METHOD AND MEANS FOR THE RAPID DETECTION OF HDV INFECTIONS

Abstract

The present invention relates to a polypeptide and a nucleic acid encoding the polypeptide for use in a method of detecting the presence of hepatitis D vims (HDV) and/or of diagnosing an HDV infection and/or of monitoring the treatment of an HDV infection. The present invention further relates to an in vitro method, an immunographic test device as well as a kit. In particular, the present invention relates to a point of care diagnostic for HDV infections.

Claims

1. A polypeptide comprising an amino acid sequence selected from an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid sequence comprising or consisting of amino acid residues 1 to 195 of SEQ ID NOs: 1 or 2, an amino acid sequence comprising amino acid residues 60 to 214 of SEQ ID NOs: 1 or 2, and an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

2. A nucleic acid encoding a polypeptide of claim 1.

3-6. (canceled)

7 An in vitro method for detecting the presence of hepatitis D virus (HDV) and/or for diagnosing an HDV infection and/or for monitoring the treatment of an HDV infection in a sample of a subject, said method comprising: providing the polypeptide of claim 1; and detecting for IgG antibodies against the Hepatitis Delta Antigen (HDAg) in said sampler.

8. The method of claim 7, wherein the sample is serum, plasma, whole blood or saliva, and/or wherein HDV and HDV infections of all genotypes can be detected, and/or wherein one or more further infections are detected, such as HBV infection.

An immunographic device for in vitro detecting the presence of Hepatitis D virus (HDV) in a sample of a subject, diagnosing an HDV infection and/or monitoring the treatment of an HDV infection, said device comprising a solid carrier coated with an anti-HDV IgG antibody binding agent, wherein the anti-HDV IgG antibody binding agent is the polypeptide of claim 1.

10. The immunographic device of claim 9, wherein the immunographic device comprises a porous membrane operably connected to (a) a sample portion/pad, (b) a conjugate portion/pad, (c) a test portion/line comprising said anti-HDV IgG antibody binding agent, (d) a control portion/line; and (e) an absorbent portion/pad.

11. The immunographic device of claim 9, wherein the immunographic device is a lateral flow assay (LFA) device.

12. The immunographic device of claim 9, wherein the sample is serum, plasma, whole blood or saliva, and/or wherein HDV and HDV infections of all genotypes can be detected, and/or wherein one or more further infections are detected.

13. The immunographic device of claim 10, wherein the conjugate portion/pad comprises a detection marker.

14. The immunographic device of claim 10, wherein the control portion/line comprises a binding agent to a detection marker or a moiety carrying the detection marker.

15. A kit for in vitro detecting the presence of Hepatitis D virus (HDV) in a sample of a subject, diagnosing an HDV infection and/or monitoring the treatment of an HDV infection, wherein the kit comprises: a) an immunographic device according to claim 9; and b) instructions for using the immunographic device to detect the presence of said anti-HDV IgG antibodies in the sample and/or HDV and HDV infections of all genotypes can be detected, and/or further infections are detected, such as HBV infection.

16. The polypeptide of claim 1, consisting of an amino acid sequence selected from an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid sequence consisting of amino acid residues 1 to 195 of SEQ ID NOs: 1 or 2, an amino acid sequence consisting of amino acid residues 60 to 214 of SEQ ID NOs: 1 or 2, and an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1

17. The method of claim 7, which is a point of care method.

18. The method of claim 8, wherein HBV is also detected.

19. The device of claim 11, which is a point of care device.

20. The device of claim 12, wherein HBV is also detected.

21. The device of claim 13, wherein the detection marker comprises a colloidal metal or latex beads.

22. The device of claim 13, wherein the detection marker is directly or indirectly bound to an antibody or an antibody-binding protein.

23. The device of claim 22, wherein the detection marker is directly or indirectly bound to an anti-human antibody, Protein A, or Protein G.

24. The kit of claim 15, wherein the sample is serum, plasma, whole blood or saliva.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG. 1. Schematic representation of HBV and HDV virions.

[0107] The RNA genome of HDV is packaged as a ribonucleoprotein together with the Hepatitis Delta Antigen (HDAg). Both virions share the same envelope proteins, the S-, M- and L-HBsAg. L-HBsAg is composed of S-HBsAg with two N-terminal elongations: preS2 and the N-terminally myristoylated preS1. From Lempp and Urban, 2017.

[0108] FIG. 2. Design of the L-HDAg consensus sequence.

[0109] A multiple sequence alignment of L-HDAg from several isolates of all eight HDV genotypes was computed using VectorNTl software and sequencing data provided in the NCBI database. The consensus sequence was determined from the multiple sequence alignment.

[0110] FIG. 3. Construct design for a recombinant HDAg.

[0111] A, Construct design for the bacterial expression of a recombinant HDAg. The Histidine-tag is fused C-terminally to the protein. Shown is also the consensus sequence.

[0112] B, Analysis of bacterial expression and purification of the construct by SDS-PAGE. Protein was expressed in E. coli (1). Bacteria were lysed by a microfluidizer method. Lysate was ultracentrifuged and the inclusion bodies containing the recombinant L-HDAg were resolubilized in 8M Urea (3). Protein was purified by nickel-affinity chromatography and eluted using a linear gradient of imidazole.

[0113] C, Summary showing the development of the recombinant pan-genotypic HDAg.

[0114] D, Coomassie stained SDS-gel samples before (left) and after (right) purification. All three protein bands after purification represent HDAg as determined by mass spectrometry.

[0115] E, Antigenicity testing in ELISA.

[0116] Purified L-HDAg or bovine serum albumin (BSA) was coated on ELISA plates and incubated with (A) a serum of an HBV/HDV co-infected patient in increasing dilutions starting from 1:1.000.000 or (B) two sera of HBV/HDV co-infected patients or the serum of a healthy patient (neg), all at 1:2.000 dilution. After washing, the plates were incubated with an HRP-labeled anti-human secondary antibody and the enzymatic reaction was started by incubation with TMB substrate. All positive patient sera highly reacted with the coated L-HDAg but not with the BSA, while the serum of the negative patient did not react with both antigens. Note, that the pos #3 serum is derived from an HBV/HDV-coinfected patient with negative HDV viremia that therefore shows a lower amount of anti-HDV antibodies but is still highly reactive to the L-HDAg.

[0117] FIG. 4. Layout of the LFA of the invention: a direct LFA for HDAg-Abs. A, A general layout of a LFA is shown. Note that the LFA outlined in the scheme detects an analyte by using antibodies, while the present invention detects antibodies against HDAg by immobilized antigen.

[0118] B, Patient serum containing anti-HDAg antibodies is placed on the sample pad (left). All human antibodies are labeled with anti-human-gold or ProteinA-gold on the conjugate pad. HDAg-specific antibodies bind to the recombinant HDAg on the test line and give a positive signal. Other non-HDAg-specific human antibodies migrate to the control line, where an antibody binds to the gold-labeled conjugate antibody and forms the control line to indicate that the test is valid.

[0119] FIG. 5. Assembly of the LFA of the invention.

[0120] Sample and conjugate pad are pretreated with buffer containing blocking agent (e.g. BSA) and surfactant (e.g. Tween-20). Pretreated conjugate pads are loaded with conjugate in buffer containing high sugar (e.g. 20% sucrose). Test and control lines are spotted on the membrane, which is subsequently blocked and dried. LFAs are assembled on a plastic backing card, cut into 0.4 cm strips and placed in a plastic housing.

[0121] FIG. 6. Performance of the LFA of the invention

[0122] The LFA was run with serum of an HBV/HDV co-infected patient (pos, left) or of a healthy patient (neg, right) or with different dilutions of the co-infected patient serum using the healthy patient serum as diluent (middle). Image was taken after 10 min of running time. The upper red line represents the control line, while the lower line represents the test line detecting anti-HDAg antibodies. Note that even in the 1:100 dilution a clear signal at the test line is visible, which is completely absent in the healthy patient serum.

[0123] FIG. 7. Screening of 16 infected and 6 healthy patients with the LFA of the invention.

[0124] The LFA was run with serum of 16 HBV/HDV co-infected (PosA1-A16) and 6 healthy patients (Neg B1-B6). Image was taken after 10min of running time.

[0125] FIG. 8. Layout of a Multiplex LFA to simultaneously detect HBsAg and anti-HDAg. The Layout of the Multiplex LFA is similar to the single anti-HDAg LFA of this invention (FIG. 4B) with the addition of a second test line with immobilized anti-HBsAg antibodies and a second anti-HBsAg coupled to gold-nanoparticles on the conjugate pad (both anti-HBsAg antibodies are of different clones).

[0126] FIG. 9. Performance of a Multiplex LFA to simultaneously detect HBsAg and anti-HDAg.

[0127] The Multiplex LFA was run with serum of an HBV mono-infected patient (left), an HBV/HDV co-infected patient (middle) or a healthy patient (right). Image taken after 10 min run time.

[0128] FIG. 10. Characterization of validation samples. For assay validation, a collection of HDV-positive and HDV-negative samples was established. HDV positivity or negativity was determined with the DiaSorin anti-HD manual plate ELISA assay.

[0129] A and B, Analysis of all validation samples for quantitative levels of anti-HDV antibodies by direct plate ELISA with coated HDAg and increasing sample dilutions.

[0130] FIG. 11. Assay validation.

[0131] All validation samples were applied to the LFA of the invention, i.e. the POC test. A, Using the DiaSorin assay as gold standard, a high sensitivity of 94.6% for the POC test was calculated.

[0132] B, to D, The quantitative anti-HDV data were plotted with the POC test results.

[0133] FIG. 12. Analysis of sera with different HDV serotypes.

[0134] The LFA (i.e. POC test) was performed with sera of patients infected with different genotypes (gt) of HDV to validate the pan-genotypic specificity of the assay. Antibodies of all tested genotypes can be detected in the LFA (i.e. POC test).

EXAMPLES

Example 1

Materials and Methods

[0135] 1.1 Protein Expression and Purification

[0136] The open reading frame of L-HDAg-consensus-His was cloned into the bacterial expression vector pET, transformed into E. coli BL21 and protein expression was induced by addition of 1 mM IPTG for 3h at 37° C. Bacteria were lysed using a microfluidizer, recombinant protein was solubilized in 8M Urea and purified using a nickel-sepharose column (HisTrap, GE Healthcare) on an Akta HPLC system. Elution was performed using a linear imidazole gradient.

[0137] 1.2 ELISA

[0138] Recombinant protein was coated on a 96-well ELISA plate (Greiner Bio-One) at 2 μg/ml and blocked with 3% BSA/PBS/0.05% Tween-20. Human serum samples were diluted in 0.1% BSA/PBS/0.05% Tween-20 buffer at the indicated dilutions and incubated on the plates for 1 h at 37° C. After washing, the plates were incubated with a secondary goat-anti-human-perodxidase antibody (Jackson Immuno, 1:5000 dilution). After washing, the enzymatic reaction was started by incubation with TMB substrate (eBioscience) for 5 min at RT and stopped by addition of 1M phosphoric acid. Absorption at 450 nm was measured using a 96-well plate reader (Tecan).

[0139] 1.3 Assembly of Lateral Flow Assay

[0140] Lateral flow assays were assembled exclusively with material of the LFA material starter kit (DCN Diagnostics). Recombinant L-HDAg-consensus-His at the test line and donkey-anti-goat (Novo Nordisk) at the control line were applied at 2 mg/ml using the applicator AS30 (biostep) with 1 μl/cm volume. Membrane was subsequently blocked with 2% BSA, washed with PBS/0.05% Tween-20 and dried. Sample and conjugate pads were pretreated with sample pad buffer and goat-anti-human-Gold (BioAssayWorks) was applied to the conjugate pad in a buffer containing 20% sucrose and 5% trehalose. After drying of the individual parts, the LFA was assembled on a plastic backing card, cut into 4 mm strips and placed into plastic cassette housings.

[0141] 1.4 DiaSorin Assay

[0142] The manual plate ELISA for anti-HDAg detection “ETI-AB-DELTAK-2 anti-HDV” (Diasorin, Italy, order no P2808) was used according to the manufacturer's instructions.

[0143] 1.5 HDV samples and patient sera/collection of serum samples from all genotypes Serum/plasma samples of HDV-infected patients were provided by the University Hospital Heidelberg, Hannover Medical School, Ankara Medical School, French blood bank (EFS) and the company Biomex (Heidelberg, Germany). All negative samples were purchased from Biomex (Heidelberg, Germany). Serum samples of patients infected with different HDV genotypes were characterized and provided by the Laboratoire de Microbiologie Clinique, Hôpital Avicenne (APHP, Bobigny, France). All serum/plasma samples were stored at −80° C. until use.

Example 2

2.1 Characterization of Validation Samples

[0144] For assay validation, a collection of HDV-positive and HDV-negative samples was established from routine clinical diagnostics, from HDV clinical studies or commercial vendors. See Table 1 below. HDV positivity or negativity was determined with the DiaSorin anti-HD manual plate ELISA assay.

TABLE-US-00002 TABLE 1 Characteristics of validation samples HDV-positive HDV-negative n = 332 n = 142 HBsAg HBsAg-positive 332 62 status HBsAg-negative 0 80 HDV- RNA-positive 121 0 RNA RNA-negative 33 0 status RNA-unknown 178 142 Sample Serum 299 42 matrix EDTA-Plasma 21 30 Citrate-Plasma 12 70

[0145] To obtain quantitative levels of anti-HDV antibodies in the validation samples, a quantitative ELISA assay was established: ELISA plates were coated with recombinant HDAg, incubated with different dilutions of the samples and bound antibodies were detected using a peroxidase-labeled antibody, as described above. See FIG. 10A and B.

[0146] 2.2 Assay Validation

[0147] All validation samples were applied to the LFA of the invention, i.e. the POC test. Using the DiaSorin assay as gold standard, a high sensitivity of 94.6% for the POC test was calculated, see FIG. 11A.

[0148] When plotting the quantitative anti-HDV data with the POC test results, it is apparent that all RNA-positive HDV sera can be detected with the POC (see FIG. 11C) and only samples with very low to undetectable levels of anti-HDV are negative in in the POC test (see FIG. 11D).

Example 3

Analysis of Sera with Different HDV Genotypes

[0149] The POC test was performed with sera of patients infected with different genotypes (gt) of HDV to validate the pan-genotypic specificity of the assay. As can be seen in FIG. 12, antibodies of all tested genotypes 1 to 8 can be detected in the POC test.

EXAMPLE 4

Multiplex LFA for Simultaneous Detection of HBsAg and anti-HDV

[0150] Rapid POC assays for HBsAg to diagnose an HBV infection are already marketed. In a proof-of-concept study, the anti-HDV assay of the invention was combined with an HBsAg assay on a multiplex LFA strip, as shown in FIG. 8.

[0151] The multiplex strips were assayed with sera of HBV/HDV co-infected, HBV-monoinfected or healthy patients. As can be seen in FIG. 9, the LFA/POC test can be multiplexed to simultaneously detect HBsAg and anti-HDV.

[0152] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

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