# US 12,029,787 B2 · Method for generating recombinant poxviruses

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- Number: US 12,029,787 B2
- Document ID: US12029787B2
- Status: Granted
- Kind: B2
- Application number: 17/365,746
- Filed: 2021-07-01
- Published: 2022-03-10
- Granted: 2024-07-09
- Earliest claimed: 2020-07-01
- Assignees: University of Connecticut
- Applicants: University of Connecticut
- Inventors: Paulo H. Verardi; Brittany Jasperse; Caitlin M. O'Connell; Yuxiang Wang
- Web page: https://patentgrep.com/patent/US12029787B2

## CPC

- A61K 39/275: Poxviridae, e.g. avipoxvirus
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/86: Viral vectors
- A61K 2039/5256: {expressing foreign proteins}
- C12N 2710/24034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/24041: Use of virus, viral particle or viral elements as a vector
- C12N 2710/24051: Methods of production or purification of viral material
- C12N 2710/24143: viral genome or elements thereof as genetic vector
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are methods for the rapid generation of recombinant poxviruses, for example, to enable vaccine development.

## Claims

### Claim 1

A method of generating recombinant poxvirus, wherein the poxvirus is vaccinia virus (VACV), and wherein a recombinant vaccina virus (rVACV) is generated expressing a gene of interest (GOI) the method comprising
homologously recombining a replication-inducible VACV (vIND) parental virus genome and a DNA shuttle vector to generate the rVACVs with the GOI,
wherein expression of one or more essential genes in the parental vIND is controlled by an inducible mechanism, wherein the one or more essential genes is required for viral replication of the vIND,
wherein the DNA shuttle vector comprises an expression cassette comprising the gene of interest operably linked to a promoter, the cassette flanked by right and left regions that are homologous to the parental vIND virus genome, wherein the right flanking region is homologous to a region in the parental vIND genome that is upstream of the inducible mechanism and the left flanking region is homologous to a region in the parental vIND genome that is downstream of the inducible gene, such that homologous recombination between vIND and the DNA shuttle vector leads to insertion of the expression cassette into the parental vIND virus genome between the homologous regions and results in loss of the inducible mechanism control on the expression of the one or more essential genes in the rVACV,
transfecting the DNA shuttle vector into parental vIND-infected cells, and
purifying the rVACVs from the parental vIND by serial passage in the absence of the parental inducer.

### Claim 2 (depends on 1)

The method of claim 1, wherein transfecting the DNA shuttle vector into parental vIND-infected cells is in the presence of the parental inducer.

### Claim 3 (depends on 2)

The method of claim 2, wherein the rVACVs further express enhanced green fluorescence protein, and the method further comprises collecting enhanced green fluorescence protein (EGFP+) plaques between a first purification step and a second purification step, and collecting the EGFP+ plaques after a purification step to obtain purified a rVACV clone and optionally amplifying the purified rVACV clone.

### Claim 4 (depends on 1)

The method of claim 1, wherein expression of the GOI is controlled by a GOI inducer such that homologous recombination renders the rVACV replication inducible, and wherein the GOI inducer is a tetracycline antibiotic or isopropyl β-D-1-thiogalactopyranoside (IPTG).

### Claim 5 (depends on 4)

The method of claim 4, wherein the tetracycline antibiotic is selected from chlortetracycline, oxytetracycline, tetracycline, demethylchlortetracycline, rolitetracycline, lymecycline, clomocycline, methacycline, doxycycline, minocycline, and tertiary-butylglycylamidominocycline.

### Claim 6 (depends on 1)

The method of claim 1, wherein the rVACV further comprises a screening marker selected from dsRed, EGFP, gusA, or lacZ.

### Claim 7 (depends on 1)

The method of claim 1, wherein the one or more essential genes required for replication of the parental vIND is one or more of D6R, A6L, F17R, A3R, E8R.

### Claim 8 (depends on 7)

The method of claim 7, wherein the parental inducer is a tet operon with a tet operator element, or a lac operon with a lac operator element.

### Claim 9 (depends on 8)

The method of claim 8, wherein the lac operator element is controlled by a P11 promoter.