ANTIBODIES TO HEPATITIS B

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 to 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 entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant no.

UL1TR001866 awarded by The National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated 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

[0004] Despite the existence of effective vaccines, hepatitis B virus (HBV) infection remains a major global health problem with an estimated 257 million people living with the infection. Whereas 95% of adults and 50-75% of children between the ages of 1 and 5 years spontaneously control HBV, only 10% of infants recover naturally. The remainder develop a chronic infection that can lead to liver cirrhosis and hepatocellular carcinoma. Although chronic infection can be suppressed with antiviral medications, there is no effective curative therapy (Dienstag, 2008; Revill et al., 2016; Thomas, 2019).

[0005] HBV is an enveloped double-stranded DNA virus of the Hepadnaviridae family. Its genome is the smallest genome among pathogenic human DNA viruses, with only four open reading frames. Infected liver cells produce both infectious HBV virions (Dane particles) and non-infectious subviral particles (Australia antigen) (Dane et al., 1970; Hu and Liu, 2017). The virion is a 42 nm-diameter particle containing the viral genome and HBV core antigen (HBcAg) encapsidated by a lipid membrane containing 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 , and ayr). Genetic analysis revealed several highly conserved domains and defined eight genotypes A-H, which are highly correlated with the 4 serotypes (Norder et ah, 2004). The HBV surface protein, HBsAg, has 4 putative transmembrane domains and can be subdivided into PreSl-, PreS2- and S-regions. The S domain is a cysteine-rich protein consisting of 226 amino acids that contain all 4 of the transmembrane domains (Abou-Jaoude and Sureau, 2007). In addition, the S-protein can be glycosylated at asparagine residue 146 (Julithe et ah, 2014).

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

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

BRIEF SUMMARY

[0009] The disclosure provides in part a description of the human humoral immune response to HBsAg in immunized and spontaneously recovered individuals that had been selected for high levels of serum neutralizing activity. The disclosure demonstrates that these individuals develop 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 to explain why certain amino acid residues are frequently mutated in escape viruses and why combinations of bNAbs may be needed to control infection. In vivo experiments in humanized mice demonstrate that the bNAbs are protective and can be therapeutic when used in combination.

[0010] Any antibody described herein can comprise at least one modification of its constant region. The modification may be made for any one or more amino acids. The modification can have any of a number of desirable effects. In certain approaches, the modification increases in vivo half-life of the antibody, or alters the ability of the antibody to bind to Fc receptors, or alters the ability of the antibody to cross placenta or to cross a blood- brain barrier or to cross a blood-testes barrier, or inhibits aggregation of the antibodies, or a combination of said modifications, or wherein the antibody is attached to a label or a substrate. In embodiments, the modification improves the manufacturability of the antibody. In embodiments, any antibody or combination thereof described herein can be present in an immunological assay, such as an enzyme-linked immunosorbent assay (ELISA) assay, or an ELISA assay control. The ELISA assay can be any of a direct ELISA assay, an indirect ELISA assay, a sandwich ELISA assay, or a competition ELISA assay.

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

[0012] In another aspect the disclosure provides vaccine formulations. In an embodiment a vaccine formulation comprises an isolated or recombinant peptide or a polynucleotide encoding the peptide, wherein the peptide is derived from an epitope that is frequently targeted by HepB immune resistance, and 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 chain and the light chain CDRs of any of the antibodies of described herein. Cells comprising the recombinant expression vectors are included, as are methods of making antibodies by culturing cells that comprise expression vectors that express the antibodies, and separating antibodies from the cells. Cell culture media containing such cells and/or antibodies is also included.

BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1. Antibody responses in HBV vaccinated and recovered individuals. (A) Donor screen. Sera from 159 volunteers were evaluated for anti-HBs binding by ELISA (x-axis) and HBV serum neutralization capacity using HepG2-NTCP cells (y-axis). Serum neutralization capacity on the y-axis was calculated as the reciprocal of the relative percentage of infected HepG2-NTCP cells. The values for unexposed naive donors are =1. Neutralization tests were performed at 1:5 serum dilution in the final assay volume. Each dot represents an individual donor. Green indicates unvaccinated and unexposed, black indicates vaccinated, and red indicates spontaneously recovered. The dashed line indicates the no serum control. Top neutralizers (serum neutralization capacity higher than 4) are indicated (top right). Boxed 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). Two assays were used to measure percent infection: ELISA to measure HBsAg protein in the medium (upper panels) and immunofluorescent staining for HBcAg in HepG2-NTCP cells (lower panels). Dashed line indicates virus-only control. (D) Schematic representation illustrating the three forms of the HBV surface protein: L-, M- and S-protein. These three forms of envelope protein all share the same S-region, with PreSl/PreS2 and PreS2 alone as the N-terminal extensions for L- and M-protein, respectively. (E) S-protein produced in Chinese hamster ovary (CHO) cells blocks serum neutralizing activity. Graphs show infection efficiency as a function of the amount of S-protein added. The concentration of polyclonal IgG antibodies (pAb) is indicated. Upper and lower panels are as in (B) and (C). A representative of at least two experiments is shown. See also Figure 8 and Table SI.

[0015] Figure 2. S-protein-specific antibodies. (A) Frequency of S-protein-specific memory B cells. Representative flow cytometry plots displaying the percentage of all IgG ⁺ memory B cells that bind to both allophycocyanin- and phycoerythrin-tagged S-protein (S- protein-APC and S-protein-PE). Flow cytometry plots from other individuals are shown in Figure 9A. Experiments were repeated two times. (B) Dot plot showing the correlation between the frequency of S-protein-binding IgG ⁺ memory B cells and the serum neutralizing activity. Spearman’s rank correlation coefficient (r _s ) and significance value (p). (C) Each pie chart represents the antibodies from an individual donor, and the total number of sequenced antibodies with paired heavy and light chains is indicated in the center. Antibodies with the same combination of IGH and IGL variable gene sequences and closely related CDR3s in each individual are shown. The slices with the same color indicate shared antibodies with the same or similar combination of IGH and IGL variable genes between individuals (Figure 9B). Grey slices indicate antibodies with closely related sequences that are unique to a single donor. In white are singlets. (D) V(D)J alignments for representative IGHV3-30/IGLV3-21, IGH V 3-33 /IGL V 3-21 and IGHV3-23/IGLV3-21 antibodies from donors #60/# 146 (H006 and H008), #146/# 13 (HO 14 and HO 12), and #13/#60/#146 (H021, H003 and H004) respectively. Boxed grey residues are shared between antibodies. See also Figure 9 and Table S2. 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).

50% effective concentration (ECso in ng/ml) required for binding of the indicated human monoclonal antibodies to the S-protein. Libivirumab (Eren et al., 2000; Eren et al., 1998) and anti -HIV antibody 10-1074 (Mouquet et al., 2012) were used as positive and negative controls, respectively. All antibodies were tested. (B) Comparative binding of the mature and unmutated common ancestor (UCA) of antibodies H006, HO 19, and H020 to S-protein by ELISA. (C) Anti-HBs antibody binding to 5 different serotypes of HBsAg. Similar to panel (A), EC50 values are color-coded: red, <50 ng/ml; orange, 50 to 100 ng/ml; yellow, 100 to 200 ng/ml; and white, > 200 ng/ml. The abbreviation b.d. indicates below detection. All antibodies were tested. All experiments were performed at least two times. See also Figure 10

[0017] Figure 4. HBsAg epitopes. (A) Competition ELISA defines 3 groups of antibodies. Results of competition ELISA shown as percent of binding by biotinylated antibodies and illustrated by colors: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >76%. Weak binders (H002, H012, H013, H014, H018) were excluded.

Representative of two experiments. (B) Results of ELISA on alanine scanning mutants of S- protein. Only the amino acids vital for antibody binding are shown. Binding to mutants relative to wild-type S-protein: black, 0-25%; dark grey, 26-50%; light grey, 51-75%; white, >75%. Additional details are provided in Figure 11. (C) Results of ELISA on human escape mutations of S-protein. Wild-type S-protein and empty vector serve as a positive and negative controls, respectively. Binding to mutants relative 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). The antibodies tested in (B and C) were selected from Group-I, -II, -III based on their neutralizing activity (Figure 5A- 5C). All experiments were performed at least two times. See also Figure 11.

[0018] Figure 5. In vitro neutralization by the monoclonal antibodies. (A and B)

In vitro neutralization assays using HepG2-NTCP cells. Percent infection in the presence of the indicated concentrations of bNAbs measured by ELISA of HBsAg in medium (A) and anti-HBcAg immunofluorescence (B). Anti -HIV antibody 10-1074 (Mouquet et ak, 2012) and libivirumab (Eren et ak, 2000; Eren et ak, 1998) were used as negative and positive controls respectively. The corresponding ICsos are shown in the left and middle column of panel (C). All experiments were repeated a minimum of two times. (C) bNAb 50% maximal inhibitory concentration (ICso) calculated based on HBsAg ELISA (left column) and HBcAg immunofluorescence (middle column) for the in vitro neutralization assays using HepG2- NTCP cells, or HBeAg ELISA (right column) for in vitro neutralization using primary human hepatocytes. The abbreviation b.d. and n.d. indicate below detection and not done respectively. (D) In vitro neutralization using primary human hepatocytes. The levels of HBeAg in medium were measured by ELISA. The calculated ICso values are shown in the right column of panel (C). Experiments were repeated three times. (E) In vitro neutralization assay using HepG2-NTCP cells. IgG antibodies were compared to their corresponding Fab fragments. Concentrations of Fab fragments were adjusted to correspond to IgG. Experiment was performed two times. See also Figure 12.

[0019] Figure 6. Crystal structure of H015 bound to its recognition motif. A single crystal was used to obtain a high resolution (1.78 A) structure. (A) Synthetic peptides (SEQ ID NOS 1452-1455, respectively, in order of appearance) spanning the antigenic loop region were subjected to ELISA for antibody binding. Among the tested antibodies, only H015 binds peptides-11 and -12. Experiments were performed three times and details are in Figure 13 A. (B and C) The peptide binds to CDR1 (R31), CDR2 (W52 and F53) and CDR3 (E99, P101, LI 03, and LI 04) of HO 15 heavy chain (green) and CDR3 (P95) of the light chain (cyan) (B). The interacting residues (C) on the heavy chain (green) are R31 (main chain), W52, F53 (main chain), E99, P101 (main chain), L103 (main chain), L104 (hydrophobic). One contact with the light chain (cyan) is with P95. (D) Electron density map of the bound peptide as seen in the 2Fo-Fc map contoured at 1 RMSD indicating high occupancy (92%). (E) The recognition motif, KPSDGN (SEQ ID NO: 1), adopts a sharp hairpin conformation due to the salt-bridge between K141 and D144 and is facilitated by kinks at P142 and G145. Glycine 145 (G145, circled) is the residue that escapes the immune system when mutated to an arginine. See also Figure 13.

[0020] Figure 7. Anti-HBs bNAbs are protective and therapeutic in vivo. (A and

E) Diagram of the prophylaxis and treatment protocols, respectively. (B) Prophylaxis with isotype control antibody 10-1074 (Mouquet et ah, 2012). (C and D) Prophylaxis with H020 and H007 respectively. The dashed line in (B-D) indicates the detection limit. (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. HBV DNA levels in serum were monitored on a weekly basis. Two independent experiments comprising a total of 5 to 8 mice were combined and displayed. (I) Mutations in the S-protein sequence from the indicated mice (red arrows) in (G), (H) and (J). S-protein sequence chromotograms are shown in Figure 14. (J-L) Treatment of viremic huFNRG mice with combination of anti-HBs bNAb H006 + H007 (J), or H017 + H019 (K), or HO 16 + HO 17 + HO 19 (L), respectively. Sequencing showed that none of the mice in (K) and (J) carried viruses with escape mutations in the S- protein. See also Figure 14.

[0021] Figure 8. Characterization of Antibody Immune Response Against HBV,

Related to Figure 1. (A) Schematic representation of different stages of HBV infection. Vaccinated or infected naturally recovered individuals were recruited for this study. (B) Sera (1:50 dilution in the final assay volume) from 159 volunteers were screened, see also Figure 1 A. (C-E) Comparison of anti-HBs ELISA titers (upper panel) and their serum neutralization capacity (lower panel) between different groups of individuals. Vaccinated or recovered individuals show statistically higher anti-HBs titers (upper panel, C) and more potent neutralizing activity (lower panel, C) than the uninfected unvaccinated individuals. Younger individuals (<45 years old) showed slightly higher antibody immune response against HBsAg (D). No difference was found between genders (E).

[0022] Figure 9. Antibody Cloning and Sequence Analysis of Anti-HBs, Related to Figure 2. (A) Frequency of S-protein-specific memory B cells in peripheral blood mononuclear cells of all twelve donors. Details are similar to Figure 2A. (B) Pie charts show the distribution of anti-HBs antibodies. Figure legends are similar to Figure 2C. VH and VL genes for each slice are shown and the 20 chosen anti-HBs antibodies are labeled. (C) Phylogenetic tree of all cloned anti-HBs antibodies based on IGH Fab region. IGH Fab regions from 244 memory B cells sorted with HBsAg were aligned followed by tree construction.

[0023] Figure 10. Autoreactivity of 20 anti-HBs antibodies, Related to Figure 3.

(A) Autoreactivity of monoclonal antibodies. Positive control antibody efficiently stained the nucleus of HEp-2 cells. Twenty anti-HBs antibodies, as well as anti-HBs antibody libivirumab and anti -HIV antibody 10-1074, were also tested. (B) Polyreactivity profiles of 20 anti-HBs antibodies. ELISA measures antibody binding to the following antigens: double- stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharides (LPS), and single-stranded DNA (ssDNA). Red and green lines represent positive control antibody ED38 and negative control antibody mG053 respectively, while dashed lines show cut-off values for positive reactivity (Gitlin et al., 2016).

[0024] Figure 11. Alanine Scanning and Peptide Screening, Related to Figure 4.

(A) Results of ELISA on 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 three times. Underlined cysteines, alanines, and amino acids known to be critical for S-protein production were not mutated (Salisse and Sureau, 2009). Figure discloses SEQ ID NO: 1456. (B) Schematic diagram of alanine scanning results. Figure discloses the primary amino acid sequence as SEQ ID NO: 1456 and the sequence containing alanine mutations as SEQ ID NO: 1457.

[0025] Figure 12. In Vitro Neutralization Assay of anti-HBs bNAb Unmutated

Common Ancestor Antibodies or Combinations, Related to Figure 5. (A-B) In vitro neutralization assay of anti-HBs bNAbs and their corresponding unmutated common ancestor (UCA) antibodies. The relative infection rates were calculated based on either HBsAg protein level in culture medium (A) or HBcAg staining intracellularly (B). (C) In vitro neutralization assay of anti-HBs bNAbs recognizing different epitopes and the same total amount of antibody combination at 1 : 1 or 1 : 1 : 1 ratio.

[0026] Figure 13. Detailed Information of Crystal Structure of H015 and Its

Linear Epitope, Related to Figure 6. (A) Synthesized peptides (SEQ ID NOS 1458-1476, respectively, in order of appearance) for antigenic loop region were subjected to ELISA for antibody binding. Among the tested antibodies, only H015 binds peptide-11 and -12. (B)

Data collection and refinement statistics for H015 Fab are summarized. Statistics for the highest-resolution shell are shown in parentheses. Refinement program PHENIX 1.16. (C) The green/red density is the unbiased omit map. Red is negative density equated to noise. (D) Table of contacts within the peptide and between Fab fragment and peptide. [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 by control antibody 10-1074, anti-HBs bNAb H020, anti-HBs bNAb H007, combination of anti-HBs bNAb (H006 + H007), (HO 17 + HO 19), and (HO 16 + HO 17 + HO 19). HBV DNA levels in mouse sera were monitored on a weekly basis. The mice without arrows bear no escape mutations at the last time point. (B) Part of the S-protein sequences from the indicated mice (arrows and numbers) are shown below as chromatograms, with mutations marked by arrowheads. (B) discloses the S-protein amino acid and nucleotide sequences as SEQ ID NOS 1477 and 1478, respectively. The sequences represented by the subsequent chromatograms that disclose amino acid residues and nucleotides are SEQ ID NOS 1479, 1480, 1480, 1480-1482, 1479, 1478, 1480, 1480, 1478, 1480, and 1483-1488, respectively, in order of columns. (C-D) HBsAg levels in mouse sera before and after antibody infusion. Mice were treated by anti-HBs combination HO 17 + HO 19 (C) (see Figure 7K) and HO 16 + HO 17 + HO 19 (D) (see Figure 7L). Each line represents a mouse with concentrations of serum HBsAg level expressed in NCU/ml (national clinical units per milliliter).

DETAILED DESCRIPTION OF THE DISCLOSURE

[0028] Unless defined otherwise, 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 given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0030] This disclosure includes every nucleotide sequence described herein, and in the tables and figures, and all sequences that are complementary to them, RNA equivalents of DNA sequences, all amino acid sequences described herein, and all polynucleotide sequences encoding the amino acid sequences. Every antibody sequence and functional fragments of them are included. Polynucleotide and amino acid sequences having from 80-99% similarity, inclusive, and including ranges of numbers there between, with the sequences provided here are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions, such as conservative substitutions, that do not adversely affect the function of the protein or polypeptide that comprises the amino acid sequences. It will be recognized that when reference herein is made to an “antibody” it does not necessarily mean a single antibody molecule. For example, “administering an antibody” includes administering a plurality of the same antibodies. Likewise, a composition comprising an “antibody” can comprise a plurality of the same antibodies.

[0031] For amino acid and polynucleotide sequences of this disclosure, contiguous segments of the sequences are included, and can range from 2 amino acids, up to full-length protein sequences. Polynucleotide sequences encoding such segments are also included. [0032] The disclosure includes DNA and RNA sequences encoding the antibodies and antigen fragments thereof, and any virus peptides described herein for use in prophylactic and therapeutic approaches as protein or DNA and/or RNA vaccines, which may be formulated and/or delivered according to known approaches, given the benefit of this disclosure. The disclosure includes a cDNA sequences encoding the antibodies, antigen binding fragments thereof, and any viral proteins or peptides described herein. Expression vectors that contain cDNAs are also included, and encode said antibodies, antigen binding fragments thereof, and viral proteins and peptides.

[0033] All sequences from the figures, text, and tables of this application or patent include every amino acid sequence associated with every Donor ID, and all possible combinations of the amino acid sequences given for all complementarity determining regions (CDRs), e.g., all combinations of heavy chain CDR1, CDR2, CDR3 sequences, and all combinations of light chain CDR1, CDR2, and CDR3 sequences, including 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. One or more antibodies may also be excluded from any combination of antibodies.

[0034] The disclosure includes antibodies described herein, which are present in an in vitro complex with one or more hepatitis B proteins.

[0035] In embodiments, the disclosure provides an isolated or recombinant antibody that binds with specificity to a hepatitis B virus epitope, and wherein the antibody optionally comprises a modification of its amino acid sequence, including but not limited to a modification of its constant region.

[0036] In embodiments, one or more antibodies described herein bind with specificity to an epitope present in the HBsAg protein or the S-protein in the unmutated, or mutated form.

[0037] In embodiments, the antibodies described herein bind to a hepatitis B protein that comprises one or more HepB escape mutations. In embodiments, the antibodies bind to a hepatitis B virus protein that comprises a mutation that is a substitution of a large positively charged residue for a small neutral residue. In embodiments, the mutation is present in the so- called “a” determinant area, which is known in the art. In embodiments, the epitope is present in the major hydrophilic region of the HBsAg protein. In embodiments, the epitope to which the antibodies bind is present 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 HBsAg L-protein, M-protein, or S-protein. In embodiments, the antibodies bind to an epitope present in the L-protein version of HBsAg, which comprises the amino acid sequence that is accessible via Accession number: AAL66340.1 as that amino acid sequence exists in the database as of the filing date of this application or patent. In an embodiment, this amino acid sequence is:

MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQ VG

AGAFGPGFTPPHGGLLGWSPQAQG1LTTVPVAPPPASTNRQSGRQPTP1SPPLRDSH PQAMQ

WNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASP1SS1FSRTGDPAPNMEST TSGFLGP

LLVLQAGFFLLTR1LT1PQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPP1 CPGYRWM

CLRRF11FLF1LLLCL1FLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTSPAQGTS MFPSCCCTKP

SDGNCTC1P1PSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSV1WMMW YWG

PCLYN1LSPFLPLLP1FFCLWVY1 (SEQ ID NO: 2).

[0039] In embodiments, the disclosure includes use of only two proteins, or at least two proteins. In an embodiment, the S proteins may be used as bait to sort 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, the amino acid sequence of which is incorporated herein as it exists in the database at the filing date of this application or patent. In an embodiment, the S protein comprises the sequence:

MENTASGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPT SNHSPTS CPP1CPGYRWMCLRRF11FLF1LLLCL1FLLVLLDYHGMLPVCPLLPGTSTTSTGPCKTC T1PAQG TSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTV WLS V1WMMWYWGPSLYN1LSPFLPLLP1FFCLWVY1 (SEQ ID NO: 3).

[0040] In non-limiting embodiments, the S polynucleotide sequence used for alanine scanning comprises: ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTC TTG

TTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAAT TTTCTA

GGGGGATCTCCCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCA CCAACC

TCCTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATA TTCCTC

TTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGATTATCAAGGTATG TTGCCC

GTTTGTCCTCTAATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACCTGC ACGACT

CCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGA AATTGC

ACCTGTATTCCCATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCC TCAGTC

CGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCC CCCACT

GTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATC GTGAG

TCCCTTTATACCGCTGTTACCAATTTTCTTTTGTCTCTGGGTATACATTTAA (SEQ ID NO:

4)·

[0041] The amino acid sequence encoded by the DNA sequence immediately above is:

MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPT SNHSPTSC PP1CPGYRWMCLRRF11FLF1LLLCL1FLLVLLDYQGMLPVCPL1PGSTTTSTGPCKTCT TPAQGN SMFPSCCCTKPTDGNCTC1P1PSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVW LS A1WMMWYWGPSLYS1VSPF1PLLP1FFCLWVY1 (SEQ ID NO: 5).

[0042] In embodiments, antibodies of this disclosure bind to an epitope present in any of the foregoing amino sequences, including linear and confirmation epitopes that may be formed by proteins comprising or consisting of said sequences.

[0043] In an embodiment, the isolated or recombinant antibody or antigen binding fragment thereof binds with specificity to an epitope comprised by a structurally defined peptide loop, as further described herein. In embodiments, the loop is as generally depicted in Figure 6, which comprises a partial structure of HepB surface protein, and demonstrates the existence of a loop that includes the most frequently targeted residue found in human escapes G145. Without intending to be bound by any particular theory, it is considered that this structure explains why this mutant can escape, and also why additional commonly found escape mutants exist. Further, the structure and the antibody peptide complex represents a new and previously undiscovered target for drug discovery. Thus, in embodiments, the disclosure provides for screening drug candidates that can interfere with formation of this structure, and thus which may also interfere with the viability of the virus. Those skilled in the art will recognize from the present disclosure how to design an assay to determine whether or not drug candidates could interfere with the complex, and how antibodies described in herein may be used in such an assay.

[0044] In embodiments, antibodies described herein bind with specificity to an amino acid sequence comprised by any peptide sequence described herein. In embodiments, the peptide comprises the sequence KPSDG (SEQ ID NO: 6), or mutants thereof. In embodiments, antibodies described herein bind with specificity to an epitope in an amino acid sequence that comprises the sequence PSSSSTKPSDGNSTS (SEQ ID NO: 7), or mutants thereof. Additional and non-limiting examples of peptides of this disclosure include those shown on Figure 6, e.g., peptide-11 and peptide-12.

[0045] In embodiments, the disclosure comprises compositions and methods that involve use of more than one distinct antibody or antigen binding fragment thereof. In embodiments, the methods of this disclosure comprise administering a combination of antibodies or antigen binding fragment thereof which bind distinct hepatitis B epitopes. In embodiments, distinct antibodies recognize epitopes present in two dominant non overlapping antigenic sites on the HBsAg, or epitopes present on the S-protein. In embodiments, the disclosure provides for use of a combination of the Group-I and Group-II antibodies described herein. Thus, the disclosure comprises co-administration or sequential administration of a combination of antibodies. In an embodiment, administration of a combination of distinct antibodies suppresses formation of viruses that are resistant to the effects of any one of the antibodies alone. In embodiments, the disclosure includes administering a combination comprising at least one Group I antibody and at least one Group II antibody, wherein at least one of the antibodies is G145R mutation resistant. In non limiting embodiments, antibodies that are provided by the present disclosure, and which can be administered to an individual in need thereof, comprise at least one of H006, H007,

H0017, H0019, or H020. Further, H005, H008 and H009 are similar to H006, and therefore may be used as alternatives to 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 this disclosure may comprise an H+L chain from one antibody, and an H+L chain from another antibody. In embodiments, the antibodies comprise modifications that are not coded for in any B cells obtained from an individual, and/or the antibodies are not produced by immune cells in an individual from which a biological sample from the individual is used at least in part to identify and/or generate and/or characterize the antibodies of this disclosure. In embodiments, antibodies provided by this disclosure can be made recombinantly, and can be expressed with a constant region of choice, which may be different from a constant region that was coded for in any sample from which the amino acid sequences of the antibodies were deduced.

[0047] As discussed above, in embodiments, the 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, a combination of antibodies of this disclosure are effective in preventing viral escape by mutation. In this regard, the disclosure includes data demonstrating that not all antibody combinations are effective in preventing escape by mutation, such as the combination of H006 and H007, which are ineffective. Thus, in embodiments, a combinations of antibodies or antigen binding fragments collectively target more than one commonly occurring escape mutation, examples of which escape mutations are known in the art and are described herein. Accordingly, combinations of antibodies and antigen binding fragments thereof of this disclosure may target non-overlapping groups of common escape mutations. In embodiments, the disclosure thus includes a proviso that excludes any combination of antibodies that collectively only target separate epitopes but have overlapping sensitivity to commonly occurring escape mutations.

[0048] In embodiments, at least one antibody or antigen binding fragment thereof included in this disclosure, and in the combinations and methods of this disclosure, has greater virus neutralizing activity than a control neutralizing activity value, such as the neutralizing capability of libivirumab. In embodiments, at least one antibody or antigen binding fragment of this disclosure exhibits a viral neutralizing activity with an ICso values that is less than 128 ng/ml, or less than 35 ng/ml, or less than 5 ng/ml, and including all integers and ranges of integers between 128 and 5 ng/ml. Such neutralizing activity can be determined using known approaches, such as by ELISA or immunofluorescence assays, and as further described in Example 5 of this disclosure. In embodiments, an antibody or antigen binding fragment thereof that is encompassed by this disclosure includes but is not limited to antibodies or antigen binding fragments selected from the HO 16, HO 17 and HO 19 antibodies, as defined by their CDRs. In an embodiment, the disclosure includes combinations of these antibodies, and can include antigen binding fragments thereof. In embodiments, the combination of antibodies comprises the HO 17 and HO 19 antibodies, and/or antigen binding fragments thereof. In an embodiment, the combination optionally further comprises the HO 16 antibody or an antigen binding fragment thereof. In embodiments, a combination of the disclosure comprises a combination that consists of only the HO 17 and HO 19 antibodies or antigen binding fragments thereof. In embodiments, a combination of the disclosure comprises a combination that consists of only the HO 16, HO 17, and HO 19 antibodies or antigen binding fragments thereof. Methods of administration of the described antibody combinations, and all other antibodies and antigen binding fragments thereof described herein, sequentially and concurrently are included within the scope of this disclosure. Thus, the disclosure includes administering to an individual in need concurrently or sequentially a combination of antibodies or antigen binding fragments thereof, which in certain embodiments comprise or consist of HO 17 and HO 19, or HO 16, HO 17, and HO 19 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 that comprise the heavy and light chain CDRs of H004, H005, and H009, and H020.

[0049] With respect to the HO 16, HO 17, and HO 19 antibodies, as can been seen from

Table S2, the HO 16 antibody comprises a heavy chain CDR1 with the amino acid sequence GFTFPSHT (SEQ ID NO: 8), a heavy chain CDR2 with the amino acid sequence ISTTSEAI (SEQ ID NO: 9), and a heavy chain CDR3 with the amino acid sequence ARV GLALTISGYWYFDL (SEQ ID NO: 10). The H016 antibody comprises a kappa light chain CDR1 with the amino acid sequence QSISSN (SEQ ID NO: 11), a kappa light chain with the CDR2 amino acid sequence RAS, and a kappa light chain with the CDR3 amino acid sequence QQYDHWPLT (SEQ ID NO: 12).

[0050] As can be seen from Table S2, the HO 17 antibody comprises a heavy chain

CDR1 with the amino acid sequence GFTFSNYW (SEQ ID NO: 13), a heavy chain CDR2 with the amino acid sequence ISTDGSST (SEQ ID NO: 14), and a heavy chain CDR3 with the amino acid sequence ARGSTYYFGSGSVDY (SEQ ID NO: 15). The H017 antibody comprises a lambda light chain with the CDR1 sequence SSDIGVYNY (SEQ ID NO: 16), a lambda light chain with the CDR2 sequence DVT, and a lambda light chain with the CDR3 sequence SSYRGSSTPYV (SEQ ID NO: 17).

[0051] As can be seen from Table S2, the HO 19 antibody comprises a heavy chain

CDR1 with the amino acid sequence GGSITTGDYY (SEQ ID NO: 18), a heavy chain CDR2 with the amino acid sequence IYYSGST (SEQ ID NO: 19), and a heavy chain CDR3 with the amino acid sequence AIYMDEAWAFE (SEQ ID NO: 20). The H019 antibody comprises a lambda light chain CDR1 with the amino acid sequence QSIGNY (SEQ ID NO: 21), a lambda light chain with the CDR2 amino acid sequence AVS, and a lambda light chain with the CDR3 amino acid sequence QQSYTISLFT (SEQ ID NO: 22). [0052] In certain embodiments, the antibodies contain one or more modifications, such as non-naturally occurring mutations. As non-limiting examples, in certain approaches the Fc region of the antibodies can be changed, and may be of any isotype, including but not limited to any IgG type, or an IgA type, etc. Antibodies of this disclosure can be modified to improve certain biological properties of the antibody, e.g., to improve stability, to modify effector functions, to improve or prevent interaction with cell-mediated immunity and transfer across tissues (placenta, blood-brain barrier, blood-testes barrier), and for improved recycling, half-life and other effects, such as manufacturability and delivery.

[0053] In embodiments, an antibody of this disclosure can be modified by using techniques known in the art, 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, pl57-159, and Ko, S-Y, et al., (2014) Nature, Vol. 514, p642-647, and Horton, H. et al., Cancer Res 2008; 68: (19), October 1, 2008, from which the descriptions are incorporated herein by reference. In certain embodiments an antibody modification increases in vivo half-life of the antibody (e.g. LS mutations), or alters the ability of the antibody to bind to Fc receptors (e.g. GRLR mutations), or alters the ability to cross the placenta or to cross the blood-brain barrier or to cross the blood-testes barrier. Thus, in certain embodiments an antibody modification comprises a change of G to R, L to R, M to L, or N to S, or any combination thereof.

[0054] In embodiments bi-specific antibodies are provided by modifying and/or combining segments of antibodies as described herein, such as by combining heavy and light chain pairs from distinct antibodies into a single antibody. Suitable methods of making bispecific antibodies are known in the art, such as in Kontermann, E. et al., Bispecific antibodies, Drug Discovery Today, Volume 20, Issue 7, July 2015, Pages 838-847, the description of which is 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, thus rendering them distinct from antibodies produced by humans. In embodiments, the modification is made in a hypervariable region, and/or in a framework region (FR).

[0056] In embodiments, mutations to an antibody described herein, including but not limited to the antibodies described, comprise modifications relative to the antibodies originally produced in humans. Such modifications include but are not necessarily limited to the heavy chain to increase the antibody half-life. [0057] In embodiments, antibodies of this disclosure have variable regions that are described herein, and may comprise or consist of any of these sequences, and may include sequences that have from 80-99% similarity, inclusive, and including ranges of numbers there between, with the sequences expressly disclosed herein, provided antibodies that have differing sequences retain the same or similar binding affinity as an antibody with an unmodified sequence. In embodiments, the sequences are at least 95%, 96%, 97%, 98% or 99% similar to an expressly disclosed sequence herein.

[0058] Antibodies comprising the sequences described in Table S2 have been isolated and characterized for at least binding affinity, and as otherwise described herein, such as for virus neutralizing activity. Thus, in embodiments the disclosure provides neutralizing antibodies. The term “neutralizing antibody” refers to an antibody or a plurality of antibodies that inhibits, reduces or completely prevents viral infection. Whether any particular antibody is a neutralizing antibody can be determined by in vitro assays described in the examples below, and as is 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] Antibodies of this 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 part of the heavy chain) fragments, Fv fragments (the two variable domains), dAb fragments, single domain fragments or single monomeric variable antibody domains, isolated CDR regions, single-chain variable fragment (scFv), and other antibody fragments that retain virus-binding capability and preferably virus neutralizing activity as further described below. In embodiments, the variable regions, including but not necessarily limited to the described CDRs, may be used as a component of a Bi-specific T-cell engager (BiTE), bispecific killer cell engager (BiKE), or a chimeric antigen receptor (CAR), such as for producing chimeric antigen receptor T cells (e.g. CAR T cells). In embodiments, the disclosure includes tri-valent antibodies, which can bind with specificity to three different epitopes.

[0060] Antibodies and antigens of this disclosure can be provided in pharmaceutical formulations. It is considered that administering a DNA or RNA polynucleotide encoding any protein described herein (including peptides and polypeptides), such as antibodies and antigens described herein, is also a method of delivering such proteins to an individual, provided the protein is expressed in the individual. Methods of delivering DNA and RNAs encoding proteins are known in the art and can be adapted to deliver the protein, particularly the described antigens, given the benefit of the present disclosure. Similarly, the antibodies of this disclosure can be administered as DNA molecules encoding for such antibodies using any suitable expression vector(s), or as RNA molecules encoding the antibodies.

[0061] Pharmaceutical formulations containing antibodies or viral antigens or polynucleotides encoding them can be prepared by mixing them with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents and the like. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents such as EDTA; salt forming counter-ions such as sodium; non-ionic surfactants such as TWEEN, PLEIRONICS or polyethylene glycol (PEG), or combinations thereof. In embodiments, a pharmaceutical/vaccine formulation exhibits an improved activity relative to a control, such as antibodies that are delivered without adding additional agents, or a particular added agent improves the activity of the antibodies.

[0062] The formulation can contain more than one antibody type or antigen, and thus mixtures of antibodies, and mixtures of antigens, and combinations thereof as described herein can be included. These components can be combined with a carrier in any suitable manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder (including lyophilized powder), syrup, suspensions that are suitable for injections, ingestions, infusion, or the like. Sustained-release preparations can also be prepared.

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

[0064] The term "treatment" of viral infection refers to effective inhibition of the viral infection so as to delay the onset, slow down the progression, reduce viral load, and/or ameliorate the symptoms caused by the infection.

[0065] The term "prevention" of viral infection means the onset of the infection is delayed, and/or the incidence or likelihood of contracting the infection is reduced or eliminated.

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

[0067] The dosage of an antibody or antigen vaccine depends on the disease state and other clinical factors, such as weight and condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration. The precise dosage to be therapeutically effective and non-detrimental can be determined by those skilled in the art. As a general rule, a suitable dose of an antibody for the administration to adult humans parenterally is in the range of about 0.1 to 20 mg/kg of patient body weight per day, once a week, or even once a month, with the typical initial range used being in the range of about 2 to 10 mg/kg. Since the antibodies will eventually be cleared from the bloodstream, re administration may be required. Alternatively, implantation or injection of the antibodies provided in a controlled release matrix can be employed.

[0068] The antibodies can be administered to the subject by standard routes, including oral, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). In addition, the antibodies and/or the antigen vaccines can be introduced into the body, by injection or by surgical implantation or attachment such that a significant amount of an antibody or the vaccine is able to enter blood stream in a controlled release fashion. In certain embodiments antibodies described herein are incorporated into one or more prophylactic compositions or devices to, for instance, neutralize a virus before it enters cells of the recipient’s body. For example, in certain embodiments a composition and/or device comprises a polymeric matrix that may be formed as a gel, and comprises at least one of hydrophilic polymers, hydrophobic polymers, poly(acrylic acids) (PAA), poly(lactic acids) (PLA), carageenans, polystyrene sulfonate, polyamides, polyethylene oxides, cellulose, poly(vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), chitosan, poly(ethylacrylate), methylmethacrylate, chlorotrimethyl ammonium methylmethacrylate, hydroxyapatite, pectin, porcine gastric mucin, poly(sebacic acid) (PSA), hydroxypropyl methylcellulose (HPMC), cellulose acetate phthalate (CAP), magnesium stearate (MS), polyethylene glycol, gum-based polymers and variants thereof, poly (D,L)-lactide (PDLL), polyvinyl acetate and povidone, carboxypolymethylene, and derivatives thereof. In certain aspects the disclosure comprises including antibodies in micro- or nano-particles 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 a gel of this disclosure comprises a carbomer, methylparaben, propylparaben, propylene glycol, sodium carboxymethylcellulose, sorbic acid, dimethicone, a sorbitol solution, or a combination thereof. In embodiments a gel of this disclosure comprises one or a combination of benzoic acid, BHA, mineral oil, peglicol 5 oleate, pegoxol 7 stearate, and purified water, and can include any combination of these compositions.

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

[0070] In embodiments, the antibodies may be isolated from cells. In embodiments, the antibodies are recombinant antibodies. “Recombinant” antibodies mean the antibodies are produced by expression within cells from one or more expression vectors.

[0071] In certain approaches the disclosure includes neutralizing antibodies as discussed above, and methods of stimulating the production of such antibodies.

[0072] In certain approaches the disclosure includes vaccinating an individual using a composition described herein, and determining the presence, absence, and/or an amount of neutralizing antibodies produced in response to the vaccination. Thus, methods of determining and monitoring efficacy of a vaccination at least in terms of neutralizing antibody production are included. In an embodiment, subsequent to determining an absence of neutralizing antibodies, and/or an amount of neutralizing antibodies below a suitable reference value, the invention includes administering a composition disclosed herein to the individual. Subsequent administrations and measurements can be made to track the treatment efficacy and make further adjustments to treatment accordingly.

[0073] Antibodies and proteins of this disclosure can be detectably labeled and/or attached to a substrate. Any substrate and detectable label conventionally used in immunological assays and/or devices is included. In embodiments the substrate comprises biotin, or a similar agent that binds specifically with another binding partner to facilitate immobilization and/or detection and/or quantification of antibodies and/or viral proteins. [0074] In embodiments any type of enzyme-linked immunosorbent (ELISA) assay can be used, and can be performed using polypeptides and/or antibodies of this disclosure for diagnostic purposes, and can include direct, indirect, and competitive ELISA assays, and adaptations thereof that will be apparent to those skilled in the art given the benefit of this disclosure.

[0075] Any diagnostic result described herein can be compared to any suitable control. Further, any diagnostic result can be fixed in a tangible medium of expression and communicated to a health care provider, or any other recipient. In one aspect the disclosure comprises diagnosing an individual as infected with hepatitis B virus and administering a composition of this invention to the individual.

[0076] In certain embodiments the disclosure includes one or more recombinant expression vectors encoding at least H and L chains of an antibody or antigen binding fragment of this disclosure, cells and cell cultures comprising the expression vectors, methods comprising culturing such cells and separating antibodies from the cell culture, the cell culture media that comprises the antibodies, antibodies that are separated from the cell culture, and kits comprising the expression vectors encoding an antibody and/or a polypeptide of this disclosure. Products containing the antibodies and/or the polypeptides are provided, wherein the antibodies and/or the polypeptides are provided as a pharmaceutical formulation contained in one or more sealed containers, which may be sterile and arranged in any manner by which such agents would be suitable for administration to a human or non human subject. The products / kits may further comprise one or more articles for use in administering the compositions.

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

Example 1

[0078] Serologic Responses Against HBV

[0079] To select individuals with outstanding antibody responses to HBsAg, we performed ELISA assays on serum obtained from 159 volunteers. These included 15 uninfected and unvaccinated controls (HBsAg-, anti-HBs-, anti-HBc-), 20 infected and spontaneously recovered (HBsAg-, anti-HBs ^+/- , anti-HBc ⁺ ), and 124 vaccinated (HBsAg-, anti-HBs ^+/- , anti-HBc-) volunteers. These individuals displayed a broad spectrum of anti-HBs titers (x-axis in Figure 1A and Figure 8B; Table SI). To determine their neutralizing activity, we tested their ability to block HBV infection in sodium taurocholate co-transporting polypeptide (NTCP)-overexpressing HepG2 cells (Michailidis et ak, 2017; Yan et al., 2012) (y-axis in Figure 1A and Figure 8B and 8C; Table SI). Sera or antibodies purified from individuals with high levels of neutralizing activity were then compared across a wide range of dilutions (Figure IB and 1C). Although anti-HBs ELISA titers positively correlated with neutralizing activity (r _s =0.492, p<0.001, Spearman’s rank correlation), there were notable exceptions as exemplified by volunteers #99 and #49, whose sera failed to neutralize HBV despite high anti-HBs ELISA titers (Figure 1 A). Thus, ELISA titers against HBsAg are not entirely predictive of neutralizing activity in vitro.

[0080] The HBV surface protein, HBsAg can be subdivided into PreSl-, PreS2- and

S-regions (Figure ID). To determine which of these regions is the dominant target of the neutralizing response in the selected top neutralizers, we used S-protein to block neutralizing activity in vitro. The neutralizing activity in volunteers that received the HBV vaccine, which is composed of S-protein, was completely blocked by S-protein (black lines in Figure IE). The same was true for the spontaneously recovered individuals in our cohort despite a reported ability of this population to produce anti -PreSl or anti-PreS2 antibodies (Coursaget et ak, 1988; Li et ah, 2017; Sankhyan et ah, 2016) (red lines in Figure IE). These results suggest that the neutralizing antibody response in the selected individuals is directed primarily against the S-protein irrespective of immunization or infection.

Example 2

[0081] Human Monoclonal Antibodies to HBV

[0082] To characterize the antibodies responsible for neutralizing activity in the selected individuals, we purified S-protein binding class-switched memory B cells (Escolano et ak, 2019; Scheid et ak, 2009a). Unexposed naive controls and vaccinated individuals with low anti-HBs ELISA titers showed background levels of S-protein specific memory B cells (Figure 2A and 9A). In contrast, individuals with high neutralizing activity displayed a distinct population of S-antigen binding B cells constituting 0.03-0.07% of the IgG ⁺ memory compartment (CD19-MicroBeads ⁺ CD20-PECy7 ⁺ IgG-Bv421 ⁺ S-protein-PE ⁺ S-protein- APC ⁺ ovalbumin-Alexa Fluor 488 ) (Figure 2A and 9A). Consistent with the findings in elite HIV-1 neutralizers (Rouers et ak, 2017), the fraction of S-protein specific cells was directly correlated to the neutralization titer of the individual (r _s =0.699, p=0.0145, Spearman's 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 ak, 2017; Scheid et ak, 2009b; von Boehmer et ak, 2016). Overall, we obtained 244 paired heavy and light chain variable regions from S-protein-binding memory B cells from eight volunteers with high anti-HBs ELISA titers (Figure 9B and 9C; Table S2). Expanded clones composed 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). Moreover, IGHV3- 30/IGLV3-21 was present in #146 and #60; IGHV3-33/IGLV3-21 in #146 and #13; and IGHV3-23/IGLV3-21 in #146, #60 and #13. The variable diversity and joining (V(D)J) region of these antibodies was approximately 80% identical at the amino acid level (Figure 2D). Antibodies with related Ig heavy and light chains were also identified between volunteer #55 (HBV infected but recovered) and vaccinated individuals (Figure 2C and 9B). We conclude that top HBV neutralizers produce clones of antigen-binding B cells that express related Ig heavy and light chains.

Example 3

[0084] Breadth of Reactivity

[0085] Twenty representative antibodies from 5 individuals, designated as H001 to

H020, were selected for expression and further testing (Figure 9B). All 20 antibodies showed reactivity to the S-protein antigen used for B cell selection (HBsAg adr CHO) by ELISA with 50% effective concentration (ECso) values ranging from 18-350 ng/ml (Figure 3 A). These antibodies carried somatic mutations that enhanced antigen binding as determined by reversion to the inferred unmutated common ancestor (UCA) (Figure 3B). Thus, affinity maturation was essential for their high binding activity.

[0086] Four major serotypes of HBV exist as defined by a constant “a” determinant and two variable and mutually exclusive determinants "d/y" and "w/r" (Bancroft et al., 1972; Le Bouvier, 1971) with a highly statistically significant association between serotypes and genotypes (Kramvis et al., 2008; Norder et al., 2004). To determine whether our antibodies cross-react to different HBsAg serotypes, we performed ELISAs with 5 additional HBsAg antigens: yeast-expressed serotype “adr”, “ adw ”, and “ayw” , as well as “ ad ’ and “ay” antigen purified from human blood (Figure 3C). Many of the antibodies tested displayed broad cross reactivity and EC5 ₀ values lower than libivirumab, a human anti-HBs monoclonal antibody that was isolated from HBV-immunized humanized mice and then tested clinically (Eren et al., 2000; Eren et al., 1998; Galun et al., 2002). These antibodies were not polyreactive or autoreactive when tested in polyreactivity ELISA and HEp-2 immunofluorescence assays respectively (Figure 10A and 10B). We conclude that the antibodies tested are broadly cross reactive with different HBV serotypes.

Example 4

[0087] Antigenic Epitopes on S-protein [0088] To determine whether the selected antibodies bind to overlapping or non overlapping epitopes, we performed competition ELISA assays, in which the S-protein was pre-incubated with a selected antibody followed by a second biotinylated antibody.

Antibodies that showed weak levels of binding in ELISA (H002, H012, H013, H014, H018) were excluded. As expected, all of the antibodies tested blocked the binding of the autologous biotinylated monoclonal (yellow boxes in Figure 4A), while control human anti-HIV antibody 10-1074 failed to block any of the anti-HBs antibodies. The competition ELISA identified three mutually exclusive groups of monoclonal antibodies, suggesting that there are at least three dominant non-overlapping antigenic sites on HBsAg (red box for Group-I, blue box for Group-II, and HO 17 in Group-Ill, Figure 4A). Each of the individuals that had 2 or more antibodies tested in the competition ELISA expressed monoclonal antibodies that targeted 2 of the 3 non-overlapping epitopes (Figure 4A and 9B).

[0089] To further define these epitopes, we produced a series of alanine mutants spanning most of the predicted extracellular domain of the S-protein with the exception of cysteines, alanines, and amino acid residues critical for S-protein production (Salisse and Sureau, 2009) (Figure ID). ELISA assays with the representative antibodies from each antibody group and the mutant proteins revealed a series of binding patterns partially corresponding to the three groups defined in the competition assays (Figure 4B and 11). For example, mutations II 10A and T148A interfered with binding by Group-I antibodies exemplified by H004, H006, HO 19, and H020, but had little measurable effect on Group-II antibodies exemplified by H007, HO 15, and HO 16 or Group-Ill antibody HO 17 (Figure 4B and 11).

[0090] However alanine scanning suggested that some residues such as D144 and

G145 are critical for binding of monoclonals in both Group-I and Group-II despite their inability to compete with each other for binding to the native antigen (Figure 4B and 11). Without intending to be constrained by any particular theory, it is considered that D144A and G145A mutations alter the overall structure of HBsAg thereby interfering with binding of antibodies that normally target independent sites on the protein.

[0091] In addition to alanine scanning, we also produced 44 common naturally occurring escape variants found in chronically infected individuals (Hsu et ak, 2015; Ijaz et ah, 2012; Ma and Wang, 2012; Salpini et ak, 2015). Whereas alanine scanning showed that some of the antibodies in Group-I and -II were resistant to G145A, the corresponding naturally occurring mutations at the same position, G145E and G145R, revealed decreased binding by most antibodies (Figure 4C). Among the antibodies tested, HO 17 and HO 19, in Groups-I and -III respectively, showed the greatest resistance to G145 mutations and the greatest breadth and complementarity (Figure 4C). We conclude that human anti-HBs monoclonals obtained from the selected individuals recognize distinct epitopes on HBsAg, most of which appear to be non-linear conformational epitopes spanning different regions of the protein.

Example 5

[0092] In Vitro Neutralizing Activity

[0093] To determine whether the new monoclonals neutralize HBV in vitro , we performed neutralization assays using HepG2-NTCP cells (Figure 5A and 5B). The 50% inhibitory concentration (ICso) values were calculated based on HBsAg/HBeAg ELISA or immunofluorescence staining for HBcAg expression (Michailidis et al., 2017) (Figure 5C). Neutralizing activity was further verified by in vitro neutralization assays using primary human hepatocytes (Michailidis et al., 2020) (Figure 5C and 5D). Fourteen of the 20 antibodies tested showed neutralizing activity with ICso values as low as 5 ng/ml (Figure 5C). By comparison, libivirumab had an ICso of 35 and 128 ng/ml in the neutralization assays based on ELISA and immunofluorescence assays respectively (Figure 5C). Somatic mutations were essential for potent neutralizing activity as illustrated by the reduced activity of the inferred UCAs (Figure 12A and 12B). In addition, optimal activity required bivalent binding since the ICso values for Fab fragments were 2 orders of magnitude higher than intact antibodies (Figure 5E). Finally, there was no overt synergy when Group-I, -II, and -III antibodies were combined (Figure 12C). We conclude that half of the new monoclonals were significantly more potent than libivirumab including Group-I H004, H005, H006, H008, H009, HO 19, and H020 and Group-II H007, HO 15, and HO 16 (Figure 5C).

Example 6

[0094] Structure of the H015 Antibody/Peptide Complex

[0095] HO 15 differed from other antibodies in that its binding was inhibited by 5 consecutive alanine mutations spanning positions K141-G145 indicating the existence of a linear epitope. This idea was verified by ELISA against a series of overlapping peptides comprising the predicted extracellular domain of S-protein (Figure 6A and 13 A). The data showed that H015 binds to KPSDGN (SEQ ID NO: 23), which is near the center of the putative extracellular domain and contains some of the most frequently mutated amino acids during natural infection. [0096] To examine the molecular basis for H015 binding, its Fab fragment was co crystallized with the target peptide epitope PSSSSTKPSDGNSTS (SEQ ID NO: 24), where all cysteine residues that flank the recognition sequence were substituted with serine to avoid non-physiological cross-linking. The 1.78 A structure (Figure 6B and 13B) revealed that the peptide is primarily bound to the immunoglobulin heavy chain (Figure 6B and 6C), interacting with residues from CDR1 (R31), CDR2 (W52, F53) and CDR3 (E99, P101, L103, LI 04) of IgH with only one contact with CDR3 (P95) of IgL. The peptide adopts a three- residue beta hairpin (class 3) of the 3:5 type involving residues K141 through G145 as only one hydrogen bond is seen, between K141 and G145 (Milner- White and Poet, 1986), and they are not part of a beta sheet. The peptide is further stabilized by a salt-bridge formed between K141 and D144 (Figure 6D and 13C). Interestingly, the distance between the Cas of the two residues (C139 and C147) flanking the recognition residues is 6.4 A and are poised to form a disulfide bond between C139 and C147 found in the native HBsAg structure (Ito et ak, 2010). The HO 15 Fab appears to stabilize the conformation of the peptide via the Fab- peptide contacts (Figure 13D) including a large binding surface (866 A ² ; antibody-antigen buried surface of 600-900 A ² (Braden and Poljak, 1995)) comprised primarily of a single salt- bridge (lysine to aspartate; 0.9± 0.3 Kcal/mol) (White et ak, 2013) and five hydrogen bonds (1-2 Kcal/mol/bond) (Sheu et ak, 2003). Moreover, the peptide further restricts loop through intra-peptide contacts (Figure 13D) even in the absence of the disulfides.

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

[0098] HBsAg can be glycosylated at N146 and this site is also strictly conserved.

However, some studies have suggested that this glycosylation site is never fully occupied, resulting in a nearly 1 : 1 ratio of glycosylated and non-glycosylated isoforms on the surface of viral envelope (Julithe et ak, 2014). The glycosylation may be either NAG-NAG-MAN or NAG-(FUC)-NAG-MAN (Hyakumura et ak, 2015). We have modeled both fucosylated and non-fucosylated options by grafting a 7mer and 1 lmer glycan conjugated at N146 of peptide in the presence of the Fab. We found that both glycosylation forms are tolerated at that location with only minimal torsional adaptations without clashes with the Fab, though the fucosylated (branched) glycan required some additional torsional angle changes to the Fab, as well.

Example 7

[0099] Protection and Therapy in Humanized Mice

[0100] HBV infection is limited 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 produced human liver chimeric Fah ^/ l\OORagr ^/ IL2rg ^mil (huFNRG) mice (de Jong et al., 2014) and injected them with control or H020 (Group-I) or H007 (Group-II) antibodies before infection with HBV (Figure 7A-7D). These two antibodies were chosen because they bind to non-overlapping sites, and have broad and potent neutralizing activity. Whereas all six control animals in two independent experiments were infected, pre exposure prophylaxis with either H007 or H020 was fully protective (Figure 7B-7D). We conclude that single anti-HBs bNAbs targeting different epitopes on the major virus surface antigen can prevent infection in vivo.

[0101] To determine whether bNAbs can also control established infections, we infused control antibody or bNAb H020 (Group-I) or H007 (Group-II) into huFNRG mice with HBV viral loads of 10 ⁶ -10 ⁸ copies/ml of serum (Figure 7E-7H and Figure 14A). Fah ¹

N OORag 1 I 2rg ^tm ^ ¹ mice are highly immunodeficient and unable to mount adaptive immune responses due to absence of T and B lymphocytes. In addition, the I 2rg ^m, mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in innate immune function including antibody-dependent cellular cytotoxicity. Thus, elimination of viremia of 10 ⁶ -10 ⁸ DNA copies/ml in huFNRG mice by antibody therapy alone would not be expected. [0102] Animals that received the control antibodies further increased viremia to as high as ~10 ^u DNA copies/ml (Figure 7F). In contrast, the 5 mice that received H020 maintained stable levels of viremia for around 30 days (Figure 7G), after which time 2 mice showed increased viremia (arrow- 1/3 in Figure 7G). A similar result was observed in the 5 mice that received H007 (Figure 7H), where only one showed a slight increase viremia at around day 50 (arrow-5 in Figure 7H).

[0103] To determine whether the animals that showed increased HBV DNA levels during antibody monotherapy developed escape mutations, we sequenced the viral DNA recovered from mouse blood. All three mice that escaped H020 (Group-I) or H007 (Group-II) monotherapy developed viruses that carried a G145R mutation in the S-protein (arrow-1/3 in Figure 7G, arrow-5 in Figure 7H, Figure 71, and Figure 14). This mutation represents a major immune escape mutation in humans (Zanetti et al., 1988). Furthermore, mutations at the same position in the S-protein were also identified in mice that maintained low level viremia (arrow-2/4 in Figure 7G, arrow-6/7 in Figure 7H, Figure 71, and Figure 14), but not in control animals (Figure 14). These results show that anti-HBs bNAb monotherapy leads to the emergence of escape mutations that are consistent with bNAb binding properties in vitro (Figure 4C).

[0104] To determine whether a combination of bNAbs targeting 2 separate epitopes would interfere with the emergence of resistant strains, we co-administered H006 + H007 (Group-I and -II, respectively) to 8 HBV-infected huFNRG mice (Figure 7J). H006 (Group-I) was chosen for this purpose because of its resistance to D144A and G145A mutation (Figure 4B). Similar to H007 monotherapy, there was only a slight increase in viremia in animals treated with the H006 + H007 anti-HBs bNAb combination during the 60-day observation period (Figure 7J and 14A). However, sequence analysis revealed that 3 of the mice developed resistance mutations including K122R/G145R, C137Y, and C137Y/D144V (arrow-8/9/10 in Figure 7J, Figure 71, and Figure 14). These mutations confer loss of binding to both H006 (Group-I) and H007 (Group-II) (Figure 4C). Thus, the combination of 2 anti- HBs bNAbs targeting separate epitopes but susceptible to the same clinical escape variants is not sufficient to inhibit emergence of escape mutations.

[0105] To attempt to block the emergence of escape mutations, we combined H017 +

HO 19 (Group-Ill and -I, respectively) bNAbs because they displayed complementary sensitivity to commonly occurring natural mutations (Figure 4C). None of 7 mice treated with the combination of showed increased viremia or escape mutations as assessed by sequence analysis (Figure 7K and 14A). Similar effects were also observed in the 9 animals treated with the HO 16, HO 17 and HO 19 (Group-II, -III, -I, respectively) triple antibody combination (Figure 7L and 14A). Moreover, both these combinations dramatically reduced the HBsAg levels in serum (Figure 14C and 14D). Altogether, these findings suggest that control of HBV infection by bNAbs requires a combination of antibodies targeting non-overlapping groups of common escape mutations.

[0106] RESOURCES TABLE

Example 8

[0107] This Example provides a description of materials, methods, and subjects used to obtain the foregoing results. [0108] EXPERIMENTAL MODELS AND SUBJECTS

[0109] Human Subjects

[0110] Volunteer recruitment and blood draws were performed at the Rockefeller

University Hospital under a protocol approved by the institutional review board (IRB QWA- 0947). Study participants ranged in age from 22-65 with a mean of 43, the female:male ratio was 81:78 (Figure 8D and 8E; Table SI).

[0111] Animals

[0112] ah^NOORag ¹ ! L2rg ^m (FNRG) female mice were produced as reported (de

Jong et al., 2014) and maintained in the AAALAC-certified facility of the Rockefeller University. Animal protocols were in accordance with NIH guidelines and approved by the Rockefeller University Institutional Animal Care and Use Committee under protocol #18063. Female littermates were randomly assigned to experimental groups.

[0113] Cell Lines

[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 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] Viruses

[0116] HBV-containing supernatant from HepDE19 cells was collected and concentrated as previously described (Michailidis et al., 2017). The concentrated virus stock was aliquoted and stored at -80°C. For in vivo experiments one aliquot of mouse-passaged genotype C HBV vims, originally launched 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 using lxlO ⁴ DNA copies per mouse.

[0117] Bacteria

[0118] E. coli DH5 -alpha were cultured at 37°C with shaking at 230 rpm.

[0119] METHODS

[0120] Collection of Human Samples

[0121] Samples of peripheral blood were collected from volunteers at the Rockefeller

University Hospital. Serum was isolated by centrifugation of coagulated whole blood, and aliquoted for storage at -80°C. PBMCs were isolated using a cell separation tube with frit barrier and cryopreserved 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 seven days, supernatant was collected every other day for two weeks and fresh medium was added. After each collection, medium was spun down to remove cell debris, passed through a 0.22 pm filter, and kept at 4°C. Collected medium was concentrated 100-fold via centrifugation using Centricon Plus-70 centrifugal filter devices (Millipore-Sigma, Billerica, MA). Mouse-passaged genotype C HBV vims (Billerbeck et al., 2016) was used for in vivo mouse experiment.

[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 seeded in 96-well collagen-coated plates in DMEM supplemented with 10% FBS and 0.1 mM NEAA. The medium was changed to DMEM with 3% FBS, 0.1 mM NEAA, and 2% DMSO the next day and cultured for an additional 24 hours before infection. The inoculation was in DMEM supplemented with 3% FBS and 0.1 mM NEAA 4% PEG and 2% DMSO. Antibodies or semm samples were incubated with the vims in the inoculation medium for one hour at 37°C before adding to cells. Semm neutralization capacity (y-axis in Figure 1 A and 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 were 100% (no semm added or the sera from unexposed naive control donors), the semm neutralization capacity would be calculated as 1; but if the relative percentage of infected cells were 50% or 10%, the serum neutralization capacity would be 2 or 10. For the blocking neutralization assay, S-protein antigen at different concentration was incubated with purified polyclonal antibodies for one hour at 37°C before incubation with HBV virus. The cells were then spinoculated for one hour by centrifugation at 1,000 g at 37°C. After a 24- hour incubation, supernatant was removed, cells were washed five times with PBS, and 100 mΐ of fresh DMEM supplemented with 3% FBS, 0.1 mM NEAA, and 2% DMSO. Both supernatant and cells were harvested 7 days after infection for analysis. Neutralization assays in primary human hepatocytes were performed as above using hepatocytes from livers of highly humanized mice that were harvested and seeded on collagen-coated plates in hepatocyte defined medium (Corning) (Michailidis et al., 2020).

[0126] Chemiluminescence Immunoassay

[0127] For quantitative analysis of secreted antigen HBsAg or HBeAg, 50 mΐ of the collected supernatant was loaded into 96-well plates of a chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China) according to the manufacturer’s instructions. Plates were 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 well in the same lane. For example, the absolute HBsAg/HBeAg level in virus-only control well (considered as reference) was 20 NCU/ml (national clinical units per milliliter), while adding one neutralizing serum sample might reduce this to 5 NCU/ml. Therefore, after normalization, the relative HBsAg/HBeAg level were calculated as 100% in control and 25% for this neutralizing serum. Since many factors (virus concentration, cell concentration, immunofluorescence reading, etc.) vary between different plates or different rounds of experiments, normalization is necessary for combining data for comparison.

[0128] Immunofluorescence

[0129] Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% goat serum, the 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 fluorescent microscope and processed with ImageJ. For high-content imaging analysis ImageXpress Micro XLS (Molecular Devices, Sunnyvale, CA) was used. The absolute HBc ⁺ percentages were obtained and the relative percentage of HBc ⁺ cells was calculated by normalizing to the virus-only control well in the same lane. For example, the absolute HBc ⁺ cell percentage in virus-only control well (considered as reference) was 40%, while adding one neutralizing serum sample might reduce this to 10%. Therefore, after normalization, the relative percentages of HBc ⁺ cells were calculated as 100% in control well and 25% for this neutralizing serum sample. Since many factors (virus concentration, cell concentration, immunofluorescence reading, etc.) vary between different plates or different rounds of experiments, normalization is necessary for combining data for comparison.

[0130] ELISA Assays

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

[0132] The binding of serum or recombinant IgG antibodies to HBsAg proteins (see

KEY RESOURCES TABLE) was measured by coating ELISA plates with 10 pg/ml of antigen in PBS. Plates were blocked with 2% BSA in PBS and incubated with antibody for one hour at room temperature. Visualization was with HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific). The 50% effective concentration (ECso) needed for maximal binding was determined by non-linear regression analysis in software PRISM.

[0133] For competition ELISAs plates were coated with 0.12 pg/ml HBsAg ( adr

CHO) and incubated with 16.7 pg/ml primary antibody for two hours, followed by directly adding 0.25 pg/ml biotinylated secondary antibody and incubation for 30 minutes all at room temperature. Detection was with streptavidin-HRP (BD Biosciences).

[0134] Autoreactivity and Polyreactivity

[0135] Autoreactivity and polyreactivity assays were performed as described (Gitlin et al., 2016; Mayer et al., 2017; Robbiani et al., 2017). For the autoreactivity assays, monoclonal antibodies were tested with the Antinuclear antibodies (HEp-2) Kit (MBL International). Antibodies were incubated at 100 pg/ml and were detected with Alexa Fluor 488 AffmiPure F(ab')2 Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch) at 10 pg/ml. Fluorescence images were taken with a wide-field fluorescence 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. For the polyreactivity ELISA assays, antibody binding to five different antigens, double-stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharides (LPS), and single-stranded DNA (ssDNA), were measured. 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 S-protein antigen were synthesized at the Proteomics Resource Center of The Rockefeller University. For peptide ELIS As plates were coated with 10 pg/ml peptide in PBS.

[0138] HBsAg-Binding Memory B cells

[0139] S-protein ( adr serotype) expressed and purified from Chinese hamster ovary

(CHO) cells (ProSpec) and ovalbumin (Sigma-Aldrich) were biotinylated using EZ-Link™ Micro NHS-PEG4-Biotinylation kit (Thermo Fisher Scientific). S-protein-PE and S-protein- APC were prepared by incubating 2-3 pg of biotin-S-protein with streptavidin-PE (eBioscience) or streptavidin-APC (BD Biosciences) in PBS respectively 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 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 CD 19 MicroBeads (Miltenyi Biotec) followed by incubation with human Fc block (BD Biosciences) and anti-CD20-PECy7 (BD Biosciences), anti-IgG- Bv421 (BD Biosciences), S-protein-PE at 10 pg/ml, S-protein- APC at 10 pg/ml, and ovalbumin- Alexa Fluor 488 at 10 pg/ml at 4°C for 20 minutes. Single CD20 ⁺ IgG ⁺ S-protein- PE ⁺ S-protein-APC ⁺ Ova-Alexa Fluor 488 memory B cells were sorted into 96-well plates using a FACSAriall (Becton Dickinson) and stored at -80°C.

[0141] Antibody Cloning, Sequencing and Production

[0142] Antibody cloning, sequencing and production were done as previously reported (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 of H006, H019 and H020 were synthesized by gBlock IDT (Table S3) and were inserted into antibody vectors for expression. V(D)J gene segment 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] Oligonucleotides fragments with the target point mutations were synthesized by gBlock IDT (Table S3), and were substituted into the antigenic loop region in plasmid pl.3xHBV-WT by Sequence and Ligation-Independent Cloning (SLIC) (Jeong et al., 2012). Mutant 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. Supernatants were collected 2 days later and stored at -80°C. Serum- free medium (50 mΐ) was directly used to coat ELISA plates.

[0145] Crystallization, X-ray Data Collection, Structure Determination and

Refinement

[0146] Antibody Fab (25 mg/ml) in 50 mM Tris 8.0, 50 mM NaCl was mixed with peptide (5 mg/ml) in the same buffer at 5:1 v/v. Molar ratio of Fab:peptide is around 1:2. Crystals were obtained upon substitution of all peptide-11 cysteine residues with serine in the peptide synthesis (Proteomics Resource Center, RU). The crystallization condition for Fab 15/peptide- 1 lSer was identified from a commercial screen (Morpheus by Molecular Dimensions) by the sitting-drop vapor-diffusion method at room temperature. The crystal used for data collection was obtained directly from the initial setup (position El) in a precipitant solution consisting of 0.12 M Ethylene glycols (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 to 1.78 A resolution. The data were integrated and scaled with the program XDS (Kabsch, 2010a, b) and other data processing utilities from the CCP4 suite (Collaborative Computational Project, 1994) using RAPD, the software available at the beam-line. 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 an initial search model in Phenix (Adams et al., 2010). Iterative model building and structural refinement were manually performed 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). All other residues were not visible and the area where they would be is fully solvent, with no crystal contacts involving any of the peptide atoms. The quality of the final model was good as noted in a Ramachandran of 96% of the observed residues within the allowable region. Data-collection and refinement statistics are summarized (Figure 13B). All molecular graphics were prepared with PyMOL (Version 2.0 Schrodinger, LLC). Atomic coordinates and experimental structure factors have been deposited in the PDB under accession code 6VJT.

[0147] Humanized Mice and In Vivo Studies

[0148] Six to eight week old Fah ^/ ODRag ^/ IL2rg ^{ml 1} (FNRG) female mice were transplanted with one million human hepatocytes from a pediatric female donor HUM4188 (Lonza Bioscience) as previously described (de Jong et al., 2014). Briefly, during isoflurane anesthesia mice underwent skin and peritoneal incision, exposing the spleen. One million hepatocytes were injected in the spleen using a 28-gauge needle. The peritoneum was then approximated using 4.0 VICRYL sutures (Johnson & Johnson), and skin was closed using MikRon Autoclip surgical clips (Becton Dickinson). Mice were cycled off the drug nitisinone (Yecuris) on the basis of weight loss and overall health. Humanization was monitored by human albumin quantification 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. The human liver chimeric (huFNRG) mice are extremely immunodeficient. The Ragl ¹ renders the mice B and T cell deficient and the IL2rg ^mil mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in functional NK cells. Moreover, the genetic background is NOD background, with suboptimal antigen presentation, defects in T and NK cell function, reduced macrophage cytokine production, suppressed wound healing, and C5 complement deficiency. Thus the mice would be unable to produce antibody-dependent effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), or passive antibody-enhanced adaptive immunity.

[0149] Mice were challenged intravenously with lxlO ⁴ genome equivalent (GE) of mouse-passaged genotype C HBV viruses diluted in PBS. For prophylaxis experiments, 500 pg of monoclonal antibody was administered intraperitoneally at 20 and again at 6 hours before infection. For therapy experiments, huFNRG mice with established HBV infections (<10 ⁸ DNA copies/ml of serum) were injected with 500 pg of each monoclonal antibody intraperitoneally 3 times per week.

[0150] DNA in mouse serum collected weekly was extracted using a QIAamp DNA

Blood Mini Kit (Qiagen). Total HBV DNA was determined by quantitative PCR (Michailidis et al., 2017). PCR was performed using a TaqMan Universal PCR Master Mix (Applied Biosystems), primers and probe (Table S3). [0151] To obtain HBV DNA from serum for sequence analysis the S domain was amplified using primers (Table S3), and Phusion DNA polymerase (Thermo Fisher Scientific). Initial denaturation was 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 at 72°C for 5 min. A -700 bp fragment was gel extracted for Sanger sequencing. Sequence alignments were performed using Mac Vector.

[0152] QUANTIFICATION AND STATISTICAL ANALYSIS

[0153] The detailed information of statistical analysis could be found in the Result and Figure Legends. Correlation was evaluated by Spearman’s rank correlation method (Figure 1 A and 2B). Statistical significance was calculated by Dunn's Kruskal-Wallis multiple comparisons with p values corrected with the Benjamini-Hochberg procedure (Figure 8C). The 50% effective concentration (ECso) values by ELISA assays (Figure 3 A and 3C) and 50% inhibitory concentration (ICso) values by neutralization assays (Figure 5C) were calculated by nonlinear regression analysis in PRISM software.

[0154] Discussion of Examples

[0155] Previous studies have identified several anti-HBs neutralizing antibodies from a small number of otherwise unselected spontaneously recovered or vaccinated individuals (Cerino et ak, 2015; Colucci et ah, 1986; Eren et ah, 1998; Heijtink et ah, 2002; Heijtink et ah, 1995; Jin et ak, 2009; Kim and Park, 2002; Li et ak, 2017; Sa'adu et ak, 1992; Sankhyan et ak, 2016; Tajiri et ak, 2007; Tokimitsu et ak, 2007; Wang et ak, 2016). In contrast, in the present disclosure, sera from 144 exposed volunteers was screened to identify elite neutralizers. Serologic activity varied greatly among the donors with a small number of individuals demonstrating high levels of neutralizing activity. To understand this activity, we isolated 244 anti-HBs antibodies from single B cells obtained from the top donors. Each of the elite donors tested showed expanded clones of memory B cells expressing bNAbs that targeted 3 non-overlapping sites on the S-protein. Moreover, the amino acid sequence of several of the bNAbs was highly similar in different individuals. These closely related antibodies target the same epitope.

[0156] The near identity of clones of HBV bNAbs in unrelated elite individuals is akin to reports for elite responders to HIV-1 (Scheid et ak, 2011; West et ak, 2012), influenza (Laursen and Wilson, 2013; Pappas et ak, 2014; Wrammert et ak, 2011), Zika (Robbiani et ak, 2017), and malaria (Tan et ak, 2018). However, none of the elite anti-HBs bNAbs shares both IgH and IgL with previously reported HBV neutralizing antibodies, the best of which have been tested in the clinic but are less potent than some of the bNAbs of this disclosure (libivirumab ICso: 35 ng/ml, tuvirumab ICso: -100 ng/ml) (Galun et al., 2002; Heijtink et al., 2001; van Nunen et al., 2001).

[0157] The described alanine scanning and competition binding analyses are consistent with the existence of at least 3 domains that can be recognized concomitantly by bNAbs (Gao et al., 2017; Tajiri et al., 2010; Zhang et al., 2016). However, the domains do not appear to be limited to either of two previously defined circular peptide epitopes, 123-137 and 139-148 (Tajiri et al., 2010; Zhang et al., 2016). Instead, residues spanning most of the external domain can contribute to binding by both Group-I and -II antibodies. For example, alanine scanning indicates that Group-I H020 binding is dependent on 1110, K141, D144, G145 and T148, while Group-II HO 16 binding depends on T 123, D 144, and G145. Thus, despite having non-overlapping binding sites some of the essential residues are shared by Group-I and II suggesting that the epitopes are conformational. Moreover, the antibody epitopes on 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 that is frequently mutated in infected humans (Ma and Wang, 2012; Tong et al., 2013), is believed to be essential for binding by all the Group-II but not all Group-I or -III antibodies tested.

[0158] Crystallization of the Group-II bNAb HO 15 and its linear epitope revealed a loop that includes P142, S/T143, D144, and G145, all of which are frequently mutated during natural infection to produce well-documented immune escape variants (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, possibly by facilitating virus interactions with cell surface glycosaminoglycans (Sureau and Salisse, 2013). Mutations in K141, PI 42 as well as Cl 39 and Cl 47, all of which contribute to the stability of the structure, decrease viral infectivity (Salisse and Sureau, 2009). Without intending to be bound by any particular theory, it is considered that drugs that destabilize the newly elucidated HO 15- peptide loop structure may also interfere with infectivity.

[0159] The G145R mutation, which is among the most frequent immune escape variants, replaces a small neutral residue with a bulky charged residue that would likely interfere with antigenicity by destroying 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), possibly because the additional charge compensates for otherwise altered interactions between HBV and cell surface glycosaminoglycans (Sureau and Salisse, 2013). Thus, the additional charge may allow G145R to function as a dominant immune escape variant while preserving infectivity.

[0160] The present disclosure describes antibodies directed at S-protein antigen in part because this is the antigen used in the currently FDA-approved vaccines, and because purified S-protein blocked nearly all of the neutralizing activity in the serum of the elite neutralizers irrespective of whether they were vaccinated or spontaneously recovered. Nevertheless, individuals who recover from infection also produce antibodies to the PreSl domain of HBsAg (Li et al., 2017; Sankhyan et ah, 2016). The PreSl domain is essential for the virus to interact with the entry factor NCTP on hepatocytes and potent neutralizing antibodies to PreSl have been described (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 engineering to enhance their neutralizing activity (Li et al., 2017). Thus, whether the human immune system also produces potent anti-PreSl bNAbs has not been determined.

[0161] Chronic HBV infection remains a major global public health problem in need of an effective curative strategy (Graber-Stiehl, 2018; Lazarus et al., 2018; Revill et al., 2016). Chronically infected individuals produce an overwhelming amount of HBsAg that is postulated to incapacitate the immune system. Consequently, immune cells, which might normally clear the virus, are unable to react to antigen, a phenomenon referred to as exhaustion or anergy (Ye et al., 2015). The appearance of anti-HBs antibodies is associated with spontaneous recovery from the disease, perhaps because they can clear the antigen and facilitate the emergence of a productive immune response (Celis and Chang, 1984; Zhang et al., 2016; Zhu et al., 2016). These findings led to the hypothesis that passively administered antibodies might be used in conjunction with antiviral drugs to further decrease the antigenic burden while enhancing immune responses that maintain long-term control of the disease. The presently described results in huFNRG mice infected with HBV indicate that antibody monotherapy with a potent bNAb 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 escape by mutation. Combinations that target separate epitopes but have overlapping sensitivity to commonly occurring escape mutations such as H006 and H007 are ineffective. In contrast, combinations with complementary sensitivity to common escape mutations prevent the emergence of escape mutations in huFNRG mice infected with HBV. Thus, as described above, the present disclosure provides immunotherapy for HB V infection with combinations of antibodies with complementary activity to avert this potential problem.

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[0163] Supplemental Table SI. Detailed information of donors, Related to Figure

1.

[0164] The anti-HBs ELISA titer (x-axis in Figure 1 A and 8B) and relative infection rate (y-axis in Figure 1A and 8B) of each donor’s serum sample were determined by ELISA assay and in vitro neutralization assay, respectively. [0165] Supplemental Table S2. Detailed information about cloned antibodies with paired heavy and light chains, Related to

Figure 2.

[0166] Variable (V), diversity (D) and joining (J) genes, mutation on the variable gene (V MUT), and CDR3 amino acid sequences of cloned immunoglobulin heavy, kappa light and lambda light chains are listed. These antibodies are grouped by their IGHV genes, with our 5 selected H001-H020 antibodies indicated. H021 antibody used for sequence alignment in Figure 2D is also indicated. The amino acid length of IGH CDR3 was between 5 and 27 amino acids, with the highest peak at 16 amino acids and the average around 15 amino acids. There are 16 of IGH CDR3 containing cysteines.

[0167] Table S3. Primers and synthesized nucleotide sequences.

[0168] It will be recognized from the foregoing that the present disclosure describes screening individuals who were either vaccinated or had spontaneously recovered from HBV infection. Antibody cloning from memory B cells revealed that all 5 of the top individuals produced clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Clones with the same immunoglobulin variable, diversity and joining heavy and light chain genes were shared among elite neutralizers. Single bNAbs protected humanized mice against infection, but selected for resistance mutations in mice with established infection. In contrast, infection was controlled in the absence of detectable escape mutations by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with a peptide epitope containing residues frequently mutated in human immune escape variants revealed a loop anchored by oppositely charged residues. The structure provides a molecular explanation for why immunotherapy for chronic HBV infection may require combinations of complementary bNAbs, as described herein.

[0169] While the disclosure has been described through 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.