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WO2015025165A1 - Épitopes de lymphocytes t du virus de la peste porcine classique - Google Patents

Épitopes de lymphocytes t du virus de la peste porcine classique Download PDF

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Publication number
WO2015025165A1
WO2015025165A1 PCT/GB2014/052560 GB2014052560W WO2015025165A1 WO 2015025165 A1 WO2015025165 A1 WO 2015025165A1 GB 2014052560 W GB2014052560 W GB 2014052560W WO 2015025165 A1 WO2015025165 A1 WO 2015025165A1
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csfv
peptide
cells
cell
ifn
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Simon Graham
Giulia FRANZONI
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UK Secretary of State for Environment Food and Rural Affairs
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UK Secretary of State for Environment Food and Rural Affairs
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A61K39/187—Hog cholera virus
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to peptides for use in the vaccination of animals against classical swine fever (CSF) and to the treatment thereof.
  • CSF classical swine fever
  • CSF Classical swine fever
  • CSFV classical swine fever virus
  • the disease is endemic in South East Asia, parts of Central and South America and the Russian Federation.
  • the virus continues to be an epizootic threat with recent outbreaks in Lithuania (2009 and 2011) and Lithuania (2012) [3].
  • CSF is amenable to control by vaccination and live attenuated C-strain vaccines are highly efficacious.
  • C-strain vaccine induced IFN- ⁇ responses have been correlated to rapid protection against the disease [5] and CSFV-specific IFN- ⁇ secreting CD8 T cells are detected in the blood early after vaccination [6]. Determining the viral proteins that are the targets of the CD8 T cell response in immune animals would provide an important step towards developing a next generation marker vaccine capable of providing rapid protection against CSFV.
  • CSFV has four structural proteins (the core protein and the envelope glycoproteins Erns, El and E2) and eight non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) [1].
  • NS3 have been described as targets of the T cell response and both proteins induce IFN- ⁇ release [6-9] and cytotoxic activity by T cells from vaccinated pigs [7-10].
  • a T cell epitope was identified on NS4 [9] and our group recently reported NS5B as a putative target of IFN- ⁇ secreting T cells from C-strain vaccinated pigs [6].
  • Epitopes may be located on other viral proteins, since peptides pooled to represent Erns, El, NS2, NS4B and NS5A were able to induce PBMC proliferation in vaccinated pigs, but their ability to elicit an IFN- ⁇ or cytotoxic response was not tested [9].
  • Most of these studies utilised inbred homozygous pigs, so were focussed on a single haplotype [7,9, 10] and the phenotype of the responding T cells/MHC restriction was not or only partially characterized [6-10].
  • a chimeric vaccine CP7_E2alf where the E2 protein of the CSFV strain Alfort 187 is inserted in the backbone of the bovine viral diarrhoea virus (BVDV) strain CP7, can fully protect pigs from challenge before the appearance of neutralizing antibodies [12]. However, it remains to be determined whether CSFV E2 specific or BVDV cross-reactive antigen specific T cells are involved in mediating this protection.
  • BVDV bovine viral diarrhoea virus
  • HCV hepatitis C virus
  • DENV dengue virus
  • Both a HCV peptide-vaccine, including 5 MHC-class I and 3 MHC class-II-restricted epitopes, a DNA vaccine encoding NS3 and NS4 and an adenovirus-based vaccine expressing NS3 of HCV induce a strong CD8 T cell response in vaccinated individuals [13-15].
  • DNA vaccines based on the NS3 protein from DENV and an adenovirus expressing DENV NS 1 induce a peptide-specific IFN- ⁇ response by CD8 T cells from vaccinated mice which correlated with protection [16, 17].
  • an isolated peptide comprising an epitope having an amino acid sequence comprising at least 8, 9, 10 or 11 contiguous amino acids taken from one of the following sequences:
  • a "variant" means a peptide in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids.
  • the variant is a functional variant, in that the functional characteristics of the peptide from which the variant is derived are maintained. For example, a similar immune response is elicited by exposure of an animal, or a sample from an animal, to the variant polypeptide.
  • any amino acid substitutions, additions or deletions preferably do not alter or significantly alter the tertiary structure of one or more epitopes contained within the peptide from which the variant is derived.
  • the skilled person is readily able to determine appropriate functional variants and to determine the tertiary structure of an epitope and any alterations thereof, without the application of inventive skill.
  • Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.
  • conservative substitution is meant the substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows:
  • Uncharged polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin
  • Acidic Asp
  • Glu Basic Lys, Arg, His.
  • variants of the 1 lmer VEYSFIFLDEY which contain conservative substitutions include include VEYSTIFLDEY and VEYSFITLDEY.
  • altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation.
  • non-conservative substitutions are possible provided that these do not disrupt the tertiary structure of an epitope within the peptide, for example, which do not interrupt the immunogenicity (for example, the antigenicity) of the peptide.
  • variants may be at least 85% identical, 87.5% identical, for example at least 88% identical, at least 88.9% identical, at least 90% identical or at least 90.9% identical to the base sequence.
  • epitope refers to the amino acids (typically 8-11 amino acids) within a peptide sequence which are essential in the generation of an immune response.
  • the epitopes referred herein immune response are those which can be detected by means of a cell- mediated immunity (CMI) assay and are recognisable by a T cell by binding of a T cell receptor to the epitope presented by an MHC class I molecule.
  • CMI cell- mediated immunity
  • the epitope may comprise consecutive amino acids, or the amino acids forming the epitope may be spaced apart from one another. In the latter case, the nature of the amino acids between the amino acids forming the epitope may not be crucial to the activity and may be varied, provided that the tertiary structure of the epitope is maintained, for example so that an immune response such as a cell-mediated immune response can occur in response to the presence of the epitope. Determination of the amino acids which form an epitope or part of an epitope can be undertaken using routine methods. For example, one of a series of small mutations such as point mutations may be made to a peptide and the mutated peptide assayed to determine whether the immunogenic or diagnostic activity has been retained. Where it has, then the variant retains the epitope. If activity has been lost, then the mutation has disrupted the epitope and so must be reversed.
  • the peptide or fragment may comprise or consist of the epitope.
  • the peptide may, therefore, be the same length, shorter or longer than the epitope.
  • the peptide may be at least 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 25, 30 or 35 amino acids in length.
  • the peptide or fragment is 9 amino acids in length.
  • the peptide or fragment may have substantial homology to the following sequence: RDNALLKF or to RVDNALLKF.
  • the peptide or fragment is longer than 9 amino acids in length, but comprises an amino acid sequence having substantial homology to or consisting of RDNALLKF or to RVDNALLKF.
  • the peptide or fragment may comprise or consist of an amino acid sequence having substantial homology to any of the sequences shown in Table A.
  • nucleic acid molecule encoding the peptide of the invention, a vector comprising such a nucleic acid molecule and a host cell comprising such a vector.
  • the host cell may be a cell other than a human embryonic stem cell.
  • variant in relation to a nucleic acid sequences means any substitution of, variation of, modification of, replacement of deletion of, or addition of one or more nucleic acid(s) from or to a polynucleotide sequence providing the resultant peptide sequence encoded by the polynucleotide exhibits at least the same properties as the peptide encoded by the basic sequence.
  • the properties to be conserved are the ability to form one or more epitopes such that an immune response is generated which is equivalent to that of the diagnostic reagent peptide or isolated peptide as defined herein.
  • allelic variants includes allelic variants and also includes a polynucleotide which substantially hybridises to the polynucleotide sequence of the present invention.
  • low stringency conditions can be defined a hybridisation in which the washing step takes place in a 0.330-0.825M NaCl buffer solution at a temperature of about 40-48°C below the calculated or actual melting temperature (T m ) of the probe sequence (for example, about ambient laboratory temperature to about 55°C), while high stringency conditions involve a wash in a 0.0165-0.0330M NaCl buffer solution at a temperature of about 5-10°C below the calculated or actual T m of the probe (for example, about 65°C).
  • T m melting temperature
  • the buffer solution may, for example, be SSC buffer (0.15M NaCl and 0.015M tri-sodium citrate), with the low stringency wash taking place in 3 x SSC buffer and the high stringency wash taking place in 0.1 x SSC buffer.
  • Nucleotide variants are provided in Table B, C, D, E and F..
  • Peptides may be prepared synthetically using conventional peptide synthesisers. Alternatively, they may be produced using recombinant DNA technology or isolated from natural sources followed by any chemical modification, if required. In these cases, a nucleic acid encoding the peptide is incorporated into suitable expression vector, which is then used to transform a suitable host cell, such as a prokaryotic cell such as E. coli. The transformed host cells are cultured and the peptide isolated therefrom. Vectors, cells and methods of this type form further aspects of the present invention. Sequence identity between nucleotide and amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid or base, then the molecules are identical at that position.
  • Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences.
  • optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences.
  • Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
  • Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include the Gap program (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453) and the FASTA program (Altschul et al, 1990, J. Mol. Biol. 215: 403-410). Gap and FASTA are available as part of the Accelrys GCG Package Version 11.1 (Accelrys, Cambridge, UK), formerly known as the GCG Wisconsin Package.
  • the peptide of the invention comprises a region that is antigenic, specifically a region that can provoke an antigenic or immune response to the CSFV.
  • the peptide may comprise more than one antigenic region and in that case, the second antigenic region may also provoke an immune response to CSFV, or may stimulate a different immune response.
  • the regions may direct or provoke an immune response to the same or different proteins from CSFV.
  • one region may relate to one of the envelope glycoproteins and the other to a different envelope glycoproteins or with a non- structural protein. Alternatively, they may both relate to a non- structural protein.
  • composition comprising a peptide or a nucleotide according to the invention.
  • the pharmaceutical composition may comprise one or more pharmaceutically or otherwise biologically active agents in addition to the peptide of the invention.
  • the composition may include another immunogenic agent or a therapeutic agent.
  • the pharmaceutical composition may include any appropriate carriers, adjuvants etc.
  • the pharmaceutical composition may contain the peptide in synthetic or recombinant form, along with one or more adjuvants.
  • the peptide may be delivered in a delivery system, such as a nanoparticle, or other particle in which the peptide may be encapsulated or otherwise delivered.
  • the pharmaceutical composition comprises a nucleotide it may be in the form of, for example, plasmid DNA, a vector, such as a viral vector or a combination of vectors
  • the pharmaceutical composition may, for example, be a vaccine.
  • it may be for administration by any appropriate route, such as orally or by injection, especially subcutaneous or intra muscular injection.
  • the peptide of the invention for use in therapy.
  • the therapy may be the treatment of or vaccination against classical swine fever.
  • the subjects may be any subject that may be infected with CSFV. In particular, it may be swine.
  • the invention will now be described in detail, by way of example only, with reference to the figures.
  • FIG. 1 Vaccination with C-strain and challenge with virulent CSFV induces predominately a virus-specific IFN- ⁇ CD8 T cell response.
  • Pigs were vaccinated on day -5 and then challenged with CSFV Brescia strain on days 0 and 28 post-challenge.
  • PBMC from vaccinated/challenged (V/C) and control animals stimulated with CSFV and IFN- ⁇ release measured by ELISpot assay.
  • Panel A shows the mean mock-virus stimulated corrected IFN- ⁇ spot-forming cells (SFC)/5xl0 5 PBMC for each group and error bars represent SEM.
  • SFC mock-virus stimulated corrected IFN- ⁇ spot-forming cells
  • Panel B shows the gating strategy used to interrogate responses in singlet, live CD8 T cells and memory CD4 T cells.
  • Plots in panel C shows the mean mock-virus stimulated corrected % IFN- ⁇ expressing CD4 T cells and CD8 T cells +/- SEM for 8 V/C animals and 3 control animals. Values of control and V/C groups were compared using a two-tailed un-paired t-test and significance is indicated by **p ⁇ 0.01, *p ⁇ 0.05.
  • CD8 T cells from pigs vaccinated with C-strain and challenged with virulent CSFV display distinct profiles of antigen reactivity.
  • PBMC from four selected pigs were stimulated with synthetic peptide pools representing the 12 CSFV proteins and IFN- ⁇ expression was assessed by flow cytometry.
  • Graphs show the mean unstimulated corrected % ⁇ FN-y + CD8 T cells for each animal in response to peptide pools +/- SEM.
  • Statistical analyses were performed using a one-way ANOVA followed by a Dunnett's multiple comparison test versus the unstimulated cells; ***p ⁇ 0.001, **p ⁇ 0.01.
  • FIG. 3 Identification of putative antigenic peptides recognised by CSFV specific CD8 T cells. Twenty one days after re-challenge, PBMC from pigs AN5, AN7, AN11 and AN13 were stimulated with synthetic peptides pooled in a 2-way matrix for proteins E2 (pig AN11), NS2 (pig AN7), NS3 (pigs AN5 and AN11) and NS5A (pigs AN5 and AN13). Peptides representing the core protein were screened individually (pig AN13). IFN- ⁇ expression by CD8 T cells was assessed by flow cytometry as described above. The mean unstimulated corrected % IFN- ⁇ expressing CD8 T cells are presented and error bars represent SEM. Statistical analyses were performed using a one-way ANOVA followed by a Dunnett's multiple comparison test versus unstimulated cells; ***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.05.
  • FIG. 4 Identification of minimal length antigenic peptides recognised by CSFV specific CD8 T cells.
  • PBMC from pigs AN5, AN7, AN11 and AN13, collected at day 28 or cryopreserved at later time-points were stimulated with the identified 15mer or consensus l lmer antigenic peptides and the truncated derivatives of NS5A LSRVDNALLKF, NS2 LISTVTGIFLI and E2 RYYEPRDSYFQ and IFN- ⁇ expression by CD 8 T cells assessed by flow cytometry.
  • Panels A, B and C shows the mean unstimulated corrected % IFN- ⁇ expressing CD8 T cells +/- SEM.
  • Panels D-H show reactivity against a logio dilution series of the identified minimal length antigenic peptides (NS5A RVDNALLKF, NS2 STVTGIFL and E2 YEPRDSYF) or antigenic regions (core PESRKKLEKALLAWA and NS3 VEYSFIFLDEY).
  • FIG. 5 Recognition of the identified epitopes by CD8 T cells from C-strain vaccinated pigs challenged with divergent CSFV strains.
  • Statistical analyses were performed using a one-way ANOVA followed by a Dunnett's multiple comparison test versus the un-stimulated control; **p ⁇ 0.01, *p ⁇ 0.05.
  • Cryopreserved PBMC from pigs AN5, AN7, ANl l and AN13 were stimulated with the identified antigenic peptides and the phenotype of IFN- ⁇ expressing CD8 T cells determined by flow cytometry. Histograms shows representative dot plots of the expression of IFN- ⁇ versus CD 107a, CD25 and CD27 by singlet, live CD 8 T cells. The table reports the mean % expression of these markers by peptide-specific ⁇ FN-y + CD8 T cells from triplicate cultures +/- SEM.
  • FIG. 7 Characterization of polyfunctional cytokine expression by CSFV epitope- specific CD8 T cells.
  • Cryopreserved PBMC from pigs AN5, AN7, ANl l and AN13 were stimulated with the identified antigenic peptides and the expression of IFN- ⁇ , TNF-a and IL- 2 by CD8 T cells was simultaneously assessed by flow cytometry.
  • Panel A shows representative dot plots of the expression of TNF-a and IL-2 by peptide specific IFN- ⁇ expressing CD8 T cells and
  • Panel B the relative proportions of IFN- ⁇ secreting CD8 T cells expressing either of the two other cytokines.
  • MFI mean fluorescence intensity
  • Experiment 1 An experimental vaccination/challenge study was performed to assess the specificity of CSFV-specific CD8 T cell IFN- ⁇ responses. Eleven Large White/Landrace pigs, 6 months of age, were utilized; eight animals were vaccinated 5 days before challenge (day -5) by intramuscular inoculation of 10 5 TCID 50 of C-strain CSFV and challenged on day 0 and 28 by intranasal inoculation of 10 5 and 10 6 TCID 50 of CSFV Brescia, respectively (1 ml divided equally between each nostril and administered using a mucosal atomization device MAD-300, Wolfe Tory Medical, Salt Lake City, USA). Three negative control pigs received similar inoculations of mock virus supernatant on each occasion. Virus back titrations of the inocula confirmed the vaccine and challenge doses were those expected.
  • Experiment 2 A second vaccination/challenge study assessed recognition of identified T cell epitopes by additional C-strain vaccinated pigs. Animals vaccinated and challenged in two independent, previously described, experiments [5] were utilised. Large White/Landrace cross male pigs, 9 weeks of age, were vaccinated intramuscularly with 2 ml of reconstituted C-strain vaccine, as described by the manufacturer (Riemser Arzneiffen AG) and after 3 or 5 days were challenged by intranasal inoculation of 10 5 TCID 50 CSFV UK2000/7.1 or CBR/93 strains as described above.
  • EDTA blood was incubated with 5 ⁇ 1 of anti-porcine CD45-FITC monoclonal antibody (mAb) (O. lmg/ml, K252-1E4, AbD Serotec, Oxford, UK) for 10 minutes at room temperature (RT) in the dark.
  • mAb anti-porcine CD45-FITC monoclonal antibody
  • FACS Lysing solution 945 ⁇ 1 (BD Biosciences, Oxford, UK) was added for 10 minutes at RT to lyse erythrocytes and fix leukocytes.
  • PBMC peripheral blood mononuclear cells
  • PBMC Heparinised venous blood was collected on days -5, 0 and every 7 days until day 56 post- challenge for Experiment 1 and every three days from day -5 to 15 post-challenge for Experiment 2.
  • PBMC were prepared by diluting 20ml of blood in 10 ml of PBS (Life Technologies), layering over 20ml of Histopaque-1077 (Sigma- Aldrich, Poole, UK) and centrifuged at 1455 x g for 30 minutes at room temperature (RT), without braking, in a rotating bucket centrifuge.
  • PBMC were aspirated from the plasma- Histopaque-1077 interface and washed three times in PBS by centrifugation at 930 x g for 5 minutes at 4°C.
  • PBMC peripheral blood mononuclear cells
  • RPMI-1640 medium Life Technologies
  • FBS forward scatter
  • SSC side scatter
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • pre-cooled (4°C) labeled cryotubes were immediately transferred to pre-cooled (4°C) Cryo 1°C Freezing Container (Nalgene, Fisher Scientific, Loughborough, UK) pre-filled with 250 ml of 100% isopropyl alcohol, which was placed in a -80°C freezer for a minimum of 4 hours to a maximum of 24 hours. Cryotubes were then transferred to a liquid nitrogen storage container.
  • a synthetic overlapping peptide library was designed which comprised pentadecamer peptides off-set by four residues.
  • the peptide sequences were designed using the predicted polyprotein of CSFV C-strain Riems (GenBank accession number AY259122.1).
  • the synthesized library of 945 peptides JPT Peptide Technologies, Berlin, Germany) were reconstituted in sterile lOmM HEPES (Life Technologies) buffered 40% acetonitrile (Sigma- Aldrich) at a concentration of 2mg/ml.
  • peptides were combined into pools representing the structural and non- structural proteins of CSFV and diluted in cRPMI and used at a final total peptide concentration of 1 ⁇ g/ml, unless otherwise stated.
  • a two-way matrix system was adopted to screen peptides representing the non- structural proteins: NS5A, NS3, NS2 and the structural protein E2.
  • Matrix pools were designed so that each peptide was uniquely present in 2 different pools.
  • the 121 NS5A peptides were prepared in 22 peptide pools (A-V), the 110 NS2 peptides in 21 peptide pools (A-U), the 168 NS3 peptides 26 peptide pools (A-Z) and the 90 E2 peptides in 19 matrix peptide pools (A-S).
  • A-V 22 peptide pools
  • A-U the 110 NS2 peptides in 21 peptide pools
  • A-Z the 168 NS3 peptides 26 peptide pools
  • A-S the 90 E2 peptides in 19 matrix peptide pools
  • ELISpot plates (96 well Multiscreen-IP Filter Plates; Millipore, Watford, UK) were prepared by pre-wetting each well with 15ml of 35% ethanol for 1 min then washing 3 times with sterile PBS.
  • the capture antibody (anti-porcine IFN- ⁇ mAb, P2G10, BD Biosciences, Oxford, UK), prepared at 0.5mg/ml in PBS, was added at 50ml/well and the plates incubated at 4°C overnight. Capture antibody was then decanted and plates washed 3 times with unsupplemented RPMI-1640 medium. Plates were blocked by addition of lOOml/well cRPMI and incubation for at least lhr at 37°C.
  • Freshly isolated PBMC were suspended at 5xl0 6 /ml in cRPMI and ⁇ of cells was added to each well.
  • CSFV Alfort 187 strain was diluted in cRPMI and added at a multiplicity of infection (MOI) of 1.
  • Concanavalin A Sigma- Aldrich
  • mock-infected PK-15 cell cryolysate supernatant were used as positive and negative controls, respectively. All conditions were tested in triplicate and plates were incubated at 37°C in a 5% C0 2 humidified atmosphere for 18 hours. Well contents were discarded and 100ml of cold water was added to each well and incubated for 5 min.
  • BCIP/NBT substrate R&D Systems, Abingdon, UK, ⁇ /well
  • spots were visualized using an automated ELISpot reader (Autolmmun Diagnostika, Strassberg, Germany). Multi-parameter cytofluorometric analysis of PBMC responses
  • cryovials were rapidly thawed in a 37°C water bath and cells transferred to tubes containing 10ml of pre-warmed (37°C) cRPMI. Cells were washed by centrifugation, 930 x g for 5 minutes at RT, and resuspended in fresh warm cRPMI. Cell densities were calculated using flow cytometry as described above and adjusted to 1 x 10 6 cells/ml and ⁇ transferred to wells of a 96-well round bottom microtitre plate (Costar, Fisher Scientific).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Mock- virus PK15 supernatants/cRPMI medium and ConA (10 ⁇ g/ml) were used as negative and positive controls, respectively.
  • Cells were incubated at 37°C for 2 (peptide-stimulation) or 14 (virus-stimulation) hours and then brefeldin A (GolgiPlug, BD Biosciences) was added (0.2 ⁇ 1 ⁇ 11) and cells were further incubated for a further 16-18 or 6 hours following stimulation with peptide or CSFV, respectively.
  • CD107a- Alexa Fluor 647 or IgGl isotype control-Alexa Fluor 647 mAbs both AbDSerotec, Oxford, UK; ⁇ /well
  • monensin Golgi Stop, BD Biosciences; 0.2 ⁇ /well
  • mAbs used for cytokine staining were: IFN-y-FITC or -Alexa Fluor 647 (CC302, AbD Serotec), TNF-a-Pacific Blue (MAbl l, Biolegend, Cambridge Bioscience, Cambridge, UK) and IL-2 (A150D 3F1, Life Technologies), labelled using Zenon Alexa Fluor 647 mouse IgG2a labelling kit (Life Technologies). IgGl-FITC or -Alexa Fluor 647 isotype control mAbs were used to control staining with IFN- ⁇ mAbs. Un-stained cells were used as control for IL-2 and T F-a.
  • the cells were given two final washes in BD Perm/Wash buffer and re-suspended in FACS buffer prior to flow cytometric analysis on a MACSQuant Analyzer (Miltenyi Biotec) or CyAn ADP (Beckman Coulter, High Wycombe, UK) flow cytometers. Cells were analyzed by exclusion of doublets, followed by gating on viable cells (Live/Dead Fixable Dead Cell Stain negative) in the lymphocyte population, then defined lymphocyte subpopulations were then gated upon and their expression of cytokines assessed.
  • Gates were set using the corresponding isotype/unstained controls and values were corrected by subtraction of the % positive events in the biological negative control (cRPMI or mock-virus supernatant stimulated).
  • the number of singlet live lymphocytes acquired for analysis was approximately 400,000.
  • the sequences of the identified T cell antigenic regions/epitopes were aligned against the predicted full-length polyprotein sequences of 14 CSFV isolates (GenBank accession numbers shown in parentheses): Genotype 1.1 - Brescia (AF091661), Alfort/187 (X87939.1), KC Vaccine (AF099102), ALD (D49532), GPE " (D49533), Alfort-A19 (U90951), cF114 (AF333000), Shimen (AF092448), Koslov (HM237795) and SWH (DQ 127910); Genotype 2.1 - Penevezys (HQ148063); Genotype 2.3 - Borken (GU233731) and Alfort Tubingen (AAA43844); the BVDV reference strain NADL (AJ133738) and the border disease virus (BDV) reference strain BD31 (U70263), using the clustal W algorithm on MegAlign (DNAStar Lasergene 9 Core Suite,
  • sequences of the identified T cell antigenic regions/epitopes were also aligned against the corresponding sequences of the CSFV isolates UK2000/7.1 and CBR/93. These were generated using RNA from CSFV strains UK2000/7.1 and CBR/93 and creating cDNA by reverse transcription as previously described [21].
  • a 5 ⁇ 1 aliquot of cDNA was used as template for PCR amplification with high fidelity Platinum Taq in a reaction mix containing: ⁇ ⁇ dNTP (lOmM of each dNTP, Promega, Victoria, UK), 5 ⁇ 1 5x PCR buffer, 1.5 ⁇ 1 MgSC-4 (50 mM), 0.2 ⁇ 1 Platinum Taq (5U/ml) (all Life Technologies) and 2 ⁇ 1 (20 ⁇ ) of each primer (Sigma- Aldrich).
  • the following primers (5 '-3') were used to amplify the selected regions: Core-F-954 AGAGCATGAGAAGGACAGYA and Core-R-1211 GTGCCRTTGTCACTYAGGTT, E2-F-2397 GTGCAAGGTGTGRTATGGC and E2-R- 3613 GTGTGGGTRATTAAGTTCCCTA, NS2-F-3840 TAGTAGTCGYYGTGATGTTR and NS2-R-4248 GCCCACATCGTAAAMACCA, NS3-F-5974
  • Pigs used in this study were genotyped for their swine leukocyte antigen (SLA) class I haplotypes by low-resolution PCR screening assays (PCR-SSP) on PBMC-derived genomic DNA as previously described [23].
  • SLA swine leukocyte antigen
  • PCR-SSP low-resolution PCR screening assays
  • PBMC from the four selected V/C pigs were stimulated in vitro with pools of overlapping 15mer peptides representing the 12 CSFV proteins and CD8 T cell reactivity was screened using IFN- ⁇ detection by flow cytometry (Fig. 2). Significant IFN- ⁇ responses were observed in all V/C animals and each animal reacted against a unique profile of antigens.
  • Pig AN5 mounted a significant IFN- ⁇ response against NS3 and NS5A peptides, pig AN7 reacted significantly against the NS2 peptide pool, pig AN11 responded to peptides representing the E2 and NS3 proteins, and pig AN13 mounted the greatest response to the core peptides with significant reactivity also observed against NS5A.
  • a two way matrix system was adopted to screen peptides representing the non- structural proteins NS5A, NS3, NS2 and the structural protein E2, whereas the peptides spanning the core protein were screened individually.
  • the matrix pools were designed so that each peptide was uniquely present in 2 defined pools.
  • PBMC from pigs AN5 and AN13 were stimulated in vitro with the NS5A matrix peptide pools and ⁇ FN-y + CD8 T cells were identified using flow cytometry. Pools C, K, N and Q induced a significant IFN- ⁇ response in CD8 T cells from pig AN5 (Fig. 3).
  • peptides #25, 33, 58 and 66 By analysing the peptide constituents of the reacting matrix pools, four potential antigenic 15mers were identified for further testing: peptides #25, 33, 58 and 66.
  • Pig AN13 did not mount a significant CD8 T cell IFN- ⁇ response to any of the NS5A matrix pools.
  • PBMC from pig AN7 were stimulated with the NS2 matrix peptide pools and a CD8 T cell IFN- ⁇ response was observed against pools B, I, O, P (Fig. 3) identifying 4 putative antigenic peptides: #35 42, 46 and 53.
  • PBMC from pigs AN5 and AN11 were stimulated in vitro with the NS3 matrix peptide pools and peptides present in the pools D, F, G, X, Y and Z induced a statistically significant CD8 T cell IFN- ⁇ response, which indicated that peptides #134, 136, 137, 147, 149, 150, 160, 162 and 163 were potentially antigenic.
  • Pig AN5 did not mount a significant response to the NS3 matrix pools.
  • E2 matrix peptide pools were screened with PBMC from pig AN11 leading to the identification of significant reactivity against pools E, G, O and P; suggesting reactivity against peptides 45, 47, 55 and 57.
  • the identified putative antigenic peptides were next screened individually to assess their recognition by CD8 T cells.
  • PBMC from pig AN5 were found to show significant reactivity to overlapping NS5A peptides 33 and 58, but not 25 and 66, suggesting that at least one epitope lay in the l lmer consensus region LSRVDNALLKF.
  • the inventors observed that the overlapping peptides 42 and 46, with the consensus sequence LISTVTGIFLI, but not 35 and 53, induced a statistically significant greater number of IFN- ⁇ expressing CD8 T cells from pig AN7 compared to un-stimulated controls.
  • PBMC from pig ANl l showed significant reactivity against two pairs of overlapping peptides, E2 peptides 47 and 55 and NS3 peptides 134 and 163 with l lmer consensus sequences of RYYEPRDSYFQ and VEYSFIFLDEY, respectively. Due to its short length, peptides spanning the core protein were screened individually using PBMC from pig AN13. Significant CD8 T cell IFN- ⁇ responses were observed against the 15mer peptide #20 (PE SRKKLEK ALL AW A) (Fig. 3).
  • the length of CD8 T cell epitopes can vary between 8 and 11 amino acids.
  • CD8 T cell epitopes are well conserved among CSFV strains Using the Clustal W protein alignment tool, the inventors investigated the conservation of the identified CD8 T cell epitopes/antigenic regions among different CSFV strains (Table 1). We observed that the antigenic region PESRKKLEKALLAWA, located on core protein, showed only one amino acid substitution in the genotype 3.3 strain CBR/93. The NS3 l lmer VEYSFIFLDEY displayed one amino acid substitution in the genotype 2.1 strain Penevezys strain and in CBR/93. The E2 epitope YEPRDSYF was 100% conserved across all the CSFV strain analysed.
  • the NS2 epitope STVTGIFL was conserved in all the CSFV strains analysed except the Penevezys strain, where there was a single amino acid substitution.
  • the NS5A epitope RVDNALLKF was conserved among all genotype 1.1 strains tested except Shimen, where a single amino acid substitution was observed. A different single amino acid substitution was observed in the genotype 2 strains and 2-3 substitutions were observed in the two genotype 3 strains.
  • the antigenic regions/epitopes on core, NS2 or E2 showed no or single amino acid substitutions 2-3 substitutions were observed in the NS2 epitope and in the NS5A epitope was poorly conserved with only three amino acids being shared.
  • the porcine swine leukocyte antigen (SLA) class I (SLA-1, SLA-2 and SLA-3) haplotypes of the four pigs were determined using a PCR-SSP-based typing assay. Each animal was heterozygous and no two haplotypes were shared between these animals (Table 2).
  • SLA porcine swine leukocyte antigen
  • SLA-1, SLA-2 and SLA-3 haplotypes of the four pigs were determined using a PCR-SSP-based typing assay. Each animal was heterozygous and no two haplotypes were shared between these animals (Table 2).
  • PBMC collected during Experiment 2 were assayed.
  • Four C-strain vaccinated/UK2000/7.1 challenged pigs (AD53, AD56, AD62 and AD65) and two C-strain vaccinated/CBR/93 challenged pigs (AE15 and AE17) reacted against the NS2 8mer STVTGIFL, with a significantly greater number of IFN-D expressing CD8 T cells compared to unstimulated cells (Fig. 5).
  • the ability of the five identified antigenic peptides to elicit cytotoxic activity was investigated by assessment of surface mobilisation of CD 107a, a marker of degranulation, by IFN-Y + CD 8 T cells. Over 90% of IFN-y + CD 8 T cells expressed CD 107a on their surface after peptide stimulation, suggesting cytotoxic activity of these cells against the peptide- presenting cells (Fig. 6). With the aim to further characterize the epitope-specific CD8 T cell populations, the expression of the activation markers CD25 and CD27 on IFN-y + CD8 T cells were investigated using flow cytometry. Interestingly, the majority of peptide-specific IFN-Y + CD8 were CD25 " and CD27 + (Fig. 6).
  • the CSFV polyprotein was screened for binding to potential restricting alleles (based on the SLA class I Lr typing results) and two of the peptides NS2i223-i23o and NS31902-1912 were predicted by NetMHCpan (www.cbs.dtu.dk/services/NetMHCpan/) to bind strongly to at least one of the class I alleles potentially present in each of the restricting haplotypes: SLA-2* 12.01 for NS2 1223-1230 (potentially present in both haplotypes Lr-38.0 and Lr-22.0) and NS3i 90 2-i9i2 was predicted to strongly bind SLA-*08.01 (Lr-07-0), SLA-3*07.01 (Lr-28.0);and SLA-2 01.01 and SLA-2 01.02 (Lr-01.0) (data not shown).
  • Immunodominance in CD8 T cell responses is thought to arise primarily as a consequence of the limitations of peptides to bind with high-affinity to available MHC class I molecules, with additional limitations in antigen processing and the CD8 T cell receptor repertoire also playing a role. Only approximately 1/2000 of the peptides within an antigen can achieve immunodominant status with a given MHC class I allele [28]. Immunodominance is thought to be critical for immunity since numerically prominent CD8 T cells have been shown to confer more effective protection than T cells specific for subdominant epitopes. It has also been shown that the efficacy of peptides in providing protection against a viral challenge is proportional to their binding affinity for the restricting MHC class I molecule [29].
  • CD8 T cell responses may limit virus replication and they are the primary targets of escape mutations [30, 31].
  • Immunodominance of CD8 T cell responses to Flaviviruses has also been described, with disparity in the degree depending on the virus/host system studied [32, 33].
  • Immunodominance may also be a key factor in determining the strain specificity of immunity if directed against polymorphic epitopes.
  • the identified epitopes in this study were well conserved amongst CSFV isolates, although for most of these variants it remains to be determined whether these substitutions could affect T cell recognition.
  • NetMHCpan predicted that the mutation of tyrosine in place of a phenylalanine at position 1906 of the NS3 l lmer VEYSFIFLDEY would not affect the binding to SLA class I alleles and we were able to show this mutation did not affect T cell reactivity.
  • the antigenic core 24 i-255 peptide and the E2 9 9 6 -ioo3 and NS31902-1912 epitopes are well conserved between CSFV, BVDV and BDV.
  • Such conserved T cell epitopes could enhance the efficacy of a BVDV/CSFV chimeric vaccine.
  • the chimeric vaccine CP7_E2alf induces rapid protection, comparable to the C-strain vaccine [12, 34]. Since this protection precedes the appearance of neutralizing antibodies, it may be that T cell responses against epitopes conserved between BVDV and CSFV are contributing to the protective effect.
  • E2 and NS3 are not necessarily the major T cell antigens and other CSFV proteins may be important targets of the CD8 T cell response.
  • E2 and NS3 are not necessarily the major T cell antigens and other CSFV proteins may be important targets of the CD8 T cell response.
  • These results may not limit the design of a marker vaccine based on only one or two immunodominant antigens.
  • a study on the related yellow fever virus showed that, in case of abrogation of the dominant CD8 T cell epitope, the frequencies of T cells recognizing the subdominant CD8 T cell epitope increased dramatically [33].
  • Translocation of lysosomal-associated membrane protein 1 (LAMP 1 /CD 107a) to the cell membrane has been validated as a marker of cytotoxic degranulation by CD8 T and K cells [38].
  • LAMP 1 /CD 107a lysosomal-associated membrane protein 1
  • the data support previous studies, which report the ability of the viral antigens E2 and NS3 to elicit cytotoxic activity in vaccinated pigs [7- 10] and suggest that these T cells possessed cytotoxic activity in addition to cytokine release.
  • the inventors also assessed the polyfunctionality of the peptide-specific CD8 T cell populations, by assessing co-expression of IFN- ⁇ , T F-a and IL-2, since this may be central to their protective capacity.
  • Studies on HCV have shown that vaccination with vectors expressing NS3 and NS4a proteins induce specific polyfunctional CD8 T cells which are associated with protection [14, 15].
  • the majority of peptide-specific CD8 T cells expressed IFN- ⁇ alone or IFN- ⁇ and T F- ⁇ , with only a small percentage co-expressing IFN- ⁇ , T F- ⁇ , and IL-2.
  • the subset expressing all three cytokines showed the highest 'quality' of response producing more IFN- ⁇ on a per cell basis.
  • T EM T effector memory
  • CD8 T cell responses of individual animals were uniquely focussed on only one or two epitopes which were mapped on the core, E2 and non- structural proteins NS2, NS3 and NS5A.
  • the individual responses were associated with the expression of distinct MHC class I haplotypes, and for two of the peptides there was evidence that they are presented by alleles present in other haplotypes.
  • the five identified antigenic peptides were highly conserved across CSFV isolates, and for some were also well conserved when aligned against the other pestiviruses.
  • the responding CD8 T cells displayed evidence of cytotoxic function, with the majority of IFN-y + cells co-expressing the cytotoxicity marker CD 107a and populations also releasing T F- ⁇ and/or IL-2.
  • the antigens and epitopes identified and characterised in this study are therefore useful in the preparation of vaccines and treatments for CSFV.
  • Essler SE Ertl W, Deutsch J, Ruetgen BC, Groiss S, Stadler M, Wysoudil B, Gerner W, Ho CS, Saalmueller A: Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs. Anim Genet 2013, 44:202-205.
  • Lunney JK, Ho CS, Wysocki M, Smith DM Molecular genetics of the swine major histocompatibility complex, the SLA complex. Dev Comp Immunol 2009, 33:362- 374.
  • Table A Conservation of identified CD8 T cell epitopes/antigenic regions among different CSFV isolates and the related pestiviruses, bovine viral diarrhoea (BVDV) and border disease virus (BDV).
  • BVDV bovine viral diarrhoea
  • BDV border disease virus
  • Table B Conservation of nucleotide sequences encoding the identified CD8 T cell antigenic region on core protein among different CSFV isolates and the rel pestiviruses, bovine viral diarrhoea virus (BVDV) and border disease virus (BDV).
  • BVDV bovine viral diarrhoea virus
  • BDV border disease virus
  • Table C Conservation of nucleotide sequences encoding the identified CD8 T cell epitope on NS2 protein among different CSFV isolates and the rel pestiviruses, bovine viral diarrhoea virus (BVDV) and border disease virus (BDV).
  • BVDV bovine viral diarrhoea virus
  • BDV border disease virus
  • Table D Consen ation of nucleotide sequences encoding the identified CD8 T cell epitope on E2 protein among different CSFV isolates and the related pesti bovine viral diarrhoea virus (BVDV) and border disease virus (BDV).
  • BVDV pesti bovine viral diarrhoea virus
  • BDV border disease virus
  • BDV BD31 1 a U70263 A . . . . A . . . . .
  • Table E Conservation of nucleotide sequences encoding the identified CD8 T antigenic region on NS3 protein among different CSFV isolates and the rel pestiviruses, bovine viral diarrhoea virus (BVDV) and border disease virus (BDV).
  • BVDV bovine viral diarrhoea virus
  • BDV border disease virus
  • Table F Conservation of nucleotide sequences encoding the identified CD8 T cell epitope on NS5A protein among different CSFV isolates and the rel pestiviruses, bovine viral diarrhoea virus (BVDV) and border disease virus (BDV).
  • BVDV bovine viral diarrhoea virus
  • BDV border disease virus
  • BDV BD31 1 a U70263 GA .. G ... C . A . ACT .. C .. G . G . A .

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Abstract

L'invention concerne des peptides pour une utilisation dans la vaccination d'animaux contre la peste porcine classique (PPC) et pour le traitement de ceux-ci. Elle concerne également des nucléotides codant pour ces peptides, des compositions pharmaceutiques et des méthodes de traitement ou de vaccination.
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WO2016176624A3 (fr) * 2015-04-30 2016-12-15 Kansas State University Research Foundation Pestivirus porcin, vaccins et dosages
CN111925416A (zh) * 2020-08-26 2020-11-13 中国农业科学院兰州兽医研究所 一类促进猪机体产生广谱获得性免疫应答的多肽及其应用
CN112209992A (zh) * 2020-08-26 2021-01-12 中国农业科学院兰州兽医研究所 一类促进猪机体产生非洲猪瘟病毒抗原特异性免疫应答的多肽及其应用

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US20120258127A1 (en) * 2010-09-10 2012-10-11 Graham Simon Paul Vaccine peptides

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WO2016176624A3 (fr) * 2015-04-30 2016-12-15 Kansas State University Research Foundation Pestivirus porcin, vaccins et dosages
US10555995B2 (en) 2015-04-30 2020-02-11 Kansas State University Research Foundation Porcine pestvirus, vaccines, and assays
CN111925416A (zh) * 2020-08-26 2020-11-13 中国农业科学院兰州兽医研究所 一类促进猪机体产生广谱获得性免疫应答的多肽及其应用
CN112209992A (zh) * 2020-08-26 2021-01-12 中国农业科学院兰州兽医研究所 一类促进猪机体产生非洲猪瘟病毒抗原特异性免疫应答的多肽及其应用
CN111925416B (zh) * 2020-08-26 2021-12-17 中国农业科学院兰州兽医研究所 一类促进猪机体产生广谱获得性免疫应答的多肽及其应用

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