POLYPEPTIDE FRAGMENTS OF THE HEPATITIS E VIRUS, THE VACCINE COMPOSITION COMPRISING SAID FRAGMENTS AND THE DIAGNOSTIC KITS

• US 20100143410A1
• Filed: 09/11/2009
• Published: 06/10/2010
• Est. Priority Date: 09/30/2000
• Status: Active Grant

• Current Assignee: Jun Zhang, Ningshao Xia, Shaowei Li, Shengxiang Ge, Ying Gu, Zhiqiang He
• Original Assignee: Jun Zhang, Ningshao Xia, Shaowei Li, Shengxiang Ge, Ying Gu, Zhiqiang He
• Inventors: ZHANG, Jun, GE, Shengxiang, XIA, Ningshao, GU, Ying, HE, Zhiqiang, LI, Shaowei
• Granted Patent: US 8,715,695 B2
• Time in Patent Office: Days
• US Class Current: 424/228.100
• CPC Class Codes: A61K 2039/55505 Inorganic adjuvants
• CPC Class Codes: A61K 2039/55566 Emulsions, e.g. Freund's adjuvant, MF59
• CPC Class Codes: A61K 2039/57 characterised by the type of response, e.g. Th1, Th2
• CPC Class Codes: A61K 2039/6075 Viral proteins
• CPC Class Codes: 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
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/29 Hepatitis virus
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 16/10 from RNA viruses
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 2760/16022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• 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 2770/28171 Demonstrated in vivo effect
• CPC Class Codes: G01N 33/576 for hepatitis

Abstract

The present invention relates to polypeptide(s) comprising the amino acid sequence as set forth in SEQ ID No. 1 of hepatitis E virus ORF 2 or its fragment, which is in the form of n-polymeric polypeptide, wherein n is an integer from 1-180; to a chimeric protein consisting of a polypeptide of the present invention and a conserved fragment of hemagglutin antigen from influenza virus; to a polypeptide of the present invention bound to a polypeptide containing epitope from hepatitis E virus ORF3 or an immunogenic fragment thereof; to a recombinant expression vector comprising the DNA molecule encoding the above polypeptides and the host cell transformed with said recombinant expression vector which is able to express polypeptide of the present invention. The present invention further relates to a vaccine composition against hepatitis E virus which comprises the above-mentioned polypeptide, or diagnostic kit for hepatitis E virus infection comprising the above-mentioned polypeptide, which includes IgG, IgM, or total antibody diagnostic kit for hepatitis E virus, and to the use of vaccine composition and diagnostic kit for prophylaxis, diagnosis and/or treatment of hepatitis E virus infection.

Claims

1. A polypeptide consisting of a fragment of an amino acid sequence as set forth in SEQ ID No. 1 of hepatitis E virus ORF 2, which fragment is selected from the group consisting of:

1) A polypeptide wherein the amino terminus starts from between amino acid residues 414 and 459, and the carboxyl terminus ends at amino acid residue 660 from SEQ ID No;

1;

2) A polypeptide wherein the amino acid sequence of amino acid residues 414 to 660 from SEQ ID No;

1, i.e., polypeptide 247;

3) A polypeptide wherein the amino acid sequence of amino acid residues 429 to 660 from SEQ ID No;

1, i.e., polypeptide 232;

4) A polypeptide wherein the amino acid sequence of amino acid residues 439 to 660 from SEQ ID NO;

1, i.e., polypeptide 222;

5) A polypeptide wherein the amino acid sequence of amino acid residues 459 to 660 from SEQ ID NO;

1, i.e., polypeptide 201;

6) A polypeptide having at least 80% homology to any one of the preceding polypeptides of the above

     1)-5) and having substantially identical antigenicity or immunogenicity.

2. A multimer of purified monomeric polypeptides, wherein said multimer is formed by 2-180 purified monomeric polypeptides via self aggregation, and each said purified monomeric polypeptide is the polypeptide according to claim 1.

3. A vaccine composition for prophylaxis and/or treatment of hepatitis E virus infection in mammals, which comprises at least one of the polypeptides according to claim 1 or the multimers according to claim 2 or any combination thereof or at least one of the multimer according to claim 2 or any combination thereof, and optionally, pharmaceutically acceptable vehicles and/or adjuvant.

4. A chimeric protein comprising a polypeptide according to claim 1 and a conserved fragment of hemagglutinin antigen from influenza virus.

5. A multimer of purified monomeric polypeptides, wherein said multimer is formed by 2-180 purified monomeric polypeptides via self aggregation, and each said purified monomeric polypeptide is the chimeric protein of claim 4.

6. A vaccine composition for prophylaxis and/or treatment of hepatitis E virus infection in mammals, which comprises chimeric protein according to claim 4 or a multimer according to claim 5, and optionally, pharmaceutically acceptable vehicles and/or adjuvant.

7. A chimeric protein comprising a polypeptide according to claim 1 and immunogenic epitope from hepatitis E virus ORF3 or an immunogenic fragment thereof.

8. A multimer of purified monomeric polypeptides, wherein said multimer is formed by 2-180 purified monomeric polypeptides via self aggregation, and each said purified monomeric polypeptide is a chimeric protein comprising a polypeptide according to claim 1 and immunogenic epitope from hepatitis E virus ORF3 or an immunogenic fragment thereof.

9. A vaccine composition for prophylaxis and/or treatment of hepatitis E virus infection in mammals, which comprises chimeric protein according to claim 7 or a multimer according to claim 8, and optionally, pharmaceutically acceptable vehicles and/or adjuvant.

10. A recombinant expression vector comprising the nucleotide sequence encoding polypeptide according to claim 1, chimeric protein according to claim 4 or chimeric protein according to claim 7.

11. A host cell transformed with the recombinant expression vector according to claim 10, which is able to express polypeptide according to claim 1, chimeric protein according to claim 4 or chimeric protein according to claim 7, respectively.

12. A diagnostic kit for the diagnosis of hepatitis E virus infection in biological sample, which comprises a diagnosis effective amount of at least one of the polypeptides according to claim 1 or any combination thereof or at least one of the multimers according to claim 2 or any combination thereof.

13. The diagnostic kit according to claim 12, which further comprises the polypeptide containing immunogenic epitope from hepatitis E virus ORF3 or an immunogenic fragment thereof, wherein said polypeptide containing immunogenic epitope from hepatitis E virus ORF3 or an immunogenic fragment thereof is optionally, covalently bound to the selected polypeptide according to claim 1.

14. The diagnostic kit according to claim 12 or 13 for determination of antibody 1gG against hepatitis E virus in the biological sample, which comprises at least one of the polypeptides according to claim 1 or the multimers according to claim 2, if desired, said polypeptide is pre-coated on the surface of a suitable support;

and further comprises detectable labeled antibody anti-IgG that is directed against IgG from biological sample to be detected, and detection agent corresponding to said detectable label, and if desired, further comprises a suitable buffer system.

15. The diagnostic kit according to claim 12 or 13 for determination of antibody IgM against hepatitis E virus in the biological sample, which comprise detectable labeled antibody anti-IgM as capture antibody that is directed against IgM from biological sample to be detected, if desired, said capture antibody is pre-coated on the surface of a suitable support;

and further comprises at least one of the detectable labeled polypeptides according to claim 1 or multimers according to claim 2, and detection agent corresponding to said detectable label;

if desired, further comprises a suitable buffer system.

16. The diagnostic kit according to claim 12 or 13 for determination of total antibodies against hepatitis E virus in the biological sample, which comprises at least one of the polypeptides according to claim 1 or the multimers according to claim 2, if desired, said polypeptide is pre-coated on the surface of a suitable support;

and further comprises at least one of the detectable labeled polypeptides according to claim 1 or multimers according to claim 2, and detection agent corresponding to said detectable label;

wherein said polypeptide or multimer for pre-coating the surface of a support and the detectable labeled polypeptide or multimer could be the same, or different one.

17. A method for detecting total antibodies against hepatitis E virus in biological samples, comprising the step of:

immobilizing at least one of the polypeptides according to claim 1 or the multimers according to claim 2 on the surface of a support;

contacting it with sample to be detected under the conditions suitable for the interaction of antigen and antibody;

washing with a suitable buffer; and

detecting antigen/antibody complex on the surface of a support by using antigen of hepatitis E virus with a detectable label and corresponding detect agent.

18. A method for detecting antibody IgG against hepatitis E virus in biological samples, comprising the step of:

immobilizing at least one of the polypeptides according to claim 1 or the multimers according to claim 2 on the surface of a support;

contacting it with sample to be detected under the conditions suitable for the interaction of antigen and antibody;

washing with a suitable buffer; and

detecting the antigen/antibody complex on the surface of a support by using detectable labeled antibody anti-IgG against hepatitis E virus and corresponding detect agent.

19. A method for detecting antibody IgM against hepatitis E virus in biological samples, comprising the step of:

immobilizing antibody anit-IgM on the surface of a support;

contacting it with sample to be detected under the conditions suitable for the interaction of antigen and antibody;

washing with a suitable buffer; and

detecting the anti-IgM/IgM complex on the surface of a support by using detectable labeled at least one of the polypeptides according to claim 1 or the multimers according to claim 2 and corresponding detect agent.

20. A method for detecting antibody IgM against hepatitis E virus in biological samples, comprising the step of:

immobilizing antibody anti-IgM on the surface of a support;

contacting it with sample to be detected under conditions suitable for the interaction of antigen and antibody;

washing with a suitable buffer; and

then contacting with at least one of polypeptides according to claim 1 or multimers according to claim 2 under conditions suitable for the interaction of antigen and antibody;

washing with a suitable buffer; and

then detecting antigen/antibody complex on the surface of a support by using detectable labeled snit-HEV polyclonal or monoclonal antibody and corresponding detect agent.

21. A diagnostic kit for the diagnosis of hepatitis E virus infection in biological sample or for the determination of antibody IgG, antibody IgM, total antibodies against hepatitis E virus in the biological sample, comprising chimeric protein according to claim 4 or multimer according to claim 5.

22. A diagnostic kit for the diagnosis of hepatitis E virus infection in biological sample or for determination of antibody IgG, antibody IgM total antibodies against hepatitis E virus in the biological sample, comprising chimeric protein according to claim 7 or multimer according to claim 8.