Human neutralizing monoclonal antibodies against hepatitis e virus and uses thereof

Abstract

The invention relates to human neutralizing monoclonal antibodies against Hepatitis E virus and their use for the diagnosis, monitoring, prevention, and treatment of Hepatitis E virus infection and associated disease.

Description

HUMAN NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST HEPATITIS E VIRUS AND USES THEREOFFIELD OF THE INVENTION

[0001] The invention relates to human neutralizing monoclonal antibodies against Hepatitis E virus (HEV) and their use for the diagnosis, monitoring, prevention, and treatment of HEV infection and associated disease.BACKGROUND OF THE INVENTION

[0002] Hepatitis E virus (HEV) infection is the leading cause of acute hepatitis, annually affecting approximately 20 million people worldwide and resulting in 30,000-40,000 deaths (World Health Organization (WHO), 2023). HEV infection is often self-limiting, but it can lead to fatal acute liver failure in pregnant women and chronic hepatitis in immunocompromised individuals, particularly solid organ transplant recipients (Ma et al., 2022; Kamar et al., 2017).

[0003] Currently, there are limited treatments available for HEV infections. Ribavirin is not universally effective and is contraindicated in pregnant women and immunocompromised patients due to its side effects. Interferon-alpha cannot be used in most transplant recipients. Therefore, there is a need for safer and more effective treatment options for HEV infections and associated disease.

[0004] HEV belongs to the Paslahepevirus genus of the Hepeviradae family and comprises eight genotypes to date (Wang et al., 2023). HEV-1 to HEV-4 are the primary genotypes responsible for human infections. HEV-1 and HEV-2 are human- restricted and transmitted by the fecal-oral route via contaminated water causing epidemics in areas with poor sanitation. In contrast, HEV-3 and HEV-4 are food-borne viruses responsible for outbreaks worldwide (Geng et al., 2023; Kamar et al., 2017).

[0005] HEV is a small non-enveloped virus with a single-stranded positive-sense RNA genome containing three major open reading frames (ORF1 to ORF3). HEV is shed into feces as a “naked” viral form, also found in the bile, but circulates in the blood as quasi-enveloped virions coated by host-derived membranes (Feng et al., 2014). The viral capsid protein (CA) is encoded by the ORF2 and assembles in a T=3 icosahedral symmetry with 90 homodimeric subunits to form the shell of the viral particle (Yamashita et al., 2009). The HEV CA monomer is composed of the protruding (P), the shell (S) and middle (M) domains. The S and M domains associate tightly around the particle's three-fold axis, while the P domain, linked tothe M domain, extends from the virion surface as a dimeric spike (Guu et al., 2009; Yamashita et al., 2009). The P domain is essential for virus-host interactions, mediating pre-attachment to heparan sulfates proteoglycans and binding to unidentified receptor(s) on the cell surface (Kalia et al., 2009; Li et al., 2009).

[0006] HEV antibodies elicited in response to infection play a crucial role in clearing the virus and protecting from liver disease (Krain et al., 2014; Walker, 2019). Long-lasting humoral immune memory induced by infection, including HEV-specific circulating antibodies and memory B cells, probably plays a key protective role against re-infections (Kulkarni et al., 2019; Zhou and Zhao, 2023). Antibodies neutralizing HEV target the viral CA protein, specifically the P domain, and inhibit de novo infection by either blocking the attachment of naked HEV virions to target cells or inducing structural disruption of the virus (Zhou and Zhao, 2023; Zhang et al., 2023). Despite existing serological data on the HEV antibody response, the molecular details, and structure-function relationships of human HEV antibodies remain elusive. HEV neutralizing monoclonal antibodies produced from immunized mice were also described (Tang et al., 2011; Gu et al., 2015; Zhao, 2015). Notably, 8G12, a murine neutralizing antibody targeting an epitope at the dimeric P interface was found to cross-react with the four human HEV genotypes (Gu et al., 2015). However, the neutralizing activity of all these monoclonal antibodies was low as evidenced by an IC50 within the pg / ml range for the best neutralizing antibodies.

[0007] Strong HEV neutralizing antibodies could potentially prevent infection or reduce infection severity and also help in cases of fulminant hepatitis. Human neutralizing antibodies against HEV would provide a targeted treatment with potentially fewer side effects. In addition, broadly neutralizing antibodies would advantageously be effective against the various HEV genotypes causing human infections.

[0008] Therefore, there is a need for human neutralizing monoclonal antibodies against HEV, in particular potent broadly neutralizing human antibodies against HEV, for the prevention and treatment of HEV infections.SUMMARY OF THE INVENTION

[0009] The inventors have identified highly potent human broadly neutralizing antibodies to HEV in a panel of anti-capsid (CA) antibodies cloned from memory B cells of HEV seroconverters. Most human neutralizing antibodies bound to the capsid protruding domain (P) and cross-reacted with all HEV genotype variants; a subset also recognized the zoonotic rat hepatitis E virus (HEV-C1). Mapping of the anti-P antibodies identified six epitope clusters(I, II, III, IV, V and VI). All antibodies in cluster VI were highly potent neutralizers (average IC50 = 13.9 [0.2-42] ng / ml). In contrast, many antibodies in the other groups (l-V) did not neutralize HEV (IC50 >1000 ng / mL) or showed only weak neutralization (IC50 100-1000 ng / mL), Figure 3G and 3H. Structural analyses of capsid protein complexed with three highly potent and broad HEV antibodies uncovered a novel neutralizing site involving two loops (RS and XY) exposed on P domain monomers at the apex of the capsid spike in a region located outside of the dimerization interface. All antibodies in cluster VI also crossreacted with all HEV genotype variants and with HEV-C1 capsid. Furthermore, the targeted epitope is conserved across HEV genotypes, indicating that the pan-genotypic reactive neutralizers are very likely to be broadly neutralizing.

[0010] In view of these results, the HEV neutralizing antibodies according to the invention represent promising immunotherapeutics and diagnostic tools for the treatment and diagnosis of HEV infection and associated disease.

[0011] The invention relates to a human neutralizing monoclonal antibody against Hepatitis E virus (HEV), which specifically binds to HEV capsid protein P domain and is a competitive inhibitor of binding to the P domain of at least one of the following reference antibodies: - the reference human antibody Es1.327 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 4;- the reference human antibody Es4.431 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:6;- the reference human antibody Es4.452 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; and- the reference human antibody Es5.127 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10.

[0012] In some embodiments, the antibody competes for binding with at least the reference human antibodies Es1.327 and Es4.431; preferably with the reference human antibodies Es1.327, Es4.431, Es4.452 and Es5.127.

[0013] In some embodiments, the antibody competes for binding with at least one reference human antibody Es1.327, Es4.431, Es4.452 or Es5.127 comprising heavy and lightchain amino acid sequences of SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; or SEQ ID NO: 17 and 18, respectively.

[0014] In some embodiments, the HEV capsid protein comprises any one of SEQ ID NO: 83 and 89-101, and / or the P domain comprises SEQ ID NO: 84.

[0015] In some embodiments, the antibody specifically binds to an epitope of HEV capsid protein P domain located within the region of the RS and XY loops, and said region consisting of the fusion of the sequences from positions 481 to 492 and 580 to 594, the indicated positions being determined by alignment with SEQ ID NO: 1; preferably wherein the region consists of the fusion of the sequences DQTTYGSSTNPM (SEQ ID NO: 105) and AISTYTTSLGAGPTS (SEQ ID NO: 106). In some particular embodiments, the antibody specifically binds at least one amino acid residue of the P domain RS loop selected from the group consisting of: Q482, T483, G486 and S488; more preferably which specifically binds the residues Q482, T483 and S488, and optionally further specifically binds the residue G486.

[0016] In some embodiments, the antibody neutralizes HEV with a half maximal inhibitory concentration (IC50) selected from 10 ng / mL or less, 5 ng / mL or less, and 1 ng / mL or less.

[0017] In some embodiments, the antibody neutralizes at least one HEV chosen from HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7, HEV-8 and HEV-C1; preferably the antibody neutralizes HEV-1, HEV-2, HEV-3, and / or HEV-4.

[0018] In some embodiments, the antibody has no predicted reactivity to human proteins.

[0019] In some embodiments, the antibody comprises:a) a heavy chain variable domain comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20 and a heavy chain CDR3 of SEQ ID NO: 21; b) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26 and a heavy chain CDR3 of SEQ ID NO: 27; c) a heavy chain variable domain comprising a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR3 of SEQ ID NO: 33; d) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38 and a heavy chain CDR3 of SEQ ID NO: 39; ore) a heavy chain variable domain comprising: heavy chain CDR1, CDR2 and CDR3 which differ from SEQ ID NO: 19 to 21, 25 to 27, 31 to 33, or 37 to 39, respectively byup to 6 (1, 2, 3, 4, 5 or 6) amino acid mutations in the sequence of 1, 2 or 3 CDRs; preferably conservative amino acid substitutions.

[0020] In some particular embodiments, the antibody comprises:a) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 21, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23 and a light chain CDR3 of SEQ ID NO: 24; b) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26 and a heavy chain CDR3 of SEQ ID NO: 27, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30; c) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR3 of SEQ ID NO: 33, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 34, a light chain CDR2 of SEQ ID NO: 35, and a light chain CDR3 of SEQ ID NO: 36; d) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38 and a heavy chain CDR3 of SEQ ID NO: 39, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 40, a light chain CDR2 of SEQ ID NO: 41, and a light chain CDR3 of SEQ ID NO: 42; or e) a heavy chain variable domain comprising heavy chain CDR1, CDR2 and CDR3 which differ from SEQ ID NO: 19 to 21, 25 to 27, 31 to 33, or 37 to 39, respectively by up to 6 (1, 2, 3, 4, 5 or 6) amino acid mutations in the sequence of 1, 2 or 3 CDRs; and a light chain variable domain comprising: light chain CDR1, CDR2, and CDR3 which differ from SEQ ID NO: 22 to 24, 28 to 30, 34 to 36 or 40 to 42, respectively by up to 6 (1, 2, 3, 4, 5 or 6) amino acid mutations in the sequence of 1, 2 or 3 CDRs; preferably conservative amino acid substitutions.

[0021] In some embodiments, the antibody comprises:a) a heavy chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 3 and a light chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 4;b) a heavy chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 5 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 6;c) a heavy chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 7 and a light chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 8;d) a heavy chain variable domain comprising an acid sequence having at least 90 % identity with SEQ ID NO: 9 and a light chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 10; ore) a heavy chain variable domain and a light chain variable domain comprising amino acid sequences having at least 90 % identity with SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO:125 and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; or SEQ ID NO: 133 and SEQ ID NO: 134.

[0022] In some particular embodiments, the heavy and light chain variable domains comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO:125 and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; or SEQ ID NO: 133 and SEQ ID NO: 134.

[0023] In some particular embodiments of the antibody according to the disclosure, the heavy chain variable domains are associated with IgG or IgA constant region; preferably I gG 1 constant region, IgA constant region further comprising a J chain and / or secretory component, and / or IgG or IgA constant region comprising mutation(s) and / or modifications that silence antibody effector functions and / or increase antibody half-life in vivo.

[0024] In some embodiments, the antibody comprises:a) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 11 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 12;b) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 13 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 14;c) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 15 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO:d) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 17 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 18.

[0025] In some embodiments, the antibody comprises a heavy and / or light chain amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences SEQ ID NO: 11 to 18.

[0026] In some embodiments, the antibody comprises a heavy chain variable domain comprising framework regions FR1, FR2, FR3 and FR4 selected from the group consisting of SEQ ID NO: 43 to 46; SEQ ID NO: 51 to 54; SEQ ID NO: 59 to 62; and SEQ ID NO: 67 to 70; and / or comprises a light chain variable domain comprising framework regions FR1, FR2, FR3 and FR4 selected from the group consisting of SEQ ID NO: 47 to 50; SEQ ID NO: 55 to 58; SEQ ID NO: 63 to 66; and SEQ ID NO: 71 to 74.

[0027] In some embodiments, the antibody is a recombinant human monoclonal antibody; preferably of IgG1 isotype or IgA isotype such as polymeric or secretory IgA; and / or preferably comprising a non-human glycosylation pattern.

[0028] In some embodiments, the antibody comprises an IgG Fc region comprising at least one substitution; preferably selected from the group consisting of substitutions that silence antibody effector functions, substitutions that enhance antibody half-life, substitutions that prevent binding to CD16 polypeptide, and substitutions that improve antiviral efficacy against pathogens.

[0029] Another aspect of the invention relates to a combination of antibodies comprising at least one anti-HEV capsid protein P domain human neutralizing antibody according to the present disclosure and at least one other anti-HEV capsid protein P domain human neutralizing antibody chosen from an antibody of any one of Groups I, II, III, IV or V as shown in Figure 3H, an antigen-binding fragment thereof or derivative thereof according to the present disclosure; preferably chosen from Es1.268, Es1.231, Es2.105, Es2.325 and Es3.108 comprising VH and VL domains of SEQ ID NO: 107-108, 109-110, 111-112, 113-114, and 115-116, respectively.

[0030] Another aspect of the invention relates to a nucleic acid molecule which encodes the antibody according to the disclosure, particularly a pair of nucleic acid molecules which encode the heavy and light chain of said antibody.

[0031] In some embodiments, the nucleic acid molecule or pair of nucleic acid molecules according to the invention is mRNA or DNA.

[0032] In some embodiments, the nucleic acid molecule or pair of nucleic acid molecules according to the invention comprises a pair of nucleic acid sequences selected from: a sequence having at least 90% identify with SEQ ID NO: 75 and a sequence having at least 90% identify with SEQ ID NO: 76 or the RNA sequences thereof; a sequence having at least 90% identify with SEQ ID NO: 77 and a sequence having at least 90% identify with SEQ ID NO: 78 or the RNA sequences thereof; a sequence having at least 90% identify with SEQ ID NO: 79 and a sequence having at least 90% identify with SEQ ID NO: 80 or the RNA sequences thereof; and a sequence having at least 90% identify with SEQ ID NO: 81 and a sequence having at least 90% identify with SEQ ID NO: 82 or the RNA sequences thereof.

[0033] Another aspect of the invention relates to a vector comprising the nucleic acid molecule(s) according to the disclosure.

[0034] In some embodiments, the vector is suitable for the expression of the nucleic acid molecule(s) into cells of a human individual; preferably the vector is a viral vector or a non-viral vector such as lipid nanoparticle.

[0035] In some embodiments, the vector is an expression vector for the production of an antibody according to the disclosure in a host cell.

[0036] In some embodiments, the expression vector is contained in a bacteria strain chosen from Es1.327_plgH, Es1.327_plgL, Es4.431_plgH, Es4.431_plgL, Es4.452_plgH, Es4.452_plgL, Es5.127_plgH and Es5.127_plgL deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6140, 1-6141, 1-6142, 1-6143, 1-6144, 1-6145, 1-6146, and 1-6147, respectively.

[0037] Another aspect of the invention relates to a host cell which has been modified with the expression vector according to the disclosure.

[0038] Another aspect of the invention relates to a pharmaceutical composition comprising the antibody according to the disclosure or an antigen-binding fragment thereof, the vector according to the disclosure, or the combination of antibodies according to the disclosure, and at least one of a pharmaceutically acceptable carrier, an adjuvant, and a preservative.

[0039] Another aspect of the invention relates to antibody according to the disclosure, antigen-binding fragment thereof, vector according to the disclosure, combination of antibodies according to the disclosure, or the pharmaceutical composition according to the disclosure, for use as a medicament; preferably for use in the prevention or treatment of HEV infection and associated disease in a subject in need thereof.

[0040] In some embodiments, the administration of the treatment reduces the risk of developing HEV-associated disease for the subject, in particular the administration of the treatment reduces the risk of hospitalization or the risk of death for the subject.

[0041] In some embodiments, the administration of the treatment reduces the likelihood of developing a severe form of HEV-associated disorder for the subject, in particular the risk of acute severe infection such as acute severe hepatitis; the risk of developing chronic HEV such as liver fibrosis and cirrhosis; or the risk of death is reduced by the treatment.

[0042] In some embodiments, the subject is not infected with HEV. In some embodiments, the subject has been exposed to HEV or is infected with HEV but has not developed HEV disease. In some embodiments, the subject is a HEV disease patient.

[0043] In some embodiments, the subject is a pregnant women or immunocompromised patient, particularly wherein the immune-compromised patient is a solid organ transplant recipient, more particularly a liver transplant recipient.

[0044] Another aspect of the invention relates to kit for the detection or diagnosis of HEV infection, or the monitoring of treatment of HEV disease, comprising at least an antibody according to the disclosure or antigen-binding fragment thereof, preferably further including a detectable label.DETAILED DESCRIPTION OF THE INVENTION

[0045] The invention provides recombinant human neutralizing monoclonal antibodies against Hepatitis E virus capsid protruding domain (P) which bind to a novel neutralizing site at the top of the P domain without involving the dimeric interface and have a broad and highly potent neutralizing activity against HEV compared to other anti-P neutralizing antibodies. The invention also provides nucleic acids, vectors encoding the antibodies, compositions, reagents, medical devices, and kits comprising the antibodies, nucleic acids, vectors according to the present disclosure. The invention encompasses methods of making and using, as well as uses of the antibodies, nucleic acids, vectors, according to the present disclosure, in particular for the detection, diagnosis, monitoring, prevention and treatment HEV infection and associated disease.Definitions

[0046] As used herein, the term “HEV capsid (CA) protein” has its general meaning in the art and refers to a protein encoded by the ORF2 gene of HEV that self-assembles to form the viral capsid (or capsid). The capsid is composed of 90 homodimeric subunits and has aT=3 icosahedral symmetry. The HEV CA monomer is composed of the shell (S), middle (M) and protruding (P) domains. The S and M domains associate tightly around the particle's three-fold axis, while the P domain, linked to the M domain, extends from the virion surface as a dimeric spike. The P domain is essential for virus-host interactions, mediating preattachment to heparan sulfates proteoglycans and binding to unidentified receptor(s) on the cell surface. A representative sequence of HEV ORF2 gene is that of HEV genotype 3 having the nucleotide sequence reported under accession number GenBank DQ079627.1 as accessed on May 31, 2006. The corresponding HEV capsid protein encoded by this gene has the canonical sequence reported under accession number GenBank AAZ30922.1 or SEQ ID NO: 1. Based on structure predictions, the S domain is from positions 126 to 319; the M domain is from positions 320 to 455; and the P domain is from positions 456 to 601 of SEQ ID NO: 1. However, the positions of the domains or sub-units may vary slightly (+1 to +15 and -1 to -15) relative to the indicated positions. The P domain which is recognized by anti-HEV-CA antibodies according to the present disclosure may typically be SEQ ID NO: 2 or SEQ ID NO: 84.

[0047] In the following description, the residues are designated by the standard one letter amino acid code and the indicated positions are determined by alignment with SEQ ID NO: 1. One skilled in the art can easily determine the positions in another HEV capsid proteins, by alignment with the reference sequence using appropriate software available in the art such as BLAST, CLUSTALW, or MAFFT (with G-INS-1 option) and others.

[0048] As used herein, HEV refers to any isolate, strain, lineage, genotype of HEV, or variant or related virus thereof, that is neutralized by the antibodies according to the invention. HEV belongs to the Paslahepevirus genus of the Hepeviradae family. HEV which may be neutralized by the antibodies according to the present invention includes in particular members of Paslahepevirus and Rocahepevirus. Paslahepevirus include the various HEV genotype variants such as HEV genotypes 1 to 8 (HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7, HEV-8), in particular the four HEV genotypes HEV-1, HEV-2, HEV-3 and HEV-4, responsible for human infections. HEV-related virus includes in particular members of other genus of the Hepeviradae capable of infecting humans such as the Rocahepevirus genus which includes Orthohepevirus C1 (or HEV-C1). HEV-C1 circulates in rodents and has been reported as an emerging zoonotic pathogen capable of infecting humans.

[0049] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term "antibody" is meantto encompass an aggregate, polymer, derivative, or conjugate of antibody or antibody fragment. As such, the term antibody encompasses not only whole antibody molecules, but also functional fragments thereof comprising at least the antigen-binding region and functional derivatives thereof.

[0050] In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (A) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. In humans there are four subclasses of IgG: IgG1, lgG2, lgG3 and lgG4 (numbered in order of decreasing concentration in serum). IgA exists in two subclasses, lgA1 and lgA2. Both lgA1 and lgA2 have been found in external secretions (secretory IgA), where lgA2 is more prominent than in the blood (serum IgA). Each chain contains distinct sequence domains. In typical IgG antibodies, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). Secretory IgA are polymeric: 2-4 IgA monomers are linked by two additional chains: the immunoglobulin joining (J) chain(s) and secretory component (SC). The J chain binds covalently to two IgA molecules through disulfide bonds between cysteine residues. The secretory component is a proteolytic cleavage product of the extracellular part of the polymeric immunoglobulin receptor (plgR) which binds to J-chain containing polymeric Ig. Polymeric IgA (mainly the secretory dimer) is produced by plasma cells in the lamina propria adjacent to mucosal surfaces. It binds to the polymetic immunoglobulin receptor on the basolateral surface of epithelial cells and is taken up into the cell via endocytosis. The receptor-lgA complex passes through the cellular compartments before being secreted on the luminal surface of the epithelial cells, still attached to the receptor. Proteolysis of the receptor occurs, and the dimeric IgA molecule, along with a portion of the receptor known as the secretory component - known as slgA, are free to diffuse throughout the lumen.

[0051] The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues thatare primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate in the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0052] The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat etal., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. The predicted CDRs of some anti-HEV antibodies, such as Es1.327, Es4.431, Es4.452 and Es5.127 are described herein.

[0053] The term “heavy-chain antibody” (HCAb) or “unique heavy chain-only” refers to antibodies that only have three regions in their structure, two fixed regions (constant domain) at the C-terminal that are homologous to the CH2 and CH3 of the human antibody, and one variable region at the N-terminal called VHH (Nanobody®) for separation from ordinary VH. Natural antibodies in camelids are unique heavy chain-only antibodies (HCAbs). The single-domain antigen-binding variable domain of HCAbs is called nanobody. Heavy chain only antibodies are preferably Heavy chain only human antibodies.

[0054] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single specificity. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant for the binding specificity.

[0055] As used herein, the term "recombinant antibody" refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies. In some embodiments a recombinant human antibody of this invention has the same amino acid sequence as a naturally-occurring human antibody but differs structurally from the naturally occurring human antibody. For example, in some embodiments the glycosylation pattern is different as a result of the recombinant production of the recombinant human antibody. In some embodiments the recombinant human antibody is chemically modified by addition or subtraction of at least one covalent chemical bond relative to the structure of the human antibody that occurs naturally in humans.

[0056] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., capsid of HEV, preferably P domain). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antigen binding region may comprise one or two variable domains, such as for example a VH domain and a VL domain or a single VHH or VNAR domain. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VHand CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb (sdAb, VHH, VHH. V-NAR or nanobody®) fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain, or any fusion proteins comprising such antigen-binding fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci.85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0057] Examples of antibody derivatives include with no limitation: modified antibodies such as chemically modified antibodies, antibody variants and immunoconjugates; constructions using the antigen-binding fragment of an antibody such as multivalent and / or multispecific (or polyspecific) antibodies. Multivalent antibodies such as bivalent antibodies bind multiple sites on one target. For example, biparatopic or bivalent antibodies can bind to two different epitopes on the target antigen and improve the therapeutic properties of the two parental antibodies or exhibit therapeutic properties, not present in a mixture of the two parental antibodies. Multispecific antibodies such as bispecific antibodies bind multiple sites on different targets. Bivalent or bispecific antibodies are advantageously based on common light chain antibodies to prevent mispairing of heavy and light chains without the need for engineering. Antibodies may also be selected or optimized to improve their developability (homogeneity, stability, solubility and specificity) by methods that are well-known in the art.

[0058] The antibody may be glycosylated. An antibody can be functional for antibodydependent cytotoxicity and / or complement-mediated cytotoxicity or may be non-functional for one or both of these activities.

[0059] Antibodies are prepared by standard methods that are well-known in the art such as hybridoma technology, selected lymphocyte antibody method (SLAM), transgenic animals, recombinant antibody libraries or synthetic production.

[0060] The expression “variable domain” or “variable region” of an antibody heavy or light chain are used interchangeably as the variable region of an antibody consists of a variable domain.

[0061] The phrases "an antibody recognizing an antigen (X)", "an antibody having specificity for an antigen (X)", “an anti-X antibody”, “an antibody against X”, and an “antibody directed against” are used interchangeably herein with the term "an antibody which binds specifically to an antigen (X)”.

[0062] As used herein, “antibody” or “nucleic acid” refers to an isolated antibody or nucleic acid.

[0063] The present disclosure encompasses the therapeutic use of both the antibody or antigen-binding fragment according to the present disclosure (antibody therapy) and a nucleic acid or vector encoding said antibody or antigen-binding fragment, in particular mRNA such as modified mRNA (nucleic acid therapy).

[0064] By the term "specifically binds" as used herein with respect to an antibody, is meant an antibody or antibody fragment, which recognizes and binds with a specific antigen, but does not substantially recognize or bind (detectably bind) other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms "specific binding" or "specifically binding," or "specifically binds," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope " A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled " A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0065] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as the HEV capsid (HEV-CA or CA) protein (monomer, dimer, HEV-like particle (HEV-LP)), particularly the HEV-CA protruding domain (P) while not detectably binding to (i.e., cross-reacting with) other epitopes. The specific binding of the antibody of the present disclosure to the HEV P domain refers to its binding to at least one of a HEV capsid (HEV-CA or CA) protein (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P (or P) protein, in particular chosen from HEV-CA (SEQ ID NO: 83 and 89-101) and HEV-P (SEQ ID NO: 84) proteins. As the P domain is present in the capsidprotein, the specificity of an antibody for the P protein also implies its specificity for the CA protein.

[0066] The specific binding of an antibody to its target antigen may be determined by standard assays such as immunoassay or Biacore assay. An antibody specifically binds to its target antigen when it has: (i) a dissociation constant (KD) of 1 pM or less for its target antigen as determined by Surface plasmon resonance using Biacore assay, and / or (ii) an EC50 (half maximal effective concentration) of 150 ng / mL or less in a direct ELISA binding assay.

[0067] For all ELISA assays, the coating step is performed overnight in PBS buffer. Washings with 0.05% Tween 20-PBS buffer are performed between each step. A blocking step of 2 h with 2% BSA, 1 mM EDTA, 0.05% Tween 20-PBS (Blocking buffer) is performed after the coating step. Antibody dilution and incubation are performed in PBS. Optical densities are measured at appropriate OD and background values given by incubation of PBS alone in coated are subtracted. OD > 0.5 (cut-off value) are considered as positive.

[0068] For direct ELISA binding assay, ELISA plates are coated with 125 ng / well of purified proteins, in particular chose from HEV-CA (SEQ ID NO: 83 and 89-101) and HEV-P (SEQ ID NO: 84). After washings, plates are blocked, washed, and incubated with serial dilutions of monoclonal antibodies for 2 hours. After washings, antigen-antibody complexes are detected using appropriate conjugate, such as goat HRP-conjugated anti-human IgG or IgA antibodies and appropriate chromogenic substrate such as ABTS (for HRP). An optical density (OD) value > 0.5 in ELISA binding assay to HEV-CA protein (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P protein (SEQ ID NO: 83, 84, 89-101) according to the present disclosure indicates the presence of binding affinity. ECso values (ng / ml) are calculated from the titration curves. Antibodies according to the present disclosure have an EC50 (half maximal effective concentration) of 150 ng / mL or less in the ELISA binding assay to HEV-CA protein (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P protein (SEQ ID NO: 83, 84 and 89-101) according to the present disclosure.

[0069] Specificity can further be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is HEV capsid (HEV-CA or CA) protein (monomer, dimer, HEV-like particle (HEV-LP)) or HEV-P (or P) protein, in particular chosen from HEV-CA (SEQ ID NO: 83 and 89 to 101) and HEV-P (SEQ ID NO: 84) proteins. Specificity demonstrated experimentally for at least the HEV-Pprotein and one non-specific antigen means that the antibody is specific to the antigen. The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope.

[0070] As used herein, the term " Avidity" refers to an informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody epitope affinity; the valence of both the antigen and antibody; and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope.

[0071] The antibodies according to the invention compete with each other for binding to HEV-CA protein (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P protein in a competition ELISA binding assay. “Cross-com pete with” is used herein interchangeably with “competitively inhibit the binding of”, or “is a competitive inhibitor of”. For competition ELISAs, ELISA plates are coated with CA protein (SEQ ID NO: 83 and 89-101) and incubated with biotinylated antibodies (at a concentration 0.33 nM) in 1:2 serially diluted solutions of antibody competitors in PBS (IgG concentration range from 1 to 133 nM), and developed as described above using HRP-conjugated streptavidin in blocking buffer.

[0072] Test antibodies may first be screened for their binding affinity to HEV-CA (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P proteins in a direct ELISA binding assay as disclosed above.

[0073] The ability of a test antibody to cross-compete with, or competitively inhibit the binding of antibodies of the present disclosure to HEV-CA (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P proteins in competitive ELISA assay as disclosed above, demonstrates that the test antibody can compete with that antibody for binding to HEV-CA (monomer, dimer, HEV-like particle (HEV-LP)) and HEV-P proteins; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on HEV-CA P domain as the antibody with which it competes.

[0074] Figure 3H shows that the antibodies of group VI which include the references antibodies Es1.327, Es4.431, Es4.452 and Es5.127 of the present disclosure competitively inhibit the binding of each other to HEV-CA protein. The percentage of competitive inhibition of the antibodies may be determined by measuring the area under curve (AUC). The antibodies according to the present disclosure inhibit at least 25%, preferably 30% or more (50%; 60%; 70%; 80%; 90%) of binding of at least one of the reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127; preferably at least the antibodies Es1.327 and Es4.431; more preferably all the reference antibodies. There is no competitive inhibition with other anti-CA antibodies of Group I, II, III and IV, and also with other anti-CA antibodies of group V when considering the four reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127 together or at least Es1.327 and Es4.431 together.

[0075] The present disclosure encompasses anti-HEV-CA P domain antibodies which inhibit at least 25% of binding to HEV-CA and / or HEV-P protein of at least one of the reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127 in the competition ELISA binding assay according to the present disclosure; preferably anti-HEV-CA P domain antibodies which inhibit at least 25% of binding to HEV-CA and / or HEV-P protein of at least the antibodies Es1.327 and Es4.431; more preferably anti-HEV-CA P domain antibodies which inhibit at least 25% of binding to HEV-CA and / or HEV-P protein of all the reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127.

[0076] The absence of polyreactivity of the antibodies according to the present disclosure is determined in ELISA binding assay as previously described (Planchais et al., 2019). ELISA plates are coated with 500 ng / well of purified double stranded (ds)-DNA, KLH, LPS, Lysozyme, Thyroglobulin, Peptidoglycan from B. subtilis, 250 ng / well of insulin, and 125 ng / well of YU2 HIV-1 Env gp140 protein in PBS. After blocking and washing steps, recombinant monoclonal IgG antibodies are tested at 4 pg / ml and 3 consecutive 1:4 dilutions in PBS. Control antibodies, mGO53 (negative) (Wardemann et al., 2003), and ED38 (high positive) (Meffre et al., 2004) are included in each experiment. ELISA binding is developed as described above. OD > 0.5 (cut-off value) are considered as positive. Results obtained with antibodies Es1.327, Es4.431, Es4.452 and Es5.127 according to the present disclosure are illustrated in Fig.4A.

[0077] The absence of reactivity to human proteins of the antibodies according to the present disclosure is determined in Protein microarray binding assay. Experiments are performed at 4°C using ProtoArray Human Protein Microarrays (Thermo Fisher Scientific). Microarrays are blocked for 1 h in blocking solution (Thermo Fisher), washed and incubated for 1h30 with IgG antibodies at 2.5 pg / ml as previously described (Planchais et al., 2019). After washings, arrays are incubated for 1h30 with AF647-conjugated goat anti-human IgG antibodies (at 1 pg / ml in PBS; Thermo Fisher Scientific), and revealed using GenePix4000B microarray scanner (Molecular Devices) and GenePix Pro 6.0 software (Molecular Devices) as previously described (Planchais et al., 2019). Fluorescence intensities are quantified using Spotxel® software (SICASYS Software GmbH, Germany), and mean fluorescence intensity (MFI) signals for each antibody (from duplicate protein spots) are plotted against the reference antibody mGO53 (non-polyreactive isotype control) using GraphPad Prismsoftware (v8.1.2, GraphPad Prism Inc.). For each antibody, Z-scores are calculated using ProtoArray® Prospector software (v5.2.3, Thermo Fisher Scientific), and deviation (o) to the diagonal, and polyreactivity index (PI) values are calculated as previously described (Planchais et al., 2019). Antibodies were defined as polyreactive when PI > 0.21. Results obtained with antibodies Es1.327, Es4.431, Es4.452 and Es5.127 according to the present disclosure are illustrated in Fig.4C.

[0078] The absence of self-reactivity of the antibodies according to the present disclosure is determined in indirect immuno-fluorescence assay (IFA) on HEp-2 cells. Recombinant HEV CA-specific and control IgG antibodies (mGO53 and ED38) at 100 pg / ml are tested in indirect immuno-fluorescence assay (IFA) on HEp-2 cells sections (ANA HEp-2 AeskuSlides®, Aesku. Diagnostics, Wendelsheim, Germany) using the kit’s controls and FITC-conjugated anti-human IgG antibodies as the tracer according to the manufacturer’ instructions. HEp-2 sections are examined using fluorescence microscope and pictures are taken at magnification x 40. Results obtained with antibodies Es1.327, Es4.431, Es4.452 and Es5.127 according to the present disclosure are illustrated in Fig.4B.

[0079] As used herein, the term “neutralizing antibody” refers to an antibody that inhibits virus infection, in particular that inhibits or blocks virus entry into host cells by competing with HEV capsid (CA) protein P domain for binding to its receptor(s) on host cells and blocking P domain interaction with HEV receptor(s) through binding to the P domain. The neutralizing activity of the monoclonal antibody is measured by HEV neutralization assay. One day before infection, 20,000 MAVS-HepG2 cells / well are seeded into 96-well plates. Serially diluted antibodies and HEV virus are incubated in duplicate or triplicate for 30 min prior to be inoculated onto the cells. After 18 h, viral suspensions are removed, and cells are washed five times with pre-warmed PBS at 37°C before adding 200 pl of fresh growth media supplemented with 2% DMSO. Cultures are kept at 37°C, 5% CO2, for 10 days. Then, the supernatants are harvested and the amount of HEV capsid is determined by sandwich ELISA using the non-overlapping HEV-CA mAbs Es1.114 and Es3.157. Plates are coated overnight with 250 ng / well of the capture antibody (Es1.114). After washings, plates are blocked 2 h with ELISA blocking buffer, PBST-washed, followed by a 2 h-incubation with 50 pl of supernatants or 1:3 serially-diluted recombinant HEV-CA as standard (3 pg / ml starting concentration). After washings, biotinylated antibody Es3.157 (at 0.5 pg / ml) is added for 1 h, followed PBST washings and a 30 min incubation with 1:1000 diluted HRP-conjugated streptavidin. After washings, plates are revealed by incubation for 1 h with goat HRP-conjugated anti-human IgG or IgA antibodies (Immunology Jackson ImmunoReseach, 0.8 pg / ml final), and by adding 100 pl of HRP chromogenic substrate (ABTS solution,Euromedex), after washing steps. After 1 h incubation, optical densities are measured at 405 nm (OD405nm). The concentration of virion-free capsid protein is interpolated from the standard curve by non-linear regression using GraphPad Prism software (v.8.2, GraphPad Prism Inc.). Neutralization percentage is calculated by applying the following formula 100-(HEV-CA IgG / HEV-CA no IgG x 100). The neutralizing activity of each monoclonal antibody is expressed as the half maximal inhibitory concentration (IC50). IC50 values (pg / ml or ng / ml) are calculated based on a reconstructed curve of the percentage neutralization at the various concentrations indicated (see Figure 3D). A neutralizing antibody has an IC50 of less than 1 pg / mL and neutralizes at least 80% of HEV in HEV neutralization assay.

[0080] As used herein, the term “ADCC” or “antibody dependent cell cytotoxicity” and “GDC” or “Complement-dependent-cytotoxicity” activity refers to cell depleting activity. ADCC and CDC activities can be measured by standard methods that are well-known in the art.

[0081] The expressions “the junction of the sequences” and “the fusion of the sequences” are equivalent and used interchangeably in the present disclosure.

[0082] As used herein “preventing” HEV infection and / or associated disease means reducing the risk of HEV infection and / or associated disease.

[0083] As used herein “HEV associated disease” refers to a disease caused by HEV infection. HEV associated disease includes in particular hepatitis, more particularly acute hepatitis or chronic hepatitis. Acute hepatitis includes in particular fatal acute liver failure. Chronic hepatitis includes in particular cirrhosis and liver cancer. HEV associated disease may also include extra-hepatic disorders such as neurological injury, renal injury, cryoglobulinaemia, pancreatitis, haematological disorders and others.

[0084] “a”, “an”, and “the” include plural referents, unless the context clearly indicates otherwise. As such, the term “a” (or “an”), “one or more” or “at least one” can be used interchangeably herein; unless specified otherwise, “or” means “and / or”.Neutralizing antibodies

[0085] Neutralizing antibodies or antigen-binding fragments thereof according to the present disclosure include the selected recombinant anti-HEV antibodies Es1.327, Es4.431, Es4.452 and Es5.127 which are structurally characterized by their heavy chain variable domain and light chain variable domain amino acid sequences as described in Table 1 below:Table 1: Variable heavy and light chain amino acid sequences of Es1.327, Es4.431, Es4.452 and Es5.127.

[0086] Neutralizing antibody or antigen-binding fragments thereof according to the present disclosure also includes human antibody which specifically binds to HEV capsid protein P domain and is a competitive inhibitor of binding to the P domain of at least one of the following reference antibodies:the reference human antibody Es1.327 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4;- the reference human antibody Es4.431 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6;the reference human antibody Es4.452 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; andthe reference human antibody Es5.127 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.

[0087] Competitive inhibition with a reference antibody is assayed in a competition ELISA binding assay according to the present disclosure (see definitions). The present disclosure encompasses anti-HEV antibodies which inhibit at least 25% of binding, preferably at least 30% of binding to HEV capsid protein and / or P domain of at least one of the reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127 in the competition ELISA binding assay according to the present disclosure. The reference antibody Es1.327, Es4.431, Es4.452 and Es5.127 comprises the above disclosed heavy and light chain variable region amino acid sequences as presented in Table 1. The reference antibody is preferably an IgG, particularly IgG1, or an IgA. The IgG1 reference antibody preferably comprises the full-length heavy and light chain amino acid sequences as presented in Table 2: Es1.327 (SEQ ID NO: 11-12); ES4.431 (SEQ ID NO: 13-14); Es4.452 (SEQ ID NO: 15-16); Es5.127 (SEQ ID NO: 17-18).

[0088] Examples of neutralizing antibodies according to the present disclosure include the antibodies of Group VI presented in Figure 3H which bind a new epitope and include all the more potent anti-HEV neutralizing antibodies. Group VI includes the reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127 and other antibodies Es1.117, Es.5.104, Es1.177, Es1.142, Es1.119, Es5.223 and Es1.346. Es1.117, Es.5.104, Es1.177, Es1.142, Es1.119, Es5.223 and Es1.346 antibodies have VH and VL domains of SEQ ID NO: 121-122; SEQ ID NO: 123-124; SEQ ID NO: 125-126; SEQ ID NO: 127-128 SEQ ID NO: 129-130; SEQ ID NO: 131-132 and SEQ ID NO: 133-134, respectively.

[0089] In some embodiments, the antibody inhibits at least 40%, 50%, 60%, 70%, 80% or 90% of binding to HEV capsid protein and / or P domain of at least one of the reference antibodies Es1.327, Es4.431, Es4.452 and Es5.127 in the competition ELISA binding assay according to the present disclosure. In particular embodiments, the antibody inhibits at least 70% of binding to HEV capsid protein or P domain of at least one of said reference antibodies, preferably at least two of said reference antibodies.

[0090] In some embodiments, the HEV capsid protein comprises any one of SEQ ID NO: 83 and 89-101, and / or the P domain comprises SEQ ID NO: 84.

[0091] In some embodiments, the HEV capsid protein and / or P domain is from HEV capable of infecting humans. In particular embodiments, the HEV capsid protein and / or P domain is from HEV genotype 1 to 8 (HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7, HEV-8), in particular HEV-1, HEV-2, HEV-3 or HEV-4 responsible for human infections. In preferred embodiments, the HEV capsid protein and / or P domain is selected from the group consisting of: SEQ ID NO: 83-84 and 89-101. In particular embodiments, the HEV capsid protein and / or P domain is from HEV-related virus capable of infecting humans such as Rocahepevirus genus, in particular Orthohepevirus C1 (or HEV-C1). In preferred embodiments, the HEV capsid protein and / or P domain is selected from the group consisting of: SEQ ID NO: 83-84 and 89-101.

[0092] In some embodiments, the antibody is a competitive inhibitor of two of the reference human antibodies disclosed herein. In some embodiments, the antibody is a competitive inhibitor of three of the reference human antibodies disclosed herein. In some embodiments, the antibody is a competitive inhibitor of all the reference human antibodies disclosed herein.

[0093] In some embodiments, the antibody is a competitive inhibitor of at least one of the reference human antibodies Es1.327, Es4.431 and Es5.127; preferably two of the reference human antibodies Es1.327, Es4.431 and Es5.127. In some particular embodiments, the antibody is a competitive inhibitor of the reference human antibodies Es1.327 and Es4.431; Es1.327 and Es5.127; or Es4.431 and Es5.127; preferably the reference human antibodies Es1.327 and Es4.431. Preferably, the reference antibody Es1.327, Es4.431 or Es5.127 is an IgG, particularly IgG1 or an IgA; the IgG1 reference antibody Es1.327, Es4.431 or Es5.127 preferably comprises the full-length heavy and light chain amino acid sequences SEQ ID NO: 11-12, SEQ ID NO: 13-14 and SEQ ID NO: 17-18, respectively.

[0094] Neutralizing antibody or antigen-binding fragments thereof according to the present disclosure also includes human antibody which specifically binds to an epitope of HEV capsid protein P domain consisting of the sequence from positions 481 to 492 and / or the sequence from positions 580 to 594, the indicated positions being determined by alignment with SEQ ID NO: 1. The indicated positions correspond to the loops between the beta-strands R and S (RS loop; positions 481-92) and the beta-strands X and Y (XY loop; positions 580 to 594), respectively. The neutralizing epitope targeted by the antibodies according to the present disclosure is a non-strictly conformational epitope. The neutralizing epitope targeted by the antibodies according to the present disclosure involves two loops (RS and XY) exposed on P domain monomers at the apex of the capsid spike in a region located outside of the dimerization interface. In some embodiments, the antibody or antigen-binding fragmentspecifically binds to a region centered around the RS loop. In some embodiments, the antibody or antigen-binding fragment specifically binds to an epitope of HEV capsid protein P domain located within the region of the RS and XY loops, said region consisting of the fusion of the sequences from positions 481 to 492 and 580 to 594; preferably, the region consists of the fusion of the sequences DQTTYGSSTNPM (SEQ ID NO: 105) and AISTYTTSLGAGPTS (SEQ ID NO: 106). In particular embodiments, the antibody or antigenbinding fragment specifically binds at least one amino acid residue of the P domain selected from the group consisting of: Q482, T483, G486 and S488; more preferably specifically binds the residues Q482, T483 and S488, and optionally further specifically binds the residue G486.

[0095] In some embodiments, the antibody neutralizes HEV with a half maximal inhibitory concentration (IC50) of 20 ng / mL to 1 ng / mL or less. In particular embodiments, the antibody neutralizes HEV with a half maximal inhibitory concentration (IC50) selected from 20 ng / mL or less, 15 ng / mL or less, 10 ng / mL or less, 5 ng / mL or less, and 1 ng / mL or less; preferably 10 ng / mL or less; more preferably 1 ng / mL or less.

[0096] In particular embodiments, the antibody neutralizes at least one HEV capable of infecting humans. In more particular embodiments, the antibody neutralizes at least one HEV chosen from HEV genotype 1 to 8 (HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7, HEV-8), preferably HEV-1, HEV-2, HEV-3, and / or HEV-4 responsible for human infections. In more particular embodiments, the antibody neutralizes at least one HEV-related virus capable of infecting humans such as Rocahepevirus genus, in particular Orthohepevirus C1 (or HEV-C1).

[0097] In particular embodiments, the antibody has no polyreactivity in the ELISA binding assay according to the present disclosure, as compared to a control antibody (see definitions). In particular embodiments, the antibody has no self-reactivity in the indirect immuno-fluorescence assay (I FA) on HEp-2 cells according to the present disclosure, as compared to a control antibody (see definitions). In particular embodiments, the antibody has no predicted reactivity to human proteins in the Protein microarray binding assay according to the present disclosure (see definitions).

[0098] In particular embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 5, 7 and 9.

[0099] In particular embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises:a) a heavy chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 3, and a light chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 4;b) a heavy chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 5, and a light chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 6;c) a heavy chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 8; ord) a heavy chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 9, and a light chain variable region comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 10;in particular embodiments, the heavy and light chain variable region in any one of a) to e) comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the above disclosed sequences.

[0100] Anti-HEV antibodies or antigen-binding fragments thereof with amino acid sequences having at least 90%, for example, at least 95%, 96%, 97%, 98%, or 99% identity to any one of the above defined amino acid sequences are part of the present disclosure, typically anti-HEV antibodies or antigen-binding fragments thereof have an IC50 of 20 ng / mL or less, preferably 10 ng / mL or less, more preferably 1 ng / mL or less, in the HEV neutralization assay according to the present disclosure.

[0101] As used herein, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

[0102] The percent identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences or nucleotide sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithmwhich has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0103] The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as the BLASTN program for nucleic acid or amino acid sequences using as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0104] Full length light and heavy chains amino acid sequences are shown in Table 2.Table 2: Full length heavy and light chain amino acid sequences of Es1.327, Es4.431, Es4.452 and Es5.127. Amino acid sequences of constant isotype regions (human IgG1) are indicated in bold.

[0105] Thus, in particular embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain amino acid sequence selected from the group consisting of: SEQ ID NO: 11, 13, 15 and 17.

[0106] In more particular embodiments, said antibody or antigen-binding fragment thereof comprises:a) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 11 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 12,b) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO:13 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 14,c) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO:15 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 16, ord) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO:17 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 18.

[0107] Anti-HEV antibodies or antigen-binding fragments thereof with amino acid sequences having at least 90%, for example, at least 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the above defined amino acid sequences are part of the present disclosure, typically anti-HEV antibodies or antigen-binding fragments thereof have an IC50 of 20 ng / mL or less, preferably 10 ng / mL or less, more preferably 1 ng / mL or less, in the HEV neutralization assay according to the present disclosure.

[0108] Other neutralizing anti-HEV antibodies or antigen-binding fragments thereof according to the present disclosure which may be used include any antibodies comprising the 6 CDRs of Es1.327, Es4.431, Es4.452 or Es5.127 as described in Table 3 below.Table 3: CDR regions of Es1.327, Es4.431, Es4.452 and Es5.127 according to Kabat numbering.

[0109] In particular embodiments, the anti-HEV antibody or antigen-binding fragment thereof according to the present disclosure comprises:a) a heavy chain variable domain comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20 and a heavy chain CDR3 of SEQ ID NO: 21, b) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26 and a heavy chain CDR3 of SEQ ID NO: 27, c) a heavy chain variable domain comprising a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR3 of SEQ ID NO: 33, or d) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38 and a heavy chain CDR3 of SEQ ID NO: 39.

[0110] In a more particular embodiment, said anti-HEV antibody or antigen-binding fragment thereof comprises:a) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 21, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23 and a light chain CDR3 of SEQ ID NO: 24, b) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26 and a heavy chain CDR3 of SEQ ID NO: 27, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30, c) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR3 of SEQ ID NO: 33,and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 34, a light chain CDR2 of SEQ ID NO: 35, and a light chain CDR3 of SEQ ID NO: 36, or d) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38 and a heavy chain CDR3 of SEQ ID NO: 39, and a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 40, a light chain CDR2 of SEQ ID NO: 41, and a light chain CDR3 of SEQ ID NO: 42.

[0111] It is further contemplated that antibodies or antigen-binding fragment thereof may be further screened or optimized for their neutralizing properties as above defined. In particular, it is contemplated that monoclonal antibodies or antigen-binding fragment thereof may have 1, 2, 3, 4, 5, 6, or more alterations in the amino acid sequence of 1, 2, 3, 4, 5, or 6 CDRs of monoclonal antibodies provided herein, in particular in the CDR of SEQ ID NO: 19-42. It is contemplated that the amino acid in position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of CDR1, CDR2, CDR3, CDR4, CDR5, or CDR6 of the VJ or VDJ region of the light or heavy variable region of antibodies may have an insertion, deletion, or substitution with a conserved or nonconserved amino acid. Such amino acids that can either be substituted or constitute the substitution are disclosed below. In particular embodiments, the monoclonal antibodies or antigen-binding fragments have 1 or 2 conservative substitutions in the amino acid sequence of 1, 2, 3, 4, 5, or 6 CDRs of monoclonal antibodies provided herein, in particular in the CDR of SEQ ID NO: 19-42.

[0112] In some embodiments, the amino acid differences are conservative substitutions, i.e., substitutions of one amino acid with another having similar chemical or physical properties (size, charge or polarity) which substitution generally does not adversely affect the biochemical, biophysical and / or biological properties of the antibody. In particular, the substitution does not disrupt the interaction of the antibody with the spike glycoprotein antigen and neutralizing properties. Said conservative substitution(s) are advantageously chosen within one of the following five groups: Group 1 -small aliphatic, non-polar or slightly polar residues (A, S, T, P, G); Group 2-polar, negatively charged residues and their amides (D, N, E, Q); Group 3-polar, positively charged residues (H, R, K); Group 4-large aliphatic, nonpolar residues (M, L, I, V, C); and Group 5-large, aromatic residues (F, Y, W).

[0113] In more preferred embodiments, said antibody or antigen-binding fragment having the CDR-H1 / H2 / H3 and CDR-L1 / L2 / L3 sequences as disclosed above further comprises:a) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO:11 and / or a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 12,b) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 13 and / or a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 14,c) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO:15 and / or a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 16, ord) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 17 and / or a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 18.

[0114] Neutralizing antibodies or antigen-binding fragments thereof according to the present disclosure defined by their CDR domains may comprise framework regions FR1, FR2, FR3 and FR4 of Es1.327, Es4.431, Es4.452 and Es5.127 antibodies as defined in the table below.Table 4: Framework regions of Es1.327, Es4.431, Es4.452 and Es5.127.

[0115] In a particular aspect of the disclosure, it is disclosed an antibody or antigen-binding fragment thereof as defined above by their CDR domains, wherein:a) the variable heavy domain comprises the amino acid sequence of the framework FR1 selected from the group consisting of: SEQ ID NO: 43, 51, 59 and 67, the amino acid sequence of the framework FR2 selected from the group consisting of: SEQ ID NO: 44, 52, 60 and 68, the amino acid sequence of the framework FR3 selected from the group consisting of: SEQ ID NO: 45, 53, 61 and 69, and / or the amino acid sequence of the framework FR4 selected from the group consisting of: SEQ ID NO: 46, 54, 62 and 70; and / or,b) the variable light domain comprises the amino acid sequence of the framework FR1 selected from the group consisting of: SEQ ID NO: 47, 55, 63 and 71, the amino acid sequence of the framework FR2 selected from the group consisting of: SEQ ID NO: 48, 56, 64 and 72, the amino acid sequence of the framework FR3 selected from the group consisting of: SEQ ID NO: 49, 57, 65 and 73 and / or the amino acid sequence of the framework FR4 selected from the group consisting of: SEQ ID NO: 50, 58, 66 and 74.

[0116] In particular, it is contemplated that monoclonal antibodies or antigen-binding fragment thereof may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more alterations in the amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8 FRs of monoclonal antibodies provided herein, in particular in the FR of SEQ ID NO: 43-74. It is contemplated that the FR sequences have an insertion, deletion, or substitution with a conserved or non-conserved amino acid. Such amino acids that can either be substituted or constitute the substitution are disclosed above. In particular embodiments, the monoclonal antibodies or antigen-binding fragment have 1, 2, 3, 4, 5; preferably 1 or 2 conservative substitutions in the amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8 FRs of monoclonal antibodies provided herein, in particular in the FR of SEQ ID NO: 43-74.

[0117] Variant antibodies according to the present disclosure are functional antibodies that specifically bind to HEV capsid protein P domain and exhibit functional properties that are substantially equal or superior to the corresponding functional properties of the corresponding reference human antibody Es1.327, Es4.431, Es4.452 and Es5.127 as described above. By substantially equal it is herein intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding functional property of the reference human antibody. In particular, variant antibodies according to the present disclosure neutralize HEV virus with an IC50 of 20 ng / mL or less, preferably 10 ng / mL or less, more preferably 1 ng / mL or less in the HEV neutralization assay according to the present disclosure.

[0118] In particular embodiments, a neutralizing antibody or antigen-binding fragments thereof according to the present comprises:a heavy chain variable domain selected from:a heavy chain variable domain comprising at least one of, preferably all three of: a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20 and a heavy chain CDR3 of SEQ ID NO: 21, or a variant with one or two conservative substitution(s); ora heavy chain variable domain comprising at least one of, preferably all three of: a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26 and a heavy chain CDR3 of SEQ ID NO: 27, or a variant with one or two conservative substitution(s); ora heavy chain variable domain comprising at least one of, preferably all three of: a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR3 of SEQ ID NO: 33, or a variant with one or two conservative substitution(s); ora heavy chain variable domain comprising at least one of, preferably all three of: a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38 and a heavy chain CDR3 of SEQ ID NO: 39, or a variant with one or two conservative substitution(s);anda light chain variable domain selected from:a light chain variable domain comprising at least one of, preferably all three of: a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23 and a light chain CDR3 of SEQ ID NO: 24, or a variant with one or two conservative substitution(s); or a light chain variable domain comprising at least one of, preferably all three of: a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29 and a light chain CDR3 of SEQ ID NO: 30, or a variant with one or two conservative substitution(s); or a light chain variable domain comprising at least one of, preferably all three of: a light chain CDR1 of SEQ ID NO: 34, a light chain CDR2 of SEQ ID NO: 35 and a light chain CDR3 of SEQ ID NO: 36, or a variant with one or two conservative substitution(s); or a light chain variable domain comprising at least one of, preferably all three of: a light chain CDR1 of SEQ ID NO: 40, a light chain CDR2 of SEQ ID NO: 41 and a light chain CDR3 of SEQ ID NO: 42, or a variant with one or two conservative substitution(s).

[0119] In some embodiments, the antibody further comprises framework regions FR1, FR2, FR3 and FR4 of Es1.327, Es4.431, Es4.452 and Es5.127 antibodies as defined in Table 4 above or variant thereof as disclosed above. In some embodiments, the antibody comprises heavy and / or light chain variable domain of Es1.327, Es4.431, Es4.452 and Es5.127 antibodies as defined in Table 1 above or variant thereof as disclosed above. The heavy and / or light chain variable domain of the antibody are preferably associated with IgG or IgA constant region that may be further modified as disclosed above. In particular embodiments, the antibody comprises a heavy and / or light chain of Es1.327, Es4.431, Es4.452 and Es5.127 antibodies as defined in Table 3 above or variant thereof as disclosed above.

[0120] In particular embodiments of the disclosure, the variable regions of the antibody as described above may be associated with antibody constant regions, like IgA, IgM, IgE, IgD or IgG such as IGg1, lgG2, lgG3, lgG4. Said variable regions of the antibody is preferably associated with IgG or IgA constant region; preferably IgG1 or IgA (lgA1, lgA2) constant regions. These constant regions are preferably human antibody constant regions. These constant regions may be further mutated or modified, by methods known in the art, in particular for modifying their binding capability towards Fc receptor or enhancing antibody half-life. The antibody comprising IgA constant region may further comprise a J chain and / or a secretory component to generate a polymeric or secretory IgA.

[0121] As used herein, the term “IgG Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acidresidue from position C226 or from P230 to the carboxyl-terminus of the IgG antibody. The numbering of residues in the Fc region is that of the Ell index of Kabat. The C-terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody. Accordingly, a composition of antibodies of the disclosure may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0122] In particular embodiments, the anti-HEV antibody according to the present disclosure is a silent antibody. As used herein, the term “silent” antibody refers to an antibody that exhibits no or low ADCC activity. Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the Art: Strohl 2009 (LALA & N297A); Baudino 2008, D265A (Baudino et al., J. Immunol. 181 (2008): 6664-69, Strohl, CO Biotechnology 20 (2009): 685-91). Examples of silent Fc IgG1 antibodies comprise N297A or L234A and L235A mutations in the IgG 1 Fc amino acid sequence.

[0123] In other particular embodiments, the antibodies or antigen-binding fragments described herein comprise a variant human Fc constant region comprising substitution(s) to enhance antibody half-life. Non-limiting examples of such variant human Fc constant region comprises the LS (M428L and N434S) or YTE (M252Y / S254T / T256E) substitutions in EU index of Kabat.

[0124] In some embodiments, the antibody of the present disclosure does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD16) polypeptide.

[0125] In some embodiments, the antibody of the present disclosure lacks an Fc domain (e.g. lacks a CH2 and / or CH3 domain).

[0126] Other mutations are G236A / A330L / I332E, herein termed “GAALIE which improves antiviral efficacy against pathogens (Bournazos et al., Nature, 2020, 588, 485-490). The M428L and N434S substitutions (LS) are advantageously combined with G236A / A330L / I332E.

[0127] Another modification of the antibodies herein that is contemplated by the present disclosure is pegylation or hesylation or related technologies. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacting with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylationcan be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (01-010) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See for example, EP 0 154 316 by Nishimura et al. and EP 0401 384 by Ishikawa et al.

[0128] Another modification of the antibodies that is herein contemplated is a conjugate or a fusion protein. The antibody or antigen-binding fragment thereof may be fused to another protein moiety of interest or conjugated to an agent of interest. The agent may be for example a therapeutic agent; a label for antibody detection or a protein which increases the half-life of the antibody. In some embodiments, at least the antigen-binding region of the antibody of the present disclosure is fused to a serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. In other embodiments, the antibody or antigen binding fragment according to the present disclosure further comprises a detectable label. Preferred labels include fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those including Europium and Terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and fluorescent derivatives thereof, a chromophore label; a luminescent label such as luminol; a radioactive label such as14C,123l,124l,125l,32P,33P,35S, or3H and others; an affinity-ligand label, such as streptavidin / biotin, avidin / biotin or anti-isotype antibody; an enzyme label, such as alkaline phosphatase, horseradish peroxidase, luciferase, p galactosidase or acetylcholinesterase; an enzyme cofactor label; a hapten conjugate label, such as digoxigenin or dinitrophenyl; a Raman signal generating label; a magnetic label; a spin label; an epitope label, such as the FLAG or HA epitope; a heavy atom label; a nanoparticle label, an electrochemical label; a light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; a spherical shell label; semiconductor nanocrystal label; as well as others known in the art, wherein the label can allow visualization with or without a secondary detection molecule.

[0129] In some embodiments said antibody is polymeric. The polymeric antibody comprises or consists of Ig polymers. The polymeric antibody is preferably a polymeric monoclonal antibody derived from a monoclonal antibody as defined above. The Ig polymers compriseor consist of dimers. The polymeric antibody usually comprises immunoglobulin joining (J) chain(s) in addition to Ig molecules. The J chain is a 137 amino acid polypeptide expressed by plasma or myeloma cells which regulate Ig polymer formation by binding covalently to two Ig molecules through disulfide bonds between cysteine residues. In particular, dimeric antibodies are formed by two monomeric Ig molecules, which covalently bind to a J chain. In a preferred embodiment, said antibody is a polymeric IgA, preferably a polymeric IgA monoclonal antibody derived from a monoclonal antibody as defined above.

[0130] In some embodiments the antibody is a secretory antibody. A secretory antibody can be transported across epithelial cells to the luminal surface of serosal tissues. The secretory antibody is usually a polymeric antibody, preferably a polymeric IgA, comprising a complex of J-chain-containing polymer of Ig and secretory component (SC). The secretory component is a proteolytic cleavage product of the extracellular part of the polymeric immunoglobulin receptor (plgR) which binds to J-chain containing polymeric Ig. The secretory antibody is preferably a secretory IgA monoclonal antibody derived from a monoclonal antibody as defined above.

[0131] In preferred embodiment, the neutralizing antibody is a recombinant human monoclonal antibody, preferably of IgG1 or IgA isotype. The IgA may be monomeric, polymeric or secretory IgA; it is preferably a polymeric or secretory IgA. In more preferred embodiments, the recombinant antibody is a silent antibody that may further comprise mutations and / or or modifications to enhance antibody half-life, as described above.

[0132] The present invention also relates to antigen binding fragment of an antibody that contain at least one variable domain comprising the CDRs domains as described above such as Fv, dsFv, scFv, Fab, Fab’, F(ab’)2. In particular, said antigen binding fragment is a F(ab')2 fragment. The F(ab')2 fragment can be produced by pepsin digestion of an antibody below the hinge disulfide; it comprises two Fab’ fragments, and additionally a portion of the hinge region of the immunoglobulin molecule. Fab fragments are monomeric fragments obtainable by papain digestion of an antibody; they comprise the entire L chain, and a VH-CH1 fragment of the H chain, bound together through a disulfide bond. The Fab' fragments are obtainable from F(ab')2 fragments by cutting a disulfide bond in the hinge region. F(ab')2 fragments are divalent, i.e. they comprise two antigen binding sites, like the native immunoglobulin molecule; on the other hand, Fv (a VHVL dimer constituting the variable part of Fab), dsFv, scFv, Fab, and Fab' fragments are monovalent, i.e. they comprise a single antigen-binding site. These basic antigen-binding fragments of the disclosure can be combined together to obtain multivalent antigen-binding fragments, such as diabodies, tribodies or tetrabodies.These multivalent antigen-binding fragments are also part of the present invention. Fv fragments consist of the VL and VH domains of an antibody associated together by hydrophobic interactions; in dsFv fragments, the VH: VL heterodimer is stabilized by a disulphide bond; in scFv fragments, the VL and VH domains are connected to one another via a flexible peptide linker thus forming a single-chain protein.

[0133] The invention also encompasses combinations of neutralizing antibodies or antigenbinding fragments thereof according to the present disclosure with each other or with other anti-HEV capsid protein P domain human neutralizing antibodies. In some embodiments, a combination of neutralizing antibodies comprises at least one anti-HEV capsid protein P domain human neutralizing antibody according to the present disclosure and at least one other anti-HEV capsid protein P domain human neutralizing antibody. In particular embodiments, the at least other antibody is chosen from an antibody of any one of Groups I, II, III, IV or V as shown in Figure 3H, an antigen-binding fragmentthereof or derivative thereof according to the present disclosure. In particular embodiments, the at least one other antibody is selected from the group consisting of: Es1.282, Es1.268, Es1.231, Es.1.416, Es.1.120, Es4.207, Es2.105, Es2.325, Es3.108, Es2.219 and antigen-binding fragment thereof or derivative thereof according to the present disclosure; preferably Es1.268, Es1.231, Es2.105, Es2.325 and Es3.108. In particular embodiments, the at least one antibody according to the present disclosure is chosen from Es1.327, Es4.431, Es4.452, Es5.127, Es.117, Es.504, Es1.177, Es1.142, Es1.119, Es5.223, Es1.346, and antigenbinding fragment thereof or derivative thereof according to the present disclosure; preferably ES1.327, Es4.431, Es4.452 and ES5.127.Nucleic acid, host cell, antibody production

[0134] Also disclosed herein are nucleic acid molecule(s) that encode(s) the anti-HEV antibody of the present disclosure.

[0135] Typically, said nucleic acid is recombinant, synthetic or semi-synthetic nucleic acid which is expressible in a host cell suitable for antibody expression or production, in particular human antibody production. The host cell may be a cell for recombinant antibody production or a patient cell for antibody production in vivo. Typically, the nucleic acid may be DNA, RNA or mixed molecule, which may further be modified and / or included in any suitable expression vector. As used herein, the terms "vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. The recombinant vector can be a vector for eukaryotic or prokaryoticexpression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of an antibody, either in vitro or in vivo.

[0136] So, a further object of the disclosure relates to a vector comprising a nucleic acid as described herein.

[0137] Examples of nucleic acid molecules are those encoding the variable light and heavy chain amino acid sequences of the anti-HEV antibody as disclosed in the previous section, and using the genetic code and, optionally taking into account the codon bias depending on the host cell species.

[0138] The nucleic acid molecule or construct sequence is advantageously codon-optimized for expression in a host cell suitable for antibody production in host cell, in particular mammalian cells. Codon optimization is used to improve protein expression level in living organism by increasing translational efficiency of target gene. Appropriate methods and softwares for codon optimization in the desired host are well-known in the art and publically available (see for example the GeneOptimizer software suite in Raab et al., Systems and Synthetic Biology, 2010, 4, (3), 215-225).

[0139] The host cell for antibody production may be eukaryote or prokaryote cell. Prokaryote cell is in particular bacteria. Eukaryote cell includes yeast, insect cell and mammalian cell.

[0140] Typically, nucleic acid encoding the variable heavy and light chain of Es1.327 antibody comprises or consists of the sequence SEQ ID NO: 75 and SEQ ID NO: 76 respectively, nucleic acid encoding the variable heavy and light chain of Es4.431 antibody comprises or consists of the sequence SEQ ID NO: 77 and SEQ ID NO: 78 respectively, nucleic acid encoding the variable heavy and light chain of Es4.452 antibody comprises or consists of the sequence SEQ ID NO: 79 and SEQ ID NO: 80 respectively, nucleic acid encoding the variable heavy and light chain of Es5.127 antibody comprises or consists of the sequence SEQ ID NO: 81 and SEQ ID NO: 82 respectively.

[0141] Nucleic acids encoding anti-HEV antibody of the disclosure with nucleotide sequences having at least 80%, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of the above defined nucleotides sequences, wherein said nucleotide sequences include DNA sequences and RNA sequences thereof, are also part of the present disclosure.

[0142] The present disclosure also pertains to nucleic acid molecules that derive from the latter sequences having been optimized for protein expression in host cells, in particular eukaryotic cells, preferably mammalian cells, for example, CHO or HEK cell lines or human cells.

[0143] In some embodiments, said nucleic acid molecule is a eukaryotic, preferably mammalian, expression cassette, wherein the antibody coding sequence(s) is operably linked to appropriate regulatory sequence(s) for their expression in an antibody producing cell or a patient cell. Such sequences which are well-known in the art include in particular a promoter, and further regulatory sequences capable of further controlling the expression of a transgene, such as without limitation, enhancer, terminator and intron. The promoter may be a tissue-specific, ubiquitous, constitutive or inducible promoter that is functional in the antibody producing cell. Such promoters are well-known in the art and their sequences are available in public sequence data bases.

[0144] In particular embodiments, the nucleic acid is RNA, preferably mRNA, wherein the coding sequence of the antibody light and / or heavy chain is operably linked to appropriate regulatory sequence(s) for their expression in an individual’s target cells or tissue(s). mRNA therapy is well-known in the art. mRNA is delivered into the host cell cytoplasm where expression generates the therapeutic protein of interest. mRNA construct comprises a cap structure, 5’ and 3’untranslated regions (UTRs), and open reading frame (ORF), and a 3’poly(A) tail. mRNA construct may be non-replicating mRNA (MRM) or self-amplifying mRNA (SAM). SAM comprises the inclusion of genetic replication machinery derived from positivestrand mRNA viruses, most commonly alphaviruses such as Sindbis and Semliki-Forest viruses. In SAM constructs, the ORF encoding viral structural protein is replaced by the transcript encoding the therapeutic protein of interest, and the viral RNA-dependent RNA polymerase is retained to direct cytoplasmic amplification of the replicon construct. Transreplicating RNA are disclosed for example in WO 2017 / 162461. RNA replicon from alphavirus suitable for gene expression are disclosed in WO 2017 / 162460. mRNA manufacturing process uses plasmid DNA (pDNA) containing a DNA-dependent RNA polymerase promoter, such as T7, and the corresponding sequence for the mRNA construct. The pDNA is linearized to serve as a template for the DNA-dependent RNA polymerase to transcribe the mRNA, and subsequently degraded by a DNase process step. The addition of the 5’ cap and the 3’poly(A) tail can be achieved during the in vitro transcription step or enzymatically after transcription. Enzymatic addition of the cap can be accomplished by using guanylyl transferase and 2’-O-methyltransferase to yield a CapO(N7MeGpppN) or Cap1 (N7MeGpppN2'oMe) structure, respectively, while the poly-A tail can be achieved through enzymatic addition via poly-Apolymerase. mRNA is then purified using standard methods suitable for mRNA purification such as high-pressure liquid chromatography (HPLC) and others. Methods for producing mRNA are disclosed for example in WO 2017 / 182524.

[0145] To improve translation efficiency in treated subject cells, the mRNA according to the invention comprises a sequence which is codon-optimized for expression in human. Further improvements of the mRNA construct according to the invention to improve its stability and translation efficiency in vivo include optimization the length and regulatory element sequences of 5’-UTR and 3’IITR; base and / or sugar modifications in the cap structure to increase ribosomal interaction and / or mRNA stability; and modified nucleosides. Modified nucleosides may be in the 5’-UTR, 3’-UTR or ORF. Examples of modified nucleosides include pseudouridine and N-1-methylpseudouridine that remove intracellular signalling triggers for protein kinase R activation. Examples of modified nucleosides that reduce RNA degradation into cells are disclosed in WO 2013 / 039857. Modified cap structures are disclosed in WO 2011 / 015347 and WO 2019 / 175356. Optimized 3’-UTR sequences are disclosed in WO 2017 / 059902. Modified polyA sequences which improve RNA stability and translation efficiency are disclosed in US 2020 / 0392518. Modified mRNA with improved stability and translation efficiency are also disclosed in WO 2007 / 036366.

[0146] The invention may use any vector suitable for the delivery and expression of nucleic acid into individual’s cells, in particular suitable for nucleic acid therapy. Such vectors that are well-known in the art include viral and non-viral vectors. Non-viral vector includes the various (non-viral) agents which are commonly used to either introduce or maintain nucleic acid into individual’s cells. Agents which are used to introduce nucleic acid into individual’s cells by various means include in particular polymer-based, particle-based, lipid-based, peptide-based delivery vehicles or combinations thereof, such as with no limitations cationic polymer, dendrimer, micelle, liposome, lipopolyplex, exosome, microparticle and nanoparticle including lipid nanoparticle (LNP) and viral-like particles; and cell penetrating peptides (CPP). Agents which are used to maintain nucleic acid into individual’s cells include in particular naked nucleic acid vectors such as plasmids, transposons and mini-circles. Viral vectors are by nature capable of penetrating into cells and delivering nucleic acid(s) of interest into cells, according to a process named as viral transduction. As used herein, the term “viral vector” refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. In viral vectors, viral genes essential for replication and virulence are replaced with an expression cassette for the transgene of interest. Thus, the viral vector genome comprises the transgene expression cassette flanked by the viral sequences required for viral vector production. As used herein, the term “recombinant virus” refers to avirus, in particular a viral vector, produced by standard recombinant DNA technology techniques that are known in the art. As used herein, the term “virus particle” or “viral particle” is intended to mean the extracellular form of a non-pathogenic virus, in particular a viral vector, composed of genetic material made from either DNA or RNA surrounded by a protein coat, called the capsid, and in some cases an envelope derived from portions of host cell membranes and including viral glycoproteins. As used herein, a viral vector refers to a viral vector particle. These vectors have minimal eukaryotic sequences to minimize the possibility of chromosomal integration. In addition, these approaches can advantageously be combined to introduce and maintain the nucleic acid of the invention into individual’s cells.

[0147] In some embodiments, a mRNA according to the present invention as disclosed above is combined with a nucleic-acid delivery agent suitable for delivery of mRNA into mammalian host cells that are well-known in the art. The mRNA delivery agent may be a polymeric carrier, polycationic protein or peptide, lipid nanoparticle or other. For example, the mRNA (non-replicating or self-amplifying) may be delivered into cells using polymers, in particular cationic polymers, such as polyethylenimine (PEI), poly-L-Lysin (PEL), polyvinylamine (PVA) or polyallylamine (PAA), wherein the mRNA is preferentially present in the form of monomers, dimers, trimers or oligomers as disclosed in WO 2021 / 001417. Alternatively, the mRNA may be combined with polyalkyleneimine in the form of polyplex particles, suitable for intramuscular administration as disclosed in WO 2019 / 137999 or WO 2018 / 011406. The mRNA may also be combined with a polycation, in particular protamine, as disclosed in WO 2016 / 000792. One or more mRNA molecules may be formulated within a cationic lipid nanoparticle (LNP); for example the formulation may comprise 20-60% cationic lipid; 5-25% non-cationic lipid, 25-55% sterol and 0.5-15% PEG-modified lipid as disclosed WO 2015 / 164674. The mRNA may also be formulated in RNA decorated particles such as RNA decorated lipid particles, preferably RNA decorated liposomes as disclosed in WO 2015 / 043613.

[0148] In particular embodiments, the vector is a particle or vesicle, in particular lipid-based micro- or nano-vesicle or particle such as liposome or lipid nanoparticle (LNP). In more particular embodiments, the nucleic acid is RNA, in particular mRNA and the vector is a particle or vesicle, in particular LNP as described above. The LNP: mRNA mass ratio can be around 10:1 to 30:1.

[0149] In other embodiments, the nucleic acid is DNA, preferably included in an expression vector such as plasmid or viral vector. The invention also relates to a vector comprising the nucleic acid according to the present disclosure. Preferably, the vector is a recombinantintegrating or non-integrating viral vector. Examples of recombinant viral vectors include, but not limited to, vectors derived from retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, poxvirus, and other virus. Retrovirus includes in particular lentivirus vector such as human immunodeficiency virus, including HIV type 1 (HIV1) and HIV type 2 (HIV2) vectors.

[0150] In preferred embodiments, the expression vector comprises a pair of nucleic acid sequences selected from: a sequence having at least 90% identity with SEQ ID NO: 75 and a sequence having at least 90% identity with SEQ ID NO: 76; a sequence having at least 90% identity with SEQ ID NO: 77 and a sequence having at least 90% identity with SEQ ID NO: 78; a sequence having at least 90% identity with SEQ ID NO: 79 and a sequence having at least 90% identity with SEQ ID NO: 80; a sequence having at least 90% identity with SEQ ID NO: 81 and a sequence having at least 90% identity with SEQ ID NO: 82.

[0151] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es1.327_plgH encoding Es1.327 antibody heavy chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es1.327_plgH. These bacteria Es1.327_plgH were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2021 under the number 1-6140.

[0152] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es1.327_plgL encoding Es1.327 antibody light chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es1.327_plgL. These bacteria Es1.327_plgL were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6141.

[0153] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es4.431_plgH encoding Es4.431 antibody heavy chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es4.431_plgH. These bacteria Es4.431_plgH were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6142.

[0154] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es4.431_plgL encoding Es4.431 antibody light chain inexpressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es4.431_plgL. These bacteria Es4.431_plgL were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6143.

[0155] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es4.452_plgH encoding Es4.452 antibody heavy chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es4.452_plgH. These bacteria Es4.452_plgH were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6144.

[0156] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es4.452_plgL encoding Es4.452 antibody light chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es4.452_plgL. These bacteria Es4.452_plgL were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6145.

[0157] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es5.127_plgH encoding Es5.127 antibody heavy chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es5.127_plgH. These bacteria Es5.127_plgH were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6146.

[0158] In preferred embodiments, the recombinant vector for expression of the antibody of the present disclosure is plasmid Es5.127_plgL encoding Es5.127 antibody light chain in expressible form, particularly as contained in the E. coli bacteria (DH10B, C3019, NEB) transformed with Es5.127_plgL. These bacteria Es5.127_plgL were deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6147.

[0159] The polynucleotide according to the disclosure is prepared by the conventional methods known in the art. For example, it is produced by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or else by total or partial chemical synthesis. The recombinant vectors are constructed and introduced into host cells by the conventional recombinant DNA and genetic engineering techniques, which are known in the art.

[0160] A further object of the present disclosure relates to a host cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention. As used herein, the term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The transformation may be transient or stable over time. Stable transformation may be by integration of the nucleic acid into the host cell genome. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

[0161] Said host cells may be prokaryotic cells such as bacteria or eukaryotic cells such as yeasts, insect cells or mammalian cells. Mammalian cells may be simian, human, dog and rodent cells. Mammalian host cells for expressing the antibodies of the disclosure include in particular Chinese Hamster Ovary (CHO cells) including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker (as described in Kaufman and Sharp, 1982), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, for example GS CHO cell lines together with GS Xceed™ gene expression system (Lonza), HEK-293 cells (ATCC CRL-1573). In a preferred embodiment, said host cells are CHO cells, or HEK-293.

[0162] The polynucleotide, vector or cell of the disclosure are useful for the production of the protein of the invention using well-known recombinant DNA techniques. The polynucleotide or vector are also useful for nucleic acid therapy as disclosed below.

[0163] Antibodies of the present disclosure can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, 1985). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. When recombinant expression vectors encoding antibody genes are introduced into host cells, in particular eukaryotic cells such as mammalian cells, the antibodies are produced by culturing the host cells for a period of time sufficient for expression of the antibody in the host cells and, optionally, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered and purified for example from the culture medium after their secretion using standard protein purification methods (Shukla et al., 2007).Pharmaceutical composition and therapeutic use

[0164] In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing an antibody, antigen-binding fragment, combination of antibodies or antigen-binding fragments, nucleic acid or vector disclosed herein, formulated together with at least one of a pharmaceutically acceptable carrier, an adjuvant, and a preservative.

[0165] In some embodiments, the composition comprises an antibody or combination of antibodies comprising at least an antibody selected from the group consisting of Es1.327, Es4.431, Es4.452, Es5.127, Es.117, Es.504, Es1.177, Es1.142 and Es1.119, their antigenbinding fragments or nucleic acid or vector encoding said antibody or antigen-binding fragment as disclosed herein; preferably an antibody selected from the group consisting of Es1.327, Es4.431, Es4.452 and Es5.127, their antigen-binding fragments or nucleic acid or vector encoding said antibody or antigen-binding fragment as disclosed herein. The composition may comprise a combination of antibodies further comprising at least one other neutralizing antibody chosen from an antibody of any one of Groups I, II, III, IV or V as shown in Figure 3H, its antigen-binding fragment or nucleic acid or vector encoding said antibody or antigen-binding fragment as disclosed herein. The at least one other antibody is preferably selected from the group consisting of: Es1.282, Es1.268, Es1.231, Es.1.416, Es.1.120, Es4.207, Es2.105, Es2.325, Es3.108, Es2.219; more preferably Es1.268, Es1.231, Es2.105, Es2.325 and Es3.108.

[0166] In some embodiments, the nucleic acid is mRNA, preferably modified mRNA as disclosed herein; the mRNA including modified mRNA is advantageously formulated in a particle or vesicle, in particular LNP, as disclosed herein.

[0167] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered.

[0168] Any suitable pharmaceutically acceptable carrier, diluent or excipient can be used in the preparation of a pharmaceutical composition (See e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997). Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as solutions (e.g. saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluids), microemulsions, liposomes, or other ordered structure suitable to accommodate a high product concentration (e.g. microparticles or nanoparticles). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0169] The pharmaceutical composition may be administered by any suitable route(s), including the enteral route, in particular oral route; the parenteral route; and / or the topical route; preferably oral or parenteral route. Parenteral administration includes in particular intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular administration. Parenteral administration may be by injection or perfusion. The pharmaceutical composition may have a systemic action, a local action, in particular on the gastrointestinal mucosa, or both systemic and local actions. The pharmaceutical composition may also be formulated for sustained delivery and / or delayed release.

[0170] In some embodiments, the composition is an injectable composition, in particular for intravenous, subcutaneous or intramuscular injection; preferably intravenous or intramuscular injection. In other embodiments, the pharmaceutical composition is an orally administered composition, in particular delayed-release composition. Antibodies may be orally delivered using appropriate carriers to protect them from gastric acid and digestive proteases. For example, antibodies may be orally administered using nanoparticle carrier or enteric-coated solid formulation, such as capsule or tablet. In some embodiments, the pharmaceutical composition is for sustained local or systemic delivery. In some embodiments, the pharmaceutical composition is formulated for delivery of the antibodies to the gastrointestinal tract for local action.

[0171] In some embodiments the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives are anti-oxidation agents and anti-bacterial agents.In some embodiments the preservative is present at a known concentration. The presence of a preservative in the composition distinguishes the composition from any composition that occurs in nature. It also imbues the composition with unique functions that are not present in any naturally occurring composition, such as in certain embodiments the ability to be used therapeutically under certain conditions in which the preservative is useful.

[0172] In some embodiments the pharmaceutical composition comprises a defined concentration of a recombinant human antibody of this invention. Such compositions do not occur naturally and have a different structure than any naturally occurring composition. The known concentration of the recombinant antibody imbues the compositions with unique functions that are not present in any naturally occurring composition, such as in certain embodiments the ability to be used therapeutically under certain conditions in which the defined concentration of the recombinant antibody is useful.

[0173] In some embodiments, the pharmaceutical composition comprises IgA, in particular polymeric or secretory IgA as disclosed herein. Such pharmaceutical composition comprising IgA is used for local action on the mucosa, in particular the gastro-intestinal mucosa. Pharmaceutical composition comprising IgA, in particular polymeric or secretory IgA are preferred as prophylactic treatment, to prevent HEV infection. In some embodiments, the pharmaceutical composition comprising IgA is for oral administration.

[0174] In other embodiments, the pharmaceutical composition comprises IgG, preferably IgG1. In particular embodiments, the pharmaceutical composition comprising IgG, preferably IgG, 1 is for injection, preferably intravenous or intramuscular injection.

[0175] These pharmaceutical compositions are exemplary only and do not limit the pharmaceutical compositions suitable for other parenteral and non-parenteral administration routes. The pharmaceutical compositions described herein can be packaged in single unit dosage or in multidosage forms.

[0176] Preferably, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Sustained release formulations, such as PLA or PLGA or other polymers, for oral administration or injection can be used.

[0177] In certain aspects, the disclosure relates to an antibody, antigen-binding fragment thereof, combination, or pharmaceutical composition according to any one of the preceding embodiments, for use as a medicament.

[0178] In certain aspects, the disclosure provides the therapeutic use of an antibody, antigen-binding fragment thereof or a composition according to any one of the preceding embodiments, preferably for treating, preventing or alleviating the symptoms of a HEV-associated or HEV-mediated disorder in a subject in need thereof.

[0179] The term “subject” or “patient” as used herein, refers to mammals. Mammalian species that can benefit from the disclosed methods of treatment include in particular humans.

[0180] As used herein, the term "treatment", "treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease, such as according to the present disclosure the reduction of the viral burden. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0181] In a further aspect, the disclosure relates to a method of treating and / or reducing the risk of developing HEV-associated disorder, in a subject in need thereof that comprises administering to the subject a therapeutically effective amount of an antibody, antigen-binding fragment thereof, an antibody combination, or a pharmaceutical composition as described above.

[0182] In some embodiments, the subject is not infected with HEV and the treatment is a prophylactic treatment.

[0183] In some embodiments, the subject has been exposed to HEV or is infected with HEV but has not developed HEV disease and the treatment is a post-exposure prophylactic treatment.

[0184] In particular embodiments, the method is a method of reducing the risk of developing HEV disease, wherein the risk of hospitalization or the risk of death is reduced by the treatment.

[0185] In some embodiments, the subject is a HEV patient and the treatment is a curative treatment.

[0186] In particular embodiments, the method is a method of treating HEV-associated disease, wherein the likelihood of developing severe disease is reduced by the treatment; in particular

[0187] wherein the risk of acute severe infection such as acute severe hepatitis; the risk of developing chronic HEV such as liver fibrosis and cirrhosis; or the risk of death is reduced by the treatment.

[0188] In some embodiments, the patient is a pregnant women or immunocompromised patient. In particular, the immune-compromised patient is a solid organ transplant recipient, more particularly liver transplant recipient.

[0189] In the context of the invention, an "effective amount" means a therapeutically effective amount. As used herein a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result, such as prophylaxis, or treatment of HEV infection and in particular the reduction of the viral burden. The therapeutically effective amount of the product of the invention, or pharmaceutical composition that comprises it may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects.

[0190] The product of the disclosure will be typically included in a pharmaceutical composition or medicament, optionally in combination with a pharmaceutical carrier, diluent and / or adjuvant. Such composition or medicinal product comprises the product of the disclosure in an effective amount, sufficient to provide a desired therapeutic effect, and a pharmaceutically acceptable carrier or excipient.

[0191] In one embodiment the antibody or antigen-binding fragment or the pharmaceutical composition for its therapeutic use is administered to the subject or patient by a parenteral route, in particularly by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular route. In another particular embodiment, the antibody or antigen-binding fragment or the pharmaceutical composition for its therapeutic use is administered to the subject or patient by oral administration.

[0192] The amount of product of the invention that is administered to the subject or patient may vary depending on the particular circumstances of the individual subject or patientincluding, age, sex, and weight of the individual; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and / or concomitant medication that has been prescribed to the subject or patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.

[0193] For any particular subject, specific dosage regimens may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

[0194] In one embodiment, the antibody or antigen-binding fragment according to the disclosure can be administered to the subject or patient for the treatment of HEV associated disease in an amount or dose comprised within a range of 2.5 mg / kg to 40 mg / kg (kg: subject’s or patient’s body weight). In a more particular embodiment, the antibody or antigenbinding fragment is administered in an amount comprised within a range of 5 to 30 mg / kg. In a more particular embodiment, the antibody or antigen-binding fragment is administered at a dosage of at least 5 mg / kg, preferably 10 mg / kg, more preferably 15 mg / kg, and more preferably 30 mg / kg.

[0195] In some embodiments, the pharmaceutical composition is included in a kit that may further comprise instructions or packaging materials that describe how to administer the product contained within the kit to a patient. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In certain embodiments, the kits may include one or more ampoules or syringes that contain the products of the invention in a suitable liquid or solution form.

[0196] Another aspect of the invention relates to a medical device, comprising the pharmaceutical composition according to the present disclosure. The medical device is in a form suitable for the administration of the composition. In some embodiments, the medical device is suitable for the injection of the composition; said medical device is advantageously chosen from a syringe, infusion bag, injection port, and others that are well-known in the art. In other embodiments, the medical device is suitable for the respiratory tract administration of the composition; said medical device is advantageously chosen from an inhaler, a nebulizer, such as small-volume nebulizer, and others that are well-known in the art.

[0197] Another aspect of the invention relates to the use of a pharmaceutical composition according to the present disclosure for the manufacture of a medicament for the prevention or treatment of HEV infection and associated disease.Diagnostic reagents, kits and methods

[0198] The antibodies or fragment thereof comprising the antigen-binding site according to the present disclosure are specific to HEV and react with various HEV genotype variants and some HEV-related viruses. Therefore, they are useful as reagent for the detection of HEV infection in biological samples from individuals suspected to be infected with HEV.

[0199] Non-limiting examples of HEV which may be diagnosed or detected by the antibodies according to the present invention include the various HEV genotype variants such as HEV genotypes 1 to 8 (HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7, HEV-8), in particular the four HEV genotypes HEV-1, HEV-2, HEV-3 and HEV-4, responsible for human infections; and HEV-related virus. HEV-related virus includes in particular members of other genus of the Hepeviradae capable of infecting humans such as the Rocahepevirus genus which includes Orthohepevirus C1 (or HEV-C1). HEV-C1 circulates in rodents and has been reported as an emerging zoonotic pathogen capable of infecting humans.

[0200] Non-limiting examples of antibodies that may be used for the diagnosis or detection of various HEV genotype variants include all include the antibodies of Group VI presented in Figure 3H: Es.117, Es.5.104, Es1.177, Es1.142, Es1.119, Es5.223, Es1.346, Es1.327, Es4.431, Es4.452 and Es5.127. All the listed antibodies react with HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7 and HEV-8; Es5.127, Es1.142 and Es1.119 react with HEC-C1.

[0201] The sample is any sample suspected of containing HEV such as in particular biological samples. A biological sample may be any tissue, body fluid or stool. Non-limiting examples of body fluids include whole-blood, serum, plasma, urine, cerebral spinal fluid (CSF), and mucosal secretions. Preferred biological samples include blood sample (wholeblood, serum, plasma) and stool.

[0202] The detection or diagnosis is performed by immunoassay technique which is well-known in the art and rely on the detection of antigen-antibody complexes using an appropriate label. The method of the invention may use any immunoassay such as with no limitations, immunoblotting, immunoprecipitation, ELISA, immunocytochemistry or immunohistochemistry, and immunofluorescence like flow cytometry assay, and FACS. Flow cytometry, also known as flow virometry, nanoscale flow cytometry or simply small-particle flow cytometry is a rapid, high-throughput, and effective method to quantify intact viral particles released by an infected cell.

[0203] The invention encompasses a method for the detection of HEV in a sample comprising: contacting said sample with an antibody according to the present disclosure and detecting the antigen-antibody complexes formed.

[0204] The method of the invention may use any appropriate label used in immunoassays such as enzymes, chemoluminescent, fluorescent dyes / proteins or radioactive agents, or others. The label may be on the antibody or fragment thereof which binds to the antigen or on a binding-partner such as secondary antibody or avidin / streptavidin conjugated to a label.

[0205] The antibody is preferably labelled, in the form of a conjugate or fusion protein, and the antigen-antibody complexes are detected by measuring the signal from the label by any appropriate means available for that purpose as disclosed above.

[0206] In some embodiments, antigen detection is performed by ELISA, lateral flow immunoassay, or bead-based immunoassay.

[0207] The detection step may be qualitative or semi-quantitative, and may comprise detecting the presence or level of viral antigen in the sample. In some embodiments, the detecting step comprises the determination of the amount of bound antigen in the mixture, and optionally, comparing the amount of bound antigen in the mixture with at least one predetermined value.

[0208] The detection of the presence or level of viral antigen in a biological sample from an individual using the methods of the invention is indicative of whether the individual is suffering from HEV infection or associated disease.

[0209] Therefore, the above method of the invention is useful for the diagnosis of HEV infection or associated disease in an individual, as well as monitoring of treatment in a HEV patient.

[0210] The treatment may be an antiviral treatment or immunotherapeutic treatment using HEV neutralizing antibodies.

[0211] In some embodiments, the above method is a method of diagnosis comprising the step of deducing therefrom whether the individual is suffering from HEV infection or associated disease.

[0212] In some embodiments, the above method is a method of monitoring of treatment in a HEV patient, comprising the step of deducing therefrom whether the treatment is efficient is not. Treatment efficacy is determined by a decrease of viral antigen level compared to previous viral antigen level determined in the patient, before treatment or during the treatment course.

[0213] In some embodiments in connection with this aspect of the invention, the above method of diagnosis comprises a further step of administering an appropriate treatment to the individual depending on whether or not the individual is diagnosed with HEV virus infection.

[0214] In some embodiments in connection with this aspect of the invention, the above method of monitoring of treatment in a HEV patient, comprises a further step of modifying the HEV treatment when said treatment is determined as not being efficient in the patient.

[0215] In another aspect, the disclosure further relates to a kit for the detection or diagnosis of SARS-CoV-2 infection or contamination, comprising at least an antibody or antigen-binding fragment thereof, preferably further including a detectable label. The kit optionally comprises reagents for the detection of the antigen / antibody complex. Reagents available for this purpose are well-known in the art and include with no limitation buffers, secondary antibody conjugated to a label, avidin / streptavidin conjugated to a label. In some preferred embodiments of the kit of the invention, the antibody, and optional reagents are in lyophilised form to allow ambient storage. The components of the kits are packaged together into any of the various containers suitable for antigen / antibody complex detection such as plates, slides, wells, dishes, beads, particles, cups, strands, chips, strips and others. The kit optionally includes instructions for performing at least one specific embodiment of the method of the invention. In some advantageous embodiments, the kit comprises micro-well plates or microtubes, preferably in a dried format, i.e., wherein the wells of the plates or microtubes comprise a dried composition containing at least the antibody, and preferably further comprising all the reagents for the detection of antigen / antibody complex. In some other advantageous embodiments, the antibody and optional reagents are included into any of the devices available for immunoassay.

[0216] The practice of the present invention will employ, unless otherwise indicated, conventional techniques, which are within the skill of the art. Such techniques are explained fully in the literature.

[0217] The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:FIGURE LEGENDS

[0218] Figure 1A-B. A. Sequence and structure diagrams of the HEV-CA protein monomer (PDB ID: 2ZTN) showing the individual domains and the surface representation of HEV VLP.B. Amino acid alignment of the selected Paslahepevirus and Rocahepevirus capsidsequences as compared to the HEV g3-2712 genotype with the shell (S), middle (M) and protruding (P) domains indicated.

[0219] Figure 2A-H. Reactivity profiles of human HEV antibodies and their corresponding immunoglobulin gene repertoire. (A) Bar graph and pie chart showing the proportion of HEV-CA IgG antibodies (n=138) recognizing the P domain for each HEV-exposed donor (left) and in total (right). (B) Pie charts comparing the distribution of H and JH gene usage of anti-CA P domain (CAP), anti-CA MS domains (CAMS) and control (IgG.mB) lgG+memory B-cell antibodies. The number of antibody sequences analyzed is indicated in the center of each pie chart. Groups were compared using 2 x 5 Fisher’s Exact test. (C) Pie charts comparing the K- VS A-lg chain usage of anti-CA P domain (CAP) and anti-CA MS domains (CAMS) IgG antibodies. Groups were compared using 2 x 2 Fisher’s Exact test.(D) Dot plots comparing plots comparing the number of somatic mutations in H, VK and VA genes of anti-CA P domain (CAP), anti-CA MS domains (CAMS) and control (IgG.mB, white) lgG+memory B-cell antibodies. Averaged mutation per group is indicated below. (E) Table categorizing HEV antibodies based on their binding to strictly conformational (no band; black), conformation-sensitive (weak-to-medium reactive band; grey) or non-conformational (strong reactive band; white) epitopes, as determined by immunoblotting. Anti-P antibodies are highlighted in bold, and group VI members are marked with an asterisk. (F) Bar graph showing the proportion of IgG antibodies recognizing conformational epitopes on the HEV-CA protein as determined by immunoblotting analysis (Fig.2E) (left). Pie chart showing the proportion of IgG antibodies binding to the HEV-CA monomers only or to both monomeric and dimeric proteins (right). (G) ELISA graphs showing the reactivity of HEV-CA-specific antibodies against purified recombinant entire HEV-CA proteins of different Paslahepevirus HEV genotypes. (H) ELISA graphs showing the binding of P-reactive (left) and non-P-reactive (right) HEV IgG antibodies against the purified recombinant Rocahepevirus HEV-C1 protein. Pie charts showing the proportion of HEV antibodies recognizing or not the HEV-C1 protein are shown on the side.

[0220] Figure 3A-L. Neutralizing activity of human HEV antibodies. (A) Dot plots comparing the percentage of HEV neutralization of human anti-CA antibodies tested in triplicate at 50 pg / ml according to targeted region (left) and donor (right). Blue-colored dots correspond to P-specific antibodies. (B) Dot plots (left) showing IC50 values of HEV neutralization for anti-P antibodies segregated according to the donors. Pie chart (right) presenting the distribution of anti-P antibodies according to their IC50 values. IC50 are colored-coded (darker colors indicate higher neutralization potencies). (C) Graph showing the neutralization curves of real naked genotype 3 HEV by human ant-CA IgG antibodies asmeasured in vitro using HepG2 / C3A MAVS cells as targets. The dotted horizontal line indicates 50% of neutralization. (D) Neutralization curves as in (C) but with a broader concentration range for highly potent antibodies Es1.327, Es4.431 and Es5.127. (E) Same as in (D) but quantifying HEV RNA copy numbers in culture supernatants. IC50 values are indicated in blue. Error bars in (C-E) indicate the SEM of duplicate values and are representative of at least two independent determinations.

[0221] (F) Dot plots (top) comparing the number of somatic mutations in H and VL genes between neutralizing and non-neutralizing anti-P antibodies. Antibodies with IC50 > 50 pg / ml were considered as non-neutralizing. Groups were compared with 2-tailed Mann-Whitney test. Correlation plot (bottom) showing the frequency of somatic mutations in H and VL genes (f(VH+VL mut.) vs IC50 values of anti-P antibodies. White dots correspond to nonneutralizing antibodies. Two-sided Spearman rho correlation coefficient and corresponding p value are shown. (G) Dot plot showing the distribution of IC50 values of anti-P antibodies according with the epitope group. Highly potent neutralizers are indicated. (H) Competition ELISA heatmap showing the level of binding inhibition to HEV-CA by biotinylated anti-P antibodies (bio-mAbs) in the presence of potential antibody competitor (comp-mAbs). Darker-and white-colored cells indicate high and no or low inhibition, respectively. Antibodies are grouped according to their epitope cluster (I- VI), with colored-coded ICso values and ability to bind strictly conformational vs non-conformational I conformation-sensitive epitopes (left). (I) Heatmap (left) comparing the reactivity of anti-HEV-CA antibodies to purified recombinant P proteins from selected Orthohepevirinae genera (Rocahepevirus, Chirohepevirus and Avihepevirus), depicted in the dendrogram (left; generated from P domain amino acid alignment). The mean ALICs from duplicate titration values are shown. Cells are color-coded according to AUC values, with darker colors indicating high binding and lighter colors representing moderate binding (white = no binding).

[0222] Binding comparison of potent human HEV neutralizing antibodies with murine antibodies 8C11 and 8H3. (J) Heatmap comparing the ELISA reactivity of selected murine (8C11 and 8H3) and human (Es5.127 and Es1.327) anti-P monoclonal IgG antibodies (mAbs) against purified HEV-CA proteins from different genotypes measured as area under the curve (AUC) values with serially-diluted IgGs. Darker- and white-colored cells indicate high and no or low binding, respectively. Means of intra-assay duplicates are shown. (K) Competition ELISA heatmap (top) showing the level of binding inhibition to HEV-CA by biotinylated anti-P antibodies (bio-mAbs) in the presence of potential antibody competitor (comp-mAbs). Darker blue and white colored cells indicate high and no or low inhibition, respectively. Pink colored cell indicates enhanced binding. Means of intra-assay duplicates are shown.Representative competition ELISA graph comparing the binding profile of biotinylated 8H3 (-bio) in the presence of selected potential anti-P antibody competitors. Means ± SD of duplicate values are shown. (L) Heatmap showing the binding assessment of murine anti-P antibodies 8C11 and 8H3 against purified recombinant P proteins from selected Orthohepevirinae genera (Rocahepevirus, Chirohepevirus and Avihepevirus). The mean ALICs from duplicate titration values are shown, and the binding profile of Es5.127 tested in parallel (Fig.3F) is displayed for comparison.

[0223] Figure 4A-D. Off-target binding characteristics of potent HEV neutralizing antibodies. (A) Heatmap showing the ELISA reactivity of selected HEV neutralizing IgG antibodies against a panel of structurally unrelated antigens. Polyreactive (ED38) and non-polyreactive (mGO53) controls are included. Color value is proportional to the reactivity level measured as the area under the curve (AUC). (B) Microscopic images showing the reactivity of selected HEV neutralizing IgG antibodies to HEp2-expressing self-antigens assayed by indirect immunofluorescence assay. The negative (mGO53), low-positive (ED38), and kit’s positive (Ctr+) controls are included. The scale bars represent 40 pm. (C) Microarray plots showing the reactivity selected HEV neutralizing IgG antibodies to human proteins. Each spot corresponds to the z-scores given on a single protein by the reference antibody (z Ref: mGO53, y axis) and test antibody (x axis). (D) Heatmap comparing the reactivity profiles of selected HEV neutralizing IgG antibodies against a panel of self-antigens as measured by a multiplex bead-based binding assay. ED38 (polyreactive), mGO53 (non-polyreactive) and kit’s negative (neg) controls antibodies are included. Color values are proportional to the median fluorescent intensity (MFI) of mean duplicates.

[0224] Figure 5A-D. Cryo-EM data processing of HEV-CA protein in complex with Es1.327, Es4.431 and Es5.127 Fabs. (A) Workflow for cryo-EM data processing performed with cryoSPARC. (B) Representative electron micrograph and 2D-classes for each complex.(C) Fourier-shell correlation (FSC) as a function of the spatial frequency indicating estimated resolutions for the final reconstructions. (D) Overall maps of the complexes colored by local resolution.

[0225] Figure 6A-D. Models of protein fitting into electron density maps. (A) Ribbon model of the ternary complex of Paslahepevirus HEV capsid with Fabs Es5.127 and Es1.114 fitted into the overall map of the complex. (B) Binding surface representation of the interaction zones of Fabs Es1.327, Es4.431 and Es5.127. (C) Same as in B but for murine antibodies 8G12, 8C11 and 8H3. Domain P is colored in shades of green. (D) close-up views of the interaction of domain P with the VH chain of Fabs Es1.327, Es4.431 and Es5.127.

[0226] Figure 7A-D. Structure of HEV capsid P domain complexes with Es1.327, Es4.431 and Es5.127. (A) Surface representation of cryo-EM models of the HEV-CA P domain in complex with Es1.327, Es4.431 and Es5.127 Fabs. Protomers of the P dimer are colored in shades of green. VH and VL chains of each Fab are highlighted with distinctive colors. The black lines indicate the approach angle. White arrows on top views of the complexes indicate the rotation angle of Es4.431 and Es5.127 Fabs relative to Es1.327 Fab.(B) Close-up in ribbon representation of the epitope and paratopes in the P domain-Fab interaction. Hydrogen bonds between chains are represented with dashed lines and the residues participating in the interactions are indicated. (C) Amino acid sequence alignment of Es1.327, Es4.431 and Es5.127 VH and corresponding germline genes. Residues participating in hydrogen bonds indicated in (B) are highlighted with red boxes. (D) Binding of Es1.327, Es4.431 and Es5.127 Fabs to HEV viral-like particle (VLP). Surface representation of the crystal structure of Paslahepevirus HEV VLP (PDB ID: 3HAG) colored by domains according to (A). 5-fold (pentagon), 3-fold (triangle) and 2-fold (oval) symmetry axes are indicated. Fabs in surface representation are shown binding to P dimers around one 5-fold and one 3-fold axes.

[0227] Figure 8A-C. Molecular determinants of cross-genotype binding by group VI anti-P HEV antibodies. (A) SPR sensorgrams (left) comparing the apparent affinity of purified neutralizing anti-P IgG antibodies (n=5) for the binding to genotype 1d (bottom) and 3a (top) HEV-CA proteins. Corresponding calculated KD values are indicated in the dot plot on the right. Rll, response units; FD, fold difference between KDs obtained with genotype 1 and 3 HEV-CA proteins. (B) Modeling of amino acid variations in the RS loop of the P domain across different HEV genotypes. Ribbon representations of Alphafold-predicted P domain structures (green) aligned to the cryo-EM model of the HEV P domain genotype 3 in complex with Es1.327 (blue) or Es4.431 Fabs (brown). Dashed lines indicate hydrogen bonds identified in the cryo-EM structures. HEV-CA UniProt accession numbers: P29326, Q03500, Q9YLQ9, A0A348BSN0, BCD83331.1, BFL88677.1, UEC95198.1, MH410174. Amino acid variants are shown as purple stick representations and highlighted with purple boxes in the sequence alignment (bottom left). (C) Heatmap comparing the ELISA binding (as AUC values) of group VI anti-P antibodies to purified recombinant genotype 3 (2712 strain) HEV-CA P domain mutant proteins Q482A / T483A / S488A, S488P and S488W. The means of assay duplicates are shown. Representative ELISA titration curves of selected antibodies are displayed on the right. Error bars represent the SD of duplicate values. Blue lines indicate the binding to wildtype HEV-CA P-domain protein as a comparator.

[0228] Figure 9A-C. Cryo-EM reconstruction of HEV-C1 P domain in complex with Es5.127. (A) Comparison of Paslahepevirus (cryo-EM) and Rocahepevirus (Alpha-fold) HEV P domains. Left and middle panels: Surface representation of P colored by electrostatic potential on a scale from -5 (negative charge) to 5 (positive charge) kT / e. Black line delimits the interaction zone of Es5.127 Fab with Paslahepevirus P domain. Right panel: close-up views of RS and XY loops in ribbon representation of Paslahepevirus and Rocahepevirus P domain. Side chains of these loops are showed in stick representation. (B) Fitting of the model of Paslahepevirus P / Es5.127 complex into the cryo-EM reconstruction of Rocahepevirus HEV P / Es5.127 complex. (C) SPR sensorgram showing the apparent affinity of the anti-HEV P-domain IgG antibody Es5.127 for binding to purified HEV-C1 protein. The corresponding calculated KD value is indicated in the graph (top left).EXAMPLESMATERIAL AND METHODSHuman samples

[0229] Already stored plasma and PBMC samples from chronically infected HCV individuals used in this study called ANTIHEV were previously obtained from the Liver Unit of Hopital Cochin (Paris, France) as part of study protocol C11-33 approved by the INSERM clinical investigation department with ethical approval from the CPP Ile-de-France II, Paris (ClinicalTrials.gov identifier: n°NCT01534728) (Alanio et al., 2015). All selected donors had positive anti-HEV IgG titers as measured using the Wantai HEV IgG ELISA kit (mean value= 11.8 [2.7-20.1]). The main clinical and immuno-virological characteristics of these donors are summarized in (Table 5). All donors gave written consent to participate in this study. Ethical issues have been monitored by the Ethics Board for European contracts, an ad hoc independent Ethics Committee in charge of reviewing periodically sensitive ethical issues in EU funded research when requested by the EU. All human sera were heat-inactivated at 56°C for 60 min. Human IgG and IgA antibodies were purified from donors’ sera by affinity chromatography using Protein G Sepharose® 4 Fast Flow (GE Healthcare) and peptide M-coupled agarose beads (Invivogen), respectively. Purified serum antibodies were dialyzed against PBS using Slide-A-Lyzer® Cassettes (30K MWCO, Thermo Fisher Scientific).Cell lines

[0230] Freestyle 293-F suspension cells (Thermo Fisher Scientific) were maintained in Freestyle Expression Medium (Thermo Fisher Scientific) and kept under constant shaking at 120 rpm. The human hepatoma-derived cell line HepG2 / C3A transfected with a MAVS-specific shRNA (El Costa et al., 2021) (MAVS-HepG2 cells) were used for HEV neutralization assay. Cells were maintained in Penicillin / Streptomycin-containing 10%-FCS DMEM (Thermo Fisher Scientific). All cell lines were cultured at 37°C, with 5% CO2 and 75% humidity.Antigens and antibody controls

[0231] Codon-optimized nucleotide fragments encoding versions of the ORF2 from HEV 3-2712 strain (DQ079627) HEV-CA [ORF2I26-601], HEV-P [OFR2455-601], HEV-PM [HEV-OFR232O-6OI], HEV-M [HEV-OFR232O-454], HEV-S [HEV-OFR2I26-319], HEV-MS [HE -OFR2I26.454], 1d Morocco (AY230202), 1e Chad T3 (AY204877), MEX-14 (M74506), 3a Meng (AF082843), 3c wbGER27 (FJ705359), 3-2712 (DQ079627), 4c JAK-Sai (AB074915), 4e IND-SW-00-01 (AY723745), 4i SwCH31 (DQ450072), HEV-C1 (ADM35750.1) and P domains from HEV-C1 (ADM35750.1), HEV-C2 HEV-4351 (AB890001.1), bat HEV-CA AYA11_F (MW249012.1) and LR3_F (MW249014.1), avian HEV-CA aHEV (NC023425.1) and VaHEV (MG976720.1), as well as HEV 3-2712 Q482A / T483A / S488A, S488P and S488W mutant proteins were synthesized and cloned into pcDNA3.1 / Zeo(+)expression vector (Thermo Fisher Scientific). All constructs included Kozak and I g K leader sequences at the N-terminus and AviTag and 10xHis C-terminal tags. Control antibodies include polyreactive and non-polyreactive ED38 (Meffre et al., 2004) and mGO53 (Wardemann, 2003), respectively. Recombinant proteins were produced by transient transfection of exponentially growing Freestyle™ 293-F suspension cells (Thermo Fisher Scientific) using the polyethylenimine (PEI)-precipitation method as previously described (Lorin and Mouquet, 2015). Proteins and antibodies were purified from culture supernatants by high-performance chromatography using the Ni Sepharose® Excel Resin or Protein G Sepharose 4 Fast Flow (GE Healthcare), respectively, according to manufacturer’s instructions dialyzed against PBS using Slide-A-Lyzer® dialysis cassettes (Thermo Fisher Scientific), quantified using NanoDrop™ One instrument (Thermo Fisher Scientific), and controlled for purity by SDS-PAGE using NuPAGE 3-12% Bis-tris gels (Life Technologies) as previously described (Lorin and Mouquet, 2015). AviTagged purified HEV-LP / T=1 CA protein (genotype 3, 2712 strain) protein was biotinylated using the Enzymatic Protein Biotinylation Kit (Sigma-Aldrich). HEV ORF220-mer overlapping peptides were synthetized by Genscript. Murine anti-P antibodies 8C11 and 8H327.28 were produced as chimeric human IgG 1 antibodies.Single B-cell sorting and expression-cloning of antibodies

[0232] Peripheral blood human B cells were isolated form donors' PBMCs by MACS using CD19 MicroBeads (Milteny Biotec). CD19+ B cells were then stained with LIVE / DEAD fixabledead cell stain kit (Thermo Fisher Scientific) and incubated for 30 min at 4°C with 1 g of biotinylated HEV-LP / T=1 CA protein. Cells were washed once with 1% FBS-PBS (FACS buffer), and incubated for 30 min at 4°C with a cocktail of mouse anti-human antibodies: CD19 A700 (HIB19, BD Biosciences, San Jose, CA), CD21 BV421 (B-ly4, BD Biosciences), CD27 PE-CF594 (M-T271, BD Biosciences), IgG BV786 (G18-145, BD Biosciences), IgA FITC (IS11-8E10, Miltenyi Biotec, Bergisch Gladbach, Germany), and streptavidin R-PE conjugate (Invitrogen, Thermo Fisher Scientific). Stained cells were washed with FACS buffer and resuspended in 1mM EDTA FACS buffer. Single lgG+and lgA+HEV-CA+B cells were sorted into 96-well using a FACS Aria III sorter (Becton Dickinson) as previously described (Planchais et al., 2022). Following a lymphocyte and single cell gating, dead cells were excluded. B-cell immunophenotyping analyses were performed using FlowJo software (v10.3, FlowJo LLC, Ashland, OR). Single-cell cDNA synthesis using SuperScript IV reverse transcription (Thermo Fisher Scientific) followed by nested-PCR amplifications of IgH, IgK, and IgA genes, and sequence analysis for Ig gene features were performed as previously described (Tiller et al., 2008). Purified digested PCR products were cloned into human Igy1-, IgK-, or IgA-expressing vectors as previously described (Tiller et al., 2008). Recombinant antibodies were produced by transient cotransfection of Freestyle 293-F suspension cells (Thermo Fisher Scientific) using the polyethylenimine (PEI)-precipitation method as previously described (Lorin and Mouquet, 2015). Recombinant human IgG antibodies were purified by protein G affinity chromatography (Protein G Sepharose 4 Fast Flow, GE Healthcare). Purified antibodies were dialyzed against PBS. For competition ELISA experiments, purified antibodies were biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific). Es1.114, Es1.327, Es4.431 and Es5.127 IgH were cloned into a 6xHis-tagged IgG1-Fab expression vector as previously described (Mouquet et al., 2012). Recombinant Fab fragments were produced by transient co-transfection of Freestyle™ 293-F suspension cells (Thermo Fisher Scientific), purified using Ni-sepharose® Excel affinity chromatography (Cytiva), and dialyzed against PBS (Gibco).ELISAs

[0233] ELISAs were performed as previously described (Mouquet et al., 2012). Briefly, high-binding 96-well ELISA plates (Costar, Corning) were coated overnight with 125 ng / well of purified proteins. After washings, plates were blocked 2 hours with 2% BSA, 1mM EDTA, 0.05% Tween 20-PBS (Blocking buffer), washed, and incubated with serial dilutions of purified serum IgG or IgA antibodies, or monoclonal antibodies in PBS for 2 hours. After washings, plates were revealed by incubation for 1 h with goat HRP-conjugated anti-human IgG or IgA antibodies (Immunology Jackson ImmunoReseach, 0.8 pg / ml final), and by adding100 l of HRP chromogenic substrate (ABTS solution, Euromedex), after washing steps. After 1 h incubation, optical densities were measured at 405 nm (OD405nm). For competition ELISAs, HEV-CA coated plates were blocked, washed, incubated for 2 h with biotinylated antibodies (at a concentration 0.33 nM) in 1:2 serially diluted solutions of antibody competitors in PBS (IgG concentration range from 1 to 133 nM), and developed as described above using HRP-conjugated streptavidin (BD Biosciences) (at 0.8 pg / ml in blocking buffer). Binding to overlapping linear peptides covering the HEV capsid and polyreactivity were performed as previously described (Planchais et al., J. Exp. Med., 2022, 219, doi: 10.1084). Experiments were performed using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan Mannedorf, Switzerland). Antibodies were tested in duplicate in at least two independent determinations, which included mGO53 or purified serum IgG and IgA antibodies from healthy donors as negative controls. Polyreactivity ELISA was performed as previously described (Planchais et al., 2019). Briefly, high-binding 96-well ELISA plates were coated overnight with 500 ng / well of purified double stranded (ds)-DNA, KLH, LPS, Lysozyme, Thyroglobulin, Peptidoglycan from B. subtilis, 250 ng / well of insulin (Sigma-Aldrich, Saint-Louis, MO), and 125 ng / well of YU2 HIV-1 Env gp140 protein in PBS. After blocking and washing steps, purified HEV IgG antibodies were tested at 4 pg / ml and 3 consecutive 1:4 dilutions in PBS. Control antibodies, mGO53 (negative), and ED38 (high positive) were included in each experiment. ELISA binding was developed as described above.Multiplex bead-based binding assay

[0234] Antibody cross- reactivity to self-antigens was evaluated by multiplex autoantigenbinding assay using the Milliplex® Human autoimmune autoantibody panel kit (Millipore) following the manufacturer’s instructions. Briefly, IgG antibodies at a final concentration of 25 pg / ml were incubated with the beads overnight at 4°C. After washings, PE-IgG conjugate was added, and mixtures were incubated at room temperature for 1 h 30. After a final washing step, readings were made on a Bio-Plex 200 instrument (BioRad). mGO53 and ED38 antibodies were used as negative and positive controls for polyreactivity, respectively.HEp-2 cell IFA

[0235] Binding of human anti-HEV and control monoclonal IgG antibodies (mGO53 and ED38) to HEp-2 cell-expressing autoantigens were analyzed at 100 pg / ml by indirect immunofluorescence assay (IFA) (ANA HEp-2 AeskuSlides®, Aesku. Diagnostics) following the manufacturer’s instructions. IFA sections were examined using the fluorescence microscope Axio Imager 2 (Zeiss, Jena, Germany), and pictures were taken at magnificationx 40 with 5000 ms-acquisition using ZEN imaging software (Zen 2.0 blue version, Zeiss) at the Photonic BioImaging platform (Institut Pasteur).Protein microarrays

[0236] All experiments were performed at 4°C using ProtoArray Human Protein Microarrays (Thermo Fisher Scientific). Microarrays were blocked for 1 h in blocking solution (Thermo Fisher), washed and incubated for 1 h 30 with IgG antibodies at 2.5 pg / ml as previously described (Planchais et al., 2019). After washings, arrays were incubated for 1 h 30 with AF647-conjugated goat anti-human IgG antibodies (at 1 pg / ml in PBS; Thermo Fisher Scientific), and revealed using GenePix 4000B microarray scanner (Molecular Devices) and GenePix Pro 6.0 software (Molecular Devices). Fluorescence intensities were quantified using Spotxel® software (SICASYS Software GmbH, Germany), and mean fluorescence intensity (MFI) signals for each antibody (from duplicate protein spots) was plotted against the reference antibody mGO53 (non-polyreactive isotype control) using GraphPad Prism software (v8.1.2, GraphPad Prism Inc.). For each antibody, Z-scores were calculated using ProtoArray® Prospector software (v5.2.3, Thermo Fisher Scientific), and deviation (o) to the diagonal, and polyreactivity index (PI) values were calculated as previously described. Antibodies were defined as polyreactive when PI > 0.21.Infrared immunoblotting

[0237] Purified recombinant HEV-CA 3-2712 protein was heat-denatured at 95°C for 5 min in LDS-containing buffer with reducing agent (Invitrogen). Denatured HEV-CA protein (50 pg total) was separated by SDS-PAGE with a NuPAGE 4-12% Bis-Tris Gel, electro-transferred onto nitrocellulose membranes and saturated in PBS-0.05% Tween 20 (PBST)-5% dry milk. Membranes were incubated with HEV-CA mAbs (1 pg / ml) and mouse anti-6xHis antibody (1 pg / ml; BD Biosciences) in PBST-5% dry milk for 2 h. After washing with PBST, membranes were incubated for 1 h with 1:25,000 diluted Alexa Fluor 680-conjugated donkey anti-human IgG (Jackson Immunoresearch) and 1:25,000-diluted IR Dye 800CW-conjugated goat antimouse IgG (Li-COR Biosciences) in PBST-5% dry milk. Membranes were washed and examined with the Odyssey Infrared Imaging system (LI-COR Biosciences).HEV neutralization assay

[0238] HEV-3c viral suspensions were generated using fecal samples from infected pigs as previously described (Sophie et al., 2014). Viral stock was prepared at 4x106GE / ml to obtain 2x105GE / well. One day before infection, 20,000 MAVS-HepG2 cells / well were seeded into 96-well plates. Serially diluted antibodies and virus were incubated in duplicate or triplicatefor 30 min prior to be inoculated onto the cells. After 18 h, viral suspensions were removed, and cells were washed five times with pre-warmed PBS at 37°C before adding 200 pl of fresh growth media supplemented with 2% DMSO. Cultures were kept at 37°C, 5% CO2, for 10 days. Supernatants were harvested and processed to quantify the viral capsid protein and the viral RNA content by ELISA and RT-qPCR, respectively. To determine the amount of HEV capsid in culture supernatants, a sandwich ELISA was performed using the nonoverlapping HEV-CA mAbs Es1.114 and Es3.157. Plates were coated overnight with 250 ng / well of the capture antibody (Es1.114). After washings, plates were blocked 2 h with ELISA blocking buffer, PBST-washed, followed by a 2 h-incubation with 50 pl of supernatants or 1:3 serially-diluted recombinant HEV-CA as standard (3 pg / ml starting concentration). After washings, biotinylated antibody Es3.157 (at 0.5 pg / ml) was added for 1 h, followed PBST washings and a 30 min incubation with 1:1000 diluted HRP-conjugated streptavidin. After washings, plates were revealed as described above. The concentration of virion-free capsid protein was interpolated from the standard curve by non-linear regression using GraphPad Prism software (v.8.2, GraphPad Prism Inc.). Neutralization percentage was calculated by applying the following formula 100-(HEV-CA IgG / HEV-CA no IgG x 100)Real time RT-qPCR

[0239] Total RNA was extracted from 150 pl of culture supernatant using the NucleoSpin® RNA virus kit (Macherey-Nagel) following manufacturer's instructions. RNA was eluted with 50 pl of pre-heated (70°C) water and immediately stored at -80°C. HEV RNA quantification was performed as previously described (Jothikumar et al., 2006). Briefly, a one-step TaqM an RT-PCR was performed on 2 pl of total RNA (final volume of 20 pl) using the QuantiTect® Probe RT-PCR Kit (Qiagen) according to manufacturer's instructions. Reverse (JVHEVR, 5’-AGGGGTTGGTTGGATGAA-3’; SEQ ID NO: 102) and forward (JVHEVF, 5’-GGTGGTTTCTGGGGTGAC-3’; SEQ ID NO: 103) primers were used at final concentrations of 0.25 pM and 0.06 pM, respectively. The probe (JVHEVP, 5’-FAM-TGATTCTCAGCCCTTCGC-MGB-3’; SEQ ID NO: 104) was used at a final concentration of 0.31 pM. Reverse transcription was performed at 50°C for 20 min followed by a denaturation step at 95°C for 15 min. DNA was amplified by 45 cycles at 95°C for 10 s and 58°C for 45 s, and a final cooling step at 40°C for 30 s. Sample processing and analysis were performed using a QuantStudio 6 Flex Real-Time PCR System (ThermoFisher Scientific). Standard quantification curves were generated using HEV RNA obtained from the pcDNA3.1+ / VHE3f / standard plasmid (Barnaud et al., 2012). After linearization, 1 pg of purified plasmid was used for RNA in vitro transcription using the mMESSAGE mMACHINE™ T7 Transcription Kit (ThermoFisher Scientific) and then purified using the NucleoSpin® RNAKit (Mackerey-Nagel). RNA was immediately aliquoted at a final concentration of 0.5x1012copies / pl and stored at -80°C until use.Surface plasmon resonance

[0240] Surface plasmon resonance (SPR)-based technology (using the Biacore 2000 system: Biacore Cytiva) was employed to assess the kinetics of interactions between anti-P monoclonal antibodies and genotype 3a and 1d HEV-CA proteins, as well as HEV-C1 proteins. Protein G sensor chips (Ref# 29179316, Biacore Cytiva) were used for these experiments. Purified IgG antibodies at a concentration of 10 pg / ml in HBS-EP buffer (10 mM HEPES pH 7.2; 150 mM NaCI; 3 mM EDTA, and 0.005 % Tween 20) were loaded onto the sensor chip channels for a contact time of 1 min. Once the baseline stabilized, HEV-CA proteins at concentrations ranging from 5 or 10 to 0.312 nM (in 2-fold dilution steps) in HBS-EP buffer were injected over the sensor surface at a flow rate of 30 pl / min. The association and dissociation phases of the anti body- protein interactions were monitored for 4 and 5 minutes, respectively. All kinetic measurements were conducted at 22 °C. Following the injection of purified capsid proteins, sensor surface was regenerated by exposure to a 10 mM glycine solution at pH 1.5 for 30 s, effectively dissociating IgG molecules from the immobilized protein G. The binding kinetics were determined by global analysis using the Langmuir binding model implemented in the BIAevaluation Software (v4.1.1, Biacore).Cryo-EM data collection, refinement, and modelling

[0241] Movies of the complexes of purified HEV-CA 3-2712 protein with Fabs Es5.127 / Es1.114, Fab Es1.327 and Fab Es4.431 were collected on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV equipped with a Gatan K3 direct electron detector and BioQuantum energy filter. Movies were acquired in tiff format in counting mode using EPU automated image acquisition software at a nominal magnification of 105,000x (0.86A / pixel), defocus list of -1.0 to -3.0 pm and a total dose of ~50e- / A2. The movies for the complex HEV-C1 protein with Fab Es5.127 were collected on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) also operating at 300 kV equipped with Falcon 4i direct electron detector and Selectris X energy filter. Movies were acquired in EER format with the EPU automated image acquisition software EPU at a nominal magnification of 215,000x (0.56A / pixel), defocus list of -1.0 to -3.0 pm and a total dose of ~40e- / A2. The raw movies were processed for patch motion correction and patch CTF estimation using cryoSPARC v4.0.3 (Punjani et al., 2017). Initial particles were picked using blob picker and selected 2D classes were used for more extensive template picking. After several iterations of 2D classification, 3 different classes of ab-initio models werereconstructed and refined using Non-Uniform Refinement. In the case of the complexes including Fab Es5.127, there was a marked preferred orientation. Picking with Topaz (Bepler et al., 2019) helped to recover set of particles with more orientations allowing reconstruction of more complete ab-initio models. The resolution was further improved with local motion correction. Models of the Fabs generated by Alphafold and the crystal structure of the P domain (PDB code: 3HAG) were fitted into the maps. The models were exanimated and manually corrected using Coot software (Emsley et al., 2010) and refined with real-space refinement in the Phenix suite (Liebschner et al., 2019).Statistical analyses

[0242] Comparison of the different immunoglobulin gene features between groups were assayed using two-sided 2x2 or 2x5 Fisher’s Exact tests with SISA online tools (http: / / www.quantitativeskills.com / sisa). The numbers of VH, VK and VA mutations were compared across groups of antibodies using two-tailed Mann-Whitney test with GraphPad Prism software (v.8.4, GraphPad Prism Inc.). Volcano plot comparing gene features (n=236 parameters) of HEV-CA B cells and control memory B cells was performed using GraphPad Prism software (v.8.4, GraphPad Prism Inc.). The y axis indicates the statistics expressed as -logic (p-values) and the x axis represents the differences between the group means for each parameter. The Barnes-Hut implementation of t-distributed stochastic neighbor embedding (t-SNE) was computed using FlowJo software (v.10.3, FlowJo LLC, Ashland, OR) with 2000 iterations and a perplexity parameter of 200. Colors represent density of surface expression markers or cell-populations varying from low (blue) to high (red). Phylogenetic tree was built using CLC Main Workbench (Qiagen) on aligned H sequences using the Neighbor-Joining method with a bootstrap analysis on 100 replicates.RESULTSHuman HEV memory B-cell antibodies

[0243] To characterize the memory B-cell antibody response to HEV in individuals who seroconverted after infection, a soluble recombinant HEV-CA protein (genotype 3 (Yamashita et al., 2009), HEV-ORF2i26-6oi) that forms homodimers having the potential to assemble into capsid viral particles (HEV-LP / T=1) (Mori and Matsuura, 2011) was produced (Fig.1). The five selected HEV-exposed donors showed serum IgG antibody binding to HEV-CA protein by ELISA (ECso range [5.3-53.7 pg / ml]), but low IgA seroreactivity (Table 5).

[0244] Table 5. Characteristics of HEV-exposed immune donors

[0245] Purified serum IgG antibodies from donors d1, d3 and d4 neutralized in vitro naked genotype 3 HEV (3c strain) with IC50 values ranging from 38 to 89 pg / ml, while no neutralization was evidenced for individuals with weaker HEV-CA IgG titers (d2 and d5) (Table 5). Binding analyses of the purified serum IgGs using recombinant protein domains (protruding (P), middle (M) and shell (S), P plus M (PM) and M plus S (MS)) showed strong antibody responses against P, PM and MS, but no or low IgG reactivity against the S or M proteins alone (data not shown). Competition experiments for P or MS antigen recognition by serum IgG antibodies in the presence of PM protein showed a decreased reactivity against P only, suggesting that most anti-MS IgGs target epitopes overlapping the M and S domains or the S domain alone (data not shown). No distinct HEV-CA immunoreactive peptides were unambiguously identified; however, two peptides located at the N- and C-termini of the M domain exhibited slightly higher antibody reactivity for some donors (data not shown). This suggests that serum HEV-CA antibodies predominantly recognize discontinuous epitopes, consistent with previous reports14. (Zhou and Zhao, 2023) (data not shown).

[0246] Peripheral blood B cells isolated from the five donors were stained with fluorescently labelled HEV-CA dimers and analyzed by flow cytometry. HEV-CA-reactive lgG+and lgA+B cells had frequencies ranging from 0.026%-0.23% and 0.035%-0.060%, respectively (Table 5), and were mainly resting memory B lymphocytes (CD19+CD27+CD21+; data not shown). From 1,065 HEV-CA-binding memory B cells captured by single-cell flow cytometric sorting, 684 heavy (IgH) and 693 light (IgL) variable domain genes were amplified by RT-PCR. From the 443 matched antibody sequences, a total of 172 unique monoclonal antibodies (157 IgG and 15 IgA) was produced by recombinant expression cloning (Tiller et al., 2008). 138 IgG (87% [63-97%]) and 6 IgA (40% [0-6]; all cloned from donor d1) antibodies strongly bound to HEV-CA protein by ELISA (mean ECso=16 [2-96] ng / ml; (Table 6). Fifty seven out of the 138 anti-HEV-CA antibodies (41%) were part of clonally expanded B-cell lineages, which were mainly found in donor d1 and d4 (75% and 65%, respectively) (data not shown). Only one clonotype was identified among HEV IgA antibodies (data not shown).Antibody repertoire and reactivity profile of HEV antibodies

[0247] Immunoglobulin genes of anti-HEV antibodies were somatically mutated at a similar level to those from lgG+memory B cells of healthy donors (IgG.mB) (Prigent et al., 2016) (20.7 vs 19.1 nucleotide mutations, p=0.20 for IgH and 11.8 vs 11, p=0.15 for IgL) (Table 6).Comparison of HEV IgG gene features with IgG.mB revealed a significant enrichment of H4 (p=0.0015) and JK1 p=0.0084) gene usage data not shown). Notably, HEV antibodies showed a strong increase in VH4-31 gene usage (15.2% vs 1.4 % for IgG.mB, p=0.0015, data not shown). A decreased use of VH3-JH6 gene rearrangements and a higher expression frequency of IgG 1 subclass were also found in HEV antibodies (data not shown). Strikingly, HEV antibodies had a strong bias towards kappa light chain (IgK) usage (83% vs 62% for IgG.mB, p=0.002), particularly those from donors d2 and d4 that all expressed an IgK (data not shown). Maximum-likelihood estimation for VH amino acid similarity revealed the diversity of the HEV antibody gene repertoire, with sequences shuffled among donors and clustering according to VH family usage (data not shown). Unlike many other antiviral antibodies (Dunand et al., 2015; Velavan et al., 2021), there was no apparent inter-individual sequence convergence indicative of a stereotyped or public antibody response.

[0248] ELISA mapping analyses identified 31 IgGs (22.5%, n=138) and one IgA (17%, n=6) binding to the P domain (Fig. 2A). Thus, except for donor d1 (53.6%, n=28), a minority of HEV antibodies targeted the P domain (d2, 15.6%, n=32; d3, 10.8%, n=37; d4, 11.7%, n=34; d5, 42.8%, n=7) (Fig.2A and Table 6). Non-P-reactive antibodies bound strongly to HEV MS protein, while only a moderate if any reactivity to M and S domains alone was detected (data not shown). This is consistent with the serum IgG mapping analyses and confirms the immunodominance of epitopes dependent on both domains. MS-reactive antibodies more frequently used VH4 genes ( / .e., VH4-31) and IgK [88% vs 64% for anti-P], whereas anti-P were enriched in VH3-30-expressing antibodies (Fig.2, B and C). HEV antibodies targeting the P domain exhibited slightly higher levels of somatic mutations compared to those binding to other regions of the CA protein, although this difference did not reach statistical significance (19.2 vs 21.1 nucleotide substitutions, p=0.15, Fig.2D). Immunoblotting analyses showed that half of the HEV IgG antibodies (52%, n=138) completely lost their reactivity against the denatured capsid protein (Fig.2E), indicating that they recognize conformational epitopes. Disruption of capsid conformation more frequently affected the reactivity of anti-MS than anti-P antibodies (61% vs 22%, respectively), indicating a higher prevalence of MS-specific antibodies targeting discontinuous epitopes (Fig.2, E and F). Most conformationindependent antibodies (75%, n=63) including Es1.327, Es4.431, Es4.452 and Es5.127, recognized both CA protein dimers and monomers (Fig.2, E and F).

[0249] HEV-5 to HEV-8 variants in the Paslahepevirus genus infect animal hosts, but can present a zoonotic potential (Velavan et al., 2021). To determine whether the human HEV antibodies recognize the different HEV genotypes their binding to purified recombinant capsid proteins from HEV-1 to HEV-8 was evaluated (data not shown). The vast majority of the HEV antibodies (92%, n=144) cross-reacted with all capsid variants with an average ECso of 13.6 ng / ml [0.1-260 ng / ml] (Fig.2G). The HEV-related virus Orthohepevirus C1 (or HEV-C1) in the Rocahepevirus genus, genetically diverging from the human genotypes (data not shown), circulates in rodents (Kinast et al., 2022). However, it has been reported as an emerging zoonotic pathogen capable of infecting humans (Qian et al., 2022). It was found that most HEV antibodies also reacted against the purified HEV-C1 capsid protein by ELISA (data not shown). Anti-MS bound more frequently than anti-P antibodies (81% vs 16% for anti-P, p<0.0001) (Fig.2H ) which is likely due to the higher degree of sequence conservation in the capsid shell protein region (MS) compared to the more variable P domain.Potent HEV neutralizing antibodies

[0250] Next, the HEV neutralizing activity of the anti-CA antibodies was evaluated with the in vitro assay using 3c strain naked virions. In line with the role of the capsid P domain in virus-host cell interactions, 78% of anti-P antibodies (n=32) exhibited neutralizing capacity, whereas only few anti-MS antibodies neutralized HEV (3%, n=106) (Fig.3A). Notably, 22% of anti-P antibodies potently neutralized HEV with half-maximum inhibitory concentration (IC50) values below 10 ng / ml [0.2-9 ng / ml], and 35% had an IC50 comprised between 13 and 56 ng / ml (Fig.3 B, C, D and E). Together, half of the anti-P antibodies cloned from all five donors efficiently neutralized HEV with IC50 < 60 ng / ml (Fig.3B). Strikingly, the 3 anti-P IgGs isolated from donor d5 were potent neutralizers, which contrast with the poor IgG seroneutralization measured in this individual (Table 5 and Fig.3B). This suggests a qualitative discordance between the serologic and memory B-cell compartments, reminiscent of the seroreversion phenomenon (Krain et al., 2014). Of note, neutralizing anti-P antibodies (IC50 < 50 pg / ml) exhibited a higher number of somatic mutations, inversely correlating with antibody IC50 values (Fig.3F). Top neutralizers (Es1.327, Es4.431 and Es5.127; Fig.3 C, D, E and G) were neither polyreactive by ELISA nor bound to self-antigens in human protein microarray and multiplex autoantigen panel, and by indirect immunofluorescence on HEp-2 cells (Fig.4). To further map the HEV antibodies, cross-competition ELISA experiments for their binding to the CA protein were next performed. Anti-P antibodies were grouped into six epitope clusters (l-VI) based on the competition profiles obtained (Fig.3H). Between groups, off-diagonal competition with cluster II, IV, V and VI antibodies were also observed (Fig.3H), suggesting physical proximity and / or overlap of the targeted epitopes resulting in stericinhibition or allosteric interferences for antibody binding. All members in the largest group (VI, n=11), which bound to non-conformational epitopes, were highly potent neutralizers (average IC50 = 13.9 [0.2-42] ng / ml) (Fig.3 G and H). In contrast, many antibodies in the other groups (l-V) did not neutralize HEV or showed only weak neutralization (Fig.3 G and H). Most members of group VI blocked the binding of antibodies in groups I, III, and IV non-reciprocally, while reciprocally inhibiting those in group V. Specifically, antibodies Es1.327, Es4.431, Es5.223, and Es1.346 from group VI blocked the binding of group V and I members only, suggesting diverse binding fingerprints and / or angles of approach on this capsid region. The four anti-P antibodies cross- reacting with the HEV-C1 capsid (Es1.119, Es1.142, Es1.346 and Es5.127) belonged to the group VI and utilized the VA1-51 gene (Table 6). However, group VI antibodies did not display any specific global antibody gene features (Table 6).Notably, only some group VI antibodies cross-reacted with both P domain proteins of Rocahepevirus (ferret and rat HEV), whereas none of the anti-P antibodies bound to P proteins from the Chirohepevirus and Avihepevirus genera (Fig. 3I). Group VI top neutralizers (Es1.327, Es4.431, Es4.452 and Es5.127) blocked HEV-CA recognition by the murine anti-P antibodies 8C11 and 8H3 (Zhang et al., 2005), and none enhanced 8H3 binding to HEV-CA, which occurs following 8C11's primary attachment (Zheng et al., 2024) (Fig.3 J, K and L). Murine anti-P antibodies 8C11 and 8H3 did not block HEV-CA recognition by group VI neutralizers (Fig.3K). Group VI neutralizing antibodies were neither polyreactive by ELISA nor bound to self-antigens in human protein microarray and multiplex autoantigen panel, as well as by indirect immunofluorescence on HEp-2 cells (Fig.4b).Structural characterization of potent HEV neutralizing antibodies

[0251] To define the neutralizing epitopes on the HEV capsid and their interactions with the most potent human antibodies, cryogenic electron microscopy (cryo-EM) of the HEV-CA in complex with antigen-binding fragments (Fab) of the group VI anti-P neutralizers Es1.327, Es4.431 and Es5.127 was performed (Fig.3 C, D, E and G, Fig.5, Tables 7 to 9). Global refinements yielded well resolved maps of 3.5A and 3.4A resolution for Es1.327 and Es4.431 Fab-HEV-CA complexes, respectively (Fig.5). Particles complexed with Es5.127 Fab adopted preferred orientations in the vitrified ice hampering the reconstruction of interpretable maps. A ternary complex with the non-overlapping Es1.114 Fab helped to obtain low-resolution reconstructions, which despite the density discontinuity, allowed fitting AlphaFold models into density and thus identify the interaction zone of Es5.127 (Fig.5, Fig.6A).Collectively, the three cryo-EM maps displayed one Fab molecule bound to each protomer of the P dimer (Fig.5, Fig.7A), whereas S and M domains were not visible due to high flexibility of the connecting linkers.

[0252] The structure of the three Fab-HEV-CA complexes revealed that most of the contacts were mediated by the IgH, representing 86 and 80% of the total interface area for Es1.327 and Es4.431, respectively, and recognizing a small, exposed region at the top of the P domain without involving the dimeric interface (Fig.7A, Fig.6B, and Table 10). This region is distinct from the epitope bound by previously disclosed murine monoclonal antibodies 8H3, 8G12 and 8C11 (Tang et al., 2011; Gu et al., 2015; Zhao, 2015; Fig.6C).

[0253] Table 10: Buried surface area calculated for the complex between HEV-CA P domain and Fabs594.2 sq A (8.5%) 586.2 sq A (7.8%)516.5 sq A (7.4%) 476.3 sq A (6.5%)T31 O Q482 NE2H100 ND1 T483 OH100 ND1 G486 OH100 O S488 NThe pair of residues forming hydrogen bonds betweenchains are indicated for each complex.

[0254] This region spans only 7.8 and 6.5% of the exposed surface of the P domain in the interaction with Es1.327 and Es4.431, respectively, and involves the loops between p-strands R and S (residues 481-492) and X and Y (residues 580-594) (Guu et al., 2009), the former embedded between the CDRH1 and CDRH3 loops supporting most of the Fab contacts (Fig.6D and Fig.7B). The CDRH3 loops of Es1.327 (12 residues) and Es4.431 (15 residues) establish hydrogen bonds with the main and side chains of a similar repertoire of P residues including Q482, T483, and S488 (Fig.6D, Fig.7B and Table 10). In contrast, the longer CDRH3 of Fabs Es4.431 (15 residues) and Es5.127 (16 residues) establish a reduced number of polar contacts (Fig.6D and Fig.7B). Alignment with VH germline genes shows that CDRH1 residues involved in RS loop interactions, located at positions H31 and T31 of Es1.327 and Es4.431, respectively, are somatically mutated (Fig.7C).

[0255] Among the three antibodies, Es1.327 exhibited the narrowest approach angle (29°), whereas Es4.431 and Es5.127 displayed wider angles (42° and 51°, respectively) (Fig.7A).Interestingly, Es5.127 lies nearly perpendicularly to the P domain dimer (Fig.7A), and the comparison of the position of CDRH1 and CDRH3 with the other two Fabs showed that they are in opposite directions relative to loops RS and XY, indicating that its Fab flips around 180° to interact with this common interaction zone (Fig.7 A and B). Modelling of Fab bindingto the viral particle showed that Es1.327 and Es4.431 can occupy all available binding sites without clashes between Fabs or affecting the organization of the virion (Fig.7D). In contrast, the wider approach angle of Fab Es5.127 would cause a clash with the Fabs binding to neighbor P dimers, therefore Es5.127 can only partially occupy the binding sites available at the surface of the virus, limiting its opsonization potential (Fig.7D). It was concluded that the region recognized by group VI mAbs constitutes a novel HEV-CA vulnerability site that differs from those previously reported for murine neutralizing monoclonal antibodies or vaccinated individuals (Zhao et al., 2015; Wen et al., 2020) (Fig.6C).Structural and molecular basis of potent HEV neutralizing antibody cross-reactivity

[0256] Despite its genetic diversity, HEV has a single serotype, consistent with conserved neutralizing sites recognized by antibodies from immunized animals and vaccinated individuals (Zhou and Zhao, 2023; Zhang et al., 2023). In line with this, most human HEV antibodies reported here, including neutralizers, exhibited pan-genotypic reactivity (Fig.2 and Table 6). To further investigate the molecular basis of antibody cross- reactivity, surface plasmon resonance was used to measure the affinities of the five most potent group VI anti-P neutralizing antibodies for HEV-CA proteins from genotype 3 and 1, the latter being among the most divergent. Four of the five antibodies (Es1.327, Es5.127, Es4.431, and Es1.117) showed high and comparable affinities for both genotype 3 and 1 HEV-CA proteins (KD = 0.16-0.81 nM), with a consistent 1.5- to 2.9-fold preference for genotype 1 (Fig. 8A and Table 11). In contrast, Es4.452 exhibited a 53-fold higher affinity for genotype 1 compared to genotype 3 (Fig. 8A and Table 11). Then, the cross- reactivity potential of the Es1.327 and Es4.431 antibodies was examined by aligning AlphaFold-generated models of P domains from HEV genotypes 1-8 with the cryo-EM structures of the genotype 3 P domain-antibody complexes (Fig.8B). It was found that the RS loop displays conservative amino acid substitutions across genotypes (e.g., Thr-to-Ser and vice versa) as well as substitutions at positions 490 / 491, which lie outside the targeted epitopes (Fig.8B). Therefore, these changes are unlikely to significantly impact antibody binding to the capsid protein in different HEV variants. The binding of group VI anti-P antibodies remained unaffected when key contact residues Q482, T483 and S488 were simultaneously replaced with Ala residues, as the interactions primarily involve the main chain of these residues (Fig.8C). However, mutation of residue S488, located at the tip of the RS loop, which inserts between the CDRH1 and CDRH3 loops of Es1.327, to either Trp or Pro drastically impaired binding (Fig.8C). In contrast, these mutations had no effect on other group VI anti-P antibodies, such as Es4.431, which also binds to the RS loop but engages a different set of interactions due to its longerCDRH3 (Fig.8C). This suggests that only substitutions introducing steric clashes or local conformational changes can disrupt the binding of group VI antibodies to the HEV-CA protein.

[0257] Table 11: Affinities of potent HEV neutralizing antibodies to HEV-CA and HEV-C1 proteinsHEV-CA |q1a| HEV-CA |q3-2712| HEV-C1ka (M'1s~1) kd (s~1) Kp (M) ka (M'1s'1) kd (s'1) Ko (M) ka (M'1s'1) kd (s'1) Ko (M) Es1.327 1.0E+06 1.9E-04 1.9E-10 3.0E+05 1.7E-04 5.6E-10 n.d. n.d. n.d. Es5.127 8.9E+05 2.4E-04 2.7E-10 6.8E+05 2.8E-04 4.1E-10 3.1E+05 4.9E-04 1.6E-09 Es4.431 8.4E+05 3.3E-04 4.0E-10 3.8E+05 3.1E-04 8.1E-10 n.d. n.d. n.d. Es4.452 1.2E+05 4.5E-04 3.7E-09 2.7E+03 5.1E-04 2.0E-07 n.d. n.d. n.d. Es1.117 1.8E+06 2.8E-04 1.6E-10 8.4E+05 2.7E-04 3.2E-10 n.d. n.d. n.d. n.d.: not determined as no significant binding detected by surface plasmon resonance.

[0258] The antibody Es5.127 was one of the group VI members cross-reacting with the zoonotic HEV-C1 (Table 6). Interestingly, amino acid sequence alignment and Alpha Fold modelling of the HEV-C1 capsid protein revealed a low level of conservation of the RS loop displaying differences in the electrostatic potential of the putative binding site of Es5.127 (Fig.9A). A 4.5A resolution cryo-EM map of the complex HEV-C1 capsid with Es5.127 Fab was obtained (Table 12).

[0259] Table 12: Cryo-EM data collection, refinement and validation statistics for the complex HEV-C1-CA P domain with Es5.127 FabData collection and processing _Magnification 105,000xVoltage (kV) 300Microscope Titan KriosElectron exposure (e- / A2) 50Defocus range (pm) -1 -0 to -3.0Pixel size (A) 0.86Initial particle images (no.) 97277Final particle images (no.) 56650Map resolution (A) 4.5FSC threshold 0.143

[0260] This analysis revealed that the HEV-CA / Es5.127 Fab model fits well into this map, providing evidence of a consistent Fab binding despite local differences in the P domain of the two capsid proteins (Fig.9B). Es5.127 showed high affinity for HEV-C1 by surface plasmon resonance, with a KD similar to genotype 3 HEV-CA (0.41 nM vs 1.6 nM) (Fig.9C and Table 12). Future investigations would be needed to determine the neutralizing capacity of Es5.127 against HEV-C1.Discussion

[0261] The molecular and functional attributes of human antibodies targeting the HEV capsid antigen have remained elusive. This word provides the characterization of 144 human anti-HEV-CA monoclonal antibodies cloned from the memory B cells of HEV seroconverted donors. It was found that despite the diversity of the HEV antibody gene repertoire and targeted epitopes, anti-capsid memory B-cell antibodies harbor certain features, such as the predominance of IgK and VH4-31 gene usage for those targeting conformational MS domain epitopes. Most antibodies recognize the MS domains, but not as strongly the individual domains, which could be explained by their tight association, and the preferential exposure of shared conformation-dependent epitopes (Guu et al., 2009). In contrast, a fraction of HEV antibodies (22%) bound to the P domain that contains the minimum antigenic region inducing neutralizing antibodies (Tang et al., 2011; Zhao et al., 2015; He et al., 2008), mainly on non-conformational epitopes. In agreement, the most potent neutralizing antibodies described here targeted the P domain, with group VI antibodies being the top neutralizers. In this diverse anti-P antibody collection, it was not observed preferential traits such as a VH1-69 gene bias and dimeric P conformational epitopes as reported in vaccinated humans (Wen et al., 2020), and immunized or infected animals (Li et al., 2009; Gu et al., 2015; Zhao et al., 2015). Anticapsid IgG antibody and HEV neutralization levels were high for all donors except for d2 and d5. Still, potent group VI neutralizing antibodies were cloned from blood memory B cells of d5. This is reminiscent of seroreversion described in convalescent HEV individuals (Krain et al., 2014; Servant-Delmas et al., 2016; Faber et al., 2018; Lopez-Lopez et al., 2022), and suggest that anamnestic responses in such immune individuals would trigger the production of protecting neutralizing antibodies. Although the inventors demonstrated high affinity, pan-genotypic binding and epitope conservation for potent anti-P neutralizers, their neutralizing activity against non-genotype 3 HEV has yet to be determined.

[0262] Neutralizing epitopes targeted by potent group VI antibodies, revealed by structural analyses, involve two loops (RS) exposed on P domain monomers at the apex of the capsid spike in a region located outside of the dimerization interface (Guu et al., 2009). Despite that these group VI antibodies recognize a common P region, mainly focused on the RS loop, they showed different binding fingerprints and adopted distinct poses. This suggests that this site can accommodate different types of potent antibodies and therefore, is a crucial component to consider for HEV vaccine design. Strikingly, the region centered around the RS loop has never been identified as a HEV neutralizing site in previous human or immunized animal studies (Zhao et al., 2015; Wen et al., 2020). The presence of neutralizing epitopes on the P domain outside of the apical dimerized surface (Li et al., 2009; Yamashita et al.,2009), suggests there are alternative ways to neutralize HEV by blocking the pre-attachment and / or receptor-binding steps. Allosteric effects resulting in conformational changes of the capsid protein or a physical disruption of the virions have also been described (Yamashita et al., 2009; Zheng et al., 2019). The extent to which anti-RS loop antibodies elicit such allosteric effects remains unknown. In contrast to monoclonal antibodies cloned from HEV vaccine recipients (Wen et al., 2020), a correlation between somatic mutation frequency and the neutralization potency of anti-P antibodies was identified, consistent with previous findings for SARS-CoV-2 and HIV-1 antibodies (Sok et al., 2013; Muecksch et al., 2021). However, the contribution of somatic mutations to neutralization potency can vary among anti-P antibodies, as demonstrated in group VI. Es1.327 and Es4.431 displayed S31-mutated antigen-contacting residues in their CDRH1, a key feature previously reported for HCV neutralizing antibodies (Weber et al., 2022). In contrast, Es5.127 antibody exhibited minimal somatic mutation loads and primarily formed contacts with the HEV P domain through germline-encoded residues. Thus, potent HEV neutralizers do not always require extensive B-cell affinity maturation and could be effective in a near-germline configuration as previously shown for antibodies neutralizing RSV, CMV, HCV and SARS-CoV-2 (Bailey et al., 2017; Kreer et al., 2020; Goodwin et al., 2018; Thomson et al., 2008). Binding modeling on HEV virion predicted that Es5.127 would not bind to all available antigenic sites due to clashes between adjacent IgG molecules, whereas Es1.327 and Es4.431 could occupy all available P domains without affecting virion organization. These distinct modes of binding may modulate the extent of target opsonization on infected cells and thus, the Fc-dependent antiviral effectiveness of anti-P neutralizing antibodies. In this regard, the Fc-dependent effector functions of HEV antibodies remains to be investigated.

[0263] The spillover of pathogens from animal hosts to humans is a major contributor to the emergence of infectious diseases as illustrated for instance with HIV-1, Ebola virus or SARS-CoV-2 (Jones et al., 2008; Mollentze and Streicker, 2020). Given the serious potential threats to global health posed by zoonotic pathogens, it is crucial to note that HEV has been ranked among the top ten infectious agents with a high risk of spillover (Grange et al., 2021). Acquired knowledge on humoral immunity in HEV-exposed humans is therefore crucial to help developing future interventional strategies and facilitating outbreak preparedness (Crowe, 2017; Pantaleo et al., 2022). In line with the proposed concept of a “common serotype” for the four main genotypes (Krain et al., 2014), it was found that HEV antibodies were broadly reactive against all Paslahepevirus HEV genotypes, also including the minor zoonotic variants HEV-5 to HEV-8 (Kinast et al., 2022). HEV-5 and HEV-8 can experimentally infect cynomolgus macaques, and a case of human infection with HEV-7 has been reported,confirming the potential risk of zoonotic HEV variants (Li et al., 2019, 2016; Wang et al., 2019; Lee et al., 2016). The zoonotic threat of rat hepatitis virus HEV-C1 was still debated until the recently described human cases in Hong Kong, central Africa and Europe (Sridhar et al., 2018; Andonov et al., 2019; Rivero-Juarez et al., 2022; Qian et al., 2022; Sridhar et al., 2021b). Although HEV and HEV-C1 capsid proteins are antigenically distinct, sharing 64% amino acid identity (Sridhar et al., 2021a; Bai et al., 2020), 66% of the HEV antibodies characterized here strongly cross-reacted with HEV-C1 capsid antigen. The vast majority bound to the more conserved viral shell (MS domains), but a fraction of them recognized the P domain. Notably, all four HEV-C1 anti- P- reactive antibodies, isolated from different donors, belonged to group VI and expressed VA1-51 gene. Comparison of cryo-EM maps of HEV and HEV-C1 capsid proteins in complex with Es5.127 revealed equivalent binding approaches. Hence, HEV and HEV-C1 capsid proteins contain common epitopes targeted by potent human neutralizing antibodies. Whether Es5.127 antibody neutralizes HEV-C1 needs to be confirmed. Together, these data highlight the HEV pan-genotypic cross- protective potential of naturally acquired antibodies to HEV upon recall with another genotype and possibly, against zoonotic transmission. Aside from advancing our understanding of the humoral immune response against hepatotropic viruses, these findings have direct implications for developing effective HEV vaccines and antibody-based immune therapies. HEV vaccination confers protection in recipients, and a vaccine based on p239 capsid protein VLPs holds approved use in China (Zhang et al., 2023). Thus, it would be interesting to explore whether vaccine-induced polyclonal serum and memory B-cell antibodies include group VI HEV-CA antibodies, particularly Es5.127-type IgGs. Murine neutralizing antibodies (Zhang et al., 2023) and the potent human HEV neutralizing antibodies reported in this study recognize the P domain, highlighting its potential as a minimal protective immunogen. However, vaccines based solely on the P protein may lack immunogenicity without a potent adjuvant (Zhang et al., 2023). Considering the healthcare benefits afforded by antibody therapies to fight infectious diseases (Crowe, 2022; Pantaleo et al., 2022), and the excellent antiviral properties of Es1.327, Es4.431 and Es5.127, these antibodies represent promising candidates for prophylactic and / or therapeutic interventions against Paslahepevirus and Rocahepevirus infections. Long-acting versions of these broadly HEV neutralizing antibodies with extended half-life could be used to provide protective immunity in immunocompromised populations and pregnant women.Table 6a. Immunoglobulin gene repertoire of human HEV memory B-cell antibodies.0 d1 1-69 / 1-69D 3-16 3 y1 13 2 4 25 8 K 2D-28 / 2-28 2 9 0 0 11 4 5 3 d1 3-48 3-16 4 a1 14 2 2 11 3 K 4-1 4 9 0 0 23 4 15 d1 4-31 6-19 5 y1 12 1 1 17 5 K 1D-39 / 1-39 2 9 0 0 10 4 1 8 d1 3-30 3-10 4 y1 15 1 2 25 7 A 2-14 3 10 2 0 16 5 7 2 d1 4-31 5-5 / 5-18 4 y1 12 2 3 19 6 K 1-5 3 9 1 0 7 3 1 2 d1 4-61 5-5 / 5-18 / 3-10 4 a1 13 4 2 36 11 K 1D-39 / 1-39 3 9 0 0 32 9 1 3 d1 4-59 2-8 4 y1 23 2 4 14 4 K 3-11 5 10 1 0 5 2 4 8 d1 4-38-2 1-26 / 3-16 4 y1 11 1 3 19 6 A 2-11 1 8 0 0 12 4 6 1 d1 3-30 3-3Z3-9 4 y1 16 2 4 21 6 K 3-11 4 9 1 0 10 4 3 6 d1 1-69 3-16 / 3-9 4 a1 10 1 2 23 7 K 2D-28 / 2-28 2 9 0 0 3 1 1 7 d1 1-18 1-14 4 y1 12 1 1 36 11 K 1D-39 / 1-39 4 10 0 1 23 8 2 9 d1 3-11 6-19 3 y1 13 0 2 27 8 K 3-15 1 9 0 0 6 2 1 3 d1 1-69 / 1-69D 3-22 4 y1 23 4 3 22 6 A 1-44 3 12 0 2 5 2 4 4 d1 4-31 3-16 5 y1 12 0 2 20 6 K 3-15 2 10 1 0 9 3 4 6 d1 3-7 2-2 4 y1 16 1 2 19 6 A 1-51 3 / 2 11 0 1 9 3 1 2 d1 1-69 / 1-69D 5-18 / 3-10 / 3-16 4 y1 10 0 1 27 8 K 2D-28 / 2-28 1 9 1 0 3 1 4 1 d1 1-8 6-19 6 y1 20 0 2 16 5 K 1-5 2 9 0 0 12 5 2 5 d1 3-30-3 6-19 3 y1 16 0 1 18 5 A 2-14 3 / 2 10 1 0 15 5 2 7 d1 3-30-3 2-21 / 1-20 / 1-7 4 a1 8 1 2 7 2 A 2-8 3 10 0 0 10 3 2 6 d1 1-69 5-24 6 y1 19 2 3 16 5 K 1-33 / 1D-33 4 9 0 0 13 5 4 1 d2 3-30 / 3-33 5-5 / 5-18 4 y1 12 1 3 24 7 K 3-11 4 9 1 0 10 4 1 5 d2 3-74 2-21 4 y1 13 1 3 8 2 K 1-27 4 9 1 0 4 1 1 7 d2 4-4 2-2 / 2-15 5 y1 14 1 2 26 8 K 4-1 1 9 0 0 11 4 1 0 d2 4-31 1-26 4 y1 11 1 1 24 7 K 1-27 2 9 0 0 9 3 2 6 d2 1-18 5-24 6 y1 20 0 2 24 7 K 3-20 1 9 0 0 14 5 1 3 d2 1-18 3-22 6 y1 18 1 3 26 7 K 4-1 1 8 0 0 19 7 1 2 d2 4-31 6-19 4 y1 10 0 3 29 9 K 3-11 1 9 1 0 11 4 14 d2 3-23 4-23 / 4-17 4 y1 11 0 3 21 6 K 3-15 1 9 1 0 12 4 3 5 d2 1-2 3-10 / 3-3 5 y1 17 1 3 22 6 K 1-5 1 10 0 0 7 3 1 3 d2 4-30-2 2-8 / 5-24 2 y1 10 0 1 18 6 K 3D-15 2 11 1 0 12 4 1 1 d2 1-18 3-10 / 4-23 / 1-7 5 y1 13 0 3 18 5 K 3-15 1 9 1 1 10 4 1 2 d2 1-3 3-22 6 y1 17 1 2 24 7 K 2D-28 / 2-28 4 8 0 0 7 3 1 8 d2 4-4 2-212-8 4 y1 16 2 1 28 8 K 1-5 4 9 1 0 5 2 3 0 d2 3-30 / 3-33 1-1 4 y1 12 0 2 24 7 K 3-11 4 9 1 0 11 4 1 2 d2 3-33 3-22 3 y1 18 0 1 23 7 K 3-20 2 10 1 0 13 5 1 7 d2 4-4 3-22 / 3-16 6 y1 21 1 3 16 4 K 1D-39 / 1-39 1 9 1 0 17 6 1 9 d2 5-Es1.1 1-14 / 2-21 / 2-15 6 y1 19 2 3 25 7 K 3-20 2 8 0 0 9 3 3 4 d2 4-39 3-10 5 / 4 y1 12 2 1 21 6 K 1-5 1 9 1 0 13 5 1 6 d2 1-18 3-22 6 y1 20 0 3 21 6 K 3-20 1 9 0 0 14 5 1 9 d2 4-4 2-21 / 3-9 6 y1 22 1 3 17 5 K 1D-39 / 1-39 1 9 1 0 21 8 4 8 d2 3-30 5-12 4 y1 12 1 3 25 8 K 3-11 2 9 1 0 16 6 2 0 d2 3-48 3-22 3 y1 16 0 2 15 4 K 3-11 2 10 1 1 11 4 2 8 d2 4-31 3-3 4 y1 10 1 3 21 6 K 3-15 2 10 2 0 11 4 1 2 d2 1-18 6-19 5 y1 13 1 2 16 5 K 3-20 5 10 0 0 13 5 1 6 d2 3-30 1-14 / 1-20 / 1-7 3 y1 12 0 3 11 3 K 3-11 2 9 1 0 6 2 1 7 d2 4-4 3-9 5 y1 16 1 2 19 6 K 1D-39 / 1-39 2 9 1 0 15 6 2 1 d2 3-74 2-21 4 y1 12 0 4 7 2 K 1-27 1 9 1 0 3 1 1 5 d2 3-23 6-19 4 y1 17 1 1 18 5 K 3-11 4 9 1 0 11 4 2 6 d2 1-2 2-15 / 2-21 6 y1 20 2 1 17 5 K 3-11 5 10 0 0 9 3 1 9 d2 4-59 2-212-8 5 y1 11 3 1 43 13 K 1D-39 / 1-39 1 9 1 0 18 7 1 6 d2 3-30 3-16 4 y1 9 1 1 14 4 K 1-5 1 10 0 1 9 3 1 2 d2 1-18 6-13 5 y1 11 0 3 22 7 K 3-20 1 9 0 0 14 5 42 d3 4-4 3-3 4 y1 14 0 2 31 9 K 1-17 1 9 2 0 11 4 1 8 d3 4-59 5-5 / 5-18 5 y2 15 2 2 19 6 K 2D-28 / 2-28 1 9 0 0 7 2 1 0 d3 3-30 / 3-30-3 1-26 / 2-8 4 y1 8 0 2 28 9 A 2-8 3 10 0 0 16 5 1 1 d3 3-30 3-3 6 y4 18 1 3 24 7 K 3-11 3 9 1 0 16 6 1 2 d3 4-4 3-10 3 y2 17 2 2 18 5 K 1D-39 / 1-39 1 9 1 0 14 5 1 5 d3 4-39 5-24 2 y2 13 2 1 18 5 A 1-51 3 11 0 1 15 5 1 6 d3 3-30 / 3-30-3 3-16 4 y2 19 1 6 8 2 K 3-20 1 9 1 0 11 4 1 7 d3 3-21 3-16 6 y1 20 2 3 21 6 A 1-40 3 11 1 1 11 4 1 8 d3 3-23 1-26 / 5-12 / 4-23 5 y1 13 1 1 20 6 K 3-20 1 9 1 0 20 7 1 0 d3 4-39 3-3 4 y1 20 2 2 19 5 K 3-20 1 10 1 0 13 5 1 3 d3 4-61 5-5 / 5-18 / 3-10 5 y3 9 0 1 20 6 K 3D-15 1 10 0 0 8 3 1 5 d3 3-21 1-1 / 7-27 3 y1 10 0 2 18 6 K 1-5 2 9 0 0 9 3 1 7 d3 4-39 5-12 4 y1 9 4 1 32 10 A 1-51 3 11 0 1 11 4 1 1 d3 3-23 6-19 4 y2 11 0 2 27 8 A 2-14 1 11 0 0 16 5 1 2 d3 1-69 3-10 6 y1 15 2 2 26 8 A 2-23 3 / 2 11 0 0 14 5 1 3 d3 4-31 7-27 4 y1 11 1 1 17 5 A 2-11 3 9 0 0 13 4 1 7 d3 4-31 1-26 4 y4 10 1 3 25 8 K 3-15 1 10 0 0 17 6 1 9 d3 4-39 3-9 4 y2 8 2 1 36 11 K 1-9 5 10 0 1 19 7 1 0 d3 1-69 / 1-69D 3-10 6 y1 15 1 2 25 7 K 2D-28 / 2-28 2 9 1 0 14 5 1 2 d3 4-61 2-15 6 y1 18 0 1 19 5 K 3-15 4 10 0 0 5 2 1 5 d3 4-31 3-9 5 / 4 y2 10 0 2 23 7 K 3D-15 3 10 1 0 16 6 1 7 d3 3-11 7-27 4 y1 7 0 2 9 3 K 1-5 1 8 0 0 4 2 3 0 d3 4-39 4-23 / 4-17 1 y1 9 1 1 30 9 K 3-15 1 9 1 0 9 3 1 3 d3 1-69 1-1 / 2-2 / 2-8 6 y1 15 0 3 17 5 A 1-40 3 12 0 1 17 6 1 7 d3 4-30-4 3-22 4 y1 21 0 3 19 5 K 3-20 1 9 0 0 10 4 20 d3 4-34 2-15 3 y4 15 2 2 24 7 K 1D-39 / 1-39 5 10 0 0 19 6 1 4 d3 3-43 6-19 6 y1 19 3 3 16 5 A 1-47 3 13 1 2 17 4 1 0 d3 1-18 3-3 / 3-22 1 y4 13 0 3 29 9 K 3-15 1 9 0 0 12 7 1 2 d3 4-39 3-16 3 y1 10 2 1 27 8 K 1-12 4 10 1 0 18 7 1 5 d3 4-4 2-15 5 y1 16 1 1 21 6 K 1D-39 / 1-39 1 9 1 0 21 7 1 7 d3 4-31 3-3 5 y2 11 0 2 33 10 K 3-20 1 9 0 0 16 6 1 9 d3 4-31 3-10 4 y1 15 0 2 24 7 K 3-11 5 11 1 0 8 3 1 0 d3 4-39 3-3 4 y1 9 2 2 23 7 K 3-20 1 8 0 0 9 3 1 2 d3 4-39 3-10 2 y2 8 1 1 23 7 A 1-40 1 10 0 1 17 6 1 2 d3 4-39 2-8 4 y1 14 3 3 22 6 K 1-5 1 8 0 1 12 5 1 3 d3 3-21 5-12 4 y1 12 1 3 15 5 K 1-5 1 10 2 0 10 4 1 1 d3 4-39 3-22 5 y1 11 1 1 12 4 A 1-51 3 / 2 11 0 1 5 2 3 5 d4 3-64D 5-12 4 y1 13 0 1 18 5 K 1-5 5 9 0 0 12 4 2 7 d4 3-74 2-21 4 y1 12 0 3 13 4 K 3-20 5 9 0 0 11 4 5 8 d4 4-59 3-16 5 y1 10 1 2 19 6 K 3-11 2 9 1 0 10 4 1 0 d4 4-59 3-3 5 y1 20 2 4 26 7 K 4-1 2 9 0 0 10 4 1 1 d4 4-31 3-3 3 y1 10 1 2 21 6 K 3-20 1 9 0 0 10 4 1 6 d4 4-59 3-3 5 y1 12 1 2 9 3 K 1-NL1 1 9 0 0 8 3 3 8 d4 3-66 3-10 6 y1 17 3 3 25 7 K 1-5 2 9 0 0 14 5 4 9 d4 4-30-4 / 4-31 6-25 4 y1 12 1 2 19 6 K 1-NL1 4 8 0 0 18 7 1 3 d4 3-33 2-8 5 / 4 y1 11 0 0 16 5 K 3-15 1 10 0 0 16 6 3 9 d4 4-59 3-3 6 y1 20 1 3 18 5 K 2D-28 / 2-28 1 9 0 0 3 1 1 7 d4 4-30-4 6-19 6 y1 25 1 3 12 3 K 1D-39 / 1-39 2 10 0 0 9 3 1 8 d4 3-33 3-22 6 y1 17 0 2 18 5 K 2D-28 / 2-28 1 10 0 0 6 2 22 d4 3-30 6-19 / 6-13 / 6-25 5 / 4 y1 12 4 2 17 5 K 3-11 4 9 1 0 14 5 2 1 d4 3-33 2-15 / 2-21 4 y1 16 1 2 20 6 K 3-15 2 8 0 0 7 3 5 3 d4 4-31 6-19 4 y1 10 1 2 19 6 K 3-20 2 10 0 0 8 3 4 8 d4 3-74 2-21 4 y1 13 1 3 21 6 K 4-1 4 9 1 0 8 3 1 9 d4 1-69 3-22 5 / 4 y1 8 0 1 21 7 K 3-11 2 10 2 0 10 4 1 1 d4 4-59 2-2 / 2-15 3 y1 10 0 1 20 6 K 3-11 1 9 1 0 8 3 1 3 d4 3-64D 2-8 4 y1 13 1 1 14 4 K 1-NL1 2 10 0 0 14 5 17 d5 5-51 2-2 5 y1 15 2 1 12 3 A 1-51 3 / 2 11 0 2 5 2 2 3 d5 4-59 3-10 6 y1 13 0 2 11 3 A 1-40 3 / 2 10 0 1 5 2 1 0 d5 3-33 3-3 4 y1 13 0 3 6 2 K 3-15 2 8 0 0 9 3 1 3 d5 3-11 3-10 6 y1 17 3 1 14 4 K 1D-39 / 1-39 3 9 1 0 25 9 1+) indicate the numbers of negatively and positively charged amminoacids in the IgH and IgL complementary determining region (CDR3), respectively. MUT, of somatic mutations; NS, no signal; ND, not determined.265] Table 6b. Immunoglobulin reactivity and antiviral activity of human HEV memory B-cell antibodies._ Reactivity HEV-CA _Reactivity HEV g3-2712 (AUG) Paslahepevirus HEV genotype (EC50 ng / ml)Es1.237 17.77 0,631 0,597 ND 14.2 0.493 9.4 6.2 7.45.46.1 5.17.16.8 23.6 17.5 30.4 18.5 ND ND ND Es1.240 15.79 0 0,403 0,579 13.22 1,16 12.1 14.4 14.1 11.3 13.9 15.9 14.2 11.2 ND ND ND ND ND 31 ND Es1.243 15.88 0,703 0,588 ND 15.4 0,42 7.18.8 6.66.68.28.9 10.1 11.512.3 14.1 17.2 22.75.1 ND ND Es1.265 14,66 0,438 0,402 1,007 11,27 5,456 9.8 9.2 13.3 12.7 8.0 9.3 10.5 6.7 31.3 35.1 34.9 37.5 4.5 14 ND Es1.268 9,963 9,319 14,41 ND 0,425 ND 8.2 12.4 10.4 7.6 14.3 9.4 7.2 7.5 115.4 18.6 NS 20.5 0.2 100 0.031 Es1.272 15.81 0.984 1,218 0,567 10,39 0,407 13.5 20.3 18.0 20.7 18.0 16.0 17.6 17.0 32.3 38.1 50.1 48.4 4.2 16 ND Es1.282 16.04 15.61 15.88 ND 0,691 ND 10.2 8.6 NS 4.7 5.3 14.7 15.2 7.6 NS 23.7 NS NS 0.2 100 0.11 Es1.283 16.92 16.31 14.74 ND 1.329 ND 10.7 11.0 12.1 10.2 35.5 16.9 13.3 15.4 21.9 34.4 13.3 26.1 0.5 42 50 Es1.308 15.42 0.409 0,399 0,497 10.01 3.657 11.2 28.8 16.1 18.8 17.6 13.1 15.6 11.4 25.5 19.9 49.6 19.1 4.3 20 ND Es1.311 15.69 14.55 14,82 ND 0.327 ND 6.2 5.9 6.2 5.1 10.0 6.3 11.6 7.8 6.7 8.4 9.7 10.3 0.1 94 0.1018 Es1.316 14.65 0.558 0,573 ND 11.93 0.385 6.7 8.8 13.3 13.0 14.2 8.3 15.9 9.3 15.0 23.3 16.2 19.5 0.1 ND ND Es1.327 16.34 15.01 14.67 ND 0.481 ND 9.1 7.2 3.7 6.4 8.0 9.7 9.2 6.5 6.9 7.9 9.9 10.3 0.1 100 0.0004 Es1.329 16.61 0.4 0.407 0,393 11.44 4.88 7.6 13.4 10.2 11.0 10.5 7.4 11.4 7.8 13.5 14.3 16.8 13.8 4.9 11 ND Es1.333 16.16 16.44 15 ND ND ND 14.0 10.6 19.8 9.4 16.3 13.7 15.6 14.0 10.1 12.4 15.2 15.2 0.1 28 50 Es1.334 14.92 0.633 0.83 ND 13.38 2.031 9.9 10.8 16.6 10.7 9.4 7.4 11.5 8.0 43.9 44.2 56.6 45.7 ND ND ND Es1.346 16.61 14.66 15.1 ND 0.482 ND 5.1 5.4 6.4 5.6 6.0 6.8 8.2 5.8 10.9 16.5 13.4 953.2 5.6 100 0.042 Es1.352 16.63 0.418 0,4 ND 15.43 1.826 15.5 11.1 15.2 13.3 11.2 15.3 18.8 14.5 10.7 11.8 14.7 14.7 0.2 37 ND Es1.371 15.3 0.371 0,404 0,373 10.42 4.133 11.0 10.5 12.9 9.7 9.4 8.1 13.1 11.9 31.6 16.2 54.6 15.8 0.1 15 ND Es1.385 15.92 15.48 14.33 ND 0.388 ND 21.8 49.2 19.9 725.4 222.9 209.2 NS 72.5 NS NS NS NS 0.2 1000.43 Es1.387 15.97 0.521 0,589 ND 14.85 0.502 6.7 8.1 7.7 5.7 7.9 6.3 7.4 5.2 9.5 12.2 11.9 13.0 4.7 ND ND Es1.416 15.49 16.45 14.47 ND 0.43 ND 7.0 12.0 7.9 8.9 11.4 10.9 7.4 8.5 11.3 14.0 20.9 27.1 0.2 0.50 Es2.101 18.58 0.343 0.436 0,358 10.78 4.771 7.8 7.4 7.47.8 5.6 5.4 8.1 8.9 13.9 13.9 19.1 16.0 4.6 ND Es2.105 17.17 16.93 14.82 ND ND ND5.4 15.9 13.5 6.5 9.7 6.4 7.08.9 9.610.9 11.0 13.4 0.1 0.056 Es2.117 18.49 0.362 0,394 0,524 10.97 0.486 9.5 7.9 8.08.9 6.5 9.0 10.6 10.0 14.2 14.9 19.8 18.0 4.5 ND Es2.130 18.74 0.464 0,519 0,893 10.31 2.21 9.5 10.0 10.9 11.39.0 14.2 17.4 14.6 54.3 25.8 76.3 44.4 2.9 ND Es2.136 18.49 0,763 0,441 0,784 10.89 4.158 11.7 10.1 13.4 21.3 10.0 10.1 15.2 17.213.9 14.2 21.1 22.4 4.4NDEs2.143 18.59 0,339 0,564 0,448 3,609 11.5 13.5 13.8 14.3 16.9 11.4 15.7 11.4 12.1 14.8 18.5 16.0 4.6 15 ND Es2.152 16.34 0,415 0,399 ND0,362 16.0 18.0 20.0 17.7 27.1 17.1 18.7 18.6 15.3 14.5 19.7 14.9 4.7 17 ND Es2.154 17.89 0,338 0,537 0,471 9,385 3,544 89.8 51.5 24.6 227.7 83.0 NS NS NS 64.6 26.5 49.5 53.1 0.6 15 ND Es2.165 18.84 0,325 0,488 0,492 10,82 1,076 21.6 17.1 22.5 32.5 32.0 35.4 22.4 18.8 46.1 28.6 89.0 39.6 3.4 ND Es2.173 18.46 1,572 3,001 3,402 11,19 1,581 10.7 12.7 17.9 12.8 17.8 20.0 15.8 11.9 27.7 30.9 35.5 39.9 4.2 ND Es2.181 18.68 0,285 0,566 0,516 12,01 0,344 12.2 12.8 20.3 14.6 20.2 105.1 18.2 20.6 9.910.7 13.1 NS 0.1 ND Es2.182 18.54 0,311 0,427 0,385 11,23 9,337 11.3 8.6 9.2 13.112.1 10.9 14.3 11.8 20.2 21.1 21.5 22.0 4.5 ND Es2.188 18.45 0,443 0,919 0,627 10,61 0,429 17.1 12.2 12.1 21.5 19.5 16.7 17.4 22.6 19.117.2 28.7 24.9 4.4 ND Es2.190 18.37 0,339 0,763 0,569 10,5 3,742 10.0 8.1 10.0 5.7 7.9 4.9 9.6 12.7 16.8 13.9 16.3 12.3 4.8 ND Es2.202 18.58 0.368 0.455 0,409 10,76 4,865 12.2 9.2 14.4 12.110.3 8.0 17.1 14.9 7.57.78.56.0 3.8 ND Es2.217 18.48 0.339 0.426 0,492 10,6 6,623 22.6 11.8 16.3 13.6 15.2 13.1 18.2 29.7 14.4 15.4 17.4 13.1 4.4 ND Es2.219 19.21 16.6 14.85 ND ND ND 7.3 8.9 14.4 8.7 11.8 8.5 7.3 7.1 10.3 32.9 17.1 17.1 0.8 0.29 Es2.224 18.44 0,548 0,453 0,556 3,548 19.2 14.8 19.4 38.0 26.9 16.2 19.8 21.7 13.4 11.5 15.6 10.2 4.4 ND Es2.236 18.65 0,318 0,332 0,418 4,964 9.5 7.8 12.1 9.4 13.0 12.7 9.6 15.3 21.4 27.4 48.7 54.7 4.4 ND Es2.239 18.45 0,348 0,302 0,485 8,172 9.6 7.8 10.9 7.7 9.3 10.3 8.9 15.6 12.6 18.8 23.5 33.0 4.8 ND Es2.278 18.72 0,373 0,316 0,5394,348 5.8 9.0 6.7 6.4 15.2 6.8 6.3 7.6 10.0 13.5 22.6 32.3 4.9 ND Es2.280 15.46 10,93 12,05 ND 0,338 ND 4.94.5 5.23.8 6.6 5.0 7.2 4.5 9.0 10.1 NS 12.2 0.1 ND Es2.288 18.44 0,325 0,354 0,967 6,007 10.5 13.9 16.4 9.164.6 14.9 9.0 24.2 10.111.6 17.9 31.4 4.8ND Es2.312 15.53 0,44 0,44 ND 0,471 260.9 92.9 16.5 21.8 23.9 NS NS NS 12.8 22.2 NS NS 0.1 0 ND Es2.316 18.72 0,334 0,342 0,466 3,219 8.2 6.5 9.49.6 7.8 7.8 11.6 8.9 7.0 9.6 15.4 20.9 4.6 ND Es2.317 18.73 0.396 0.434 0,3910,333 10.0 8.2 14.4 25.7 9.5 8.8 26.9 11.3 11.1 30.0 26.1 38.3 4.8 ND Es2.321 16.4 16.51 16.16 ND 0,674 ND 6.3 5.8 13.9 5.5 9.4 7.4 8.1 8.0 7.321.2 10.6 10.2 0.1 50 Es2.325 15.05 10,68 11,19 ND 0,38 ND4.2 4.2 7.8 4.5 8.2 4.7 3.87.5 11.7 16.4 19.2 22.6 0.1 0.037 Es2.326 18.4 0,381 0,404 0,41 0,397 10.0 6.7 13.2 7.2 12.0 8.5 26.4 14.1 8.3 12.1 17.6 25.0 3.6 ND Es2.329 18.85 0,487 0,342 0,472 0,528 8.5 7.6 12.1 10.1 10.1 9.8 24.9 15.9 6.2 7.1 14.8 19.4 4.9 ND Es2.336 18.49 0,451 0,389 0,4310,391 7.0 5.1 11.7 6.711.2 5.3 15.2 9.34.87.19.7 15.7 4.6NDEs2.342 18.58 0,384 0,4 0,496 13.76 0.38 15.0 9.0 25.7 14.3 19.3 25.2 24.8 21.09.712.0 18.4 NS 0.1 100 ND Es3.102 13.13 2,066 2,495 ND 10.64 ND 47.5 66.5 69.6 113.2 51.9 53.5 53.6 118.0 648.4 994.0 NS NS 0.1 ND ND Es3.108 14.33 13.96 13.64 ND ND ND 8.3 8.6 12.1 11.5 8.8 9.6 7.0 6.8 9.0 13.8 16.4 17.9 0.2 99 0.044 Es3.110 10.84 0,335 0,504 0,33 14.57 6.628 8.1 7.9 7.5 11.4 6.7 8.5 7.5 9.8 12.8 13.3 20.7 29.7 4.3 13 ND Es3.111 12.53 0,363 0,322 0,589 13.75 5.448 7.97.2 7.6 12.3 8.1 7.4 7.28.5 20.6 17.3 34.6 29.2 4.6 4 ND Es3.112 12.25 0,379 0,355 0,502 13.81 7.392 10.1 10.5 9.2 13.511.2 7.3 10.2 10.3 21.5 30.0 41.0 54.7 4.4 33 ND Es3.125 11.39 0,354 0,434 0,463 14.37 3.958 10.8 11.3 22.6 10.4 16.8 9.7 9.4 14.7 24.5 30.3 29.0 61.4 4.4 14 ND Es3.126 9,652 0,302 0,461 0,413 13.12 0.738 14.2 15.6 19.3 29.0 20.4 15.5 19.6 18.1 NS NS NS 69.0 0.1 2 ND Es3.127 11,76 0,332 0,331 0,456 15.01 3.884 10.3 8.6 14.0 8.1 11.1 8.5 14.0 12.1 10.7 10.2 14.7 20.3 4.5 11 ND Es3.128 11,83 0,348 0,306 0,496 14.75 0.405 11.7 11.7 9.3 10.0 9.6 7.4 11.3 9.9 12.5 15.6 34.0 37.5 0.8 16 ND Es3.130 11,95 0,687 0,821 1,45 15.31 1.103 6.59.4 10.4 6.39.2 10.1 12.8 14.1 14.8 16.1 25.2 34.9 4.4 23 ND Es3.133 11,24 0,337 0,474 0,406 13.03 2.953 16.7 15.9 22.9 13.7 13.8 13.7 21.8 19.3 22.3 41.9 66.1 83.2 4.5 16 ND Es3.135 11,89 0,293 0,338 0,923 13.73 4.029 10.9 12.6 15.8 10.1 7.8 14.7 8.8 13.3 33.7 30.2 62.6 70.2 ND 0 ND Es3.137 10,58 0,301 0,332 0,443 12.72 3.577 5.8 5.5 3.7 5.3 10.9 4.8 5.2 5.4 22.3 24.6 32.4 57.1 4.2 ND Es3.141 12,5 0,393 0,446 0,473 14.44 2.416 7.38.6 5.15.6 9.0 5.5 6.8 6.1 11.7 11.6 15.8 23.4 5.0 ND Es3.142 12,4 0,386 0,941 0,573 13.46 4.669 12.8 15.2 10.7 6.118.6 6.4 7.711.019.0 19.1 34.4 40.4 4.3 ND Es3.143 13,68 2,076 1,634 ND 11.99 ND 12.4 10.5 18.7 17.8 24.6 20.1 15.1 11.6 46.2 35.5 50.6 64.6 4.4 ND Es3.147 12,59 0,378 0,335 0,448 12.19 4.113 11.3 10.9 8.1 6.7 13.5 7.9 8.7 10.7 14.4 15.4 22.9 35.9 4.7 ND Es3.149 12,42 0,524 0,446 0,415 13.44 3.426 9.5 12.0 7.4 6.0 18.9 8.1 8.1 21.3 10.9 10.0 18.9 27.5 ND ND Es3.150 13,84 0,408 0,789 1,032 13.76 5.892 1.1 0.9 0.1 0.4 0.4 0.80.7 60.8 10.8 12.6 20.0 26.5 3.8 ND Es3.152 9849 0397 0522 0,504 12.5 2.808 10.1 7.6 9.7 9.6 9.2 12.5 11.6 7.6 15.117.7 25.4 32.8 4.3 ND Es3.155 ND 13.37 3.698 5.1 5.2 6.56.8 7.8 6.5 15.7 6.3 14.2 15.7 26.1 32.6 4.4 ND Es3.157 ND ND ND 8.8 9.5 12.4 7.2 8.7 9.0 8.4 8.7 8.310.5 11.3 11.2 0.1 50 Es3.160 0,54 14.76 0.384 6.7 8.6 7.7 8.512.9 7.4 18.7 8.5 14.7 15.0 18.1 26.2 4.8 ND Es3.163 0,637 14.12 5.942 7.9 7.3 14.1 12.4 14.3 11.8 14.2 12.6 20.1 24.2 35.1 50.5 4.6 ND Es3.167 ND 15.36 1.399 18.4 7.7 24.6 19.0 20.9 21.8 18.1 14.414.9 16.9 24.8 30.1 4.7 ND Es3.170ND 1.097 ND 21.9 21.3 28.2 23.4 31.5 27.2 39.0 183.7 13.6 118.7 22.3 22.5 0.59.78Es5.223 10,93 10,2 15,47 ND ND ND 7.8 6.3 7.9 6.1 9.2 5.3 5.2 4.7 9.5 11.6 9.9 12.4 0,2 100 0.0405) and (+) indicate the numbers of negatively and positively charged amminoacids in the IgH and IgL complementary determining region (CDR3), respectively. MUT, umber of somatic mutations; NS, no signal; ND, not determined.

[0266] Table 7: Cryo-EM data collection, refinement and validation statistics for the complex HEV-CA P domain with Es1.327 FabData collection and processingMagnification 105,000xVoltage (kV) 300Microscope Titan KriosElectron exposure (e- / A2) 50Defocus range (pm) -1 -0 to -3.0Pixel size (A) 0.86Initial particle images (no.) 171045Final particle images (no.) 106903Map resolution (A) 3,5FSC threshold 0.143RefinementInitial model used (PDB code) 2ZTNModel compositionNon-hydrogen atoms (no.) 5538Protein residues (no.) 727Ligands (no.) 2B factorsProtein (A2) 80.86Ligands (A2) 78.08R.m.s. deviationsBond lengths (A) 0.007Bond angles (°) 1.487ValidationMolProbity score 2.38Clashscore 19.33Poor rotamers (%) 3-21Ramachandran plotFavored (%) 96.64Allowed (%) 3.36Disallowed (%) 0-0

[0267] Table 8: Cryo-EM data collection, refinement and validation statistics for the complex HEV-CA P domain with Es4.431 FabData collection and processingMagnification 105,000xVoltage (kV) 300Microscope Titan KriosElectron exposure (e- / A2) 50Defocus range (pm) -1 -0 to -3.0Pixel size (A) 0-86Initial particle images (no.) 171045Final particle images (no.) 106903Map resolution (A) 3.5FSC threshold 0.143RefinementInitial model used (PDB code) 2ZTNModel compositionNon-hydrogen atoms (no.) 5531 Protein residues (no.) 735Ligands (no.) 2B factorsProtein (A2) 68.01 Ligands (A2) 64.21 R.m.s. deviationsBond lengths (A) 0.008 Bond angles (°) 1.621 ValidationMolProbity score 2.01 Clashscore 8.88Poor rotamers (%) 3.70Ramachandran plotFavored (%) 97.51 Allowed (%) 2.35Disallowed (%) 0.14

[0268] Table 9: Cryo-EM data collection, refinement and validation statistics for the complex HEV-CA P domain with Es5.127 Fab and Es1.114 Data collection and processingMagnification 105,000xVoltage (kV) 300Microscope Titan Krios Electron exposure (e- / A2) 50Defocus range (pm) -1 -0 to -3.0Pixel size (A) 0-86Initial particle images (no.) 611380Final particle images (no.) 207869Map resolution (A) 3,8FSC threshold 0.143RefinementInitial model used (PDB code) 2ZTNModel compositionNon-hydrogen atoms (no.) 8766 Protein residues (no.) 1172 Ligands (no.) 0B factorsProtein ( 2) 31.18 Ligands (A2)R.m.s. deviationsBond lengths (A) 0.005 Bond angles (°) 1.046 ValidationMolProbity score 2.25 Clashscore 12.61 Poor rotamers (%) 2.04 Ramachandran plotFavored (%) 94.06 Allowed (%) 5.24 Disallowed (%) 0.7

[0269] Table 13: Sequences disclosed in the present applicationREFERENCES

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Vaccine 23, 2881-2892. doi: 10.1016 / j. vaccine.2004.11.065.Zhao, M., X. J. Li, Z. M. Tang, F. Yang, S. L. Wang, W. Cai, K. Zhang, N. S. Xia, and Z. Z. Zheng.2015. A comprehensive study of neutralizing antigenic sites on the Hepatitis E Virus (HEV) capsid by constructing, clustering, and characterizing a tool box. J. Biol. Chem. 290:19910-19922. doi: 10.1074 / jbc. M 115.649764.Zheng, Q., J. Jiang, M. He, Z. Zheng, H. Yu, T. Li, and W. Xue. 2019. Viral neutralization by antibody-imposed physical disruption. 1-8. doi: 10.1073 / pnas.1916028116.Zheng, M. et al. Structural basis for the synergetic neutralization of hepatitis E virus by antibody-antibody interaction. 2024. Proceedings of the National Academy of Sciences 121, e2408585121.Zhou, Y.-H., and H. Zhao. 2023. Immunobiology and Host Response to HEV. Adv. Exp. Med. Biol. 1417:93-118. doi:10.1007 / 978-981-99-1304-6_7.B24-4578QT1 / 5PCT(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application)B24-4578QT2 / 5PCT(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application)B24-4578QT3 / 5PCT(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application)B24-4578QT4 / 5PCT(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application)B24-4578QT5 / 5PCT(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application)FOR RECEIVING OFFICE USE ONLYFOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. A human neutralizing monoclonal antibody against Hepatitis E virus (HEV), which specifically binds to HEV capsid protein P domain and is a competitive inhibitor of binding to the P domain of at least one of the following reference antibodies:the reference human antibody Es1.327 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and (ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 4; the reference human antibody Es4.431 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:6; the reference human antibody Es4.452 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8; and the reference human antibody Es5.127 comprising (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10.

2. The antibody according to claim 1, which competes for binding with at least the reference human antibodies Es1.327 and Es4.431; preferably with the reference human antibodies Es1.327, Es4.431, Es4.452 and Es5.127.

3. The antibody according to claim 1 or claim 2, which competes for binding with at least one reference human antibody Es1.327, Es4.431, Es4.452 or Es5.127 comprising heavy and light chain amino acid sequences of SEQ ID NO: 11 and 12; SEQ ID NO: 13 and 14; SEQ ID NO: 15 and 16; or SEQ ID NO: 17 and 18, respectively.

4. The antibody according to any one of claims 1 to 3, wherein the HEV capsid protein comprises any one of SEQ ID NO: 83 and 89-101, and / or the P domain comprises SEQ ID NO: 84.

5. The antibody according to any one of claims 1 to 4, which specifically binds to an epitope of HEV capsid protein P domain located within the region of the RS and XY loops, and said region consisting of the fusion of the sequences from positions 481 to 492 and 580 to 594, the indicated positions being determined by alignment withSEQ I D NO: 1; preferably wherein the region consists of the fusion of the sequences DQTTYGSSTNPM (SEQ ID NO: 105) and AISTYTTSLGAGPTS (SEQ ID NO: 106).

6. The antibody according to claim 5, which specifically binds at least one amino acid residue of the P domain RS loop selected from the group consisting of: Q482, T483, G486 and S488; more preferably which specifically binds the residues Q482, T483 and S488, and optionally further specifically binds the residue G486.

7. The antibody according to any one of claims 1 to 6, which neutralizes HEV with a half maximal inhibitory concentration (IC50) selected from 10 ng / mL or less, 5 ng / mL or less, and 1 ng / mL or less.

8. The antibody according to any one of claims 1 to 7, which neutralizes at least one HEV chosen from HEV-1, HEV-2, HEV-3, HEV-4, HEV-5, HEV-6, HEV-7, HEV-8 and HEV-C1; preferably which neutralizes HEV-1, HEV-2, HEV-3, and / or HEV-4.

9. The antibody according to any one of claims 1 to 8, which has no predicted reactivity to human proteins.

10. The antibody according to any one of claims 1 to 9, which comprises:a) a heavy chain variable domain comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20 and a heavy chain CDR3 of SEQ ID NO: 21; preferably which further comprises a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23 and a light chain CDR3 of SEQ ID NO: 24; b) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26 and a heavy chain CDR3 of SEQ ID NO: 27; preferably which further comprises a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30;c) a heavy chain variable domain comprising a heavy chain CDR1 of SEQ ID NO: 31, a heavy chain CDR2 of SEQ ID NO: 32 and a heavy chain CDR3 of SEQ ID NO: 33; preferably which further comprises a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 34, a light chain CDR2 of SEQ ID NO: 35, and a light chain CDR3 of SEQ ID NO: 36;105d) a heavy chain variable domain comprising: a heavy chain CDR1 of SEQ ID NO: 37, a heavy chain CDR2 of SEQ ID NO: 38 and a heavy chain CDR3 of SEQ ID NO: 39; preferably which further comprises a light chain variable domain comprising: a light chain CDR1 of SEQ ID NO: 40, a light chain CDR2 of SEQ ID NO: 41, and a light chain CDR3 of SEQ ID NO: 42; or e) a heavy chain variable domain comprising: heavy chain CDR1, CDR2 and CDR3 which differ from SEQ ID NO: 19 to 21, 25 to 27, 31 to 33, or 37 to 39, respectively by up to 6 (1, 2, 3, 4, 5 or 6) amino acid mutations in the sequence of 1, 2 or 3 CDRs; preferably which further comprises a light chain variable domain comprising: light chain CDR1, CDR2, and CDR3 which differ from SEQ ID NO: 22 to 24, 28 to 30, 34 to 36 or 40 to 42, respectively by up to 6 (1, 2, 3, 4, 5 or 6) amino acid mutations in the sequence of 1, 2 or 3 CDRs; and / or preferably wherein the amino acid mutations are conservative amino acid substitutions.

11. The antibody according to any one of claims 1 to 10, which comprises:a) a heavy chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 3 and a light chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 4;b) a heavy chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 5 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 6;c) a heavy chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 7 and a light chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 8;d) a heavy chain variable domain comprising an acid sequence having at least 90 % identity with SEQ ID NO: 9 and a light chain variable domain comprising an amino acid sequence having at least 90 % identity with SEQ ID NO: 10; ore) a heavy chain variable domain and a light chain variable domain comprising amino acid sequences having at least 90 % identity with SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO:125106and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; or SEQ ID NO: 133 and SEQ ID NO: 134.

12. The antibody according to claim 11, wherein the heavy and light chain variable domains comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO:125 and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; or SEQ ID NO: 133 and SEQ ID NO: 134.

13. The antibody according to any one of claims 1 to 12, which comprises:a) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 11 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 12;b) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 13 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 14;c) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 15 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 16; ord) a heavy chain amino acid sequence having at least 90 % identity with SEQ ID NO: 17 and a light chain amino acid sequence having at least 90 % identity with SEQ ID NO: 18.

14. The antibody according to claim 13, which comprises a heavy and / or light chain amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences SEQ ID NO: 11 to 18.

15. The antibody according to any one of claims 1 to 14, which comprises a heavy chain variable domain comprising framework regions FR1, FR2, FR3 and FR4 selected from the group consisting of SEQ ID NO: 43 to 46; SEQ ID NO: 51 to 54; SEQ ID NO: 59 to 62; and SEQ ID NO: 67 to 70; and / or which comprises a light chain variable domain comprising framework regions FR1, FR2, FR3 and FR4 selected107from the group consisting of SEQ ID NO: 47 to 50; SEQ ID NO: 55 to 58; SEQ ID NO: 63 to 66; and SEQ ID NO: 71 to 74.

16. The antibody according to any one of claims 1 to 15, which is a recombinant human monoclonal antibody; preferably of I gG 1 isotype or IgA isotype such as polymeric or secretory IgA; and / or preferably comprising a non-human glycosylation pattern.

17. The antibody according to any one of claims 1 to 16, which comprises an IgG Fc region comprising at least one substitution; preferably selected from the group consisting of substitutions that silence antibody effector functions, substitutions that enhance antibody half-life, substitutions that prevent binding to CD16 polypeptide, and substitutions that improve antiviral efficacy against pathogens.

18. A combination of antibodies comprising at least one antibody according to anyone of claims 1 to 17, and at least one other anti-HEV capsid protein P domain human neutralizing antibody chosen from an antibody of any one of Groups I, II, III, IV or V as shown in Figure 3H, an antigen-binding fragment thereof or derivative thereof; preferably chosen from Es1.268, Es1.231, Es2.105, Es2.325 and Es3.108 comprising VH and VL domains of SEQ ID NO: 107-108, 109-110, 111-112, US- 114, and 115-116, respectively.

19. A nucleic acid molecule which encodes the antibody according to any one of claims 1 to 17, particularly a pair of nucleic acid molecules which encode the heavy and light chain of said antibody.

20. The nucleic acid molecule or pair of nucleic acid molecules according to claim 19, which is mRNA or DNA.

21. The pair of nucleic acid molecules according to claim 19 or 20, which comprises a pair of nucleic acid sequences selected from: a sequence having at least 90% identify with SEQ ID NO: 75 and a sequence having at least 90% identify with SEQ ID NO: 76 or the RNA sequences thereof; a sequence having at least 90% identify with SEQ ID NO: 77 and a sequence having at least 90% identify with SEQ ID NO: 78 or the RNA sequences thereof; a sequence having at least 90% identify with SEQ ID NO: 79 and a sequence having at least 90% identify with SEQ ID NO: 80 or the RNA sequences thereof; and a sequence having at least 90% identify with SEQ ID NO: 81 and a sequence having at least 90% identify with SEQ ID NO: 82 or the RNA sequences thereof.10822. A vector comprising the nucleic acid molecule(s) according to any one of claims 19 to 21.

23. The vector according to claim 22, which is suitable for the expression of the nucleic acid molecule(s) into cells of a human individual; preferably a viral vector or a non- viral vector such as lipid nanoparticle.

24. The vector according to claim 22, which is an expression vector for the production of a recombinant antibody in a host cell.

25. The expression vector according to claim 24, which is contained in a bacteria strain chosen from Es1.327_plgH, Es1.327_plgL, Es4.431_plgH, Es4.431_plgL, Es4.452_plgH, Es4.452_plgL, Es5.127_plgH and Es5.127_plgL deposited under the terms of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 13, 2024 under the number 1-6140, 1-6141, 1-6142, 1-6143, 1-6144, 1-6145, 1-6146, and 1-6147, respectively.

26. A host cell which has been modified with the expression vector of claim 24 or 25.

27. A pharmaceutical composition, comprising the antibody according to any one of claims 1 to 17 or an antigen-binding fragment thereof, the vector according to claim 22 or 23, or the combination of antibodies according to claim 18, and at least one of a pharmaceutically acceptable carrier, an adjuvant, and a preservative.

28. The antibody according to any one of claims 1 to 17 or antigen-binding fragment thereof, the vector according to claim 22 or 23, the combination of antibodies according to claim 18, or the pharmaceutical composition according to claim 27, for use as a medicament; preferably for use in the prevention or treatment of HEV infection and associated disease in a subject in need thereof.

29. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to claim 28, wherein the administration of the treatment reduces the risk of developing HEV- associated disease for the subject.

30. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to claim 29,109wherein the administration of the treatment reduces the risk of hospitalization or the risk of death for the subject.

31. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to claim 28, wherein the administration of the treatment reduces the likelihood of developing a severe form of HEV-associated disorder for the subject.

32. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to claim 31, wherein the risk of acute severe infection such as acute severe hepatitis; the risk of developing chronic HEV such as liver fibrosis and cirrhosis; or the risk of death is reduced by the treatment.

33. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to any one of claims 28 to 32, wherein the subject is selected from the group consisting of: a subject which is not infected with HEV, a subject which has been exposed to HEV or is infected with HEV but has not developed HEV disease, and a HEV disease patient.

34. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to any one of claims 28 to 32, wherein the subject is a pregnant women or immunocompromised patient.

35. The antibody or antigen-binding fragment thereof, the vector, the combination of antibodies or the pharmaceutical composition for the use according to claim 34, wherein the immune-compromised patient is a solid organ transplant recipient, particularly liver transplant recipient.

36. A kit for the detection or diagnosis of HEV infection, or the monitoring of treatment of HEV disease, comprising at least an antibody according to any one of claims 1 to 17 or antigen-binding fragment thereof, preferably further including a detectable label.

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