METHOD FOR REDUCING HEPATITIS B VIRUS SURFACE ANTIGEN (HBsAg) AND HEPATITIS B DRUG FOR USE IN THE METHOD

Abstract

There is provided a method of curing chronic hepatitis B infection in a human, the method comprising administering to a subject in need thereof an effective amount of one or both of an exogenous anti-HBs antibody or an anti-HBs antibody producing carrier, the present invention relates to a method for reducing cellular and blood hepatitis B surface antigen (HBsAg) by providing continuously increased levels of anti-HBs antibodies in the subject.

Description

[0001] Cross-references to related applications

[0002] This application relates to and claims priority to U.S. Provisional Application No. 63 / 506,582, filed June 6, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0003] Statement on Federally Funded Research

[0004] This invention was carried out with government support under Contract No. 75N930220C00042 granted by the National Institutes of Health. The government enjoys certain rights in this invention. Technical Field

[0005] This invention relates to a method for reducing cellular and serum hepatitis B virus surface antigen (HBsAg) levels in chronic hepatitis B virus (HBV) infection by maintaining high levels of anti-HBs antibodies and blocking new infection-mediated cccDNA supplementation. Background Technology

[0006] Hepatitis B virus (HBV) chronically infects 316 million people worldwide, and nearly one million people die from HBV-related diseases each year. Current HBV drugs rarely deliver durable HBV replication suppression after years of treatment, let alone functional cure.

[0007] Currently, to establish HBV functional cure, both serum HBsAg and HBV DNA markers need to become undetectable. In other words, HBV functional cure is defined as the undetectable presence of serum HBsAg and HBV DNA after a limited period of HBV treatment (Alter et al., Hepatology 67, 1127-1131 (2018)). While serum HBV DNA can be suppressed and reduced to undetectable levels after long-term treatment with approved HBV drugs using nucleoside / nucleotide analogs (NAs), the most challenging aspect is effectively reducing serum HBsAg to undetectable levels.

[0008] Current strategies for lowering serum HBsAg levels involve directly inhibiting intracellular HBsAg synthesis using siRNA or antisense oligonucleotide (ASO) drugs. However, the efficacy of this strategy and siRNA / ASO-based therapies is limited; both preclinical and clinical evaluations show an average reduction of <2 log in serum HBsAg, and serum HBsAg levels rebound after treatment cessation.

[0009] A more efficient method to reduce HBsAg levels is needed to establish an effective functional cure for HBV. Summary of the Invention

[0010] This invention discloses how to effectively reduce cellular and serum HBsAg levels, and includes the following elements:

[0011] 1. In contrast to current strategies that reduce serum HBsAg by directly inhibiting intracellular HBsAg synthesis, the method of the present invention does not require direct inhibition of intracellular HBsAg synthesis.

[0012] 2. cccDNA is the primary HBsAg transcription template, but it is frequently spontaneously deleted in infected cells. HBV-infected cells continue to secrete HBsAg into the bloodstream, and if the missing cccDNA library is not replenished, HBsAg depletion in infected cells can occur. Therefore, the method of this invention aims to reduce cellular and serum HBsAg levels by using sustained high levels of anti-HBs antibodies to block new infection-mediated cccDNA replenishment, in the presence or absence of other anti-HBV drugs.

[0013] 3. This invention provides a method for expressing sustained high levels of anti-HBs antibodies using an AAV anti-HBs vector after a single injection, and can be used to block new infection-mediated cccDNA supplementation.

[0014] The advantages of this invention (whether alone or in combination) can be achieved by a method for curing chronic hepatitis B infection in humans, said method comprising:

[0015] By providing subjects with sustained elevated levels of anti-HBs antibodies, administering an effective amount of exogenous anti-HBs antibodies or a vector that produces anti-HBs antibodies, or both, to subjects in need, to reduce cellular and blood hepatitis B surface antigen (HBsAg), wherein the reduction in cellular and blood HBsAg occurs if: (i) new infection-mediated cccDNA supplementation is blocked, or (ii) HBsAg synthesis is not directly inhibited, or both (i) and (ii). Attached Figure Description

[0016] A more complete understanding of the invention and its many accompanying advantages will readily come into view, and it will be better understood when considered in conjunction with the accompanying drawings by referring to the following detailed description, in which:

[0017] Figure 1A-1D The dynamic HBsAg levels in the blood of HBV-infected uPA / SCID chimeric mice with humanized livers are shown (days 14 to 162 post-infection). Figure 1A The unprocessed group 1 (G1) is shown. Figure 1B-1D This indicates the treatment of groups 2 and 3 (G2-G4) with HBVZ10 and entecavir for 12 weeks, but starting at different time points to achieve different HBsAg levels. HBVZ10 was administered as follows: in G2, at a dose of 2.5E11 copies on day 11 (d11) and at a dose of 4E11 copies on day 58. Figure 1B In G3, a dose of 1.8E12 copies was administered on day 22. Figure 1C ), and in G4, on day 44, at a dose of 7.2E12 copies ( Figure 1D In mice that died before day 162, the time point was truncated. The acceptable lower limit for HBsAg was 0.05 IU / ml (HBsAg EIA kit, Bio-rad).

[0018] Figure 2 The copy number of HBsAg and the number of influent cells per cell are shown for determination of HBsAg in liver lysates from HBV-infected uPA / SCID chimeric mice with humanized livers. Cellular HBsAg levels were titrated with sequential dilutions of 1 / 1000, 1 / 100, 1 / 10, 1 / 2, 1 / 3.3, and 1 / 2.5. Inhibition was detected at a 1 / 2.5 dilution, and a dilution of 1 / 3.3 was permissible. Four total lysates from each of animals 825, 838, and 831 were analyzed and numbered .1, .2, .3, and .4, respectively.

[0019] Figure 3 shows Figure 2 The numerical relationship between cellular HBsAg and rcDNA in each cell of the mouse. Figure 3A A graphical representation is provided, showing the copy number of HBsAg and rcDNA in each cell of two mice in which serum HBsAg was undetectable and an untreated mouse control. Figure 3B Provided Figure 3A The ratio of HBsAg to rcDNA in the cells of mice.

[0020] Figures 4A-4C A graphical representation is provided, showing serum HBsAg levels, serum HBV DNA levels, and intracellular HBsAg, rcDNA, and cccDNA levels during the HBV infection phase.

[0021] Figures 5A-5C A graphical representation is provided showing the cccDNA levels in the analyzed mouse liver samples.

[0022] Figure 6A graphical representation is provided showing the correlation between mean cccDNA and HBV RNA levels in HBV infection.

[0023] Figures 7A-7C A graphical representation is provided to illustrate the effect of treatment with the AAV anti-HBs vector HBVZ10 according to one embodiment of the present invention on the mean cccDNA and rcDNA levels in mice.

[0024] Figures 8A-8H A graphical representation is provided showing the effects on mean cccDNA levels, kinetic serum HBsAg levels, and kinetic serum HBeAg, demonstrating the progressive clearance of cccDNA after treatment using the embodiments of the present invention.

[0025] Figures 9A-9B A graphical representation is provided, showing that subjects treated using embodiments of the present invention showed no significant difference in mean Ki67 RNA levels, but a significant difference in mean cccDNA levels, compared to untreated subjects.

[0026] Figure 10A-10J Graphical representations are provided showing kinetic serum HBsAg levels and intracellular HBsAg levels, which are also confirmed by the Western blot analysis shown in these figures.

[0027] Figure 11A-11B A schematic representation of cccDNA deletion driven by replication through spontaneous clearance or cell disruption pathways is provided. Detailed Implementation

[0028] The embodiments identified as exemplary herein are intended to be illustrative and not limiting. Table 1 summarizes some key features of the invention compared to current conventional therapies for reducing HBsAg, and these key features are further elaborated in the description below.

[0029] Table 1.

[0030]

[0031] Hepatitis B virus (HBV) is a hepatotropic DNA virus that, if contracted in infancy, can cause persistent and largely non-cytopathic infection [see, for example, Summers J., Hepatology, 1981;1(2):179-83; and Seeger C et al., The Liver: Biology and Pathobiology, 2020:793-820]. Establishment and maintenance of HBV infection in hepatocytes require the formation of a free, covalently closed circular DNA (cccDNA) molecule, which serves as a template for viral transcription in the cell nucleus [see, for example, Summers J et al., Cell, 1982;29(2):403-15; and Tuttleman JS et al., Cell, 1986;47(3):451-60]. Therefore, a single copy of cccDNA in infected cells is the minimum required. cccDNA molecules are considered long-lived [Alter H et al. Hepatology. 2018;67(3):1127-31], as chronic HBV infection often persists for years or decades [Seto WK et al., The Lancet, 2018;392(10161):2313-24]. Chronic HBV infection is thought to be due to the host's immune system's failure to clear an established infection [Guidotti LG et al., Annual Review of Pathology. 2006;1:23-61]; therefore, chronic HBV infection is often considered a continuation of an established initial infection. Conventional HBV cure strategies aim to directly eliminate or permanently silence cccDNA [Alter H et al. Hepatology. 2018;67(3):1127-31], or clear infected cells from the liver [Fanning GC et al., Nature Reviews Drug discovery, 2019;18(11):827-44].

[0032] HBV replicates robustly in infected human hepatocytes, such as through elevated serum hepatitis B surface antigen (HBsAg), HBV DNA levels [see, for example, Keating SM et al., The Journal of Infectious Diseases, 2014;209(6):845-54; Jaroszewicz J et al., Journal of Hepatology, 2010;52(4):514-22; and Nguyen T et al., Journal of Hepatology, 2010;52(4):508-13], and the accumulation of HBsAg and hepatitis B core antigen (HBcAg) proteins in infected cells during chronic HBV infection [see, for example, Naoumov NV et al., Gastroenterology, 1990;99(4):1248-5312; and Chu CM et al., Journal of Clinical Pathology, 1995;48(5):470-3] or in vitro infection [see, for example, Ko C et al., Journal of Hepatology, 2014;209(6):845-54; Jaroszewicz J et al., Journal of Hepatology, 2014;209(6):845-54 ... As demonstrated in *Journal of Hepatology*, 2018;69(6):1231-41; and König A et al., *Journal of Hepatology*, 2019;71(2):289-300]. Intracellular accumulation of viral products can indicate that the ability of infected cells to secrete viral particles lags behind the ability to replicate HBV, which, if not stopped, can lead to cytopathic changes. Retention of the L protein in hepatocytes of HBV transgenic mice causes a range of pathologies, including necrosis and persistently elevated ALT levels, and the severity of the pathology is correlated with the concentration of intracellular envelope proteins [Chisari FV et al., *Proceedings of the National Academy of Sciences of the United States of America*, 1987;84(19):6909-13]. The accumulation of intracellular HBsAg in the smooth endoplasmic reticulum (ER) causes ER proliferation and displaces other organelles to the periphery, giving some hepatocytes a "frosted glass" appearance in chronically infected cells [Hadziyannis S et al., ArchPathol, 1973;96(5):327-30], [Gerber MA et al., The American Journal of Pathology, 1974;75(3):489].However, most HBsAg-positive cells in liver sections did not show a ground-glass appearance [Deodhar K et al., Journal of Clinical Pathology, 1975;28(1):66-70]. In the early 1990s, Summers et al. discussed the following principles regarding the replication and persistent infection of hepatotropic DNA viruses: i. Persistent infection with hepatotropic DNA viruses depends on the suppression of replication in the late stages of infection; ii. Control of cccDNA copy number is required to maintain persistent non-cytopathic infection, as high levels of cccDNA are always associated with cytopathic effects in infected hepatocytes; and iii. Intracellular accumulation of L protein acts as an overall inhibitor of replication and allows for persistent infection [see, for example, Summers J et al., Journal of Virology, 1991;65(3):1310-7; and Lenhoff RJ et al., Journal of Virology, 1994;68(9):5706-13].

[0033] Clinical evidence suggests the dynamic evolution of cccDNA populations. During chronic hepatitis B infection, wild-type viral populations in serum or cccDNA in the liver can be cleared and replaced by mutant populations [see, for example, Brunetto MR et al., Proceedings of the National Academy of Sciences of the United States of America, 1991;88(10):4186-90; Carman WF et al., Lancet, 1989;2(8663):588-91; Jiang B et al., Alimentary Pharmacology & Therapeutics, 2019;50(8):940-54; Chen QY et al., Infection, Genetics and Evolution, 2021:105184; and Huang Q et al., Hepatology (Baltimore, Md), 2020]. In patients with chronic hepatitis B who received nucleoside analogue (NA) therapy, complete cccDNA turnover occurred within a duration of as short as 24 weeks [Huang Q et al., Hepatology (Baltimore, MD), 2020].

[0034] Quantitative detection of cccDNA levels in serial liver tissue supported the pre-existing deletion of cccDNA, indicating that cccDNA levels were progressively reduced by 20 to 100 times during NA treatment in prairie dogs chronically infected with prairie hepatitis virus (an animal model very similar to chronic HBV infection in humans) [Zhu Y et al., Journal of Virology, 2001;75(1):311-22]. It has been reported that cccDNA levels were also reduced by 1–2.9 log in human patients treated with NA [see, for example, Werle-Lapostolle B et al., Gastroenterology, 2004;126(7):1750–8; Wong DK et al., Antiviral Therapy, 2006;11(7):909–16; Wursthorn K et al., Hepatology, 2006;44(3):675–84; Lutgehetmann M et al., Antiviral Therapy, 2008;13(1):57–66; Boyd A et al., Journal of Hepatology, 2016;65(4):683–91; and Lai CL et al., Journal of Hepatology, 2017;66(2):275–81]. The detected reduction in cccDNA increases the likelihood that cccDNA molecules can be spontaneously cleared from infected cells.

[0035] In summary, the inventors hypothesize that HBV-infected cells spontaneously clear cccDNA in vivo. Testing this hypothesis in uPA / SCID chimeric mice with humanized livers that support robust persistent HBV infection in the absence of functional T and B cell immunity [Tateno C et al., PloS ONE, 2015;10(11):e0142145] provides evidence supporting the concept of spontaneous cccDNA loss in HBV-infected cells, and a cccDNA elimination strategy that transforms spontaneous cccDNA clearance into progressive cccDNA elimination by blocking cccDNA replenishment. Therefore, this invention relates to the treatment of persistent HBV infection by blocking cccDNA replenishment and providing highly effective cccDNA elimination therapy.

[0036] The anti-hepatitis B drug used in this invention

[0037] Any existing or newly developed anti-hepatitis B drug that can block cccDNA and / or rcDNA supplementation and / or synthesis can be used in the treatment methods described above. Such drugs include, but are not limited to, exogenously or endogenously expressed antibody drugs, small molecule drugs, peptide drugs, and carrier drugs that inhibit any step of HBV infection and replication. The following AAV anti-HBs vector-based HBV drugs were designed and tested in this invention. These AAV anti-HBs vector-based HBV drugs endogenously express anti-HBs antibodies after a single injection, and it was found that when used in the treatment methods described above, these AAV anti-HBs vector-based HBV drugs are effective against chronic hepatitis infection by blocking cccDNA supplementation in cells. A total of nine AAV anti-HBs vectors contain the following sequences:

[0038] Nucleic acid sequence ID NO: 1 encodes the amino acid sequence of sequence ID NO: 2. Sequence ID NO: 2 is the variable region of the heavy chain of HBVZ10 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0039] Nucleic acid sequence ID NO: 3 encodes the amino acid sequence of sequence ID NO: 4. Sequence ID NO: 4 is the variable region of the light chain of HBVZ10 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0040] Nucleic acid sequence ID NO: 5 encodes the amino acid sequence of sequence ID NO: 6. Sequence ID NO: 6 is the variable region of the heavy chain of HBVZ20 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0041] Nucleic acid sequence ID NO: 7 encodes the amino acid sequence of sequence ID NO: 8. Sequence ID NO: 8 is the variable region of the light chain of HBVZ20 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0042] Nucleic acid sequence ID NO: 9 encodes the amino acid sequence of sequence ID NO: 10. Sequence ID NO: 10 is the variable region of the heavy chain of HBVZ30 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0043] Nucleic acid sequence ID NO: 11 encodes the amino acid sequence of sequence ID NO: 12. Sequence ID NO: 12 is the variable region of the light chain of HBVZ30 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0044] Nucleic acid sequence ID NO: 13 encodes the amino acid sequence of sequence ID NO: 14. Sequence ID NO: 14 is the variable region of the heavy chain of HBVZ40 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0045] Nucleic acid sequence ID NO: 15 encodes the amino acid sequence of sequence ID NO: 16. Sequence ID NO: 16 is the variable region of the light chain of HBVZ40 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0046] Nucleic acid sequence ID NO: 17 encodes the amino acid sequence of sequence ID NO: 18. Sequence ID NO: 18 is the variable region of the heavy chain of HBVZ50 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0047] Nucleic acid sequence ID NO: 19 encodes the amino acid sequence of sequence ID NO: 20. Sequence ID NO: 20 is the variable region of the light chain of HBVZ50 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0048] Nucleic acid sequence ID NO: 21 encodes the amino acid sequence of sequence ID NO: 22. Sequence ID NO: 22 is the variable region of the heavy chain of HBVZ60 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0049] Nucleic acid sequence ID NO: 23 encodes the amino acid sequence of sequence ID NO: 24. Sequence ID NO: 24 is the variable region of the light chain of HBVZ60 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0050] Nucleic acid sequence ID NO: 25 encodes the amino acid sequence of sequence ID NO: 26. Sequence ID NO: 26 is the variable region of the heavy chain of HBVZ70 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0051] Nucleic acid sequence ID NO: 27 encodes the amino acid sequence of sequence ID NO: 28. Sequence ID NO: 28 is the variable region of the light chain of HBVZ70 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0052] Nucleic acid sequence ID NO: 29 encodes the amino acid sequence of sequence ID NO: 30. Sequence ID NO: 30 is the variable region of the heavy chain of HBVZ80 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0053] Nucleic acid sequence ID NO: 31 encodes the amino acid sequence of sequence ID NO: 32. Sequence ID NO: 32 is the variable region of the light chain of HBVZ80 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0054] Nucleic acid sequence ID NO: 33 encodes the amino acid sequence of sequence ID NO: 34. Sequence ID NO: 34 is the variable region of the heavy chain of HBVZ90 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0055] Nucleic acid sequence ID NO: 35 encodes the amino acid sequence of sequence ID NO: 36. Sequence ID NO: 36 is the variable region of the light chain of HBVZ90 human anti-HBs monoclonal IgG1 antibody against four HBsAg serotypes.

[0056] Nucleic acid sequence ID NO: 37 is the nucleic acid sequence of the AAV vector, consisting of 3758 bp (including two ITRs (inverted terminal repeats from AAV)), a chicken β-actin promoter, constant regions of the human IgG1 heavy and light chains, WPRE (post-transcriptional regulatory element of marmot hepatitis virus), and an SV40 polyadenylation signal. This AAV vector (Sequence ID NO: 37) allows cloning of two variable regions of both the heavy and light chains to express a complete human IgG1 monoclonal anti-HBs antibody.

[0057] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 2 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0058] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 6 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0059] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 10 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0060] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 14 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0061] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 18 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0062] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 22 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0063] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 26 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0064] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 30 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0065] In one embodiment, the present invention relates to a separate binding molecule or antigen-binding fragment thereof that specifically binds to HBV viral particles and / or HBsAg subviral particles containing antibody VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 34 and antibody VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least 95% sequence homology. In one embodiment, the present invention relates to a nucleic acid molecule encoding the above-described separate binding molecule or antigen-binding fragment. In one embodiment, the present invention relates to a vector comprising the above-described nucleic acid molecule.

[0066] In one embodiment, the present invention relates to a method for curing chronic hepatitis B infection in a human, the method comprising administering an effective amount of a carrier for generating anti-HBs antibodies to a subject in need to reduce cellular and blood hepatitis B surface antigen (HBsAg) by providing a sustained-rise level of anti-HBs antibodies in the subject. In some embodiments, the sustained-rise level of anti-HBs antibodies is 100 mIU / ml, 1000 mIU / ml, 10,000 mIU / ml, 100,000 mIU / ml or higher, for a period of 3 months or longer. In a preferred embodiment, these levels of anti-HBs antibodies are generated during the period by a single administration of the carrier for generating anti-HBs antibodies.

[0067] In a preferred embodiment, a single dose of the vector that generates anti-HBs antibodies is 1E11 copies or more, more preferably 2E11 copies or more, still more preferably 1E12 copies or more, and most preferably 3E12 copies or more.

[0068] If the concept of spontaneous cccDNA clearance from infected cells is valid, then continuous cccDNA replenishment is required to maintain cccDNA levels. The method of this invention provides a therapeutic intervention designed to block cccDNA replenishment.

[0069] Two known pathways facilitate cccDNA replenishment: intracellular recycling and neo-infection. The intracellular recycling pathway involves the delivery of newly synthesized rcDNA molecules to the nucleus for cccDNA conversion [see, for example, Tuttleman JS et al., Cell, 1986;47(3):451-60; and Nassal M., Gut, 2015;64(12):1972-84]. However, this pathway functions primarily during the early stages of replication and is impaired by the accumulation of envelope proteins during the later stages of replication [Lenhoff RJ et al., Journal of Virology, 1994;68(9):5706-13]. Previous studies have shown that new infection is the main pathway for cccDNA replenishment [see, for example, König A et al., Journal of Hepatology, 2019;71(2):289-300; Allweiss L et al., Gut, 2018;67(3):542-52; and Volz T et al., Journal of Hepatology, 2013;58(5):861-7].

[0070] This invention rethinks cccDNA elimination strategies. Directly targeting cccDNA or killing infected cells is generally recommended for cccDNA elimination and complete cure of chronic HBV infection. Based on this invention, such strategies may not be necessary. Instead, this invention provides an unconventional cccDNA elimination strategy that does not require direct targeting of the cccDNA molecule, but rather aims to transform spontaneous cccDNA loss into progressive cccDNA elimination by blocking cccDNA replenishment.

[0071] In some embodiments of the method of the present invention, the reduction of HBsAg in cells and blood occurs when: (i) the new infection-mediated cccDNA supplementation is blocked, or (ii) HBsAg synthesis is not directly inhibited, or both (i) and (ii).

[0072] In some embodiments, the anti-HBs antibody provides blocking of neonatal infection-mediated cccDNA supplementation. In some embodiments, the anti-HBs antibody blocks neonatal infection by blocking the attachment of HBV particles (viral particles and subviral particles) to human hepatocytes. In some embodiments, the anti-HBs antibody is specific for the "a" determinant of HBsAg, and preferably specific for the human hepatocyte attachment site within the "a" determinant of HBsAg.

[0073] In some embodiments, anti-HBs antibodies are provided by one or more administrations, wherein said administration is performed via a procedure selected from: injection, infusion, oral administration, or transdermal administration. In embodiments of the invention, sustained high levels of anti-HBs antibodies are provided endogenously or exogenously. In some embodiments, anti-HBs antibodies can be expressed by administration of a virus, non-viral vector, or nanoparticles that deliver a human anti-HBs antibody gene or mRNA to express anti-HBs antibodies or a vaccine that expresses or directly delivers HBsAg protein or peptide to elicit an anti-HBs antibody response in a receptor.

[0074] In some embodiments, anti-HBs antibodies are expressed by administering a viral vector (including, but not limited to, AAV vector-based therapies) to express sustained high levels of anti-HBs antibodies. In a preferred embodiment, the AAV vector-based therapy comprises administering one or more members selected from the group consisting of HBVZ10, HBVZ20, HBVZ30, HBVZ40, HBVZ50, HBVZ60, HBVZ70, HBVZ80, and HBVZ90. In some embodiments, exogenous anti-HBs antibodies are infused or human anti-HBs antibodies, nanobodies, or antibody fragments are injected.

[0075] In the method of the present invention, the reduction of HBsAg in the blood can be gradual or abrupt, both of which reduce blood HBsAg by 1-5 log, preferably 3-5 log, or to an undetectable level or < 0.05 IU / ml.

[0076] In one implementation, treating chronic HBV infection includes treating newborns / children who are already infected with HBV.

[0077] In one implementation, treating chronic HBV infection includes treating adults who are already infected with HBV.

[0078] In one implementation, an HBV-infected human patient is an individual with chronic HBV infection who has been HBsAg positive for more than 6 months and has normal or elevated alanine aminotransferase (ALT) levels.

[0079] In one implementation, the human patient infected with HBV is an HBV-positive pregnant woman, or an organ transplant recipient who is HBsAg-positive or HBsAg-negative / anti-hepatitis B core antibody (anti-HBc) positive after transplantation and is prone to recurrent HBV infection.

[0080] In one implementation, the HBV neutralizing antibody or antibody fragment is generated from an HBV therapeutic vector.

[0081] In one embodiment, the HBV therapeutic vector comprises a mixed population of vectors, wherein each vector encodes a specific anti-HBs antibody or antibody fragment that binds to one or more epitopes of the HBV envelope protein, or comprises a single vector that encodes an HBV neutralizing antibody or antibody fragment that binds to one or more epitopes of the HBV envelope protein.

[0082] In some embodiments, the method is a monotherapy. In other embodiments, the method is a combination therapy with one or more additional HBV drugs that inhibit the synthesis of intracellular HBV DNA, HBV RNA, and / or viral proteins. In such combination therapy embodiments, the one or more additional HBV drugs are preferably members of the group consisting of: reverse transcription inhibitors, capsid inhibitors, cccDNA inhibitors, RNA transcription inhibitors, viral protein synthesis inhibitors, entry inhibitors, interferons, therapeutic vaccines, immune checkpoint inhibitors, and immunomodulatory drugs.

[0083] In one key embodiment, the present invention utilizes the discovery of the cccDNA molecule, which is the primary transcriptional template directing HBsAg synthesis in cells and is frequently spontaneously deleted from infected cells. HBsAg is continuously secreted from infected cells into the bloodstream. The deletion of cccDNA and the continuous secretion of HBsAg will lead to the depletion of HBsAg in infected cells. Therefore, if the replenishment of the depleted cccDNA library is prevented, HBsAg can be cleared from infected cells through secretion alone. This understanding forms the basis of the present invention.

[0084] In a preferred embodiment, the present invention recognizes that if the cccDNA library in infected cells remains replenished, HBsAg cannot be cleared even if HBsAg secretion or HBsAg synthesis is inhibited, because, as demonstrated by preclinical evaluations and clinical trials, inhibiting HBsAg synthesis alone cannot address the root cause of HBsAg production in infected cells.

[0085] In another embodiment, the present invention utilizes the function of HBsAg secretion, the natural process of HBV-infected cells, and the properties of HBsAg protein to clear HBsAg from infected cells.

[0086] There are two pathways for replenishing the cccDNA library: a recycling pathway, which transports newly synthesized rcDNA molecules to the cell nucleus for cccDNA conversion, and a novel infection pathway, in which viral particles circulating in the blood attach to and enter hepatocytes, initiating a new round of infection by establishing a cccDNA library in the nucleus of the infected hepatocytes. However, these two cccDNA replenishment pathways are not utilized equally. The inventors have found that the recycling pathway is generally limited, and novel infection is the primary pathway for replenishing the cccDNA library. Therefore, an extended embodiment of the present invention aims to block novel infection to effectively prevent cccDNA replenishment.

[0087] To further demonstrate the invention, durable blocking of new infections can be achieved by using an AAV anti-HBs vector that expresses sustained high levels of anti-HBs antibodies after a single intramuscular injection. This not only delivers durable efficacy without causing HBsAg relapse, but also simplifies therapies for HBsAg reduction.

[0088] In one combined implementation, contrary to current methods of reducing serum HBsAg levels by directly inhibiting intracellular HBsAg synthesis, the present invention reduces cellular and serum HBsAg by blocking cccDNA supplementation (which eliminates the root cause of HBsAg production). Furthermore, this method does not require direct inhibition of cellular HBsAg synthesis.

[0089] Example

[0090] Example 1. Experimental procedure for HBV infection and treatment using uPA / SCID chimeric mice.

[0091] A total of 49 HBV-infected uPA / SCID chimeric mice were divided into 7 groups, as shown in Table 2 below.

[0092] Table 2. Animal Experiment Design

[0093]

[0094]

[0095] Blood samples are taken every two or three weeks until the process is terminated, at which point liver tissue is collected and rapidly frozen.

[0096] Example 2. Analysis of HBV infection in blood and liver samples

[0097] Quantitative analysis was performed on the levels of HBsAg, HBeAg, HBV DNA, anti-HBs antibody, and human albumin in serial blood samples.

[0098] Quantitative analysis of 20 samples from each liver revealed the average intracellular levels of HBsAg, rcDNA, and cccDNA.

[0099] Western blotting of HBsAg in liver lysates and immunohistochemical staining of HBsAg and HBcAg in liver sections were also performed.

[0100] Figure 1A-1D A graphical representation of the dynamic HBsAg levels in the blood of HBV-infected uPA / SCID chimeric mice with humanized livers (day 14 to day 162 post-infection) is provided, in which... Figure 1A The unprocessed group 1 (G1) is shown; and Figure 1B-1D This indicates the treatment of groups 2 and 4 (G2-G4) with HBVZ10 and entecavir for 12 weeks, but starting at different time points to achieve different HBsAg levels. HBVZ10 was administered as follows: in G2, at a dose of 2.5E11 copies on day 11 (d11) and at a dose of 4E11 copies on day 58. Figure 1B In G3, a dose of 1.8E12 copies was administered on day 22. Figure 1C ), and in G4, on day 44, at a dose of 7.2E12 copies ( Figure 1D In mice that died before day 162, the time point was truncated. The acceptable lower limit for HBsAg was 0.05 IU / ml (HBsAg EIA kit, Bio-rad).

[0101] Figure 2 A graphical representation of the copy number of HBsAg per cell and the number of input cells in liver lysates from HBV-infected uPA / SCID chimeric mice with humanized livers is provided. Cellular HBsAg levels were titrated with sequential dilutions of 1 / 1000, 1 / 100, 1 / 10, 1 / 2, 1 / 3.3, and 1 / 2.5. Inhibition was detected at a 1 / 2.5 dilution, and a dilution of 1 / 3.3 was permissible. Four total lysates from each of animals 825, 838, and 831 were analyzed and numbered .1, .2, .3, and .4, respectively.

[0102] Figures 3A-3B It shows Figure 2 The numerical relationship between cellular HBsAg and rcDNA in each cell of the mouse. Figure 3A A graphical representation is provided, showing the copy number of HBsAg and rcDNA in each cell of two mice in which serum HBsAg was undetectable and an untreated mouse control. Figure 3B Provided Figure 3AThe ratio of HBsAg to rcDNA in the cells of mice.

[0103] Example 3. The method of the present invention shows a more effective reduction in serum HBsAg than that reported using siRNA or ASO drugs that directly inhibit intracellular HBsAg synthesis, particularly in the following aspects:

[0104] 1. All mice treated using the method of this invention responded with a reduction in serum HBsAg. Furthermore, a progressive reduction of 3-5 log was observed in serum HBsAg, and by 162 days post-inoculation, serum HBsAg became undetectable in 8 out of 11 mice. Note: In both preclinical evaluation and clinical trials, direct inhibition of intracellular HBsAg synthesis showed an average reduction of < 2 log.

[0105] 2. The gradual decrease in serum HBsAg closely resembles the gradual decrease in serum HBeAg levels, which means that the observed gradual decrease in serum HBsAg reflects the gradual clearance of HBV in the liver.

[0106] 3. Compared with untreated control mice, mice with undetectable serum HBsAg had 3-4 log lower levels of HBsAg, rcDNA, and cccDNA in the liver.

[0107] Materials and Methods

[0108] Animals and HBV infection

[0109] All animal experiments were conducted at Noble Life Sciences Inc. (Sykesville, MD), a preclinical research contract service provider. The selection of Noble Life Sciences Inc. as a subcontractor for animal experiments was approved by the NIH's NIAID Office of Contracts (NIH Approved Animal Welfare Protection Number, A4633-01). All animal studies were approved by Noble Life Sciences' Institutional Animal Care and Use Committee (IACUC) protocol NLS-614. All animals received humane care.

[0110] Immunoactive female mice (CD1) were purchased from Charles River Laboratories (Boston, MA, USA), and immunodeficient male mice (uPA / SCID chimeric mice) were provided by PhoenixBio USA (New York, NY, USA). All mice were housed in BSL-2 cages (TP107, One Corporation, Osaka, Japan) at 23°C with a 12-hour light / dark cycle. All animals were randomly fed gamma-irradiated CRF1 food and autoclaved water. HBV inoculum (HBsAg ADR subtype / genotype C) was prepared from mouse serum (Project No. H01-108 Animal 4) by diluting viremia (5E9 HBV DNA copies / mL) to 2E7 HBV DNA copies with 100 μl of PBS and administered intravenously (tail vein) to each chimeric mouse.

[0111] AAV anti-HBs vector (HBVZ10)

[0112] This invention provides novel HBV therapy candidates known as AAV anti-HBs vectors, which utilize optimized adeno-associated virus (AAV) vectors [see, for example, Balazs A et al., Nature Medicine, 2014;20(3):296-300; Balazs A et al., Nature Biotechnology, 2013;31(7):647-52; de Jong YP et al., ScienceTranslational Medicine, 2014;6(254):254ra129; and Deal C et al., Proceedings of the National Academy of Sciences of the United States of America, 2014;111(34):12528-32] to deliver human anti-hepatitis B surface antigen (anti-HBs) antibody genes. The AAV anti-HBs vectors of the preferred embodiments of this invention express sustained high levels of anti-HBs antibodies after a single injection. By using these AAV anti-HBs vectors as novel HBV therapy candidates, the method of this invention compensates for the inadequacy of anti-HBs antibody production in chronic HBV infection. The preferred AAV anti-HBs vector is HBVZ10. In chimeric mice, intramuscular administration of HBVZ10 to endogenously express human anti-HBs antibodies blocked new infections in the presence or absence of entecavir.

[0113] Production of AAV anti-HBs or anti-malarial antibody vectors

[0114] In summary, 293 cells were co-transfected with an AAV vector encoding anti-HBs or anti-malarial antibodies and the plasmid pDP8.ape (Plasmid Factory, Bielefeld, Germany), which provides pHELP plasmid function and trans-encodes the AAV2 rep and AAV8 cap proteins for packaging the AAV vector. The resulting AAV vectors contained no viral open reading frames (ORFs). AAV was purified by PEG precipitation and cesium chloride ultracentrifugation. The infectivity of AAV aliquots was confirmed in vitro by transducing 293 cells and quantifying antibody concentrations in the medium using ELISA. A total of 1E14 genomic copies were obtained for each vector after production, purification, and concentration.

[0115] HBVZ10 dosage

[0116] In animal experiments 1 and 2, a small dose of HBVZ10 containing 1E11 genome copies was administered intramuscularly, and in animal experiment 3, a higher dose of HBVZ10 containing 2.5E11, 1.8E12, or 7E12 genome copies was administered.

[0117] Monitor HBV infection and human albumin levels in the blood.

[0118] Blood was collected every three weeks to quantify serum HBV DNA (qPCR, see below), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), and anti-HBs antibody (MONOLISA anti-HBs EIA 25200, Bio-Rad) levels by ELISA according to the instructions using calibrators (MONOLISA anti-HBs 20 Calibration Kit 25219, Bio-Rad) and human albumin (Human Albumin ELISA Kit E-80AL, Immunology Consultants Laboratory).

[0119] In addition, serum HBsAg in the selected samples was analyzed by Western blotting.

[0120] Serum alanine aminotransferase (ALT) activity

[0121] Serum ALT activity was assessed using the Alanine Aminotransferase Colorimetric Assay Kit (Cayman Chemical, trade number 700260) according to the test manual. Absorbance was measured at 340 nm every minute for 10 minutes, and the resulting 10 absorbance values ​​were plotted against time. Due to the limited serum volume, modifications were made: the 20 μl serum sample was adjusted to 10 μl, and compensated with 10 μl of H₂O. Therefore, in the calculation formula, 0.02 ml of serum sample was adjusted to 0.01 ml accordingly.

[0122] Analysis of intrahepatic HBV DNA

[0123] Each liver was randomly sampled 20-40 times by cutting 20-40 mg of liver tissue (weighed and recorded), and placed in a disposable microhomogenizer (BioMasher, Takara catalog number: 9790B) in 500 μl of isotonic buffer (154 mM Tris-HCl, pH 7.5, 1 mM EDTA and 0.05% Triton X-100) for 10 strokes. The homogenized tissue suspension was spun at 14,000 rpm for 2 minutes, and 100 μl of the lysate was reserved for Western blotting or ELISA of intracellular HBsAg, while the remaining 400 μl was transferred to a new microtube for the isolation of replication intermediates (RI), and the nuclear precipitate was left in the tube for cccDNA isolation.

[0124] Each extraction round includes two negative controls, one placed in the first sample location and the other in the last location, to monitor for any contamination during extraction.

[0125] rcDNA was extracted from 400 μl of supernatant using the following procedure: [Zhang YY et al., Journal of Virology, 2004;78(3):1195-201]

[0126] 1. Add 110 μl of proteinase K (final 0.5 mg / mL) and 1% SDS, and incubate at 50 °C for one hour.

[0127] 2. Add 500 μl of phenol, vortex and cool on ice for 3 minutes, and centrifuge at 14,000 rpm for 2 minutes.

[0128] 3. Transfer the supernatant to a new tube and add 1000 μl of 100% ethanol to precipitate the sample. Centrifuge the sample at 14,000 rpm for 15 minutes.

[0129] 4. Wash the precipitate with 1000 μl of 100% ethanol at 14,000 rpm for 10 minutes.

[0130] 5. Remove any residual ethanol and air dry for 5 minutes.

[0131] 6. Dissolve the precipitate in 200 μl of 10:1 TE buffer (pH 7.4), and then the rcDNA is ready for qPCR.

[0132] cccDNA was extracted from nuclear precipitates using the following procedure: [Zhang YY et al., Proceedings of the National Academy of Sciences of the United States of America, 2003;100(21):12372-7]

[0133] 1. Resuspend the precipitate in 200 μl of 10:1 TE (containing 0.05% Triton-X100, pH 7.4).

[0134] 2. Add 200 μl of 6% SDS-0.1M NaOH solution and incubate at 37°C for 15 minutes.

[0135] 3. Add 100 μl of 3M KAc (pH 5.07) and mix thoroughly. Cool on ice for 5 minutes, and then microcentrifuge at 14,000 rpm for 2 minutes to remove the KSDS-protein-ssDNA complex (precipitate).

[0136] 4. Transfer the supernatant to a new tube, add 500 μl of phenol, and centrifuge at 14,000 rpm for 2 minutes.

[0137] 5. Recover the supernatant and add 5 μl of glycogen (4 μg / μl, 20 μg total).

[0138] 6. Add 1000 μl of ethanol and centrifuge at 14,000 rpm for 15 minutes.

[0139] 7. Wash with 1000 μl of ethanol and centrifuge at 14,000 rpm for 10 minutes.

[0140] 8. Dissolve in 50 μl EcoR I buffer at 37°C for 15 minutes, and then inactivate at 80°C for 20 minutes. The cccDNA sample is now ready for qPCR.

[0141] RT-qPCR detection of total HBV RNA in cccDNA and rcDNA samples

[0142] Total HBV RNA levels were determined in each of 120 cccDNA and 120 rcDNA samples prepared from untreated mice 842 and 836 (representing the amplification phase), 831 and 987 (representing the maintenance phase), and 38 and 813 (representing treated mice that achieved a progressive reduction in serum HBsAg to undetectable levels while cccDNA was reduced >100-fold). Specifically, 20 cccDNA and 20 rcDNA samples from each liver were tested. The mean RNA concentration in the cccDNA samples was approximately 1.5 μg / μl, and the mean RNA concentration in the rcDNA samples was 0.5 μg / μl. The A260 / 280 ratio varied minimally between 1.98 and 2.08. All RNA samples were diluted 10-fold and then RT-qPCR was performed using 2 μl of the TaqMan™ Rapid Virus 1-Step Master Mixture (Thermo Fisher 4444432) and primers / probes located on the S gene (rcDNA for qPCR in Table 3). In addition to cccDNA detection in the same cccDNA sample, rcDNA was also detected in the same cccDNA sample and un-RT-tested rcDNA sample on the same plate using the same primers / probes. The detected HBV RNA level in the same sample was approximately 10 times that of rcDNA. Net RNA copies were plotted after subtracting rcDNA copies from the same sample. The ratio of RNA copy number / cell to cccDNA copy number / cell was calculated using the total net RNA copy number (nuclear RNA copy number + cytoplasmic RNA copy number).

[0143] RT-qPCR detection of human Ki67 RNA levels in 840 cccDNA samples

[0144] A pre-stored human Ki67 RNA primer / probe system (FAM-MGB, Hs01032435_g1) was purchased from ThermoFisher Scientific. This detection system produces 179 bp amplicons, which are isolated for the preparation of qPCR standards. Human Ki67 RNA was detected by RT-qPCR using two μL of total nuclear RNA containing cccDNA, with the same TaqMan™ Rapid Virus 1-Step Master Mixture (ThermoFis 4444432). To first establish the full picture of Ki67 RNA expression in humanized livers of chimeric mice, 840 cccDNA samples from 42 livers were analyzed, including 15 untreated livers, 11 livers treated with anti-HBs antibody or a combination of HBVZ10 and entecavir but with detectable serum HBsAg (HBsAg+), and 16 treated livers with progressively decreased serum HBsAg levels to undetectable levels (HBsAg-). Subsequently, scatter plots were used to analyze the correlation between Ki67 RNA and cccDNA in the same sample, generating a correlation trend line and R. 2 value.

[0145] cccDNA samples were always stored at -20°C, and all processing procedures were performed in a biosafety cabinet with the blower turned on. All tips, plates, tubes, and solutions used were nuclease-free. Analysis of the differences in Ki67 RNA levels between existing cccDNA and freshly isolated cccDNA samples from five livers (mouse 907, 471, 987, 831, and 805) revealed no significant results (data not shown), indicating that RNA in existing cccDNA samples was not significantly degraded.

[0146] qPCR of serum HBV DNA, liver rcDNA and cccDNA

[0147] Table 3 lists the primer and probe sequences for detecting serum HBV DNA and intracellular rcDNA by qPCR, while the primer sequence for detecting cccDNA is located on the flanking side of the gap region, and the probe is placed immediately after the DR1 sequence (Table 3).

[0148] Table 3. Location and sequence of cccDNA and rcDNA primers and probes

[0149]

[0150]

[0151] The listed cccDNA primers and probes are specific enough to distinguish rcDNA amplifications from 300 to 6000 times. qPCR was performed using the Taqman Rapid Advanced Master Mixture (ThermoFisher catalog number: 4444558) on a QuantStudio 3 instrument (ThermoFisher catalog number: A28136) that accommodates 0.1 ml 96-well hard-shell plates.

[0152] All standards used for qPCR were calibrated using Absolute Q digital PCR.

[0153] Absolute Q (ABQ) digital PCR of cccDNA

[0154] The cccDNA copy number / cell ratio was initially calculated based on qPCR and then retested using Absolute Q digital PCR (ThermoFisher catalog number: A52864). In short, the procedure includes the following steps:

[0155] 1. Prepare 9.1 μl of reaction mixture, which consists of 1.8 μl of 5x DNA dPCR mixture (ThermoFisher catalog number: A52490), 0.5 μl of 20x primer / probe mixture (the final concentration of each primer and 250 nM probe is 900 nM), 1 μl of cccDNA sample, and 5.8 μl of DNase and RNase-free H2O.

[0156] 2. Load 9 μl of the reaction mixture into one well of a microfluidic array plate (MAP, ThermoFisher catalog number: A53301).

[0157] 3. Run dPCR, which consists of the following: preheating at 96°C for 10 minutes, and 40 cycles of 5 seconds at 96°C and 15 seconds at 60°C.

[0158] 4. Use QuantStudio Absolute Q digital PCR software to generate a data report.

[0159] The sensitivity of dPCR is single copy per microcompartment, and the result is considered valid if >19,000 of the 20,480 microcompartments per sample read the Rox fluorescence signal.

[0160] Procedure and principle for simultaneous detection of cccDNA and rcDNA in the same cell nucleus using ABQ double digital PCR

[0161] The main procedures for detecting cccDNA and rcDNA in the same cell nucleus are as follows:

[0162] 1. Homogenize 20–30 mg of liver tissue in 500 μl homogenization buffer (10 mM Tris-HCl (pH 7.5), 3 mM MgCl2, 0.25 M sucrose, and 0.05% Triton X-100). Centrifuge to precipitate cell nuclei and resuspend in homogenization buffer containing 2 μg / ml ethidium bromide.

[0163] 2. Individual cell nuclei were individually sorted and deposited in the wells of a 96-well plate.

[0164] 3. Digest the deposited cell nuclei with proteinase K at a concentration of 0.5 mg / ml for 60 minutes, and then inactivate them at 80°C for 15 minutes.

[0165] 4. The released HBV DNA was linearized using NcoI digestion.

[0166] 5. Linearized HBV DNA was subjected to ABQ dPCR detection using both cccDNA and rcDNA.

[0167] The principle of simultaneous detection of cccDNA and rcDNA in the same cell nucleus using ABQ double digital PCR

[0168] The ABQ digital PCR instrument can simultaneously detect four fluorescence signals: FAM, VIC, ABY, and JUN / Cy5. This allows for the detection of four different targets in the same reaction (multiplex PCR). FAM and Cy5 have emission wavelengths of 517 nm and 670 nm, respectively, and their wavelength spectra do not overlap. Therefore, selecting FAM to label a cccDNA probe and Cy5 to label an rcDNA probe to detect two molecules in the same reaction is called duplex dPCR.

[0169] The specificity of cccDNA detection is provided by cccDNA-specific primers located on the flanking side of the gap region in the HBV genome and cccDNA-specific probes placed immediately after the DRI sequence (Table 3).

[0170] Linearized cccDNA templates cannot generate fluorescent signals using rcDNA primer / probe detection systems.

[0171] HBV DNA released from each deposited cell nucleus will undergo NcoI digestion to exclude cccDNA from detection using rcDNA primers / probes. The positive strand of rcDNA is only partially synthesized, containing a single-stranded gap of 600–2100 nucleotides at the 3' end [Summers J et al., Proceedings of the National Academy of Sciences of the United States of America, 1975;72(11):4597–601]. Since NcoI is located between nt1372 and 1376, close to the 3' end of the positive strand, it is most likely present in the single-stranded sequence of the rcDNA molecule [Summers J et al., Proceedings of the National Academy of Sciences of the United States of America, 1975;72(11):4597–601]. Therefore, NcoI will linearize cccDNA but will not cleave rcDNA.

[0172] The NcoI-linearized cccDNA sequence begins at C at nt1373 (5') and ends at C at nt1372 (3'). The rcDNA forward primer binds to the 3' end of the linearized cccDNA, but the rcDNA probe binds to its 5' end. Taq DNA polymerase, which binds to the forward primer at the 3' end, cannot reach the probe at the 5' end and therefore cannot cleave the first C base with Cy5 dye via its 5'-3' exonuclease activity, nor can it generate a Cy5 fluorescent signal. If both the rcDNA forward primer and probe bind to the sequential template containing nt1345 to nt1454, that is, if the F primer binds upstream of the probe binding site, a Cy5 fluorescent signal will be generated, which only occurs in the NcoI-cleaved rcDNA. Therefore, after NcoI cleavage, rcDNA (not cccDNA) will be specifically detected by the rcDNA primer / probe.

[0173] HBV DNA plasmids (ADW isotype monomers cloned into the Psp65 vector) were used as substitutes for cccDNA molecules (linearized via NcoI) to verify that the rcDNA probe / primers did not detect cccDNA. NcoI-digested plasmids were serially diluted and tested using ABQ dual dPCR containing both cccDNA and rcDNA probes / primers. Serially diluted cccDNA molecules were detected using the FAM-labeled cccDNA probe; however, no positive signal was detected using the Cy5-labeled rcDNA probe, which detects extracted rcDNA.

[0174] Tests for simultaneous detection of both cccDNA and rcDNA.

[0175] cccDNA samples extracted using the modified Hirt method [Zhang YY et al., Journal of Virology, 2005;79(15):9896-903] contained rcDNA molecules. Deproteinized rcDNA molecules were detected in the extracted cccDNA samples using DNA blotting [see, for example, Tuttleman JS et al., Cell, 1986;47(3):451-60; Gao W et al., Journal of Virology, 2007;81(12):6164-74; and Blondot ML et al., Journal of Hepatology, 2016;64(1):S49-S59]. Therefore, the extracted cccDNA samples were used to evaluate the ability of dual dPCR to detect cccDNA and rcDNA. rcDNA was detected in the extracted cccDNA samples by conventional qPCR using a FAM-labeled rcDNA probe. Both cccDNA and rcDNA were detected in the extracted cccDNA samples by ABQ dual dPCR. These results not only demonstrate the ability of dual dPCR to detect both cccDNA and rcDNA molecules, but also support the view that nuclear rcDNA molecules can be used as biomarkers for HBV infection.

[0176] The sorted cell nuclei in each well are digested with NcoI. DNA samples from individual cell nuclei are mixed with a dPCR solution containing both ccc and rcDNA primers / probes and loaded into a microfluidic array plate (MAP) for dPCR detection. dPCR results are generated using QuantStudio absolute Q digital PCR software 6.0.

[0177] Thresholds of FAM and Cy5 positive fluorescence

[0178] After extensively evaluating fluorescence intensity and distribution patterns between cccDNA-positive and uninfected samples, a 500 value for both FAM and Cy5 fluorescence intensities was set as the threshold for a positive signal. However, approximately 5% FAM-positive signal and 1% Cy5-positive signal were detected in 576 cell nuclei prepared from three uninfected human livers (two purchased from PheonixBio, and one collected pre-infection). This is because, despite the standard HLPC-based purification of the FAM and Cy5-labeled probes, some free fluorescent molecules remained in each probe. These free fluorescent molecules are independent of quenchers, and if a large number of free fluorescent molecules are distributed within a microcompartment, they can be detected at high intensity without amplification. For example, ROX fluorescence (unlabeled and in free form) was included in the ABQ dPCR master mix and distributed to each microcompartment for quality control. However, the number of ROX molecules distributed to each of the 20,480 microcompartments varied, and high intensity was generated if many molecules were distributed in a single microcompartment. Using findings from three uninfected livers as a reference, the false positive rates for cccDNA and rcDNA by dual dPCR were assumed to be approximately 5% and 1%, respectively. This means that the true number of cccDNA-positive cell nuclei in the three infected liver samples may be 5% lower than the detected number. However, they did not significantly affect the primary finding of cccDNA- / rcDNA+ cell nuclei.

[0179] The rcDNA molecules detected in the cell nucleus did not bind nonspecifically to the cell nucleus.

[0180] To evaluate the likelihood of nonspecific binding of rcDNA, detected in individual sorted nuclei, to the nuclear membrane during the homogenization preparation of nuclear suspensions by releasing viral particles and capsids into lysates, nuclear suspensions were prepared from two livers of two uninfected chimeric mice (Animal IDs HKB-043-020 or B20 and HKB-043-046 or B46, purchased from PheonixBio). Each nuclear suspension was aliquoted into two vials: one for direct sorting and the other mixed with lysates from Mouse 987 (containing 0.05% Triton-X100) for 20 minutes (the mice were untreated controls with an average of 870 copies of rcDNA / cell). The lysates were then removed and dissolved in isotonic buffer (154:1 TE, containing 0.05% Triton-X100) for sorting. Sorted nuclei from four vials were subjected to double ABQ dPCR. Table 4 shows that there was no significant difference between the two nuclear suspensions mixed with mouse 987 lysate and the two unmixed nuclear suspensions in detecting nuclei with Cy5 intensity ≥ 500. This suggests that the rcDNA molecules detected in sorted nuclei are unlikely to originate from released viral particles and capsids that bind nonspecifically to the nucleus. This is consistent with the concept that the HBV capsid primarily utilizes cellular transport mechanisms rather than diffusion or passive capture to reach the nuclear membrane, where nuclear localization signals on the capsid interact with nuclear import receptors [see, for example, Blondot ML et al., Journal of Hepatology, 2016;64(1):S49-S59; and Gallucci L et al., Viruses, 2017;9(1):21].

[0181] Western blot analysis of serum HBsAg and liver lysate HBsAg and HBc proteins

[0182] Serum and liver samples were analyzed using SDS-PAGE, and HBV surface proteins were detected by Western blot analysis using rabbit polyclonal anti-HBs antibody (Virostat) [see, for example, Hong X et al., Journal of Virology, 2021;95(3):10.1128 / jvi.01695-20; and Xi J et al., Journal of Virology, 2022;96(1):e01305-21].

[0183] Immunohistochemical staining of HBsAg on tissue sections

[0184] In summary, formalin-fixed paraffin-embedded liver sections were cut to a thickness of 5 μM and, after dewaxing, digestion with proteinase K, and inactivation of endogenous peroxidase with 3% hydrogen peroxide, were used for HBsAg staining. Rabbit anti-HBs antibody (LS-C683282, LSBio) and HRP-conjugated goat anti-rabbit IgG (LS-C316062, LSBio) were used as primary and secondary antibodies, respectively. A DAB staining kit (ACH500-IFU, CP Lab Chemicals) was used for staining.

[0185] Statistical analysis

[0186] HBsAg (IU / ml), HBV DNA (copy number / ml), and antibody levels (μg / mL) are expressed as mean ± standard deviation (SD). Mean intracellular rcDNA and cccDNA levels are expressed as copy number / cell. The number of cells sampled was calculated by multiplying the sample weight (mg) by 1.39E5 cells per mg of liver tissue [Sohlenius-Sternbeck AK, Toxicology in vitro, 2006;20(8):1582-6], and then normalized to a factor of 0.7, taking into account that 70% of human hepatocytes are hepatocytes [Mason WS et al., Journal of Virology, 2010;84(16):8308-15. Epub2010 / 06 / 04]. The formula for calculating copy number / cell is shown below:

[0187]

[0188] The procedure for demonstrating the method of the present invention comprises three components:

[0189] 1. Evaluate the impact of efficient HBV replication on the presence of cccDNA in infected cells.

[0190] 2. Analysis of cccDNA levels at the somatic cell and single-cell nucleus levels.

[0191] 3. Evaluate the therapeutic impact on cccDNA levels by blocking cccDNA supplementation.

[0192] In vivo replication kinetics suggest that inhibition of HBV replication may be mediated by cccDNA clearance.

[0193] To understand the kinetics of HBV replication in vivo, untreated chimeric mice infected with HBV were euthanized on days 18, 45, 50, 52, 82, 99, 141, and 212 post-inoculation (pi) to measure kinetic serum HBsAg and HBV DNA levels and intrahepatic HBV markers. There were two infection phases (…). Figure 4A and 4B The first phase is the spread of infection to all infectable cells, during which both serum HBsAg and HBV DNA levels rise rapidly after inoculation and peak around day 82. The second phase is the persistent infection phase, during which HBV infection remains at a stable level. The kinetics of serum HBsAg and HBV DNA observed in this model recapture the typical acute HBV infection that persists in humans [Keating SM et al., The Journal of Infectious Diseases, 2014;209(6):845-54].

[0194] The kinetics of intracellular accumulation of viral products also involve two phases, such as... Figure 4C As shown. The first phase is a gradual increase in the accumulation of viral products. For example, intrahepatic HBsAg levels increased from 230 copies / cell on day 18 post-inoculation to 110,000 copies / cell on day 82 post-inoculation, reflecting robust HBV replication, and during the accumulation phase, the secretion of viral particles and subviral particles lagged behind unrestricted HBV replication. The second phase is the phase where the accumulation stops increasing after reaching a peak on day 82 post-inoculation. For example, intracellular HBsAg levels remained at approximately 100,000 copies / cell after the peak. Average cccDNA levels fluctuated by 2-fold but also stopped rising over the next 130 days. Serum HBsAg and HBV DNA levels remained stable. Figure 4A and 4B The cessation of accumulation is unlikely to be caused by an increase in viral particle secretion, but rather by the inhibition of replication required to establish a persistent non-cytopathic infection [see, for example, Summers J et al., Journal of Virology, 1991;65(3):1310-7; and Lenhoff RJ et al., Journal of Virology, 1994;68(9):5706-13].

[0195] The direct cytopathic effect of HBV infection in this model was reported

[35] . Of the 18 HBV-infected livers, 4 showed extensive confluent hepatic necrosis with extensive infiltration involving up to 50% of the parenchyma on the sections, but this was not observed in the remaining 14 liver sections.

[0196] As mentioned above, in vivo cccDNA dynamics consist of two phases ( Figure 4C Furthermore, cccDNA may be missing in both of these stages:

[0197] i. Amplification Phase. Total cccDNA levels in the liver are primarily amplified by expanding infection within the liver. In the early stages of infection, the cccDNA library in individually infected cells is amplified via intracellular recycling pathways [see, for example, Tuttleman JS et al., Cell, 1986;47(3):451-60; Ko C et al., Journal of Hepatology, 2018;69(6):1231-41; Summers J et al., Journal of Virology, 1990;64(6):2819-24; and Tuttleman JS et al., Journal of Virology, 1986;58(1):17-25]. cccDNA levels increased from 0.00001 copies / cell (day 18 post-inoculation) to 0.35 copies / cell on day 82 post-inoculation. This is not the only event that occurs. Reaching peak infection means that all infectable cells must be infected, as evidenced by the detection of HBsAg and HBcAg in almost all hepatocytes. The expected cccDNA level is ≥ 1 copy / cell, as at least one copy of cccDNA is required in each infected cell. However, the mean cccDNA level at peak infection (day 82 post-inoculation) was 0.35 copies / cell, which is approximately 1 copy of cccDNA per 3 infected cells. This suggests that cccDNA may be absent in some infected cells after initial establishment.

[0198] ii. Maintenance Phase. cccDNA is maintained at a stable level (0.35–0.6 copies / cell) to maintain HBV infection at a stable level when peak infection is reached. When the average cccDNA level is < 1 copy / cell, the Poisson distribution predicts that some cells may contain > 1 copy / cell, and other cells may not contain cccDNA. This indicates that cccDNA is spontaneously cleared from some cells during the maintenance phase. Therefore, in the persistent infection phase, a stable HBV infection level may be achieved by establishing a balance between the number of infected cells with cccDNA that maintains HBV replication and the number of cells lacking cccDNA and ceasing viral replication. This means that infected cells primarily regulate HBV replication by clearing cccDNA.

[0199] The average cccDNA level after peak infection was < 1 copy / cell.

[0200] Analysis of cccDNA levels in untreated mice can be extended to obtain a range of cccDNA levels in the liver. To avoid unrepresentative findings from single or multiple samplings, each liver was routinely sampled 20 times, resulting in 220 cccDNA samples from 11 livers between day 82 and day 253 post-inoculation. Among the 220 cccDNA samples, the highest mean cccDNA level was 2.5 copies / cell, while the lowest mean cccDNA level was 0.003 copies / cell. Figure 5A ).

[0201] Of the 220 cccDNA samples, 28 (12.7%) had an average cccDNA level > 1 copy / cell, while the remaining 192 (87.3%) had an average cccDNA level < 1 copy / cell. Figure 5C This indicates that some cells may not contain cccDNA molecules at different time points.

[0202] Among the 11 livers, the levels of cccDNA detected varied considerably, ranging from 1.2 copies / cell to 0.16 copies / cell. Figure 5B Of the 11 livers, only 1 liver had an average cccDNA level > 1 copy / cell (1.2 copies / cell).

[0203] Total HBV RNA levels were measured in four untreated livers to compare relative RNA transcription efficiency between the two infection phases. Mice 842 and 836 were sacrificed on day 50 and day 52 post-inoculation (representing the amplification phase, respectively). Mice 831 and 987 were sacrificed on day 141 and day 218 post-inoculation (representing the maintenance phase, respectively). Total HBV RNA levels in 20 cccDNA samples and 20 rcDNA samples from each liver were determined using RT-qPCR.

[0204] HBV RNA was detected in both cccDNA and rcDNA samples, and it was more abundant in the cytoplasm (ranging from several hundred to eight thousand copies / cell) than in the nucleus (ranging from a few to several hundred copies / cell). Typically, the total HBV RNA copy number in the cytoplasm was 10–20 times that of rcDNA copy number / cell. HBV DNA integration in chronically infected human liver has been reported at a frequency of approximately once per 100 cells [Mason WS et al., Gastroenterology, 2016;151(5):986–98. e4], and HBV DNA integration was detected in this model, but transcriptional silencing was observed [Allweiss L et al., Gut, 2018;67(3):542–52], therefore, the detected HBV RNA was likely primarily transcribed from cccDNA. The ratio of RNA copy number / cell to cccDNA copy number / cell was used to measure the relative efficiency of RNA transcription from cccDNA. In the same samples, a high ratio of mean RNA copy number / cell to mean cccDNA copy number / cell was evident, ranging from 6107 to 7518 in mice 842 and 836 (representing the amplification phase), and from 4837 to 9187 in mice 831 (representing the maintenance phase). These findings suggest that a single copy of cccDNA can undergo more than 1000 transcriptions, indicating that efficient RNA transcription can be carried out by reusing single or several copies of cccDNA in HBV-infected cells. Notably, no significant difference in relative transcription efficiency was observed between the two phases of infection.

[0205] To investigate whether RNA transcription could be affected by reduced cccDNA levels, total HBV RNA levels were examined in two additional livers (mice 813 and 838) in response to treatment that blocked cccDNA supplementation, resulting in a >100-fold reduction in cccDNA and the presence of only residual cccDNA. The mean HBV RNA level was 5 copies / cell, less than 1 / 100th of the mean HBV RNA level observed in four untreated mice, indicating a direct correlation between the reduction in HBV RNA and the reduction in cccDNA. However, the ratio of RNA copy number / cell to cccDNA copy number / cell remained above 1000 (mean 7000 to 10000), indicating that relative transcriptional efficiency remained high even in residual infected cells. Furthermore, a positive correlation (R0.05) was found between the mean cccDNA level and HBV RNA level (copy number / cell) in the six mice. 2 = 0.93, Figure 6 This indicates that the total HBV RNA level largely depends on the cccDNA level in infected cells in this model. Unlike the latent phase of HIV infection, during which viral RNA transcription in stored cells is suppressed, the efficient RNA transcription from cccDNA suggests that the suppression of RNA transcription is an unlikely mechanism to prevent HBV replication and maintain non-cytopathic effects in HBV-infected cells.

[0206] cccDNA deletion was detected at the single-cell nucleus level.

[0207] One of the criteria used by supplier PhoenixBio to select uPA / SCID chimeric mice with human livers was a liver replacement index (RI) > 70% [Tateno C et al., PloS ONE, 2015;10(11):e0142145]. Of the 57 mice received, only four had an RI of 76–78%, and the remaining 53 mice had an RI of 80–93%, with approximately 76–93% of the hepatocytes being human hepatocytes. Since native cells are routinely used for cccDNA quantification in our assays, an average of 30% non-human (mouse) hepatocytes was used to normalize the calculated cccDNA copy number / cell ratio. The actual number of non-human hepatocytes in each sample varies, which may affect the calculated copy number / cell ratio. The quantitative detection of cccDNA copies at the single-cell nuclear level [Zhang YY et al., Proceedings of the National Academy of Sciences of the United States of America, 2003;100(21):12372-7] aimed to confirm the absence of cccDNA in some infected cells.

[0208] HBV rcDNA is 100-1000 times more abundant than cccDNA and is also expected to be delivered to the nucleus for cccDNA transformation [Tuttleman JS et al., Journal of Virology, 1986;58(1):17-25]. Absolute Q dual digital PCR (ABQ dual dPCR) was used to simultaneously detect cccDNA and rcDNA in each cell nucleus, and the detected rcDNA was used as a biomarker for HBV infection. This article details the strategy, principles, and controls for the simultaneous detection of cccDNA and rcDNA.

[0209] Using BD FACSAria II, cell nuclei from three livers collected from untreated mice 831, 987, and 907 at day 141, day 218, and day 253 post-inoculation, respectively, were deposited in 96-well plates at a rate of one nucleus per well. Table 4 lists the total number of analyses performed.

[0210] Table 4. Percentage of HBV-positive cell nuclei identified by dual dPCR

[0211]

[0212] cccDNA and rcDNA detected at the single-cell nucleus level

[0213] cccDNA was detected as either cccDNA alone or coexisting with rcDNA, and rcDNA was detected as either coexisting with cccDNA or being rcDNA alone.

[0214] cccDNA copy number / nucleus

[0215] In most cccDNA-positive cell nuclei, cccDNA was detected as a single copy. In mouse 831, twenty (66.7%) of the 30 cccDNA-positive cell nuclei contained only a single copy, while the remaining ten nuclei had >1 copy, ranging from two to eight copies. In mouse 987, 41 (75%) of the 55 cccDNA-positive cell nuclei contained a single copy of cccDNA, while 14 (25%) nuclei had >1 copy. In mouse 907, cccDNA was detected as a single copy in 34 (77%) of the 44 cccDNA-positive cell nuclei, while the remaining ten nuclei had >1 copy, ranging from 2 to 6 copies / nucleus. Therefore, ≥ 2 / 3 of the detected cccDNA-positive cell nuclei contained only a single copy of cccDNA.

[0216] rcDNA copy number / nucleus

[0217] In mouse 831, a single copy of rcDNA was detected in 24 (57%) of the 42 rcDNA-positive nuclei, with the remaining 18 (43%) nuclei containing 2–11 copies / nucleus. In mouse 987, a single copy of rcDNA was detected in 41 (60%) of the 66 rcDNA-positive nuclei, with the remaining 25 containing 2–8 copies / nucleus. In mouse 907, a single copy of rcDNA was detected in 13 (52%) of the 25 rcDNA-positive nuclei, with the remaining 12 (48%) nuclei containing >1 copy of rcDNA, ranging from 2 to 19 copies / nucleus.

[0218] cccDNA- / rcDNA+ cell nucleus

[0219] In three livers, cells infected in portions of the nuclei from 27%, 47%, to 55% (Table 5) showed no detectable cccDNA, while rcDNA was detectable in the same nuclei. Infection kinetics data ( Figure 4A and 4B ) and published data on HBV infection kinetics in this model [Ishida Y et al., Hepatology, 2018;68(2):473-84] indicate that peak infection can be reached between 82 and 90 days post-inoculation, meaning that all infectable human hepatocytes may be infected approximately 90 days post-inoculation. HBsAg and HBcAg staining showed that most cells in mouse sections 831, 987, and 907 were positive. Therefore, cccDNA- / rcDNA+ cells may indicate the absence of cccDNA in infected cells, or the absence of cccDNA followed by the formation of rcDNA delivered by newly infected cells from recently generated uninfected cells.

[0220] The detection of cccDNA- / rcDNA+ nuclei confirmed the findings based on somatic cells, namely that cccDNA may have been spontaneously lost from some of the infected cells.

[0221] Table 5. Percentage of cell nuclei positive for cccDNA and rcDNA

[0222]

[0223] After blocking the two cccDNA supplementation pathways, the average cccDNA level decreased by >100-fold.

[0224] The therapeutic effect of blocking new infections with anti-HBs antibodies at the cccDNA level was evaluated.

[0225] Two sources of anti-HBs antibodies were used, both targeting the "ad" and "ay" subtypes: an exogenous mouse anti-HBs antibody (AM31509 PU-N OriGene) and an endogenous anti-HBs antibody expressed by the AAV anti-HBs vector HBVZ10 (described in detail in the "Methods" section). HBVZ10 could express high levels (up to 500 μg / mL) of anti-HBs antibodies and maintain >100 μg / mL for at least 252 days after a single injection in both immunocompetent and immunodeficient mice, or >100,000 mIU / mL if measured using a WHO-referenced calibrator (as a standard for clinical reporting).

[0226] A total of 15 mice received anti-HBs treatment. Thirteen mice were injected with the AAV anti-HBs vector HBVZ10 at a dose of 1E11 genome copies at week 7 post-inoculation, while the remaining two mice were injected with mouse anti-HBs antibodies nine times every three weeks starting day 74 post-inoculation, with each injection containing 250 μg. Anti-HBs antibodies were detectable in all 15 chimeric mice after treatment. However, serum HBsAg remained positive, indicating that not all viral particles were neutralized and that new infections were only partially blocked.

[0227] The first group consisted of six livers, and the second group consisted of nine livers, collected on day 204 and day 253 post-inoculation, respectively. Each of the 15 livers was randomly sampled 20 times, and a second round of sampling was performed on seven of the 15 livers, resulting in a total of 440 cccDNA samples. Figure 7A The average level of cccDNA in 440 samples is shown. Figure 5B The mean cccDNA level per liver or per 20 samples (intermediate samples) is presented. Only five (1.1%) of the 440 samples had cccDNA levels > 1 copy / cell, significantly lower than the 13% in untreated mouse cccDNA samples. The mean cccDNA level in the 15 partially blocked new infections was 0.2 copies / cell, significantly lower (p = 0.012) than the 0.5 copies / cell level in untreated mice. Figure 7B The lower cccDNA levels in this group were supported by a proportionally lower rcDNA level. Figure 7C These results indicate that cccDNA levels are sensitive to partially blocking new infections.

[0228] Then, the effects of completely blocking new infections or blocking the two cccDNA supplementation pathways on cccDNA levels were evaluated.

[0229] The hallmark of complete blocking of new infection is the transition from HBsAg positivity to HBsAg negative / anti-HBs positive (HBsAg- / anti-HBs+). Blocking new rounds of infection was achieved by combining anti-HB antibodies with 9-12 weeks of entecavir therapy to reduce cccDNA rcDNA synthesis and intracellular recycling, thereby blocking two cccDNA supplementation pathways. CccDNA analysis was performed on 17 mice that achieved HBsAg negative / anti-HBs positive status. Two mice (mice 970, which received nine injections of anti-HBs antibodies every three weeks, and mice 819, which received a single dose of 1.8E12 copies of HBVZ10) underwent anti-HBs antibody monotherapy, while the remaining 15 mice were treated with a combination of anti-HBs and entecavir.

[0230] Each of the 17 livers collected after peak infection between day 123 and day 253 post-vaccination was randomly sampled 20 times, and two livers were sampled a second time for 20 times, resulting in a total of 380 cccDNA samples. Figure 5B The rightmost group). 140 cccDNA samples from 7 mice were analyzed using qPCR and ABQ dPCR. All cccDNA levels were < 1 copy / cell, and most were < 0.01 copies / cell. Figure 8A The mean cccDNA level in the 17 mice was 0.0028 copies / cell, which was less than 1 / 100 of the 0.5 copies / cell level in untreated mice (p = 0.0001) and significantly lower than the 0.2 copies / cell level in mice with partially blocked neopre-infection (p = 4E-4). Figure 5B and 8B Furthermore, after 20 samplings of each liver, cccDNA was not detected in either mouse. This indicates that complete blocking of neo-infection is crucial for cccDNA elimination. Adding entecavir to the anti-HBs antibody blocked the recycling pathway and made the anti-HBs antibody more effective at blocking neo-infection, as the reduction in viral particle production lowered the likelihood of neo-infection. These results further support the hypothesis that cccDNA replenishment is necessary to maintain cccDNA levels, highlighting the spontaneous clearance of cccDNA from infected cells.

[0231] The kinetic human albumin levels were similar between untreated and treated mice, suggesting that cccDNA elimination was primarily due to the blockade of cccDNA replenishment, rather than the absence of human hepatocytes in the humanized liver.

[0232] When the complementary pathway in both stages of infection is blocked, cccDNA is progressively eliminated.

[0233] Further experiments were conducted to demonstrate the effect of progressive cccDNA clearance on blocking the cccDNA replenishment pathway.

[0234] In chimeric mice infected with HBV, the increase in serum HBsAg levels after inoculation paralleled the increase in viremia. Figure 4A and 4B This indicates that HBV replication is primarily driven by cccDNA. Entecavir treatment is known to reduce serum HBV DNA levels, but has no parallel effect on serum HBsAg levels, especially over a short period [Chang TT et al., Hepatology, 2010;52(3):886-93]. Therefore, serum HBsAg levels in entecavir-treated mice, rather than HBV DNA levels, can be used as a substitute for intrahepatic cccDNA levels. Serum HBsAg levels in all 16 mice (…) Figure 8C and 8D Both showed a gradual decrease of 3-5 log and became undetectable upon blocking the two cccDNA supplementation pathways. Blockage either occurred before peak infection (i.e., during the cccDNA amplification phase) or... Figure 8C It begins during the period ( ) or during the cccDNA maintenance phase ( ) Figure 8D The process began with administration of additional doses of mouse anti-HBs antibodies to increase anti-HBs antibody levels, transitioning from partial blockade to complete blockade after peak infection. The progressive reduction in serum HBsAg was associated with and likely caused by the progressive elimination of cccDNA. The progressive decrease in serum HBeAg in 16 mice further supports this finding. Figure 8E and 8F (Green in the text). HBeAg is synthesized from precore mRNA, which is transcribed from cccDNA molecules [Yuh CH et al., Journal of Virology, 1992;66(7):4073-84]. In chronic HBV infection, cccDNA levels typically decrease 10-100-fold when HBV infection transitions from an HBeAg-positive to an HBeAg-negative phase [see, for example, Werle-Lapostolle B et al., Gastroenterology, 2004;126(7):1750-8; and Laras A et al., Hepatology, 2006;44(3):694-702]. Therefore, a gradual decrease in serum HBeAg ( Figure 8E and 8FThis reflects a gradual reduction in cccDNA in the liver. Both anti-HBs antibodies and entecavir primarily block cccDNA replenishment rather than directly eliminating cccDNA molecules; therefore, the observed cccDNA elimination is likely mediated by spontaneous clearance (non-treatment mediated) occurring in two phases.

[0235] A decrease of >100-fold in cccDNA levels was observed within 80 days.

[0236] uPA / SCID chimeric mice with human livers are fragile and cannot withstand stressful procedures such as successive hepatectomies, which poses a challenge to establishing baseline cccDNA levels before treatment. Therefore, cccDNA levels from different mice with comparable serum HBsAg levels were used as a reference. Figure 5F shows that mouse 838 (treated with anti-HBs antibody expressed with HBVZ10 and entecavir) reached a baseline HBsAg level of approximately 5000 IU / mL on days 54 and 82, which then gradually decreased. This is comparable to mouse 833, which received HBVZ10 monotherapy on day 44. Mouse 833 showed serum HBsAg ( Figure 8G The mouse 833 showed a double positivity for both anti-HBs antibodies and anti-HBs antibodies; therefore, neonatal infection in mouse 833 was considered partially blocked. Serum HBsAg levels in mouse 833 remained stable at approximately 5000 IU / mL from day 82 to day 162 (the termination day). cccDNA levels in mouse 833 were used as a reference for baseline cccDNA levels prior to HBsAg clearance in mouse 838. Compared to intracellular HBsAg and cccDNA levels in mouse 833, intracellular HBsAg and cccDNA levels in mouse 838 decreased by >100-fold over the 80-day period from day 82 to day 162 post-inoculation. Figure 8H The mean decrease in cellular HBsAg levels was 423 copies / day. The cccDNA results further support the use of kinetic serum HBsAg levels as a substitute for cccDNA levels, and the observed spontaneous cccDNA clearance was efficient and could be converted into gradual cccDNA elimination upon blocking both cccDNA supplementation pathways.

[0237] Human Ki67 RNA levels in 840 cccDNA samples

[0238] Human Ki67 was chosen as a biomarker to examine the proliferation of human hepatocytes and its correlation with the cccDNA deletion observed in humanized livers of chimeric mice. This choice was based on previous proliferation studies using human Ki67 within the same model [Allweiss L et al., Gut, 2018;67(3):542-52]. Ki67 RNA levels were measured in existing cccDNA samples containing nuclear RNA using RT-qPCR. Ki67 RNA levels were determined in 840 cccDNA samples isolated from 42 livers, including 15 untreated livers, 11 livers treated with detectable serum HBsAg, and 16 livers treated with progressively decreasing serum HBsAg to undetectable levels, accompanied by a >100-fold reduction in cccDNA levels. In the majority of samples (76%, 639 out of 840 samples), Ki67 RNA levels were <0.01 copies / cell. This means that only one copy of Ki67 RNA was detected in approximately >100 cells, or that less than 1% of the cells in these samples may express Ki67 RNA. Figure 9A Of the 840 samples, 9 (1.1%) showed the highest Ki67 expression levels, ranging from 0.1 to 0.28 copies / cell. This indicates that 10 to 28 copies of Ki67 RNA were detected in 100 cells, or that 10–28% of cells may express Ki67 RNA, although some cells may contain more than one copy. Therefore, the actual percentage of cells expressing Ki67 RNA is likely lower than 10–28%.

[0239] There were no significant differences in the mean Ki67 RNA levels per liver among the three groups: untreated, treated with detectable HBsAg, and treated with undetectable HBsAg, while there were significant differences in the mean cccDNA levels among the three groups. Figure 9B ).

[0240] If cell proliferation is the primary driver of cccDNA loss, a negative correlation between Ki67 RNA and cccDNA levels could be expected. However, no correlation was detected between Ki67 RNA and cccDNA kinetics during both the cccDNA amplification and maintenance phases, between treated and untreated mice, or between three different experiments using different batches of chimeric mice. The maximum Ki67 RNA level reached 0.08 copies / cell per liver, indicating that approximately 8% of cells in one mouse (ID: 823) treated with combination therapy expressed detectable Ki67 RNA. Ki67 RNA levels were detected in 6 out of 20 samples, ranging from 0.1 to 0.21 copies / cell. However, no correlation was observed between Ki67 RNA and cccDNA levels in this specific mouse.

[0241] Serum alanine aminotransferase (ALT) activity

[0242] ALT activity was assessed in nine consecutive serum samples, comprising three from untreated mice and six from treated mice (which achieved a progressive reduction in serum HBsAg to undetectable levels and a >100-fold reduction in cccDNA compared to untreated mice). ALT activity fluctuated over a six-month period (from day 21 to day 207 post-vaccination), with 84 of the 88 samples remaining below 40 U / L. In the remaining four samples collected from the treated group at day 35 post-vaccination, prior to the initiation of entecavir (ETV) treatment, ALT levels were borderline or slightly elevated, ranging from 40 U / L to 54 U / L. However, these levels subsequently decreased and remained below 40 U / L during the treatment and follow-up period (from day 60 to day 207 post-vaccination). Slight elevations in ALT levels at individual time points likely reflect only the upper limit of the ALT fluctuation range and are considered insignificant.

[0243] The reduction of HBsAg in both serum and liver was confirmed by ELISA, Western blotting, and immunohistochemical staining.

[0244] Two consecutive serum samples from untreated mice and two serum samples from treated mice were subjected to Western blot analysis, in which serum HBsAg experienced a progressive decrease and became undetectable. Figure 10A and 10B This demonstrates a kinetic similar to that detected by ELISA, confirming a gradual decrease in serum HBsAg in two consecutive serum samples treated with anti-HBs. Figure 10C , 10D and 10E).

[0245] The intrahepatic HBsAg levels in seven mice were also analyzed, with serum HBsAg progressively decreasing. Intrahepatic HBsAg was either undetectable or detectable at low levels by ELISA. Figure 10F This was confirmed by Western blot analysis. Figure 10G In addition, Western blotting analysis was performed on the intrahepatic HBcAg levels in 20 liver lysates (n = 4 lysates per liver) from five mice, showing a progressive decrease in serum HBsAg. Cellular HBcAg was not detected in any of the 20 liver lysates. Figure 10H and 10J This supports the concept that a gradual decrease in serum HBsAg reflects intracellular HBV clearance when cccDNA replenishment is blocked.

[0246] Consistent with ELISA and Western blot data, immunohistochemical staining of HBsAg in the sections showed that intracellular HBsAg was reduced to undetectable or nearly undetectable levels in the seven mice that achieved HBsAg- / anti-HBs positivity.

[0247] Contrary to the prevailing view that HBV cccDNA molecules are stable in infected cells [Alter H et al. Hepatology. 2018;67(3):1127-31], spontaneous cccDNA loss was observed in this study using chimeric mice with humanized livers during both the infection transmission phase and the persistent HBV infection phase. Furthermore, cccDNA replenishment was required to maintain cccDNA levels and persistence.

[0248] Analysis of cccDNA copies at the single-nuclear level indicated that most infected cells contained a single copy of cccDNA; however, at peak levels, the average intracellular HBsAg level accumulated to approximately 100,000 copies / cell, highlighting the remarkable efficiency of both RNA transcription and viral protein synthesis. Two possible scenarios exist. In scenario 1, the persistent presence of cccDNA after peak levels in infected cells is expected to continuously drive transcription and further increase the accumulation of viral products to intolerable levels, leading to cytopathic destruction through which cccDNA will be lost. Furthermore, unlike HIV, HBV has no known latent infection phase, meaning there is no significant repression of RNA transcription, supported by high serum HBsAg levels in both HBeAg-positive and HBeAg-negative phases [see, for example, Jaroszewicz J et al., Journal of Hepatology, 2010;52(4):514-22; and Nguyen T et al., Journal of Hepatology, 2010;52(4):508-13]. Therefore, given such highly efficient RNA transcription and viral protein synthesis, long-term persistence of cccDNA in infected cells may be impractical.

[0249] However, HBV is largely non-cytopathic. In scenario 2, infected cells may respond well to stress caused by the accumulation of high levels of intracellular viral products. As reported during the second phase of hepatotropic DNA virus replication, viral envelope proteins accumulate in infected cells. The accumulated L protein, alone or in combination with the M and / or S proteins, inhibits further cccDNA amplification or reduces cccDNA levels [see, for example, Lenhoff RJ et al., Journal of Virology, 1994;68(7):4565-71; Gao W et al., Journal of Virology, 2007;81(12):6164-74; and Lentz TB et al., Journal of Virology, 2011;85(22):11916-27]. Given the highly efficient RNA transcription and viral protein synthesis, which suggest that there is no efficient inhibition of these steps, clearing cccDNA appears to be the only effective option to prevent HBV replication and protect cells from damage.

[0250] Therefore, this type of replication-driven cccDNA deletion can be spontaneously cleared ( Figure 11A ) or cell damage ( Figure 11BThe occurrence of this phenomenon is also observed in infected primary hepatocytes and acute liver injury during in vivo infection with the duck hepatitis B virus (DHBV, a member of the Hepadnaviridae family) L protein mutant G133E, which causes defects in enveloped virus production and increases intracellular levels of cccDNA, RNA, capsid and rcDNA [see, for example, Lenhoff RJ et al., Journal of Virology, 1994;68(9):5706-13; and Lenhoff RJ et al., Hepatology, 1999;29(2):563-71].

[0251] The time span from cccDNA establishment to its deletion in infected cells represents one cycle of infection. The duration of cccDNA presence in infected cells can be influenced by both HBV replication efficiency and the infected cells' ability to secrete viral particles. Slower replication or more efficient viral particle secretion may prolong cccDNA presence in infected cells, while more efficient HBV replication or less efficient viral particle secretion may accelerate cccDNA deletion. A new cycle of infection begins when cccDNA is re-established through neo-infection of cccDNA-negative cells. This cyclical characteristic of cccDNA indicates that persistent HBV infection is maintained by multiple cycles of neo-infection. Figure 11A ).

[0252] Published studies on the dynamics of intracellular viral product levels have shown that in HBV-infected HepG2-NTCP cells, intracellular HBcAg peaks at approximately 2–4 weeks, depending on the efficiency of viral particle secretion from infected cells [see, for example, Ko C et al., Journal of Hepatology, 2018;69(6):1231-41; and König A et al., Journal of Hepatology, 2019;71(2):289-300]. In in vivo DHBV infection, intracellular L protein peaks around day 5 post-inoculation [Zhang YY et al., Journal of Virology, 2004;78(3):1195-201], suggesting that intracellular hepatotropic DNA virus infection may peak on average 1–4 weeks post-inoculation. Within this timeframe (after which cccDNA clearance may occur), each cycle of HBV infection may end within a few weeks with individual cellular-level cccDNA loss. However, in the absence of sufficient anti-HBs antibodies, HBV infection at the liver level can persist for years or even decades because there is always a new cycle of infection, while the early cycle of infection ends after cccDNA is lost.

[0253] Previous studies have shown that cccDNA molecules may be lost during cell division [see, for example, Allweiss L et al., Gut, 2018;67(3):542-52; and Zhang YY et al., Proceedings of the National Academy of Sciences of the United States of America, 2003;100(21):12372-7]. Therefore, spontaneous cccDNA loss could also be caused by random human hepatocyte proliferation. However, our findings indicate that human Ki67 RNA expression is uncommon, with levels <0.01 copies / cell detected in 76% of 840 samples. Furthermore, no correlation was observed between Ki67 RNA levels and cccDNA levels. These results suggest low human hepatocyte turnover. These findings regarding Ki67 RNA levels are consistent with observations by the mouse provider (PhoenixBio, Personal Communications) that human hepatocyte proliferation is negligible after human liver growth is complete, and are also consistent with Ki67 immunostaining results that revealed 3% Ki67-positive cells in liver sections prepared 100 days after hepatocyte transplantation in the same uPA / SCID mouse model [Allweiss L et al., Gut, 2018;67(3):542-52]. The Ki67 RNA data are consistent with normal serum ALT levels and stable serum human albumin levels in both treated and untreated mice. Therefore, the low-level proliferation of human hepatocytes observed in humanized mice is unlikely to be a significant driver of cccDNA loss in these data.

[0254] The following are exemplary embodiments of the present invention:

[0255] Implementation Scheme 1. A method for curing chronic hepatitis B infection in humans, the method comprising:

[0256] Administer an effective amount of exogenous anti-HBs antibody or a carrier that produces anti-HBs antibody to subjects in need to reduce cellular and blood hepatitis B surface antigen (HBsAg) by providing sustained elevated levels of anti-HBs antibody in said subjects.

[0257] Implementation Scheme 2. The method according to Implementation Scheme 1, wherein the reduction of HBsAg in cells and blood occurs when: (i) the new infection-mediated cccDNA supplementation is blocked, or (ii) HBsAg synthesis is not directly inhibited, or both (i) and (ii).

[0258] Implementation Scheme 3. The method according to Implementation Scheme 1 or 2, wherein the anti-HBs antibody is specific for the "a" determinant of HBsAg.

[0259] Implementation Scheme 4. The method according to Implementation Scheme 3, wherein the anti-HBs antibody is specific for the human hepatocyte attachment site in the "a" determinant of HBsAg.

[0260] Implementation Scheme 5. The method according to Implementation Scheme 4, wherein the anti-HBs antibody blocks new infection by blocking HBV particles (viral particles and subviral particles) from attaching to human hepatocytes.

[0261] Implementation Scheme 6. The method according to Implementation Scheme 1, wherein the continuously elevated anti-HBs antibody level is 100 mIU / ml or higher, lasting for 3 months or longer.

[0262] Implementation Scheme 7. The method according to Implementation Scheme 6, wherein the persistently elevated anti-HBs antibody level is 1,000 mIU / ml or higher, lasting for 3 months or longer.

[0263] Implementation Scheme 8. The method according to Implementation Scheme 7, wherein the persistently elevated anti-HBs antibody level is 10,000 mIU / ml or higher, lasting for 3 months or longer.

[0264] Implementation Scheme 9. The method according to Implementation Scheme 8, wherein the persistently elevated anti-HBs antibody level is 100,000 mIU / ml or higher, lasting for 3 months or longer.

[0265] Implementation Scheme 10. The method according to any one of Implementation Schemes 1 to 9, wherein the application is a single or multiple application of the exogenous anti-HBs antibody, or a single application of the vector that generates the anti-HBs antibody.

[0266] Implementation Scheme 11. The method according to any one of Implementation Schemes 1 to 10, wherein cellular and blood HBsAg is reduced by blocking new infection-mediated cccDNA supplementation.

[0267] Implementation Scheme 12. The method according to Implementation Scheme 12, wherein the effective amount of exogenous anti-HBs antibody or the vector that generates anti-HBs antibody is an amount sufficient to maintain a level of anti-HBs antibody sufficient to effectively and completely block new infection-mediated cccDNA supplementation, in the presence or absence of other anti-HBV drugs.

[0268] Implementation Scheme 13. The method according to any one of Implementation Schemes 1 to 12, wherein the application is the application of an effective amount of the anti-HBs antibody.

[0269] Implementation Scheme 14. The method according to any one of Implementation Schemes 1 to 12, wherein the administration is the administration of an effective amount of the carrier for generating anti-HBs antibodies, the carrier for generating anti-HBs antibodies providing endogenous production of anti-HBs antibodies in the subject.

[0270] Implementation Scheme 15. The method according to Implementation Scheme 14, wherein the administration is a single dose of the vector that generates anti-HBs antibodies, administered in an amount of 1E11 copies or more.

[0271] Implementation Scheme 16. The method according to Implementation Scheme 15, wherein the single-dose administration of the vector generating anti-HBs antibodies is administered in an amount of 2E11 copies or more.

[0272] Implementation Scheme 17. The method according to Implementation Scheme 16, wherein the single-dose administration of the vector generating anti-HBs antibodies is administered in an amount of 1E12 copies or more.

[0273] Implementation Scheme 18. The method according to Implementation Scheme 16, wherein the single-dose administration of the vector generating anti-HBs antibodies is administered in an amount of 3E12 copies or more.

[0274] Implementation Scheme 19. The method according to any one of Implementation Schemes 14 to 18, wherein the vector for generating anti-HBs antibodies is an AAV vector selected from the group consisting of: HBVZ10, HBVZ20, HBVZ30, HBVZ40, HBVZ50, HBVZ60, HBVZ70, HBVZ80 and HBVZ90.

[0275] Implementation Scheme 20. The method according to any one of Implementation Schemes 14 to 18, wherein the vector for generating anti-HBs antibodies is a viral vector, a non-viral vector, or nanoparticles.

[0276] Implementation Scheme 21. The method according to Implementation Scheme 20, wherein the vector that generates anti-HBs antibodies can be administered alone or in combination thereof.

[0277] Implementation Scheme 22. The method according to Implementation Scheme 1, wherein the exogenous anti-HBs antibody is co-administered with human anti-HBs antibody, nanobody or antibody fragment.

[0278] Implementation Scheme 23. The method according to Implementation Scheme 1, wherein the administration results in a reduction of blood HBsAg, the reduction being either gradual or abrupt, and in either case, the reduction of blood HBsAg is at least 1-5 log.

[0279] Implementation Scheme 24. The method according to Implementation Scheme 23, wherein the administration results in a reduction of blood HBsAg, the reduction being either gradual or abrupt, in either case, the blood HBsAg being reduced to a level of < 0.05 IU / ml.

[0280] Implementation Scheme 25. The method according to any one of Implementation Schemes 1 to 24, wherein the method is a monotherapy.

[0281] Implementation Scheme 26. The method according to any one of Implementation Schemes 1 to 24, wherein the method is a combination therapy with one or more additional HBV drugs that inhibit the synthesis of intracellular HBV DNA, HBV RNA and / or viral proteins.

[0282] Implementation Scheme 27. The method according to Implementation Scheme 26, wherein the one or more additional HBV drugs are members of one or more selected from the group consisting of: reverse transcription inhibitors, capsid inhibitors, cccDNA inhibitors, RNA transcription inhibitors, viral protein synthesis inhibitors, entry inhibitors, interferons, therapeutic vaccines, immune checkpoint inhibitors, and immunomodulatory drugs.

[0283] In view of the above teachings, many modifications and variations of the invention are possible. Therefore, it should be understood that, within the scope of the appended claims, the invention may be practiced in ways different from those specifically described herein.

Claims

1. A method for curing chronic hepatitis B infection in humans, the method comprising: Administer an effective amount of exogenous anti-HBs antibody or a carrier that produces anti-HBs antibody to subjects in need to reduce cellular and blood hepatitis B surface antigen (HBsAg) by providing sustained elevated levels of anti-HBs antibody in said subjects.

2. The method according to claim 1, wherein the reduction of HBsAg in cells and blood occurs when: (i) the novel infection-mediated cccDNA supplementation is blocked, or (ii) HBsAg synthesis is not directly inhibited, or both (i) and (ii).

3. The method according to claim 1 or 2, wherein the anti-HBs antibody is specific for the "a" determinant of HBsAg.

4. The method according to claim 3, wherein the anti-HBs antibody is specific for the human hepatocyte attachment site in the "a" determinant cluster of HBsAg.

5. The method according to claim 4, wherein the anti-HBs antibody blocks new infection by blocking HBV particles (viral particles and subviral particles) from attaching to human hepatocytes.

6. The method of claim 1, wherein the continuously elevated level of anti-HBs antibody is 100 mIU / ml or higher, and lasts for 3 months or longer.

7. The method of claim 6, wherein the continuously elevated anti-HBs antibody level is 1,000 mIU / ml or higher, and lasts for 3 months or longer.

8. The method of claim 7, wherein the continuously elevated anti-HBs antibody level is 10,000 mIU / ml or higher, and lasts for 3 months or longer.

9. The method of claim 8, wherein the continuously elevated anti-HBs antibody level is 100,000 mIU / ml or higher, and lasts for 3 months or longer.

10. The method according to any one of claims 1 to 9, wherein the application is a single or multiple application of the exogenous anti-HBs antibody, or a single application of the carrier that generates the anti-HBs antibody.

11. The method according to any one of claims 1 to 10, wherein cellular and blood HBsAg is reduced by blocking new infection-mediated cccDNA supplementation.

12. The method of claim 12, wherein the effective amount of exogenous anti-HBs antibody or the vector that generates anti-HBs antibody is an amount sufficient to maintain a level of anti-HBs antibody sufficient to effectively and completely block new infection-mediated cccDNA supplementation, in the presence or absence of other anti-HBV drugs.

13. The method according to any one of claims 1 to 12, wherein the application is the application of an effective amount of the anti-HBs antibody.

14. The method according to any one of claims 1 to 12, wherein the administration is the administration of an effective amount of the carrier for generating anti-HBs antibodies, the carrier for generating anti-HBs antibodies providing endogenous production of anti-HBs antibodies in the subject.

15. The method of claim 14, wherein the administration is a single dose of the vector that generates anti-HBs antibodies, administered in an amount of 1E11 copies or more.

16. The method of claim 15, wherein the single-dose administration of the vector generating the anti-HBs antibody is administered in an amount of 2E11 copies or more.

17. The method of claim 16, wherein the single-dose administration of the vector generating the anti-HBs antibody is administered in an amount of 1E12 copies or more.

18. The method of claim 16, wherein the single-dose administration of the vector generating the anti-HBs antibody is administered in an amount of 3E12 copies or more.

19. The method according to any one of claims 14 to 18, wherein the vector for generating anti-HBs antibodies is an AAV vector selected from the group consisting of: HBVZ10, HBVZ20, HBVZ30, HBVZ40, HBVZ50, HBVZ60, HBVZ70, HBVZ80 and HBVZ90.

20. The method according to any one of claims 14 to 18, wherein the carrier for generating anti-HBs antibodies is a viral vector, a non-viral vector, or nanoparticles.

21. The method of claim 20, wherein the vector for generating anti-HBs antibodies may be administered alone or in combination thereof.

22. The method of claim 1, wherein the exogenous anti-HBs antibody is administered in combination with human anti-HBs antibody, nanobody, or antibody fragment.

23. The method of claim 1, wherein the administration results in a reduction of blood HBsAg, the reduction being either gradual or abrupt, and in either case, a reduction of blood HBsAg of at least 1-5 log.

24. The method of claim 23, wherein the administration results in a reduction of blood HBsAg, the reduction being gradual or abrupt, in either case, the reduction of blood HBsAg to a level < 0.05 IU / ml.

25. The method according to any one of claims 1 to 24, wherein the method is a monotherapy.

26. The method according to any one of claims 1 to 24, wherein the method is a combination therapy with one or more additional HBV drugs that inhibit the synthesis of intracellular HBV DNA, HBV RNA, or / and viral proteins.

27. The method of claim 26, wherein the one or more additional HBV drugs are members of one or more selected from the group consisting of: reverse transcription inhibitors, capsid inhibitors, cccDNA inhibitors, RNA transcription inhibitors, viral protein synthesis inhibitors, entry inhibitors, interferons, therapeutic vaccines, immune checkpoint inhibitors, and immunomodulatory drugs.

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