INFECTIOUS HEPATITIS E VIRUS GENOTYPE 3 RECOMBINANTS

• US 20130302790A1
• Filed: 01/10/2012
• Published: 11/14/2013
• Est. Priority Date: 01/10/2011
• Status: Active Grant

• Current Assignee: Royal Cornwall Hospital Trust, United States Secretary of Health and Human Services is the head of the United States Department of Health and Human Services
• Original Assignee: Royal Cornwall Hospital Trust, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Emerson, Suzanne U., Purcell, Robert H., Shukla, Priyanka, Nguyen, Hanh T., Dalton, Harry R., Bendall, Richard
• Granted Patent: US 9,181,530 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2770/28121 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2770/28122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/28134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/28143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2770/28151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2770/28152 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D

Abstract

The invention relates to the discovery of an HEV strain from a chronically infected patient. The virus grow unusually well in numerous cell cultures. Thus, the invention provides cell cultures, vectors, and vaccine compositions based on the virus. The invention relates, in part, on the identification of a new strain of HEV genotype 3 virus. Strain Kernow-C1 (genotype 3) of HEV, which was isolated from a chronically infected patient, was used to identify human, pig and deer cell lines permissive for infection. Adaptation of the Kernow-C 1 strain to growth in human hepatoma cells selected for a rare virus recombinant that contained an insertion of 174 ribonucleotides (58 amino acids) of a human ribosomal protein gene and additional mutations.

Claims

1. An infectious hepatitis E virus (HEV) type 3 cDNA clone, wherein the infectious clone comprises an insert, relative to the HEV nucleic acid sequence set forth in SEQ ID NO:

5, in the region of the nucleic acid sequence that encodes the hypervariable region of ORF1, wherein the insert encodes a polypeptide sequence having at least 85% identity to SEQ ID NO;

9.

2. The infectious HEV type 3 cDNA clone of claim 1, wherein the insert encodes a polypeptide sequence that comprises SEQ ID NO:

9.

3. The infectious HEV type 3 cDNA clone of claim 1, wherein the insert is positioned in the hypervariable region of ORF1 such that the first amino acid encoded by the insert occurs at position 750 relative to SEQ ID NO:

6.

21. A cell culture system comprising cells that comprise a cDNA clone of claim 1.

22. A method of producing a vaccine, the method comprising introducing an RNA obtained from a cDNA clone of claim 1 into cells and obtaining virus produced by the RNA.

23. The method of claim 22, wherein the RNA is introduced into the cells as virus particles.

24. The method of claim 22, wherein the cells are MRC 5 lung cells.

25. A method of producing a vaccine, the method comprising:

introducing an RNA obtained from a cDNA clone of claim 1 into a cell line, wherein the RNA is incapable of producing ORF3; and

obtaining virus produced by the RNA.

26. The method of claim 25, wherein the insert encodes an in-frame polypeptide sequence that has at least 80% identity, or at least 90% identity, to the amino acid sequence set forth in SEQ ID NO:

9;

or encodes a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO;

9.

29. The method of claim 25, wherein the insert is positioned in the hypervariable region of ORF1 such that the first amino acid encoded by the insert occurs at position 750 relative to SEQ ID NO:

6.

30. A method of assessing the virus status of a product of interest subjected to a virus treatment process, the method comprising:

adding HEV virus produced using a cDNA clone of claim 1 into the product;

subjecting the product to a virus treatment process that removes and/or inactivates virus present in the product; and

determining the level of added virus in the product remaining after or during the virus treatment process.

4. An infectious hepatitis E virus (HEV) cDNA clone, wherein the cDNA clone has at least 75% sequence identity to SEQ ID NO:

1 and comprises an insert in the region of the nucleic acid sequence that encodes the hypervariable region of ORF1, relative to the HEV nucleic acid sequence set forth in SEQ ID NO;

5.

5. The HEV cDNA clone of claim 4, wherein the cDNA clone has at least 85% identity, or at least 95% identity, to SEQ ID NO:

1;

or comprises the nucleic acid sequence of SEQ ID NO;

1.

8. The HEV cDNA clone of claim 4, wherein the insert encodes an in-frame amino acid sequence of 30 to 100 amino acids in length.

9. The HEV cDNA clone of claim 8, wherein the insert encodes an amino acid sequence that has at least 80% identity, at least 90% identity, or at least 95% identity to SEQ ID NO:

9;

or encodes the amino acid sequence set forth in SEQ ID NO;

9.

13. The HEV cDNA clone of claim 4, wherein the insert has at least 90% nucleic acid sequence identity to the underlined portion of SEQ ID NO:

10.

14. The HEV cDNA clone of claim 13, wherein the insert comprises the underlined portion of SEQ ID NO:

10.

6. (canceled)

7. (canceled)

10-12. -12. (canceled)

15. An infectious cDNA clone comprising a hepatitis E virus (HEV) nucleic acid sequence, wherein the infectious clone comprises an insert in the region of the nucleic acid sequence that encodes the hypervariable region of ORF1 and wherein the insert encodes an amino acid sequence of at least 10 amino acids in length.

16. The cDNA clone of claim 15, wherein the insert encodes an in-frame polypeptide sequence that has at least 80% identity, or at least 90% identity, to the amino acid sequence set forth in SEQ ID NO:

9;

or comprises the amino acid sequence of SEQ ID NO;

9.

19. The cDNA clone of claim 15, wherein the cDNA clone is a genotype 3 HEV or genotype 1 HeV.

17. (canceled)

18. (canceled)

20. (canceled)

27. (canceled)

28. (canceled)

31. (canceled)

32. (canceled)