Development of HBV-and/or HDV-susceptible cells, cell lines and non-human animals

Abstract

The present invention relates to a novel Hepatitis B virus (HBV) and/or Hepatitis D virus (HDV) receptor and its use for the development of cells, cell lines and non-human animals that are susceptible to HBV and/or HDV infection and can be used for immunological studies and/or for the screening of drugs, post-entry restriction factors and host dependency factors. It further relates to the use of the receptor for the identification of compounds useful in the treatment of HBV and/or HDV infection.

Claims

1. A method for producing a cell that is susceptible to Hepatitis B virus (HBV) and/or Hepatitis D Virus (HDV) infection, or has an increased susceptibility to HBV and/or HDV infection, or is able to bind HBV and/or HDV, said method comprising the steps of: A) providing a cell that is non-susceptible to HBV and/or HDV infection or has a low susceptibility to HBV and/or HDV infection or is unable to bind HBV and/or HDV: B) transducing said cell with a viral vector comprising a nucleic acid sequence encoding: an amino acid sequence comprising SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8, and an amino acid sequence consisting of the general formula Pro-Tyr-X-Gly-Ile, wherein X is selected from Lys, Arg and Val, C) knocking-down one or more endogenous genes of said cell, wherein one of said endogenous genes is the gene encoding the natural sodium taurocholate cotransporter polypeptide (NTCP/SCL10A1) polypcptide of said cell, and said knocking-down one or more endogenous genes of said cell is achieved by means of an shRNA vector; and D) culturing the transduced cell in the presence of DMSO when the transduced cell is contacted with HBV and/or HDV.

2. A method for producing a cell that is susceptible to Hepatitis B virus (HBV) and/or Hepatitis D Virus (HDV) infection, or has an increased susceptibility to HBV and/or HDV infection, or is able to bind HBV and/or HDV, said method comprising the steps of: A) providing a cell that is non-susceptible to HBV and/or HDV infection or has a low susceptibility to HBV and/or HDV infection or is unable to bind HBV and/or HDV; B) transducing said cell with a viral vector comprising a nucleic acid sequence encoding: an amino acid sequence consisting of SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and an amino acid sequence comprising the sequence of SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8, C) knocking-down one or more endogenous genes of said cell, wherein one of said endogenous genes is the gene encoding the natural sodium taurocholate cotransporter polypeptide (NTCP/SCL10A1) polypeptide of said cell, and said knocking-down one or more endogenous genes of said cell is achieved by means of an shRNA vector; and D) culturing the transduced cell in the presence of DMSO when the transduced cell is contacted with HBV and/or HDV.

3. A method for producing a cell that is susceptible to Hepatitis B virus (HBV) and/or Hepatitis D Virus (HDV) infection, or has an increased susceptibility to HBV and/or HDV infection, or is able to bind HBV and/or HDV, said method comprising the steps of: A) providing a cell that is non-susceptible to HBV and/or HDV infection or has a low susceptibility to HBV and/or HDV infection or is unable to bind HBV and/or HDV; B) introducing into said cell a nucleic acid sequence encoding: an amino acid sequence consisting of SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, and an amino acid sequence comprising the sequence of SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8 or an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8.

4. A method for producing a cell that is susceptible to Hepatitis B virus (HBV) and/or Hepatitis D Virus (HDV) infection, or has an increased susceptibility to HBV and/or HDV infection, or is able to bind HBV and/or HDV, said method comprising the steps of: A) providing a cell that is non-susceptible to HBV and/or HDV infection or has a low susceptibility to HBV and/or HDV infection or is unable to bind HBV and/or HDV; B) introducing into said cell a nucleic acid sequence encoding: an amino acid sequence comprising the sequence of SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8, and an amino acid sequence consisting of the general formula Pro-Tyr-X-Gly-Ile, wherein X is selected from Lys, Arg and Val.

Description

(1) Reference is now made to the figures, wherein

(2) FIG. 1 shows a sequence alignment of the sodium taurocholate co-transporter polypeptide NTCP/SLC10A1 from different species (Human (SEQ ID NO:1); Chimpanzee (SEQ ID NO:3); Orangutan (SEQ ID NO:4); Three shrew (SEQ ID NO:5); Mouse (SEQ ID NO:6); Rat (SEQ ID NO:7); Dog (SEQ ID NO:8); Cynomolgus (SEQ ID NO:9); and Pig (SEQ ID NO: 10)). Species supporting peptide binding and HBV infection (human, chimpanzee, orang-utan, and Tupaia belangeri), species that are competent in binding HBV-preS-derived lipopeptides without supporting infection (mouse, rat, dog) and species that are unable to bind and do not support infection are depicted. Identical amino acids are highlighted in yellow. Non conserved amino acid changes are shown without shading. The two amino acids (157 and 158) that differ in the non-binding species cynomolgus and pig (Meier et al., Hepatology 2012; Schieck et al., Hepatology 2012 in press) indicate the essential binding site (highlighted by the box);

(3) FIG. 2 shows that transient transfection of human NTCP and mouse NTCP into HuH7 cells confers binding of an Atto645-labeled HBV preS-lipopeptide (referred to as Myrcludex B, MyrB). HuH7 cells where transiently transfected with a plasmid encoding GFP (left), a plasmids encoding GFP together with human NTCP (middle) and a plasmid encoding GFP and mouse NTCP. 3 days post transfection, cells were incubated with a fluorescently labeled HBV preS-lipopeptide, washed and analyzed by fluorescent microscopy. GFP-fluorescence is shown in the upper left, cells are shown in the lower left, peptide binding is shown in the upper right panel; the merged pictures of transfected and binding competent cells is shown in the lower right panel;

(4) FIG. 3 shows that stably transduced HepG2 cells expressing human or mouse NTCP specifically bind an HBV preS-lipopeptide. HepG2 cells were stably transduced with hNTCP (upper pictures) or mNTCP (lower pictures) and incubated with 500 nM of an Atto-labelled wildtype HBV preS-lipopeptide (left pictures) or the same concentration of a respective mutant peptide with amino acid exchanges in the essential HBV-receptor binding domain (right pictures). Binding of the peptides was visualized by fluorescence microscopy;

(5) FIG. 4 shows that stably transduced mouse hepatoma cells (Hep56.1D and Hepa1-6) specifically bind an HBV preS-lipopeptide. HepG2, Hep56.1D and Hepa1-6 cells were stably transduced with hNTCP and incubated with 500 nM of an Atto-labelled wildtype HBV preS-lipopeptide. Binding of the peptide was visualized by fluorescence microscopy;

(6) FIG. 5 shows that HuH7 cells transfected with human NTCP are susceptible to HDV infection. HuH7 inoculated with a HDV-containing human serum do not show any marker of HDV infection 4 days after inoculation (left picture). Following transfection with a human NTCP expression plasmid an HDV delta antigen-specific staining was observed (right picture);

(7) FIG. 6 shows that endogenous expression of human NTCP in Hep56.1D mouse cell lines renders them susceptible to HDV infection. Hep56.1D mouse hepatoma cell lines alone (mock) or transfected with human NTCP (hNTCP) were infected with an HDV containing serum (right picture). Hepatitis delta antigen expressing cells were counted 5 days post infection. As a second control, human HuH7 cells were transfected with human NTCP or mouse NTCP and infected with HDV (left picture);

(8) FIG. 7 shows that transfection of human but not mouse NTCP renders HuH7 cells susceptible to infection with hepatitis delta virus (HDV). HuH7 cells were transiently transfected with expression vectors encoding mouse NTCP (left panels) or human NTCP (right 4 panels in 2 different magnifications). At confluence, cells were incubated with a patient's serum containing HDV. 4 days after infection cells were stained with an antiserum detecting nuclear delta antigen;

(9) FIG. 8 shows immunofluorescence analysis of NTCP (human) transfected mouse Hep56.1D cells after infection with hepatitis delta virus. Hep56.1D mouse hepatoma cell lines were transfected with mouse NTCP (2 panels on the left) or human NTCP (hNTCP) (4 panels on the right in 2 different magnifications) were infected with an HDV-containing serum and stained with a hepatitis delta antigen specific antibody.

(10) FIG. 9 shows that HepG2 and HuH7 cells stably expressing human NTCP become susceptible to Hepatitis B Virus (HBV) infection. Stably hNTCP transduced HepG2 and HuH7 cell lines were inoculated with HBV at different concentrations of DMSO to induce differentiation processes. Medium was collected on day-5 post infection and HBeAg was quantified. As a specific control for infection an HBV preS-derived lipopeptide (MyrB) was used (left bars). In comparison to HepG2 cells, HuH7 cells produce lower amounts of viral replication markers indicating the presence of a restriction step.

(11) The present invention is now further described by means of the following examples, which are meant to illustrate the present invention, and not to limit its scope.

EXAMPLES

(12) Material & Methods

(13) Sequence of NTCP

(14) The protein sequences of NTCP from different species were obtained from Ensemble (www.ensemble.org).

(15) Alignment

(16) The alignment of NTCP proteins from different species was created by using Vector NTI 9.0 (Invitrogen).

(17) Plasmids and Peptides

(18) The human NTCP (hNTCP) containing construct (pCMV6-XL4-hNTCP) was bought from Origene (USA). The open reading frames of hNTCP and NTCP were amplified by PCR and inserted into pWPIlentiviral vector for transient (pWPI-GFP) or stable expression (pWPI-puro).

(19) The peptide used for inhibition of HBV infection has been described previously as Myrcludex B (MyrB). It is a N-myristoylated peptide comprising the 47 amino acid of HBV L protein. ATTO 645 and ATTO 488 (ATTO-TEC, Germany) are fluorescent dyes used to label the peptide for the binding assay. A mutant peptide with an alanine substitution in the essential binding site (amino acids 11-15) was used as control of the binding specificity.

(20) TABLE-US-00007 MyrB SEQIDNO:12 Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEAN KVG-amide mutantMyrB.sup.Ala11-15 SEQIDNO:13 Myr-GTNLSVPNPAAAAADHQLDPAFGANSNNPDWDFNPNKDHWPEAN KVG-amide
Lentivirus Transduction

(21) To produce recombinant lentiviruses, HEK 293T cells were seeded one day prior transfection. 3 g of the envelope protein expression construct pczVSV-G, 9 g of the HIV Gag-Pol expression construct pCMVAR8.74 and 9 g of the lentiviral vector pWPI were mixed with 25 g polyethyleneimine before adding to 293T cells. The supernatant containing lentiviral pseudoparticles were harvested and concentrated by ultracentrifugation. The precipitated lentiviral particles were resuspended in cell medium.

(22) For transient expression, hepatic cells were incubated with lentiviruses in the presence of 4% PEG8000. The inoculum was removed after overnight incubation. The cells were washed once with PBS and cultivated for 3 days for expression of the target proteins. For the establishment of stable cell lines, 2.5 g/ml puromycin was added to select stably transduced cells. Generally, 90% of hepatic cells survived the selection without significant morphological difference compared to untransduced cells.

(23) Cells

(24) Four hepatic cells were used in this work. Two of them are derived from human (HuH-7 and HepG2) and the other two from mouse (Hep56.1D and Hepa1-6). HEK 293T cell were used for lentiviral production.

(25) Binding Assay with Fluorescently Labeled Peptides

(26) To determine the binding competence of hepatocytes, transiently or stably transduced cells were incubated with 200-500 nM fluorescence-labeled peptides in cell medium for 15-60 minutes. Then cells were washed with PBS for 3 times and analyzed by fluorescence microscopy.

(27) HBV and HDV Infection Assay

(28) HBV particles were obtained from HepAD38 cells. For HBV or HDV infection, cells were inoculated with medium containing 4% PEG 8000 and 10-20 l virus (100 virus stock) overnight at 37 C. Afterwards, cells were washed three times with PBS and further cultivated for 5 days. Presence of the Hepatitis B virus-antigen (HBeAg) secreted into the culture supernatant was determined by Abbott HBeAg assay (Abbott Laboratories). HDV infection was determined by immuno-staining of HDV infected cells with an anti-HDV sera.

(29) Results

(30) The invention of the HepaRG cell line lead to the identification of peptidic receptor ligands derived from the N-terminal preS1-domain of the large (L) viral surface protein, which specifically bind to HBV-susceptible cells and efficiently block infection. Mapping of essential sites within the peptides revealed the requirement of the lipid moiety and the integrity of a conserved sequence 9-NPLGFFP-15 [SEQ 1D NO: 14]. Radioactively and fluorescently labelled peptidic ligands where applied to analyse the bio distribution of the preS/receptor complex mice, rats, dogs, cynomolgus and chimpanzees and the expression patterns and turnover kinetics primary hepatocytes of the respective species or hepatoma cell lines. The results revealed that the receptor: (i) is specifically expressed in liver (ii) becomes induced during differentiation of HepaRG cells, (ii) is down-modulated upon dedifferentiation of PMH and PRH, (iii) shows association with the cytoskeleton allowing little lateral movement within the plasmamembrane, (iv) shows a limited rate of endocytosis (v) is exclusively sorted to the basolateral membrane (vi) conserved binding domain in human, mouse, rat, dog, chimpanzee, but not pig and cynomolgus monkey.

(31) Based on these result the inventors performed a differential affimetrix based expression screen. Up regulated genes in HepaRG-cells undergoing DMSO-induced differentiation were subtracted from to down-regulated genes in PMH during dedifferentiation in the absence of DMSO. The most prominent hits of both screens were combined and subjected to the criteria defined above. Sodium taurocholate cotransporting polypeptide (NTCP, SLC10A1) was the only appropriate candidate meeting these criteria: NTCP, an integral multi-transmembrane protein is exclusively expressed on the basolateral membrane of differentiated hepatocytes. It is scarcely expressed on HepG2, HuH7 and many other hepatoma cell lines. NTCP is instantly induced in HepaRG cells upon DMSO treatment at levels that correspond to the saturation levels of Myrcludex B. It is associated with the cytoskeleton and undergoes slow and regulated (PKC-dependent) endocytosis.

(32) By sequence alignment of NTCP from three groups of hosts (FIG. 1), which differ in their infection and binding competency, the inventors defined two critical amino acids of NTCP (amino acids 157 to 158). The consensus sequence (KG) is present in most susceptible and binding-competent hosts. In contrast, the binding incompetent hosts like cynomolgus monkey and pig do not contain this motif.

(33) The inventors transduced hNTCP or mNTCP into HuH-7 cells and performed a peptide-binding assay (FIG. 2). In comparison to the control with an empty vector (Mock), both human and mouse derived NTCP bind to the peptide. These signals of bound peptides are correlated to the amount of co-expressed GFP, which indicate the expression level of NTCP.

(34) The inventors further generated four hepatic cells (HuH-7, HepG2, Hep56.1D and Hepa1-6) stably expressing hNTCP or mNTCP. The cells show homogenous binding with the wildtype (WT) peptide but not the mutant peptide (FIG. 3). The mouse hepatoma cells expressing hNTCP or mNTCP show a strong binding to the peptide as well (FIG. 4).

(35) Although the transfection efficacy is low (20%), HuH-7 cells transfected with hNTCP can be infected by HDV (FIG. 5). The gained susceptibility to HDV infection by hNTCP could also be observed in both human and mouse cells (FIG. 6). Transient transduction of hNTCP confers susceptibility of HuH-7 cell to HDV infection (FIG. 7), whereas the mNTCP protein supporting peptide-binding does not support HDV infection. The mouse cell line Hep56.1D supports HDV infection after transduction with hNTCP (FIG. 8).

(36) The gained susceptibility to HBV infection by NTCP could be observed in HepG2 cells stably expressing hNTCP (FIG. 9). This infection could be specifically inhibited by the peptide Myrcludex B (MyrB) and enhanced by adding DMSO to the cultivation medium. HuH-7 cells expressing hNTCP seem to support HBV infection at a lower level, indicating that an unknown co-factor supporting HBV infection is absent in HuH-7 cells in comparison to HepG2 cells.

(37) The features of the present invention disclosed in the specification, the claims, and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.