# US 2018/0008693 A1 · Development of mutations useful for attenuating dengue viruses and chimeric dengue viruses

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- Number: US 2018/0008693 A1
- Document ID: US20180008693A1
- Status: Published application
- Kind: A1
- Application number: 15/650,482
- Filed: 2017-07-14
- Published: 2018-01-11
- Earliest claimed: 2001-05-22
- Assignees: The United States of America, as represented by the Secretary, Dept. of Health and Human Services
- Applicants: The United States of America, as represented by the Secretary, Dept. of Health and Human Services
- Inventors: Stephen S. Whitehead; Brian R. Murphy; Kathryn A. Hanley; Joseph E. Blaney; Ching-Juh Lai
- Web page: https://patentgrep.com/patent/US20180008693A1

## CPC

- A61K 39/12: Viral antigens
- A61P 31/14: for RNA viruses
- A61P 37/04: Immunostimulants
- 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 7/04: Inactivation or attenuation; Producing viral sub-units
- A61K 39/295: Polyvalent viral antigens (vaccinia virus or variola virus A61K39/285); Mixtures of viral and bacterial antigens
- A61K 2039/5252: {inactivated (killed)}
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/5256: {expressing foreign proteins}
- A61K 2039/54: {characterised by the route of administration}
- A61K 2039/575: {humoral response}
- A61K 2039/70: {Multivalent vaccine}
- C12N 2770/24121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- 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 2770/24161: Methods of inactivation or attenuation
- C12N 2770/24162: by genetic engineering
- C12N 2770/24163: by chemical treatment
- C12N 2770/24171: Demonstrated in vivo effect
- C12Q 1/701: {Specific hybridization probes}
- C12Q 2600/156: Polymorphic or mutational markers
- G01N 2333/18: Togaviridae; Flaviviridae
- G01N 2500/10: involving cells
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A menu of mutations was developed that is useful in fine-tuning the attenuation and growth characteristics of dengue virus vaccines.

## Claims

### Claim 1

A flavivirus having a phenotype in which the viral genome is modified by the introduction of a mutation, singly or in combination, taken from the group consisting of the mutations of any of Table 1-37, preferably Table 37.

### Claim 2 (depends on 1)

The flavivirus of claim 1, further comprising the Δ30 mutation.

### Claim 3 (depends on 1)

The flavivirus of claim 1, wherein the flavivirus is a dengue virus type 1, a dengue virus type 2, a dengue virus type 3, or a dengue virus type 4.

### Claim at position 4

4-6. (canceled)

### Claim 7 (depends on 1)

The flavivirus of claim 1, wherein the flavivirus is a chimeric virus.

### Claim 8 (depends on 7)

The chimeric virus of claim 7 having a dengue 1 backbone, a dengue 2 backbone, a dengue 3 backbone, or a dengue 4 backbone.

### Claim at position 7

9-11. (canceled)

### Claim 12 (depends on 1)

The flavivirus of claim 1, wherein the phenotype is temperature sensitivity in Vero cells or the human liver cell line HuH-7.

### Claim 13 (depends on 1)

The flavivirus of claim 1, wherein the phenotype is host-cell restriction in mosquito cells or the human liver cell line HuH-7.

### Claim 14 (depends on 1)

The flavivirus of claim 1, wherein the phenotype is host-cell adaptation for improved replication in Vero cells.

### Claim 15 (depends on 1)

The flavivirus of claim 1, wherein the phenotype is attenuation in mice.

### Claim 16 (depends on 1)

A pharmaceutical composition comprising a pharmacologically acceptable vehicle and a flavivirus according to claim 1.

### Claim 17 (depends on 16)

A kit comprising a pharmaceutical composition according to claim 16 in a pack or dispenser device and instructions for administration.

### Claim 18 (depends on 1)

A method of producing neutralizing antibodies against dengue virus comprising the administration of a therapeutically effective amount of a pharmaceutical composition comprising a pharmacologically acceptable vehicle and a flavivirus according to claim 1.

### Claim 19 (depends on 18)

The method of claim 18, wherein administration is by subcutaneous, intradermal, or intramuscular injection.

### Claim 20 (depends on 1)

A tetravalent vaccine comprising a pharmacologically acceptable vehicle and a flavivirus according to claim 1.

### Claim 21 (depends on 1)

An live attenuated vaccine comprising a pharmacologically acceptable vehicle and a flavivirus according to claim 1.

### Claim 22 (depends on 21)

The live attenuated vaccine of claim 21 in unit dosage form having from about 102-109 plaque forming units (PFU)/ml.

### Claim 23 (depends on 1)

An inactivated vaccine comprising a pharmacologically acceptable vehicle and a flavivirus according to claim 1.

### Claim 24 (depends on 23)

The inactivated vaccine of claim 23 in unit dosage form having from about 0.1 to 50 μg of E protein/ml.

### Claim 25 (depends on 1)

A cDNA molecule encoding a flavivirus according to claim 1.

### Claim 26 (depends on 1)

An RNA molecule encoding a flavivirus according to claim 1.

### Claim 27 (depends on 1)

A method of preparing a flavivirus comprising:
(a) synthesizing full-length viral genomic RNA in vitro using a cDNA molecule that encodes a flavivirus according to claim 1;
(b) transfecting cultured cells with the viral genomic RNA to produce virus; and
(c) isolating the virus from the cultured cells.

### Claim 28 (depends on 1)

A method of making a pharmaceutical composition comprising combining a pharmacologically acceptable vehicle and a flavivirus according to claim 1.

### Claim 29

A method of identifying a mutation that restricts replication in human liver cells or a mutation that promotes growth in Vero cells comprising:
(a) introducing mutations into a dengue virus genome to make mutant viruses;
(b) screening the mutant viruses for a phenotype characterized by host-cell restriction in human liver cells or host-cell adaptation for improved replication in Vero cells; and
(c) determining the genetic basis for the phenotype by direct sequence analysis of the virus genome.

### Claim 30

(canceled)

### Claim 31

A method of assembling a menu of mutations for use in fine-tuning the attenuation and growth characteristics of recombinant dengue viruses comprising (a) introducing mutations into a dengue virus genome to make mutant viruses; (b) screening the mutant viruses for a phenotype characterized by temperature sensitivity in Vero cells or human liver cells, host cell restriction in mosquito cells or human liver cells, host-cell adaptation for improved replication in Vero cells, or attenuation in mice; (c) determining the genetic basis for the phenotype by direct sequence analysis of the virus genome; and (d) performing multiple iterations of steps (a)-(c), whereby a menu of mutations is assembled.

### Claim 32 (depends on 29)

The method of any of claim 29, further comprising introducing the mutation identified by said method into a recombinant dengue virus, and characterizing the resulting phenotype.