Tagged hepadnavirus E antigen and its use in screening antiviral substances

Patent metadata

Field	Value
Publication number	US-10214567-B2
Application number	US-201815981316-A
Country	US
Kind code	B2
Filing date	May 16, 2018
Priority date	Jun 20, 2014
Publication date	Feb 26, 2019
Grant date	Feb 26, 2019

Abstract

The present invention relates to methods and uses for screening anti-hepadnaviral substances, wherein the substances are screened for the capacity to inhibit covalently closed circular (ccc) DNA of a hepadnavirus, like hepatitis B virus. The methods and uses take advantage of cells comprising a nucleic sequence encoding a tagged hepadnavirus e antigen, like Hepatitis B virus e antigen (HBeAg). Furthermore, the present invention provides nucleic acid sequences encoding a tagged hepadnavirus e antigen and proteins encoded thereby. Also kits for use in the screening methods are provided.

First claim

The invention claimed is:

1. A nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen, wherein the nucleic acid molecule comprises a sequence encoding one or more tags, wherein the sequence is inserted into an epsilon structure as encoded by a hepadnavirus genome, wherein said nucleic acid molecule comprising a sequence encoding the one or more tag is inserted between nucleotides corresponding to position C1902 and position A1903 of the HBV genome, wherein said nucleic acid molecule comprises 5′ of the sequence encoding the one or more tag a sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome and wherein the sequence that is capable of forming base pairs with said lower stem of the epsilon structure as encoded by a hepadnavirus genome is capable of forming base pairs with nucleotides corresponding to positions T1849 to A1854 of the HBV genome.

2. The nucleic acid molecule of claim 1 , wherein said hepadnavirus is Hepatitis B virus (HBV) and wherein said hepadnavirus e antigen is Hepatitis B virus e antigen (HBeAg).

3. The nucleic acid molecule of claim 1 , wherein said tagged hepadnavirus e antigen contains only one tag; or wherein said tagged hepadnavirus e antigen contains two or more tags.

4. The nucleic acid molecule of claim 3 , wherein said tag is selected from the group consisting of a hemagglutinin (HA) tag, His-tag, Flag-tag (like 1×Flag-tag or a 3×Flag-tag), c-myc-tag, V5-tag, and C9-tag.

5. The nucleic acid molecule of claim 4 ,
wherein the nucleic acid sequence encoding the HA tag is shown in SEQ ID NO: 1;
wherein the nucleic acid sequence encoding the His-tag is shown in SEQ ID NO: 2;
wherein the nucleic acid sequence encoding the 1×Flag-tag is shown in SEQ ID NO: 3;
wherein the nucleic acid sequence encoding the 3×Flag-tag is shown in SEQ ID NO: 7;
wherein the nucleic acid sequence encoding the c-myc-tag is shown in SEQ ID NO: 4;
wherein the nucleic acid sequence encoding the V5-tag is shown in SEQ ID NO: 5; and/or
wherein the nucleic acid sequence encoding the C9-tag is shown in SEQ ID NO: 6; or
wherein the amino acid sequence of the HA tag is shown in SEQ ID NO: 8;
wherein the amino acid sequence of the His-tag is shown in SEQ ID NO: 9;
wherein the amino acid sequence of the 1×Flag-tag is shown in SEQ ID NO: 10;
wherein the amino acid sequence of the 3×Flag-tag is shown in SEQ ID NO: 14;
wherein the amino acid sequence of the c-myc-tag is shown in SEQ ID NO: 11;
wherein the amino acid sequence of the V5-tag is shown in SEQ ID NO: 12; and/or
wherein the amino acid sequence of the C9-tag is shown in SEQ ID NO: 13.

6. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a hepadnavirus precore protein.

7. The nucleic acid molecule of claim 6 , wherein the nucleic acid sequence encoding a hepadnavirus precore protein is shown in SEQ ID NO: 15; or
wherein the amino acid sequence of the hepadnavirus precore protein is shown in SEQ ID NO: 17.

8. The nucleic acid molecule of claim 6 , wherein said nucleic acid sequence encoding the one or more tag is 3′ downstream of the nucleic acid sequence encoding the N-terminal 29 amino acids of a hepatitis B virus precore protein.

9. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule comprises a hepadnavirus genome, such as a Hepatitis B virus (HBV) genome as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33 or 34.

10. The nucleic acid molecule of claim 9 , wherein said HBV genome is a genome of HBV subgenotype ayw.

11. The nucleic acid molecule of claim 1 , wherein the nucleic acid sequence of the epsilon structure as encoded by a HBV genome is shown in SEQ ID NO: 25.

12. The nucleic acid molecule of claim 1 , wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome consists of the sequence shown in SEQ ID NO: 26; or
wherein the sequence that is capable of forming base pairs with the lower stem of the epsilon structure as encoded by a hepadnavirus genome encodes a polypeptide as shown in SEQ ID NO: 40.

13. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence as shown in SEQ ID NO: 41; or
wherein the nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen comprises a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO: 42.

14. The nucleic acid molecule of claim 2 , wherein the nucleic acid sequence encoding the tagged HBeAg is shown in SEQ ID NO: 20; or
wherein the amino acid sequence of the tagged HBeAg is shown in SEQ ID NO: 22.

15. The nucleic acid molecule of claim 2 , wherein said tagged hepadnavirus e antigen contains only one tag; or wherein said tagged hepadnavirus e antigen contains two or more tags.

16. The nucleic acid molecule of claim 7 , wherein said nucleic acid sequence encoding the one or more tag is 3′ downstream of the nucleic acid sequence encoding the N-terminal 29 amino acids of a hepatitis B virus precore protein.

17. The nucleic acid molecule of claim 6 , wherein the nucleic acid sequence encoding the tagged HBV precore protein is shown in SEQ ID NO: 19; or
wherein the amino acid sequence of the tagged HBV precore protein is shown in SEQ ID NO: 21.

18. The nucleic acid molecule of claim 2 , wherein the nucleic acid sequence encoding the HBeAg is shown in SEQ ID NO: 16; or
wherein the amino acid sequence of the HBeAg is shown in SEQ ID NO: 18.

19. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule comprises 3′ of the sequence encoding the one or more tag a sequence encoding a linker.

20. The nucleic acid molecule of claim 19 , wherein said sequence encoding a linker consists of the sequence GGC; or wherein said sequence encodes a glycine residue.

Assignees

• Univ Drexel
• Baruch S Blumberg Inst
• Hoffmann La Roche

Inventors

• Guo Haitao
• Cai Dawei
• Cuconati Andrea
• Ji Changhua

Classifications

• G01N2333/02

  Hepadnaviridae, e.g. hepatitis B virus · CPC title

• C12N2830/006

  tet repressible · CPC title

• G01N33/576

  for hepatitis · CPC title

• C07K2319/40

  containing a tag for immunodetection, or an epitope for immunisation · CPC title

• C07K14/005 Primary

  from viruses · CPC title