Recombinant LSDV vector foot-and-mouth disease antigen constructs

Abstract

The present invention relates to a recombinant nodular dermatosis virus (LSDV) vector comprising an expression cassette encoding a foot and mouth disease virus (FMDV) polyprotein and a modified FMDV 3C protease. The invention also relates to a composition comprising the recombinant LSDV vector and an expression cassette. The invention further relates to a dual vaccine against LSDV and FMDV.

Description

Background Art

[0001] Foot-and-mouth disease (FMD) and lumpy skin disease (LSD) are two highly contagious diseases in cattle and other farm animals, causing serious economic losses. The strategy of using inactivated vaccines to control these two diseases has proven to be costly and limited in effectiveness. Attenuated live recombinant vaccines have the potential to enhance the immune response and coverage, while reducing vaccine costs and alleviating the economic burden imposed by these diseases. A dual vaccine against FMD and LSD would also bring economic benefits.

[0002] Foot-and-mouth disease (FMD) is caused by foot-and-mouth disease virus (FMDV), which belongs to the family Picornaviridae, genus Aphthoviridae, and species Foot-and-mouth disease virus. It is a highly contagious disease in cattle and other farm animals. The virus is prevalent in Africa, Asia, and South America, and the disease has caused serious economic losses to affected countries due to productivity losses and trade embargoes to control its spread. The associated FMD results in blisters in the mouth and feet of cattle, pigs, sheep, goats, and other cloven-hoofed animals. The virus is highly contagious and is a major plague in the livestock industry. Currently, strict import restrictions and sanitary control measures have been implemented to isolate the disease. These measures include culling of diseased animals and vaccination programs using inactivated vaccines; however, current chemically inactivated vaccine formulations have several limitations, including thermal instability, high cost implications, slow production speed, and limited effectiveness.

[0003] The foot-and-mouth disease virus consists of a single-stranded positive-sense RNA genome approximately 8,400 nucleotides (nt) in length. The genome is encapsulated in an icosahedral viral protein capsid approximately 25 - 30 nanometers in diameter, making it a non-enveloped virus. The capsid is composed of 60 copies of capsomers. Each capsomer is composed of four structural polypeptides, VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 are exposed on the virus surface, while VP4 is located internally ( Figure 1 ). Empty capsid particles without the RNA genome can also self-assemble, thus forming virus-like particles with high immunogenicity.

[0004] The coding region of the viral genome is approximately 7000 nucleotides in length and encodes a large polyprotein, which is subsequently cleaved by viral proteases to form four different structural proteins and eleven different non-structural proteins, as well as various precursors, some of which have unique functions. After translation, four major products are initially formed, namely Lpro, P1-2A, P2, and P3. The leader protease (Lpro) is the N-terminal component of the polyprotein. The L coding region contains two independent AUG start codons (usually separated by 84 nucleotides), resulting in the production of two different L proteins, designated La and Lb (La / b). Lpro is responsible for inhibiting the synthesis of the host cell protein eIF4G by inducing the cleavage of the host protein eIF4G, which is a translation initiation factor required for the translation of capped cellular mRNAs. The P1-2A capsid precursor is cleaved by the 3C protease (3Cpro) to generate 1AB (VP0), 1C (VP3), and 1D (VP1) (as well as 2A), and during the genome encapsidation process, VP0 is cleaved to form VP4 and VP2 (note: 2A is a very short peptide, <20 residues). VP4 is entirely located inside the virus particle, while VP1, VP2, and VP3 are exposed on the surface and are involved in the antigenic properties of the virus.

[0005] Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus in the family Poxviridae and is the causative agent of lumpy skin disease (LSD) in cattle. Lactating cows have been found to be most susceptible to the disease. During an outbreak, the incidence is usually high and can rise up to 85%. LSD in cattle can range from subclinical to acute infections and usually lasts 2-5 weeks. Acute infections are characterized by fever and the appearance of local or disseminated nodules on the skin. Lesions usually occur in the upper respiratory tract and are often accompanied by secondary bacterial infections. Nodules have been reported to appear on skeletal muscles as well as on the oral and upper respiratory mucosa. Systemic effects include fever, anorexia, lactation disorders, and pneumonia. Cows may lose their reproductive and milk-producing capabilities for several months. Fever, anorexia, and abortion are also common. The economic impact of the disease is exacerbated by the decrease in milk production in lactating cows, temporary or permanent infertility in cows and bulls, and the skin lesions caused by the virus.

[0006] The culling campaigns and trade restrictions imposed on live animals and animal products have further exacerbated the economic losses during the outbreak. In Africa, the economic losses caused by LSD are comparable to those of foot-and-mouth disease. This has led the World Organization for Animal Health (OIE) to list LSD as a notifiable agricultural disease. Although both FMD and LSD are important agricultural diseases, there is currently no dual vaccine that is effective against both diseases simultaneously. Developing a single vaccine for the control of these two viral diseases would be attractive as it could reduce costs and the number of vaccine doses, which would be beneficial for both cattle owners and vaccine manufacturers. Summary of the Invention

[0007] The present invention relates to a recombinant LSDV vector, comprising or consisting of an expression cassette encoding a foot-and-mouth disease virus (FMDV) polyprotein and a modified FMDV 3C protease. The present invention also relates to a composition containing the rLSDV vector and the expression cassette, and a dual vaccine against LSDV and FMDV.

[0008] In a first aspect of the present invention, there is provided a recombinant lumpy skin disease virus (rLSDV) vector, which comprises or consists of an expression cassette that comprises or consists of a nucleic acid encoding a foot-and-mouth disease virus (FMDV) polyprotein and a nucleic acid encoding a modified FMDV 3C protease. The FMDV polyprotein comprises or consists of FMDV structural proteins VP1, VP2, VP3, VP4 and 2A peptide.

[0009] In a first embodiment of the present invention, the rLSDV comprises or consists of a stabilized SOD homolog (SOD-is) gene. Preferably, the SOD-is gene encodes the amino acid sequence of SEQ ID NO: 57.

[0010] In a second embodiment of the present invention, the FMDV is or can be a SAT-1, SAT-2 or SAT-3 FMDV strain.

[0011] In a third embodiment of the present invention, the modified FMDV 3C protease comprises or consists of an L127P mutation.

[0012] In a fourth embodiment of the present invention, the nucleic acid encoding the FMDV polyprotein is (operably) operably linked to a regulatory sequence that enables the expression of the FMDV polyprotein. It should be understood that the nucleic acid encoding the FMDV polyprotein can be controlled by any suitable promoter to express the fusion polypeptide. Preferably, the nucleic acid encoding the FMDV polyprotein is (operably) controlled by the mH5 poxvirus promoter that enables the expression of the FMDV polyprotein.

[0013] In a fifth embodiment of the present invention, the nucleic acid encoding the FMDV 3C protease is (operably) operably linked to a regulatory sequence that enables the expression of the FMDV 3C protease. It should be understood that the nucleic acid encoding the FMDV 3C protease can be controlled by any suitable promoter to express the fusion polypeptide. Preferably, the nucleic acid encoding the FMDV 3C protease is (operably) controlled by the MVA 95 poxvirus promoter that enables the expression of the FMDV 3C protease.

[0014] In the sixth embodiment of the present invention, it should be understood that when the FMDV polyprotein and the FMDV 3C protease are expressed, the FMDV polyprotein can be proteolytically cleaved by the 3C protease into FMDV VP1, VP2, VP3, and VP4 polypeptides, as well as the foot-and-mouth disease virus 2A peptide.

[0015] In the seventh embodiment of the present invention, an expression cassette encoding the foot-and-mouth disease virus (FMDV) polyprotein and a modified FMDV 3C protease is inserted between ORF 49 and ORF 50 of the LSDV-SODis genome.

[0016] In a second aspect of the present invention, there is provided a composition comprising or consisting of an rLSDV vector, wherein the rLSDV vector comprises or consists of an expression cassette, and the expression cassette comprises or consists of a nucleic acid encoding the foot-and-mouth disease virus (FMDV) polyprotein and a nucleic acid encoding a modified FMDV 3C protease. Those skilled in the art should understand that the FMDV polyprotein comprises or consists of the FMDV structural proteins VP1, VP2, VP3, VP4, and the FMDV 2A peptide.

[0017] In a first embodiment of the second aspect of the present invention, the rLSDV vector comprises or consists of a stabilized SOD homolog (SOD-is) gene. Preferably, the stabilized SOD-is gene encodes the amino acid sequence of SEQ ID NO: 57.

[0018] In a second embodiment of the second aspect of the present invention, the FMDV can be the SAT-1, SAT-2, or SAT-3 FMDV strain.

[0019] In a third embodiment of the second aspect of the present invention, the modified FMDV 3C protease comprises or consists of the L127P mutation.

[0020] In a fourth embodiment of the second aspect of the present invention, the nucleic acid encoding the FMDV polyprotein can be operably linked to a regulatory sequence that enables the expression of the FMDV polyprotein. It should be understood that the nucleic acid encoding the FMDV polyprotein can be controlled by the mH5 poxvirus promoter that enables the expression of the FMDV polyprotein.

[0021] In a fifth embodiment of the second aspect of the present invention, the nucleic acid encoding the FMDV 3C protease can be operably linked to a regulatory sequence that enables the expression of the FMDV 3C protease. It should be understood that the nucleic acid encoding the FMDV 3C protease can be controlled by the MVA 95 poxvirus promoter that enables the expression of the 3C protease.

[0022] In the sixth embodiment of the second aspect of the present invention, when the FMDV polyprotein and the FMDV 3C protease are expressed, the FMDV polyprotein can be proteolytically cleaved by the 3C protease into VP1, VP2, VP3, and VP4 polypeptides, as well as the FMDV 2A peptide.

[0023] In the seventh embodiment of the second aspect of the present invention, an expression cassette encoding the foot-and-mouth disease virus (FMDV) polyprotein and a modified FMDV 3C protease is inserted between ORF 49 and ORF 50 of the LSDV-SODis genome.

[0024] In the third aspect of the present invention, there is provided a vaccine comprising or consisting of the rLSDV vector described herein, or the composition described herein, and a pharmaceutically acceptable carrier or adjuvant.

[0025] In the fourth aspect of the present invention, there is provided the rLSDV vector, composition or vaccine described herein for use in a method of inducing an immune response against foot-and-mouth disease in a subject. Wherein, the method comprises or consists of the step of administering to the subject an immunologically effective amount of the vector, composition or vaccine.

[0026] In the first embodiment of the fourth aspect of the present invention, the rLSDV vector, composition or vaccine additionally induces an immunologically effective response in the subject against foot-and-mouth disease virus and / or LSDV.

[0027] In the second embodiment of the fourth aspect of the present invention, it should be understood that the subject is or (may be) selected from cattle, pigs, sheep, goats, deer, antelopes, giraffes, camels, and buffaloes.

[0028] In the fifth aspect of the present invention, there is provided a method of inducing an immune response against foot-and-mouth disease in a subject, the method comprising or consisting of administering to the subject an immunologically effective amount of the rLSDV vector, composition or vaccine described herein.

[0029] In the first embodiment of the fifth aspect of the present invention, the rLSDV vector, composition or vaccine additionally induces an immunologically effective response in the subject against foot-and-mouth disease virus and / or LSDV.

[0030] In the second embodiment of the fifth aspect of the present invention, it should be understood that the subject is or (may be) selected from cattle, pigs, sheep, goats, deer, antelopes, giraffes, camels, and buffaloes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following non-limiting embodiments of the present invention will be described only by way of example and with reference to the accompanying drawings:

[0032] Figure 1: Genome structure of FMDV and virus structure. The FMDV genome consists of a single open reading frame (ORF) encoding a large polyprotein, as shown by the shaded rectangle. Regions within the rectangle denote the respective proteins. The 5'UTR contains multiple distinct structural elements, including: poly(C) tracts (Cn), three or four pseudoknot structures (PK), and an internal ribosome entry site (IRES). The VPg peptide is covalently linked to the RNA genome. The figure shows the process of assembling virus particles from protomer subunits and pentamer subunits. The assembled virus particles contain a single copy of the viral RNA and 60 copies of four different capsid proteins (VP1-VP4). The image is taken from (Jamal and Belsham (2013).

[0033] Figure 2 : Schematic diagram of the expression cassette in the transfer vector pFMDV:SAT2-Red for recombinant LSDV construction. The P1 gene (SEQ ID NO:1, SEQ ID NO:11, SEQ ID NO:21 or SEQ ID NO:31), 2A gene (SEQ ID NO:42), 3C protease gene (SEQ ID NO:45), and mCherry gene (SEQ ID NO:48) are controlled by the poxvirus promoters mH5 (SEQID NO:41), MVA 95 (SEQ ID NO:44), and mFP (SEQ ID NO:47), respectively. The target gene is flanked by regions of the LSDV open reading frames ORF 49 and ORF 50. The relevant restriction enzyme sites are marked in the figure. The L127P mutation used to reduce the cytotoxicity of the 3C protease has been marked in the figure.

[0034] Figure 3 : A) Lamb testis cells at passage 0 were infected with LSDV (SODis) BEFV-Gb (green) and transfected with pFMDV:SAT2-Red (red). B) BHK cells at passage 1 (P1) were infected with the P0 cell lysate to screen for red foci. C) Foci at passage 2 (P2) were picked from P1 and further passaged in BHK cells. D) Red foci at passage 2 (P2) were picked for passage 3 (P3), serially diluted in 96-well plates and passaged to screen for wells containing only single red foci and no green foci.

[0035] Figure 4 : Comparison of the recombinant LSDV (SODis) FMDV:SAT2 vaccine candidate gene sequence with the parental nLSDV SODis-UCT at the L49-R50 gene locus.

[0036] Figure 5: PCR amplification results of the region between open reading frames 49 and 50 of LSDV. DNA was extracted from 5 single red lesions (S1 - 5) of LSDV(SODis)FMDV - SAT2; -ve = H2O.

[0037] Figure 6 : Western blot analysis of MDBK cell lysates infected with the LSDV(SODis)FMDV - SAT2 recombinant vaccine candidate or the parental virus LSDV(SODis)BEFV - Gb. The primary antibody used was rabbit anti - FMDV SAT2 serum, and the secondary antibody was goat anti - rabbit IgG conjugated to alkaline phosphatase. White arrows indicate the positions of VP1, VP2, and VP3 proteins.

[0038] Figure 7 : Transmission electron microscopy observations: A) Uninfected MDBK cell culture medium; B) MDBK cell culture medium 6 days after infection with LSDV(SODis)FMDV:SAT2.

[0039] Figure 8 : Codon - optimized nucleic acid sequence encoding the FMDV SAT - 2 (Kenya strain) P1 polypeptide (SEQ ID NO:1).

[0040] Figure 9 : Amino acid sequence of the FMDV SAT - 2 (Kenya strain) P1 polypeptide (SEQ ID NO:2).

[0041] Figure 10 : Codon - optimized nucleic acid sequence encoding the FMDV SAT - 1 (Kruger strain, KNP) P1 polypeptide (SEQ ID NO:11).

[0042] Figure 11 : Amino acid sequence of the FMDV SAT - 1 (Kruger strain, KNP) P1 polypeptide (SEQ ID NO:12).

[0043] Figure 12 : Codon - optimized nucleic acid sequence encoding the FMDV SAT - 2 (Kruger strain, KNP) P1 polypeptide (SEQ ID NO:21).

[0044] Figure 13 : Amino acid sequence of the FMDV SAT - 2 (Kruger strain, KNP) P1 polypeptide (SEQ ID NO:22).

[0045] Figure 14 : Codon - optimized nucleic acid sequence encoding the FMDV SAT - 3 (Kruger strain, KNP) P1 polypeptide (SEQ ID NO:31).

[0046] Figure 15: Amino acid sequence of the P1 polypeptide of FMDV SAT-3 (Kruger strain, KNP) (SEQ ID NO: 32).

[0047] Figure 16 : Codon-optimized nucleic acid sequence of the 2A peptide used in all constructs (SEQ ID NO: 42).

[0048] Figure 17 : Amino acid sequence of the 2A peptide used in all constructs (SEQ ID NO: 43).

[0049] Figure 18 : Nucleic acid sequence encoding the modified FMDV 3C protease used in all constructs (SEQ ID NO: 45).

[0050] Figure 19 : Amino acid sequence of the modified FMDV 3C protease (SEQ ID NO: 46).

[0051] Figure 20 : Full-length codon-optimized nucleic acid expression cassette encoding the P1-2A fragment and modified 3C protease of FMDV SAT-2 (Kenya strain) (SEQ ID NO: 50).

[0052] Figure 21 : Full-length codon-optimized nucleic acid expression cassette inserted into the recombinant LSDV vector (SEQ ID NO: 51).

[0053] Figure 22 : Full-length codon-optimized nucleic acid expression cassette inserted into the recombinant LSDV vector (SEQ ID NO: 52).

[0054] Figure 23 : Full-length codon-optimized nucleic acid inserted into the recombinant LSDV vector (SEQ ID NO: 53).

[0055] Figure 24 : Codon-optimized nucleic acid sequence of the P1-2A polyprotein of FMDV (SAT-2, Kenya isolate SAT2 / KEN / K137 / 2014) (SEQ ID NO: 54).

[0056] Figure 25 : Amino acid sequence of the P1-2A polyprotein of FMDV (SAT-2, Kenya isolate SAT2 / KEN / K137 / 2014) (SEQ ID NO: 55).

[0057] Sequence Listing

[0058] The nucleic acid and amino acid sequences listed in this Sequence Listing are represented by the standard letter abbreviations of nucleotide bases and the standard three-letter abbreviations of amino acids. Those skilled in the art should understand that although the listed nucleic acid sequences show only a single strand, any reference to the shown strand includes its complementary strand. In the accompanying Sequence Listing:

[0059] SEQ ID NO:1 - Codon-optimized nucleic acid sequence of FMDV SAT-2 (Kenya strain) P1 polypeptide.

[0060] SEQ ID NO:2 - Amino acid sequence of FMDV SAT-2 (Kenya strain) P1 polypeptide.

[0061] SEQ ID NO:3 - Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kenya strain) VP1 polypeptide.

[0062] SEQ ID NO:4 - Amino acid sequence of FMDV SAT-2 (Kenya strain) VP1 polypeptide.

[0063] SEQ ID NO:5 - Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kenya strain) VP2 polypeptide.

[0064] SEQ ID NO:6 - Amino acid sequence of FMDV SAT-2 (Kenya strain) VP2 polypeptide.

[0065] SEQ ID NO:7 - Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kenya strain) VP3 polypeptide.

[0066] SEQ ID NO:8 - Amino acid sequence of FMDV SAT-2 (Kenya strain) VP3 polypeptide.

[0067] SEQ ID NO:9 - Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kenya strain) VP4 polypeptide.

[0068] SEQ ID NO:10 - Amino acid sequence of FMDV SAT-2 (Kenya strain) VP4 polypeptide.

[0069] SEQ ID NO:11 - Codon-optimized nucleic acid sequence of FMDV SAT-1 (Kruger strain) P1 polypeptide.

[0070] SEQ ID NO:12 - Amino acid sequence of FMDV SAT-1 (Kruger strain) P1 polypeptide.

[0071] SEQ ID NO:13 - Codon-optimized nucleic acid sequence encoding FMDV SAT-1 (Kruger strain) VP1 polypeptide.

[0072] Amino acid sequence of SEQ ID NO:14 - FMDV SAT-1 (Kruger strain) VP1 polypeptide.

[0073] Codon-optimized nucleic acid sequence encoding FMDV SAT-1 (Kruger strain) VP2 polypeptide of SEQ ID NO:15.

[0074] Amino acid sequence of SEQ ID NO:16 - FMDV SAT-1 (Kruger strain) VP2 polypeptide.

[0075] Codon-optimized nucleic acid sequence encoding FMDV SAT-1 (Kruger strain) VP3 polypeptide of SEQ ID NO:17.

[0076] Amino acid sequence of SEQ ID NO:18 - FMDV SAT-1 (Kruger strain) VP3 polypeptide.

[0077] Codon-optimized nucleic acid sequence encoding FMDV SAT-1 (Kruger strain) VP4 polypeptide of SEQ ID NO:19.

[0078] Amino acid sequence of SEQ ID NO:20 - FMDV SAT-1 (Kruger strain) VP4 polypeptide.

[0079] Codon-optimized nucleic acid sequence of SEQ ID NO:21 - FMDV SAT-2 (Kruger strain) P1 polypeptide.

[0080] Amino acid sequence of SEQ ID NO:22 - FMDV SAT-2 (Kruger strain) P1 polypeptide.

[0081] Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kruger strain) VP1 polypeptide of SEQ ID NO:23.

[0082] Amino acid sequence of SEQ ID NO:24 - FMDV SAT-2 (Kruger strain) VP1 polypeptide.

[0083] Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kruger strain) VP2 polypeptide of SEQ ID NO:25.

[0084] Amino acid sequence of SEQ ID NO:26 - FMDV SAT-2 (Kruger strain) VP2 polypeptide.

[0085] Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kruger strain) VP3 polypeptide of SEQ ID NO:27.

[0086] Amino acid sequence of SEQ ID NO:28 - FMDV SAT-2 (Kruger strain) VP3 polypeptide.

[0087] SEQ ID NO:29 - Codon-optimized nucleic acid sequence encoding FMDV SAT-2 (Kruger strain) VP4 polypeptide.

[0088] Amino acid sequence of SEQ ID NO:30 - FMDV SAT-2 (Kruger strain) VP4 polypeptide.

[0089] SEQ ID NO:31 - Codon-optimized nucleic acid sequence encoding FMDV SAT-3 (Kruger strain) P1 polypeptide.

[0090] Amino acid sequence of SEQ ID NO:32 - FMDV SAT-3 (Kruger strain) P1 polypeptide.

[0091] SEQ ID NO:33 - Codon-optimized nucleic acid sequence encoding FMDV SAT-3 (Kruger strain) VP1 polypeptide.

[0092] Amino acid sequence of SEQ ID NO:34 - FMDV SAT-3 (Kruger strain) VP1 polypeptide.

[0093] SEQ ID NO:35 - Codon-optimized nucleic acid sequence encoding FMDV SAT-3 (Kruger strain) VP2 polypeptide.

[0094] Amino acid sequence of SEQ ID NO:36 - FMDV SAT-3 (Kruger strain) VP2 polypeptide.

[0095] SEQ ID NO:37 - Codon-optimized nucleic acid sequence encoding FMDV SAT-3 (Kruger strain) VP3 polypeptide.

[0096] Amino acid sequence of SEQ ID NO:38 - FMDV SAT-3 (Kruger strain) VP3 polypeptide.

[0097] SEQ ID NO:39 - Codon-optimized nucleic acid sequence encoding FMDV SAT-3 (Kruger strain) VP4 polypeptide.

[0098] Amino acid sequence of SEQ ID NO:40 - FMDV SAT-3 (Kruger strain) VP4 polypeptide.

[0099] SEQ ID NO:41 - Nucleic acid sequence of mH5 promoter.

[0100] SEQ ID NO:42 - Codon-optimized nucleic acid sequence of 2A peptide used in all constructs.

[0101] Amino acid sequence of SEQ ID NO:43 - 2A peptide.

[0102] Nucleic acid sequence of SEQ ID NO:44 - MVA095 promoter.

[0103] Nucleic acid sequence of SEQ ID NO:45 - modified 3C protease used in all constructs.

[0104] Amino acid sequence of SEQ ID NO:46 - modified 3C protease.

[0105] Nucleic acid sequence of SEQ ID NO:47 - mFP promoter.

[0106] Nucleic acid sequence encoding mCherry polypeptide of SEQ ID NO:48.

[0107] Amino acid sequence of SEQ ID NO:49 - mCherry polypeptide.

[0108] Full - length nucleic acid sequence of the SAT - 2 (Kenya strain) gene expression cassette inserted into the recombinant LSDV vector of SEQ ID NO:50.

[0109] Full - length nucleic acid sequence of the SAT - 1 (Kruger strain) gene expression cassette inserted into the recombinant LSDV vector of SEQ ID NO:51.

[0110] Full - length nucleic acid sequence of the SAT - 2 (Kruger strain) gene expression cassette inserted into the recombinant LSDV vector of SEQ ID NO:52.

[0111] Full - length nucleic acid sequence of the SAT - 3 (Kruger strain) gene expression cassette inserted into the recombinant LSDV vector of SEQ ID NO:53.

[0112] Codon - optimized nucleic acid sequence of the SAT - 2 (Kenya isolate SAT2 / KEN / K137 / 2014) P1 - 2A fragment of SEQ ID NO:54.

[0113] Amino acid sequence of the SAT - 2 (Kenya isolate SAT2 / KEN / K137 / 2014) P1 - 2A fragment of SEQ ID NO:55. Detailed implementation manners

[0114] The present invention will be described more fully hereinafter with reference to the accompanying drawings, which show some (but not all) embodiments of the present invention.

[0115] The described invention should not be limited to the specific embodiments disclosed, and various modifications and other embodiments should also be included within the scope of the invention. Although specific terms are used herein, these terms are used only in a general and descriptive sense and not for purposes of limitation.

[0116] In this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include the plural forms.

[0117] The terms and expressions used herein are for descriptive purposes only and should not be regarded as restrictive. The use of the terms "comprising", "including", "having", and "containing" and their variants in this document is intended to cover the items listed hereinafter and their equivalents as well as other additional items.

[0118] The inventors modified, optimized, and developed the FMDV SAT-2 strain sequence previously reported from Kenya (Palinski et al., 2019). First, after downloading the wild-type DNA sequence, the inventors developed an FMDV SAT-2 expression cassette and integrated it into a transfer vector to generate a recombinant LSDV capable of expressing genes encoding FMDV structural proteins and a stabilized 3C protease.

[0119] Although there are existing methods for constructing recombinant LSDV in the prior art, the difference of the present invention lies in the site selection for inserting the FMDV expression cassette into the LSDV SODis genome - this insertion site is within the gene between LSDV ORF 49 and ORF 50 and does not cause inactivation of any LSDV gene function. Other recombinant LSDV candidate vaccines usually use the thymidine kinase gene or the ribonucleotide reductase gene as the foreign gene insertion site. Inserting a foreign gene at such sites will lead to further attenuation of the LSDV strain.

[0120] The term "LSDV vector" used herein refers to the LSDV vector backbone containing the stabilized SOD homologous gene, as described in International Publication No. WO 2019 / 220403.

[0121] "Protein", "peptide", or "polypeptide" refers to a chain composed of two or more amino acids, including natural or unnatural amino acids and amino acid analogs, and is not restricted by post-translational modifications such as glycosylation or phosphorylation.

[0122] "Antigen" refers to a compound, composition, or substance that can stimulate the production of antibodies and / or a CD4+ or CD8+ T cell response in an animal, including a composition injected or absorbed into the animal. An antigen can react with specific humoral or cellular immune products, including immune products induced by a heterologous immunogen.

[0123] The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" as used herein are used interchangeably and encompass ribonucleotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA and synthetic DNA. The nucleic acid can be double-stranded or single-stranded. When the nucleic acid is single-stranded, it can be the sense strand or the antisense strand. A nucleic acid molecule can be any chain composed of two or more nucleotides linked by covalent bonds, including natural or unnatural nucleotides, nucleotide analogs or derivatives. The term "RNA" refers to a sequence composed of two or more natural or modified ribonucleotides linked by covalent bonds. The term "DNA" refers to a sequence composed of two or more natural or modified deoxyribonucleotides linked by covalent bonds. The term "DNA" refers to a sequence composed of two or more deoxyribonucleotides that are naturally occurring or modified and linked by covalent bonds. The term "cDNA" refers to complementary DNA or copy DNA produced using RNA as a template by the action of RNA-dependent DNA polymerase (reverse transcriptase).

[0124] The term "isolated" as used herein means that it has been removed from its natural environment.

[0125] The term "purified" means that a molecule or compound is isolated in a form substantially free of contaminants or impurities. These contaminants are usually associated with the molecule or compound in its natural environment, so "purified" means that the purity is increased by separation from other components of the original composition. The term "purified nucleic acid" describes a nucleic acid sequence that has been separated from other compounds, including but not limited to polypeptides, lipids and carbohydrates that are usually associated with it in its natural state.

[0126] The term "complementary" means that two nucleic acid molecules (such as DNA or RNA) are capable of forming a double-stranded region between the two nucleic acid molecules through Watson-Crick base pairing. Those skilled in the art should understand that not every nucleotide in a nucleic acid molecule needs to form a matching Watson-Crick base pair with a nucleotide on the opposite complementary strand to form a duplex. A nucleic acid molecule is considered "complementary" to a second nucleic acid molecule if it can hybridize to the second nucleic acid molecule under high stringency conditions. The nucleic acid molecules according to the present invention include both of these complementary molecules.

[0127] As used herein, a "substantially identical" sequence refers to an amino acid or nucleotide sequence that differs from a reference sequence only in the following respects: one or more conservative substitutions; one or more non-conservative substitutions, deletions or insertions, provided that these changes are at sequence positions that do not disrupt or significantly reduce the antigenicity of one or more polypeptides expressed or of one or more polypeptides encoded by a nucleic acid molecule. Sequence alignments for determining the percentage sequence identity can be achieved in a variety of ways well known to those skilled in the art. These include using computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software, etc. Those skilled in the art can readily determine the appropriate parameters for measuring the alignment, including all algorithms required to achieve the maximum alignment of the full length of the sequences being compared. In one embodiment of the present invention, the provided polypeptide or polynucleotide sequence has at least about 80% sequence identity, at least about 90% sequence identity, or even higher sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the sequences described herein.

[0128] Alternatively, or additionally, two nucleic acid sequences may be considered "substantially identical" if they are capable of hybridizing under high stringency conditions. The "stringency" of a hybridization reaction can be readily determined by those of ordinary skill in the art, typically by empirical calculations based on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures to achieve correct annealing, while shorter probes require lower temperatures. The hybridization process typically depends on the ability of denatured DNA to re-anneal in an environment below its melting temperature in the presence of complementary strands. Typical examples of such "stringent" hybridization conditions are: hybridization with gentle shaking at 65 °C for 18 hours, followed by a first wash with wash buffer A (0.5% SDS; 2×SSC) at 65 °C for 12 minutes, and then a second wash with wash buffer B (0.1% SDS; 0.5×SSC) at 65 °C for 10 minutes.

[0129] Those skilled in the art will appreciate that polypeptides, peptides or peptide analogs can be synthesized using standard chemical techniques, such as by automated synthesis using liquid or solid phase synthesis methods. Automated peptide synthesizers are commercially available and employ techniques known in the art. Polypeptides, peptides and peptide analogs can also be prepared from their corresponding nucleic acid molecules by recombinant DNA technology.

[0130] As used herein, the term "gene" refers to a nucleic acid encoding a functional product (such as RNA, polypeptide or protein). A gene may contain regulatory sequences located upstream or downstream of the sequence encoding the functional product.

[0131] As used herein, the term "coding sequence" refers to a nucleic acid sequence that encodes a specific amino acid sequence. On the other hand, a "regulatory sequence" refers to a nucleotide sequence located upstream, downstream, or within a coding sequence. Typically, regulatory sequences affect the transcription, RNA processing, or stability of the associated coding sequence, or the translation process. Regulatory sequences include, but are not limited to: effector binding sites, enhancers, introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures, and translation leader sequences.

[0132] In certain embodiments, the genes used in the methods of the present invention can be operably linked to other sequences. The term "operably linked" means that a nucleic acid molecule encoding a recombinant polypeptide of the present invention is linked to a regulatory sequence in such a way that expression of the protein is permitted when an appropriate molecule binds to the regulatory sequence. Such operably linked sequences can be contained in a vector or an expression construct, which can be transformed or transfected into a host cell for expression. It should be understood that any vector can be used for the purpose of expressing the recombinant antigen polypeptide of the present invention.

[0133] The term "promoter" refers to a DNA sequence capable of controlling the expression of a nucleic acid coding sequence or a functional RNA. A promoter can be based entirely on a native gene or can consist of elements from different promoters in nature. Different promoters are capable of directing gene expression in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. A "constitutive promoter" refers to a promoter that directs the expression of a gene of interest in most host cell types in most cases.

[0134] The term "recombinant" refers to a substance that has been recombined. When used in reference to a nucleic acid construct, the term denotes a molecule prepared or ligated by molecular biology techniques from nucleic acid sequences. When referring to a protein or polypeptide, "recombinant" denotes a protein or polypeptide molecule produced by the expression of a recombinant nucleic acid constructed by molecular biology techniques. A recombinant nucleic acid construct can contain sequences that are ligated or otherwise manipulated to be linked to nucleic acid sequences that are not linked in their natural state, or are linked to nucleic acid sequences at different positions in their natural state. Thus, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated by genetic engineering (i.e., human intervention). A recombinant nucleic acid construct can be introduced into a host cell by transformation. Such a recombinant nucleic acid construct can contain sequences from the same host cell species or different host cell species.

[0135] As used herein, the term "chimeric" means that a sequence consists of sequences that have been "recombined". Exemplarily, these sequences have been recombined and do not exist in the same molecule in their natural state. The term "recombinant" refers to any method of joining two or more polynucleotides, and the term includes end-to-end joining as well as the operation of inserting one sequence into another. The term encompasses physical joining techniques such as sticky-end ligation and blunt-end ligation. Sequences can also be obtained recombinantly by synthetic means. In addition, the term also includes integrating one sequence into another by, for example, homologous recombination.

[0136] The term "vector" refers to a tool for introducing a polynucleotide or gene sequence into a cell. There are various types of vectors known in the art, including plasmids, viruses, bacteriophages, and cosmids, etc. Generally, a polynucleotide or gene sequence is inserted into a vector in the form of an expression cassette. The term "expression cassette" refers to one or more gene sequences inserted into a vector. In certain embodiments, the expression cassette provides regulatory elements for expressing the polynucleotide or gene sequence. In other embodiments, the vector provides the regulatory sequences required for expressing the polypeptide of the present invention. In still other embodiments, the vector provides some of the regulatory sequences, while the nucleotide or gene sequence provides other regulatory sequences. "Regulatory sequences" include, but are not limited to: promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, polyadenylation (poly(A)) tailing sequences, and / or replication origins.

[0137] The expression cassette of the present invention contains the following genes controlled by poxvirus promoters: the FMDV P1-2A (FMDV polyprotein) gene, the FMDV 3C protease gene, and the mCherry reporter gene, which are controlled by the mH5, MVA95, and mFP promoters, respectively. These target genes are flanked by the LSDV ORF 49 and ORF 50 regions. To reduce the cytotoxicity of the 3C protease, the L127P mutation has been introduced into the 3C protease.

[0138] Those skilled in the art should understand that the gene sequence can be derived from the SAT-1, SAT-2, or SAT-3 strains of FMDV.

[0139] It should also be understood that the 3C protease cleaves the FMDV polyprotein into its individual components, namely the FMDV VP1, VP2, VP3, and VP4 polypeptides, and the FMDV 2A peptide.

[0140] The recombinant LSDV vector or composition encoding the recombinant FMDV expression cassette of the present invention can be provided alone or used in combination with other compounds (such as nucleic acid molecules, small molecules, polypeptides or peptide analogs), and can include liposomes, adjuvants or any carrier (such as a pharmaceutically acceptable carrier), and its dosage form is suitable for administration to mammals (such as humans, cattle, sheep, etc.). Preferably, the recombinant FMDV SAT-2 expression cassette is carried by the LSDV vector.

[0141] In one embodiment, the LSDV vector containing the recombinant FMDV expression cassette is co-formulated with an adjuvant for immunization. The adjuvants well-known to those skilled in the field of vaccine development are not limited to the types specifically exemplified herein.

[0142] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes all physiologically compatible antibacterial agents, antifungal agents, coating materials, dispersion media, solvents, isotonic agents, absorption delaying agents, etc. The "pharmaceutically acceptable carrier" can include solid or liquid fillers, diluents or encapsulating substances that can be safely used to administer the recombinant antigen or vaccine composition to a subject. The pharmaceutically acceptable carrier is suitable for intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, oral or sublingual administration. The pharmaceutically acceptable carrier includes sterile aqueous solutions, dispersions and sterile powders for formulating sterile solutions. The uses of media and reagents well-known in the art for preparing pharmaceutically active substances are known. If any conventional medium or reagent is incompatible with the active ingredient, it is not considered for use in the pharmaceutical composition of the present invention. In addition, supplementary active ingredients can also be incorporated into the composition.

[0143] Preparations or compositions containing the LSDV vector with the recombinant FMDV expression cassette suitable for administration to subjects suffering from foot-and-mouth disease or lumpy skin disease or to subjects in the pre-symptomatic stage of conditions related to foot-and-mouth disease or lumpy skin disease also fall within the scope of the present invention. Any suitable route of administration can be employed, such as parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, topical or oral administration. It should be understood that the compositions of the present invention can include the LSDV vector containing the recombinant FMDV expression cassette.

[0144] For vaccine formulations and pharmaceutical compositions, an effective amount of a composition comprising a recombinant FMDV expression cassette can be provided, which can be used alone or in combination with other compounds, i.e., in combination with an immunoadjuvant (such as aluminum hydroxide, dimethyldioctadecylammonium hydroxide, or incomplete Freund's adjuvant). The LSDV vector comprising the recombinant FMDV expression cassette can also be conjugated with a suitable carrier and / or other molecules (such as bovine serum albumin or keyhole limpet hemocyanin) to enhance immunogenicity. The vaccine formulations and compositions useful in the present invention comprise an LSDV vector containing a recombinant FMDV expression cassette, which is capable of initiating and / or enhancing an immune response against FMD and / or LSDV.

[0145] In one embodiment, the recombinant FMDV expression cassette is capable of "initiating" an immune response against foot-and-mouth disease, and / or the LSDV vector is capable of producing polypeptides and / or virus-like particles (VLPs) to "initiate" an immune response against lumpy skin disease. Examples of such initiating compositions include the compositions and / or virus-like particles of the present invention.

[0146] It should also be understood that a "booster" composition can include an LSDV vector comprising a recombinant FMDV expression cassette. The booster composition can include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten subsequent administrations of the LSDV vector containing the recombinant FMDV expression cassette.

[0147] In some embodiments, the LSDV vector containing the recombinant FMDV expression cassette of the present invention can be provided in the form of a kit, optionally together with a carrier and / or an adjuvant, and accompanied by instructions for use.

[0148] An "effective amount" of the LSDV vector containing the recombinant FMDV expression cassette includes a therapeutically effective amount, an immunologically effective amount, or a prophylactically effective amount. A "therapeutically effective amount" refers to an effective amount that can achieve the desired therapeutic effect (such as treating an infection or a condition associated with the infection) at a necessary dose and administration period. The therapeutic effect can be evaluated by the following indicators: reduction of viremia, inhibition of viral gene expression, delay of the pathological process associated with foot-and-mouth disease and / or lumpy skin disease infection, activation of the immune system, or any other detection method that can determine the therapeutic benefit. The therapeutically effective amount of a compound can vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compound to elicit the desired response in the individual. The dosage regimen can be adjusted to obtain the optimal therapeutic effect. The therapeutically effective amount also needs to ensure that the therapeutic beneficial effect of the compound exceeds any toxic or harmful effects.

[0149] The dosage of the LSDV vector containing the recombinant FMDV expression cassette or the composition of the present invention will vary depending on the symptoms, age and weight of the subject, the nature and severity of the disease to be treated or prevented, the route of administration, and the form of the composition. Any composition of the present invention can be administered as a single dose or multiple doses. The dosage of the composition of the present invention can be easily determined by techniques known to those skilled in the art or by the methods described herein.

[0150] "Immunologically effective amount" refers to an effective amount that can achieve the desired immune response at the necessary dosage and administration period. The desired immune response may include stimulating or eliciting an immune response, such as a T cell response.

[0151] "Prophylactically effective amount" refers to an effective amount that can achieve the desired prophylactic effect (such as preventing the occurrence of diseases associated with foot-and-mouth disease and / or lumpy skin disease infection) at the necessary dosage and administration period. Generally, prophylactic doses are used in subjects before the onset of the disease or in the early stages of the disease, so the prophylactically effective amount may be less than the therapeutically effective amount.

[0152] The dosage values of the LSDV vector containing the recombinant FMDV expression cassette of the present invention can vary and can be adjusted over time according to individual needs and the judgment of the person administering or supervising the administration. The dosage ranges described herein are merely exemplary and do not limit the dosage ranges that can be selected. The amount of the active compound in the composition can vary depending on factors such as the disease state, individual age, sex and weight. The dosage regimen can be adjusted to obtain the best therapeutic effect. For example, a single dose can be administered, or multiple doses can be administered over time. It may be advantageous to formulate the composition in unit dosage form to facilitate administration and ensure dose uniformity.

[0153] The vaccination regimens defined herein for eliciting an immune response against foot-and-mouth disease and / or lumpy skin disease in a subject generally comprise a series of single doses of the antigens or compositions described herein. As used herein, a single dose or dose refers, respectively, to a priming dose (i.e., the initial first or second dose using the same antigen) and any subsequent dose, preferably administered such that these doses “boost” the immune response. In this context, each single dose comprises the administration of one antigen or composition of the invention, wherein the interval between the administrations of two single doses can vary, being at least one week apart, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks apart. Most preferably, the antigens or compositions of the invention are administered at intervals of 4 weeks or 8 weeks. It should be understood that the interval between single doses can be constant or can vary during the course of the immunization regimen, e.g., the interval can be shorter at the start (such as 4 weeks apart) and longer during the later part of the regimen (such as 8 weeks apart). Additionally, depending on the total number of single doses and the interval between single doses, the immunization regimen can last for a period of time, preferably at least one week, more preferably for several weeks, and even more preferably for several months (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months). Each single dose comprises the administration of one LSDV vector comprising a recombinant FMDV expression cassette as described herein.

[0154] When used in the context of an infectious disease or other medical disease or condition, the term “prevention” is well known in the art and includes the administration of a composition that, relative to a subject who has not received the composition, reduces the frequency of occurrence of symptoms or delays the onset of symptoms in the subject. Prevention of a disease includes, for example, reducing the number of diagnoses of the infection in the treated population compared to an untreated control population; and / or delaying the onset of symptoms of the infection in the treated population compared to an untreated control population.

[0155] “Prophylactic or therapeutic” treatment, well known to those of skill in the art, includes the administration of one or more compositions of the invention to a subject. The treatment is prophylactic if the composition is administered before the manifestation of clinical symptoms of an adverse condition (such as a disease or other adverse state of the subject), i.e., it protects the host from developing the adverse condition; while the treatment is therapeutic if the composition is administered after the manifestation of the adverse condition (i.e., it is intended to alleviate, ameliorate, or stabilize the existing adverse condition or its side effects).

[0156] The toxicity and therapeutic efficacy of the compositions of the invention can be determined by standard pharmaceutical methods in cell cultures or experimental animals, e.g., by determining the median lethal dose (LD 50 ) and the median effective dose (ED 50 ). Data obtained from cell culture and / or animal studies can be used to formulate a dosage range suitable for a subject. The dosage of any composition of the invention is preferably within a range that encompasses the ED 50(Median Effective Dose) within a circulating concentration range with essentially no toxicity or completely non-toxic. The actual dose can be adjusted within this range according to the dosage form and administration route employed. For the compositions of the present invention, the therapeutically effective dose can be first estimated through cell culture assays.

[0157] The following examples are for illustrative purposes only and are not intended to limit the present invention.

[0158] Example

[0159] Design of Foot-and-Mouth Disease Virus (FMDV) Transfer Vector

[0160] Since the correct processing and assembly of the FMDV vaccine target antigens VP1-4 to form immunogenic virus-like particles (VLPs) requires a complex proteolytic process mediated by the cytotoxic viral 3C protease, the construction of a stable FMDV recombinant vaccine has always been challenging.

[0161] To ensure that all necessary proteolytic steps proceed correctly and to enable the production of immunogenic VLPs without affecting the stability of the recombinant virus due to the cytotoxic FMDV 3C protease, several important genetic modifications need to be introduced.

[0162] The recently reported full-genome sequence of the Kenyan FMDV SAT-2 type (Kenyan strain) was selected for modification, optimization, and the development of an FMDV SAT-2 gene transfer vector. First, the wild-type DNA sequence (MN116692.1) (Palinski et al, (2019)) was downloaded from the GenBank database. This sequence was isolated from vesicular epithelial tissues collected during an outbreak of bovine vesicular disease at the Kenyan Foot-and-Mouth Disease Laboratory between 2014 and 2016. This novel FMDV SAT2 strain was named isolate SAT2 / KEN / K137 / 2014 after cloning and sequencing.

[0163] Upon analysis, the VP1 nucleotide sequence of this SAT-2 (Kenya) isolate showed the highest similarity (94.1% identity) to the SAT2 / TAN / 6 / 2004 (KF561723) isolate reported in Tanzania in 2004. Subsequently, the sequence was translated to obtain the FMDV polyprotein amino acid sequence (SEQ ID NO: 2). The FMDV polyprotein is encoded by a codon-optimized nucleic acid sequence (SEQ ID NO: 1). Through analysis with CLC Main Workbench v7.9.1 software, viral capsid peptide fragments were identified and annotated from the amino acid sequence. At the same time, proteolytic cleavage sites were identified with reference to the cleavage site comparison table. The structural proteins VP4-VP2-VP3-VP1 and the 2A peptide were further identified (DNA annotation Figure 1The amino acid sequence of P1-2A in (SEQ ID NO:55) was reverse-translated into a DNA sequence (SEQ ID NO:54) using Bos taurus codon optimization to enhance the expression efficiency of this recombinant antigen in cattle. An ATG start codon was added at the start of the sequence, and a TAG stop codon was added at the end. Subsequently, the poxvirus termination signal sequence TTTTTAT was ligated. Subsequently, an MfeI restriction enzyme site was introduced at the end of this sequence to replace the SAT-2 (Kenya strain) gene with a gene encoding the P1 polyprotein of other SAT strains (such as SAT-1, SAT-2, or SAT-3). Then, the native intermediate poxvirus promoter sequence (SEQ ID NO:44) from the MVA virus 95 gene was added, followed by ligation of the start codon and the unoptimized FMDV 3C protease DNA sequence (SEQ ID NO:45) identified and isolated from the same MN116692.1 DNA sequence. The 3C protease sequence was modified by introducing the L127P mutation that has been reported to reduce the cytotoxicity of this protease. A TAG stop codon and a poxvirus termination signal sequence were added at the end of the sequence. HindIII and EcoRI restriction enzyme sites were added on both sides of the synthetic DNA sequence to subclone this synthetic gene fragment into a novel LSDV DNA transfer vector. The full-length expression cassette sequence encoding SAT-2 (Kenya strain) P1-2A and 3C protease was synthesized by GenScript and subcloned by HindIII and EcoRI digestion to construct a transfer vector named pFMDV:SAT2-Red, which was verified on a 0.7% agarose gel using the same restriction endonucleases ( Figure 2 ). The inserted gene expression cassette was confirmed by Sanger sequencing.

[0164] Recombinant Vaccine Development

[0165] After primary lamb testis (LT) cells were inoculated and co-infected with LSDV (SODis) BEFV-Gb (which shows green fluorescence due to the expression of eGFP fluorescent protein), pFMDV:SAT2-Red was transfected according to the standard procedure. After the cells were cultured in an incubator at 37 °C and 5% CO2 for 3 days, imaging was performed at passage 0 (P0) ( Figure 3 ). In the P0 passage, the cells that were infected and successfully transfected showed both red and green fluorescence, indicating the presence of the parental virus and the transfected DNA expressed by the parental virus ( Figure 3 ).

[0166] After the above cells were subjected to two freeze-thaw cycles, the lysate was used to infect BHK21 cells as passage 1 (P1). By picking red fluorescent foci ( Figure 3)Recombinant LSDV was screened. After repeatedly picking red fluorescent foci and continuous passage until only red fluorescent foci were visible.

[0167] After obtaining single wells containing only red (recombinant) virus by isolation, virus amplification was carried out by further culturing in LT cells. DNA of infected cells was extracted for PCR reaction to evaluate the DNA fragment size of the viral L49 R50 locus ( Figure 4 ), so as to conduct screening.

[0168] The region between open reading frames 49 and 50 of LSDV was amplified by PCR. DNA fragments of 5.4 kb were amplified from the DNA isolated from 5 red fluorescent foci, confirming the successful insertion of the recombinant FMDV gene ( Figure 5 ). The 5.4 kb DNA fragment amplified by PCR ( Figure 5 ) was sequenced to confirm that the inserted sequence was correct and there were no mutations (data not shown).

[0169] Western blot detection showed the expression of viral proteins VP1, VP2, and VP3 (all with a molecular weight of approximately 22 kDa) ( Figure 6 ). Transmission electron microscopy (TEM) observation confirmed the presence of FMDV virus-like particles (VLPs) of 25 - 30 nm ( Figure 7 ). LSDV(SODis)FMDV:SAT2 infected and uninfected MDBK cell cultures were prepared according to standard procedures for TEM detection ( Figure 7 ).

[0170] Development of Recombinant LSDV Vectors Containing Other Strains

[0171] Sequences of FMDV SAT-1 (KNP) (GenBank accession number AF283429.1), FMDV SAT-2 (KNP) (GenBank accession number KJ999935), and FMDV SAT-3 (KNP) (accession number DQ009739) encoding the P1 polyprotein were also obtained from GenBank and modified, optimized, and developed in the same manner as the FMDV SAT-2 (Kenya strain) sequence. The resulting modified sequences were: SAT-1 (KNP) P1 polyprotein (SEQ ID NO:11), SAT-2 (KNP) P1 polyprotein (SEQ ID NO:21), and SAT-3 (KNP) P1 polyprotein (SEQ ID NO:31). The above SAT-1, SAT-2, and SAT-3 genes encode polypeptides: SEQ ID NO:12 (SAT-1 (Kruger strain)), SEQ ID NO:22 (SAT-2 (Kruger strain)), and SEQ ID NO:32 (SAT-3 (Kruger strain)), respectively. The codon-optimized 2A gene (SEQ ID NO:42) was added to the 3' end of the three P1 polyprotein-encoding genes.

[0172] The nucleic acid sequences encoding the P1-2A polyprotein were cloned into the pFMDV:SAT2-Red transfer vector through the HindIII and MfeI restriction sites ( Figure 2 ) to generate three novel transfer vectors. Subsequently, three additional recombinant LSDVs were prepared using the above transfer vectors in the same method as that for the LSDV (SODis) FMDV:SAT2.

[0173] References

[0174] Jamal SM, Belsham GJ. 2013. Foot-and-mouth disease: past, present and future. Veterinary Research 44:116.

[0175] Palinski RM, Sangula A, Gakuya F, Bertram MR, Pauszek SJ, Hartwig EJ, Smoliga GR, Obanda V, Omondi G, VanderWaal K, Arzt J. 2019. Genome Sequences of Foot-and-Mouth Disease Virus SAT1 and SAT2 Strains from Kenya in 2014 to 2016. Microbiology Resource Announcements 8: e00809-19.

[0176] Thomas AA, Woortmeijer RJ, Puijk W, Barteling SJ. 1988. Antigenic sites on foot-and-mouth disease virus type A10. J Virol 62: 2782-9.

Claims

1. A recombinant lumpy skin disease virus (rLSDV) vector, said vector comprising an expression cassette, said expression cassette comprising a nucleic acid encoding a foot-and-mouth disease virus (FMDV) polyprotein and a nucleic acid encoding a modified FMDV 3C protease, Among them, Said FMDV polyprotein comprises FMDV structural proteins VP1, VP2, VP3, VP4 and 2A peptide.

2. The rLSDV vector according to claim 1, wherein said rLSDV comprises a stabilized SOD homolog (SOD-is) gene.

3. The rLSDV vector according to claim 1 or 2, wherein said FMDV is a SAT-1, SAT-2 or SAT-3 FMDV strain.

4. The rLSDV vector according to any one of claims 1 to 3, wherein said modified FMDV 3C protease comprises an L127P mutation.

5. The rLSDV vector according to any one of claims 1 to 4, wherein the nucleic acid encoding said FMDV polyprotein is operably linked to regulatory sequences such that said FMDV polyprotein is expressed.

6. The rLSDV vector according to claim 5, wherein the nucleic acid encoding said FMDV polyprotein is controlled by an mH5 poxvirus promoter that causes expression of said FMDV polyprotein.

7. The rLSDV vector according to any one of claims 1 to 6, wherein the nucleic acid encoding said FMDV 3C protease is operably linked to regulatory sequences such that said FMDV 3C protease is expressed.

8. The rLSDV vector according to claim 7, wherein the nucleic acid encoding said FMDV 3C protease is controlled by an MVA 95 poxvirus promoter that causes expression of said FMDV 3C protease.

9. The rLSDV vector according to any one of claims 1 to 8, wherein when the FMDV polyprotein and the FMDV 3C protease are expressed, the FMDV polyprotein is proteolytically cleaved by the 3C protease into FMDV VP1, VP2, VP3 and VP4 polypeptides and the FMDV 2A peptide.

10. A composition, said composition comprising an rLSDV vector, said vector comprising an expression cassette, said expression cassette comprising a nucleic acid encoding a foot-and-mouth disease virus (FMDV) polyprotein and a nucleic acid encoding a modified FMDV 3C protease, Said FMDV polyprotein comprises FMDV VP1, VP2, VP3, VP4 and FMDV 2A peptide.

11. The composition according to claim 10, wherein said rLSDV vector comprises a stabilized SOD homolog (SOD-is) gene.

12. The composition according to claim 10 or 11, wherein said FMDV is a SAT-1, SAT-2 or SAT-3 FMDV strain.

13. The composition according to any one of claims 10 to 12, wherein said modified FMDV 3C protease comprises an L127P mutation.

14. The composition according to any one of claims 10 to 13, wherein the nucleic acid encoding said FMDV polyprotein is operably linked to regulatory sequences such that said FMDV polyprotein is expressed.

15. The composition according to claim 14, wherein the nucleic acid encoding the FMDV polyprotein is controlled by the mH5 poxvirus promoter such that the FMDV polyprotein is expressed.

16. The composition according to any one of claims 10 to 15, wherein the nucleic acid encoding the FMDV 3C protease is operably linked to a regulatory sequence such that the FMDV 3C protease is expressed.

17. The composition according to claim 16, wherein the nucleic acid encoding the FMDV 3C protease is controlled by the MVA 95 poxvirus promoter such that the 3C protease is expressed.

18. The composition according to any one of claims 10 to 17, wherein when the FMDV polyprotein and the FMDV 3C protease are expressed, the FMDV polyprotein is proteolytically cleaved by the 3C protease into VP1, VP2, VP3, and VP4 polypeptides and the FMDV 2A peptide.

19. A vaccine comprising the rLSDV vector according to any one of claims 1 to 9 or the composition according to any one of claims 10 to 18, and a pharmaceutically acceptable carrier or adjuvant.

20. A method for inducing an immune response against foot-and-mouth disease in a subject using the rLSDV vector according to any one of claims 1 to 9, the composition according to any one of claims 10 to 18, or the vaccine according to claim 19, the method comprising administering to the subject an immunologically effective amount of the vector, composition, or vaccine.

21. The rLSDV vector, composition, or vaccine for use according to claim 20, wherein the rLSDV vector, composition, or vaccine additionally induces an immunologically effective response against foot-and-mouth disease virus in the subject.

22. The recombinant LSDV vector, composition, or vaccine for use according to claim 20 or 21, wherein the subject is selected from cattle, pigs, sheep, goats, deer, antelopes, giraffes, camels, and buffalo.

23. A method for inducing an immune response against foot-and-mouth disease in a subject, the method comprising administering to the subject an immunologically effective amount of the rLSDV vector according to any one of claims 1 to 9, the composition according to any one of claims 10 to 18, or the vaccine according to claim 19.

24. The method according to claim 23, wherein the rLSDV vector, composition, or vaccine additionally induces an immunologically effective response against foot-and-mouth disease virus in the subject.

25. The rLSDV vector, composition, or vaccine for use according to claim 20, wherein the subject is selected from cattle, pigs, sheep, goats, deer, antelopes, giraffes, camels, and buffalo.

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