Hoof disease virus (FMDV) consensus protein, its coding sequence, and vaccines made therefrom

Abstract

Synthetic consensus foot-and-mouth disease virus (FMDV) immunogenic proteins and nucleic acid molecules encoding such proteins, providing vaccines against FMDV The present invention relates to synthetic consensus foot-and-mouth disease virus (FMDV) immunogenic proteins and nucleic acid molecules encoding such proteins, to the provision of vaccines against FMDV, to methods of inducing an immune response against FMDV, to methods of distinguishing between individuals infected with FMDV and individuals vaccinated against FMDV, and to methods of prophylactically and / or therapeutically immunizing individuals against FMDV.

Description

[Technical Field]

[0001] The present invention relates to synthetic consensus foot-and-mouth disease virus (FMDV) immunogenic proteins and nucleic acid molecules encoding such proteins, vaccines against FMDV, methods for inducing an immune response against FMDV, methods for distinguishing between individuals infected with FMDV and individuals vaccinated against FMDV, and methods for prophylactically and / or therapeutically immunizing individuals against FMDV. [Background technology]

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

[0003] The causative agent of FMD is the foot-and-mouth disease virus (FMDV), a group 4 (+)ssRNA virus of the Aphthovirus genus in the Picornaviridae family. FMDV occurs in seven major serotypes: O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1. These serotypes are regionally restricted, with the O serotype being the most common worldwide. The single-stranded, positive-sense RNA genome of FMDV is approximately 8,500 bases enclosed by an icosahedral capsid containing 60 copies of each of the four structural proteins VP1–VP4. Viral proteins exhibit high antigenic diversity within its several subtypes, including A, Asia-1, O, C, SAT1, SAT2, and SAT3.

[0004] FMD is economically devastating, and infection of cloven-hoofed livestock can cause significant losses. Recent outbreaks have resulted in billions of dollars in losses. Recent outbreaks in several previously disease-free countries, including Taiwan in 1997 and the United Kingdom and the Netherlands in 2001, as well as outbreaks in several South American countries, highlight the economically devastating nature of the virus. Furthermore, economic terrorists could use FMDV to target nations with large livestock industries, such as the US$100 billion annual livestock industry, making it a global problem.

[0005] Previous measures to control FMDV include culling infected or contact animals and decontamination. Countries that have culled livestock due to FMDV outbreaks can only resume livestock production if they have been FMDV-free for three months since the last outbreak. Countries that vaccinated animals and did not cull would have to wait a full year to regain FMD-free status, so countries typically use animal vaccination to deal with FMDV outbreaks as a last resort. However, countries hope to vaccinate animals before an FMDV outbreak and remain FMD-free.

[0006] Previous FMDV vaccines contained chemically inactivated whole virus antigens with adjuvants, but this approach has drawbacks, such as the need for expensive, highly enclosed manufacturing facilities. Over the past 25–30 years, researchers have attempted to develop vaccines that confer protection after a single inoculation. These efforts have included the use of purified VP1 from virus particles, bioengineered VP1, VP1 peptides, chemically synthesized VP1 peptides, live vectors expressing VP1 epitopes, inoculation with DNA encoding the VP1 epitope, and the use of whole capsid proteins VP1–VP4 generated from FMDV-infected cultures or delivery of VP1–VP4 capsids via replication-deficient human adenovirus type 5 (Ad5) vectors. All of these approaches provide inoculated animals with only a small number of epitopes spanning all FMDV subtypes.

[0007] Thus, there is a need in the art for vaccines suitable for conferring protection against multiple FMDV epitopes across various subtypes of FDMV, and for methods of diagnosing mammals infected with FMDV. Summary of the Invention

[0008] Disclosed is a nucleic acid molecule comprising a sequence encoding a viral protein, wherein viral protein VP4 is linked at its C-terminus to a protease cleavage site, viral protein VP2 is linked at its C-terminus to a protease cleavage site, viral protein VP3 is linked at its C-terminus to a protease cleavage site, a sequence encoding viral protein VP1 is linked at its C-terminus to a protease cleavage site, and viral protein 2A is linked at its C-terminus to a nucleic acid molecule encoding a leader sequence at the 5' end of the coding sequence for viral protein VP4. In some embodiments, the coding sequence for viral protein VP4 is omitted. In some embodiments, the coding sequence for 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 containing nucleic acid molecules are provided, including plasmids in which the viral proteins are derived from an FMDV subtype selected from the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3. 。 Vaccines are provided that include four plasmids, wherein the viral protein encoding nucleic acid sequences are from each of the FMDV subtypes in the group consisting of A, Asia1, C, and O. In some embodiments, vaccines are also provided that include seven plasmids, wherein the viral protein encoding nucleic acid sequences are from each of the FMDV subtypes in 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 include fewer than seven, ie, 1, 2, 3, 4, 5, or 6, plasmids encoding nucleic acid sequences.

[0010] Disclosed herein is a nucleic acid molecule comprising a sequence encoding a viral protein, wherein the sequence encoding viral protein VP4 is linked at its C-terminus to a sequence encoding a protease cleavage site, linked at its C-terminus to a sequence encoding viral protein VP2, linked at its C-terminus to a sequence encoding a protease cleavage site, linked at its C-terminus to a sequence encoding viral protein VP3, linked at its C-terminus to a sequence encoding a protease cleavage site, linked at its C-terminus to a sequence encoding viral protein VP1, linked at its C-terminus to a sequence encoding a protease cleavage site, and linked at its C-terminus to a sequence encoding viral protein 2A, said nucleic acid molecule being referred to as the long version or "long." Disclosed herein is a nucleic acid molecule comprising a sequence encoding a viral protein, in which the sequence encoding viral protein VP2 is linked at its C-terminus to a sequence encoding a protease cleavage site, to a sequence encoding viral protein VP3, to a sequence encoding a protease cleavage site, to a sequence encoding viral protein VP1, and to a sequence encoding viral protein 2A; the nucleic acid molecule is referred to as a short version or "short." In both the long and short versions, the coding sequence for the protease cleavage site linked to the 3' end of the coding sequence encoding viral protein VP1 and the coding sequence for viral protein 2A may be omitted. In both the long and short versions, the coding sequence for the N-terminal leader sequence is linked to the N-terminus of the coding sequence for viral protein VP4 in the long version and to the coding sequence for viral protein VP2 in the long sequence. The N-terminal leader is preferably an 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 comprising nucleic acid molecules are provided, including plasmids in which viral proteins are from an FMDV subtype selected from the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3. In some embodiments, vaccines are provided that comprise four plasmids in which viral protein encoding nucleic acid sequences are from each of the FMDV subtypes in the group consisting of A, Asia1, C, and O. In some embodiments, vaccines are also provided that comprise seven plasmids in which viral proteins encode nucleic acid sequences from each of the FMDV subtypes in the group consisting of A, Asia1, C, O, SAT1, SAT2, and SAT3.

[0012] Methods are provided for generating an immune response to FMDV in an individual by administering one of the disclosed vaccines to the individual.

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

[0014] Provided herein are isolated nucleic acids comprising a sequence encoding at least VP1-VP3, and preferably the consensus amino acid sequence of VP1-VP4, of foot-and-mouth disease virus that elicit a cross-reactive immune response in subjects vaccinated against multiple FMD subtypes, including A, Asia 1, C, O, SAT1, SAT2, SAT3, and SAT4. The nucleic acids include (a) a construct derived from FMDV-A24cruzeiro comprising the nucleotide sequence set forth in SEQ ID NO:1, encoding VP-4-VP2-VP3-VP1 (long) as set forth in SEQ ID NO:2; (b) a construct derived from FMDV-A24cruzeiro comprising the nucleotide sequence set forth in SEQ ID NO:3, encoding VP2-VP3-VP1 (short) as set forth in SEQ ID NO:4; (c) a construct derived from FMDV-As1-Shamir89 comprising the nucleotide sequence set forth in SEQ ID NO:5, encoding VP-4-VP2-VP3-VP1 (long) as set forth in SEQ ID NO:6; (d) a construct derived from FMDV-As1-Shamir89 comprising the nucleotide sequence set forth in SEQ ID NO:7, which encodes VP2-VP3-VP1 (short) as set forth in SEQ ID NO:8; (e) a construct derived from FMDV-SAT2 comprising the nucleotide sequence set forth in SEQ ID NO:9, which encodes VP-4-VP2-VP3-VP1 (long) as set forth in SEQ ID NO:10; (f) a construct derived from FMDV-STA2 comprising the nucleotide sequence set forth in SEQ ID NO:11, which encodes VP2-VP3-VP1 (short) as set forth 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, as set forth in SEQ ID NO: 1 (FMDV-A24cruzeiro-long), inserted into a plasmid such as pVAX having the sequence set forth in SEQ ID NO: 13; b) a modified nucleotide sequence derived from FMDV-A24cruzeiro, as set forth in SEQ ID NO: 3 (FMDV-A24cruzeiro-short), inserted into a plasmid such as pVAX having the sequence set forth in SEQ ID NO: 14; c) a modified nucleotide sequence derived from FMDV-As1-Shamir89, as set forth in SEQ ID NO: 5 (FMDV-As1-Shamir89-long), inserted into a plasmid such as pVAX having the sequence set forth in SEQ ID NO: 15; and d) a modified nucleotide sequence derived from FMDV-As1-Shamir89, as set forth in SEQ ID NO: 7 (FMDV-As1-Shamir89-long), inserted into a plasmid such as pVAX having the sequence set forth in SEQ ID NO: 16.

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

[0017] The amino acid sequence of the cleavage site recognized by the 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) that encodes a C3 protease consensus protein (SEQ ID NO:29).

[0019] Also provided herein is a vaccine capable of raising an immune response in a mammal against multiple foot-and-mouth disease virus (FMDV) subtypes, 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, the DNA plasmid being capable of expressing the consensus FMDV antigen in mammalian cells in an amount effective to elicit a broadly cross-reactive immune response in the mammal. The vaccine can raise an immune response against FMDV subtypes A, Asia 1, C, O, SAT1, SAT2, SAT3, or a combination thereof.

[0020] Also provided herein is a vaccine capable of generating an immune response in a mammal against multiple foot-and-mouth disease virus (FMDV) subtypes. The vaccine comprises 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 a combination thereof, and a pharmaceutically acceptable excipient therefor. The DNA plasmid is capable of expressing the consensus FMDV antigen in mammalian cells in an amount effective to elicit an immune response in the mammal. The vaccine may be administered to a mammal, such as a pig, ruminant, human, or primate. The vaccine may elicit an immune response in the mammal, such as a humoral response, a cellular response, or both a humoral and a cellular response.

[0021] Also provided herein is a vaccine capable of generating an immune response against multiple FDMV subtypes in a mammal. The vaccine comprises 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 therefor. 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-enhancing agent. The transfection-enhancing agent may be a polyanion, polycation, or lipid, e.g., poly-L-glutamate, at a concentration of less than 6 mg / ml. The vaccine may be administered to a mammal, such as a pig, ruminant, human, or primate. The vaccine may elicit an immune response in the mammal, e.g., a humoral response, a cellular response, or both a humoral and a cellular response.

[0022] Also provided herein is a method for eliciting an immune response against multiple FMDV virus subtypes in a mammal, comprising delivering the DNA plasmid vaccine described herein to mammalian tissue and electroporating the cells of the tissue with a constant current energy pulse effective to allow the DNA plasmid to enter the cells. Delivery of the DNA plasmid vaccine described herein can be achieved by a method that may include injecting the DNA plasmid vaccine into interdermal, subcutaneous, or muscle tissue. The DNA plasmid of the method may be delivered by presetting the current and energy pulse at a constant current equal to the existing current. 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 based on the measured impedance to maintain a constant current in the electroporated cells, wherein the measuring and adjusting steps are performed within the life of the energy pulse. The electroporation step may further include delivering energy pulses to multiple electrodes using a pulse sequence pattern that delivers the energy pulses in a distributed pattern.

[0023] Also provided is a method for diagnosing a mammal infected with FMDV, the method comprising isolating a fluid sample from the mammal, isolating antibodies from the mammalian fluid sample, and comparing the isolated antibodies to a control mammal vaccinated as described herein, wherein the control mammal has only antibodies to FMDV VP1-VP4 proteins, and the FMDV-infected mammal has antibodies to FMDV VP1-VP4 proteins and FMDV nonstructural proteins, which may be FMDV 2C, 3A, and 3D polymerases.

[0024] Methods for eliciting an immune response against one or more FMDV virus subtypes in a mammal are provided. In some embodiments, the methods, including using the vaccines described herein, may include administering to a mammalian tissue a nucleic acid molecule encoding a protein having an FMDV immunogenic sequence, and electroporating cells of the tissue with a constant current energy pulse effective to allow the DNA plasmid to enter the cells.

[0025] Also provided is a method for diagnosing FMDV infection in a mammal vaccinated by the processes disclosed herein. The method comprises isolating a fluid sample from the vaccinated mammal and detecting the presence of FMDV proteins not included in the vaccine and / or antibodies to FMDV proteins not included in the vaccine. The presence of such FMDV proteins and / or antibodies to such FMDV proteins indicates that the vaccinated mammal is infected with FMDV. [Brief explanation of the drawings]

[0026] [Figure 1]1 is a schematic diagram of the FMDV-As1-Shamir-89 DNA vaccine construct for serotype Asia 1, showing the As1 Shamir-89 insertion cloned into the BamH1 and Xho-1 sites. The plasmid map is based on the plasmid pVAX. An example of the FMDV-As1-Shamir insertion can be in the long form, which is shown in FIG. 1 as pFMDV-As1 Shamir-89-L, or in the short form, which is shown in FIG. 1 as pFMDV-As1 Shamir-89-S. [Figure 2] A pair of stained gels showing the cloning of As1-Shamir89-S (left—SEQ ID NO: 7) and As1-Shamir89-L (right—SEQ ID NO: 5) and the amino acid sequence of the FMDV-As1-Shamir89-L long form (SEQ ID NO: 6 is the FMDV-As1-Shamir89-L long form sequence). The sequence contained the shaded N-terminal IgE leader sequence, proteolytic cleavage sites written in lowercase, and the bolded VP4 sequence between the IgE leader and the first proteolytic cleavage site. The VP1 sequence is shown in bold between the third and fourth proteolytic cleavage sites, and the 2a sequence is between the last (fourth) proteolytic cleavage site and the termination. [Figure 3] 3 is a schematic representation of the FMDV-A24cruzeiro DNA vaccine construct, showing that the A24cruzeiro insert is cloned into the BamH1 and Xho-1 sites. The plasmid map is based on the plasmid pVAX. An example of the FMDV-A24cruzeiro insert can be in the long form, which is shown in FIG. 3 as pFMDV-A24cruzeiro-L, or in the short form, which is shown in FIG. 3 as pFMDV-A24cruzeiro-S. [Figure 4]A pair of stained gels showing the cloning of A24cruzeiro-S (left - SEQ ID NO: 3) and A24cruzeiro-L (right - SEQ ID NO: 1) and the amino acid sequence of FMDV-A24cruzeiro-L long form (SEQ ID NO: 2 is the FMDV-A24cruzeiro-L long form sequence). The sequence includes the IgE leader sequence at the N-terminus shaded, the proteolytic cleavage sites written in lowercase, and the VP4 sequence is shown between the IgE leader and the first proteolytic cleavage site. The VP1 sequence is shown between the third and fourth proteolytic cleavage sites, and the 2a sequence is between the last (fourth) proteolytic cleavage site and the termination. [Figure 5] 5 is a schematic diagram of the FMDV-Sat2 DNA vaccine construct, showing that the Sat2 insertion is cloned into the BamH1 and Xho-1 sites. The plasmid map is based on the plasmid pVAX. An example of an FMDV-Sat insertion can be in the long form, which is shown in FIG. 5 as pFMDV-As1-Sat2-L, or in the short form, which is shown in FIG. 5 as pFMDV-Sat2-S. [Figure 6] Figure 1 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) and the amino acid sequence of the FMDV-Sat2-L long form (SEQ ID NO: 10 is the FMDV-Sat2-L long form sequence). The sequence includes the IgE leader sequence at the N-terminus shaded, the proteolytic cleavage sites written in lowercase, and the VP4 sequence is shown between the IgE leader and the first proteolytic cleavage site. The VP1 sequence is shown between the third and fourth proteolytic cleavage sites, and the 2a sequence is between the last (fourth) proteolytic cleavage site and the termination. [Figure 7] 1 shows the experimental results of protein expression. [Figure 8]The experimental protocol for 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, and 5) FMDV-Shamir89-S, FMDV-Sat2-L, and FMDV-Sat2-S compared to untreated control is shown. [Figure 9] 1 shows data on cellular immune responses induced by FMDV-A24cruzeiro-L and FMDV-A24cruzeiro-S vaccines. [Figure 10] 1 shows data on cellular immune responses elicited by FMDV-As1-Sharma89-L and FMDV-As1-Sharma89-S vaccines. [Figure 11] 1 shows data on cellular immune responses elicited by FMDV-Sat2-L and FMDV-Sat2-S vaccines. [Figure 12] The experimental protocol for DNA transfection and cell lysate preparation for ELISA analysis is shown. [Figure 13] 1 shows data on antibody induction in mice elicited by FMDV-A24cruzeiro-L and FMDV-A24cruzeiro-S vaccines and by FMDV-As1-Sharma89-L and FMDV-As1-Sharma89-S vaccines. [Figure 14] Shown are data from ELISA analysis of antibody binding using protein lysates prepared from FMDV-A24cruzeiro-L and FMDV-As1-Sharma89-L transfected cells. [Figure 15] Amino acid sequence comparisons of the Shamir and Cruzeiro sequences are shown. The Shamir VP4 sequence (SEQ ID NO: 17) is shown relative to the Cruzeiro VP4 sequence (SEQ ID NO: 18), the Shamir VP2 sequence (SEQ ID NO: 19) is shown relative to the Cruzeiro VP2 sequence (SEQ ID NO: 20), and the Shamir 2A sequence (SEQ ID NO: 21) is shown relative to the Cruzeiro 2A sequence (SEQ ID NO: 22). [Figure 16] Amino acid sequence comparisons of the Shamir and Cruzeiro sequences are shown. The Shamir VP3 sequence (SEQ ID NO: 23) is shown relative to the Cruzeiro VP3 sequence (SEQ ID NO: 24), and the Shamir VP1 sequence (SEQ ID NO: 25) is shown relative to the Cruzeiro VP1 sequence (SEQ ID NO: 26). [Figure 17] 1 shows a schematic diagram of the generic FMDV DNA vaccine construct, showing that the insert is cloned into the BamH1 and Xho-1 sites. The plasmid map of the generic FMDV vaccine is based on the plasmid pVAX. Examples of FMDV inserts can be in the long form, shown in FIG. 17 as the long form insert, or in the short form, shown in FIG. 7 as the short form insert. The IgE leader shown in each form is indicated as optional or may be replaced with a different leader. The 2A sequence is indicated as optional, and the furin cleavage site (rgrkrrs - SEQ ID NO: 27) is indicated as replaceable. DETAILED DESCRIPTION OF THE INVENTION

[0027] Consensus amino acid sequences have been generated for fusion proteins containing multiple FMDV proteins and individual FMDV proteins from various serotypes, and nucleic acid molecules encoding the proteins have also been generated.

[0028] In one aspect of the invention, there are fusion proteins comprising FMDV proteins VP1, VP2, VP3, VP4, and / or 2A and / or 3C and nucleic acid sequences encoding these proteins that can be produced and used in vaccines 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. Preferably, the VP1 gene is consensus for a selected subtype of FMDV, e.g., described herein is FMDV-Sat2, in which the VP1 is the Sat2 consensus VP1.

[0029] Without being bound by scientific theory, a vaccine directed to the consensus amino acid sequence of VP1, VP2, VP3, and / or VP4 for one or more subtypes of FMDV would present a large repertoire of epitopes that would be effective in eliciting an effective immune response (either humoral, cellular, or both) against most species within each subtype of FMDV. Without being bound by scientific theory, VP1 is an excellent immunogenic target for a vaccine directed to the consensus amino acid sequence of VP1. VP1 is the primary immunogen.

[0030] Constructs of some embodiments include long and short formats. Constructs of some embodiments provide 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, 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, or FMDV protease 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.

[0031] In one aspect of the present 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 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.

[0032] In another embodiment of the present invention, there is a fusion protein comprising the consensus FMDV protein VP1 and a nucleic acid sequence encoding this protein from two different subtypes that 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.

[0033] In another embodiment of the present invention, there is a consensus FMDV protein VP1 and a nucleic acid sequence encoding it that can be generated and used in vaccines to provide protection in 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.Definition The terminology used herein is for the purpose of describing particular embodiments only and is 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] Where numerical ranges are recited herein, each intervening number therebetween, to the same degree of precision, is expressly contemplated. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

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

[0037] b.Antibodies "Antibody" may refer to antibodies of the classes IgG, IgM, IgA, IgD, or IgE, or fragments, including Fab, F(ab'), Fd, and single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof, or fragments or derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0038] c. coding sequence As used herein, a "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0039] d. complementary sequence As used herein, "complementary sequence" or "complementary" may refer to a nucleic acid that can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

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

[0041] f. Constant current As used herein, "constant current" defines the current received or experienced by a tissue, or cells defining the tissue, for 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 a feedback element with instantaneous feedback so that the current remains constant in the tissue for the life of the electrical pulse. The feedback element measures the resistance of the tissue (or cells) throughout the duration of the pulse and can vary the electrical energy output of the electroporation device (e.g., increase the voltage) so that the current in the same tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element comprises a controller.

[0042] g. Current feedback or feedback As used herein, "current feedback" or "feedback" may be used interchangeably and may refer to the active response of a provided electroporation device, including measuring the tissue current between the electrodes and correspondingly varying the energy output delivered by the EP device to maintain the current at a constant level. This constant level is preset by the user prior to the initiation of a pulse sequence or electrical treatment. Feedback may be accomplished by the electroporation components of the electroporation device, e.g., a controller, such that an electrical circuit within the device can continuously monitor the tissue current between the electrodes, compare the monitored current (or tissue current) to a preset current, and continuously make energy output adjustments to maintain the monitored current at the preset level. The feedback loop may be analog, closed-circuit feedback, and therefore instantaneous.

[0043] h. Distributed current As used herein, "distributed current" may refer to a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein that minimizes or preferably eliminates the occurrence of electroporation-related thermal stress in any region of the tissue being electroporated.

[0044] i. Electroporation "Electroporation," "electropermeabilization," or "electrokinetics" ("EP"), as used interchangeably herein, may refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows the passage of biomolecules, such as plasmids, oligonucleotides, siRNA, drugs, ions, and water, from one side of the cell membrane to the other.

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

[0046] k. Fragment As used herein, a "fragment" may refer to a portion or nucleic acid encoding a polypeptide capable of eliciting an immune response in a mammal substantially similar to that of the non-fragment for at least one FMDV subtype, e.g., 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 sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11. The DNA fragment may be 30 or more nucleotides 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, 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, 1400 or more, 1500 or more, 1600 or more, 1700 or more, 1800 or more, 1900 or more, 2000 or more, 2100 or more, 2200 or more, 2300 or more, 2400 or more, 2500 or more, 2600 or more, 2700 or more, 2800 or more, 2900 or more, 3000 or more, 3100 or more, 3200 or more, 3300 or more, 3400 or more, 3500 or more, 3600 or more, 3700 or more, 3800 or more, 3900 or more, 4000 or more, 4200 or more, 4800 or more, 5400 or more, 6000 or more, 6600 or more, 7200 or more, 7800 or more, 8400 or more, 9000 or more, It may be 40 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 nucleotides in length.

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

[0048] DNA fragments are those containing 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] A "fragment" may refer to a polypeptide fragment capable of eliciting an immune response in a mammal substantially similar to that of the non-fragment for at least one FMDV subtype, e.g., A, Asia 1, C, O, SAT1, SAT2, or SAT3. The fragment may be a polypeptide fragment selected from at least one of the various encoded polypeptide sequences of the present invention, including SEQ ID NOs: 2, 4, 6, 8, 10, and 12. The polypeptide fragment may be analyzed for contact with at least one antigenic epitope provided by a publicly available database, such as the FMDV Sequence Database of Los Alamos National Laboratory. A 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 set forth in the polyprotein set forth in SEQ ID NOs: 2, 4, 6, 8, 10, or 12. The polypeptide may also comprise an amino acid sequence for an immunoglobulin leader, e.g., IgE or IgG. A 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. A 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 Multiple sequence alignments of homology may be generated using ClustalW (http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html).

[0051] m. identical As used herein, "identical" or "identity," in the context of two or more nucleic acid or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are identical in a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences in a specified region, determining the number of positions where 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 specified 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 staggered ends and the specified region being compared contains only a single sequence, the residues of the single sequence are included in the denominator rather than 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 a computer sequence algorithm such as BLAST or BLAST 2.0.

[0052] Impedance "Impedance" as used herein may be used when discussing feedback mechanisms and can be converted to a current value by Ohm's law and then compared to a preset current.

[0053] Immune response As used herein, "immune response" may refer to the activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of an FMDV consensus antigen via a provided DNA plasmid vaccine. The immune response may be in the form of a cellular response or a humoral response, or both.

[0054] p.Nucleic acid As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may refer to at least two nucleotides covalently linked to each other. The designation of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a 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. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0055] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequence. Nucleic acids may be DNA, both genomic and cDNA, RNA, or hybrids, 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. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0056] Nucleic acids generally contain phosphodiester linkages, but may also include nucleic acid analogs that may have at least one different linkage, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkages, as well as peptide nucleic acid backbones and linkages. Other similar nucleic acids include those with straight backbones; non-ionic backbones; and non-ribose backbones, such as those described in U.S. Patent Nos. 5,235,033 and 5,034,506, which are incorporated by reference. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included in the definition of nucleic acid. Modified nucleotide analogs may be located, for example, at the 5' and / or 3' ends of a nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. However, it should be noted that nucleobase-modified ribonucleotides, i.e., ribonucleotides containing non-naturally occurring nucleobases instead of naturally occurring nucleobases, such as uridine or cytidine modified at the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and guanosine modified at the 8-position, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deazaadenosine; O- and N-alkylated nucleotides, e.g., 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 (wherein R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I). Modified nucleotides may include nucleotides conjugated to cholesterol via 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 include locked nucleic acids (LNA), as described in U.S. Patent Application Publication 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 performed for a variety of reasons, for example, to improve the stability and half-life of such molecules in physiological environments, to facilitate diffusion through cell membranes, or as probes in biochips. Mixtures of naturally occurring nucleic acids and analogs may be produced, or mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs may be produced.

[0057] q. Functionally linked As used herein, "operably linked" may mean that the expression of a gene is under the control of a spatially connected 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 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 refer to a synthetic or naturally occurring molecule capable of providing, activating, or promoting expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which can be located thousands of base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may constitutively regulate expression of genetic components or may be variable, depending on the cell, tissue, or organ in which expression occurs, the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or transducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

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

[0060] t. substantially complementary As used herein, "substantially complementary" can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over 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 that the two sequences hybridize under stringent hybridization conditions.

[0061] u. Substantially identical As used herein, "substantially identical" can mean that a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over 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, where the first sequence is substantially complementary to the complement of the second sequence.

[0062] v. Subtype or serotype "Subtype" or "serotype," as used interchangeably herein and in reference to FMDV virus, refer to genetic variants of FMDV viral antigens such that one subtype is recognized by the immune system separately from a different subtype.

[0063] w. mutant As used herein, a "variant" with respect to a nucleic acid may mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) a 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" refers to a peptide or polypeptide that differs in amino acid sequence due to amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. Variant may refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the peptide's maximum local average hydrophilicity, a useful index that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by reference in its entirety, is incorporated herein by reference. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions may be made with amino acids with hydrophilicity values within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0065] x.vector As used herein, "vector" may refer to a nucleic acid sequence containing a replication origin. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either an autonomously replicating extrachromosomal vector or a vector that integrates into a host genome.

[0066] 2. FMDV Proteins and Coding Sequences The genomes of each of the subtypes A, C, O, Asia, SAT1, SAT2, and SAT3 can be 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 location 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 noted above, WO 2011 / 054011 discloses an FMDV vaccine having VP1, VP2, VP3, and VP4 from FMDV subtypes A, C, O, Asia, SAT1, SAT2, and SAT3, albeit using a different design. One skilled in the art can use WO 2011 / 054011 and the information in GenBank to identify the coding sequences for each of the FMDV proteins VP1, VP2, VP3, and 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 homologous 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 having 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 for these FMDV proteins, homologous proteins, fragments of FMDV proteins, and fragments of homologous proteins may be used in the constructs.

[0071] Natural proteolytic cleavage sites may be present between each of the consensus antigen sequences, such as the amino acid sequence: RGRKRRS.

[0072] Provided herein are antigens capable of eliciting an immune response in mammals against one or more foot-and-mouth disease virus (FMDV) subtypes. The antigen may be an FMDV antigen including capsid proteins VP1, VP2, VP3, VP4, a consensus thereof, a variant thereof, a fragment thereof, or a combination thereof. The FMDV antigen may be derived from FMDV subtype A, Asia 1, C, O, SAT1, SAT2, or SAT3. The FMDV antigen may contain 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 to VP4 of the FMDV antigen provide the full repertoire of immunogenic sites and epitopes present in intact FMDV viruses. A consensus FMDV antigen sequence may be obtained from FMDV antigenic sequences of multiple FMDV viruses of a single 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, VP2, VP3, and VP4 sequences of the consensus VP1-VP4 protein. Cleavage of the consensus VP1-VP4 protein by protein 3C may cleave the consensus VP1-VP4 protein to generate consensus VP1-, consensus VP2-, consensus VP3-, and consensus VP4-proteins. Alternatively, a native proteolytic cleavage site 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% homologous. In some embodiments, the proteins are 90% homologous. In some embodiments, the proteins are 95% homologous. In some embodiments, the proteins are 96% homologous. In some embodiments, the proteins are 97% homologous. In some embodiments, the proteins are 98% homologous. In some embodiments, the proteins are 99% homologous.

[0074] Provided herein are coding sequences for antigens capable of eliciting an immune response in mammals against one or more foot-and-mouth disease virus (FMDV) subtypes. The antigen may be an FMDV antigen including capsid proteins VP1, VP2, VP3, VP4, a consensus thereof, a variant thereof, a fragment thereof, or a combination thereof. The FMDV antigen may be derived from FMDV subtype A, Asia 1, C, O, SAT1, SAT2, or SAT3. The FMDV antigen may contain 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 full repertoire of immunogenic sites and epitopes present in intact FMDV viruses. A consensus FMDV antigen sequence may be obtained from FMDV antigenic sequences of multiple FMDV viruses of a single 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, a native proteolytic cleavage site may be present between each of the consensus antigen sequences, such as the amino acid sequence: RGRKRRS. Coding sequences for fusion proteins containing a consensus protease 3C sequence are provided.

[0075] In addition, the coding sequence may encode a protein that may be a fragment of a 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 may encode a protein that is homologous to the proteins 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 a protein described herein. In some embodiments, the coding sequence encodes a protein that is 20% homologous. In some embodiments, the coding sequence encodes a protein that is 30% homologous. In some embodiments, the coding sequence encodes a protein that is 40% homologous. In some embodiments, the coding sequence encodes a protein that is 50% homologous. In some embodiments, the coding sequence encodes a protein that is 60% homologous. In some embodiments, the coding sequence encodes a protein that is 70% homologous. 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 to a protein.

[0078] 3. Plasmids 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 the mammal. The vector may contain a heterologous nucleic acid encoding the FMDV antigen. The vector may be a plasmid. The plasmid may be useful for transfecting cells with the nucleic acid encoding the FMDV antigen, and the transformed host cells are cultured and maintained under conditions that allow expression of the FMDV antigen.

[0079] The plasmid may contain a nucleic acid encoding an FMDV antigen selected from the proteins provided herein, fragments thereof, homologous sequences thereof, and homologous fragments. The plasmid may further contain an initiation codon or leader sequence, which may be upstream of the coding sequence, and a termination codon, which may be downstream of the coding sequence. The initiation and termination codons may be in-frame with 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), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may be from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may be a natural or synthetic tissue-specific promoter, such as a muscle- or skin-specific promoter. Examples of such promoters are described in US 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 present 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 a 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 human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as CMV, FMDV, RSV, or EBV. Enhanced polynucleotide function is described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, which are incorporated herein by reference in their entirety.

[0083] The plasmid may contain a mammalian origin of replication to maintain the plasmid extrachromosomally 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 Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, allowing for high copy number 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 sufficient for gene expression in a cell 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 5' to the coding sequence. The consensus protein encoded by this sequence may include an N-terminal Ig leader followed by a consensus protein. The N-terminal Ig leader may be IgE or IgG.

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

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

[0088] In some embodiments, the plasmid comprises 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 coding sequences for 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 contains coding sequences for two different consensus FMDV antigen VP1s from two of subtypes Asia, A, O, and C, e.g., 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 a coding sequence for the 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 controlled by operably linked promoters, e.g., a DNA plasmid having coding sequences containing multiple consensus FMDV antigens controlled 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 mutations were found in the sequence of pVAX1, which was used as the backbone for plasmids 1-6 described herein: [Table 1]

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

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

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

[0098] The coding sequence may include an Ig leader sequence. The leader sequence may be 5' to the coding sequence. The consensus antigen encoded by this sequence may include an N-terminal Ig leader followed by the consensus antigen protein. The N-terminal Ig leader may be IgE or IgG.

[0099] The plasmid may be pSE420 (Invitrogen, San Diego, CA), which may be used for protein production in Escherichia coli (E. coli). The plasmid may be pYES2 (Invitrogen, San Diego, CA), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may be the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, CA), which may be used for protein production in insect cells. The plasmid may be pcDNA I or pcDNA3 (Invitrogen, San Diego, CA), which may be used for protein production in mammalian cells, such as Chinese hamster ovary (CHO) cells.

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

[0101] This includes: An isolated nucleic acid comprising a sequence encoding at least VP1-VP3, and preferably the consensus amino acid sequence of VP1-VP4, of foot-and-mouth disease virus, which elicits a cross-reactive immune response in a subject vaccinated against multiple subtypes of FMD, including A, Asia 1, C, O, SAT1, SAT2, SAT3, and SAT4. The nucleic acid may comprise a sequence selected from the group consisting of: (a) a nucleotide sequence encoding SEQ ID NO:1; SEQ ID NO:2, (b) a nucleotide sequence encoding SEQ ID NO:3; SEQ ID NO:4, (c) a nucleotide sequence encoding SEQ ID NO:5; SEQ ID NO:6, d) a nucleotide sequence encoding SEQ ID NO:7; SEQ ID NO:8, e) a nucleotide sequence encoding SEQ ID NO:9; SEQ ID NO:10, and f) a nucleotide sequence encoding SEQ ID NO:11; SEQ ID NO:12.

[0102] Provided herein is a vaccine capable of generating an immune response in a mammal against one or more FMDV subtypes. The vaccine may comprise the plasmids discussed above. The vaccine may comprise multiple plasmids, each directed against one or more FMDV subtypes, such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or a combination thereof. The vaccine may comprise FMDV antigens, each directed against one or more FMDV subtypes, such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or a combination thereof. The vaccine may comprise a plasmid directed against an FMDV subtype from a particular region of the world, e.g., Asia, Europe, or sub-Africa. Alternatively, or in addition, the vaccine may comprise one or more proteins from an FMDV subtype, such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or a combination thereof. The vaccine may comprise an FMDV antigen, which itself is directed against one or more FMDV subtypes, such as A, Asia 1, C, O, SAT1, SAT2, SAT3, or a combination thereof. The vaccine may comprise plasmids and / or proteins directed against FMDV subtypes from specific regions of the world, e.g., Asia, Europe, and sub-Africa. The vaccine may be provided to induce a therapeutic or prophylactic immune response.

[0103] Provided herein are pharmaceutical compositions according to the invention comprising about 1 nanogram to about 10 mg of DNA. In some embodiments, pharmaceutical compositions according to the invention comprise: 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, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1550, 1600, 1700, 18 65, 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, 4 40, 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, 8 15, 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 10 mg or more; 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, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620 5, 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, 8 20, 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 1,000 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.In some embodiments, the pharmaceutical composition according to the present invention comprises between about 5 nanograms and about 10 mg of DNA. In some embodiments, the pharmaceutical composition according to the present invention comprises between about 25 nanograms and about 5 mg of DNA. In some embodiments, the pharmaceutical composition comprises between about 50 nanograms and about 1 mg of DNA. In some embodiments, the pharmaceutical composition comprises between about 0.1 and about 500 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 1 and about 350 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 5 and about 250 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 10 and about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 15 and about 150 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 20 and about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 25 and about 75 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises between about 30 and about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 to about 40 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.The pharmaceutical composition contains 1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains 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 sterile, 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 cases, 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 multiple identical or different plasmids containing one or more consensus prostate antigen nucleic acid coding sequences. The DNA vaccine may contain one or more nucleic acid sequences encoding one or more consensus prostate antigens. When the DNA vaccine contains more than one consensus prostate antigen coding sequence, all of these sequences may be present on a single plasmid, or each of these sequences may be present on a different plasmid.

[0107] In some embodiments, the vaccine may comprise a nucleic acid sequence 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 fully incorporated herein by reference. DNA vaccines can further comprise elements or reagents that inhibit chromosomal integration. Vaccines can also be prostate antigen RNA. RNA vaccines 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 a live attenuated vaccine, a killed vaccine, and a vaccine that uses a recombinant vector to deliver a foreign gene encoding one or more consensus prostate antigens, a subunit vaccine, and a protein vaccine. Examples of live attenuated vaccines, live attenuated 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, No. 5,310,668, No. 5,387,744, No. 5,389,368, No. 5,424,065, No. 5,451,499, No. 5,4 No. 53,364, No. 5,462,734, No. 5,470,734, No. 5,474,935, No. 5,482,713, No. 5,591,43 Nos. 9, 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. Vaccines may also include other vaccine components, such as a plasmid in combination with an expression vector encoding an FMDV protein or protein.

[0110] The provided vaccines may be used to induce immune responses, including therapeutic or prophylactic immune responses. Antibodies and / or killer T cells directed against consensus prostate antigens 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 such as a vehicle, adjuvant, carrier, or diluent. The pharmaceutically acceptable excipient may be a transfection-enhancing agent, such as surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, endoplasmic reticulum such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-enhancing agents.

[0112] The transfection-facilitating agent may be a polyanion, polycation, or lipid, such as poly-L-glutamate (LGS). The transfection-facilitating agent is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg / ml. Transfection-facilitating agents may include surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs, such as monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles, such as squalene and squalene, or may be administered in conjunction with the gene construct using hyaluronic acid. In some embodiments, the DNA plasmid vaccine may contain a transfection-facilitating agent, such as a lipid, a liposome, including lecithin liposomes or other liposomes known in the art as DNA liposome mixtures (see, e.g., WO 09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-facilitating agents. Preferably, the transfection facilitating agent 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] The pharmaceutically acceptable excipient may be an adjuvant. The adjuvant may be another gene expressed in an alternative plasmid or delivered as a protein in combination with the previous plasmid in the vaccine. The adjuvant may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 containing IL-15 with a deleted signal sequence, and optionally containing a signal peptide derived from IgE. 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-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, mutant forms of IL-18, CD40, CD40L, angiogenesis 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, and Caspase. These include genes encoding 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 functional fragments thereof.

[0115] The vaccine may further comprise a genetic vaccine facilitator as described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated 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 sterile, 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 also contain a vasoconstrictor. The isotonic solution may include phosphate-buffered saline. The vaccine may further contain stabilizers, including gelatin and albumin. Stabilization, such as LGS or polycations or polyanions, in the vaccine formulation may allow the formulation to remain stable for extended periods at room or ambient temperature.

[0117] 5. Methods of vaccine delivery Provided herein are methods of vaccine delivery for providing genetic constructs and proteins of FMDV antigens containing epitopes that are particularly effective against FMDV immunogens capable of inducing an immune response. Vaccine delivery methods or vaccination methods can be provided for inducing therapeutic and prophylactic immune responses. The vaccination process can generate an immune response against multiple FMDV subtypes in a mammal. Vaccines can be delivered to individuals to modulate the activity of the mammal's immune system and promote an immune response. Vaccine delivery can be via transfection of FMDV antigens as nucleic acid molecules that are expressed in cells and delivered to the cell surface, where they are recognized by the immune system to induce a cellular response, a humoral response, or a cellular and humoral response. Vaccine delivery can be used to induce or elicit an immune response in a mammal against multiple FMDV viruses by administering the vaccines described above to a mammal.

[0118] Upon delivery of the vaccine and plasmids into mammalian cells, the transfected cells express and secrete the consensus capsid of each of the injected plasmids from the vaccine. These secreted capsid proteins are recognized as foreign by the immune system, and antibodies are generated against them. These antibodies are retained by the immune system, allowing rapid clearance of subsequent FMDV infection.

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

[0120] a. Combination treatment The vaccine may be administered to a patient receiving a vaccine containing α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 containing IL-15 with a deleted signal sequence and optionally containing a 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-1a, MIP-1p, IL-8, RANTES, L-selectin, P-secretin, or IL-1. Lectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular epithelial growth factor 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 The vaccine may also be administered in combination with other proteins or genes encoding K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof or combinations thereof. The vaccine may also 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 a combination thereof. For veterinary use, the composition may be administered in an appropriately tolerated formulation according to standard veterinary practice. A veterinarian can easily determine the most suitable administration regimen and route for a particular animal. The vaccine may be administered by traditional syringe, needleless injection device, "microparticle bombardment gene gun", or other physical methods, such as electroporation ("EP"), "hydrodynamic method", or ultrasound.

[0122] Vaccine plasmids may be delivered to mammals by several 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 adenovirus-associated virus, and recombinant vaccinia. FMDV antigens may also be delivered via DNA injection in conjunction with in vivo electroporation.

[0123] B. Electroporation Administration of a vaccine via electroporation of a vaccine plasmid may be accomplished using an electroporation device that can be configured to deliver an energy pulse to a desired mammalian tissue, generating a constant current similar to a preset current input by a user. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include or incorporate one or more of the various elements of an electroporation device, including a controller, 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 supply, and a power switch. Electroporation may be accomplished using a VGXP Cellectra™ system to facilitate transfection of cells with the plasmid.

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

[0125] The plurality of electrodes may deliver energy pulses in a distributed pattern. The plurality of electrodes may deliver energy pulses in a distributed pattern via electrode control under a programmed sequence, the programmed sequence being 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, including one neutral electrode that measures impedance, and a subsequent pulse of the plurality of pulses is delivered by a different one of the at least two active electrodes, including 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 circuit. 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 neutral electrode may measure the impedance in the desired tissue, which is communicated to the feedback mechanism, which adjusts the energy pulse in response to the impedance to maintain a constant current similar to the preset current. The feedback mechanism may maintain a constant current continuously and instantaneously during delivery of the energy pulse.

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

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

[0129] U.S. Patent Application Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues in a body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between an array of electrodes based on user control and pulse parameter input, and allows for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk with an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of U.S. Patent Application Publication No. 2005 / 0052630 are 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 deeply penetrate tissues such as muscle, as well as other tissues or organs. Depending on the configuration of the electrode array, an injection needle (delivering a selected biomolecule) may be fully inserted into the target organ and administered by injection perpendicular to the target tissue in the area previously designated by the electrode. 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] Additionally, as contemplated in some embodiments incorporating electroporation devices and their use, there are electroporation devices described in the following patents: 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. Additionally, patents addressing subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, which relates to delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, which focuses on methods of DNA injection, are contemplated herein. The foregoing patents are incorporated by reference in their entirety.

[0132] c. Methods for preparing vaccines Provided herein are methods for preparing vaccines. In some embodiments, the methods are for preparing vaccines comprising DNA plasmids. The DNA plasmids, after a final subcloning step into a mammalian expression plasmid, can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art. The plasmids are transformed into compatible host cells and cultured under conditions that allow expression of FMDV antigens. FMDV antigens can be recovered from the culture by lysing the cells or recovered from the medium and isolated. Isolated VP1-4 consensus proteins can be used as a natural source of antibodies in vaccines. FMDV antigens can also be produced by recombinant techniques using automated synthesizers that can be used to produce isolated, essentially pure FMDV antigens. These techniques can be useful for introducing variants of FMDV antigens of specific FMDV subtypes.

[0133] The DNA plasmids used with the EP devices of the present invention can be formulated or manufactured using a combination of known equipment 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, filed May 23, 2007, which is subject to licensing. In some examples, the DNA plasmids used in these studies can be formulated at concentrations of 10 mg / mL or greater. The manufacturing techniques include or incorporate a variety of equipment and protocols generally known to those skilled in the art, including those described in U.S. Provisional Patent Application No. 60 / 939,792, as well as those described in U.S. Patent Application No. 7,238,522, which issued July 3, 2007, which is subject to licensing. The previously referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are incorporated herein in their entireties. Example Example 1

[0134] Constructs of several embodiments have been made and tested, as shown in Figures 1-17, which show that vaccines have been made and data generated from their use.

[0135] Figure 17 shows a schematic diagram of the generic FMDV DNA vaccine construct, showing that the insert is cloned into the BamH1 and Xho-1 sites. The plasmid map of the generic FMDV vaccine is based on the plasmid pVAX. Examples of FMDV inserts can be in the long form, shown in Figure 17 as the long form insert, or in the short form, shown in Figure 7 as the short form insert. The IgE leader shown in each form is indicated as optional or may be replaced with a different leader. The 2A sequence is indicated as optional, and the furin cleavage site (rgrkrrs - SEQ ID NO: 27) is indicated as 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 insertion is cloned into the BamH1 and Xho-1 sites. The FMDV-A24cruzeiro DNA vaccine construct shown in Figure 3 is cloned into the BamH1 and Xho-1 sites. The FMDV-SAT DNA vaccine construct shown in Figure 5 is cloned into the BamH1 and Xho-1 sites. In each of Figures 1, 3, and 5, the plasmid maps are based on the plasmid pVAX. An example of an FMDV-As1-Shamir insertion can 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. An example of the insertion can 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. An example of an FMDV-SAT2 insertion can be in the long form, which is shown in Figure 5 as 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), and 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). 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 inserts were properly integrated 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 contained the shaded N-terminal IgE leader sequence, the proteolytic cleavage sites in lowercase, and the bolded VP4 sequence between the IgE leader and the first proteolytic cleavage site. Between the first and second proteolytic cleavage sites is the coding sequence for VP2. Between the second and third proteolytic cleavage sites is the coding sequence for VP3. Between the third and fourth proteolytic cleavage sites is the coding sequence for VP1. Between the final (fourth) proteolytic cleavage site and the termination is the 2A sequence.

[0138] Figure 7 shows the results of a protein expression experiment. Western blots of proteins on SDS gels compared protein expression from samples produced from FMDV-A24cruzeiro-S short format, FMDV-A24cruzeiro-L long format, pVAX, FMDV-As1-Shamir89-S short format, and FMDV-As1-Shamir89-L long format. The blots were probed with anti-A24 antiserum.

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

[0140] Figure 9 shows data on cellular immune responses induced by the FMDV-A24cruzeiro-L and FMDV-A24cruzeiro-S vaccines. Figure 10 shows data on cellular immune responses induced by the FMDV-As1-Sharma89-L and FMDV-As1-Sharma89-S vaccines. Figure 11 shows data on cellular immune responses induced by the FMDV-Sat2-L and FMDV-Sat2-S vaccines. Figure 12 shows the experimental protocol for DNA transfection and cell lysate preparation for ELISA analysis. Figure 13 shows data on antibody induction in mice induced by the FMDV-A24cruzeiro-L and FMDV-A24cruzeiro-S vaccines and by the FMDV-As1-Sharma89-L and FMDV-As1-Sharma89-S vaccines. Figure 14 shows data from ELISA analysis of antibody binding using protein lysates prepared from FMDV-A24cruzeiro-L transfected cells and FMDV-As1-Sharma89-L transfected cells. The FMDV vaccine was immunogenic in mice. Seroconversion was observed in all immunized animals. The long form of the vaccine was more efficacious than the short form. Humoral responses appeared to be most efficacious for the Shamir vaccine compared to the Creuzeiro vaccine, but both vaccines were efficacious. Cellular responses were more cross-reactive with the Shamir vaccine compared to the Creuzeiro vaccine. Comparison of bovine sera with positive sera, showing reasonable levels of immunoreactivity, was induced by the vaccine.

[0141] Figure 15 shows an amino acid sequence comparison of the Shamir and Cruzeiro sequences. The Shamir VP4 sequence (SEQ ID NO: 17) is shown relative to the Cruzeiro VP4 sequence (SEQ ID NO: 18), the Shamir VP2 sequence (SEQ ID NO: 19) is shown relative to the Cruzeiro VP2 sequence (SEQ ID NO: 20), and the Shamir 2A sequence (SEQ ID NO: 21) is shown relative to the Cruzeiro 2A sequence (SEQ ID NO: 22).

[0142] Figure 16 shows an amino acid sequence comparison of the Shamir and Cruzeiro sequences. The Shamir VP3 sequence (SEQ ID NO: 23) is shown relative to the Cruzeiro VP3 sequence (SEQ ID NO: 24), and the Shamir VP1 sequence (SEQ ID NO: 25) is shown relative to the Cruzeiro VP1 sequence (SEQ ID NO: 26). Example 2

[0143] Fourteen constructs have been designed to prepare an FMDV vaccine. Sequences from seven FMD virus subtypes, A, Asia 1, C, O, SAT1, SAT2, SAT3, and SAT4, are used. Two construct designs, a long and a 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, and SAT4, yielding 14 constructs. A vaccine can be produced using as few as four, and typically seven, constructs.

[0144] The generic long format is shown in Figure 17. The immunogen coding sequences are arranged in the order VP4, VP2, VP3, and VP1. Coding sequences for protease cleavage sites separate each of the four viral proteins. 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 format is shown in Figure 17. The immunogen coding sequences are arranged in the order VP2, VP3, and VP1. Coding sequences for protease cleavage sites separate each of the four viral proteins. Coding sequences can be provided for any IgE leader sequence provided. Similarly, a 16 amino acid 2A tail is provided at the end containing the protease cleavage site.

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

[0147] In some embodiments, the vaccine comprises an A-long format, an Asia 1-long format, a C-long format, an O-long format, a SAT1-long format, a SAT2-long format, a SAT3-long format, and a SAT4-long format.

[0148] In some embodiments, the vaccine comprises an A-short format, an Asia 1-short format, a C-short format, an O-short format, a SAT1-short format, a SAT2-short format, a SAT3-short format, and a SAT4-short format.

[0149] In some embodiments, the vaccine comprises an A-long format, an Asia 1-long format, a C-long format, and an O-long format.

[0150] In some embodiments, the vaccine comprises an A-short format, an Asia 1-short format, a C-short format, and an O-short format.

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

[0152] The individual viral proteins are separated from one another by proteases that are often present in the cells in which 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 embodiment of the present invention, there is a fusion protein comprising the consensus FMDV protein VP1 and a nucleic acid sequence encoding this protein from two different subtypes that 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.

[0157] In another embodiment of the present invention, there is a consensus FMDV protein VP1 and its encoding nucleic acid sequence that can be generated and used in vaccines 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

1. A nucleic acid molecule encoding the following sequence: a), b), c), or d): a) a leader sequence, viral protein VP4 (VP4), viral protein VP3 (VP3), viral protein VP2 (VP2), viral protein VP1 (VP1), viral protein 2A (2A), and four protease cleavage sites (CS), wherein the leader sequence, VP4, VP3, VP2, VP1, 2A, and CS are linked in the following order: leader sequence-VP4-CS-VP2-CS-VP3-CS-VP1-CS-2A; b) a leader sequence, viral protein VP3 (VP3), viral protein VP2 (VP2), viral protein VP1 (VP1), viral protein 2A (2A), and three protease cleavage sites (CS), wherein the leader sequences, VP3, VP2, VP1, 2A, and CS are linked in the following order: leader sequence-VP2-CS-VP3-CS-VP1-CS-2A; c) a leader sequence, viral protein VP4 (VP4), viral protein VP3 (VP3), viral protein VP2 (VP2), viral protein VP1 (VP1), and three protease cleavage sites (CS), wherein the leader sequence, VP4, VP3, VP2, VP1, and CS are linked in the following order: leader sequence-VP4-CS-VP2-CS-VP3-CS-VP1; or d) a leader sequence, viral protein VP3 (VP3), viral protein VP2 (VP2), viral protein VP1 (VP1), and two protease cleavage sites (CS), wherein the leader sequence, VP3, VP2, VP1, and CS are linked in the following order: leader sequence-VP2-CS-VP3-CS-VP1; wherein the coding sequence for viral protein VP4 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, and amino acids 19-103 of SEQ ID NO:10; wherein the coding sequence for viral protein VP2 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, and amino acids 111-329 of SEQ ID NO:10; wherein the coding sequence for viral protein 2A encodes an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, and amino acids 784-799 of SEQ ID NO:10; wherein the coding sequence for viral protein VP3 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, and amino acids 337-558 of SEQ ID NO:10; and wherein the coding sequence of viral protein VP1 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and amino acids 566-776 of SEQ ID NO:10; The nucleic acid molecule characterized in that:

2. A nucleic acid molecule as described in claim 1, wherein the leader sequence is an IgE sequence.

3. A nucleic acid molecule described in claim 1 or 2, wherein the cleavage site is rgrkrrs of sequence number 27, which is recognized by furin.

4. A plasmid comprising a nucleic acid molecule described in any one of claims 1 to 3, wherein the viral protein is derived from an FMDV subtype selected from the group consisting of A, Asia1, C, O, SAT1, SAT2 and SAT3.

5. A vaccine comprising the plasmid 1, 2, 3, 4, 5, 6 or 7 described in claim 4, wherein the nucleic acid sequence encoding the viral protein is derived from an FMDV subtype 1 to 7 selected from the group consisting of FMDV subtypes A, Asia 1, C, O, SAT1, SAT2, and SAT3.

6. The vaccine described in claim 5, comprising four plasmids, the nucleic acid sequences encoding the viral proteins being derived from each of the FMDV subtypes in the group consisting of A, Asia1, C, and O.

7. The vaccine described in claim 5, wherein the nucleic acid sequence encoding the viral protein is derived from each of FMDV subtypes A, Asia 1, C, O, SAT1, SAT2, and SAT3.

8. A nucleic acid molecule described in 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 comprising the nucleic acid molecule of claim 8, wherein the plasmid comprises the nucleic acid sequence of SEQ ID NO: 1, the plasmid comprises the nucleic acid sequence of SEQ ID NO: 3, the plasmid comprises the nucleic acid sequence of SEQ ID NO: 5, the plasmid comprises the nucleic acid sequence of SEQ ID NO: 7, 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. A vaccine comprising one or more of the plasmids described in claim 9.

11. A composition for generating an immune response against FMDV in an individual, comprising the vaccine of claim 5, 6, 7 or 10.

12. A composition for preventing FMDV infection in an individual, comprising the vaccine of claim 5, 6, 7 or 10.

13. A composition for treating an individual infected with FMDV, comprising the vaccine of claim 5, 6, 7 or 10.

14. A nucleic acid molecule comprising one or more sequences encoding one or more FMDV proteins selected from the group consisting of VP1, VP2, VP3, VP4, and 2A, the coding sequence for viral protein VP4 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, and amino acids 19-103 of SEQ ID NO:10; the coding sequence for viral protein VP2 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, and amino acids 111-329 of SEQ ID NO:10; the coding sequence for viral protein 2A encodes an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, and amino acids 784-799 of SEQ ID NO:10; The coding sequence for viral protein VP3 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, and amino acids 337-558 of SEQ ID NO:10; and The coding sequence of viral protein VP1 encodes an amino acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, and amino acids 566-776 of SEQ ID NO:10; The nucleic acid molecule characterized in that:

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