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WO2017068352A1 - Procédés - Google Patents

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Publication number
WO2017068352A1
WO2017068352A1 PCT/GB2016/053278 GB2016053278W WO2017068352A1 WO 2017068352 A1 WO2017068352 A1 WO 2017068352A1 GB 2016053278 W GB2016053278 W GB 2016053278W WO 2017068352 A1 WO2017068352 A1 WO 2017068352A1
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Prior art keywords
yeast
viral
epitope
preparation
optionally
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English (en)
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Dirk Werling
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Royal Veterinary College
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Royal Veterinary College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to yeast-based vaccines and their use in pigs.
  • PCV2 porcine circovirus type 2
  • PCVAD porcine circovirus associated diseases
  • Vaccination is effective in reducing symptoms and increasing production parameters, but does not prevent spread of the virus.
  • 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.
  • 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, 1].
  • GRAS general regarded as safe
  • Yeast itself possess adjuvant like properties, activating both inflammatory and phagocytic receptors expressed on APC [12].
  • freeze- drying of S.c. expressing PCV2 Cap protein on its surface renders it completely non-viable, without affecting expression of the Cap protein.
  • GMO genetically modified organism
  • 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].
  • 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.
  • a yeast cell-derived preparation or composition is considered to have significant practical value.
  • 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.
  • 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.
  • the physical proximity of the viral antigen and the yeast surface 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.
  • antigen may leak (at low levels) from the MHC II pathway into the MHC I pathway, and this is called cross-presentation.
  • the inactivated, typically freeze-dried, nature of the yeast preparation 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 horringer 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.
  • the invention provides a preparation of an inactivated orfreeze-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.
  • 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.
  • 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.
  • 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 Kiuyveromyces, 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.
  • 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.
  • the vector is the pYD1 vector, as described in http://img55.chem17.eom/5/20130809/6351 1650621 1258776521 .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.
  • the yeast expresses the relevant viral epitope (or epitopes) from a genomic locus.
  • 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.
  • 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.
  • the yeast cell may express a viral epitope from both a vector and from a genomic location.
  • 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 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.
  • the promoter may be any suitable promoter known in the art.
  • the promoter is an inducible promoter, for example a promoter inducible by galactose.
  • the promoter is a constitutive promoter.
  • 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.
  • 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.
  • the epitope is or comprises a polypeptide epitope.
  • the viral epitope is from a Coronavirus, for example from PEDV, TGEV, porcine hemagglutinating encephalomyelitis virus or PRCV.
  • the viral epitope is the full length Spike protein of PEDV (for example as indicated in SEQ ID NO: 1 , encoded by SEQ ID NO: 2) or a fragment thereof.
  • the viral epitope is a truncated version of the Spike protein of PEDV termed S1 (for example as indicated inSEQ ID NO: 3, encoded by SEQ ID NO: 4). This truncated region is considered to contain the most immunogenic sites as well as those sites necessary to induce viral neutralising antibody production.
  • Underline 2 amino acid changes relative to the closest genbank sequence (accession KM052365), which, for example, may also be used.
  • PEDV1303 S FULL nucleotide (SEQ ID NO: 2)
  • PEDV1303 S ecto protein (note: no ATG1 (SEQ ID NO: 31
  • the viral epitope is from a virus of the Circoviridae family, for example PCV2.
  • the viral epitope is ORF2 from PCV2.
  • the ORF2 is from a PCV2b strain, for example with the sequence detailed in GenBank accession number JX193799.
  • 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.
  • 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.
  • the epitope is a viral cap protein or is derived from a viral cap protein.
  • the viral epitope is a viral envelope protein or is derived from a viral envelope protein.
  • the viral epitope is a spike protein or is derived from a spike protein.
  • the viral epitope is a non-structural protein or is derived from a non-structural protein.
  • the epitope comprises the Cap protein of PCV2, or fragment thereof.
  • the epitope comprises the spike protein of PEDV, or fragment thereof, for example a truncated version of the Spike protein termed S1.
  • envelope proteins in enveloped viruses such as PEDV, a coronavirus
  • 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.
  • 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.
  • 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.
  • the epitope comprises the entire full length protein.
  • 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 non- consecutive amino acids become spatially arranged in close proximity to each other, or in another way that provokes the generation of an antibody.
  • conformational epitopes may not be limited to a single molecule, but may span two or more molecules.
  • epitopes are formed at the interface of protein dimers, for example dimers of viral envelope proteins.
  • 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.
  • 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.
  • the Cap protein of PCV2 may comprise several epitopes.
  • a benefit of the yeast system is the ability express more than one molecule containing a viral epitope simultaneously.
  • 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.
  • 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.
  • 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.
  • protection against a variety of strains of the same virus may be achieved.
  • 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.
  • 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.
  • 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.
  • 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.
  • protection against an array of porcine viral species may be obtained from the production of a single type of yeast cell.
  • 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 epitopes of PEDV, for example the full length Spike protein or a truncated Spike protein, for example S1.
  • 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.
  • 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 Immunopathol. 2009 Oct 15;131(3-4):238-45. doi: 10. 016/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.
  • Jores J1 Meens J, Buettner FF, Linz B, Naessens J, Gerlach GF.
  • 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.
  • 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.
  • modifications may be engineered into the cell, such that they are added for example postranslationally to the polypeptide.
  • 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.
  • 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.
  • 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.
  • yeast cell surface display for example employing members of the glycosylphosphatidylinositol (GPl) family of cell wall proteins.
  • GPl glycosylphosphatidylinositol
  • the desired epitope is located at the cell surface, using routine techniques such as immunomicroscopy or immunoflowcytometry.
  • 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.
  • the polypeptide will be localised at the cell surface.
  • 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.
  • 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.
  • the yeast cell is non-viable. This may be as a result of freeze- drying, 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.
  • yeast cell also expresses other antigens or factors that enhance the immune response.
  • yeast can bind to, for example, TLR2 and Dectin-1 expressed by immune cells through native yeast beta- glucans, 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.
  • the yeast cell may also be engineered to have increased expression of these native yeast beta-glucans.
  • 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.
  • 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.
  • 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.
  • 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 pathogen associated molecular patterns
  • DAMPs damage associated molecular patterns
  • PRRs pattern recognition receptors
  • 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 Iigands, such as R-848 and imiquimod; and unmethylated CpG islands found in bacterial and viral DNA, such as CpG ODNs.
  • LPS lipopolysaccharide
  • LTA lipoteichoic acid
  • flagellin such as flagellin
  • double stranded RNA double stranded RNA
  • single stranded RNA from viruses such as synthetic Iigands, such as R-848 and imiquimod
  • 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).
  • 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 cell- surface-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.
  • 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.
  • the one or more PAMPs or DAMPs is chemically added to the yeast preparation.
  • versican and/or flagellin are chemically added to the yeast preparation.
  • 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.
  • any combination of the epitopes, native yeast proteins or PAMPs or DAMPs may be co-expressed in the same cell.
  • yeast cell as defined in any of the embodiments above also expresses one or more bacterial-derived epitopes.
  • the yeast cell as defined in any of the embodiments above also expresses one or more bacterial-derived epitopes.
  • the yeast may express one or more Rotavirus epitopes, and/or one or more Corona virus epitopes, and one or more Salmonella epitopes.
  • 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.
  • 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.
  • 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.
  • 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.
  • the yeast cell expresses or comprises one or more moieties that target the yeast cell to the M cells.
  • moieties that target the yeast cell to the M cells.
  • 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.
  • the yeast cell comprises one or more of fucose-targeting lectins, N-acetyl- galactosamine and N-acetyl-neuramin of Psophocarpus tetragonolobus (WBA, winged bean).
  • WBA winged bean
  • 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.
  • 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.
  • 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.
  • the preparation is stored at between 10°C and 40°C.
  • the yeast is stored at between 10°C and 35°C.
  • the preparation is stored at between 10°C and 30°C.
  • the preparation is stored at between 10°C and 25°C.
  • the preparation of the invention 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.
  • 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.
  • 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):261 - 9.
  • vaccination with one particular epitope may give rise to protection against closely related species.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the preparation of the invention 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.
  • 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 1 1 fold higher, for example may be between 7 and 10 fold higher, for example may be between 8 and 9 fold higher.
  • 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.
  • 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.
  • lymphoid tissue for example is tonsil, mesenteric lymph node, inguinal lymph node, jejunal lymph node or ileum tissue.
  • 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.
  • 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 preparation of the invention results in an increase of at least 2- fold 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 9- fold or 10-fold IgA in the mucosa or faeces compared to an unvaccinated subject.
  • vaccination with the preparation of the invention does not affect the levels of the pro-inflammatory cytokines.
  • pro-inflammatory cytokines examples 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.
  • 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.
  • 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.
  • I FN are generally considered to be anti-viral cytokines.
  • yeast cell may comprise any combination and any number of the different epitopes described.
  • 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.
  • 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.
  • the composition comprises preparations of yeast cells that express different epitopes.
  • 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.
  • 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.
  • 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.
  • compositions as described above further comprise one or more additional agents.
  • 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.
  • the composition may also comprise standard animal feedstuff.
  • the invention provides an animal foodstuff comprising the preparation or composition according to any of the embodiments described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 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.
  • 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 yeast cell of the invention may comprise any number of, and any combination of, the antigens described herein at the yeast cell surface.
  • 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 5 Mean inflammatory cytokine levels in the serum of pigs in different groups over the 12 weeks of the study.
  • FIG. 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.
  • FIG. 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 Infection or mock infection
  • PM Post mortem collection of tissues. No sampling was performed on weeks 8, 10 or 1 1 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 1 1 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 1 x 10 7 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.
  • 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.
  • FIG. 13 Recombinant S. cerevisiae particles were produced expressing either the full length or Spike protein of a porcine epidemic diarrhoea virus (SEQ ID NO: 1) or a shortened version of the spike protein (termed S1 ) (SEQ ID NO: 3) containing the most immunogenic sites as well as those sites necessary to induce viral neutralising antibody production.
  • Pigs were vaccinated orally as before on day 0 and 14, blood samples drawn weekly and analysed for PEDV specific IgG antibodies or faeces analysed for total IgA. Control was yeast transfected with the empty plasmid.
  • Example 1 Oral vaccination reduces PCV2 copy numbers in the serum
  • Example 3 Oral vaccination does not seem to impact on serum Cap-specific antibody concentration
  • 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 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
  • Example 6 Oral vaccination reduces PCV2 DNA copy number as well as Cap protein expression in tissues
  • 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
  • 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 ll-restricted T cells [10].
  • 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.
  • IFNy levels in vaccinated animals increased earlier compared to unvaccinated animals (Fig. 5D).
  • 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].
  • 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]
  • 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
  • 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 freeze- dried using a MicroModulyo Freeze Dryer (Therm o-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.
  • MicroModulyo Freeze Dryer Therm o-scientific
  • PCV2b strain used for the challenging experiments [17] was propagated as described [2].
  • the titre of PCV2 was 1.5 x 10 9 copies per ml, as determined by qPCR, equalling a titre of 10 6 - 05 TCI Dso ml "1 , analysed as described [2].
  • Mock samples consisted of supernatant of uninfected cells and was PCV2 free as analysed by qPCR.
  • 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.
  • pigs were inoculated intra-nasally with 7.5x10 9 PCV2 particles in 5ml of media, which has been shown to result in a successful PCV2 infection [9].
  • Control pigs were inoculated intra- nasally with 5ml of media from uninfected cells (mock). Temperature of infected pigs was monitored daily for 7 days post infection.
  • 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.
  • 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.
  • DNA was isolated from 200 ⁇ cell-free serum collected weekly using the QiAMPMinElute Virus spin kit (Qiagen) according the manufacturer's instructions.
  • 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.
  • 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 20 ⁇ per well in MicroAmp Fast Optical 48-microtiter well plates (Applied Biosystems).
  • Each well contained 2 ⁇ DNA (standard or test sample), 10 ⁇ 2x Taq an Universal Master Mix II (Applied Biosystems), 50pmol primers (Forward: 5'-GCTCTYTATCGGAGGATTAC-3', Reverse: 5'- ATAAAAACCATTACGAWGTGATA-3') (MWG) and 2.5 ⁇ TaqMan probe (5'FAM- CCATG CCCTG AATTTCCATATG AAAT-3TAM RA) (Applied Biosystems), targeting a 137bp fragment of the PCV2 ORF1 [20].
  • 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.
  • Serum samples collected on a weekly basis were assayed for TNFa, IL- ⁇ ⁇ , IFNa and IFNy by cytometric bead array by Affymetrix bioscience (Affymetrix, Vienna).
  • Viral antigen labelling was determined by immunohistochemistry using a Cap protein specific PCV2 monoclonal antibody (Ingenasa). Briefly, once tissue sections were de- waxed in xylene and rehyd rated 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 / 0.05% 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 (3 x 5 min), incubated for 30 min with Envision * System-HRP labelled polymer (Dako), followed by 3 x washing.
  • PBST phosphate buffered saline / 0.05% Tween20 solution
  • 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 lgG2 a ; 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].
  • PEDV specific IgG as well as total soluble IgA was increased in both treatment groups receiving either the yeast expressing the whole spike protein or its truncated form.

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Abstract

La présente invention concerne une préparation d'une cellule de levure inactivée ou lyophilisée (ou d'une partie de celle-ci contenant la surface cellulaire) exprimant un épitope viral porcin, l'épitope étant exprimé sur la surface de la cellule de levure. La levure n'est typiquement pas viable. La levure est typiquement du genre Saccharomyces, et de préférence Saccharomyces cerevisiae, mais peut être du genre Pichia, par exemple, Pichia pastoris, ou peut être du genre Kluyveromyces, par exemple K. lactis. L'épitope viral peut provenir d'un Coronavirus, éventuellement du PEDV, du TGEV, du virus de l'encéphalomyélite hémagglutinante porcine ou du PRCV; d'un rotavirus porcin, ou d'un virus de la famille Circoviridae, facultativement du PCV2.
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