# US 9,655,961 B2 · Recombinant measles virus expressing chikungunya virus polypeptides and their applications

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- Number: US 9,655,961 B2
- Document ID: US9655961B2
- Status: Granted
- Kind: B2
- Application number: 14/430,975
- Filed: 2013-09-26
- Published: 2015-08-27
- Granted: 2017-05-23
- Earliest claimed: 2012-09-27
- Assignees: Institut Pasteur; Themis Bioscience GmbH; Centre National De La Recherche Scientifique
- Applicants: Institut Pasteur; Themis Bioscience GMBH; Centre National De La Recherche Scientifique
- Inventors: Frederic Tangy; Samantha Brandler; Philippe Despres; Andre Habel
- Web page: https://patentgrep.com/patent/US9655961B2

## CPC

- A61K 39/12: Viral antigens
- A61P 31/14: for RNA viruses
- C07K 14/005: from 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)
- C12N 15/86: Viral vectors
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/5256: {expressing foreign proteins}
- A61K 2039/5258: {Virus-like particles}
- A61K 2039/70: {Multivalent vaccine}
- C12N 2760/18421: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/18434: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18443: viral genome or elements thereof as genetic vector
- C12N 2760/18444: Chimeric viral vector comprising heterologous viral elements for production of another viral vector
- C12N 2770/36122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/36123: Virus like particles [VLP]
- C12N 2770/36134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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 invention relates to recombinant Measles virus expressing Chikungunya virus polypeptides, and concerns in particular virus like particles (VLP) that contain envelope and capsid proteins of a Chikungunya virus at their surface. These particles are recombinant infectious particles able to replicate in a host after an administration. The invention provides means, in particular nucleic acids, vectors, cells and rescue systems to produce these recombinant infectious particles. The invention also relates to the use of these recombinant infectious particles, in particular under the form of a composition, more particularly in a vaccine formulation, for the treatment or prevention of an infection by Chikungunya virus.

## Claims

### Claim 1

A nucleic acid construct which comprises:
(1) a polynucleotide encoding the C-E3-E2-6K-E1 structural proteins of a Chikungunya virus (CHIKV); and
(2) a cDNA molecule which encodes a full-length, infectious antigenomic (+) RNA strand of a measles virus (MV);
wherein the polynucleotide encoding the C-E3-E2-6K-E1 structural proteins and the cDNA molecule are operatively linked.

### Claim 2 (depends on 1)

The nucleic acid construct according to claim 1, wherein said cDNA molecule consists of a number of nucleotides that is a multiple of six.

### Claim 3 (depends on 1)

The nucleic acid construct according to claim 1, comprising the following polynucleotides from 5′ to 3′:
(a) a polynucleotide encoding a full length of N protein of the MV,
(b) a polynucleotide encoding a full length of P protein of the MV,
(c) the polynucleotide encoding the C-E3-E2-6K-E1 structural proteins of CHIKV,
(d) a polynucleotide encoding a full length of M protein of the MV,
(e) a polynucleotide encoding a full length of F protein of the MV,
(f) a polynucleotide encoding a full length of H protein of the MV, and
(g) a polynucleotide encoding a full length of L protein of the MV,
wherein said polynucleotides are operably linked in the nucleic acid construct and under a control of viral replication and transcription regulatory sequences.

### Claim 4 (depends on 1)

The nucleic acid construct according to claim 1, wherein said Measles virus is an attenuated virus strain selected from the group consisting of the Schwarz strain, the Zagreb strain, the AIK-C strain and the Moraten strain.

### Claim 5 (depends on 1)

The nucleic acid construct according to claim 1, wherein said polynucleotide encoding the C-E3-E2-6K-E1 structural proteins of CHIKV has been optimized for a Macacca codon usage or has been optimized for a human codon usage.

### Claim 6 (depends on 1)

The nucleic acid construct according to claim 1, wherein said polynucleotide encoding the C-E3-E2-6K-E1 structural proteins comprises at least one measles editing-like sequence selected from AAAGGG, AAAAGG, GGGAAA, and GGGGAA that has been mutated.

### Claim 7 (depends on 1)

The nucleic acid construct according to claim 1, wherein said Chikungunya virus is from the strain designated 06-49 strain.

### Claim 8 (depends on 1)

The nucleic acid construct according to claim 1, wherein the encoded E2 structural protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15.

### Claim 9 (depends on 1)

The nucleic acid construct according to claim 1, wherein said nucleic acid construct encodes C-E3-E2-6K-E1 structural proteins having a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 28.

### Claim 10 (depends on 1)

The nucleic acid construct according to claim 1, wherein said nucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 27, and SEQ ID NO: 31.

### Claim 11 (depends on 1)

A transfer vector plasmid, comprising the nucleic acid construct of claim 1.

### Claim 12 (depends on 11)

The vector according to claim 11, wherein said vector is pMV-CHIKV.

### Claim 13 (depends on 1)

Transformed eukaryotic cells comprising the nucleic acid construct according to claim 1.

### Claim 14 (depends on 1)

Recombinant infectious replicating MV-CHIKV particles produced by a method comprising expressing the nucleic acid construct according to claim 1 in a host cell comprising an RNA polymerase recognized by said host cell, a nucleoprotein (N) of a MV, and a phosphoprotein (P) of a MV.

### Claim 15 (depends on 14)

The recombinant infectious replicating MV-CHIKV particles of claim 14, wherein said virus particles are encoded by a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 31.

### Claim 16 (depends on 14)

A composition comprising the recombinant infectious replicating MV-CHIKV particles according to claim 14, CHIKV-C-E3-E2-6K-E1 Virus Like Particles (VLPs) and a pharmaceutically acceptable vehicle.

### Claim 17 (depends on 16)

A method of inducing a protective immune response against CHIKV in a host, comprising administering the composition according to claim 16 to the host.

### Claim 18 (depends on 16)

A method of treating or preventing an infection by CHIKV in a host, comprising administering the composition according to claim 16 to the host.

### Claim 19 (depends on 14)

A process to rescue recombinant measles virus (MV) expressing the C-E3-E2-6K-E1 structural proteins of a Chikungunya virus (CHIKV) and CHIKV-C-E3-E2-6K-E1 Virus Like Particles (VLPs), comprising:
1) cotransfecting helper cells that stably express T7 RNA polymerase, and measles N and P proteins with (i) a transfer vector plasmid according to claim 14, and (ii) a vector, encoding the MV L polymerase;
2) cultivating said cotransfected helper cells in conditions enabling the production of MV-CHIKV recombinant virus;
3) propagating the thus produced recombinant virus by co-cultivating said helper cells of step 2) with cells enabling said propagation; and
4) recovering replicating MV-CHIKV recombinant virus expressing the C-E3-E2-6K-E1 structural proteins of CHIKV and CHIKV-C-E3-E2-6K-E1 VLPs.

### Claim 20 (depends on 19)

The process according to claim 19, wherein the transfer vector plasmid comprises a sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 31.