MUTANT PORCINE EPIZOOTIC DIARRHEA VIRUS FOR USE IN A VACCINE

Abstract

The invention relates to a mutant Porcine Epizootic Diarrhea Virus (PEDVdN), to methods of producing said PEDVdN, and to compositions comprising the PEDVdN. The invention further relates to methods of stimulating an immune response in a pig comprising administering the PEDVdN, to vaccines comprising the PEDVdN, and to methods for preventing or ameliorating porcine epizootic diarrhoea in a pig.

Claims

1. A mutant Porcine Epizootic Diarrhea Virus (PEDVdN), comprising a deletion of three or more amino acids residues within a region corresponding to a region from about amino acid residue 19 to about amino acid residue 233 of SEQ ID NO:1.

2. The PEDVdN of claim 1, which functionally expresses a C-terminal domain of the S1 subunit of spike protein.

3. The PEDVdN of claim 1, wherein the deletion encompasses amino acid residues 194-196, having the amino acid sequence NKR, of SEQ ID NO:1, or corresponding amino acid residues in other PEDV sequences.

4. The PEDVdN of claim 1, wherein all amino acid residues are deleted within the region corresponding to the region from about amino acid residue 19 to about amino acid residue 233 of SEQ ID NO:1.

5. The PEDVdN of claim 1, wherein the viral genomic sequences are of a virulent PEDV.

6. The PEDVdN of claim 1, wherein the virus is attenuated by a deletion in ORF3, preferably a deletion of ORF3, and/or by a rearranged gene order.

7. The PEDVdN of claim 1, further comprising a marker gene.

8. The PEDVdN of claim 1, which is a live, infectious virus.

9. A cell comprising the PEDVdN of claim 1.

10. A method of producing PEDVdN of claim 1, comprising providing a cell with a RNA molecule encoding the PEDVdN.

11. A composition comprising the PEDVdN of claim 1 and a pharmacologically and/or veterinary acceptable carrier.

12. A method of stimulating an immune response in a pig comprising administering the composition of claim 11 to the pig in an amount effective to induce an immune response.

13. A vaccine, preferably a DIVA vaccine, comprising an effective immunizing amount of the composition of claim 11.

14. A method for preventing or ameliorating porcine epizootic diarrhea in a pig, comprising administering the PEDVdN according to claim 1 to the pig, preferably wherein the pig is a pregnant sow.

Description

FIGURE LEGENDS

[0053] FIG. 1. Spike protein sequences of the PEDV-Sca_flag and PEDV-ScaΔN_flag viruses.

[0054] 1A: Sca_flag protein sequence. The 18 most N-terminal amino acids are in bold, while the N-terminal amino acids A-A-N-V-F of the C-terminal part of the S1 subunit are underlined, the Flag-tag containing sequence is shown in italics.

[0055] 1B: ScaΔN flag protein sequence. The 18 most N-terminal amino acids are in bold, while the N-terminal amino acids A-A-N-V-F of the C-terminal part of the S1 subunit are underlined, the Flag-tag containing sequence is shown in italics.

[0056] FIG. 2. Spike nucleotide sequences of the PEDV-Sca_flag and PEDV-ScaΔN_flag viruses.

[0057] 2A: Sca_flag_nucleotide_sequence. The sequence encoding the 18 most N-terminal amino acids is in bold, while that of the N-terminal amino acids A-A-N-V-F of the C-terminal part of the S1 subunit is underlined, the Flag-tag containing sequence is shown in italics.

[0058] 2B: ScaΔN_flag_nucleotide_sequence. The sequence encoding the 18 most N-terminal amino acids are in bold, while that of the N-terminal amino acids A-A-N-V-F of the C-terminal part of the S1 subunit are underlined, the Flag-tag containing sequence is shown in italics.

[0059] FIG. 3. Nucleotide sequence of part of the PEDV-ScaΔN_flag virus genome covering the Spike N-domain deletion.

[0060] PEDV-ScaΔN_flag_nucleotide_sequence. The sequence encoding the 18 most N-terminal amino acids of the Spike protein are in bold, while that of the N-terminal amino acids A-A-N-V-F of the C-terminal part of the S1 subunit are underlined.

[0061] FIG. 4. Outline of spike protein and of ΔN spike protein. Potential glycosylation sites are indicated (Ψ).

[0062] FIG. 5. Recombinant PEDV carrying S lacking N-domain is viable. Fluorescence images of cells infected with the GFP-expressing recombinant PEDV-Sca_flag and PEDV-ScaΔN_flag virus.

[0063] FIG. 6. Recombinant PEDV carrying S lacking N-domain is viable. Comparative growth curve of PEDV-Sca_flag and PEDV-ScaΔN_flag viruses. Vero cells were inoculated with the indicated viruses. The tissue culture infective dose that produces a pathological change in 50% of cell cultures inoculated (TCID50) was determined in the culture medium at the indicated times.

[0064] FIG. 7: Western blot analysis of the S proteins on the PEDV-Sca_flag and PEDV-ScaΔN_flag recombinant viruses. Semi-purified virions were subjected to Western blotting and S proteins were detected by ECL chemiluminescence imaging using an antibody directed against the C-terminally appended flag-tag. Position and size (in kDa) of the protein molecular weight markers are indicated on the left.

EXAMPLES

Example 1

Construction of Recombinant Viruses

[0065] The transfer vector for the PEDV-ScaΔN_flag virus was a derivative of the previously described PEDV-Sca_flag transfer vector, the latter containing i) a BamHI restriction site between ORF1b and the S gene, ii) a gene encoding the green fluorescent protein as a replacement of the ORFS gene and iii) a FLAG peptide (DYKDDDDK)-encoding gene fragment as a C-terminal extension of the S gene (Li et al., 2013. PlosOne 8: e6997).

[0066] The PEDV-ScaΔN_flag transfer vector with the S gene lacking the sequence encoding the S-N-domain (residues 19-233, FIGS. 1, 2, 3 and 4) was constructed by in-fusion cloning using primer pair 8206 and 8207 (5′-GCTGCCAATGTATTTGCC-3′ and 5′-GCTAAGTGTTAGAAGTACTG-3′, resp.), using the PEDV-Sca_flag transfer vector as a template.

[0067] The PEDV-ScaΔN_flag virus was generated as described by Li et al. (Li et al., 2013. PlosOne 8: e6997) and Wicht et al (Wicht et al. JVI 2014). Viral RNA was extracted from the PEDV-ScaΔN_flag virus at two to three passages after plaque selection, and the genotype was confirmed by sequencing using primer 5109 (5′- GACGGCAACACCATGCATGCC-3′).

[0068] Vero cells were transiently transfected with expression plasmids encoding the PEDV-Sca_flag and PEDV-ScaΔN_flag protein for 48 h. Cells were treated with trypsin or soybean trypsin inhibitor (SBTI) for 1 h and subsequently examined by immunostaining against S protein. As is shown in FIG. 5, PEDV-ScaΔN_flag protein has the capacity to mediate cell-cell fusion upon exposure to trypsin.

Example 2

[0069] Vero cells were inoculated for 1 hour at an MOI of 0.1 with recombinant strain DR13-based PEDV in which the ORFS gene had been replaced by a GFP gene and which carried a Flag-tagged S protein (rPEDV-SDR13_Flag-dORF3/GFP) (see Li et al., 2013. PlosOne 8: e6997). In the virus rPEDV-SDR13-dN_Flag-dORF3/GFP, the N-domain of the S protein had been deleted.

[0070] Small aliquots were taken from the culture medium every 12 hours and the infectivity released from the cells into the medium was determined by titration (TCID50) of the virus in these samples on Vero cells. As is indicated in FIG. 6, the growth curve of rPEDV-SDR13-dN_Flag-dORF3/GFP is comparable to the growth curve of rPEDV-SDR13_Flag-dORF3/GFP and both viruses grow to high titers.

Example 3

Material and Methods

[0071] Vero cells were inoculated for 1 hour at an MOI of 1 with PEDV-Sca_flag and PEDV-ScaΔN_flag. At 24 h post infection, virus containing cell culture supernatants were collected and virus particles in the supernatant were concentrated by sedimentation through a 20% cushion of sucrose in HCN buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2) at 100,000×g for 3 h at 4° C. A similar purification procedure was done with culture medium from mock infected Vero cells (Mock). Virus particles were handled on ice and resuspended in HCN buffer. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous gel with 8% acryl amide in the separating gel. Next, samples were transferred to a polyvinylidene fluoride membrane (BioRad, 162-0176) and blocked with bovine serum. The flag-tagged PEDV S proteins were detected using a mouse monoclonal anti-FLAG conjugated to horseradish peroxidase (Sigma, A8592) with a swine anti-rabbit immunoglobulin G conjugated horseradish peroxidase (Dako, P0217) as a secondary antibody. Protein bands were visualized by Chemiluminescent autoradiography using the Amersham ECL Western Blotting Analysis System (GE healthcare, RPN2109) in combination with X-Omat LS films (Kodak, Sigma F1149).

Results

[0072] The S proteins of PEDV-Sca_flag and PEDV-ScaΔN_flag recombinant viruses were analysed biochemically by Western Blotting using an antibody that recognizes the C-terminal flag-tag. The results indicate a faster gel mobility of the S protein of the PEDV-ScaΔN_flag virus compared to that of the PEDV-Sca_flag virus, which is consistent with the deletion of most of the N-domain of the spike protein.