Processing engineered FMDV P1 polypeptide using an alternative TEV protease

Patent metadata

Field	Value
Publication number	US-11008564-B2
Application number	US-201916364633-A
Country	US
Kind code	B2
Filing date	Mar 26, 2019
Priority date	Jan 17, 2017
Publication date	May 18, 2021
Grant date	May 18, 2021

Abstract

Polynucleotide constructs that express an engineered foot-and-mouth disease (FMDV) P1 precursor protein and a non-FMDV TEV protease and methods for safe and efficient recombinant production of FMDV antigens and immunogens. Recombinant production of FMDV antigens avoids the need to culture highly-infectious FMDV, while conventional culture methods for producing FMDV antigens rely on the native FMDV 3C protease which exerts toxic effects on host cells. The inventors have developed a new system that efficiently and safely processes FMDV P1 precursor without the FMDV 3C protease, thus avoiding the toxic effects associated with use of the 3C protease. The invention is also directed to the FMDV antigens and virus-like particles produced by this system as well as to FMDV vaccines, diagnostics and other biologics.

First claim

What is claimed is:

1. A pharmaceutical composition, comprising:
at least one selected from the group consisting of VP0, VP1, VP2, VP3 and VP4, in the form of virus-like particles (VLPs); and
a pharmaceutically acceptable buffer,
wherein the VLPs are produced from proteolytic processing of at least one engineered Foot-and-mouth disease virus (FMDV) P1 precursor polypeptide that comprises at least one Tobacco etch virus (TEV) protease cleavage site, and
wherein the at least one TEV protease cleavage site comprises E 1 -D 2 -A 3 -Y 4 -T 5 -Q 6 -S 7 (SEQ ID NO: 39), E 1 -F 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 40), E 1 -D 2 -L 3 -Y 4 -F 5 -Q 6 -S 7 (SEQ ID NO: 41), E 1 -K 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 42), E 1 -L 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 43) or E 1 -A 2 -L 3 -Y 4 -K 5 -Q 6 -S 7 (SEQ ID NO: 44) in at least one junction between VP4 and VP2, VP2 and VP3, VP3 and VP1, or VP1 and 2A components of the engineered FMDV P1 precursor polypeptide.

2. The pharmaceutical composition of claim 1 , comprising VP0, VP1 and VP3 in the form of VLPs, or VP4, VP2, VP3 and VP1 in the form of VLPs.

3. The pharmaceutical composition of claim 1 , wherein one or more of the VLPs comprise empty capsids not containing RNA.

4. The pharmaceutical composition of claim 1 , comprising a pharmaceutically acceptable adjuvant.

5. The pharmaceutical composition of claim 1 , further comprising at least one other active immunogen, drug or antiviral agent.

6. The pharmaceutical composition of claim 1 , formulated for parenteral, intramuscular, intranasal, intravenous, intrapulmonary, intratracheal, oral, mucous membrane or alimentary canal administration.

7. The pharmaceutical composition of claim 1 , wherein the composition is a vaccine.

8. The pharmaceutical composition of claim 1 , wherein said proteolytic processing comprises:
culturing a host cell comprising a polynucleotide that encodes the at least one engineered FMDV P1 precursor polypeptide that comprises at least one Tobacco etch virus (TEV) protease cleavage site, and a polynucleotide encoding a TEV protease; and
recovering cleavage products of the at least one engineered FMDV P1 precursor polypeptide produced by cleavage of the at least one TEV protease cleavage site.

9. A method for preventing or treating Foot-and-mouth disease virus (FMDV) infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising:
at least one selected from the group consisting of VP0, VP1, VP2, VP3 and VP4, in the form of virus-like particles (VLPs); and
a pharmaceutically acceptable buffer,
wherein the VLPs are produced from proteolytic processing of at least one engineered Foot-and-mouth disease virus (FMDV) P1 precursor polypeptide that comprises at least one Tobacco etch virus (TEV) protease cleavage site, and
wherein the at least one TEV protease cleavage site comprises E 1 -D 2 -A 3 -Y 4 -T 5 -Q 6 -S 7 (SEQ ID NO: 39), E 1 -F 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 40), E 1 -D 2 -L 3 -Y 4 -F 5 -Q 6 -S 7 (SEQ ID NO: 41), E 1 -K 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 42), E 1 -L 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 43) or E 1 -A 2 -L 3 -Y 4 -K 5 -Q 6 -S 7 (SEQ ID NO: 44) in at least one junction between VP4 and VP2, VP2 and VP3, VP3 and VP1, or VP1 and 2A components of the engineered FMDV P1 precursor polypeptide.

10. The method of claim 9 , wherein the subject is uninfected with FMDV.

11. The method of claim 9 , wherein the subject is infected with FMDV.

12. The method of claim 9 , wherein the subject is at risk of having foot-and-mouth disease (FMD).

13. The method of claim 9 , wherein the subject has FMD.

14. The method of claim 9 , wherein the subject is a bovine, caprine, ovine or swine.

15. The method of claim 9 , wherein the pharmaceutical composition is administered parenterally, intramuscularly, intranasally, intravenously, intrapulmonarily, intratracheally, orally, on to a mucous membrane or into an alimentary canal.

16. A method for preventing or treating Foot-and-mouth disease virus (FMDV) infection in a subject, comprising administering to the subject an effective amount of a host cell that expresses:
an engineered FMDV P1 precursor polypeptide that comprises at least one Tobacco etch virus (TEV) protease cleavage site, wherein the at least one TEV protease cleavage site comprises E 1 -D 2 -A 3 -Y 4 -T 5 -Q 6 -S 7 (SEQ ID NO: 39), E 1 -F 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 40), E 1 -D 2 -L 3 -Y 4 -F 5 -Q 6 -S 7 (SEQ ID NO: 41), E 1 -K 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 42), E 1 -L 2 -L 3 -Y 4 -K 5 -Q 6 -G 7 (SEQ ID NO: 43) or E 1 -A 2 -L 3 -Y 4 -K 5 -Q 6 -S 7 (SEQ ID NO: 44) in at least one junction between VP4 and VP2, VP2 and VP3, VP3 and VP1, or VP1 and 2A components of the engineered FMDV P1 precursor polypeptide; and
a TEV protease.

17. The method of claim 16 , wherein the at least one TEV protease cleavage site in the engineered FMDV P1 precursor polypeptide is positioned at junction(s) that when cleaved by a TEV protease produce VP0, VP3, and VP1, or produce VP2, VP4, VP3 and VP1.

18. The method of claim 16 , wherein the host cell is autologous.

19. The method of claim 16 , wherein the host cell expresses a FMDV 3C protease.

20. The method of claim 19 , wherein the FMDV 3C protease contains at least one of a V28K, L127P, V141T, C142T and C163A substitution.

Assignees

• The Government Of The Us Secretary Of Homeland Security

Inventors

• Puckette Michael
• Rasmussen Max

Classifications

• C12N2770/32151

  Methods of production or purification of viral material · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2770/32122

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

• C07K14/005

  from viruses · CPC title

• A61K39/12

  Viral antigens · CPC title