RU2846473C1 - Baculovirus expression vector - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to biotechnology and medicine, in particular to a baculovirus expression vector for recombinant expression of a FMDV capsid precursor protein under the control of a promoter, the expression vector comprising a nucleic acid sequence encoding a capsid precursor protein FMDV and translation enhancers Syn21 and p10UTR, as well as to a host cell comprising a baculovirus expression vector, a method for producing FMDV virus-like particles (VLP) and a method for producing a vaccine.

EFFECT: invention provides enhanced expression of a capsid precursor protein FMDV by means of a baculovirus expression system.

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Description

The invention relates to a baculovirus expression vector for recombinant expression of a capsid precursor protein of foot-and-mouth disease virus (FMDV) under the control of a promoter, wherein the expression vector comprises a nucleic acid sequence encoding a capsid precursor protein of FMDV and a translation enhancer. In addition, the invention relates to a host cell containing the baculovirus expression vector, to a method for producing FMDV virus-like particles (VLPs) and to a method for producing a vaccine.

State of the art of the invention

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 ungulate species 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 affects the entire livestock industry with loss of income for local farmers.

Current vaccines are prepared from inactivated virus. Live FMD virus is produced in high-containment laboratories prior to virus inactivation, which limits FMD production. Effective vaccination against FMD requires the presence of intact FMDV capsids instead of capsid structural elements, which have been shown to be insufficiently immunogenic (Doel and Chong, 1982, Archives of Virology). Inactivated FMD viruses are fragile structures that readily disintegrate into capsid structural elements at acidic pH or elevated temperatures. Thus, a cold chain is required to deliver effective FMD vaccines to livestock producers.

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

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. The advantages of VLP technology over currently used technology include, for example, greater product stability, greater freedom regarding the production site (low-contaminant production) and faster responses to outbreaks caused by new strains. VLP-based vaccines are typically developed as marker vaccines, which reduce the need for production steps that remove non-structural proteins.

The FMDV genome encodes a single open reading frame (ORF) that produces a precursor polyprotein that is processed into twelve mature viral proteins, Fig. 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 the FMDV protease 3C into 2A, VP0 (also known as 1AB), VP3 (1C), and VP1 (1D). The VP0 protein is separated into VP4 and VP2 during encapsidation. FMDV virions are formed by self-assembly from the processed viral structural proteins.

VLPs for use in VLP-based vaccines can be produced by recombinant expression of FMDV precursor proteins in suitable host cells in a manner analogous to the self-assembly of FMDV virions. Similarly, FMDV VLPs are formed by self-assembly from processed viral structural proteins.

Thermal stability and sensitivity of VLPs to low pH can be improved by introducing covalent linkages between capsid proteins, such as cysteine bridges, or by introducing other mutations through rational design (Porta et al. (2013) PLoS Pathog).

Since the initial yield of FMDV VLPs per milliliter of cell culture is generally low even using established successful expression systems, there is a need to increase expression levels to make a VLP-based FMD vaccine a cost-effective alternative to the classical FMD vaccines currently commercialized. Therefore, a baculovirus expression vector was optimized to increase the yield of VLPs and to achieve a large-scale production process that is at least equal to or even superior to the established FMD vaccine production process in terms of antigen yield.

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

The essence of the invention

In the context of the present invention, it has been surprisingly found that the expression of the FMDV capsid precursor protein by a baculovirus expression system can be enhanced using a combination of two translation enhancers: Syn21 and P10UTR. Surprisingly, it has been possible to show that the combination of these two translation enhancers provides higher expression yields than other translation enhancers used in prior art baculovirus expression systems.

Thus, in a first aspect, the present invention relates to a baculovirus expression vector capable of recombinantly expressing the FMDV capsid precursor protein under the control of a promoter, wherein the expression vector comprises:

(i) a nucleic acid sequence encoding the FMDV capsid precursor protein,

(ii) the Syn21 translation enhancer located in the 5' untranslated region (UTR) of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein, and

(iii) the P10UTR translation enhancer located in the 3'-UTR of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein.

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

In a third aspect, the invention relates to a method for producing FMDV VLPs, the method comprising the steps of: infecting a host cell with a baculovirus expression vector as described herein, and collecting the VLPs produced by the host cell.

In addition, the invention relates to the use of a baculovirus expression vector for recombinant expression of the capsid precursor protein of FMDV.

The invention further relates to a method for producing a vaccine by producing FMDV VLPs and incorporating the FMDV VLPs into the vaccine by adding a pharmaceutically acceptable carrier.

The invention further relates to a method for protecting a subject from FMDV infection by expressing an FMDV capsid precursor protein from a baculovirus expression vector of the present invention in a host cell to produce a VLP, incorporating the VLP into a vaccine by adding a pharmaceutically acceptable carrier, and administering the VLP to the subject.

Furthermore, the invention relates to a baculovirus expression vector according to the first aspect of the invention for protecting a subject from FMDV infection.

The invention further relates to a baculovirus expression vector as described herein for use in the manufacture of a medicament for protecting a subject from FMDV infection.

Detailed description of the invention

DEFINITION OF TERMS

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

An "expression vector" (synonymous with "expression construct" ) generally 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 it 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 a POI, the expression vector typically contains at least a promoter to initiate expression of the gene, and may further contain one or more translation enhancers.

A "baculovirus expression vector" is a baculovirus-based expression vector that is 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 by a recombinant gene or cDNA. These genes are often under the control of the baculovirus polyhedrin or p10 promoters.

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 parental virus (such as AcNPV) genome.

A "translation enhancer" is a nucleotide sequence that forms an element that can promote translation and thereby increase protein production. Typically, a translation enhancer can be found in the 5' and 3' untranslated regions (UTR) of an 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.

The "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.

"A capsid precursor protein" is a precursor of one or more structural proteins, also referred to as capsid proteins, that participate in the formation of a viral capsid or a structural element thereof. FMDV capsid precursor proteins typically comprise the P1 structural protein. Since the P1 protein is processed by FMDV 3C protease (3Cpro) into the 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 precursor protein typically comprises at least P1, including the VP1, VP2, VP3, and VP4 proteins. Alternatively, an FMDV capsid precursor protein may comprise one or more of the VP1, VP2, VP3, and VP4 proteins. An FMDV capsid precursor protein may also comprise a VP0 protein, which includes the VP2 and VP4 proteins. Most preferably, the FMDV capsid precursor protein comprises at least the P1 and 2A proteins (also referred to herein as the P1-2A capsid precursor).

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 that lacks the RNA or DNA genome. A VLP should be antigenic and immunogenic similar to the wild-type virus, since it retains the same structural epitopes, but it should not cause infection due to the absence of a viral genome.

FMDV VLPs are typically formed from the capsid precursor P1-2A. As described above, protease 2A cleaves itself at its C-terminus, releasing P1-2A from P2. The capsid precursor P1-2A is processed by protease 3C, producing 2A and the capsid proteins VP0, VP3, and VP1. The VLP is formed by self-assembly from these capsid proteins.

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 (Abrahams et al (1995)). 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 is preserved.

The term "vaccine" as used herein refers to a preparation that, when administered to a subject, induces or stimulates a protective immune response. A vaccine may provide immunity to a particular disease.

"Protect an animal against infection with FMDV" means contributing to the prevention, mitigation or cure of a pathogenic infection with FMDV, or contributing to the prevention, mitigation or cure of a disorder resulting from that infection, such as to prevent or reduce one or more clinical signs resulting from infection with FMDV following treatment (i.e. following vaccination).

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

BACULOVIRAL EXPRESSING VECTOR

The baculovirus expression vector according to the first aspect of the present invention is capable of recombinantly expressing the capsid precursor protein of FMDV under the control of a promoter. The expression vector comprises two translation enhancers: Syn21 and p10UTR, which enhance the recombinant expression of the capsid precursor protein of FMDV.

The "Syn21" translation enhancer is an AT-rich synthetic sequence of 21 nucleotides (nt) obtained by combining the Cavener consensus sequence with elements from the polyhedrin gene of Malacosoma Neustria nuclear polyhedrosis virus (MnNPV), as described in "BD Pfeiffer et al, PNAS (2012), Vol. 109(17), p. 6626-6631". The nucleic acid sequence of the Syn21 translation 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.1). In the context of the present invention, the Syn21 translation enhancer is located in the 5'-untranslated region (UTR) of the nucleic acid sequence encoding the FMDV precursor capsid protein. In a preferred embodiment, the Syn21 sequence is located in close proximity 5' to the ATG initiation codon of the nucleic acid sequence encoding the FMDV capsid precursor protein.

The translation enhancer "P10UTR" is located in the 3'-UTR of a nucleic acid sequence encoding the FMDV precursor capsid protein. The term "P10UTR" as used herein refers to the 3'-UTR of the p10 gene of AcNPV 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. 2.

The terms "Syn21" and "P10UTR" encompass nucleic acid sequences corresponding to SEQ ID NOs. 1 and 2, but including conservative modifications such as mutation and/or natural variation of one or more nucleic acids. A modification may be a deletion or insertion of one or more nucleotides, or a substitution of one or more nucleotides with one or more other nucleotides. A conservative modification is typically one that does not substantially alter the function of the sequence as a translation enhancer, i.e., the modified sequence is still capable of enhancing expression under the control of the promoter of the expression cassette.

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 of recombinantly expressing the FMDV capsid precursor protein in a baculovirus expression system. Preferred promoters for use in the baculovirus expression system of the present invention are the polyhedrin (polh) promoter (described in Ayres MD 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 orf46 viral gene promoter of S. exigua nuclear polyhedrosis virus (SeNPV) (described in M. Martínez-Solís et al., PeerJ (2016), DOI 10.7717/peerj.2183).

Baculovirus expression vectors for use in baculovirus expression systems for recombinant expression of proteins are commercially available and are widely used in the art for the production of proteins and virus-like particles. The systems may comprise, 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), the pFastBac ^® vector (Thermo Fisher Scientific, Germany), the flashBAC ^® vector (Oxford Expression Technologies Ltd, UK) and the BestBac ^® vector (EXPRESSION SYSTEMS, CA).

The baculovirus expression vector of the present invention comprises an expression cassette comprising a nucleic acid sequence encoding the FMDV capsid precursor protein, which is expressed in a host cell under the control of a functional promoter and includes the Syn21 and P10UTR translation enhancers.

Thus, the expression cassette for recombinant expression of the FMDV capsid precursor protein essentially has the schematic structure shown in Fig. 1B.

This structure may contain additional elements, in particular other cis-acting elements, such as additional translation enhancers, which may be used in combination with the Syn21 and p10UTR translation enhancers in the baculovirus expression vector of the present invention.

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

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

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 the FMDV capsid precursor protein. 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 empty FMDV capsids. As mentioned above, for FMDV, the proteolytic processing of the P1 precursor into VP0 (VP2 plus VP4), VP3 and VP1 occurs by the viral 3C protease or its 3CD precursor. Thus, the protease is preferably the FMDV 3C protease. The wild-type FMDV 3C protease sequence from the FMDV type A strain 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 containing one or more mutations that reduce its proteolytic activity, such as a mutation at cysteine 142.

In a further preferred embodiment, the baculovirus expression vector according to the first aspect may be a nucleotide sequence which comprises (i) a nucleic acid sequence of an FMDV capsid precursor, (ii) a Syn21 translation enhancer, (iii) a P10UTR translation enhancer, and (iv) a protease nucleic acid sequence. The nucleotide sequences of (i) the FMDV capsid precursor and (iv) the protease are preferably located adjacent to each other. Between the nucleic acid sequences of (i) and (iv) there may be a nucleic acid sequence. A control element may be present in this sequence, such as a control element as described in WO 2011/048353, which is incorporated herein by reference, such that it controls the expression of the protease, but does not control or affect the expression of the capsid precursor protein.

The capsid precursor protein can be cleaved by a protease to form an empty capsid (or part of it). The precursor protein may contain all the proteins needed to form an empty capsid.

The capsid precursor protein may be P1, which is cleaved by protease 3C into VP0, VP3, and VP1. Alternatively, the capsid precursor protein may be P1-2A. Peptide 2A cleaves itself at its C-terminus, releasing 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 P1, 2A, and 3C proteins. Expression of the 3C enzyme in the P1-2A-3C cassette allows expression and processing of the P1-2A precursor into structural proteins that assemble into VLPs. The capsid precursor protein and protease may be expressed under the control of separate promoters or under the control of the same promoter. For example, the capsid precursor protein may be expressed under the control of a first promoter, as described herein, wherein gene expression is regulated by the Syn21 and p10UTR translation enhancers, and the protease is expressed under the control of a separate (second) promoter, which may be different from the first promoter.

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 separated by gel electrophoresis and the separated proteins 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 VP3-1 (~47 kDa), VP0 (~33 kDa), VP2 (~22 kDa), VP3 (~24 kDa), and/or VP1 (~24 kDa).

HOST CELL

In a second aspect, the invention relates to a host cell comprising a 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 is preferably capable of producing FMDV VLPs.

The host cell may, for example, be a bacterial cell, an insect cell, a plant cell, or a mammalian cell. Preferably, the host cell is an insect cell, such as an Sf9 cell (a clinical isolate of Spodoptera frugiperda cells Sf21), 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.

METHOD FOR PRODUCING VIRUS-LIKE PARTICLES

In a third aspect, the invention relates to a method for producing a capsid precursor protein of FMDV. Alternatively, the invention relates to a method for producing a VLP of FMDV. The method according to the third aspect comprises the steps of:

(i) infecting a host cell according to the second aspect with a baculovirus expression vector according to the first aspect, and

(iia) collecting the FMDV capsid precursor protein produced by the host cell, or

(iib) collecting FMDV VLPs produced by the host cell.

Thus, the method comprises culturing a host cell under conditions suitable for the expression in the host cell of a capsid precursor protein from a baculovirus expression vector to produce the capsid precursor protein. In the case where the baculovirus expression construct further expresses a protease capable of cleaving the capsid precursor protein as described above, FMDV VLPs can be produced by the host cell. If the VLPs are released by the host cell, they can be collected from the cell culture medium. If empty viral capsids remain within the cell, they can be collected by, for example,

(i) lysis of host cells (e.g. by freeze-thawing), and optionally

(ii) concentration (e.g. by precipitation with PEG), and/or

(iii) cleaning.

VACCINES AND THEIR PRODUCTION

Furthermore, the present invention relates to the production of FMDV VLPs that are used to produce a vaccine.

Thus, the present invention also relates to a method for producing a vaccine, which comprises the step of producing FMDV VLPs by the method according to the third aspect and incorporating the FMDV VLPs into the vaccine, for example by adding a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art. By way of example only, such a carrier may be simply sterile water or a buffer solution such as PBS. The vaccine may comprise one 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, an active agent that can stimulate early protection prior to the adaptive immune response induced by the vaccine component. The agent may be an antiviral agent such as type I interferon. Alternatively or additionally, the agent may be granulocyte-macrophage colony-stimulating factor (GM-CSF).

The vaccine may 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 to prevent or reduce the likelihood of spread of the disease.

Many commercially available FMD vaccines are polyvalent to provide protection against different FMD serotypes. Similarly, the vaccine of the present invention may contain multiple vaccinating components, each directed to a separate serotype and/or separate subtypes within a given serotype.

TREATMENT

The present invention also relates to a method for protecting a subject from FMDV infection by administering an effective amount of a vaccine of the present invention.

In the case of FMD, the subject 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). Some wild animals such as hedgehogs, nutria, and any wild even-toed ungulates such as deer and zoo animals including elephants are also susceptible to FMD.

INTRODUCTION

The present invention provides at least one administration to an animal of an effective amount of a vaccine according to the invention. The vaccine can be administered by any method 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 a body cavity (intraperitoneal, i/c), orally, anal, etc. For this vaccine, administration i/m, i/d and s/c is preferred.

DRAWINGS

Fig. 1 schematically shows DNA structures for use in accordance with the invention.

Fig. 2 shows the output in different ways when using the invention.

Fig. 3 shows a Western blot to demonstrate the yield when using the invention.

EXAMPLES

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.

Baculovirus construct "Opt1"

Transcription of the gene of interest (GOI) in the commercially available standard baculovirus transfer vector ^pFastBac® (Thermo Fisher Scientific, Germany) is controlled by the polyhedrin (polh) promoter. The resulting mRNA contains the SV40 3'UTR.

To investigate whether translation of this mRNA could be improved, the SV40 3'UTR was replaced with the 3'UTR from the p10 gene (P10UTR) of Autographa californica nuclear polyhedrosis virus (AcNPV). In addition, the Syn21 translation enhancer was placed in the 5'UTR of the mRNA and immediately upstream of the open reading frame (ORF) encoding the FMDV capsid precursor protein. The resulting modification of the expression cassette was designated "Opt1" (Fig. 1B). Expression of FMDV capsid precursor protein from the standard expression cassette was compared with expression of FMDV capsid precursor protein from the "Opt1" expression cassette.

Cloning of the O/TUR/5/09 VP2-S93F expression cassette used in Example 1 and the A/IRN/7/13 VP2-H93F expression cassette used in Example 2 was performed by standard cloning procedures well known in the art. The nucleotide sequence of the O/TUR/5/09 VP2-S93F expression cassette corresponds to the nucleic acid sequence of SEQ ID NO. 3. The nucleotide sequence of the A/IRN/7/13 VP2-H93F expression cassette corresponds to the nucleic acid sequence of SEQ ID NO. 4.

Example 1

In this example, the expression level of FMDV serotype O capsid precursor protein from the Opt1 expression cassette is compared to expression from a standard expression cassette in terms of achieving high yields.

Erlenmeyer flasks with 100 ml containing 3.2× ¹⁰⁵ Tni cells/ml were infected at MOI=1 with recombinant baculoviruses containing the O/TUR/5/09 VP2-S93F-based P1-2A-3Cpro expression cassette. The culture was harvested at 4 days post infection (dpi) and the cells were collected by centrifugation and then sonicated in Tris-KCl buffer, pH8.0, at one-tenth of the original culture volume.

Samples were analyzed by Western blotting using a monoclonal antibody against VP0 (Loureiro et al. , 2018, https://wellcomeopenresearch.org/articles/3-88). Visual inspection of the Western blot indicates that the Opt1 version of the baculovirus vector has slightly better performance than the standard version in terms of yield of FMDV-related proteins.

To quantify the differences between the two baculovirus constructs, an ELISA was performed using the monoclonal antibody INT-FMA-01-08, which detects both intact capsids (75S/146S) and pentameric capsid structural elements (12S). For this, serially diluted samples were incubated for 1 h at 37°C in microtiter plates, which were coated with antibody overnight at 4°C. After sample removal and three washes with PBS-Tween, a fixed amount of biotinylated INT-FMA-01-08 antibody 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.

The Western blot results are shown in Fig. 2A. The ELISA results are shown in Fig. 2B. The ELISA showed a 1.4-fold increase in the expression of FMDV serotype O-related proteins for the Opt1 expression cassette compared to the standard expression cassette. This may seem insignificant, but given that a round of production typically takes 5 days, this means that the same amount of antigen can be produced in 10 days (2 rounds) using the Opt1 expression cassette and in approximately 15 days (3 rounds) using the other expression cassette.

Example 2

In this example, the expression level of FMDV serotype A capsid precursor protein from the Opt1 expression cassette is compared to expression from a standard expression cassette.

Cell infection and cell culture were performed as described in Example 1, except that an A/IRN/7/13 VP2-H93F based expression cassette was used.

Samples were analyzed by Western blotting as described in Example 1. Visual inspection of the Western blot indicates that the Opt1 version of baculovirus has superior performance to the standard version in terms of yield of FMDV-related proteins.

The Western blot results are shown in Fig. 3. Based on the band intensities, the expression of FMDV serotype A related proteins was estimated to be more than 3-fold higher for the Opt1 expression cassette compared to the standard expression cassette.

Example 3

In this example, the expression level associated with the Syn21 and P10UTR translation enhancers in the Opt1 expression cassette was compared with a commercial system (TopBac ^® , Algenex). In the TopBac ^® expression vector, expression was achieved under the control of the polyhedron (polh) promoter and the homologously repeated (hr) transcriptional enhancer sequence operably linked in cis to the chimeric p10 promoters. The TopBac ^® expression vector has been reported to achieve up to a 4-fold increase in the production yield of a recombinant model protein (green fluorescent protein, GFP) relative to a standard baculovirus vector (López-Vidal, J. et al, PLoS ONE 10(10): e0140039).

Infection of cells and cell culture was performed as described in Example 1.

Cells and supernatant were analyzed by Western blotting as described in Example 1. Visual inspection of the Western blot indicates that the Opt1 version of the baculovirus vector is superior to the commercial ^TopBac® vector in terms of yield of FMDV-related proteins.

To better quantify the differences between the 2 constructs, ELISA was performed as described in Example 1.

The ELISA data confirmed the Western blot results in that the Opt1 expression vector provided overall higher protein yields than the ^TopBac® vector. In fact, the ^TopBac® vector, although successful for many other proteins such as GFP, did not achieve high levels of expression of the FMDV P1-2A-3Cpro cassette from the Opt1 expression vector (see Table 1 below).

Table 1: ELISA results showing expression levels for the Opt1 construct compared to the commercial system. Expression levels achieved by Opt1 were taken as 100%.

Baculovirus expression vector ELISA (%) Cells Cell culture medium Opt1 100 100 Top-Bac 27 20

CONCLUSIONS

For two FMDV strains belonging to two different serotypes, O and A, increased levels of FMDV protein expression were achieved using the Opt1 baculovirus construct containing the Syn21 and P10UTR translation enhancers compared to a standard expression system. Surprisingly, increased expression levels were achieved using the Opt1 baculovirus expression vector even compared to a commercial system containing other translation enhancers, which has been described in the art to provide increased protein expression and yield.

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SEQUENCE LIST

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tgtataacaa atgctgtaaa cgccacaatt gtgtttgttg caaataaacc cagtattatt 180

tgattaaaat tgttgttttc tttgttcata gacaatagtg tgttttgcct aaacgtgtac 240

tgcataaact ccatgcgagt gtatagcgag ctagtggcta acgcttgccc caccaaagta 300

gattcgtcaa aatcctcaat ttcatcaccc tcctccaagt ttaacatttg gccgtcggaa 360

ttaacttcta aagatgccac ataatctaat aaatgaaata gagattcaaa cgtggcgtca 420

tcgtccgttt cgaccatttc cgaaaagaac tcgggcataa actctatgat ttctctggac 480

gtggtgttgt cgaaactctc aaagtacgca gtcaggaacg tgcgcgacat gtcgtcggga 540

aactcgcgcg gaaacatgtt gttgtaaccg aacgggtccc atagcgccaa aaccaaatct 600

gccagcgtca atagaatgag cacgatgccg acaatggagc tggcttggat agcgattcga 660

GTTAAC 666

<210> 3

<211> 3781

<212> DNA

<213> Artificial sequence

<220>

<223> expression construct syn21-O/TUR/5/09-P10UTR

<400> 3

aacttaaaaa aaaaaatcaa aggatccggt aaccaaatat gggggcgggt caatcgtcac 60

cagcaacggg gtctcagaat caaagcggga acacggggtc tattatcaac aactactaca 120

tgcagcaata ccaaaacagc atggacaccc aactgggcga caacgctatc tctggcggtt 180

caaacgaggg ctcaactgac accaccagca accacacgac caatacccaa aacaatgact 240

ggttctctaa gctcgccagc agcgctttca gcggcctgtt cggtgctctt cttgcagaca 300

agaaaaccga ggaaactacg ctgctggagg accgtatcct gaccactagg aacggccaca 360

cgaccagcac tacgcaaagc agcgttggcg tgacctacgg ttatgctacc actgaagact 420

tcgtttccgg tcccaacacc agcggcctgg agactcgtgt tgtgcaggct gaacgtttct 480

tcaagacca tctgttcgac tgggttacta gcgatagctt cggacgttgc cacctgctgg 540

agctgccaac tgaccataaa ggagtgtacg gcttcctgac cgattcctac gcctatatgc 600

gtaacggctg ggacgtcgaa gtaaccgctg ttggcaacca attcaatgga gggtgcctac 660

tagttgctat ggtgcccgag ctgtgtagca tcaacaagcg tgaactgtac caattaactc 720

tgttcccgca ccagttcatc aacccacgta ccaatatgac tgctcatatt accgttccct 780

tcgttggcgt gaaccgttac gaccaatata aggtgcacaa accctggacc ctggttgtta 840

tggttgtggc ccccctcacc gtgaacactg aaggcgctcc ccaaatcaag gtctacgcga 900

acattgctcc caccaacgtc catgtagccg gagagttccc cagcaaagaa ggcatcttcc 960

ctgttgcttg cagcgacgga tacggcggtc tggtgacgac cgaccccaag accgctgacc 1020

ccgcatacgg taaagttttc aaccccccta ggaacatgct gcccggacgt ttcactaact 1080

tccttgacgt ggccgaggct tgccctacct tccttcactt cgaaggcgat gtcccctacg 1140

taactacgaa gactgacagc gatcgtatcc tcgctcaatt cgaccttagc ctcgctgcca 1200

aacacatgag caacaccttc ctggctggac ttgcccaata ctatacccag tacagcggca 1260

ctatcaacct gcatttcatg ttcaccggtc ccactgacgc gaaggctcgt tacatgatcg 1320

cctatgcacc gcccggtatg gagcccccta aaacccccga agctgctgct cactgcatcc 1380

atgctgagtg ggacaccggc ctgaactcta agttcacttt ctcaattccc taccttagcg 1440

ctgctgacta cgcatatacc gcttctgata ctgctgaaac cactaacgtc caaggctggg 1500

tttgtctgtt ccagatcacc cacggaaagg ctgacggcga cgctctggtt gtactagcta 1560

gcgccggcaa agacttcgag ctgcgcttac ccgttgacgc taggacccaa acgaccagcg 1620

ctggcgaatc agctgacccc gtgaccgcta ctgtcgagaa ctacggaggg gaaacccaag 1680

tccagcgtcg tcaacatact gacgtttctt tcatcctgga ccgtttcgtt aaagtgaccc 1740

ccaaagacca aatcaacgtt ctggacctga tgcagacccc agctcacact ctggtgggcg 1800

ctctgttacg taccgctact tactatttcg ctgacctgga ggtcgctgta aagcatgaag 1860

gtaatctgac ctgggttccc aacggcgctc ccgagactgc tctggacaac actacgaatc 1920

cgaccgctta ccacaaggcc cctctgaccc gtctcgccct accgtacact gctccacatc 1980

gcgtgcttgc caccgcatac aacggcaatt gcaagtacgg tgaatctcac accactaacg 2040

ttcgtggaga cctgcaagtg cttgctcaga aagcggctcg caccctgccc acttcattca 2100

actacggcgc catcaaggca acccgtgtta ctgagctgct ttacaggatg aaacgtgctg 2160

aaacctactg tccccgtccc ctcctagcca tccacccctc agaggctcgc cataagcaaa 2220

aaattgttgc tcctgtgaag caattattaa atttcgacct actgaagtta gctggggacg 2280

ttgaatcgaa cccgggaccc agcggccgcg gacctttttt agggaagatc tggccttcct 2340

acaagggaag gccagggaat tttcttacga gggacctgtg aagaagcctg tcgctttgaa 2400

agtgaaagct aagaacttga ttgtcactga gagtggagcc ccaccgaccg acttgcaaaa 2460

gatggtcatg ggcaacacca agcctgttga gcttatcctc gacgggaaga cggtggccat 2520

ttgttgtgct accggagtgt ttggcactgc gtacctcgtg cctcgtcatc tttttgcaga 2580

aaaatatgac aagatcatgc tggacggcag agccatgaca gacagtgact acagagtgtt 2640

tgaattcgag attaaagtaa aaggacagga catgctctca gacgctgcgc tcatggtact 2700

ccaccgtggg aatcgcgtga gagacatcac gaaacacttt cgtgacacag caagaatgaa 2760

gaaaggcacc cctgttgtcg gagtgatcaa caatgccgac gttggggagac tgatcttctc 2820

tggtgaggcc ttaacctaca aggacattgt agtgactatg gatggagaca ccatgcctgg 2880

cctgtttgcc tacaaagccg ccaccaaggc tggctactgt gggggagccg ttcttgctaa 2940

ggacggagct gacacattca tcgttggtac ccactccgca ggcggcaatg gagttggata 3000

ctgctcatgc gtctcgaggt ccatgttgct gaaaatgaag gcgcacatcg accccgaacc 3060

acaccacgag taatctagag gatcccctca ggaagcttgt cgagaagtac tagagatgaa 3120

tcgtttttaa aataacaaat caattgtttt ataatattcg tacgattctt tgattatgta 3180

ataaaatgtg atcattagga agattacgaa aaatataaaa aatatgagtt ctgtgtgtat 3240

aacaaatgct gtaaacgcca caattgtgtt tgttgcaaat aaacccagta ttatttgatt 3300

aaaattgttg ttttctttgt tcatagacaa tagtgtgttt tgcctaaacg tgtactgcat 3360

aaactccatg cgagtgtata gcgagctagt ggctaacgct tgccccacca aagtagattc 3420

gtcaaaatcc tcaatttcat caccctcctc caagtttaac atttggccgt cggaattaac 3480

ttctaaagat gccacataat ctaataaatg aaatagagat tcaaacgtgg cgtcatcgtc 3540

cgtttcgacc atttccgaaa agaactcggg cataaactct atgatttctc tggacgtggt 3600

gttgtcgaaa ctctcaaagt acgcagtcag gaacgtgcgc gacatgtcgt cgggaaactc 3660

gcgcggaaac atgttgttgt aaccgaacgg gtcccatagc gccaaaacca aatctgccag 3720

cgtcaataga atgagcacga tgccgacaat ggagctggct tggatagcga ttcgagttaa 3780

c 3781

<210> 4

<211> 3784

<212> DNA

<213> Artificial sequence

<220>

<223> syn21-A/IRN/7/13-P10UTR expression construct

<400> 4

aacttaaaaa aaaaaatcaa aggatccggt aaccaaatat gggagcggga caatcgtcgc 60

ctgccacggg gagccaaaac caatctggga acactggaag catcatcaat aactactata 120

tgcaacaata ccaaaacagc atggacaccc aactgggcga caacgctatc agcggcggtt 180

ctaacgaggg atctaccgac accacttcaa ctcacacgac caacacccaa aacaatgact 240

ggttcagcaa gctcgctagc tctgccttca gcggcctgtt cggcgctctg cttgcggaca 300

agaaaaccga ggaaactacg ctgctggagg accgtatcct caccactcgc aacggccaca 360

cgaccagcac tacgcaaagc agcgtcggag taacttacgg gtattctacc ggagaagacc 420

atgtttcggg accaaacacc agcggcctgg agacccgtgt tgtgcaggct gaacgtttct 480

tcaagaaaca cctgttcgac tggaccactg ataaggcttt cggccatctt gagaaacttg 540

aactccccac cgagcacaag ggagtttacg ggttcctggt ggactctttc gcttacatgc 600

gtaacggctg ggacgtcgaa gtgaccgccg ttggcaacca attcaatgga gggtgcctac 660

tagttgctat ggtgcccgag tggaaggagt tcacgacccg tgaaaaatac caactaacct 720

tattcccgca ccagttcatc aaccctcgta ccaatatgac tgctcatatt accgttccct 780

accttggcgt taaccgttac gaccaatata agcagcacaa accctggacc ctggttgtta 840

tggttgtgag ccctcttact accagcaaca tcggcgcttc acaaatcaag gtttacgcta 900

acatcgcccc caccttcgtc catgtagctg gcgagctgcc ctcaaaagaa ggaatcgttc 960

cagtggcttg cagcgacgga tacggcggtc tggttactac tgaccccaag acagctgacc 1020

ccgtctacgg catggtatat aacccccctc gtactaatta ccccggtcgc ttcaccaacc 1080

ttctcgacgt ggctgaggct tgccccacct tcctgtgttt cgacggaggc aagccctacg 1140

ttgaaaccag gactgacgct caacgtctat tagccaagtt cgatgtgtca ctcgctgcca 1200

aacacatgtc gaacacctac ctgagcggaa tcgctcaata ctatactcag tacagcggca 1260

ccatcaacct tcatttcatg ttcaccggtt caactgactc gaaggcacgt tacatggttg 1320

cctatatccc gccaggaatg gacacccccc ctgatactcc tgagaaggca gcgcactgca 1380

tccatgctga atgggacact ggtctcaact ctaaattcac cttctcaatc ccctacgtta 1440

gcgctgccga ctacgcatat actgccagcg atgaggcaga agcgaccaac gttcaaggat 1500

gggtgtgtat ctaccaaatt actcacggca aggctgagca agacaccctg gttgttagcg 1560

ccagcgctgg caaggacttc gaactgaggc ttcccatcga tccccgtgct caaacgacta 1620

ctgcaggaga gtcagccgac cccgtcacta ccactgttga gaactacggc ggtgaaactc 1680

aagttcagcg tcgtcaccat accgacgtgg gattcatcat ggatcgtttc gtgaagatca 1740

gcccggtcgg cccaactcac gtgatcgacc tcatgcaaac ccaccaacac gctctggttg 1800

gcgctctgct tcgtgctgcg acctactatt tcagcgacct tgagatcgtt gtgcgtcacg 1860

aaggtcatct cacctggggtg cccaacggcg ctcccgtggg cgctctggtt aacacctcta 1920

atcctactgc ttactgcaag gagcccttca ctcgtctcgc tctaccgtac accgccccac 1980

accgcgttct tgctaccgtg tacaacggcg ttagcaaata cagcaccact ggaggcgaca 2040

ggcgtggcga cctgggtagc cttgctgccc gtgttgcagc gcaccttccc agcagcttca 2100

acttcggcgc catcaaggca accaacattc atgagctgct ggttcgtatg aaaagggctg 2160

aactctactg tccccgtccc ttactggccg tcgaggttag cagccaagac cgccacaagc 2220

agaaaatcat tgcacccgcg aagcaattac tgaacttcga cctactgaaa ttagcggggag 2280

acgttgaatc gaaccccgga cccagcggcc gcggaccttt tttagggaag atctggcctt 2340

cctacaaggg aaggccagg aattttctta cgagggacct gtgaagaagc ctgtcgcttt 2400

gaaagtgaaa gctaagaact tgattgtcac tgagagtgga gccccaccga ccgacttgca 2460

aaagatggtc atgggcaaca ccaagcctgt tgagcttatc ctcgacggga agacggtggc 2520

catttgttgt gctaccggag tgtttggcac tgcgtacctc gtgcctcgtc atctttttgc 2580

agaaaaatat gacaagatca tgctggacgg cagagccatg acagacagtg actacagagt 2640

gtttgaattc gagattaaag taaaaggaca ggacatgctc tcagacgctg cgctcatggt 2700

actccaccgt gggaatcgcg tgagagacat cacgaaacac tttcgtgaca cagcaagaat 2760

gaagaaaggc acccctgttg tcggagtgat caacaatgcc gacgttggga gactgatctt 2820

ctctggtgag gccttaacct acaaggacat tgtagtgact atggatggag acaccatgcc 2880

tggcctgttt gcctacaaag ccgccaccaa ggctggctac tgtgggggag ccgttcttgc 2940

taaggacgga gctgacacat tcatcgttgg tacccactcc gcaggcggca atggagttgg 3000

atactgctca tgcgtctcga ggtccatgtt gctgaaaatg aaggcgcaca tcgaccccga 3060

accacaccac gagtaatcta gaggatcccc tcaggaagct tgtcgagaag tactagagat 3120

gaatcgtttt taaaataaca aatcaattgt tttataatat tcgtacgatt ctttgattat 3180

gtaataaaat gtgatcatta ggaagattac gaaaaatata aaaaatatga gttctgtgtg 3240

tataacaaat gctgtaaacg ccacaattgt gtttgttgca aataaaccca gtattatttg 3300

attaaaattg ttgttttctt tgttcataga caatagtgtg ttttgcctaa acgtgtactg 3360

cataaactcc atgcgagtgt atagcgagct agtggctaac gcttgcccca ccaaagtaga 3420

ttcgtcaaaa tcctcaattt catcaccctc ctccaagttt aacatttggc cgtcggaatt 3480

aacttctaaa gatgccacat aatctaataa atgaaataga gattcaaacg tggcgtcatc 3540

gtccgtttcg accatttccg aaaagaactc gggcataaac tctatgattt ctctggacgt 3600

ggtgttgtcg aaactctcaa agtacgcagt caggaacgtg cgcgacatgt cgtcgggaaa 3660

ctcgcgcgga aacatgttgt tgtaaccgaa cgggtcccat agcgccaaaa ccaaatctgc 3720

cagcgtcaat agaatgagca cgatgccgac aatggagctg gcttggatag cgattcgagt 3780

taac 3784

<---

Claims (29)

1. A baculovirus expression vector capable of recombinantly expressing the capsid precursor protein of foot-and-mouth disease virus (FMDV) under the control of a promoter, wherein the expression vector comprises: (i) a nucleic acid sequence encoding the FMDV capsid precursor protein, (ii) the Syn21 translation enhancer located in the 5' untranslated region (UTR) of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein, and (iii) the P10UTR translation enhancer located in the 3'UTR of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein. 2. The baculovirus expression vector of claim 1, wherein the Syn21 translation enhancer has a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO. 1. 3. A baculovirus expression vector according to claim 1 or 2, wherein the P10UTR translation enhancer has a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO. 2. 4. A baculovirus expression vector according to claims 1-3, wherein FMDV is of serotype A. 5. A baculovirus expression vector according to claims 1-3, wherein FMDV is of serotype O. 6. A baculovirus expression vector according to any one of claims 1 to 5, wherein the capsid precursor protein comprises a P1 capsid precursor. 7. A baculovirus expression vector according to any one of claims 1 to 6, wherein the vector further comprises: (iv) a nucleic acid sequence encoding a protease capable of cleaving a capsid precursor protein into one or more capsid proteins. 8. The baculovirus expression vector of claim 7, wherein the capsid precursor protein comprises a P1 capsid precursor and a 2A peptide and the protease is 3C. 9. A host cell for recombinant expression of the capsid precursor protein of foot-and-mouth disease virus (FMDV), comprising a baculovirus expression vector according to any one of claims 1 to 8. 10. The host cell according to claim 9, which is an insect cell. 11. A method for producing a capsid precursor protein of FMDV, comprising the steps of: (i) infecting a host cell with a baculovirus expression vector according to any one of claims 1 to 6, and (ii) collecting the FMDV capsid precursor protein produced by the host cell. 12. A method for producing FMDV virus-like particles (VLPs), comprising the steps of: (i) infecting a host cell with a baculovirus expression vector according to claim 7 or 8, and (ii) collecting FMDV VLPs produced by the host cell. 13. Use of a baculovirus expression vector for recombinant expression of a capsid precursor protein of FMDV, wherein the expression vector comprises: (i) a nucleic acid sequence encoding the FMDV capsid precursor protein, (ii) the Syn21 translation enhancer located in the 5'UTR of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein, and (iii) the P10UTR translation enhancer located in the 3'UTR of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein. 14. Use of a baculovirus expression vector according to claim 13, wherein expression is achieved in an insect cell. 15. A method for producing a vaccine against FMDV infection, which comprises the steps of: (i) producing FMDV VLPs by the method of claim 12, and (ii) incorporating FMDV VLPs into a vaccine by adding a pharmaceutically acceptable carrier. 16. A method for protecting a subject from FMDV infection, which comprises the step of expressing an FMDV capsid precursor protein from a baculovirus expression vector according to claim 7 or 8 in a host cell to produce a VLP, incorporating the VLP into a vaccine by adding a pharmaceutically acceptable carrier, and administering the vaccine to the subject. 17. Use of a baculovirus expression vector according to any one of claims 1 to 8 for producing a medicament for protection against FMDV infection.