Hepatitis B virus pre-S1 derived synthetic polypeptides and uses thereof

Abstract

The invention relates to a group of synthetic polypeptides, derived from the pre-S1 region of HBV, that efficiently interfere with early steps of an HBV infection. The peptides of the invention can be used in diagnostics for the detection of antigens and/or antibodies.

Claims

1. A method for treating a subject having a HBV infection, comprising administering to the subject a synthetic polypeptide of formula (I)
XYZ(I) wherein X is an amino acid, or absent; Y is the amino acid sequence consisting of the amino acid starting at position 2 and ending at an amino acid between position 28 and position 48 of a pre-S1 region of HBV large (L) envelope protein corresponding to SEQ ID NO:1 or a variant thereof, wherein the variant has a homologous sequence in viral species, strains or subtypes of HBV large (L) envelope protein; Z, linked to the CO group of the last residue of Y, is absent, or is the amino acid sequence consisting of the amino acid starting at position 49 and ending at an amino acid between position 49 and position 78 of pre-S1 region of HBV L envelope protein corresponding to SEQ ID NO:1 or a variant thereof, wherein the variant has a homologous sequence in viral species, strains or subtypes of HBV large (L) envelope protein, if the amino acid sequence of Y includes the amino acid at position 48; and wherein said polypeptide is chemically attached to a hydrophobic moiety that is a fatty acid or a cholesterol.

2. The method according to claim 1, wherein the hydrophobic moiety is a saturated or unsaturated fatty acid having at least 4 carbon atoms.

3. The method according to claim 1, wherein the hydrophobic moiety is myristic acid or stearic acid.

4. The method according to claim 1, wherein the first amino acid of said polypeptide is chemically modified to bear the hydrophobic moiety.

5. The method according to claim 1, wherein said HBV L envelope protein is selected from the group consisting of HBV L envelope protein of human HBV, chimpanzee HBV strain LSH, woodchuck HBV, and Woolly Monkey HBV.

6. The method according to claim 5, wherein said human HBV is HBV strain alpha1.

7. The method according to claim 5, wherein said human HBV is HBV subtypes adr, ad, adw, adyw, ar or ayw.

8. A method for treating a subject having a HBV infection, comprising the step of administering to the subject a synthetic polypeptide that is chemically modified to bear a hydrophobic moiety that is a fatty acid or a cholesterol, wherein the synthetic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 14.

9. A method for treating a subject having a HBV infection, comprising the step of administering to the subject a synthetic polypeptide consisting of SEQ ID NO: 2 that is chemically modified to bear a hydrophobic moiety that is a fatty acid or a cholesterol.

10. The method according to claim 9, wherein the first amino acid of the polypeptide of SEQ ID NO: 2 is chemically modified to bear myristic acid or stearic acid.

11. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 1.

12. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 4.

13. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 5.

14. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 6.

15. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 7.

16. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 8.

17. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 9.

18. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 10.

19. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 13.

20. The method of claim 8, wherein the amino acid sequence consists of SEQ ID NO: 14.

21. The method of claim 1, wherein X is absent and myristic acid is attached to the first amino acid of Y.

22. The method of claim 1, wherein the synthetic polypeptide consists of Y chemically attached to a fatty acid or a cholesterol.

23. The method of claim 1, wherein the synthetic polypeptide consists of Y chemically attached to a fatty acid or a cholesterol and Y consists of the amino acid starting at position 2 and ending at position 48.

24. The method of claim 1, wherein the synthetic polypeptide consists of Y chemically attached to myristic acid, wherein Y consists of amino acids starting at position 2 and ending at position 48.

Description

(1) The invention will be further understood in view of the following examples and the annexed figures wherein:

(2) FIG. 1 is the comparison of HBV infectivity of human hepatocytes cultured in the presence of a myristoylated or non myristoylated peptide corresponding to a pre-S1 region domain (1-78). The infection efficiency was evaluated by measuring HBsAg in the supernatant of infected cells, 14 days post-infection. Values are expressed as a % of the control (no peptide).

(3) FIG. 2 illustrates the comparison of inhibitory activity of C-terminally truncated myristoylated peptides. Human hepatocytes were HBV infected in the presence of myristoylated or non-myristoylated peptides corresponding to parts of the Pre-S1 domain of the HBV L protein. The concentration of peptides ranged from 0.8 to 800 nM. Infection efficiency was evaluated by measuring HBsAg secretion 7 days after infection and expressed as a % of the positive control.

(4) FIG. 3 is the comparison of the inhibitory activity of C-terminally truncated peptides shorter than Myr 2-48. The concentration of peptides ranged from 0.8 to 8 M. Infection efficiency was evaluated by measuring HBsAg secretion 8 days after infection and expressed in ng/ml.

(5) FIG. 4 illustrates the comparative inhibitory activity of WMHBV and HBV derived myristoylated 2-48 peptides. Infection of human primary human hepatocytes with HBV was performed in the presence of decreasing concentration of peptides. The supernatant of infected cells was collected at day 10 post-infection and analysed for the presence of HbsAg.

(6) FIG. 5 is a Dot Blot analysis of duck HBV (DHBV) in the serum of ducks treated with duck preS Myr 2-41 (DpreS2-41.sup.myr), heron preS Myr 2-44 (HepreS2-44.sup.myr), human preS Myr 2-68 (HupreS2-68.sup.myr), duck preS Myr 2-21 (DpreS2-21.sup.myr) or ddH.sub.2O, 5, 9, 15 and 28 days post-infection (p.i.) with DHBV.

(7) FIG. 6 is a Western Blot analysis of DHBV L protein in the serum of ducks 35 days post infection.

EXAMPLE 1: MATERIAL AND METHODS FOR THE INHIBITION OF HBV INFECTION WITH PRE-S1-HBV SYNTHETIC POLYPEPTIDES

(8) a) Establishment of HBV-Infectable Cell Culture

(9) Fragments of normal adult human liver were obtained from patients undergoing hepatic resection for liver metastases (the fragments were taken at a distance from the metastasis in macroscopically normal liver). Access to this biopsy material was in agreement with French laws and satisfied the requirements of the French National Ethics Committee. Hepatocytes were isolated by the procedure of Guguen-Guillouzo and Guillouzo and cultured in H medium supplemented with 3.510.sup.6 M hydrocortisone hemisuccinate, 2 mM L-glutamine, 50 mg of gentamicin per liter, 2% dimethyl sulfoxide, 5% adult human serum, and 5% FCS. Three days after seeding, the cells were infected.

(10) Alternatively, for some experiments, instead of using primary cultures, we made use of a new hepatoma derived cell line, called HepaRG, which is also susceptible to HBV infection (patent application FR 0109044). Before the infection procedure cells were allowed to differentiate, allowing cells to gain an hepatocyte-like morphology (patent application FR 0109044). Cells were then infected.

(11) b) HBV Infection of Cell Culture

(12) As an infectious inoculum, a 50-fold concentrated culture medium of HepG2 clone 2.2.15 cells was used, because of an unlimited supply and a constant quality.

(13) It was prepared as described previously. Differentiated cells were incubated with the concentrated infectious source, 10-fold diluted in culture medium supplemented with 5% PEG 8000 (Sigma), for 20 h at 37 C. as described previously (Gripon et al., 1988; Gripon et al., 1993). Control cultures were incubated with 5% PEG and 25% FCS diluted in phosphate-buffered saline (PBS) instead of the infectious source. At the end of the incubation, cells were washed three times with the culture medium and maintained in the presence of 2% DMSO and 510.sup.5 M hydrocortisone hemisuccinate and harvested at indicated times.

(14) c) Polypeptide Competition Assays

(15) Polypeptide competition assays were performed by pre-incubating cells with the analyzed polypeptide for 30 min, at 37 C., prior to the addition of the infectious source.

(16) d) Inhibition of HBV Infection Assessment

(17) HBsAg assay

(18) HBsAg was detected in the medium by an ELISA kit (Monolisa AgHBs plus) obtained from Bio-Rad Laboratories. Values are expressed in ng/ml of supernatant or percent of control (absence of peptide).

(19) RNA Extraction and Analysis

(20) Total cellular RNA was extracted by Total SV RNA kit (Promega, France), fractionated on a 1.5% agarose gel and analyzed by standard Northern blot procedure (Sambrook et al., 1989). Control of the RNA amount transferred onto filters was performed after methylene blue staining. Hybridization was performed with P.sup.32 labeled HBV DNA.

EXAMPLE 2: RESULTS OF HBV INFECTION INHIBITION WITH PRE-S1-HBV SYNTHETIC POLYPEPTIDES

(21) a) Influence of the Myristoylation of Pre-S1 Synthetic Peptides on HBV Infection Inhibition

(22) Mutagenesis experiments have previously shown that a part (amino acids (AA) 3-77) of the pre-S1 region was essential for HBV infectivity (Le Seyec et al., 1999). In addition we have also demonstrated that myristoylation of the AA 2, a glycine residue, associated with the removal of AA 1, a methionine residue, was also critical (Gripon et al., 1995). We have therefore postulated that a peptide comprising amino acids 2-77, with a myristoylated glycin, could interfere with the HBV infection process. To evaluate this hypothesis two peptides were synthesized: PreS 1-78 and Myr PreS 2-78. These peptides were then added prior and during the infection process of human hepatocyte cultures, the infection level was evaluated by measuring the HBsAg secretion of infected cells. FIG. 1 displays that although the non-myristoylated peptide has only a faint effect on HBV infectivity at 1 M, the same amount of the myristoylated peptide did almost completely abolish it, lower doses were partly inhibitory. These results were confirmed by RNA analysis.

(23) Other experiments conducted with higher peptide concentrations (up to 100 M) indicate that myristoylation is not absolutely required for the inhibition of HBV infection but strongly enhances the activity of the peptides by a factor of about 100 fold.

(24) b) Activity of C-Terminally Truncated Pre-S1 Synthetic Peptides

(25) Human hepatocytes were HBV infected in the presence of myristoylated or non-myristoylated peptides corresponding to parts of the Pre-S1 domain of the HBV L protein. The concentration of peptides ranged from 0.8 to 800 nM. FIG. 2 shows the inhibition activity of myristoylated truncated peptides. Results obtained with non-myristoylated peptides are displayed in Table 1 (infra).

(26) It appears that peptide Myr 2-48 shows the highest inhibitory activity. The larger peptides, Myr 2-68 and Myr 2-78, although very efficient at 800 nM are less active at lower doses. The smaller peptide Myr 2-28 is largely less active although a 50% inhibition is observed at 800 nM.

(27) As some activity still persists for the peptide smaller than 2-48, in order to evaluate the contribution of the N-terminal amino acids, we have produced and evaluated a new set of short peptides. The results are shown on FIG. 3. From this figure it is obvious that Myr 2-38 and Myr 2-28 peptides still retain a significant inhibitory activity as they almost completely blocks HBV infection at 8 M. By contrast the 2 shorter peptides are no longer active and the shorter one tends to increase HBV infectivity, for unknown reasons.

(28) The effect of myristoylation and C-terminal truncation of pre-S1 peptides was also studied through RNA analysis. The results confirm that myristoylated truncated peptides displayed enhanced inhibitory activity as compared to the corresponding non myristoylated peptides, and that the highest inhibitory activity is obtained with Myr 2-48.

(29) c) HBV Infection Inhibitory Activity of Pre-S1 Homologous Sequences

(30) A recently discovered primate hepadnavirus, the Wolly Monkey Hepatitis B virus (WMHBV) has been shown very poorly infectious for Chimpanzee and for human hepatocyte primary cultures. The WMHBV pre-S 1-78 polypeptidic sequence shows 66% sequence identity to the original HBV derived peptide. We have investigated the inhibitory activity of a WMHBV derived Myr 2-48 peptide (SEQ ID NO: 14) towards HBV infection of human cells.

(31) FIG. 4 illustrates the comparative inhibitory activity of WMHBV and HBV derived myristoylated 2-48 peptides (64% sequence identity). This experiment clearly shows that the WMHBV derived peptide is surprisingly nearly as efficient as the HBV derived peptide in inhibiting the HBV infection. This result is in contrast with the complete absence of activity of a DHBV derived peptide (PreS Myr 2-41 (SEQ ID NO: 16), see table 1) on HBV infection although this peptide is a strong inhibitor of DHBV infection.

(32) From these results we can conclude that it is possible to efficiently inhibit the HBV infection. The tolerance of up to 46% variations in the peptide sequence suggests that it will be possible to inhibit the infection of HBV viruses of all genotypes with a single peptide.

(33) TABLE-US-00001 TABLE1 summaryoftheresultsofthecompetition experiments. HBV HBV infec- infec- tivity tivity of of human human Non hepato- hepato- myristoylated cytes Myristoylated cytes polypeptides inhi- polypeptides inhi- (100M) bition (1M) bition HBVPreS1-78 ND HBVMyrPreS2-78 ++++ (SEQIDNO:1) (SEQIDNO:4) HBVPreS1-68 ND HBVMyrPreS2-68 ++++ (SEQIDNO:5) HBVPreS1-48 +++ HBVMyrPreS2-48 +++++ (SEQIDNO:6) (SEQIDNO:7) HBVPreS1-38 ND HBVMyrPreS2-38 +++ (SEQIDNO:8) HBVPreS1-28 + HBVMyrPreS2-28 ++ (SEQIDNO:9) (SEQIDNO:10) HBVPreS1-18 ND HBVMyrPreS2-18 (SEQIDNO:11) HBVPreS1-8 ND HBVMyrPreS2-8 (SEQIDNO:12) HBVPreS19-48 HBVMyrPreS19-48 (SEQIDNO:17) (SEQIDNO:18) WMHBVPreS1-48 +++ WMHBVMyrPreS2-48 +++++ (SEQIDNO:13) (SEQIDNO:14) DHBVPreS1-41 DHBVMyrPreS2-41 (SEQIDNO:15) (SEQIDNO:16) ND: Not Determined

EXAMPLE 3: IN VIVO DHBV INFECTION INHIBITION WITH PRE-S-HBV SYNTHETIC POLYPEPTIDES

(34) Ducks are simultaneously injected with DHBV and duck preS Myr 2-41 (DpreS2-41.sup.myr), heron preS Myr 2-44 (HepreS2-44.sup.myr), human preS Myr 2-68 (HupreS2-68.sup.myr), duck preS Myr 2-21 (DpreS2-21.sup.myr) or ddH.sub.2O. The viremia of infected animals is assessed by Dot Blot analysis of viral DNA, 5, 9, 15 and 28 days post-infection. The results displayed in FIG. 5 show that viral DNA is detected in control animals treated with ddH.sub.2O, indicated a successful infection of these animals. On the contrary, a transitory viremia can be observed at days 9 and 15 post-infection in ducks treated with either DpreS2-41.sup.myr or DpreS2-21.sup.myr. At day 28, viral DNA is decreased or no longer detectable which suggests that the animals eliminated the virus. This analysis is further supported by the Western Blot analysis shown in FIG. 6 that shows the L protein of DHBV is not detected in the serum of ducks treated with either DpreS2-41.sup.myr or DpreS2-21.sup.myr 35 days post-infection.

(35) On the contrary, the human peptide HupreS2-68.sup.myr seems to delay but do not prevent infection of ducks with DHBV (FIGS. 5 and 6).

(36) Since peptide DpreS2-21.sup.myr was shown to have no in vitro inhibitory activity towards DHBV infection of primary duck hepatocytes, the in vivo protection observed with this peptide would result from an indirect effect of the peptide, i.e. enhancement of the immune response through eliciting antibodies directed against DpreS2-21.sup.myr.

(37) This experiment illustrates that myristoylated synthetic peptides can confer protection against hepatitis B virus infection.

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