BROADLY NEUTRALIZING ANTIBODIES AGAINST HEPATITIS E VIRUS

FIELD OF THE INVENTION

The present invention relates to broadly neutralizing monoclonal antibodies against infectious hepatitis E virus particles. The present invention relates to antibody constructs capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2, e.g., to monoclonal antibody p60.1 or monoclonal antibody p60.12 or single chains, derivatives or antigen-binding fragments thereof, as specified herein. It also provides heavy chain constructs and/or a light chain constructs of said antibody constructs. The antibody constructs, expression vectors encoding them and host cells capable of expressing them are useful in medicine, e.g., for preventing, treating and diagnosing hepatitis E virus infection.

BACKGROUND OF THE INVENTION

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide and is an emerging problem in industrialized countries. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. It is estimated that one third of the population in the world may be infected with HEV during their lifetime. This virus infects approximately 20 million people every year and is responsible for an estimated 3.4 million symptomatic cases and 70,000 deaths, mainly in developing countries. While HEV infection is asymptomatic for most patients, some human populations including pregnant women and immunocompromised patients have higher risk to develop severe forms and chronic infections, respectively. Currently, there is neither a specific treatment nor a universal vaccine against HEV.

HEV strains infecting humans have been classified into 4 main distinct genotypes (gt) belonging to a single serotype. Genotypes gt1 and gt2 that infect humans only, are primarily transmitted through contaminated drinking water and are responsible for waterborne hepatitis outbreaks in developing countries. In contrast, gt3 and gt4 are zoonotic and are largely circulating in industrialized countries. They are mainly transmitted by contact with swine and consumption of inadequately heated pork products. Recently, further HEV genotypes, subgenotypes and strains have been identified in rabbit, hare, wild boar, moose, camels, and other animals. In addition to human pathogenic HEV, related viruses have been identified in fowl, rats, bats, carnivores and even in fish. After its initial description in Germany, rat HEV has been detected in many regions of the world suggesting a worldwide distribution. Recently, several rat HEV-caused human disease cases have been reported, and rat HEV is thought to pose a considerable zoonotic threat.

HEV is a quasi-enveloped, positive-sense RNA virus expressing three open reading frames (ORFs): ORF1 , ORF2 and ORF3. ORF1 encodes the non-structural polyprotein that is the viral replicase. ORF2 encodes the viral capsid protein which is involved in particle assembly, binding to host cells and eliciting neutralizing antibodies. ORF3 encodes a small protein that is involved in virion morphogenesis and egress.

ORF2 is the best characterized of the three ORFs. Its protein, pORF2, is the main structural component of the virion, carrying the receptor binding domain, the major target for neutralizing antibodies. Three potential N-linked glycosylation sites at positions N137, N310, and N562 have been identified in pORF2, but N-linked glycosylation has only been confirmed to occur at positions N137 and N562. During its lifecycle, HEV produces at least 3 forms of the pORF2 capsid protein: infectious pORF2 (pORF2i), glycosylated pORF2 (pORF2g), and cleaved pORF2 (pORF2c). The pORF2i protein, the non-glycosylated form of pORF2, is the structural component of infectious particles. pORF2g and pORF2c, on the other hand, are highly glycosylated, and are not associated with the infectious particle. Notably, these secreted forms are very stable, exist as dimers, and are the most abundant forms of pORF2 in sera of HEV infected patients. These proteins probably do not play an important role in the life cycle of HEV, but may inhibit antibody mediated neutralization, thus serving as an immune evasion mechanism for HEV. pORF2g and pORF2c likely act as a humoral immune decoy that inhibits antibody-mediated neutralization.

The use of monoclonal antibodies targeting pORF2 for diagnosis, prophylaxis and/or treatment of HEV infections has often been proposed. Examples thereof are the families of WO9517501A1 “Monoclonal antibodies against HEV ORF-2 and methods for using the same”, US5830636A “HEV ORF-2 monoclonal antibodies and methods for using same”, or EP1452541 A1 “Hepatitis E virus monoclonal antibodies for the binding of it and the use thereof’. However, none of these families teach monoclonal antibodies specific for the nonglycosylated form of pORF2.

The family of WO2018138344A1 “Hepatitis E virus ORF2 capsid polypeptides and uses thereof’ concerns hepatitis E virus pORF2 polypeptides which may be both glycosylated or not and antibodies specific for either the non-glycosylated pORF2i or the glycosylated pORF2g polypeptides for use in HEV diagnosis. However, WO2018138344A1 does not teach the therapeutic use of antibodies specific for the non-glycosylated pORF2i.

The family of W02020011755A1 “Antibodies having specificity for the ORF2i protein of hepatitis E virus and uses thereof for diagnostic purposes” concern antibodies which specifically bind to pORF2i and not to pORF2g or pORF2c, to a linear epitope around glycosylation site N3 562NTT. However, W02020011755A1 does not teach the therapeutic use of antibodies specific for the non-glycosylated pORF2i. In addition, detection of infectious virus particles under non-denaturing and non-reducing conditions (e.g., in stool or plasma samples from HEV infected individuals) by ELISA using antibodies described in W02020011755A1 has not been shown. This is likely due to the linear character of the targeted epitope described therein that is not accessible in natively folded pORF2 dimers constituting infectious particles.

In view of the continuing threat to human health, there is an urgent need for preventive and therapeutic antiviral therapies for HEV control. In particular, antibodies that specifically bind to infectious HEV forms with high affinity, that effectively inhibit virus infectivity and that can be used in the diagnosis, prevention and/or treatment of HEV are needed. The solution to this technical problem is achieved by providing the embodiments characterized in the claims and described further below.

SUMMARY OF THE INVENTION

The present invention in one aspect relates to a heavy chain construct and/or a light chain construct of an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2, a) wherein the heavy chain construct comprises a CDR1 having at least 90% sequence identity to SEQ ID NO: 2, a CDR2 having at least 88% sequence identity to SEQ ID NO: 3, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the light chain construct comprises a CDR1 having at least 83% sequence identity to SEQ ID NO: 6, a CDR2 having at least 66% sequence identity to the sequence AAS, and a CDR3 having at least 85% sequence identity to SEQ ID NO: 8; or b) wherein the heavy chain construct comprises a CDR1 having at least 87% sequence identity to SEQ ID NO: 10, a CDR2 having at least 87% sequence identity to SEQ ID NO: 11 , and a CDR3 having at least 93% sequence identity to SEQ ID NO: 12 and/or the light chain construct comprises a CDR1 having at least 88% sequence identity to SEQ ID NO: 14, a CDR2 having at least 66% sequence identity to the sequence DVT, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 16.

Preferably, said heavy chain construct and/or light chain construct of the invention comprise a) a heavy chain variable region having at least 90% sequence identity to amino acids 1-

121 of SEQ ID NO: 1 and/or and light chain variable region having at least 90% sequence identity to amino acids 1-106 of SEQ ID NO: 5; or b) a heavy chain variable region having at least 90% sequence identity to amino acids 1-

122 of SEQ ID NO: 9 and/or and light chain variable region having at least 90% sequence identity to amino acids 1-111 of SEQ ID NO: 13.

The antibody construct may be a Fab fragment, a Fab ₂ fragment or an scFv or a single chain antibody. The antibody construct may be a human antibody, optionally, a human antibody comprising a constant region.

In the heavy chain construct and/or light chain construct of the invention, the sequence identity in the CDR1 , CDR2, and/or CDR3 may be 100% (to the respective defined CDRs), wherein, preferably, all sequence identity in the CDRs is 100%. Optionally, sequence identity in the variable regions also is 100% to the respective defined variable regions.

In a preferred embodiment, the invention provides an antibody construct comprising the heavy chain construct and light chain construct of the invention, i.e. , an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2.

The present invention also relates to a nucleic acid molecule encoding a heavy chain construct and/or light chain construct of the invention or an antibody construct of the invention, as well as to a recombinant expression vector comprising said nucleic acid molecule, preferably, under the control of a heterologous promoter, and to a host cell comprising said recombinant expression vector, which is able to express the heavy chain construct and/or light chain construct or the antibody construct of the invention. The cell may be, e.g., a B cell, a T cell or an NK cell.

The present invention also relates to said antibody construct for use in the diagnosis of hepatitis E virus infection.

The present invention also relates to a diagnostic kit for the diagnosis of hepatitis E virus infection, comprising at least one of the antibody constructs of the invention, , or any combination thereof, in an amount effective for diagnosis, and, optionally, detection agents suitable for detection of the interaction of antigen and antibody construct.

The present invention also relates to the use of said antibody constructs as a medicament for prophylaxis and/or treatment of hepatitis E virus infection.

Also provided is a pharmaceutical composition comprising a) the antibody construct of the invention, b) the expression vector of the invention, wherein the nucleic acid encodes the antibody construct of the invention and/or c) the host cell of the invention, wherein the nucleic acid encodes the antibody construct of the invention, and, optionally, a pharmaceutically acceptable vehicle and/or excipient.

The present invention also relates to said pharmaceutical composition for use in preventing or treating hepatitis E virus infection.

Further embodiments of the present invention will be apparent from the description, the examples and the figures that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic illustration of HEV pORF2 domains (A) and expression constructs for the P domain fused to either mNeon or mRuby (B). pGS38: intracellular P domain; pGS39: secreted P domain. The secreted construct carried a BiP signal sequence to allow translocation into the endoplasmic reticulum. Numbers indicate amino acid positions within HEV gt3 pORF2 (SEQ ID NO: 17).

Figure 2 shows the results of neutralization assays of HEV Kernow C1 p6 G1634R viral strain. Neutralization of naked viral particles by p60.1 (A) and p60.12 (B) as well as neutralization of pseudo-enveloped viral particles by p60.1 (C) and p60.12 (D) is shown. Each panel shows the titration results (left) and the respective dose response fitted curve used to calculate the IC50 (right). Panel E shows neutralization results of p60.1 and p60.12 against the gt3 HEV viral strain 83-2-27. FFU/well normalized to the control are depicted.

Figure 3 illustrates monoclonal antibody expression and purification: (A) Size exclusion chromatography profiles representing the general behavior of the antibodies p60.1 and p60.12 after separation on SD200 increase 10/300 GL column equilibrated in 1x PBS at a flow rate of 0.5 ml/min. (B) A 12 % Coomassie-stained SDS-PAGE gel with the antibodies p60.1 and p60.12 loaded under reducing conditions. The estimated molecular weight for the heavy chain is ca. 55 kDa and ca. 25 kDa for the light chain.

Figure 4 A illustrates the results of cross-genotype ELISA experiments showing the binding activity of the monoclonal antibodies p60.1 or p60.12 against P domains from HEV gt1 , gt2, gt3, gt4, and rat-HEV. Error bars indicate standard deviation (N=2). Graph plotted using GraphPad Prism. Panel B shows the results of binding of the depicted antibody to proteins present in hepatoma cells transfected with the indicated viral strain. The GLUC reporter replicon was used as negative control as it does not contain ORF2. As a positive staining control, convalescent patient serum was used. p60.1 shows binding to the gt1 viral strain SAR55.

Figure 5 depicts ELISA results showing the ability of the monoclonal antibodies of the invention to differentiate between the secreted and intracellular forms of the P domain from HEV gt3. Error bars indicate standard deviation (N=2). Graph plotted using GraphPad Prism.

Figure 6 shows ELISA results using the anti-HEV monoclonal antibodies p60.1 or p60.12 together with sera from infected patients. The indicated antibodies were used to capture antigens present in HEV RNA positive patient plasma. As control, negative plasma was used.

Figure 7 illustrates the differential binding activity of the anti-HEV monoclonal antibodies of the invention as tested by size exclusion chromatography. The elution profiles demonstrate that scFv-p60.1 and scFv-p60.12 only bind to the non-secreted P domain (pGS99) and not to the secreted P domain (pGS100) from HEV gt3.

Figure 8 illustrates the determination of P domain-monoclonal antibody binding properties: (A) Sensorgrams showing association and dissociation phases of the interaction between non-secreted HEV gt3 P domain and the neutralizing antibodies of the invention. (B) Sensorgrams showing association and dissociation phases of the interaction between secreted HEV gt3 P domain and the neutralizing antibodies of the invention. Analyte concentrations (mAbs) were 5, 10, 25, 50 and 75 nM. Binding data are shown as black lines and fitting is shown overlaid in gray lines.

Figure 9 shows the size exclusion chromatography profiles obtained from complexes of anti-HEV scFvs with the HEV gt3 P domain for scFv-p60.1 and pGS99 and scFv-p60.12 and pGS99.

Figure 10 shows the crystals obtained from complexes of anti-HEV scFvs with the HEV gt3 P domain (pGS99) in complex with the neutralizing mAbs (A) scFv-p60.1 and (B) Fab- p60.12.

Figure 11 shows the crystal structure of the HEV gt3 P domain (pGS99) in complex with the neutralizing mAb scFv-p60.1 , shown in cartoon representation with the heavy (VH) and light chains (VL). The P domain dimer has one protomer colored black and the other grey. One complex in the asymmetric unit shows the P domain with surface representation, while the other is a cartoon representation.

Figure 12 depicts an overview (top panels) and close-up view (lower panels) of antibodyantigen interfaces of p60.1 (A) and p60.12 (B) in complex with the dimeric HEV gt3 P domain. The antibody (heavy chain in dark grey, light chain in light grey) contacts the two protomers of the P domain (grey, shown at the bottom) around the asparagine side chain of N562 (shown in sticks) which serves as attachment site for an N-linked glycan in the secreted pORF2 dimer.

Figure 13 shows a comparison of the complexes between the HEV gt3 P domain dimer with human (scFv-p60.1) and mouse (scFv from Fab 8C11 and scFv from Fab 8G12) anti- HEV antibodies.

Figure 14 illustrates the fitting of the crystal structures of the HEV gt3 P domain in complex with mouse and human scFvs to HEV virus-like particle (VLP) structure. The VLP (PDB 2ztn) is shown as surface representation while the scFvs are shown as cartoons. (A) scFv- 8C11 (PDB 3rkd); (B) scFv-p60.1 . The P domain was used to fit the complexes to the VLP.

Figure 15 shows the amino acid sequence alignment of the P domains from the five HEV genotypes studied (SEQ ID NO: 18-22). Contact residues for the mAbs are indicated as follows: ▲ : contact residues shared between p60.1 and p60.12 epitope. •: contact residues for p60.12 and ★: contact residues for p60.1. Secondary structural elements shown are for PDB 3rkd (gt1). Sequence conservation is marked as: black boxes = conserved across all genotypes; black letters = non-conserved residues; grey letters = conserved within genotypes. Figure generated using the ESPript server.

DETAILED DESCRIPTION OF THE INVENTION

Highly potent neutralizing antibodies have been gaining importance as treatment options for viral infections. Neutralizing antibodies can be classified into two groups, the first group comprising antibodies that inhibit only the infecting viral variant and not any other virus strains or variants. These antibodies are known as autologous antibodies and their neutralization is considered transient and highly affected by viral evolution. The second group of antibodies comprises broadly neutralizing antibodies (bNAbs). These have the ability to neutralize several variants of the same virus or members of the same viral family. Such antibodies often target epitopes that are highly conserved across diverse strains, virus subtypes and families.

The present invention provides highly potent bNAbs, which may be used for the diagnosis, the prophylaxis and/or the therapy of HEV, showing high binding affinity, potent neutralization activity, as well as broad reactivity across HEV genotypes, including human and rat HEV. Said antibodies or antibody constructs specifically target a glycan-dependent conformational neutralization epitope in pORF2 that is only accessible in the infectious (non-glycosylated) HEV particle, so that their neutralization activity is specific for the non- glycosylated form of pORF2, and cannot be evaded by the soluble secreted (glycosylated) forms of pORF2.

Accordingly, the first object of the present invention is a heavy chain construct and/or a light chain construct of an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2, a) wherein the heavy chain construct comprises a CDR1 having at least 90% sequence identity to SEQ ID NO: 2, a CDR2 having at least 88% sequence identity to SEQ ID NO: 3, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the light chain construct comprises a CDR1 having at least 83% sequence identity to SEQ ID NO: 6, a CDR2 having at least 66% sequence identity to the sequence AAS, and a CDR3 having at least 85% sequence identity to SEQ ID NO: 8 (i.e., the CDRs of monoclonal antibody p60.1); or b) wherein the heavy chain construct comprises a CDR1 having at least 87% sequence identity to SEQ ID NO: 10, a CDR2 having at least 87% sequence identity to SEQ ID NO: 11 , and a CDR3 having at least 93% sequence identity to SEQ ID NO: 12 and/or the light chain construct comprises a CDR1 having at least 88% sequence identity to SEQ ID NO: 14, a CDR2 having at least 66% sequence identity to the sequence DVT, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 16 (i.e., the CDRs of monoclonal antibody p60.12).

Preferably, said heavy chain construct and/or light chain construct of the invention comprise a) a heavy chain variable region having at least 90% sequence identity to amino acids 1-

121 of SEQ ID NO: 1 and/or and light chain variable region having at least 90% sequence identity to amino acids 1-106 of SEQ ID NO: 5 (i.e., the variable regions of monoclonal antibody p60.1); or b) a heavy chain variable region having at least 90% sequence identity to amino acids 1-

122 of SEQ ID NO: 9 and/or and light chain variable region having at least 90% sequence identity to amino acids 1-111 of SEQ ID NO: 13 (i.e., the variable regions of monoclonal antibody p60.12).

In one embodiment, the invention provides a heavy chain construct and/or light chain construct of the invention, wherein a) the heavy chain construct comprises a CDR1 having at least 90% sequence identity to SEQ ID NO: 2, a CDR2 having at least 88% sequence identity to SEQ ID NO: 3, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the light chain construct comprises a CDR1 having at least 83% sequence identity to SEQ ID NO: 6, a CDR2 having at least 66% sequence identity to the sequence AAS, and a CDR3 having at least 85% sequence identity to SEQ ID NO: 8.

In said embodiment, preferably, the heavy chain variable region has at least 90% sequence identity to amino acids 1-121 of SEQ ID NO: 1 and/or and the light chain variable region has at least 90% sequence identity to amino acids 1-106 of SEQ ID NO: 5, and, optionally, the heavy chain has at least 90% sequence identity to SEQ ID NO: 1 and/or and light chain has at least 90% sequence identity to SEQ ID NO: 5. Antibody constructs comprising said CDRs, said variable regions or said heavy and light chain constructs are designated p60.1. The preferred scFv p.60.1 comprises a heavy chain construct of SEQ ID NO: 1 and a light chain construct of SEQ ID NO: 5.

In an alternative embodiment, the invention provides a heavy chain construct and/or light chain construct of the invention, wherein a) the heavy chain construct comprises a CDR1 having at least 87% sequence identity to SEQ ID NO: 10, a CDR2 having at least 87% sequence identity to SEQ ID NO: 11 , and a CDR3 having at least 93% sequence identity to SEQ ID NO: 12 and/or the light chain construct comprises a CDR1 having at least 88% sequence identity to SEQ ID NO: 14, a CDR2 having at least 66% sequence identity to the sequence DVT, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 16.

In said embodiment, preferably, the heavy chain variable region has at least 90% sequence identity to amino acids 1-122 of SEQ ID NO: 9 and/or and the light chain variable region has at least 90% sequence identity to amino acids 1-111 of SEQ ID NO: 13, and, optionally, the heavy chain has at least 90% sequence identity to SEQ ID NO: 9 and/or and light chain has at least 90% sequence identity to SEQ ID NO: 13. Antibody constructs comprising said CDRs, said variable regions or said heavy and light chain constructs are designated p60.12. The preferred scFv p.60.12 comprises a heavy chain construct of SEQ ID NO: 9 and a light chain construct of SEQ ID NO: 13.

As used herein, the term "antibody construct" or "antibody", also “immunoglobulin”, has its general meaning in the art and refers to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, and single domain antibodies (DABs). Chimeric antigen receptors (CARs) are also considered antibody constructs.

Antibody constructs may, as in natural antibodies, e.g., of the IgG class, comprise two heavy chains linked to each other by disulfide bonds, wherein each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, 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. Antibody constructs of the invention may comprise constant regions, and may belong to each isotype. For example antibodies of the invention may be of the IgG isotype, e.g. IgGi or lgG4. Each chain contains distinct sequence domains. The light chain typically includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain typically includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). Structurally a natural antibody is also partitioned into two antigen-binding fragments (Fab), containing one VL, VH, CL, and CH1 domain each, as well as the crystallizable fragment (Fc), composed of the constant domains from the heavy chains. 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. The Fv fragment is the N-terminal part of the Fab fragment of an antibody 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 that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR, meaning amino acid sequences interposed between CDRs) influence the overall domain structure. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native antibody binding site. The light and heavy chains of an antibody each have three CDRs, designated L-CDR1 , L-CDR2, L- CDR3 and H-CDR1 , H- CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.

The term "antibody fragment" refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single chain antibody molecules, in particular scFv (single chain variable fragments), disulfide-l inked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as, for example, sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as, for example, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III.

Accordingly, in a further embodiment, the antibody construct of the invention may be a Fab fragment or a single chain antibody.

In some embodiments, the antibody construct is a monoclonal antibody. The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody. Although historically a monoclonal antibody was produced by immortalization of a clonally pure immunoglobulin secreting cell line, a monoclonally pure population of antibody molecules can also be prepared by other methods known in the art.

A heavy chain construct or a light chain construct of the invention is able to form an antibody construct of the invention, together with the respective other chain. The exact structure of the heavy and light chain constructs thus depends on the desired structure of the antibody construct, e.g., as detailed herein. The heavy chain construct and the light chain construct can also be part of the same amino acid chain, e.g., in a single chain antibody such as an scFv.

In relation to a particular pathogen, a "neutralizing antibody" or "broadly neutralizing antibody", is one that can neutralize the ability of that pathogen to initiate and/or perpetuate an infection in a host. In some embodiments, the antibodies produced in accordance with the present invention have neutralizing activity, where the antibody can neutralize at a concentration of 10’ ⁸ M or lower (e.g., 10’ ⁹ M, 10’ ¹⁰ M, 10’ ¹¹ M, 10’ ¹² M or lower).

As used herein, the term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.

The antibody construct of the present invention has specificity for the ORF2i protein. As used herein, the term “specificity” refers to the ability of an antibody to detectably bind to a conformational epitope presented on the ORF2i protein, while having relatively little detectable reactivity with the ORF2g protein and the ORF2c protein. Specificity can be relatively determined by binding or competitive binding assays as known from the art. Specificity can 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. The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. Binding affinity can be described by an antibody's equilibrium dissociation constant (KD), which is defined as the ratio Kd/Ka at equilibrium. Ka is the antibody's association rate constant and Kd is the antibody's dissociation rate constant. Binding affinity is determined by both the association and the dissociation and alone neither high association nor low dissociation can ensure high affinity. The association rate constant (Ka), or on-rate constant (Kon), measures the number of binding events per unit time, or the propensity of the antibody and the antigen to associate reversibly into its antibody-antigen complex. The association rate constant is expressed in M ^-1 s ^-1 , and is symbolized as follows: [Ab]x[Ag]xKon. The larger the association rate constant, the more rapidly the antibody binds to its antigen, or the higher the binding affinity between antibody and antigen. The dissociation rate constant (Kd), or off-rate constant (Koff), measures the number of dissociation events per unit time propensity of an antibody-antigen complex to separate (dissociate) reversibly into its component molecules, namely the antibody and the antigen. The dissociation rate constant is expressed in s ^-1 , and is symbolized as follows: [Ab+Ag]xKoff. The smaller the dissociation rate constant, the more tightly bound the antibody is to its antigen, or the higher the binding affinity between antibody and antigen. The equilibrium dissociation constant (KD) measures the rate at which new antibody-antigen complexes formed equals the rate at which antibody-antigen complexes dissociate at equilibrium. The equilibrium dissociation constant is expressed in M, and is defined as Koff/Kon=[Ab]x[Ag]/[Ab+Ag], where [Ab] is the molar concentration of the antibody, [Ag] is the molar concentration of the antigen, and [Ab+Ag] is the of molar concentration of the antibody-antigen complex, where all concentrations are of such components when the system is at equilibrium. The smaller the equilibrium dissociation constant, the more tightly bound the antibody is to its antigen, or the higher the binding affinity between antibody and antigen. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments, e.g., as described in the example below.

In a further aspect of the invention, the antibody construct of the invention has an equilibrium dissociation constant KD for non-glycosylated form of pORF2 of less than 100 nM, less than 50 nM less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM or preferably less than 0.5 nM.

As used herein, the term “epitope” refers to a specific arrangement of amino acids located on a protein to which an antibody binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be linear or conformational, i.e. , involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous. The term “conformational epitope” refers to amino acid residues that are, at least in part, discontinuous in the epitope protein sequence yet come within close proximity to form an antigenic surface on the protein's three-dimensional structure.

The terms "peptide", "polypeptide", and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A polypeptide is not limited to a specific length: it must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a polypeptide's sequence. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. In one embodiment, as used herein, the term “peptides” refers to a linear polymer of amino acids linked together by peptide bonds, preferably having a chain length of less than about 50 amino acids residues; a "polypeptide" refers to a linear polymer of at least 50 amino acids linked together by peptide bonds, and a protein specifically refers to a functional entity formed of one or more peptides or polypeptides, optionally of non-polypeptides cofactors. This term also includes post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

As used herein, the term "glycosylated" with respect to a protein means that a carbohydrate moiety is present at one or more sites of the protein molecule. In particular, a glycosylated protein refers to a protein that is typically modified by N-glycan or O-glycan addition.

As used herein, “ORF2” refers to the open reading frame encoding the HEV ORF2 viral capsid protein (“pORF2”). As illustrated in Figure 1 A, the ORF2 protein sequence contains 660 amino acids, has a signal peptide at its N-terminus (that can shuttle it to the extracellular compartment, depending on the start codon), and is further organized into three domains designated as shell (S, amino acids 129 - 319), middle (M, amino acids 320 - 455), and protruding domain (P, amino acids 456 - 606). Three highly conserved potential N-glycosylation sites represented by the sequon Asn-X-Ser/Thr (N-X-S/T) have been identified in pORF2, but N-linked glycosylation has only been confirmed to occur at positions N137 and N562.

HEV infection leads to the production of at least 3 forms of pORF2 capsid protein: infectious ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c), as shown by Montpellier et al. (2018. “Hepatitis E virus lifecycle and identification of 3 forms of the ORF2 capsid protein” Gastroenterology 154 (1): 211-223). The ORF2i protein is the structural component of infectious particles, and it is not glycosylated. In contrast, ORF2g and ORF2c proteins are secreted in large amounts in culture supernatant and sera of infected patients, are sialylated, N- and O-glycosylated but are not associated with infectious virions. Importantly, ORF2g and ORF2c proteins, the most abundant antigens detected in patient sera, might inhibit antibody-mediated neutralization.

The present invention provides antigen-binding proteins, antibody constructs, such as antibodies and antigen-binding fragments thereof, preferably monoclonal antibodies, that specifically bind to a non-glycosylated (infectious) HEV particle, or an antigenic fragment thereof, but not to a glycosylated (non-infectious) HEV particle. Preferably, the present invention provides antibody constructs, such as antibodies and antigen-binding fragments thereof, preferably monoclonal antibodies, that specifically bind to a conformational epitope of non-glycosylated pORF2, preferably that specifically bind to a non-glycosylated conformational epitope at the dimerization interface at the tip of the P domain dimer of pORF2 containing glycosylation site N562.

In one embodiment of the present invention, said antibody construct, e.g., said monoclonal antibody is monoclonal antibody p60.1 against HEV, which can specifically bind and neutralize non-glycosylated (infectious) HEV particles. It can also be a derivative thereof, preferably, sharing the CDRs and, optionally, the variable regions thereof .

In another embodiment of the present invention, said antibody constructs, e.g., said monoclonal antibody is monoclonal antibody p60.12 against HEV, which can specifically bind and neutralize non-glycosylated (infectious) HEV particles. It can also be a derivative thereof, preferably, sharing the CDRs and, optionally, the variable regions thereof .

In a further embodiment, the antibody constructs of the invention recognize a quaternary epitope that assembles at the dimerization interface at the tip of the P domain dimer of pORF2. The broad neutralizing antibodies of the invention bind predominantly to one protomer, but make contacts also with the other one, resulting in an asymmetric binding mode. Thus, a correctly folded pORF2 dimer is required for antigen-antibody interaction of said antibodies with HEV according to the invention. The antibodies of the invention recognize conformation-sensitive epitopes comprising three segments distant in the amino acid sequence of pORF2: 1) amino acids 480-490, 2) amino acids 555-565 and 3) amino acids 580-590. The epitopes recognized by p60.1 and p60.12 largely overlap with each other, sharing contact residues Q482, R488, Y557, Y561 , N562, Y584. Importantly, N562 of both protomers, which serve as attachment points for the N-linked glycans in the secreted pORF2 dimer, form hydrogen bonds with both antibodies, so that such an attached glycan would preclude binding of these bnAbs via steric clashes and thereby causes glycan sensitivity of the antibodies.

According to the invention, for monoclonal antibody p60.1 the CDRs of the heavy chain variable region (SEQ ID NO: 1) correspond to the amino acid sequences as set forth in SEQ ID NO:2 (p60.1-HC CDR1), SEQ ID NO:3 (p60.1-HC CDR2) and SEQ ID NO:4 (p60.1 -HC CDR3). The CDRs of the light chain variable region (SEQ I D NO: 5) correspond to the amino acid sequences as set forth in SEQ ID NO:6 (p60.1-LC CDR1), the sequence AAS (p60.1-LC CDR2) and SEQ ID NO:8 (p60.1-LC CDR3).

According to the invention, for monoclonal antibody p60.12 the CDRs of the heavy chain variable region (SEQ ID NO: 9) correspond to the amino acid sequences as set forth in SEQ ID NO:10 (p60.12-HC CDR1), SEQ ID NO:11 (p60.12-HC CDR2) and SEQ ID NO:12 (p60.12-HC CDR3). The CDRs of the light chain variable region (SEQ ID NO: 13) correspond to the amino acid sequences as set forth in SEQ ID NO: 14 (p60.1-LC CDR1), the sequence DVT (p60.12-LC CDR2) and SEQ ID NO: 16 (p60.12-LC CDR3).

Particularly, in a further embodiment, the antibodies of the invention may be human antibodies.

The term “human” antibodies, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. However, the term human antibody, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).

The antibodies may alternatively be non-human antibodies, e.g., murinized antibodies or rat antibodies, e.g., for in vivo experiments in mice or rats. The present invention also relates to conserved variants or active fragments of any one of the antibody constructs of the invention sharing the CDRs of p60.1 or p60.12, wherein one or more of amino acid residues in said variant or active fragment thereof have been conservatively substituted, added or deleted, wherein the variant or fragment still retains the capability of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2.

Term "conserved variants" used herein means that the variants substantially retain the parent's properties, such as basic immunological properties, structural properties, regulating properties or biochemical properties. Generally, the amino acid sequence of the conserved variants of the polypeptide is limitedly different from the parent polypeptide such that the conserved variants and the parent polypeptide are closely similar as a whole and are identical in a lot of regions. The difference of amino acid sequence between the conserved variants and parent polypeptide can be replacement, addition and deletion of one or more amino acid residues or any combination thereof. The replaced or added amino acid residues may or may not be encoded by genetic code. The conserved variants of the polypeptide may be variants produced spontaneously or not spontaneously. The polypeptide's conserved variants produced not spontaneously may be produced by induced mutation techniques or by direct synthesis. According to the disclosed content of the invention, a person skilled in the art would appreciate that the fragment of said antibody of the invention may be modified to substantially preserve the property of specifically binding to hepatitis E virus of the generated antibody variants.

Thus, in the heavy chain construct and/or light chain construct of the invention, any deviations from the defined SEQ ID NO: may be conservative substitutions, wherein, preferably, there is at most one conservative substitution per chain. Conservative substitutions are known in the art, wherein an amino acid is exchanged for a different amino acid with similar biochemical properties, e.g., charge, hydrophobicity and/or size. To this end, preferably, substitutions are selected from the same class of amino acids, as defined herein: In the heavy chain construct and/or light chain construct of the invention, the sequence identity in the CDR1 , CDR2, and/or CDR3 may be 100% (to the respective defined CDRs). Optionally, sequence identity in all CDRs or a heavy chain consruct and/or a light chain construct, preferably, in both, is 100%. Optionally, sequence identity in the variable regions also is 100%, i.e., the variable regions of the antibody construct or the inventions correspond to the variable regions of p60.1 or p60.2.

In a preferred embodiment, the invention does not only provide the heavy and light chain constructs, but also an antibody construct comprising the heavy chain construct and light chain construct of the invention, i.e., an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2. As described herein, as broadly neutralizing antibodies, said antibody constructs have been found to be particularly advantageous for diagnostic, therapeutic and prophylactic uses.

Another aspect of the present invention also relates to a nucleic acid molecule encoding a heavy chain construct and/or light chain construct of the invention or an antibody construct of the invention. In this context, it is noted that, in the context of the invention, unless explicitly stated otherwise, "a" is understood to mean "at least one", i.e., a nucleic acid molecule of the invention may also encode a heavy chain construct of the invention and a light chain construct of the invention, and preferably encodes an antibody constructs of the invention. Said nucleic acid molecule may be a recombinant expression vector comprising said nucleic acid molecule, preferably, under the control of a heterologous promoter. The promotor preferably is able to mediate expression in a host cell, e.g., a bacterial cell, a yeast cell, a eukaryotic cell, an insect cell, a mammalian cell such as a CHO cell or a human cell, e.g., a B cell, a T cell or an NK cell. The promotor can be a constitutive promotor or an inducible promotor.

According to the amino acid sequences of the antibodies of the present invention or of their active fragments, the skilled in the art can get the nucleotide sequences encoding the same and get their variants according to codon degeneracy. Preferably, the nucleic acid is codon-optimized for expression in the desired host cell. According to the disclosed content in the present invention, the skilled in the art can get recombinant expression vectors comprising the nucleotide sequences described above, and host cells transformed with the expression vectors by many methods. Selection of host cells and transformation technologies that can be used to express said antibodies or their active fragments are well known from the art. Therefore, the present invention also relates to a recombinant expression vector comprising the nucleic acid molecule encoding said antibodies or active fragments. By "vector" is meant any genetic element, such as a plasmid, minicircle, YAC, phage, transposon, e.g., suitable for being transposed by Sleeping Beauty, cosmid, chromosome, a viral vector or virus, e.g., a retroviral or adenoviral vector etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. The antibodies of the invention and/or active fragments thereof can be expressed in proper host cells by genetic engineering methods known in the art. Thus, the invention further relates to host cells transformed with said recombinant expression vectors. Many expression host cells can be used in the present invention, for example, prokaryotic cells including but not limited to Escherichia coli, Bacillus, Streptomyces, eukaryotic cells including but not limited to Aspergillus, Saccharomycetes, as well as mammalian cells, plant cells and so on. The expression of the antibody constructs of the invention is not limited to any specific expression vectors or host cells, as long as they can be used to express said antibodies.

The invention thus also provides a host cell comprising said recombinant expression vector, which is able to express the heavy chain construct and/or light chain construct or the antibody construct of the invention. Preferably, the host cell expresses said construct, e.g., said antibody construct, on its cell surface (e.g., in case of a CAR or a transmembrane immunoglobulin), or it secretes said construct (typically, in the case of an antibody, e.g., a single chain antibody). The cell may be a human cell, e.g., a B cell, a T cell or an NK cell.

The antibody constructs of the present invention bind to antigens present in sera of patients infected with HEV. Thus, the present invention also relates to said antibody constructs for use in diagnosis of hepatitis E virus infection.

Accordingly, a further object of the present invention relates to a method for detecting the presence of native, non-glycosylated HEV pORF2 in a sample, e.g., a sample from a patient, comprising contacting the sample with the antibody constructs of the present invention under conditions that allow an immunocomplex of the protein and the antibodies to form, wherein detection of the immunocomplex indicates the presence of native, nonglycosylated HEV pORF2 in the sample (for instance: immunoprecipitation, immunofluorescence or western blotting).

In general, infectious HEV particles may be detected using the antibody constructs of the present invention because these antibody constructs, e.g., monoclonal antibodies, recognize specific antigenic determinants for infectious HEV particles located on the surface of HEV. Thus, more particularly, a further object of the present invention relates to a method for detecting the presence of infectious HEV particles in a sample comprising contacting the sample with the antibodies of the invention under conditions that allow an immunocomplex of the antibody and the infectious particles to form, wherein detection of the immunocomplex indicates the presence of the infectious particles in the sample. As used herein, the term "sample" includes any solid or fluid sample, liable to contain infectious particles of hepatitis E virus. In some embodiments, the sample is selected from the group consisting of ascites, urine, saliva, sweat, milk, synovial fluid, peritoneal fluid, amniotic fluid, percerebrospinal fluid, lymph fluid, lung embolism, cerebrospinal fluid, and pericardial fluid. In some embodiments, the sample is a feces sample. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a blood sample. As used herein the term “blood sample” means any blood sample derived from a subject.

The detecting methods of the present invention are particularly suitable for diagnosing acute HEV infection, recent HEV infection, chronic HEV infection, weak active HEV infection or cleared HEV infection. Assays and conditions for the detection of immunocomplexes are known to those of skill in the art. Such assays include, for example, competition assays, direct reaction assays sandwich-type assays and immunoassays (e.g., ELISA). The assays may be quantitative or qualitative. There are a number of different conventional assays for detecting formation of an antibody-peptide complex comprising an antibody of the present invention. For example, the detecting step can comprise performing an ELISA assay, performing a lateral flow immunoassay, performing an agglutination assay, analyzing the sample in an analytical rotor, or analyzing the sample with an electrochemical, optical, oropto-electronic sensor. These different assays are well- known to those skilled in the art.

In particular, the antibody constructs of the present invention may be used in highly sensitive methods for screening and identifying individuals carrying HEV and/or infected with HEV, as well as for screening for HEV-contaminated samples. The antibody constructs of the present invention may also be used in assays for monitoring the progress of anti-HEV therapies in treated individuals, or for monitoring the growth rate of HEV cultures used in research and investigation of the HEV agent.

Also described herein is a diagnostic kit for the diagnosis of hepatitis E virus infection, comprising at least one of the antibody constructs of the invention, or any combination thereof (e.g., an antibody construct based on p60.1 and an antibody construct based on p60.12). The kit may comprise the antibody construct in an amount effective for diagnosis. It optionally comprises detection agents suitable for detection of the interaction of antigen and antibody construct corresponding to the adopted detection method. The kit may also comprise suitable buffers.

Those skilled in the art would be competent for the design of an appropriate immunological method, and relevant reagent, and buffer system suitable for the preceding diagnostic kit according to specific interactions of antigen and antibody construct, immunochemical test methods as commonly used and the present disclosure.

The term "amount effective for diagnosis" is intended to mean the antibody construct of the invention, conserved variants or active fragments thereof at the amount effective for detection of HEV in a biological sample. According to the known immunochemical methods, those skilled in the art recognize that the amount of the above-mentioned material would be variable depending on the different immunochemical methods used. Under the teachings of the art, they know how to select an appropriate amount of the antibody construct of the invention as described herein so as to diagnose HEV in a biological sample. And they also know that the diagnostic kit, where appropriate, should further include suitable absorbent carriers, buffer reagent/solutions, reagents used to produce visible signals fortesting and instructions for use. Examples of kits include but are not limited to ELISA assay kits, and kits comprising test strips and dipsticks.

The antibody constructs of the invention specifically recognize antigenic determinants located on the surface of infectious HEV particles, showing high binding affinity, potent neutralization activity, as well as broad reactivity across HEV genotypes. The antibodies of the invention efficiently neutralize naked and quasi-enveloped viral particles. Said antibodies may therefore be used for the prophylaxis and/or for the passive immunization treatment of HEV infections, especially for chronical and immunocompromised HEV patients.

Thus, the invention also provides a pharmaceutical composition comprising a) an antibody construct of the invention, b) an expression vector of the invention, wherein the nucleic acid encodes the antibody construct of the invention and/or c) a host cell of the invention, wherein the nucleic acid encodes the antibody construct of the invention, and, optionally, a pharmaceutically acceptable vehicle and/or excipient.

Said pharmaceutical composition may be for use in preventing or treating hepatitis E virus infection. They may comprise a pharmaceutically effective amount of said antibody construct.

Thus, in another aspect, the present invention relates to the antibody constructs of the invention for use as a medicament for prophylaxis and/or treatment of HEV infection.

"Prevention" and "prophylaxis" are used synonymously herein. When supplied prophylactically , the pharmaceutical composition of the invention is provided in advance of any exposure to any one or more of the HEV strains or in advance of any symptoms due to infection of the viruses. The prophylactic administration of the pharmaceutical composition of the invention serves to prevent, reduce the risk or likelihood of or attenuate any subsequent infection of these viruses in a mammal, preferably in a human subject. For therapeutic use, the pharmaceutical composition of the invention is provided at or after (preferably, shortly after) the onset of infection or at the onset of any symptom of infection or any disease or deleterious effects caused by these viruses. The therapeutic administration of the pharmaceutical composition of the invention serves to attenuate the infection or disease. The pharmaceutical composition of the present invention may, thus, be provided either prior to the anticipated exposure to HEV or after the initiation of infection. According to the specific conditions of the subject to be treated, the skilled in the art know how to select proper doses and administration routes. The pharmaceutical composition of the invention may also comprise a therapeutically effective amount of the antibodies of the invention combined with at least one other anti-viral agent as an additional active ingredient. Such agents may include but are not limited to interferons, other anti HEV monoclonal antibodies, anti HEV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, immunomodulators, antisense compounds and ribozymes.

In another aspect of the present invention, there is provided a method of treating a subject infected with HEV or of reducing the likelihood of infection of a subject at risk of contracting HEV, comprising delivering to said subject a prophylactically effective amount or a therapeutically effective amount of at least one of the antibody constructs of the invention.

The term "prophylactically effective amount" can be replaced with the term "immunologically effective amount", which means the amount sufficient to elicit immune prevention for a subject. It is well known that the "prophylactically effective amount" may vary according for example to the administration way, the characteristics of the individual subject, and the antibody construct used. According to the published references, teachings and corresponding clinical criteria in the art, the "prophylactically effective amount" of an antibody construct can be determined. The preferable prophylactically I immunologically effective amount is 0.0001 mg - 0.1 mg per dose.

Similarly, the term "therapeutically effective amount" means the amount sufficient to elicit effective protection for the subjects and to neutralize HEV, or an amount effective in alleviating the symptoms of the HEV infection or reducing the number of circulating viral particles in an individual. It is well known that a therapeutically effective amount of an antibody may vary depending on different treatment schemes, illness courses, characteristics of the individual subject, and the antibodies used. According to the published references, teachings and corresponding clinical criteria in the art, a clinician can determine the "therapeutically effective amount" of an antibody construct based on their own experience. The preferable therapeutically effective amount is 0.001 mg - 20 mg per kg weight.

As the inventors have shown that the antibody constructs of the invention based on p60.1 are particularly effective in neutralizing HEV g3, g4 and g2, and the constructs based on p60.12 are particularly effective in neutralizing HEV g3, g4 and rat-HEV, they are preferably used for diagnosis, treatment or prevention of said virus subtypes.

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

The invention will be further illustrated by the following examples and figures. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. References are fully incorporated herein.

EXAMPLES

MATERIALS AND METHODS

1.1 Cloning, expression, and purification of HEV P domains

1.1.1 Construct design for the different HEV P domains

The P domains starting at amino acid 456 of the HEV pORF2, from human infective genotypes 1 , 2, 3, 4, and the rat genotype, were used. The P domain serves as the receptor-binding domain of HEV and is thus a primary target for neutralizing antibodies. pORF2 exhibits both secreted and non-secreted forms during infections. The former are present as glycosylated dimers in sera of infected patients, while the later are components of the infectious particle. To identify broadly neutralizing antibodies targeting the infectious particle, constructs for all genotypes were designed to represent the non-secreted form of the P domain. As a control, a construct representing the secreted form was designed using HEV genotype 3 P domain (Table 1).

Table 1 : Constructs for HEV P domains

Construct Description Form pGS38 HEV gt3 P domain (aa 455-660) fused to mNeon intracellular pGS39 HEV gt3 P domain (aa 455-660) fused to mRuby secreted pGS99 HEV gt3 P domain (aa 455-660) intracellular pGS100 HEV gt3 P domain (aa 455-660) secreted pGS118 HEV gt1 P domain (aa 455-660) intracellular pGS120 HEV gt2 P domain (aa 455-660) intracellular pGS122 HEV gt4 P domain (aa 455-660) intracellular pGS135 Rat HEV P domain (aa 455-660) intracellular

1.1.2 Cloning of HEV P domains into a pMT vector

All genes were introduced into the pMT vector backbone (Krey et al., 2010. The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule. PLoS Pathog 6(2): e1000762. doi: 10.1371 /journal. ppat.1000762) using the restriction-free cloning method. The inserts were amplified from the codon-optimized synthetic genes (Twist Bioscience) listed in Table 2.

Table 2: Codon-optimized HEV ORF2 synthetic genes

ORF2 Origin UniprotKB

HEV gt1 lsolate/human/Pakistan/Sar55 P33426

HEV gt2 Isolate/human/Mexico Q03500

HEV gt3 Isolate/human/Japan C4B4T9

HEV gt4 Isolate/human/China Q9IVZ8

Rat HEV R68/DEU/2009 E0XL23

1.1.3 Transfection and expression of recombinant HEV P domains in Drosophila S2 cells

The pGS plasmids according to Table 1 verified by sequencing to carry the desired inserts were transfected into Drosophila S2 cells essentially as described previously (Johansson et al. 2012. Production of Recombinant Antibodies in Drosophila mela nogaster S2 Cells. Patrick Chames (ed.), Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, 907). Briefly, stable cell lines were generated by cotransfecting the plasmids coding for the gene of interest with a puromycin-resistance selection plasmid. Following selection with puromycin and adaptation of the cells to insect- Xpress media, cells were induced with CdCh for large-scale protein production. On day five post-induction, the intracellular and the secreted P domains were affinity purified from cell lysates and culture supernatants, respectively. Protein purity assessment was performed by SDS-PAGE analysis.

1.2 Memory B cell isolation and sequencing

In order to obtain P domain specific monoclonal antibodies, antigen-specific memory B cells were isolated from peripheral blood mononuclear cells (PBMCs) from two convalescent HEV patients (N1960 (p60), 4100869889, 8.5 x 10 ⁷ cells and N1961 (p61), 1707407510, 1.44 x 10 ⁸ cells) by magnetic activated cell sorting (MACs). MACs-sorted cells were then stained with fluorescently labeled P domains and analyzed for the frequency and phenotype of antigen-specific B cells by flow cytometry prior to sorting of antigen-specific B cells. This was then followed by single B cell RNA sequencing as described in the next sections.

1.2.1 B-cell sorting

PBMCs from two convalescent HEV patients (p60 and p61), were isolated using density gradient separation medium (Histopaque, Sigma Aldrich) according to the manufacturer’s instructions and stored at -150 °C in 10% DMSO and 90% (v/v) fetal bovine serum. B cells were obtained from PBMCs by magnetic separation using CD19 microbeads and stained with anti-human CD20-Alexa Fluor 700, anti-human IgG-APC, LIVE/DEAD™ Fixable Near-IR Dead Cell Stain, and recombinant differentially fluorescently labelled HEV gt3 P domains (pGS38 and pGS39) with the aim of obtaining antibodies that bind the nonglycosylated form but not the glycosylated form. Approximately 4,170 and 19,760 live cells were sorted for p60 and p61 , respectively, using band pass filters R 780/60, R 730/45, R 670/30, YG 610/20 and B 530/30. Antigen-specific memory B cells were single-cell sorted using 10xGenomics technology and B cell receptor sequences obtained by Next Generation Sequencing using Illumina technology. The obtained FASTQ files were used to generate single cell V(D)J sequences and annotations using Cell ranger vdj (version 3.1.0). Output files were loaded and analyzed with the Loupe V(D)J Browser (10x Genomics).

1.2.2 Single-chain fragments of the variable region (scFv)

The variable regions of antibodies were selected after analysis of the sequences using the Loupe V(D)J browser based on a criterion described in the result section below. In order to assemble single-chain variable fragments, the variable region of the heavy chain was covalently linked via its C-terminus to the N-terminus of the respective light chain variable region using a 3x glycine-serine linker (GS-linker). Codon-optimized synthetic genes of the covalently linked paired sequences cloned into a pMT vector were purchased from Twist biosciences. All constructs carried a Drosophila BiP signal sequence at the N-terminus as well as an EK cleavage site and a double strep-tag at the C-terminus. The name of the individual scFvs was defined by scFv as a prefix, followed by the donor ID and the clone number. Expression and purification of all scFvs followed the protocols in sections 1.1.3.

1.3 Crystallization, data collection, and structure determination

1 .3.1 Proteolytic removal of the double strep-tag Protein preps intended for crystallization were digested to remove the affinity purification tag. A TEV cleavage site at the N-terminus of the P domains and the enterokinase (EK) cleavage site at the C-terminus of the scFvs were used for this purpose.

For TEV digestion, the P domains and TEV protease were mixed in a 1 :4 ratio and digestion done in SEC buffer at 28 °C for 4 hours. On the other hand, a reaction mix for EK digestion was set up containing 2 mg protein, 300pl 10x EK buffer (500 mM NaCI, 20 mM CaCh, 200 mM Tris pH 8), and 100 pl EKMax™ enterokinase (Thermo Scientific) diluted 1 :25 in 1x EK buffer. This was then topped up to 3 ml with Milli-Q water and incubated at 28 °C overnight, after which 30 pl 100 mM PMSF was added to inactivate the enterokinase. Since crystallization requires large amounts of protein, the quantity of protein digested was adjusted accordingly.

To separate the digested protein from the undigested protein, the digestion product was flown through a strep-tactin column, thereby retaining the undigested protein still carrying the strep tag. To remove the histidine tagged TEV protease from the digested proteins, the digestion product was applied onto a 1 ml HisTrap™ excel column (Cytiva), thereby retaining the TEV in the column. The flow-through and wash fractions that contained the digested proteins were pooled and further purified by size exclusion chromatography. The elution fractions containing the proteins of interest were pooled, concentrated, and stored at -80°C.

1.3.2 Crystallization of HEV gt3 P domain (pGS99) in complex with the best neutralizing scFvs

Crystallization experiments of HEV gt3 P domain in complex with the best neutralizing scFvs involved mixing pGS99 with scFvs (1 :1.2 molar ratio, respectively) and incubating the mixture overnight at 16 °C. The complexes were purified on a superdex 200 increase 10/300 GL column (Cytiva) equilibrated in gel filtration buffer. The purified complexes were then concentrated to 9.3 mg/ml (scFv-p60.1-pGS99) and 6.7 mg/ml (Fab-p60.12-pGS99). Five crystallization screens per complex (Wizard 1&2, Wizard 3&4, Classic 1-4, Classic 5- 8 and PACT++, Jena Bioscience) each with 96 different conditions, were set up using the Crystal Gryphon (Art Robbins Instruments). The crystals were grown at 293 K using the sitting-drop vapor diffusion method in drops containing 0.15 pl of the complex solution mixed with 0.15 pl of reservoir solution. Of the 480 conditions screened, more than 20 conditions resulted in crystals of similar shape for scFv-p60.1 , and 3 conditions resulted in crystals of similar shape for the complex of Fab-p60.12.

1 .3.3 Data collection and structure determination of HEV gt3 P domain in complex with the best neutralizing scFvs For the scFv-p60.1-pGS99 and Fab-p60.12-pGS99 complexes the crystals were flash- frozen in mother liquor containing 30 % ethylene glycol and diffraction data were collected at P11 (DESY-Hamburg) and PX1 (Swiss Light source-SLS), respectively. Data were processed, scaled, and reduced with XDS (Kabsch W. 2010. Xds. Acta Crystallogr D Biol Crystallogr. 66 (2): 125-32), Pointless (Evans PR. 2011. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr. 67 (4): 282-92), and programs from the CCP4 suite (Winn et al. 2011. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 67 (4): 235-42). Phaser (Bunkoczi et al., 2013. Phaser. MRage: automated molecular replacement. Acta Crystallogr D Biol Crystallogr. 69 (11): 2276-86) was used for molecular replacement with PDB 3rkc (Tang et al., 2011. Structural basis for the neutralization and genotype specificity of hepatitis E virus. PNAS 108 (25): 10266-10271) and PDB 6dsi (Rouet et al., 2019. Anti-recombinant prolactin receptor scFv. doi: 10.2210/pdb6DSI/pdb) as search models to overcome the phase problem. This was followed by model building using Coot (Emsley et al., 2010. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 66 (4): 486-501) with several rounds of refinement using AutoBuster (Smart et al., 2012. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr D Biol Crystallogr. 68 (4): 368-80), and validation with MolProbity (Chen et al., 2010. MolProbity: all atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 66 (1): 12-21). Figures were generated using PYMOL (http://www.pymol.Org/2/ support.html).

1.4 IgG cloning, expression, and purification

1.4.1 Cloning of IgGs into a HEK cell expression vector

Paired heavy and light chain variable regions of antibodies were amplified from the respective top neutralizing scFvs. These were inserted into a pcDNA3.1 expression vector. The heavy chain variable regions were cloned into the IgG 1 expression vector, while their respective kappa or lambda pairs were cloned into expression vectors carrying the corresponding constant regions. All genes were cloned downstream a CD5 signal peptide to allow the secretion of mature IgGs. Plasmid DNA was amplified, isolated and sequenced.

1.4.2 Transient IgG expression and purification in HEK cells

HEK Expi293F™ cell lines were thawed and maintained in Expi293™ expression medium (Gibco) according to the supplier’s manual and cultured at 37 °C, 8 % CO2, with shaking at 125 rpm. For transfection, the ExpiFectamine™ 293 transfection kit (Gibco) was used with slight modifications. A day before transfection, 2.5 x 10 ⁶ viable cells/ml were seeded and cultured overnight in a 500 ml Erlenmeyer flask. Cell density and viability were determined by trypan blue exclusion on a Countess II FL cell counter (Life technologies). The required volume of cells was centrifuged for 5 minutes at 300x g before carefully resuspending the cells in fresh media. On the day of transfection, the overnight culture with a viability > 90 % was diluted to 2 x 10 ⁶ viable cells/ml in 85 ml pre-warmed medium, and cells were returned to the incubator. The plasmid DNA cocktail was prepared in 5 ml Opti-MEM (Gibco) at ratios listed in Table 3, resulting in total plasmid DNA of 1 pg per ml of culture volume transfected. Of note, addition of p21 , p27 and SV40 serves to enhance protein expression in HEK cells. The ExpiFectamine™ 293 transfection reagent was then prepared by mixing 266.7 pl of the reagent with 4733.3 pl Opti-MEM™ and allowed to incubate for 5 minutes at room temperature. Following the 5-minute incubation, the diluted reagent was mixed with the plasmid DNA cocktail by inversion. The ExpiFectamine™ 293/DNA complexes were incubated for 20 minutes at room temperature, after which they were added to the cells dropwise, and the cells were subsequently incubated for 16-22 hours. This was followed by dropwise addition of 500 pl ExpiFectamine™ 293 transfection Enhancer 1 and 5 ml ExpiFectamine™ 293 transfection Enhancer 2 to the transfected cells, and the cells were returned to the incubator.

Table 3: Plasmid DNA cocktail

Plasmids Amount (pq/ml of transfection) pq/100 ml transfection volume

Heavy chain 0.345 34.5

Light chain 0.345 34.5 pT1242 (p21) 0.05 5 pT1243 (p27) 0.25 25 pT1244 (SV40) 0.01 1

On day five post-transfection, the cells were harvested by adding 10 ml 10x PBS and centrifugation at 70,000x g for 20 minutes. The supernatant was filtered through a 0.22 pm syringe filter prior to loading onto a 1 ml HiTrap Protein-G column (Cytiva) connected to the AKTA™ pure 25 system. Unbound proteins were washed off with 1x PBS and IgGs eluted in 0.1 M Glycine pH 2.7, which was then neutralized by adding 100 pl 1 M Tris-HCI pH 8. The eluted neutralized IgGs were buffer exchanged to PBS using a superdex 200 increase 10/300 GL column. The elution fractions containing the protein of interest were pooled, concentrated and stored at 4 °C until needed for further experiments (neutralization assays, ELISA and Biacore).

1.5 Enzyme-Linked Immunosorbent Assay (ELISA)

An indirect ELISA analysis was performed to screen human anti-HEV antibodies for cross genotype binding potential, as well as for their ability to differentiate between secreted and non-secreted forms of the HEV g3 P domain. Nunc 96 well ELISA plates (Thermo Scientific) were coated with 100 ng of antigen per well in PBS overnight at 4°C. Plates were washed 3x with 300 pl PBS-T and blocked with 100 pl blocking buffer (PBS-T and 5 % skimmed milk) per well for 2 hours at room temperature. The plates were then washed once with PBS-T and the wells were loaded with 50 pl 2 pg/ml antibody diluted in blocking buffer. Following incubation for 1 hour at room temperature, the plates were washed 4x with 300 pl PBS-T and incubated for 30 minutes at room temperature with 50 pl per well of horse-radish peroxidase conjugated goat anti-human antibody diluted 1 :40,000 in blocking buffer. The plates were then washed 4x with PBS-T, and the reaction was developed by adding 100 pl TMB substrate (BioLegend) per well. The reaction was stopped after 10 minutes by the addition of 50 pl 1 M H3PO4 and absorbance was measured at 450 nm with 630 nm as reference using the ELx808 absorbance plate reader (BioTek). Data were analyzed using GraphPad prism (GraphPad).

For capture ELISA of patient sera, Nunc 96 well ELISA plates were coated with 125ng of the depicted antibody in PBS for 1 hour at 37°C. Plates were washed 3x using TBS-T (Tris buffered saline supplemented with 0.1% Tween 20) followed by blocking with 10OpI /well TBS-T supplemented with 5% horse serum for 2 hours (blocking buffer) at 37°C. Patient sera were diluted 1 :10 in blocking buffer and 50pl/well were incubated for 1 hour at 37°C. After another three washes, 50pl/well anti-pGS99 rabbit polyclonal serum was added in a 1 :100 dilution for 1 hour at 37°C followed by incubation with horse-radish peroxidase conjugated goat anti-rabbit antibody (Thermo Fisher) diluted 1 :10,000 in blocking buffer for 30minutes at 37°C. In between all incubation steps plates were washed three times by submersion in TBS-T. 50pl of TMB solution were added and after 5 minutes the reaction was stopped by adding 50pl of 1 M sulfuric acid. Optical density was measured at 450 nm with 630 nm as reference. Data were analyzed using GraphPad prism 9. HEV Antigen positivity of depicted sera was confirmed by performing WANTAI HEV Antigen ELISA according to the manufacturers protocol (WANTAI BioPharm). HEV-RNA positivity was tested by the central laboratory at Hannover Medical School using PCR.

1.6 Biacore analysis

Strep-TactinXT (I BA Life sciences) was amine coupled to a carboxyl-derivatized CM-5 sensor chip (Cytiva) using a Biacore 3000 (GE Healthcare) following the protocol from the Twin-Strep-tag capture kit for surface plasmon resonance (I BA Life sciences). Firstly, the chip surfaces were prepared by pre-treatment with three consecutive 1 -minute pulse injections of 50 mM NaOH. The surfaces were then activated for 10 minutes with a 1 :1 mixture of NHS and EDC at a flow rate of 10 pl/min. 35 pg/ml Strep-TactinXT in 10 mM sodium acetate pH 4.5 was immobilized for 10 minutes at a density of 3922 RU on all flow cells. Following immobilization, surfaces were washed with 1x HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCI, 0.003 M EDTA, 0.05 % Tween-20) until a stable baseline was obtained. After washing, deactivation I blocking for 10 minutes with ethanolamine was performed, followed by conditioning with three consecutive 30-second pulse injections using 10 mM NaOH.

To collect kinetic data for HEV antibodies, 25 nM ligand (26 kDa, HEV gt3 secreted and non-secreted P domains) in HBS-EP buffer was injected for 1 minute over flow cell 2 at a flow rate of 10 pl/min. Then, the analytes (lgG-p60.1 and p60.12) in HBS-EP buffer were injected over two flow cells (1 and 2) at concentrations of 75, 50, 25, 10 and 5 nM at a flow rate of 30 pl/min and a temperature of 25 °C. The complex was allowed to associate and dissociate for 3.33 and 7 minutes, respectively. The surfaces were then regenerated with a 45-second injection of 3 M guanidine hydrochloride. Measurements were performed twice per sample, and the individual curves were used to produce the mean affinity constant by global fitting to a 1 :1 binding model using the BiaEvaluation software.

RESULTS

2.1 Expression and purification of HEV gt3 P domain fused to fluorescent proteins

For expression of HEV gt3 P domain comprising amino acid 456-660 (UniProtKB accession number: C4B4T9; SEQ ID NO: 17) fused to fluorescent proteins, the constructs carried either mNeonGreen or mRuby at the N-terminus for the non-secreted (pGS38) and secreted form (pGS39), respectively (Figure 1 B). The secreted form was cloned into a pMT vector carrying a BiP signal sequence while the non-secreted form was inserted into the same vector without the signal peptide for expression in Drosophila S2 cells as described above. A stable S2 transfectant was established per construct and the proteins produced as described above.

The retention volume of both proteins was approximately 190 ml on a HiLoad 26/600 superdex 200 pg (Cytiva), but the profile of the secreted P domain showed an additional peak eluting slightly earlier. Since the P domains form dimers, the elution volume was expected, but the additional peak observed for the secreted P domain suggested a tendency to form higher oligomers. Both intracellular and secreted proteins showed > 90 % purity as judged from analysis by SDS-PAGE using a 12 % gel under reducing conditions followed by Coomassie staining. Interestingly, the apparent molecular weight of the secreted P domain was slightly higher than the intracellular protein, a difference that can be attributed to the attached glycan at position 562 in the secreted construct.

2.2 Analysis of B cell sequenced data To obtain antibodies targeting the HEV P domain, P domain-specific memory B cells from two HEV infected patients were sorted as described above. In total, 3824 heavy and light chain pairs were obtained from the sorted B-cells of the two patients (456 and 3359 from p60 and p61 , respectively). High clonal diversity and a similar pattern of germline gene activation were observed from both patients, with heavy-chain genes originating mainly from IGHV3 (40 genes), IGHV1 (20 genes), and IGHV4 (16 genes).

In total, 91 sequence pairs, 50 from donor p60 and 41 from donor p61 , were selected for expression in Drosophila S2 cells. The sequences represented 6 germline genes, and varied in heavy-chain complementarity determining region-3 (CDRH3) amino acid lengths (8-25 aa long), according to IMGT numbering, as well as somatic hypermutations (2-37 nucleotide substitutions per VH sequence).

2.3 Expression and purification of anti-HEV scFvs

Synthetic genes of the selected 91 -paired sequences, cloned into a pMT vector as singlechain fragments of the variable region (scFv) were purchased from Twist biosciences. The name was defined by scFv as a prefix, followed by the donor ID and the clone number. All scFvs carried a C-terminal EK site and a double strep-tag to aid purification. Furthermore, they were cloned downstream a BiP signal sequence to allow expression through the secretory pathway. A stable S2 cell line was generated per scFv as described above, and a similar protocol was followed for expression and purification of the scFvs. The majority of scFvs eluted as single monomeric peaks with a retention volume of approximately 16 ml on a superdex 200 increase 10/300 GL column. Some scFvs displayed a second peak eluting earlier, likely corresponding to the elution volume of a diabody, a noncovalent dimer of a scFv. Overall, samples were collected only from the monomeric peak. The yields from a 250 ml-S2 culture ranged between 0.04 and 7 mg/l for the worst- and best-producing scFvs, respectively.

2.4 Neutralizing activity of isolated anti-HEV mAbs against HEV-gt3

Briefly, in vitro assays using HEV gt3 Kemow-C1 p6 clone produced as previously described (Todt et al. 2020. Robust hepatitis E virus infection and transcriptional response in human hepatocytes PNAS 117 (3): 1731-1741) were performed. HepG2 cells were transfected with the viral genome by electroporation. Four days after transfection, virus was harvested. To generate viral stocks containing naked viral particles, transfected cells were lysed by three freeze-thaw cycles using liquid nitrogen. For virus stocks containing pseudo-enveloped particles, the transfected cells supernatants were used after filtration (0.45pm). Indicated virus stocks were incubated with different mAb concentrations for 1 hour at room temperature before infecting HepG2/C3a Hepatoma cell lines. Cells were then incubated for 24 hours before the addition of new media. After four days, cells were fixed with paraformaldehyde and treated with 0.2 % Triton X-100 solution for 5 minutes at room temperature for permeabilization. Subsequently, samples were washed and stained overnight with an anti-ORF2 rabbit polyclonal antibody. Infected cells were visualized following staining with Alexa Fluor 488-labeled goat anti-rabbit antibody, and focus forming units (FFU) counted using the Elispot CTL system (Immunospot). The half-maximal inhibitory concentration (IC50) was determined by a titration of mAbs (an example is shown in Figure 2 A), and dose-response curves were fitted using a non-linear regression model from GraphPad prism. The majority of mAbs showed little or no neutralization to the naked viral particles with IC50 > 5 pg/ml, while the IC50 values of the best neutralizers were < 0.01 pg/ml. Results for mAb p60.1 and p60.12 are shown in figures 2 A and B (IC50 values for p60.1 and p60.12 are 0.15 ng/ml and 0.89 ng/ml, respectively). Figures 2 C and D show neutralization results with pseudo-enveloped viral particles (IC50 values for p60.1 and p60.12 are 0.27 pg/ml and 0.12 pg/ml). Neutralization of naked viral particles of another genotype 3 viral strain (HEV83-2-27) at a concentration of 10pg/ml is shown in figure 2 E. mAb p60.1 showed neutralization of more than 80% whereas p60.12 neutralized more than 50% of the virus.

2.5 Characterizing anti-HEV mAbs

2.5.1 Expression and purification of anti-HEV scFvs as mAbs

To further characterize the isolated scFvs as well as to establish a final ranking, the variable region genes for 15 scFvs with IC50 < 0.5pg/ml were cloned into plasmids for expression of human lgG1 , and expressed in Expi293F cells. All purified antibodies eluted as homogenous single heterodimeric peaks (Figure 3 A) with > 95 % purity upon analysis on a Coomassie-stained reducing SDS-PAGE gel (Figure 3 B).

2.5.2 Expression of P domains from different HEV genotypes in Drosophila S2 cells

To facilitate the cross-binding analysis of a-HEV neutralizing antibodies to different HEV genotypes, genes encoding the P domains (amino acid residues 456-660) from the four human pathogenic genotypes and a rat-HEV isolate (Table 2) were cloned into a pMT vector for expression in Drosophila S2 cells. Unlike the fluorescently labeled P domains described in section 2.1 , the proteins carried both a TEV and EK protease cleavage sites in tandem, in addition to an N-terminal double strep-tag. For all genotypes, only the intracellular form of the P domains was used except in the case of genotype 3. Here, both the secreted and intracellular forms were cloned. Stably transfected S2 cells were generated, protein expression induced, and purified as described above. The retention volume for the dimers was approximately 200 ml, however, the rat intracellular (pGS135) and genotype 3 secreted (pGS100) P domains showed a similar tendency to form higher oligomers. Nevertheless, all proteins showed high purity upon analysis on a Coomassie- stained 15 % SDS-PAGE gel loaded under reducing conditions. The HEV gt3 secreted P domain (pGS100) migrated slightly slower than the intracellular domains. Fractions considered in this study were obtained from the dimeric peak.

2.5.3 Characterizing anti-HEV mAbs for cross genotype binding activity by ELISA and immunofluorescence microscopy

Human pathogenic HEV genotypes have a single serotype and the level of amino acid sequence variation ranges between 89 and 93 % across the four genotypes, suggesting high level of conservation. The amino acid conservation between the rat-HEV P domain and the human genotypes reaches approximately 31.5 %. The level of conservation suggests that it is possible to isolate cross-binding and potentially cross-neutralizing mAbs. Therefore, to explore the cross-genotype binding activity of the mAbs, the purified P domains from section 2.5.2 were used in an indirect ELISA assay as described above. All mAbs demonstrated moderate-to-strong binding to HEV gt3 P domain, while the majority of them also showed cross-binding activity to at least two genotypes (as shown in Figure 4 A for p60.1 and p60.12). Immunofluorescent staining of transfected hepatoma cells confirmed binding of p60.1 to two gt3 viral strains (Kernow C1 p6 and HEV83-2-27) as well as to the gt1 viral strain SAR55. p60.12 only showed binding to the Kernow C1 p6 viral strain in this assay (Figure 4 B).

2.5.4 Ability of anti-HEV mAbs to distinguish between corresponding secreted and intracellular P domains using ELISA

In a second step, the antibodies were tested for their ability to distinguish between the secreted and intracellular P domains. The goal was to identify antibodies that targeted only the non-secreted form, a component of the infectious particle, but not a secreted soluble pORF2 dimer that is present in the serum at high levels. Forthis purpose, an indirect ELISA was set-up using HEV gt3 secreted (pGS100) and non-secreted (pGS99) P domains as antigens. Altogether, four mAbs did not differentiate between the two forms, whereas the rest of the antibodies tested demonstrated ability to differentiate between the two forms, but differences in signal were more pronounced especially with mAbs p60.1 and p60.12 (Figure 5), which also show broad reactivity with at least two human infective genotypes, as shown in Figure 4 A.

2.5.5 mAb p60.1 and mAb p60.12 bind to antigens present in sera of patients chronically infected with HEV

A sandwich ELISA confirmed binding of both mAb p60.1 and p60.12 to antigens present in sera of three different patients, chronically infected with HEV (Figure 6). All positive samples were highly HEV-RNA positive (between 1x10 ⁵ and 9x10 ⁶ lU/ml) as determined by the central laboratory of Hannover Medical School. Presence of HEV antigens was confirmed by using the commercial WANTAI HEV Ag ELISA. As control, a negative serum as determined by HEV RNA PCR and WANTAI HEV Ag ELISA was used.

2.5.6 ScFv-p60.1 and scFv-p60.12 show no binding to secreted HEV gt3 P domain

(pGS100) in size exclusion chromatography

As a confirmation for the ELISA data, the ability of p60.1 and p60.12 to distinguish between secreted and non-secreted forms was analyzed by size exclusion chromatography (SEC). The two mAbs were considered for further analysis in SEC because of their IC50 0.01 g/ml. For SEC analysis, the antigen was incubated overnight at 16 °C with a 1 :3 molar ratio (pGS100: scFv) of the respective scFvs. The mixture was centrifuged at maximum speed in a tabletop centrifuge (Eppendorf) for 10 min at 4 °C to remove any precipitate. Cleared samples were then loaded on a SD200 increase 10/300 column using 20 mM HEPES pH 7.4 and 150 mM NaCI as running buffer at a flow rate of 0.5 ml/min. While a considerable part of the mixtures containing other scFvs eluted earlier from the column, suggesting complex formation of those scFvs with both secreted (pGS100) and non-secreted (pGS99) P domains, such a peak did not appear in SEC analysis of mixtures containing scFv-p60.1 and scFv-p60.12 together with the secreted form (pGS100), indicating that no complex formation was possible (Figure 7). Thus, the results confirmed that indeed p60.1 and p60.12 distinguish between the two antigenic forms.

2.5.7 Biophysical characterization of anti-HEV mAbs with differential binding potential

In order to further analyze the differences in signal strength observed in the ELISA studies, the antibodies were further characterized by surface plasmon resonance (SPR) using a Biacore 3000 (GE Healthcare). Experiments were performed at 25 °C using a Strep- TactinXT CM-5 chip prepared as described above. Briefly, the ligand (HEV gt3 P domains, pGS99 or pGS100) was injected over one flow cell followed by injecting different concentrations of the analytes (mAbs p60.1 , p60.12) over two flow cells. Measurements were fitted to a 1 :1 binding model using the BiaEvaluation software resulting in the sensorgrams of Figure 8 A and B. SPR data showed that the binding of the antibodies towards the intracellular P domain was in the picomolar range, with p60.1 showing the highest affinity binding (Table 5).

The association (Ka) and dissociation (Kd) rates for all antibodies using the non-secreted P domain (pGS99) were similar, but the absolute binding (as judged from the number of response units) obtained for p60.1 and p60.12 were lower than for the other antibodies. On the other hand, the kinetic parameters for p60.1 and p60.12 using the secreted P domain (pGS100) were not calculated because data was below the threshold recommended in the kit manual (I BA Lifesciences twin-strep-Tag capture kit). Overall, the data show that the antibodies of the invention isolated from HEV-infected patients interact with high affinity with the HEV P domains.

Table 5: Binding parameters of the antibodies of the invention

2.6 Crystallization and structure determination of HEV gt3 P domain (pGS99) in complex with anti-HEV scFvs

2.6.1 Purification of complexes between HEV gt3 P domain (pGS99) and scFvs for crystallization

Protein crystallization was carried out using pGS99 in complex with scFvs or Fabs derived from the mAbs of the invention in order to structurally characterize the antibody-antigen interaction. Prior to crystallization, strep-tags were proteolytically removed from pGS99 and the scFvs/Fabs using TEV protease and enterokinase, respectively. Thereafter, complex formation was performed by mixing pGS99 and the antibodies (scFv p60.1 and Fab p60.12) in 1 :1.2 molar ratio, and incubating at 16°C overnight. Purification of the complexes by gel filtration showed that the antibody fragments formed complexes with pGS99 (Figure 9, black arrows), with excess of the antibody fragments observed in both complexes (Figure 9).

2.6.2 Crystallization and structure determination of gt3 P domain (pGS99) in complex with antibody fragments

To facilitate structure determination, crystallization drops were set-up using the purified complexes described above. Crystals for complexes with scFv-p60.1 and Fab-p60.12 were obtained at 293K using sitting drop vapor diffusion method (Figure 10). The conditions that produced diffraction quality crystals per complex are presented in Table 6. The crystals of the complexes were obtained from initial screening conditions and required no further optimization. For data collection, all crystals were flush frozen in mother liquor containing 30 % ethylene glycol. The diffraction capacity of the crystals is listed in Table 6. Table 6: Crystallographic data collection and statistics

2.6.3 HEV gt3 P domain epitope and interacting residues of scFv-p60.1 and Fab-p60.12 - crystal structure of pGS99 in complex with scFv-p60.1 and Fab-p60.12

The crystal structures of the two complexes containing the HEV gt3 P domain and either the p60.1 antibody or the p60.12 antibody facilitated a precise view on the epitope and the interacting residues. These three-dimensional structures accurately describe the recognized epitopes on the P domain, as epitope mapping by structure determination is still considered the gold-standard for the identification of epitopes recognized by monoclonal antibodies.

Precise atomic bonds between p60.1 and the P domain were identified using the available crystal structure, the Protein Interactions Calculator (http://pic.mbu.iisc.ernet.in/) and the PISA server (Krissinel and Henrick 2007. Inference of macromolecular assemblies from crystalline state, J. Mol. Biol. 372: 774-797), and are listed below:

Hydrophobic interactions

480 TYR D 60 TYR G

559 TYR D 105 PRO G

561 TYR D 105 PRO G

594 TYR D 105 PRO G

Protein-Protein-Main chain - Side chain hydrogen bonds

489 D THR OG1 92 H PHE O

561 D TYR OH 103 G ARG O

585 D THR OG1 32 G ILE O

590 D ALA N 57 G SER OG

33 G ASN ND2 562 F ASN O 33 G ASN OD1 562 F ASN O

52 G ARG NH2 488 D SER O

52 G ARG NH2 488 D SER O

49 H TYR OH 562 D ASN O

Protein-Protein-Side chain - Side chain hydrogen bonds

482 D GLN NE2 54 G TYR OH

488 D SER OG 62 G GLU OE1

488 D SER OG 62 G GLU OE2

557 D TYR OH 33 G ASN OD1

562 D ASN ND2 106 G THR OG1

33 G ASN OD1 557 D TYR OH

54 G TYR OH 482 D GLN NE2

56 G ARG NH2 584 D TYR OH

103 G ARG NH1 561 D TYR OH

103 G ARG NH1 561 F TYR OH

106 G THR OG1 562 D ASN ND2

56 H SER OG 584 F TYR OH

Protein-Protein Aromatic-Aromatic interactions

480 TYR D 60 TYR G 6.82 98.52

Protein-Protein Cation-Pi Interactions

561 TYR F 103 ARG G 5.88 99.97

584 TYR D 56 ARG G 5.47 47.18

Precise atomic bonds between p60.12 and the P domain were identified using the available crystal structure, the Protein Interactions Calculator (http://pic.mbu.iisc.ernet.in/) and the PISA server (Krissi nel and Henrick 2007. Inference of macromolecular assemblies from crystalline state, J. Mol. Biol. 372: 774-797) and are listed below and are listed below:

Hydrophobic interactions

480 TYR D 52 ILE H

480 TYR D 55 ILE H

559 TYR D 34 PHE L

561 TYR D 108 TRP H

561 TYR D 34 PHE L

590 ALA D 54 ILE H

590 ALA D 55 ILE H Protein-Protein-Main chain - Main chain hydrogen bonds

105 H ARG N 562 C ASN O

Protein-Protein-Main chain - Side chain hydrogen bonds

557 D TYR OH 105 H ARG O

562 D ASN ND2 33 L ASN O

562 D ASN ND2 33 L ASN O

584 D TYR OH 106 H GLY O

31 H SER OG 586 D THR O

104 H ARG NH1 559 C TYR O

104 H ARG NH1 559 C TYR O

104 H ARG NH2 559 C TYR O

104 H ARG NH2 559 C TYR O

107 H ASN N 561 D TYR OH

33 L ASN ND2 562 D ASN O

33 L ASN ND2 562 D ASN O

55 L ASN N 584 C TYR OH

Protein-Protein-Side chain - Side chain hydrogen bonds

561 C TYR OH 52 L ASP OD2

561 C TYR OH 55 L ASN ND2

482 D GLN NE2 57 H THR OG1

482 D GLN NE2 57 H THR OG1

488 D SER OG 59 H ASN OD1

559 D TYR OH 107 H ASN ND2

561 D TYR OH 52 L ASP OD1

584 D TYR OH 99 H ASN ND2

586 D THR OG1 31 H SER OG

57 H THR OG1 482 D GLN NE2

59 H ASN OD1 488 D SER OG

59 H ASN OD1 488 D SER OG

99 H ASN ND2 584 D TYR OH

99 H ASN ND2 584 D TYR OH

104 H ARG NH1 562 C ASN OD1

104 H ARG NH1 562 C ASN OD1

107 H ASN ND2 559 D TYR OH

107 H ASN ND2 559 D TYR OH

54 L THR OG1 584 C TYR OH 55 L ASN ND2 561 C TYR OH

55 L ASN ND2 561 C TYR OH

Protein-Protein Aromatic-Aromatic interactions

559 TYR D 34 PHE L 5.07 112.20

561 TYR D 108 TRP H 7.00 110.92

561 TYR D 34 PHE L 5.53 112.24

Protein-Protein Cation-Pi Interactions

559 TYR C 104 ARG H 5.25 20.98

561 TYR C 104 ARG H 4.62 19.26

The complex pGS99_scFv-p60.1 crystallized as two P domain dimers each bound by the scFv at the dimer interface per asymmetric unit (Figure 11). Since the P domain is known to form dimers, the presence of these dimers in the structures of scFv-p60.1 was expected. The electron density map of the P domain was well defined and superposition of the complex to the E2s dimer (PDB 3rkc) yielded a root mean square deviation of 0.234 A for all Ca atoms of the E2s domain. This suggests that the conformation of the P domain remains unaltered upon binding of the scFv. In the structure, the linker region connecting the variable heavy and light chains of the scFv was not defined in the electron density map as expected, since this region is known to be intrinsically disordered.

Both p60.1 and p60.12 recognize a quaternary epitope that assembles at the dimerization interface at the tip of the P domain dimer (Figure 12). Both broad neutralizing antibodies bind predominantly to one protomer, but make contacts also with the other one, resulting in an asymmetric binding mode and indicating that a correctly folded pORF2 dimer is required for antigen-antibody interaction. Scrutiny of the interfaces reveals three segments distant in the amino acid sequence that constitute the two epitopes including 1) aa 480- 490, 2) aa 555-565 and 3) aa 580-590, confirming that both antibodies recognize conformation-sensitive epitopes. The interactions of the P domain with p60.1 and p60.12 bury a total of 789.7 A2 and 885.6 A2 of the surface area, respectively, as calculated by PISA, with approximately 70.7% and 58.6% (or 558 A2 and 519 A2, respectively) buried by the heavy chain. Both epitopes largely overlap with each other, sharing contact residues Q482, R488, Y557, Y561 , N562, Y584. Importantly, N562 of both protomers, which serve as attachment points for the N-linked glycans in the secreted pORF2 dimer, form hydrogen bonds with both antibodies, demonstrating that such an attached glycan would preclude binding of these bnAbs via steric clashes and thereby causing the observed glycan sensitivity. This explains why no binding was observed between scFv-p60.1 or scFv- p60.12 and pGS100 (secreted P domain) in the ELISA and SEC analyses.

2.6.6 Comparison of human anti-HEV scFv structures with mouse anti-HEV Fab structures

After identifying the epitopes of the isolated cross-binding human antibodies, their structures were compared with available crystal structures in complex with mouse antibodies. Of the 12 anti-HEV mouse antibodies available, only the structures of 8G12 and 8C11 (Gu et al., 2015. Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody. Cell Research 25 (5): 604-620; Tang et al., 2011. Structural basis for the neutralization and genotype specificity of hepatitis E virus. PNAS 108 (25): 10266-10271) have been determined in complex with the P domain (E2s). A comparison of the human anti-HEV structures to the 2 available mouse antibody structures revealed that 8G12 (PDB 4plj) binds at the dimer interface partially overlapping with scFv-p60.1 (Figure 12). However, although 8G12 partially overlaps with scFv-p60.1 , it makes no contact with the glycan at position N562.

In order to gain insight into the biological relevance of the interactions observed in the complexes of the present study, the crystal structure of scFv-p60.1 was superposed onto HEV VLP (PDB 2ztn) (Figure 13). The superpositions shows that scFv-p60.1 occupies the apex of the dimer. Thus, scFv-p60.1 may act by blocking attachment to the receptor, since its epitope is located in the region proposed to contain the receptor binding site (Mori and Matsuura, 2011. Structure of hepatitis E viral particle. Virus Research 161 (1): 59-64; Xu et al., 2016. Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein. Infection, Genetics and Evolution 37: 99-107).

2.6.4 Determinants for cross-binding activity of human anti-HEV antibodies

Using a cutoff criterion of an OD ₄ 5onm > 0.150 the ELISA data described above and illustrated in Figure 4 A suggested that p60.1 binds to 3 of the 4 human genotypes available, whereas p60.12 binds to 2 of the 4 human genotypes plus the rat-HEV genotype used in this study. The four human pathogenic genotypes represent a single serotype and therefore cross-binding activity would point to sequence conservation within the antibody epitopes across the genotypes. Amino acid sequence alignment of the P domains from the 5 genotypes in this study revealed low conservation with the rat-HEV, but high conservation for the human genotypes as reported previously. The contact residues for both antibodies are well conserved across the genotypes (Figure 14). Interestingly, these observations do not entirely agree with the ELISA data, suggesting that there may be other intrinsic determinants of cross binding. DISCUSSION

Using HEV gt3 P domains fused to fluorescent proteins for antigen-specific single B cell sorting and sequencing, sequences of roughly 3800 antibodies from two HEV convalescent patients were obtained. All the most efficient antibodies in the neutralization assay except one were obtained from patient p60 suggesting differences in response to infection. The variation between patients is not unusual as a number of factors such as age, sex, and comorbidities are known to influence response to infection and disease. The isolated antibodies were highly potent in the neutralization assays with HEV gt3, blocking viral infection at concentrations as low as < 0.01 pg/ml and therefore provide a potential option for treatment of HEV infection. They were mainly derived from IGHV1 and IGHV3 germline genes that are frequently used during viral infections and were also predominantly used by antibodies isolated from Hecolin vaccinated patients (Wen et al., 2020. Quantitative evaluation of protective antibody response induced by hepatitis E vaccine in humans. Nature Communications 11(1)). The presence of normal CDRH3 length and moderate somatic hypermutation (SHM) in these antibodies suggests that extensive antibody maturation is not required to achieve effective neutralization. In fact, this is mirrored by data from Wen et al. (2020), showing that antibodies from vaccinated donors with moderate SHM and CDRH3 lengths were more potent neutralizers than from donors with opposite antibody patterns (i.e., very high SHM and short CDRH3 loops or very low SHM and long CDHRH3 loops).

The majority of the antibodies showed cross-binding activity to all P domains, but with varying degrees of signal strength correlating with differences in binding affinities. The P domain of the human infective genotypes is highly conserved, which explains the antibody cross reactivity. Although the neutralization potential against other genotypes was not tested, it is hypothesized that these antibodies could confer cross-genotype protection and preliminary experiments support this notion. Such an expectation is supported by the direct correlation between binding in ELISA and neutralization observed for HEV gt3, which is in line with the findings of Wen et al. (2020), where all antibodies reactive to the vaccine antigen in the ELISA exhibited detectable HEV neutralization. It was also observed that a fraction of the antibodies in the present study was capable of distinguishing between the secreted and non-secreted forms of the P domain. The non-secreted form is the component of the infectious particle (Ankavay et al., 2019. New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle. Scientific Reports, 9(1)), whereas the secreted form is a soluble dimer that is present in the serum at high levels even after infection is cleared (Behrendt etal., 2016. Hepatitis e Virus (HEV) ORF2 Antigen Levels Differentiate between Acute and Chronic HEV Infection. Journal of Infectious Diseases 214 (3): 361-368). The secreted form is glycosylated at the conserved glycan position N562 (Xu et al., 2016. Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein. Infection, Genetics and Evolution 37: 99-107; Ankavay et al., 2019). This position is located at the apex of the P domain, which is the receptor-binding region and a binding site for some neutralizing antibodies (Guu et al., 2009. Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. PNAS 106 (31): 12992-12997; Mori and Matsuura, 2011. Structure of hepatitis E viral particle. Virus Research 161 (1): 59-64; Zhao et al., 2015. A comprehensive study of neutralizing antigenic sites on the Hepatitis E Virus (HEV) capsid by constructing, clustering, and characterizing a tool box. Journal of Biological Chemistry 290 (32): 19910-19922; Wen et al., 2020). Although the precise role of the secreted form remains to be determined, it is tempting to speculate that the soluble pORF2 dimer serves as decoy to reduce effective antibodies from circulation. In view of this hypothesis, it is likely that all antibodies that do not bind the secreted form either make direct contact with residue N562 or bind in close proximity to the glycosylation site, and their interaction is sterically hindered by the presence of a glycan molecule. For example, mAb 8G12 is a mouse derived cross-genotype antibody against HEV (Gu et al., 2015. Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody. Cell Research 25 (5): 604-620) that binds at the dimer interface. Residues T563 and T564 are among the contact residues of 8G12 and are located next to N562. The close proximity of the glycan therefore may sterically block the binding of 8G12. Indeed, structural studies of the best neutralizing antibodies of the present study revealed that p60.1 and p60.12 bind at the apex of the P domain, directly making contact with the two-asparagine sidechain that serve as attachment site for N-linked glycans at the dimer interface. Interestingly, p60.1 makes contact with residues T489, N560, Y561 , N562, T564, T585, and T586 which have been implicated in HEV receptor-interaction (Mori and Matsuura, 2011. Structure of hepatitis E viral particle. In Virus Research 161 (1):59-64. doi.org/10.1016/j.virusres.2011.03.015), and are highly conserved across genotypes. This suggests that p60.1 and p60.12 act by directly blocking receptor interaction of the HEV particle. Since the secreted form does not interact with broadly neutralizing antibodies like p60.1 or p60.12, it does not affect their potency in an infection model. Of the 19 residues involved in interaction between the P domain and the p60.12 antibody, 14 and 16 residues are conserved in the rat and human infective genotypes, respectively. On the other hand, of the 20 residues involved in p60.1 antibody interaction, 14 and 17 residues are conserved in the rat and human infective genotypes, respectively. The level of conservation suggests that these residues play an important role in HEV infection. However, as the receptor required for HEV entry is still unknown, the role of these residues remains elusive to date.

Six antigenic cluster sites (C1-C6) targeted by anti-HEV antibodies have been mapped on the P domain by alanine scanning and antibody competition assays (Zhao et al., 2015; Wen et al., 2020). A comparison of the epitopes targeted by p60.1 and p60.12 shows that both antibodies occupy antigenic site C3, which is located at the apex of the P domain and is the cell attachment region. In the studies of Zhao et al. (2015) and Wen et al. (2020), C2 and C6 are described as the target for the majority of the neutralizing mAbs, with the C6- directed mAbs being the most potent. C6 is associated with high neutralization potency and limited or no cross-genotype reactivity, whereas clusters C5 and C3 demonstrate weak or no neutralization potential. This is in contrast to the present invention in which it is shown that surprisingly antibodies targeting antigenic cluster 03, p60.1 and p60.12, are the most potent neutralizers. p60.1 and p60.12 are highly potent neutralizers, demonstrate cross-genotype binding potential and cross-neutralization and may act via blocking virusreceptor interactions and a direct binding competition with the receptor.

Overall, potent neutralizing anti-HEV antibodies from HEV convalescent patients have been isolated. These antibodies bind with picomolar affinities to HEV gt3 P domain, show potent neutralization activity in in vitro assays, and demonstrate broad reactivity across HEV genotypes. The most potent antibodies target only the non-secreted form of the P domain, which is the component of the infectious particle. So far, it is hypothesized that the antibodies of the present study likely act by blocking receptor interaction. Currently, the best antibodies are being tested in an HEV infection model to evaluate their potency in vivo.

Of note, HEV circulates as quasi-enveloped virions in the blood and such virions have been so far reported to be resistant to antibody neutralization (Yin et al., 2016. Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses. Journal of Virology 90 (8): 4232-4242). Most importantly, the antibodies isolated in the present invention efficiently neutralize also quasi-enveloped virions and therefore demonstrate their potential therapeutic benefits for chronical and immunocompromised HEV patients.

SEQUENCE LISTING

SEQ ID NO: 1 P60.1-HC

SEQ ID NO: 2 P60.1-HC CDR1

SEQ ID NO: 3 P60.1-HC CDR2

SEQ ID NO: 4 P60.1-HC CDR3

SEQ ID NO: 5 P60.1-LC (Kappa)

SEQ ID NO: 6 P60.1-LC CDR1

AAS P60.1-LC CDR2

SEQ ID NO: 8 P60.1-LC CDR3

SEQ ID NO: 9 P60.12-HC

SEQ ID NO: 10 P60.12-HC CDR1 SEQ ID NO: 11 P60.12-HC CDR2

SEQ ID NO: 12 P60.12-HC CDR3

SEQ ID NO: 13 P60.12-LC (Lambda)

SEQ ID NO: 14 P60.12-LC CDR1

DVT P60.12-LC CDR2

SEQ ID NO: 16 P60.12-LC CDR3

SEQ ID NO: 17 HEV gt3 pORF2 (Uniprot C4B4T9)

SEQ ID NO: 18 HEV pORF2 gt1_(lsolate/human/Pakistan/Sar-55)_Taxon ID: 33774

(UniProtKB: P33426)

SEQ ID NO: 19 HEV pORF2 gt2_(lsolate/human/Mexico)_Taxon ID: 31768

(UniProtKB: Q03500)

SEQ ID NO: 20 HEV pORF2 gt3_(lsolate/human/Japan)_Taxon ID: 12461

(BAH59609.1):(UniProtKB: C4B4T9)

SEQ ID NO: 21 HEV pORF2 gt4_(lsolate/human/China/T1)_Taxon ID: 509627

(UniprotKB: Q9IVZ8)

SEQ ID NO: 22 rat HEV pORF2 (R68/DEU/2009)_Taxon ID: 879096

(UniprotKB: E0XL23)