PESTIVIRUS MARKER VACCINE

Abstract

The present invention provides a mutant Pestivirus comprising a chimeric Erns gene, which provides the mutant Pestivirus with the capacity to evade serologic detection, but maintains good vaccine properties, and viral replication.

Claims

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21. (canceled)

22. A mutant Pestivirus comprising a genome wherein an Erns gene is mutated, wherein said mutated Erns gene is a chimeric Erns gene, wherein said chimeric Erns gene comprises a 5 part and a 3 part, wherein said 5 part comprises 60-95% of the chimeric Erns gene, wherein said 3 part comprises the remainder of the chimeric Erns gene, wherein said 5 part comprises the corresponding part of an Erns gene from a Pestivirus that is genetically distant from the mutant Pestivirus, and wherein said 3 part comprises the corresponding part of an Erns gene from a Pestivirus that is genetically close to the mutant Pestivirus.

23. The mutant Pestivirus of claim 22, wherein the mutant Pestivirus is a Pestivirus selected from the group consisting of: bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and border disease virus.

24. The mutant Pestivirus of claim 22, wherein the Erns gene from a Pestivirus that is genetically distant from the mutant Pestivirus, is an Erns gene from a Pestivirus selected from the group consisting of: Antelope Pestivirus, Bungowannah virus, Norway rat Pestivirus, atypical porcine Pestivirus (APPV), and Rhinolophus affinis Pestivirus.

25. The mutant Pestivirus of claim 22, wherein the Erns gene from a Pestivirus that is genetically close from the mutant Pestivirus, is an Erns gene from a Pestivirus selected from the group consisting of: bovine viral diarrhoea virus type 1 (BVDV-1), bovine viral diarrhoea virus type 2 (BVDV-2), CSFV, border disease virus, Reindeer Pestivirus, Giraffe Pestivirus, and HoBi Pestivirus.

26. The mutant Pestivirus of claim 22, wherein the mutant Pestivirus is an attenuated Pestivirus.

27. The mutant Pestivirus of claim 26, wherein the attenuated Pestivirus comprises a genome wherein the Npro gene is mutated.

28. The mutant Pestivirus of claim 22, wherein the mutant Pestivirus is a bovine viral diarrhoea virus (BVDV), wherein the BVDV is of the cytopathogenic biotype.

29. A method for the construction of the mutant Pestivirus of claim 22, wherein said method comprises mutating the Erns gene in a Pestivirus genome into a chimeric Erns gene as defined in claim 22.

30. A host cell comprising the mutant Pestivirus of claim 22.

31. A host cell constructed by the method of claim 29.

32. A vaccine for animals comprising the mutant Pestivirus according to claim 22 and a pharmaceutically acceptable carrier.

33. A vaccine for animals comprising the host cell of claim 30 and a pharmaceutically acceptable carrier.

34. A vaccine for animals comprising the host cell of claim 31 and a pharmaceutically acceptable carrier.

35. A method for the preparation of a vaccine for animals comprising the mutant Pestivirus according to claim 22 and a pharmaceutically acceptable carrier, wherein said method comprises: a. infecting a culture of host cells with said mutant Pestivirus of claim 22, b. incubating said infected culture of host cells, c. harvesting said culture of host cells or a part thereof, and d. admixing the culture of host cells or the part thereof, with a pharmaceutically acceptable carrier.

36. The mutant Pestivirus of claim 22 for use in a vaccine for animals.

37. The host cell of claim 30 for use in a vaccine for animals.

38. The host cell of claim 31 for use in a vaccine for animals.

39. A method for the prevention or reduction of an infection by a Pestivirus or of associated signs of disease in animals, wherein said method comprises administering the vaccine of claim 32 to said animals.

40. A method of vaccinating animals for the prevention or reduction of an infection by a Pestivirus or of associated signs of disease, wherein said method comprises administering the vaccine of claim 32 to said animals.

41. A method for differentiating animals vaccinated with the vaccine according to claim 32, from animals infected with a Pestivirus other than a mutant Pestivirus comprised in the vaccine, the method comprising the use of an antibody against an Erns protein, which antibody does not bind specifically with the chimeric Erns protein expressed by a mutant Pestivirus that is comprised in the vaccine.

42. A method of claim 41, wherein said antibody binds specifically with an Erns protein from a Pestivirus selected from the group consisting of: BVDV-1, BVDV-2, CSFV, and border disease virus.

43. A method for diagnosing an animal that was vaccinated with the vaccine according to claim 32, for an infection with a Pestivirus other than a mutant Pestivirus comprised in the vaccine, the method comprising the steps of: a. obtaining a sample from said vaccinated animal, and b. testing said sample for the presence of an antibody against a Pestivirus other than a mutant Pestivirus comprised in the vaccine, by using a mutant Pestivirus comprised in the vaccine, or a chimeric Erns protein as expressed by the mutant Pestivirus comprised in the vaccine, in an immuno-assay.

44. A diagnostic test kit comprising the mutant Pestivirus according to claim 22, or a chimeric Erns protein as expressed by the mutant Pestivirus.

45. A method for controlling an infection with a Pestivirus in a population of animals from the order of the Artiodactyla, comprising the use of the vaccine according to claim 32.

46. A method for controlling an infection with a Pestivirus in a population of animals from the order of the Artiodactyla, comprising the use of the diagnostic test kit according to claim 44.

Description

LEGEND TO THE FIGURES

[0324] FIG. 1: Multistep growth curve kinetics of BVDV-Ib_synth_N.sup.pro_E.sup.rns_Bungowannah (Cp7_Npro_Erns Bungo) clones 1 and 10, at P23, in comparison to recombinant parent viruses BVDV-Ib (Cp7) and BVDV-Ib_synth_N.sup.pro (Cp7_Npro). KOP-R cells were infected with an m.o.i. of 0.1.

[0325] FIG. 2: Results of serum-neutralisation assays following inactivated vaccinations. Numbers (352, 353, 365, and 321) are calf ear-tag numbers. Broad arrows indicate days of vaccination/booster.

[0326] FIG. 3: Immunofluorescence inhibition (IFI) assay. Median fluorescence intensity values were normalized to the control staining of uninfected cells (control WB210). Mean values and standard derivations of two experiments are shown. PC is a cattle serum immunized with BVDV-Id, and NC is a serum negative for BVDV-specific antibodies.

[0327] FIG. 4: Results of ELISA assays for BVDV-specific antibodies in cattle sera from inactivated-antigen vaccination trial.

[0328] Panel A: competition Elisa for BVDV Erns antibodies: in the presence of BVDV_Erns specific antibodies in cattle sera, the binding of WB210 (BVDV Erns specific antibody) to immobilized BVDV is inhibited and detected in the competition ELISA. Sera from cattle immunized with constructs carrying a Bungo-Erns were not able to block binding of BVDV Erns-antibody WB210.

[0329] Panel B: total anti-BVDV antibodies: BVDV total Ab indirect ELISA (Idexx) was used to detect total BVDV-specific antibodies in the cattle sera. Samples were considered positive if S/P values were greater than 0.3.

[0330] 0d=initial bleeding before first immunization; 49dpi=49 days post first immunization.

[0331] FIG. 5: Results of serum neutralization assays, to determine neutralizing antibodies against BVDV and against Bungowannah virus in sera of cattle vaccinated with live mutant Pestiviruses according to the invention: clone 1 clone 10. The broad arrows indicate the days of immunization.

[0332] FIG. 6: Detection of NS3-specific antibodies induced by live virus vaccination with live mutant Pestiviruses according to the invention: clone 1 and clone 10. Detection was by using the BVDV p80 antibody competition ELISA kit (IDvet) over time. The broad arrows indicate the days of immunization. Samples were considered positive if S/N % values were lower than 40%.

[0333] FIG. 7: Results of the detection of BVDV-specific antibodies in cattle sera at 49 days post vaccination with live mutant Pestiviruses according to the invention: clone 1 and clone 10.

[0334] Panel A: In the presence of BVDV Erns specific antibodies in cattle sera, the binding of moab WB210 (BVDV Erns specific) to immobilized BVDV is inhibited, as detected in a competition ELISA. Both clones 1 and 10 did not induce detectable BVDV Erns-specific antibodies, using moab WB210 as detector.

[0335] Panel B: Results of the BVDV total Ab ELISA, detecting total anti-BVDV-specific antibodies in the cattle sera. Samples were considered positive if S/P values were greater than 0.3. Control cattle sera: BVDV positive (PC), Bungowannah positive (PC Bungo), and BVDV negative (NC).