Tagged hepadnavirus e antigen and its use in screening antiviral substances

Patent metadata

Field	Value
Publication number	US-10072047-B2
Application number	US-201515309680-A
Country	US
Kind code	B2
Filing date	Jun 19, 2015
Priority date	Jun 20, 2014
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

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 method for assessing the capacity of a candidate molecule to inhibit covalently closed circular (ccc) DNA of a hepadnavirus comprising the steps of
(a) contacting a cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen with said candidate molecule; wherein the nucleic acid molecule comprises a sequence encoding one or more tags, wherein the sequence is inserted into the 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;
(b) assessing the level of the tagged hepadnavirus e antigen; and
(c) selecting a candidate molecule when the level of tagged hepadnavirus e antigen is decreased compared to a control.

2. The method 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 method 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 method of claim 3 , wherein said tag is selected from the group consisting of hemagglutinin (HA)-tag, His-tag, Flag-tag (like 1×Flag-tag or 3×Flag-tag), c-myc-tag, V5-tag and/or C9-tag.

5. The method 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: 2; and/or
wherein the amino acid sequence of the C9-tag is shown in SEQ ID NO: 13.

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

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

8. The method 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 method 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 method of claim 9 , wherein said HBV genome is a genome of HBV subgenotype ayw.

11. The method of claim 1 , wherein the epsilon structure as encoded by a hepadnavirus genome is the epsilon structure as encoded by an HBV genome as shown in SEQ ID NO: 25.

12. The method 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 method 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 method 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 method of claim 1 , wherein said step (a) further comprises a step (aa) which comprises culturing a cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a tagged hepadnavirus e antigen in conditions allowing
(i) the synthesis of hepadnavirus pregenomic (pg) RNA;
(ii) the reverse transcription of said synthesized pgRNA into a minus strand DNA;
(iii) the synthesis of a second plus strand DNA so that said minus strand DNA and said plus strand DNA form a double stranded relaxed circular DNA;
(iv) formation of cccDNA from said relaxed circular double stranded DNA;
(v) optionally restoration of conditions allowing the translation of the tagged hepadnavirus e antigen;
(vi) transcription of an mRNA encoding a tagged hepadnavirus e antigen;
(vii) translation of a tagged hepadnavirus e antigen,
wherein the restoration of conditions allowing the translation of the tagged hepadnavirus e antigen is the restoration of the start codon.

16. The method of claim 1 , wherein assessing the level of the tagged hepadnavirus e antigen according to step (b) is performed by ELISA, CLIA, or AlphaLISA.

17. The method of claim 1 , wherein assessing the level of the tagged hepadnavirus e antigen according to step (b) comprises the use of an antibody specifically recognizing said hepadnavirus e antigen and one or more antibodies specifically recognizing the one or more tags.

18. The method 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.

19. The method 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.

20. The method 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.

Assignees

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

Inventors

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

Classifications

• C07K2319/42

  containing a HA(hemagglutinin)-tag · CPC title

• G01N33/576

  for hepatitis · CPC title

• C12N2830/006

  tet repressible · CPC title

• C12N2730/10151

  Methods of production or purification of viral material · CPC title

• C12N2730/10122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title