Methods for improved poxvirus yields

Patent metadata

Field	Value
Publication number	US-12398375-B2
Application number	US-201917266499-A
Country	US
Kind code	B2
Filing date	Aug 7, 2019
Priority date	Aug 8, 2018
Publication date	Aug 26, 2025
Grant date	Aug 26, 2025

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 of producing a poxvirus at an increased growth rate or titer, the method comprising:
contacting a host cell with a nucleocytoplasmic transport inhibitor at a concentration of at least 0.01 μM; wherein the host cell is a transformed cell;
contacting the host cell with a poxvirus, wherein the host cell is contacted with the poxvirus at a multiplicity of infection (MOI) less than one; and
culturing the host cell to produce progeny of the poxvirus at an increased growth rate compared to the growth rate in identical conditions lacking the nucleocytoplasmic transport inhibitor or an at least 3-fold increase in titer compared to that produced in identical conditions lacking the nucleocytoplasmic transport inhibitor,
wherein the nucleocytoplasmic transport inhibitor is one or more selected from the group consisting of: Leptomycin A, Leptomycin B, Ratjadone A, Ratjadone B, Ratjadone C, Ratjadone D, and Anguinomycin A; and
wherein the poxvirus is myxoma virus.

2. The method of claim 1 , further comprising harvesting the progeny of the poxvirus.

3. The method of claim 1 , wherein the host cell is contacted with the nucleocytoplasmic transport inhibitor prior to contacting the host cell with the poxvirus.

4. The method of claim 1 , wherein the host cell is contacted with the nucleocytoplasmic transport inhibitor after contacting the host cell with the poxvirus.

5. The method of claim 1 , wherein the host cell is contacted with the nucleocytoplasmic transport inhibitor and the poxvirus at the same time.

6. The method of claim 1 , wherein the poxvirus is genetically modified.

7. The method of claim 1 , wherein the culturing is conducted under good manufacturing practices (GMP) for manufacture of the poxvirus.

8. The method of claim 1 , wherein the host cell is contacted with the poxvirus at a multiplicity of infection (MOI) of between about 0.01 and about 0.1.

9. The method of claim 1 , wherein the increase in viral titer is evaluated 24 hours or 48 hours after contacting the host cell with the poxvirus.

10. A method for replicating a leporipoxvirus, the method comprising:
exposing a host cell to a nucleocytoplasmic transport inhibitor at a concentration of at least 0.01 μM; wherein the host cell is a transformed cell;
infecting the host cell with a leporipoxvirus at a multiplicity of infection (MOI) less than 1, thereby generating an infected host cell; and
incubating the infected host cell in a culture medium under conditions that allow for replication of the leporipoxvirus,
wherein the rate of replication is greater than the rate of replication in identical conditions lacking the nucleocytoplasmic transport inhibitor or an at least 3-fold increase in titer compared to that produced in identical conditions lacking the nucleocytoplasmic transport inhibitor
wherein the nucleocytoplasmic transport inhibitor is one or more selected from the group consisting of: Leptomycin A, Leptomycin B, Ratjadone A, Ratjadone B, Ratjadone C, Ratjadone D, and Anguinomycin A; and
wherein the leporipoxvirus is a myxoma virus.

11. The method of claim 1 , wherein the host cell is A549 cell.

12. The method of claim 10 , wherein the host cell is A549 cell.

Assignees

• Univ Arizona State

Inventors

• Mcfadden Douglas Grant
• Rahman Mohammed Masmudur

Classifications

• A61P31/00

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

• C12N2710/24151

  Methods of production or purification of viral material · CPC title

• C12N2710/24051

  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