Methods for improved poxvirus yields

Patent metadata

Field	Value
Publication number	US-2021301263-A1
Application number	US-201917266499-A
Country	US
Kind code	A1
Filing date	Aug 7, 2019
Priority date	Aug 8, 2018
Publication date	Sep 30, 2021
Grant date	—

Abstract

A method of producing poxviruses at an increased growth rate and/or progeny virus titer in cells, the method including: contacting a host cell with an effective amount of nucleocytoplasmic transport inhibitor; contacting the host cell with a poxvirus of interest under conditions that permit the poxvirus of interest to adsorb to the surface of the host cell; and culturing the host cell to produce progeny poxvirus of interest.

First claim

We claim:

1 . A method for replicating a poxvirus, comprising:
exposing a host cell to an effective amount of a nucleocytoplasmic transport inhibitor and the poxvirus under conditions that permit the poxvirus to adsorb to the surface of the host cell; and incubating the infected host cell in a culture medium to allow for replication of the poxvirus.

2 . The method of claim 1 , further comprising harvesting the poxvirus produced in the host cell.

3 . The method of claim 1 or claim 2 , wherein the poxvirus is genetically modified.

4 . The method of any one of claims 1 - 3 , wherein the poxvirus is a Leporipoxvirus.

5 . The method of claim 4 , wherein the Leporipoxvirus is a myxoma virus.

6 . The method of any one of claims 1 - 3 , wherein the poxvirus is an Orthopoxvirus.

7 . The method of claim 6 , wherein the Orthopoxvirus is a vaccinia virus.

8 . The method of claim 7 , wherein the vaccinia virus is a vaccinia virus strain selected from the group consisting of Lister, Wyeth, Western Reserve, Modified Vaccinia virus Ankara, and LC16m series.

9 . The method of claim 6 , wherein the Orthopoxvirus is a Raccoonpox virus.

10 . The method of any one of claims 1 - 3 , wherein the poxvirus is a Capripox virus.

11 . The method of claim 10 , wherein the Capripox virus is an Orf virus.

12 . The method of any one of claims 1 - 11 , wherein the nucleocytoplasmic transport inhibitor is selected from the group consisting of Leptomycin A, Leptomycin B, Ratjadone A, Ratjadone B, Ratjadone C, Ratjadone D, Anguinomycin A, Goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthol, KOS 2464, PKF050-638, CBS9106, and Selinexor.

13 . The method of any one of claims 1 - 12 , further comprises contacting the host cell with the nucleocytoplasmic transport inhibitor prior to infecting the host cell with the poxvirus.

14 . The method of any one of claims 1 - 13 , further comprises contacting the host cell with the nucleocytoplasmic transport inhibitor after infecting the host cell with the poxvirus.

15 . The method of any one of claims 1 - 14 , further comprises exposing the host cell to the nucleocytoplasmic transport inhibitor and the poxvirus at the same time.

16 . The method of any one of claims 1 - 15 , wherein the poxvirus is replicated at a rate that is at least 30% faster than a replication rate in an absence of the nucleocytoplasmic transport inhibitor.

17 . The method of any one of claims 1 - 16 , wherein the nucleocytoplasmic transport inhibitor increases the replication of the poxvirus by at least 3 fold after 24 hours post infection.

18 . The method of any one of claims 1 - 17 , wherein the host cell is contacted with the poxvirus at a multiplicity of infection (MOI) lower than 2.0.

19 . The method of any one of claims 1 - 18 , wherein the effective amount of the nucleocytoplasmic transport inhibitor is in the range of about 0.0005 μM to about 0.5 μM.

20 . The method of any one of claims 1 - 19 , wherein the culture medium comprises the nucleocytoplasmic transport inhibitor.

21 . The method of any one of claims 1 - 20 , wherein the host cell is an immortalized human or primate cell.

22 . The method of any one of claims 1 - 21 , wherein the host cell is from a cell line used in good manufacturing practices (GMP) for manufacture of the poxvirus.

23 . The method of any one of claims 1 - 22 , wherein the host cell is a Human A549 cell, a HeLa cell, a 239 cell, or a primate Vero cell.

24 . The method of any one of claims 1 - 23 , further comprising removing un-adsorbed poxvirus prior to incubating the infected host cell in the culture medium.

25 . A method of producing poxviruses at an increased growth rate and/or titer in cells, the method comprising:
contacting a host cell with an effective amount of a nucleocytoplasmic transport inhibitor; contacting the host cell with a poxvirus of interest under conditions that permit the poxvirus of interest to adsorb to a surface of the host cell; and culturing the host cell to produce progeny of the poxvirus of interest.

26 . The method of claim 25 , further comprising harvesting the progeny of the poxvirus of interest.

27 . The method of claim 25 or claim 26 , wherein the poxvirus of interest is genetically modified.

28 . The method of any one of claims 25 - 27 , wherein the nucleocytoplasmic transport inhibitor comprises one or more of Leptomycin A, Leptomycin B, Ratjadone A, Ratjadone B, Ratjadone C, Ratjadone D, Anguinomycin A, Goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthol, KOS 2464, PKF050-638, CBS9106, or Selinexor.

29 . The method of any one of claims 25 - 28 , wherein the host cell is cultured in a presence of an effective amount of the nucleocytoplasmic transport inhibitor.

30 . The method of any one of claims 25 - 29 , wherein the poxvirus of interest is an oncolytic virus candidate.

31 . The method of any one of claims 25 - 30 , wherein the poxvirus of interest is a Leporipoxvirus.

32 . The method of claim 31 , wherein the Leporipoxvirus is a myxoma virus.

33 . The method of any one of claims 25 - 30 , wherein the poxvirus of interest is an Orthopoxvirus.

34 . The method of claim 33 , wherein the Orthopoxvirus is a vaccinia virus.

35 . The method of claim 34 , wherein the vaccinia virus is a vaccinia virus strain selected from the group consisting of Lister, Wyeth, Western Reserve, Modified Vaccinia virus Ankara, and LC16m series.

36 . The method of claim 33 , wherein the Orthopoxvirus is a Raccoonpox virus.

37 . The method of any one of claims 25 - 30 , wherein the poxvirus of interest is a Capripox virus.

38 . The method of claim 37 , wherein the Capripox virus is an Orf virus.

39 . The method of any one of claims 25 - 38 , wherein the host cell is an immortalized human or primate cell.

40 . The method of any one of claims 25 - 39 , wherein the host cell is from a cell line used in good manufacturing practices (GMP) for manufacture of the poxvirus of interest.

41 . The method of any one of claims 25 - 40 , wherein the host cell is a Human A549 cell, a HeLa cell, a 239 cell, or a primate Vero cell.

42 . The method of any one of claims 25 - 41 , wherein the host cell is contacted with the poxvirus of interest at a multiplicity of infection (MOI) of between about 0.01 and 1.0.

43 . The method of any one of claims 25 - 42 , further comprising removing un-adsorbed poxvirus of interest prior to the culturing.

44 . The method of any one of claims 25 - 43 , wherein the effective amount of the nucleocytoplasmic transport inhibitor is between about 0.0005 μM and about 0.5 μM.

Assignees

• Univ Arizona State

Inventors

• Mcfadden Douglas Grant
• Rahman Mohammed Masmudur

Classifications

• C12N2710/24151

  Methods of production or purification of viral material · CPC title

• C12N7/00 Primary

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

• C12N2710/24051

  Methods of production or purification of viral material · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title