ANTIBODIES HAVING SPECIFICITY FOR THE ORF2I PROTEIN OF HEPATITIS E VIRUS AND USES THEREOF FOR DIAGNOSTIC PURPOSES

Abstract

Hepatitis E virus (HEV) is annually responsible for 20 million infections with 3.4 million symptomatic cases and 70,000 deaths mainly occurring in less developed regions of the world. HEV is a non-enveloped virus containing a linear, single-stranded, positive-sense RNA genome that contains three open reading frames (ORFs), namely, ORF1, ORF2 and ORF3. ORF2 encodes the ORF2 viral capsid protein, which is involved in particle assembly, binding to host cells and eliciting neutralizing antibodies. Recently, 3 different forms of the ORF2 capsid protein were identified: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein, for which the precise sequence has been identified, is the form that is associated with infectious particles and thus antibodies having specificity for the ORF2i protein would be suitable for the diagnosis of HEV. The present fulfills this need by providing an antibody which binds to the ORF2i protein of hepatitis E virus and wherein said antibody does not bind to the ORF2g protein nor to the ORF2c of hepatitis E virus, and wherein the epitope of said antibody comprises at least one amino acid residue from amino acid residues 542 to 555 of SEQ ID NO: 1.

Claims

1. An antibody which binds to the ORF2i protein of hepatitis E virus and wherein said antibody does not bind to the ORF2g protein nor to the ORF2c of hepatitis E virus, and wherein the epitope of said antibody comprises at least one amino acid residue from amino acid residues 542 to 555 of SEQ ID NO: 1 (SEQ ID NO:4).

2. The antibody of claim 1 wherein the antibody binds to an epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues from SEQ ID NO:4, or from a sequence sharing at least 90% of identity over SEQ ID NO: 4.

3. The antibody of claim 1 wherein the antibody binds to an epitope comprising the amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence sharing at least 90% of identity over SEQ ID NO: 4.

4. The antibody of claim 1 which is a polyclonal antibody or a monoclonal antibody.

5. The antibody of claim 1 which is a Fab′, Fab, F(ab′)2, scFv or a single domain antibody.

6. The antibody of claim 1 which is conjugated with a detectable label.

7. The antibody of claim 6 wherein the detectable label is a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, or a bio luminescent label.

8. The antibody of claim 6 wherein the detectable label is selected from the group consisting of β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.

9. (canceled)

10. A method for detecting the presence of the ORF2i protein in a sample comprising contacting the sample with the antibody of claim 1 under conditions that allow an immunocomplex of the protein and antibody to form wherein detection of the immunocomplex indicates the presence of the ORF2i protein in the sample.

11. A method for detecting the presence of infectious particles of hepatitis E virus in a sample comprising contacting the sample with the antibody of claim 1 under conditions that allow an immunocomplex of the antibody and the infectious particles to form wherein detection of the immunocomplex indicates the presence of the infectious particles in the sample.

12. The method of claim 10 wherein the sample is selected from the group consisting of faeces, blood, ascites; urine; saliva; sweat; milk; synovial fluid; peritoneal fluid; amniotic fluid; cerebrospinal fluid; lymph fluid; lung embolism; cerebrospinal fluid; and pericardial fluid.

13. A method for diagnosing and treating an acute HEV infection, a recent HEV infection, a chronic HEV infection, a weak active HEV infection or a cleared HEV infection in a subject in need thereof, comprising contacting a sample from the subject with the antibody of claim 1, wherein the step of contacting is performed under conditions that allow formation of immunocomplexes of the antibody with i) ORF2i protein; and/or ii) infectious particles of hepatitis E virus; and treating the subject when immunocomplexes are detected.

14. A kit or device for identifying the presence of infectious hepatitis E viral particles in a sample, the kit or device comprising at least one antibody of claim 1.

15. The kit or device of claim 14 which is a flow immunoassay device.

16. The kit or device of claim 14, wherein the at least one antibody is immobilized on a solid support.

17. The method of claim 11 wherein the sample is selected from the group consisting of faeces, blood, ascites; urine; saliva; sweat; milk; synovial fluid; peritoneal fluid; amniotic fluid; cerebrospinal fluid; lymph fluid; lung embolism; cerebrospinal fluid; and pericardial fluid.

18. The antibody of claim 8, wherein the peroxidase is horseradish perodixase.

Description

FIGURES

[0041] FIG. 1: Generation of specific antibodies directed against the ORF2i protein using the peptide AGYPYNYNTTASDQ (SEQ ID NO:4) Reactivity and specificity of P2S2 and control (CTL) mouse sera and 1E6 antibody were analyzed (A) by immunofluorescence on fixed HEV-infected cells and (B) by western blotting on a mixture of ORF2 proteins (ORF2i and ORF2g/ORF2c). The asterisk indicates a non-specific band. Reactivity and specificity of P2H1, P2H2 and P2H3 hybridoma supernatants and 1E6 antibody were analyzed (C) by immunofluorescence on fixed HEV-infected cells and by western blotting on a mixture of ORF2 proteins (D) or an extract of HEV-infected cells (E).

RESULTS: GENERATION OF SPECIFIC ANTIBODIES DIRECTED AGAINST THE ORF2I PROTEIN USING THE PEPTIDE AGYPYNYNTTASDQ (SEQ ID NO:4)

[0042] The ORF2 protein sequence contains three highly conserved N-glycosylation sites represented by the sequon Asn-X-Ser/Thr (N—X—S/T). The ORF2i protein is not glycosylated whereas the ORF2g and ORF2c proteins are highly N-glycosylated (Montpellier et al. Gastroenterology, 2018). Among the three sites, the third site (.sup.549NTT, N3) is highly (at least 95%) occupied by N-glycans in ORF2g and ORF2c proteins (Ankavay et al., submitted). Therefore, a peptide covering the N3 site can represent a good strategy for obtaining highly specific antibodies of the ORF2i form. Such antibodies will recognize the non-glycosylated ORF2i protein but not the glycosylated ORF2g and ORF2c proteins. In order to verify the specificity between strains/genotypes, sequence alignment between amino acids (a.a) 510 to 580 was carried out (data not shown). The prediction of secondary structure, prediction of immunogenicity, hydrophilicity, and accessibility were performed to define the best sequence for the immunogen. Finally, the peptide AGYPYNYNTTASDQ (SEQ ID NO:4) was selected for the immunization. In order to make this sequence more immunogenic during murine immunizations, a coupling to the protein carrier KLH was carried out via a maleimide function by adding a cysteine at the N-terminal position. Immunization was performed according a routine protocol. Five mice were immunized three times at three weeks intervals with the peptide (SEQ ID NO:4). Freund's complete and incomplete adjuvants were used during immunisation. The animals were immunized by subcutaneous and intraperitoneal routes. Ten days after the third immunisation, mice have been bleeded and their sera tested for immunoreactivity. Sera were first assayed by indirect ELISA on plates coated with the peptide (SEQ ID NO:4) (data not shown). Their reactivity was analyzed by immunofluorescence (IF) on fixed HEV-infected cells (FIG. 1A). The 1E6 monoclonal antibody (antibody registry #AB-827236), which recognizes the three forms of ORF2 proteins (Montpellier et al., Gastroenterology, 2018), was used as a positive control. The serum of a PBS-immunized mouse was used as a negative control (CTL mouse). As shown in FIG. 1A, serum of the P2S2 mouse showed a specific reactivity in IF. Specificity of the P2S2 serum was next analyzed in western blotting experiments with a mixture of ORF2 proteins (ORF2i and ORF2g/ORF2c) as antigens (FIG. 1B). The P2S2 serum showed a highly specific recognition of the ORF2i protein, with no cross-reaction with the ORF2g/c proteins, as compared to the 1E6 antibody that recognizes the three forms.

[0043] After a final boost, the P2S2 mouse was sacrificed. Lymphocytes were isolated from the spleen and fused with a myeloma cell line using polyethylene glycol to form a hydridoma. Following fusion, cells were placed in media permissive for growth of hybridomas. Following culture of the hybridomas, cell supernatants were first screened by indirect ELISA on plates coated with the peptide (SEQ ID NO:4) (data not shown) and then by immunofluorescence. As shown in FIG. 1C, the P2H1, P2H2 and P2H3 hybridoma supernatants displayed an ORF2i-specific staining. Specificity of these hybridomas was next analyzed in western blotting experiments with a mixture of ORF2 proteins (ORF2i and ORF2g/ORF2c) (FIG. 1D) or an extract of HEV-infected cells (FIG. 1E), as antigens. The P2H1, P2H2 and P2H3 clones showed a highly specific recognition of the ORF2i protein, with no cross-reaction with the ORF2g/c proteins, as compared to the 1E6 antibody that recognizes the three forms.

[0044] Together, these results indicate that antibodies specifically directed against the ORF2i polypeptides (SEQ ID NO:4) can be produced. Such antibodies will be very suitable for determining presence of infectious particles of hepatitis E virus in a sample. More particularly, detection of the ORF2i polypeptide of the present invention is suitable for diagnosing hepatitis E virus infection in a subject.

REFERENCES

[0045] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.