Method for obtaining virus-like particle of foot-and-mouth disease virus

Abstract

FIELD: biotechnology.SUBSTANCE: method for producing a virus-like particle (VLP) of foot-and-mouth disease virus (FMDV) strain Asia1 in a baculovirus expression system is described, wherein the method comprises: (i) infecting an insect cell with a baculovirus expression vector, wherein the insect cell is capable of recombinantly producing FMDV VLP, (ii) culturing the insect cell in a cell culture medium under conditions in which the insect cells produce FMDV VLP, wherein the culturing is carried out for 5 days or more after infection, (iii) collecting FMDV VLP produced by the insect cells from the cell culture medium. A method for producing a virus-like particle (VLP) of foot-and-mouth disease virus (FMDV) strain SAT2 in a baculovirus expression system is disclosed, wherein the method comprises: (i) infecting an insect cell with a baculovirus expression vector, wherein the insect cell is capable of recombinantly producing FMDV VLP, (ii) culturing the insect cell in a cell culture medium under conditions in which the insect cells produce FMDV VLP, wherein the culturing is carried out for 5 days or more after infection, (iii) collecting FMDV VLP produced by the insect cells from the cell culture medium. Also a method for protecting an individual from FMDV infection is disclosed, which comprises the stage of producing FMDV VLPs by the disclosed method, incorporating the VLPs into a vaccine, adding a pharmaceutically acceptable carrier, and administering the vaccine to the individual.EFFECT: expanding the range of means for protecting individuals from FMDV infection.8 cl, 12 dwg, 1 tbl, 9 ex

Description

[0001] The present invention relates to the field of veterinary medicine and virology. In particular, the invention relates to a method for producing a virus-like particle (VLP) of foot-and-mouth disease virus (FMDV) strains Asia1 and SAT2 in a baculovirus expression system, wherein the method comprises the steps of (i) infecting an insect cell with a baculovirus expression vector, (ii) culturing the insect cell in a cell culture medium for 5 days or more after infection, and (iii) collecting the FMDV VLP from the cell culture medium. Furthermore, the invention relates to a vaccine for use in protecting an individual from FMDV infection, wherein the vaccine is obtained by the method of the invention.

[0002] Background of the invention

[0003] Foot-and-mouth disease (FMD) is a highly contagious acute viral disease of domestic and wild ungulates. It is classified as a transboundary animal disease by the Food and Agriculture Organization of the United Nations (FAO). It is also a notifiable disease. FMD is endemic in large areas of Africa, South America, the Middle East, and Asia and is generally the most economically significant infectious disease of domestic animals, affecting cattle, pigs, sheep, goats, and other ungulates such as buffalo and deer. FMD once spread worldwide but has been eradicated from some regions, including North America and Eastern Europe. In endemic countries, FMD imposes economic restrictions on international trade in livestock products and can easily re-enter disease-free areas unless strict precautions are taken.FMD impacts the entire livestock industry with loss of income for local farmers.

[0004] Modern vaccines are made from inactivated viruses. Before virus inactivation, live FMD virus is produced in high-containment laboratories, which limits FMD production. The cost of building and maintaining such facilities is higher than for conventional facilities, and due to containment restrictions, operating costs are also higher.

[0005] Effective vaccination against FMD requires the presence of intact FMDV capsids (also known as 146S particles) instead of capsid structural elements, which have been shown to be insufficiently immunogenic (Doel and Chong, 1982, Archives of Virology). Inactivated FMD viruses are unstable structures that easily disintegrate into capsid structural elements at acidic pH or elevated temperatures. Therefore, a cold chain is required to deliver effective FMD vaccines to livestock producers. Consequently, there is a significant vaccine shortage worldwide, particularly in Africa. Thus, a new vaccine technology is needed for commercial FMD vaccines that can overcome many of the shortcomings of current classical viral vaccines.

[0006] A new vaccine technology is needed for commercial FMD vaccines that can overcome many of the shortcomings of current inactivated virus-based vaccines.

[0007] Virus-like particle (VLP) technology is currently considered one of several technologies that have the potential to become an effective alternative to conventional inactivated vaccines. Compared to currently used technology, VLP technology offers advantages such as improved product stability, greater flexibility in production location (low-contamination production), and faster responses to outbreaks caused by new strains. VLP-based vaccines are being developed as marker vaccines, reducing the need for production steps that remove non-structural proteins.

[0008] The FMDV genome encodes a single open reading frame (ORF) that produces a precursor polyprotein that is processed into twelve mature viral proteins, Fig. 1A (from: Balinda et al. Virology Journal 2010, 7:199). The intermediate form of the P1 polyprotein contains four capsid structural proteins, VP1-VP4, located immediately upstream of the 2A protein, which causes non-proteolytic separation of the P1 and P2 polyproteins during translation, releasing P1-2A from P2. The P1-2A polyprotein is then processed by FMDV protease 3C into 2A, VP0 (also known as 1AB), VP3 (1C), and VP1 (1D). The VP0 protein is thought to be separated into VP4 and VP2 during encapsidation. FMDV virions are formed by self-assembly from processed viral structural proteins.

[0009] VLPs for use in VLP-based vaccines can be produced by recombinant expression of FMDV precursor proteins in suitable host cells in analogy to the self-assembly of FMDV virions.

[0010] The baculovirus expression vector platform is currently used as one of the preferred platforms for VLP production. For example, recombinant expression can be performed in a baculovirus expression system using a modified 3C protease, which is less toxic to insect cells (Porta et al (2013) J Virol Methods). VLPs self-assemble from processed viral structural proteins VP0, VP3, and VP1, which are released from the P1-2A structural precursor protein by the virally encoded 3C protease. The moderate and non-toxic activity of the 3C enzyme in the P1-2A-3C cassette allows for the expression and processing of the P1-2A precursor into structural proteins that assemble into empty capsids.Thermal stability and resistance of VLPs to low pH can be improved by introducing covalent linkages between capsid proteins, such as cysteine ​​bridges (WO2002 / 000251) or by introducing other mutations through rational engineering (Porta et al. (2013) PLoS Pathog).

[0011] However, the relatively low expression levels of FMDV VLPs enabled by the baculovirus expression platform limit the development of a VLP-based FMD vaccine. Furthermore, the inventors found that FMDV VLPs collected from insect cell culture exhibit only moderate thermal stability, particularly for certain serotypes.

[0012] Proteins produced by baculovirus expression systems typically enter insect cells unless the proteins contain a signal sequence that directs them to the extracellular environment. Recombinant proteins taken up by insect cells can be released by cell disruption methods known in the art. The resulting cell lysate contains all cellular components and debris and often requires labor-intensive purification to obtain the recombinant protein in purer form. Furthermore, cell disruption methods also release numerous unwanted cellular proteins, such as proteases, which can degrade the desired proteins, thereby reducing protein yield and quality. Therefore, targeting sequences are often intentionally inserted into protein sequences if the protein of interest needs to be directed to the supernatant, from which it can be easily recovered.Furthermore, the authors found that FMDV VLPs purified from lysed insect cells had only moderate thermal stability, particularly for certain serotypes.

[0013] Therefore, there is a need in the art for improved methods for producing FMDV VLPs in insect cells that provide high yields with good thermal stability of the produced VLPs.

[0014] Essence of the invention

[0015] It was unexpectedly found within the scope of the present invention that FMDV VLPs of the Asia1 or SAT2 strain have increased stability when cultured for at least 5 days, i.e., by collecting the VLPs at the earliest 5 days post infection (dpi). It was found that, although not engineered with signal sequences, a significant portion of the VLPs were transported into the cell culture medium, which appears to be an active process, since VLP levels were found to increase in the cell culture medium before cells ruptured as a result of baculovirus infection. It is believed that VLPs mature as they move into the extracellular matrix. Since a significant portion of the VLPs are ultimately transported into the extracellular matrix, this explains the unexpected observation within the scope of the present invention that VLPs obtained from the cell culture medium at least 5 days post infection, in general (i.e.,(optionally including VLPs from cells, e.g., released into the environment after cell lysis) are more stable than those collected at an earlier time point (4 dpi or earlier).

[0016] Notably, it was possible to observe that VLPs appear to mature as they move toward the extracellular matrix. This may explain the unexpected observation in the present invention that VLPs obtained from cell culture medium are more stable than those obtained from cell lysates.

[0017] Thus, in a first aspect, the present invention relates to a method for producing a virus-like particle (VLP) of foot-and-mouth disease virus (FMDV) strain Asia1 or SAT2 in a baculovirus expression system, comprising the steps of:

[0018] (i) infecting an insect cell with a baculovirus expression vector, wherein the insect cell is capable of recombinantly producing FMDV VLP,

[0019] (ii) culturing the insect cell in a cell culture medium under conditions in which the insect cell produces the FMDV VLP, wherein the culturing is carried out for 5 days or more after infection,

[0020] (iii) collecting FMDV VLPs produced by insect cells from the cell culture medium (including the supernatant and optionally the contents of the cells, such as after lysis of those cells).

[0021] In a second aspect, the invention relates to a vaccine for use in protecting an individual from an FMDV infection, the vaccine being produced by the method of the present invention.

[0022] In a third aspect, the invention relates to a method for protecting an individual from an FMDV infection, which comprises the step of producing an FMDV VLP by the method of the present invention, incorporating the VLP into a vaccine by adding a pharmaceutically acceptable carrier, and administering the vaccine to the individual.

[0023] Detailed description of the invention

[0024] DEFINITION OF TERMS

[0025] The term "capsid" of a virus is often used in this field to refer to the protein shell of the virus, which typically contains its genetic material.

[0026] A "capsid protein precursor" is a structural protein that participates in the formation of a viral capsid or a structural element thereof. FMDV capsid protein precursors typically contain the P1 structural protein. Since the P1 protein is processed by FMDV 3C protease (3Cpro) into mature VP0, VP3, and VP1 proteins, the P1 protein may also be referred to as a polyprotein or proprotein. In the context of the present invention, an FMDV capsid protein precursor typically contains at least P1, including the VP1, VP2, VP3, and VP4 proteins. Alternatively, an FMDV capsid protein precursor may contain one or more of the VP1, VP2, VP3, and VP4 proteins. An FMDV capsid protein precursor may also contain the VP0 protein, which contains the VP2 and VP4 proteins. Most preferably, the FMDV capsid protein precursor comprises at least the P1 and 2A proteins (also referred to herein as the P1-2A capsid precursor).

[0027] A "virus-like particle" (VLP), which may also be referred to in the art as an "empty capsid," is a structure that contains the protein coat of a virus but lacks the RNA or DNA genome. A VLP should be antigenic and immunogenic similar to the wild-type virus, as it retains the same structural epitopes, but it should not cause infection due to the absence of a viral genome.

[0028] FMDV VLP is typically formed from the capsid precursor P1-2A. As described above, protease 2A cleaves itself at its C-terminus, releasing P1-2A from P2. Protease 3C processes the capsid precursor P1-2A, producing 2A and the capsid proteins VP0, VP3, and VP1. The VLP is formed through self-assembly from these capsid proteins.

[0029] VLPs can also be produced in the baculovirus expression system of the present invention using a modified 3C protease that is less toxic to insect cells (Porta et al. (2013) J Virol Methods). The intermediate and non-toxic activity of the 3C enzyme in the P1-2A-3C expression cassette allows for recombinant expression and processing of the P1-2A precursor into the structural proteins VP0, VP1, and VP3, which are assembled into VLPs. VLP production can be examined and confirmed using methods known in the art, such as sucrose density gradient centrifugation or electron microscopy. Monoclonal antibodies specific for conformational epitopes on the wild-type virus can be used to examine whether the structure and antigenicity of the empty capsid are preserved.

[0030] The term "vaccine," as used herein, refers to a preparation that, when administered to an individual, induces or stimulates a protective immune response. A vaccine can provide immunity to a specific disease.

[0031] "To protect an animal against infection with FMDV" means to contribute to the prevention, mitigation or cure of a pathogenic infection with FMDV, or to contribute to the prevention, mitigation or cure of a disorder resulting from this infection, for example, to prevent or reduce one or more clinical signs resulting from infection with FMDV after treatment (i.e. after vaccination).

[0032] The term "prevention" or "preventive" is intended to refer to the prevention, slowing, inhibition, or retardation of FMDV infection through prophylactic treatment. A vaccine can, for example, prevent or reduce the likelihood of infectious FMDV entering a host cell.

[0033] The term "nucleic acid sequence" includes a sequence of RNA or DNA. It can be single-stranded or double-stranded. It can be, for example, genomic, recombinant, mRNA, or cDNA.

[0034] "Expression vector" (synonymous with "expression construct") typically refers to a plasmid or virus designed to express a recombinant gene in cells. The vector is used to introduce a specific gene into a target cell and can recruit the cellular protein synthesis machinery to produce the protein of interest (POI) encoded by the gene. To express a recombinant gene to produce the POI, the expression vector typically contains at least a promoter to initiate expression of the gene of interest (GOI) and may also contain one or more translation enhancers to increase the yield of the POI.

[0035] A "baculovirus expression vector" is a baculovirus-based expression vector used to express a recombinant gene in a host cell, such as an insect cell. Baculovirus expression systems are recognized 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 of the wild-type baculovirus genome is typically replaced with a recombinant gene or cDNA. These genes are often under the control of the baculovirus polyhedrin or p10 promoters.

[0036] The most common baculovirus used for gene expression is Autographa californica nucleopolyhedrovirus (AcNPV). AcNPV has a large (130 kbp), circular, double-stranded DNA genome. The gene of interest (GOI) is cloned into a transfer vector containing a baculovirus promoter flanked by baculovirus DNA derived from a nonessential locus, such as the polyhedrin gene. Recombinant baculovirus containing the GOI is produced by homologous recombination in insect cells between the transfer vector and the genome of the parent virus (such as AcNPV).

[0037] A "translation enhancer" is a nucleotide sequence that forms an element that can promote translation and thereby increase protein production. Translation enhancers are typically found in the 5' and 3' untranslated regions (UTRs) of mRNA. In particular, nucleotides in the 5' UTR immediately upstream of the ATG GOI initiation codon can have a significant effect on the level of translation initiation.

[0038] BACULOVIRUS EXPRESSING SYSTEM

[0039] In the method of the present invention, FMDV VLPs are produced in a baculovirus expression vector system (BEVS) using a baculovirus expression vector.

[0040] The baculovirus expression vector may be any baculovirus expression vector capable of recombinantly expressing the FMDV capsid protein precursor under the control of a promoter. The promoter is not particularly limited, but may be any promoter capable of recombinantly expressing the FMDV capsid protein precursor 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 MD 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 orf46 viral gene promoter of S. exigua nucleopolysaccharide virus (SeNPV) (described in M. Martínez-Solís et al. (2016) PeerJ, DOI 10.7717 / peerj.2183).

[0041] The expression vector may further comprise one or more translation enhancers that enhance recombinant expression of the FMDV capsid protein precursor. For example, the baculovirus expression vector may comprise two translation enhancers, Syn21 and p10UTR, as described in EP 20 203 373, incorporated herein by reference in its entirety.

[0042] Baculovirus expression vectors for use in baculovirus expression systems for recombinant protein expression are commercially available and are widely used in the art to produce proteins and virus-like particles. The systems may include, 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 expanded. Commercially available baculovirus expression vectors include, but are not limited to, the Top-Bac vector.® (ALGENEX, Spain), pFastBac vector ® (Thermo Fisher Scientific, Germany), flashBAC vector ® (Oxford Expression Technologies Ltd, UK) and BestBac vector ® (EXPRESSION SYSTEMS, CA).

[0043] The baculovirus expression vector used in the method of the present invention may thus comprise an expression cassette comprising a nucleic acid sequence encoding a precursor of the FMDV capsid protein, which is expressed in an insect cell under the control of a functional promoter, and preferably one or more translation enhancers and / or other cis-acting elements.

[0044] The nucleic acid sequence encoding the FMDV capsid protein precursor is not specifically limited to a certain strain and may be any FMDV strain belonging to serotype Asia1 or SAT2.

[0045] In the method of the present invention, the FMDV capsid protein precursor may contain all the elements necessary for VLP processing and assembly. Thus, the FMDV capsid protein precursor typically contains at least the capsid P1 precursor and preferably additionally contains peptide 2A. Peptide 2A is capable of releasing P1-2A from any protein sequence downstream of its C-terminus.

[0046] In a further preferred embodiment, the baculovirus expression vector further comprises a nucleic acid sequence encoding a protease capable of cleaving the FMDV capsid protein precursor. The protease may be any protease capable of cleaving the FMDV capsid protein precursor as a step in the production and assembly of FMDV VLPs. As mentioned above, for FMDV, the proteolytic processing of the P1 precursor into VP0 (VP2 plus VP4), VP3, and VP1 occurs via the viral protease 3C or its precursor 3CD. Thus, the protease is preferably the FMDV 3C protease. The sequence of the wild-type FMDV protease 3C from an FMDV strain (type A, but conserved among different FMDV strains) is described in the art and is disclosed in WO 2011 / 048353, which is incorporated herein by reference in its entirety.Protease 3C can also be a functional derivative incorporating one or more mutations that reduce its proteolytic activity, such as a mutation at cysteine ​​142.

[0047] The capsid protein precursor may be P1, which is cleaved by protease 3C into VP0, 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 P1, 2A, and 3C proteins. Expression of the 3C enzyme in the P1-2A-3C cassette enables the expression and processing of the P1-2A precursor into structural proteins that assemble into VLPs. The capsid protein precursor and protease can be expressed under the control of separate promoters or under the control of the same promoter. Furthermore, alternatively, the capsid protein precursors required for FMDV VLP assembly can be separated into multiple expression elements and expressed separately, such as by recombinant production of VP1, VP2, VP3, and VP4, or recombinant production of VP0, VP1, and VP3.In this alternative embodiment, proteolytic cleavage of the capsid protein precursor by protease 3C may not be necessary.

[0048] Cleavage of the capsid precursor protein or VLP can be analyzed using methods known in the art. For example, extracts from baculovirus-infected host cells can be analyzed by gel electrophoresis, and the separated proteins can be transferred to a nitrocellulose membrane for Western blotting. Western blotting with protein-specific antibodies should reveal the extent of protease-mediated cleavage. For example, for FMDV, the unprocessed capsid precursor protein (P1-2A) will appear as a band of approximately 81 kDa, and cleavage can result in VP3-VP1 (~47 kDa), VP0 (~33 kDa), VP2 (~22 kDa), VP3 (~24 kDa), and / or VP1 (~24 kDa).

[0049] METHOD FOR PRODUCING VIRUS-LIKE PARTICLES

[0050] The method of the present invention comprises culturing a host cell, which within the scope of the invention is an insect cell, under conditions suitable for the cell to express a capsid protein precursor from a baculovirus expression vector for the production of VLPs. The term "insect cell capable of recombinantly producing FMDV VLPs" thus means that the insect cell can be used as a host cell for the production of recombinant capsid protein precursors that assemble into VLPs.

[0051] The first step of the method of the invention comprises infecting an insect cell with a baculovirus expression vector (step (i) of the method of the invention). The insect cell may be any insect cell that is capable of producing FMDV VLPs in cell culture. In particular, the insect cell may be an Sf9 cell (a clonal isolate of Sf21 cells of Spodoptera frugiperda), an Sf21 cell, a High-Five® cell (BTI-TN-5B1-4), or a Tni cell (ovary 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.

[0052] Methods for infecting an insect cell with a baculovirus expression vector for recombinant expression of proteins are well known to those skilled in the art.

[0053] In the method of the invention, the cultivation of the insect cell is carried out in a cell culture medium (step (ii) of the method of the invention), such as a suspension cell culture in a serum-free medium.

[0054] Culturing infected insect cells under conditions in which the insect cell produces FMDV VLPs 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), pp. 306-320). General techniques for expressing recombinant proteins using BEVS in insect cell cultures are known in the art and are described, for example, in "Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques", Invitrogen®, Instruction Manual; L. King, The Baculovirus Expression System, A laboratory guide; Springer, 1992; Baculovirus and Insect Cell Expression Protocols, Humana Press, DW Murhammer (ed.) 2007; Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press, DR O'Reilly, 1993.

[0055] Traditionally, supplemented Grace's medium (TNM-FH) has been the preferred medium for culturing insect cells. However, since the advent of Grace's medium, other serum / hemolymph-dependent and serum-free formulations have been developed. The optimal range for growing and infecting most cultured insect cells is generally between 25°C and 30°C with a pH of 6.0 to 6.4.

[0056] In the method of the present invention, the infected cells are cultured for 4 or more days post infection (dpi). In the context of the present invention, it was unexpectedly observed that VP0 protein was present in the cell culture medium on days 4 and 5 after baculovirus infection and was absent on days 6 and 7 after infection. Simultaneously with the disappearance of VP0, VP2 protein appeared in the cell culture, especially at 5 dpi. Thus, it was possible to show that the VP0 protein in the culture medium was cleaved into VP2 and VP4 proteins. It is believed that the cleavage of VP0 into VP2 and VP4 occurs at the final stage of maturation of the viral particle (Curry et al., 1997, J. Virol. 71:9743-9752). Thus, the results obtained by the inventors indicate that mature VLPs appear at 5 dpi.

[0057] Thus, even though the recombinant capsid protein precursor produced by an insect cell may lack the signal sequence, VLPs formed from the recombinant capsid protein precursor are released by the cell into the cell culture medium. Therefore, using the supernatant as a source of vaccine antigen (optionally in addition to cells as the antigen source) increases the number of mature VLPs compared to using VLPs from cells alone, thus enhancing the observed stability.

[0058] Thus, culturing is carried out for five or more days after infection, such as five, six, or seven days, preferably five or six days, and most preferably five days. Although culturing for more than five days achieves a higher yield, the additional culturing time required is unfavorable for large-scale production from a cost standpoint. Therefore, culturing for five days was found to be optimal.

[0059] After culturing, insect cells can be separated from the cell culture to obtain a cell-free cell culture medium (also referred to as a supernatant; step (iii) of the method of the invention). Although the overall stability of the collected VLPs is higher in this case, the overall yield is lower, depending, of course, on the amount of VLPs still present in the cells at the time of collection. The term "supernatant" refers to a cell culture from which insect cells have been removed.

[0060] In one embodiment, the VLP is obtained only from the supernatant. Thus, cells are removed from the cell culture to obtain a cell-free cell culture medium, also referred to herein as the supernatant, which is substantially free of insect cells. "Substantially free" of insect cells means that only residual cells that are nonessential for the production of the VLP of the present invention may be present. Most preferably, the supernatant does not contain any residual cells.

[0061] Conventional methods for separating cells from small-scale or large-scale cell cultures are well known in the art and include one or more of membrane filtration such as ultrafiltration, centrifugation, and sedimentation.

[0062] VLP in the supernatant can be concentrated by dialysis, membrane filtration, or precipitation followed by centrifugation.

[0063] In step (iii) of the method of the present invention, FMDV VLPs produced by insect cells are collected from the cell culture medium, typically including collection of the supernatant and cells (by adding a lysis step), as is widely known in the art. Collection may include separation of the VLPs from the culture medium and, if necessary, further purification of the VLPs. Collection can be carried out by precipitation of the VLPs, for example, with polyethylene glycol (PEG). Chromatographic methods, such as affinity chromatography or ion exchange chromatography, can also be used to purify and concentrate the VLPs. Collection may also include ultrafiltration to concentrate the VLPs in the cell culture medium or diafiltration to concentrate the VLPs by replacing the cell culture medium with a preferred liquid or buffer.If the concentration and purity of the VLPs in the cell culture medium are high enough to produce a vaccine, step (iii) of the present invention may not involve any purification steps.

[0064] During collection, concentration and / or purification procedures, the presence of protease inhibitors may reduce unwanted proteolytic activity.

[0065] VACCINES AND THEIR OBTAINING

[0066] As described above, embodiments of the present invention are preferably applicable in the field of veterinary medicine, in particular for vaccination against FMD. Thus, the present invention further relates to the production of FMDV VLPs that are used to produce a vaccine.

[0067] In particular, the VLPs collected from the cell culture medium in step (iii) of the method of the invention can be used as an antigen for vaccinating individuals. Preferably, the VLPs are included in a composition comprising the VLPs and one or more pharmaceutically acceptable carriers.

[0068] Thus, the present invention also relates to a method for producing a vaccine, which comprises the step of producing FMDV VLP by the method as described above and incorporating FMDV VLP into the vaccine, for example, by adding a pharmaceutically acceptable carrier.

[0069] Pharmaceutically acceptable carriers are well known in the art. By way of example only; such a carrier may be as simple as sterile water or a buffered solution such as PBS. The vaccine may contain a single carrier or a combination of two or more carriers. The vaccine may also contain one or more pharmaceutically acceptable diluents, adjuvants, and / or excipients. The vaccine may also contain or be capable of expressing another active substance, for example, substances that can stimulate early protection before the VLP-induced adaptive immune response. The substance may be an antiviral agent such as type I interferon. Alternatively or additionally, the agent may be granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0070] The vaccine can be used therapeutically to treat existing FMDV infection (especially in herds or regions where the virus is endemic), but is preferably used prophylactically to block or reduce the likelihood of FMDV infection and / or prevent or reduce the likelihood of spread of the disease.

[0071] Many commercially available FMD vaccines are multivalent to provide protection against different FMD serotypes. Similarly, the vaccine of the present invention may contain multiple different VLPs directed against different serotypes and / or different subtypes within a given serotype.

[0072] Thus, in a further preferred embodiment, the method of the invention further comprises the step (iv) of incorporating the FMDV VLP into the vaccine by adding a pharmaceutically acceptable carrier.

[0073] The vaccine produced by the method described above can be used to protect an individual from FMDV infection.

[0074] The present invention also relates to a method for protecting an individual from FMDV infection by administering an effective amount of the vaccine of the present invention. The method for protecting an individual from FMDV infection comprises the step of obtaining FMDV VLPs by the method described above, incorporating the VLPs into a vaccine by adding a pharmaceutically acceptable carrier, and administering the vaccine to the individual.

[0075] In the case of FMD, the individual may be an even-toed ungulate. Animals susceptible to FMD include cattle, sheep, pigs, and goats among farm animals, as well as camelids (camels, llamas, alpacas, guanacos, and vicuñas). Also susceptible to FMD are some wild animals, such as hedgehogs, nutria, and any wild even-toed ungulates, such as deer, and zoo animals, including elephants.

[0076] INTRODUCTION

[0077] The present invention provides at least one administration to an animal of an effective amount of the vaccine according to the invention. The vaccine can be administered by any route known in the art, including any local or systemic route of administration. The administration can be carried out, for example, by introducing antigens into muscle tissue (intramuscular, i / m), into the skin (intradermal, i / c), under the skin (subcutaneous, s / c), under the mucous membrane (submucosal, s / m), into the veins (intravenous, i / v), into the body cavity (intraperitoneal, i / j), orally, anal, etc. For this vaccine, administration by i / m, i / d and s / c is preferred.

[0078] EXAMPLES

[0079] The invention is further described by means of the following non-limiting examples, which are intended to facilitate the invention by one skilled in the art.

[0080] BRIEF DESCRIPTION OF DRAWINGS

[0081] Fig. 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.

[0082] Fig. 2: The result of the experiment to determine the time-dependent O / TUR / 5 / 2009 VLPs, which were collected at 4, 5, 6, or 7 dpi. FMD proteins in cells (C) or cell culture supernatant (S) were visualized by Western blotting.

[0083] Fig. 3: Quantification of O / TUR / 5 / 2009 protein in cell culture supernatant collected at different time points after infection by ELISA.

[0084] Fig.4: Quantitative determination of O / TUR / 5 / 2009 protein in different samples by ELISA.

[0085] Figure 5: Western blot analysis of fractions derived from a 20-40% sucrose gradient. Bands were visualized using both anti-VP0 and anti-VP2 antibodies. The percentage of sucrose in the fraction is indicated below the graph.

[0086] Fig. 6: Western blot analysis of culture samples collected at either 4 or 7 dpi. Bands were visualized using polyclonal bovine serum.

[0087] Fig. 7: Percentage dissociation of Asia1 / Shamir / 89 VLPs incubated at 56°C for 20 min.

[0088] Fig. 8: Virus neutralizing titers induced after vaccination of cattle with O / TUR / 5 / 2009 VLPs obtained either from insect cells or from cell culture supernatant.

[0089] Fig. 9: Quantification by ELISA of the concentration of SAT2 / SAU / 6 / 2000 VLPs in ultrasonicated insect cell cultures collected at different time points after baculovirus infection (Example 8).

[0090] Fig. 10: Thermal stability of SAT2 / SAU / 6 / 2000 VLP after incubation at 46°C for 20 minutes (Example 8).

[0091] Fig. 11: Quantitative determination of the concentration of A / SAU / 1 / 2015 VLP in purified culture fluid of insect cells collected at different time points after baculovirus infection by ELISA (Example 9).

[0092] Fig. 12: Thermal stability of A / SAU / 1 / 2015 VLP after incubation at 56°C for 20 minutes (Example 9).

[0093] Obtaining baculovirus constructs

[0094] Cloning of baculovirus expression constructs was performed using standard cloning techniques well known in the art. Recombinant baculoviruses were produced using the ProEasy system. TMfrom AB Vector. They had the P1-2A-3Cpro expression cassette as described by Porta et al., 2013, J Virol Methods. To increase expression levels, the so-called Syn21 translation enhancer was placed upstream of the P1-2A-3Cpro open reading frame and the 3'-UTR from the p10 gene (P10UTR) of Autographa californica nucleopolysaccharide virus (AcNPV) was inserted downstream of the P1-2A-3Cpro coding region (Liuet al., 2015, Biotechnol Lett). Amino acid modifications were introduced using synthetic cDNA, which was placed into the transfer vector used to produce recombinant baculoviruses. The following baculovirus expression constructs were used in the examples described below for recombinant VLP production in insect cells:

[0095] i) Expression construct O / TUR / 5 / 2009, containing the P1-2A-3Cpro FMDV expression cassette of strain O / TUR / 5 / 2009, not stabilized by any mutation;

[0096] ii) An expression construct O / TUR / 5 / 2009-VP2-S93F comprising the P1-2A-3Cpro FMDV expression cassette of strain O / TUR / 5 / 2009 stabilized by the VP2-S93F mutation as described in WO 2014 / 154655 A1;

[0097] iii) Expression construct A / IRN / 7 / 2013-VP2-H93F, comprising the P1-2A-3Cpro FMDV expression cassette of strain A / IRN / 7 / 2013, stabilized by the VP2-H93F mutation, as described in WO 2014 / 154655 A1;

[0098] iv) The expression construct SAT2 / SAU / 6 / 2000-VP1-T12N-VP4-D53G, containing the P1-2A-3Cpro FMDV expression cassette of the SAT2 / SAU / 6 / 2000 strain, stabilized by a mutation in VP1 (T12N) and VP4 (D53G); the VP1-T12N mutation refers to an amino acid mutation of threonine (T) to asparagine (N) amino acid at position 12 in VP1. The VP4-D53G mutation refers to an amino acid mutation of aspartate (D) to glycine (G) at position 53 in VP4.

[0099] v) An Asia1 / Shamir-VP2-S93C expression construct comprising the P1-2A-3Cpro expression cassette based on the FMDV strain Asia1 / Shamir / 89, stabilized by the VP2-S93C mutation, as described in WO 2014 / 154655 A1.

[0100] vi) Expression construct A / SAU / 1 / 2015-VP2-H93C, containing the P1-2A-3Cpro expression cassette based on FMDV strain A / SAU / 1 / 2015. The VP2-H93C mutation refers to an amino acid mutation of histidine (H) to cysteine ​​(C) at position 093 in VP2 and is as described in WO 2002 / 000251.

[0101] Baculovirus expression system was used to recombinantly express VLP.

[0102] Example 1

[0103] 100ml Tni cell culture with a concentration of 3.2×10 5Cells / ml were infected at MOI 1 with recombinant baculoviruses containing the O / TUR / 5 / 2009 expression cassette. After incubation at 27°C, cells were harvested at 4, 5, 6, or 7 days post infection (dpi) by centrifugation and resuspended in 10% of the original cell culture volume, achieving a concentration factor of 10x. Cell lysis was not used. The cell culture supernatant, i.e., the cell-free cell culture medium from which insect cells were removed by centrifugation, was left untreated. Supernatant and cell samples were analyzed by Western blotting using both the anti-VP0 monoclonal antibody (Loureiro et al., 2018, https: / / wellcomeopenresearch.org / articles / 3-88) and the polyclonal bovine serum FMD13.70.445 (MSD Animal Health).

[0104] The results shown in Figure 2 demonstrated that FMDV proteins were detectable in the cell culture supernatant as early as 4 dpi. Over time, and most noticeably on the VP0 Western blot, the amount of FMDV proteins in the cells decreased, while the amount of FMDV proteins in the cell culture supernatant increased, demonstrating that recombinant FMDV proteins are efficiently released from the cells into the culture medium.

[0105] The polyclonal serum blot revealed another interesting observation that is likely related to capsid maturation. On days 4 and 5, VP0 protein was present in the cell culture medium and was absent on days 6 and 7 post-infection. Simultaneously with the disappearance of the VP0 band, a band appeared on the polyclonal serum blot that may correspond to the VP2 protein. In this case, the Western blot indicates that the VP0 protein in the culture medium was cleaved into VP2 and VP4 proteins. It is believed that cleavage of VP0 into VP2 and VP4 occurs at the final stage of viral particle maturation (Curry et al., 1997, J. Virol. 71:9743-9752). This is an unexpected finding, since empty capsids do not contain the RNA genome and usually do not contain cleaved VP0. However, it has become possible to show that the culture medium is a good source of vaccine antigen because it contains mature VLPs, as opposed to cells.

[0106] To quantify differences in FMDV protein concentrations in cell culture supernatant, ELISA was performed using the monoclonal antibody INT-FMA-01-08 (MSD Animal Health), which detects both intact capsids (75S / 146S) and pentameric capsid structural elements (12S). For this purpose, serially diluted samples were incubated for 1 h at 37°C on microtiter plates, which were coated with antibody overnight at 4°C. After removing the samples and washing three times with PBS-Tween, a fixed amount of biotinylated INT-FMA-01-08 was added to the plates and incubated for 1 h at 37°C. The biotinylated antibody was removed and the plates were washed three times with PBS-Tween, after which peroxidase-conjugated streptavidin was added to the plates, followed by chromophore detection.

[0107] The graph in Fig. 3 is a visual representation of the ELISA results, demonstrating that the amount of VLPs in the cell culture medium increased by up to 3.4-fold at 7 dpi compared to 4 dpi. By comparing the ELISA data for untreated samples with those for samples that were heat-treated at 56°C for 50 min to convert 75S capsids to 12S pentamers, it was estimated that the VLP integrity (i.e., the amount of 75S) in the supernatant for unstabilized wild-type O / Tur / 5 / 2009 was 54%, indicating that intact capsids were indeed released into the cell culture medium.

[0108] This example shows that recombinant FMDV proteins are efficiently released from cells into the culture medium, with the number of VLPs in the cell culture medium increasing over time to form mature VLPs.

[0109] Example 2

[0110] Two 100ml Tni cell cultures with 3.2×105 Cells / ml were infected at MOI = 2 with recombinant baculoviruses containing the O / TUR / 5 / 2009-VP2-S93F expression cassette. After incubation at 27°C, cells from one culture were harvested at 4 dpi by centrifugation, and the cell pellet was then sonicated in 50 mM Tris buffer pH 8.0-100 mM KCl in 10% of the infected culture volume. The supernatant of the second cell culture was obtained by centrifugation at 7 dpi.

[0111] The different collection times for each fraction were based on the data presented in Example 1, which showed that the amount of recombinant proteins in cells was highest at 4 dpi, while in cell culture medium it was highest at 7 dpi. To test whether the cell culture supernatant could be concentrated in a simple manner, the material was concentrated using an ultrafiltration (UF) step using a membrane system with a molecular weight cutoff of 100 kDa. To quantify the recombinant FMDV proteins in the samples, ELISA was performed using the INT-FMA-01-08 monoclonal antibody, as described in Example 1. A reference sample with a known concentration (in units / mL ELISA or IU / mL) was included in the ELISA to estimate the concentration in the samples.

[0112] Figure 4 shows the individual ELISA graphs, while the obtained values ​​are shown in Table 1. From these data, it can be concluded that significantly more FMDV VLPs could be collected from the supernatant of the cell-based baculovirus expression system compared with cells (approximately 6x), and the supernatant could be concentrated in a 1-step method, which can be easily used in large-scale production.

[0113] Table 1. Quantitative determination of O / TUR / 5 / 2009 protein in different samples by ELISA

[0114] Sample Concentration in sample (IU / ml) Calculated concentration in the original cell culture (IU / ml) 4 dpi - cells (10x) 64 6,4 7dpi - supernatant (1x) 40 40 7dpi - supernatant, UV-concentrated (~18x) 570 32

[0115] This example shows that more O strain VLPs can be collected from the culture supernatant than from the cells.

[0116] Example 3

[0117] Two 100ml Tni cell cultures with 3.2×10 5Cells / ml were infected at MOI=1 with recombinant baculoviruses containing the O / TUR / 5 / 2009-VP2-S93F expression cassette. After incubation at 27°C, cells from one culture were harvested at 4 dpi by centrifugation and the cell pellet was sonicated in 50 mM Tris buffer pH 8.0-100 mM KCl in 10% of the infection culture volume. Cell culture supernatant was obtained by centrifugation at 7 dpi. Samples containing lysed cells and supernatant were subjected to zonal density gradient centrifugation. The gradient consisted of 20%-40% sucrose, and samples were loaded on top of the gradient and then centrifuged at 50,000×g for 50 min at 20°C. Gradient fractions were analyzed by Western blotting using the anti-VP2 monoclonal antibody F1412SA (Yanget al., 2007, Vet Immunol Immunopathol).

[0118] Western blot analysis showed that VP0 and / or VP2 proteins were detected in the gradient at a sucrose concentration level of approximately 35% where 75S particles were expected, indicating that intact VLPs were present in both the cells and the supernatant (Fig. 5). Western blot analysis also revealed that in the VLPs in the supernatant, their VP0 was partially processed to VP4 and VP2, as indicated by the presence of a relatively prominent VP2 band compared to the VP0 precursor band. This result confirms our earlier observation described in Example 1.

[0119] In this example, it was possible to show that the cell culture supernatant contains intact VLPs of the O-strain FMDV.

[0120] Example 4

[0121] Two 100ml Tni cell cultures with 3.2×10 5Cells / ml were infected at MOI 1 with recombinant baculoviruses containing the A / IRN / 7 / 2013-VP2-H93F expression cassette and then incubated at 27°C. One of the two cultures was harvested at 4 dpi, and the second was harvested at 7 dpi. Cells and cell culture supernatant fractions were separated by centrifugation. The resulting cell pellet was sonicated in 50 mM Tris buffer pH 8.0-100 mM KCl in 10% of the infected culture volume. The cell culture supernatant was left untreated.

[0122] Cell and supernatant samples were analyzed by Western blotting using polyclonal bovine serum FMD13.70.445 (Fig. 6). Visual inspection of the Western blots revealed that the VP2 band intensity was higher in the supernatant samples than in the cell lysate samples. Because the cell lysates were concentrated 10-fold, it could be concluded that at least 10-fold more VP2 protein was present in the extracellular medium at both 4 and 7 days post-infection. Western blotting also demonstrated that the cell culture supernatant contained significantly fewer P1 polyprotein processing intermediates and that the majority of the VP0 protein was cleaved to VP2 (and VP4). This again indicates that mature capsids are predominantly present in the cell culture supernatant, as discussed in Examples 1 and 3.

[0123] In this example, it was possible to show that there were more FMDV A-strain VLPs in the cell culture supernatant than in the cells at both 4 and 7 dpi.

[0124] Example 5

[0125] Tnao38 insect cells at a concentration of 2.2×10 6 Cells / mL in a 2 L bioreactor were infected at an MOI of 1 with recombinant baculoviruses containing the SAT2 / SAU / 6 / 2000-VP1-T12N-VP4-D53G expression construct. After incubation at 28°C, at 5 dpi, the cells were collected by centrifugation and the cell pellet was sonicated in 50 mM buffer pH 8.0-100 mM in 5% of the infected culture volume. The culture supernatant from the centrifugation step was further concentrated 18.6-fold by ultrafiltration using a membrane with a molecular weight cutoff of 30 kilodaltons (kDa).

[0126] The concentration of intact virus-like particles was determined by ELISA using the M377F VHH (Harmse et al., 2017, Front. Immunol. 8:960, doi: 10.3389 / fimmu.2017.00960). For this, serially diluted samples were incubated for 1 h at room temperature (rt) on microtiter plates that were coated with M377F overnight at 4°C. After removing the samples and washing three times with PBS-Tween, a fixed amount of biotinylated M377F was added to the plates and incubated for 1 h at rt. The biotinylated antibody was removed, and the plates were washed three times with PBS-Tween. Peroxidase-conjugated streptavidin was then added to the plates, followed by chromophore detection. According to ELISA, the 20x cell lysate contained 117 IU / ml of intact VLPs, while the concentrated culture supernatant contained 92 IU / ml.Thus, 46% of all SAT2 / SAU / 6 / 2000 VLPs were present in the cell culture supernatant at 5 dpi.

[0127] As a result, it was possible to show that FMDV SAT2 VLPs accumulate in cell culture supernatant.

[0128] Example 6

[0129] Erlenmeyer flasks containing 40 ml 1×10 6 Tnao38 insect cells per ml were inoculated with 3 ml of the P1 stock culture containing recombinant baculoviruses containing the Asia1 / Shamir-VP2-S93C expression construct. After incubation at 27.5°C, at 4 or 6 dpi, the cells were collected by centrifugation for 5 min at 3000 rpm. The resulting cell pellet was resuspended in 50 mM HEPES pH 8.0-100 mM KCl at a volume of 1 / 10 of the original culture volume, and the cells were lysed by sonication. The cell culture supernatant was also collected after centrifugation.

[0130] The resulting material was heat-treated at 56°C for 20 min, and the number of intact VLPs was determined before and after heat treatment by homologous ELISA using the M332F antibody (Harmse et al., 2017, Front. Immunol. 8:960) according to the method described in Example 5 but with incubation at 37°C instead of rt.

[0131] The percentage of capsids that survived after incubation at 56°C is shown in Figure 7. The results demonstrate that supernatant-derived VLPs are more thermostable than cell-derived VLPs, and a longer culture time (i.e., 6 days instead of 4) appears to enhance thermostability. This observation is not believed to be due to a stabilizing effect of the insect cell culture medium on these VLPs, as no stabilizing effect was detected in additional experiments designed to determine the effect of cell culture medium on VLP thermostability. A possible explanation is that VLPs in cell culture supernatant are more mature, as they are actively transported into the extracellular environment, similar to FMDV capsids in naturally infected cells.Consistent with the VLP maturation theory (as mentioned in Example 1), the data show that VLPs become more resistant to heat over time: the thermal stability of VLPs collected at 6 dpi is higher than that at 4 dpi.

[0132] In this example, it was possible to demonstrate that the thermal stability of FMDV VLPs of the Asia1 / Shamir / 89 strain obtained from cell culture supernatant is higher than that of VLPs obtained from cells.

[0133] Example 7

[0134] A test was conducted to demonstrate that VLPs obtained from cell culture supernatant are at least as immunogenic as cell-derived VLPs. Ten calves aged 4-6 months were divided into 2 groups of 5 calves each. On day 0, the calves were vaccinated intramuscularly (i.m.) with 2 ml of a vaccine formulated with 8 μg of FMDV VLPs of the O / TUR / 5 / 2009 strain and the in-house adjuvant SVEA-E. One group received VLPs obtained from insect cells, while the other group received VLPs obtained from cell culture supernatant. Blood samples were collected at 0, 7, 14, and 21 days post-vaccination (dpv). Serum was obtained from the clotted blood and then tested in a virus neutralization assay (VNT) using O / TUR / 5 / 2009.

[0135] O / TUR / 5 / 2009 VP2-S93C VLPs were produced at 30°C in 2-liter bioreactors containing 2×10 6Tnao38 insect cells per ml were infected at an MOI of 1. The cell culture supernatant and cells were collected at 5 dpi by centrifugation at 200×g. VLPs were released by sonication. The concentration of intact VLPs was determined by ELISA using VHH C1 (Wang et al., 2015, BMC Veterinary Research 11:120, DOI 10.1186 / s12917-015-0437-2) according to the method described in Example 5, but with incubation at 37°C instead of RT.

[0136] In all animals in both groups, high levels of neutralizing antibodies to FMDV were detectable at 7 dpv, resulting in group averages of 2.26 log 10 for a group of cells and 2.39 log 10 for the cell culture supernatant group (Fig. 8). Titers increased slightly to 2.30 log 10 and 2.53 log 10, respectively, on day 21 post-vaccination. The results indicate that both sources, cells and cell culture supernatant, provided immunogenic VLPs.

[0137] As a result, it was possible to show that VLPs obtained from cells or cell culture supernatant are immunogenic in both cases.

[0138] Example 8

[0139] In this example, we assessed whether harvesting SAT2 / SAU / 6 / 2000 VLPs at later time points improves capsid yield and stability.

[0140] To study the effect of harvest time after baculovirus infection on the yield and thermal stability of VLPs, a 2-liter bioreactor containing Tnao38 insect cells was inoculated at an MOI of 0.1 with recombinant baculoviruses containing the SAT2 / SAU / 6 / 2000-VP1-T12N+VP4-D53G expression cassette. The baculovirus-infected insect cell culture was incubated at 30°C, and a portion of the culture was harvested at 4, 5, and 6 days post-infection. The harvested materials were sonicated to lyse the insect cells and release intracellular VLPs. The resulting material (i.e., lysed cells in cell culture fluid) was then clarified by centrifugation at 3000×g for 10 min.

[0141] The amount of intact VLPs in the material was determined by ELISA using VHH M377F as described in Example 5.

[0142] It was found that harvesting at 5 or 6 days post-baculovirus infection (dpi) resulted in significantly higher VLP yield than harvesting at 4 dpi (see Fig. 9).

[0143] The purified material was heat-treated at 46°C for 20 minutes, and the number of intact VLPs was determined by ELISA before and after heat treatment. From these ELISA data, the percentage of capsids that survived after incubation at 46°C could be calculated (Fig. 10). The thermal stability of SAT2 VLPs collected at 5 or 6 dpi was higher than that at 4 dpi.

[0144] Overall, the data shown in this example indicate that the yield and thermal stability of SAT2 / SAU / 6 / 2000 VP1-T12N+VP4-D53G VLPs are optimal when VLPs are harvested at 5 dpi or later.

[0145] Example 9

[0146] In this example, the time point after infection at which the yield and thermal stability of A / SAU / 1 / 2015 VLP are most optimal was assessed.

[0147] To investigate whether harvest time also affects the yield and thermal stability of VLPs from a strain belonging to another FMDV serotype, a new group of recombinant baculoviruses with the P1-2A-3Cpro expression cassette was generated based on the A / SAU / 1 / 2015 strain according to the method described above. A / SAU / 1 / 2015 VLPs were stabilized by the VP2-H93C mutation.

[0148] VLPs were produced in a 2-liter bioreactor containing Tnao38 insect cells inoculated at an MOI of 0.1 with recombinant baculoviruses containing the A / SAU / 1 / 2015-VP2-H93C expression cassette. The baculovirus-infected insect cell culture was incubated at 28°C, and a portion of the culture was harvested at 3, 4, 5, 6, and 7 days postinfection. The collected material was centrifuged at 3000×g for 10 min to obtain clarified cell culture fluid.

[0149] The number of intact VLPs in the material was determined by ELISA using VHH M702F (Liet al., 2021, Vaccines: 9, 620, doi.org / 10.3390 / vaccines9060620) according to the method described in Example 5, except that the incubations were carried out at 37°C.

[0150] It was found that the concentration of VLPs in the cell culture fluid increased over time and was highest at 7 dpi (see Fig. 11).

[0151] The purified material was heat-treated at 56°C for 20 minutes, and the number of intact VLPs was determined by ELISA before and after heat treatment. From these ELISA data, the percentage of capsids that survived after incubation at 46°C could be calculated (Fig. 12). The thermal stability of A VLPs improved at 5 dpi compared to 3 dpi, but did not improve thereafter, indicating that the best thermal stability was already achieved at 5 dpi.

[0152] Overall, the data obtained for A / SAU / 1 / 2015 VLPs indicate that for best thermal stability, VLPs should not be collected earlier than 5 dpi.

[0153] CONCLUSION

[0154] The present invention demonstrated that recombinant FMDV proteins from strains Asia1 and SAT2 can be efficiently produced in a baculovirus / insect cell expression system when harvested at 5 dpi or later. It is believed that release from cells into the culture supernatant results in more mature VLPs. These VLPs exhibit higher thermal stability compared to VLPs obtained solely from cells. VLPs obtained from cell culture according to the present invention are immunogenic and can be used to vaccinate individuals, providing protection against FMDV infection.

Claims

1. A method for producing a virus-like particle (VLP) of foot-and-mouth disease virus (FMDV) strain Asia1 in a baculovirus expression system, the method comprising: (i) infecting an insect cell with a baculovirus expression vector, wherein the insect cell is capable of recombinantly producing FMDV VLP, (ii) culturing the insect cell in a cell culture medium under conditions in which the insect cell produces the FMDV VLP, wherein the culturing is carried out for 5 days or more after infection, (iii) collection of FMDV VLPs produced by insect cells from cell culture medium.

2. A method for producing a virus-like particle (VLP) of foot-and-mouth disease virus (FMDV) strain SAT2 in a baculovirus expression system, the method comprising: (i) infecting an insect cell with a baculovirus expression vector, wherein the insect cell is capable of recombinantly producing FMDV VLP, (ii) culturing the insect cell in a cell culture medium under conditions in which the insect cell produces the FMDV VLP, wherein the culturing is carried out for 5 days or more after infection, (iii) collection of FMDV VLPs produced by insect cells from cell culture medium.

3. The method according to paragraph 1 or 2, wherein the cultivation is carried out for five days after infection.

4. The method according to any one of the preceding claims, wherein the baculovirus expression vector comprises a nucleic acid sequence encoding an FMDV capsid protein precursor.

5. The method according to claim 4, wherein the baculovirus expression vector further comprises a nucleic acid sequence encoding a protease capable of cleaving the FMDV capsid protein precursor into one or more capsid proteins.

6. The method of claim 5, wherein the capsid protein precursor comprises the FMDV P1 capsid precursor and the 2A peptide, and the protease is 3C.

7. The method according to any of the preceding paragraphs, wherein the method further comprises: (iv) incorporating FMDV VLP into a vaccine by adding a pharmaceutically acceptable carrier.

8. A method for protecting an individual from FMDV infection, which comprises the step of producing FMDV VLPs by the method of any one of claims 1-6, incorporating the VLPs into a vaccine, adding a pharmaceutically acceptable carrier, and administering the vaccine to the individual.

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