received by the International Bureau on 26 September 2016 (26.09.2016)

We claim:

1. A method, comprising:

incubating porcine epidemic diarrhea virus (PEDV) infected cells for an effective period of time to result in one or more replicated PEDV viral particles being released from the infected cells; isolating cells infected with PEDV away from cell-free PEDV virus particles to obtain isolated cells containing cell-associated PEDV proteins and antigens;

separating the PEDV proteins and antigens from the isolated cells; and

combining the separated PEDV proteins and antigens with an adjuvant to form an immunogenic composition comprising isolated PEDV proteins and antigens and the adjuvant.

2. The method of claim 1, wherein separating inactivates viral particles released from the infected cells.

3. The method of claim 1, wherein the adjuvant is selected to stimulate a mucosal antibody response.

4. The method of claim 3, wherein the adjuvant is selected for intranasal

administration.

5. The method of claim 3, wherein the adjuvant is selected for intravaginal

administration.

6. The method of claim 3, wherein the adjuvant adheres to the mucous membranes.

The method of claim 4, wherein the adjuvant comprises polyacrylic acid.

The method of claim 1 , wherein separating comprises extracting PEDV proteins and

9. The method of claim 1, wherein separating comprises eluting PEDV proteins and antigens.

10. The method of claim 1, wherein separating comprises a freeze-thaw cycle.

11. The method of claim 1, wherein separating PEDV proteins and antigens comprises breaking open, extracting, rupturing, freezing and thawing, lysing, centrifuging, filtering the cells, or any combination thereof to release the PEDV proteins and antigens from the cells. 12. The method of claim 1, further comprising adding an inactivating agent.

13. The method of claim 12, wherein the inactivating agent comprises binary ethyleneimine. 14. The method of claim 1, wherein separating the PEDV proteins and antigens from the isolated cells comprises contacting the isolated cells with a detergent.

15. The method of claim 14, wherein contacting forms inactivated viral particles. 16. The method of claim 14, wherein the detergent is Triton X-100.

17. The method of claim 14, wherein separating further comprising adding an inactivating agent. 18. The method of claim 17, wherein the inactivating agent comprises binary ethyleneimine.

19. The method of claim 1, wherein the immunogenic composition is a vaccine. 20. The method of claim 1, comprising:

incubating PEDV infected cells for a period of from 24 to 60 hours; isolating cells infected with PEDV away from cell-free PEDV virus particles to obtain isolated cells containing cell-associated PEDV proteins and antigens;

lysing the isolated cells to form a first composition comprising isolated PEDV proteins and antigens;

inactivating viral particles in the first composition to produce a second composition; and adding an adjuvant comprising polyacrylic acid to the second composition to form an immunogenic composition formulated for intranasal administration.

21. The method of claim 19, comprising incubating the PEDV infected cells for from 24 to 48 hours.

22. The method of claim 1, wherein incubating PEDV infected cells comprises incubating cells infected with PEDV having at least 90% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

23. The method of claim 1, wherein incubating PEDV infected cells comprises incubating cells infected with PEDV having at least 99% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

24. The method of claim 1, wherein incubating PEDV infected cells comprises incubating cells infected with PEDV having at least 99.5% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

25. The method of claim 1, wherein incubating PEDV infected cells comprises incubating cells infected with PEDV having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

26. The method of claim 1 , wherein the isolated PEDV proteins and antigens comprise an amount of S protein.

27. The method of claim 26, wherein the amount of S protein is sufficient to produce an immune response in a subject receiving the immunogenic composition.

28. The method of claim 26, wherein the S protein is an intact S protein.

29. The method of claim 26, wherein the S protein has at least 90% sequence identity to at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. 30. The method of claim 26, wherein the S protein has at least 99% sequence identity to at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

31. The method of claim 26, wherein the S protein has a sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

32. The method of claim 1, comprising:

incubating PEDV infected cells for a period of from 24 to 60 hours sufficient to produce to tissue culture infective doses/ml (TCIDso/ml) of from 10¹ to 10¹⁰, the cells infected with a PEDV strain that has at least 99% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6;

isolating cells infected with PEDV away from cell-free PEDV virus particles to obtain isolated cells containing cell-associated PEDV proteins and antigens;

lysing the isolated cells to form a first composition comprising isolated PEDV proteins and antigens comprising S protein having a molecular weight of about 152 kDa based on deduced amino acid sequences, and a molecular weight of from 180 kDa to at least 350 kDa after post- translational modifications;

adding an inactivating agent comprising binary ethyleneimine to the first composition to produce a second composition; and

adding an adjuvant comprising polyacrylic acid to the second composition to form an immunogenic composition formulated for intranasal administration.

33. An immunogenic composition, comprising:

a first antigenic component comprising isolated PEDV proteins and/or antigens from a first PEDV strain; and

an adjuvant.

34. The immunogenic composition of claim 33, comprising an S protein.

35. The immunogenic composition of claim 34, comprising an amount of S protein sufficient to produce an immune response in a subject receiving the immunogenic composition.

36. The immunogenic composition of claim 34, wherein the S protein is an intact S protein.

37. The immunogenic composition of claim 34, wherein the S protein has at least 90% sequence identity to at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:

13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

38. The immunogenic composition of claim 34, wherein the S protein has at least 99% sequence identity to at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

39. The immunogenic composition of claim 38, wherein the S protein has a sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

40. The immunogenic composition of claim 33, wherein the first PEDV strain has at least 90% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

41. The immunogenic composition of claim 33, wherein the first PEDV strain has at least 99% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

42. The immunogenic composition of claim 33, wherein the first PEDV strain has at least 99.5% sequence identity to at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

43. The immunogenic composition of claim 33, wherein the first PEDV strain has a sequence identity selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. 44. The immunogenic composition of claim 33, comprising a second antigenic component.

45. The immunogenic composition of claim 44, wherein the second antigenic component comprises isolated PEDV proteins and/or antigens from a second PEDV strain different from the first PEDV strain.

46. The immunogenic compositions of claim 44, wherein the second antigenic component comprises isolated proteins and/or antigens from a second pathogen other than PEDV. 47. The immunogenic composition of claim 46, wherein the second pathogen is porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae, Mycoplasma hyorhinis, Clostridium tetani, , Clostridium perfringens, porcine parvovirus, Erysipelothrix rhusiopathiae , Leptospira pomona, Leptospira grippotyphosa,

Leptospira hardjo, Leptospira canicola, Leptospira icterohaemorrhagiae, Leptospira bratislava, porcine circovirus, Lawsonia intracellularis, Escherchia coli, Actinobacillus pleuropneumoniae , Haemophilus parasuis, Salmonella choleraesuis, Salmonella typhimurium, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Serpulina hyodysenteriae, encephalomyocarditis virus, swine influenza virus, transmissible gastroenteritis virus, swine delta coronavirus, rotavirus diarrhea, foot and mouth disease virus, classical swine fever virus, pseudorabies virus, Japanese encephalitis virus (JEV), encephalomyocarditis virus, or a combination thereof.

48. The immunogenic composition of claim 47, wherein the second pathogen is porcine reproductive and respiratory syndrome virus.

49. The immunogenic composition of claim 47, wherein the second pathogen is not Mycoplasma hyopneumoniae.

50. The immunogenic composition of claim 33, wherein the immunogenic composition is a vaccine. 51. The immunogenic composition of any one of claims 33-50, wherein the adjuvant is selected to stimulate a mucosal antibody response.

52. The immunogenic composition of any one of claims 33-50, wherein the adjuvant adheres to the mucous membranes.

53. The immunogenic composition of any one of claims 33-50, wherein the adjuvant comprises emulsified oil-in-water adjuvant.

54. The immunogenic composition of claim 53, wherein the adjuvant comprises an ammonium salt.

55. The immunogenic composition of claim 53, wherein the ammonium salt is a tetraalkylammonium salt. 56. The immunogenic composition of claim 53, wherein the tetraalkylammonium salt is dimethyldioctadecylammonium bromide .

57. The immunogenic composition of any one of claims 33-50, wherein the adjuvant comprises polyacrylic acid.

58. A method, comprising administering to a first pig an effective amount of a first immunogenic composition according to claim 33. 60

59. The method of claim 58, wherein the adjuvant is selected to stimulate a mucosal antibody response. 60. The method of claim 59, wherein the adjuvant adheres to the mucous membranes.

61. The method of claim 58, wherein the adjuvant comprises an emulsified oil-in-water adjuvant. 62. The method of claim 61, wherein the adjuvant comprises an ammonium salt.

63. The method of claim 62, wherein the ammonium salt is a tetralkylammonium salt.

64. The method of claim 63, wherein the tetralkylammonium salt is

dimethyldioctadecylammonium bromide .

65. The method of claim 58, wherein the adjuvant comprises a polyacrylic acid.

66. The method of claim 58, wherein the first pig is less than 7 days old.

67. The method of claim 58, wherein the first pig is 5 days old or less.

68. The method of claim 58, wherein the first pig is 2 days old or less. 69. The method of any one of claims 58-68, wherein administering comprises administering orally, intramuscularly, or subcutaneously.

70. The method of any one of claims 58-68, wherein administering comprises administering intranasally.

71. The method of claim 58, wherein the immunogenic composition further comprises a second antigenic component different from the first antigenic component. 61

72. The method of claim 71, wherein the second antigenic component comprises isolated PEDV proteins and/or antigens from a second PEDV strain. 73. The method of claim 71, wherein the second antigenic component comprises isolated proteins and/or antigens from a second pathogen other than PEDV.

74. The method of claim 73, wherein the second pathogen is porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae, Mycoplasma hyorhinis, Clostridium tetani, Clostridium perfringens, porcine parvovirus, Erysipelothrix rhusiopathiae , Leptospira pomona, Leptospira grippotyphosa, Leptospira hardjo, Leptospira canicola, Leptospira icterohaemorrhagiae, Leptospira bratislava, porcine circovirus, Lawsonia intracellularis, Escherchia coli, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Salmonella choleraesuis, Salmonella typhimurium, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Serpulina hyodysenteriae, encephalomyocarditis virus, swine influenza virus, transmissible gastroenteritis virus (TGE), swine delta coronavirus, rotavirus diarrhea, foot and mouth disease virus, classical swine fever virus, pseudorabies virus, Japanese encephalitis virus (JEV), encephalomyocarditis virus or a combination thereof.

75. The method of claim 73, wherein the second pathogen is porcine reproductive and respiratory syndrome virus.

76. The method of claim 73, wherein the second pathogen is not Mycoplasma hyopneumoniae.

77. The method of claim 58, comprising administering a second immunogenic composition to a second pig, the second pig being a sow and the first pig being a piglet farrowed from the sow.

78. The method of claim 77, wherein the first immunogenic composition is administered intranasally.

62

79. The method of claim 77, wherein the second immunogenic composition is administered intramuscularly.

80. The method of claim 77, wherein the second immunogenic composition comprises an immunogenic composition according to claim 33.

81. The method of claim 77, wherein the sow is a pregnant sow.

82. The method of claim 77, wherein the sow is a sow expected to become pregnant subsequent to administration of the second immunogenic composition.

83. The method of claim 77, wherein administering the second immunogenic composition comprises administering the second immunogenic composition at a time point prior to the sow becoming pregnant such that, when the sow becomes pregnant, the sow has a greater immunity to PEDV compared to a pig not administered the immunogenic composition.

84. A use of an immunogenic composition made by the method of any one of claims 1- 32 in the manufacture of a medicament for administration to a swine.