Bacillus based delivery system and methods of use

Patent metadata

Field	Value
Publication number	US-9956277-B2
Application number	US-201514849123-A
Country	US
Kind code	B2
Filing date	Sep 9, 2015
Priority date	Feb 22, 2008
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

Herein a Bacillus exosporium antigen delivery (BEAD) system that provides a means to introduce recombinant proteins or small molecules into the exosporium of members of the B. cereus family of bacteria, i.e. B. anthracis, B. cereus , and B. thuringiensis , is disclosed. The system results in the surface display of recombinant proteins or small molecules such that they can stimulate an immune response. In addition, methods of making and using the system are described.

First claim

What is claimed is:

1. A Bacillus exosporium molecule delivery (BEMD) system comprising a recombinant Bacillus cereus family member expressing a fusion construct, wherein the fusion construct comprises at least one molecule of interest (MOI) and:
(a) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 10;
(b) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 10;
(c) a targeting sequence comprising SEQ ID NO: 10; or
(d) a targeting sequence having at least 50% identity with amino acids 13-28 of SEQ ID NO: 10, wherein the identity with amino acids 18-28 of SEQ ID NO: 10 is at least 63%;
and wherein expression of the fusion construct results in display of the MOI on the exosporium of the recombinant Bacillus cereus family member.

2. The BEMD system of claim 1 , wherein the recombinant Bacillus cereus family member is selected from the group consisting of strains of B. anthracis, B. cereus, B. thuringiensis , and combinations thereof.

3. The BEMD system of claim 1 , wherein the MOI is an immunogenic molecule.

4. The BEMD system of claim 3 , wherein the immunogenic molecule of the fusion construct is physically oriented in the exosporium to be able to stimulate an immune response.

5. A method of making a delivery system comprising:
a) constructing a fusion construct, wherein the fusion construct comprises at least one MOI and:
(i) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 10;
(ii) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 10;
(iii) a targeting sequence comprising SEQ ID NO: 10; or
(iv) a targeting sequence having at least 50% identity with amino acids 13-28 of SEQ ID NO: 10, wherein the identity with amino acids 18-28 of SEQ ID NO: 10 is at least 63%;

b) cloning the fusion construct into a shuttle plasmid; and
c) electroporating the shuttle plasmid containing the fusion construct into a Bacillus cereus family member, wherein expression of the fusion construct results in display of the MOI on the exosporium of the Bacillus cereus family member.

6. The method of claim 5 , wherein the Bacillus cereus family member is selected from the group consisting of strains of B. anthracis, B. cereus, B. thuringiensis , and combinations thereof.

7. The method of claim 5 , wherein the MOI is an immunogenic molecule.

8. The method of claim 7 , wherein the immunogenic molecule of the fusion construct is physically oriented in the exosporium to be able to stimulate an immune response.

9. A kit comprising the delivery system of claim 1 .

10. The kit of claim 9 , wherein the recombinant Bacillus cereus family member is selected from the group consisting of strains of B. anthracis, B. cereus, B. thuringiensis , and combinations thereof.

11. The kit of claim 9 , wherein the MOI is an immunogenic molecule.

12. The kit of claim 11 , wherein the immunogenic molecule of the fusion construct is physically oriented in the exosporium to be able to stimulate an immune response.

13. A method of using a delivery system comprising administering to a subject the recombinant a recombinant Bacillus cereus family member of claim 1 .

14. The method of claim 13 , wherein the recombinant Bacillus cereus family member is selected from the group consisting of strains of B. anthracis, B. cereus, B. thuringiensis , and combinations thereof.

15. The method of claim 13 , wherein the MOI is an immunogenic molecule.

16. The method of claim 15 , wherein the immunogenic molecule of the fusion construct is physically oriented in the exosporium to be able to stimulate an immune response.

17. The BEMD system of claim 2 , wherein the recombinant Bacillus cereus family member comprises B. thuringiensis.

18. The method of claim 6 , wherein the recombinant Bacillus cereus family member comprises B. thuringiensis.

19. The kit of claim 10 , wherein the recombinant Bacillus cereus family member comprises B. thuringiensis.

20. The method of claim 14 , wherein the recombinant Bacillus cereus family member comprises B. thuringiensis.

21. The BEMD system of claim 1 , wherein the fusion construct is expressed under the control of a BclA promoter.

22. The BEMD system of claim 1 , wherein the Bacillus cereus family member is inactivated.

23. The method of claim 13 , further comprising inactivating the Bacillus cereus family member prior to administration.

Assignees

• Univ Missouri

Inventors

• Stewart George C
• Thompson Brian Matthew

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C12N2770/10022

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• B09C1/10

  microbiologically, {biologically} or by using enzymes · CPC title

• A61K39/12

  Viral antigens · CPC title

• A61K2039/523

  expressing foreign proteins · CPC title