Method for eliminating hepatitis B virus cccDNA and rcDNA and hepatitis B drug for use in said method

Abstract

Disclosed herein is a method for treating human chronic Hepatitis B virus infection by reducing or eliminating cccDNA in liver cells of HBV-infected patients for three months or more.

Description

[Technical field]

[0001] [Background technology]

[0002] Hepatitis B virus (HBV) chronically infects 302 million people worldwide, with approximately 1 million deaths from HBV-related disease each year. Current HBV drugs rarely provide sustained efficacy after years of medication, let alone cure HBV. HBV covalently closed circular DNA (cccDNA) is formed in the nucleus of infected cells upon infection and serves as a transcription template (Nassal M, Gut 64, 1972-1984 (2015); Tuttleman et al., Cell 47, 451-460 (1986)). The presence of cccDNA is considered the root cause of persistent HBV infection. Current HBV cure strategies aim to directly eliminate or permanently silence cccDNA (Alter et al., Hepatology 67, 1127-1131 (2018)). Drugs that can directly eliminate or permanently silence cccDNA remain overlooked.

[0003] Clinical evidence indicates frequent serovirus population switching in chronic HBV infection, e.g., preC, core, preS, S, or drug-resistant mutants frequently replace wild-type (WT) virus (Brunetto et al., Proc Natl Acad Sci USA 88, 4186-4190 (1991); Carman et al., Lancet 2, 588-591 (1989); Lok et al., Proc Natl Acad Sci USA 91, 4077-4081 (1994); Okamoto et al., J Virol 68, 8102-8110 (1994); Zoulim and Locarnini, Proc Natl Acad Sci USA 91, 4077-4081 (1994)). (1994)), suggesting frequent cccDNA loss and replacement (conversion) in the liver, further suggesting that: 1. the initial viral population is frequently cleared, and 2. once the initial viral population in the infected liver is cleared, there is a new round of infection.

[0004] There is a need for an effective method for treating chronic HBV infection. In the prior invention of the present invention (US Pat. No. 11136378B2), it was disclosed that chronic HBV infection can be treated by administering to an HBV-infected human patient an HBV neutralizing antibody or an HBV therapeutic vector expressing such an HBV neutralizing antibody at a level higher than the level of serum HBV particles in the HBV-infected human patient, such that the level of HBV neutralizing antibody results in undetectable levels of HBV particles in serum or complete HBsAg seroconversion from HBsAg positive to anti-HBs antibody positive. Further research has been conducted and new findings are described in this application. Summary of the Invention

[0005] The present invention discloses a method for effectively curing chronic Hepatitis B infection, and a method for simplifying curative treatment and shortening the course of treatment. Some of the discoveries on which the present invention is built include the following elements: 1. In contrast to the current consensus in the HBV field, clearing HBV infection does not require directly targeting or permanently silencing HBV cccDNA; 2. Furthermore, contrary to the consensus in the HBV field, clearing HBV infection does not require specific HBV cellular immunity; 3. Reducing and eliminating cccDNA from infected livers of HBV-infected patients by blocking the recruitment of HBV rcDNA pools; 4. Blocking rcDNA replenishment by blocking new rounds of infection with sustained high levels of anti-HBs antibodies; 5. To provide an AAV vector-based HBV drug that endogenously expresses high levels of sustained anti-HBs antibodies after injection into muscle cells that are independent of the host's adaptive immunity; 6. Most patients with chronic HBV infection can be treated with a single injection of an AAV vector-based HBV drug; 7. Complete and sustained anti-HBs seroconversion, a change from HBsAg positive and anti-HBs antibody negative (HBsAg+ / anti-HBs-) to HBsAg negative and anti-HBs positive (HBsAg- / anti-HBs+), is necessary to achieve a more effective HBV cure; 8. The most effective way to reduce serum HBsAg to undetectable levels (HBsAg seroclearance) is to achieve complete and sustained anti-HBs seroconversion; 9. The current lifelong daily medication-based HBV treatment can be shortened to a finite period when it is added to or combined with an AAV-anti-HBs vector that expresses sustained high levels of anti-HBs antibodies; and 10. Current therapies that mediate HBsAg seroclearance or HBV functional cure, or / and the natural clearance that induces it, are often counterproductive, i.e., non-sustainable. However, this can be made sustained by adding AAV-anti-HBs vectors or by providing exogenous anti-HBs antibodies. [Brief description of the drawings]

[0006] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the features, advantages, and principles of the invention. [Figure 1] Dynamic serum antibody levels in the malaria group (A, n=5) and the anti-HBs group (B, n=15) are shown. AAV vectors expressing either malaria or anti-HBs antibodies were injected intramuscularly at a dose of 10 genome copies on day 49 post-infection (pi), and the dynamic serum antibody levels determined by ELISA are shown from day 49 to day 183 pi. Note that two test animals No. 970 and 909 in the anti-HBs group were injected intraperitoneally with mouse anti-HBs as a control (250 mg per injection, 9 times). No antibodies were detected on day 49, and 0.1 mg / ml was assigned in order to plot the curves on a logarithmic scale. [Diagram 2] Viremia and serum HBsAg levels were significantly reduced in the anti-HBs treated groups. Figure 2A shows the different viremia kinetics between malaria antibodies and the three anti-HBs groups. Anti-HBs-total includes all 15 mice treated with anti-HBs antibodies. The 15 mice were further divided into two subgroups: the anti-HBs-A group consisted of 9 mice whose infection peaked before day 183, and the anti-HBs-B group consisted of 6 animals whose infection was significantly delayed and did not peak. Viremia levels in the anti-HBs-B group were reduced >100-fold at several time points compared to the malaria group. Figure 2B shows that the mean viremia at day 183 (black bars) was significantly reduced in both the anti-HBs-total and anti-HBs-B groups. Mean serum HBsAg levels (grey curves) were significantly reduced in all three anti-HBs groups compared to the malaria group at day 183. [Diagram 3]HBV functional cure in animal 970. Figure 3A shows that after anti-HBs treatment was started, both viremia and serum HBsAg became undetectable. The lower limit of detection of serum HBV DNA is 100 copies / ml and 1 ng / ml for serum HBsAg and anti-HBs antibodies, respectively. Equivalent viremia to 970's pretreatment levels from animal 973 is also plotted as a reference for pretreatment levels of intrahepatic HBV DNA in Figure 3B. Figure 3B shows that the average intrahepatic rcDNA levels (copies / cell) from two rounds of 20 liver samplings in 970 were significantly reduced by 500-1,800-fold (p=1.33E-9 and 1.29E-9), and the average cccDNA levels were reduced by 100-675-fold (p=5.27E-7 and 4.69E-7) than animal 973, whose rcDNA and cccDNA levels serve as pretreatment level controls. Figure 3C shows that rcDNA ≦1 copy / cell was detected in 7 of 20 liver samples from the first round and 20 of 20 samples from the second round (Figure 3C2). No cccDNA was detected in 3 of 20 samples from the first round and 7 of 20 samples from the second round. Very low cccDNA levels were detected in most cccDNA samples, and no cccDNA amplification was detected in any of the 40 samples (Figures 3C1 and C2). [Figure 4]Figure 4 shows various cccDNA levels and cccDNA loss in a fraction of infected cells in HBV-infected human livers of chimeric mice. Up to 60-fold differences in cccDNA levels between different samplings were detected despite comparable rcDNA levels between corresponding liver samplings of 907 liver (Figure 4A). More than 30-fold differences in cccDNA levels were detected between different samples despite comparable rcDNA levels between corresponding liver samplings of animal 909 liver (Figure 4B). cccDNA levels of ≦1 copy / cell were detected in 5 of 20 samplings of animal 907 (Figure 4A) and 17 of 20 samplings of animal 909 (Figure 4B), and in 260 of the total 566 cccDNA samplings (46%) (Figure 4C). The number of liver samplings with a mean cccDNA level of ≦1 copy / cell was significantly higher in the anti-HBs group than in the malaria antibody group (X2=25.2 and p<0.001) (Figure 4D). [Diagram 5] Figure 5 shows that the mean intrahepatic rcDNA level was reduced by 4,000 to 10,000 copies / cell in all nine liver samples treated with anti-HBs antibodies compared to animal 907 treated with malaria antibodies. The mean rcDNA levels were calculated from 20 samplings of each liver sample. The efficiency of reducing rcDNA levels depends mainly on the viremia level (Figure 5A). P values ​​ranged from E-9 to E-16 (Figure 5B). [Figure 6] Figure 6 illustrates three components involved in the reduction of intrahepatic rcDNA levels by anti-HBs therapy. Figure 6A shows virion secretion, which expels rcDNA from infected cells, while pre-existing cccDNA replenishes rcDNA. Figure 6B illustrates spontaneous cccDNA loss. The combination of virion secretion with cccDNA loss can clear HBV from infected cells, but not sustainably in the absence of anti-HBs. Figure 6C illustrates that blocking new rounds of infection with anti-HBs establishes sustained HBV clearance. [Figure 7] The main animal experimental procedures, timing, and duration are shown. N is the number of animals at the three time points. [Figure 8]Figure 8 shows the difference in HBV infection levels between animals 907 and 973. Animal 907 was treated with an AAV vector expressing a malaria antibody, and animal 973 was treated with an AAV vector expressing an anti-HBs antibody. Figure 8A shows that the difference in viremia is >100-fold at the late time points. Figure 8B shows the significant difference in rcDNA and cccDNA levels detected between the two animals. [Figure 9] The main experimental procedures and timeline of HBV-infected uPA / SCID chimeric mice treated with entecavir for 9 weeks followed by AAV-anti-HBs vector administration are shown. [Figure 10] Addition of AAV-anti-HBs vector to 9 weeks of ETV treatment prevents HBV recurrence and increases the level of elevated anti-HBs antibodies to HBsAg loss / complete anti-HBs seroconversion and HBV functional cure. Figure 10A shows the kinetic viremia curves in untreated (blue) and ETV monotherapy (orange) groups. Figure 10B shows the kinetic serum HBsAg curves in untreated and ETV only groups. Figures 10C and 10D show the kinetic viremia and serum HBsAg curves in 10 animals treated with ETV with add-on. Figure 10E shows the number of animals with no HBV recurrence and undetectable HBsAg. Figure 10F shows the number of animals with HBV functional cure. [Figure 11] Figures 11A-E show that HBV functional cure occurred at or after complete and sustained anti-HBs seroconversion, which was achieved by increasing anti-HBs antibody levels at different time points. Figures 11A-E show HBV functional cure markers established at or after anti-HBs seroconversion. Figure 11F shows that serum HBV DNA remained detectable at the end of the experiment, characterized by HBsAg- / HBV DNA+ / anti-HBs+ or pre-cure phase. [Figure 12]HBV recurrence is caused by a new round of infection. Figure 12A shows that HBV recurrence cannot be prevented by anti-HBs treatment if cccDNA persists in infected cells. Figure 12B shows that HBV recurrence was prevented with anti-HBs treatment because of the spontaneous loss of cccDNA and the existence of a new round of infection after ETV withdrawal. Figure 12C shows that it takes years / decades to reach complete anti-HBs seroconversion in most naturally cured cases, which is mainly driven by the reduction of HBsAg levels. Figure 12D shows that sufficient anti-HBs antibody therapy can rapidly achieve complete anti-HBsAg seroconversion, shortening the period required for HBV functional cure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The embodiments specified herein are intended to be illustrative and not limiting. Tables 1 and 2 outline some of the main inventive features of the present invention, which are further detailed in the following description. [Table 1] The present invention further describes a method of altering the HBV infection status by mediating an increase in serum anti-HBs antibody levels, as outlined in Table 2. [Table 2]

[0008] In one embodiment, in contrast to the established consensus in the HBV field that exhausted specific cellular immunity is the cause of chronic HBV infection, the present invention emphasizes that high levels of specific humoral immunity to HBsAg are required to cure hepatitis B.

[0009] In one embodiment, the present invention distinguishes between the current ultimate therapeutic targets, which aim to directly inhibit HBV cccDNA synthesis or kill HBV infected cells, through specific HBV cellular immunity for cccDNA elimination. The present invention devise a method for reducing and eliminating HBV cccDNA without directly inhibiting cccDNA synthesis or killing infected cells.

[0010] In one embodiment, the present invention is based on two principles in devising a method for eliminating cccDNA: 1. by taking advantage of the natural clearance of HBV, which involves the natural loss of cccDNA from HBV-infected cells, and 2. by blocking cccDNA replenishment.

[0011] In one embodiment, the present invention emphasizes that blocking cccDNA replenishment should be mediated by depleting HBV rcDNA.

[0012] In one embodiment, the present invention emphasizes that depleting HBV rcDNA includes blocking rcDNA replenishment.

[0013] In one embodiment, the present invention emphasizes that blocking rcDNA replenishment is primarily required to block new rounds of infection.

[0014] Current HBV treatment methods for HBV drugs approved or in development focus on inhibiting intracellular HBV replication and do not aim to directly increase HBV neutralizing antibody levels as disclosed in the present invention.

[0015] Several biopharmaceutical companies have developed exogenous HBV neutralizing antibodies. However, they attribute the observed efficacy to Fc receptor-mediated cellular functions rather than direct neutralization functions, even though regular anti-HBs antibodies can provide comparable therapeutic efficacy in uPA / SCID chimeric mice without introducing mutations into the Fc portion of human IgG (Lampp, Hepatology 74, 513A (2021); Zhang et al., Gut 65, 658-671 (2016).). Furthermore, these exogenous antibodies cannot provide sustained therapeutic effects because repeated injections are required to maintain therapeutic levels.

[0016] In one embodiment, the present invention emphasizes the function of direct neutralization by HBV neutralizing antibodies that directly bind to the attachment sites of whole and subviral particles, which prevent viral particles from attaching to hepatocytes in the human liver. Absence of viral particle attachment to hepatocytes results in absence of viral entry to initiate new rounds of infection. Thus, HBV antibodies against the attachment sites of viral particles provide the most effective neutralization of HBV infectivity. Such antibody binding specificity is important for blocking rcDNA and cccDNA recruitment.

[0017] In one embodiment, the present invention emphasizes that increasing the endogenous capacity to express HBV neutralizing antibodies is most effective in resulting in sustained cccDNA loss and clearance and HBV cure.

[0018] Some existing HBV therapeutic vaccines show disappointing results in clinical trials. The function of therapeutic vaccines depends on host immune cells from antigen processing and presentation to signal for T cell and B cell activation to exert antiviral function. Furthermore, the number of specific T cells or B cells is limited and may not have sufficient capacity to produce high levels of HBV neutralizing antibodies to block persistently high levels of subviral particles (HBsAg) in HBV-infected patients.

[0019] However, in the present invention, it is found that endogenous expression of HBV neutralizing antibodies should be independent of the host's adaptive immune system, and that non-traditional antibody-expressing cells are necessary to expand the endogenous capacity to express HBV neutralizing antibodies.

[0020] In one embodiment, the present application invents an AAV-anti-HBs vector as an HBV drug that converts muscle cells into anti-HBs antibody-producing cells after a single injection, which leads to sustained high-level endogenous expression of anti-HBs antibodies, and thus has a sustained therapeutic efficacy.

[0021] Current mainstream HBV treatments have significant inefficiencies, requiring lifelong daily dosing, as treatment withdrawal often leads to recurrence of HBV infection and recurrent liver damage, which can have fatal consequences. In sequential embodiments, the present invention shortens the current open-ended Hepatitis B treatment period to a limited course of treatment, primarily requiring a single injection.

[0022] In one embodiment, the present invention allows current HBV treatment to be safely weaned without recurrence of HBV infection and recurrence of liver damage.

[0023] Unfortunately, with existing antiviral treatment methods, the achieved clearance of viral particles is often counterproductive, as cells cleared from HBV infection are not protected from viral particle attachment and are therefore frequently exposed to new rounds of infection.

[0024] In one embodiment, the present invention protects cells that have cleared HBV infection from recurrent HBV infection with sustained high levels of HBV neutralizing antibodies, such that the clearance achieved is sustained and gradually extends until a complete HBV cure is established.

[0025] In one embodiment, the present invention shifts HBV treatment from inhibiting current intracellular HBV replication to blocking new rounds of infection. To block new rounds of HBV infection, sustained high levels of anti-HBs antibodies are required. There are several approaches that can be explored to increase anti-HBs levels. The first is a therapeutic vaccine. However, the performance of therapeutic vaccines has been disappointing in clinical trials (Fontaine et al., Gut 64, 139-147 (2015)., Godon et al., Molecular therapy: the journal of the American Society of Gene Therapy 22, 675-684 (2014), Michel et al., Journal of hepatology 54, 1286-1296 (2011), Zoulim et al., Human vaccines & immunotherapeutics, (2019)). The second is repeated injections of exogenous anti-HBs antibodies, which are not sustained (Galun et al., Hepatology 35, 673-679 (2002), Sneha V. Gupta et al EASL 2021 International Liver Congress. (2021), pp. PO_43_ILC2021), or Myrcludex Daily injections of entry inhibitors such as B (Bogomolov et al., Journal of hepatology 65, 490-498 (2016)), which do not react with viral particles and cannot prevent HBV particles from attaching to hepatocytes, are less effective in blocking new rounds of infection in established HBV infections in both HBV-infected uPA / SCID chimeric mice and chronically infected humans. The inventors of the present invention choose to utilize an engineered humoral immunological approach to develop a new HBV cure drug that can persistently treat anti-HBs deficiency in chronic HBV-infected patients. Specifically, an optimized AAV vector is used to carry the human anti-HBs gene, and the resulting drug is called AAV-anti-HBs vector, and one of the leading candidates is HBVZ10 as described in this application.HBVZ10 can endogenously express persistently high levels of anti-HBs antibodies after a single injection into skeletal muscle cells. This engineered humoral immunological approach does not rely on adaptive immunity, avoiding the significant loss in both HBV-specific T (Gehring and Protzer, Gastroenterology 156, 325-337 (2019)) and B cells (Burton et al., The Journal of Clinical Investigation, (2018)) during chronic HBV infection.

[0026] Table 3 summarizes the main advantages of the present invention over current HBV treatments and drugs. [Table 3]

[0027] Based on the above discoveries, the inventors of the present application have developed several methods for effectively treating chronic Hepatitis B infection.

[0028] In one embodiment, the present invention is directed to a method of treating Hepatitis B virus (HBV) infection by reducing or eliminating cccDNA (covalently closed circular DNA) and / or rcDNA (relaxed circular DNA) in liver cells of a human chronically infected HBV patient for three months or more.

[0029] In one embodiment, the HBV cccDNA level in liver cells of an HBV-infected patient is reduced to <1 copy / cell, <1 copy / 10 cells, <1 copy / 100 cells, <1 copy / 1,000 cells, or <1 copy / 10,000 cells.

[0030] In another embodiment, the level of rcDNA in the liver of an infected patient is reduced to <1 copy / cell, <1 copy / 10 cells, <1 copy / 100 cells, <1 copy / 1,000 cells, or <1 copy / 10,000 cells.

[0031] In another embodiment, HBV cccDNA and rcDNA levels in liver cells of HBV-infected patients are sustainably reduced by administering exogenous anti-HBs antibodies and / or viral or non-viral vectors or nanoparticles that endogenously express anti-HBs antibodies to maintain high levels of anti-HBs antibodies in the blood.

[0032] In another embodiment, the reduction or elimination of cccDNA in liver cells of an HBV-infected patient is manifested by maintaining high levels of HBV neutralizing antibodies in the patient's blood, thereby reducing or rendering undetectable levels of HBV DNA, HBeAg, and HBsAg in the patient's blood.

[0033] In another embodiment, maintaining high levels of HBV neutralizing antibodies in the patient's blood is represented by complete and sustained anti-HBs seroconversion achieved by endogenous or exogenous infusion of sufficient amounts of anti-HBs antibodies to persistently convert serum HBsAg positive and anti-HBs negative (HBsAg+ / anti-HBs-) to serum HBsAg negative and anti-HBs positive (HBsAg- / anti-HBs+).

[0034] In another embodiment, achieving complete and sustained anti-HBs seroconversion with sufficient anti-HBs antibodies is one of the most effective ways to induce serum HBsAg loss or serum clearance.

[0035] In another embodiment, complete and sustained anti-HBs seroconversion is the primary goal of HBV curative treatment and is required to result in a more effective functional cure of HBV.

[0036] In another embodiment, producing a more effective HBV functional cure requires first removing serum HBsAg or rendering serum HBsAg undetectable by developing or administering sustained high levels of anti-HBs antibodies.

[0037] In another embodiment, complete and sustained anti-HBs seroconversion is required to establish sustained HBsAg serum clearance or functional cure among HBV drug-treated patients who have transiently cleared serum HBsAg or achieved a functional cure with HBV drugs such as nucleotide analogues, interferon, and / or NAP or RNAi.

[0038] In another embodiment, high levels of HBV neutralizing antibodies in the patient's blood are maintained for 1, 2, 3, 4, 5, 6, 7, or 8 months or more.

[0039] In another embodiment, an HBV neutralizing antibody is an antibody that can prevent viral particles from attaching to hepatocytes, thus effectively blocking the entry of HBV virions into hepatocytes.

[0040] In another embodiment, the high level of HBV neutralizing antibodies is at a level of >1, >10, >100, >200, or >300 μg / ml, or >10, >100, >1,000, >10,000, >20,000, or >30,000 mIU / ml in the patient's blood.

[0041] In another embodiment, HBV neutralizing antibodies are administered to the patient by injecting one, two, or three exogenous human HBV neutralizing antibodies at a dose of about 0.1 mg / kg, 0.5 mg / / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, or ≧30 mg / kg body weight.

[0042] In another embodiment, infusions of exogenous human neutralizing antibodies comprising single, multiple or repeated infusions to maintain levels of antibody in the patient's blood of >1, >10, >100, >200, or >300 μg / ml, or >10, >100, >1,000, >10,000, >20,000, or >30,000 mIU / ml for three months or more.

[0043] In another embodiment, the HBV neutralizing antibodies are one, two, or three endogenously expressed human neutralizing antibodies.

[0044] In another embodiment, endogenous human HBV neutralizing antibodies are expressed by viral or non-viral vectors comprising nanoparticles such as lipid nanoparticles (LNPs) or GalNAc particles.

[0045] In another embodiment, the viral or non-viral vector contains a DNA or mRNA sequence encoding an HBV neutralizing antibody or antibody fragment. In one embodiment, the viral vector is or comprises an adeno-associated virus (AAV) vector. In one embodiment, the AAV vector comprises a nucleic acid sequence encoding an HBV neutralizing antibody or antibody fragment thereof, thus becoming an AAV-anti-HBV vector.

[0046] In one embodiment, the AAV-anti-HBV vectors are each about 1×10 9 Genome copies / kg, 1×10 10 Genome copies / kg, 1×10 11 Genome copies / kg, 1×10 12 Genome copies / kg, 2 x 10 12 Genome copies / kg, 2.5 × 10 12 It is injected into muscle cells of HBV patients at a dose of genome copies / kg or more.

[0047] In one embodiment, the viral or non-viral vector is injected into the patient, either once or multiple times as needed.

[0048] In one embodiment, depletion of rcDNA through the combination of an HBV neutralizing antibody with an inhibitor of intracellular HBV replication results in significant, near complete, or complete cccDNA loss.

[0049] In one embodiment, intracellular HBV inhibitors include RT inhibitors, capsid inhibitors, RNAi drugs, nucleic acid polymers (NAPs), interferons, innate immune agonists, and entry inhibitors.

[0050] In one embodiment, AAV anti-HBV vectors expressing HBV neutralizing antibodies are used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and more effectively cure HBV in HBV treatment-naive chronically infected individuals.

[0051] In one embodiment, AAV anti-HBV vectors expressing HBV neutralizing antibodies are used to block cccDNA, induce HBsAg seroclearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure supplementation to reduce and eliminate cccDNA, induce HBsAg seroclearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure in chronic HBV infected individuals undergoing HBV treatment but unable to safely wean off antiviral drugs.

[0052] In one embodiment, AAV anti-HBV vectors expressing HBV neutralizing antibodies are used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure in chronically HBV infected individuals who wish to become HBV infection-free, regardless of their infection status / stage or their treatment status (naive or treated).

[0053] In one embodiment, AAV anti-HBV vectors expressing HBV neutralizing antibodies are used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure in HBV-infected pregnant women or patients with liver transplants.

[0054] In one embodiment, AAV anti-HBV vectors expressing HBV neutralizing antibodies are used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and more effectively cure HBV in people who have clinically resolved HBV infection but are at high risk of HBV recurrence.

[0055] In one embodiment, the present invention is directed to a method of treating chronic HBV infection and / or providing protection against recurrence of HBV infection in a human patient, comprising administering to an HBV-infected human patient a sufficient amount of an HBV neutralizing antibody or antibody fragment, wherein the amount of HBV neutralizing antibody is at a level that blocks the recruitment of both rcDNA and cccDNA, allowing the reduction and depletion of rcDNA to <1 copy / cell, and promoting the reduction and elimination of cccDNA to <1 copy / cell.

[0056] In one embodiment, the treatment of chronic HBV infection includes treating newborns / children infected with HBV, hi another embodiment, the treatment of chronic HBV infection includes treating adults infected with HBV.

[0057] In one embodiment, the HBV-infected human patient is a chronically HBV-infected individual who has been HBsAg positive for more than six months and has normal or elevated alanine aminotransferase (ALT) levels. In another embodiment, the HBV-infected human patient 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 and susceptible to recurrence of HBV infection after transplantation.

[0058] In one embodiment, HBV neutralizing antibodies or antibody fragments are produced by the HBV therapeutic vector.

[0059] In one embodiment, the HBV therapeutic vector comprises a mixed population of vectors, each of which encodes one specific anti-HBs antibody or antibody fragment that binds to one or more epitopes of the HBV envelope protein, or a single vector that encodes one HBV neutralizing antibody or antibody fragment that binds to one or more epitopes of the HBV envelope protein. In one embodiment, the HBV neutralizing antibody or antibody fragment is one of the antibodies described below.

[0060] Anti-Hepatitis B Drugs Used in the Present Invention Any existing or newly developed anti-hepatitis B drug that can eliminate or deplete cccDNA and / or rcDNA can be used in the above treatment method. Such drugs include, but are not limited to, exogenously or endogenously expressed antibody drugs, small molecule drugs, peptide drugs, and vector drugs. The following AAV-anti-HBs vector-based HBV drugs that endogenously express anti-HBs antibodies after a single injection were designed and tested in the present invention and found to be effective against chronic hepatitis infection when used in the above treatment method. A total of nine AAV-anti-HBs vectors contain the following sequences:

[0061] Nucleic acid SEQ ID NO:1 encodes the amino acid sequence of SEQ ID NO:2, which is the variable region of the heavy chain of the HBVZ10 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0062] Nucleic acid SEQ ID NO:3 encodes the amino acid sequence of SEQ ID NO:4, which is the variable region of the light chain of the HBVZ10 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0063] Nucleic acid SEQ ID NO:5 encodes the amino acid sequence of SEQ ID NO:6, which is the variable region of the heavy chain of the HBVZ20 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0064] Nucleic acid SEQ ID NO:7 encodes the amino acid sequence of SEQ ID NO:8, which is the variable region of the light chain of the HBVZ20 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0065] Nucleic acid SEQ ID NO:9 encodes the amino acid sequence of SEQ ID NO:10, which is the variable region of the heavy chain of the HBVZ30 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0066] The nucleic acid SEQ ID NO:11 encodes the amino acid sequence of SEQ ID NO:12. SEQ ID NO: 12 is the variable region of the light chain of the HBVZ30 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0067] The nucleic acid SEQ ID NO:13 encodes the amino acid sequence of SEQ ID NO:14. SEQ ID NO: 14 is the variable region of the heavy chain of the HBVZ40 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0068] The nucleic acid SEQ ID NO:15 encodes the amino acid sequence of SEQ ID NO:16. SEQ ID NO: 16 is the variable region of the light chain of the HBVZ40 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0069] The nucleic acid SEQ ID NO:17 encodes the amino acid sequence of SEQ ID NO:18. SEQ ID NO: 18 is the variable region of the heavy chain of the HBVZ50 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0070] The nucleic acid SEQ ID NO:19 encodes the amino acid sequence of SEQ ID NO:20. SEQ ID NO:20 is the variable region of the light chain of the HBVZ50 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0071] The nucleic acid SEQ ID NO:21 encodes the amino acid sequence of SEQ ID NO:22. SEQ ID NO:22 is the variable region of the heavy chain of the HBVZ60 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0072] Nucleic acid SEQ ID NO:23 encodes the amino acid sequence of SEQ ID NO:24, which is the variable region of the light chain of the HBVZ60 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0073] The nucleic acid SEQ ID NO:25 encodes the amino acid sequence of SEQ ID NO:26. SEQ ID NO:26 is the variable region of the heavy chain of the HBVZ70 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0074] The nucleic acid SEQ ID NO:27 encodes the amino acid sequence of SEQ ID NO:28. SEQ ID NO:28 is the variable region of the light chain of the HBVZ70 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0075] The nucleic acid sequence SEQ ID NO:29 encodes the amino acid sequence of SEQ ID NO:30. SEQ ID NO:30 is the variable region of the heavy chain of the HBVZ80 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0076] Nucleic acid SEQ ID NO:31 encodes the amino acid sequence of SEQ ID NO:32, which is the variable region of the light chain of the HBVZ80 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0077] The nucleic acid SEQ ID NO:33 encodes the amino acid sequence of SEQ ID NO:34. SEQ ID NO:34 is the variable region of the heavy chain of the HBVZ90 human anti-HBs monoclonal IgG1 antibody against HBsAg of four serotypes.

[0078] Nucleic acid SEQ ID NO:35 encodes the amino acid sequence of SEQ ID NO:36, which is the variable region of the light chain of the HBVZ90 human anti-HBs monoclonal IgG1 antibody against HBsAg of the four serotypes.

[0079] Nucleic acid SEQ ID NO:37 is the nucleic acid sequence of an AAV vector consisting of 3758 bp, including two ITRs (inverted terminal repeats from AAV), a chicken beta actin promoter, constant regions of human IgG1 heavy and light chains, a WPRE (woodchuck hepatitis virus posttranscriptional regulatory element), and an SV40 polyadenylation signal. This AAV vector (SEQ ID NO:37) allows for cloning of two variable regions of both heavy and light chains to express a complete human IgG1 monoclonal anti-HBs antibody. In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, the VH comprising the amino acid sequence of SEQ ID NO:2, and an antibody VL, the VL comprising the amino acid sequence of SEQ ID NO:4, or variants thereof having at least 95% sequence homology. In one embodiment, the present invention is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising the above-described nucleic acid molecule.

[0080] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO:6, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0081] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 10, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0082] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 14, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0083] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 18, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0084] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 22, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0085] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 26, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0086] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 30, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0087] In one embodiment, the present invention is directed to an isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 34, and an antibody VL, wherein the 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 is directed to a nucleic acid molecule encoding said isolated binding molecule or antigen-binding fragment. In one embodiment, the present invention is directed to a vector comprising said nucleic acid molecule.

[0088] In one embodiment, the invention is directed to a composition comprising one or more of the above-described isolated binding molecules or antigen-binding fragments.

[0089] In one embodiment, the invention relates to a pharmaceutical composition comprising one or more of the above antibodies and a pharma- ceutically acceptable carrier.

[0090] In one embodiment, complete and sustained anti-HBs seroconversion is the most effective way to induce serum HBsAg loss.

[0091] There are several drug candidates, including RNAi drugs (siRNA or antisense RNA) and nucleic acid polymers (NAPs), which directly inhibit the synthesis of HBsAg or / and block HBsAg secretion from infected cells, leading to a reduction in serum HBsAg that takes several months. The average serum HBsAg reduction by RNAi drugs is about 1.5 log. Available data show that patients with high HBsAg levels >10,000 IU / ml respond poorly to RNAi drugs. NAP-induced serum HBsAg reduction is usually accompanied by liver damage, which raises safety concerns.

[0092] The present invention represents the most effective way to reduce serum HBsAg to undetectable levels, i.e., to induce complete and sustained anti-HBs seroconversion by providing sufficient amounts of anti-HBs antibodies, either expressed or infused, or a combination of both. As shown in Figure 7, complete anti-HBs seroconversion reduced serum HBsAg levels from 3-5 logs depending on baseline levels, much more effective than the average of 1.5 logs by RNAi drugs. If sufficient anti-HBs antibodies are provided that are >serum HBsAg levels, it only takes a few days for serum HBsAg levels to become undetectable. Maintaining anti-HBs levels >serum HBsAg levels results in sustained HBsAg loss.

[0093] In one embodiment, with the addition or combination of an AAV-anti-HBs vector or anti-HBs antibody, the current lifelong daily dosing can be shortened to a finite period as short as 9 weeks.

[0094] The main reason for the need for lifelong medication is that current nucleotide analogue therapy does not address new rounds of infection occurring in chronic HBV infection, does not improve insufficient anti-HBs levels, and requires lifelong treatment to suppress rebound viral replication when therapy is stopped. As demonstrated in Figure 7, delayed addition of AAV-anti-HBs vector and exogenous anti-HBs antibody to 9 weeks of entecavir treatment achieved HBV functional cure in 5 out of 8 mice with complete anti-HBs seroconversion, establishing a proof-of-concept that the current lifelong HBV treatment can be shortened to a finite period as short as 9 weeks. Therefore, a key way to shorten HBV treatment is to add sufficient AAV-anti-HBs vector and / or exogenous anti-HBs antibody to the current therapy, and anti-HBs antibody levels should always be higher than serum HBsAg levels.

[0095] In one embodiment, non-sustained serum HBsAg loss or functional cure mediated by current therapy or by natural clearance of HBV can be prevented and converted to sustained HBsAg loss and functional cure.

[0096] Although serum HBsAg loss or HBV functional cure can be achieved naturally or through current therapy, such cure or HBsAg loss may be rare and non-sustainable. The main reason for non-sustainable HBsAg loss or functional cure is lack of sufficient anti-HBs antibodies in chronic HBV infected patients. As demonstrated in FIG. 7, serum HBsAg loss or HBV functional cure became sustained when a sufficiently high level of anti-HBs antibodies was provided and maintained. Therefore, the key method to achieve sustained serum HBsAg loss and / or sustained HBV functional cure is to provide and maintain anti-HBs levels that should always be higher than serum HBsAg levels.

[0097] In one embodiment, the present invention is directed to a method of treating chronic HBV infection in a human patient and providing protection against recurrent new rounds of HBV infection, comprising administering to an HBV-infected human patient a sufficient amount of an HBV neutralizing antibody or antibody fragment, the amount of HBV neutralizing antibody being at a level that blocks the recruitment of both rcDNA and cccDNA, allowing the reduction and depletion of rcDNA to <1 copy / cell, promoting the reduction and elimination of cccDNA to <1 copy / cell, inducing HBsAg serum clearance, achieving complete anti-HBs seroconversion and resulting in a more effective HBV cure.

[0098] In one embodiment, treatment of chronic HBV infection includes treatment of newborns / children infected with HBV.

[0099] In one embodiment, treating a chronic HBV infection includes treating an adult infected with HBV.

[0100] In one embodiment, the HBV-infected human patient is a chronically HBV-infected individual who has been HBsAg positive for more than six months and has normal or elevated alanine aminotransferase (ALT) levels.

[0101] In one embodiment, the HBV-infected human patient 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 and susceptible to recurrence of HBV infection after transplantation.

[0102] In one embodiment, HBV neutralizing antibodies or antibody fragments are produced by the HBV therapeutic vector.

[0103] In one embodiment, the HBV therapeutic vector comprises a mixed population of vectors, each of which encodes one specific anti-HBs antibody or antibody fragment that binds to one or more epitopes of the HBV envelope protein, or a single vector encoding one HBV neutralizing antibody or antibody fragment that binds to one or more epitopes of the HBV envelope protein. EXAMPLES

[0104] Example 1. Experimental procedure using uPA / SCID chimeric mice for HBV infection and treatment. A total of 32 male uPA / SCID chimeric mice were cultured at 5 × 10 8 Mice were infected with an inoculum of 1 × 10 HBV DNA copies 7 weeks post-infection (pi). AAV vectors expressing malaria antibodies (antibody specificity control) or anti-HBs antibodies were then administered to mice. 11 Animals were injected intramuscularly into the right thigh muscle at a dose of 100 genome copies and then monitored for an additional 30 weeks (Figure 7). By day 183, 20 animals remained, and by day 253 (end date), 10 animals remained.

[0105] To have a sufficient number of animals for statistical analysis, serological data were reported up to day 183. Intrahepatic HBV DNA data from 18 livers, 8 of which were collected around day 204 and the remaining 10 were collected on day 253. Each liver was randomly sampled at least 20 times by cutting 20-30 mg of tissue for each sampling and analyzed. HBV levels in each sampling were quantitatively determined individually and the mean HBV level was calculated from the 20 samplings of each liver.

[0106] Example 2. Dynamic serum antibody levels in the two groups As shown in Figure 1A, five mice were injected 49 days pi with an AAV vector expressing a malaria antibody as an expression control, and expressed antibody levels remained above 100 mg / ml throughout the observation period (approximately 200 days; 183 days time point not shown), suggesting that this optimized AAV vector is capable of expressing sustained high levels of antibody after a single injection.

[0107] Of the 15 animals that underwent anti-HBs treatment (Fig. 1B), 13 were injected with AAV-anti-HBs vectors expressing anti-HBs antibodies, and the remaining 2 animals (no. 970 and no. 909) were injected every 3 weeks with exogenous mouse anti-HBs antibodies at a dose of 250 mg per injection starting on day 74 pi as controls. Kinetic anti-HBs levels in most mice were around 100 mg / ml, comparable to malaria antibody levels, but were also detected in some animals at around 10 mg / ml or less.

[0108] Example 3. HBV infection levels were significantly lower in animals treated with anti-HBs antibodies The mean viremia for all animals was 1E on day 49 p.i. 8 ~1E 9 Because hepadnaviruses take <24 h to establish in vivo or in vitro infection in susceptible cells, their livers were infected with >1 × 107 This suggests that all infectious cells must have been infected because they were exposed to HBV DNA copies / ml of viremia. The mean viremia in the 15 mice treated with anti-HBs antibody (anti-HBs-total) was lower than the mean viremia in the malaria antibody group before AAV injection on day 49. Viremia in the anti-HBs-total group decreased after AAV vector or anti-HBs injection and was lower than that in the malaria antibody group, demonstrating a response to anti-HBs treatment. Thereafter, viremia in both groups increased sharply and peaked. The reduced viremia in the anti-HBs-total group became detectable again after the peak, whereas viremia in the malaria group remained stable (Figure 2A).

[0109] The 15 mice in the anti-HBs group were further divided into anti-HBs-A (n=9) and anti-HBs-B (n=6) groups based on whether HBV infection reached the typical peak viremia. Viremia in the anti-HBs-A group reached the typical peak (serum HBV DNA level >1E 10 After reaching 100 copies / ml, a reduction in viremia continued and HBV infection in the anti-HBs-B group was delayed but had not yet peaked. The difference in viremia between the malaria antibody and anti-HBs-B groups was >100-fold at some time points.

[0110] Mean viremia on day 183 was significantly lower in both the anti-HBs-total and anti-HBs-B groups compared to the malaria group (p=0.029 and p=0.012, respectively, see Figure 2B). Mean serum HBsAg levels on day 183 in all three anti-HBs groups were significantly lower compared to the malaria group (Figure 2B; p=0.0009 for anti-HBs-total, p=0.0128 for anti-HBs-A, and p=0.0008 for anti-HBs-B).

[0111] Both viremia and HBsAg data suggest that blocking new rounds of infection after all infectious cells are infected can significantly reduce the level of HBV infection, in other words, the level of HBV infection is determined by the level of new rounds of infection.

[0112] Example 4. Functional cure of HBV in test animal No. 970 Baseline viremia in test animal No. 970 was 4.6 × 10 before anti-HBs treatment, similar to the high viremia in CHB patients. 8 Anti-HBs therapy reduced viremia to 1×10 8 From 1×10 4 The results showed a 4-log reduction in serum erythrocyte serum levels, which then spiked to pretreatment levels briefly and then became undetectable for 4 weeks until termination (Figure 3A).

[0113] Although serum HBsAg in animal No. 970 remained undetectable for 6 consecutive months after anti-HBs treatment, anti-HBs levels reached 60 mg / ml on day 77 p.i. and persisted at >100 mg / ml for most time points during the observation period (Fig. 3A ), representing complete and sustained anti-HBs seroconversion.

[0114] To provide a reference for the pretreatment levels of intrahepatic rcDNA and cccDNA in Figure 3B, test animal number 973, which had baseline viremia equivalent to the pretreatment levels of test animal number 970, was used as a reference plotted in Figure 3A.

[0115] Serological data suggest that a functional cure of HBV infection was achieved in test animal No. 970. This HBV cure process took approximately 6 months (from day 74 with the first anti-HBs injection to day 239 with undetectable HBV DNA).

[0116] To the best of our knowledge, test animal number 970 represents the first case of successful HBV functional cure with the tested intervention in an HBV-infected chimeric mouse model that supports robust HBV infection.

[0117] The mean intracellular rcDNA levels in the 20 liver samplings in the first and second rounds of test animal No. 970 were 0.66 and 0.18 copies / cell, respectively, which were significantly higher than those of animal 973, whose liver served as the pretreatment level control (p=1.33x10 -9 and 1.29x10 -9 The mean cccDNA levels were 1 copy per 200 cells (0.005 copies / cell) and 1 copy per 1250 cells (0.0008 / cell) in the 20 rounds of sampling in animal 970, respectively, i.e., cccDNA was 100-675 times lower compared to test animal no. 973 (p=5.27x10 -7 and 4.69x10 -7 , FIG. 3B). The results show that both rcDNA and cccDNA underwent significant loss in test animal No. 970.

[0118] The average rcDNA level in 7 of 20 samples from the first round and 20 of 20 samples from the second round was ≦1 copy / cell, and no cccDNA was detected in 3 and 8 of 20 cccDNA samples from the first and second rounds, respectively, but the cccDNA levels detected in most samples were very low, ranging from 1 copy per 77 cells to 11,100 cells (Fig. 3C1 and C2), suggesting that the vast majority of infected cells had lost cccDNA in this liver by day 253.

[0119] Anti-HBs antibodies functioned primarily extracellularly, and cccDNA elimination was the result of spontaneous loss of cccDNA. The cccDNA data from test animal No. 970 also suggest that anti-HBs monotherapy may result in complete cccDNA elimination by blocking rcDNA recruitment by sustained high levels of anti-HBs antibodies.

[0120] Example 5. Dynamic cccDNA status and spontaneous loss of cccDNA Various cccDNA levels were observed in different samplings of the same liver, for example the lowest cccDNA level was 0.5 copies / cell (sampling 17) and the highest cccDNA level was 31 copies / cell (sampling 18), a difference of 60-fold between the samplings of animal 907 treated with malaria antibodies (Figure 4A). In test animal No. 909 treated with anti-HBs antibodies, the highest level was 6.6 copies / cell and the lowest level was 0.18 copies / cell (Figure 4B), a difference of >30-fold. Various cccDNA levels in both test animals No. 909 and No. 907 indicate a dynamic cccDNA status, i.e. cccDNA was amplified in some cells while disappeared in other cells. It should be noted that the average rcDNA levels among the 20 samples from test animals No. 907 and No. 909 were 10,467 (range 6,992-14,610 copies / cell) and 3,583 copies / cell (range 2,304-11,145 copies / cell), respectively, suggesting that most of the cells sampled were HBV-infected cells.

[0121] In this experiment, it was also observed that cccDNA molecules were already lost in a proportion of infected cells.

[0122] A mean cccDNA level of ≦1 copy / cell was detected in 5 of 20 samplings in test animal No. 907 treated with malaria antibodies and in 17 of 20 samplings in test animal No. 909 treated with anti-HBs antibodies (FIGS. 4A and B). A total of 566 cccDNA samples were analyzed, and 260 of them (46%) had cccDNA levels ≦1 copy / cell (FIG. 4C). When the mean cccDNA level was ≦1 copy / cell, it suggested that cccDNA in some cells was >1 copy but not present in others, and that cccDNA had already disappeared in a portion of infected cells in HBV-infected human hepatocytes in chimeric mice. Note that all HBV-infected cells must have infected human liver cells by 253 days pi when the livers were harvested.

[0123] The proportion of liver samplings with mean cccDNA levels ≤1 copy / cell was significantly higher in the anti-HBs group than in the malaria antibody group (p<0.001, Figure 4D), suggesting that blocking new rounds of infection is likely to block de novo infection-mediated cccDNA replenishment in cells that have naturally lost cccDNA and expand the number of cells with cccDNA ≤1 copy / cell, i.e., blocking new rounds of infection establishes and expands the net cccDNA loss that can be replenished in the absence of sufficient anti-HBs antibodies.

[0124] The observed spontaneous loss of cccDNA in a fraction of infected cells is an important finding not only for understanding cccDNA biology but also for HBV curative therapy, providing evidence that cccDNA can be naturally eliminated from infected cells, which forms the basis for establishing HBV cure.

[0125] Example 6. rcDNA levels significantly reduced by blocking new rounds of infection Intrahepatic rcDNA levels from HBV-infected uPA / SCID chimeric mice were analyzed in two sets of liver samples. The first set of samples included eight livers, three of which were treated with AAV vector expressing malaria antibody as a treatment control, and the remaining five livers were treated with anti-HBs antibody, including four with AAV vector expressing anti-HBs antibody and one injected with mouse anti-HBs antibody. Seven of the eight livers were collected around day 204 pi, and one (animal no. 969) was collected on day 183. Treatment with expressed or injected anti-HBs antibody reduced viremia by 2- to 100-fold (Figure 5A). The mean rcDNA levels from 20 samples from each of the three livers treated with malaria antibodies ranged from approximately 2,000 to 2,700 copies / cell, whereas in the five livers treated with anti-HBs antibodies, an average of 219 to 454 copies / cell was detected, i.e., the rcDNA levels were significantly reduced by at least 1,500 copies / cell during the 130 days of anti-HBs treatment (see Figure 5B). P values ​​are E -23 ~E -25 The range was.

[0126] The second set of livers was collected on day 253 and included 10 livers. One liver was treated with AAV vector-expressed malaria antibody, and the other 9 livers were treated with anti-HBs antibody (7 with anti-HBs antibody expressed by AAV-anti-HBs vector, and the other 2 livers of animals no. 970 and 909 were treated with injected mouse anti-HBs antibody). The average rcDNA level from 20 samples in animal no. 907 treated with malaria antibody was 10,467 copies / cell, and the average rcDNA level was significantly lower across all 9 livers treated with anti-HBs antibody (see Figure 5C). The efficiency of reducing rcDNA levels was mainly dependent on the peak viremia level, assuming comparable virion secretion rates between different animals. For example, 1 × 10 8 ~1×10 9 The rcDNA level in animal 970 with HBV DNA copies / ml viremia was 0.66 copies / cell, 1 × 10 9 ~1×10 10The rcDNA level in animal 959, with HBV DNA copies / ml viremia, was 661 copies / cell, a reduction of nearly 10,000 copies / cell compared to animal 907. The mean rcDNA level was 1×10 10 ~1×10 11 In the remaining seven livers with viremia, the viremia was either reduced by 4,000–8,000 copies / cell or >1 × 10 9 There was a delayed peak in HBV DNA copies / ml. P values ​​were -9 ~E -16 (See FIG. 5D).

[0127] Example 7. Add-on of AAV-anti-HBs vector to 9-week entecavir (ETV) treatment In another round of experiments, HBV-infected uPA / SCID chimeric mice were divided into three groups: i) untreated; ii) ETV monotherapy; and iii) ETV with add-on of AAV-anti-HBs vector. ETV treatment was started at 6 weeks pi (post-infection) and was injected intraperitoneally at a dose of 0.3 mg / kg three times a week for 9 weeks. In the add-on group, AAV-anti-HBs vector expressing human monoclonal anti-HBs antibody was administered by intramuscular injection at a dose of 1E11 genome copies at 6 weeks pi or 11 weeks pi of ETV treatment. ETV was weaned at 14 weeks pi, and HBV infection was monitored until 30 weeks.

[0128] HBV infection in untreated mice HBV infection reached peak levels around day 81 p.i., as evidenced by viremia ranging from 3E9 to 2E10 HBV DNA copies / ml and serum HBsAg ranging from 5,000 to 10,000 IU / ml, after which serum HBV DNA and HBsAg remained at stable levels (see Fig. 6A and B).

[0129] HBV infection recurred after discontinuation of ETV in the ETV monotherapy group. Baseline viremia on day 35 (treatment initiation) ranged from 8E7 to 1E9 in this group. As expected, ETV treatment reduced viremia by 2-3 logs, but had no detectable effect on serum HBsAg levels. After ETV withdrawal, HBV DNA levels gradually recovered, eventually reaching levels comparable to untreated animals, whereas serum HBsAg levels remained stable (Figure 6A and B).

[0130] HBV recurrence was prevented in animals treated with ETV and the AAV-anti-HBs vector add-on.

[0131] Baseline viremia on day 35 ranged from 3E7 to 1E9 in this group. Serum HBV DNA levels were reduced by 2-3 logs to below 1E6 during ETV treatment. HBV recurrence in both serum HBV DNA and HBsAg levels was not observed in all 10 animals with add-on therapy throughout the observation period after ETV withdrawal (Figure 6C and D). The successful prevention of HBV recurrence by anti-HBs antibody monotherapy confirmed that HBV recurrence is mainly caused by a new round of infection.

[0132] Successful prevention of HBV recurrence reflects the lack of spread of HBV infection after ETV withdrawal. It also shows no significant reduction of HBV, i.e., HBV infection is maintained in an equilibrium state characterized by double HBsAg and anti-HBs positivity. Such a coexisting serological profile shows relatively insufficient anti-HBs levels that could not completely bind all serum HBsAg products or did not completely block new rounds of infection.

[0133] Complete anti-HBs seroconversion is required for functional cure of HBV To test whether increasing anti-HBs levels would alter the treatment outcome, eight of the ten mice received 3-week injections of 250 mg of exogenous mouse anti-HBs antibodies starting at different time points (six of them are shown in Figure 7A-F). As a result of increasing anti-HBs levels, serum HBsAg loss and complete anti-HBs seroconversion were established in all eight mice (Figure 6C). Furthermore, HBV functional cure was achieved in five of the eight mice by the end of the experiment (Figure 7A-E).

[0134] This experiment shows that different anti-HBs levels determine different outcomes: 1. Absence of anti-HBs caused HBV relapse characterized by HBsAg positivity only. 2. Suboptimal anti-HBs levels, i.e. anti-HBs levels ≦ serum HBsAg levels characterized by double positivity of HBsAg and anti-HBs, could suppress significant HBV relapse but could not completely block new rounds of infection, resulting in stable infection levels. 3. Sufficient anti-HBs levels, i.e. anti-HBs levels > serum HBsAg levels that resulted in HBsAg loss and complete anti-HBs seroconversion, could completely block new rounds of infection and lead to subsequent functional cure characterized by HBsAg- / anti-HBs+. All HBV functional cures in six mice occurred at or after complete anti-HBs seroconversion (Figure 3 and Figures 7A-7E). HBV functional cure had not yet occurred in the remaining three mice after anti-HBs seroconversion due to insufficient observation period; two died 1 or 3 weeks after anti-HBs seroconversion; and the last one, 971, had both high baseline viremia of 1E9 copies / mL and serum HBsAg level of 11,000 IU / mL and achieved anti-HBs seroconversion on day 165, but HBV DNA remained positive until termination (day 218, Figure 7F).

[0135] Example 8. Complete anti-HBs seroconversion results in a more effective HBV functional cure characterized by a higher HBV cure rate achieved in a few months compared to spontaneous clearance, which usually takes years or decades, or the rarity of HBV treatment mediated by current therapies. As shown in Figure 7, HBV functional cure was achieved in four of five HBV-infected chimeric mice with complete anti-HBs seroconversion during the 16-week observation period. HBV cure had not yet occurred in the last animal, which had the highest baseline HBsAg and HBV DNA levels and achieved complete anti-HBs seroconversion on day 162. However, this animal was followed for only 56 days and the experiment was terminated on day 218.

[0136] Such results are consistent with the observation that anti-HBs seroconversion indicates resolution of acute HBV infection. 21 Indeed, complete anti-HBs seroconversion indicates that anti-HBs levels already exceed serum HBsAg levels, which leads to completely blocking new rounds of infection and establishing progressive HBV clearance. Therefore, the main goal of HBV curative treatment should be to establish complete anti-HBs seroconversion with sufficient anti-HBs antibodies as early as possible.

[0137] HBV spontaneous cure occurs in chronic HBV infection and is mainly caused by the gradual reduction of HBsAg levels, but anti-HBs seroconversion cannot be achieved immediately and completely, and new rounds of infection continue. This is why it takes several years / decades to achieve HBV functional spontaneous cure (Figure 8C). However, complete anti-HBs seroconversion can be achieved quickly by expressing sustained and high levels of anti-HBs antibodies, which significantly shortens the HBV cure period compared with the HBV spontaneous cure course (Figure 8D).

Claims

1. A composition for treating hepatitis B virus (HBV) infection by reducing or eliminating cccDNA (covalently closed circular DNA) and / or rcDNA (relaxed circular DNA) in liver cells of human patients with chronic HBV infection for more than three months, the composition comprising a viral or non-viral vector comprising a nanoparticle, such as a lipid nanoparticle (LNP) or a GalNAc particle, the viral or non-viral vector containing a DNA or mRNA sequence encoding an HBV-neutralizing antibody or antibody fragment.

2. 2. The composition of claim 1, wherein the HBV cccDNA level in the liver cells of the HBV-infected patient is reduced to <1 copy / cell, <1 copy / 10 cells, <1 copy / 100 cells, <1 copy / 1,000 cells, <1 copy / 10,000 cells, or undetectable.

3. 3. The composition of claim 1 or 2, wherein the level of rcDNA in the liver of the infected patient is reduced to undetectable <1 copy / cell, <1 copy / 10 cells, <1 copy / 100 cells, <1 copy / 1,000 cells, <1 copy / 10,000 cells.

4. The composition according to any one of claims 1 to 3, wherein the HBV cccDNA and rcDNA levels in the liver cells of the HBV-infected patient are sustainably reduced by administering exogenous anti-HBs antibodies and / or viral or non-viral vectors or nanoparticles that endogenously express anti-HBs antibodies to maintain high levels of anti-HBs antibodies in the blood.

5. 5. The composition of claim 4, wherein the reduction or elimination of cccDNA in liver cells of the HBV-infected patient is manifested by maintaining high levels of HBV-neutralizing antibodies in the patient's blood, thereby reducing or rendering undetectable HBV DNA, HBeAg, and HBsAg levels in the patient's blood.

6. 6. The composition of claim 5, wherein said maintaining a high level of HBV neutralizing antibodies in the patient's blood is represented by complete and sustained anti-HBs seroconversion achieved by endogenously expressing or exogenously infusing sufficient amounts of anti-HBs antibodies to sustainably convert serum HBsAg positive and anti-HBs negative (HBsAg+ / anti-HBs-) to serum HBsAg negative and anti-HBs positive (HBsAg- / anti-HBs+).

7. The composition of claim 6, wherein achieving complete and sustained anti-HBs seroconversion with sufficient anti-HBs antibodies is one of the most effective ways to induce serum HBsAg loss or serum clearance.

8. The composition of claim 6, wherein complete and sustained anti-HBs seroconversion is the primary goal of HBV curative therapy and is required to result in a more effective functional cure of HBV.

9. The composition of claim 8, wherein producing a more effective HBV functional cure requires first removing serum HBsAg or rendering serum HBsAg undetectable by developing or administering sustained high levels of anti-HBs antibodies.

10. 9. The composition of claim 8, wherein complete and sustained anti-HBs seroconversion is required to establish sustained HBsAg serum clearance or functional cure among HBV drug-treated patients who have transiently cleared serum HBsAg or achieved functional cure with HBV drugs such as nucleotide analogs, interferon, and / or NAP or RNAi.

11. 6. The composition of claim 5, wherein high levels of HBV neutralizing antibodies in the patient's blood are maintained for 1, 2, 3, 4, 5, 6, 7, or 8 months or more.

12. The composition of claim 10, wherein the HBV neutralizing antibody is an antibody that can prevent viral particles from attaching to hepatocytes, and thus can effectively block HBV virions from entering hepatocytes.

13. 12. The composition of claim 11, wherein the high level of HBV neutralizing antibodies is at a level of >1, >10, >100, >200, or >300 μg / ml, or >10, >100, >1,000, >10,000, >20,000, or >30,000 mIU / ml in the patient's blood.

14. 13. The composition of claim 12, wherein the HBV neutralizing antibodies are administered to the patient by injecting one, two, or three exogenous human HBV neutralizing antibodies at a dose of about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, or ≧30 mg / kg body weight.

15. 14. The composition of claim 13, comprising single, multiple, or repeated infusions of exogenous human neutralizing antibody to maintain a level of antibody in the patient's blood of >1, >10, >100, >200, or >300 μg / ml, or >10, >100, >1,000, >10,000, >20,000, or >30,000 mIU / ml for three months or more.

16. 15. The composition of claim 14, wherein the HBV neutralizing antibodies are one, two, or three endogenously expressed human neutralizing antibodies.

17. The composition of claim 1 , wherein the viral vector comprises an adeno-associated viral (AAV) vector.

18. The composition of claim 17, wherein the AAV vector comprises a nucleic acid sequence encoding the HBV-neutralizing antibody or antibody fragment thereof, thus becoming an AAV-anti-HBV vector.

19. The AAV-anti-HBV vectors were each about 1 x 10 ９ Genome copies / kg, 1 x 10 １０ Genome copies / kg, 1 x 10 １１ Genome copies / kg, 1 x 10 １２ Genome copies / kg, 2 x 10 １２ Genome copies / kg, 2.5 x 10 １２ 19. The composition of claim 18, which is injected into muscle cells of an HBV patient at a dose of genome copies / kg or more.

20. 10. The composition of claim 1, wherein the viral or non-viral vector is injected into the patient one or more times as needed.

21. 10. The composition of claim 1, wherein depleting rcDNA through the combination of an HBV neutralizing antibody with an intracellular HBV replication inhibitor results in significant, near-complete, or complete cccDNA loss.

22. 22. The composition of claim 21, wherein the intracellular HBV inhibitor comprises an RT inhibitor, a capsid inhibitor, an RNAi drug, a nucleic acid polymer (NAP), an interferon, an innate immune agonist, and an entry inhibitor.

23. 19. The composition of claim 18, wherein the AAV anti-HBV vector expressing HBV neutralizing antibodies is used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and more effectively cure HBV in HBV treatment-naive chronically HBV-infected individuals.

24. 24. The composition of claim 23, wherein the AAV anti-HBV vector expressing HBV neutralizing antibodies is used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and more effectively cure HBV in chronically HBV-infected individuals who are undergoing HBV treatment but who cannot safely wean off the antiviral drug.

25. 24. The composition of claim 23, wherein the AAV anti-HBV vector expressing HBV neutralizing antibodies is used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure in chronically HBV-infected individuals who wish to become HBV infection-free, regardless of their infection status / stage or their treatment status (naive or treated).

26. 24. The composition of claim 23, wherein the AAV anti-HBV vector expressing HBV neutralizing antibodies is used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure in HBV-infected pregnant women or patients with liver transplants.

27. 24. The composition of claim 23, wherein the AAV anti-HBV vector expressing HBV neutralizing antibodies is used to block cccDNA replenishment to reduce and eliminate cccDNA, induce HBsAg serum clearance, achieve complete anti-HBs seroconversion, and for more effective HBV cure in people whose HBV infection has clinically resolved but who are at high risk of HBV recurrence or reactivation.

28. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 2, and an antibody VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 4, or a variant thereof having at least 95% sequence homology.

29. 29. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 28.

30. A vector comprising the nucleic acid molecule of claim 29.

31. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 6, and an antibody VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 95% sequence homology.

32. 32. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 31.

33. A vector comprising the nucleic acid molecule of claim 32.

34. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 10, and an antibody VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 12, or a variant thereof having at least 95% sequence homology.

35. 35. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 34.

36. A vector comprising the nucleic acid molecule of claim 35.

37. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 14, and an antibody VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least 95% sequence homology.

38. 38. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 37.

39. A vector comprising the nucleic acid molecule of claim 38.

40. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH comprising the amino acid sequence of SEQ ID NO: 18, and an antibody VL comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 95% sequence homology.

41. 41. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 40.

42. A vector comprising the nucleic acid molecule of claim 41.

43. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH comprising the amino acid sequence of SEQ ID NO: 22, and an antibody VL comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least 95% sequence homology.

44. 44. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 43.

45. A vector comprising the nucleic acid molecule of claim 44.

46. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 26, and an antibody VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least 95% sequence homology.

47. 47. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 46.

48. A vector comprising the nucleic acid molecule of claim 47.

49. 1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH comprising the amino acid sequence of SEQ ID NO: 30, and an antibody VL comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least 95% sequence homology.

50. 50. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 49.

51. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to HBV virions and / or HBsAg subviral particles, comprising an antibody VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 34, and an antibody VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least 95% sequence homology.

52. 52. A nucleic acid molecule encoding the isolated binding molecule or antigen-binding fragment of claim 51.

53. A vector comprising the nucleic acid molecule of claim 52.

54. 52. A composition comprising the isolated binding molecule or antigen-binding fragment of any one of claims 28, 31, 34, 38, 40, 43, 46, 49, and 51.

55. 55. A pharmaceutical composition comprising the composition of claim 54 and a pharmaceutically acceptable carrier.

56. A composition for treating chronic HBV infection in a human patient and providing protection against new rounds of HBV infection, said treatment comprising administering to said HBV-infected human patient a sufficient amount of an HBV neutralizing antibody or antibody fragment, said amount of HBV neutralizing antibody being at a level that blocks the recruitment of both rcDNA and cccDNA, enables the reduction and depletion of rcDNA to <1 copy / cell, promotes the reduction and elimination of cccDNA to <1 copy / cell, induces HBsAg serum clearance, achieves complete anti-HBs seroconversion, and results in a more effective HBV cure, and said composition comprises a viral or non-viral vector containing a DNA or mRNA sequence encoding the HBV neutralizing antibody or antibody fragment, or the HBV neutralizing antibody or antibody fragment encoded thereby.

57. 57. The composition of claim 56, wherein said treating chronic HBV infection comprises treating newborns / children infected with HBV.

58. 57. The composition of claim 56, wherein said treating chronic HBV infection comprises treating an adult infected with HBV.

59. The composition described in claim 56, wherein the HBV-infected human patient is a chronically HBV-infected individual who has been HBsAg positive for more than 6 months and has normal or elevated alanine aminotransferase (ALT) levels.

60. The composition described in claim 56, wherein the HBV-infected human patient 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 and is susceptible to recurrence of HBV infection after transplantation.

61. 57. The composition of claim 56, wherein the HBV neutralizing antibody or antibody fragment is produced by an HBV therapeutic vector.

62. The composition described in claim 56, wherein the HBV therapeutic vector comprises a mixed population of vectors, each of which encodes one specific anti-HBs antibody or antibody fragment that binds to one or more epitopes of an HBV envelope protein, or a single vector encoding one HBV neutralizing antibody or antibody fragment that binds to one or more epitopes of an HBV envelope protein.

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