Targeted gene disruption methods and immunogenic compositions

Patent metadata

Field	Value
Publication number	US-11541108-B2
Application number	US-201816609097-A
Country	US
Kind code	B2
Filing date	Apr 30, 2018
Priority date	Apr 28, 2017
Publication date	Jan 3, 2023
Grant date	Jan 3, 2023

Abstract

Targeted disruption of a specific gene and its subsequent restoration in obligate intracellular bacteria remains extremely challenging due to their absolute requirement for residence inside a host cell to replicate. Here, targeted allelic exchange mutations were created to inactivate two genes and then to restore one of the two genes of a rickettsial pathogen, Ehrlichia chaffeensis. These methods were then also successfully utilized in Ehrlichia canis and Anaplasma phagocyophilum. The resultant mutated pathogens are useful in immunogenic compositions for reducing the incidence of or severity of infection with ricksettsial pathogens.

First claim

What is claimed is:

1. An immunogenic composition comprising:
a Rickettsiale bacteria having a targeted allelic exchange mutation therein, wherein said targeted allelic exchange mutation comprises a disrupted gene that has had the integrity thereof restored; and
a component selected from the group consisting of a veterinary-acceptable carrier, a pharmaceutical-acceptable carrier, an adjuvant, a preservative, a buffer, an antibiotic, cell culture supernatant, an immunomodulatory agent, and any combination thereof.

2. The immunogenic composition of claim 1 , wherein said Rickettsiale bacteria is selected from the group consisting of species of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia , and Orientia.

3. The immunogenic composition of claim 2 , wherein the Ehrlichia bacteria species is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia ruminatium , and Ehrlichia canis.

4. The immunogenic composition of claim 2 , wherein the Anaplasma bacteria is selected from the group consisting of Anaplasma phagocytophilum, Anaplasma platys , and Anaplasma marginale.

5. The immunogenic composition of claim 1 , wherein the targeted allelic mutation attenuates the bacteria and/or inactivates a gene.

6. The immunogenic composition of claim 5 , wherein the gene functions as an aid to replication.

7. The immunogenic composition of claim 2 , wherein the targeted allelic exchange mutation is in a location selected from the group consisting of:
a) Ech_0379 or Ech_0660 in Ehrlichia chaffeensis;
b) Ecaj_0381 in Ehrlichia canis;
c) APH_0634 in Anaplasma phagocytophilum
d) Erum_3930 in E. ruminatium;
e) AMH_581 in Anaplasma marginale ; or
f) EMUR_02070 in Ehrlichia muris AS145.

8. The immunogenic composition of claim 1 , wherein said component is an adjuvant selected from the group consisting of a saponin, a cyclic GMP-AMP, montanide gel, or any combination thereof.

9. The immunogenic composition of claim 1 , further comprising an antigen from another disease causing organism.

10. The immunogenic composition of claim 1 , wherein said bacteria includes a sequence with at least 70% sequence identity with SEQ ID NO. 35, 54, or 55.

11. An immunogenic composition comprising:
a Rickettsiale or Chlamydiale bacteria having a targeted allelic exchange mutation therein and includes a sequence with at least 70% sequence identity with SEQ ID NO. 35, 54, or 55; and
a component selected from the group consisting of a veterinary-acceptable carrier, a pharmaceutical-acceptable carrier, an adjuvant, a preservative, a buffer, an antibiotic, cell culture supernatant, an immunomodulatory agent, and any combination thereof.

12. A method of reducing the incidence of or severity of at least one clinical sign caused by a Rickettsiale bacteria comprising the step of:
administering an immunogenic composition at least once to an animal in need thereof, wherein said immunogenic composition comprises a Rickettsiale bacteria having a targeted allelic exchange mutation therein, wherein said targeted allelic exchange mutation comprises a disrupted gene that has had the integrity thereof restored, and a component selected from the group consisting of a veterinary-acceptable carrier, a pharmaceutical-acceptable carrier, an adjuvant, a preservative, a buffer, a stabilizer, an antibiotic, cell culture supernatant, an immunomodulatory agent, and any combination thereof.

13. The method of claim 12 , wherein said immunogenic composition is administered using an administration mode selected from the group consisting of intravenously, intramuscularly, intranasally, intradermally, intratracheally, intravaginally, intravenously, intravascularly, intraarterially, intraperitoneally, orally, intrathecally, by direct injection into any target tissue, or any combination thereof.

14. The method of claim 12 , wherein said reduction in incidence is at least 10% and is in comparison to a group of animals that have not received an administration of the immunogenic composition.

15. The method of claim 12 , wherein said reduction in severity is at least 10% in comparison to a group of animals that have not received an administration of the immunogenic composition.

16. The method of claim 12 , wherein the Rickettsiale bacteria is selected from the group consisting of species of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, and Orientia.

17. The method of claim 16 , wherein the Ehrlichia bacteria is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia ruminatium , and Ehrlichia canis.

18. The method of claim 16 , wherein the Anaplasma bacteria is selected from the group consisting of Anaplasma phagocytophium, Anaplasma platys , and Anaplasma marginale.

19. The method of claim 12 , wherein the targeted allelic exchange mutation inactivates a gene.

20. The method of claim 12 , wherein the targeted allelic exchange mutation is in a location selected from the group consisting of:
a) Ech_0379, or Ech_0660 in Ehrlichia chaffeensis;
b) Ecaj_0381 in Ehrilichia canis;
c). APH_0634 in Anaplasma phagocytophilum;
d) Erum_3930 in E. ruminatium;
e) AMH_581 in Anaplasma marginale ; or
f) EMUR_02070 in Ehrlichia muris AS145.

21. The method of claim 12 , wherein said bacteria includes a sequence with at least 70% sequence identity with SEQ ID NO. 35, 54, or 55.

22. The method of claim 12 , wherein said animal is selected from the group consisting of pigs, cattle, goats, horses, dogs, deer, coyote, cats, and poultry.

23. The method of claim 12 , wherein said animal is between 3 weeks and 6 months of age when receiving said administration.

Assignees

• Univ Kansas State

Inventors

• Ganta Roman R
• Wang Ying

Classifications

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• A61K39/118 Primary

  Chlamydiaceae, e.g. Chlamydia trachomatis or Chlamydia psittaci · CPC title

• A61K39/0233 Primary

  Rickettsiales, e.g. Anaplasma · CPC title

• A61K2039/522

  avirulent or attenuated · CPC title