FMDV virus-like particle with stabilizing mutations

Abstract

The invention concerns a recombinant foot-and-mouth disease virus (FMDV) capsid precursor protein comprising at least the virus proteins VP1, VP3 and VP0 (VP0 may be cleaved to produce VP2 and VP4), wherein the amino acid sequence of the capsid precursor protein is modified. The invention further relates to an isolated nucleic acid molecule and an expression vector comprising the nucleic acid molecule for recombinant expression of the modified capsid precursor protein. In further aspects, the invention relates to a virus-like particle (VLP) obtained from the modified capsid precursor protein and a vaccine produced from the VLP for use in the protection of a subject against an infection with FMDV.

Description

[0001] FMDV Virus-like Particle with Stabilizing Mutations

[0002] TECHNICAL FIELD

[0003] The present invention relates to the fields of veterinary medicine and virology. The invention concerns a recombinant Foot-and-mouth disease virus (FMDV) capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4, wherein the amino acid sequence of the capsid precursor protein is modified. The invention further relates to an isolated nucleic acid molecule and an expression vector comprising the nucleic acid molecule for recombinant expression of the modified capsid precursor protein. In further aspects, the invention relates to a virus-like particle (VLP) obtained from the modified capsid precursor protein and a vaccine for use in the protection of a subject against an infection with FMDV.

[0004] BACKGROUND

[0005] Foot-and-mouth disease (FMD) is a highly contagious, acute viral disease of cloven-hoofed, domesticated and wild animals. It is classified as a transboundary animal disease by the Food and Agriculture Organisation of the United Nations (FAO). It is also a notifiable disease. Foot-and-mouth disease is endemic in large parts of Africa, South America, The Middle East and Asia and is, globally, the most economically important infectious disease of livestock, affecting cattle, pigs, sheep, goats and other artiodactyl species like buffalo and deer. FMD was once distributed worldwide but has been eradicated in some regions, including North America and Western Europe. In endemic countries, FMD places economic constraints on the international livestock trade and can be easily reintroduced into disease-free areas unless strict precautions are in place. FMD impacts on the whole livestock industry with loss of income for local farmers.

[0006] In respect of prevalence, serotypes A and 0 have an almost worldwide presence, whereas serotype C has not given any outbreak since 2004. The three SAT serotypes occur in several regions of Africa and the Middle East, and serotype Asial in Asia and the Middle East.The seven serotypes also differ in biophysical properties, mainly in their stability. This is relevant as FMDV, next to being highly contagious, is also quite unstable, and is readily inactivated by heat, acidity, etc. As a result, all FMD vaccines need to be shipped and stored under strict cold-chain logistics. This is a special handicap in the (sub-) tropical- and developing regions of the world where FMD is endemic. In this respect the virions of serotype A are relatively more stable than those of other serotypes and have a workable shelf-life of 6 months or more.

[0007] However, serotype 0 vaccines have a more limited biological half-life, typically only a few months. Even worse is the situation for the three SAT serotypes, for which the notoriously low stability only yields vaccines of low protective capacity, even when administered multiple times.

[0008] Current vaccines are made of inactivated virus. Before the virus is inactivated, live FMD virus is grown during production in high containment facilities, limiting FMD vaccine production. An effective vaccination against FMD requires the presence of intact FMDV capsids rather than the capsid building blocks that have been proven to be insufficiently immunogenic (Doel and Chong, 1982, Archives of Virology). The inactivated FMD viruses are fragile structures that at acidic pH or at elevated temperatures easily fall apart in the capsid building blocks. Hence, a cold chain is required to deliver effective FMD vaccines to livestock keepers.

[0009] A new vaccine technology for commercial FMD vaccines that can overcome many of the drawbacks of the current inactivated virus vaccines is needed.

[0010] The virus-like particle (VLP) technology is currently considered one of the few technologies with the potential to be a viable alternative to conventional inactivated vaccines. The benefits of the VLP technology as compared to the current technology are for example higher product stability, greater flexibility in production location (low-containment production), and quicker responses to outbreaks of new strains. VLP-based vaccines are per definition marker vaccines which relieves the necessity of implementing production steps to remove non-structural proteins.In the FMD virion, virus capsids are formed via self-assembly from virus structural proteins. The FMDV genome encodes a single open reading frame (ORF) that produces a precursor polyprotein that is processed into twelve mature viral proteins, Fig. 1 (from: Puckette M. et al., Journal of Biotechnology, Vol. 275, 2018, p. 7-12). The P1 polyprotein intermediate is comprised of four capsid structural proteins, VPs 1-4, sited immediately upstream of the 2A protein which causes non-proteolytic separation of the P1 and P2 polyproteins during translation to release P1-2Afrom P2. The P1-2A polyprotein is subsequently processed by the FMDV 3C protease into mature VPO (also known as 1AB), VP3 (1 C), and VP1 (1 D) proteins. It is believed that VPO (1 AB) protein separates into VP4 and VP2 during genome encapsidation but varying levels of cleavage are seen in VLPs. For the avoidance of doubt, residue numbers for the mutations in VPO defined below refer to the position in the cleaved VP4 and VP2 chains. Virus capsids are formed by self-assembly from the processed virus structural proteins.

[0011] VLPs for use in VLP-based vaccines can be produced by recombinantly expressing FMDV precursor proteins in suitable host cells in analogy to the selfassembly of virus capsids in the FMD virion. For example, by the expression of FMDV subunits or -epitopes in a variety of systems, such as cell-free expression, or cell-based expression in prokaryotic or eukaryotic cells, including plant cells. Another option is the use of VLP (also called empty FMDV capsids) which are safer to produce than whole virus and were found to be effective immunogens (A.C. Mignaqui et al., 2019, Crit. Rev. Biotechnol., vol. 39(3), p. 306-320). Such empty capsids can be produced efficiently in a recombinant expression system, such as recombinant Baculovirus using insect cells (Cao et al., 2009, Vet.

[0012] Microbiol., vol. 137, p. 1; B.M. Subramanian et al., 2012, Antiviral Res. vol. 96(3), p. 288-95; S.A. Bhat et al., 2013, Vet. Sci. Res. J., vol. 95(3), p. 1217-23).

[0013] Wild-type (unmodified) VLPs, however, cannot be efficiently produced because of their intrinsic instability. They were often found to be even less stable than virions; apparently the viral RNA genome provides some stabilising effect to an FMDV capsid structure. The FMDV capsid rapidly dissociates into pentamers (known as 12S particles) above physiological temperatures and below physiological pH. Forvaccine use this is unfavourable, as the 12S pentamers are immunogenic but are not able to efficiently induce strong virus-neutralizing antibody titres like intact capsids (known as 146S particles if containing genome, 75S if empty). An approach to improve the thermo- and / or the acid stability of an FMDV capsid is to introduce capsid-stabilizing mutations into one or more of the viral structural proteins.

[0014] In particular, the thermostability and resistance to low pH of VLPs can be improved by the introduction of covalent links between the capsid proteins, such as cysteine bridges (WO 2002 / 000251), or by the introduction of other rationally designed mutations (Porta et al. (2013) PLoS Pathog 9(3): e1003255). Due to the FMDV capsid stabilization that is linked to the VLP technology, the inclusion of SAT strains into FMD vaccines is possible, something that is not straightforward with the conventional vaccine technology due to the labile nature of SAT capsids.

[0015] Consequently, the development and improvement of safe, stable and effective FMD vaccines is a continued need.

[0016] WO 2002 / 000251 in particular relates to a modified FMDV P1 antigen comprising a stabilizing mutation, i.e. a substitution of a serine (S) in the wild-type sequence of an FMDV A10 strain to cysteine (C) in the modified sequence at amino acid position 179 (corresponding to amino acid position 93 of VP2) of wild-type P1. The modification can also be described as VP2-S093C with 093 corresponding to the amino acid position in the VP2 amino acid sequence at which S is mutated to C. However, despite this mutation, VLPs based on some serotypes, such as the SAT1 , SAT2, SAT3 or A strain, that harbor this substitution (VP2-K093C in most SAT2 strains), are still not sufficiently (heat) stable. In addition, yield obtained with VLPs based on these strains harboring the mutation is relatively low.

[0017] WO 2023 / 020737 and WO 2023 / 020738 relate to stabilizing mutations for providing improved production yield and stability compared to prior art strains by introducing amino acid modifications in the FMDV capsid precursor protein.WO 2023 / 020737 in particular relates to a modified FMDV P1 antigen comprising a stabilizing mutation, i.e. an amino acid modification from threonine to asparagine at position 12 of the VP1 protein, i.e. having the modification VP1-T012N in the amino acid sequence of the FMDV capsid precursor protein. This mutation results in FMDV VLPs, in particular those of the SAT2 strain, that have improved thermostability and that are produced at higher levels than VLPs including the prior art modification at position 93 of the VP2 protein. The VLP stability and VLP production yield can be further improved in case the VLP is produced from a capsid precursor protein harboring the modification VP1-T012N in combination with an additional amino acid modification from aspartate to glycine at position 53 of the VP4 protein, i.e. having the additional modification VP4-D053G.

[0018] WO 2023 / 020738 provides a recombinant FMDV VP2 protein, in particular of the Asia1 / Shamir / ISR / 89 strain or A / SAU / 1 / 2015 strain, wherein the amino acid sequence of the VP2 protein is modified: (i) by replacement of amino acid 93 of the VP2 amino acid sequence by a cysteine, i.e. having modification VP2-S093C and (ii) by replacement of amino acid 190 of the VP2 amino acid sequence by an asparagine, i.e. having modification VP2-K190N, relative to the amino acid sequence of the wild-type strain FMDV Asia1 / Shamir / ISR / 89.

[0019] Despite this prior art providing some improvement in VLP production yield and VLP stability, there is still a need in the art for improved FMDV capsid precursor proteins, which assemble into VLPs with improved stability, in particular thermostability, and which can be obtained with high yield.

[0020] Further, the introduction of additional amino acid modifications can be problematic, since these modifications might be detrimental for the antigenic properties of the VLP, thereby diminishing its suitability for use as a vaccine. Thus, it is an additional object of the present invention to provide effective and safe vaccines against foot-and-mouth disease.

[0021] SUMMARY OF INVENTIONIn the present invention, it has surprisingly been found that VLP stability, in particular thermostability and storage stability, can be improved by providing a recombinant foot-and-mouth disease virus (FMDV) capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4, wherein the amino acid sequence of the capsid precursor protein as set forth in SEQ ID NO:1 is modified at certain specific positions of the P1 protein sequence.

[0022] It could also be surprisingly shown that the FMDV capsid precursor protein of the invention can be produced at higher levels compared to a wild-type strain or a strain having only a single mutation, such as the prior art modification at position 93 of the VP2 protein disclosed in WO 2002 / 000251.

[0023] It could be surprisingly shown that the modifications (also termed herein as “mutations”) introduced into the virus proteins enhance the structural integrity of the VLPs formed from these modified virus proteins, leading to beneficial effects of improved thermostability and storage stability.

[0024] The VLPs derived from these modified virus proteins are immunogenic and can beneficially be used for the vaccination of subjects to provide protection against the infection with FMDV.

[0025] Thus, in a first aspect the present invention provides a FMDV capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4, wherein the amino acid sequence of the capsid precursor protein is modified

[0026] by replacement of amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C),

[0027] and characterized in that the amino acid sequence of the capsid precursor protein further comprises at least one of the following modifications (I) to (V):

[0028] (I) a replacement of amino acid 194 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 194 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C), (II) a replacement of amino acid 196 of the VP3 amino acid sequence asset forth in SEQ ID N0:1 or of the amino acid corresponding to amino acid 196 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C), (III) a replacement of amino acid 143 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 143 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 by aspartic acid (D), (IV) a replacement of amino acid 12 of the VP1 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 12 of the VP1 amino acid sequence as set forth in SEQ ID NO:1 by asparagine (N), or (V) a replacement of amino acid 53 of the VP4 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 53 of the VP4 amino acid sequence as set forth in SEQ ID NO:1 by glycine (G).

[0029] A modification “by replacement of amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C)” means changing the amino acid lysine (K) in SEQ ID NO: 1, which is the amino acid sequence of the P1 protein of FMDV strain SAT2 / ETH / 65 / 2009, to cysteine (C).

[0030] A modification “by replacement of the amino acid corresponding to amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1” means changing any amino acid in an amino acid sequence that is aligned with SEQ ID NO: 1 , at the position that is equal to position 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 , to cysteine (C). “Aligned” in this sense means that the sequence identity in the two FMDV VP2 antigens (the one according to SEQ ID NO:1 and the corresponding sequence), using the BLAST® program with default settings, is at least 55%, preferably at least 60%, or even above such as 65%, 70%, 75%, 80%, 85%, 90%, 95% up to 100%.

[0031] Thus, the modification will be designated herein as “VP2-K093C”, with “K” and “C” identifying the amino acid change in the VP2 protein in SEQ ID NO:1, which is the P1 protein of FMDV strain SAT2 / ETH / 65 / 2009, from lysine to cysteine, and the digit “093” identifying the position of the modification in the VP2 amino acid sequence. The amino acid numbering thus designates the respective position in the capsid protein.The modification of the amino acid sequence of the capsid precursor protein by replacement of amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 (or of the amino acid corresponding to amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C)) corresponds to the modification described in the prior art WO 2002 / 000251.

[0032] In addition to this modification as described in the prior art, the FMDV capsid precursor protein of the present invention comprises one or more additional modifications. It has surprisingly been found that the introduction of these additional modification(s) beneficially improves the stability of VLPS derived from these modified capsid proteins.

[0033] Therefore, the amino acid sequence of the capsid precursor protein of the first aspect further comprises at least one of the modifications (I) to (V), which can also be abbreviated as follows:

[0034] (I): VP2-N194C

[0035] (II): VP3-X196C (X is referring to any amino acid)

[0036] (III): VP3-H143D

[0037] (IV): VP1-T012N

[0038] (V): VP4-D053G

[0039] Thus, the present invention in the first aspect provides a recombinant FMDV capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4 harboring the amino acid modification VP2-K093C as described in the prior art, and further comprising at least one of the modifications (I) to (V) as shown above.

[0040] “SEQ ID NO: 1" describes the amino acid sequence of the P1 protein of the wildtype strain FMDV SAT2 / ETH / 65 / 2009, thus comprising the amino acid sequences of the capsid proteins VP1 , VP2, VP3 and VP4. However, the invention is not limited to a capsid protein of this particular strain, which is merely used to identifythe position of the amino acid modifications in the amino acid sequences of the VP1 , VP2, VP3 and VP4 proteins. Due to natural sequence variation between different FMDV strains, these positions can be different in other FMDV strains, as will be described herein below. Hence, the invention also covers an FMDV capsid precursor protein comprising the VP1 , VP2, VP3 and VP4 proteins of other FMDV strains, even though the position of the modifications might differ from the position according to SEQ ID NO: 1. The corresponding position of the amino acid modifications in other FMDV strains can be found, for example, via aligning the amino acid sequences of capsid precursor proteins of different FMDV strains.

[0041] Since the amino acid sequences, such as the amino acid sequences of the P1 protein, may vary between different FMDV strains, one or more of the modifications described herein may be naturally present in an FMDV strain. Thus, the present invention encompasses an FMDV capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4 of an FMDV strain, wherein the amino acid sequence of the capsid precursor protein is modified as described herein, but in which one or more of the modifications (I) to (V) are naturally present, i.e. the amino acid in the respective position of the “modification” does not differ from that present in the FMDV wild-type strain.

[0042] In addition, since the amino acid sequences, such as the amino acid sequences of the P1 protein, may vary between different FMDV strains, also the modified amino acid may vary between different FMDV strains. For example, the modification “VP2-N194C” modification in strain SAT2 / ETH / 65 / 2009 corresponds to a modification “VP2-Q194C” in FMDV SAT3-strains, e.g. strain SAT3 / ZAM / 3 / 2015.

[0043] Therefore, encompassed by the present invention according to the first aspect is a recombinant FMDV capsid precursor protein having:

[0044] a cysteine (C) at position 093 of VP2,

[0045] and further having one or more of:

[0046] (I) a cysteine (C) at position 194 of VP2,

[0047] (II) a cysteine (C) at position 196 of VP3,

[0048] (III) an aspartic acid (D) at position 143 of VP3,(IV) an asparagine (N) at position 012 of VP1 , and

[0049] (V) a glycine (G) at position 053 of VP4,

[0050] wherein the position of the respective amino acid corresponds to that of SEQ ID NO:1.

[0051] In a further (second) aspect, the present invention provides an isolated nucleic acid encoding recombinant FMDV capsid precursor protein according to the first aspect.

[0052] In a further (third) aspect, the present invention provides an expression vector comprising an expression cassette comprising the nucleic acid sequence according to the second aspect operably linked to a promoter.

[0053] In a further (fourth) aspect, the present invention provides a method of producing FMDV virus-like particles (VLPs) in a recombinant expression system, the method comprising:

[0054] (a) infecting a host cell with an expression vector according to the third aspect, wherein the host cell is capable of recombinantly producing the FMDV VLP,

[0055] (b) culturing the host cell under conditions under which the host cell produces the FMDV VLP, and

[0056] (c) harvesting FMDV VLP produced by the host cell from the cell culture.

[0057] In a further (fifth) aspect, the present invention provides a vaccine for use in the protection of a subject against an infection with FMDV.

[0058] In a further (sixth) aspect, the present invention provides a method of protecting a subject against an infection with FMDV, which comprises the step of producing a FMDV VLP, incorporating the VLP into a vaccine by addition of a pharmaceutically acceptable carrier, and administering the vaccine to the subject.

[0059] In a further (seventh) aspect, the present invention provides a vaccine comprising a FMDV VLP produced from a recombinant capsid precursor protein according tothe first aspect.

[0060] In a further (eighth) aspect, the present invention provides a separate method of protecting a subject against an infection with FMDV, the method comprising the steps of:

[0061] (a) expressing an FMDV capsid precursor protein from the expression vector according to the third aspect in a host cell to produce a VLP,

[0062] (b) incorporating the VLP into a vaccine by addition of a pharmaceutically acceptable carrier and

[0063] (c) administering the vaccine to the subject.

[0064] In a further (ninth) aspect, the present invention provides a baculovirus expression vector according to the third aspect for use in the manufacture of a medicament for the protection of a subject against an infection with FMDV.

[0065] DEFINITIONS

[0066] A "capsid precursor protein" is a structural protein, which takes part in the formation of a virus capsid or of a building block thereof. FMDV capsid precursor proteins typically comprise at least the structural protein P1. Most preferably, the FMDV capsid precursor protein at least comprises the P1 and 2A proteins (also referred to herein as P1-2A capsid precursor).

[0067] A “capsid precursor protein PT’ of the invention refers to the FMDV structural protein processed by the FMDV 3C protease (3Cpro) into the mature VPO, VP3, and VP1 proteins. The capsid precursor protein P1 may also be referred to as polyprotein or proprotein. In the context of the present invention, the FMDV capsid precursor protein P1 typically comprises at least the proteins VP1 , VP2, VP3 and VP4.

[0068] A “VPO protein", “VP1 protein", “VP2 protein", “VP3 protein", and “VP4 protein" of the invention refers to the viral protein number 0, 1 , 2, 3 or 4 of FMDV, which areknown as structural proteins of an FMDV capsid. As the skilled person readily appreciates, the variability that is inherent to FMDV means that variations in size and amino acid sequence of these structural proteins will occur in nature. The amino acid sequences of these structural proteins from a large number of FMDV isolates are publicly available from sequence databases such as GenBank™, or Swiss Prot™.

[0069] A “modification"" is a replacement of one element for another; for the invention this is a mutation which regards the replacement of one amino acid or nucleic acid base by another, depending on whether the subject is a protein, a DNA or an RNA molecule. The element that is replaced is the element that occurs in the unmodified parental, or wild-type version of the protein or nucleic acid. As a result, a modification according to the invention leads to a capsid precursor protein P1 that differs from its parental, or wild-type form.

[0070] To serve as a reference for the invention, "SEQ ID NO: 1" presents the amino acid sequence of the capsid precursor protein P1 of FMDV strain SAT2 / ETH / 65 / 2009, a partial amino acid sequence can be found in GenBank under accession no. UDL09771. The capsid precursor protein P1 is provided at the N-terminus with a methionine (M) to reflect the recombinantly produced version of this protein described in the invention.

[0071] The following numbering includes the added M as amino acid 1 and specifies the protein sections of VP proteins for FMDV strain SAT2 / ETH / 65 / 2009. The VPO protein is the section of amino acids no. 1 - 305 of the complete P1 polyprotein, and which is processed into the separate proteins VP4 and VP2. The VP1 protein is the section of amino acids no. 528 - 741 of the complete P1 polyprotein. VP2 protein is the section of amino acids no. 87 - 305 of the complete P1 polyprotein. The VP3 protein is the section of amino acids no. 306 - 527 of the complete P1 polyprotein. The VP4 protein is the section of amino acids no. 1 - 86 of the complete P1 polyprotein.

[0072] The amino acid position given for each of the modifications in the presentinvention is identified relative to its position in the VP1 protein, VP2 protein, VP3 protein and VP4 protein as separate proteins, i.e. amino acid no. 1 of VP1 relates to the amino acid at position 528 of the entire P1 polyprotein. A modification “VP1-T012N” designates a mutation of the amino acid at position no. 12 of the VP1 protein, corresponding to amino acid 539 of the entire P1 polyprotein.

[0073] In the same way, amino acid no. 1 of VP2 relates to the amino acid at position 87 of the entire P1 polyprotein. A modification “VP2-K093C” designates a mutation of the amino acid at position no. 93 of the VP2 protein, corresponding to amino acid 179 of the entire P1 polyprotein.

[0074] In the same way, amino acid no. 1 of VP3 relates to the amino acid at position 306 of the entire PI polyprotein. A modification “VP3-H143D” designates a mutation of the amino acid at position no. 143 of the VP3 protein, corresponding to amino acid 448 of the entire P1 polyprotein.

[0075] In the same way, amino acid no. 1 of VP4 relates to the amino acid at position 1 of the entire PI polyprotein. A modification “VP4-D053G” designates a mutation of the amino acid at position no. 53 of the VP4 protein, corresponding to amino acid 53 of the entire P1 polyprotein.

[0076] The inherent variability of FMDV means that the position of the modifications within the capsid precursor protein P1 of other FMDV isolates or serotypes is not in the exact same position, e.g. it can be offset by one or more amino acids, in either the N-terminal or C-terminal direction. Nevertheless, the exact position within the nucleic acid or amino acid sequence can be easily identified using, for example, a standard computer program for molecular-biological analysis such as sequence alignment tools. Consequently, for the invention the amino acid position numbers of the capsid precursor protein P1 are specified relative to SEQ ID NO: 1, but in different FMDV isolates these may be located at different position numbers, and may be, for example, one or two amino acids upstream or downstream relative to the position identified herein for each modification.Thus, the term “corresponding to" (e.g. amino acid 093 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C)), as used herein, means that the position of a respective amino acid modification is identified herein relative to its position in the reference sequence SEQ ID NO:1 as the amino acid sequence of the capsid precursor protein P1 of FMDV strain SAT2 / ETH / 65 / 2009, but the position may vary in the amino acid numbering of the P1 proteins of other FMDV strains. For the various wild-type FMDV strains, the VP1 , VP2, VP3 and VP4 regions of the capsid precursor protein are conserved to a high level. This means that the amino acid sequence of other strains can be unambiguously aligned with the sequence SEQ ID NO:1 of FMDV strain SAT2 / ETH / 65 / 2009, for example using programs such as MUSCLE (Nucleic Acids Res. 2004; 32(5):

[0077] 1792-1797) orT-Coffee (Nucleic Acids Res. 2011 Jul 1; 39 (Web Server issue): W13-W17). After the alignment, the corresponding amino acids in the other strains are found by going to the same position as the position identified here above for the mutations in SEQ ID NO:1 of FMDV strain SAT2 / ETH / 65 / 2009.

[0078] FMDV for use in the invention are one or more FMDV strains of the A, 0, C, SAT1 , SAT2, SAT3, or Asial serotype(s); preferably FMDV for use in the invention are one or more FMDV strains that are circulating in the field at a certain time. More preferred are one or more FMDV strains from O, SAT1 , SAT2, or SAT3 serotypes, as for these serotypes lack of stability issues have the most impact in the field. It has surprisingly been observed that the present invention is most beneficial in FMDV strains from the SAT serotype(s). Without being limited thereto, FMDV strains for use in the invention are thus preferably FMDV strains from SAT1 , SAT2, or SAT3 serotype(s). Most preferably, the FMDV strain is of the SAT2 or SAT3 serotype.

[0079] Preferred FMDV strains are those that are recommended by the World Reference Laboratory for Foot-and-Mouth Disease (WRL-FMD) as high priority vaccine candidates. The recommendations are published by WRL-FMD on a quarterly basis. A "virus-like particle" (VLP), which may also be referred to in the art as "empty capsid', is an entity which comprises the protein shell of a virus but lacks the RNA or DNA genome. A VLP should be antigenic and immunogenic in thesame way as the wild-type virus because it retains the same structural epitopes, but it should produce no infection, due to the lack of the virus genome.

[0080] An FMDV VLP is typically formed from the P1-2A capsid precursor. As described above, the 2A protease cleaves itself at its C-terminus to release P1-2A from P2. Processing of the P1-2A capsid precursor is affected by the 3C protease to produce 2A and the capsid proteins VPO, VP3 and VP1. The VLP is formed by self-assembly of these capsid proteins.

[0081] A "virus-like particle" (VLP), which may also be referred to in the art as “empty capsid”, is an entity which comprises the protein shell of a virus but lacks the RNA or DNA genome. A VLP should be antigenic and immunogenic in the same way as the wild-type virus because it retains the same structural epitopes, but it should produce no infection, due to the lack of the virus genome.

[0082] A FMD VLP is typically formed from the P1-2A capsid precursor. As described above, the 2A protease cleaves itself at its C terminus to release P1-2A from P2. Processing of the P1-2A capsid precursor is affected by the 3C protease to produce the capsid proteins 1AB (VPO), 1C (VP3) and 1D (VP1). The VLP is formed by self-assembly from the processed virus structural proteins.

[0083] VLPs may also be produced in a recombinant expression system, such as baculovirus expression system using a modified 3C protease that is less toxic to the insect cells (Porta et al. (2013) J Virol Methods, 187(2):406-12). Intermediate and non-toxic activity of the 3C enzyme in a P1-2A-3C expression cassette allows recombinant expression and processing of the P1-2A precursor into the structural proteins, VPO, VP1 , and VP3, which assemble into VLPs (see also EP 2491118). The production of VLPs may be investigated or verified using techniques known in the art such as sucrose density centrifugation or electron microscopy (Abrahams et al, J Gen Virol (1995), 76, 3089-98). Monoclonal antibodies may be used specific for conformational epitopes on the virus capsid in order to investigate whether the structure and antigenicity of the empty capsid is retained.The term "nucleic acid sequence" includes an RNA or DNA sequence. It may be single or double stranded. It may, for example, be genomic, recombinant, mRNA or cDNA.

[0084] The term "isolated1is to be interpreted as: isolated from its natural context, by deliberate action or human intervention; e.g. by an in vitro procedure for biochemical purification.

[0085] Typically, a "nucleic acid’ or "nucleic acid molecule" encoding a protein, in the context of the present invention, refers to an open reading frame (ORF) encoding a modified FMDV capsid precursor protein as defined herein, indicating that no undesired stop-codons are present that would prematurely terminate the translation of an ORF into protein. For the invention the nucleic acid molecule typically encodes the complete capsid precursor protein P1. In an alternative embodiment of the invention, the P1 coding sequence may be split up into multiple expression units, which are expressed as separate recombinant proteins for the assembly of VLPs. For example, the capsid precursor proteins required for the assembly of FMDV VLPs may be expressed separately, for example by recombinantly producing VP1 , VP2, VP3 and VP4, or recombinantly producing VPO, VP1 and VP3. Thus, in the present invention FMDV VLPs may be obtained either from the recombinant P1 capsid precursor protein, which comprises at least the modifications described herein, or may be obtained by separately expressing the VP1 , VP2, VP3 and VP4 recombinant proteins necessary for the assembly of VLPs as separate entities, and which comprise the respective modifications.

[0086] For the present invention, the exact nucleotide sequence of a nucleic acid molecule according to the invention is not critical, provided the nucleotide sequence allows the expression of the desired amino acid sequence, here the desired FMDV VP1 , VP2, VP3 and VP4 proteins. However, as is well known in the art, different nucleic acids can encode the same protein due to the 'degeneracy of the genetic code'.

[0087] For the present invention, a nucleic acid molecule can be a DNA or an RNAmolecule. This depends on the source material used for its isolation, and on the intended use. The skilled person is well aware of methods to isolate one or the other type of molecule from a variety of starting materials, and of methods to convert one type into the other.

[0088] An “isolated nucleic acid molecule" according to the invention can conveniently be manipulated in the context of a vector, such as a DNA plasmid, when it is in DNA form. To allow an isolated nucleic acid molecule according to the invention to actually express the modified FMDV capsid precursor protein P1 according to the invention, it will require proper expression control signals and a suitable environment. For example, a nucleic acid molecule needs to be operatively linked to an upstream promoter element and needs to contain a translation start at the beginning of the coding sequence and a translation stop at the end of the coding sequence. In addition, translational enhancers can be included upstream and / or downstream of the coding region to increase expression levels. Typically, the plasmids and vectors used in the context of a particular expression system will provide for such elements and enhancers. Also, the bio-molecular machinery for transcription and translation is typically provided by a host cell used for such expression. By modifying the various elements and enhancers, the expression of the capsid precursor protein P1 according to the invention can be optimised in e.g. timing, level, and quality; all this is within the routine capabilities of the skilled person. Therefore, in a preferred embodiment, the isolated nucleic acid molecule according to the invention in addition comprises expression control signals. A recombinant expression system for use in the invention typically employs a host cell, which can be cultured in vitro. Well known in the art are host cells from bacterial, yeast, fungal, plant, insect, or vertebrate cell expression systems.

[0089] Alternatively, in vivo expression systems may be used, employing (transgenic) animals, plants or insects for recombinant gene expression (He et al., 2020, Arch Virol 165: 2301-2309, doi.org / 10.1007 / s00705-020-04754-9).

[0090] An “expression vector"’ (syn. “expression construct’), is usually a plasmid or virus designed for recombinant gene expression in cells. The vector is used to introduce a specific gene into a target cell and can commandeer the cell's mechanism forprotein synthesis to produce the protein of interest (POI) encoded by the gene. In order to express the recombinant gene to produce the POI, the expression vector typically comprises at least a promotor for initiating gene expression and may further comprise one or more translational enhancers.

[0091] A “baculovirus expression vector"’ is an expression vector based on a baculovirus, which is used for recombinant gene expression in a host cell, such as an insect cell. Baculovirus expression systems are established in the art and are commercially available, such as the Bac-to-Bac expression system (ThermoFisher Scientific, Germany). In these baculovirus expression systems, the naturally occurring polyhedrin gene within the wild-type baculovirus genome is typically replaced with a recombinant gene or cDNA. These genes are commonly under the control of polyhedrin and p10 promoters.

[0092] The most common baculovirus used for gene expression is Autographa californica nucleopolyhedrovirus (AcNPV). AcNPV has a large (130kb), circular, doublestranded DNA genome. The gene of interest (GOI) is cloned into a transfer vector containing a baculovirus promoter flanked by baculovirus DNA, e.g. derived from a nonessential locus, such as the polyhedrin gene. The GOI is inserted into the genome of the parent virus (such as AcNPV) by homologous recombination after transfection into insect cells.

[0093] A “translational enhancer"’ is a nucleotide sequence forming an element, which can promote translation and, thereby, increase protein production. Typically, a translational enhancer may be found in the 5' and 3' untranslated regions (UTRs) of mRNAs. In particular, nucleotides in the 5'-UTR immediately upstream of the initiating ATG codon of the GOI may have a profound effect on the level of translation initiation.

[0094] The term “vaccine" as used herein refers to a preparation which, when administered to a subject, induces or stimulates a protective immune response. A vaccine can render an organism immune to a particular disease.“Protection" or “protecting" a subject (e.g. an animal, such as pig) against an infection with FMDV, as used herein, encompasses the prevention and the treatment of the disease. To “protect a pig against an infection with FMDV” means aiding in preventing, ameliorating or curing a pathogenic infection with FMDV, or aiding in preventing, ameliorating or curing a disorder arising from that infection, for example by avoiding or reducing one or more clinical signs resulting from the infection with FMDV.

[0095] The term “prevention" or “preventing" is intended to refer to averting, delaying, impeding or hindering the FMDV infection by a prophylactic treatment. The vaccine may, for example, prevent or reduce the likelihood of an infectious FMDV entering a host cell.

[0096] “Treatment’ or “treating" as used herein refers to caring for a subject suffering from FMDV infection, in order to ameliorate, cure or reduce the symptoms of the disease, or reduce or halt the progression of the disease. It also refers to treatment which renders the virally-infected subject non-infectious to other subjects.

[0097] DESCRIPTION OF EMBODIMENTS

[0098] In a first aspect, the invention provides a recombinant FMDV capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4, wherein the amino acid sequence of the capsid precursor protein comprises the modification VP2-093C, and further comprises at least one of the modifications (I) VP1-012N, (II) VP4-053G, (III) VP2-194C, (IV) VP3-196C and (V) VP3-143D described herein.

[0099] It has surprisingly been found in the present invention that the modification(s) (I) to (V), when combined with the modification VP2-093C, provide an improved capsid stability resulting in improved thermostability, storage stability and higher expression levels resulting in more VLPs produced compared to a wild-type strain or to a comparative strain harboring only the prior art modification, which changes the amino acid at position 93 of the VP2 amino acid sequence in the wild-typestrain to cysteine, as described in WO 2002 / 000251.

[0100] In particular, it has surprisingly been found in the present invention that a combination of at least one of the amino acid modifications (I) to (V), whether introduced or naturally occurring, improves the modification VP2-093C in terms of thermostability and yield. Thus, a recombinant FMDV VLP assembled from the VP1 , VP2, VP3 and VP4 virus proteins described herein is particularly suitable for the development of a vaccine against FMD.

[0101] To identify or confirm the amino acid which is to be modified, the amino acid sequence of this region of several FMDV strains may be aligned with the corresponding region (for example of the order of about ten or slightly more - e.g.

[0102] 10 to 20 - amino acids) of the sequence in SEQ ID NO: 1 , taking into account the fact that the sequences are well conserved in structure among the different foot-and-mouth viruses.

[0103] In a preferred embodiment of the first aspect, the amino acid sequence of the capsid precursor protein comprises more than one of the modifications (I) to (V), such as two, three, four or five of the modifications (I) to (V).

[0104] Preferred combinations of the modifications (I) to (V) are the following:

[0105] (a) the modifications (I) and (II); or

[0106] (b) the modifications (III), (IV) and (V); or

[0107] (c) the modifications (I), (II), (IV) and (V); or

[0108] (d) the modifications (I), (II), (III), (IV) and (V).

[0109] In abbreviated form, the above combinations (a) to (c) may also be described as follows, each including the prior art modification VP2-093C:

[0110] (a) VP2-K093C+VP2-N194C+VP3-X196C;

[0111] (b) VP2-K093C+VP1 -T012N+VP4-D053G+VP3-H143D;

[0112] (c) VP2-K093C+VP1 -T012N+VP4-D053G+VP2-N194C+VP3-X196C; (d) VP2-K093C+VP1-T012N+VP4-D053G+VP2-N194C+VP3- X196C+VP3-H143D.Particularly preferred among (a) to (d) is the combination (c) of modifications (I), (II), (IV) and (V).

[0113] In a further preferred embodiment, the capsid precursor protein comprising the VP1 , VP2, VP3 and VP4 proteins of the invention comprising the modifications described herein is part of the full-length capsid precursor protein P1.

[0114] In a preferred embodiment of the first aspect, the recombinant FMDV capsid precursor protein of the invention comprises, or may consist of, the amino acid sequence of SEQ ID NO: 2, which is based on the amino acid sequence of the P1 protein of FMDV strain SAT2 / ETH / 65 / 2009 (SEQ ID NO:1) and including, in addition to the modification VP2-093C, the modifications (I), (II), (IV) and (V) described above.

[0115] In another preferred embodiment of the first aspect, the recombinant FMDV capsid precursor protein of the invention comprises, or may consist of, the amino acid sequence of SEQ ID NO: 3, which is based on the amino acid sequence of the P1 protein of FMDV strain SAT3 / ZAM / 3 / 2015 (SEQ ID NO: 3) and including, in addition to the modification VP2-093C, the modifications (I), (II), (III), (IV) and (V) described above.

[0116] In another preferred embodiment of the first aspect, the recombinant FMDV capsid precursor protein of the invention comprises, or may consist of, the amino acid sequence of SEQ ID NO: 4, which is based on the amino acid sequence of the P1 protein of FMDV strain SAT2 / SUD / 3 / 2013 (SEQ ID NO: 4) and including, in addition to the modification VP2-093C, the modifications (I), (II), (IV) and (V) described above.

[0117] The present invention also relates to the nucleic acid sequences, notably the cDNA incorporating the modifications as described in the first aspect. In particular, the invention relates to the cDNA, and expression vectors incorporating them,comprising the sequence coding for the VP1 , VP2, VP3 and VP4 proteins, or the full-length capsid precursor protein P1 comprising the VP1 , VP2, VP3 and the VP4 proteins as described above, and which incorporate these modifications as described in the first aspect, for example cDNA sequences coding for P1-2A, and the sequences incorporating them, for example sequences incorporating them with the sequences allowing their recombinant expression, thus being operably linked to a promoter.

[0118] The present invention also relates to the amino acid sequences encoded by these nucleic acid sequences. In a further aspect, the invention relates to an expression vector for the recombinant expression of the nucleic acid sequence of the invention and in which the nucleic acid sequence encoding the capsid precursor protein comprising the modified VP1 , VP2, VP3 and VP4 protein is operably linked to a promoter.

[0119] In the following, the recombinant capsid precursor protein comprising the modified VP1-4 proteins of the invention and including the modifications described above is designated as “recombinant FMDV capsid precursor protein according to the invention". The “recombinant FMDV capsid precursor protein according to the invention" may either be expressed as single entity including all structural proteins necessary for the formation of VLPs or may be expressed as separate entities, such as by separately expressing the structural VP proteins, including the VP1 , VP2, VP3 and VP4 proteins of the invention.

[0120] In vitro recombinant DNA methods known to the skilled person can be used to generate a recombinant nucleic acid molecule that encodes the capsid precursor protein according to the invention, comprising the amino acid modifications described herein. Conveniently, this can be done by making and sub-cloning PCR fragments, or by de novo gene synthesis techniques and may include site-directed mutagenesis.

[0121] A recombinant FMDV capsid precursor protein according to the invention can beobtained in a variety of ways. A variety of in vivo and in vitro expression systems are well known in the art. For example, a recombinant FMDV capsid precursor protein according to the invention can be generated by manipulation of FMDV genetic material, transfection of cDNA into appropriate host cells, or amplification of infectious FMDV virus in an appropriate host cell, e.g. BHK-21 cells.

[0122] Alternatively, a recombinant FMDV capsid precursor protein according to the invention can be produced via an in vitro cell-based expression system, as this provides advantages in respect of yields and safety. The expression system can be based on prokaryotic or eukaryotic cells; if eukaryotic, it can be based on host cells from a yeast, a mammal, an insect, or a plant, all as described in the prior art.

[0123] A preferred in vitro expression system for the expression of a recombinant FMDV capsid precursor protein according to the invention is the Baculovirus expression vector system (BEVS). This system uses a baculovirus expression vector, which is capable of recombinantly expressing the gene of interest in insect cells, which in the present invention is the modified FMDV capsid precursor protein.

[0124] The baculovirus expression vector can be any baculovirus expression vector capable of recombinantly expressing an FMDV capsid precursor protein under control of a promoter. The promoter is not particularly limited but may be any promoter capable of recombinantly expressing the FMDV capsid precursor protein in a baculovirus expression system. Preferred promoters for use in the baculovirus expression system of the present invention are the polyhedrin (polh) promoter (described in: Ayres M.D. et al. (1994) Virology, Vol. 2020, p. 586-605) and the p10 promoter (described in: Knebel D. et al. (1985) EMBO J. Vol. 4(5), 1301-1306) of AcNPV. Another preferred promoter is the promoter of the orf46 viral gene of Spodoptera exigua nucleopolyhedrovirus (SeNPV) (described in M. Martinez-Solis et al. (2016) PeerJ, DOI 10.7717 / peeq.2183).

[0125] The expression vector may further comprise one or more translational enhancers, which enhance the recombinant expression of the FMDV capsid precursor protein. For example, the baculovirus expression vector may comprise the twotranslational enhancers Syn21 and plOUTR as described in WO 2022 / 084426, which is incorporated herewith by reference in its entirety.

[0126] Baculovirus expression vectors for use in baculovirus expression systems for the recombinant expression of proteins are commercially available and are extensively used in the art for the production of proteins and virus-like particles. The systems may encompass, for example, one or more transfer plasmids used to transform cells, such as E. coli cells or insect cells, in which the baculovirus expression vector is propagated. Commercially available baculovirus expression vectors include, but are not limited to, Top-Bac® vector (ALGENEX, Spain), pFastBac® vector (Thermo Fisher Scientific, Germany), flashBAC® vector (Oxford Expression Technologies Ltd, UK) and BestBac® vector (EXPRESSION SYSTEMS, CA).

[0127] The baculovirus expression vector for use in the present invention thus may contain an expression cassette comprising the nucleic acid sequence encoding the FMDV capsid precursor protein, which is expressed in the insect cell under control of a functional promoter, and preferably including one or more translational enhancers and / or other cis-acting elements.

[0128] The nucleic acid sequence encoding the FMDV capsid precursor protein is not particularly limited to a certain strain and may be of any FMDV strain belonging to serotype A, 0, Asial , SAT1 , SAT2, SAT3 or C. In a particularly preferred embodiment, the FMDV capsid precursor protein according to the invention is from the SAT serotype, such as SAT1, SAT2, and SAT3 serotype, and most preferably from the SAT2 or SAT3 serotype.

[0129] In the present invention, the FMDV capsid precursor protein may comprise all elements necessary for the processing and assembly of VLPs. Hence, the FMDV capsid precursor protein typically comprises at least the capsid precursor P1 and preferably further comprises the 2A peptide. The 2A peptide is able to release P1-2A from any downstream protein sequence. In a further preferred embodiment, the baculovirus expression vector further comprises a nucleic acid sequence encoding a protease capable of cleaving an FMDV capsid precursor protein. The proteasemay be any protease capable of cleaving the FMDV capsid precursor protein as a step in the production and assembly of FMDV VLP. As mentioned above, for FMDV, proteolytic processing of the capsid precursor P1 according to the invention into VPO (VP2 plus VP4), VP3 and VP1 occurs by means of the picornavirus 3C protease or its precursor 3CD. Hence, the protease is preferably the 3C protease of FMDV. The sequence of FMDV wild-type 3C protease from an FMDV serotype A strain is described in the art and is disclosed in WO 2011 / 048353, which is hereby incorporated by reference in its entirety. The 3C protease may also be a functional derivative including one or more mutations, which reduce its proteolytic activity, for example a mutation at cysteine 142.

[0130] The capsid precursor protein of the invention is typically cleaved by the 3C protease into VPO, VP3 and VP1. Most preferably, the baculovirus expression system expresses a P1-2A-3C cassette, i.e. it simultaneously expresses the coding regions for the proteins P1 , 2A and 3C. Expression of the 3C enzyme in a P1-2A-3C cassette allows expression and processing of the P1-2A precursor into the structural proteins which assemble into VLPs. The capsid precursor protein and the protease may be expressed under control of individual promotors or under control of the same promoter. As described above, the capsid precursor proteins required for the assembly of FMDV VLPs may be split up into multiple expression units and expressed separately, for example by recombinantly producing VP1 , VP2, VP3 and VP4, or recombinantly producing VPO, VP1 and VP3. In this alternative embodiment, a proteolytic cleavage of a capsid precursor protein by a 3C protease may not be necessary.

[0131] Cleavage of the capsid precursor protein may be analysed using techniques known in the art. For example, extracts from baculovirus-infected host cells may be analyzed by gel electrophoresis and the separated proteins transferred onto a nitrocellulose membrane for western blotting. Western blotting with protein-specific antibodies should reveal the degree of protease-mediated cleavage. For example, for FMDV, the unprocessed capsid precursor protein (P1-2A) would appear as a band of around 81 kDa, and cleavage may produce VP3-VP1 (~47kDa), VP3 (~24kDa), VP1 (~24 kDa) and VPO (~33kDa). VPO may be cleaved to produceVP2 (~22 kDa) and VP4 (8 kDa) as has been observed to occur in VLPs and is not medidated by 3C.

[0132] METHOD OF PRODUCING VIRUS LIKE PARTICLES

[0133] The method for recombinantly producing the modified capsid precursor protein of the invention includes the culturing of host cells under conditions suitable for the host cell to recombinantly express the capsid precursor protein from the expression vector in order to produce VLPs. In case of using BEVS, the host cell may be an insect cell and the expression vector is a baculovirus expression vector. The term “the host cell is capable of recombinantly producing the FMDV VLP” thus means that the insect cell can be used as a host cell for the production of recombinant capsid precursor proteins, which assemble into VLPs.

[0134] The first step of the method of the invention comprises infecting a host cell, for example an insect cell, with the expression vector, for example a baculovirus expression vector (step (a) of the method of the invention). In the preferred embodiment, the insect cell may be any insect cell, which is capable of producing FMDV VLPs in cell culture. In particular, the insect cell may be a Sf9 cell (a clonal isolate of Spodoptera frugiperda Sf21 cells), or a Tni cell (ovarian cells isolated from Trichoplusia ni). Most preferably, the host cell is a Tni cell, or a Tni-derived cell line, such as a Tnao38 cell.

[0135] Methods of infecting an insect cell with a baculovirus expression vector for the recombinant expression of proteins are known to the skilled person and are described, for example, in L. King, The Baculovirus Expression System, A laboratory guide; Springer, 1992; Baculovirus and Insect Cell Expression Protocols, Humana Press, D.W. Murhammer (ed.) 2007; Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press, D.R. O'Reilly, 1993. In the method of the invention, culturing of the insect cell is performed in cell culture medium (step (b) of the method of the invention). Cell culture of infected insectcells under conditions under which the insect cell produces the FMDV VLP is established in the art and can be performed, for example, as described in (Porta et al., 2013, J. Virol. Methods, vol. 187, p. 406; A.C. Mignaqui et al., 2019, Critical Reviews in Biotechnology, vol. 39(3), p. 306-320).

[0136] After culturing, the cells may optionally be separated from the cell culture to obtain culture supernatant. The term “supernatant” thus relates to the cell culture medium from which the insect cells have been removed. Recombinant proteins that are trapped inside insect cells can be released by cell disruption techniques known in the art. The obtained cell lysate contains all the cellular components and debris, and often requires laborious purification to obtain the recombinant protein in a purer form. Further, cell disruption techniques also release a lot of unwanted cellular proteins, such as proteases, which can degrade the desired proteins, thereby reducing protein yield and quality.

[0137] Conventional techniques for separation of the cells from the cell culture medium are well known in the art and include one or more of filtration, centrifugation, and sedimentation.

[0138] In step (c) of the method of the present invention, the FMDV VLPs produced by the host cells are harvested from the cell culture and optionally are further purified. Harvesting may include the separation of the VLPs from the cells and / or culture medium and, if necessary, further purification of the VLPs. Harvesting can be performed by one or more techniques including precipitation of the VLPs with for example polyethylene glycol (PEG), affinity chromatography, or molecular sieve chromatography.

[0139] VACCINES AND PRODUCTION THEREOF

[0140] As described above, the preferred utility of the embodiments of the present invention is in veterinary medical use, in particular for vaccination against FMD. The present invention thus further relates to the production of FMDV VLPsobtained from the modified capsid precursor protein of the invention, and which are used in the production of a vaccine. In a preferred embodiment, the vaccine of the invention comprises FMDV VLPs produced from the modified capsid precursor protein, which is from a FMDV strain of the SAT2 serotype.

[0141] In particular, the VLPs obtained from the modified capsid precursor protein and produced by the method according to the invention may be used as antigen for vaccination of subjects. Preferably, the VLPs are incorporated into a composition comprising the VLPs and one or more pharmaceutically acceptable carriers.

[0142] The present invention thus also provides a method for the production of a vaccine, which comprises the step of producing FMDV VLPs by a method as described above and incorporating the FMDV VLPs in a vaccine, such as by the addition of a pharmaceutically acceptable carrier.

[0143] Pharmaceutically acceptable carriers are well-known in the art. Merely as an example; such a carrier can be as simple as sterile water or a buffer solution such as PBS. The vaccine may comprise a single carrier or a combination of two or more carriers. The vaccine may also comprise one or more pharmaceutically acceptable diluents, adjuvants and / or excipients.

[0144] Preferably, the adjuvant is based on emulsions of water and oil. A preferred adjuvant composition for use in a vaccine of the invention comprises an emulsion of water, a tocopherol or a pharmaceutically acceptable ester thereof, and a polyethoxy ethylene cetostearyl ether. In said composition, the tocopherol or the pharmaceutically acceptable ester thereof acts as an oily adjuvant, and the polyethoxy ethylene cetostearyl ether acts as an emulsifier. An example of such an adjuvant is described in WO 2023 / 118553, which is hereby incorporated in its entirety.

[0145] The vaccine may also comprise, or be capable of expressing, another active agent, for example one which may stimulate early protection prior to the VLP-induced adaptive immune response. The agent may be an antiviral agent, such astype I interferon. Alternatively, or in addition, the agent may be granulocytemacrophage colony stimulating factor (GM-CSF).

[0146] The vaccine may be used therapeutically, to treat an existing FMDV infection (especially in herds or regions where the virus is endemic), but preferably is used prophylactically, to block or reduce the likelihood of FMDV infection and / or prevent or reduce the likelihood of spreading the disease. Many commercially available FMD vaccines are multivalent to provide protection against the different FMD serotypes. By the same token, the vaccine of the present invention may comprise a plurality of different VLPs, each directed at a different serotype and / or different subtypes within a given serotype.

[0147] Thus, in a further preferred embodiment, the method of the invention further comprises the step (d) of incorporating the FMDV VLPs into a vaccine by addition of a pharmaceutically acceptable carrier.

[0148] The vaccine obtained by the method as described above may be used in the protection of a subject against an infection with FMDV.

[0149] The present invention also provides a method of protecting a subject against an infection with FMDV by administration of an effective amount of a vaccine of the present invention. A method of protecting a subject against an infection with FMDV comprises the step of producing an FMDV VLP by a method as described above, incorporating the VLP into a vaccine by addition of a pharmaceutically acceptable carrier, and administering the vaccine to the subject.

[0150] For FMD the subject may be a cloven-hoofed animal. FMD susceptible animals include cattle, sheep, pigs, and goats among farm stock, as well as camelids (camels, llamas, alpacas, guanaco and vicuna). Some wild animals such as buffalo, hedgehogs, coypu, and any wild cloven-footed animals such as deer and zoo animals including elephants are also susceptible to FMD.ADMINISTRATION

[0151] The present invention contemplates at least one administration to an animal of an efficient amount of the vaccine according to the invention. A vaccine can be administered in any art-known method, including any local or systemic method of administration. Administration can be performed e.g. by administering the antigens into muscle tissue (intramuscular, IM), into the dermis (intradermal, ID), underneath the skin (subcutaneous, SC), underneath the mucosa (submucosal, SM), in the veins (intravenous, IV), into the body cavity (intraperitoneal, IP), orally, anally etc. For the current vaccine IM, ID and SC administration are preferred.

[0152] Thus, in a further aspect, the present invention provides a method of protecting a subject (preferably animal, most preferably pig) against an infection with FMDV comprising the steps (a) to (d) as described above to obtain a vaccine, and further comprising the step (e) of administering the vaccine to the subject.

[0153] EXAMPLES

[0154] The invention will be further described by way of the following non-limiting examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention.

[0155] DESCRIPTION OF DRAWINGS

[0156] Figure 1: Schematic representation of the FMDV genome encoding a single open reading frame (ORF) that produces a precursor polyprotein that is processed into twelve mature viral proteins.

[0157] Figure 2: Quantification by ELISA of the SAT2 / ETH / 65 / 2009 VLP concentration in insect cell culture supernatants in Example 1. Error bars represent standard error.

[0158] Figure 3: Heat stability of the different SAT2 / ETH / 65 / 2009 VLP mutants inExample 1.

[0159] Figure 4: Quantification by ELISA of the SAT2 / SUD / 3 / 2013 VLP concentration in insect cell culture supernatants in Example 2. Error bars represent standard error.

[0160] Figure 5: Heat stability of the different SAT2 / SUD / 3 / 2013 VLP mutants in Example 2.

[0161] Figure 6: Cryo-EM electron potential map for SAT2 / SU D / 3 / 2013-5xmut VLPs in Example 2.

[0162] Figure 7: Quantification by ELISA of the SAT3 / ZAM / 3 / 2015 VLP concentration in purified insect cell culture supernatants in Example 5.

[0163] Figure 8: Cryo-EM electron potential map for SAT3 / ZAM / 3 / 2015-‘6xmut-mimic’ VLPs in Example 5.

[0164] Preparation of baculovirus constructs

[0165] Recombinant baculoviruses were generated using the ProEasy™ system from AB Vector. They were equipped with the P1-2A-3Cpro expression cassette as described by Porta et al., 2013, J Virol Methods. To increase expression levels the so-called Syn21 translational enhancer was placed in front of the P1-2A-3Cpro open reading frame, and downstream of the P1-2A-3Cpro coding region the 3’-UTR from the Autographa califomica nucleopolyhedrovirus (AcNPV) p10 gene (P10UTR) was inserted (Liu et al., 2015, Biotechnol Lett).

[0166] Since wild-type capsids cannot be expressed due to their inherent instability, the previously described modification VP2-S093C in VP2 was introduced in the P1 coding sequence as described in WO 2002 / 000251 (corresponding to VP2-K093C in SAT2 strains). As an alternative to this previously described mutation, four novel sets of mutations were introduced in P1 , each containing VP2-K093C as the basic mutation and several other additional mutations:Table 1: Mutations used in FMDV strain expression constructs

[0167]

[0168] The VP2-K093C mutation refers to a lysine (K) to cysteine (C) amino acid mutation at position 93 in VP2 and is as described in WO 2002 / 000251. Similarly, the other mutations refer to a change of one amino acid at a certain position in the indicated protein to another amino acid at the indicated position. X is referring to any amino acid.

[0169] The amino acid modifications were introduced using synthetic cDNA which was placed in a transfer vector used for producing the recombinant baculoviruses.

[0170] The following baculovirus expression constructs were used in the following examples for the recombinant production of VLPs in insect cells:

[0171] i) Expression construct SAT2 / ETH / 65 / 2009_VP2-K093C containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / ETH / 65 / 2009.

[0172] ii) Expression construct SAT2 / ETH / 65 / 2009_VP2-K093C+VP2-N194C+VP3-A196C (3xmut) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / ETH / 65 / 2009.

[0173] iii) Expression construct SAT2 / ETH / 65 / 2009_VP2-K093C+VP1 -T012N+VP4-D053G+VP2-N194C+VP3-A196C (5xmut) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / ETH / 65 / 2009 (SEQ ID NO: 2).iv) Expression construct SAT2 / ETH / 65 / 2009_VP2-K093C+VP1 -T012N+VP4-D053G+VP2-N194C+VP3-A196C+VP3-H143D (6xmut) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / ETH / 65 / 2009 (SEQ ID NO: 3).

[0174] v) Expression construct SAT2 / SUD / 3 / 2013_VP2-K093C containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / SU D / 3 / 2013.

[0175] vi) Expression construct SAT2 / SUD / 3 / 2013_VP2-K093C+VP1 -T012N+VP4-D053G+VP3-H143D (4xmut) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / SU D / 3 / 2013.

[0176] vii) Expression construct SAT2 / SUD / 3 / 2013_VP2-K093C+VP1 -T012N+VP4-D053G+VP2-N194C+VP3-S196C (5xmut) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / SU D / 3 / 2013 (SEQ ID NO: 4).

[0177] viii) Expression construct SAT2 / SUD / 3 / 2013_VP2-K093C+VP1 -T012N+VP4-D053G+VP2-N194C+VP3-S196C+VP3-H143D (6xmut) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT2 / SU D / 3 / 2013.

[0178] ix) Expression construct SAT3 / ZAM / 3 / 2015_wild-type containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT3 / ZAM / 3 / 2015.

[0179] x) Expression construct SAT3 / ZAM / 3 / 2015_VP2-A093C containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT3 / ZAM / 3 / 2015.

[0180] xi) Expression construct SAT3 / ZAM / 3 / 2015_VP2-A093C+VP2-Q194C+VP3-H194C+VP1-T012N+VP4-D053G+VP3-H142D (6xmut-mimic) containing the P1-2A-3Cpro expression cassette based on FMDV strain SAT3 / ZAM / 3 / 2015.

[0181] The baculovirus expression system was used to recombinantly express the SAT2 and SAT3 VLPs.Example 1: Stabilization of SAT2 / ETH / 65 / 2009 VLPs

[0182] Erlenmeyer shaker flasks containing T106Tnao38 insect cells per ml were inoculated with P2 baculovirus stocks and incubated at 30.0°C and 150 revolutions per minute (rpm) for 5 days post infection (dpi). The cell culture supernatant was collected by removing the insect cells by centrifugation for 5 min at 3000xg.

[0183] A part of the harvested supernatant was heat treated at 56°C for 20 minutes and the amount of intact VLPs before and after heat treatment was determined by ELISA using VHH M377F (Harmsen etal., 2017, Front. Immunol. 8:960, doi:

[0184] 10.3389 / fimmu.2017.00960) specific for intact capsids. Serially diluted samples were incubated for 1h at room temperature (RT) on microtiter plates coated overnight at 4°C with VHH M377F. After removing the samples and three washes with PBS-Tween, a fixed amount of biotinylated VHH M377F was added to plates and incubated for 1h at RT. The biotinylated antibody was removed and plates were washed three times with PBS-Tween, after which peroxidase-conjugated streptavidin was added to the plates followed by chromophoric detection.

[0185] The yields of non-treated capsids are presented in Fig. 2. VLPs harbouring the prior art mutation VP2-K093C were not detected, indicating a very low level of VLP production possibly due to the inherent instability of these VLPs. In contrast, the other mutants (i.e. 3xmut, 5xmut, 6xmut) could be detected in the cell culture supernatant.

[0186] The percentage of VLPs that survived the 20-m inute incubation at 56°C is presented in Fig. 3. The prior art mutant VP2-K093C was not expressed and, therefore, its thermostability could not be assessed in this experiment and the thermostability was set to 0% in the graph. A large proportion of the new mutants (i.e. 3xmut, 5xmut, and 6xmut) showed good thermostability, an important parameter for the development of a stable vaccine against FMD.Example 2: Stabilization of SAT2 / SUD / 3 / 2013 VLPs

[0187] Supernatant containing the SAT2 / SUD / 3 / 2013 VLP was produced according to the method described in Example 1. The amount of intact VLPs was determined by ELISA as described in Example 1 and presented in Fig. 4. The results demonstrated that the 5xmut VLPs are produced at a somewhat higher levels than VP2-K093C VLP, while the other 2 mutants, i.e. 4xmut and 6xmut, did not produce well.

[0188] Next, a part of the harvested supernatant was heat treated at 56°C for 20 minutes and the amount of intact VLPs before and after heat treatment was determined by VHH M377F ELISA as described in Example 1. The percentage of capsids that survived the incubation at 56°C is presented in Fig. 5, demonstrating that the VP2-K093C VLP and the 5xmut VLP are the most stable VLPs after the heat treatment. From this data it can be concluded that the 5xmut VLP of the SAT2 / SU D / 3 / 2013 strain is produced at higher levels and shows a similar or improved thermostability as compared to the other mutant capsids tested.

[0189] Example 3: Capsid structure of SAT2 / SUD / 3 / 2013 VLPs

[0190] Grids (Quantifoil copper 2 / 1 200 mesh with a 2 nm carbon layer) of 5xmut-stabilised SAT2 / SUD / 3 / 2013 VLPs were prepared using a Vitrobot Mark IV with a total blot time of 6s, 30s wait and +6 force before plunge freezing into liquid ethane. Data were collected from a total of 8,958 exposures using a Titan Krios3 at eBIC (UK National Cryo-EM facility, visit ID: bi34631-4 on 21 / 11 / 23) equipped with a Falcon4i / SelectrisX detector (calibrated pixel size 0.723 A / pix) operating at 300 kV. The data were acquired in EER format at a magnification of 165 kX and total dose of 30 e-lf 2. Using the CryoSPARC™ live pre-processing interface, particles were picked using a template from a previous SAT2 data collection and a total of 188,469 were selected, classified in 3D (3 classes, heterogeneous refinement) from which 170,765 were selected. This particle set was then reextracted in the full box size, homogenously refined imposing I2 icosahedralsymmetry, yielding a volume with gold-standard-Fourier shell correlation of 2.8 A resolution (CryoSPARC). CTF parameters globally and locally were then refined in two rounds, with a positive Ewald sphere curvature sign, yielding a final reconstruction to 2.3 A global resolution (gold-standard FSC, CryoSPARC).

[0191] Shown is the final volume (Fig. 6).

[0192] The analysis reveals that the inventive VLPs are correctly assembled and resemble the published and deposited SAT2 / ZIM / 7 / 83 mutant VP2-S093Y virus (PDB:5ACA). The position of the mutations in the capsid is such that they do not contribute to the major antigenic surfaces of the virus. This is because two mutations (VP1-T012N and VP4-D053G) are internal and function by stabilising the network of internal interactions, while the other 3 mutations each to a cysteine residue (VP2-K093C (Porta et al., 2013, PLoS Pathog) and VP2-N194C + VP3-S196C) are designed to function by forming covalent interactions at the interface between the pentameric structural building blocks of the capsid. Thus, this beneficial combination of amino acid modifications achieves a VLP design, which is minimally disruptive of the antigenic properties while improving the chemical and structural integrity of the VLPs.

[0193] In conclusion, the capsid structural analysis surprisingly revealed that the beneficial combination of amino acid modifications is suitable to impart beneficial capsid stability, confirming the experimental results.

[0194] Example 4: Immunogenicity of SAT2 / SUD / 3 / 2013 VLPs

[0195] A prime-boost vaccination study was performed to evaluate the immunogenicity of four mutant SAT2 / SUD / 3 / 2013 VLPs (i.e. VP2-K093C, 4xmut, 5xmut, and 6xmut). The four vaccines were produced based on the SAT2 / SUD / 3 / 2013 VLP antigens produced in Example 2. Prior vaccine formulation, cell culture supernatant containing the antigen was concentrated 20 times by polyethylene glycol (PEG) precipitation using 5% PEG8000. The vaccines were subsequently formulated at 5 pg / ml with a proprietary oil-in-water adjuvant (i.e. SVEA-E). The water phase ofthe vaccine contained 50 mM HEPES pH 7.5 - 100 mM KCI buffer.

[0196] Four groups of cattle, each comprising 5 animals, were available for this study. Each group received a different SAT2 / SU D / 3 / 2013 vaccine that was administered intramuscularly as a 2-ml dose. Twenty-eight days later, animals received a booster vaccination with the same vaccine. Blood samples were taking regularly during the study. The virus neutralization titre (VNT) in blood against the SAT2 / SUD / 3 / 2013-related SAT2 / SU D / 4 / 2010 strain was determined by VN assay.

[0197] All animals developed high levels of VNT antibodies, as shown in Table 2:

[0198] Table 2

[0199] <

[0200] <

[0201] <

[0202]

[0203] <

[0204] Specify what the cut-off is for a positive VNT assay.

[0205] From the animal study it can be concluded that the recombinant SAT2 / SUD / 3 / 2013 VLPs containing the 5 or 6 modifications of the present invention (5xmut- and 6xmut-stabilized VLPs) can induce high levels of neutralizing antibodies similar to that of the prior art mutant VP2-K093C.

[0206] Example 5: Stabilization of SAT3 / ZAM / 3 / 2015 VLP

[0207] Mutations in SAT3 / ZAM / 3 / 2015 (a strain isolated in 2015 from cattle in Zimbabwe) were designed to be functional orthologs of those in SAT2: VP3_H142D in SAT3 is the same as VP3-H143D in SAT2, and the potential disulphide bridge forming mutations in SAT3 (VP3-H194C and VP2-Q194C) are the functional ortholog of VP3-A196C and VP2-N-194C in SAT2 / ETH / 65 / 2009.Thus, SAT3 / ZAM / 3 / 2015-6xmut mimic (VP2-A093C+VP2-Q194C+VP3-H194C+VP1-T012N+VP4-D053G+VP3-H142D) is equiv. to SAT2 / ETH / 65 / 2009-6xmut: (VP2-K093C+VP1 -T012N+VP4-D053G+VP2-N194C+VP3-A196C+VP3-H 143 D ) . There is no SAT3 equivalent of SAT2-5xmut.

[0208] Wild-type, VP2-A093C, and the 6xmut mimic were expressed in a transient gene expression (TGE) system as previously described (Meyer et al., 2019, PLoS ONE; 14 https: / / doi.org / 10.1371 / journal.pone.0068674 P ). The TGE system avoids the generation of recombinant baculoviruses and allows the rapid screening of multiple VLP mutants. Expression of VLPs was performed in Tnao38 insect cells that were transfected with plasmids containing the P1-2A-3C expression cassette based on the SAT3 / ZAM / 3 / 2015 strain. After incubation to allow for VLP expression, cell culture supernatants were harvested and subjected to sucrose gradient centrifugation with the gradient containing 10-50% sucrose in HEPES 50 mM, NaCI 0.2 M, pH 8.0. Fraction(s) with the highest VLP level based on FMDV protein content were collected and subjected to VLP quantification using a homologous sandwich ELISA similar to the one described in Example 1 , with integrin av[36 as the capture ligand and HRP-labeled integrin av[36 as the detection ligand. The integrin-av[36-based ELISA can be used as a tool for the quantification and detection of intact FMDV capsids (unpublished).

[0209] The results suggest that the prior art VP2-A093C mutation improves the expression level of intact VLPs as compared to wild-type, while the novel 6xmut-mimic VLPs display an even further improvement of VLP yields (Fig. 7).

[0210] Example 6: Capsid structure of SAT3 / ZAM / 3 / 2015 VLPs

[0211] Grids (Quantifoil copper 2 / 1 200 mesh with a 2 nm carbon layer) of 6xmut-mimic stabilised SAT3 / ZAM / 3 / 2015 VLPs were prepared using a Vitrobot Mark IV with a total blot time of 6s, 30s wait and +6 force before plunge freezing into liquid ethane. Data were collected at the Oxford Particle Imaging centre (OPIC) on theTitan Krios G3i equipped with Falcon4i-SelectrisX. A total dose of 30 e7A2was applied at a magnification of 165kX, corresponding to a calibrated pixel size of 0.7303 A / pix. Pre-processing was performed in CryoSPARC live with particles picked using a low pass filtered template of a previous FMDV VLP reconstructed volume. Picked particles were binned 4 times and a total of 93,962 selected particles were first aligned with I2 symmetry before 3D classification into 20 classes. The class with clear secondary structure was then selected (58,746 particles), extracted to a bigger box size (2-fold binned) and then further classified in 3D before a final particle set was selected (17,083 particles), refined with I2 symmetry to 2.45 A resolution before CTF refinement, taking into account Ewald sphere curvature, resulting in a final reconstruction to 2.13 A resolution (gold-standard FSC, CryoSPARC), Fig 8.

[0212] The analysis reveals that the inventive VLPs are correctly assembled. The position of the mutations in the capsid is such that they do not contribute to the major antigenic surfaces of the virus. This is because two mutations (VP1-T012N and VP4-D053G) are internal and function by stabilising the network of internal interactions, while 3 of the other 4 mutations each to a cysteine residue (VP2-A093C (Porta et al., 2013, PLoS Pathog) and VP2-Q194C+VP3-H194C) are designed to function by forming covalent interactions at the interface between the pentameric structural building blocks of the capsid. The VP3-H142D mutation is likewise close to an interface and intended to moderate the trigger of capsid dissociation by low pH. Thus, this combination of amino acid modifications achieves a VLP design, which is minimally disruptive of the antigenic properties while improving the pH and thermal stability of the VLPs.

[0213] Example 7: PD50 study with SAT2 / SU D / 3 / 20135xmut VLPs

[0214] To determine the efficacy of a FMD vaccine, challenge PD50 trials (50% protective dose) in target animals must be performed, as described in the WOAH terrestrial manual chapter 3.1.8 and European Pharmacopoeia (Ph. Eur.) Monograph 0063 on FMD (ruminants) vaccine (inactivated). For determining the PD50 value of avaccine containing the SAT2 / SU D / 3 / 20135xmut VLPs, 4 groups of animals were available. Animals in groups 1 , 2 and 3 were vaccinated with the

[0215] SAT2 / SU D / 03 / 2013 VLP-5xmut vaccine by decreasing the volume, i.e. 2.0 ml, 0.5 ml and 0.125 ml, respectively. The 2 animals in Group 4 served as unvaccinated challenge control animals.

[0216] For producing the SAT2 / SU D / 3 / 20135xmut VLP antigen a bioreactor containing approximately 2 106Tnao38 insect cells per ml was inoculated with recombinant baculovirus at low MOI and incubated at 32.0°C for 5 days. After incubation, the antigen was clarified through high g-force centrifugation for 10 min. Subsequently, the antigen was further clarified and concentrated using filtration. The baculoviruses in the antigen were then inactivated with binary ethylenimine (BEI) for 72 hours and remaining BEI was neutralized with sodium-thiosulfate. After inactivation, the antigen was clarified further by another round of high g-force centrifugation. After buffer exchange to 50 mM HEPES-100 mM KCI pH 7.5 with 15% (v / v) glycerol, the antigen was stored frozen. The vaccine for group 1, 2, and 3 was formulated using this frozen antigen as described under Example 4.

[0217] All animals were challenged via the intradermolingual route at 21 days post vaccination using the homologous FMDV strain SAT2 / SU D / 03 / 2013 at a dose equivalent to 10,000 cattle ID50. Blood samples were taken on days 0 -8 after challenge. The level of FMDV challenge virus in blood was determined by realtime RT-PCR. FMD-specific lesions on the feet were scored daily after challenge for 8 days.

[0218] Viremia post challenge was detected on a single day (Ct-values >35.5) for the protected animals in group 1 , 2 and 3. Two unprotected animals in group 3 were positive (Ct-values >33.4) for two days (on 2 dpc the scientific endpoint was reached for these animals and were euthanized). The unvaccinated control animals were viraemic (Ct-value <29.5) on day 1 , 2, and 3 post challenge (on 3 dpc the control animals reached the scientific endpoint and were euthanized). At the end of the study (i.e. day 8 post challenge) all vaccinated animals in group 2, 4 out 5 of the vaccinated animals in group 1 and 2 out 5 of the vaccinated animals in group 3 were protected against challenge, whereas both control animals were not.Study results are summarized in Table 3.

[0219] Table 3. Animal groups, treatment, and results of the SAT2 / SU D / 03 / 2013 PD50 study.

[0220]

[0221] * PD50 calculation with method of Karber (Anonymous, 2017, Statistical analysis of results of biological assays and tests (chapter 5.3), European Pharmacopoeia 9.0, EDQM: 635-664).

[0222] From the PD50 trial it can be concluded that a vaccine harbouring the recombinantly produced SAT2 / SUD / 03 / 2013 VLPs containing the 5x modifications of the present invention (‘5xmut’ -stabilized VLPs) was of high potency (i.e. 10.6 PD50).

[0223] Conclusions

[0224] In the present invention, it could be shown that the amino acid modifications in the amino acid sequence of the capsid precursor protein in addition to the prior art VP2-X093C mutant result in virus-like particle mutants that are significantly more thermostable than the wild-type or the prior art VP2-K093C mutant and can be produced at high level, in particular for SAT2 and SAT3 serotypes. The VLPs derived from these mutant capsid precursor proteins are immunogenic and can be used for the vaccination of subjects to provide protection against an infection with FMDV.

Claims

CLAIMS1. A recombinant foot-and-mouth disease virus (FMDV) capsid precursor protein comprising at least the virus proteins VP1 , VP2, VP3 and VP4, wherein the amino acid sequence of the capsid precursor protein is modified by replacement of amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 93 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C),and characterized in that the amino acid sequence of the capsid precursor protein further comprises at least one of the following modifications (I) to (V):(I) a replacement of amino acid 194 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 194 of the VP2 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C), (II) a replacement of amino acid 196 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 196 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 by cysteine (C), (III) a replacement of amino acid 143 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 143 of the VP3 amino acid sequence as set forth in SEQ ID NO:1 by aspartic acid (D), (IV) a replacement of amino acid 12 of the VP1 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 12 of the VP1 amino acid sequence as set forth in SEQ ID NO:1 by asparagine (N), or (V) a replacement of amino acid 53 of the VP4 amino acid sequence as set forth in SEQ ID NO:1 or of the amino acid corresponding to amino acid 53 of the VP4 amino acid sequence as set forth in SEQ ID NO:1 by glycine (G).

2. The recombinant FMDV capsid precursor protein according to claim 1 , wherein the amino acid sequence of the capsid precursor protein comprises at least:(a) the modifications (I) and (II); or(b) the modifications (III), (IV) and (V); or(c) the modifications (I), (II), (IV) and (V); or(d) the modifications (I), (II), (III), (IV) and (V).

3. The recombinant FMDV capsid precursor protein according to claim 2, wherein the amino acid sequence of the capsid precursor protein comprises at least:(c) the modifications (I), (II), (IV) and (V).

4. The recombinant FMDV capsid precursor protein according to any one of the preceding claims, wherein the amino acid sequence of the capsid precursor protein comprises the amino acid sequence of SEQ ID NO. 2, the amino acid sequence of SEQ ID NO. 3, or the amino acid sequence of SEQ ID NO. 4.

5. The recombinant FMDV capsid precursor protein according to any one of the preceding claims, which comprises at least the capsid precursor protein P1.

6. An isolated nucleic acid encoding recombinant FMDV capsid precursor protein according to any one of claims 1 to 5.

7. An expression vector comprising an expression cassette comprising the nucleic acid sequence according to claim 6 operably linked to a promoter.

8. The expression vector according to claim 7, which is a baculovirus expression vector.

9. The expression vector according to claim 7 or 8, wherein the FMDV is of the SAT2 or SAT3 serotype.

10. The expression vector according to claim 9, wherein the FMDV is of the SAT2 serotype.

11. The expression vector according to any one of claims 8 to 10, the vector comprising an expression cassette for expressing a nucleic acid sequenceencoding a protease capable of cleaving the capsid precursor protein.

12. The expression vector according to claim 11 , wherein the capsid precursor protein comprises the capsid precursor P1 and the 2A peptide and the protease is 3C.

13. A method of producing FMD virus-like particles (VLP) in a recombinant expression system, the method comprising:(a) infecting a host cell with an expression vector according to any one of claims 7 to 12, wherein the host cell is capable of recombinantly producing the VLP,(b) culturing the host cell under conditions under which the host cell produces the FMDV VLP, and(c) harvesting FMDV VLP produced by the host cell from the cell culture.

14. The method according to claim 13, wherein the host cell is an insect cell.

15. The method according to claim 13 or 14 the method further comprising:(d) incorporating the FMDV VLP into a vaccine by addition of a pharmaceutically acceptable carrier.

16. A vaccine for use in the protection of a subject against an infection with FMDV, the vaccine being obtainable by a method according to claim 15.

17. A method of protecting a subject against an infection with FMDV, which comprises the step of producing an FMDV VLP by a method according to claims 13 or 14, incorporating the VLP into a vaccine by addition of a pharmaceutically acceptable carrier, and administering the vaccine to the subject.

18. A vaccine comprising a FMDV VLP produced from a recombinant capsid precursor protein according to any one of claims 1 to 5.

19. The vaccine according to claim 18, wherein the recombinant capsidprecursor protein is from a FMDV of the SAT2 or SAT3 serotype.

20. A method of protecting a subject against an infection with FMDV, the method comprising the steps (a) to (d) according to claims 13 to 15 to obtain a vaccine, and(e) administering the vaccine to the subject.

21. A baculovirus expression vector according to any one of claims 7 to 12 for use in the manufacture of a medicament for the protection of a subject against an infection with FMDV.

22. Vaccine for the protection of a subject against an infection with FMDV, the vaccine comprising as antigen an effective amount of FMDV VLPs, the FMDV VLPs being obtainable by the method according to claim 15.

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