average titers of the IM + EP group and almost three times the average titers of the IM group alone (* P &lt;0.03). Untreated controls do not exhibit any of the HI ratings.

EXAMPLE 5 I] V1 PT: a '' '\ n «μ-. ·

Cross protection in primates

Using delivery method - ID injection - followed by electroporation (EP)

Studies in nonhuman primates with the influenza vaccine (including H5, NA and M2eNP consensual antigens, see above) indicate that ID injection followed by electroporation elicits higher antibody responses to vaccine antigens than IM injections. In one of the non-human primate study animals (NHP) they are vaccinated per table 7.

TABLE 7

<td>Group</td><td>n / group</td><td>Antigen</td><td>Supply</td><td>Ion concentration (mg / plasmid)</td><td>EP Conditions</td>

<td> 1</td><td> 5</td><td>pVax (sham)</td><td>IM</td><td>1 mg / construct</td><td>0.5 Amps, 3 pulses, 52 msec, 1 sec between pulses</td>

<td> 2</td><td> 5</td><td>H5, NA, M2e-NP</td><td>IM</td><td>1 mg / construct</td><td>0.5 Amps, 3 pulses, 52 msec, 1 sec between pulses</td>

<td> 3</td><td> 5</td><td>M2e-NP</td><td>IM</td><td>1 mg / construct</td><td>0.5 Amps, 3 pulses, 52 msec, 1 sec between pulses</td>

<td> 4</td><td> 5</td><td>H5, NA, M2e-NP</td><td>ID</td><td>1 mg / construct</td><td>0.2 Amps, 2X2 pulses, 52 msec, 1 sec between pulses</td>

Designed study and conditions. Rhesus macaques are immunized at weeks 0, 4, and 8

Each animal receives three vaccines, and HAI and microneutralization assessments are performed for both subtypes and cross-subtypes. As shown, the consensual vaccine offers broad protection not only within the same subtype, but also subtypes-crossovers. The results are included in Table 8.

<img file="MX339241B_D0034.tif" />

TABLE 8

<td></td><td>Subtype 1 A / Vietnam</td><td>Subtype 2.1 A / Indonesia</td><td>Subtype 2.2 To turquia</td><td>Subtype 2.3.4 A / Anhui</td>

<td>HAI Assay 2nd Immunization VGX-3400 IM VGX-3400ID</td><td> 160 (80-320) 664 (40-1280)</td><td> 36 (20-80) 120 (20-320)</td><td> 110 (0-320)^{4 / b} 205 (0-320)^{4 / s}</td><td> 80 (40-160) 592 (40-1280)</td>

<td>3rd immunization VGX-3400 IM VGX-3400ID</td><td> 288 (160-640) 416 (160-640)</td><td> 32 (0-80)^{J / b} 64 (0-160)^{2 / s}</td><td> 36 (20-80) 145 (20-320)</td><td> 84 (20-160) 275 (20-640)</td>

<td>Microneutralization 3rd immunization VGX-3400 IM VGX-3400ID</td><td> 144 (40-360) 740 (20-2560)</td><td> 8 (0-40)^1/5 96 (0-320)^3/5</td><td> 32 (0-80)^{2 / b} 296 (0-1280)^{2 / s}</td><td> 88 (0-160)^4/51172 (20- 2560)</td>

Table 8. The results of hemagglutination tests (HAI) and microneutralization. The values ​​presented indicate the average rating, the interval (in parentheses) and the number of responses if it is less than 5/5 (in overwrite). Note: HAI titrations&gt; 1:20 are generally considered seroprotective in the NHP model.

The needles in the ID electroporation device are much shorter (~ 5mm), lower calculus, and elicit no visible muscle contractions or pain responses in the animals tested to date. Furthermore, the electric field required for effective ID EP is less than that required for optimal IM supply. The ID injection has been shown to elicit better immune responses to influenza vaccine antigens. (Holland D. et al., (2008).

<img file="MX339241B_D0035.tif" />

inferior in ID supply to achieve similar humoral responses.

It is noted that in relation to this date, the best method known to the applicant for putting the aforementioned invention into practice.

is what is clear from the present description of the invention.

I f