# US 11,028,133 B2 · Modified flavivirus envelope sequences comprising unique glycosylation sites

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- Number: US 11,028,133 B2
- Document ID: US11028133B2
- Status: Granted
- Kind: B2
- Application number: 16/303,588
- Filed: 2017-05-22
- Published: 2019-10-03
- Granted: 2021-06-08
- Earliest claimed: 2016-05-20
- Assignees: Excivion LTD
- Applicants: Excivion Ltd.
- Inventors: Peter Laing
- Web page: https://patentgrep.com/patent/US11028133B2

## CPC

- C07K 14/1825: {Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue}
- A61K 39/12: Viral antigens
- A61P 31/14: for RNA viruses
- C07K 14/005: from viruses
- G01N 33/56983: {Viruses}
- A61K 2039/5254: {avirulent or attenuated}
- A61K 2039/5258: {Virus-like particles}
- A61K 2039/53: {DNA (RNA) vaccination}
- C12N 2770/24122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- G01N 2333/185: Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
- 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
- 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)
- 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
- 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)
- 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 isolated recombinant analogues of flavivirus E-protein fusion loops comprising at least one glycosylation site for an N-linked glycan that is not present in the natural flavivirus E-protein fusion loop sequence, wherein the at least one glycosylation site is an N-linked glycosylation sequon (Asn-X-Ser/Thr) and the Asn (N) residue of the sequon occupies any of positions 98-110 (SEQ ID NO: 1) (DRGWGNGCGLFGK) of the natural flavivirus E-protein fusion loop amino acid sequence, wherein X is any amino acid residue except proline and Ser/Thr denotes a serine or threonine residue.

## Claims

### Claim 1

An isolated recombinant glycosylation site modified flavivirus E-protein fusion loop epitope, wherein the epitope comprises at least one glycosylation site for an N-linked glycan that is not present in a natural flavivirus E-protein fusion loop sequence, wherein the at least one glycosylation site is an N-linked glycosylation sequon (Asn-X-Ser/Thr) and the Asn (N) residue of the sequon occupies any of positions 98-110 (DRGWGNGCGLFGK) SEQ ID NO: 1 of the natural flavivirus E-protein fusion loop amino acid sequence, wherein X is any amino acid residue except proline and Ser/Thr denotes a serine or threonine residue.

### Claim 2 (depends on 1)

The isolated recombinant flavivirus E-protein fusion loop epitope according to claim 1 comprising two glycosylation sites that are not present in a natural flavivirus E-protein fusion loop sequence.

### Claim 3

An isolated recombinant flavivirus E-protein comprising a glycosylation site modified flavivirus E-protein fusion loop epitope, wherein the epitope comprises at least one glycosylation site for an N-linked glycan that is not present in a natural flavivirus E-protein fusion loop sequence, wherein the at least one glycosylation site is an N-linked glycosylation sequon (Asn-X-Ser/Thr) and the Asn (N) residue of the sequon occupies any of positions 98-110 (DRGWGNGCGLFGK) SEQ ID NO: 1 of the natural flavivirus E-protein fusion loop amino acid sequence, wherein X is any amino acid residue except proline and Ser/Thr denotes a serine or threonine residue.

### Claim 4 (depends on 3)

The isolated recombinant protein of claim 3, further comprising at least one additional N-linked glycan attached thereto.

### Claim 5 (depends on 3)

The isolated recombinant protein of claim 3, which is the product of expression of a recombinant DNA or RNA sequence.

### Claim 6 (depends on 3)

The isolated recombinant protein of claim 3, comprising an N-linked glycosylation sequon (Asn-X-Ser/Thr) such that an Asn (N) residue of the sequon occupies any of positions 98-101 and/or 106-110.

### Claim 7 (depends on 3)

The isolated recombinant protein of claim 3, wherein X is selected from the following 13 amino acid residues of Gly, His, Asn, Gln, Tyr, Val, Ala, Met, Ile, Lys, Arg, Thr or Ser.

### Claim 8 (depends on 3)

The isolated recombinant protein of claim 3, wherein the flavivirus E-protein is a dengue virus E-protein and the Asn (N) residue of the sequon occupies position 101, 108 or both 101 and 108 of the amino acid sequence of the flavivirus E-protein fusion loop or the flavivirus E-protein is a Zika E-protein and the Asn (N) residue of the sequon occupies position 100 of the amino acid sequence of the flavivirus E-protein fusion loop.

### Claim 9 (depends on 8)

The isolated recombinant protein of claim 8, wherein the flavivirus is a dengue virus and the amino acid sequence of the analogue flavivirus E-protein fusion loop 98-110 is selected from: DRGNGSGCGLNGS (SEQ ID NO: 2), DRGNGSGCGLFGK (SEQ ID NO: 3) and DRGWGNGCGLNGS (SEQ ID NO: 4).

### Claim 10 (depends on 8)

The isolated recombinant protein of claim 8, wherein the flavivirus is a Zika virus and the amino acid sequence of the analogue flavivirus E-protein fusion loop 98-110 is DRNHTNGCGLFGK (SEQ ID NO: 5).

### Claim 11 (depends on 1, 3)

A composition comprising the isolated recombinant glycosylation site modified flavivirus E-protein fusion loop epitope according to claim 1 or the isolated recombinant protein according to claim 3 and a diluent.

### Claim 12 (depends on 11)

The composition of claim 11, further comprising a pharmaceutically acceptable diluent, adjuvant and/or carrier.

### Claim 13 (depends on 12)

The composition of claim 12 comprising one or more flavivirus analogues selected from an analogue of DEN-1, an analogue of DEN-2, an analogue of DEN-3, an analogue of DEN-4 and an analogue of Zika.

### Claim 14 (depends on 12)

The composition of claim 12 comprising four dengue analogues representing each of the four dengue virus serotypes DEN-1 DEN-2 DEN-3 and DEN-4.

### Claim 15 (depends on 12)

The composition of claim 12 comprising a zika virus analogue.

### Claim 16 (depends on 12)

The composition of claim 12 comprising four dengue analogues representing each of the four dengue serotypes DEN-1 DEN-2 DEN-3 and DEN-4 and a zika virus analogue.

### Claim 17 (depends on 12)

The composition of claim 12, wherein the composition further comprises an adjuvant, and is prepared for administration to a subject in need thereof for prophylactic or therapeutic treatment of a flavivirus infection.

### Claim 18 (depends on 12)

The composition of claim 12 combined with and further comprising sera from a subject to determine if the subject is seropositive or seronegative for the flavivirus.