METHODS FOR IMPROVING IMMUNOLOGICAL RESPONSE IN VACCINATED ANIMALS

• US 20160051668A1
• Filed: 08/20/2015
• Published: 02/25/2016
• Est. Priority Date: 08/21/2014
• Status: Active Grant

• Current Assignee: Braasch Biotech LLC
• Original Assignee: Braasch Biotech LLC
• Inventors: Haffer, Keith N.
• Granted Patent: US 10,441,652 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/552 Veterinary vaccine
• CPC Class Codes: A61K 2039/6031 Proteins
• CPC Class Codes: A61K 2039/627 characterised by the linker
• CPC Class Codes: A61K 39/0005 Vertebrate antigens from snakes A61K39/38
• CPC Class Codes: A61K 39/385 Haptens or antigens, bound to carriers
• CPC Class Codes: C07K 14/655 Somatostatins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C12N 9/1033 Chloramphenicol O-acetyltransferase (2.3.1.28)

Abstract

A method is provided for increasing an immunological response to a target antigen in an animal by administering an immunogenic amount of a vaccine comprising a polypeptide conjugate comprising the target antigen conjugated to a carrier polypeptide by means of a linker polypeptide which is rich in predicted linear B-cell epitopes.

Claims

1. A method for stimulating an improved immunological response to a target antigen in an animal, the method comprising administering an immunogenic amount of a vaccine to an animal, the vaccine comprising a polypeptide conjugate comprising (a) a carrier polypeptide comprising an inactivated chloramphenicol acetyl transferase (CAT) enzyme;

(b) a linker polypeptide, and (c) a target antigen, wherein the carrier polypeptide does not stimulate a substantial T cell response.

2. The method according to claim 1, wherein the target antigen exhibits low % predicted linear B-cell epitopes.

3. The method according to claim 2, wherein the target antigen exhibits low % predicted linear B-cell epitopes selected from <

10%, <

7%, <

5%, <

4%, <

3%, <

2%, <

1%, <

0.1% or 0% predicted linear B-cell epitopes, wherein the % predicted linear B-cell epitopes for the target antigen is determined by

(1) generating a BepiPred 1.0 predicted Linear B-cell epitope score for each amino acid in the amino acid sequence of the target antigen;

(2) counting the amino acids for which the BepiPred 1.0 predicted Linear B-cell epitope score is above a threshold value;

(3) dividing the number of amino acids above the threshold value by the number of amino acids in the target antigen to get a fraction; and

(4) multiplying the fraction by 100 to obtain the % predicted linear B-cell epitopes for the target antigen.

4. The method according to claim 1, wherein the linker polypeptide exhibits high % predicted linear B-cell epitopes.

5. The method according to claim 4, wherein the linker polypeptide exhibits high % predicted linear B-cell epitopes selected from >

50%, >

60%, >

70% or >

80% predicted linear B-cell epitopes, wherein the % predicted linear B-cell epitopes in the linker polypeptide is determined by

(1) generating a BepiPred 1.0 predicted Linear B-cell epitope score for each amino acid in the amino acid sequence of the linker polypeptide;

(2) counting the amino acids in the linker polypeptide for which the BepiPred 1.0 predicted Linear B-cell epitope score is above a threshold value;

(3) dividing the number of amino acids above the threshold value by the total number of amino acids in the linker polypeptide to get a fraction; and

(4) multiplying the fraction by 100 to get the % predicted Linear B-cell Epitopes for the linker polypeptide.

6. The method according to claim 1, wherein the carrier polypeptide comprises an inactivated chloramphenicol acetyl transferase (CAT) enzyme comprising an amino acid sequence selected from SEQ ID NO:

3, 7, 8, 26, 27, 28, or 29.

7. The method according to claim 4, wherein the linker polypeptide comprises an amino acid sequence selected from SEQ ID NO:

10, 11, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 53, 54, 55, 56 or 57.

8. The method according to claim 1, wherein the improved immunological response comprises one or more of (1) a statistically significant increase in serum anti-target antigen IgG or serum anti-carrier IgG from the animal observed after the first or subsequent administration of the vaccine comprising the polypeptide conjugate, compared to control immunized with target antigen and/or carrier without the linker polypeptide, (2) a statistically significant increase or decrease in a specific effect in the animal following administration of the vaccine comprising the polypeptide conjugate compared to a control, or (3) a statistically significant faster statistically significant immunological response or specific effect in the animal following administration of the vaccine comprising the polypeptide conjugate compared to a control.

9. The method according to claim 8, wherein the improved immunological response in the animal comprises a statistically significant faster immunological response or specific effect compared to a control, wherein the response or effect occurs within six days, five days, four days, three days, two days, or within one day following the first or subsequent administrations to the animal of the vaccine comprising the polypeptide conjugate.

10. The method according to claim 9, wherein the specific effect is selected from increased milk production, increased body weight, increased lean body weight, decreased percent body-fat, or decreased body weight in the animal compared to a control without immunization.

11. The method according to claim 1, wherein the immunogenicity of said target antigen in the polypeptide conjugate is increased as compared to the target antigen in the absence of the polypeptide conjugate.

12. The method according to claim 3, wherein the target antigen is selected from somatostatin 14, gonadotropin releasing hormone (GnRH), luteinizing hormone releasing factor, calcitonin neuropeptide, myostatin, HIV envelope protein, tuberculosis outer membrane protein, Neisseria meningitides outer membrane protein complex;

or a fragment thereof.

13. The method according to claim 3, wherein the fragment is a polypeptide from 5 to 40 amino acids in length.

14. The method according to claim 3, wherein the target antigen comprises an amino acid sequence consisting of one or more of SEQ ID NOs:

1, 32, 33, 34, 35, 36, 37, 38, 40, 41, 43, 44, 46, 47, 48, 49, 50, 51, or 52.

15. The method according to claim 13, wherein the target antigen comprises an amino acid sequence consisting of SEQ ID NO:

1.

16. The method according to claim 3, wherein the target antigen is not somatostatin 14.

17. The method according to claim 1, wherein the CAT enzyme has one or more 192 or 193 wild type histidine residues is independently replaced with an amino acid selected from alanine, glycine or other like amino acid.

18. The method according to claim 1, wherein the chloramphenicol acetyl transferase polypeptide is truncated at the C-terminus by 10 amino acids relative to the wild type polypeptide.

19. The method according to claim 1, wherein the vaccine is substantially endotoxin free.

20. The method according to claim 1, wherein the vaccine is at least 80% endotoxin free.

35. The method according to claim 3, wherein the BepiPred 1.0 predicted Linear B-cell epitope score threshold is 0.2.

36. The method according to claim 5, wherein the BepiPred 1.0 predicted Linear B-cell epitope score threshold is 0.2.

37. The method according to claim 1, wherein the linker polypeptide is heterologous to the target antigen.

21. A method for rapidly increasing milk production in an animal, the method comprising:

(1) administering an immunogenic amount of a vaccine to an animal, the vaccine comprising a polypeptide conjugate comprising a target antigen that is somatostatin-14 attached to an inactivated chloramphenicol acetyl transferase (CAT) enzyme by a linker polypeptide, wherein milk production is significantly increased relative to that of a control animal that does not receive administration of said vaccine.

22. The method according to claim 21, wherein the animal is a dairy cow, beef cow, goat, or sow.

23. The method according to claim 21, wherein milk production is increased within 4 days of the administration of the vaccine.

24. The method according to claim 21, wherein peak milk production is achieved within 8-14 days of the administration of the vaccine.

25. The method according to claim 21, wherein the increase in milk production persists for at least 21 days.

26. The method according to claim 21, further comprising administering an additional immunogenic amount of the vaccine to the animal, wherein the additional administration is performed after peak milk production has been observed.

27. The method of claim 26, wherein the additional administration is performed after a return to baseline milk production has been observed.

28. The method of claim 27, wherein the additional administration is performed at intervals of approximately 21 days.

29. A method for rapidly increasing lean meat production in an animal, the method comprising:

(1) administering an immunogenic amount of a vaccine to the animal, the vaccine comprising (a) a polypeptide conjugate comprising somatostatin-14 covalently attached to an inactivated CAT enzyme by a linker polypeptide, and (2) subsequently administering at least one additional immunogenic amount of the vaccine, wherein subsequent administration does not produce an anamnestic response to the carrier, and wherein lean meat production is increased relative to that of an animal that does not receive said administration.

30. The method according to claim 29, wherein the vaccine is administered at least 3 times.

31. The method of claim 29, wherein the vaccine is administered at intervals of 21 days.

32. The method of claim 29 wherein the animal is a pig or cow.

33. A method for developing a conjugated vaccine, comprising

selecting a target antigen exhibiting low % predicted B-cell epitopes;

and covalently attaching the target antigen to a carrier polypeptide via a linker polypeptide exhibiting high % predicted linear B-cell epitopes to form a polypeptide conjugate;

wherein the carrier polypeptide does not stimulate a substantial T cell response.

34. The method according to claim 33, wherein the target antigen exhibits lower % predicted B-cell epitopes than the linker polypeptide.