WO1994006913B1

WO1994006913B1 - Recombinant proteins of a pakistani strain of hepatitis e and their use in diagnostic methods and vaccines

Info

Publication number: WO1994006913B1
Application number: PCT/US1993/008849
Authority: WO
Filing date: 1993-09-17
Publication date: 1994-08-04
Anticipated expiration: 1995-03-18

Abstract

A strain of hepatitis E virus from Pakistan (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, is disclosed. The invention relates to the expression of the whole structural region of SAR-55, designated open reading frame 2 (ORF-2), in a eukaryotic expression system. The expressed protein is capable of forming HEV virus-like particles which can serve as an antigen in diagnostic immunoassays and as an immunogen or vaccine to protect against infection by hepatitis E.

Claims

- 95 -AMENDED CLAIMS[received by the International Bureau on 27 June 1994 (27.06.94); original claims 1-31 replaced by amended claims 1-35 (5 pages)]

1. A cDNA of isolated and purified hepatitis E virus strain SAR-55.

2. The cDNA of claim 1, wherein the cDNA has a cDNA sequence according to SEQ ID No. 4.

3. A synthetic DNA sequence comprising a sequence capable of directing host organism synthesis of at least one complete open reading frame protein, said protein having substantial homology to a protein from HEV strain SAR-55, or a variant of said protein.

4. A DNA sequence of Claim 3, wherein said DNA encodes a protein comprising the amino acid sequence of SEQ ID NO. 2 or a variant thereof.

5. A recombinant protein derived from the cDNA sequence of claim 2.

6. The recombinant protein of claim 5 wherein the protein is encoded by cDNA sequence comprising a complete ORF.

7. The recombinant protein of claim 6, wherein said protein comprises an amino acid sequence of SEQ ID NO. 2 or substantially homologous sequence thereof.

8. A recombinant protein derived from a complete open-reading frame of a hepatitis E virus.

9. The recombinant protein of claim 8, wherein said protein is derived from complete open reading frame 2 of a hepatitis E virus.

10. A method of producing a recombinant HEV protein, - 96 -

comprising:

(a) cloning a cDNA sequence of HEV strain SAR-55;

(b) inserting the cDNA sequence into an expression vector;

(c) transferring the expression vector into a host organism;

(d) culturing the host organism under conditions appropriate for amplification of the vector and expression of the protein; and

(e) harvesting the protein.

11. The method of claim 10, wherein the expression vector is a eukaryotic expression vector.

12. The method of claim 10, wherein the expression vector is a baculovirus vector.

13. The method of claim 10, wherein the host organism is a eukaryotic cell.

14. The method of claim 13, wherein the eukaryotic cell is an insect cell.

15. The method of claim 10, wherein the cDNA sequence encodes a complete open-reading frame.

16. The method of claim 15, wherein the cDNA sequence encodes complete open-reading frame 2.

17. The method of claim 16, wherein the protein comprises an amino acid sequence according to SEQ ID NO:2 or a variant thereof.

18. A recombinant expression vector comprising a cDNA sequence according to claim 2. - 97 -

19. The recombinant vector of claim 17, wherein said vector comprises a cDNA sequence encoding a complete open- reading frame.

20. The recombinant expression vector of claim 18, wherein the cDNA sequence encodes a complete open-reading frame 2.

21. The recombinant expression vector of claim 19, wherein the vector is p63-2.

22. A host organism transformed or transfected with a recombinant expression vector according to claim 18, 19, 20 or 21.

23. A method for the recombinant DNA synthesis of at least one complete open reading frame protein of HEV comprising:

(a) culturing a transformed or transfected host organism containing a DNA sequence capable of directing the host organism to produce an open reading frame protein of a hepatitis E virus under conditions such that the protein is produced;

(b) harvesting the protein.

24. The method of claim 23, wherein the protein produced in step (a) comprises open reading frame 2.

25. A method of detecting antibodies to Hepatitis E in a biological sample suspected of containing said anti¬ bodies comprising:

(a) contacting the sample with the recombinant HEV protein of claims 5 or 8 to form an immune complex with the antibodies; and (b) detecting the presence of the immune - 98 -

complex.

26. The method of claim 25, wherein the biological sample is whole blood, plasma, serum, cerebrospinal fluid, tissue, urine and pleural fluid.

27. The method of claim 25, wherein Ig or IgG anti¬ body is detected.

28. The method of claim 25, wherein the recombinant HEV protein is bound to a solid support.

29-. The method of claim 25, wherein the immune complex is detected using a labeled antibody.

30. A kit for use in a method of claim 25 comprising: a recombinant HEV protein comprising an amino acid sequence of SEQ ID No. 2 or a substantially homologous sequence thereof.

31. The kit of claim 30 further comprising a labeled secondary antibody.

32. A pharmaceutical composition comprising the recombinant protein of claims 5 or 8 and a suitable excipient, diluent or carrier.

33. A method of preventing hepatitis E, comprising administering the pharmaceutical composition of claim 32 to a mammal in an effective amount to stimulate the production of protective antibody.

34. A vaccine for immunizing a mammal against hepatitis E infection, comprising a recombinant protein according to claims 5 or 8 in a pharmacologically acceptable carrier. - 99 -

35. The vaccine of claim 34, wherein the recombinant protein is derived from a complete open reading frame 2.