WO2018213912A1 - Porcine epidemic diarrhea virus-like particles - Google Patents

Abstract

This application pertains to Porcine Epidemic Diarrhea virus (PEDv) virus like particles (VLPs), methods of producing them, and expression constructs for making them. In particular, this application relates to VLPs produced with the PEDv M protein alone and in combination with PEDv E. protein.

Description

PORCINE EPIDEMIC DIARRHEA VIRUS-LIKE PARTICLES

BACKGROUND OF THE INVENTION

1. Field of Invention

The presently disclosed technology relates to recombinant expression of virus-like particles. More particularly, the presently disclosed technology relates to the production of Porcine Epidemic Diarrhea virus-like particles comprising the M protein.

2. Description of Related Art

Porcine epidemic diarrhea virus (PEDv) is an enveloped alphacoronavirus that causes porcine epidemic diarrhea (PED), a disease which affects pigs, and in particular, newly born piglets around the world. PEDv is encoded by a 28 kilobase single-stranded, positive-sense RNA genome (Song and Park, 2012). PEDv causes the destruction of villus enterocytes and atrophy of intestinal villi. The disease is 95% fatal for neonatal piglets in naive unvaccinated herds (Stevenson et al., Journal of Veterinary Diagnostic Investigation 25, 649-654). The death of millions of suckling piglets and diarrhea-derived weight loss in fattening pigs has caused severe economic losses in the U.S. and Canada (Chen et al., Archives of Virology 155, 1471- 1476). An effective vaccine is needed.

Virus-like particles (VLPs) represent advancements in subunit vaccine development, showing higher immunogenicity. VLPs are structures made up of assembled viral proteins that resemble the morphology of their respective pathogen (reviewed by Kushnir et al., Vaccine 31 , 58-83). Like other subunit vaccines, they benefit from being non-replicative and non-infectious due to not having any genetic material. In addition, VLPs display viral protein epitopes in correct conformations, and as exogenous, particulate antigens, are processed and presented on antigen presenting cells (APC) by MHC class I or II molecules. This means that they can effectively stimulate humoral and cellular immune responses as the native viruses would, and do not require the use of adjuvants (reviewed by Grgacic and Anderson, Methods 40, 60-65). It is also important that VLPs can target dendritic cells, APCs involved in innate and adaptive immunity. Dendritic cell stimulation for cytokine production requires an intact virion. VLPs have an advantage over live attenuated and inactivated viruses, as both are shown to interfere with dendritic cell activation (reviewed by Grgacic and Anderson).

To produce subunit vaccines including VLPs, a suitable platform must be chosen for recombinant protein production. Enveloped VLPs acquire lipid envelopes, characteristically with embedded immunogenic glycoproteins, when they bud off. Therefore, they are only produced in eukaryotic systems (Lua et al., Biotechnology and Bioengineering 1 1 1 , 425-440). Mammalian cell lines such as Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are currently the gold standard for biopharmaceutical production, but are expensive and bear the risk of harboring mammalian pathogens (Fischer et al., Biotechnology Advances 30, 434-439).

Research has shown that plants can be an efficient platform for the production of recombinant proteins and provide numerous advantages (reviewed by Rybicki, Virology Journal 1 1 , 205). Plant platforms can be easily scalable, and are safe from mammalian and bacterial pathogens (Menassa et al., 2012). Plants have the capability to fold and glycosylate complex proteins. Plants can be grown in greenhouses using current farming techniques (Fischer et al., 2012; Menassa et al., Molecular Farming in Plants: Recent Advances and Future Prospects pp. 183-198. Dordrecht, Netherlands: Springer). Plants may also allow for easy delivery of vaccines, as plants can be fed without processing or extracting the protein. Plant components, including the plant cell matrix, may act as adjuvants, stimulating antigen-specific and nonspecific immune responses (Bae et al., Vaccine 21 , 4052-4058). The plant cell wall may also protects the antigen from degradation in the gastro-intestinal tract.

Enveloped plasma membrane-derived VLPs for influenza have been produced through transient expression in N. benthamiana, and were able to confer complete protection to mice against a lethal challenge (D'Aoust et al., 2008).

Referring to Figure 1 , the PEDv genome has a 5' cap, a 3' polyadenylated tail and seven open reading frames (ORFs), which code for three non-structural proteins (ORF 1 a, ORF1 b, and ORF3), and four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) (Song and Park, Virus Genes 44, 167-175). The two overlapping open reading frames, ORF1 a and ORF1 b code for two polyproteins. These are processed by three virus- encoded proteases, a 3C-like proteinase (3CLpro) and two papain-like proteinases (PLP) which results in 16 non-structural proteins required for genome replication and mRNA transcription (John et al., Scientific Reports 6, 25961 ; reviewed by Prentice et al., Journal of Virology 78, 9977-9986). The accessory protein ORF3 is a potassium ion channel, and its role is not well defined (Wang et al., FEBS Letters 586, 384-391 ).

Referring to Figure 2A, an assembled PEDv virus is depicted generally at 20. Nucleocapsid protein (N) 22 forms a ribonucleoprotein complex with viral RNA 24. Envelope protein (E) 26 is embedded in the membrane 28 as is membrane protein (M) 30, encompassing an amino-terminal domain 30 outside the virus, three transmembrane segments 32, and a longer carboxy- terminal domain 34 inside the virus. Spike protein (S) 36 also embeds in the membrane, and forms surface projections 38, or 'spikes'. Of the four structural proteins, S and M are the proteins believed to be most important for antigenicity.

M is an N-glycosylated transmembrane protein, and is the most abundant component of the viral envelope (Neuman et al. , Journal of Structural Biology 174, 1 1 -22; Utiger et al., Virus Genes 10, 137-148). M is predicted to have three transmembrane segments, with two flanking domains one short, and one long (The UniProt Consortium, 2015). The shorter domain lies outside the virion on the amino terminus, while the longer carboxyl tail is found inside. In contrast to S, the sequence of M is not prone to variation or adaptation (Chen et al.; Sato et al., Virus Genes 43, 72-78). Various coronavirus M proteins have been shown to induce both humoral and cellular immune responses. M- M interactions drive the formation of the envelope, and thus play a key role in coronavirus assembly. Thus, the M protein may be useful in the production of VLPs.

The requirements for coronavirus- VLP formation are not well understood, and appear to vary depending on the virus. In Mouse hepatitis virus (MHV), it is believed that M and E are the minimal requirements to form VLPs. However, conflicting studies have emerged for Severe Acute Respiratory Syndrome- associated coronavirus (SARS-CoV). An initial study found M and E were sufficient for VLP formation (Ho et al., Biochemical and Biophysical Research Communications 318, 833-838), aligning with previous coronavirus research, but a subsequent study instead found that M and N were both sufficient and necessary to form VLPs (Huang et al., Journal of Virology 78, 12557-12565). Another study has shown that SARS CoVLPs can be produced with only M, or M and N (Tseng et al., Journal of Biological Chemistry 285, 12862-12872). Further, VLPs for Avian infectious bronchitis virus (IBV) were produced by co- expressing M and S (Liu et al., Vaccine 31 , 5524-5530). The requirements to produce PEDv VLPs are not known, as they have never been reported in the literature. As a membrane protein, coronavirus M protein can be difficult to express.

Some groups have attempted to express full length PEDv M in E. coli only to resort to expressing different fragments of the protein (Zhang et al., Chinese Veterinary Science 1 , 008), or just expressing a truncated version of the protein (Shenyang et al., Veterinary Microbiology 123, 187-193). Recombinant expression of M has mostly utilized prokaryotic platforms, and M is almost always expressed as a fusion protein. Two groups have produced M in eukaryotic platforms (Ren et al., Journal of Animal and Veterinary Advances 1 1 , 3234-3241 ; Utiger et al. , Virus Genes 10, 137-148). While some of the produced M proteins have induced antibodies, none of these antibodies have been tested for virus neutralizing activity. Table 1 lists all known studies that have produced recombinant PEDv M protein.

Table 1. Production of S-CE in plants¹.

Plant Host Transient Fusions Yield Promoter Reference or and/or

Transgenic Enhancer

Used

Nicotian a Transgenic 10 2x35S, TOL (Bae et al., tabacum mg/kg of 2003)³

protein

per

fresh

weight²

N. Transient 5% TSP TMV RNA (Kang et tabacum al., Protein

Expression and

Purification 38, 129-135)

No- Transgenic 2.1 % 2x35S, TOL (Kang et al. , nicotine N. TSP Vaccine 23, tabacum 2294-2297)

N. Transgenic 0.1 % 2x35S, TOL (Kang et al., tabacum TSP Protein

Expression and

Purification 41 , 378-38)

Solanum Transgenic 0.1 % 2x35S, TOL (Kim et al., tuberosum TSP Plant Cell,

Tissue and Organ Culture 82, 125-130)

Lemna Transgenic Not 35S (Ko et al., minor reported Lemna minor.

Horticulture, Environment, and

Biotechnology 52, 511-515)

Ipomoea Transgenic Not 35S (Yang et al., batatas reported Journal of

Plant

Biotechnology 32, 263-268)

Zea mays Transgenic 0.122% 2x35S, (Kun et al., seed TSP maize intron Journal of

Hsp70 Agricultural

Science and Technology (Beijing) 16, 28-35)*

Daucus Transgenic Not 2x35S, TOL (Kim et al., carota reported Plant

Resources 6, 108-1 13)

Lactuca Transgenic LTB 0.048% Ubiquitin (Huy et al., sativa TSP promoter Biotechnology and

Bioprocess Engineering 14, 731 -737)

Oryza Transgenic 1.3% (Oszvald et sativa TSP p, Act1-i al., Molecular endosperm Biotechnology

35, 215-22)

Oryza Transgenic 1.9% HMW-Bx17- (Tamas, Acta sativa TSP p, Act1-i Agronomica endosperm Hungarica 58,

55-64)

N. Transgenic 1.6% Ubiquitin (Kang et al., tabacum TSP promoter Protein

Expression and

Purification 46, 16-22.)

Oryza Transgenic Co1 0.083% R Amy 3D (Huy et al., sativa calli TSP Plant Cell

Reports 31 , 1933-1942)³

¹2x, duplicated; 35S, cauliflower mosaic virus (CaMV) 35S promoter; Act1 -I, rice actin first intron; Co1 , M cell-targeting ligand; HMW-Bx17-p, wheat high molecular weight glutenin subunit Bx17 endosperm-specific promoter; LTB, heat-labile enterotoxin B subunit of Escherichia, coli; RAmy3D, rice a-amylase 3D promoter; TOL, TMV Omega-prime leader, containing transcriptional and translational enhancer from the coat protein gene of TMV; TSP, total soluble protein; all yield values are highest levels reported. ²Total soluble protein levels were not reported in this study. ³Study also showed antibody production against protein. SUMMARY OF THE INVENTION

Various embodiments of the claimed invention relate to a eukaryotic organism or eukaryotic cell comprising a first recombinant polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus sufficient to form a virus-like particle (VLP) in the organism or cell, wherein the M protein has an amino acid at least 75% identical to the amino acid sequence of SEQ ID NO: 1.

Various embodiments of the claimed invention relate to an expression construct comprising a polynucleotide molecule encoding a polypeptide, wherein the polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus sufficient to form a virus-like particle (VLP) in a cell, wherein the polynucleotide molecule is operably linked to a promoter for initiating transcription of the polynucleotide molecule in the cell, wherein the M protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1.

Various embodiments of the claimed invention relate to a method of producing a virus-like particle (VLP) comprising a portion of the membrane (M) protein of Porcine Epidemic Diarrhea virus, the method comprising expressing an expression cassette as claimed.

Various embodiments of the claimed invention relate to a virus-like particle (VLP) comprising a polypeptide, wherein the polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus, wherein the

M protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1. Such VLPs may be useful for vaccinating a porcine against infection by Porcine Epidemic Diarrhea virus. Such VLPs, or plant materials comprising such VLPs, may be useful for inducing mucosal immunity against Porcine Epidemic Diarrhea virus in a porcine. Other aspects and features of the presently claimed invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention,

Figure 1 is a schematic diagram of the genome of PEDv.

Figure 2 is a schematic diagram of an assembled PEDv virus (A), and a hypothetical PEDv VLP (B).

Figure 3 is a schematic diagram of the expression cassette used to express M Polypeptide, E Polypeptide, N Polypeptide, and S

Polypeptide in N. benthamiana leaves. "GOI" refers to "gene of interest" and may include coding sequences for any of M, E, N, and S proteins of PEDv.

Figure 4 is a Western blot of M Construct expressed in N. benthamiana leaves using an expression construct as depicted in Figure 3. Figure 5 is a Western blot of extracts from N. benthamiana leaves in which the M Construct, E Construct, N Construct, and S Construct were individually expressed.

Figure 6 is a Western blot of extracts from N. benthamiana leaves in which, from left to right, the M Construct, E Construct, N Construct, and S Construct were individually expressed, and in which: M Construct is co-expressed with E Construct; M Construct is co-expressed with E Construct and N Construct; and M Construct is co- expressed with E Construct, N Construct, and S Construct.

Figure 7 is a Western blot of extracts from, from left to right, wild-type N.

benthamiana leaves (first to third lanes) and N. benthamiana leaves in which: M Construct is co-expressed with E Construct (fourth to sixth lanes); M Construct is co-expressed with E Construct, N Construct, and S Construct (seventh to ninth lanes); and M Construct is expressed alone (tenth to twelfth lanes).

Figure 8 is a Western blot of sucrose gradient fractions obtained for plants expressing (A) M Construct alone or (B) in combination with E Construct.

Figure 9 is TEM analysis of 40% sucrose gradient fractions from (A) wild type leaves, (B) leaves expressing M Construct alone and (C) M

Construct in combination with E Construct

Figure 10 is Immungold TEM analysis using anti-c-Myc primary antibody of

40% sucrose gradient fractions from leaves expressing M

Construct alone.

DETAILED DESCRIPTION

The inventor Menassa is a public servant within the meaning of the Public Servants Inventions Act, R.S.C., 1985, c. P-32.

The present disclosure relates generally to the production of PEDv VLPs. More particularly, the disclosure relates to the production of VLPs comprising the M protein of PEDv. Particular aspects of the disclosure relate to the use of plants to produce such VLPs.

Unless otherwise indicated to the contrary, all technical terms used herein have the meaning commonly understood by the skilled person to whom this disclosure is directed. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprises", "comprised", "comprising" mean "includes", "included", and "including". "Operably linked" refers to a functional linkage between a promoter and a second DNA sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second DNA sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous.

As used herein, the term "polypeptide" encompasses any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100 or more amino acids) or post-translational modification (e.g., glycosylation or phosphorylation) or the presence of e.g. one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to the peptide, and includes, for example, natural proteins, synthetic or recombinant polypeptides and peptides, hybrid molecules, peptoids, peptidomimetics, etc. As used herein, the terms "polypeptide", "peptide" and "protein" may be used interchangeably.

"Nucleotide sequence", "polynucleotide sequence", "nucleic acid" or "nucleic acid molecule" as used herein refers to a polymer of DNA or RNA which can be single or double stranded and optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. "Nucleic acid", "nucleic acid sequence", "polynucleotide sequence" or "nucleic acid molecule" encompasses genes, cDNA, DNA and RNA encoded by a gene. Nucleic acids, nucleic acid sequences, polynucleotide sequence and nucleic acid molecule may comprise at least 3, at least 10, at least 100, at least 1000, at least 5000, or at least 10000 nucleotides or base pairs.

"Wildtype" as used herein refers to an organism, or material derived therefrom, e.g. plant or plant material, that was not transformed with a nucleic acid molecule or construct as described herein. The term "identity" as used herein refers to sequence similarity between two polypeptide or polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid or nucleic acid sequences is a function of the number of identical or matching amino acids or nucleic acids at positions shared by the sequences, for example, over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the Clustal W™ program, available at http://clustalw.genome.ad.jp, the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.).

Sequence identity may also be determined using the BLAST algorithm (e.g. BLASTn and BLASTp), described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis is available through the National Center for Biotechnology Information (through the Internet at http://www.ncbi.nlm.nih.gov/). For instance, sequence identity between two nucleic acid sequences can be determined using the BLASTn algorithm at the following default settings: expect threshold 10; word size 11 ; match/mismatch scores 2, -3; gap costs existence 5, extension 2. Sequence identity between two amino acid sequences may be determined using the BLASTp algorithm at the following default settings: expect threshold 10; word size 3; matrix BLOSUM 62; gap costs existence 11 , extension 1. In another embodiment, the person skilled in the art can readily and properly align any given sequence and deduce sequence identity/homology by mere visual inspection.

"Expression" or "expressing", as used herein refers to the process by which information from a gene is used in the synthesis of a functional gene product, and may relate to production of any detectable level of a product, or activity of a product, encoded by a gene. Gene expression may be modulated (i.e. initiated, increased, decreased, terminated, maintained or precluded) at many levels including transcription, RNA processing, translation, post-translational modification, protein degradation.

As used herein, a "construct" may refer to any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, vector, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source. A "construct" may comprise a promoter, a polyadenylation site, an enhancer or silencer and a transcription terminator, in addition to a nucleotide sequence encoding a gene or a gene fragment of interest.

"Expression construct" as used herein refers to any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. An expression construct of the disclosure nucleic acid molecule may further comprise a promoter and other regulatory elements, for example, an enhancer, a silencer, a polyadenylation site, a transcription terminator, a selectable marker or a screenable marker.

As used herein, a "promoter" refers to a nucleotide sequence that directs the initiation and rate of transcription of a coding sequence (reviewed in Roeder,

Trends Biochem Sci, 16: 402, 1991). The promoter contains the site at which RNA polymerase binds and also contains sites for the binding of other regulatory elements (such as transcription factors). Promoters may be naturally occurring or synthetic (see Datla et al. Biotech Ann. Rev 3:269, 1997 for review of plant promoters). Further, promoters may be species specific (for example, active only in B. napus); tissue specific (for example, the napin, phaseolin, zein, globulin, dlec2, γ-kafirin seed specific promoters); developmentally specific (for example, active only during embryogenesis); constitutive (for example maize ubiquitin, rice ubiquitin, rice actin, Arabidopsis actin, sugarcane bacilliform virus, CsVMV and CaMV 35S, Arabidopsis polyubiquitin, Solanum bulbocastanum polyubiquitin, Agrobacterium tumefaciens-derived nopaline synthase, octopine synthase, and mannopine synthase gene promoters); or inducible (for example the stilbene synthase promoter and promoters induced by light, heat, cold, drought, wounding, hormones, stress and chemicals). A promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box or an Inr element, and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. A promoter may also refer to a nucleotide sequence that includes a minimal promoter plus DNA elements that regulates the expression of a coding sequence, such as enhancers and silencers. Thus in one aspect, the expression of the constructs of the present disclosure may be regulated by selecting a species specific, a tissue specific, a development specific or an inducible promoter.

"Constitutive promoter" as used herein refers to a promoter which drives the expression of the downstream-located coding region in a plurality of or all tissues irrespective of environmental or developmental factors.

Enhancers and silencers are DNA elements that affect transcription of a linked promoter positively or negatively, respectively (reviewed in Blackwood and Kadonaga, Science, 281 : 61 , 1998).

Polyadenylation site refers to a DNA sequence that signals the RNA transcription machinery to add a series of the nucleotide A at about 30 bp downstream from the polyadenylation site. Transcription terminators are DNA sequences that signal the termination of transcription. Transcription terminators are known in the art. The transcription terminator may be derived from Agrobacterium tumefaciens, such as those isolated from the nopaline synthase, mannopine synthase, octopine synthase genes and other open reading frame from Ti plasmids. Other terminators may include, without limitation, those isolated from CaMV and other DNA viruses, dlec2, zein, phaseolin, lipase, osmotin, peroxidase, Pinll and ubiquitin genes, for example, from Solanum tuberosum.

In the context of the disclosure the nucleic acid construct may further comprise a selectable marker. Selectable markers may be used to select for organisms or cells that contain the exogenous genetic material. The exogenous genetic material may include, but is not limited to, an enzyme that confers resistance to an agent such as a herbicide or an antibiotic, or a protein that reports the presence of the construct.

Eukaryotic Organisms and Eukaryotic Cells

One aspect of the present disclosure relates a eukaryotic organism or eukaryotic cell comprising a first recombinant polynucleotide sequence encoding a recombinant polypeptide. The polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus that is sufficient to form a VLP in the organism or cell.

The skilled person will understand that the sequence of the M protein may vary between isolates of PEDv. Thus, in various embodiments, the M protein may have an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1. In various embodiments, the M protein may have an amino acid at least 80% identical to the amino acid sequence of SEQ ID

NO: 1. In various embodiments, the M protein may have an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 1. In various embodiments, the M protein has an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1. In various embodiments, the M protein has an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 1. In various embodiments, the M protein has an amino acid identical to the amino acid sequence of SEQ ID NO: 1.

The skilled person will understand that the portion of the M protein sufficient to form a VLP in the organism or cell may not include the entirety of the M protein. Moreover, the skilled person will be able to determine whether or not any particular portion is sufficient to form a VLP in the organism or cell. In particular embodiments, however, the portion of the M protein will include the entirety of the M protein.

In various embodiments, the polypeptide may further include a polypeptide tag to facilitate purification from, or recombinant protein accumulation in the organism or cell. In particular embodiments, the polypeptide tag may include an elastin-like polypeptide. Elastin-like polypeptides (ELPs) are pentapeptide repeat polymers of Val-Pro-Gly-Xaa-Gly, where the guest residue Xaa can be any amino acid except proline. ELPs have been explored as fusion partners for an inexpensive non-chromatographic method for protein purification. ELP fusions have also been shown to increase accumulation levels of several heterologous proteins. In various embodiments, the elastin-like polypeptide includes the amino acid sequence of SEQ ID NO: 2. However, the skilled person will understand that the number of repeats of the polymer may vary.

In various embodiments disclosed herein, the recombinant polypeptide comprises the sequence of SEQ ID NO: 3.

In various embodiments, the organism or cell further comprises a second recombinant polynucleotide sequence encoding a portion of an envelope (E) protein of PEDv. The skilled person will understand that the sequence of the E protein may vary between isolates of PEDv. Thus, in various embodiments, the E protein may have an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 4. In various embodiments, the E protein may have an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 4. In various embodiments, the E protein may have an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 4. In various embodiments, the E protein has an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In various embodiments, the E protein has an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 4. In various embodiments, the E protein has an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 4.

In the exemplary embodiments disclosure herein, the organism or cell is a plant or a plant cell. More particularly, the plants and plant cells exemplified herein are tobacco plants and tobacco plant cells. Even more particularly, the tobacco plants and plant cells are Nicotiana benthamiana plants and plant cells. However, the skilled person will reasonably expect that, while the plants producing PEDv VLPs disclosed herein are Nicotiana benthamiana plants, any plant that can be transformed to express recombinant polypeptides could be used to produce such VLPs comprising the PEDv M protein.

The introduction of DNA into plant cells by Agrobacterium mediated transfer is well known to those skilled in the art. If, for example, the Ti or Ri plasmids are used for the transformation of the plant cell, at least the right border, although more often both the right and the left border of the T-DNA contained in the Ti or Ri plasmid must be linked to the genes to be inserted as flanking region. If agrobacteria are used for the transformation, the DNA to be integrated must be cloned into special plasmids and specifically either into an intermediate or a binary vector. The intermediate vectors may be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination due to sequences, which are homologous to sequences in the T-DNA. This also contains the vir- region, which is required for T-DNA transfer. Intermediate vectors cannot replicate in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors are able to replicate in E. coli as well as in agrobacteria. They contain a selection marker gene and a linker or polylinker framed by the right and left T-DNA border region. They can be transformed directly into agrobacteria. The agrobacterium acting as host cell should contain a plasmid carrying a vir-region. The vir-region is required for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. Such a transformed agrobacterium is used for the transformation of plant cells. The use of T-DNA for the transformation of plant cells has been intensively studied and has been adequately described in standard review articles and manuals on plant transformation. Plant explants cultivated for this purpose with Agrobacterium tumefaciens or Agrobacterium rhizogenes can be used for the transfer of DNA into the plant cell.

Nevertheless, the present invention is not limited to any particular method for transforming plant cells, and the skilled person will readily understand that any other suitable method of DNA transfer into plant may be used. Methods for introducing nucleic acids into cells (also referred to herein as "transformation") are known in the art and include, but are not limited to: Viral methods (Clapp. Clin Perinatol, 20: 155-168, 1993; Lu et al. J Exp Med, 178: 2089-2096, 1993; Eglitis and Anderson. Biotechniques, 6: 608-614, 1988; Eglitis et al, Avd Exp Med Biol, 241 : 19-27, 1988); physical methods such as microinjection

(Capecchi. Cell, 22: 479-488, 1980), electroporation (Wong and Neumann. Biochim Biophys Res Commun, 107: 584-587, 1982; Fromm et al, Proc Natl Acad Sci USA, 82: 5824-5828, 1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang. Methods Cell Biol, 43: 353-365, 1994; Fynan et al. Proc Natl Acad Sci USA, 90: 11478-11482, 1993); chemical methods

(Graham and van der Eb. Virology, 54: 536-539, 1973; Zatloukal et al. Ann NY Acad Sci, 660: 136-153, 1992); and receptor mediated methods (Curiel et al. Proc Natl Acad Sci USA, 88: 8850-8854, 1991 ; Curiel et al. Hum Gen Ther, 3: 147-154, 1992; Wagner et al. Proc Natl Acad Sci USA, 89: 6099-6103, 1992). Another method for introducing DNA into plant cells is by biolistics. This method involves the bombardment of plant cells with microscopic particles (such as gold or tungsten particles) coated with DNA. The particles are rapidly accelerated, typically by gas or electrical discharge, through the cell wall and membranes, whereby the DNA is released into the cell and incorporated into the genome of the cell. This method is used for transformation of many crops, including corn, wheat, barley, rice, woody tree species and others. Biolistic bombardment has been proven effective in transfecting a wide variety of animal tissues as well as in both eukaryotic and prokaryotic microbes, mitochondria, and microbial and plant chloroplasts (Johnston. Nature, 346: 776-777, 1990; Klein et al. Bio Technol, 10: 286-291 , 1992; Pecorino and Lo. Curr Biol, 2: 30-32, 1992; Jiao et al, Bio/Technol, 1 1 : 497-502, 1993).

Another method for introducing DNA into plant cells is by electroporation. This method involves a pulse of high voltage applied to protoplasts/cells/tissues resulting in transient pores in the plasma membrane which facilitates the uptake of foreign DNA. The foreign DNA enter through the holes into the cytoplasm and then to the nucleus.

Plant cells may be transformed by liposome mediated gene transfer. This method refers to the use of liposomes, circular lipid molecules withr an aqueous interior, to deliver nucleic acids into cells. Liposomes encapsulate DNA fragments and then adhere to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus.

Other well-known methods for transforming plant cells which are consistent with the present invention include, but are not limited to, pollen transformation (See University of Toledo 1993 U.S. Pat. No. 5,177,010); Whiskers technology (See U.S. Pat. Nos. 5,464,765 and 5,302,523). The nucleic acid constructs of the present invention may be introduced into plant protoplasts. Plant protoplasts are cells in which its cell wall is completely or partially removed using either mechanical or enzymatic means, and may be transformed with known methods including, calcium phosphate based precipitation, polyethylene glycol treatment and electroporation (see for example Potrykus et al., Mol. Gen. Genet, 199: 183, 1985; Marcotte et al., Nature, 335: 454, 1988). Polyethylene glycol (PEG) is a polymer of ethylene oxide. It is widely used as a polymeric gene carrier to induce DNA uptake into plant protoplasts. PEG may be used in combination with divalent cations to precipitate DNA and effect cellular uptake. Alternatively, PEG may be complexed with other polymers, such as poly(ethylene imine) and poly L lysine.

The skilled person will understand that, while the methods of producing PEDv VLPs are herein exemplified for the first time using plants, the eukaryotic and organisms useful in the production of PEDv VLPs comprising the M protein may not be limited to plants. Now that the present inventors have demonstrated that PEDv VLPs can be produced, the skilled person will reasonably expect that the PEDv M protein may be expressed in other eukaryotic organisms to produce VLPs.

The skilled person will further understand that, while the exemplary embodiments disclosed herein employ transient expression, the methods for producing VLPs will work with organisms that are stably transformed with expression constructs as described herein.

Expression Constructs

Another aspect of the present disclosure relates to an expression construct comprising a polynucleotide molecule encoding a recombinant polypeptide. The polypeptide will include a portion of a membrane (M) protein of PEDv sufficient to form a VLP in a cell. The polynucleotide molecule is operably linked to a promoter for initiating transcription of the polynucleotide molecule in the cell.

Again, the skilled person understands that the sequence of the M protein may vary between isolates of PEDv. Thus, in various embodiments, the M protein may have an amino acid sequence that varies from SEQ ID NO: 1 as described above. In various embodiments, however, the M protein will have the amino acid sequence of SEQ ID NO: .

Furthermore, as discussed above, the skilled person will understand that the portion of the M protein sufficient to form a VLP in the organism or cell may not necessarily include the entirety of the M protein. Moreover, the skilled person will be able to determine whether or not any particular portion is sufficient to form a VLP in the organism or cell. In particular embodiments, however, the portion of the M protein will include the entirety of the M protein.

The polypeptide may further include a polypeptide tag to facilitate purification from, or recombinant protein accumulation in the organism or cell. In particular embodiments, the polypeptide tag may include an elastin-like polypeptide as describe above.

In various embodiments disclosed herein, the recombinant polypeptide comprises the sequence of SEQ ID NO: 3.

Another aspect of the present disclosure relates to an expression construct comprising a polynucleotide molecule encoding a recombinant polypeptide. The polypeptide will include a portion of a envelope (E) protein of PEDv useful in forming a VLP in a cell. The polynucleotide molecule is operably linked to a promoter for initiating transcription of the polynucleotide molecule in the cell.

Again, the skilled person understands that the sequence of the E protein may vary between isolates of PEDv. Thus, in various embodiments, the E protein may have an amino acid sequence that varies from SEQ ID NO: 4 as described above. In various embodiments, however, the E protein will have the amino acid sequence of SEQ ID NO: 4. Furthermore, as discussed above, the skilled person will understand that the portion of the E protein useful in forming a VLP in the organism or cell may not necessarily include the entirety of the E protein. Moreover, the skilled person will be able to determine whether or not any particular portion is useful in forming a VLP in the organism or cell. In particular embodiments, however, the portion of the E protein will include the entirety of the E protein.

The polypeptides may further include a polypeptide tag to facilitate purification from, or recombinant protein accumulation in the organism or cell. In particular embodiments, the polypeptide tag may include an elastin-like polypeptide as describe above.

In various embodiments, the cell is a eukaryotic cell. In the particular embodiments exemplified herein, the eukaryotic cell is a plant cell and, more particularly, a Nicotiana benthamiana plant cell. Accordingly, in embodiments involving a plant cell, the promoter will be a promoter operable to initiate transcription in a plant cell. In the expression constructs exemplified herein, the Cauliflower Mosaic Virus 35S, a promoter generally considered to be a constitutive promoter operable to initiate transcription in most plant tissues, is used to initiate expression of the polynucleotides. However, an abundance of promoters operable to initiate transcription in plants cells are known to the skilled person, and thus the skilled person will be able to choose an appropriate promoter.

As discussed above, the skilled person will reasonably expect that such expression constructs can be used with any plant that can be transformed with such constructs vectors. Furthermore, as discussed above, the skilled person will reasonably expect that the PEDv M protein, alone or in combination with the E protein, may be expressed in other eukaryotic organisms other than plants to produce VLPs, e.g. mammalian cells or insect cells, and the skilled person will be able to choose appropriate promoters for initiating expression in such eukaryotic cells, as well as appropriate means for delivering such expression constructs to the cells.

Methods of Producing VLPs

In another aspect, the disclosure relates to a method of producing a VLP comprising a portion of the membrane (M) protein of Porcine Epidemic Diarrhea virus. The method includes the step of expressing an expression construct as defined above in a cell. The method may further include expressing a portion of the envelope (E) protein of porcine Epidemic Diarrhea virus as described above. In the exemplary embodiments disclosed herein, the cell is a plant cell and, more particularly a Nicotiana benthamiana plant cell. However, the skilled person understands that the method could be performed with a variety of plant species. The skilled person further understands that the methods could be performed with other eukaryotic cells including yeast, mammalian cells, or insect cells.

In various embodiments, the method further includes a step of purifying the VLP from the cell. "Purified" as used herein does not require absolute purity. For the purposes of this disclosure, a "purified" or "isolated" VLP is one that has been separated from other cellular components to some extent. In various embodiments, the VLP will be purified to the extent that at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or substantially all of the cellular components have been removed. The VLPs disclosed herein can be purified by any of the means known to the skilled person Virus-Like Particles

In another aspect, the disclosure relates to a VLP comprising a recombinant polypeptide. The recombinant polypeptide comprises a portion of an M protein of PEDv. Again, the skilled person understands that the sequence of the M protein may vary between isolates of PEDv. Thus, in various embodiments, the M protein may have an amino acid sequence that varies from SEQ ID NO: 1 as described above. In various embodiments, however, the M protein will have the amino acid sequence of SEQ ID NO: 1. Furthermore, as discussed above, the skilled person will understand that the portion of the M protein may not necessarily include the entirety of the M protein. In particular embodiments, however, the portion of the M protein will include the entirety of the M protein. The VLP may further include a polypeptide tag to facilitate purification from a cell used to produce the VLP, or to facilitate VLP accumulation in such cell. In particular embodiments, the polypeptide tag may include an elastin-like polypeptide as describe above. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

In various embodiments, the VLP consists of the polypeptide comprising the portion of the M protein. In other embodiments, however, the VLP further comprises a polypeptide comprising a portion of an E protein of PEDv. Again, the skilled person understands that the sequence of the E protein may vary between isolates of PEDv. Thus, in various embodiments, the E protein may have an amino acid sequence that varies from SEQ ID NO: 4 as described above. In various embodiments, however, the E protein will have the amino acid sequence of SEQ ID NO: 4.

Referring to Figure 2B, a hypothetical PEDv VLP is shown generally at 40. PEDv VLP 40 is smaller in diameter, and forms with the M and E proteins. Envelope protein (E) 26 is embedded in the membrane 28 as is membrane protein (M) 30, encompassing an amino-terminal domain 30 outside the virus, three transmembrane segments 32, and a longer carboxy-terminal domain 34 inside the virus..

Differences in N-linked glycan processing in plant and mammalian cells occur during endomembrane transport as proteins transit through the Golgi apparatus (Downing et al. Plant biotechnology journal 4: 169-181 ). Within this compartment, enzymes convert the original high-mannose glycans of proteins to complex glycans by a series of sequential reactions that rely on the accessibility of the glycan chain(s) to the Golgi processing machinery. In mammalian cells, for example, core a(1 ,6)-fucose residues and terminal sialic acid residues are added to the trimmed N-linked glycans. However, bisecting (1 ,2)-xylose and core a(1 ,3)-fucose residues are assembled on to the trimmed N-linked glycans of plant-synthesized proteins; core a(1 ,6)-fucose residues and terminal sialic acid residues are added in mammalian cells. Thus typically processed N-linked glycans of proteins produced in plants are mostly of a Man₃GlcNAc₂ structure with or without -1 ,2-xylose and/or a-1 ,3-fucose residues. Accordingly, in embodiments involving VLPs produced in plant cells, the VLP will have N-linked glycans having bisecting (1 ,2)-xylose, core a(1 ,3)-fucose residues, or both. Moreover, since the sugars associated with plant-specific complex glycans have been shown to be immunogenic in several animal species, plant-derived VLPs could be particularly useful as vaccines. Methods of determining the glycosylation patterns of a glycoprotein are disclosed in Downing et al.

Alternatively, VLPs with altered glycosylation products could be produced using plants in which the N-linked glycosylation pathway has been modified. For example, the cgl mutant of Arabidopsis thaliana ls blocked in the synthesis of complex N-glycans because of a deficiency of N- acetylglucosaminyl transferase I (GnT I), the first enzyme in the pathway of complex glycan biosynthesis. With no addition of N-acetylglucosamine to the trimmed glycan, xylosyl- and fucosyl-transferases are unable to add xylose and fucose, respectively. Thus, the N-glycans on proteins synthesized in this mutant are in the high-mannose form, predominantly Man₅-GlcNAc₂, with minor amounts of Man₆, Man₇ and Man₈ (reviewed in Downing et al).

Accordingly, plant genetic backgrounds devoid of GNT I activity can be used to produce recombinant proteins, including VLPs, devoid of complex glycans.

In various embodiments, the VLP will be an isolated VLP that has been purified from the cell used to produce it. "Isolated" as used herein refers to being substantially separated or purified away from other biological components of the cell in which the VLP is produced. As discussed above, "purified" as used herein does not require absolute purity. For the purposes of this disclosure, a purified VLP is one that has been separated from other cellular components to some extent. The VLPs disclosed herein can be purified by any of the means known to the skilled person

Immunogenic Compositions and Vaccines In another aspect, the disclosure relates to a vaccine for vaccinating a porcine against infection by Porcine Epidemic Diarrhea virus. The vaccine comprises a VLP as described above. Because VLP is particulate, it may be possible to administer the vaccine without the use of adjuvants. Nevertheless, the vaccine may further include an adjuvant. Suitable adjuvants for use in the vaccine are known to the skilled person and include those described in WO

2015/179412.

In another aspect, the disclosure relates to a method of inducing mucosal immunity against Porcine Epidemic Diarrhea virus in a porcine. The method comprises feeding a VLP as described herein to the porcine. Alternatively, the method comprises feeding a plant expressing a VLP as described herein, or plant material harvested from such a plant, to the porcine. Examples

Constructs

Peptide sequences for the M, E, N, and Sproteins were obtained from the National Center for Biotechnology Information (NCBI) database (accession number KF650373). This sequence is from a PEDv strain isolated in the U.S. that showed >99.7% nucleotide identity with nine other U.S. strains that have been sequenced (Chen et al., 2014). The M, E, N, and S nucleotide sequences were synthesized by BioBasic Inc. (Markham, Ontario), optimized for nuclear expression in Nicotiana tabacum with a C-terminal fusion to a

Strepll tag (Schmidt et al., Journal of Molecular Biology 255, 753-766) for protein purification.

Gateway® cloning technology (Invitrogen, Thermo Fisher Scientific, Waltham, U.S.A.) was used to recombine the sequences encoding the M, E, N, and S proteins into pCaMGate expression vectors (Pereira et al., BMC Biotechnology 14, 59). Gene constructs that were synthesized with flanking attB sites underwent a BP reaction to be cloned into the pDONR vector. This was followed by an LR reaction to introduce the inserts into various expression vectors.

Referring to Figure 3, the gene sequences encoding M, E, N, and S were cloned into an expression cassette including both N- and C-terminal tags. An N-terminal tobacco pathogenesis-related-1 b signal peptide (PR1 b; Huub and Van Loon, Critical Reviews in Plant Sciences 10, 123-150) was included for subcellular targeting. An Xpress tag (DLYDDDDK) and a C-terminal c-Myc tag were included to facilitate immunodetection of the recombinant protein. An Elastin-like Polypeptide sequence (ELP) was included to potentially enhance protein accumulation (Conley et al., BMC Biology 7, 48-48). A further C-terminal KDEL peptide was included to direct the recombinant M protein to the endoplasmic reticulum (ER). For the purposes of this disclosure, the expression construct comprising the coding sequences for the M protein is referred to as the "M Construct" and the encoded polypeptide is referred to as "M Polypeptide". Similarly, for the purposes of this disclosure, the expression construct comprising the coding sequences for the E protein is referred to as the Έ Construct" and the encoded polypeptide is referred to as Έ Polypeptide". For the purposes of this disclosure, the expression construct comprising the coding sequences for the N protein is referred to as the "N Construct" and the encoded polypeptide is referred to as "N Polypeptide". For the purposes of this disclosure, the expression construct comprising the coding sequences for the S protein is referred to as the "S Construct" and the encoded polypeptide is referred to as "S Polypeptide". The skilled person will understand that the signal peptide is ultimately removed during cellular processing to result in a mature polypeptide. The coding sequences for the M Construct and E Construct, the amino acid sequences (both full and mature) for the M Polypeptide and E Polypeptide, and the amino acid sequences of the M, E, N, and S proteins are provided in the list of sequences at the end of the description.

Transient Expression in Nicotiana benthamiana

Cultures of Agrobacterium tumefaciens EHA 105 cells transformed with the M Construct, the E Construct, the N Construct, and the S Construct were used to infiltrate 5-7 week old N. benthamiana plants that were grown in a growth room with the following conditions: 16 hours of light/8 hours of dark, 21 -22°C, 55% humidity, and receiving approximately 100 μΓηοΙ/photons m-2s-1 of photosynthetically active radiation (PAR). If syringe infiltrations were performed, a 3 ml syringe was used to infiltrate the A. tumefaciens suspensions into the underside of the leaves, occasionally with the help of a needle to make a cut. Three different biological replicates were used, with constructs having equal representation on the lower, middle, and upper leaves of the plants. After infiltration, plants were returned to the growth chamber until tissue was collected two to eight days later. A. tumefaciens cultures carrying the M Construct, the E Construct, the N Construct, the S Construct, and combinations thereof, were infiltrated into N. benthamiana leaves with a tumefaciens carrying a p19 expression construct, a suppressor of gene silencing (Silhavy et al., EMBO Journal 21 , 3070-3080). Samples were collected from plant leaves four days post infiltration (DPI).

Total soluble protein (TSP) was then extracted with either PEB or FEB (see Table 2), separated on 12% SDS-PAGE gels, and analyzed by Western blotting to detect the recombinant protein. Samples were collected from three different biological replicates consisting of different plants. Table 2. Composition of various buffers used for protein extraction.

Extraction Detergent Protease Inhibitors Buffer Buffer

Plant 0.1 % (v/v) Tween-20 2% (PVPP) (w/v), 1 mM 1xPBS, pH

Extraction EDTA, 1 mM PMSF, 1 7.8

Buffer (PEB) μg/ml leupeptin, 100

mM sodium L- abscorbate

ProteoExtract® 0.2% digitonin (Buffer 1) Protease Inhibitor 300 mM

Native 0.5% Triton X-100 (Buffer 2) Cocktail (Calbiochem Saccharose

Membrane 539134; 100 mM 15 mM

Protein AEBSF, 80 μΜ NaCI

Extraction Kit³ aprotinin, 5 mM 10 mM bestatin, 1.5 mM E-64, Pipes pH

2 mM leupeptin, 1 mM 7.2 pepstatin A, in DMSO)

Final Lab 1.5% (v/v) Triton X-100 2% PVPP, 1 mM 100 mM

Extraction EDTA, 1 mM PMSF, 1 Tris pH 8,

Buffer (FEB) mg/ml leupeptin, 100 300 mM mM sodium L- sucrose, 15 abscorbate mM NaCI

VLP Extraction 0.1% Triton X-100 2% PVPP, 1 mM 50 mM

Buffer (VEB) EDTA, 100 mM sodium Tris-HCI pH

L-abscorbate 7.5

140 mM

NaCI

Figure 4 is a Western blot of protein extracts from three N. benthamiana plants in which the M Construct was transiently expressed in leaves, using the anti-c-Myc primary antibody as a probe to detect the recombinant protein. A band of the expected size for the M Polypeptide, i.e. 43 kDa, was observed.

Total recombinant protein was quantified with dot blots using Totallab TL100 software (Nonlinear Dynamics, Durham, USA). Sample extracts and negative controls from the same biological replicates were spotted on to nitrocellulose membranes (Bio-Rad Laboratories, Inc.) in a dilution series. Known amounts of a cellulose binding domain (CBD) synthetic protein standard (GenScript) were also spotted in two sets of dilutions for densitometry analysis. Two sets of CBD dilutions were used to develop a standard curve which was used to determine the amount of protein in samples. The amount of protein in mg per g was then calculated using the tissue weight of each sample. Final accumulation amount was determined by subtracting any detected protein in negative control dilutions from the amount of protein detected in sample dilutions. A balanced analysis of variance (ANOVA), followed by Tukey Pairwise Comparisons was performed with Minitab (State College,

Pennsylvania, USA) to analyze differences in mean accumulation levels. Statistical significance level for all the tests was defined as 0.05 or lower. M Polypeptide production in N. benthamiana leaves was determined to be above 0.7 mg/g of fresh leaf weight.

Figure 5 is a Western blot of extracts from N. benthamiana leaves in which the M Construct, E Construct, N Construct, and S Construct were individually expressed. The anti-c-Myc primary antibody was used as the probe to detect the recombinant proteins. M Polypeptide and E Polypeptide monomers are clearly detected in the first and third lanes from the left, respectively. The E

Construct showed a major band of the predicted size as well as a putative dimer (26 kDa and slightly larger than 50 kDa, respectively).

Mass spectrometry analysis of the putative E Polypeptide dimer band confirmed the presence of E Polypeptide, indicating that E Polypeptide was forming a dimer The bands detected in extracts from leaves transformed with the N Construct (the second lane from the left) were smaller than the expected size of 66.6 kDa, i.e. 26 kDa and slightly larger than 50 kDa. None of the bands detected in extracts from leaves transformed with the S Construct (the fourth lane from the left) corresponded to the predicted 169.4 kDa monomer. Neither full size S Polypeptide nor a breakdown product were observed. All lanes showed a band smaller than 37 kDa thought to be plant peroxidase, due to its presence in the wild type tissue extract (fifth lane from the left).

Three combinations of A. tumefaciens carrying expression constructs were co-infiltrated: M Construct with E construct; M Construct with E Construct and N Construct; and M Construct with E Construct, N Construct, and S Construct. Figure 6 is a Western blot of extracts from N. benthamiana leaves in which, from left to right, the M Construct, E Construct, N Construct, and S Construct were individually expressed, and in which: M Construct is co-expressed with E Construct; M Construct is co-expressed with E Construct and N Construct; and M Construct is co-expressed with E Construct, N Construct, and S Construct. Protein was extracted from pooled leaf tissue from three plants with FEB. The M Construct, N Construct, E Construct, S Construct and wild type (non- infiltrated) tissue lanes (the first to fifth lanes from the left, respectively) show generally the same banding patterns as in Figure 5. When proteins were co- expressed, the banding patterns of the respective proteins were all found. Co- expression of Construct and E Construct resulted in detection of the E Polypeptide monomer and dimer, and the M Polypeptide monomer (sixth lane from the left). Co-expression of N Construct (seventh lane from the left), or N Construct and S Construct (eighth lane from the left), with M Construct and E Construct resulted in the detection of N Polypeptide fragments, with the N Polypeptide fragments detected most strongly when all four proteins were co- expressed. In contrast, M Polypeptide and E Polypeptide bands were weaker with the co-expression of N Construct or N Construct and S Construct. Figure 7 is a Western blot of extracts from, from left to right, wild-type Nicotiana benthamiana leaves (first to third lanes) and N. benthamiana leaves in which: M Construct is co-expressed with E Construct (fourth to sixth lanes); M Construct is co-expressed with E Construct, N Construct, and S Construct (eighth to tenth lanes); and M Construct is expressed alone (twelfth to fourteenth lanes). "B" indicates an empty lane. Proteins were extracted with a commercial kit, i.e. Protein was extracted with ProteoExtract™ Native Membrane Protein Extraction Kit, as opposed to FEB (see Table 2). When proteins were extracted with the commercial kit, a laddering effect was observed. In extracts corresponding to co-expression of M Construct and E Construct, bands the size of the E Polypeptide monomer and dimer were observed (at the positions marked on the right with one and two squares, respectively). When M Construct was expressed alone or together with E Construct, the M Polypeptide monomer was not observed (at the expected position marked by a single diamond on the right). Rather, an approximately 80 kDa band was observed (at the position marked by two diamonds on the left) that might correspond to a dimer of M Polypeptide. This dimer was also the only recombinant protein observed in the M Construct-only lanes. The higher molecular weight bands observed on this blot likely indicate multimerization and VLP formation.

The higher molecular weight bands observed in Figure 7 indicated that the proteins are assembling into higher order structures, and possibly VLPs.

Accordingly, M Construct was expressed alone or in combination with E Construct in leaves, and 8 g of infiltrated and wild type tissue was harvested four days postinfiltration.

To extract protein, infiltrated leaf tissue was ground into a fine powder with a mortar and pestle. Three parts (v/w) VLP Extraction Buffer (see Table 2), were added to the mortar, and the mixture was ground in to a paste. Using a serological pipette the solution was then transferred to a conical tube. The solution was centrifuged twice for ten minutes at 4,200 x g. To concentrate any produced VLPs, 22 ml of supernatant was applied to a discontinuous sucrose gradient ranging from 30% to 60% in 10% 3 ml steps. The gradient was then ultracentrifuged using an Optima™ L-100 (Beckman Coulter) and a SW28 rotor at 86,329 x g at 4°C. Ultracentrifuge tubes had a hole pierced in the bottom through which each fraction was collected into 1 ml aliquots.

Samples were analyzed by immunoblot. Again, the anti-c-Myc primary antibody was used as the probe to detect the recombinant protein..

Referring to Figure 8A, the full-length M Polypeptide was detected in all fractions, with the highest amount of protein in the 40% fraction. A 25 kDa cleavage product previously observed was detected less in the heavier sucrose gradient fractions, which would contain sedimented VLPs (Figure 8A). Truncated M Polypeptide not being detected in heavier sucrose fractions suggests that the truncated protein may not be incorporated into VLPs.

Referring to Figure 8B, analyzing the sucrose gradient fractions of co- expressed M Construct and E Construct showed the combined banding pattern of the M Polypeptide monomer and the E Polypeptide monomer and dimer. The highest amount of total detected protein in this protein extract is also in the 40% fraction. The E Polypeptide and M Polypeptide monomers are heaviest at 40% sucrose, and the putative E Polypeptide dimer is heaviest at 30% sucrose (Figure 8B). The smallest band of the M construct/ E construct sucrose gradient was analyzed, and found to contain both E polypeptide and the truncated M Polypeptide. As both M Construct and M Construct + E Construct extracts showed the highest amount of protein at 40% sucrose, i.e. the same density where PEDv virions are expected to be found (Hofmann and Wyler, Veterinary Microbiology 20, 131 -142), these fractions were chosen for TEM analysis

VLP analysis

The 40% fraction from the sucrose gradients of both treatments and the wild type were analyzed by transmission electron microscopy TEM. A droplet of extract was placed on carbon grids (Electron Microscopy Sciences, Hartfield, U.S.), and allowed to sit for two minutes. Liquid was then drawn off, and the grid was washed in three consecutive drops of water for two minutes each. Finally, a drop of 2% uranyl acetate was placed on the grid, and allowed to sit for a minute before being drawn off. The negatively stained grids were examined with a CM-10 transmission electron microscope (Philips, Amsterdam, Netherlands) equipped with a digital camera (Advanced Microscopy Techniques, MA) at 80 kV.

A droplet of protein extract from the 40% sucrose fraction from either M Construct, co-expressed M Construct and E Construct, or wild type tissue was placed on a carbon grid (Electron Microscopy Sciences), and allowed to sit for two minutes. Liquid was then drawn off. Grids were blocked by placing them specimen-side down in a drop of goat normal serum (25596; Aurion,

Netherlands) for fifty minutes. Grids were washed in two consecutive drops of dilution buffer (1% bovine serum albumin (BSA), 0.2% BSA-c™ (Aurion), 0.05% Tween-20 in PBS pH 7.35) for two minutes each. One set of grids was incubated specimen-side down in a droplet of mouse anti-c-Myc primary antibody diluted 1 :10 with dilution buffer , while a second set of grids was simultaneously incubated with just dilution buffer as a negative control. All grids were then washed in three consecutive drops of dilution buffer for two minutes. All grids were incubated for one hour in a drop of secondary antibody diluted 1 :10 in dilution buffer. Secondary antibody was goat antimouse IgG conjugated to 10 nm gold particles (Aurion). Grids were washed in three consecutive drops of dilution buffer for ten minutes each, and then in four consecutive drops of Milli-Q water for three minutes each. Once dry, grids were stained with 2% uranyl acetate and examined under TEM as described above.

The pixel count of TEM scale bars were measured to deduce the nm:pixel ratio for each image. The pixel count of VLP envelope thickness was measured in three areas for each VLP, and the measurements for all VLPs were averaged. This average was multiplied by the nm:pixel ratio to obtain an estimate of VLP envelope thickness. Referring to Figure 9A, TEM revealed no VLP structures in wild type tissue.

Referring to Figure 9B, TEM revealed that the 40% fraction from extracts of leaves expressing M Construct alone contained circular particles. While native virions, excluding spike projections are just larger than 100 nm, M VLPs for SARS were observed to be 50 nm in diameter (Tseng et al., 2010). The particles observed in Figure 9a were also about 50 nm in diameter. A distinct feature of coronaviruses is that M forms a lattice in the viral envelope, resulting in a membrane twice the thickness of typical biological membranes (4 nm) (Barcena et al., Proceedings of the National Academy of Sciences of the United States of America 106, 582-587). The observed particles have an envelope thickness of approximately 9.3 nm. Thus, the size and envelope thickness of the particles indicates that these are VLPs formed with only the M protein component of PEDv.

Referring to Figure 9C, TEM revealed that extracts from leaves co-expressing M Construct and E Construct showed similar particles. Figure 9c shows that when the two proteins were co-expressed, the VLPs were larger than with M Polypeptide alone. The VLPs observed in Figure 9c have a thick membrane of 10.1 nm, and are approximately 80 nm in diameter, again indicating that they are VLPs.

To confirm that the observed VLP structures in Figures 9B and 9C contained M Polypeptide, immunogold TEM was performed on the with same fraction as depicted in 9B above. Again, the primary antibody used for detecting M polypeptide was the c-Myc tag antibody. As the c-Myc tag is at the C-terminus the M Polypeptide, which should fold to the inside a VLP, the inventors predicted that immunogold TEM should reveal no binding of the antibody when virions are intact, but that binding would be observed on burst virions. Consistent with this hypothesis, Figure 0 reveals that the anti-c-Myc antibody was able to bind to burst but not intact structures, giving further evidence that these VLPs are formed with the M Polypeptide, and that protein is folding in the correct conformation. Thus, the present disclosure represents the first reported instance of PEDv VLP production.

A list of the sequences relevant to the M, E, N, and S Constructs and Polypeptides are as follows: SEQ ID NO: 1 Amino acid sequence of M in M Polypeptide

SNGSIPVDEVIQHLRNWNFTWNIILTILLVVLQYGHYKYSAFLYGVKMAILWILWPLVLALSLFDAWAS FQVNWVFFAFSILMACITLMLWIMYFVNSIRLWRRTHSWWSFNPETDALLTTSVMGRQVCIPVLGAPTG VTLTLLSGTLLVEGYKVATGVQVSQLPNFVTVAKATTTIVYGRVGRSVNASSGTG AFYVRSKHGDYSA VSNPSSVLTDSEKVLHLV

SEQ ID NO: 2 Amino acid sequence of ELP in M Polypeptide, E Polypeptide, N Polypeptide, and S Polypeptide

VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VG

SEQ ID NO: 3 Amino Acid Sequence of expected mature M Polypeptide DLYDDDDKSNGSIPVDEVIQHLRN NFT NI ILTILLVVLQYGHYKYSAFLYGVK AILWILWPLVLAL

SLFDAWASFQVN VFFAFSILMACITLML IMYFVNSIRL RRTHSWWSFNPETDALLTTSVMGRQVCI PVLGAPTGVTLTLLSGTLLVEGYKVATGVQVSQLPNFVTVAKATTTIVYGRVGRSVNASSGTGWAFYVR SKHGDYSAVSNPSSVLTDSE VLHLVWSHPQFEKDPAFLYKVVITVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGEQKLISEEDLKDEL

SEQ ID NO: 4 Amino acid sequence of E in E Polypeptide

LQLVNDNGLVVNVIL LFVLFFLLIISITFVQLVNLCFTCHRLCNSAVYTPIGRLYRVYKSYMQIDPLP STVIDV SEQ ID NO: 5 Coding sequence for M Construct (with, from the 5' end to the 3' end: the PR1 b signal peptide; Xpress tag, attB1 site; M protein; STREPII tag and attB2 site; ELP;c-Myc tag; and KDEL tag)

ATGGGATTTTTTCTCTTTTCACAAATGCCCTCATTTTTTCTTGTCTCTACACTTCTCTTATTCCTAATA ATATCTCACTCTTCTCATGCCGATCTCTATGATGACGATGACAAAGTTATCACAAGTTTGTACAAAAAA GCAGGCTTAAGTAACGGTAGTATCCCTGTCGATGAAGTCATCCAACATCTGAGAAACTGGAACTTCACC TGGAATATCATACTGACCATTCTACTGGTCGTCTTGCAATACGGTCATTACAAGTACAGTGCATTCCTC TATGGTGTGAAGATGGCTATACTATGGATTCTATGGCCTCTCGTACTCGCTTTGAGTCTTTTCGATGCA TGGGCATCATTTCAAGTAAACTGGGTTTTCTTTGCTTTTAGCATTCTCATGGCCTGCATTACCTTGATG CTTTGGATTATGTATTTTGTTAATTCCATTCGACTTTGGAGGCGTACCCACAGCTGGTGGTCATTCAAT CCAGAGACTGATGCATTGCTTACAACTAGCGTGATGGGTAGACAGGTTTGTATTCCAGTGTTGGGTGCT CCAACTGGAGTTACTTTAACACTTTTGTCTGGAACATTGCTTGTGGAAGGATACAAGGTTGCAACAGGA GTTCAGGTTTCACAGTTACCCAATTTTGTGACTGTGGCTAAAGCTACAACTACTATAGTATATGGGCGT GTTGGAAGGTCTGTTAATGCTTCCTCTGGCACTGGCTGGGCCTTTTATGTTAGATCTAAACATGGGGAC TATTCTGCCGTATCAAATCCGTCATCTGTTTTAACAGACTCCGAGAAAGTTCTTCACTTAGTATGGTCA CATCCTCAATTTGAGAAAGACCCAGCTTTCTTGTACAAAGTGGTGATAACTGTTCCAGGGGTAGGTGTC CCTGGTGTCGGTGTACCGGGTGTCGGAGTGCCTGGCGTAGGGGTTCCGGGAGTGGGTGTCCCAGGTGTC GGCGTACCAGGAGTCGGAGTCCCCGGAGTAGGAGTTCCAGGGGTGGGAGTTCCAGGAGTAGGAGTACCT GGCGTGGGTGTACCTGGTGTTGGTGTCCCAGGAGTGGGAGTTCCTGGAGTTGGAGTCCCTGGAGTCGGA GTGCCAGGAGTGGGTGTACCCGGTGTAGGCGTGCCTGGTGTTGGTGTACCCGGAGTTGGAGTGCCCGGC GTAGGGGTTCCAGGTGTGGGGGTGCCCGGAGTCGGTGTCCCTGGTGTAGGGGTTCCAGGAGTCGGCGTG CCGGGTGTTGGAGTACCTGGTGTGGGGGTCCCCGGAGTAGGGGTGCCTGGTGTCGGCGAACAAAAGTTG ATCTCTGAGGAAGACCTCAAGGATGAGCTTTGA

SEQ ID NO: 6 Complete amino acid sequence of M Construct (translated from SEQ ID NO: 5)

GFFLFSQMPSFFLVSTLLLFLI ISHSSHADLYDDDDKVI SLYKKAGLSNGSIPVDEVIQHLRNWNFT WNIILTILLVVLQYGHYKYSAFLYGVKMAILWILWPLVLALSLFDAWASFQVN VFFAFSILMACITLM LWIMYFVNSIRL RRTHSW SFNPETDALLTTSVMGRQVCIPVLGAPTGVTLTLLSGTLLVEGYKVATG VQVSQLPNFVTVAKATTTIVYGRVGRSVNASSGTGWAFYVRS HGDYSAVSNPSSVLTDSEKVLHLVWS HPQFEKDPAFLYKVVITVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVP GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGV PGVGVPGVGVPGVGVPGVGEQKLISEEDLKDEL

SEQ ID NO 7: Coding sequence for expected mature M Polypeptide:

AGTAACGGTAGTATCCCTGTCGATGAAGTCATCCAACATCTGAGAAACTGGAACTTCACCTGGAATATC ATACTGACCATTCTACTGGTCGTCTTGCAATACGGTCATTACAAGTACAGTGCATTCCTCTATGGTGTG AAGATGGCTATACTATGGATTCTATGGCCTCTCGTACTCGCTTTGAGTCTTTTCGATGCATGGGCATCA TTTCAAGTAAACTGGGTTTTCTTTGCTTTTAGCATTCTCATGGCCTGCATTACCTTGATGCTTTGGATT ATGTATTTTGTTAATTCCATTCGACTTTGGAGGCGTACCCACAGCTGGTGGTCATTCAATCCAGAGACT GATGCATTGCTTACAACTAGCGTGATGGGTAGACAGGTTTGTATTCCAGTGTTGGGTGCTCCAACTGGA GTTACTTTAACACTTTTGTCTGGAACATTGCTTGTGGAAGGATACAAGGTTGCAACAGGAGTTCAGGTT TCACAGTTACCCAATTTTGTGACTGTGGCTAAAGCTACAACTACTATAGTATATGGGCGTGTTGGAAGG TCTGTTAATGCTTCCTCTGGCACTGGCTGGGCCTTTTATGTTAGATCTAAACATGGGGACTATTCTGCC GTATCAAATCCGTCATCTGTTTTAACAGACTCCGAGAAAGTTCTTCACTTAGTATGGTCACATCCTCAA TTTGAGAAAGACCCAGCTTTCTTGTACAAAGTGGTGATAACTGTTCCAGGGGTAGGTGTCCCTGGTGTC GGTGTACCGGGTGTCGGAGTGCCTGGCGTAGGGGTTCCGGGAGTGGGTGTCCCAGGTGTCGGCGTACCA GGAGTCGGAGTCCCCGGAGTAGGAGTTCCAGGGGTGGGAGTTCCAGGAGTAGGAGTACCTGGCGTGGGT GTACCTGGTGTTGGTGTCCCAGGAGTGGGAGTTCCTGGAGTTGGAGTCCCTGGAGTCGGAGTGCCAGGA GTGGGTGTACCCGGTGTAGGCGTGCCTGGTGTTGGTGTACCCGGAGTTGGAGTGCCCGGCGTAGGGGTT CCAGGTGTGGGGGTGCCCGGAGTCGGTGTCCCTGGTGTAGGGGTTCCAGGAGTCGGCGTGCCGGGTGTT GGAGTACCTGGTGTGGGGGTCCCCGGAGTAGGGGTGCCTGGTGTCGGCGAACAAAAGTTGATCTCTGAG GAAGACCTCAAGGATGAGCTTT

SEQ ID NO: 8 Amino Acid Sequence of expected mature M Polypeptide

DLYDDDDKSNGSIPVDEVIQHLRNWNFT NIILTILLVVLQYGHYKYSAFLYGVKMAILWILWPLVLAL SLFDAWASFQVNWVFFAFSILMACITLMLWIMYFVNSIRL RRTHSWWSFNPETDALLTTSVMGRQVCI PVLGAPTGVTLTLLSGTLLVEGYKVATGVQVSQLPNFVTVAKATTTIVYGRVGRSVNASSGTGWAFYVR SKHGDYSAVSNPSSVLTDSEKVLHLVWSHPQFEKDPAFLY VVITVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGEQKLISEEDLKDEL

SEQ ID NO: 9 Coding sequence for M in M Construct

AGTAACGGTAGTATCCCTGTCGATGAAGTCATCCAACATCTGAGAAACTGGAACTTCACCTGGAATATC ATACTGACCATTCTACTGGTCGTCTTGCAATACGGTCATTACAAGTACAGTGCATTCCTCTATGGTGTG AAGATGGCTATACTATGGATTCTATGGCCTCTCGTACTCGCTTTGAGTCTTTTCGATGCATGGGCATCA TTTCAAGTAAACTGGGTTTTCTTTGCTTTTAGCATTCTCATGGCCTGCATTACCTTGATGCTTTGGATT ATGTATTTTGTTAATTCCATTCGACTTTGGAGGCGTACCCACAGCTGGTGGTCATTCAATCCAGAGACT GATGCATTGCTTACAACTAGCGTGATGGGTAGACAGGTTTGTATTCCAGTGTTGGGTGCTCCAACTGGA GTTACTTTAACACTTTTGTCTGGAACATTGCTTGTGGAAGGATACAAGGTTGCAACAGGAGTTCAGGTT TCACAGTTACCCAATTTTGTGACTGTGGCTAAAGCTACAACTACTATAGTATATGGGCGTGTTGGAAGG TCTGTTAATGCTTCCTCTGGCACTGGCTGGGCCTTTTATGTTAGATCTAAACATGGGGACTATTCTGCC GTATCAAATCCGTCATCTGTTTTAACAGACTCCGAGAAAGTTCTTCACTTAGTA SEQ ID NO: 10 Coding sequence of ELP in M Construct, E Construct, N Construct, and S Construct

GTTCCAGGGGTAGGTGTCCCTGGTGTCGGTGTACCGGGTGTCGGAGTGCCTGGCGTAGGGGTTCCGGGA GTGGGTGTCCCAGGTGTCGGCGTACCAGGAGTCGGAGTCCCCGGAGTAGGAGTTCCAGGGGTGGGAGTT CCAGGAGTAGGAGTACCTGGCGTGGGTGTACCTGGTGTTGGTGTCCCAGGAGTGGGAGTTCCTGGAGTT GGAGTCCCTGGAGTCGGAGTGCCAGGAGTGGGTGTACCCGGTGTAGGCGTGCCTGGTGTTGGTGTACCC GGAGTTGGAGTGCCCGGCGTAGGGGTTCCAGGTGTGGGGGTGCCCGGAGTCGGTGTCCCTGGTGTAGGG GTTCCAGGAGTCGGCGTGCCGGGTGTTGGAGTACCTGGTGTGGGGGTCCCCGGAGTAGGGGTGCCTGGT GTCGGC

SEQ ID NO: 11 Complete coding sequence of E Construct

ATGGGATTTTTTCTCTTTTCACAAATGCCCTCATTTTTTCTTGTCTCTACACTTCTCTTATTCCTAATA ATATCTCACTCTTCTCATGCCGATCTCTATGATGACGATGACAAAGTTATCACAAGTTTGTACAAAAAA GCAGGCTTATTACAGCTGGTAAACGATAACGGTCTTGTAGTTAATGTCATCTTGTGGCTATTCGTTTTG TTCTTTTTACTGATCATATCCATTACCTTTGTGCAACTCGTCAATCTCTGCTTTACTTGTCATAGGCTT TGTAATTCAGCTGTTTACACTCCAATTGGAAGATTGTACCGTGTTTATAAGTCTTATATGCAAATAGAC CCTCTTCCCAGTACAGTGATTGATGTGTGGTCACATCCTCAATTTGAGAAAGACCCAGCTTTCTTGTAC AAAGTGGTGATAACTGTTCCAGGGGTAGGTGTCCCTGGTGTCGGTGTACCGGGTGTCGGAGTGCCTGGC GTAGGGGTTCCGGGAGTGGGTGTCCCAGGTGTCGGCGTACCAGGAGTCGGAGTCCCCGGAGTAGGAGTT CCAGGGGTGGGAGTTCCAGGAGTAGGAGTACCTGGCGTGGGTGTACCTGGTGTTGGTGTCCCAGGAGTG GGAGTTCCTGGAGTTGGAGTCCCTGGAGTCGGAGTGCCAGGAGTGGGTGTACCCGGTGTAGGCGTGCCT GGTGTTGGTGTACCCGGAGTTGGAGTGCCCGGCGTAGGGGTTCCAGGTGTGGGGGTGCCCGGAGTCGGT GTCCCTGGTGTAGGGGTTCCAGGAGTCGGCGTGCCGGGTGTTGGAGTACCTGGTGTGGGGGTCCCCGGA GTAGGGGTGCCTGGTGTCGGCGAACAAAAGTTGATCTCTGAGGAAGACCTCAAGGATGAGCTTTGA

SEQ ID NO: 12 Complete amino acid sequence of E Construct

GFFLFSQMPSFFLVSTLLLFLIISHSSHADLYDDDDKVITSLYKKAGLLQLVNDNGLVVNVILWLFVL FFLLIISITFVQLVNLCFTCHRLCNSAVYTPIGRLYRVYKSYMQIDPLPSTVIDVWSHPQFEKDPAFLY KVVITVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VGVPGVGEQKLISEEDLKDEL .

SEQ ID NO: 13 Coding sequence for expected mature E Polypeptide

TTACAGCTGGTAAACGATAACGGTCTTGTAGTTAATGTCATCTTGTGGCTATTCGTTTTGTTCTTTTTA CTGATCATATCCATTACCTTTGTGCAACTCGTCAATCTCTGCTTTACTTGTCATAGGCTTTGTAATTCA GCTGTTTACACTCCAATTGGAAGATTGTACCGTGTTTATAAGTCTTATATGCAAATAGACCCTCTTCCC AGTACAGTGATTGATGTGTGGTCACATCCTCAATTTGAGAAAGACCCAGCTTTCTTGTACAAAGTGGTG ATAACTGTTCCAGGGGTAGGTGTCCCTGGTGTCGGTGTACCGGGTGTCGGAGTGCCTGGCGTAGGGGTT CCGGGAGTGGGTGTCCCAGGTGTCGGCGTACCAGGAGTCGGAGTCCCCGGAGTAGGAGTTCCAGGGGTG GGAGTTCCAGGAGTAGGAGTACCTGGCGTGGGTGTACCTGGTGTTGGTGTCCCAGGAGTGGGAGTTCCT GGAGTTGGAGTCCCTGGAGTCGGAGTGCCAGGAGTGGGTGTACCCGGTGTAGGCGTGCCTGGTGTTGGT GTACCCGGAGTTGGAGTGCCCGGCGTAGGGGTTCCAGGTGTGGGGGTGCCCGGAGTCGGTGTCCCTGGT GTAGGGGTTCCAGGAGTCGGCGTGCCGGGTGTTGGAGTACCTGGTGTGGGGGTCCCCGGAGTAGGGGTG CCTGGTGTCGGCGAACAAAAGTTGATCTCTGAGGAAGACCTCAAGGATGAGCTT

SEQ ID NO: 14 Amino acid sequence of expected mature E Polypeptide:

LQLVNDNGLVVNVILWLFVLFFLLIISITFVQLVNLCFTCHRLCNSAVYTPIGRLYRVYKSYMQIDPLP STVIDVWSHPQFEKDPAFLYKVVITVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VGVPGVGVPGVGVPGVGVPGVGVPGVGEQKLISEEDLKDEL

SEQ ID NO: 15 Coding sequence of E in E Construct:

TTACAGCTGGTAAACGATAACGGTCTTGTAGTTAATGTCATCTTGTGGCTATTCGTTTTGTTCTTTTTA CTGATCATATCCATTACCTTTGTGCAACTCGTCAATCTCTGCTTTACTTGTCATAGGCTTTGTAATTCA GCTGTTTACACTCCAATTGGAAGATTGTACCGTGTTTATAAGTCTTATATGCAAATAGACCCTCTTCCC AGTACAGTGATTGATGTG

SEQ ID NO: 16 Amino acid sequence of M protein from NCBI accession number KF650373

1 msngsipvde viqhlrnwnf twniiltill vlqyghyky saflygvkma il ilwplvl 61 alslfdawas fqvnwvffaf silmacitlm Iwiinyfvnsi rlwrrthsw sfnpetdall 121 ttsvmgrqvc ipvlgaptgv tltllsgtll vegykvatgv qvsqlpnfvt vakatttivy 181 grvgrsvnas sgtgwafyvr skhgdysavs npssvltdse kvlhlv

SEQ ID NO: 17 Amino acid sequence of E protein from NCBI accession number KF650373

1 mlqlvndngl vnvilwlfv lfflliisit fvqlvnlcft chrlcnsavy tpigrlyrvy 61 ksymqidplp stvidv

SEQ ID NO: 18 Amino acid sequence of N protein from NCBI accession number KF650373

1 masvsfqdrg rkrvplslya plrvtndkpl skvlannavp tnkgnkdqqi gywneqirwr 61 mrrgerieqp sn hfyylgt gphadlryrt rtegvfwvak egakteptnl gvrkasekpi 121 ipnfsqqlps vveivepntp ptsransrsr srgngnnrsr spsnnrgnnq srgnsqnrgn 181 nqgrgasqnr ggnnnnnnks rnqsknrnqs ndrggvtsrd dlvaavkdal kslgigenpd 241 klkqqqkpkq ersdssgknt pkknksrats kerdlkdxpe wrrxpkgens vaacfgprgg 301 fknfgdaefv ekgvdasgya qiaslapnva allfggnvav reladsyeit ynykmtvpks 361 dpnvellvsq vdafktgnak pqrkkekknk rettqqlnee aiyddvgvps dvthanlewd 421 tavdggdtav eiineifdtg n

SEQ ID NO: 19 Amino acid sequence of S protein from NCBI accession number KF650373

lmksltyfwlf lpvlstlslp qdvtrcsant nfrrffskfn vqapa vvlg gylpigenqg

61vnstwycagq hptasgvhgi fvshirgghg feigisqepf dpsgyqlylh katngntnat

121arlricqfps xktlgptann dvttgrnclf nkaipahmse hsvvgitwdn drvtvfsdki

181yyfyfknd s rvatkcynsg gcamqyvyep tyymlnvtsa gedgisyqpc tancigyaan

241vfatepnghi pegfsfnnwf llsndstlvh gkvvsnqpll vncllaipki yglgqffsfn

301qtidgvcnga avqrapealr fnindtsvil aegsivlhta Igtnfsfvcs nssnphlatf

361aiplgatqvp yycflkvdty nstvykflav Ipptvreivx tkygdvyvng fgylhlglld

421avtinftghg tdddvsgfwt iastnfvdal xevqgtaiqr ilycddpvsq lkcsqvafdl

481ddgfypxssr nllsheqpxs fvtlpsfndh sfvnitvsas fgghsganli asdttingfs

541sfcvdtrqft islfynvtns ygyvsksqds ncpftlqsvn dylsfskfcv stsllasact

601idlfgypefg sgvkftslyf qftkgelitg tpkplegvtd vsfmtldvct tytiygfkge

661giitltnssf lagvyytsds gqllafknvt sgavysvtpc sfseqaayvd ddivgvissl

721ssstfnstre lpgffyhsnd gsnctepvlv ysnigvcksg sigyvpsqsg qvkiaptvtg

781nisiptnfsm sirteylqly ntpvsvdcat yvcngnsrck qlltqytaac ktiesalqls

841arlesvevns mltiseealq latissfnqd gynftnvlgv svydpasgrv vqkrsfiedl

9011fnkvvtngl gtvdedykrc sngrsvadlv caqyysgvmv lpg vdaekl hmysasligg

961mvlggftsaa alpfsyavqa rlnylalqtd vlqrnqqlla esfnsaigni tsafesvkea

1021isqtskglnt vahaltkvqe vvnsqgaalt qltvqlqhnf qaisssidd ysrldilsad

1081vqvdrlitgr Isalnafvaq tltkytevqa srklaqqkvn ecvksqsqry gfcggdgehi

1141fslvqaapqg llflhtvlvp sdfvdviaia glcvndeial tlrepglvlf thelqnhtat

1201eyfvssrrmf eprkptvsdf vqiesc vty vnltrdqlpd vipdyxdvnk tldeilaslp

1261nrtgpslpld vfnatylnlt geiadleqrs eslrntteel qsliyninnt lvdlewlnrv

1321etyikwpwwv wliifivlif vvsllvfcci stgccgccgc ccacfsgccr gprlqpyevf 1381ekvhvq

Operation

While specific embodiments of the disclosure have been described and illustrated, such embodiments should be considered illustrative only and not as limiting the invention as claimed.

Claims

What is claimed is:

1. A eukaryotic organism or eukaryotic cell comprising a first recombinant polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus sufficient to form a virus-like particle (VLP) in the organism or cell, wherein the M protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1.

2. The organism or cell of claim 1 , wherein the portion of the M protein has the amino acid sequence of SEQ ID NO: 1.

3. The organism or cell of claim 1 or 2, wherein the polypeptide further comprises an elastin-like tag.

4. The organism or cell of claim 3, wherein the elastin-like tag comprises the amino acid sequence of SEQ ID NO: 2.

5. The organism or cell of any one of claims 1 to 4, wherein the polypeptide comprises the sequence of SEQ ID NO: 3.

6. The organism or cell of any one of claims 1 to 5, further comprising a second recombinant polynucleotide sequence encoding a portion of an envelope (E) protein of porcine Epidemic Diarrhea virus, wherein the E protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 4.

7. The organism or cell of claim 6, wherein the portion of the E protein has the amino acid sequence of SEQ ID NO: 4.

8. The organism or cell of any one of claims 1 to 7, wherein the organism or cell is a plant or a plant cell.

9. The organism or cell of claim 8, wherein the plant or plant cell is a tobacco plant or a tobacco plant cell.

10. The organism or cell of claim 9, wherein the tobacco plant or a tobacco plant cell is an Nicotiana benthamiana plant or Nicotiana benthamiana plant cell.

11. An expression construct comprising a polynucleotide molecule encoding a polypeptide, wherein the polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus sufficient to form a virus-like particle (VLP) in a cell, wherein the polynucleotide molecule is operably linked to a promoter for initiating transcription of the polynucleotide molecule in the cell, wherein the M protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1.

12. The expression construct of claim 1 1 , wherein the portion of the M protein has the amino acid sequence of SEQ ID NO: 1.

13. The expression construct of claim 11 or 12, wherein the polypeptide further comprises an elastin-like tag.

14. The expression construct of claim 13, wherein the elastin-like tag comprises the amino acid sequence of SEQ ID NO: 2.

15. The expression construct of any one of claims 1 to 14, wherein the polypeptide comprises the sequence of SEQ ID NO: 3.

16. The expression construct of any one of claims 1 to 15, wherein the cell is a eukaryotic cell.

17. The expression construct of claim 16, wherein the eukaryotic cell is a plant cell.

18. A method of producing a virus-like particle (VLP) comprising a portion of the membrane (M) protein of Porcine Epidemic Diarrhea virus, the method comprising expressing an expression cassette as defined in any one of claims 11 to 17 in a cell.

19. The method of claim 18, further comprising expressing a portion of the envelope (E) protein of porcine Epidemic Diarrhea virus, wherein the E protein has the amino acid sequence of SEQ ID NO: 4.

20. The method of claim 19, wherein the portion of the E protein has the sequence of SEQ ID NO:4.

21. The method of claim 18, 19, or 20, wherein the cell is a plant cell.

22. A virus-like particle (VLP) comprising a polypeptide, wherein the polypeptide comprises a portion of an membrane (M) protein of Porcine Epidemic Diarrhea virus, wherein the M protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1.

23. The VLP of claim 22, wherein in the portion of the M protein has the sequence of SEQ ID NO: 1.

24. The VLP of claim 19 or 20, wherein the polypeptide further comprises an elastin-like tag.

25. The VLP of claim 24, wherein the elastin-like tag comprises the amino acid sequence of SEQ ID NO: 2.

26. The VLP of any one of claims 22 to 25, wherein the polypeptide comprises the sequence of SEQ ID NO: 3.

27. The VLP of any one of claims 22 to 26 consisting of the polypeptide.

28. The VLP of any one of claims 22 to 26, further comprising a portion of an envelope (E) protein of Porcine Epidemic Diarrhea virus, wherein the E protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 4.

29. The VLP of claim 28, wherein the portion of the E protein has the amino acid sequence of SEQ ID NO: 4.

30. The VLP of any one of claims 22 to 29, comprising N-linked glycans having bisecting P(1 ,2)-xylose and core a(1 ,3)-fucose residues.

31. The VLP of any one of claims 22 to 30, wherein the VLP is an isolated VLP.

32. A vaccine for vaccinating a porcine against infection by Porcine Epidemic Diarrhea virus, the vaccine comprising a virus-like particle (VLP) as defined in any one of claims 22 to 31.

33. A method of vaccinating a porcine against infection by Porcine Epidemic Diarrhea virus comprising administering a virus-like particle (VLP) as defined in any one of claims 22 to 31 to the porcine.

34. Use of a virus-like particle as defined in any one of claims 22 to 31 for vaccinating a porcine against infection by Porcine Epidemic Diarrhea virus.

35. Use of a virus-like particle as defined in any one of claims 22 to 31 in the preparation of a vaccine for vaccinating a porcine against infection by by Porcine Epidemic Diarrhea virus.

36. A vaccine for infection by Porcine Epidemic Diarrhea virus, the vaccine comprising a virus-like particle as defined in any one of claims 22 to 31 and an adjuvant.

37. A method of inducing mucosal immunity against Porcine Epidemic Diarrhea virus in a porcine, the method comprising feeding a virus-like particle as defined in any one of claims 22 to 31 to the porcine.

38. A method of inducing mucosal immunity against Porcine Epidemic Diarrhea virus in a porcine, the method comprising feeding a plant as defined in claim 8, 9 or 10, or plant material harvested from a plant as defined in claim 8, 9, or 10 to the porcine.

39. Use of a virus-like particle as defined in any one of claims 22 to 31 for inducing mucosal immunity against Porcine Epidemic Diarrhea virus in a porcine.

40. Use of an organism as defined in claim 8, 9, or 10, or plant material harvested from an organism as defined in claim 8, 9, or 10, for inducing mucosal immunity against Porcine Epidemic Diarrhea virus in a porcine.

41. An expression construct for use in producing a virus-like particle (VLP) in a cell, the expression construct comprising a polynucleotide molecule encoding a polypeptide, wherein the polypeptide comprises a portion of an envelope (E) protein of Porcine Epidemic Diarrhea virus, wherein the polynucleotide molecule is operably linked to a promoter for initiating transcription of the polynucleotide molecule in the cell, wherein the E protein has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 4.

42. The expression construct of claim 41 , wherein the portion of the E protein has the amino acid sequence of SEQ ID NO: 4.

43. The expression construct of claim 41 or 42, wherein the polypeptide further comprises an elastin-like polypeptide (ELP) tag.

44. The expression construct of claim 43, wherein the ELP tag comprises the amino acid sequence of SEQ ID NO: 2.

45. The expression construct of any one of claims 41 to 44, wherein the polypeptide comprises the sequence of SEQ ID NO: 3.

46. The expression construct of any one of claims 41 to 45, wherein the cell is a eukaryotic cell.

47. The expression construct of claim 46, wherein the eukaryotic cell is a plant cell.