# US 11,478,541 B2 · Method for inactivating Zika virus and for determining the completeness of inactivation

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- Number: US 11,478,541 B2
- Document ID: US11478541B2
- Status: Granted
- Kind: B2
- Application number: 16/761,340
- Filed: 2018-11-05
- Published: 2021-06-17
- Granted: 2022-10-25
- Earliest claimed: 2017-11-03
- Assignees: Takeda Vaccines, Inc.
- Applicants: Takeda Vaccines, Inc.
- Inventors: Jill A. Livengood; Holli Giebler; Hansi Dean; Tatsuki Satou; Raman Rao; Jackie Marks; Mark Lyons; Asae Shintani; James Gifford
- Web page: https://patentgrep.com/patent/US11478541B2

## CPC

- A61K 39/12: Viral antigens
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61P 31/14: for RNA viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 2039/5252: {inactivated (killed)}
- A61K 2039/54: {characterised by the route of administration}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- A61K 2039/55505: {Inorganic adjuvants}
- C12N 2770/00011: Details
- C12N 2770/24034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2770/24071: Demonstrated in vivo effect
- C12N 2770/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2770/24151: Methods of production or purification of viral material
- C12N 2770/24163: by chemical treatment
- C12N 2770/24164: by serial passage
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present disclosure relates to methods for inactivating a Zika virus which can be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation.

## Claims

### Claim 1

A method for determining the completeness of inactivation of an arbovirus preparation, comprising, the steps of: (i) inoculating, cultured insect cells with an arbovirus preparation which was subjected to an inactivation step and incubating the insect cells for a first period of time, thereby producing an insect cell supernatant; (ii) inoculating cultured mammalian cells with the insect cell supernatant produced in (i) and incubating the mammalian cells for a second period of time; and (iii) determining whether the arbovirus preparation contains a residual replicating virus that produces a cytopathic effect on the mammalian cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the insect cells are selected from CCL-125 cells, Aag-2 cells, RML-12 cells, C6/36 cells, C7-10 cells, AP-61 cells, A.t. GRIP-1 cells, A.t. GRIP-2 cells, A.t. GRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells and TRA-171 cells.

### Claim 3 (depends on 2)

The method of claim 2, wherein the insect cells are C6/36 cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first period of time is 3 to 7 days.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mammalian cells are selected from Vero cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (deposit number DSM ACC 2219), BHK21-F cells, HKCC cells and Chinese hamster ovary cells (CHO cells).

### Claim 6 (depends on 5)

The method of claim 5, wherein the mammalian cells are Vero cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the second period of time is 3 to 14 days.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method is capable of detecting less than 1.0 TCID50 of the arbovirus.

### Claim 9 (depends on 1)

The method of claim 1, wherein the arbovirus preparation was subjected to a treatment with detergent, formaldehyde, hydrogen peroxide, beta-propiolactone (BPL), binary ethylamine (BET), acetyl ethyleneimine, methylene blue, or psoralen.

### Claim 10 (depends on 1)

The method of claim 1, wherein the arbovirus is a flavivirus or an alphavirus.

### Claim 11 (depends on 10)

The method of claim 10, wherein the arbovirus is a Zika virus, a West Nile virus, a Yellow Fever virus, a Japanese Encephalitis virus, a tick borne-encephalitis virus, a dengue virus, a St. Louis Encephalitis virus, a Chikungunya virus, a O′nyong′nyong virus or a Mayarovirus.

### Claim 12 (depends on 11)

The method of claim 11, wherein the arbovirus is a Zika virus.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the method for determining the completeness of inactivation of a Zika virus preparation, comprises:
(i) inoculating C6/36 cells with a Zika virus preparation which was subjected to an inactivation step and incubating the C6/36 cells for a first period of tune, thereby producing a C6/36 cell supernatant;
(ii) inoculating Vero cells with the C6/36 cell supernatant produced in (i) and incubating the Vero cells for a second period of time, and
(iii) determining whether the Zika virus preparation contains a residual replicating virus that produces a cytopathic effect on the Vero cells.

### Claim 14 (depends on 13)

The method of claim 13, wherein the first period of time is 3 to 7 days.

### Claim 15 (depends on 13)

The method of claim 13, wherein the second period of time is 3 to 14 days.

### Claim 16 (depends on 1)

A pharmaceutical composition comprising an inactivated Zika virus preparation, wherein the inactivated Zika virus preparation comprises less than 1.0 TCID50 of residual replicating virus as determined by the method of claim 1.

### Claim 17 (depends on 16)

The pharmaceutical composition according to claim 16, having a residual formaldehyde content of less than 0.5 μg/mL.