US 12,697,381 B2Grant

Baculovirus expression vector

Issue Date:2026-08-04

•13 Claims

•3 Drawing Sheets

Abstract

The invention concerns a baculovirus expression vector for recombinantly expressing a Foot-and-mouth disease virus (FMDV) capsid precursor protein under control of a promoter, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein, wherein the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein is preceded at position −4 to −1 by the nucleic acid sequence 5′-AAAT-3′. The invention further relates to a host cell comprising the baculovirus expression vector, a method of producing FMDV virus-like particles (VLPs), and a method of producing a vaccine.

Metadata

Assignee

Intervet, Inc.

Inventor

Erwin Van Den Born

Application Information

Application Number:US 18/249,431

Filing Date:2021-10-21

Priority Date:2020-10-22

Art Unit:1671

Classifications

IPC:

A61K39/135C12N15/866

Patent Drawings (3 sheets)

Description

SEQUENCE LISTING

[0001] The instant application contains an electronic Sequence Listing which has been submitted in txt format via the Patent Center, the entire content of which is hereby incorporated by reference in its entirety. The Sequence Listing txt file submitted via the Patent Center is entitled “25115WO-SEQL.txt” and was created on Oct. 21, 2020, and is 61,440 bytes in size.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a National Stage application of International Patent Application No. PCT/EP2021/079180, filed Oct. 21, 2021, which claims priority to European Patent Application No. 20203379.1, filed Oct. 22, 2020.

[0003] The invention concerns a baculovirus expression vector for recombinantly expressing a Foot-and-mouth disease virus (FMDV) capsid precursor protein under control of a promoter, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein. The invention further relates to a host cell comprising the baculovirus expression vector, a method of producing FMDV virus-like particles (VLPs), and a method of producing a vaccine.

BACKGROUND OF THE INVENTION

[0004] 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.

[0005] Current vaccines are made of inactivated virus. Before the virus is inactivated, live FMD virus is produced in high containment facilities, limiting FMD vaccine production. 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.

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

[0007] 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 typically designed as marker vaccines which relieves the necessity of implementing production steps to 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 P1 polyprotein intermediate is comprised of four capsid structural proteins, VP1-VP4, sited immediately upstream of the 2A protein which causes non-proteolytic separation of the P1 and P2 polyproteins during translation to release P1-2A from P2. The P1-2A polyprotein is subsequently processed by the FMDV 3C protease into 2A, VP0 (also known as 1AB), VP3 (1C), and VP1 (1D). The VP0 protein separates into VP4 and VP2 during encapsulation. FMDV virions are formed by self-assembly from the processed virus structural proteins.

[0009] VLPs for use in VLP-based vaccines can be produced by recombinantly expressing FMDV precursor proteins in suitable host cells in analogy to the self-assembly of FMDV virions. FMDV VLP's are likewise formed by self-assembly from the processed virus structural proteins.

[0010] The thermostability and sensitivity to low pH of VLPs can be improved by the introduction of covalent links between the capsid proteins, such as cysteine bridges, or by the introduction of other rationally designed mutations (Porta et al. (2013) PLoS Pathog).

[0011] Because the initial yield of FMDV VLPs per milliliter cell culture is typically low, even when using common successful expression systems, there is a need to increase expression levels to make the VLP-based FMD vaccine a cost-effective alternative to the classic FMD vaccines currently on the market. Therefore, the baculovirus expression vector was optimized to improve the yield of VLPs and to achieve a large-scale production process that at least equals or even outperforms the conventional FMD vaccine production process in terms of antigen yield.

[0012] The baculovirus expression vector platform is currently used as one of the preferred platforms for the production of VLPs. However, the relatively low expression levels of FMDV VLPs provided by the baculovirus expression platform limits the development of a VLP-based FMD vaccine.

SUMMARY OF THE INVENTION

[0013] In the present invention, it has surprisingly been found that expression of an FMDV capsid precursor protein via the baculovirus expression system can be enhanced by optimizing the nucleic acid initiation sequence preceding the start codon of the open reading frame (ORF) encoding the FMDV capsid precursor protein. It could surprisingly be shown that in case the initiation sequence has the nucleic acid sequence at position −4 to −1 of 5′-AAAT-3′, the expression yields can be enhanced compared to baculovirus expression systems used in the art.

[0014] Thus, in a first aspect the present invention provides a baculovirus expression vector capable of recombinantly expressing a Foot and mouth disease virus (FMDV) capsid precursor protein under control of a promoter, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein, wherein the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein is preceded at position −4 to −1 by the nucleic acid sequence 5″-AAAT-3″.

[0015] In a second aspect of the invention, there is provided a host cell comprising the baculovirus expression vector of the present invention. Such a host cell can be used in vitro, in a tissue culture, the host cell typically being an immortalized cell.

[0016] In a third aspect, the invention provides a method of producing FMDV capsid precursor proteins, the method comprising the steps of: infecting a host cell with the baculovirus expression vector as described herein, and harvesting FMDV capsid precursor proteins produced by the host cell.

[0017] The invention further provides a method of producing FMDV VLPs, the method comprising the steps of: infecting a host cell with the baculovirus expression vector as described herein, wherein the expression vector further comprises a nucleic acid sequence encoding a protease capable of cleaving a capsid precursor protein into one or more capsid proteins to assemble into VLPs, and harvesting the VLPs produced by the host cell.

[0018] The invention further relates to the use of the baculovirus expression vector for the recombinant expression of a FMDV capsid precursor protein.

[0019] The invention further relates to a method of producing a vaccine by producing FMDV VLPs and incorporating the FMDV VLPs into a vaccine by addition of a pharmaceutically acceptable carrier.

[0020] The invention further relates to a method of protecting a subject against an infection with FMDV by expressing an FMDV capsid precursor protein from the baculovirus expression vector of the present invention in a host cell to produce VLPs, incorporating the VLPs into a vaccine by addition of a pharmaceutically acceptable carrier and administering the VLPs to the subject.

[0021] The invention further relates to a baculovirus expression vector according to the first aspect of the invention for use in the protection of a subject against an infection with FMDV.

[0022] The invention further relates to a baculovirus expression vector as described herein for use in the manufacture of a medicament for the protection of a subject against an infection with FMDV.

[0023] FIG. 1 is a Western Blot using anti-VP0 monoclonal antibody showing the level of expression of the Asia1/Irn/49/2011 VP2-S93Y FMDV capsid precursor protein of Asial serotype in Tni cells transfected with a baculovirus expression cassette comprising the AAAT sequence (lane 1) as compared to expression levels of Tni cells transfected with a baculovirus expression cassette without the AAAT sequence (lane 2).

[0024] FIG. 2 is a Western Blot using a cocktail of 5 SAT2-specific monoclonal antibodies: 1D5, DA10, GE11, GD12, and GG1 showing the level of expression of the SAT2/Egy/2/12 VP2-K93Y FMDV capsid precursor protein of serotype SAT2 serotype in Tni cells after transfection with a baculovirus expression cassette comprising the AAAT sequence (lane 1), as compared to expression levels in Tni cells after transfection with a baculovirus expression cassette without the AAAT sequence (lane 2).

[0025] FIG. 3 is a Western Blot using an anti-VP0 monoclonal antibody showing the level of expression of the O/Tur/5/09 VP2-S93F FMDV capsid precursor protein of FMDV O serotype Tni cells (lane 1) and supernatant (lane 2) after transfection with a baculovirus expression cassette comprising the AAAT sequence, as compared to expression levels in Tni cells (lane 3) and supernatant (lane 4) after transfection with a baculovirus expression cassette without the AAAT sequence.

[0026] FIG. 4 is a Western Blot using polyclonal cattle serum showing the level of expression of the A/Irn/7/13 VP2-H93F FMDV capsid precursor protein of FMDV A serotype in Tni cells (lane 1) and supernatant (lane 2) after transfection with a baculovirus expression cassette comprising the AAAT sequence, as compared to expression levels in Tni cells (lane 3) and supernatant (lane 4) after transfection with a baculovirus expression cassette without the AAAT sequence.

DETAILED DESCRIPTION OF THE INVENTION

Definition of Terms

[0027] 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.

[0028] The term “initiation sequence” herein refers to the nucleic acid sequence in direct proximity upstream, i.e. in 5′ direction, of the ATG start codon of an open reading frame (ORF) encoding an FMDV capsid precursor protein. In consistency with conventional nomenclature, the “A” nucleotide of this ATG start codon is enumerated as “+1”. More specifically, the term “initiation sequence” in the present invention refers to the nucleic acid sequence 5 ‘-AAAT-3’ at position −4 to −1 relative to this ATG start codon.

[0029] 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 for protein 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.

[0030] 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 the polyhedrin or p10 baculovirus promoters.

[0031] The most common baculovirus used for gene expression is Autographa californica nucleopolyhedrovirus (AcNPV). AcNPV has a large (130 kb), 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. The 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).

[0032] 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.

[0033] A virus “capsid” is commonly understood in the art as the protein shell of a virus, typically enclosing its genetic material.

[0034] A “capsid precursor protein” is a precursor of one or more structural proteins, also called capsid proteins, which takes part in the formation of a virus capsid or of a building block thereof. FMDV capsid precursor proteins typically comprise the structural protein P1. Since the protein P1 is processed by the FMDV 3C protease (3Cpro) into the mature VP0, VP3, and VP1 proteins, the P1 protein may also be referred to as polyprotein or proprotein. In the context of the present invention, the FMDV capsid precursor protein typically comprises at least P1 including the proteins VP1, VP2, VP3 and VP4. Alternatively, the FMDV capsid precursor protein may comprise one or more of the proteins VP1, VP2, VP3 and VP4. The FMDV capsid precursor protein may also comprise the protein VP0 comprising the proteins VP2 and VP4. Most preferably, the FMDV capsid precursor protein at least comprises the P1 and 2A proteins (also referred to herein as P1-2A capsid precursor).

[0035] 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. 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 VP0, VP3 and VP1. The VLP is formed by self-assembly from these capsid proteins.

[0036] VLPs may also be produced in the baculovirus expression system of the present invention using a modified 3C protease that is less toxic to the insect cells (Porta et al. (2013) J Virol Methods). 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, VP0, VP1, and VP3, which assemble into VLPs. 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 (1995)). Monoclonal antibodies may be used specific for conformational epitopes on the wild-type virus in order to investigate whether the structure and antigenicity of the empty capsid is retained.

[0037] 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.

[0038] To “protect an animal 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 to prevent or reduce one or more clinical signs resulting from a post treatment (i.e. post vaccination) infection with FMDV.

[0039] 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 cell.

Baculovirus Expression Vector

[0040] The baculovirus expression vector of the first aspect of the present invention is capable of recombinantly expressing an FMDV capsid precursor protein under control of a promoter. In particular, the baculovirus expression vector of the present invention contains an expression cassette including the initiation sequence as described herein and comprising a nucleic acid sequence encoding an FMDV capsid precursor protein, which is expressed in a host cell under control of a functional promoter.

[0041] The baculovirus expression vector of the first aspect of the present invention is capable of recombinantly expressing an FMDV capsid precursor protein under control of a promoter. The expression vector comprises the nucleic acid sequence at position −4 to −1 of 5″-AAAT-3″. It has surprisingly been found in the present invention that the nucleic acid sequence 5″-AAAT-3″ (also referred herein as “initiation sequence” can enhance the recombinant expression of the FMDV capsid precursor protein in a host cell.

[0042] The baculovirus expression vector of the invention may further comprise one or more restriction sites for cleavage by one or more restriction enzymes between the nucleic acid sequence at position −4 to −1 of 5′-AAAT-3′ and the promoter. The one or more restriction site is not particularly limited but may be any restriction site conventionally used in the art for cloning procedures, such as a BstEII restriction site. Suitable restriction sites for cleavage by one or more restriction enzymes are well known to the skilled person and are available, for example, via the REBASE® Restriction Enzyme Database (rebase.neb.com/).

[0043] In a preferred embodiment of the invention, the nucleic acid sequence preceding the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein at position −11 to −1 has the nucleic acid sequence 5″-GGTAACCAAAT-3′ (SEQ ID NO. 1).

[0044] The baculovirus expression vector of the invention may further comprise one or more cis-acting elements, such as translational enhancers. The translational enhancers may be selected from one or more translational enhancers known in the art and commonly used in the field for enhancing the recombinant expression of proteins, such as FMDV capsid precursor proteins.

[0045] In a preferred embodiment, the translational enhancer comprises the translational enhancer “Syn21”, which is an AT-rich synthetic sequence of 21 nucleotides (nt) made by combining the Cavener consensus sequence with elements from the Malacosoma neustria nucleopolyhedrovirus (MnNPV) polyhedrin gene as described in “B. D. Pfeiffer et al, PNAS (2012), Vol. 109(17), p. 6626-6631”. The nucleic acid sequence of the Syn21 translational enhancer may have a nucleic acid sequence corresponding to the nucleic acid sequence 5′-AAC TTA AAA AAA AAA ATC AAA-3′ (SEQ ID NO.2). In this invention, the translational enhancer Syn21 is typically located within the 5′ untranslated region (UTR) of the nucleic acid sequence encoding the FMDV capsid precursor protein. In a further preferred embodiment, the nucleic acid sequence of SEQ ID NO. 2 precedes the nucleic acid sequence of SEQ ID NO. 1. Hence, the nucleic acid sequence in the baculovirus expression vector of the present invention may have the order of:
5′-SEQ ID NO.2-SEQ ID NO.1-ATG-ORF(FMDV coding region)-3′

[0046] In a further preferred embodiment, the translational enhancer comprises the “P10UTR”, which is typically located within the 3′ UTR of the nucleic acid sequence encoding the FMDV capsid precursor protein. The term “P10UTR” as used herein relates to the 3′ UTR from the AcNPV p10 gene as described in “Y. Liu et al., Biotechnol. Lett. (2015), Vol. 37, p. 1765-1771”. Preferably, the P10UTR has a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO. 3.

[0047] Further preferably, the baculovirus expression vector of the invention comprises both the Syn21 and the P10UTR translational enhancers. In this preferred embodiment, the nucleic acid sequence in the baculovirus expression vector of the present invention may have the order of:
5′-SEQ ID NO.2-SEQ ID NO.1-ATG-ORF(FMDV coding region)-SEQ ID NO.3-3′

[0048] Encompassed by the terms “Syn21” and “P10UTR” are nucleic acid sequences corresponding to those of SEQ ID NO. 2 and 3, but including conservative modifications, such as mutation and/or natural variation, of one or more nucleic acids. A modification may be a deletion or addition of one or more nucleotides or a replacement of one or more nucleotides by one or more other nucleotides. A conservative modification is typically a modification that does not substantially alter the function of the sequence as translational enhancer, i.e. the modified sequence is still capable of enhancing expression under control of the promotor of the expression cassette.

[0049] The FMDV capsid precursor protein is recombinantly expressed under the control of a suitable promoter. The promoter is not particularly limited but may be any promoter capable for the recombinant expression of 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., Virology (1994), Vol. 2020, p. 586-605) and the p10 promoter (described in: Knebel D. et al., EMBO J. (1985) Vol. 4(5), 1301-1306) of AcNPV. Another preferred promoter is the promoter of the orf46 viral gene of S. exigua nucleopolyhedrovirus (SeNPV) (described in M. Martinez-Solis et al., PeerJ (2016), DOI 10.7717/peerj.2183).

[0050] 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).

[0051] The nucleic acid sequence encoding the FMDV capsid precursor protein is not particular limited and may be of any FMDV serotype, such as of serotypes A, O, Asia1, SAT1, SAT2, SAT3 and C. In a particularly preferred embodiment, the FMDV capsid precursor protein is from the Asia1 or SAT2 serotype.

[0052] In the baculovirus expression vector of the present invention, the capsid precursor protein typically comprises at least the capsid precursor P 1. More preferably, the capsid precursor protein comprises the capsid precursor P1 and the 2A peptide.

[0053] In a further preferred embodiment, the baculovirus expression vector of the present invention further comprises a nucleic acid sequence encoding a protease capable of cleaving an FMDV capsid precursor protein into one or more capsid proteins. The protease may be any protease capable of cleaving the FMDV capsid precursor protein as a step in the production and assembly of capsids to produce FMDV VLPs. As mentioned above, for FMDV, proteolytic processing of the precursor P1 into VP0 (VP2 plus VP4), VP3 and VP1 occurs by means of the viral 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 type A strain is described in the art and is disclosed, e.g., 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.

[0054] In a further preferred embodiment, the baculovirus expression vector of the first aspect thus may be a nucleic acid sequence which further comprises a nucleic acid sequence encoding a protease. The nucleic acid sequences of the FMDV capsid precursor protein and the protease are preferably arranged in a contiguous manner. There may be a nucleic acid sequence between nucleic acid sequences encoding the FMDV capsid precursor protein and the protease. A control element, such as a control element as described in WO 2011/048353, may be present in that sequence, such that it controls expression of the protease but does not control or affect expression of the capsid precursor protein.

[0055] The capsid precursor protein may be cleavable by the protease into one or more capsid proteins to form (part of) a virus-like particle (VLP). The precursor protein may comprise all proteins necessary to form a VLP.

[0056] The capsid precursor protein may be P1, which is cleaved by the 3C protease into VP0, VP3 and VP1. Alternatively, the capsid precursor protein may be P1-2A. The 2A peptide cleaves itself at its C terminus to release P1-2A from any downstream protein sequence. 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. For example, the capsid precursor protein may be expressed under control of a first promoter as described herein and wherein gene expression is enhanced by the initiation sequence as described herein, and the protease is expressed under control of a separate (second) promoter, which may be different from the first promoter.

[0057] Cleavage of the capsid precursor protein or VLP may be analysed using techniques known in the art. For example, extracts from baculovirus-infected host cells may be separated 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 VP3-1 (~47 kDa), VP0 (~33 kDa), VP2 (~22 kDa), VP3 (~24 kDa) and/or VP1 (~24 kDa).

Host Cell

[0058] In a second aspect, the invention provides a host cell comprising the baculovirus expression vector according to the first aspect of the invention. In a further embodiment, the host cell is capable of producing capsid precursor proteins, and preferably is capable of producing FMDV VLPs.

[0059] The host cell may, for example, be a bacterial cell, an insect cell, plant cell or a mammalian cell. Preferably, the host cell is an insect cell, such as 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.

[0060] Infection of host cells, such as insect cells, can be performed by standard methods well known to the skilled person. For example, commercial suppliers of baculovirus expression systems typically provide suitable instruction manuals, which can be followed, such as the Invitrogen ° “Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques”.

Method of Producing Virus-Like Particles

[0061] In a third aspect, the invention provides a method of producing FMDV capsid precursor protein. Alternatively, the invention provides a method of producing FMDV VLPs. The method according to the third aspect comprises the steps of:

- [0062] (i) infecting a host cell according to the second aspect with the baculovirus expression vector according to the first aspect, and
- [0063] (iia) harvesting FMDV capsid precursor protein produced by the host cell, or
- [0064] (iib) harvesting FMDV VLPs produced by the host cell.

[0065] The method thus includes the culturing of the host cell under conditions suitable for the host cell to express the capsid precursor protein from the baculovirus expression vector in order to produce capsid precursor protein.

[0066] In case the baculovirus expression construct further expresses a protease capable of cleavage of the capsid precursor protein into one or more capsid proteins, which assemble into VLPs, as described above, FMDV VLPs may be produced by the host cell.

[0067] If the capsid precursor protein or the VLPs are released by the host cell, they may be harvested from the cell culture medium. If the capsid precursor protein or the VLPs are retained inside the host cell, they may be harvested by, for example,

- [0068] (i) lysis of the host cells (for example by freeze-thawing); and optionally
- [0069] (ii) concentration (e.g. by PEG-precipitation), and/or
- [0070] (iii) purification.
  Vaccines and Production Thereof

[0071] The present invention further relates to the production of FMDV VLPs, which are used in the production of a vaccine.

[0072] 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 according to the third aspect and incorporating the FMDV VLPs in a vaccine, such as by the addition of a pharmaceutically acceptable carrier.

[0073] 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. The vaccine may also comprise, or be capable of expressing, another active agent, for example one which may stimulate early protection prior to the vaccinating entity-induced adaptive immune response. The agent may be an antiviral agent, such as type I interferon. Alternatively, or in addition, the agent may be granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0074] 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.

[0075] 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 vaccinating entities, each directed at a different serotype and/or different subtypes within a given serotype.

Treatment

[0076] 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.

[0077] 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 hedgehogs, coypu, and any wild cloven-footed animals such as deer and zoo animals including elephants are also susceptible to FMD.

Administration

[0078] 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.

EXAMPLES

[0079] 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.

[0080] The standard baculovirus expression vector as described by Porta et al (2013; J Virol Methods) contains the BstEII restriction enzyme site upstream of the ATG start codon of the P1-2A-3C expression cassette. It was evaluated if the 5′-AAAT-3′ sequence that precedes the highly expressed polyhedrin gene of AcNPV can improve translation of the P1-2A-3C expression cassette.

[0081] Two versions of the initiation sequence upstream of the ATG start codon were compared. The version used as the comparative Example does not contain a special sequence upstream of the ATG start codon, just the BstEII restriction enzyme site (GGTTACCATGGG; designated in the following as “Standard”, SEQ ID NO. 4). The version according to the invention has the 5′-AAAT-3′ sequence between the BstEII site and the ATG codon and contains a single nucleotide change in the BstEII site (GGTAACCAAATATGGG; designated in the following as “AAAT”, SEQ ID NO. 5).

[0082] Baculovirus expression plasmids used in Examples 1-4 were based on the standard baculovirus transfer vector pFastBac® (Thermo Fisher Scientific, Germany), in which expression is driven by the polyhedrin (polh) promoter. The expression plasmids comprising a nucleic acid sequence encoding a P1-2A-3C capsid precursor protein were obtained by using standard cloning procedures well known in the art. The nucleic acid sequences of the expression plasmids are according to SEQ ID NO. 6-9.

Example 1—Expression of P1-2A-3C Expression Cassette Based on FMDV Asia1 Serotype

[0083] In this Example, the expression level of an FMDV capsid precursor protein of an Asia1 serotype from the “AAAT” expression cassette is compared to the expression from the “standard” expression cassette without the AAAT sequence.

[0084] Erlenmeyer flasks with 100 ml containing 3.2×10⁵cells/ml of Tni cells were infected at MOI=1 with recombinant baculoviruses containing the P1-2A-3C expression cassette based on Asia1/Irn/49/11 VP2-S93Y of FMDV Asia1 serotype. The culture was harvested at 4 dpi and the cells were collected by centrifugation and subsequently sonicated in Tris-KCl pH8.0 buffer at one-tenth of the original 35 culture volume.

[0085] Samples were analyzed by Western blotting using the anti-VP0 monoclonal antibody as described by: Loureiro S. et al., Wellcome Open Res 2018, 3:88.

[0086] Visual inspection of the Western blot shows that the standard baculovirus vector performs less than the AAAT-containing vector in terms of yield of FMDV-related proteins, and the difference is estimated at 2-fold (FIG. 1).

Example 2—Expression of P1-2A-3C Expression Cassette Based on FMDV SAT2 Serotype

[0087] In this Example, the expression level of an FMDV capsid precursor protein of a SAT2 serotype from the “AAAT” expression cassette is compared to the expression from the “standard” expression cassette without the AAAT sequence.

[0088] Tni cells infected with recombinant baculoviruses containing the P1-2A-3C expression cassette based on SAT2/Egy/2/12 VP2-K93Y of FMDV SAT2 serotype were cultivated and the culture was harvested as described in Example 1.

[0089] Samples were analyzed by Western blotting using a cocktail of 5 SAT2-specific monoclonal antibodies: 1D5, DA10, GE11, GD12, and GG1 as described by: Opperman et al., 2014, J Virol. Visual inspection of the Western blot shows that the standard baculovirus vector performs less than the AAAT-containing vector in terms of yield of FMDV-related proteins, and the difference is estimated at 2-fold (FIG. 2; note that the 2 proteins on the blot, VP1 and VP1+VP3 are indicated based on size and reaction pattern, but that it has not been validated which FMDV proteins are detected by the cocktail of 5 monoclonal antibodies).

Example 3—Expression of P1-2A-3C Expression Cassette Based on FMDV 0 Serotype

[0090] In this Example, the expression level of an FMDV capsid precursor protein of an O serotype from the “AAAT” expression cassette is compared to the expression from the “standard” expression cassette without the AAAT sequence.

[0091] Tni cells infected with recombinant baculoviruses containing the P1-2A-3C expression cassette based on O/Tur/5/09 VP2-S93F of FMDV O serotype were cultivated and the culture was harvested as described in Example 1. In addition to the cells, supernatant samples were collected after the centrifugation step described in Example 1. Supernatant samples represent the cell culture fluid without the cells.

[0092] Cell and supernatant samples were analyzed by Western blotting using the anti-VP0 monoclonal antibody as described by: Loureiro S. et al., Wellcome Open Res 2018, 3:88. Visual inspection of the Western blot shows that the standard baculovirus vector performs better than the AAAT-containing one in terms of yield of FMDV-related proteins (FIG. 3).

Example 4—Expression of P1-2A-3C Expression Cassette Based on FMDV a Serotype

[0093] In this Example, the expression level of an FMDV capsid precursor protein of an A serotype from the “AAAT” expression cassette is compared to the expression from the “standard” expression cassette without the AAAT sequence.

[0094] Tni cells infected with recombinant baculoviruses containing the P1-2A-3C expression cassette based on A/Irn/7/13 VP2-H93F of FMDV A serotype were cultivated and the culture was harvested as described in Example 3.

[0095] Cell and supernatant samples were analyzed by Western blotting using polyclonal cattle serum (MSD Animal Health, NL). Visual inspection of the Western blot suggests that the standard baculovirus vector performs equally well as or maybe slightly better than the AAAT-containing one in terms of yield of FMDV-related proteins (FIG. 4).

Summary of Results:

[0096]

TABLE 1

Results of recombinant expression of capsid precursor protein
		Relative expression
		Approx. fold
Example	Capsid precursor protein	(AAAT/standard)

1	Asia1/Irn/49/2011 VP2-S93Y	2.0×
2	SAT2/Egy/02/2012 VP2-K93Y	2.0×
3	O/Tur/05/2009 VP2-S93F	0.4×
4	A/Irn/07/2013 VP2-H93F	1.0×

[0097] Examples 1-4 show that recombinant expression of capsid precursor proteins originating from Asia1 and SAT2 serotypes was surprisingly higher for expression constructs with the AAAT sequence included compared to expression constructs including the standard ATG initiation sequence (see Table 1 for a summary).

Claims

The invention claimed is:

1. A baculovirus expression vector for recombinantly expressing a Foot and mouth disease virus (FMDV) capsid precursor protein, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein under the control of a promoter, wherein the ATG start codon of an open reading frame encoding the FMDV capsid precursor protein is preceded at position −4 to −1 by the nucleic acid sequence 5′-AAAT-3′ and wherein the FMDV is of the Asia1 or SAT2 serotype.

2. The baculovirus expression vector according to claim 1, the vector further comprising one or more restriction sites for cleavage by one or more restriction enzymes between the nucleic acid sequence at position −4 to −1 of 5′-AAAT-3′ and the promoter.

3. The baculovirus expression vector according to claim 1, having the nucleic acid sequence 5′-GGTAACCAAAT-3′ (SEQ ID NO. 1) at position −11 to −1.

4. The baculovirus expression vector according to claim 1, wherein expression of the FMDV capsid precursor protein is achieved under the control of the baculovirus polyhedrin promoter (polh) or p10 promoter.

5. The baculovirus expression vector according to claim 1, wherein the capsid precursor protein comprises the capsid precursor P1.

6. The baculovirus expression vector according to claim 1, the vector further comprising a nucleic acid sequence encoding a protease capable of cleaving the capsid precursor protein into one or more capsid proteins.

7. The baculovirus expression vector according to claim 6, wherein the capsid precursor protein comprises the capsid precursor P1 and the peptide 2A and the protease is 3C.

8. A host cell comprising the baculovirus expression vector according to claim 1.

9. The host cell according to claim 8, which is an insect cell.

10. A method of producing FMDV capsid precursor protein, the method comprising the steps of:

(i) infecting a host cell with the baculovirus expression vector according to claim 1, and

(ii) harvesting FMDV capsid precursor protein produced by the host cell.

11. A method of producing FMDV virus-like particles (VLPs), the method comprising the steps of:

(i) infecting a host cell with the baculovirus expression vector according to claim 6, and

(ii) harvesting FMDV VLPs produced by the host cell.

12. A method of producing a vaccine, which comprises the steps of:

(i) producing FMDV virus-like particles (VLPs) by the method according to claim 11 and

(ii) incorporating the FMDV VLPs into a vaccine by addition of a pharmaceutically acceptable carrier.

13. A method of protecting a subject against an infection with FMDV, which comprises the step of expressing an FMDV capsid precursor protein from the baculovirus expression vector according to claim 1 in a host cell to produce a VLPs, incorporating the VLPs into a vaccine by addition of a pharmaceutically acceptable carrier and administering the vaccine to the subject.

Patent Citations (6)

Patent	Date	Inventor	Cited By
US2016/0052973(A1)	2016-02-01	Kotecha	Examiner
US2023/0390380(A1)	2023-12-01	Van Den Born	Examiner
CA1330425(C)	1994-06-01		Applicant
CN105056227(B)	2017-12-01		Applicant
WO2016049209(A1)	2016-03-01		Applicant
WO2017132666(A1)	2017-08-01		Applicant

Non-Patent Literature (13)

Porta et al. (Journal of Virological Methods. 2013; 187: 406-412).Examiner
Lautenberger et al. (Gene. 1980: 171-174).Examiner
Li et al. (PLoS One. 2008; 3 (5): e2273).Examiner
Badri, H. et al., Optimization of radiation dosing schedules forproneural glioblastoma, J Math Bio, 72(5), 1301-1336 (Abstract Only), 2016.Applicant
Baylot, Virginie et al., TCTP Has a Crucial Role in theDifferent Stages of Prostate CancerMalignant Progression, Results Probl Cell Differ, 64, 255-261 (Abstract Only), 2017.Applicant
Ruiz, V. et al., Comparison of Strategies for the Production of FMDV Empty Capsids Using the Baculovirus Vector System, Mol Biotechnol, 56, 963-970, 2014.Applicant
Shaikh, Hanif et al., Current and future cholera vaccines, Vaccine, 38, A118-A126, 2020.Applicant
Li et al., 2016, “Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge,” Vet. Microbiol., 183:92-96 (Epub 2015).Applicant
Martinez-Solis et al., 2019, “Engineering of the baculovirus expression system for optimized protein production,” Appl. Microbiol. Biotechnol., 103(1):113-123 (Epub 2018).Applicant
Subramanian et al., 2012, “Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency,” Antiviral Res., 96(3):288-295.Applicant
Deepak, P.R. et al., Generation of acid resistant virus like particles of vaccine strains of foot-and mouth disease virus (FMDV), Biologicals, 60, 28-35, 2019.Applicant
Kang, C Yong, Expression of Human Immunodeficiency Virus Genes Using Baculovirus Expression System, Mol Biotechnol, 8, 173-187, 1997.Applicant
Lin, Qinying et al., Improved Heterologous Expression of the White-Rot Fungal Ligninase H8 by Crossover Linker Mutagenesis, Appl Biochemi Biotechnol, 66, 269-279, 1997.Applicant