OA21072A - Live attenuated strains of foot and mouth disease modified by deoptimization and uses thereof. - Google Patents

Abstract

The present disclosure describes deoptimized foot and mouth viruses and their use for prophylactic and therapeutic treatment of mammalian subjects. The recombinant viruses provided herein include alterations in several genomic regions as well as Differentiating Infected from Vaccinated Animals (DIVA) markers.

Description

LIVE ATTENUATED STRAINS OF FOOT AND MOUTH DISEASE MODIFIED BY DEOPTIMIZATION AND USES THEREOF

CROSS-REFERENCE

[0001] The present application claims priority to U .S. Provisional Patent Application Ser. No. 63/030,431 filed on May 27, 2020 the content of which is expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

FIELD OF INVENTION

[0002] This invention relates to attenuated viruses and the prophylactic and therapeutic treatment of viral disease.

BACKGROUND

[0003] Foot-and-mouth disease (FMD) îs one of the most highly contagious viral diseases of cloven-hoofed animais and it is caused by the FMD virus (FMDV), a member of the Picornaviridae family. The virus can infect over 70 species of livestock and wild animais including cattle, swine, sheep, goat, and deer. FMD îs listed by the International Organization of Animal Health (OIE) as a reportable disease and severe trading restrictions are imposed upon notification of an outbreak. Disease outbreaks in previously FMD-free countries are înitially controlled by culling of infected and in-contact animais, restriction of susceptible animal movement, disinfection of infected premises and occasionally, vaccination with an inactivated whole virus antigen préparation. In countries where the disease is enzootic animais are prophylactically vaccinated. While not hazardous to human health, an FMD outbreak carries severe économie costs. For instance, the recent UK outbreak of 2001, afforded économie losses that surpassed US$12 billion, seriously impacting the overall economy of the affected areas.

[0004] In addition to the inactivated whole antigen vaccine formulation, a recombinant vaccine involving a réplication defective human adenovirus 5 that delivers empty FMDV capsids (Ad5FMD) has been successfully tested in recent years, however thus far this vaccine has only been granted aconditional license in the U.S. and its production could be costly. Both, the inactivated vaccine and the Ad5-FMD vaccine, require approximately 7 daysto induce protective immunity in swine and cattle and the duration of immunity is shorter than that conferred by natural infection. As a resuit, vaccinated animais are susceptible to disease if exposed to FMDV prior to 7 days or after approximately 6 months post vaccination.

[0005] The Global Foot-and-Mouth Disease Research Alliance (GFRA), an international organization launched in 2003, has set as part of their five main goals, the development of next génération control measures and strategies including improved vaccines and biotherapeutics. [0006] It has been reported that rapid and long-lasting protection against viral infection is usually best achieved by vaccination with live attenuated vaccines (LAVs). Indeed, using attenuated viral vaccines, smallpox and rînderpest viruses hâve been eradicated and measles has been elimînated from some parts of the world. Due to full or partial virulence în animais or réversion to virulence of experimental modified attenuated FMDV candidates, no LAV has been successfully developed or implemented to control FMDV. Previously described efforts examining genetically engîneered leaderless FMDV strains, carrying a délétion of the nonstructural viral protein L^pro coding région, showed reduced pathogenicity in swine and cattle. However, animais inoculated with a leaderless virus were not completely protected when challenged with certain wîld type (WT) viruses. More recently, a highly attenuated marker leaderless FMDV termed “FMDLL3B3D” (Uddowla et al, J. Virol. (2012) 86:11675-11685) was developed showing no signs of disease in the natural host, when administered live. In fact, the additional modifications including négative antigenic (3B and 3D) marker introduced in the FMDLL3B3D backbone, and a délétion of one of three 3B copies in the viral genome, rendered this mutant virus very stable and further restrîcted its réplication in cattle or swine (Uddowla et al, supra; Eschbaumer et al, Pathogens, (2020) 9:129). The atténuation of this novel vaccine candidate is such that the FMD-LL3B3D A₂₄ Cruzeîro vaccine platform strain and a large number of capsid coding cassettes were excluded from the United States Select Agent Program régulations in April 2018 (Select Agent Program. (2020). Foot and Mouth Disease. www.selectagents.gov/exclusion s-usda.html#footmouth) and that currentiy, it has been license in the US for manufacturing as a chemically inactivated safe FMDV marker vaccine (not as an LAV). This vaccine platform encodes two unique restriction sites to flank the capsid coding région to accommodate swapping capsid coding cassettes. Other experimental LAV vaccines carrying L^pro mutations introduced in a conserved protein motif, SAF-A/B, Acinus and PIAS (SAP) domain (de los Santos et al, J. Virol., (2009) 83:1800-1810), hâve also been described. Specificaily, substitutions of two conserved amino acid residues in this domain in FMDV A12 generated an attenuated mutant virus în cell culture and in swine. Interestingly, when the modified attenuated strain was tested as a vaccine candidate in swine, animais were completely protected even when challenged as early as two days post vaccination. However, since only two amino acid residues were substituted, reversion to virulence of the SAP mutant poses a considérable risk.

[0007] Codon usage bias îs characteristic of ail biological Systems since the frequencies of synonymous codon use for each amîno acid are unequal. Despite the low codon usage bias în RNA viruses deoptimization of the Pl/capsid région has proven to be an effective genetic engineering technique to attenuate poliovirus, which like FMDV belongs to the Picomaviridae family of viruses. Independently of the “codon bias” concept, some synonymous codon pairs are used more or less frequently than statistically predicted, resulting in a particular “codon pair bias” in every examined species.

SUMMARY OF THE INVENTION

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0009] The present disclosure provides, in one embodiment, a deoptimîzed foot and mouth disease virus (FMDV) containing a substituted genomic région, where the substituted genomic région is a nucleic acid at least 95% identical to SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and encodes the same polypeptide as SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively, or encodes the same polypeptide as SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO:4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively, with up to 10 amino acid replacements, délétions or additions. In some embodiments, the deoptimîzed FMDV has a substituted genomic région comprising a nucleic acid at least 99% identical to, or !00% identical to, SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, deoptimîzed FMDV also contain a DIVA marker, such as mutations in the 3B and 3D coding régions.

[0010] The present disclosure further provides, in an additional embodiment, a deoptimîzed modified foot and mouth disease virus (FMDV) constructed by substituting the P2 domain, or the P3 domain with a codon deoptimîzed or codon-pair deoptimîzed région encoding the same protein sequence, or encoding a protein sequence with up to 10 amino acid replacements, délétions or additions, where the codon pair bias ofthe modified sequence îs less than the codon pair bias ofthe parent FMDV, and is reduced by 0.05, 0.1, or 0.2. In particular embodiments, the deoptimîzed genomic région is the P2 domain, the P3 domain, or both the P2 and P3 domain. In spécifie embodiments, the deoptimîzed FMDV is A24-P2-3B3D, A24-P3-3B3D, or A2421072

P2/P3-3B3D.

[0011] The present disclosure further provides process embodiments, including a method of eiicitîng an immune response to foot and mouth disease, by administering any of the deoptimized modified viruses described herein to a mammalian subject. In some embodiments, administering the deoptimized modified virus is donc by administering 10², 10³, 10⁴ or 10⁵ pfu/mammalian subject of the deoptimized modified virus. In some embodiments, the subject is a bovid or a suid. In additional embodiments, administering the deoptimized modified virus entaîls administering a prime dose, and one or more boost doses, to the mammalian subject. In some embodiments of this method, the prime dose is administered when the mammalian subject does not hâve foot and mouth disease. in some embodiments of this method, the one or more boost doses are administered when the mammalian subject does not hâve foot and mouth disease. In some embodiments of this method, the one or more boost doses are administered when the mammalian subject has been exposed to foot and mouth disease. In some embodiments, the immune response elîcited by this methodology is a protective immune response.

[0012] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjonction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

INCORPORATION BY REFERENCE

[0013] Ail publications, patents and patent applications mentioned in this spécification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The novel features of the invention are set forth with partîcularity in the claims. Features and advantages ofthe present invention are referred to in the following detailed description, and the accompanying drawings of which:

[0015] FIG. IA, FIG. IB and FIG. IC depîct sequences of A24Cru with deoptimized P2 and/or P3 and 3B3D DIVA markers. FIG. IA depicts SEQ ID NO: l. FIG. IB depicts SEQ ID NO: 2. FIG. IC depicts SEQ ID NO: 3.

[0016] FIG. 2A, FIG. 2B and FIG. 2C depict the génération of deoptimized FMDV. FIG. 2A: Schematics of A₂4Cru wild type (WT) infections clone (Rieder et al 2005) with a unique added N/wI site. Relevant restriction sites used for cloning are depicted <NAeI, M/êl and BamHI). (FIG. 2B) NAel/M/êl, M/èl/Bu/nHl of NAel/ BomHI fragments containing deoptimized codons (Burns et al., 2006) were synthesized and respectively replaced in pAziCruNAel. Synthesized fragments contained DIVA markers including a small délétion in 3B and amino acid substitutions in 3B and 3D (Uddowla et al. 2012). FIG. 2C: Plaque morphology of WT and deoptimized viruses was analyzed in BHK-21 cells. Plaques were developed at 48 hpi. (see Example 1).

[0017] FIG. 3 depicts kinetics of growth and plaque morphology in cell culture. BHK-21 and IBRS2 cells Unes or PK and EBK primary cells were înfected with FMDV A24Cru wild type (A24-WT) or deoptimized variants, A24-P2Deopt3B3D, A24-P3Deopt3B3D and A24P2/3Deopt3B3D at MOI-2. After 1 h incubation, unabsorbed virus was removed by washing with 150 mM NaCi, 20 mM MES (pH=6.0) followed by addition of MEM complété media and incubation at 37° C. Samples were taken at 1, 3, 6 and 24 hpi and virus titers were determined by plaque assay on BHK-21 cells.

[0018] FIG. 4A, FIG. 4B and FIG. 4C depicts deoptimized viruses are attenuated in vivo in mice. 6 to7 weeks old female C57BL/6 mice (n = 6/group) were subcutaneously inoculated in the footpad with FMDV A24-deoptimized mutants, A24-P2/P3Deopt, A24-P2Deopt or A24P3Deopt at the indicated doses (plaque forming units -pfu-/animal). One group was inoculated with IxUfpfu/animal of A24-wild type (WT) as control. FIG. 4A: Survival rates determined daily post inoculation. FIG. 4B. Virus titers were measured in sérum samples collected for 7 days post inoculation (dpi). FIG. 4C: FMDV spécifie antibody neutralizing titers were measured in sérum samples collected at 0, 5, 7, 14 and 21 dpi with deoptimized variants and after 7- and 14-days post challenge (dpc) in ail animais that survived the initial inoculation.

[0019] FIG. 5 depicts clinical outcome in animais inoculated with A24-WT. 18-23 kg castrated male Yorkshîre swine (n ⁼ 3/group) were inoculated with 10^ plaque forming units (pfu) of FMDV A24-WT. Animais were monitored for 7 days and samples of heparinized blood, sérum and nasal swabs were collected daily. Clinical score (blue bars) and % of lymphocytes (green line) for each animal are represented in the top panels; Virus isolation in sérum (red line) and nasal sécrétion (blue line) and presence of viral copy numbers (GCN) per ml of sérum (red dashed line) and nasal sécrétion (blue dashed line) are presented in the bottom panels.

[0020] FIG. 6 depicts clinical outcome in animais inoculated with A24-P2Deopt3B3D. 18-23 kg castrated male Yorkshîre swine (n = 3/group) were inoculated with 10⁶ plaque forming units (pfu) [animais 8 to 10] or 10⁷pfu [animais 12 to 14] of FMDV A24-P2Deopt_ÎBîD. One naïve animal [#11 and #15] was housed in contact with each group throughout the duration ofthe experiment. Animais were monitored for 7 days and samples of heparinized blood, sérum and nasal swabs were collected daily. Clinical score (blue bars) and % of lymphocytes (green line) for each animal are represented in the top panels; Virus isolation in sérum (red line) and nasal sécrétion (blue line) and presence of viral copy numbers (GCN) per ml of sérum (red dashed line) and nasal sécrétion (blue dashed line) are presented in the bottom panels.

[0021] FIG. 7 depicts clinicai outcome in animais inoculated with A24-P3DeoptiB3D- 18-23 kg castrated male Yorkshire swine (n = 3/group) were inoculated with 10⁶ plaque torming units (pfu) of FMDV A24-P3Deopt3B3D. One naïve animal [#7] was in contact with the inoculated pigs throughout the duration ofthe experiment. Animais were monitored for 7 days and samples of heparinized blood, sérum and nasal swabs were collected daily. Clinicai score (blue bars) and % of lymphocytes (green line) for each animal are represented in the top panels; Virus isolation in sérum (red line) and nasal sécrétion (blue line) and presence of viral copy numbers (GCN) per ml of sérum (red dashed line) and nasal sécrétion (blue dashed line) are presented in the bottom panels.

[0022] FIG. 8 depicts clinicai outcome in animais inoculated with A24-P2/P3Deopt3B3D. 1823 kg castrated male Yorkshire swine (n = 3/group) were inoculated with 10^â plaque forming units (pfu) [animais 16 to 18] or 10⁷pfu [animais 20 to 22] of FMDV A24-P2/P3Deopt3B3DOne naïve animal [# 19 and #23] was housed in contact with each group throughout the duration ofthe experiment. Animais were monitored for 7 days and samples ofheparinized blood, sérum and nasal swabs were collected daily. Clinicai score (blue bars) and % of lymphocytes (green line) for each animal are represented in the top panels; Virus isolation in sérum (red line) and nasal sécrétion (blue line) and presence of viral copy numbers (GCN) per ml of sérum (red dashed line) and nasal sécrétion (blue dashed line) are presented in the bottom panels.

[0023] FIG. 9 depicts détermination of FMDV neutralizing antibodies in the sérum of animais inoculated with A24-P2, P3 or P2/P3Deopt3B3DOr A₂₄Cru wild type (A24-WT).

[0024] FIG. 10 depicts clinicai outcome in swine inoculated with low doses of A24P2/P3Deopt3B3D. 18-23 kg castrated male Yorkshire swine (n = 4/group) were inoculated with 10²· 10³, or 10⁵ pfu/animal of FMDV A24-P2/P3Deopt3B3D. An additional group was inoculated with 10³ pfu/animal of FMDV A24 WT as control. Animais were monitored for 7 days. Clinicai score (blue bars) and % of lymphocytes (green line) for each animal was determined daily.

[0025] FIG. 11 depicts détermination of virus or viral RNA in sérum and nasal sécrétions of animais inoculated with low doses A24-P2/P3 Deopt3B3D. The amount of virus was detected by virus isolation in sérum (red line) and nasal sécrétions (blue line). The presence of viral RNA was measured by qPCR and expressed as genome copy numbers (GCN) per ml of sérum (red dashed line) or per ml of nasal sécrétions (blue dashed line).

[0026] FIG. 12 depicts détermination of FMDV neutralizing antibodies in the sérum of animais inoculated with A24-P2/P3Deopt3B3D or A?4Cru wild type (A24-WT) Presence of FMDV neutralizîng antibodies was evaluated by a microtiter neutralization assay on BHK-21 cells in sera of animais inoculated with different doses of A24-P2/P3Deopt3B3Dor WT at the indicated time points after inoculation. Titers are reported as the logio of the reciprocal of the highest dilution of sérum that neutralîzed the virus in50% of the wells. Each data point represents the mean ± standard déviation (SD) of each group.

[0027] FIG. 13 depicts sequences of A24Cru with deoptimized Pl région in the A24Cru IC containing 3B3D DIVA markers and denoting restriction sites (Fsseland Nhel) used for cloning (SEQ IDNO:4).

[0028] FIG. 14 depicts sequences of Asial deoptimized Pl. LJnderlined are Fsel and Nhet restriction sites (SEQ ID NO: 5).

[0029] FIG. 15A and FIG. 15B depict génération of A24-P1 Deopt_3B3D virus. (FIG. 15A) Cloning strategy to generate A24-P1 Deopt3B3D infectious clone. (FIG. 15B) Plaque morphology of A24-WT3B3D and A24-P1 Deopt3B3D- Plaques were developed at 48 hpi in

BHK-21 cells.

[0030] FIG. 16A and FIG. 16B depict génération of ASIA-P1 Deopt3B3D virus. (FIG. 16A) Cloning strategy to generate ASIA-P1 Deopt3B3D infectious clone. (FIG. 16B) Plaque morphology of A24-WT3B3D and ASIA-P1 DeoptjæD- Plaques were developed at 48 hpi in

BHK-21 cells.

[0031] FIG. 17A and FIG. 17B depict kinetics of growth of A24 and Asia 1 Pl deoptimized FMDV: (FIG. 17A) BHK-21 and (FIG. 17B) SK6 cells were infected with the indicated viruses at MOI=5. After 1 h incubation, unabsorbed virus was removed by washing with 150 mM NaCl, 20 mM MES (pH=6.0) followed by addition of'MEM complété media and incubation at 37°C. Samples were taken at 1,2,4, 7 and 24 hpi and virus titers were determined by plaque assay on

BHK-21 cells.

[0032] FIG. ISA, FIG. 18B, and FIG. 18C depict A24-P1 Deopt3B3D virus atténuation in vivo in mice. 6 to7 weeks old female C57BL/6 mice (n = 6/group) were subcutaneously inoculated in the footpad with A24-P i Deopt_3B3D, at the indicated doses (pfu/animal). (FIG. 18A) Survival rates were calculated as (number of surviving animals/number of animais per group) x 100, daily post inoculation. (FIG. 18B) Virus titers were measured in sérum samples collected for 7 days post inoculation (dpi). (FIG. 18C) Presence of FMDV neutralizing antibodies was evaluated by a microtiter neutralization assay on BHK-21 cells in sera of animais inoculated with different doses of A24-P1 Deopt_3B3D at the indicated time points after inoculation or challenge. Control animais died due to disease before 7 dpi. Titers are reported as the loglO of the reciprocal of the highest dilution of sérum that neutralized the virus in 50% of the wells. Each data point represents the mean ± standard déviation (SD) of each group.

[0033] FIG. 19 depicts clinical outcome in animais inoculated with A24-P1 Deopt3B3D. 18-23 kg castrated male Yorkshire swine (n = 4/group) were inoculated with 10⁶ plaque forming units (pfu) [animais 28 to 31 ], 1 O^pfu [animais 32 to 35], 10⁴pfu [animais 36 to 39] or 10³pfu [animais 40 to 43] of FMDV Α24-ΡΙ Deopt3B3D. Animais were monitored for 7 days and samples of heparinized blood, sérum and nasal swabs were collected every other day. Clinical score (blue bars) and % of lymphocytes (green line) for each animal are represented

[0034] FIG. 20 depicts the détermination of virus in sérum and nasal sécrétions of animais inoculated with A24-P1 Deopt3B3D. 18-23 kg castrated male Yorkshire swine (n = 4/group) were inoculated with 10³, LO⁴,10⁵, or 10⁶ pfu of FMDV A24-P1 Deopt3B3D- Animais were monitored for 7 days and samples of sérum and nasal swabs were collected daily. The amount of virus was detected by virus isolation in sérum (triangle) and nasal sécrétions (circle). Détermination of FMDV neutralizing antibodies in the sérum of animais inoculated with A24-P1 Deopt3B3D. Presence of FMDV neutralizing antibodies was evaluated by a microtiter neutralization assay on BHK-21 cells in sera of animais inoculated with different doses of A24-P1 Deopt3B3D or with A24Cru wild type (control) at the indîcated time points after inoculation. Titers are reported as the loglO ofthe reciprocal of the highest dilution of sérum that neutralized the virus in 50% ofthe wells. Each data point represents the mean ± standard déviation (SD) of each group.

[0035| FIG. 21 depicts détermination of FMDV neutralizing antibodies in the sérum of animais inoculated with A24-P1 Deopt_3B3D. Presence of FMDV neutralizing antibodies was evaluated by a microtiter neutralization assay on BHK.-21 cells in sera of animais inoculated with different doses of A24-P1 Deopt3B3DOr with A₂4Cru wild type (control) at the indicated time points after inoculation. Titers are reported as the logw ofthe reciprocal ofthe highest dilution of sérum that neutralized the virus in 50% of the wells. Each data point represents the mean ± standard déviation (SD) of each group.

[0036] FIG. 22A, FIG. 22B, and FIG. 22C depict deoptimized viruses are attenuated in vivo in mice. 6 to 7 weeks old female C57BL/6 mice (n = 6/group) were subcutaneously inoculated in the footpad with FMDV AS1A-P1 Deopt_3B3oat the indicated doses (plaque forming units pfu-/animal) or Asial at 4x10⁵ pfti/animal (control). (FIG. 22A) Survival rates determined daily post inoculation. (FIG. 22A) Virus titers were measured in sérum samples collected for 7 days post inoculation (dpi). (FIG. 22A) FMDV spécifie antibody neutralizing titers were measured in sérum samples collected at 0, 5, 7, 14 and 21 dpi with the different viruses and doses.

[0037] FIG. 23 depicts clinical outcome in animais inoculated with ASIA-Pl Deopt3B3D or ASIA-WT. 18-23 kg castrated male Yorkshire swine (n = 4/group) were inoculated with l O⁶ plaque fonning units (pfu) [animais 81 to 84], l0⁵pfu [animais 85 to 88] or lO³pfu [animais 89 to 92] of FMDV ASIA-Pl Deopt3B3D or lO’pfu [animais 93, 95 & 96] of FMDV ASIA-WT. Animais were monîtored for 7 days and samples of heparinized blood, sérum and nasal swabs were collected every other day. Clinical score (blue bars) and % of lymphocytes (green line) for each animal are represented.

[0038] FIG. 24 depicts détermination of virus in sérum and nasal sécrétions of animais inoculated with ASIA-Pl Deopt3B3D or ASIA-WT. 18-23 kg castrated male Yorkshire swine (n = 4/group) were inoculated with lΟ⁵, ΙΟ²*, or 10⁶ pfu of FMDV ASIA-Pl Deopt3B3D or HFpfu of FMDV ASIA-WT. Animais were monîtored for 14 days and samples of sérum and nasal swabs were collected daîly. The amount of virus was detected by virus isolation in sérum (triangle) and nasal sécrétions (circle).

[0039] FIG. 25 depicts détermination of FMDV neutralizing antibodies in the sérum of animais inoculated with ASIA-Pl Deopt_3B3D or ASIA-WT. Presence of FMDV neutralizing antibodies was evaluated by a microtîter neutral ization assay on BHK-21 cells in sera of animais inoculated with different doses of ASIA-Pl Deopt3B3D at the indicated time points after inoculation. Titers are reported as the loglO of the reciprocal of the highest dilution of sérum that neutralized the virus in 50% of the wells. Each data point represents the mean ± standard déviation (SD) of each group.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Foot-and-mouth disease (FMD) is one of the most feared viral diseases that can affect livestock. Although this disease appearedto be contained in developed nations, recent outbreaks hâve demonstrated that infection can spread rapidly, causing devastating économie and social conséquences. To develop novel countermeasures, “synonymous codon deoptimization” of certain coding régions of the FMDV genome was performed to produce stable modified attenuated viral strains. Mutant viruses were also engineered to contain spécifie mutations in the 3B and 3D coding régions that confer markers for différentiation of infected from vaccinated animais (DIVA), and convenîent restriction endonuclease cleavage sites for capsid swapping. Deoptimization of selected coding régions, including DIVA markers, resulted in viable progeny that exhibited atténuation in cell culture, in mice and in swine, a natural host. Dur work demonstrates that codon deoptimization technologies can be applied to FMDV to obtain modified live attenuated strains of FMDV containing DIVA markers, reduced risk for reversion and recombination with virulent cîrculating strains for potential development as modified vaccine candidates.

[0041] Herein we démon strate that stable, viable strains of FMDV can be produced by deoptimization of genomic régions other than the capsid régions. Deoptimization of conserved P2/P3 FMDV régions resulted in attenuated strains that can grow to end point titers similar to those of the parental WT strain in tissue culture. Furthermore, P2/P3 deoptimized variants tolerated the introduction of: A) DIVA (différentiation of infected from vaccinated animais) markers în the 3B and 3D régions, and B) restriction sites for easy capsid exchange.

[0042] Ail together, these results demonstrate that codon/codon pair bias deoptimization is applicable to multiple targets as a means to dérivé viable strains of reduced virulence, and decreased probability of recombination, while allowîng for maintenance of genetic markers for differential diagnostics, highlighting their potential for development into modified live attenuated FMDV vaccine candidates.

[0043] Ail references cîted herein are incorporated by reference in their entïrety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3^,d ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7^ih ed, J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4^lk ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to préparé antibodies, see D. Lane, Antibodies: A Laboratory Manual 2^nd ed. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. ImmunoL 6: 511; Queen et al. U. S. Patent No. 5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep;23(9):l 126-36).

[0044] One skilled în the art will recognize many methods and materials similar or équivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is în no way limited to the methods and materials described. For purposes ofthe present invention, the following tenus are defined below.

[0045] As used in the spécification and claims, use of the singular ’‘a”, “an”, and “the” include plural references unless the context clearly dictâtes otherwise.

[0046] The terms isolated, purified, or biologically pure as used herein, refer to material that is substantially or essentially free from components that normally accompany the referenced material in its native State.

[0047] The term “about” is defined as plus or minus ten percent of a recited value. For example, about 1.0g means 0.9g to 1.1g and ail values within that range, whether specifically stated or not.

[0048] The term “a nucleic acid consisting essentially ofand grammatical variations thereof, means nucleic acids that differ from a reference nucleic acid sequence by 20 or fewer nucleic acid residues and also perform the function of the reference nucleic acid sequence. Such variants include sequences which are shorter or longer than the reference nucleic acid sequence, hâve different residues at particular positions, or a combination thereof.

[0049] “Coding sequence” as used herein refers to a nucleic acid sequence encompassing an open reading frame or part thereof, which encodes a viral or host cell protein sequence or part thereof.

[0050] Modified sequences (nucleic acids, proteins) of the present invention also include nucleic acids with high identity to a reference sequence. For example, nucleic acids having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any of the sequences provided herein. As a practical matter, percentge identity to a given reference sequence can be determined conventionally using known computer programs to find the best segment of homology between two sequences. When using sequence alignment program(s) to détermine whether a particular sequence is, for instance, 96% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference sequence.

[00511 The identity or simîlarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more dîstantly related (such as human and C. elegans sequences).

[0052] “Deoptimized modîfied vîruses” as used herein refer to vîruses in which their genome, in whole or in part, has synonymous codons and/or codon rearrangements and variation of codon pair bias. Deoptimized modîfied vîruses of the present invention can be used to for prophylactic and therapeutic treatment of viral infections.

[0053] “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees, and other apes and monkey species; farm animais such as cattle, buffalo, sheep, pigs, goats and horses; domestic maminals such as dogs and cats; laboratory animais including rodents such as mice, rats and guînea pigs, and the like. The term does not dénoté a particular âge or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be including within the scope of this term.

[0054] We believe that codon and codon-pair bias deoptîmization can also be a useful tool to develop attenuated vaccine candidates against any RNA virus. In various studies we hâve recently demonstrated that, codon-pair bias deoptîmization îs also tolerated by FMDV. Deoptîmization of the Pl structural région resulted in a FMDV strain (A12-P1 deopt) that was highly attenuated in mice, and that induced a protective immune response against léthal FMDV WT challenge at a relative low dose offering an approximately-10,000 “safety margin” (ratio between the dose required to kill animais and the dose required to induce protection) (Diaz-San Segundo étal, J. Virok, (2015) 90:1298-1310). Furthermore, virulence studies in swine showed that the Al 2-P1 deopt virus was also attenuated in the naturel host since a dose ~l00 fold higher than the dose of homologous WT virus was required to cause équivalent disease. Interestingly, high levels of neutralizing antibodies (Abs) were detected in sera suggesting that swine vaccinated with A12-P1 deopt virus could be protected against FMD.

[0055] FMDV structural proteins are involved in capsid assembly and stability, virus binding to target cells, and antigenic specificîty, influencing significant aspects of virus infection and immunity. The high level of variability in FMDV capsid proteins reflects sélective pressures and virus adaptation, as represented by the multiple serotypes and subtypes of this virus. In contrast, other régions of the viral genome are more conserved and can be substituted for each other in infectious FMDV clones without affecting viability. In fact, we hâve recently shown that a master seed vaccine strain could be constructed with the AzÆru IC backbone and spécifie capsids could be exchanged without significantly affecting the virus growth properties.

[0056] Described herein, we demonstrate that stable viable strains of FMDV can be produced by deoptîmization of other than the capsid régions. Deoptîmization of conserved P2/P3 régions resulted in attenuated strains that in tissue culture can grow to end point titers simîlarly to those ofthe parental WT strain. Furthermore, introduction of DIVA markers in the 3B and 3D régions and restriction sites for easy capsid exchange were well tolerated in the P2/P3 deoptimized variants. Among the different mutants obtained and evaluated, studies in mice and swine demonstrated that the resulting A24-P2/P3Deopt3B3D virus was attenuated in vivo and induced an adaptive immunity. These results highlight the potential of codon deoptîmization as a strategy to reduce the virulence of FMDV and decrease the probability of recombination with circulating strains, making them attractive candidates for further development into modified live attenuated FMDV vaccines. Various embodiments of the present invention are based, in part, on these finding, as well as those further described herein.

[0057] Alternative Encoding

[0058] A given peptide can be encoded by a large number of nucleic acid sequences. For example, even a typical short 10-mer oligopeptide can be encoded by approximately 4^lû(about 10⁶) different nucleic acids, and the PI capsid protein of poliovirus (881 amino acid long) can be encoded by about lO⁴⁴² different nucleic acids. Natural sélection has ultimately chosen one of these possible l O⁴⁴² nucleic acids as the PV genome. Whereas the primary amino acid sequence is the most important level of information encoded by a given mRNA, there are additional kînds of information within different kinds of RNA sequences. These include RNA structural éléments of distinct function (e.g., for PV, the cis-acting réplication element, or CRE, translational kinetic signais (pause sites, frame shift sites, etc.), polyadenylatîon signais, splice signais, enzymatic functions (ribozyme) and, quite likely, other as yet unidentified information and signais).

[0059] Even with the caveat that signais such as the CRE must be preserved, l O⁴⁴³ possible encoding sequences provide tremendous flexibilîty to make drastic changes in the RNA genome of FMD virus (FMDV), a virus of similar size to polivirus, while preservîng the capacity to encode the same exact protein. Changes can be made in codon bias or codon pair bias, and nucleic acid signais and secondary structures in the RNA can be added or removed. Additional or novel proteins can even be simultaneously encoded in alternative frames.

[0060] Codon Pair Bias

[0061] A distinct feature of coding sequences is their codon pair bias. This may be illustrated by considering the amino acid pair Ala-Glu, which can be encoded by 8 different codon pairs and this pairing can hâve a bias that effects translation of human and viral genes in human cells (Table 1). If no factors other than the frequency ofeach individual codon (as shown in Table

2) are responsable for the frequency ofthe codon pair, the expected frequency of each ofthe 8 encodings can be calculated by multiplyîng the frequencies of the two relevant codons. For example, by this calculation the codon pair GCA-GAA would be expected to occur at a frequency of 0.097 out of ail Ala-Glu coding pairs (0.23x0.42; based on the frequencies in Table 2). In order to relate the expected (hypothetical) frequency of each codon pair to the actually observed frequency in the human genome the Consensus CDS (CCDS) database of consistently annotated human coding régions, containing a total of 14,795 human genes, was used. This set of genes is a comprehensive représentation of human coding sequences. Using this set of genes, the frequencies of codon usage were re-calculated by dividing the number of occurrences of a codon by the number of ail synonymous codons coding for the same amino acid. As expected, the frequencies correlated closely with previously published ones such as the ones given in Table 2. Slight frequency variations are possibly due to an oversamplîng effect in the data provîded by the codon usage database at Kazusa DNA Research Institute (www.kazusa.or.jp/codon/codon.html) where 84949 human coding sequences were included in the calculation (far more than the actual number of human genes). The codon frequencies thus calculated were then used to calculate the expected codon-pair frequencies by first multiplyîng the frequencies ofthe two relevant codons with each other (see Table 1 expected frequency), and then multiplyîng this resuit with the observed frequency (în the entire CCDS data set) with which the amino acid pair encoded by the codon pair in question occurs. In the example of codon pair GCA-GAA, this second calculation gives an expected frequency of0.098 (compared to 0.97 in the first calculation using the Kazusa dataset). Finally, the actual codon pair frequencies as observed in a set of 14,795 human genes was determîned by counting the total number of occurrences of each codon pair în the set and dividing it by the number of ail synonymous coding pairs în the set coding for the same amino acid pair (Table 2; observed frequency). Frequency and observed/expected values for the amino acid pair Ala-Glu, which can be encoded by 8 different codon pairs is seen in Table 1 and the complété set of 3721 (61²) codon pairs, based on the set of 14,795 human genes, (Coleman et al. 2008)

Table 1. Codon pair scores for the amino acid pair Alanine-Glutamine

Amino acid pair	codûn pair	expected frequency	observed frequency	obstop ratio
AE	GCAGAA	Œ09K	0. J 63	1.65
AE	GCAGAG	il 132	0.I9S	______151
AE	GCCGAA	Chl7i	0.021	(HH
AE	GCCGAG	0,229	0.142	062
AE	GCGGAA	0.046	0.027	0,57 _____
AE	GCGGAO	0.062	0.0K9	1.44________
AE	GCTGAA	0.112	ÜJ45	1.29
AE	GCTGAG	0.150	(1206	1.37
Total		LÜOO	1,tW

Table 2. Codon Bias in Human Genes

Amino Acid	Codon	Number	/1000	Fraction
Gly	GGG	636457.00	16.45	0.25
Gly	GGA	637120.00	16.47	0.25
Gly	GGT	416131.00	10.76	0.16
Gly	GGC	862557.00	22.29	0.34
Glu	GAG	1532589.00	39.61	0.58
Glu	GAA	1116000.00	28.84	0.42
Asp	GAT	842504.00	21.78	0.46
Asp	GAC	973377.00	25.16	0.54
Val	GTG	1091853.00	28.22	0.46
Val	GTA	273515.00	7.07	0.12
Val	GTT	426252.00	11.02	0.18
Val	GTC	562086.00	14.53	0.24
Ala	GCG	286975.00	7.42	0.11
Ala	GCA	614754.00	15.89	0.23
Ala	GCT	715079.00	18.48	0.27
Ala	GCC	1079491.00	27.90	0.40
Arg	AGG	461676.00	11.93	0.21
Arg	AGA	466435.00	12.06	0.21
Ser	AGT	469641.00	12.14	0.15
Ser	AGC	753597.00	19.48	0.24
Lys_______	AAG	1236148.00	31.95	0.57
Lys	AAA	940312.00	24.30	0.43
Asn	AAT	653566.00	16.89	0.47
Asn	AAC	739007.00	19.10	0.53
Met	ATG	853648.00	22.06	1.00
Ile	ATA	288118.00	7.45	0.17
Ile	ATT	615699.00	15.91	0.36
Ile	ATC	808306.00	20.89	0.47
Thr	ACG	234532.00	6.06	0.11
Thr	ACA	580580.00	15.01	0.28
Thr	ACT	506277.00	13.09	0.25
Thr	ACC	732313.00	18.93	0.36
Trp	TGG	510256.00	13.19	1.00
End	TGA	59528.00	1.54	0.47
Cys_______	TGT	407020.00	10.52	0.45
Cys	TGC	487907.00	12.61	0.55
End	TAG	30104.00	0.78	0.24
End	TAA	38222.00	0.99	0.30
Tyr_______	TAT	470083.00	12.15	0.44
Tyr	TAC	592163.00	15.30	0.56
Leu	TTG	498920.00	12.89	0.13
Leu	TTA	294684.00	7.62	0.08____
Phe	TTT	676381.00	17.48	0.46
Phe	TTC	789374.00	20.40	0.54
Ser	TCG	171428.00	4.43	0.05
Ser	TCA	471469.00	12.19	0.15

Ser	TCT	585967.00	15.14	0.19
Ser	TCC	684663.00	17.70	0.22
Arg	CGG	443753.00	11.47	0.20
Arg	CGA	239573.00	6.19	0.11
Arg	CGT	176691.00	4.57	0.08
Arg	CGC	405748.00	10.49	0.18
Gin	CAG	1323614,00	34.21	0.74
Gin	CAA	473648,00	12.24	0.26
His	CAT	419726.00	10.85	0.42
His	CAC	583620.00	15.08	0.58
Leu	CTG	1539118.00	39.78	0.40
Leu	CTA	276799.00	7.15	0.07
Leu	CTT	508151.00	13.13	0.13
Leu	CTC	759527.00	19.63	0.20
Pro	CCG	268884.00	6.95	0.11
Pro	CCA	653281.00	16.88	0.28
Pro	CCT	676401.00	17.48	0.29
Pro	CCC	767793.00	19.84	0.32

[0062] If the ratio of observed frequency/expected frequency of the codon pair is greater than one the codon pair is said to be overrepresented. If the ratio is smaller than one, it is said to be underrepresented. In the example the codon pair GCA-GAA is overrepresented 1.65-fold while the coding pair GCC-GAA is more than 5-fold underrepresented.

[0063] Many other codon pairs show very strong bias; some pairs are under-represented, while other pairs are over-represented. For instance, the codon pairs GCCGAA (AlaGlu) and GATCTG (AspLeu) are three- to six-fold under-represented (the preferred pairs being GCAGAG and GACCTG, respectiveîy), while the codon pairs GCCAAG (AlaLys) and AATGAA (AsnGlu) are about two-fold over-represented. It is noteworthy that codon pair bias has nothing to do with the frequency of pairs of amino acids, nor with the frequency of individual codons. For instance, the under-represented pair GATCTG (AspLeu) happens to use the most frequent Leu codon, (CTG).

[0064] Codon pair bias was discovered in prokaryotic cells, but has since been seen in ail other examined species, including humans. The effect has a very high statîstical significance, and is certainly not just noise. However, its functional significance, if any, is a mystery. One proposai is that some pairs of tRNAs interact well when they are brought together on the ribosome, while other pairs interact poorly. Since different codons are usually read by different tRNAs, codon pairs might be biased to avoid putting together pairs of incompatible tRNAs. Another idea is that many (but not ail) under-represented pairs hâve a central CG dînucleotide (e.g., GCCGAA, encoding AlaGlu), and the CG dînucleotide is systematically under-represented in mammals.

Thus, the effects of codon pair bias could be of two kinds—one an indirect effect of the underrepresentation of CG in the mammalian genome, and the other having to do with the efficiency, speed and/or accuracy of translation. It is emphasized that the present invention is not limited to any particular molecular mechanism underlying codon pair bias.

[0065] Calculation of Codon Pair Bias

[0066] Every individual codon pair of the possible 3721 non-“STOP” containing codon pairs (e.g., GTT-GCT) carries an assigned “codon pair score,” or “CPS” that is spécifie for a given “traîning set” of genes. The CPS of a given codon pair is defined as the log ratio of the observed number of occurrences over the number that would hâve been expected in this set of genes (in this example the human genome). Determinîng the actual number of occurrences of a particular codon pair (or in other words the likelihood of a particular amino acid pair being encoded by a particular codon pair) îs simply a matter of counting the actual number of occurrences of a codon pair in a particular set of coding sequences. Determinîng the expected number, however, requires additional calculations. The expected number is calculated so as to be independent of both amino acîd frequency and codon bias similariy to Gutman and Hatfield. That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a spécifie codon. A positive CPS value signifies that the given codon pair is statistically over-represented, and a négative CPS indicates the pair is statistically underrepresented in the human genome.

[0067] To perform these calculations within the human context, the most recent Consensus CDS (CCDS) database of consistently annotated human coding régions, containing a total of 14,795 genes, was used. This data set provided codon and codon pair, and thus amino acid and amino-acid pair frequencies on a genomic scale. Although exemplified here by the human example, this can be applied to any animal (e.g., cow, swine, and other domesticated animais). [0068] The paradigm of Federov et al. (2002), was used to further enhanced the approach of Gutman and Hatfield (1989). This allowed calculation of the expected frequency of a given codon pair independent of codon frequency and non-random associations of neighboring codons encoding a particular amino acid pair. The detailed équations used to calculate CPB are disclosed in WO 2008/121992 and WO 2011/044561, which are incorporated by reference.

z x _ . (N^P^ _ ( N₀(P^

WAV

[0069] In the calculation, Pîj is a codon pair occurrîng with a frequency of N₀(Pij) in its synonymous group. Ci and Cjare the two codons comprising Py, occurrîng with frequencies

F(C,) and F(Cj) in their synonymous groups respectively. More explicîtly, F(C,) isthe frequency that corresponding amino acid X, is coded by codon C, throughout ail coding régions and F(Ci)=No(Cj)/No(Xi), where No(Ci) and No(Xi) are the observed number of occurrences of codon Ci and amino acid X, respectively. F(Cj) is calculated accordingly. Further, Νο(Χή) is the number of occurrences of amino acid pair Xÿ throughout ail coding régions. The codon pair bias score S(Pjj) of Pij was calculated as the log-odds ratio of the observed frequency N₀(Pij) over the expected number of occurrences of N_t(Pij).

[0070] Using the formula above, it was then determined whether individual codon pairs in individual coding sequences are over- or under-represented when compared to the corresponding genomic N_e(Pÿ) values that were calculated by using the entire human CCDS data set. This calculation resulted in positive S(Pij) score values for over-represented and négative values for under-represented codon pairs in the human coding régions.

[0071] The “combined” codon pair bias of an individual coding sequence was calculated by averaging ail codon pair scores according to the following formula:

i = l

[0072] The codon pair bias of an entire coding région is thus calculated by adding ail of the individual codon pair scores comprisîng the région and dividîng this sum by the length ofthe coding sequence.

[0073] Calculation of Codon Pair Bias, Implémentation of Algorithm to Alter Codon-Pair Bias. [0074] An algorithm was developed to quantîfy codon pair bias. Every possible individual codon pair was given a “codon pair score”, or “CPS”. CPS is defined as the natural log ofthe ratio of the observed over the expected number of occurrences of each codon pair over ail human coding régions, where humans represent the host species of the instant vaccine virus to be recoded.

/ F(AB)o \

- ^ln FQQ x F® . . I XFW x F(r) 7

[0075] Although the calculation of the observed occurrences of a particular codon pair is straightforward (the actual count within the gene set), the expected number of occurrences of a codon pairrequires additional calculation. We calculate this expected number to be independent both of amino acid frequency and of codon bias, similar to Gutman and Hatfield. That is, the expected frequency îs calculated based on the relative proportion of the number of times an amino acid îs encoded by a spécifie codon. A positive CPS value signifies that the given codon pair is statistically over-represented, and a négative CPS indicates the pair is statistically under represented in the human genome

[0076] Using these calculated CPSs, any codîng région can then be rated as using over- or under-represented codon pairs by taking the average of the codon pair scores, thus giving a

Codon Pair Bias (CPB) for the entire gene.

CPB

CPSi k - 1

[0077] As discussed further below, codon pair bias takes into account the score for each codon pair in a coding sequence averaged over the entire length of the codîng sequence. According to the invention, codon pair bias is determined by cpb=y i=i çpsi K-l

[0078] Accordingly, similar codon pair bias for a coding sequence can be obtained, for example, by minimized codon pair scores over a subsequence or moderately diminished codon pair scores over the full length of the coding sequence.

[0079] Since ail 61 sense codons and ail 3721 sense codon pairs can certaînly be used (and are used) in naturally occurring coding sequences, it would not be expected that substîtuting a single rare codon for a frequent codon, or a rare codon pair for a frequent codon pair, would hâve much effect. Irrespective of the précisé mechanism, the data indîcate that the large-scale substitution of synonymous deoptîmized codons înto a viral genome results in severely attenuated viruses. This procedure for producing modified viruses has been dubbed SAVE (Synthetic Attenuated Virus Engineering).

[0080] According to aspects of the invention, viral modification can be accomplished by changes in codon pair bias as well as codon bias in one or more portions of the virus s genome. However, it is expected that adjusting codon pair bias îs particularly advantageous. For example, attenuatîng a virus through codon bias generally requîtes élimination of common codons, and so the complexity of the nucieotide sequence is reduced. In contrast, codon pair bias réduction or minimization can be accomplished whîle maintaining far greater sequence diversity, and consequently greater control over nucleic acid secondary structure, annealing température, and other physical and biochemical properties. The work disclosed herein includes modified codon pair bias-reduced or -minimized sequences in which codons are shuffled, but the codon usage profile is unchanged.

[0081] The effects of our virus modification can be confirmed in ways that are well known to one of ordinary skill in the art. Non-limiting examples induce plaque assays, growth measurements, and reduced lethalîty in test animais. The instant application demonstrates that the modified viruses are capable of inducing protective immune responses in a host.

[0082] Various embodiments of the present invention provide for deoptimized modified foot and mouth virus derived from a wild-type foot and mouth virus, or a previously modified foot and mouth virus, by substitutîng at least one genomic région of the wild-type foot and mouth virus, or the previously modified foot and mouth virus, with a codon deoptimized région encoding the protein sequence, or encoding a protein sequence with up to 10 amino acid replacements, additions, or délétions.

[0083] Various embodiments of the present invention provide for deoptimized modified foot and mouth virus derived from a wild wild-type foot and mouth virus or a previously modified foot and mouth virus, by substitutîng at least one genomic région of the wild-type foot and mouth virus or the previously modified foot and mouth virus with a codon-pair deoptimized région encoding the protein sequence, or encoding a protein sequence with up to 10 amino acid replacements, additions, or délétions.

[0084] Various embodiments of the present invention provide for deoptimized modified foot and mouth virus derived from a wild-type foot and mouth virus, or a previously modified foot and mouth virus, by substitutîng at least one genomic région of the wild-type foot and mouth virus, or the previously modified foot and mouth virus, with a codon deoptimized and/or codonpair deoptimized région encoding the protein sequence, or encoding a protein sequence with up to 10 amino acid replacements, additions, or délétions.

[0085] In various embodiments, codon deoptimized foot and mouth virus comprises at least 10 deoptimized codons in the at least one genomic région, wherein the at least 10 deoptimized codons are each a synonymous codon less frequently used in the foot and mouth virus. In various embodiments, the codon deoptimized foot and mouth virus comprises at least 20, 30, 40, 50, 60, 70, 80, 90, I00, 125, 150, 200, 250, 300, 400, 500, 600, or 700 deoptimized codons in in the at least one genomic région, wherein the at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, or 700 deoptimized codons are each a synonymous codon less frequently used in the in the foot and mouth virus. The synonymous codon less frequently used in the in the at least one genomic région is a codon that encodes the same amino acid, but the codon is an unpreferred codon by the foot and mouth virus for the amino acid.

[0086] In various embodiments, codon deoptimized foot and mouth virus comprises at least 10 deoptimized codons in the at least one genomic région, wherein the at least 10 deoptimized codons are each a synonymous codon less frequently used in the mammalian host. In various embodiments, the codon deoptimized foot and mouth virus comprises at least 20, 30, 40, 50,

60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, or 700, deoptimized codons in in the at least one genomic région, wherein the at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, or 700 deoptimized codons are each a synonymous codon less frequent!y used în the in the the mammalian host. The synonymous codon less frequently used în the in the at least one genomic région is a codon that encodes the saine amino acid, but the codon is an unpreferred codon by the mammalian host for the amino acid.

[0087] In various embodiments, the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus, and is reduced by at least about 0.01, at least about 0.02, at least about 0.03, at least about 0.04, at least about 0.05, at least about 0.10, at least about 0.15, at least about 0.20, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.40, or at least about 0.50.

[0088] In various embodiments, the at least one genomic région is the P2 or P3 domain of a foot and mouth virus. A foot and mouth virus utilized in practicîng the present disclosure can be of any strain or serotype, such as A24, Al2, Asia, Ol Manîsa and Ol Campos, for example. In some embodiments, such modified viruses also contain a DIVA région, such as 3B3D régions detailed herein.

[0089] Administration

[0090] The modified FMDV described herein can be administered to a mammalian subject, such as a suid or a bovid. In various eipbodiments, administering the deoptimized modified virus comprises administering a dose of ΙΟ², ΙΟ³, 10⁴ or 10⁵ pfu/mammalian subject of the deoptimized modified virus. In some instances, administration comprises administering a prime dose to the mammalian subject; and administering one or more boost doses. The skilled artisan is able to détermine an effective dosing regimen, but typically the prime dose and/or the one or more boost doses (if any) are administered when the mammalian subject does not hâve foot and mouth disease. One or more boost doses are administered when the mammalian subject does not hâve foot and mouth disease. In various embodiments, the one or more boost dose is administered when the mammalian subject has been exposed to foot and mouth disease.

[0091 ] Modified FMDV described herein can be formulated for and administered via any route of administration known in the art, including arerosol, subcutaneous, intraoral, intranasal, intram uscular, transdermal, transmucosal and/or intradermal route(s). “Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parentéral” refers to a route of administration that îs generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapu Imonary, intraspînal, intrasternaî, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parentéral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0092] Via the enterai route, the pharmaceutical compositions can be in the form of tablets, gel 5 capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, émulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.

Via the parentéral route, the compositions may be in the form of solutions or suspensions for infusion or for injection.

[0093] Via the topical route, the pharmaceutical compositions based on compounds according 10 to the invention may be formulated for treating the skin and mucous membranes and are în the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending 15 on the clinical indication. Via the ocular route, they may be in the form of eye drops.

[0094] The pharmaceutical/veterinary compositions according to the invention can also contain any pharmaceutically/veterinarally acceptable carrier. “Pharmaceutically acceptable carrier” and “veterïnarally acceptable carrier” as used herein refers to acceptable materials, compositions, or vehicles that are involved in carrying or transporting a compound of interest 20 from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.

For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be acceptable in that it must be compatible with the other ingrédients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may corne in contact, 25 meaning that it must not carry a risk, or has a very low risk, of toxicity, irritation, allergie response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0095] The pharmaceutical and veterinary compositions provided herein can also be encapsulated, tableted or prepared in an émulsion or syrup for oral administration. Solid or 30 liquid carriers may be added to enhance or stabilize the composition, or to facilitate préparation ofthe composition. Liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include, but are not limited to, starch, lactose, calcium sulfate, dihydrate, terra alba, magnésium stéarate or stearîc acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0096] The pharmaceutical/veterinary préparations are made following the conventional techniques of pharmacy involving milling. mixing, granulation, and compressîng, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the préparation will be in the form of a syrup, élixir, émulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0097] The pharmaceutical/veterinary compositions according to the invention may be delivered in a therapeutically effective amount. The précisé therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamies, and bioavailability), the physiological condition ofthe subject (including âge, sex, disease type and stage, general physical condition, responsîveness to a given dosage, and type of médication), the nature ofthe pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical, veterinary, and pharmacological arts will be able to détermine a therapeutically effective amount through routine expérimentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly.

[0098] Typical dosages of an effective deoptimized modified foot and mouth virus can be in the ranges indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activîty. Thus, the actual dosage will dépend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsîveness of the relevant primary cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models, as previously described.

[0099] Kits

[0100] The present disclosure is also directed to a kit to prophylactically and therapeutically treat subjects in need of treatment for foot and mouth disease. The kit is useful for practîcîng the inventive method of eliciting a protective immune response against foot and mouth virus, reducing the likelihood of having foot and mouth virus, and treating foot and mouth virus. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including the deoptimized modîfied as described above.

loioi] The exact nature of the components configured in the inventive kit dépends on its intended purpose. For example, some embodiments are configured for the purpose of method of elîciting a protective immune response against foot and mouth virus, other embodiments are configured for the purpose of reducing the likelihood of having foot and mouth virus, and still other embodiments are configured for the purpose of treating foot and mouth virus. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animais, domestic animais, and laboratory animais.

[0102] Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to elîciting a protective immune response against foot and mouth virus, reducing the likelihood of having foot and mouth virus, and treating foot and mouth virus. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, cathéters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0103] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen températures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a stérile, contaminant-free environment. The packaging materials employed in the kit can be those customarily utîlized in vaccine thérapies. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a syringe or glass vial used to contain suitable quantities of an inventive composition containing deoptimized modîfied foot and mouth virus. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components. 101041 Having generally described this invention, the same will be better understood by reference to certain spécifie examples, which are included herein to further îllustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

EXAMPLES

[0105] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that spécifie materials are mentioned, it is merely for purposes of illustration and is not întended to 1 imit the invention. One skilled in the art may develop équivalent means or reactants without the exercise of inventive capacity and without départing from the scope of the invention.

[0106] Example 1

[0107] Cells

[0108] Porcine kidney cell lines (LF-PK and IBRS-2) were obtained from the Foreign Animal Disease Diagnostic Laboratory (FADDL), Animal, Plant, and Health Inspection Service (APHIS) at the P1ADC. Secondary porcine kidney (PK) cells were provided by the APHIS National Veterinary Service Laboratory, Ames, lowa. BHK-21 cells (baby hamster kidney cells strain 21, clone 13, ATCC CL 10), were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Ail cells were maintained as previously reported (De los Santos et al., 2009).

[0109] Viruses

[0110] FMDV A24-WT was generated from the full-length serotype A24 Cruzeiro infections clone (pA₂4-WT) as previously described (Rieder et al 2005). A dérivative of this plasmîd was constructed to contain a Nftel unique site in the 2A coding région (pA24 Cru-Nhel). Deoptimized P2, P3 or P2/P3 clones were derived by subcloning codon modified sequences desîgned using the method described by Burns et al (2006) into pA24 Cru-NAel backbone. Specifically, a 1,517 bp ΝΛβΙ/Μ/eI, a 2,001 bp M/el/Baw/fl or a 3512 bp N/7eI/B«wÆI fragments containing P2 and/or P3 modified sequences and DIVA markers in the 3B and 3Dpol régions (Uddowla et al., 2012) were used to substitute WT sequences in pA24-Nhel. cDNAs were linearized with Swal and viral RNA were derived by in vitro transcription with T7 polymerase using MEGAscript T7 kit (Ambion) purified with an RNeasy (Qiagen) kit following the manufacturer’s directions. 10-20 ug of viral RNAs were electroporated in BHK21 cells as previously described (Rieder 2005) and after 24h incubation at 37°C, cells were frozen for subséquent virus release and passage. Recovered viruses were sequenced and used for large scale préparation. Virus stocks were purified and concentrated by density gradient centrifugation in sucrose 10-50% (W/V).

[OUI] A summary of ail deoptimized virus designs including the method of deoptimization, the final % of CG dinucleotides and overall level of atténuation in vivo is depicted in Table 3.

Table 3. Deoptimized virus designs

Name	Serotype	Deoptimization method	Atténuation	%CpG
A24-WT	A24	N/A	N/A	5.73
A24-P2 de0pt3B3D	A24-DIVA	CD	++ (mouse) + (pig)	6.50
A24-P3 deOpt3B3D	A24-DIVA	CD	+ (mouse) + (pig)	7.01
A24 P2/P3 deOpt3B3D	A24-DIVA	CD	-H-H- (mouse) ++ (Pig)	7.79
A24P1 deOpt3B3D	A24-DIVA	CPB (SAVE)	-H-H- (mouse) ++ (pig)	7.44
ASIA PI deOpt3B3D	Asial-DIVA	CPB (SAVE)	-h· 1 1 i (mouse) ++ (pig)	7.44

[0112] FMDV cell infections

[0113] Cultured cell monolayers were infected with FMDV at a multiplicity of infection (moi) of 10. After 1 h adsorption at 37°C, unabsorbed virus was removed by washing the cells with a solution containing ISOmM NaCi in 20mM morpholineethanesulfonic acid (MES) pH=6.0, before adding MEM and proceeding with incubation at 37°C in 5% CO2. Infected cells were frozen at 1, 3, 6 and 24 h and virus tîters were determined after thawing by plaque assay on BHK-21 cells. WT plaques were counted at 30 hpi and P2-P3 deopt plaques were counted at 48 hpi.

[0114] The number of VP/ml was determined by qRT-PCR using primers and probe targeting a conserved and herein unmodified 3D région of the FMDV genome and standard cycling conditions (Callahan et al 2002). Cycle threshold (Ct) values were converted to RNA copies per milliliter or milligram using the équation derived from analysis of serial 10-fold dilutions of in vitro synthesized FMDV RNA of known concentration. The number pfu/ml was determined by conventional plaque assay staining at 48 hpi (Rieder et al 1993)

[0115] Anima! experiments

[0116] Animal experiments were performed in the high-containment facilities of the Plum Island Animal Disease Center, conducted in compliance with the Animal Welfare Act (AWA), the 2011 Guide for Care and Use of Laboratory Animais, 2002 PHS Policy for the Humane Care and Use of Laboratory Animais, and U.S. Government Principles for Utilization and Care of Vertebrates Animal Used in Testing, Research and Training (IRAC 1985), as well as spécifie animal protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Plum Island Animal Disease Center (USDA/APHIS/AC Certificate number:

l-F-OOOl ; Protocol 204-14R for mice and 151-13R for swine).

[0117] Mice experiment

[0118] C57BL/6 6-7-week-old female mice were purchased from Jackson Labs (Bar Harbor, ME) and were acclimated for one week. To evaluate virulence, a comparison of A24P2Deopt₃B3D, A24-P3Deopt3B3D and A24-P2/P3Deopt3B3D vs A24 WT. Groups of C57BL/6 mice (n=6) were anesthetîzed with îsofluorane (Webster Veterînary, Devens, MA) and immediately infected subcutaneously (SC) in the left rear footpad with 50-100 ul of different doses of FMDV A24-P2Deopt3B3D deopt, A24-P3Deopt3B3D, and A24-P2/P3Deopt3B3D, or reference FMDV A24. Animais were monitored for 8 days. Vîremîa was determined by plaque assay or end point dilution on BHK-21 cells and sérum samples were collected weekly to assess neutralizing antibody response.

[0119] Swine experiments

[0120] In a first experiment, 23 Yorkshire gilts (five weeks old and weighing approximately 18-23 kg each) were acclimated for 1 week and were subsequently divided in 5 groups of 4 animais each and one group of 3 animais. In the 5 groups of 4 animais each, 3 animais were inoculated intradermally in the heel bulb (IDHB) of the right hind foot with 10⁶ or 10⁷ pfu/animal of FMDV A24-P2DeoptjB3D or A24-P3Deopt3B3D or A24-P2/P3Deopt3B3D. The remainîng animal of the group was a naïve animal for évaluation of contact transmission from dîrectly inoculated animais. The 6^,h group comprised 3 animais and was inoculated with 10³ of FMDV A24 WT.

[0121] In a second experiment, 16 swine were divided in 4 groups of 4 animais each. Three groups were IDHB inoculated as described above but at lower doses of ΙΟ², 10³ or I0³ pfu/animal ofFMDV A24 P2/P3 deopt. The remainîng group was inoculated with 10³ of FMDV WT.

[0122] Following each FMDV inoculation, clinical scores were evaluated daily for 7 days by determining the number of toes presenting FMD lésions and the presence of lésions in the snout and/or mouth. The maximum score consîdered was 17, and lésions restricted to the site of inoculation were not counted. The % of lymphocytes in the white cell population from whole blood collected in EDTA was measured for the first 7 days using a Hemavet cell counter (Drew Scientific-Erba Diagnostics, Miami Lakes, FL). Samples of sérum and nasal swab were collected the day of inoculation (baseline) and daily for 7 days after inoculation.

[0123] Détection of virus in sera and nasal swabs

[0124] Mice and swine sera and swine nasal swabs were assayed for the presence of virus by plaque titration on BHK-21 cells. Virus titers were expressed as logto pfu/ml of sérum or nasal swab sécrétions. The détection level of this assay is 5 pfu/ml. In addition, FMDV RNA was detected by real-tîme RT-PCR (rRT-PCR) as previously described (Alejo et al., 2013). Cycle threshold (Ct) values were converted to RNA copies per miliiliter using the équation derived from analysis of sériai I0-fold dilutions of in vitro synthesized FMDV RNA of known concentration and expressed as the genome copy number per ml of sérum or nasal swab.

[0125] Evaluation of humoral immune response

[0126] Neutrahzing antibody titers were determined in mice or swine sera samples by endpoint titration according to the method of Karber (OIE 2012). Antibody titers were expressed as the logio value of the recîprocal of the dilution that neutralîzed 100 TCID50 in 50% of the wells.

[0127] Data analyses

[0128] Data handling, analysis and graphie représentations were performed using Prism 5.0 (GraphPad Software, San Diego, CA) or Microsoft Excel (Microsoft, Redmond, WA). Statistical signifîcance was determined using Student's t test.

[0129] Example 2

[0130] Synonymous deoptimization of P2, P3 or P2/P3 genomic régions résulte in viable FMDV

[0131] Previous studies hâve shown that sequence deoptimization ofthe Pl genomic région is tolerated by FMDV strain A12 (Diaz san Segundo et al, supra). The Pl région is variable from strain to strain, so in order to test if such a strategy would work for the highly conserved P2 and/or P3 régions of FMDV, we desîgned viral genomes in which codon usage was deoptimized by replacement with non-preferred codon. Deoptimized sequences contained 320 and/or 367 nucléotide substitutions, respectively, throughout P2 and P3 coding régions, in addition to mutations that provided négative antigenic markers in the 3B and 3Dpol viral proteins, (FIG. 1). Modified sequences were synthetically obtained from a commercial supplier and subsequently replaced into the A24 Cruzeiro FMDV infectious cDNA clone (FIG. 2A and FIG. 2B). Electroporation of in vitro synthetized RNAs ofthe modified FMDV A24 derived clones in BHK-21 cells, rendered viable viruses with a yield of approximately 10⁷ pfu/ml, similarly to the WT parent. High titer viral stocks (10⁸-lO⁹ pfu/ml) were obtained after concentration with PEG and/or purification through sucrose gradients. Repeated passage in BHK-21 cells indicated that modified viral sequences remained unchanged for at least 7 passages and no extra substitutions were detected in the original unmodified viral genome, at least as determined by consensus sequencing. In BHK-21 cells, viruses with P2 or P3 deoptimized sequences displayed a plaque morphology similar to WT, however viruses containing both, deoptimized

P2 and P3, displayed a small plaque phenotype (FIG. 2C). It is known that FMDV has a relatively low spécifie infectivity (SI) since the ratio VP/pfu is relatively high and mostly dépends on serotype and experimental purification procedures. In fact, we had previously demonstrated that deoptimîzatîon of the Pl région of FMDV Al2 reduces the SI by approximately 5-fold. Analyses of the SI of A24- P2, P3 or P2/P3 deoptimîzed virus showed no significant différences with respect to the parental WT. SI ranged from 2,580-9,900 VP/pfu for the deoptimîzed viruses while a value of 4,350 VP/pfu was obtained for the WT virus grown and purified under identical experimental conditions (FIG. 2C).

[0132] FMDVA24 with deoptimîzed P2 and/or P3 codîng régions is attenuated in primary cell cultures

[0133] The phenotype of A24-deoptimized viruses was analyzed by following kinetics of growth on cell culture. Conventional cell lines used to propagate FMDV, including BHK-21 or 1BRS-2, and primary porcine kidney (PK) or embryonic bovine kidney (EBK) cells, were infected with the different viruses and samples were frozen at different times post infection. Infected cells where then thawed, and tîters of released viruses were determined by plaque assay on BHK-21 cells. Plaques were staîned at 48 hpi to facilitate counting. As seen in FIG. 3A, by 24 hpi, the three deoptimîzed viruses reached end point titers of~ I0⁷ pfu/ml in BHK-21 cells, similarly to WT virus, although deoptimîzed A24-P2/P3DeoptjBJD grew at somewhat slower growth rate. A similar phenotype was detected în IBRS-2 cells but the end point titer of the A24-P2/P3Deopt3B3D virus was about one log lower ( 10⁶ pfu/ml) in comparison to the titers of A24-WT or A24-P2Deopt3B3D or A24-P3DeoptîBîD deoptimîzed viruses (FIG. 3B). Interestingly ail deoptimîzed viruses were attenuated in cells that posed sélective innate pressure such as primary kidney cultures derived from swine or bovines (FIG. 3C and FIG. 3D). In these cells, the yield of ail three deoptimîzed viruses was between 2-4 logs lower than the yield attaîned by WT virus. These results indicated that P2-P3 deoptimîzed FMDV were attenuated in cell culture and behaved similarly to other attenuated FMDV strains previously reported.

[0134] Synonymous deoptimîzatîon ofP2 and/or P3 codîng régions results in atténuation of FMD V in mice

[0135] We hâve previously confirmed that an FMD mouse model developed for FMDV serotype C is also an efficient tooi for FMDV serotype A and O. To examine the virulence of A24-P2- P3 deopt viruses 6-7week-old female C57BL/6 mîce were inoculated with different doses of FMDV A24-P2Deopt_3B3D, A24-P3Deopt3B3D or A24-P2/P3Deopt3B3D or FMDV A24WT. Clinical signs, survival rate and the presence of virus or viral RNA in blood were monitored for a week post infection. As expected, animais inoculated with l O⁴ pfu of WT FMDV A24 developed clinical signs, including lethargy and rough fur (data not shown), and died by 24-48 h post inoculation (FIG. 4A). In contrast, animais inoculated with deoptimized A24 viruses displayed different levels of survival depending on the virus variant and the dose. Interestingly, ail animais inoculated with A24-P2/P3Deopt3B3D, independently ofthe dose used, animais inoculated with A24-P2Deopt3B3D at I0⁶ pfu, and 80% of the animais inoculated with l0⁷pfu of A24-P2Deopt3B3D did not show clinical signs or died by one week post inoculation (FIG. 4A), while the group ofmice inoculated with A24-P3Deopt3B3Ddid not survive, although disease progression was significantly slower than inoculation with WT FMDV A24. Consîstently with the survival data, animais inoculated with A24-P2/P3Deopt3B3D virus developed the iowest viremia levels (in the order of 10⁴ pfu/ml), followed by A24-P2Deopt3B3D (10⁶ pfu/ml), A24-P3Deopt3B3D (l O⁷ pfu/ml) and A24-WT (10⁸ pfu/ml) (FIG. 4B). Ail inoculated animais inoculated with the deoptimized variants developed high levels of neutralizing antibody titers, about 2 log 10 by 7-15 dpi that were boosted after challenge with WT virus at 21 dpi (FIG. 4C). Directly correlated with the seroneutralization data, ail animais that survived the initial inoculation with the A24 deoptimized mutants were complétély protected after challenge with a léthal dose of A24 WT virus, and did not develop détectable viremia (data not shown).These results indicate that FMDV A24-P2/and/or P3 deoptimized viruses were attenuated in mice and eiicited a strong protective humoral response against challenge with WT virus.

[0136] Synonymous deoptimization ofP2 and/or P3 coding régions results in atténuation of FMD V in swine

[0137] The promising results obtained in mice prompted us to evaluate virulence of A24P2Deopt3B3D, A24-P3Deopt3B3D and A24-P2/P3Deopt3B3D in swine, FMDV natural host. Groups of three pîgs were inoculated IDHB in the rear heel bulb with 10⁶ or 10⁷ pfu/animal of A24-P2Deopt3B3D and A24-P2/P3Deopt3B3D. Given the relatively low atténuation observed in vitro and in mice for the A24-P3Deopt3B3D virus, only a group inoculated with 10⁶ pfu/animal was included. A naïve contact animal was included in each group and remain in contact for the duration of the experiment. We also included one extra group inoculated with I0⁵ pfu of A24WT virus as control.

[0138] Ail animais inoculated with lO^pfu of A24-WT virus developed clinical signs by 2-3 dpi reaching high scores (10-17 lésions) between days 3-7 post inoculation, and virus was detected in sérum and nasal sécrétion either by virus isolation or by real time PCR. (FIG. 5).

Animais inoculated with 10x more virus, 10⁶ pfu/animal, of A24-P3DeoptîB3D behaved similarly to WT (FIG. 6). However, a delay of one day în the appearance of disease was observed in animais inoculated with A24-P2Deopt3B3D virus, in which animais did not show clinical disease until day 3 when inoculated with I O⁶ pfu/pig (FIG. 7). Interestingly, animais inoculated with A24-P2/P3Deopt3B3odisplayed a signîficantly reduced severity of disease (2-7 lésions) and one animal did not develop any clinical sign (FIG. 8). Remarkably, naïve animais maintained in contact with the animais directly inoculated with l O⁶ pfu/anîmal of either one of the three deoptimized FMDV variants did not develop clinical signs of disease. Consistently, viremia was detected in ail animais that develop lésions but only in the naïve contact anima! that was cohoused with A24-P3Deopt3B3D· Virus was detected in nasal sécrétions of al! animais, inoculated or in contact, but at significantly low levels in animais that did not display lésions (FIGS. 5-8). These results indicated that deoptimization of P2 or P3 coding régions results în viruses that are attenuated in swine but with highest levels of atténuation are achieved when deoptimization of both, P2 and P3 régions, is included simultaneously in the modified strain. [0139] Analysis of spécifie neutralizing antibodies throughout the experiment showed a strong response in ail animais inoculated with 10⁶ or 10⁷ pfu of A24-P2Deopt3B3D, 10⁶ or 10⁷ pfu of A24-P3Deopt3B3D and 10⁷ pfu of A24-P2/P3Deopt3B3D, and similar to the group of pigs inoculated with 10⁵ pfu of A24 WT, consistent with slow réplication of the deoptimized virus in the animal host (FIG. 9).

[0140] Given that the highest levels of atténuation were achieved in the A24-P2/P3Deopt3B3D variant, a second animal experiment was performed to détermine the minimum infectious dose. Groups of animais were inoculated with 10², 10³ of 10⁵ of A24-P2/P3DeoptjB3D virus and one group was inoculated with 10³ pfu/animal of A24-WT as control. As seen if FIG. 10, none of the animais inoculated with A24-P2/P3Deopt3B3D virus developed clinical signs by 7 days IDHB inoculation. In contrast, ail swine inoculated with 10³ pfu/animal of A24 WT developed disease. Despite the relative low amount of inoculated A24 WT, three of the four inoculated animais of this group reached high scores (15-17 lésions. Max=l 7) by 5-7 dpc and one animal had a score of 10 lésions by day 7, resembling the kinetics observed in the first swine experiment using a dose lOx higher (10⁴ pfu/animal). A consistent lymphopenia was observed in ail animais that developed lésions. However, although none of the animais inoculated with A24-P2/P3Deopt3B3D developed vesicular lésions, one animal (#66) had détectable lymphopenia by 7dpi but no lésions had appeared by 14 dpi (data not shown). Consistently, virus or viral RNA were detected în sérum and nasal swabs of ail animais inoculated with A24WT virus, while no virus or viral RNA were detected in sérum or nasal sécrétions in ail animais inoculated with A24-P2/P3Deopt3B3D(FIG. 11), except from animal #66 that showed lymphopenia at 7dpi, as indicated above. However, live virus was not isoiated from sérum of this animal, consistent with the lack of lésions détection.

[0141] Analysis of spécifie neutralizîng antibody response throughout the experiment in these animais showed a mild response in the group of pigs inoculated with I0⁵ pfu of A245 P2/P3Deopt3B3D, while presence of neutralizîng antibodies was undetectable in the animais inoculated with lower doses (FIG. 12). As expected, animais inoculated with FMDV A24 WT developed neutralizîng antibodies starting at 7 dpi showing a slight delay as compare to animais inoculated with higher doses of WT virus (data not shown). Our data indicate that there is a dose response induction of adaptive immune response.

[0142] Example 3

[0143] Synonymous deoptimization of PI can be extended to multiple FMDV serotypes/subtypes.

[0144] We had previously demonstrated that codon pair bias deoptimizafion of Pt could be applied to FMDV serotype A (A12) rendering a viable virus attenuated in mice and swine 15 (Diaz-San Segundo et al, supra). In order to détermine whether this approach could be extended to other FMDV serotypes/subtypes, we applied the same algorithm to deoptimize the PI régions of FMDV A24 (FIG 13) and FMDV Asia 1 (FIG. 14). Synthetic A24 and Asial PI deopt sequences were procured and cloned in pA24Cru3B3D using the conveniently engineered Fsel/Nhel restriction sites (FIG. 15A and FIG. 16A) Virus was rescued in BHK-21 cells 20 showing a relatively small plaque size as compared to the respective A24 or Asia 1 wîld type viruses. (FIG. 15B and FIG. 16B). Analysis of growth kinetics suggested that both viruses were attenuated in BHK-21 and in porcine SK6 cells (FIG. 17A and FIG. 17B) suggesting that deoptimization of PI caused a delay in virus production, presumably due to an effect on virus réplication or translation (Lauring et al, Cell Host Microbe., (2012) 12:623-632.; Yu et ai., 25 2015, Molecular Cell (2015) 59:744-754).

[0145] Synonymous deoptimization of A24cru WT 3B3D PI coding région results in atténuation of FMDVin mice

[0146] To examine the întraserotype reproducibilîty of FMDV virulence in vivo, we first performed a mice experiment with A24-PI deopt3B3D FMDV. Clinical signs, survivai rate and 30 the presence of virus or viral RNA în blood were monitored for a week post infection. As expected and consistent with previous experiments (Diaz-San Segundo 2012, J Virol. 87:54475460 -), 83% of mice inoculated with l0^?pfu of FMDV A24 WT, died byi-2 dpi; one animai died at 6dpi (FIG. 18A). In contrast, ail animais inocuiated with A24 Pldeopt3B3D survived for 7 days even those inoculated with i0⁷ pfu. Interestingly, only the control animais and those inoculated with 10⁶and 10⁷ pfu developed viremia butât levels lower than those detected in the control group which had been inoculated with 10⁵ pfu of FMDV A24WT (FIG. 18B). Ail inoculated animais inoculated A24 Pldeopt3B3D developed détectable levels of neutralizing antibody with titers oscilating between 0.2 to 1.2 log 10 depending on the amount of inoculated virus (FIG. Ï8C). Highest titers were detected for the control group inoculated with A24WT virus. These results were consistent with previous results obtaîned for FMDV A12 Pl deopt (Diaz-San Segundo et al 2015. J Virol. 90:1298-1310) indicating that Pl deoptîmization of FMDV A24 results in similar atténuation in mice elîciting a détectable humoral response.

[0147] Synonymous deoptîmization of A24cru WT 3B3D Pl coding région results in atténuation of FMDVin swine (0148] The virulence of FMDV A24 Pldeopt3B3D was further evaluated in swine. Groups of 4 pigs were inoculated IDHB in the rear heel bulb with doses varying from 10³ to 10⁶ pfu/anîmal of A24-PlDeopt3B3D. Except for one, ail animais inoculated with 10³ and 10⁴ pfu/animal did not develop clinical signs. Animais inoculated with 10⁶ pfu of A24-PlDeopt3B3D virus did develop clinical signs by 2-4 dpi but with lower scores (1-10 lésions) (FIG. 19) than those observed for A24 WT virus (10-17 lésions) (FIG. 5). ïnterestingly, in the group inoculated with 10⁵ pfu, one animal did not develop lésions. Overall, low viremia was detected in some animais but not in nasal sécrétions (FIG. 20) Analysis of neutralizing antibody titers indicated that significant levels (> 2 log 10 TCID50/ml) were elîcited in the animais inoculated with the highest doses of virus (10⁵ and I0⁶ pfu) (FIG. 21).

[0149] Synonymous deoptîmization of Asîa 3B3D Pl coding région results in atténuation of FMD V în mice

[0150] Similar animal experiments were performed with Asîa PlDeopt3B3D. Groups of mice were inoculated with 10² to 10⁷ of Asial-PlDeopt3B3D virus and one group was inoculated with 5x10⁴ pfu/animal of Asia-WT as control. This dose had been earlier established as approprîate to cause 100 % léthality in C57/BL6 mice. As seen if FIG. 22, none of the animais inoculated with Asial Pl Deopt3B3D virus died by 7 dpi. In contrast, ail mice inoculated with Asial WT dîed by 1-2 dpi. None of the animais inoculated with Asial Pl Deopt3B3D, even those that received 10⁷ pfu/animal developed viremia while the control animais did. ïnterestingly, high levels of neutralizing antibodies were induced by Asial-PlDeopt3B3D, mainly at the higher doses.

[0151] Synonymous deoptîmization of Asia 3B3D Pl coding région results în atténuation of

FMDVin swine

[0152] A follow up experiment was performed in swine. Three groups of 4 animais each were inoculated wîth AsîalPlDeopt3B3D at ΙΟ³, I0⁵, and I0⁶ pfu/animal, respectively; one group was inoculated with 10⁵pfu/animal of Asial WT as control. Remarkably, eleven out of twelve AsîalPlDeopt3B3D inoculated animais, dîd not develop clinical signs (FIG. 23). One of the animais inoculated with 10⁶pfu had a low score (4 out of possible 17) about one-week post inoculation. No viremia or shedding was detected in any of the AsialPlDeoptsB3D inoculated animais (FIG. 24). In contrast, ail animais in the group inoculated with Asia I WT virus developed clinical signs includîng lésions and lymphopenia between 2 and 3 dpi and one animal died by 2 dpi. Consistently, viremia and virus shedding was only detected in animais inoculated with the Asia 1 WT virus. Only animais inoculated with the two highest doses (I0⁵ and 10⁶ pfu/animal) of Asial PI Deopt3B3D developed a neutralizing antibody response although levels were lower than those elicited by Asial WT virus. Overall, the antibody titers were lower for AsialPIDeoptîBîD than those elicited for AsialPIDeoptjB3D (FIG. 25).

[0153] Ail together these results indicate that deoptîmization of the FMDV Pl région causes consistent atténuation independently of the serotype or subtype.

[0154] While the invention has been described wîth reference to details of the illustrated embodiments, these details are not intended to limit the scope of the invention as defined in the appended claims. The embodiment of the invention in which exclusive property or privilège îs claimed is defined as follows:

Claims (14)

1. l. A deoptimized modified foot and mouth disease virus (FMDV) comprising a substituted genomic région, wherein the substituted genomic région comprises a nucleic acid at least 95% identical to SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and encodes the same polypeptide as SEQ ID NO: l, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively, or encodes the same polypeptide as SEQ ID NO: I, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, respectively, with up to I0 amino acid replacements, délétions or additions.
2. 2. The deoptimized modified FMDV of ciaim l, wherein the substituted genomic région comprises a nucleic acid at least 99% identical to SEQ ID NO: l, or wherein the substituted genomic région comprises SEQ ID NO: l.
3. 3. The deoptimized modified FMDV of claim l, wherein the substituted genomic région comprises a nucleic acid at least 99% identical to SEQ ID NO: 2, or wherein the substituted genomic région comprises SEQ ID NO: 2.
4. 4. The deoptimized modified FMDV of claim l, wherein the substituted genomic région comprises a nucleic acid at least 99% identical to SEQ ID NO: 3, or wherein the substituted genomic région comprises SEQ ID NO: 3.
5. 5. The deoptimized modified FMDV of claim l, wherein the substituted genomic région comprises a nucleic acid at least 99% identical to SEQ ID NO: 4, or wherein the substituted genomic région comprises SEQ ID NO: 4.
6. 6. The deoptimized modified FMDV of claim 1, wherein the substituted genomic région comprises a nucleic acid at least 99% identical to SEQ ID NO: 5, or wherein the substituted genomic région comprises SEQ ID NO: 5.
7. 7. The deoptimized modified FMDV of claim 1, wherein the substituted genomic région comprises a nucleic acid at least 99% identical to SEQ ID NO: 6, or wherein the substituted genomic région comprises SEQ ID NO: 6.
8. 8. The deoptimized modified FMDV of claim 1, wherein the substituted genomic région comprises a nucleic acid at least 99% identîcal to SEQ JD NO: 7, or wherein the substituted genomîc région comprises SEQ ID NO: 7.
9. 9. The deoptîmized modified FMDV of any of claims l -15, further comprising a DIVA marker, wherein the DIVA marker comprises mutations in the 3B and 3D coding régions.

   I0. A deoptîmized modified foot and mouth disease virus (FMDV) constructed by substîtuting the P2 domain, or the P3 domain with a codon deoptîmized or codon-pair deoptîmized région encoding the same protein sequence, orencoding a protein sequence with up to 10 amino acid replacements, délétions or additions, wherein the codon pair bias of the modified sequence is less than the codon pair bîas of the parent FMDV, and is reduced by 0.05, 0.1, or 0.2.
10. 11. The deoptîmized modified foot and mouth virus of claim 18, wherein the deoptîmized genomîc région is the P2 domain and wherein the deoptîmized modified virus is A24P2-3B3D deoptîmized foot and mouth virus.
11. 12. The deoptîmized modified foot and mouth virus of claim 18, wherein the deoptîmized genomîc région îs the P3 domain and wherein the deoptîmized modified virus is A24P3-3B3D deoptîmized foot and mouth virus.
12. 13. The deoptîmized modified foot and mouth virus of claim 18, wherein the deoptîmized genomîc région is the P2 domain and the P3 domain and wherein the deoptîmized modified virus is A24-P2/P3-3B3D deoptîmized foot and mouth virus.
13. 14. Use of a deoptîmized modified virus of any of claims l-24 in the manufacture of a médicament for use in a method of eliciting an immune response to foot and mouth disease in a mammalian subject.
14. 15. The use of claim 14, wherein the subject is a bovid or a suid.