Foot and mouth disease virus (FMDV) consensus proteins, coding sequences therefor, and vaccines made therefrom

Abstract

To provide synthetic, consensus foot-and-mouth disease virus (FMDV) immunogenic proteins, nucleic acid molecules encoding such proteins, and vaccines against FMDV.SOLUTION: The present invention relates to synthetic, consensus foot-and-mouth disease virus (FMDV) immunogenic proteins and nucleic acid molecules encoding such proteins, to vaccines against FMDV, to methods for inducing immune responses against FMDV, to methods for distinguishing individuals infected with FMDV from those vaccinated against FMDV, and to methods of prophylactically and / or therapeutically immunizing individuals against FMDV.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a synthetic consensus foot-and-mouth disease virus (FMDV) immunogenic protein and a nucleic acid molecule encoding such a protein, a vaccine against FMDV, a method of inducing an immune response against FMDV, a method of distinguishing an individual infected with FMDV from an individual vaccinated with a vaccine against FMDV, and a method of prophylactically and / or therapeutically immunizing an individual against FMDV.

Background Art

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild cloven-hoofed animals such as cattle, pigs, goats, and deer, and replicates rapidly in the host and spreads to susceptible animals in contact. The disease is characterized by fever, lameness, and vesicular lesions on the tongue, feet, nose, and teats, with a high morbidity rate but a low mortality rate in adult animals. FMDV infection causes severe blister disease in cattle, buffalo, sheep, goats, and pigs, which can progress to persistent infection (except in pigs). FMDV can infect many other mammalian species, including, among others, antelopes, elephants, and hamsters. However, the original natural host of FMDV may be African buffalo, because i) African buffalo are persistently infected and ii) the disease is rarely observed.

[0003] The causative agent of FMD is a picornavirus, Foot-and-Mouth Disease Virus (FMDV), which belongs to the Aphthovirus genus of the Picornaviridae family and is a Group 4 (+) ssRNA virus. FMDV occurs in seven main serotypes: O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1. These serotypes are geographically restricted, and the most common globally is the O serotype. The single-stranded positive-sense RNA genome of FMDV is approximately 8,500 bases surrounded by an icosahedral capsid with 60 copies each of four structural proteins, VP1 to VP4. The viral proteins have high antigenic diversity within some of its subtypes, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

[0004] FMD is economically devastating, and infection in cloven-hoofed livestock can cause significant losses. Recent outbreaks have resulted in losses of billions of dollars. In recent years, it has occurred in multiple countries where the disease did not occur in the past, such as Taiwan in 1997, the UK and the Netherlands in 2001, and in multiple countries in South America, which has recognized that it is an economically destructive virus. Furthermore, there is a possibility that economic terrorists could use FMDV to target countries with large livestock industries, such as the $100 billion-a-year livestock industry, making it a global problem.

[0005] Previous means of controlling FMDV included culling infected or exposed animals and decontamination. In countries where livestock were culled due to FMDV outbreaks, the livestock industry could only be restarted if it had been free of FMDV for three months after the last outbreak. In countries where animals were vaccinated and not culled, it took a full year to regain a FMD-free status, so countries usually used vaccination of animals to deal with FMDV outbreaks as a last resort. However, countries are hoping to vaccinate animals before FMDV outbreaks to maintain a FMD-free status.

[0006] In the past, FMDV vaccines contained chemically inactivated whole-virus antigen together with an adjuvant, but this had drawbacks such as the need for expensive, highly contained manufacturing facilities for vaccine production. For the past 25 to 30 years, researchers have attempted to develop vaccines that provide protection after a single vaccination. These efforts have included the use of VP1 purified from virus particles, biotechnologically produced VP1, VP1 peptides, chemically synthesized VP1 peptides, the use of live vectors expressing VP1 epitopes, the inoculation of DNA encoding VP1 epitopes, and the use of whole capsid proteins VP1-VP4 produced from FMDV-infected cultures or the delivery of VP1-VP4 capsids via a replication-deficient human adenovirus type 5 (Ad5) vector. All of these approaches provide only a small number of epitopes spanning all subtypes of the FMDV virus to the inoculated animals.

[0007] Accordingly, there is a need in the art for a vaccine suitable for providing protection against multiple FMDV epitopes spanning the various subtypes of FDMV, and a method for diagnosing mammals infected with FMDV. SUMMARY OF THE INVENTION

[0008] A nucleic acid molecule comprising an array encoding a viral protein, wherein the viral protein VP4 is ligated at its C-terminus to a protease cleavage site, ligated at its C-terminus to the viral protein VP2, ligated at its C-terminus to a protease cleavage site, ligated at its C-terminus to the viral protein VP3, ligated at its C-terminus to a protease cleavage site, ligated at its C-terminus to the array encoding the viral protein VP1, ligated at its C-terminus to a protease cleavage site, and ligated at its C-terminus to the viral protein 2A. The nucleic acid molecule may further comprise a nucleic acid sequence encoding a leader sequence at the 5' end of the coding sequence of the viral protein VP4. In some embodiments, the coding sequence of the viral protein VP4 is omitted. In some embodiments, the coding sequence of the viral protein 2A is omitted. In some embodiments, the coding sequence encoding the N-terminal leader sequence is omitted. In some embodiments, the coding sequence encoding the N-terminal leader sequence is an Ig leader sequence such as an IgG or IgE leader sequence. In some embodiments, the cleavage site is recognized by furin.

[0009] Plasmids are provided that contain nucleic acid molecules, including plasmids in which the viral protein is from an FMDV subtype selected from the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3. 。 Vaccines are provided that contain four plasmids in which the viral protein encoding the nucleic acid sequence is from each FMDV subtype of the group consisting of A, Asia1, C, and O. In some embodiments, vaccines are also provided that contain seven plasmids in which the viral protein encodes nucleic acid sequences from each FMDV subtype of the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3. In some embodiments, the viral protein is from an FMDV subtype selected from the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3 ofVaccines are provided that encode a nucleic acid sequence and contain fewer than 7, i.e., 1, 2, 3, 4, 5, or 6 plasmids.

[0010] The nucleic acid molecules disclosed herein that contain sequences encoding viral proteins are such that the sequence encoding viral protein VP4 is ligated at its C-terminus to a sequence encoding a protease cleavage site, at its C-terminus to a sequence encoding viral protein VP2, at its C-terminus to a sequence encoding a protease cleavage site, at its C-terminus to a sequence encoding viral protein VP3, at its C-terminus to a sequence encoding a protease cleavage site, at its C-terminus to a sequence encoding viral protein VP1, at its C-terminus to a sequence encoding a protease cleavage site, at its C-terminus to a sequence encoding viral protein 2A, and the nucleic acid molecule is referred to as the long version or "long". The nucleic acid molecules disclosed herein that contain sequences encoding viral proteins are such that the sequence encoding viral protein VP2 is ligated at its C-terminus to a sequence encoding a protease cleavage site, at its C-terminus to a sequence encoding viral protein VP3, at its C-terminus to a sequence encoding a protease cleavage site, at its C-terminus to a sequence encoding viral protein VP1, at its C-terminus to a sequence encoding viral protein 2A, and the nucleic acid molecule is referred to as the short version or "short". In both the long and short versions, the coding sequence of the protease cleavage site ligated to the 3'-end of the coding sequence encoding viral protein VP1, and the coding sequence of viral protein 2A ligated thereto may be omitted. In both the long and short versions, the coding sequence of the N-terminal leader sequence is ligated to the N-terminus of the coding sequence of viral protein VP4 in the case of the long version, and to the coding sequence of viral protein VP2 in the case of the long sequence. The N-terminal leader is a preferred Ig leader such as an IgG or IgE signal sequence. In some embodiments, the cleavage site is recognized by furin.

[0011] In some embodiments, plasmids are provided that contain nucleic acid molecules, including plasmids encoding FMDV subtypes in which the viral protein is selected from the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3. In some embodiments, a vaccine is provided that contains four plasmids in which the viral protein encoding the nucleic acid sequence is derived from each FMDV subtype of the group consisting of A, Asia1, C, and O. In some embodiments, a vaccine is also provided that contains seven plasmids in which the viral protein encodes a nucleic acid sequence from each FMDV subtype of the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3.

[0012] A method is provided for eliciting an immune response against FMDV in an individual by administering to the individual one of the disclosed vaccines.

[0013] A method is provided for preventing FMDV infection in an individual by administering to the individual one of the disclosed vaccines.

[0014] Provided herein is an isolated nucleic acid comprising a sequence encoding the consensus amino acid sequence of at least VP1-VP3, and preferably VP1-VP4, of foot-and-mouth disease virus that induces a cross-reactive immune response in a subject vaccinated against multiple subtypes of FMD, including A, Asia 1, C, O, SAT1, SAT2, SAT3, SAT4. The nucleic acid may comprise a sequence selected from the group consisting of: (a) a construct derived from FMDV-A24cruzeiro comprising the nucleotide sequence shown in SEQ ID NO: 1 encoding VP-4-VP2-VP3-VP1 (long) shown in SEQ ID NO: 2; (b) a construct derived from FMDV-A24cruzeiro comprising the nucleotide sequence shown in SEQ ID NO: 3 encoding VP2-VP3-VP1 (short) shown in SEQ ID NO: 4; (c) a construct derived from FMDV-As1-Shamir89 comprising the nucleotide sequence shown in SEQ ID NO: 5 encoding VP-4-VP2-VP3-VP1 (long) shown in SEQ ID NO: 6; (d) a construct derived from FMDV-As1-Shamir89 comprising the nucleotide sequence shown in SEQ ID NO: 7 encoding VP2-VP3-VP1 (short) shown in SEQ ID NO: 8; (e) a construct derived from FMDV-SAT2 comprising the nucleotide sequence shown in SEQ ID NO: 9 encoding VP-4-VP2-VP3-VP1 (long) shown in SEQ ID NO: 10; (f) a construct derived from FMDV-STA2 comprising the nucleotide sequence shown in SEQ ID NO: 11 encoding VP2-VP3-VP1 (short) shown in SEQ ID NO: 12.

[0015] Provided herein are nucleic acid molecules such as those selected from the group consisting of: a) a modified nucleotide sequence derived from FMDV-A24cruzeiro, such as that shown in SEQ ID NO: 1 (FMDV-A24cruzeiro-long), inserted into a plasmid such as pVAX having the sequence shown in SEQ ID NO: 13; b) a modified nucleotide sequence derived from FMDV-A24cruzeiro, such as that shown in SEQ ID NO: 3 (FMDV-A24cruzeiro-short), inserted into a plasmid such as pVAX having the sequence shown in SEQ ID NO: 14; c) a modified nucleotide sequence derived from FMDV-As1-Shamir89, such as that shown in SEQ ID NO: 5 (FMDV-As1-Shamir89-long), inserted into a plasmid such as pVAX having the sequence shown in SEQ ID NO: 15; and d) a modified nucleotide sequence derived from FMDV-As1-Shamir89, such as that shown in SEQ ID NO: 7 (FMDV-As1-Shamir89-long), inserted into a plasmid such as pVAX having the sequence shown in SEQ ID NO: 16.

[0016] The nucleic acid molecules in the composition may include the following nucleic acid sequences, and / or their fragments, and / or sequences homologous to those sequences, and / or fragments of such homologous sequences: a) a nucleic acid sequence derived from FMDV-As1-Shamir89 encoding VP4 as shown in SEQ ID NO: 17; b) a nucleic acid sequence derived from FMDV-A24cruzeiro encoding VP4 as shown in SEQ ID NO: 18; c) a nucleic acid sequence derived from FMDV-As1-Shamir89 encoding VP2 as shown in SEQ ID NO: 19; d) a nucleic acid sequence derived from FMDV-A24cruzeiro encoding VP2 as shown in SEQ ID NO: 20; e) a nucleic acid sequence derived from FMDV-As1-Shamir89 encoding 2A as shown in SEQ ID NO: 21; f) a nucleic acid sequence derived from FMDV-A24cruzeiro encoding 2A as shown in SEQ ID NO: 21; g) a nucleic acid sequence derived from FMDV-As1-Shamir89 encoding VP3 as shown in SEQ ID NO: 23; h) a nucleic acid sequence derived from FMDV-A24cruzeiro encoding VP3 as shown in SEQ ID NO: 24; i) a nucleic acid sequence derived from FMDV-As1-Shamir89 encoding VP1 as shown in SEQ ID NO: 25; j) a nucleic acid sequence derived from FMDV-A24cruzeiro encoding VP2 as shown in SEQ ID NO: 26.

[0017] The amino acid sequence of the cleavage site recognized by protease furin is the sequence shown in SEQ ID NO: 27.

[0018] In some embodiments, the construct may include a C3 consensus coding sequence (SEQ ID NO: 28) encoding a C3 protease consensus protein (SEQ ID NO: 29).

[0019] Also provided herein is a vaccine capable of eliciting an immune response against multiple foot-and-mouth disease virus (FMDV) subtypes in a mammal, the vaccine comprising a DNA plasmid comprising a promoter operably linked to a coding sequence encoding a consensus FMDV antigen comprising capsid proteins VP1-VP4 from one or more FMDV subtypes, and a pharmaceutically acceptable excipient, wherein the DNA plasmid is capable of expressing the consensus FMDV antigen in mammalian cells in an amount effective to induce a broad cross-reactive immune response in the mammal. The vaccine can elicit an immune response against FMDV subtypes A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof.

[0020] Also provided herein is a vaccine capable of eliciting an immune response against multiple foot-and-mouth disease virus (FMDV) subtypes in a mammal, the vaccine comprising one or more DNA plasmids comprising a promoter operably linked to a coding sequence encoding a consensus FMDV antigen comprising capsid proteins VP1-VP4 from one or more FMDV subtypes selected from the group consisting of subtypes A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof, and its pharmaceutically acceptable excipient, wherein the DNA plasmid is capable of expressing the consensus FMDV antigen in mammalian cells in an amount effective to induce an immune response in the mammal. The vaccine may be administered to a mammal, such as a pig, ruminant, human, or primate. The vaccine can elicit an immune response in a mammal, such as a humoral response, a cellular response, or both a humoral response and a cellular response.

[0021] Also provided herein is a vaccine capable of eliciting an immune response against multiple FDMV subtypes in a mammal, the vaccine comprising an antigen comprising one or more consensus amino acid sequences encoding capsid proteins VP1-VP4 of foot-and-mouth disease virus (FMDV) subtypes A, Asia 1, C, O, SAT1, SAT2, or SAT3, and a pharmaceutically acceptable excipient thereof. The pharmaceutically acceptable excipient may be an adjuvant selected from the group consisting of IL-2 and IL-15. The pharmaceutically acceptable excipient of the vaccine may be a transfection promoter. The transfection promoter may be a polyanion, polycation or lipid at a concentration of less than 6 mg / ml, such as poly-L-glutamate. The present vaccine may be administered to a mammal, such as a pig, ruminant, human or primate. The present vaccine may elicit an immune response in a mammal, such as a humoral response, a cellular response, or both a humoral response and a cellular response.

[0022] Also provided herein is a method of eliciting an immune response against multiple FMDV virus subtypes in a mammal, comprising delivering the DNA plasmid vaccine described herein to a mammalian tissue and electroporating the cells of the tissue with a constant current energy pulse effective to cause the DNA plasmid to enter the cells. Delivery of the DNA plasmid vaccine described herein may be accomplished by a method that may include injecting the DNA plasmid vaccine into an intradermal, subcutaneous, or muscular tissue. The current and energy pulse may be preset at a constant current equal to an existing current to deliver the DNA plasmid of the method. The electroporation step of the method may further include measuring the impedance in the electroporated cells and adjusting the energy level of the energy pulse relative to the measured impedance to maintain a constant current in the electroporated cells, wherein the measuring and adjusting steps are performed within the lifetime of the energy pulse. The electroporation step may further include delivering the energy pulse to a plurality of electrodes by a pulse sequence pattern that delivers the energy pulse in a dispersed pattern.

[0023] Also provided is a method of diagnosing a mammal infected with FMDV, the method comprising isolating a liquid sample from the mammal, isolating an antibody from the liquid sample of the mammal, and comparing the isolated antibody to a vaccinated control mammal described herein, the control mammal having antibodies only to FMDV VP1-VP4 proteins, and the mammal infected with FMDV having antibodies to FMDV VP1-V4 proteins and FMDV non-structural proteins. The non-structural proteins may be FMDV 2C, 3A, and 3D polymerases.

[0024] A method of inducing an immune response against one or more FMDV virus subtypes in a mammal is provided. The method, which in some embodiments includes administering a nucleic acid molecule encoding a protein having an FMDV immunogenic sequence to a tissue of the mammal, may include the step of electroporating the cells of the tissue with an energy pulse of a constant electric current effective to cause the DNA plasmid to penetrate the cells.

[0025] A method of diagnosing a mammal infected with FMDV in a mammal vaccinated by the process disclosed herein is also provided. The method includes isolating a liquid sample from the vaccinated mammal and detecting the presence of an FMDV protein not included in the vaccine and / or an antibody to an FMDV protein not included in the vaccine. The presence of such an FMDV protein and / or an antibody to such an FMDV protein indicates that the vaccinated mammal is infected with FMDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Mode for Carrying Out the Invention

[0027] The consensus amino acid sequence has been generated for a fusion protein comprising multiple FMDV proteins and individual FMDV proteins from various serotypes. Nucleic acid molecules encoding the protein have also been generated.

[0028] In one aspect of the invention, for protecting mammals from foot-and-mouth disease across one or more subtypes of FMDV including A, Asia 1, O, C, SAT1, SAT2, and SAT3, there is a fusion protein comprising FMDV proteins VP1, VP2, VP3, VP4, and / or 2A, and / or 3C, and nucleic acid sequences encoding these proteins, which can be generated and used in a vaccine. Preferably, the VP1 gene is a consensus for a selected subtype of FMDV, for example, as described herein, it is FMDV - Sat2 where VP1 is the Sat2 consensus VP1.

[0029] Although not bound by scientific theory, a vaccine targeting the consensus amino acid sequences of VP1, VP2, VP3, and / or VP4 for one or more subtypes of FMDV will present a large repertoire of epitopes effective in inducing an immune response (either humoral, cellular, or both) effective against most species within each subtype of FMDV. Although not bound by scientific theory, VP1 is an excellent immunogenic target for vaccines targeting the consensus amino acid sequence of VP1. VP1 is the major immunogen.

[0030] Constructs of some embodiments include a long form and a short form. Constructs of some embodiments provide the viral proteins VP1, VP2, VP3, and VP4 in the following specific order: VP4 - VP2 - VP3 - VP1. An optional tail, 2A, is also provided. The construct has an optional IgE leader sequence. When present, the proteolytic cleavage site "CS" is provided between each of VP4, VP2, VP3, VP1, and 2A when present. The protease capable of processing the site may be furin in some embodiments, or the FMDV protease in some embodiments. Other protease sites may also be used. The site must be recognized by proteases commonly found within the cells in which the vaccine is expressed.

[0031] In one aspect of the invention, there are fusion proteins comprising consensus FMDV proteins VP1, VP2, VP3, VP4, and / or 2A, and / or 3C, and nucleic acid sequences encoding these proteins, which can be generated and used in a vaccine to protect mammals from foot - and - mouth disease across one or more subtypes of FMDV, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

[0032] In another aspect of the invention, there is a fusion protein, derived from two different subtypes, comprising a consensus FMDV protein VP1 and a nucleic acid sequence encoding this protein, which is produced and can be used in a vaccine to protect mammals from foot-and-mouth disease across one or more subtypes of FMDV, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

[0033] In another aspect of the invention, there is a consensus FMDV protein VP1 and a nucleic acid sequence encoding it, which are produced and used in a vaccine to provide protection for mammals against foot-and-mouth disease across one or more subtypes of FMDV, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

[0034] 1. Definitions The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0035] In the citation of numerical ranges herein, each intervening number, including the same degree of precision, is clearly contemplated. For example, in the range of 6 to 9, in addition to 6 and 9, the numerical values 7 and 8 are contemplated, and in the range of 6.0 to 7.0, the numerical values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are clearly contemplated.

[0036] a. Adjuvant As used herein, "adjuvant" may mean any molecule that is added to the DNA plasmid vaccine described herein to enhance the antigenicity of the foot-and-mouth disease virus (FMDV) antigen encoded by the DNA plasmid and the coding nucleic acid sequence described below.

[0037] b. Antibody "Antibody" may mean a classified antibody such as IgG, IgM, IgA, IgD or IgE, or a fragment thereof, including Fab, F(ab’)2, Fd and single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof, or a fragment or derivative of such an antibody. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0038] c. Coding sequence As used herein, "coding sequence" or "coding nucleic acid" may mean a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include start and stop signals operably linked to regulatory elements including a promoter and a polyadenylation signal capable of directing expression in a cell of an individual or mammal to which the nucleic acid is administered.

[0039] d. Complementary sequence As used herein, "complementary sequence" or "complementary" may mean a nucleic acid capable of meaning Watson-Crick type (e.g., A-T / U and C-G) or Hoogsteen type base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0040] e. Consensus or consensus sequence As used herein, "consensus" or "consensus sequence" may mean a synthetic nucleic acid sequence or corresponding polypeptide sequence constructed based on the analysis of the alignment of multiple subtypes of a particular influenza antigen and which can be used to induce a broad range of immunity against multiple subtypes or serotypes of a particular influenza antigen. The consensus FMDV antigen may include VP1, VP2, VP3, VP4, and C2 protease nucleotide and amino acid sequences. Similarly, synthetic antigens, such as fusion proteins, may be engineered into the consensus sequence (or consensus antigen).

[0041] f. Constant current As used herein, "constant current" is defined as the current received or accepted by a tissue, or the cells that define the tissue, over the duration of an electrical pulse delivered to the same tissue. The electrical pulse is delivered from an electroporation device described herein. The electroporation devices provided herein preferably have instantaneous feedback and have a feedback element, so this current maintains a constant ampere in the tissue within the lifetime of the electrical pulse. The feedback element can measure the resistance of the tissue (or cell) through the duration of the pulse and vary the electrical energy output of the electroporation device (e.g., increase the voltage), whereby the current within the same tissue remains constant through the electrical pulse (in units of microseconds) and between pulses. In some embodiments, the feedback element includes a control device.

[0042] g. Current feedback or feedback As used herein, "current feedback" or "feedback" may be used interchangeably and may mean the active response of a provided electroporation device that includes measuring the in-tissue current between electrodes and varying the energy output delivered by the EP device to hold that current at a constant level accordingly. This constant level is preset by the user prior to the start of a pulse sequence or an electrical treatment. In the electrical circuit within the device, the in-tissue current between the electrodes can be continuously monitored to compare the monitored current (or in-tissue current) to a preset current, and the energy output adjustment can be continuously performed to hold the monitored current at the preset level, so the feedback may be performed by an electroporation component of the electroporation device, such as a control device. Since the feedback loop is an analog closed-loop feedback, it may be instantaneous.

[0043] h. Distributed current As used herein, "distributed current" may mean the pattern of current delivered from the various needle electrode arrays of the electroporation device described herein, and by that pattern, the generation of electroporation-related thermal stress in any region of the tissue being electroporated is minimized or preferably eliminated.

[0044] i. Electroporation As used interchangeably herein, "electroporation", "electropermeabilization", or "electrokinetic enhancement" ("EP") may refer to inducing microscopic pathways (pores) in a biological membrane by the use of transmembrane electric field pulses, and by their presence, biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water pass from one side of the cell membrane to the other.

[0045] j. Feedback mechanism As used herein, "feedback mechanism" may refer to a process implemented by software or hardware (or firmware) that receives the impedance of the desired tissue (before, during, and / or after delivery of the energy pulse), compares it to an existing value, preferably current, and adjusts the delivered energy pulse to execute a preset value. The feedback mechanism may be implemented by an analog closed loop.

[0046] k. Fragment As used herein, "fragment" may mean a portion or nucleic acid encoding a polypeptide capable of inducing an immune response in a mammal that is substantially similar to the immune response of a non-fragment for at least one FMDV subtype, such as A, Asia 1, C, O, SAT1, SAT2, or SAT3. The fragment may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the FMDV protein encoded by the nucleic acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, or 11. The DNA fragment may be of a nucleotide length of 30 or more, 45 or more, 60 or more, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210 or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 or more, 480 or more, 540 or more, 600 or more, 660 or more, 720 or more, 780 or more, 840 or more, 900 or more, 960 or more, 1020 or more, 1080 or more, 1140 or more, 1200 or more, 1260 or more, 1320 or more, 1380 or more, 1440 or more, 1500 or more, 1560 or more, 1620 or more, 1680 or more, 1740 or more, 1800 or more, 1860 or more, 1820 or more, 1880 or more, 1940 or more, 2000 or more, 2600 or more, 2700 or more, 2800 or more, 2900 or more, 2910 or more, 2920 or more, 2930 or more, 2931 or more, 2932 or more, 2933 or more, 2934 or more, 2935 or more, 2936 or more, 2937 or more, or 2938 or more.

[0047] The DNA fragment may contain a coding sequence for an immunoglobulin leader, such as an IgE or IgG sequence.

[0048] The DNA fragment may be less than 10 nucleotides, less than 20, less than 30, less than 40, less than 50, less than 60, less than 75, less than 90, less than 120, less than 150, less than 180, less than 210, less than 240, 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, less than 1800, less than 1860, less than 1820, less than 1880, less than 1940, less than 2000, less than 2600, less than 2700, less than 2800, less than 2900, less than 2910, less than 2920, less than 2930, less than 2931, less than 2932, less than 2933, less than 2934, less than 2935, less than 2936, less than 2937, or less than 2938.

[0049] "Fragment" may mean a polypeptide fragment capable of inducing an immune response in mammals that is substantially similar to the immune response of non-fragmented for at least one FMDV subtype, such as A, Asia 1, C, O, SAT1, SAT2, or SAT3. The fragment may be a polypeptide fragment selected from at least one of the various coding polypeptide sequences of the present invention including SEQ ID NO: 2, 4, 6, 8, 10, 12. The polypeptide fragment may be analyzed to contact at least one antigenic epitope provided by a publicly available database such as the FMDV Sequence Database of the Los Alamos National Laboratory. The protein fragment may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the FMDV protein shown in the polyprotein shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12. The polypeptide may comprise an amino acid sequence for an immunoglobulin leader, such as IgE or IgG. The polypeptide fragment may be 30 or more amino acids in length, 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, or 710 or more amino acids in length. The polypeptide fragment may be less than 10 amino acids, less than 20, less than 30, less than 40, less than 50, less than 60, less than 75, less than 90, less than 120, less than 150, less than 180, less than 210, less than 240, 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 700, less than 701, less than 702, less than 703, less than 704, less than 705, less than 706, less than 707, less than 708, less than 709, or less than 710 amino acids in length.

[0050] l. Homology The homology of multiple sequence alignments may be generated using ClustalW (http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html).

[0051] m. identical In the context of two or more nucleic acid or polypeptide sequences, as used herein, "identical" or "identity" may mean that a sequence has a specified percentage of residues that are the same in a particular region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences in the particular region, determining the number of positions at which the identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the particular region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment generates one or more gapped ends and the particular region being compared includes only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using computer sequence algorithms such as BLAST or BLAST 2.0.

[0052] n. impedance As used herein, "impedance" may be used when considering feedback mechanisms and can be converted to a current value by Ohm's law and thus compared to a preset current.

[0053] o. immune response As used herein, "immune response" may mean the activation of the host immune system, such as that of a mammal, in response to the introduction of an FMDV consensus antigen via the provided DNA plasmid vaccine. The immune response can be in the form of a cellular response, a humoral response, or both.

[0054] p. nucleic acid As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked to each other. A single-stranded representation also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the described single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complementary sequences. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. That is, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0055] A nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. A nucleic acid may be DNA, both genomic DNA and cDNA, RNA, or a hybrid, and may contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. A nucleic acid may be obtained by chemical synthesis or recombinant methods.

[0056] Nucleic acids generally contain phosphodiester linkages, but may also include at least one different linkage, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages, and nucleic acid analogs that may have a peptide nucleic acid backbone and linkages. Other similar nucleic acids include those having a positive backbone, a non-ionic backbone, and a non-ribose backbone, such as those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated herein by reference. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within the definition of nucleic acids. Modified nucleotide analogs may be located, for example, at the 5' and / or 3' termini of a nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar-modified or backbone-modified ribonucleotides. However, it should be noted that ribonucleotides modified with nucleobases, i.e., ribonucleotides containing non-naturally occurring nucleobases instead of naturally occurring nucleobases, such as uridine or cytidine modified at the 5-position, for example, 5-(2-amino)propyluridine, 5-bromouridine; adenosine and guanosine modified at the 8-position, for example 8-bromoguanosine; deazapurines, for example 7-deazaadenosine; O- and N-alkylated nucleotides, for example N6-methyladenosine, are also suitable. The 2'-OH group may be substituted with a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Modified nucleotides may include nucleotides conjugated to cholesterol by cleaving a hydroxyprolinol bond, as described, for example, in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Application Publication No. 20050107325, which are incorporated herein by reference. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference.Additional modified nucleotides and nucleic acids are described in U.S. Patent Application Publication No. 20050182005, which is incorporated herein by reference. Modifications of the ribose-phosphate backbone may be carried out for various reasons, for example, to improve the stability and half-life of such molecules in a physiological environment, to facilitate diffusion through cell membranes, or as probes for biochips. Mixtures of naturally derived nucleic acids and analogs may be produced, or mixtures of different nucleic acid analogs as well as mixtures of naturally derived nucleic acids and analogs may be produced.

[0057] q. operably linked As used herein, "operably linked" may mean that the expression of a gene is under the control of a spatially linked promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls within the gene from which the promoter is derived. As is known to those skilled in the art, variations in this distance can be accommodated without loss of promoter function.

[0058] r. promoter As used herein, "promoter" may mean a synthetic or naturally-derived molecule capable of conferring, activating or promoting the expression of nucleic acids in a cell. The promoter may contain one or more specific transcriptional regulatory sequences in order to further promote expression and / or to change its spatial and / or temporal expression. The promoter may contain distal enhancer or repressor elements, which can be located thousands of base pairs from the transcription start site. The promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter may constitutively regulate the expression of genetic components, or may regulate variably in response to the cell, tissue or organ in which expression occurs, or the developmental stage at which expression occurs, or external stimuli such as physiological stress, pathogens, metal ions or introduced agents. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0059] s. stringent hybridization conditions As used herein, "stringent hybridization conditions" can mean conditions under which a first nucleic acid sequence (e.g., a probe), such as in a complex mixture of nucleic acids, hybridizes to a second nucleic acid sequence (e.g., a target). Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10 °C lower than the thermal melting point (T m ) of a particular sequence at a defined ionic strength and pH. T m may be the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess, T mIn this case, 50% of the probes are occupied in the equilibrium state. Stringent conditions may be such that the salt concentration is less than about 1.0 M sodium ions, for example, a sodium ion (or other salt) concentration of 0.01 - 1.0 M at pH 7.0 - 8.3, and the temperature is at least about 30 °C for short probes (e.g., about 10 - 50 nucleotides) and at least about 60 °C for long probes (e.g., more than about 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least 2 - 10 times that of the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C, or 5×SSC, 1% SDS, incubated at 65 °C, washed with 0.2×SSC, and 0.1% SDS at 65 °C.

[0060] t. Substantially complementary As used herein, "substantially complementary" means that in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complementary sequence of the second sequence, or the two sequences hybridize under stringent hybridization conditions.

[0061] u. Substantially identical As used herein, "substantially identical" may mean that, when a first sequence is substantially complementary to the complementary sequence of a second sequence, the first and second sequences are in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or, with respect to nucleic acids, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical.

[0062] v. subtype or serotype As used interchangeably herein and in connection with FMDV virus, "subtype" or "serotype" means a genetic variant of an FMDV virus antigen such that one subtype is recognized by an immune system distinct from that of a different subtype.

[0063] w. variant As used herein with respect to nucleic acids, "variant" may mean: (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complementary sequence of a referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complementary sequence, or a sequence substantially identical thereto.

[0064] "Variant" with respect to a peptide or polypeptide whose amino acid sequence differs by amino acid insertions, deletions, or conservative substitutions but retains at least one biological activity. The variant may mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacing one amino acid with another having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is typically recognized in the art as involving small-scale changes. These small-scale changes can be identified, in part, by considering the hydrophobic-hydrophilic index of the amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobic-hydrophilic index of an amino acid is based on its hydrophobicity and charge considerations. It is known in the art that amino acids with similar hydrophobic-hydrophilic indices can be substituted and the function of the protein can still be retained. In one embodiment, amino acids having a hydrophobic-hydrophilic index of ±2 are substituted. It is also possible to identify substitutions that result in a protein retaining its biological function by taking advantage of the hydrophilicity of the amino acids. By considering the hydrophilicity of amino acids in the context of a peptide, it is possible to calculate a useful metric that has been reported to correlate well with the maximum local average hydrophilicity of the peptide, i.e., antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is hereby incorporated by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilic values can result in a peptide retaining its biological activity, e.g., immunogenicity. The substitution may be carried out using amino acids having hydrophilic values within ±2 of each other. Both the hydrophobic index and the hydrophilic value of an amino acid are affected by its particular side chain. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0065] x. Vector As used herein, "vector" may mean a nucleic acid sequence containing an origin of replication. The vector may be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be either an extrachromosomal vector that self-replicates or a vector integrated into the host genome.

[0066] 2. FMDV Proteins and Coding Sequences The genomes of each of subtypes A, C, O, Asia, SAT1, SAT2, and SAT3 are found in GenBank under the following accession numbers. A: JF749843 C: NC_002554 O: JF749851 Asia: DQ533483 SAT-1: JF749860 SAT-2: JF749862 SAT-3: NC_011452. These can be used to identify the positions of the coding sequences for each of VP1, VP2, VP3, and VP4 for each of subtypes A, C, O, Asia, SAT1, SAT2, and SAT3. Similarly, as described above, International Publication No. WO 2011 / 054011 discloses FMDV vaccines having VP1, VP2, VP3, VP4 from FMDV subtypes A, C, O, Asia, SAT1, SAT2, and SAT3, although different designs are used. One of ordinary skill in the art can use the information in International Publication No. WO 2011 / 054011 and GenBank to identify the coding sequences for each of the FMDV proteins VP1, VP2, VP3, VP4 from subtypes A, C, O, Asia, SAT1, SAT2, and SAT3.

[0067] Homologous proteins that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to FMDV proteins VP1, VP2, VP3, or VP4 from subtypes A, C, O, Asia, SAT1, SAT2, or SAT3 may be used in some constructs.

[0068] Fragments of FMDV proteins VP1, VP2, VP3, or VP4 from subtypes A, C, O, Asia, SAT1, SAT2, or SAT3 that have 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the full-length sequence may be used in some constructs.

[0069] Fragments of proteins that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to FMDV proteins VP1, VP2, VP3, or VP4 from subtypes A, C, O, Asia, SAT1, SAT2, or SAT3 and have 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the full-length sequence may be used in some constructs.

[0070] The coding sequences of these FMDV proteins, homologous proteins, fragments of FMDV proteins, and fragments of homologous proteins may be used in constructs.

[0071] The original proteolytic cleavage site may be present between each of the consensus antigen sequences of the amino acid sequence: RGRKRRS, etc.

[0072] Provided herein are antigens capable of inducing an immune response in a mammal against one or more foot-and-mouth disease virus (FMDV) subtypes. The antigen may be an FMDV antigen comprising capsid proteins VP1, VP2, VP3, VP4, their consensus, variants, fragments thereof, or combinations thereof. The FMDV antigen may be from FMDV subtypes A, Asia 1, C, O, SAT1, SAT2, or SAT3. The FMDV antigen may comprise at least one antigenic epitope that may be effective against a specific FMDV immunogen capable of inducing an immune response. The empty virus capsid proteins VP1-VP4 of the FMDV antigen provide the entire repertoire of immunogenic sites and epitopes present in the intact FMDV virus. The consensus FMDV antigen sequence may be obtained from the FMDV antigen sequences of a plurality of FMDV viruses of one FMDV subtype. The consensus FMDV antigen may comprise VP1, VP2, VP3, and VP4 FMDV subtype consensus protein sequences, which may be consensus VP1-VP4 proteins. The consensus VP1-VP4 proteins may comprise at least one FMDV protein 3C cleavage site. The protein 3C cleavage site may be present between each of the consensus VP1-VP4 sequences of the consensus VP1-4 proteins. Cleavage of the consensus VP1-VP4 proteins by protein 3C may cleave the consensus VP1-VP4 proteins to produce consensus VP1-, consensus VP2-, consensus VP3-, and consensus VP4-proteins. Alternatively, native proteolytic cleavage sites may be present between each of the consensus antigen sequences such as the amino acid sequence: RGRKRRS.

[0073] In some embodiments, the proteins are 80% identical. In some embodiments, the proteins are 90% identical. In some embodiments, the proteins are 95% identical. In some embodiments, the proteins are 96% identical. In some embodiments, the proteins are 97% identical. In some embodiments, the proteins are 98% identical. In some embodiments, the proteins are 99% identical.

[0074] Provided herein is a coding sequence of an antigen capable of inducing an immune response in a mammal against one or more foot-and-mouth disease virus (FMDV) subtypes. The antigen may be an FMDV antigen comprising capsid proteins VP1, VP2, VP3, VP4, their consensus, variants, fragments or combinations thereof. The FMDV antigen may be derived from FMDV subtypes A, Asia 1, C, O, SAT1, SAT2, or SAT3. The FMDV antigen may comprise at least one antigenic epitope that may be effective against a specific FMDV immunogen capable of inducing an immune response. The empty virus capsid proteins VP1-4 of the FMDV antigen provide the entire repertoire of immunogenic sites and epitopes present in the intact FMDV virus. The consensus FMDV antigen sequence may be obtained from the FMDV antigen sequences of multiple FMDV viruses of one FMDV subtype. The consensus FMDV antigen may comprise VP1, VP2, VP3, and VP4 FMDV subtype consensus protein sequences, which may be consensus VP1-4 proteins. The consensus VP1-4 proteins may comprise at least one FMDV protein 3C cleavage site. The protein 3C cleavage site may be present between each of the consensus VP1, VP2, VP3, and VP4 sequences of the consensus VP1-4 proteins. Cleavage of the consensus VP1-4 proteins by protein 3C may cleave the consensus VP1-4 proteins to generate consensus VP1-, consensus VP2-, consensus VP3-, and consensus VP4-proteins. Alternatively, the native proteolytic cleavage site may be present between each of the consensus antigen sequences such as the amino acid sequence: RGRKRRS. A coding sequence of a fusion protein comprising a consensus of protease 3C is provided.

[0075] In addition, the coding sequence can encode a protein that can be a fragment of the protein described herein. In some embodiments, the coding sequence encodes a protein that is 20% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 30% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 40% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 50% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 60% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 70% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 85% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 90% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 95% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 96% of the consensus protein. In some embodiments, the coding sequence encodes a protein that is 97% of the consensus protein. I

[0076] In addition, the coding sequence can encode a protein that is homologous to the protein provided herein. In some embodiments, the coding sequence encodes a protein that is 80% homologous. In some embodiments, the coding sequence encodes a protein that is 90% homologous. In some embodiments, the coding sequence encodes a protein that is 95% homologous. In some embodiments, the coding sequence encodes a protein that is 96% homologous. In some embodiments, the coding sequence encodes a protein that is 97% homologous. In some embodiments, the coding sequence encodes a protein that is 98% homologous. In some embodiments, the coding sequence encodes a protein that is 99% homologous.

[0077] In addition, the coding sequence encodes a protein that is a fragment of a protein homologous to the proteins described herein. In some embodiments, the coding sequence encodes a protein that is 20% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 30% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 40% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 50% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 60% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 70% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 80% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 90% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 95% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 96% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 97% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 98% of the homologous protein. In some embodiments, the coding sequence encodes a protein that is 99% of the homologous protein.

[0078] 3. Plasmid Provided herein is a vector capable of expressing one or more FMDV antigens in mammalian cells in an amount effective to induce an immune response in a mammal. The vector may contain a heterologous nucleic acid encoding an FMDV antigen. The vector may be a plasmid. The plasmid may be useful for transfecting cells with a nucleic acid encoding an FMDV antigen, and the host cells to be transformed are cultured and maintained under conditions under which expression of the FMDV antigen occurs.

[0079] The plasmid may contain an FMDV antigen selected from the proteins provided herein, a fragment thereof, a homologous sequence thereof, and a nucleic acid encoding a homologous fragment. The plasmid may further contain a start codon or leader sequence that may be present upstream of the coding sequence and a stop codon that may be present downstream of the coding sequence. The start codon and stop codon may be within the frame of the coding sequence.

[0080] The plasmid may contain a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence may be a promoter from Simian virus 40 (SV40), Mouse mammary tumor virus (MMTV) promoter, Human immunodeficiency virus (HIV) promoter, for example, Bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, Avian leukosis virus (ALV) promoter, Cytomegalovirus (CMV) promoter, for example, CMV immediate early promoter, Epstein - Barr virus (EBV) promoter, or Rous sarcoma virus (RSV) promoter. The promoter may be a promoter from a human gene, for example, a promoter from human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may be a natural or synthetic tissue - specific promoter, for example, a muscle or skin - specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. 20040175727, the contents of which are incorporated herein by reference in their entirety.

[0081] The plasmid may contain a polyadenylation signal, which may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0082] The plasmid may contain an enhancer upstream of the coding sequence. The enhancer may be a human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as those of CMV, FMDV, RSV or EBV. Enhancement of the function of the polynucleotide is described in U.S. Patent Nos. 5,593,972, 5,962,428 and International Publication No. 94 / 016737, which are hereby incorporated by reference in their entirety.

[0083] The plasmid may contain a mammalian origin of replication to maintain the plasmid episomally and generate multiple copies of the plasmid within the cell. The plasmid may be pVAX1, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may contain the origin of replication of Epstein-Barr virus and the coding region of the nuclear antigen EBNA-1 and may generate high-copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0084] The plasmid may contain regulatory sequences, which may be well-suited for gene expression in the cells to which the plasmid is administered. The coding sequence may contain codons, which may allow for more efficient transcription of the coding sequence in the host cell.

[0085] The coding sequence may include an Ig leader sequence. The leader sequence may be at the 5' of the coding sequence. The consensus protein encoded by this sequence may include a consensus protein following an N-terminal Ig leader. The N-terminal Ig leader may be IgE or IgG.

[0086] The plasmid may be pSE420 (Invitrogen, San Diego, CA), and it may be used for protein production in Escherichia coli (E. coli). The plasmid may be pYES2 (Invitrogen, San Diego, CA), and it may be used for protein production in the Saccharomyces cerevisiae strain of yeast. The plasmid may be of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, CA), and it may be used for protein production in insect cells. The plasmid may be pcDNA I or pcDNA3 (Invitrogen, San Diego, CA), and it may be used for protein production in mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0087] The plasmid may include one or more coding sequences encoding one or more of VP1, VP2, VP3, VP4, and 3C from one or more subtypes, such as Asia, A, O, C, SAT1, SAT2, and SAT3.

[0088] In some embodiments, the plasmid includes coding sequences for multiple different consensus FMDV antigens VP1, VP2, VP3, VP4, and 3C from subtypes Asia, A, O, C, SAT1, SAT2, or SAT3.

[0089] In some embodiments, the plasmid comprises the coding sequences of multiple different consensus FMDV antigens VP1, VP2, VP3, and VP4 from subtypes Asia, A, O, C, SAT1, SAT2, or SAT3.

[0090] In some embodiments, the plasmid comprises the coding sequences of two different consensus FMDV antigens VP1 from two of subtypes Asia, A, O, and C, for example, VP1 from subtype Asia and VP1 from subtype O, or VP1 from subtype A and VP1 from subtype C.

[0091] In some embodiments, the plasmid comprises the coding sequence of a consensus FMDV antigen VP1, for example, VP1 subtype Asia, VP1 subtype A, VP1 subtype O or VP1 subtype C.

[0092] The coding sequences can be encoded by different DNA plasmids all regulated by a functionally linked promoter, for example, a DNA plasmid having coding sequences containing multiple consensus FMDV antigens regulated by one or more promoters.

[0093] The vector can be pVAX1 or a pVax1 variant with changes, such as the mutant plasmids described herein. The mutant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVAX1 (Invitrogen, Carlsbad, CA). The CMV promoter is located at bases 137 - 724. The T7 promoter / priming site is at bases 664 - 683. The multiple cloning site is at bases 696 - 811. The bovine GH polyadenylation signal is at bases 829 - 1053. The kanamycin resistance gene is at bases 1226 - 2020. The pUC origin is at bases 2320 - 2993.

[0094] Based on the sequence of pVAX1 available from Invitrogen, the following mutants were found in the sequence of pVAX1 used as the backbone of plasmids 1 - 6 described herein:

Table 1

[0095] Base pairs 2, 3, and 4 are changed from ACT to CTG upstream of the backbone and the CMV promoter.

[0096] The backbone of the vector can be pAV0242. The vector can be a replication - defective adenovirus type 5 (Ad5) vector.

[0097] The plasmid may contain regulatory sequences, which may be well - suited for gene expression in the cells to which the plasmid is administered. The coding sequence may contain codons that allow for more efficient transcription of the coding sequence in the host cell.

[0098] The coding sequence may contain an Ig leader sequence. The leader sequence may be at the 5’ of the coding sequence. The consensus antigen encoded by this sequence may contain a consensus antigen protein following the N - terminal Ig leader. The N - terminal Ig leader may be IgE or IgG.

[0099] The plasmid may be pSE420 (Invitrogen, San Diego, California), and it may be used for protein production in Escherichia coli (E. coli). The plasmid may be pYES2 (Invitrogen, San Diego, California), and it may be used for protein production in the yeast Saccharomyces cerevisiae strain. The plasmid may be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, California), and it may be used for protein production in insect cells. The plasmid may be pcDNA I or pcDNA3 (Invitrogen, San Diego, California), and it may be used for protein production in mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0100] 4. Vaccine Without being bound by scientific theory, a vaccine that can be used to induce a broad immune response (humoral, cellular, or both) against FMDV may contain one or more of the previously shown coding sequences, i.e., nucleic acid sequences encoding one or more of the proteins VP1, VP2, VP3, VP4, and 2A from subtypes selected from the group consisting of FMDV subtypes such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof. In some embodiments, the vaccine may contain a nucleic acid encoding FMDV C3 protease, which may be a consensus C3 protease nucleic acid.

[0101] This includes the following: An isolated nucleic acid comprising a sequence encoding at least VP1-VP3, and preferably VP1-4 consensus amino acid sequences of foot-and-mouth disease virus that induce cross-reactive immune responses in subjects vaccinated against multiple subtypes of FMD, including A, Asia 1, C, O, SAT1, SAT2, and SAT3. The nucleic acid may comprise a sequence selected from the group consisting of: (a) nucleotide sequences encoding SEQ ID NO:1; SEQ ID NO:2, (b) nucleotide sequences encoding SEQ ID NO:3; SEQ ID NO:4, (c) nucleotide sequences encoding SEQ ID NO:5; SEQ ID NO:6, (d) nucleotide sequences encoding SEQ ID NO:7; SEQ ID NO:8, (e) nucleotide sequences encoding SEQ ID NO:9; SEQ ID NO:10, and (f) nucleotide sequences encoding SEQ ID NO:11; SEQ ID NO:12.

[0102] Provided herein is a vaccine capable of eliciting an immune response against one or more FMDV subtypes in a mammal. The vaccine may comprise the plasmid(s) discussed previously. The vaccine may comprise multiple plasmids, each targeting one or more FMDV subtypes such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof. The vaccine may comprise an FMDV antigen that itself targets one or more FMDV subtypes such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof. The vaccine may comprise plasmids targeting FMDV subtypes from specific regions of the world, such as Asia, Europe, and sub-Saharan Africa. Alternatively, or in addition, the vaccine may comprise one or more proteins of FMDV subtypes such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof. The vaccine may comprise an FMDV antigen that itself targets one or more FMDV subtypes such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or combinations thereof. The vaccine may comprise plasmids and / or proteins targeting FMDV subtypes from specific regions of the world, such as Asia, Europe, and sub-Saharan Africa. The vaccine may be provided to induce a therapeutic or prophylactic immune response.

[0103] Provided herein is a pharmaceutical composition according to the present invention containing from about 1 nanogram to about 10 mg of DNA. In some embodiments, the pharmaceutical composition according to the present invention contains 1) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms, or at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or more than 10 mg; and 2) 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms or less, or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms or less, or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.Contain between 5 or 10 mg or less. In some embodiments, the pharmaceutical composition according to the present invention contains from about 5 nanograms to about 10 mg of DNA. In some embodiments, the pharmaceutical composition according to the present invention contains from about 25 nanograms to about 5 mg of DNA. In some embodiments, the pharmaceutical composition contains from about 50 nanograms to about 1 mg of DNA. In some embodiments, the pharmaceutical composition contains from about 0.1 to about 500 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 1 to about 350 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 5 to about 250 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 10 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 15 to about 150 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 20 to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 25 to about 75 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 30 to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 35 to about 40 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains from about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.It contains from 1 to about 500 micrograms. In some preferred embodiments, the pharmaceutical composition contains from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains from about 100 to about 200 micrograms of DNA.

[0104] The pharmaceutical composition according to the present invention is formulated according to the mode of administration used. When the pharmaceutical composition is an injectable pharmaceutical composition, it is sterilized, pyrogen-free and particulate-free. Preferably, an isotonic formulation is used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some examples, an isotonic solution, such as phosphate buffered saline, is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0105] Preferably, the pharmaceutical composition is a vaccine, more preferably a DNA vaccine.

[0106] The vaccine may be a DNA vaccine. The DNA vaccine may contain a plurality of identical or different plasmids containing the nucleic acid coding sequences of one or more consensus prostate antigens. The DNA vaccine may contain one or more nucleic acid sequences encoding one or more consensus prostate antigens. When the DNA vaccine contains the coding sequences of more than one consensus prostate antigen, such sequences may all be present on a single plasmid, or each of such sequences may be present on a different plasmid.

[0107] In some embodiments, the vaccine may contain nucleic acid sequences encoding one or more consensus prostate antigens in combination with one or more consensus prostate antigens.

[0108] DNA vaccines are disclosed in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are hereby incorporated by reference in their entirety. The DNA vaccine can further include an element or reagent that inhibits integration into the chromosome. The vaccine can be RNA of a prostate antigen. The RNA vaccine can be introduced into cells.

[0109] The vaccine may be a recombinant vaccine containing the gene construct or antigen described above. The vaccine may also contain one or more consensus prostate antigens in the form of one or more protein subunits, or one or more attenuated virus particles containing one or more consensus antigens. The attenuated vaccine may be an attenuated live vaccine, a killed vaccine, and a vaccine that delivers a foreign gene encoding one or more consensus prostate antigens using a recombinant vector, a subunit vaccine, and a protein vaccine. Examples of attenuated live vaccines, attenuated live vaccines using recombinant vectors to deliver prostate antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. Patent Nos. 4,510,245, 4,797,368, 4,722,848, 4,790,987, 4,920,209, 5,017,487, 5,077,044, 5,110,587, 5,112,749, 5,174,993, 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, 5,294,548, 5,310,668, 5,387,744, 5,389,368, 5,424,065, 5,451,499, 5,453,364, 5,462,734, 5,470,734, 5,474,935, 5,482,713, 5,591,439, 5,643,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference. The vaccine may contain other vaccine components, for example, a plasmid combined with an FMDV protein or an expression vector encoding a protein.

[0110] The provided vaccine may be used to induce an immune response, including a therapeutic or prophylactic immune response. Antibodies and / or killer T cells targeting consensus prostate antigen may be generated. Such antibodies and cells may be isolated.

[0111] The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule acting as a vehicle, adjuvant, carrier, or diluent. The pharmaceutically acceptable excipient may be a transfection promoter, which includes surfactants such as immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters.

[0112] The transfection promoter is a polyanion, polycation, or lipid including poly-L-glutamate (LGS). The transfection promoter is poly-L-glutamate, and more preferably the poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg / ml. The transfection promoter may include a surfactant, such as an immunostimulating complex (ISCOMs), Freund's incomplete adjuvant, an LPS analog including monophosphoryl lipid A, muramyl peptide, quinone analog, and vesicles such as squalene and squalene, and may be administered in combination with hyaluronic acid integrated with the gene construct. In some embodiments, the DNA plasmid vaccine may include a transfection promoter, such as a lipid, lecithin liposome or other liposomes known in the art such as a DNA liposome mixture (see, for example, WO 93 / 24640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters. Preferably, the transfection promoter is a polyanion, polycation, or lipid including poly-L-glutamate (LGS). The concentration of the transfection agent in the 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.

[0113] A pharmaceutically acceptable excipient may be an adjuvant. The adjuvant may be expressed in an alternative plasmid or another gene delivered as a protein combined with the previous plasmid in the vaccine. The adjuvant can be selected from the group consisting of alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine in epithelium (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 lacking a signal sequence and optionally including an IgE-derived signal peptide. The adjuvant may be IL-12, IL-15, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0114] Other genes that may be useful adjuvants include MCP-1, MIP-1a, MIP-1b, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factors, fibroblast growth factors, IL-7, nerve growth factors, vascular endothelial growth factors, 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, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and genes encoding functional fragments thereof.

[0115] The vaccine may further contain a gene vaccine adjuvant as described in U.S. Patent Application No. 021,579, filed April 1, 1994, which is incorporated herein by reference in its entirety.

[0116] The vaccine may be formulated according to the mode of administration used. The injectable vaccine pharmaceutical composition may be sterilized and may be pyrogen-free and particulate-free. An isotonic formulation or solution may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may contain a vasoconstrictor. The isotonic solution may contain phosphate buffered saline. The vaccine may further contain stabilizers including gelatin and albumin. Stabilization such as LGS, polycation, or polyanion to the vaccine formulation may allow the formulation to be stable for a long time at room temperature or ambient temperature.

[0117] 5. Method of delivering the vaccine Provided herein is a method of delivering a vaccine for providing a gene construct and a protein of an FMDV antigen comprising an epitope that is particularly effective against an immunogen of FMDV capable of inducing an immune response. The method of delivering the vaccine or the method of vaccination may be provided to induce therapeutic and prophylactic immune responses. By the vaccination process, an immune response against multiple FMDV subtypes can be elicited in mammals. The vaccine can be delivered to an individual to modulate the activity of the mammalian immune system and promote an immune response. Delivery of the vaccine can be transfection of the FMDV antigen as a nucleic acid molecule that is expressed in cells and delivered to the cell surface, against which the immune system recognizes and induces a cellular response, a humoral response, or both a cellular and a humoral response. Vaccine delivery can be used to induce or evoke an immune response against multiple FMDV viruses in a mammal by administering the previously described vaccine to the mammal.

[0118] When delivering a vaccine and a plasmid into mammalian cells, the transfected cells express and secrete the consensus capsid of each plasmid injected from the vaccine. These secreted capsid proteins are recognized as foreign substances by the immune system, and antibodies are generated against them. These antibodies are retained by the immune system and rapidly clear subsequent FMDV infections.

[0119] A vaccine may be administered to a mammal to induce an immune response in the mammal. The mammal may be a human, a primate, a non-human primate, a cow, a beef cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, a bovine, a deer, a hamster, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0120] a. Combination treatment The vaccine is administered in combination with α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 lacking the signal sequence and optionally containing the signal peptide from IgE, IL-12, IL-15, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-18 mutant forms, CD40, CD40L, vascular endothelial 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, inactivated NIK, SAP K, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, O×40, O×40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and other proteins or genes encoding their functional fragments or combinations thereof. The vaccine may be administered in combination with CTACK protein, TECK protein, MEC protein or functional fragments thereof.

[0121] The vaccine may be administered by different routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or combinations thereof. In veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the most suitable dosage regimen and route of administration for a particular animal. The vaccine may be administered by traditional syringes, needleless injection devices, "particle bombardment gene guns", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

[0122] The plasmid of the vaccine may be delivered to mammals by a plurality of well-known technologies including DNA injection (also called DNA vaccination) with or without in vivo electroporation, liposome-mediated vectors, nanoparticle-facilitated vectors, recombinant vectors such as recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia. The FMDV antigen may be delivered via DNA injection together with in vivo electroporation.

[0123] b. Electroporation Administration of the vaccine via electroporation of the plasmid of the vaccine may be accomplished using an electroporation device configured to deliver energy pulses that generate a constant current similar to a user-presettable current input to the desired tissue of the mammal. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation device including a control device, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. Electroporation may be accomplished using the VGXP Cellectra™ system to facilitate transfection of cells by the plasmid.

[0124] The electroporation component may function as an element of an electroporation device, and other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component may function as more than one element of the electroporation device, and it may also communicate with other elements of an electroporation device that are separate from the electroporation component. Elements of an electroporation device that exist as part of one electromechanical or mechanical device need not be limited such that the elements function as one device or as separate elements that communicate with each other. The electroporation component may be capable of delivering energy pulses that generate a constant current within a desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes within a spatial arrangement, where the electrode assembly receives an energy pulse from the electroporation component and delivers it to the desired tissue via the electrodes. At least one of the plurality of electrodes is neutral during delivery of the energy pulse and measures the impedance within the desired tissue and communicates that impedance to the electroporation component. The feedback mechanism may receive the measured impedance and can adjust the energy pulse delivered by the electroporation component to maintain a constant current.

[0125] The plurality of electrodes may deliver energy pulses in a dispersed pattern. The plurality of electrodes may deliver energy pulses in a dispersed pattern via electrode control under a programmed sequence, and the programmed sequence is input into the electroporation component by a user. The programmed sequence may include a plurality of pulses delivered in sequence, where each pulse of the plurality of pulses is delivered by at least two active electrodes that include one neutral electrode that measures impedance, and the next pulse of the plurality of pulses is delivered by a different one of the at least two active electrodes that includes one neutral electrode that measures impedance.

[0126] The feedback mechanism may be implemented by either hardware or software. The feedback mechanism may be implemented by an analog closed loop. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The impedance in the desired tissue may be measured with a neutral electrode, and that impedance may be communicated to the feedback mechanism, which adjusts the energy pulse in response to the impedance and maintains a constant current at a value similar to the preset current. The feedback mechanism may continuously and instantaneously maintain a constant current upon delivery of the energy pulse.

[0127] Examples of electroporation devices and electroporation methods that can facilitate delivery of the DNA vaccine of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al. and U.S. Patent Application Publication No. 2005 / 0052630 filed by Smith et al., the entire contents of which are hereby incorporated by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate delivery of the DNA vaccine include those provided in U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, co-pending and commonly assigned, which claims the benefit under 35 USC 119(e) to U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, the entire contents of which are hereby incorporated by reference in their entirety.

[0128] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues within the body or in plants. The modular electrode may include a plurality of needle electrodes; a hypodermic needle; an electrical connector providing electrical connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue within the body or in a plant. Thereafter, the biomolecules are delivered to the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into cells between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is incorporated herein by reference.

[0129] U.S. Patent Application Publication No. 2005 / 0052630 by Smith et al. describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues within the body or in plants. The electroporation device includes an electrokinetic device (the "EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between the electrodes of an array based on user control and pulse parameter input, and enables the storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Application Publication No. 2005 / 0052630 is incorporated herein by reference.

[0130] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 0052630 may be configured to penetrate deeply not only into tissues such as muscle, but also into other tissues or organs. Depending on the form of the electrode array, an injection needle (for delivering a selected biomolecule) is fully inserted into the target organ and administered by injection perpendicular to the target tissue in the region pre-marked by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0131] In addition, as contemplated in some embodiments incorporating an electroporation device and its use, the following patents exist: U.S. Patent No. 5,273,525, issued December 28, 1993; U.S. Patent No. 6,110,161, issued August 29, 2000; U.S. Patent No. 6,261,281, issued July 17, 2001; U.S. Patent No. 6,958,060, issued October 25, 2005; and U.S. Patent No. 6,939,862, issued September 6, 2005, which describe electroporation devices. Further, patents dealing with the subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, relating to the delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, which noted a method of DNA injection, are contemplated herein. The foregoing patents are incorporated by reference in their entirety.

[0132] c. Method of preparing a vaccine Provided herein is a method for preparing a vaccine. In some embodiments, the method is a method for preparing a vaccine comprising a DNA plasmid. The DNA plasmid can be used to inoculate a cell culture in a large-scale fermentation tank using methods known in the art after the final subcloning step into a mammalian expression plasmid. The plasmid is transformed into a compatible host cell and cultured and maintained under conditions in which expression of the FMDV antigen occurs. The FMDV antigen can be recovered from the culture by lysing the cells or recovered from the medium and isolated. The isolated VP1-4 consensus protein may be used as a natural source of antibodies in the vaccine. The FMDV antigen may be produced by recombinant techniques using an automated synthesizer that can be used to produce an isolated, essentially pure FMDV antigen. These techniques can be useful for introducing variants of FMDV antigens of specific subtypes of FMDV.

[0133] The DNA plasmids used with the EP device of the present invention can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using the optimized plasmid manufacturing techniques described in co-pending U.S. Provisional Patent Application No. 60 / 939,792, which is the subject of a license application filed on May 23, 2007. In some examples, the DNA plasmids used in these studies can be formulated at a concentration of 10 mg / mL or greater. The manufacturing techniques include, or incorporate, various devices and protocols generally known to those skilled in the art, including those described in U.S. Patent No. 7,238,522, which is the subject of a license patent issued on July 3, 2007, in addition to those described in U.S. Patent Application No. 60 / 939,792. The previously referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are hereby incorporated by reference in their entirety. Examples Example 1

[0134] As shown in FIGS. 1 to 17, constructs of several embodiments have been made and tested. These figures show that the vaccines have been made and data have been generated by their use.

[0135] FIG. 17 shows a schematic of a generic FMDV DNA vaccine construct, showing that the insert is a clone into the BamH1 and Xho-1 sites. The plasmid map of the generic FMDV vaccine is based on plasmid pVAX. The example of the FMDV insert may be in long form, which is shown in FIG. 17 as a long form insert, or in short form, which is shown in FIG. 7 as a short form insert. The IgE leader shown in each form is shown to be optional or may be replaced with a different leader. The 2A sequence is shown to be optional and the furin cleavage site (rgrkrrs - SEQ ID NO: 27) is shown to be replaceable.

[0136] Figure 1 is the FMDV-As1-Shamir-89 version of the generic FMDV DNA vaccine shown in Figure 17. Figure 3 is the FMDV-A24cruzeiro DNA version of the generic FMDV DNA vaccine shown in Figure 17. Figure 5 is the FMDV-SAT2 DNA version of the generic FMDV DNA vaccine shown in Figure 17. Figure 1 is a schematic diagram of the FMDV-As1-Shamir-89 DNA vaccine construct for serotype Asia 1, showing that the As1 Shamir89 insert is a clone into the BamH1 and Xho-1 sites. The FMDV-A24cruzeiro DNA vaccine construct shown in Figure 3 has the clone BamH1 and Xho-1 sites. The FMDV-SAT DNA vaccine construct shown in Figure 5 has the clone BamH1 and Xho-1 sites. In each of Figures 1, 3, and 5, the plasmid map is based on plasmid pVAX. Examples of the FMDV-As1-Shamir insert may be in the long form, which is shown in Figure 1 as pFMDV-As1 Shamir-89-L, or in the short form, which is shown in Figure 1 as pFMDV-As1 Shamir-89-S. Examples of the insert may be in the long form, which is shown in Figure 3 as pFMDV-A24cruzeiro-L, or in the short form, which is shown in Figure 3 as pFMDV-A24cruzeiro-S. Examples of the FMDV-SAT2 insert may be in the long form, which is shown in Figure 5 as the pFMDV-As1 Sat2 long form, or in the short form, which is shown in Figure 5 as pFMDV-Sat2.

[0137] Figure 2 shows a pair of stained gels demonstrating the cloning of As1-Shamir89-S (left - SEQ ID NO: 7) and As1-Shamir89-L (right - SEQ ID NO: 5), Figure 4 shows a pair of stained gels demonstrating the cloning of A24cruzeiro-S (left - SEQ ID NO: 3) and A24cruzeiro-L (right - SEQ ID NO: 1), and Figure 6 shows a pair of stained gels demonstrating the cloning of Sat2-S (left - SEQ ID NO: 11) and Sat2-L (right - SEQ ID NO: 9). These data indicate that the insertions were properly incorporated into their respective plasmids. Figure 2 shows the amino acid sequence of the FMDV-As1-Shamir89-L long form. Figure 4 shows the amino acid sequence of the FMDV-A24cruzeiro-L long form. Figure 6 shows the amino acid sequence of the FMDV-Sat2 long form. In each long form, the sequence included an IgE leader sequence at the shaded N-terminus, a proteolytic cleavage site written in lowercase, and a VP4 sequence in bold between the IgE leader and the first proteolytic cleavage site. Between the first and the second proteolytic cleavage sites is the coding sequence of VP2. Between the second and the third proteolytic cleavage sites is the coding sequence of VP3. Between the third and the fourth proteolytic cleavage sites is the coding sequence of VP1. The 2A sequence between the last (fourth) proteolytic cleavage site and the termination.

[0138] Figure 7 shows the experimental results of protein expression. Western blot of the proteins on the SDS gel compared protein expression from samples produced from FMDV-A24cruzeiro-S short form, FMDV-A24cruzeiro-L long form, pVAX, FMDV-As1-Shamir89-S short form and FMDV-As1-Shamir89-L long form. The blot was probed with anti-A24 antiserum.

[0139] Figure 8 shows the experimental protocol of an immunization experiment using electroporation to evaluate the immune response following administration of 1) pVAX, 2) FMDV-A24cruzeiro-L, 3) FMDV-A24cruzeiro-S, 4) FMDV-Shamir89-L, 5) FMDV-Shamir89-S, FMDV-Sat2-L, and FMDV-Sat2-S, compared to the untreated group.

[0140] Figure 9 shows the data of the cellular immune response induced by the FMDV-A24cruzeiro-L and FMDV-A24cruzeiro-S vaccines. Figure 10 shows the data of the cellular immune response induced by the FMDV-As1-Sharma89-L and FMDV-As1-Sharma89-S vaccines. Figure 11 shows the data of the cellular immune response induced by the FMDV-Sat2-L and FMDV-Sat2-S vaccines. Figure 12 shows the experimental protocol for DNA transfection for ELISA analysis and preparation of cell lysates. Figure 13 shows the data of antibody induction in mice induced by the FMDV-A24cruzeiro-L and FMDV-A24cruzeiro-S vaccines, as well as the FMDV-As1-Sharma89-L and FMDV-As1-Sharma89-S vaccines. Figure 14 shows the data of ELISA analysis of antibody binding using protein lysates prepared from FMDV-A24cruzeiro-L transfected cells and FMDV-As1-Sharma89-L transfected cells. The FMDV vaccines were immunogenic in mice. Seroconversion was observed in all immunized animals. The long form of the vaccine was more effective than the short form. The humoral response seems to be most effective against the Shamir vaccine compared to the Creuzeiro vaccine, but both vaccines were effective. The cellular response was more cross-reactive with the Shamir vaccine compared to the Creuzeiro vaccine. A comparison with positive bovine serum showing a proper level of immunoreactivity was induced by the vaccine.

[0141] Figure 15 shows the amino acid sequence comparison between the sharir and cruzeiro arrays. The Shamir VP4 sequence (SEQ ID NO: 17) is shown in comparison with the cruzeiro VP4 sequence (SEQ ID NO: 18), the Shamir VP2 sequence (SEQ ID NO: 19) is shown in comparison with the cruzeiro VP2 sequence (SEQ ID NO: 20), and the Shamir 2A sequence (SEQ ID NO: 21) is shown in comparison with the cruzeiro 2A (SEQ ID NO: 22).

[0142] Figure 16 shows the amino acid sequence comparison between the sharir and cruzeiro arrays. The Shamir VP3 sequence (SEQ ID NO: 23) is shown in comparison with the cruzeiro VP3 sequence (SEQ ID NO: 24), and the Shamir VP1 sequence (SEQ ID NO: 25) is shown in comparison with the cruzeiro VP1 sequence (SEQ ID NO: 26). Example 2

[0143] Fourteen constructs were designed to prepare the FMDV vaccine. Sequences from seven foot-and-mouth disease virus subtypes A, Asia 1, C, O, SAT1, SAT2, SAT3, SAT4 were used. Two construct designs, long form and short form, can be used. Thus, there are long and short forms of each construct for subtypes A, Asia 1, C, O, SAT1, SAT2, SAT3, SAT4, resulting in fourteen constructs. The vaccine can be generated using only four, typically seven, constructs.

[0144] The generic long form is shown in Figure 17. The immunogen coding sequences are arranged in the order of VP4, VP2, VP3, VP1. The coding sequences of the protease cleavage sites separate each of the four viral proteins. The coding sequences can be provided for any optional IgE leader sequence provided. Similarly, the FMDV peptide 2A tail is provided at the end containing the protease cleavage site.

[0145] The generic short form is shown in Figure 17. The immunogen coding sequences are arranged in the order of VP2, VP3, and VP1. The coding sequences of the protease cleavage sites separate each of the four viral proteins. The coding sequences can be provided for any optional IgE leader sequence provided. Similarly, the 16-amino acid 2A tail is provided at the end containing the protease cleavage site.

[0146] The construct is inserted into a plasmid expression vector, resulting in 14 plasmids.

[0147] In some embodiments, the vaccine includes the A-long form, Asia 1-long, C-long form, O-long form, SAT1-long form, SAT2-long form, SAT3-long form, and SAT4-long form.

[0148] In some embodiments, the vaccine includes the A-short form, Asia 1-short, C-short form, O-short form, SAT1-short form, SAT2-short form, SAT3-short form, and SAT4-short form.

[0149] In some embodiments, the vaccine includes the A-long form, Asia 1-long, C-long form, and O-long form.

[0150] In some embodiments, the vaccine includes the A-short form, Asia 1-short, C-short form, and O-short form.

[0151] The N-terminus can be a leader sequence such as IgE or IgG, or there can be no leader.

[0152] The individual viral proteins are separated from each other by proteases that are commonly present in the cells where expression is desired.

[0153] WO 2011 / 054011 discloses an FMDV vaccine. Included in the disclosure are amino acid sequences and coding sequences for 28 sequences that can be included in various embodiments. The 14 viral sequences are VP1, VP2, VP3, and VP4 of FMDV subtypes A, Asia 1, O, C, SAT1, SAT2, and SAT3, respectively. The sequences disclosed therein can be used to generate constructs that can be included in vaccines.

[0154] Constructs include long and short formats. Figure 1 shows a partial generic format for each. In the present invention, the construct provides viral proteins VP1, VP2, VP3, and VP4 in a specific order: VP4-VP2-VP3-VP1, as shown in Figure 17. An optional tail, 2A, is also provided. The construct has an optional IgE leader sequence. When present, a proteolytic cleavage site "CS" is provided between each of VP4, VP2, VP3, VP1, and, when present, 2A. The protease capable of processing the site may be furin in some embodiments. Other protease sites may also be used. The site must be recognized by a protease commonly found in the cells in which the vaccine is expressed.

[0155] In one aspect of the present invention, there are fusion proteins comprising consensus FMDV proteins VP1, VP2, VP3, VP4 and / or 3C and nucleic acid sequences encoding these proteins that can be generated and used in vaccines to protect mammals against foot-and-mouth disease across one or more subtypes of FMDV, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

[0156] In another aspect of the invention, there is a fusion protein comprising a consensus FMDV protein VP1 and a nucleic acid sequence encoding this protein, derived from two different subtypes, which is produced and can be used in a vaccine to protect mammals from foot-and-mouth disease across one or more subtypes of FMDV, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

[0157] In another aspect of the invention, there is a consensus FMDV protein VP1 and a nucleic acid sequence encoding it, which is produced and can be used in a vaccine to protect mammals from foot-and-mouth disease across one or more subtypes of FMDV, including A, Asia 1, O, C, SAT1, SAT2, and SAT3.

Claims

**Claim 1** A nucleic acid molecule comprising: a) a leader sequence linked to a coding sequence of a protease cleavage site linked to a coding sequence linked to the C-terminus of viral protein VP1, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein 2A, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP3, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP2, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP4; and b) a leader sequence linked to a coding sequence of a protease cleavage site linked to a coding sequence linked to the C-terminus of viral protein VP1, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein 2A, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP3, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP2; and c) a leader sequence linked to a coding sequence of a protease cleavage site linked to a coding sequence linked to the C-terminus of viral protein VP3, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP1, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP2, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP4; and d) a leader sequence linked to a coding sequence of a protease cleavage site linked to a coding sequence linked to the C-terminus of viral protein VP3, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP1, said protease cleavage site being linked to a coding sequence linked to the C-terminus of viral protein VP2; **Claim 2** The nucleic acid molecule according to claim 1, wherein the leader sequence is an IgE sequence. **Claim 3** The nucleic acid molecule according to any one of claims 1 or 2, wherein the cleavage site is rgrkrrs (SEQ ID NO: 27) recognized by furin. **Claim 4** The plasmid according to any one of claims 1 to 3, wherein the viral protein encoding the be nucleic acid sequence is derived from an FMDV subtype selected from the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3.

5. The vaccine comprising one, two, three, four, five, six, or seven plasmids according to claim 4, wherein the viral protein encoding the be nucleic acid sequence is derived from one to seven FMDV subtypes of the group consisting of FMDV subtypes A, Asia1, C, O, SAT1, SAT2, and SAT3.

6. The vaccine according to claim 5, comprising four plasmids, wherein the viral protein encoding the be nucleic acid sequence is derived from each FMDV subtype of the group consisting of A, Asia1, C, and O.

7. The vaccine according to claim 5, comprising seven plasmids, wherein the viral protein encoding the be nucleic acid sequence is derived from each of the FMDV subtypes A, Asia1, C, O, SAT1, SAT2, and SAT3.

8. The nucleic acid molecule according to claim 1, having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

9. A plasmid selected from the group consisting of the nucleic acid molecules according to claim 8, wherein the plasmid comprises the nucleic acid sequence of SEQ ID NO: 1, or the plasmid comprises the nucleic acid sequence of SEQ ID NO: 3, or the plasmid comprises the nucleic acid sequence of SEQ ID NO: 5, or the plasmid comprises the nucleic acid sequence of SEQ ID NO: 7, or the plasmid comprises the nucleic acid sequence of SEQ ID NO: 9, or the plasmid comprises the nucleic acid sequence of SEQ ID NO:

11.

10. The vaccine comprising one or more of the plasmids according to claim 9.

11. A method of eliciting an immune response against FMDV in an individual, comprising administering to the individual the vaccine according to claim 5, 6, 7, or 10.

12. A method of preventing infection of an individual with FMDV, comprising administering to the individual the vaccine according to claim 5, 6, 7, or 12.

13. A method of treating an individual infected with FMDV, comprising administering to the individual the vaccine according to claim 5, 6, 7, or 12.

14. Anucleic acid molecule comprising one or more sequences selected from the group consisting of the FMDV VP1, VP2, VP3, or VP4 protein coding sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11; fragments thereof that encode at least 70% of the FMDV VP1, VP2, VP3, or VP4 protein encoded by the sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11; nucleic acid sequences that are 90% homologous to the coding sequences encoding the FMDV VP1, VP2, VP3, or VP4 protein at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11; and fragments that encode at least 70% of the FMDV VP1, VP2, VP3, or VP4 protein encoded by a nucleic acid sequence that is 90% homologous to the coding sequence encoding the FMDV VP1, VP2, VP3, or VP4 protein at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

15. The nucleic acid molecule according to claim 14, comprising one or more sequences selected from the group consisting of the FMDV VP1, VP2, VP3, or VP4 protein coding sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

16. The nucleic acid molecule according to claim 14, comprising one or more sequences selected from the group consisting of fragments of the FMDV VP1, VP2, VP3, or VP4 protein coding sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, wherein the fragment encodes at least 80% of the FMDV VP1, VP2, VP3, or VP4 protein encoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

17. The nucleic acid molecule according to claim 14, comprising one or more sequences selected from the group consisting of fragments of the FMDV VP1, VP2, VP3, or VP4 protein coding sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, wherein said fragment encodes at least 90% of the FMDV VP1, VP2, VP3, or VP4 protein encoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

18. The nucleic acid molecule according to claim 14, comprising one or more sequences selected from the group consisting of fragments of the FMDV VP1, VP2, VP3, or VP4 protein coding sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, wherein said fragment encodes at least 95% of the FMDV VP1, VP2, VP3, or VP4 protein encoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

19. The nucleic acid molecule according to claim 14, comprising one or more sequences selected from the group consisting of nucleic acid sequences that are 95% homologous to the coding sequences encoding the FMDV VP1, VP2, VP3, or VP4 proteins encoded by the sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:

11.

20. The nucleic acid molecule according to claim 14, comprising one or more sequences selected from the group consisting of nucleic acid sequences that are 98% homologous to the coding sequences encoding the FMDV VP1, VP2, VP3, or VP4 proteins encoded by the sequences at SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11.

Images