Compositions and methods related to human neutralizing antibodies against hepatitis B

Abstract

Broadly neutralizing antibodies (bNAbs) and antigen-binding fragments thereof that specifically bind to epitopes expressed by hepatitis B virus (HBV) are provided. bNAbs target non-overlapping epitopes on HBV S antigen (HBsAg). Pharmaceutical compositions containing bNAbs or modified bNAbs are provided. Combinations of bNAbs are included, which are useful for preventing and treating HBV infection and for suppressing the occurrence of HBV escape mutations in infected individuals. Expression vectors encoding bNAbs and their antigenic fragments, and methods for producing bNAbs and their antigenic fragments are included. HBV peptides for use as vaccines are provided, which contain at least two non-overlapping epitopes derived from HBsAg. Diagnostic reagents containing bNAbs or antigenic fragments thereof are provided, as are methods for detecting HBV and diagnosing HBV infection.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 898,735, filed September 11, 2019, and U.S. Provisional Patent Application No. 62 / 982,276, filed February 27, 2020, the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. UL1TR001866 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on September 9, 2020, is named 076091_00092_SL.txt and is 307,317 bytes in size. [Background technology]

[0004] Despite the existence of an effective vaccine, hepatitis B virus (HBV) infection remains a major global health problem, with an estimated 257 million people living with infection. Approximately 95% of adults and 50-75% of children (aged 1-5 years) naturally control HBV, and only 10% of infants and young children recover naturally. The remainder develop chronic infection that can lead to cirrhosis and hepatocellular carcinoma. Chronic infection can be suppressed with antiviral drugs, but no effective curative treatment exists (Dienstag, 2008; Revill et al., 2016; Thomas, 2019).

[0005] HBV is an enveloped double-stranded DNA virus of the hepadnavirus family. Its genome is the smallest of any pathogenic human DNA virus with only four open reading frames. Infected hepatocytes produce both infectious HBV virions (Dane particles) and non-infectious subviral particles (Australia antigen) (Dane et al., 1970; Hu and Liu, 2017). Virions are particles with a diameter of 42 nm and contain the viral genome and HBV core antigen (HBcAg), which are encapsidated by a lipid membrane that contains the hepatitis B surface antigen (HBsAg) (Blumberg, 1964; Venkatakrishnan and Zlotnick, 2016). Subviral particles lack the viral genome.

[0006] HBV strains were originally grouped into four HBsAg serotypes (adr, adw, ayw, ayr). Genetic analysis revealed several highly conserved domains and defined eight genotypes AH, which are highly correlated with the four serotypes (Norder et al., 2004). The HBV surface protein, HBsAg, has four putative transmembrane domains and can be subdivided into PreSl-, PreS2-, and S-domains. The S-domain is a cysteine-rich protein of 226 amino acids that contains all four transmembrane domains (Abou-Jaoude and Sureau, 2007). The S-protein can also be glycosylated at asparagine residue 146 (Julithe et al., 2014).

[0007] Antibodies to HBsAg (anti-HBs) are associated with successful vaccination and recovery from acute infection, whereas antibodies to HBcAg (anti-HBc) indicate past or current HBV infection (Ganem, 1982). Indeed, the most striking difference between chronically infected and naturally recovered individuals is the robust antibody response to HBsAg (Ganem, 1982). Conversely, the inability to produce these antibodies during acute infection is associated with chronicity (Trepo et al., 2014). Whether these associations reflect a pathogenic role played by anti-HBs antibodies in protection from or clearance of established infection is not known. However, depletion of antibody-producing B lymphocytes in exposed humans by anti-CD20 therapy (e.g., rituximab) is associated with HBV reactivation, indicating that B cells and / or their antibody products play a key role in controlling infection (Loomba and Liang, 2017).

[0008] Several human antibodies against HBsAg have been obtained using various methods, including phage display (Kim and Park, 2002; Li et al., 2017; Sankhyan et al., 2016; Wang et al., 2016); humanized mice (Eren et al., 1998); Epstein-Barr virus-induced B cell transformation (Heijtink et al., 2002; Heijtink et al., 1995; Sa'adu et al., 1992); hybridoma technology (Colucci et al., 1986); human B cell culture (Cerino et al., 2015); and microwell array chips (Jin et al., 2009; Tajiri et al., 2010). However, donors in these studies were not selected for serum neutralizing activity. Thus, there is a need for improved approaches and compositions to combat HBV infection. This disclosure addresses this need. Summary of the Invention

[0009] This disclosure provides, in part, a description of the human humoral immune response to HBsAg in immunized and naturally recovered individuals (individuals selected for high levels of serum neutralizing activity). This disclosure demonstrates that these individuals develop multiple closely related bNAbs that target shared non-overlapping epitopes in HBsAg. The crystal structure of one of the antibodies with its peptide target reveals a loop that helps explain why certain amino acid residues are frequently mutated in escape viruses and why combinations of bNAbs are required to control infection. In vivo experiments in humanized mice demonstrate that bNAbs are protective and may be a cure when used in combination.

[0010] Any antibody described herein may include at least one modification of its constant region. The modification may be made to any one or more amino acids. The modification may have any of several desired effects. In certain approaches, the modification increases the in vivo half-life of the antibody, or alters the ability of the antibody to bind to an Fc receptor, or alters the ability of the antibody to cross the placenta, or cross the blood-brain barrier, or cross the blood-testis barrier, or inhibits aggregation of the antibody, or a combination of the above modifications, and wherein the antibody is attached to a label or substrate. In some embodiments, the modification improves the manufacturability of the antibody. In some embodiments, any antibody described herein or combinations thereof may be present in an immunological assay, such as an enzyme-linked immunosorbent assay (ELISA assay), or an ELISA assay control. The ELISA assay may be a direct ELISA assay, an indirect ELISA assay, a sandwich ELISA assay, or a competitive ELISA assay.

[0011] In another aspect, the present disclosure provides a method for preventing or treating hepatitis virus infection, comprising administering an effective amount of at least one antibody or antigen-binding fragment thereof described herein to an individual in need thereof. The antibody may comprise at least one modification of the constant region. In embodiments, the composition is administered to an individual infected with or at risk of being infected with Hepatitis B virus. In one approach, at least two antibodies are administered, optionally wherein the two antibodies recognize distinct HBV epitopes. In one embodiment, administering at least two distinct antibodies inhibits the formation of viruses resistant to the antibodies.

[0012] In another aspect, the present disclosure provides a vaccine formulation. In one embodiment, the vaccine formulation comprises an isolated or recombinant peptide, or a polynucleotide encoding said peptide, wherein said peptide is derived from an epitope frequently targeted by HepB immune resistance, which is located in a loop anchored by oppositely charged residues (as further described herein).

[0013] In another aspect, the disclosure provides one or more recombinant expression vectors, and kits comprising the expression vectors. The expression vectors encode at least the heavy and light chain CDRs of any of the antibodies described herein. Cells comprising the recombinant expression vectors are also included, as are methods of producing antibodies by culturing cells comprising the expression vector that express the antibody and isolating the antibody from the cells. Also included are cell culture media comprising such cells and / or antibodies. [Brief description of the drawings]

[0014] [Figure 1] Antibody responses in HBV vaccinated and convalescent individuals. (A) Donor screen. Sera from 159 volunteers were assessed for anti-HBs binding by ELISA (x-axis) and for HBV serum neutralization potency (y-axis) using HepG2-NTCP cells. Serum neutralization potency on the y-axis was calculated as the reciprocal of the relative percentage of infected HepG2-NTCP cells. Values ​​for unexposed naïve donors are approximately equal to 1. Neutralization tests were performed at a serum dilution of 1:5 in the final assay volume. Each dot represents an individual donor. Green indicates unvaccinated / unexposed, black indicates vaccinated, and red indicates spontaneous recovery. The dashed line indicates serum-free control. Top neutralizers (serum neutralization potency higher than 4) are indicated (top right). Boxes are representative samples shown in Figure 2A. Spearman's rank correlation coefficient (r s ) and significance value (p). (B and C) Dose-dependent HBV neutralization by serum (B) or by purified IgG (C). Percent infection was measured using two assays: HBsAg protein in the medium by ELISA (top panel) and immunofluorescence staining of HBcAg in HepG2-NTCP (bottom panel). The dashed line indicates the virus-only control. (D) Schematic diagram showing the three forms of HBV surface proteins: L-, M-, and S-proteins. All three forms of the envelope protein share the same S region, with PreS1 / PreS2 and PreS2 alone being N-terminal extensions for the L- and M-proteins, respectively. (E) S-protein produced in Chinese Hamster Ovary (CHO) cells inhibits serum neutralizing activity. The graph shows the infection efficiency as a function of the amount of S-protein added. Concentrations of polyclonal IgG antibodies (pAb) are shown. Top and bottom panels are as in (B) and (C). A representative of at least two experiments is shown. See also Figure 8 and Table S1.

[0015] [Figure 2] S-protein-specific antibodies. (A) Frequency of S-protein-specific memory B cells. Representative flow cytometry plots show total IgG binding to both allophycocyanin- and phycoerythrin-labeled S-proteins (S-protein-APC and S-protein-PE). + The percentage of memory B cells is shown. Flow cytometry plots from other individuals are shown in Figure 9A. The experiment was repeated twice. (B) S-protein-bound IgG + Dot plot showing the correlation between memory B cell frequency and serum neutralizing activity. Spearman's rank correlation coefficient (r s ) and significance value (p). (C) Each pie chart represents antibodies from an individual donor, with the total number of sequenced antibodies with paired heavy and light chains shown in the center of the circle. Antibodies with the same combination of IGH and IGL variable gene sequences and closely related CDR3s in each individual are shown. Areas of the same color indicate shared antibodies with the same or similar combination of IGH and IGL variable genes between individuals (Figure 9B). Areas of grey indicate antibodies with closely related sequences that are unique to a single donor. White is singlets. (D) V(D)J alignment of representative IGHV3-30 / IGLV3-21, IGHV3-33 / IGLV3-21, and IGHV3-23 / IGLV3-21 antibodies from donors #60 / #146 (H006 and H008), #146 / #13 (H014 and H012), and #13 / #60 / #146 (H021, H003, and H004), respectively. Boxed grey residues are shared between the antibodies. See also FIG. 9 and Table S2. The figure discloses SEQ ID NOs: 1438-1451, respectively, in order of appearance.

[0016] [Figure 3] Broad cross-reactivity (A) Binding to S-protein (adr serotype). The 50% effective concentration (EC) required for binding of the indicated human monoclonal antibodies to S-protein. 50(ng / ml)). Ribivirumab (Eren et al., 2000; Eren et al., 1998) and the anti-HIV antibody 10-1074 (Mouquet et al., 2012) were used as positive and negative controls, respectively. All antibodies were tested. (B) Comparison of binding of the mature and unmutated common ancestor (UCA) of antibodies H006, H019, and H020 to S-protein (by ELISA). (C) Anti-HBs antibody binding to five different serotypes of HBsAg. As in panel (A), EC 50 Values ​​are color coded: red, ≦50 ng / ml; orange, 50-100 ng / ml; yellow, 100-200 ng / ml; white, >200 ng / ml. The abbreviation bd indicates below the limit of detection. All antibodies were tested. All experiments were performed at least in duplicate. See also Figure 10.

[0017] [Figure 4] HBsAg epitope (A) Competitive ELISA defines three groups of antibodies. Competitive ELISA results are shown as % binding by biotinylated antibodies and are interpreted by color: black, 0-25%; dark gray, 26-50%; light gray, 51-75%; white, >76%. Weak binders (H002, H012, H013, H014, H01) were excluded. Representative of two experiments. (B) ELISA results for alanine scanning mutants of the S-protein. Only amino acids essential for antibody binding are shown. Binding to mutants compared to wild-type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >75%. Further details are shown in FIG. 11. (C) ELISA results for human escape mutations of S-protein. Wild-type S-protein and empty vector served as positive and negative controls, respectively. Binding to mutants compared to wild-type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >75%. Amino acid mutations in bold represent frequently observed mutations in humans (Ma and Wang, 2012). Antibodies tested in (B and C) were selected from groups-I, -II, -III based on their neutralizing activity (Figures 5A-5C). All experiments were performed at least twice. See also Figure 11.

[0018] [Figure 5] In vitro neutralization by monoclonal antibodies (A and B) In vitro neutralization assays using HepG2-NTCP cells. Infection rates in the presence of the indicated concentrations of bNAbs; measured by ELISA; HBsAg (A) and anti-HBcAg immunofluorescence (B) in the medium. Anti-HIV antibodies 10-1074 (Mouquet et al., 2012) and ribivirumab (Eren et al., 2000; Eren et al., 1998) were used as negative and positive controls, respectively. Corresponding IC 50 are shown in the left and center columns of panel (C). All experiments were repeated at least twice. (C) Calculated bNAb 50% maximal inhibitory concentrations (IC50) based on HBsAg ELISA (left column), HBcAg immunofluorescence for in vitro neutralization assay with HepG2-NTCP cells (middle column), or HBeAg ELISA for in vitro neutralization with primary human hepatocytes (right column), where the abbreviations bd (below detection) and nd (not done) indicate below the detection limit and experiment not performed, respectively. (D) In ​​vitro neutralization using primary human hepatocytes. Levels of HBeAg in the culture medium were measured by ELISA. Calculated IC 50 Values ​​are shown in the right column of the panel (C). The experiment was repeated three times. (E) In vitro neutralization assay using HepG2-NTCP cells. IgG antibodies were compared with the corresponding Fab fragments. The concentration of the Fab fragments was adjusted to correspond to IgG. Experiments were performed in duplicate. See also Figure 12.

[0019] [Figure 6] Crystal structure of H015 bound to the recognition motif. A single crystal was used to obtain a high-resolution (1.78 Å) structure. (A) Synthetic peptides spanning the antigen loop regions (SEQ ID NOs: 1452-1455, respectively, in order of appearance) were subjected to ELISA for antibody binding. Among the antibodies tested, only H015 binds peptides -11 and -12. The experiment was performed in triplicate and details are shown in FIG. 13. (B and C) The peptide binds to CDR1 (R31), CDR2 (W52 and F53), and CDR3 (E99, P101, L103, and L104) of the H015 heavy chain (green) and CDR3 (P95) of the light chain (cyan) (B). The interacting residues (C) of the heavy chain (green) are R31 (main chain), W52, F53 (main chain), E99, P101 (main chain), L103 (main chain), L104 (hydrophobic). Making contact with the light chain (cyan) is P95. (D) Electron density map of the bound peptide, as seen in the 2Fo-Fc map, is contoured with 1 RMSD, suggesting high occupancy (92%). (E) The recognition motif, KPSDGN (SEQ ID NO: 1), adopts a sharp hairpin structure due to a salt bridge between K141 and D144, facilitated by kinks at P142 and G145. Glycine 145 (G145, circled) is a residue that escapes from the immune system when mutated to arginine. See also FIG. 13.

[0020] [Figure 7] Anti-HBs bNAbs exhibit protective and therapeutic effects in vivo. (A and E) Diagram of the prophylactic and therapeutic protocols, respectively. (B) Protection with isotype control antibody 10-1074 ( Mouquet et al., 2012 ). (C and D) Protection by H020 and H007, respectively. The dashed line in (BD) indicates the limit of detection. (F) Treatment of viremic huFNRG mice with control antibody 10-1074. (G and H) Treatment of viremic huFNRG mice with H020 alone or H007 alone, respectively. Serum HBV DNA levels were monitored on a weekly basis. A combination of two independent experiments consisting of a total of 5-8 mice is shown. (I) Mutations in the S-protein sequence from mouse (red arrows) indicated in (G), (H) and (J). The S-protein sequence chromatogram is shown in FIG. (JL) Treatment of viremic huFNRG mice with combinations of anti-HBs bNAbs H006+H007 (J), or H017+H019 (K), or H016+H017+H019 (L), respectively. Sequencing showed that none of the mice in (K) and (J) harbored virus with an escape mutation in the S-protein. See also Figure 14.

[0021] [Figure 8] Characterization of antibody immune responses to HBV (related to Figure 1) (A) Schematic diagram of different stages of HBV infection. Vaccinated individuals or those infected but recovered naturally were recruited for this study. (B) Sera (1:50 dilution in final assay volume) from 159 volunteers were screened (see also Fig. 1A ). (C-E) Comparison of anti-HBs ELISA titers (upper panel) and their serum neutralizing activity (lower panel) among different individual groups. Vaccinated or recovered individuals show statistically higher anti-HBs titers (upper panel, C) and stronger neutralizing activity (lower panel, C) than uninfected / unvaccinated individuals. Younger individuals (<45 years) showed a slightly higher antibody immune response to HBsAg (D). No differences were observed between the sexes (E).

[0022] [Figure 9] Anti-HBs antibody cloning and sequence analysis (related to Figure 2) (A) Frequency of S-protein-specific memory B cells in peripheral blood mononuclear cells from all 12 donors. Details are the same as in Fig. 2A. (B) Pie chart shows the distribution of anti-HBs antibodies. Figure legend is the same as in Fig. 2C. For each section, the VH and VL genes are shown and 20 selected anti-HBs antibodies are labeled. (C) Phylogenetic tree of all cloned anti-HBs antibodies based on IGH Fab regions. The IGH Fab regions from 244 HBsAg-selected memory B cells were aligned into a tree.

[0023] [Figure 10] Autoreactivity of 20 types of anti-HBs antibodies (related to Figure 3) (A) Autoreactivity of monoclonal antibodies. The positive control antibody efficiently stains the nuclei of HEp-2 cells. Twenty anti-HBs antibodies were also tested, as well as the anti-HBs antibody ribivirumab and the anti-HIV antibody 10-1074. (B) Polyreactivity profile of 20 anti-HBs antibodies. The ELISA measures antibodies that bind to the following antigens: double-stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), and single-stranded DNA (ssDNA). The red and green lines represent the positive control antibody ED38 and the negative control antibody mGO53, respectively, and the dashed line indicates the cutoff value for positive reactivity (Gitlin et al., 2016).

[0024] [Figure 11] Alanine scanning and peptide screening (related to Figure 4) (A) ELISA results for alanine scanning mutants of HBsAg. Binding to mutants was normalized to wild type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%, white, >76%. Experiments were performed in triplicate. Underlined cysteines, alanines, and amino acids known to be important for S-protein production were not mutated (Salisse and Sureau, 2009). The figure discloses SEQ ID NO: 1456. (B) Schematic representation of the alanine scanning results. The figure discloses the primary amino acid sequence as SEQ ID NO:1456 and the sequence containing the alanine mutations as SEQ ID NO:1457.

[0025] [Figure 12] In vitro neutralization assay of unmutated common ancestor antibodies or combinations of anti-HBs bNAbs (related to Figure 5) (AB) In vitro neutralization assays of anti-HBs bNAbs and their corresponding unmutated common ancestor (UCA) antibodies. Relative infection rates were calculated based on either HBsAg protein levels in the medium (A) or intracellular HBcAg staining (B). (C) In vitro neutralization assay of anti-HBs bNAbs recognizing different epitopes and combinations of the same total amount of antibody (1:1 or 1:1:1 ratio).

[0026] [Figure 13] Detailed information on the crystal structure of H015 and its linear epitope (related to Figure 6) (A) Synthetic peptides for antigen loop regions (SEQ ID NOs: 1458-1476, respectively, in order of appearance) were subjected to ELISA for antibody binding. Of the antibodies tested, only H015 binds to peptides -11 and -12. (B) Data collection and refinement statistics for H015 Fab are summarized. Statistics for the highest resolution shell are shown in brackets. Refinement program PHENIX 1.16 (C) Green / red congestion is an unbiased omission map. Red is negative congestion, which is equivalent to noise. (D) Table of intrapeptide contacts and contacts between Fab fragments and peptides.

[0027] [Figure 14] HBV DNA levels and S-protein sequences in antibody-treated huFNRG mice (related to Figure 7) (A) HBV DNA levels in representative individual huFNRG mice treated with control antibody 10-1074, anti-HBs bNAb H020, anti-HBs bNAb H007, anti-HBs bNAb combinations (H006+H007), (H017+H019), and (HO16+HO17+HO 19). HBV DNA levels in mouse serum were monitored on a weekly basis. Mice without arrows do not have escape mutations at the final time point. (B) A portion of the S-protein sequence from the indicated mice (arrows and numbers) is shown below as a chromatogram (with mutations indicated by arrowheads). (B) discloses the amino acid and nucleotide sequences of the S-protein as SEQ ID NOs: 1477 and 1478, respectively. The sequences represented by the subsequent chromatograms disclosing the amino acid residues and nucleotides are, in column order, SEQ ID NOs: 1479, 1480, 1480, 1480-1482, 1479, 1478, 1480, 1480, 1478, 1480, and 1483-1488, respectively. (C-D) HBsAg levels in mouse serum before and after antibody injection. Mice were treated with the anti-HBs combinations H017+H019 (C) (see FIG. 7K) and H016+H017+H019 (D) (see FIG. 7L). Each line represents an individual mouse with the concentration of serum HBsAg levels expressed in NCU / ml (national clinical units per milliliter). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] Every numerical range described herein includes its upper and lower limits, and every narrower numerical range subsumed within that range, as if such narrower numerical ranges were all expressly written herein.

[0030] The present disclosure includes all nucleotide sequences described herein, in the tables and figures, and all sequences complementary thereto, the RNA equivalents of DNA sequences, all amino acid sequences described herein, and all polynucleotide sequences encoding said amino acid sequences. All antibody sequences and functional fragments thereof are included. Polynucleotide and amino acid sequences having 80%-99% similarity (inclusive and including the numerical ranges therebetween) with the sequences provided herein are included in the present invention. All amino acid sequences described herein may include amino acid substitutions (such as conservative substitutions) that do not adversely affect the function of the protein or polypeptide that contains said amino acid sequence. It will be recognized that an "antibody" as referred to herein does not necessarily mean a single antibody molecule. For example, "administering an antibody" includes administering multiple identical antibodies. Similarly, a composition that includes an "antibody" may include multiple identical antibodies.

[0031] The amino acid and polynucleotide sequences of the present disclosure include contiguous segments of sequences, which can range from two amino acids to the full length protein sequence, and also include polynucleotide sequences that encode such segments.

[0032] The present disclosure includes DNA and RNA sequences encoding the antibodies and antigenic fragments thereof, as well as any viral peptides, described herein for use as protein or DNA and / or RNA vaccines in prophylactic and therapeutic approaches, which may be formulated and / or delivered according to known approaches, with the benefit of the present disclosure. The present disclosure includes cDNA sequences encoding the antibodies, antigen-binding fragments thereof, and any viral proteins or peptides, as described herein. Also included are expression vectors containing cDNA, encoding said antibodies, antigen-binding fragments thereof, and viral proteins and peptides.

[0033] All sequences in the figures, text, and tables of this application or patent include every amino acid sequence associated with every donor ID, and every possible combination of amino acid sequences for every complementarity determining region (CDR) (e.g., every combination of heavy chain CDR1, CDR2, and CDR3 sequences, and every combination of light chain CDR1, CDR2, and CDR3 sequences, and also heavy chain sequences, and light chain sequences that are either lambda or kappa light chain sequences). The disclosure includes all combinations of antibodies described herein. Also, one or more antibodies may be excluded from any combination of antibodies.

[0034] The present disclosure includes the antibodies described herein in an in vitro complex with one or more Hepatitis B proteins.

[0035] In embodiments, the present disclosure provides isolated or recombinant antibodies that specifically bind to a Hepatitis B virus epitope, wherein the antibody may contain amino acid sequence modifications, including, but not limited to, constant region modifications.

[0036] In embodiments, one or more of the antibodies described herein specifically bind to an epitope present in the native or mutated HBsAg protein or S-protein.

[0037] In embodiments, the antibodies described herein bind to a Hepatitis B protein that contains one or more HepB escape mutations. In embodiments, the antibodies bind to a Hepatitis B virus protein that contains a mutation in which a small neutral residue is replaced with a large positively charged residue. In embodiments, the mutation is in the so-called "a" determinant region, known in the art. In embodiments, the epitope is in a major hydrophilic region of the HBsAg protein. In embodiments, the epitope to which the antibody binds is in the S-protein (including, but not necessarily limited to, the predicted or actual extracellular domain of the S-protein).

[0038] In embodiments, the epitope to which the described antibodies bind is common to the HBsAg L-protein, M-protein, or S-protein. In embodiments, the antibody binds to an epitope present in the L-protein version of HBsAg, which comprises an amino acid sequence accessible via accession number AAL66340.1, which amino acid sequence is present in a database at the filing date of this application or patent. In one embodiment, the amino acid sequence is as follows: MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPVAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPNMESTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTSPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPCLYNILSPFLPLLPIFFCLWVYI (SEQ ID NO: 2)

[0039] In embodiments, the disclosure includes using only two proteins or using at least two proteins. In embodiments, the S-protein may be used as a bait to select B cells purified from Chinese Hamster Ovary (CHO) cells, or any other suitable cell type, including but not limited to human cell cultures. In embodiments, the S-protein comprises or consists of the amino acid sequence available under Uniprot ID_P30019, which is included herein as it exists in the database as of the filing date of this application or patent. In one embodiment, the S-protein comprises the following sequence; MENTASGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYHGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI (SEQ ID NO: 3)

[0040] In a non-limiting embodiment, the S polynucleotide sequence used for alanine scanning comprises the following sequence: ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCTCCCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAAC CTCCAATCACTCACCAACCTCCTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAA CAACAACCAGTACGGGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTC TCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCGTGAGTCCCTTTATACCGCTGTTACCAATTTTCTTTTGTCTCTGGGTATACATTTAA (Sequence number 4)

[0041] The amino acid sequence encoded by the DNA sequence above is: MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI (SEQ ID NO:5)

[0042] In embodiments, the antibodies of the present disclosure bind to an epitope present in any of the above amino acid sequences (including linear and conformational epitopes that may be formed by proteins that include or consist of the above sequences).

[0043] In one embodiment, the isolated or recombinant antibody or antigen-binding fragment thereof specifically binds to an epitope constituted by a structurally defined peptide loop, as further described herein. In some embodiments, the loop comprises a partial structure of the HepB surface protein, as generally shown in FIG. 6, demonstrating the presence of a loop that includes the most frequently targeted residue (G145) found in human escape. Without intending to be bound by any particular theory, it is believed that this structure explains why this mutant is able to escape and also explains why there are additional escape mutants that are common. Furthermore, this structure and the antibody-peptide complex represent a novel and previously undiscovered target for drug discovery. Thus, in some embodiments, the present disclosure provides for screening drug candidates that can disrupt the formation of this structure and therefore also disrupt the viability of the virus. Those skilled in the art will recognize from the present disclosure how to design an assay to determine whether a drug candidate disrupts the complex and how the antibodies described herein can be used in such an assay.

[0044] In some embodiments, the antibodies described herein specifically bind to an amino acid sequence contained in any of the peptide sequences described herein. In some embodiments, the peptide comprises the sequence KPSDG (SEQ ID NO: 6) or a variant thereof. In some embodiments, the antibodies described herein specifically bind to an epitope in an amino acid sequence comprising the sequence PSSSSTKPSDGNSTS (SEQ ID NO: 7) or a variant thereof. Further non-limiting examples of peptides of the present disclosure include those depicted in FIG. 6, e.g., peptide-11 and peptide-12.

[0045] In embodiments, the disclosure includes compositions and methods involving the use of two or more distinct antibodies or antigen-binding fragments thereof. In embodiments, the methods of the disclosure include administering a combination of antibodies or antigen-binding fragments thereof that bind distinct Hepatitis B epitopes. In embodiments, the distinct antibodies recognize epitopes present at two dominant non-overlapping antigenic sites on HBsAg or epitopes present on the S-protein. In embodiments, the disclosure provides for the use of a combination of Group-I and Group-II antibodies as described herein. Thus, the disclosure includes the simultaneous or sequential administration of a combination of antibodies. In one embodiment, the combined administration of the distinct antibodies suppresses the formation of viruses that are resistant to the effects of any one antibody alone. In embodiments, the disclosure includes administering a combination including at least one Group I antibody and at least one Group II antibody, where at least one of the antibodies is G145R mutation resistant. In non-limiting embodiments, antibodies provided by the present disclosure that can be administered to an individual in need thereof include at least one of H006, H007, H0017, H0019, or H020. Also, H005, H008, and H009 are similar to H006 and can be used as substitutes for H006.

[0046] All combinations of H and L chains described herein are included, including all kappa and lambda light chains. In embodiments, a single antibody of the disclosure can include H+L chains from one antibody and H+L chains from another antibody. In embodiments, the antibody includes modifications not encoded in B cells obtained from an individual and / or the antibody is not produced by immune cells in the individual (a biological sample from which is used at least in part to identify and / or produce and / or characterize the antibody of the disclosure). In embodiments, the antibodies provided by the disclosure can be recombinantly produced and expressed with a selected constant region, which may be different from the constant region encoded in any sample from which the amino acid sequence of the antibody is deduced.

[0047] As mentioned above, in embodiments, the present disclosure includes a combination of antibodies or antigen-binding fragments thereof, or a composition comprising or consisting of said antibodies or antigen-binding fragments thereof. In embodiments, the antibody combination of the present disclosure is effective in preventing mutational virus escape. In this regard, the present disclosure includes data demonstrating that not all antibody combinations are effective in preventing mutational escape (such as the combination of H006 and H007, which is not effective). Thus, in embodiments, the antibody or antigen-binding fragment combination collectively targets two or more commonly occurring escape mutations, examples of which are known in the art and described herein. Thus, the antibody and antigen-binding fragment combination of the present disclosure may target non-overlapping groups of common escape mutations. In embodiments, the present disclosure includes the proviso to exclude certain antibody combinations, which only collectively target distinct epitopes, but have overlapping susceptibility to commonly occurring escape mutations.

[0048] In some embodiments, at least one antibody or antigen-binding fragment thereof included in the present disclosure and in the combinations and methods of the present disclosure has a virus-neutralizing activity greater than a control neutralizing activity value (e.g., the neutralizing capacity of ribivirumab). In some embodiments, at least one antibody or antigen-binding fragment of the present disclosure has an IC of less than 128 ng / ml, or less than 35 ng / ml, or less than 5 ng / ml. 50The antibody or antigen-binding fragment thereof includes a viral neutralizing activity with a value of 0.1 to 0.5, including all integers and ranges of all integers between 128 ng / ml and 5 ng / ml. Such neutralizing activity can be determined using known approaches, such as ELISA or immunofluorescence assays, and can be determined as described in Example 5 of the present disclosure. In embodiments, the antibody or antigen-binding fragment thereof encompassed by the present disclosure includes, but is not limited to, an antibody or antigen-binding fragment selected from H016, H017, and H019 antibodies defined by their CDRs. In one embodiment, the present disclosure includes a combination of these antibodies and can include antigen-binding fragments thereof. In embodiments, the antibody combination includes H017 and H019 antibodies, and / or antigen-binding fragments thereof. In one embodiment, the combination optionally further includes an H016 antibody or antigen-binding fragment thereof. In embodiments, the combination of the present disclosure includes a combination consisting of only H017 and H019 antibodies or antigen-binding fragments thereof. In some embodiments, the combinations disclosed herein include combinations consisting of only HO16, H017, and H019 antibodies or antigen-binding fragments thereof. The administration methods of the described antibody combinations, and all other antibodies and antigen-binding fragments thereof described herein, whether sequential or simultaneous, are included within the scope of the present disclosure. Thus, the present disclosure includes administering to an individual in need thereof a combination of antibodies or antigen-binding fragments thereof, either simultaneously or sequentially, and in certain embodiments, the combinations comprise or consist of H017 and H019, or H016, H017, and H019 (and antigen-binding fragments thereof). Additional antibodies and antibody combinations (including antigen-binding fragments thereof) include, but are not limited to, antibodies and antigen-binding fragments thereof comprising the heavy and light chain CDRs of H004, H005, and H009, and H020.

[0049] As can be seen from Table S2 for the H016, H017, and H019 antibodies, the H016 antibody comprises a heavy chain CDR1 having the amino acid sequence GFTFPSHT (SEQ ID NO:8), a heavy chain CDR2 having the amino acid sequence ISTTSEAI (SEQ ID NO:9), and a heavy chain CDR3 having the amino acid sequence ARVGLALTISGYWYFDL (SEQ ID NO:10). The H016 antibody comprises a kappa light chain CDR1 having the amino acid sequence QSISSN (SEQ ID NO:11), a kappa light chain having the CDR2 amino acid sequence RAS, and a kappa light chain having the CDR3 amino acid sequence QQYDHWPLT (SEQ ID NO:12).

[0050] As can be seen from Table S2, the H017 antibody comprises a heavy chain CDR1 having the amino acid sequence GFTFSNYW (SEQ ID NO: 13), a heavy chain CDR2 having the amino acid sequence ISTDGSST (SEQ ID NO: 14), and a heavy chain CDR3 having the amino acid sequence ARGSTYYFGSGSVDY (SEQ ID NO: 15). The H017 antibody comprises a lambda light chain having the CDR1 sequence SSDIGVYNY (SEQ ID NO: 16), a lambda light chain having the CDR2 sequence DVT, and a lambda light chain having the CDR3 sequence SSYRGSSTPYV (SEQ ID NO: 17).

[0051] As can be seen from Table S2, the H019 antibody comprises a heavy chain CDR1 having the amino acid sequence GGSITTGDYY (SEQ ID NO: 18), a heavy chain CDR2 having the amino acid sequence IYYSGST (SEQ ID NO: 19), and a heavy chain CDR3 having the amino acid sequence AIYMDEAWAFE (SEQ ID NO: 20). The H019 antibody comprises a lambda light chain CDR1 having the amino acid sequence QSIGNY (SEQ ID NO: 21), a lambda light chain having the CDR2 amino acid sequence AVS, and a lambda light chain having the CDR3 amino acid sequence QQSYTISLFT (SEQ ID NO: 22).

[0052] In certain embodiments, the antibody includes one or more modifications, such as a non-naturally occurring mutation. As a non-limiting example, in one approach, the Fc region of the antibody may be altered and may be of any isotype, including but not limited to IgG or IgA. The antibodies of the present disclosure may be modified to improve certain biological properties of the antibody, such as to improve stability, modify effector function, improve or prevent interaction with cellular immunity and passage through tissues (placenta, blood-brain barrier, blood-testis barrier), and improve recirculation, half-life, and other effects (e.g., manufacturability, deliverability, etc.).

[0053] In some embodiments, the antibodies of the disclosure may be modified using techniques known in the art (e.g., such as those described in Buchanan, et al., Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression mAbs 5:2, 255-262; March / April 2013, and in Zalevsky J. et al., (2010) Nature Biotechnology, Vol. 28, No.2, p157-159, and Ko, SY, et al., (2014) Nature, Vol. 514, p642-647, and Horton, H. et al., Cancer Res 2008; 68: (19), October 1, 2008), which are incorporated herein by reference. In certain embodiments, the antibody modification increases the in vivo half-life of the antibody (e.g., an LS mutation), or alters the antibody's ability to bind to an Fc receptor (e.g., a GRLR mutation), or alters its ability to cross the placenta, the blood-brain barrier, or the blood-testis barrier. Thus, in certain embodiments, the antibody modification comprises a G to R change, an L to R change, an M to L change, or an N to S change, or any combination thereof.

[0054] In some embodiments, bispecific antibodies are provided by modifying and / or combining segments of the antibodies described herein (e.g., by combining pairs of heavy and light chains from separate antibodies into a single antibody). Methods suitable for making bispecific antibodies are known in the art (e.g., Kontermann, E. et al., Bispecific antibodies, Drug Discovery Today, Volume 20, Issue 7, July 2015, Pages 838-847), the disclosures of which are incorporated herein by reference.

[0055] In embodiments, any antibody described herein comprises a modified heavy chain, a modified light chain, a modified constant region, or a combination thereof, thereby distinguishing it from an antibody produced by a human, hi embodiments, the modifications are made in the hypervariable and / or framework regions (FR).

[0056] In embodiments, mutations to the antibodies described herein (including but not limited to the described antibodies) include modifications to antibodies originally produced in humans, including, but not necessarily limited to, modifications of the heavy chain to increase antibody half-life.

[0057] In embodiments, the antibodies of the disclosure have variable regions as described herein and may comprise or consist of any of these sequences, and may include sequences having 80-99% similarity (inclusive and including any numerical range therebetween) to the sequences explicitly disclosed herein, with antibodies having different sequences that retain the same or similar binding affinity as antibodies having unmodified sequences being provided. In embodiments, the sequences have at least 95%, 96%, 97%, 98% or 99% similarity to the sequences explicitly disclosed herein.

[0058] Antibodies comprising sequences set forth in Table S2 have been isolated and characterized for at least binding affinity, and, unless otherwise stated herein, for virus neutralizing activity, etc. Thus, in embodiments, the present disclosure provides neutralizing antibodies. The term "neutralizing antibody" refers to an antibody or antibodies that inhibit, reduce, or completely prevent viral infection. Whether a particular antibody is a neutralizing antibody can be determined by in vitro assays as described in the Examples below, or as otherwise known in the art. The term "broadly neutralizing" antibody refers to an antibody that can neutralize more than one strain or serotype of a virus.

[0059] The antibodies of the present disclosure can be provided as intact immunoglobulins or as antigen-binding fragments of immunoglobulins, including, but not necessarily limited to, antigen-binding (Fab) fragments, Fab' fragments, (Fab')2 fragments, Fd (N-terminal portion of the heavy chain) fragments, Fv fragments (two variable domains), dAb fragments, single domain fragments, or single monomeric variable antibody domains, isolated CDR regions, single chain variable fragments (scFv), and other antibody fragments that retain virus-binding ability, and preferably virus-neutralizing activity as further described below. In embodiments, the variable regions (including, but not necessarily limited to, the CDRs described) can be used as components of bispecific T cell-based combinations (BiTEs), bispecific killer cell-based combinations (BiKEs), or chimeric antigen receptors (CARs), such as to generate chimeric antigen receptor T cells (e.g., CAR T cells). In embodiments, the present disclosure includes trivalent antibodies capable of specifically binding to three different epitopes.

[0060] The antibodies and antigens of the present disclosure can be provided in pharmaceutical formulations. Administering DNA or RNA polynucleotides encoding any of the proteins (including peptides and polypeptides) described herein, such as the antibodies and antigens described herein, is also considered a method of delivering such proteins to an individual, if the protein is expressed in the individual. Methods of delivering DNA and RNA encoding proteins are known in the art and can be adapted to deliver proteins, particularly the antigens described, with the benefit of the present disclosure. Similarly, the antibodies of the present disclosure can be administered as DNA molecules encoding such antibodies, or as RNA molecules encoding antibodies, using any suitable expression vector.

[0061] Pharmaceutical formulations containing antibodies or viral antigens or polynucleotides encoding them can be prepared by mixing them with a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonicity agents, and the like. The carrier may be a liquid, semi-solid (e.g., paste), or solid carrier. Examples of carriers include water, saline or other buffers (e.g., phosphate, citrate buffers), oils, alcohols, proteins (e.g., serum albumin, gelatin), carbohydrates (e.g., monosaccharides, disaccharides, and other carbohydrates, including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, or dextrins), gels, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents (e.g., EDTA); counterions that form salts (such as sodium); non-ionic surfactants, such as TWEEN, PLURONICS, or polyethylene glycol (PEG), or combinations thereof. In several embodiments, the pharmaceutical / vaccine formulation exhibits improved activity compared to a control (e.g., an antibody delivered without the addition of an additional agent), or alternatively, a particular added agent improves the activity of the antibody.

[0062] The formulation may contain more than one antibody or antigen, and thus may include mixtures of antibodies and mixtures of antigens, as well as combinations thereof, as described herein. These components may be combined with the carrier by any suitable method, such as mixing, dissolving, suspending, emulsifying, encapsulating, absorbing, etc., and may be produced as a formulation, such as a tablet, capsule, powder (including lyophilized powder), syrup, suspension, etc., suitable for injection, oral ingestion, infusion, etc. Sustained release formulations may also be prepared.

[0063] The antibodies and vaccine components of the present disclosure are used for the treatment and / or prevention of Hepatitis B virus infection in a subject, as well as for the inhibition and / or prevention of their transmission from one individual to another.

[0064] The term "treatment" of a viral infection means the effective suppression of the viral infection, thereby delaying the onset, slowing the progression, reducing the viral load, and / or ameliorating the symptoms caused by the infection.

[0065] The term "prevention" of a viral infection means that the onset of infection is delayed and / or the frequency or likelihood of contracting an infectious disease is reduced or eliminated.

[0066] In embodiments, a therapeutic amount of an antibody or antigen vaccine disclosed herein is administered to a subject in need thereof to treat and / or prevent a viral infection. The term "therapeutically effective amount" refers to the dose required to inhibit infection and to treat and / or prevent an infectious disease.

[0067] The dosage of antibody or antigen vaccine depends on the disease state and other clinical factors, such as the weight and condition of the subject, the subject's response to treatment, the type of formulation and the route of administration. The exact dosage that is therapeutically effective and non-toxic can be determined by one skilled in the art. As a general rule, suitable doses of antibodies for administration to adult humans range from about 0.1 to 20 mg / kg of patient body weight parenterally, daily, weekly, or monthly, with a typical initial range used being in the range of about 2 to 10 mg / kg. The antibody is eventually cleared from the bloodstream and may require re-administration. Alternatively, implants or injections of the antibody (antibody contained in a controlled release matrix) can be used.

[0068] Antibodies can be administered to a subject by standard routes, including oral, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, or intramuscular). Additionally, antibodies and / or antigen vaccines can be introduced into the body by injection or by surgical implantation or attachment, such that a significant amount of the antibody or vaccine can enter the bloodstream in a controlled release manner. In certain embodiments, the antibodies described herein are incorporated into one or more prophylactic compositions or devices, for example, to neutralize the virus before it enters the recipient's body cells. For example, in certain embodiments, the compositions and / or devices comprise a polymer matrix that may be formed as a gel and include at least one of a hydrophilic polymer, a hydrophobic polymer, poly(acrylic acid) (PAA), poly(lactic acid) (PLA), carrageenan, polystyrene sulfonic acid, polyamide, polyethylene oxide, cellulose, poly(vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), chitosan, poly(ethyl acrylate), methyl methacrylate, chlorotrimethylammonium methyl methacrylate, hydroxyapatite, pectin, porcine gastric mucin, poly(sebacic acid) (PSA), hydroxypropyl methylcellulose (HPMC), cellulose acetate phthalate (CAP), magnesium stearate (MS), polyethylene glycol, gum base polymers and variants thereof, poly(D,L)-lactide (PDLL), polyvinyl acetate and povidone, carboxypolymethylene, and derivatives thereof. In certain aspects, the present disclosure includes antibodies in microparticles or nanoparticles formed from any suitable biocompatible material, including, but not necessarily limited to, poly(lactic-co-glycolic acid) (PLGA). Liposomal and microsomal compositions are also included. In certain aspects, the gels of the present disclosure include carbomer, methylparaben, propylparaben, propylene glycol, sodium carboxymethylcellulose, sorbic acid, dimethicone, sorbitol solution, or combinations thereof.In embodiments, the gels of the present disclosure include one or a combination of benzoic acid, BHA, mineral oil, pegcol 5 oleate, pegoxol 7 stearate, and purified water, and may include any combination of these compositions.

[0069] The antibodies of the present disclosure can be produced by utilizing techniques available to those skilled in the art. For example, one or separate DNA molecules encoding one or both of the heavy and light chains of the antibody can be constructed based on the coding sequences using standard molecular cloning techniques. The resulting DNA can be placed into a variety of suitable expression vectors known in the art, which are then transfected into host cells (preferably human cells cultured in vitro, but which can also be E. coli or yeast cells, monkey COS cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney 293 cells, etc.). The antibodies can be produced from one or separate expression vectors, including but not limited to separate vectors for the heavy and light chains, and may optionally include separate vectors for the kappa and lambda light chains.

[0070] In embodiments, the antibody may be isolated from a cell. In embodiments, the antibody is a recombinant antibody. By "recombinant" antibody is meant an antibody produced by expression in a cell from one or more expression vectors.

[0071] In one approach, the disclosure includes neutralizing antibodies as described above, and methods for stimulating the production of such antibodies.

[0072] In one approach, the disclosure includes vaccinating an individual using a composition described herein and determining the presence, absence, and / or amount of neutralizing antibodies produced in response to vaccination. Thus, methods are included for determining and monitoring the effectiveness of vaccination, at least in terms of neutralizing antibody production. In one embodiment, following determining the absence of neutralizing antibodies and / or the amount of neutralizing antibodies below an appropriate baseline, the invention includes administering a composition disclosed herein to the individual. Subsequent administrations and measurements can be performed to track the effectiveness of treatment and further treatment adjustments can be made accordingly.

[0073] The antibodies and proteins of the present disclosure can be detectably labeled and / or attached to a substrate, including any substrate and detectable label conventionally used in immunological assays and / or devices. In some embodiments, the substrate comprises biotin or a similar agent that specifically binds to another binding partner to facilitate immobilization and / or detection and / or quantification of the antibody and / or viral protein.

[0074] In embodiments, any type of enzyme-linked immunosorbent (ELISA) assay may be used and performed for diagnostic purposes using the polypeptides and / or antibodies of the present disclosure, including direct, indirect, and competitive ELISA assays, and adaptations thereof that will be apparent to those of skill in the art given the benefit of this disclosure.

[0075] The diagnostic results described herein can be compared to any suitable control. Additionally, any diagnostic results can be fixed in a tangible medium and communicated to a health care provider or any other recipient. In one aspect, the present disclosure includes diagnosing an individual infected with Hepatitis B virus and administering a composition of the present invention to the individual.

[0076] In certain embodiments, the disclosure includes one or more recombinant expression vectors encoding at least the heavy and light chains of an antibody or antigen-binding fragment of the disclosure, cells and cell cultures comprising said expression vectors, methods comprising culturing such cells and isolating the antibody from the cell culture, cell culture medium comprising the antibody, antibodies isolated from the cell culture, and kits comprising expression vectors encoding the antibodies and / or polypeptides of the disclosure. Articles of manufacture containing the antibody and / or polypeptide are provided, where the antibody and / or polypeptide are provided as pharmaceutical formulations contained in one or more sealed containers, which may be sterilized and prepared in any manner that renders such agents suitable for administration to a human or non-human subject. The articles of manufacture / kits may further comprise one or more items for use in administering the composition.

[0077] The following examples are intended to illustrate, but not limit, the invention.

[0078] [Example 1] Serological response to HBV

[0079] To select individuals with a good antibody response to HBsAg, we performed ELISA assays on sera obtained from 159 volunteers, including 15 uninfected and unvaccinated control volunteers (HBsAg - , anti-HBs - , anti-HBc - ), 20 infected and spontaneously recovered volunteers (HBsAg - , anti-HBs + / - , anti-HBc + ), and 124 vaccinated volunteers (HBsAg - , anti-HBs + / - , anti-HBc -). These individuals showed broad-spectrum anti-HBs titers (x-axis in Fig. 1A and Fig. 8B, Table S1). To determine their neutralizing activity, we tested their ability to block HBV infection in sodium-taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells (Michailidis et al., 2017; Yan et al., 2012) (y-axis in Fig. 1A and Fig. 8B and 8C; Table S1). Purified sera or antibodies from individuals with high levels of neutralizing activity were then compared across a wide range of dilutions (Fig. 1B and 1C). Anti-HBs ELISA titers positively correlated with neutralizing activity (r s = 0.492, p < 0.001, Spearman rank correlation), there were clear exceptions as exemplified by volunteers #99 and #49 whose sera failed to neutralize HBV despite high anti-HBs ELISA titers (Figure 1A). Thus, ELISA titers against HBsAg are not perfectly predictive of in vitro neutralizing activity.

[0080] HBsAg, an HBV surface protein, can be subdivided into PreS1-, PreS2-, and S-regions (Figure 1D). To determine which of these regions is the predominant target of the neutralizing response in the selected top neutralizers, we used the S-protein to block neutralizing activity in vitro. Neutralizing activity in volunteers who received an HBV vaccine consisting of the S-protein was completely blocked by the S-protein (black line in Figure 1E). The same was true for spontaneously recovered individuals in our cohort, despite the reported ability of this population to generate anti-PreS1 or anti-PreS2 antibodies (Coursaget et al., 1988; Li et al., 2017; Sankhyan et al., 2016) (red line in Figure 1E). These results suggest that the neutralizing antibody response in the selected individuals is primarily directed against the S-protein, regardless of immunity or infection.

[0081] [Example 2] Human monoclonal antibodies against HBV

[0082] To characterize the antibodies responsible for neutralizing activity in selected individuals, we purified S-protein binding class-switched memory B cells (Escolano et al., 2019; Scheid et al., 2009a). Unexposed naive controls and vaccinated individuals with low anti-HBs ELISA titers showed background levels of S-protein specific memory B cells (Figures 2A and 9A). In contrast, individuals with high neutralizing activity showed IgG + We displayed a distinct population of S-antigen-binding B cells that constituted 0.03–0.07% of the memory compartment (CD19-MicroBeads + CD20-PECy7 + IgG-Bv421 + S-Protein-PE + S-protein-APC + Ovalbumin-Alexa Fluor 488 - ) (Figures 2A and ​ and9A). Consistent with findings in elite HIV-1 neutralizers (Rouers et al., 2017), the fraction of S-protein-specific cells directly correlated with individual neutralization titers (r s = 0.699, p = 0.0145, Spearman rank correlation) (Figure 2B).

[0083] Immunoglobulin heavy (IGH) and light (IGL or IGK) chain genes were amplified from single memory B cells by PCR (Robbiani et al., 2017; Scheid et al., 2009b; von Boehmer et al., 2016). Overall, we obtained 244 pairs of heavy and light chain variable regions from S-protein-binding memory B cells of eight volunteers with high anti-HBs ELISA titers (Figures 9B and 9C; Table S2). Expanded clones consisting of cells producing antibodies encoded by the same Ig variable gene segments with closely related CDR3s were found in each of the top neutralizers #146, #60, and #13 (Figure 2C). Furthermore, IGHV3-30 / IGLV3-21 is present in #146 and #60; IGHV3-33 / IGLV3-21 is present in #146 and #13; IGHV3-23 / IGLV3-21 is present in #146, #60 and #13. The variable diversity and joining (V(D)J) regions of these antibodies were approximately 80% identical at the amino acid level (Figure 2D). Antibodies with related Ig heavy and light chains were also identified between volunteer #55 (who was infected with HBV but recovered) and vaccinated individuals (Figures 2C and 9B). We conclude that top HBV neutralizers produce clones of antigen-binding B cells expressing related Ig heavy and light chains.

[0084] [Example 3] Responsiveness range

[0085] Twenty representative antibodies from five individuals, designated H001–H020, were selected for expression and further testing (Figure 9B). All 20 antibodies showed reactivity to the S-protein antigen (HBsAg adr CHO) used for B cell selection by ELISA and showed 50% effective concentrations (EC 50 ) values ​​(Figure 3A). These antibodies harbored somatic mutations that enhanced antigen binding, as determined by reversion to a putative unmutated common ancestor (UCA) (Figure 3B). Thus, affinity maturation was essential for their high avidity.

[0086] There are four major serotypes of HBV, as defined by a constant "one" determinant and two variable, mutually exclusive determinants "d / y" and "w / y" (Bancroft et al., 1972; Le Bouvier, 1971), with highly statistically significant associations between serotype and genotype (Kramvis et al., 2008; Norder et al., 2004). To determine whether our antibodies cross-react with different HBsAg serotypes, we performed ELISA with five additional HBsAg antigens: yeast-expressed serotype "adr", "adw", and "ayw" antigens, as well as "ad" and "ay" antigens purified from human blood (Figure 3C). Many of the antibodies tested showed broad cross-reactivity and were significantly associated with EC 50 The values ​​were lower than those of ribivirumab, a human anti-HBs monoclonal antibody isolated from HBV-immunized humanized mice and subsequently tested clinically (Eren et al., 2000; Eren et al., 1998; Galun et al., 2002). These antibodies were not polyreactive or autoreactive when tested by polyreactive ELISA and HEp-2 immunofluorescence assay, respectively (Figures 10A and 10B). We conclude that the tested antibodies are broadly cross-reactive with different HBV serotypes.

[0087] [Example 4] Antigenic epitopes on the S-protein

[0088] To determine whether the selected antibodies bind overlapping or non-overlapping epitopes, a competitive ELISA assay was performed in which S-protein was pre-incubated with the selected antibodies followed by a second biotinylated antibody. Antibodies that showed weak levels of binding in the ELISA (H002, H012, H013, H014, H018) were excluded. As expected, all antibodies tested blocked the binding of the self-biotinylated monoclonals (yellow boxes in Figure 4A), whereas the control human anti-HIV antibody 10-1074 did not block any of the anti-HBs antibodies. The competitive ELISA identified three mutually exclusive groups of monoclonal antibodies, suggesting the presence of at least three dominant non-overlapping antigenic sites on HBsAg (red boxes for group-I, blue boxes for group-II, and H017 for group-III, Figure 4A). Each individual with more than one antibody tested in the competitive ELISA expressed monoclonal antibodies targeting two of the three non-overlapping epitopes (Figures 4A and 9B).

[0089] To further define these epitopes, we generated a series of alanine mutants spanning most of the predicted extracellular domain of the S-protein, excluding cysteines, alanines, and amino acid residues important for S-protein production (Salisse and Sureau, 2009) (Figure ID). ELISA assays with representative antibodies from each antibody group and mutant proteins revealed a series of binding patterns that partially corresponded to the three groups defined in the competition assay (Figures 4B and 11). For example, mutations I110A and T148A disrupted binding by group-I antibodies exemplified by H004, H006, H019, and H020, but had little measurable effect on group-II antibodies exemplified by H007, H015, and H016, or on the group-III antibody H017 (Figures 4B and 11).

[0090] However, alanine scanning suggests that some residues such as D144 and G145 are important for binding of monoclonals in both group-I and group-II, even though they cannot compete with each other for binding to the native antigen (Figures 4B and 11). Without intending to be bound by any particular theory, it is believed that the D144A and G145A mutations alter the overall structure of HBsAg, thereby interfering with the binding of antibodies that normally target independent sites on the protein.

[0091] In addition to alanine scanning, we also generated 44 common naturally occurring escape mutants found in chronically infected individuals (Hsu et al., 2015; Ijaz et al., 2012; Ma and Wang, 2012; Salpini et al., 2015). Alanine scanning showed that some of the antibodies in groups-I and -II were resistant to G145A, whereas the corresponding naturally occurring mutations at the same position, G145E and G145R, revealed reduced binding by most of the antibodies (Figure 4C). Among the antibodies tested, H017 and H019 from groups-I and -III, respectively, showed the greatest resistance and the greatest breadth and complementation to the G145 mutation (Figure 4C). We conclude that the human anti-HBs monoclones obtained from selected individuals recognize distinct epitopes on HBsAg, most of which appear to be nonlinear conformational epitopes spanning different regions of the protein.

[0092] [Example 5] In vitro neutralizing activity

[0093] To determine whether the new monoclonal antibodies neutralized HBV in vitro, we performed neutralization assays using HepG2-NTCP cells (Figures 5A and 5B). 50) values ​​were calculated based on HBsAg / HBeAg ELISA or immunofluorescence staining for HBcAg expression (Michailidis et al., 2017) (Figure 5C). Neutralizing activity was further validated by an in vitro neutralization assay using primary human hepatocytes (Michailidis et al., 2020) (Figures 5C and 5D). Fourteen of the 20 antibodies tested had IC values ​​as low as 5 ng / ml. 50 In comparison, ribivirumab showed neutralizing activity with IC values ​​of 35 and 128 ng / ml in ELISA- and immunofluorescence-based neutralization assays, respectively (Figure 5C). 50 The somatic mutations were essential for the potent neutralizing activity, as indicated by the reduced activity of the putative UCA (Figs. 12A and 12B). Furthermore, the IC 50 Optimal activity required bivalent binding, since values ​​were two orders of magnitude higher than for the intact antibodies (Figure 5E). Finally, there was no apparent synergy when combining antibodies from groups-I, -II, and -III (Figure 12C). We conclude that half of the new monoclonals were significantly more potent than ribivirumab, including H004, H005, H006, H008, H009, H019, and H020 from group-I, and H007, H015, and H016 from group-II (Figure 5C).

[0094] [Example 6] H015 Structure of antibody / peptide complex

[0095] H015 differed from the other antibodies in that its binding was inhibited by five consecutive alanine mutations spanning positions K141 to G145, indicating the presence of a linear epitope. This idea was confirmed by ELISA against a series of overlapping peptides that encompass the predicted extracellular domain of the S-protein (Figures 6A and 13A). The data showed that H015 bound to KPSDGN (SEQ ID NO: 23), a sequence that is close to the center of the predicted extracellular domain and contains some of the most frequently mutated amino acids during natural infection.

[0096] To investigate the molecular basis for H015 binding, the Fab fragment was co-crystallized with the target peptide epitope PSSSSTKPSDGNSTS (SEQ ID NO: 24), where all cysteine ​​residues flanking the recognition sequence were replaced with serine to avoid non-physiological cross-linking. The 1.78 Å structure (Figures 6B and 13B) revealed that the peptide binds primarily to the immunoglobulin heavy chain (Figures 6B and 6C), interacting with residues in CDR1 (R31), CDR2 (W52, F53), and CDR3 (E99, P101, L103, L104) of IgH, and only one contact with CDR3 (P95) of IgL. Since only one hydrogen bond is found between K141 and G145 (Milner-White and Poet, 1986), and they are not part of a β-sheet, the peptide adopts a three-residue β-hairpin (class 3) of the 3:5 type, including residues K145 to G145. The peptide is further stabilized by a salt bridge formed between K141 and D144 (Figures 6D and 13C). Interestingly, the distance between the Cαs of the two residues (C139 and C147) adjacent to the recognition residues is 6.4 Å, poised to form a disulfide bond between C139 and C147 found in the native HBsAg structure (Ito et al., 2010). H015Fab appears to stabilize the peptide conformation through Fab-peptide contacts (Figure 13D), which are formed over a large binding surface (866 Å). 2 ;Antibody-antigen buried surface 600~900Å 2( Braden and Poljak, 1995)), which consists mainly of one salt bridge (lysine-aspartic acid; 0.9 ± 0.3 Kcal / mol) (White et al., 2013), and five hydrogen bonds (1–2 Kcal / mol / bond) (Sheu et al., 2003). Furthermore, the peptide further constrains the loop through the intrapeptide contacts, even in the absence of disulfides (Figure S13D).

[0097] The residues that form the hairpin are important for anti-HBs antibody recognition as determined by alanine scanning (Figures 4B and 11). Furthermore, each of these residues has been identified as important for immune recognition during natural infection (Ma and Wang, 2012). G145R is the most common naturally occurring S-protein escape mutation and replaces a small neutral residue (circled residue in Figure 6E) with a large positively charged residue, potentially altering the antigen-binding surface. G145 adopts a positive phi angle of 77.9 and, in doing so, introduces a kink in the β-strand, a structure that is disrupted upon substitution with arginine.

[0098] HBsAg can be glycosylated at N146, and this site is also strictly conserved. However, some studies suggest that this glycosylation site is not fully occupied, resulting in an almost 1:1 ratio of glycosylated and non-glycosylated isoforms on the surface of the viral envelope (Julithe et al., 2014). Glycosylation can be either NAG-NAG-MAN or NAG-(FUC)-NAG-MAN (Hyakumura et al., 2015). We modeled both fucosylated and non-fucosylated options by grafting 7-mer and 11-mer glycans attached to N146 of the peptide in the presence of Fab. We also found that both glycosylated forms are tolerated at that position with only minimal torsional accommodation without clashes with Fab, even though fucosylated (branched) glycans require some additional torsion angle changes in the Fab.

[0099] [Example 7] Protection and therapy in humanized mice

[0100] HBV infection is restricted to humans, chimpanzees, tree shrews, and human liver chimeric mice (Sun and Li, 2017). To determine whether our anti-HBs bNAbs prevent infection in vivo, we used human liver chimeric Fah - / - NODRag1 - / -IL2rg null We generated human FNRG (huFNRG) mice (de Jong et al., 2014) and injected them with control or H020 (group-I) or H007 (group-II) antibodies prior to infection with HBV (Figures 7A-7D). These two antibodies were selected because they bind to non-overlapping sites and have broad and potent neutralizing activity. We infected all six control animals in two independent experiments, and pre-exposure prophylaxis with either H007 or H020 was fully protective (Figures 7B-7D). We conclude that a single anti-HBs bNAb targeting distinct epitopes on the major viral surface antigen can prevent infection in vivo.

[0101] To determine whether bNAbs could also control established infection, we administered control antibodies or bNAbs H020 (group-I) or H007 (group-II) at 10 6 ~10 8 HBV was injected into huFNRG mice carrying a viral load of 10 copies (Figures 7E-7H and 14A). - / - NODRag1 - / - IL2rg null Mice are severely immunodeficient and are unable to mount an adaptive immune response due to the absence of T and B lymphocytes. null The mutations prevent cytokine signaling through multiple receptors, resulting in defects in innate immune function, including antibody-dependent cellular cytotoxicity. Thus, antibody therapy alone can reduce the 10 6 -10 8 It is not expected to eliminate viremia in DNA copies / ml.

[0102] Animals receiving control antibody developed viremia at ~10 11The viremia levels further increased to as high as 1000 DNA copies / ml (Figure 7F). In contrast, the five mice that received H020 maintained a stable level of viremia for approximately 30 days (Figure 7G), after which two mice showed increased viremia (arrows 1 and 3 in Figure 7G). Similar results were observed in the five mice that received H007 (Figure 7H), although only one showed a slightly increased viremia at approximately day 50 (arrow 5 in Figure 7H).

[0103] To determine whether animals that showed increased HBV DNA levels during antibody monotherapy developed escape mutations, we sequenced viral DNA recovered from mouse blood. All three mice that escaped H020 (group-I) or H007 (group-II) monotherapy developed viruses carrying the G145R mutation in the S-protein (arrows 1 and 3 in Fig. 7G, arrow 5 in Fig. 7H, Fig. 7I and Fig. 14). This mutation represents a major immune escape mutation in humans (Zanetti et al., 1988). Furthermore, a mutation at the same position in the S-protein was also identified in mice that maintained low levels of hepatitis (arrows 2 and 4 in Fig. 7G, arrows 6 and 7 in Fig. 7H, Fig. 7I and Fig. 14), but not in control animals (Fig. 14). These results indicate that anti-HBs bNAb monotherapy results in the emergence of escape mutations consistent with bNAb binding properties in vitro (Fig. 4C).

[0104] To determine whether the combination of bNAbs targeting two distinct epitopes would prevent the emergence of resistant strains, we co-administered H006+H007 (groups-I and -II, respectively) to eight HBV-infected huFNRG mice (Figure 7J). H006 (group-I) was selected for this purpose because of its resistance to D144A and G145A mutations (Figure 4B). Similar to H007 monotherapy, only a slight increase in viremia was observed in animals treated with the H006+H007 anti-HBs bNAb combination during the 60-day observation period (Figures 7J and 14a). However, sequence analysis revealed that three of the mice had developed resistance mutations, including K122R / G145R, C137Y, and C137Y / D144V (arrows 8, 9, and 10 in Figure 7J, Figure 7I, and Figure 14). These mutations abolish binding to both H006 (group-I) and H007 (group-II) (Figure 4C). Thus, the combination of two anti-HBs bNAbs targeting separate epitopes but sensitive to the same clinical escape mutants is not sufficient to inhibit the emergence of escape mutants.

[0105] To prevent the emergence of escape mutations, we combined H017 and H019 (groups-III and -I, respectively) bNAbs because they display complementary susceptibility to commonly occurring spontaneous mutations (Figure 4C). None of the seven mice treated with this combination showed increased viremia or escape mutations as assessed by sequence analysis (Figures 7K and 14A). Similar effects were also observed in nine animals treated with the triple antibody combination of H016, H017, and H019 (groups-II, -III, and -I, respectively) (Figures 7L and 14A). Moreover, both of these combinations dramatically reduced serum HBsAg levels (Figures 14C and 14D). Taken together, these findings suggest that the control of HBV infection by bNAbs requires a combination of antibodies targeting non-overlapping groups of common escape mutations.

[0106] [Table 1-1] [Table 1-2] [Table 1-3]

[0107] [Example 8] This example provides a description of the materials, methods, and subjects used to obtain the aforementioned results.

[0108] Experimental Models and Subjects

[0109] Human Subjects

[0110] Volunteer recruitment and blood collection were conducted at Rockefeller University Hospital under a protocol approved by the Institutional Review Board (IRB QWA-0947). Study participants ranged in age from 22 to 65 years (mean 43 years) with a female:male ratio of 81:78 (Figures 8D and 8E; Table S1).

[0111] animal

[0112] Fah - / - NODRag1 - / - IL2rg null (FNRG) female mice were generated as previously described (de Jong et al., 2014) and maintained in the Rockefeller University AAALAC-accredited facility. Animal protocols conformed to NIH guidelines and were approved by the Rockefeller University Institutional Animal Care and Use Committee under protocol #18063. Female littermates were randomly assigned to experimental groups.

[0113] cell line

[0114] HepG2-NTCP cells (Michailidis et al., 2017) and HepDE19 cells (Cai et al., 2012) were maintained in collagen-coated flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% or 3% fetal bovine serum (FBS) and 0.1 mM non-essential amino acids (NEAA). Huh7.5-NTCP cells were maintained in DMEM supplemented with 10% FBS and 0.1 mM NEAA. All liver cell lines were cultured at 37°C in 5% CO2. Human embryonic kidney HEK293-6E suspension cells were cultured at 37°C in 8% CO2 with shaking at 120 rpm.

[0115] virus

[0116] HBV-containing supernatants were harvested and concentrated from HepDE19 cells as previously described (Michailidis et al., 2017). Concentrated virus stocks were aliquoted and stored at -80°C. For in vivo experiments, one aliquot of mouse-passaged genotype C HBV virus originally generated from patient serum (Billerbeck et al., 2016) was stored at -80°C and thawed for mouse infection experiments. For protection and treatment experiments, animals were challenged intravenously (1 × 10 per mouse). 4 (using a DNA copy of the

[0117] bacteria

[0118] E. coli DH5-α was grown at 37°C with shaking at 230 rpm.

[0119] method

[0120] Collection of human samples

[0121] Peripheral blood samples were collected from volunteers at Rockefeller University Hospital. Serum was isolated by centrifugation of clotted whole blood and aliquoted for storage at -80°C. PBMCs were isolated using cell separator tubes with fritted barriers and stored frozen in liquid nitrogen in 90% heat-inactivated FBS supplemented with 10% dimethylsulfoxide (DMSO).

[0122] HBV stock

[0123] HepDE19 cells (Cai et al., 2012) were cultured in the absence of tetracycline to induce HBV replication. After 7 days, supernatants were collected every other day for 2 weeks and fresh medium was added. After each collection, the medium was sedimented to remove cell debris, passed through a 0.22 μm filter, and kept at 4 °C. The collected medium was concentrated 100-fold by centrifugation using a Centricon Plus-70 centrifugal filter device (Millipore-Sigma, Billerica, MA). Mouse-passaged genotype C HBV virus (Billerbeck et al., 2016) was used for in vivo mouse experiments.

[0124] In vitro HBV neutralization assay

[0125] In vitro HBV infection was performed as previously described (Michailidis et al., 2017). Briefly, HepG2-NTCP cells were plated in 96-well collagen-coated plates in DMEM supplemented with 10% FBS and 0.1 mM NEAA. The medium was replaced the next day with DMEM containing 3% FBS, 0.1 mM NEAA, and 2% DMSO and cultured for an additional 24 h before infection. Cells were seeded in DMEM supplemented with 3% FBS, 0.1 mM NEAA, 4% PEG, and 2% DMSO. Antibody or serum samples were incubated with virus in the seeding medium for 1 h at 37°C before being added to the cells. Serum neutralizing capacity (y-axis in Figure 1A and Figure 8B) was calculated as the reciprocal of the relative percentage of infected HepG2-NTCP cells immunostained by rabbit anti-HBV core antibody (AUSTRAL Biologicals). For example, if the relative percentage of infected cells is 100% (no serum added or serum from unexposed naive control donors), the serum neutralizing capacity is calculated as 1, whereas if the relative percentage of infected cells is 50% or 10%, the serum neutralizing capacity is 2 or 10. In the blocking neutralization assay, different concentrations of S-protein antigen were incubated with purified polyclonal antibodies for 1 h at 37°C prior to incubation with HBV virus. The cells were then spun down for 1 h by centrifugation at 37°C and 1000g. After 24 h of incubation, the supernatant was removed and the cells were washed five times with 100 μl of fresh DMEM supplemented with PBS, 3% FBS, 0.1 mM NEAA and 2% DMSO. Both the supernatant and cells were collected 7 days after infection for analysis. Neutralization assays in primary human hepatocytes were performed as described above using hepatocytes from highly humanized mouse livers, which were harvested and plated on collagen-coated plates in hepatocyte defined medium (Corning) ( Michailidis et al., 2020 ).

[0126] Chemiluminescence immunoassay

[0127] For quantitative analysis of secretory antigens HBsAg or HBeAg, 50 μl of the collected supernatant was added to a 96-well plate of a chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China) according to the manufacturer's instructions. The plate was read using a FLUOstar Omega luminometer (BMG Labtech). The absolute concentrations were measured and the relative values ​​were calculated by normalizing to the virus-only control wells in the same lane. For example, the absolute HBsAg / HBeAg level in the virus-only control wells (considered as the reference) was 20 NCU / ml (national clinical units per milliliter), while the addition of one neutralizing serum sample can reduce this to 5 NCU / ml. Thus, after normalization, the relative HBsAg / HBeAg levels were calculated as 100% for the control and 25% for the neutralizing serum. Since many factors (virus concentration, cell concentration, immunofluorescence readings, etc.) vary between different plates or between different experiments of a round, normalization is necessary to combine data for comparison.

[0128] Immunofluorescence

[0129] Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% goat serum, cells were incubated with rabbit anti-HBV core antibody (AUSTRAL Biologicals) overnight at 4°C and visualized with goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific). Nuclei were stained with DAPI. Cells were imaged using a Nikon Eclipse TE300 fluorescence microscope and processed with ImageJ. For high content imaging analysis, ImageXpress Micro XLS (Molecular Devices, Sunnyvale, CA) was used. Absolute HBc +The percentage was calculated and normalized to the virus-only control well in the same lane to determine the HBc + The relative percentage of cells was calculated, e.g., absolute HBc in virus-only control wells (considered as the baseline). + The percentage of HBc cells is 40%, whereas the addition of one neutralizing serum sample can reduce this to 10%. Thus, after normalization, + The relative percentage of cells was calculated as 100% for control wells and 25% for the neutralizing serum samples. Because many factors (virus concentration, cell concentration, immunofluorescence readings, etc.) vary between different plates or between different rounds of experiments, normalization is necessary to combine data for comparison.

[0130] ELISA assay

[0131] Blood samples were submitted to Memorial Sloan Kettering Cancer Center for clinical testing. The presence of HBsAg protein and anti-HBc antibodies, as well as anti-HBs titers, were determined by ELISA (Abbott Laboratories) according to the manufacturer's instructions.

[0132] Binding of serum or recombinant IgG antibodies to HBsAg protein (see Table of Key Resources) was measured by coating ELISA plates with antigen at 10 μg / ml (in PBS). Plates were blocked with 2% BSA (in PBS) and incubated with antibodies for 1 h at room temperature. Visualization was performed using HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific). The 50% effective concentration (EC) required for maximal binding was determined. 50 ) was determined by nonlinear regression analysis in the software PRISM.

[0133] Competitive ELISA plates were coated with HBsAg(adr CHO) at 0.12 μg / ml and incubated with primary antibody at 16.7 μg / ml for 2 h, followed by direct addition of biotinylated secondary antibody at 0.25 μg / ml and incubation at room temperature for 30 min. Detection was performed with streptavidin-HRP (BD Biosciences).

[0134] Autoreactivity and polyreactivity

[0135] Autoreactivity and polyreactivity assays were performed as previously described (Gitlin et al., 2016; Mayer et al., 2017; Robbiani et al., 2017). For autoreactivity assays, monoclonal antibodies were tested using an antinuclear antibody (HEp-2) kit (MBL International). Antibodies were incubated at 100 μg / ml and detected with Alexa Fluor 488 AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) (Jackson ImmunoResearch) · 10 μg / ml. Fluorescent images were captured with a wide-field fluorescent microscope (Axioplan 2, Zeiss), a 40x dry objective, and a Hamamatsu Orca ER B / W digital camera. Images were analyzed with Image J. Human serum containing antinuclear antibodies (MBL International) was used as a positive control. A multireactive ELISA assay measured antibody binding to five different antigens (double-stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS), and single-stranded DNA (ssDNA)). ED38 (Wardemann et al., 2003) and mG053 (Yurasov et al., 2005) antibodies were used as positive and negative controls, respectively.

[0136] Synthetic peptides

[0137] Eighteen peptides spanning the antigenic loop region of the S-protein antigen were synthesized at the Proteomics Resource Center at Rockefeller University. Peptide ELISA plates were coated with peptides at 10 μg / ml in PBS.

[0138] HBsAg-binding memory B cells

[0139] S-protein (adr serotype) and ovalbumin (Sigma-Aldrich) expressed and purified from Chinese hamster ovary (CHO) cells (ProSpec) were used with the EZ-Link TM Biotinylation was performed using a Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher Scientific). S-Protein-PE and S-Protein-APC were prepared by incubating 2-3 μg of biotin-S-Protein with streptavidin-PE (eBioscience) or streptavidin-APC (BD Biosciences), respectively, in PBS overnight at 4°C in the dark. Ovalbumin-Alexa Fluor 488 was generated by incubating biotin-ovalbumin with streptavidin-Alexa Fluor 488 (Thermo Fisher Scientific).

[0140] B cell purification, labeling, and sorting were performed as previously described (Escolano et al., 2019; Robbiani et al., 2017; Tiller et al., 2008; von Boehmer et al., 2016). Briefly, PBMCs were thawed and washed with RPMI medium at 37°C. B lymphocytes were positively selected using CD19 MicroBeads (Miltenyi Biotec) and subsequently incubated with human Fc block (BD Biosciences) and anti-CD20-PECy7 (BD Biosciences), anti-IgG-Bv421 (BD Biosciences), 10 μg / ml S-protein-PE, 10 μg / ml S-protein-APC, 10 μg / ml ovalbumin-Alexa Fluor 488 for 20 min at 4°C. Single CD20 + IgG + S-Protein-PE + S-protein-APC + Ova-Alexa Fluor 488 - Memory B cells were sorted into 96-well plates using a FACSAriaII (Becton Dickinson) and stored at -80°C.

[0141] Antibody cloning, sequencing and production

[0142] Antibody cloning, sequencing and production were performed as previously described (Robbiani et al., 2017; Tiller et al., 2008; von Boehmer et al., 2016). Primers are listed in Table S3. Unmutated common ancestor (UCA) antibody sequences H006, H019, H020 were synthesized by gBlock IDT (Table S3) and inserted into antibody vectors for expression. V(D)J gene segments and CDR3 sequences were determined by IgBlast (Ye et al., 2013) and / or IMGT / V-QUEST (Brochet et al., 2008).

[0143] S-protein mutagenesis

[0144] Oligonucleotide fragments carrying the target point mutations were synthesized by gBlock IDT (Table S3) and substituted into the antigen loop region in the plasmid p1.3xHBV-WT by Sequence and Ligation-Independent Cloning (SLIC) (Jeong et al., 2012). The mutated plasmids were transfected into Huh-7.5-NTCP cells using X-tremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich), and the culture medium was changed to serum-free DMEM after 24 hours. The supernatants were harvested after 2 days and stored at -80°C. Serum-free medium (50 μl) was directly used to coat the ELISA plates.

[0145] Crystallization, X-ray data collection, structure determination, refinement

[0146] Antibody Fab (25 mg / ml) in 50 mM Tris 8.0, 50 mM NaCl was mixed 5:1 (v / v) with peptide (5 mg / ml) in the same buffer. The molar ratio of Fab:peptide was approximately 1:2. Crystals were obtained by substituting all peptide-11 cysteine ​​residues with serine during peptide synthesis (Proteomics Resource Center, RU). Crystallization conditions for Fab15 / peptide-11Ser were identified from a commercial screen (Morpheus from Molecular Dimensions) by sitting drop vapor diffusion at room temperature. The crystals used for data collection were obtained directly from the initial setup (position E1) in a precipitant solution consisting of 0.12 M ethylene glycol (di-, tri-, tetra-, and penta-ethylene glycol), 0.1 M Buffer Mix 1 (imidazole / MES) at pH 6.5, and 30% Precipitant Mix 1 (20% v / v PEG 500* MME; 10% w / v PEG 20000). The crystals were flash-cooled in liquid nitrogen (directly from the mother liquor without additional cryoprotectant). X-ray diffraction data were collected from a single crystal on the Advanced Photon Source (APS) beamline 24-ID-E (1.78 Å resolution). The data were integrated and scaled using the program XDS (Kabsch, 2010a, b) and other data processing utilities from the CCP4 suite (Collaborative Computational Project, 1994) (using RAPD), software available at the beamline. Initial phase estimates and electron density maps were obtained by molecular replacement with Phaser (McCoy et al., 2007), using a single FAB molecule from (PDB:5GGU) as the initial search model in Phenix (Adams et al., 2010). Iterative model building and structural refinement were performed manually using COOT (Emsley et al., 2010) and Phenix, respectively. The peptide density was well defined and refined to 90% occupancy for residues STKPSDGNST (SEQ ID NO:25).Not all other residues are visible, and the regions that are completely solvent have no crystal contacts involving peptide atoms. The quality of the final model was good (96% of observed residues in allowed regions as described by Ramachandran). Data collection and refinement statistics are summarized in Figure 13B. All molecular graphics were generated using PyMOL (Version 2.0 Schrodinger, LLC). The atomic coordinates and experimental structure factors have been deposited in the PDB under the accession code 6VJT.

[0147] Humanized mice and in vivo studies

[0148] 6-8 week old Fah - / - NODRag1 - / - IL2rg null (FNRG) female mice were transplanted with 1 million human hepatocytes from a pediatric female donor HUM4188 (Lonza Bioscience) as previously described (de Jong et al., 2014). Briefly, under isoflurane anesthesia, mice underwent incision of the skin and peritoneum to expose the spleen. One million hepatocytes were injected into the spleen using a 28-gauge needle. The peritoneum was then approximated using 4.0 VICRYL sutures (Johnson & Johnson) and the skin was closed using MikRon Autoclip surgical clips (Becton Dickinson). Mice were cycled off the drug nitisinone (Yecuris) based on weight loss and overall health. Humanization was monitored by quantifying human albumin in mouse serum using a human-specific ELISA (Bethyl Labs). Humanized FNRG mice with human albumin values ​​greater than 1 mg / ml were used for infection experiments. Human liver chimeric (huFNRG) mice are severely immunodeficient. - / - Depletes mouse B and T cells and inhibits IL2rg nullThe mutations prevent cytokine signaling through multiple receptors, leading to a lack of functional NK cells. Furthermore, the genetic background is a NOD background, with suboptimal antigen presentation, defects in T and NK cell function, reduced macrophage cytokine production, inhibited wound healing, and C5 complement deficiency. Thus, the mice will be unable to generate antibody-dependent effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), or passive antibody-enhanced adaptive immunity.

[0149] Mice were administered 1 x 10 4 Genome equivalents (GE) of mouse-passaged genotype C HBV virus were administered intravenously. For prophylactic experiments, 500 μg of monoclonal antibody was administered intraperitoneally 20 h and again 6 h before infection. For therapeutic experiments, huFNRG mice with established HBV infection (<10 per mL of serum) were administered intravenously. 8 The mice (DNA copies) were intraperitoneally injected with 500 μL of each monoclonal antibody three times a week.

[0150] DNA from mouse serum collected weekly was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). Total HBV DNA was determined by quantitative PCR (Michailidis et al., 2017). PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems), primers and probes (Table S3).

[0151] To obtain HBV DNA for sequence analysis from serum, the S domain was amplified using primers (Table S3) and Phusion DNA polymerase (Thermo Fisher Scientific). Initial denaturation was performed at 98°C for 30 s, followed by 40 amplification cycles (98°C for 10 s, 60°C for 30 s, and 72°C for 30 s) followed by one cycle (72°C for 5 min). A ∼700 bp fragment was gel extracted for Sanger sequencing. Sequence alignment was performed using MacVector.

[0152] Quantification and statistical analysis

[0153] Detailed information on statistical analysis can be found in the Results and figure legends. Correlations were evaluated by Spearman's rank correlation method (Figures 1A and 2B). Statistical significance was calculated by Dunn's Kruskal-Wallis multiple comparisons with p-values ​​corrected by the Benjamini-Hochberg procedure (Figure 8C). Median effective concentration (EC 50 ) values ​​(Figures 3A and 3C) and the 50% inhibitory concentration (IC 50 ) values ​​(FIG. 5C) were calculated by nonlinear regression analysis in PRISM software.

[0154] Experimental Considerations

[0155] Previous studies have identified some anti-HBs neutralizing antibodies from a small number of randomly selected spontaneously recovered or vaccinated individuals (Cerino et al., 2015; Colucci et al., 1986; Eren et al., 1998; Heijtink et al., 2002; Heijtink et al., 1995; Jin et al., 2009; Kim and Park, 2002; Li et al., 2017; Sa'adu et al., 1992; Sankhyan et al., 2016; Tajiri et al., 2007; Tokimitsu et al., 2007; Wang et al., 2016). In contrast, in this disclosure, we screened sera from 144 exposed volunteers to identify highly potent neutralizers. Serological activity varied widely among donors, with a few individuals showing high levels of neutralizing activity. To understand this activity, we isolated 244 anti-HBs antibodies from single B cells obtained from top donors. Each of the elite donors tested showed expanded clones of memory B cells expressing bNAbs targeting three non-overlapping sites on the S-protein. Furthermore, the amino acid sequences of some bNAbs were highly similar in different individuals. These closely related antibodies target the same epitope.

[0156] The approximate clonal identity of HBV bNAbs in unrelated elite individuals is similar to reports of elite responders to HIV-1 (Scheid et al., 2011; West et al., 2012), influenza (Laursen and Wilson, 2013; Pappas et al., 2014; Wrammert et al., 2011), Zika (Robbiani et al., 2017), and malaria (Tan et al., 2018). However, none of the elite anti-HBs bNAbs share both IgH and IgL with previously reported HBV neutralizing antibodies, and the best previously reported HBV neutralizing antibodies have been tested in the clinic but are not as potent as some of the bNAbs disclosed herein (ribivirumab IC 50 : 35ng / ml, tuvilumab IC 50 :~100ng / ml) (Galun et al., 2002; Heijtink et al., 2001; van Nunen et al., 2001).

[0157] The described alanine scanning and competitive binding analyses are consistent with the presence of at least three domains that can be simultaneously recognized by bNAbs (Gao et al., 2017; Tajiri et al., 2010; Zhang et al., 2016). However, the domains do not appear to be restricted to either of the two previously defined cyclic peptide epitopes 123-137 and 139-148 (Tajiri et al., 2010; Zhang et al., 2016). Instead, residues across most of the ectodomain can contribute to binding by both group-I and -II antibodies. For example, alanine scanning shows that group-I H020 binding is dependent on I110, K141, D144, G145 and T148, while group-II H016 binding is dependent on T123, D144 and G145. Therefore, despite having non-overlapping binding sites, some of the essential residues are shared by groups-I and -II, suggesting that the epitopes are conformational. Furthermore, the antibody epitopes on the S-protein identified using mouse and human antibodies may be distinct (Chen et al., 1996; Ijaz et al., 2003; Paulij et al., 1999; Zhang et al., 2019; Zhang et al., 2016). Finally, G145, a residue frequently mutated in infected humans (Ma and Wang, 2012; Tong et al., 2013), appears to be essential for binding by all group-II antibodies tested (versus not all group-I or -III antibodies tested).

[0158] Crystallization of group-II bNAb H015 and its linear epitope revealed a loop containing P142, S / T143, D144, and G145, all of which frequently mutate during natural infection to generate well-documented immune escape mutants (Hsu et al., 2015; Ijaz et al., 2012; Ma and Wang, 2012; Salpini et al., 2015). In addition to immune escape, the residues that form this structure are also essential for infectivity, likely by facilitating viral interactions with cell surface glycosaminoglycans (Sureau and Salisse, 2013). Mutations at K141, P142, and C139 and C147 all contribute to the stability of the structure and reduce viral infectivity (Salisse and Sureau, 2009). Without intending to be bound by any particular theory, it is believed that agents that destabilize the newly elucidated H015-peptide loop structure may also interfere with infectivity.

[0159] The G145R mutation, one of the most frequent immune escape mutants, replaces a small neutral residue with a bulky charged residue, which may interfere with antigenicity by disrupting the salt bridge between K141 and D144 that anchors the peptide loop. However, this drastic structural change does not alter infectivity (Salisse and Sureau, 2009), presumably because the additional charge compensates for the altered interaction between HBV and cell surface glycosaminoglycans (Sureau and Salisse, 2013). Thus, the additional charge may allow G145R to function as a dominant immune escape mutant while maintaining infectivity.

[0160] This disclosure describes antibodies directed in part to the S-protein antigen because this antigen is the antigen used in current FDA-approved vaccines and because purified S-protein blocked almost all neutralizing activity in the sera of elite neutralizers (irrespective of whether they were vaccinated or recovered naturally). Nevertheless, individuals who recover from infection produce antibodies against the PreS1 domain of HBsAg (Li et al., 2017; Sankhyan et al., 2016). The PreS1 domain is essential for the virus to interact with the entry factor NCTP on hepatocytes, and potent neutralizing antibodies against PreS1 have been reported (Li et al., 2017). However, these are not naturally occurring antibodies, but rather randomly paired IgH and IgL chains derived from phage libraries obtained from unexposed or vaccinated healthy donors (Li et al., 2017). Moreover, the phage antibodies required further genetic engineering to enhance their neutralizing activity (Li et al., 2017). Thus, it remains to be seen whether the human immune system can produce potent anti-PreS1 bNAbs.

[0161] Chronic HBV infection is a major global public health problem that requires effective treatment strategies (Graber-Stiehl, 2018; Lazarus et al., 2018; Revill et al., 2016). Chronically infected individuals produce overwhelming amounts of HBsAg that are hypothesized to disable the immune system. As a result, immune cells that would normally be able to clear the virus are unable to react with antigens (a phenomenon called exhaustion or anergy) (Ye et al., 2015). The appearance of anti-HBs antibodies is associated with spontaneous recovery from the disease, presumably because they can clear antigens and promote the emergence of a proliferative immune response (Celis and Chang, 1984; Zhang et al., 2016; Zhu et al., 2016). These findings lead to the hypothesis that passively administered antibodies may be combined with antiviral drugs to further reduce antigenic load while enhancing immune responses that maintain long-term control of the disease. The currently described results in HBV-infected huFNRG mice show that antibody monotherapy with potent bNAbs can lead to the emergence of the very same escape mutations commonly found in chronically infected individuals. Moreover, not all bNAb combinations are effective in preventing mutational escape. Combinations that target distinct epitopes but have overlapping susceptibility to commonly occurring escape mutations (such as H006 and H007) are ineffective. In contrast, combinations with complementary susceptibility to common escape mutations prevent the emergence of escape mutations in HBV-infected huFNRG mice. Thus, as described above, the present disclosure provides immunotherapy of HBV infection with a combination of antibodies with complementary activities to circumvent this potential problem.

[0162] The following list of references is not intended to indicate that any particular reference is patentable material. [Reference List-1] TIFF0007629649000004.tif195157[Reference List-2] TIFF0007629649000005.tif231156[Reference List-3] TIFF0007629649000006.tif239158[Reference List-4] TIFF0007629649000007.tif230158[Reference List-5] TIFF0007629649000008.tif233157[Reference List-6] TIFF0007629649000009.tif239159[Reference List-7] TIFF0007629649000010.tif235160[Reference List-8] TIFF0007629649000011.tif227156[Reference List-9] TIFF0007629649000012.tif228159[Reference List-10] TIFF0007629649000013.tif241156[Reference List-11] TIFF0007629649000014.tif30157

[0163] Supplementary Table S1. Donor details; related to Figure 1

[0164] The anti-HBs ELISA titers (x-axis in Figs. 1A and 8B) and relative infection rates (y-axis in Figs. 1A and 8B) of serum samples from each donor were determined by ELISA assay and in vitro neutralization assay, respectively. [Table S1-1] TIFF0007629649000015.tif163159[Table S1-2] TIFF0007629649000016.tif237158[Table S1-3] TIFF0007629649000017.tif233157[Table S1-4] TIFF0007629649000018.tif233156[Table S1-5] TIFF0007629649000019.tif236158[Table S1-6] TIFF0007629649000020.tif237162[Table S1-7] TIFF0007629649000021.tif237161 [Table S1-8] TIFF0007629649000022.tif235162 [Table S1-9] TIFF0007629649000023.tif186158

[0165] Supplementary Table S2. Detailed information on cloned antibodies with paired heavy and light chains; related to Figure 2

[0166] Listed are the variable (V), diversity (D), and joining (J) genes, mutations on the variable genes (V MUT), and CDR3 amino acid sequences of the cloned immunoglobulin heavy, kappa, and lambda light chains. These antibodies are grouped by their IGHV genes and are shown with our selected antibodies H001-H020. The H021 antibody used for sequence alignment in Figure 2D is also shown. The amino acid length of the IGH CDR3 ranges between 5 and 27 amino acids, with 16 being the most common and the average being about 15 amino acids. There are 16 IGH CDR3s that contain cysteines. [Table S2-1] TIFF0007629649000024.tif244107[Table S2-2] TIFF0007629649000025.tif244150[Table S2-3] TIFF0007629649000026.tif240148[Table S2-4] TIFF0007629649000027.tif243143[Table S2-5] TIFF0007629649000028.tif244149[Table S2-6] TIFF0007629649000029.tif243151[Table S2-7] TIFF0007629649000030.tif244146[Table S2-8] TIFF0007629649000031.tif244143[Table S2-9] TIFF0007629649000032.tif243150[Table S2-10] TIFF0007629649000033.tif244150[Table S2-11] TIFF0007629649000034.tif243149[Table S2-12] TIFF0007629649000035.tif243149[Table S2-13] TIFF0007629649000036.tif244149[Table S2-14] TIFF0007629649000037.tif243149[Table S2-15] TIFF0007629649000038.tif243149[Table S2-16] TIFF0007629649000039.tif243142[Table S2-17] TIFF0007629649000040.tif244149[Table S2-18] TIFF0007629649000041.tif243149[Table S2-19] TIFF0007629649000042.tif243150[Table S2-20] TIFF0007629649000043.tif243149[Table S2-21] TIFF0007629649000044.tif244149[Table S2-22] TIFF0007629649000045.tif243150[Table S2-23] TIFF0007629649000046.tif243150[Table S2-24] TIFF0007629649000047.tif243143[Table S2-25] TIFF0007629649000048.tif244147[Table S2-26] TIFF0007629649000049.tif244149[Table S2-27] TIFF0007629649000050.tif243149[Table S2-28] TIFF0007629649000051.tif243150[Table S2-29] TIFF0007629649000052.tif244151 [Table S2-30] TIFF0007629649000053.tif244150[Table S2-31] TIFF0007629649000054.tif243150[Table S2-32] TIFF0007629649000055.tif244149[Table S2-33] TIFF0007629649000056.tif244151 [Table S2-34] TIFF0007629649000057.tif244151[Table S2-35] TIFF0007629649000058.tif244151 [Table S2-36] TIFF0007629649000059.tif241144[Table S2-37] TIFF0007629649000060.tif244149 [Table S2-38] TIFF0007629649000061.tif244149 [Table S2-39] TIFF0007629649000062.tif245149 [Table S2-40] TIFF0007629649000063.tif243151[Table S2-41] TIFF0007629649000064.tif244150 [Table S2-42] TIFF0007629649000065.tif244150 [Table S2-43] TIFF0007629649000066.tif243150[Table S2-44] TIFF0007629649000067.tif244151 [Table S2-45] TIFF0007629649000068.tif244142 [Table S2-46] TIFF0007629649000069.tif244146 [Table S2-47] TIFF0007629649000070.tif243146[Table S2-48] TIFF0007629649000071.tif244152[Table S2-49] TIFF0007629649000072.tif244150[Table S2-50] TIFF0007629649000073.tif244132

[0167] Table S3. Primer and synthetic nucleotide sequences [Table S3-1] TIFF0007629649000074.tif228158[Table S3-2] TIFF0007629649000075.tif232157[Table S3-3] TIFF0007629649000076.tif232157[Table S3-4] TIFF0007629649000077.tif238158[Table S3-5] TIFF0007629649000078.tif218157[Table S3-6] TIFF0007629649000079.tif199156[Table S3-7] TIFF0007629649000080.tif200156[Table S3-8] TIFF0007629649000081.tif200156[Table S3-9] TIFF0007629649000082.tif200156[Table S3-10] TIFF0007629649000083.tif200156[Table S3-11] TIFF0007629649000084.tif200156[Table S3-12] TIFF0007629649000085.tif200156[Table S3-13] TIFF0007629649000086.tif201156[Table S3-14] TIFF0007629649000087.tif200156[Table S3-15] TIFF0007629649000088.tif200157 [Table S3-16] TIFF0007629649000089.tif200158 [Table S3-17] TIFF0007629649000090.tif202156 [Table S3-18] TIFF0007629649000091.tif201158 [Table S3-19] TIFF0007629649000092.tif204161 [Table S3-20] TIFF0007629649000093.tif202158 [Table S3-21] TIFF0007629649000094.tif201157 [Table S3-22] TIFF0007629649000095.tif200157 [Table S3-23] TIFF0007629649000096.tif201159 [Table S3-24] TIFF0007629649000097.tif200156 [Table S3-25] TIFF0007629649000098.tif211169 [Table S3-26] TIFF0007629649000099.tif233158[Table S3-27] TIFF0007629649000100.tif59161

[0168] From the above, it will be appreciated that the present disclosure describes screening individuals who were vaccinated or spontaneously recovered from HBV infection. Antibody cloning from memory B cells revealed that all five of the top individuals produced clones of broadly neutralizing antibodies (bNAbs) targeting three non-overlapping epitopes on the HBV S antigen (HBsAg). Clones with the same immunoglobulin variability, diversity, and joining heavy and light chain genes were shared among the elite neutralizers. A single bNAb protected humanized mice from infection but selected for resistance mutations in mice with established infection. In contrast, a combination of bNAbs targeting non-overlapping epitopes and with complementary sensitivity to mutations commonly emerging during human infection controlled infection in the absence of detectable escape mutations. The co-crystal structure of one of the bNAbs with a peptide epitope containing residues frequently mutated in human immune escape mutants revealed a loop anchored by oppositely charged residues. This structure provides a molecular explanation for why immunotherapy for chronic HBV infection may require a combination of complementary bNAbs as described herein.

[0169] While the present disclosure has been described via specific embodiments, routine modifications will be apparent to those skilled in the art and such modifications are intended to be within the scope of the present disclosure.

Claims

1. A combination of isolated or recombinant antibodies or antigen-binding fragments thereof administered simultaneously or sequentially to a subject to prevent or treat Hepatitis B virus infection, comprising: The combination is an H019 antibody or an antigen-binding fragment thereof that is an anti-HBs antibody; and The antibody H017 or an antigen-binding fragment thereof is an anti-HBs antibody, The H019 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 that is GGSITTGDYY (SEQ ID NO: 1010), a heavy chain CDR2 that is IYYSGST (SEQ ID NO: 1011), and a heavy chain CDR3 that is AIYMDEAWAFEI (SEQ ID NO: 1012); and a light chain CDR1 that is QSVGNY (SEQ ID NO: 1013), a light chain CDR2 that is AVS, and a light chain CDR3 that is QQSYTISLFT (SEQ ID NO: 1014), The H017 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 that is GFTFSNYW (SEQ ID NO: 965), a heavy chain CDR2 that is ISTDGSST (SEQ ID NO: 966), and a heavy chain CDR3 that is ARGSTYYFGSGSVDY (SEQ ID NO: 967); and a light chain CDR1 that is SSDIGVYNY (SEQ ID NO: 968), a light chain CDR2 that is DVT, and a light chain CDR3 that is SSYRGSSTPYV (SEQ ID NO: 969). combination.

2. A combination of isolated or recombinant antibodies or antigen-binding fragments thereof administered simultaneously or sequentially to a subject to prevent or treat Hepatitis B virus infection, comprising: The combination is H019 antibody or an antigen-binding fragment thereof which is an anti-HBs antibody; an anti-HBs antibody, H017 antibody or an antigen-binding fragment thereof; and The antibody H016 or an antigen-binding fragment thereof is an anti-HBs antibody, The H019 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 that is GGSITTGDYY (SEQ ID NO: 1010), a heavy chain CDR2 that is IYYSGST (SEQ ID NO: 1011), and a heavy chain CDR3 that is AIYMDEAWAFEI (SEQ ID NO: 1012); and a light chain CDR1 that is QSVGNY (SEQ ID NO: 1013), a light chain CDR2 that is AVS, and a light chain CDR3 that is QQSYTISLFT (SEQ ID NO: 1014), The H017 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 that is GFTFSNYW (SEQ ID NO: 965), a heavy chain CDR2 that is ISTDGSST (SEQ ID NO: 966), and a heavy chain CDR3 that is ARGSTYYFGSGSVDY (SEQ ID NO: 967); and a light chain CDR1 that is SSDIGVYNY (SEQ ID NO: 968), a light chain CDR2 that is DVT, and a light chain CDR3 that is SSYRGSSTPYV (SEQ ID NO: 969), The H016 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 that is GFTFPSHT (SEQ ID NO: 855), a heavy chain CDR2 that is ISTTSEAI (SEQ ID NO: 856), and a heavy chain CDR3 that is ASVGLDSKISGYWYFDL (SEQ ID NO: 857); and a light chain CDR1 that is QSISSN (SEQ ID NO: 858), a light chain CDR2 that is RAS, and a light chain CDR3 that is QQYDHWPLT (SEQ ID NO: 859). combination.

3. A combination as described in claim 1 or 2, administered in an effective amount to an individual in need thereof.

4. A combination described in any one of claims 1 to 3 for use in providing a therapeutic and / or prophylactic effect against infection with Hepatitis B virus containing one or more escape mutations in at least one of the HBsAg or S proteins of the Hepatitis B virus.

5. A pharmaceutical composition for use in the prevention or treatment of hepatitis B virus infection, comprising a combination according to any one of claims 1 to 4.

6. A combination of recombinant expression vectors used to produce the combination described in any one of claims 1 to 4, wherein the vectors encode the heavy and light chains of any one of the antibodies or antigen-binding fragments thereof described in any one of claims 1 to 4.

7. A plurality of cells comprising the combination of recombinant expression vectors described in claim 6.

8. A method for producing a combination of antibodies described in any one of claims 1 to 4, comprising culturing a plurality of cells described in claim 7, and isolating antibodies from the plurality of cells.

9. A kit for producing a combination described in any one of claims 1 to 4, comprising a combination of recombinant expression vectors described in claim 6.

10. A method for detecting hepatitis B virus, comprising: A method comprising contacting a biological sample from an individual with a combination of antibodies according to any one of claims 1 to 4 and detecting the presence of a complex comprising at least one of the antibodies and a Hepatitis B virus protein.

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