GB2552441A - Methods - Google Patents

Abstract

A preparation of an inactivated or freeze-dried yeast cell or cell-surface-containing portion thereof expressing a porcine viral epitope, wherein the epitope is expressed on the surface of the yeast cell. The yeast is typically is non-viable. The yeast is typically from the genus Saccharomyces, and is preferably Saccharomyces cerevisiae, but may be from the genus Pichia, for example Pichia pastoris, or may be from the genus Kluyveramyces, for example K. lactis. The viral epitope may be from a Coronavirus, optionally from PEDV, TGEV, porcine hemagglutinating encephalomyelitis virus or PRCV; a porcine to Rotavirus; or a virus of the Circoviridae family, optionally from PCV2. The viral epitope expressed by the yeast is preferably PCV2 ORF2 (Cap protein).

Description

(71) Applicant(s):

Royal Veterinary College (Incorporated in the United Kingdom)

University of London, Royal College Street, LONDON, NW1 OTU, United Kingdom (72) Inventor(s):

Dirk Werling (74) Agent and/or Address for Service:

Potter Clarkson LLP

The Belgrave Centre, Talbot Street, NOTTINGHAM, NG1 5GG, United Kingdom (51) INT CL:

A61K39/12 (2006.01) A23K 10/16 (2016.01) (56) Documents Cited:

EP 2789346 A1 WO 2017/068352 A1

WO 2014/142515 A1 WO 2012/036391 A2 Vaccine, Vol. 33, epub-21.10.2015, Patterson R., et al., Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo., pp. 6199-6205. Available at:

Vaccine, Vol. 27, 2009, Bucarey, S.A., The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. , pp. 5781-5790.

Appl. Microbiol. Biotechnol., Vol. 97, 2013, Tu Y, High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris., pp. 2867-2875. Available at:

Braz. J. Microbiol., Vol. 45, 2014, da Silva, A.J., et al., Live bacterial vaccine vectors: An overview. , pp. 1117-1129. Available at: https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC4323283/

Dev. Comp. Immunol., Vol. 37, 2012, Patterson R., Yeast-surface expressed BVDV E2 protein induces a Th1/Th2 response in naive T cells. , pp. 107-114. Available at: https://www.ncbi.nlm.nih.gov/ pubmed/22067741 (58) Field of Search:

INT CLA61K, C12N

Other: WPI, EPODOC, BIOSIS, MEDLINE (54) Title of the Invention: Methods

Abstract Title: PCV2 Cap Yeast Vaccine Vector (57) A preparation of an inactivated or freeze-dried yeast cell or cell-surface-containing portion thereof expressing a porcine viral epitope, wherein the epitope is expressed on the surface of the yeast cell. The yeast is typically is nonviable. The yeast is typically from the genus Saccharomyces, and is preferably Saccharomyces cerevisiae, but may be from the genus Pichia, for example Pichia pastoris, or may be from the genus Kluyveramyces, for example K. lactis. The viral epitope may be from a Coronavirus, optionally from PEDV, TGEV, porcine hemagglutinating encephalomyelitis virus or PRCV; a porcine to Rotavirus; or a virus of the Circoviridae family, optionally from PCV2. The viral epitope expressed by the yeast is preferably PCV2 ORF2 (Cap protein).

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Methods

Field of the invention

The invention relates to yeast-based vaccines and their use in pigs.

Background

Due to the increase in multi-drug resistant pathogens, anti-microbial abuse, increased customer awareness for antimicrobial residues in food products, and the looming ban of antimicrobial food additives, there is a heightened need to protect farm animals by vaccination. These vaccines should be easy to administer, should not contain genetically modified organisms, should be cheap to produce, induce a local and systemic immune response and should potentially allow for the discrimination of vaccinated versus naturally infected animals.

Infection of pigs with porcine circovirus type 2 (PCV2), a non-enveloped ssDNA virus causes porcine circovirus associated diseases (PCVAD) [1-3]. Vaccination is effective in reducing symptoms and increasing production parameters, but does not prevent spread of the virus. Many commercial PCV2 vaccines are PCV2 capsid (Cap) protein subunit vaccines, which is encoded by the second open reading frame (ORF2) [4], Vaccines are administered by injection which can be time consuming on large pig farms. As the driving factor behind the development of new farm-animal vaccines is often the economic viability, vaccines that do not need to be administered individually, are cheap to produce and can be stored without a cooling chain are an attractive option. Here, oral vaccination provides an ideal target. These are often employed to achieve a systemic IgG as well as mucosal IgA response following antigen uptake by micro-fold (M) cells [5, 6]. Vaccines administered via the oral route cause in general less stress and associated immune-suppression for the recipient [7], both of which are risk factor for developing PCVAD [8, 9].

Saccharomyces cerevisiae (S.c.) is commonly used to produce recombinant proteins and has a “generally regarded as safe” (GRAS) status. Recently, S.c. has been used as delivery system for cancer vaccines, resulting in humoral and cellular immune responses [10, 11]. Yeast itself possess adjuvant like properties, activating both inflammatory and phagocytic receptors expressed on APC [12], We have demonstrated herein that freezedrying of S.c. expressing PCV2 Cap protein on its surface renders it completely non-viable, without affecting expression of the Cap protein. This has interesting implications as inactivated S.c. is no longer considered a genetically modified organism (GMO) according

Directive 2001/18/EC. Additionally, inactive yeast would not require refrigeration, cutting down on storage costs. Previous in vivo studies have demonstrated some success in oral vaccination using recombinant S. cerevisiae for viral [13], parasitic [14] and bacterial infections [15] in mice. Additionally, oral vaccination of mice with S.c. secreting the PCV2 Cap protein, potentially resulting in VLP formation, induced Cap specific antibodies [7].

Park et al (2007) Biotechnology and Bioprocess Engineering 2007,12: 690-695 describes yeast surface displayed expression of a Neutralizing Epitope of Spike Protein from a Korean strain of Porcine Epidemic Diarrhea Virus, but does not investigate effects of using the yeast.

Brief description of the invention

The present invention relates to a preparation of an inactivated or freeze-dried yeast cell (or cell-surface-containing portion thereof), or composition comprising a preparation of an inactivated or freeze-dried yeast cell (or cell-surface-containing portion thereof), expressing a porcine viral epitope, wherein the epitope is expressed on the surface of the yeast cell; and to the oral vaccination of pigs with such a yeast cell-derived preparation or composition. Such a yeast cell-derived preparation or composition is considered to have significant practical value. For example, the yeast cell-surface localisation of the viral epitope is considered to provide advantages over yeast production of virus-like particles because the yeast (whether intact or fragmented, for example as a result of cell inactivation method(s)) can act as delivery system and adjuvant, binding to receptors such as TLR2 and Dectin-1 expressed by immune cells. The result is an increased phagocytosis with stimulation of a pro-inflammatory cytokine response. After oral application, yeast is sampled from the gut lumen by dendritic cells and M cells, leading to a response that may include IgA and systemic IgG production, cell mediated immunity and/or stimulation of a pro-inflammatory cytokine response; which may be particularly beneficial in providing a protective response against a virus that targets the gastrointestinal tract.

Thus it is envisaged that the physical proximity of the viral antigen and the yeast surface (whether intact or fragmented) promotes enhanced phagocytosis, thus ensuring a high concentration of antigenic peptides being subsequently presented on MHC class II molecules, and potentially via cross-presentation on MHC class I molecules. Yeast is an exogenous antigen, thus being phagocytosed and presented via MHC II. However, antigen may leak (at low levels) from the MHC II pathway into the MHC I pathway, and this is called cross-presentation.

Furthermore, the inactivated, typically freeze-dried, nature of the yeast preparation (whilst retaining structural integrity of the viral epitope) allows storage of the vaccine at normal food storage temperatures, meaning that the vaccine can be incorporated into animal food stuffs in bulk and stored for lengthy periods of time, facilitating the vaccination of a herd of pigs en masse, rather than individually.

Evidence that points to a lack of correlation between antibodies produced to the viral Cap protein (for example the Cap protein of PCV2) and protection (see Fachinger et al (2008) Vaccine 26, 1488-1499, particularly Figure 2 and Charreyre et al (2004) Merial 18^th IPVC Germany June 2004 would, on the face of it, make the expression of viral epitopes on the surface of yeast cells a pointless endeavour.

Further, the skilled person would also not expect a yeast approach to work with viral antigens because viruses are normally endogenous antigens (free in cytoplasm) and their epitopes are normally presented on MHC I molecules. In contrast, bacteria are exogenous antigens, which are phagocytosed, similar to what happens with yeast, and peptides are presented on MHC II molecules. Thus, even if a yeast approach were to work with bacterial antigens, the skilled person would not consider that that would mean that it would be likely to work with a viral antigen.

However, as demonstrated herein, such an approach with viral antigen does have a protective/therapeutic effect.

We have demonstrated that such a yeast preparation, delivered by the oral route, is capable of resulting in a reduced viral load in both the serum and the faeces of subsequently challenged subjects. We consider that the invention provides an efficacious vaccine composition directed against porcine viral disease, particularly porcine gastrointestinal viral disease. Without wishing to be bound by any theory, the inventors consider it likely that the anti-viral activity of the present compositions arises principally through the simulation of a mucosal IgA response, rather than an IgG response.

Detailed description of the invention

In a first aspect, the invention provides a preparation of an inactivated or freeze-dried yeast cell (or cell-surface-containing portion thereof) expressing a porcine viral epitope, wherein the epitope is expressed on the surface of the yeast cell.

The yeast cell may be of any species suitable for administration to a pig. In one embodiment, the yeast is non-toxic, although it is considered that the freeze-drying process renders the yeast inviable, so in other embodiments the yeast may be such that when viable it is toxic. However, it will be appreciated that it is preferred if the yeast is non-toxic. In one embodiment the yeast is of a species that is routinely used for protein expression, such that there are numerous genetic and molecular tools available to express the epitope of interest in the yeast. In a preferred embodiment, the yeast is from the genus Saccharomyces, and is preferably Saccharomyces cerevisiae, or the yeast is from the genus Pichia, and is preferably Pichia pastoris, or the yeast is from the genus Kluyveromyces, and the yeast is K. lactis.

The yeast may be a naturally occurring yeast into which the relevant constructs have been introduced to express the viral epitope. It is more likely however that the yeast will be, for example, a lab strain of yeast such as Saccharomyces cerevisiae. In any event, the yeast will have been modified to an extent where it recombinantly expresses the relevant viral epitope (or epitopes) on the cell surface. For example, the yeast may express the relevant viral epitope (or epitopes) from a plasmid vector. The vector may express the relevant viral epitope(or epitopes) as a fusion polypeptide, for example as a fusion polypeptide comprising a portion that directs the fusion polypeptide to the cell surface, as will be well known to those skilled in the art. As noted below, the fusion may be with all or part of the Aga2 polypeptide, for example.

The vector may be a single copy number vector, such that it segregates faithfully during mitosis, or alternatively, the vector may be a multicopy plasmid such that one yeast cell harbours many copies of the plasmid. In one embodiment, the vector is the pYD1 vector, as described in http://img55.chem17.eom/5/20130809/635116506211258776521 .pdf and comprises the Aga2 gene. Correct cloning of the sequence encoding the epitope results in an in-frame protein fusion, wherein the Aga 2 protein targets the epitope to the yeast cell surface.

The vector may also contain a marker, such as an antibiotic resistance conferring gene, or a visual marker such as GFP. The vector may comprise any suitable promoter, and the promoter may be a yeast promoter or any other promoter such as those routinely used in the art to drive expression of proteins within yeast.

In another embodiment, the yeast expresses the relevant viral epitope (or epitopes) from a genomic locus. For example, homologous recombination may be used to target a cassette comprising the viral epitope under the control of a promoter to a region of the yeast genome, to form a stable expression system, in which case selection pressure need not be maintained. The cassette may also contain a marker, such as an antibiotic resistance conferring gene, or a visual marker such as GFP. As above, the relevant viral epitope(or epitopes) may be expressed as a fusion polypeptide, for example as a fusion polypeptide comprising a portion that directs the fusion polypeptide to the cell surface, as will be well known to those skilled in the art.

In another embodiment the yeast cell may express a viral epitope from both a vector and from a genomic location. In such an embodiment, the viral epitope may be the same in both cases, or the viral epitope may be different, such that the vector expresses one viral epitope, and the genomic locus expresses a different viral epitope.

The viral epitope (or epitopes) may be under the expression of a homologous promoter, i.e. a promoter with a sequence that is native to the yeast cell, or may be under the control of a heterologous promoter, i.e. a promoter with a sequence that is not native to the yeast cell. For example, the promoter may be any suitable promoter known in the art. In one embodiment the promoter is an inducible promoter, for example a promoter inducible by galactose. In another embodiment the promoter is a constitutive promoter.

By viral epitope we include the meaning of any fragment of a virus which is capable of raising a specific antibody response to that virus (or to a closely related virus) following exposure of a pig to the fragment. For example, the epitope may comprise a polypeptide, an RNA molecule, or a DNA molecule. The epitope may comprise a carbohydrate or lipid moiety, for example in the form of a glycoprotein or lipoprotein. In a preferred embodiment, the epitope is or comprises a polypeptide epitope.

In one embodiment the viral epitope is from a Coronavirus, for example from PEDV, TGEV, porcine hemagglutinating encephalomyelitis virus or PRCV. In another embodiment the viral epitope is from a porcine Rotavirus. In one embodiment the viral epitope is from a virus of the Circoviridae family, for example PCV2. In a particular embodiment, the viral epitope is ORF2 from PCV2. In a further particular embodiment the ORF2 is from a PCV2b strain, for example with the sequence detailed in GenBank accession number JX193799.

In a further embodiment, the virus is one that causes gastrointestinal symptoms, for example diarrhoea. The virus may be, for example, a Rotavirus, Coronavirus (this includes PEDV and TGEV), Norovirus, Parvovirus, PRRSV (Porcine reproductive and respiratory syndrome virus).

The preparation of the invention is considered to be particularly advantageous when the yeast expresses a viral epitope at the cell surface derived from a virus that causes diarrhoea in the first week of life of the pig, and/or where an IgA and IgG response would be advantageous.

Preferably, the epitope is one that is presented to the immune system on natural infection with the virus, such that antibodies raised in response to the epitope expressed by the yeast cell are capable of binding to the same epitope on the virus particle, for example the epitope may be located on the external surface of the virus. In one embodiment the epitope is a viral cap protein or is derived from a viral cap protein. In another embodiment the viral epitope is a viral envelope protein or is derived from a viral envelope protein. In a further embodiment the viral epitope is a spike protein or is derived from a spike protein. In yet another embodiment, the viral epitope is a non-structural protein or is derived from a non-structural protein.

In particular embodiments, the epitope comprises the Cap protein of PCV2, or fragment thereof. In another embodiment the epitope comprises the spike protein of PEDV, or fragment thereof. In general, envelope proteins in enveloped viruses (such as PEDV, a coronavirus) are good targets for the immune response. In PEDV, the main protein to aim for would be the Spike protein (major protein in envelope). An appropriate epitope can typically be established by establishing from in vivo infections to which protein(s)/sequences(s) (or other epitope(s)) antibodies are made.

The skilled person will appreciate that an epitope may be linear, i.e. the epitope is formed from consecutive amino acids, or the epitope may be conformational, in which case it is the particular arrangement of amino acids in space that is important.

Where the epitope is a linear epitope, the epitope may comprise a small fragment of a polypeptide, for example may comprise between 5 amino acids and 300 amino acids, for example between 10 amino acids and 250 amino acids, for example between 15 amino acids and 200 amino acids, for example between 20 amino acids and 150 amino acids, for example between 25 amino acids and 100 amino acids, for example between 30 amino acids and 90 amino acids, for example between 40 amino acids and 80 amino acids, for example between 50 amino acids and 70 amino acids, for example 60 amino acids. In another embodiment, the epitope comprises the entire full length protein. For example, it is feasible to express polypeptides with at least 800 amino acids on the yeast cell surface.

Conformational epitopes are formed from the spatial arrangement of amino acids, which may be, for example, non-consecutive amino acids, yet once the protein is folded, the nonconsecutive amino acids become spatially arranged in close proximity to each other, or in another way that provokes the generation of an antibody. Furthermore, such conformational epitopes may not be limited to a single molecule, but may span two or more molecules. For example it is known that epitopes are formed at the interface of protein dimers, for example dimers of viral envelope proteins. Thus, the term epitope also encompasses such three-dimensional epitopes. The skilled person will be well aware of techniques to ensure that a particular three-dimensional structure is expressed within a yeast cell and preserved at the cell surface. For example, using computer modelling, the skilled person may engineer additional cysteine residues to form stabilising disulphide bonds. Alternatively or additionally the skilled person may incorporate the known three dimensional epitope into a protein scaffold, which following translation folds into the correct shape so as to present the desired three-dimensional epitope.

It will be appreciated that where for example a polypeptide comprises a relative large sequence, for example greater than 10 amino acids, the polypeptide is likely to comprise more than 1 epitope, i.e. there is likely to be more than one particular site of the polypeptide capable of raising a specific antibody. Reference to expression of “a viral epitope” includes the meaning of expression of more than one epitope located on the same molecule, or formed by the same combination of molecules if, for example the desired epitope is formed on a dimer of proteins. For example, the Cap protein of PCV2 may comprise several epitopes.

In addition to expressing a viral epitope of a particular virus (or more than one epitope if more than one epitope is located on the same molecule), a benefit of the yeast system is the ability express more than one molecule containing a viral epitope simultaneously. For example, in one embodiment, the yeast may express one molecule comprising a particular epitope (or epitopes), but may also simultaneously express another molecule comprising a different epitope (or epitopes). This approach is considered to give wider protection, particularly if the different molecules comprising the epitope (or epitopes) are derived from different viral strains, or species. For example, in one embodiment, the same yeast cell may express an epitope, for example a polypeptide, derived from virus 1 strain 1, and an epitope , for example a polypeptide derived from virus 1 strain 2. It is clear that the number of epitopes expressed may increase to for example 10 different molecules comprising 1 or more epitopes, for example between 1 and 10 different molecules comprising 1 or more epitopes, for example between 2 and 9 different molecules comprising 1 or more epitopes, for example between 3 and 8 different molecules comprising 1 or more epitopes, for example between 4 and 7 different molecules comprising 1 or more epitopes, for example 5 or 6 different molecules comprising 1 or more epitopes. In this way, protection against a variety of strains of the same virus may be achieved.

However, it will be realised that a further advantage of the present invention is the ability to express an epitope, for example a polypeptide, derived from virus 1, and an epitope , for example a polypeptide derived from virus 2. For example, the same yeast cell may express one molecule, for example a polypeptide, comprising an epitope (or epitopes) derived from virus 1, and may also express one molecule, for example a polypeptide, comprising an epitope (or epitopes) derived from virus 2. It will be appreciated that the number of molecules, for example polypeptides, comprising an epitope (or epitopes) derived from different viral species may increase to for example 10 different molecules. For example the same yeast cell may express, at its cell surface, between 1 and 10 different molecules comprising epitopes from different viral species, for example between 2 and 9 different molecules comprising epitopes from different viral species, for example between 3 and 8 different molecules comprising epitopes from different viral species, for example between 4 and 7 different molecules comprising epitopes from different viral species, for example 5 or 6 different molecules comprising epitopes from different viral species.

In this way, protection against an array of porcine viral species may be obtained from the production of a single type of yeast cell. In one embodiment the yeast cell may express epitopes from porcine parvovirus and/or PRRS virus and/or PCV2, as concurrent infection with porcine parvovirus or PRRS virus with PCV2 leads to increased replication of PCV2 and more severe disease in PCV2 infected pigs (Ellis 2014 Veterinary Pathology 51 (2): 315-327).

The yeast may also or alternatively express one or more epitope of PEDV. The yeast may also or alternatively express one or more epitope from a Rotavirus, Coronavirus (this includes PEDV and TGEV), Norovirus, Parvovirus, PRRSV.

The yeast may additionally express one or more bacterial or mycoplasma epitopes. Examples of bacterial epitopes that it may be useful to include are: toxins of Actinobacillus pleuropneumoniae, toxins, capsid and structural proteins of Mycoplasma subspecies, such as L-a-glycerol-3-phosphate oxidase or galactofuranose components of Mycoplasma mycoides subs, mycoides or relevant counterparts in other Mycoplasma (see Vet Immunol Immunopathoi. 2009 Oct 15; 131 (3-4):238-45. doi: 10.1016/j.vetimm.2009.04.016. Epub 2009 May 4. Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species. JoresJl, MeensJ, Buettner FF, Linz B, Naessens J, Gerlach GF. ), as well as Salmonella subspecies.

Alternatively, more than one type of yeast cell of the invention (for example displaying different viral epitopes) may be used together in a composition, for example in a foodstuff, between them displaying more than one epitope.

In either case, as noted above the epitope, for example a polypeptide, may also comprise one or more modifications, for example one or more carbohydrate moieties, or one or more lipid moieties. Such modifications may be engineered into the cell, such that they are added for example postranslationally to the polypeptide. Alternatively, the modifications may be added for example chemically following freeze drying, for example one modification that is considered to be useful is the addition of particular lectins, described in more detail below.

It is considered that the epitope or epitopes should be expressed at the cell surface of the yeast cell, i.e. localised to the cell surface, rather than internally localised or secreted. The skilled person will be well aware of techniques to ensure that such an epitope, for example a polypeptide, is targeted to the cell surface. For example, as disclosed in the examples the desired polypeptide epitope may be engineered such that it is expressed in frame with the yeast Aga2 protein, for example by using the pYD1 vector. The Aga2 protein then binds to Aga1 on the yeast cell surface via two disulphide bonds, resulting in cell surface localisation of the desired epitope. This is of course but one example and the skilled person, should they wish to, is capable of devising an alternative system to ensure cell surface localisation of the required epitope. There are many alternative systems for yeast cell surface display, for example employing members of the glycosylphosphatidylinositol (GPI) family of cell wall proteins. Other such systems are reviewed in Pepper et al 2008 Comb Chem High Throughput Screen 11(2): 127-134.

Furthermore, it is well within the skills of the skilled person to determine whether or not the desired epitope is located at the cell surface, using routine techniques such as immunomicroscopy or immunoflowcytometry. For example a fluorescently labelled antibody could be used in conjunction with a cell sorter to identify cells that are positive for cell surface epitope, as will be known to those skilled in the art.

It will be appreciated that not 100% of the epitope, for example the polypeptide, will be localised at the cell surface. For example a portion of the polypeptide may be being trafficked to the cell surface, or become trapped in the cytosol. A portion of the polypeptide may also be secreted. However, it is considered that the yeast cell preparation is useful if, for example, yeast cell surface epitope can be detected using a routine technique such as one of those indicated in the preceding paragraph.

In a preferred embodiment, the yeast cell is non-viable. This may be as a result of freezedrying, or through other means such as heat inactivation, or through a combination of such treatments. It is not considered useful if the inactivation process affects the conformation of the epitope at the cell surface. Accordingly, chemical inactivation of the yeast cell is not generally considered to be suitable for use in the invention, unless that chemical inactivation does not affect the conformation of the epitope or epitopes at the yeast cell surface, including useful epitopes natively expressed by the yeast.

In a preferred embodiment, the inactivation renders the yeast completely inviable. However, it will be appreciated that in some scenarios, for example when a population of yeast cells are inactivated through, for example freeze-drying or heat inactivation, there may be some cells that are not entirely inactivated. The skilled person will be capable of performing routine quality controls to ensure that the yeast cells have been rendered sufficiently inviable. See, for example, Figure 10 and the legend thereto below. Thus, as an example, plating out the inactivated preparation and checking that nothing grows under conditions where cell growth would normally be expected, may be used as a test.

It will be appreciated that viable yeast cells may still be useful in producing an immune response, though the profile of the response may be different. However, in practical terms viable yeast cells modified as described herein may be considered as genetically modified organisms (GMOs) and therefore subject to different regulations that may make them more burdensome to use in practice.

It is considered useful if, in addition to expressing an epitope (or epitopes) of a porcine viral antigen, as described above, the yeast cell also expresses other antigens or factors that enhance the immune response. For example, it is known that yeast can bind to, for example, TLR2 and Dectin-1 expressed by immune cells through native yeast betaglucans, which are recognised by Dectin-1 and TLR2, leading to enhanced phagocytosis via dectin-1 and induction of pro-inflammatory cytokines via TLR2 through stimulation of the kinase Syk and the transcription factor NfKB, respectively.

Accordingly, the yeast cell may also be engineered to have increased expression of these native yeast beta-glucans. In such a situation, the yeast cell has been modified to increase expression of these beta-glucans. Polysaccharides may also be chemically linked to the surface of the yeast.

In addition to native yeast polypeptides and factors, the yeast cell may be engineered to express, in addition to the porcine viral epitope or epitopes, other polypeptides or factors that are considered to enhance the immune response. Preferably these other polypeptides or factors are expressed in the yeast and localised at the yeast cell surface. Steps similar to those described above to ensure that the viral epitope or epitopes are located at the cell surface may be taken.

For example in one embodiment the yeast cell may express one or more of pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) which bind to pattern recognition receptors (PRRs) on immune cells and stimulate intracellular signalling, gene expression and activation of antimicrobial and inflammatory activities, which include the phagocytosis by macrophages and dendritic cells of the yeast cell (which also expresses the viral epitope or epitopes).

PAMPs include: lipopolysaccharide (LPS) from Gram-negative bacteria; components from Gram-positive bacteria, including lipoteichoic acid (LTA); component from Gram-positive bacteria, for example LTA; flagellin; double stranded RNA; single stranded RNA from viruses; synthetic ligands, such as R-848 and imiquimod; and unmethylated CpG islands found in bacterial and viral DNA, such as CpG ODNs.

DAMPs include a HSP; HMGB1; ATP; mitochondrial formyl peptides; mitochondrial DNA; uric acid; NY-ESO-1; Hyaluron; heparan sulfate fragments; S100 family proteins, for example S100A8 (MRP8, calgranulin A) and S100A9 (MRP14, calgranulin B); fibronectin; surfactant protein A; biglycan; versican; mitochondrial DNA; and Serum amyloid A (SAA).

Where the PAMP or DAMP is not a polypeptide, the PAMP or DAMP may be added to the surface of the yeast cell by, for example, chemical means.

One embodiment therefore provides a preparation of a freeze-dried yeast cell (or cellsurface-containing portion thereof) that expresses a porcine viral epitope, or epitopes (as defined in earlier embodiments), at the cell surface, and which also expresses one or more PAMPs or DAMPs. In one embodiment the one or more PAMPs or DAMPs is a polypeptide, for example is flagellin, HSP, HMGB1, mitochondrial formyl peptides, S100 family proteins for example S100A8 (MRP8, calgranulin A) and S100A9 (MRP14, calgranulin B), fibronectin, surfactant protein A, versican or serum amyloid A.

In one embodiment, the one or more PAMPs or DAMPs is chemically added to the yeast preparation. In a further embodiment, versican and/or flagellin are chemically added to the yeast preparation.

It will be appreciated that as with the viral epitopes, the cell may express more than one PAMP or DAMP. The cell may also overexpress one or more of the native yeast proteins as described above. The skilled person will realise that any combination of the epitopes, native yeast proteins or PAMPs or DAMPs may be co-expressed in the same cell.

The skilled person will appreciate that the nature of the yeast system also allows the same yeast cell to express one or more bacterial-derived epitopes, as well as the viral epitopes. Thus in one embodiment the yeast cell as defined in any of the embodiments above also expresses one or more bacterial-derived epitopes. For example, it is considered useful to vaccination pigs against rotavirus, and/or corona virus along with vaccination against Salmonella. Accordingly, in one embodiment, the yeast may express one or more Rotavirus epitopes, and/or one or more Corona virus epitopes, and one or more Salmonella epitopes.

Without wishing to be bound by any theory, it is considered that the microfold membranous or microvillous cells (M cells) are responsible, at least in part, for the uptake and sampling of the yeast cell of the invention. M cells are located within the epithelia lining the various mucosa associated lymphoid tissues (MALT). They rank amongst the most important epithelial cell types that play a role in the adhesion, uptake and sampling of foreign antigens at mucosal surfaces. More specifically, M cells endocytose luminal soluble macromolecules, particles and entire microorganisms at their apical membranes and exocytose these to their basolateral membranes, where both T and B lymphocytes and macrophages are present in a basolateral pocket. Little or no endocytosed material is directed to lysosomes, and does therefore not get degraded. As such, M cells represent the first cells involved in the initiation of immune responses against harmful antigens at the inductive sites of MALT. M cells are considered to be important factors to consider in the development of mucosal vaccines.

Accordingly, a further embodiment of the invention provides a preparation according to any the above aspects of the invention, wherein the yeast cell expresses or comprises one or more moieties that target the yeast cell to the M cells. For example, fucose-targeting lectins, N-acetyl-galactosamine and N-acetyl-neuramin of Psophocarpus tetragonolobus (WBA, winged bean) are all known to bind selective to the M cells of pigs. Thus in one embodiment the yeast cell comprises one or more of fucose-targeting lectins, N-acetylgalactosamine and N-acetyl-neuramin of Psophocarpus tetragonolobus (WBA, winged bean). The skilled person will be well aware of suitable methods to incorporate such agents into or onto a yeast cell. In one embodiment, the agents such as fucose-targeting lectins, N-acetyl-galactosamine and N-acetyl-neuramin of Psophocarpus tetragonolobus (WBA, winged bean) are chemically linked to the yeast after freeze-drying has taken place.

In a further embodiment, the preparation as defined by any of the embodiments detailed above is in a powdered form. In one embodiment this is achieved by the use of a pestle and mortar. However, on a large scale, it will be appreciated that industrial equipment will be required. The skilled person will be well aware of such methods, and in one embodiment this may involve spray freeze-drying. Providing the yeast preparation in a powdered form is considered to provide better uptake of the relevant epitope or epitopes or other factors expressed by the yeast cell, or applied to the yeast cell.

In one embodiment, the preparation may be stored at -80°C. However, due to the freezedried and inactivated nature of the preparation, it is considered that the preparation may be stored at ambient temperature, for example from between 10°C to 40°C, without the conformation of any of the epitopes being affected. Thus in one embodiment the preparation is stored at between 10°C and 40°C. In another embodiment, the yeast is stored at between 10°C and 35°C. In a further embodiment, the preparation is stored at between 10°C and 30°C. In yet a further embodiment, the preparation is stored at between 10°C and 25°C.

As will be apparent, the preparation of the invention, as described above, is suitable for use in vaccinating pigs against the virus from which the viral epitope expressed on the cell surface of the yeast is derived. For example, where the viral epitope is derived from PCV2, oral vaccination of a pig with that yeast causes a reduction in the copy number of the PCV2 virus in the serum of the subject following exposure to the same virus, compared to unvaccinated pigs. The skilled person will appreciate that some epitopes may give rise to cross protection, for example a cap protein from PCV2 strain PCV2a also affords protection against strain PCV2b (Patterson et al 2015 Vet Microbiol Jun 12;177(3-4):2619. doi: 10.1016/j.vetmic.2O15.03.010. Epub 2015 Mar 20) and strain PCV2d (Xiao et al 2015 J Gen Virol. Jul;96(Pt 7):1830-41. doi: 10.1099/vir.0.000100. Epub 2015 Feb 23).

In some situations vaccination with one particular epitope may give rise to protection against closely related species.

The skilled person will also realise that some epitopes are strain specific, and may give rise only to protection against infection with that particular strain of the same virus.

The skilled person will be aware of methods to determine the protection afforded by a particular yeast cell preparation and some of these methods are disclosed in the examples. For example following vaccination the skilled person may expose the vaccinated subjects to the same viral strain that was used in the yeast vaccination, a closely related strain, and a closely related species in order to determine the level of protection afforded. The skilled person will be aware of what species are considered to be closely related.

Thus in one embodiment, oral vaccination of a subject with the yeast preparation causes a reduction in the copy number of the same viral strain in the serum of the subject following subsequent exposure to the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

In a further embodiment, oral vaccination of a subject with the preparation causes a reduction in the copy number of a similar viral strain in the serum of the subject following subsequent exposure to a similar viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

In yet another embodiment, oral vaccination of a subject with the preparation causes a reduction in the copy number of a closely related virus in the serum of the subject following subsequent exposure to a closely related viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

By “similar viral strain” we include the meaning of viral strains which show at least 90% sequence identity to each other across the length of the genome, for example at least 91 % sequence identity, at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5% sequence identity to each other.

By “closely related species” we include the meaning of viral species which show at least 80% sequence identity to each other, 90% sequence identity to each other across the length of the genome, for example at least 91% sequence identity, at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5% sequence identity to each other.

In a further embodiment, oral vaccination of a subject with the preparation causes a reduction in the viral load in faeces of the same viral strain in the serum of the subject following subsequent exposure to the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

In a further embodiment, oral vaccination of a subject with the preparation causes a reduction in the viral load in faeces of a similar viral strain in the serum of the subject following subsequent exposure to a similar viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

In yet another embodiment, oral vaccination of a subject with the preparation causes a reduction in the viral load in faeces of a closely related virus in the serum of the subject following subsequent exposure to a closely related viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

In one embodiment, the preparation of the invention, as described in any of the embodiments above, results in a reduction in viral load in the tissues of a subject following subsequent exposure to the virus, compared to unvaccinated subjects. The skilled person will be equipped to determine the effects of vaccination on viral load, and some methods are detailed in the examples. For example, in one embodiment, vaccination with the yeast cell of the invention cause a reduction in viral DNA copy number in the tissues of a subject following subsequent exposure to the virus, compared to unvaccinated subjects, as detailed in Example 6. The DNA copy number may be between 2 and 15 fold higher in the unvaccinated subjects, for example may be between 3 and 14 fold higher, for example may be between 4 and 13 fold higher, for example may be between 5 and 12 fold higher, for example may be between 6 and 11 fold higher, for example may be between 7 and 10 fold higher, for example may be between 8 and 9 fold higher. In a further embodiment, viral load may be assessed by determination of the level of the epitope which was expressed on the yeast cell surface in the tissue, i.e. determination of the same epitope but expressed by the virus indicates the amount of the virus in the tissue.

In an embodiment, the tissue in which the viral load is decreased by vaccination with the yeast cell of the invention, upon subsequent exposure to the virus, compared to unvaccinated subjects is lymphoid tissue, for example is tonsil, mesenteric lymph node, inguinal lymph node, jejunal lymph node or ileum tissue.

As detailed above and in the examples, it is not considered necessary for oral vaccination with a preparation as defined above to affect the level of serum antibodies. For example, in one embodiment a vaccinated and unvaccinated subject may show the same change in the level of serum antibodies upon subsequent exposure to either the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, or upon exposure to a different viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, or upon exposure of a closely related viral species to that from which the viral epitope expressed on the cell surface of the yeast was derived.

One factor that is considered to be important however is that vaccination with the preparation as described above is capable of inducing an increase in the level of IgA antibody, for example the level of IgA antibody in the mucosa or in the faeces of the subject upon subsequent exposure to either the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, or upon subsequent exposure to a different viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, or upon subsequent exposure of a closely related viral species to that from which the viral epitope expressed on the cell surface of the yeast was derived.

The skilled person will be able to determine whether a particular yeast celi/preparation possesses such properties through application of routine techniques in the art. Some of these techniques are disclosed in the examples.

In one embodiment, the preparation of the invention results in an increase of at least 2fold IgA in the mucosa or faeces compared to an unvaccinated subject, for example may result in an increase of at least 3-fold or 4-fold or 5-fold or 6-fold or 7-fold or 8-fold or 9fold or 10-fold IgA in the mucosa or faeces compared to an unvaccinated subject.

In one embodiment, vaccination with the preparation of the invention does not affect the levels of the pro-inflammatory cytokines. Examples of pro-inflammatory cytokines include IL-1 b, TNF, IL-6, IL-2, and also include reactive oxygen species and reactive nitrogen intermediates. Accordingly, in one embodiment, vaccination with the preparation of the invention does not affect the levels of IL-1 b, TNF, IL-6, IL-2, reactive oxygen species and reactive nitrogen intermediates following exposure to the virus, compared to unvaccinated subjects. In a further embodiment vaccination with the preparation of the invention does not affect the levels of IL-1 b and TNFa following exposure to the virus, compared to unvaccinated subjects.

In an alternative or additional embodiment, vaccination with the preparation of the invention causes an increase in the levels of anti-viral components, such as interferon levels, for example in the levels of INFa and INFy and IFNtau and Mx proteins following exposure to the virus, compared to unvaccinated subjects. In one embodiment vaccination with the preparation of the invention causes an increase in the levels of INFa and INFy following exposure to the virus, compared to unvaccinated subjects.

IFN are generally considered to be anti-viral cytokines.

It is considered that excess levels of pro-inflammatory cytokines (including reactive oxygen species and reactive nitrogen intermediates) may damage tissue, and be harmful rather than beneficial. No such effect of excess IFNs is known.

It will be appreciated that the embodiments detailed above are not to be taken in isolation, but that the yeast cell may comprise any combination and any number of the different epitopes described. For example, the yeast cell may express at the cell surface 2 viral epitopes from virus 1, 1 viral epitope from virus 2, and 1 epitope from bacterial species 1.

It will be appreciated that the yeast cell or preparation may be part of a composition, comprising other yeast cells and/or other various factors and agents. Accordingly, a second aspect of the invention provides a composition comprising a preparation as defined in any of the embodiments above.

In one embodiment, the composition comprises preparations of yeast cells that express different epitopes. For example the composition may comprise preparations of 2 different types of yeast cell expressing 2 different epitopes, or may comprise 3 different types of yeast cell expressing 3 different epitopes, or may comprise 4 different types of yeast cell expressing 4 different epitopes, or may comprise 5 different types of yeast cell expressing 5 different epitopes, or may comprise 6 different types of yeast cell expressing 6 different epitopes, or more.

It will be appreciated that as described above, a single yeast cell may express more than one epitope, for example, more than one epitope on the same molecule, for example on a polypeptide; or more than one epitope on different molecules. Also as described above the epitopes may be from the same viral strain, different viral strains, different viral species, bacterial species, PAMPs, DAMPs, or native yeast proteins engineered to be overexpressed at the yeast cell surface. Accordingly, each yeast cell may express a different set of epitopes. The invention therefore provides a composition that may comprise preparations of 1 type of yeast expressing 1 set of epitopes, or may comprise 2 different types of yeast cell each type expressing a unique set of epitopes, or may comprise 3 different types of yeast cell each type expressing a unique set of epitopes, or may comprise 4 different types of yeast cell each type expressing a unique set of epitopes, or may comprise 5 different types of yeast cell each type expressing a unique set of epitopes, or may comprise 6 different types of yeast cell each type expressing a unique set of epitopes, or more.

In one embodiment, the compositions as described above further comprise one or more additional agents. These agents may be, for example, agents that give a particular taste to the composition to aid in oral administration, or the agents may be, for example, agents that have other health benefits. In one embodiment the composition may also comprise standard animal feedstuff.

Therefore, in another aspect of the invention, the invention provides an animal foodstuff comprising the preparation or composition according to any of the embodiments described above. In a preferred embodiment the animal foodstuff is a pig foodstuff. The preparation or composition may be added to the animal foodstuff during manufacture of the animal foodstuff, or may be added subsequently to the manufacture of the animal foodstuff, for example just prior to feeding to the pigs.

The animal foodstuff of the invention may take any form, for example may be a solid, and may for example be a powder or a pellet, or the animal foodstuff may be a liquid.

As will be apparent from the disclosure above, the preparation of the invention is considered to be useful in the vaccination of pigs. Accordingly, a further aspect of the invention provides a preparation, a composition, or an animal foodstuff as described above for use in the vaccination of pigs. A method of vaccination of pigs is also provided, wherein the method comprises administering the preparation, the composition, or the animal food stuff as described above. The invention also provides the use of the preparation, the composition, or the animal food stuff in the manufacture of a medicament for vaccinating pigs.

Relevant vaccination schedules will be apparent to the skilled person. One such schedule is described in the examples and comprises administration of the preparation, composition or animal food stuff at weekly intervals. In one embodiment the preparation, composition or animal food stuff is administered at weekly intervals for 3 weeks. The preparation, composition or animal food stuff may be administered at weekly intervals for 1, 2, 3, 4, 5 weeks.

Vaccination may be carried out at any time during the life of the pig. However, it is considered beneficial if vaccination is commenced soon after birth, for example between 1 hour and 14 days after birth, for example between 6 hours and 12 days after birth, for example between 12 hours and 10 days after birth, for example between 18 hours and 8 days after birth, for example between 24 hours and 7 days after birth, for example between 36 hours and 6 days after birth, for example between 48 hours and 5 days after birth, for example between 3 and 4 days after birth.

The preparation, composition or animal food stuff may be administered at any time points. The preparation, composition or animal food stuff may be administered at regular intervals, or irregular intervals. Vaccination with the freeze-dried yeast, composition or animal food stuff may occur more than once, for example a particular vaccination schedule in which the vaccination is administered one or more times may be repeated at a particular time point, for example 6 months after administration of the first vaccine schedule, or for example 1 year, or 1.5 years, or 2 or more years later.

The amount of yeast cell preparation, composition or animal foodstuff to deliver to the subject will be apparent to the skilled person. In one embodiment, the equivalent of 7g of yeast (i.e. the physical amount would be more where the yeast is part of a composition or a foodstuff) is administered to each pig on each administration. For example, 7g of yeast is considered to be an appropriate amount for a pig that is 2 weeks old at the first administration and weighs approximately 4 kg. Accordingly, an appropriate amount of yeast of the invention to administer as a vaccine may be considered to be 1.75g yeast / kg pig. Accordingly, the amount that is administered may be increased where for example the pig is larger and may be reduced where the pig is smaller.

The inventors surprisingly found that pigs that had been administered the yeast preparation of the invention showed an increased body mass following subsequent exposure to the virus, compared to unvaccinated pigs. The invention therefore also provides the yeast preparation, the composition, or the animal foodstuff of the invention for use in increasing body mass in pigs. We also provide a method of increasing body mass in pigs comprising administering the yeast preparation, the composition, or the animal food stuff of the invention. Also provided is the use of the yeast preparation, the composition, or the animal foodstuff of the invention for use in the manufacture of a medicament for increasing body mass in pigs.

The invention also provides a kit of parts comprising in separate containers an animal food stuff, for example a pig foodstuff; and the yeast preparation or composition according to the invention.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, the yeast cell of the invention may comprise any number of, and any combination of, the antigens described herein at the yeast cell surface.

The invention will now be further illustrated by the non-limiting figures and examples given below.

Figure legends

Figure 1: Mean PCV2 copy number detected in the serum of pigs in different groups over the 12 weeks of the study. DNA was isolated from serum collected from each pig on a weekly basis and the number of PCV2 copies was determined by qPCR and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by * = p < 0.05; ** = p < 0.01

Figure 2: Mean PCV2 copy number detected in the faeces of pigs in different groups over the 12 weeks of the study. DNA was isolated from faeces samples collected from each pig on a weekly basis and the number of PCV2 copies was determined by qPCR and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by * = p < 0.05; ** = p < 0.01

Figure 3: Mean PCV2 antibody titre in the serum of pigs in different groups over the 12 weeks of the study. Diluted serum samples collected from each pig on a weekly basis was tested for the presence of anti-PCV2 antibodies by ELISA and comparison to known standards. Arrow indicates time-point of last vaccination and challenge.

Figure 4: Mean IgA antibody titre in the faeces of pigs in different groups over the 12 weeks of the study. Faeces samples collected from each pig on a weekly basis were diluted 8-fold (w/v) in PBS, 0.05% Tween 20 and supernatant containing antibodies was harvested. The antibody concentration was determined by ELISA and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by * = p < 0.05.

Figure 5; Mean inflammatory cytokine levels in the serum of pigs in different groups over the 12 weeks of the study. Diluted serum samples collected from each pig on a weekly basis was tested for the presence of TNFa (A), IL-1 β (B), IFNa (C) and IFNy (D) by cytometric bead array and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by * = p<0.05; ** = p < 0.01

Figure 6: PCV2 copies in the mesenteric lymph node (A), jejunal lymph node (B), tonsil (C) and ileum (D) as well as IHC scores of pigs in the vaccinated and unvaccinated groups at week 12 of the study. Tissue was collected from each pig post mortem and DNA was isolated from a known mass. The number of PCV2 copies was determined by qPCR and comparison to known standards. Tissue was collected post mortem and sectioned in paraffin after which sections were stained for the capsid protein of PCV2. Examples of positive (Ei) and isotype control staining (Eii) are shown. IHC scoring (F) and overall scoring (G) are shown for pigs in the vaccinated and unvaccinated groups for all tissues collected.

Figure 7: A representation of the study timeline showing the procedures performed on each week of the study. VD = Vaccine dose, S = Collection of temperature readings, serum and faeces, I or Ml = Infection or mock infection, PM = Post mortem collection of tissues. No sampling was performed on weeks 8, 10 or 11 of the study.

Figure 8: Mean pig weight at the start and end of the study (A). Mean rectal temperatures of pigs in different groups over the 12 weeks of the study (B) and mean rectal temperatures of pigs in both experimentally infected groups for the first 7 days post infection (C). Arrow indicates time-point of last vaccination/challenge.

Figure 9: Anti-PCV2 antibody titre found in the serum of each individual pig during weeks 0-5 (A) and weeks 6-12 (B) of the study. No serum samples were collected on week 8, 10 or 11 of the study. Arrow indicates time-point of last vaccination/challenge.

Figure 10: Freeze dried yeast is non-viable. Photos showing YPD plates spread with 1x 10⁷ live, freeze-dried, heat-killed or both freeze dried and heat killed EBY100-pYD1-Cap cells suspended in PBS. Plates were incubated at 30°C for 3 days. Only the live yeast was viable, a) PBS control b) Freeze dried EBY100-pYD1-Cap c) EBY100-pYD1-Cap d) Freeze dried and heat killed EBY100-pYD1-Cap e) Heat killed EBY100-pYD1-Cap.

Figure 11: Freeze dried recombinant yeast still expresses the Cap protein at its surface. A) Fluorescent Microscopy of live and freeze-dried EBY100 cells grown in galactose containing media for 60 hours. Cells were stained with PCV2-Cap antibody to show expression of the Cap protein at the yeast surface. B) Fluorescent Microscope picture of staining of live and freeze-dried EBY 100 cells. Cells were stained using an isotype control antibody.

Figure 12: a) Map of the plasmid used to confer cell surface expression of the Cap protein, b) Scheme showing how the desired protein is expressed at the cell surface.

Examples

Example 1 - Oral vaccination reduces PCV2 copy numbers in the serum

All pigs remained PCV2 free until challenge at week 2 of the study. At week 3, PCV2 DNA was detected in the serum of all challenged pigs (Fig. 1), but not in the control group (data not shown). In vaccinated pigs, the highest viral load was detected in week 3, and viral load constantly declined thereafter. By week 9 of the study, PCV2 was undetectable in the sera of two of the vaccinated pigs. In contrast, PCV2 viral load in sera of unvaccinated pigs showed an increase from week 3 to week 7. It has to be mentioned that sera of control animals remained PCV2-free until week 7 of the study when one pig tested positive for PCV2. By week 9 of the study, all of the pigs in the control group tested positive for PCV2 by qPCR indicating a breakdown in biosecurity in the pen housing the control group (data not shown).

Example 2 - Oral vaccination reduces viral load in faeces

Both groups challenged with PCV2 at week 2 of the study had detectable levels of PCV2 in the faeces by week 4. The PCV2 copy number in the faeces of the vaccinated group was 3.5 x 10⁴ copies mg'¹ compared to 8.5 x 10⁴ copies mg'¹ in the unvaccinated group (Fig. 2). By week 5 of the study, PCV2 copy number found in faeces of the vaccinated group had reduced (Fig. 2). Faeces of the control pigs remained negative until week 7 when one animal tested positive.

Example 3 - Oral vaccination does not seem to impact on serum Cap-specific antibody concentration

We next assessed whether differences in PCV2 Cap specific antibody production could be detected. With the exception of one pig in the vaccinated group, all pigs remained Cap antibody-negative before infection at week 2. (Fig. 3; Fig. S3A). At week 4, PCV2 Cap antibodies were detectable in vaccinated and unvaccinated pigs (Fig. 3). Overall, antibody titres in both groups seemed to follow a very similar pattern (Fig. 3). Additionally, anti23

PCV2 antibodies developed between week 9 and 12 in all control pigs (Fig. S3B). The presence of antibodies in the control pigs again confirmed PCV2 infection in these animals.

Example 4 - Oral vaccination leads to an increased IgA antibody peak in faeces

Faecal samples collected weekly were tested for their concentration of total slgA. As large variations in slgA were observed between animals of the same group only the mean for each group at each week are shown. The mean slgA levels in the faeces of unvaccinated pigs peaked at week 3 and then rapidly declined (Fig. 4). In comparison, the mean slgA levels in the vaccinated pigs showed a first increase at week 1 and peaked at week 5, and seemed to stay slightly elevated for the rest of the study (Fig. 4). The slgA peak in faeces of vaccinated pigs was 3-fold higher and 2 weeks later compared to the unvaccinated pigs. No slgA was detected in faeces of control animals (data not shown).

Example 5 - Oral vaccination prevents proinflammatory cytokine production, but alters IFN response

For both pro-inflammatory cytokines (ΙΙ_-1β, TNFa), a clear increase in cytokine concentrations was seen in unvaccinated pigs from week 5 onwards, reaching its peak at week 6 before declining (Fig 5A and 5B), whereas sera of vaccinated pigs remained negative. Due to the large variation between pigs of the same group, none of the observed differences reached a level of statistical significance.

IFNa concentration in sera of vaccinated pigs started to rise at week 1 and peaked at week 5 of the study, before declining to baseline levels by week 6 (Fig. 5C). In contrast, there was a small increase in IFNa in sera of unvaccinated pigs at week 3 and 4, before IFNa level peaked at week 5 and declined thereafter (Fig. 5C). Serum IFNy concentration in the sera of vaccinated pigs started to rise three weeks after challenge (week 4), peaked at week 7 and declined thereafter (Fig. 5D). In contrast, IFNy was only detected at week 6 and 7 in unvaccinated pigs, and the concentration of IFNy in sera of unvaccinated pigs stayed lower compared to sera of vaccinated pigs (Fig. 5D). Neither IFN was detected in sera of control pigs (data not shown).

Example 6 - Oral vaccination reduces PCV2 DNA copy number as well as Cap protein expression in tissues

PCV2 copy number in tissues of vaccinated pigs was lower in samples taken from the MLn (Fig. 6A), JLn (Fig. 6B), tonsils (Fig. 6C) and ileum (Fig. 6D), compared to the unvaccinated group. The difference between groups was most pronounced in the ileum (Fig. 6D), with differences varying between 2-fold to almost 10-fold higher copy number in the unvaccinated group. However, none of these differences reached statistical significance due to large variation in PCV2 copy number between animals of the same group.

Immunohistochemistry was performed on tissue sections to potentially confirm qPCR for ORF1 would also be reflected on the protein level for ORF2. Tissue sections were scored in a blinded fashion for lymphoid depletion, signs of inflammation and PCV2 immunolabelling according to previously described methods [21]. A representative difference in labelling between the positive and negative staining is shown (Fig. 6Ei and 6Eii). The mean IHC score and overall score for the unvaccinated group were consistently higher, albeit not statistically different than that of the vaccinated group (Fig. 6F and 6G).

Example 7 - Rectal temperature and weight gain is not affected by oral yeast application

To assess potential adverse effects of yeast application, rectal temperature of all pigs in the study was measured as described and each pig weight was measured at the beginning and end of the experiment. The mean body weight of all pigs showed no significant differences between pigs in the different groups (Fig. 8A); however, there was a tendency of vaccinated pigs to gain more weight compared to unvaccinated pigs. After challenge, there was a tendency for the mean rectal temperature (MRT) of pigs in the vaccinated group to be lower than that of the pigs in the unvaccinated (Fig. 8B). In the week after challenge, the MRT of the vaccinated pigs mirrored that of the unvaccinated pigs for days 0 to 3 post challenge (Fig. S2C), but dropped markedly after this in the vaccinated pigs, remaining below that of the unvaccinated pigs for the rest of this week (Fig. 8C).

Example 8 - Discussion

Recombinant yeast has been used in a variety of mouse and human cancer- as well as viral infection models [12, 23-26], even in its live form [10], resulting in a strong adjuvant effect, augmenting antigen presentation to MHC class I- and class Il-restricted T cells [10],

In the present study, we assessed the potential of a powdered freeze-dried S.c. stably expressing the PCV2 Cap protein on its surface as an oral vaccine against challenge with PCV2. Vaccinated pigs had reduced PCV2 DNA copy numbers in all samples analysed. Interestingly however, no significant difference for anti-PCV2 antibodies were seen between the two groups (Fig. 4). This result is in-line showing the absence of significant differences in antibody titres between vaccinated and non-vaccinated pigs [27], leading to the assumption that these do not correlate with protection/immunity during PCV2 infection [28-30], However, there were clear differences in slgA production between both groups (Fig. 5). Whereas we cannot exclude that the increase in slgA at the early time-point in vaccinated animals may be a response to the yeast particles, our combined data indicates that the oral application also induces a PCV2-specific IgA response.

Whereas high titres of neutralising antibodies seem to be inversely correlated with PCV2 load [31], correlates of cellular immune responses are less well understood [32], Koinig et al recently described a CD4 T-cell subset that was vaccine antigen specific and coproduced IFNy as well as TNFa in response to PCV2 [33], In the present study, proinflammatory cytokine and IFN response differed between the vaccinated and unvaccinated group. Throughout the study, we were unable to detect TNFa and IL-1 β in vaccinated animals whereas the levels in unvaccinated animals peaked at week 5 and 6 (Fig. 5A and 5B). These data are in line with the findings of others describing a TNFa peak at 21 days post infection [34, 35]. Furthermore, the absence of TNFa in the vaccinated group may be due to the yeast supplementation itself as a recent study showed decreased serum TNFa levels in mice feed S. cerevisiae and infected with PCV2 [36], However, in contrast to our results, no difference in IL-Ιβ between the two dietary groups were observed in this study [36].

Of interest are the different kinetics in the systemic IFN responses in the two different groups. In vaccinated animals, IFNa levels increased from the second week of the experiment (Fig. 5C), with a clear increase after challenge. We believe that the oral vaccination already induced an IFNa response, which contributed to the reduced PCV2 replication/level of DNA and subsequently contributed to the protection of the animals. In contrast, IFNa levels in the unvaccinated group started to increase after challenge (Fig. 5C), before peaking at week 5, at the same time a rise in pro-inflammatory cytokines was seen, potentially reflecting the response to the replicating virus. Similarly, IFNy levels in vaccinated animals increased earlier compared to unvaccinated animals (Fig. 5D). For the unvaccinated group, the peak in IFNy concentration correlates with the peak in the presence of IFNy producing cells in response to challenge demonstrated by others at 24 days post infection [33], The fact that an IFNy response was seen a week earlier in the vaccinated group may be due to the sensitisation of the immune system to the Cap protein expressed on the yeast surface which has been shown to induce the development of IFNy secreting cells [37] as well as a potential positive feedback loop with IFND. In addition to the earlier rise, the mean concentration of IFNy was also higher in the vaccinated compared to the unvaccinated group (Fig. 5D). The presence of IFNy in PCV2 infection is considered advantageous for controlling disease with an inverse correlation observed between IFNy secreting cells and PCV2 copies [38, 39],

Similar to the qPCR results for ORF1, the IHC scoring demonstrated a reduced viral ORF2 expression in the tissues of vaccinated pigs. This was most evident in the ileum (Fig. 6D), the site where one would expect to find an orally applied antigen to induce an immune response. Our data could potentially indicate that the yeast particles are necessary to stimulate a strong enough immune response to affect PCV2 viral replication.

Although this study was only a proof of concept study, we believe that yeast-particles expressing one or multiple vaccine antigens on their surface could provide a new and cheap option for mass vaccination of farm animals. The success of this vaccination approach can further be enhanced by including molecules specifically targeting yeast particles to M cells [40]

Example 9 - Materials and Methods

Ethics statement

All animal studies were performed according to the regulations and guidance provided under the UK Home Office Animals (Scientific Procedures) Act 1986, under project licence number (70/7291), as well as regulation of the RVC Ethics and Welfare Committee.

Cloning

ORF2 was amplified from a recently cloned PCV2b strain (GenBank accession number JX193799; [17]), using Forward (5-GGTACCAATGACGTATCCA-3’) and Reverse (5’CTCGAGAGGGTTAAGTGG-3’) primers designed to add a Kpnl and Xhol restriction sites PCR conditions were 95°C for 1 min, 55°C for 1 min, 72°C for 1 min for 34 cycles, followed by a final extension at 72°C for 7mins. Bands representing the amplified ORF2 were excised, eluted, digested and ligated into the linearized pYD1 plasmid [18] using T4 DNA ligase (Promega). The correct insert in the resulting pYD1-ORF2 plasmid was confirmed by PCR and sequencing.

Production of freeze-dried Cap expressing yeast particles

Preparation of competent yeast cells and transformation with 1 pg of pYD1 or pYD1-ORF2 was done as described [18]. After growing on selective plates, one colony of EBY100pYD1-ORF2 was inoculated into 400 ml of YNB-CAA with 2% glucose, and incubated for 48 hr at 30°C and 200rpm. Thereafter, the content pelleted in 50ml Falcon tubes (Fisher) by centrifugation at 300 x g, 10 min at RT, the resulting pellet re-suspended in YNB-CAA with 2% galactose, and transferred into a 2L Erlenmeyer conical tissue culture flask (Fisher) filled to 1.5L with YNB-CAA with 2% galactose. Culture was incubated at 20°C at

200rpm for 48 hr and resulting yeast now expressing the PCV2b Cap protein at its surface was termed EBY100-pYD1-Cap. After pelleting at 300gfor 10mins at RT, yeast pellets were re-suspended in YNB-CAA with 2% galactose and 5% glycerol, and aliquots stored at -80°C. Each batch of recombinant S.c. was tested for Cap expression by flow cytometry and all batches showed between 50 and 60% expression. Subsequently, yeast was freezedried using a MicroModuIyo Freeze Dryer (Thermo-scientific) with the following cycle conditions: -40°C for 60 min with no vacuum, -30°C for 300 min under vacuum, -10°C for 300 min under vacuum, 20°C for 420 min under vacuum and 20°C for 60 min with no vacuum. Freeze dried yeast was powdered using a sterile pestle and mortar and 7g batches were stored at -80°C.

Virus preparation for infection

The PCV2b strain used for the challenging experiments [17] was propagated as described [2], The titre of PCV2 was 1.5 x 10⁹ copies per ml, as determined by qPCR, equalling a titre of 10⁸⁰⁵ TCID₅₀ ml¹, analysed as described [2]. Mock samples consisted of supernatant of uninfected cells and was PCV2 free as analysed by qPCR.

Experimental design

Seventeen two week old “Babraham pigs” [19] were purchased from The Pirbright Institute (UK). All pigs were certified free of swine influenza strains H1N1, H1N3 and H1N5 as well as Mycoplasma hyopneumoniae, PCV1 and PCV2. Pigs were randomly allocated to three groups (Control (n=5), Vaccinated (n=6) and Unvaccinated (n=6)), and housed in separate rooms with controlled environment, separated air-flow or sewage draining, and ad lib access to food and water. Samples as well as rectal temperatures were taken from all pigs on a weekly basis, and weight was measured at the beginning and end of the study. For the first 3 weeks of the study, pigs were vaccinated weekly by oral administration of 7g of freeze dried EBY100-pYD1-Cap, suspended in 20ml of sterile PBS (Sigma). Unvaccinated pigs group received 3 x 20ml PBS instead. On the same day as the third vaccination, pigs were inoculated intra-nasally with 7.5x10⁹ PCV2 particles in 5ml of media, which has been shown to result in a successful PCV2 infection [9], Control pigs were inoculated intranasally with 5ml of media from uninfected cells (mock). Temperature of infected pigs was monitored daily for 7 days post infection. At the conclusion of the study, pigs were humanly killed. Samples of tonsil, mesenteric (MLn), inguinal (ILn), and jejunal lymph node (JLn) as well as ileum were taken and either stored in 10% buffer formalin for microscopic analysis or stored at -80°C for subsequent nucleic acid extraction. The study is represented in a timeline diagram in Fig. S1.

Isolation of total DNA from tissue and serum samples

Total DNA was isolated from 25 to 50mg of tissue sample collected at post-mortem using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. In addition, DNA was isolated from 200μΙ cell-free serum collected weekly using the QiAMPMinElute Virus spin kit (Qiagen) according the manufacturer’s instructions. In both cases, isolated DNA was eluted in sterile, nucleic acid-free water (Sigma-Aldrich) and DNA concentration was determined using an Infinite 200 Nanoquant spectrophotometer (Tecan). Eluted DNA was stored at -20°C.

Quantification of PCV2 in tissue or serum samples

The number of PCV2 copies in tissue, faeces and serum was determined by comparison to known known concentrations of plasmid containing the whole PCV2b genome using qPCR. Sample were measured in triplicate in a final volume of 20μΙ per well in MicroAmp Fast Optical 48-microtiter well plates (Applied Biosystems). Each well contained 2μΙ DNA (standard or test sample), 10μΙ 2x TaqMan Universal Master Mix II (Applied Biosystems), 50pmol primers (Forward: 5’-GCTCTYTATCGGAGGATTAC-3’, Reverse: 5'ATAAAAACCATTACGAWGTGATA-3’) (MWG) and 2.5μΜ TaqMan probe (5’FAMCCATGCCCTGAATTTCCATATGAAAT-3TAMRA) (Applied Biosystems), targeting a 137bp fragment of the PCV2 ORF1 [20], Standard measurements were performed in a 10fold serial dilution from 10⁹ PCV2 plasmid copies to 0 copies using a StepOne Real-time PCR machine with a StepOne software (Version 2.2.2; both Applied Biosystems). The cycle conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 55°C for 1 min. Data was analysed using either StepOne software or Excel 2010 (Microsoft). Mean Ct values were used to calculate PCV2 ORF1 copy number in samples, which was then divided by the original weight of tissue or faeces in mg or the volume of serum in μΙ.

PCV2 Cap ELISA

Sera were assayed for anti-Cap PCV2 antibodies using the PCV2 Ab mono blocking SERILSA kit (Synbiotics) according to the manufacturer’s instructions. Samples were read using a SpectraMax M2 plate reader with dual reading (450nm/630nm; Molecular Devices), and PCV2 antibody titre was calculated from the corrected OD values.

IgA ELISA

Faeces samples taken at weekly intervals throughout the study were thawed at room temperature. 125 to 400mg of each sample was placed in a separate Eppendorf tube, and 8x the volume (w/v) of PBST with 0.001% NaN3 was added to each tube and vortexed. Samples were clarified by centrifugation at 10,000gfor 10 min at RT. Supernatants were collected and stored at -20°C until ELISA. The IgA content was determined using a Pig IgA ELISA kit (Bethyl Laboratories) according to manufacturer’s instructions. Samples were read at 450nm using a SpectraMax M2 plate reader (Molecular Devices) and the concentration of IgA antibodies in samples determined against a 4-PL standard curve.

Quantification of inflammatory cytokines in serum samples

Serum samples collected on a weekly basis were assayed for TNFa, IL-1 β, IFNa and IFNy by cytometric bead array by Affymetrix bioscience (Affymetrix, Vienna).

Immunohistochemistry

Viral antigen labelling was determined by immunohistochemistry using a Cap protein specific PCV2 monoclonal antibody (Ingenasa). Briefly, once tissue sections were dewaxed in xylene and rehydrated with graded alcohols, antigen retrieval was performed by immersion in a 0.05% protease (Streptomyces griseus, Sigma, UK) with an adjusted pH of 7.8 at 37°C, for 15 min. After rinsing, endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in 100% methanol for 20 min. Sections were then washed with a phosphate buffered saline 10.05% Tween20 solution (PBST), followed by incubation with 25% normal goat serum in PBST for 1 h, and subsequent incubation for 18 h, 4°C in a humidified chamber with the primary antibody (1:200 in Dako Antibody Diluent). After overnight incubation, sections were washed in PBST (3x5 min), incubated for 30 min with Envision¹ System-HRP labelled polymer (Dako), followed by 3 x washing. Sections were finally incubated with liquid DAB* Substrate Chromogen System (Dako) for 3 min, rinsed, counterstained with haematoxylin, dehydrated, mounted in DPX, covered by a coverslip and examined. Sections of a study pig that developed clinical signs of PCVAD served as positive controls and sections omitting the primary antibody or incubated with an isotype-matched negative control antibody (murine lgG2a! Dako) served as negative controls. Inflammation, lymphoid depletion and PCV2 staining of tissue sections was assessed and scored in a blinded fashion by a resident pathologist according described methods [21, 22]. Each pig in the study was given an IHC score which was the sum of the scores for all tissues with regards to PCV2 staining as well as an overall score which was the sum of the IHC score, the lymphoid depletion score and the inflammation score. Images were taken using an Olympus BX60 microscope at x200 magnification with a QICAM FAST Cooled Mono 12-bit camera and Image Pro Plus 5.0 software.

Statistical analysis

Analyses were performed using Instat software (Graph Pad Software, San Diego, CA, USA). Data are expressed as average +/-SEM. All samples were tested for normal distribution. Time-dependent differences in the parameters assessed were analysed by a repeated-measure analysis of variance. In case of significant differences, the Dunn multiple analysis was used. For figures 1, 2 and 4, data were Iog10-transformed, re-tested for normal distribution using a Kolmogorov-Smirnov test, and weekly values compared using an unpaired t-test.

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Claims (40)

Claims

1. A preparation of an inactivated or freeze-dried yeast cell (or cell-surface-containing portion thereof) expressing a porcine viral epitope, wherein the epitope is expressed on the surface of the yeast cell.

2. The preparation according to claim 1 wherein the yeast is non-viable.

3. The preparation according to claim 1 or 2 wherein the yeast cell expresses more than 1 separate viral epitope, optionally 2 separate viral epitopes, optionally 3 separate viral epitopes, optionally 4 viral separate epitopes.

4. The preparation according to claim 3 wherein the yeast cell expresses viral epitopes from more than one viral species or from more than one viral strain, optionally 2 viral species or 2 viral strains, optionally 3 viral species or 3 viral strains, optionally 4 viral species or 4 viral strains.

5. The preparation according to any of claims 1-4 wherein the epitope comprises a polypeptide, RNA ,or DNA, optionally wherein the epitope further comprises one or more carbohydrate and/or lipid moieties.

6. The preparation according to any of claims 1-5 wherein the epitope is a capsid protein, an envelope protein, a spike protein or a non-structural protein, or is derived from a capsid protein, an envelope protein, a spike protein or a non-structural protein

7. The preparation according to any of claims 1-6 wherein the epitope is a stabilised dimer of proteins.

8. The preparation according to any of claims 1-7 wherein the epitope is expressed as part of a heterologous scaffold protein which stabilises the epitope in its relevant conformation.

9. The preparation according to any of claims 1 -8 wherein the yeast is from the genus Saccharomyces, and is preferably Saccharomyces cerevisiae, or the yeast is from the genus Pichia, and is preferably Pichia pastoris, or the yeast is from the genus Kluyveromyces, and the yeast is K. lactis.

10. The preparation according to any of claims 1-9 wherein the epitope is expressed from a plasmid.

11. The preparation according to any of claims 1-10 wherein the epitope is expressed from a genomically integrated DNA fragment.

12. The preparation according to any of claims 1-11 wherein the epitope comprises one or more modifications, optionally wherein the epitope comprises one or more carbohydrate moieties, or one or more lipid moieties.

13. The preparation according to any of claims 1-12 wherein the yeast cell also expresses a further recombinant antigen that enhances the immune response, optionally wherein the further recombinant is a pathogen associated molecular pattern, optionally selected from the group consisting of lipopolysaccharide (LPS) from Gram-negative bacteria; components from Gram-positive bacteria, including lipoteichoic acid (LTA); component from Gram-positive bacteria, for example LTA; flagellin; double stranded RNA; single stranded RNA from viruses; synthetic ligands, such as R-848 and imiquimod; and unmethylated CpG islands found in bacterial and viral DNA, such as CpG ODNs; and/or damage associated molecular pattern, optionally selected from the group consisting of a HSP; HMGB1; ATP; mitochondrial formyl peptides; mitochondrial DNA; uric acid; NY-ESO-1; Hyaluron; heparan sulfate fragments; S100 family proteins, for example S100A8 (MRP8, calgranulin A) and S100A9 (MRP14, calgranulin B); fibronectin; surfactant protein A; biglycan; versican; mitochondrial DNA; and Serum amyloid A (SAA).

14. The preparation according to any of claims 1-13 wherein the yeast cell expresses molecules that target the yeast cell to microfold cells, and/or wherein the yeast cell comprises molecules at the yeast cell surface that target the yeast cell to a microfold cell, optionally wherein the yeast cell comprises one or more of fucose-targeting lectins, Nacetyl-galactosamine and N-acetyl-neuramin of Psophocarpus tetragonolobus (WBA, winged bean) optionally wherein the yeast cell.

15. The preparation according to any of claims 1-14 wherein the yeast cell or cellsurface-containing portion thereof is powdered, optionally powdered using a pestle and mortar or spray-freeze drying.

16. The preparation according to any of claims 1-15 wherein the viral epitope is from a Coronavirus, optionally from PEDV, TGEV, porcine hemagglutinating encephalomyelitis virus or PRCV; a porcine Rotavirus;

a virus of the Circoviridae family, optionally from PCV2.

17. The preparation according to claim 16 wherein the viral epitope is the ORF2 from a PCV2, optionally from a PCV2b strain, optionally from GenBank accession number JX193799.

18. The preparation according to any of the preceding claims wherein the preparation is stored at ambient temperatures, optionally stored at between about 10°C and 25°C 80C.

19. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation causes a reduction in the copy number of the same viral strain in the serum of the subject following subsequent exposure to the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects;

of a similar viral strain in the serum of the subject following subsequent exposure to a similar viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects; and/or of a closely related virus in the serum of the subject following subsequent exposure to a closely related viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

20. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation causes a reduction in the viral load in faeces of the same viral strain in the serum of the subject following subsequent exposure to the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects;

of a similar viral strain in the serum of the subject following subsequent exposure to a similar viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects; and/or of a closely related virus in the serum of the subject following subsequent exposure to a closely related viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, compared to unvaccinated subjects.

21. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation does not affect the level of serum antibodies raised in the subject directed specifically towards the epitope upon subsequent exposure to either the same viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, or upon exposure to a different viral strain from which the viral epitope expressed on the cell surface of the yeast was derived, or upon exposure of a closely related viral species to that from which the viral epitope expressed on the cell surface of the yeast was derived.

22. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation causes an increase in IgA antibody levels, optionally an increase in IgA in the mucosa or in the faeces of the subject following exposure to the virus, compared to unvaccinated subjects, optionally an increase of at least 2-fold, or 3fold or 4-fold or 5-fold or 6-fold or 7-fold or 8-fold or 9-fold or 10-fold IgA in the mucosa or faeces compared to an unvaccinated subject.

23. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation causes no increase in pro-inflammatory cytokines following exposure to the virus, optionally causes no increase in any one or more of IL-1 b, TNF, IL-6, IL-2, reactive oxygen species or reactive nitrogen intermediates compared to unvaccinated subjects, optionally causes no increase in any one or more of IL-1 b and TNFa, compared to unvaccinated subjects.

24. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation causes an increase in interferon levels, optionally an increase in any one or more of INFa, INFy, IFNtau and Mx levels, following exposure to the virus compared to unvaccinated subjects, optionally causes an increase in any one or more of INFa or INFy levels, following exposure to the virus compared to unvaccinated subjects.

25. The preparation according to any of the preceding claims wherein oral vaccination of a subject with the preparation causes a reduction in viral DNA copy number and/or the expression of the epitope in tissues following exposure to the virus, compared to unvaccinated subjects, optionally wherein the tissue is one or more tissues selected from the group consisting of tonsil, mesenteric lymph node, inguinal lymph node, jejunal lymph node and ileum, optionally wherein the viral DNA copy number is between 2-fold and 10-fold higher in the unvaccinated subject.

26. A composition comprising the preparation according to any of the preceding claims, optionally wherein the composition is a powder.

27. The composition according to claim 26 wherein the composition comprises preparations from different yeast cells expressing different epitopes, optionally comprises preparations of 2, 3, 4, 5, 6 or more different yeast cells expressing different epitopes.

28. The composition according to any of claims 26 and 27 wherein the composition further comprises preparations of one or more yeast cells expressing one or more bacterial or mycoplasma epitopes.

29. The composition according to any of claims 26-28 further comprising one or more agents optionally wherein the agents:

aid in oral administration, and/or agents with other health benefits.

30. An animal foodstuff comprising the preparation or composition according to any of the preceding claims, optionally wherein the animal foodstuff is a pig foodstuff.

31. The animal food stuff of claim 30 wherein the foodstuff is a solid, optionally a powder.

32. The animal food stuff of claim 30 wherein the foodstuff is a liquid.

33. The preparation, the composition, or the animal foodstuff for use in the vaccination of pigs.

34. A method of vaccination of pigs comprising administering the preparation, the

5 composition, or the animal food stuff according to any of claims 1-32.

35. Use of the preparation, the composition, or the animal foodstuff according to any of claims 1-32 for use in the manufacture of a medicament for vaccinating pigs.

10

36. The preparation, the composition, orthe animal foodstuff for use in increasing body mass in pigs.

37. A method of increasing body mass in pigs comprising administering the preparation, the composition, or the animal food stuff according to any of claims 1-32.

38. Use of the preparation, the composition, or the animal foodstuff according to any of claims 1-32 for use in the manufacture of a medicament for increasing body mass in pigs.

20

39. The method, use, preparation or composition according to any of claims 33-38 wherein the preparation or composition is administered weekly, optionally for 1, 2, 3, 4, 5 weeks, optionally wherein

7g of preparation or freeze-dried yeast, or composition comprising freeze-dried yeast or preparation is administered per pig, or

25 1.75g of preparation or freeze-dried yeast is administered per kg of pig.

40. A kit of parts comprising in separate containers an animal food stuff, for example a pig foodstuff; and the preparation or composition according to any of the preceding claims.

Intellectual

Property

Office

Application No: Claims searched:

GB1518762.8 1-40