All experiments were performed under Biosafety level 2 (BSL2) conditions.

Animals

Experiments with animals were performed in accordance with national and European regulations. Authors complied with the ARRIVE guidelines 2.0 for reporting of conditions and findings of animal-connected experiments.

Two BALB/c mice originated from the Friedrich-Loeffler-Institute’s internal mouse breeding program and were housed in local facilities. The experiment was conducted in line with the national and European legislation (EURL 63/2010), with approval by the s Federal State of Mecklenburg-Western Pomerania, Germany (file number: 7221.3-2-042/17).

Additionally, 14 commercial hybrid pigs were procured from a local breeder (Landboden Glasin, Glasin, Germany) at the age of 10–14 weeks and a weight of 25–35 kg. The swine experiment and sampling protocol was approved by the competent authority of the Federal State of Mecklenburg-Vorpommern, Germany, prior to its implementation based on national and European legislation, EURL 63/2010 for the protection of laboratory animals (reference 7221.3-1-010/22). No additional preregistration was arranged. A statistical evaluation was carried out to determine the group size in accordance with national legislation section 31, Paragraph 1 of the Tierschutzversuchstierverordnung (Animal Welfare Ordinance). The treatment group size was determined using G*Power 3.1.9.4⁵⁸ to achieve a statistical power of 80% with a significance level of 5%.

Animals were acclimated for three days in the local husbandry of the Friedrich-Loeffler-Institut prior to infection. Throughout the experiment, all possible measures were implemented to enhance overall well-being of the pigs, to create an enriched environment for the pigs and to keep the number of animals in the experiment to the required minimum.

Proteins used in the study: synthesis and purification

To generate MAbs, a recombinant bacterial protein, derived from a plasmid encoding the partial HEV-3 capsid protein p239, was used as an immunogen. The plasmid, synthetized as part of an earlier study²¹, encodes a 239 amino acid (aa) segment of the HEV-3 capsid protein, corresponding to nucleotide positions 6300–7016 of strain KP294371. The protein was also used as an antigen for antibody detection and to determine binding specificity.

The recombinant capsid protein was expressed in E. coli BL21(DE3) (Thermo Fisher Scientific, Life Technologies GmbH, Darmstadt, Germany) and subsequently purified via Ni-NTA columns, following previously established procedures⁵⁹ (Qiagen manual, Qiagen, Hilden, Germany), under denaturation conditions, with an elution buffer pH adjusted to 5.9 and 4.5. Following purification, proteins underwent dialysis in a 0.05 M carbonate-bicarbonate buffer (pH 10.3).

Purity of the recombinant protein was determined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as previously described²¹ (Supplemental Fig. 1B).

Quantification of protein concentration was performed using Roti^®Quant universal kit following the manufacturer’s instructions (Carl Roth GmbH + Co. KG, Ebersberg, Germany). The resulting protein was stored at − 20 °C.

Mouse immunization and cell fusion

Fifty µg of bacterially expressed protein p239 was mixed with an equal volume of GERBU Adjuvant MM (v/v) (Biotechnik Gerbu, Heidelberg, Germany) and administered intraperitoneally into BALB/c mice at 4-week intervals. After four vaccinations, the mice were euthanized by inhalation of an isoflurane overdose followed by intracardial exsanguination. Spleen cells from the mice were fused with SP2/0 myeloma cells, and the resulting hybridoma cells were screened for monoclonal antibodies against HEV-3 capsid protein using an indirect enzyme linked immunosorbent assay (ELISA). Stabile hybridoma clones were established as described elsewhere⁶⁰.

Purification of antibodies

The antibodies were purified using HiTrap Protein G antibody purification columns (GE Healthcare Bio-Sciences, Uppsala, Sweden) according to the manufacturer’s instructions. The elution fractions were dialyzed against phosphate buffered saline (PBS) pH 7.4, and the protein concentration was determined colorimetrically using Roti^®Quant universal (Carl Roth GmbH + Co. KG, Ebersberg, Germany) as outlined in the user’s manual. Purity of the MAb before and after dialysis was determined by SDS-PAGE (Supplemental Fig. 1A). The MAb for the treatment experiment was classified according to the Mouse Monoclonal Antibody Isotyping Test Kit (Bio-Rad, Feldkirchen, Germany) following the manufacturer’s instructions. The MAbs were centrifugated and supernatant was aliquoted into 0.88 ml portions, with a concentration of 2.65 mg/ml, and stored at − 20 °C until use.

ELISA

An indirect antigen ELISA was performed according to standard protocol⁵⁹ using recombinant partial HEV capsid protein HEV p239³³. Nunc MaxiSorp 96-well plates (Invitrogen by Thermo Fisher Scientific, Life Technologies GmbH, Darmstadt, Germany) were coated with the corresponding antigen in a concentration of 1 µg/ml in 0.05 M carbonate-hydrogen carbonate buffer pH 10.3. Following a blocking step with 10% skimmed milk in PBS, cell culture supernatants, containing MAbs, were added 1:2 in 2% skimmed milk powder, or serum samples were added 1:25, and incubated for one hour at 37 °C.Two MAbs (7C3 and 6A2) against the HEV capsid protein were used as positive controls (Laboratory Eiden, Friedrich-Loeffler-Institute, Greifswald – Insel Riems, Germany, data unpublished). After washing with PBS/0.1% Tween, horseradish peroxidase (HRP) conjugated polyclonal Rabbit Anti-Mouse Immunoglobulins (1:2000, Dako, Hamburg, Germany) or Protein G, HRP conjugate (1:5000, EMD Millipore Corp., Darmstadt, Germany) was applied as secondary antibody. A solution of 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) was then added and the plates were incubated for 30 min at room temperature. The reaction was then stopped with 1% sodium dodecyl sulfate (SDS), and the optical density (OD) at 405 nm was measured in an ELISA-reader (Tecan, Männedorf, Switzerland). The MAb used in the treatment experiment was classified according to the Mouse Monoclonal Antibody Isotyping Test Kit (Bio-Rad, Feldkirchen, Germany) following the manufacturer’s instructions.

A commercial ELISA (ID Screen^® Hepatitis E Indirect Multi-species, ID.vet Innovative Diagnostics, Grabels, France) was also performed following the ID Screen manual.

Peptide ELISA

An ELISA assay using biotinylated oligopeptides, each 20 amino acids long with a 12-amino acid overlap (produced by JPT Peptide Technologies GmbH, Berlin, Germany), was used to identify linear epitopes of the newly generated antibodies. Corresponding sequences, covering 239 amino acids of the central ORF 2 capsid protein, are depicted in the supplementary material (Supplemental Table 1). The peptides were dissolved in 100 µl dimethylsulfoxide (DMSO) and stored at − 20 °C. The peptides were then diluted 1:250 in coating buffer (PBS-0.05% Tween, 40% DMSO) and 100 µl of the solution was added to 96-well streptavidin plates (Nunc Immobilizer Streptavidin, ThermoScientific, Life Technologies GmbH, Darmstadt, Germany) and incubated for one hour. After washing the plates four times with PBS-0.05% Tween, a two-step blocking procedure was performed: First, a 30-minute blocking with blocking buffer I (PBS, 20% sucrose, 0.4% biotin) at room temperature, followed by a one hour blocking with blocking buffer II (3% Bovine serum albumin (BSA) in Tris buffered saline (TBS) 0.1% Tween) at 30 °C. Subsequently, undiluted antibody-containing cell culture supernatants were incubated on the plate for one hour at 30 °C. After three washes with TBS-0.05% Tween, mouse anti-rabbit-HRP (diluted 1:2000 in blocking buffer II) was added and incubated for one hour at 30 °C. The plate was washed three times with TBS-0.05% Tween and ABTS was added. After 30 min the reaction was stopped with 50 µl 1% SDS and the OD was measured (as above). The two HEV capsid directed MAbs 6A2 and 7C3 (above) and serum from a rabbit vaccinated with a partial HEV capsid protein (p429, Supplemental Data 1) were used as positive control. The rabbit serum was diluted 1:25, and Protein G, functioning as a detection reagent, was used at a 1:5000 dilution.

SDS-PAGE and Western blot

SDS-PAGE were made using 13% or 16% bis/acrylamide gel as described earlier⁵⁹. Staining was conducted using ROTI^® Blue quick (Carl Roth GmbH + Co. KG, Ebersberg, Germany) according to manufacturer’s instructions. For Western blot analyses, protein samples were transferred from SDS-PAGE to Polyvinylidene fluoride (PVDF) membranes (Immobilon^®, Carl Roth GmbH + Co. KG, Ebersberg, Germany) by semi-dry electroblotting. After blocking with blocking buffer (PBS, 0.1% Teen, 5% sim milk powder) at room temperature for 30 min, membranes were incubated for one hour in the corresponding primary antibody, diluted 1:20 in blocking buffer. As negative controls, three monoclonal antibodies, directed against HEV-ORF 3 (Laboratory Eiden, Friedrich-Loeffler-Institute, Greifswald – Insel Riems, Germany, data unpublished), or a Rift Valley fever – virus (RVFV) directed MAb⁶¹ were used. 6A2 and 7C3, two in-house HEV capsid protein directed antibodies (data unpublished) were utilized as positive controls (Supplemental Fig. 2). After washing with PBS-0.1% Tween, the membrane was incubated in HRP-conjugated polyclonal Rabbit Anti-Mouse Immunoglobulins (1:2000), and visualized using chemiluminescent substrate SuperSignal™ West Pico Plus (ThermoScientific, Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer’s instructions on a Versa.Doc Imaging System (Bio-Rad Laboratories, Hercules, California, USA).

Indirect Immunofluorescence assay (IFA)

Sterile 96-well tissue culture plates (Falcon, Fisher Scientific, Life Technologies GmbH, Darmstadt, Germany) were seeded with 3 × 10⁵ PLC/PRF/5 cells in Eagle’s minimum essential medium (EMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM l-glutamine, 1% non-essential amino acids (NEAA), 100 U/mL penicillin G, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B (Sigma laboratories, Mumbai, India) and 30 mM MgCl₂ (MEMM) at 37 °C and 5% CO₂⁶². After 24 h incubation the confluency reached about 80–90%. Medium was replaced by Opti-MEM (Zellbank, Friedrich-Loeffler-Institut, Greifswald – Insel Riems, Germany) medium containing 100 U/mL penicillin G, 100 µg/mL streptomycin and 2.5 µg/ml amphotericin B and cells were transfected with the pVax1-ub HEV-SMP plasmid, containing the HEV-3 ORF 2 S, M and P domains (GenBank accession number KP294371, Supplemental Data 1). Transfection was performed using Lipofectamine^®2000 (Invitrogen by Thermo Fisher Scientific, Life Technologies GmbH, Darmstadt, Germany) according to manufacturer’s instructions. After a growth period of 3 days at 37 °C and 5% CO₂, cells were fixed with 100 µl 4% paraformaldehyde (PFA, Carl Roth, Ebersberg, Germany) for 30 min at room temperature and permeabilized with 50 µl Triton X 0.1% for ten minutes. Blocking was performed in two consecutive steps: Incubation with 0.1 M 50 µL Glycin solution 0.1 M (Carl Roth GmbH + Co. KG, Ebersberg, Germany) for ten minutes, and with 50 µl blocking reagent (2% BSA, 0.2% Tween, 3% glycerin and 0.5% sodium acid) for 30 min.

For staining, wells were incubated with cell culture supernatants containing monoclonal antibodies (0.5 mg/ml, diluted 1:10 in blocking reagent) for one hour, washed and finally incubated with Cy3™-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, Pensilvania, USA) and DAPI (1:20000, Invitrogen by Thermo Fisher Scientific, Life Technologies GmbH, Darmstadt, Germany) for one hour. The two MAbs, directed against the HEV capsid protein (7C3 and 6A2), were used as positive controls, as described before. Wells were washed three times with 50 µl PBS and staining was evaluated by fluorescence microscopy (Nikon, Tokyo, Japan) using the 40 × 0.6 objective and exposure times of 40 ms (DAPI) or 140 ms (Cy3).

To evaluate the binding of MAbs to different HEV-3 strains, human hepatoma HepG2/C3A cells were transfected with HEV constructs according to the protocol described before⁶³. Two different plasmids were employed for the transfection, one encoding the full-length HEV-3 “Kernow-C1 clone p6” (GenBank accession Nr.: JQ679013), and the other encoding the “HEV83-2-27 clone” (GenBank accession Nr.: AB740232). Additionally, a plasmid containing the subgenomic sequence of Kernow-C1 p6 HEV linked to a Gaussia luciferase reporter gene, and the HEV-1 strain (“Sar55”, GenBank accession Nr.: AF444002.1) were used as controls. The transfection was carried out via electroporation. Five days after transfection, the cells were fixed with 3% PFA in PBS and permeabilized with 0.2% Triton. Cells were then incubated overnight at room temperature with MAb. In this assay, antibodies were used from hybridoma culture supernatant. A monoclonal anti-hepatitis C virus (HCV) antibody served as negative control. The following day the cells were stained using Alexa Fluor 488-conjugated anti-mouse IgG. Images were captured at 10x magnification using an Olympus IX2 inverted microscope (Olympus, Tokyo, Japan), and analyzed using ImageJ software⁶⁴ to calculate the MFI.

Neutralisation assay

MAbs were titrated in duplicate in ten-fold serial dilutions, starting from a concentration of 5 µg/ml, in EMEM low IgG FCS medium. Naked HEV genotype 3 viral strain Kernow C1p6 G1634R was prepared according to the previously established protocol⁶³. Each MAb dilution (40 µl) was combined with 40 µl of medium containing the naked HEV-3 and incubated at 37 °C for one hour. Parallel assays were performed with PBS and an anti-HCV antibody as negative controls. The MAb-virus mixtures were then applied to human hepatoma cells (HepG2/C3A) that had been seeded in 96-well plates, and incubation was continued at 37 °C with 5% CO₂. After 24 h, 100 µl of fresh medium was added. Four days post-infection, the cells were fixed with 3% PFA in PBS and permeabilized with 0.2% Triton. Cells were subsequently stained for the ORF2-encoded capsid protein. Focus-forming units (FFUs) were counted using an ELISpot reader (Immunospot, Shaker Hights, Cleveland, UA). The endpoint FFU in the presence of MAb was calculated as: 100 × [average FFU in the presence of MAb / average FFU in the presence of PBS].

Inoculum and infection procedure

A 25% (w/v) liver inoculum was prepared from a liver sample sourced from a wild boar experimentally infected with HEV-3³⁷ (GeneBank Accession Nr. KP294371.1). The inoculum contained not only naked but mostly enveloped virus particles, which was experimentally confirmed by sucrose density gradient fractionation⁶⁵. Liver tissue was homogenized in PBS using a TissueLyser II (Qiagen, Hilden, Germany), followed by centrifugation at 7,459xg for five minutes. The supernatant was pooled and filtered twice through syringe filters (0.22 µl Millex^®-GP 33 mm filter unit, Carrigtwohill, Ireland). The infectious homogenates were aliquoted into 2 ml portions and stored at − 80 °C until use. The corresponding inocula were thawed over night at 4 °C and acclimated to room temperature shortly before infection. In pigs, the infection was administered intravenously into the cranial vena cava. Intravenous inoculation provides a standard infection model, that is used also in other pig infection studies with similar infection courses and patterns^37,66,67.

Molecular analysis

From the fecal samples, a 25% (w/v) suspension was prepared using a 0.89% NaCl solution. The suspension was thoroughly mixed then centrifuged at 7,500 x g at 4 °C for ten minutes. 100 µg of tissue samples were homogenized in 500 µl of ZB5 cell culture medium, containing MEM and NEAA (Zellbank, Friedrich-Loeffler-Institut, Greifswald – Insel Riems, Germany) using a TissueLyser II (Qiagen, Hilden, Germany), followed by centrifugation. Serum samples were obtained by centrifugation of blood samples at 833 x g for 12 min. The pellets were discarded.

Genomic material was extracted from 100 µl of serum, inoculum, or supernatant from homogenized tissue or feces, using the NucleoMag VET kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions, in combination with the KingFisher Flex extraction robot (ThermoScientific, Life Technologies GmbH, Darmstadt, Germany).

Viral RNA quantification was performed using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) for the ORF 2/3 overlapping region of HEV-3 following an established protocol⁶⁸ on a BioRad CFX96™ Real-Time System. β-actin and MS2⁶⁹ were used as RNA extraction controls. For quantification, standards were generated through digital droplet PCR (ddPCR) using the One-Step RT-ddPCR Advanced Kit for Probes 200rxns (Bio-Rad Laboratories, Hercules, California, USA) and the Bio-Rad QX-200^® Droplet Reader (Bio-Rad Laboratories, Hercules, California, USA). The HEV-3 RNA used for the standards was obtained from the liver tissue of an experimentally infected wild boar³⁷. The samples were classified as HEV-negative if the PCR results showed a copy number per microliter (copies/µl) below 1 compared to the standards.

Experimental design of the pig treatment study

Fourteen piglets (10–14 weeks, 25–30 kg) were randomly selected by animal keepers from a commercial fattening farm in Germany (Landboden Glasin, Glasin, Germany). Before infection, fecal samples and serum from all piglets were tested for previous exposure to the HEV-3 pathogen using RT-qPCR and ELISA. During the experiment, daily monitoring of clinical parameters, general condition and behavior was conducted, with a clinical score recorded. The evaluation form, the list of observed parameters, and the criteria for termination of the examination are listed in Supplemental Data 2.

The experiment design, including infection procedure and sampling, was based on previously established protocols^33,37. The animals were divided into four groups: uninfected control (n = 2), infected control without antibody treatment (n = 4), uninfected control with antibody control (n = 2), and an infected treatment group (n = 6) (Table 2). The animals were housed in separate stable units containing two to four pigs of the same group. Organic chemistry of blood samples, taken before infection and 28 days post-infection, was conducted using the Vetscan V2 device and the comprehensive diagnostic profile (Abraxis, Zoetis, Parsippany-Troy Hills Township, New Jersey, USA, Supplemental Table 2).

The pigs in the treatment group and the infection control group were intravenously infected with 2 ml inoculum (9.6 × 10³ copies/µl) on day zero. Subsequently, piglets in the treatment group, as well as the animals in the uninfected antibody control group, received doses of purified monoclonal antibodies (2.46 g/dose) on day one and day seven.

Blood and fecal samples were collected before infection and every 2–3 days post-infection. From day ten to day 20 post-infection, fecal samples were obtained on a daily basis.

Four-weeks post-infection, the animals were euthanized by electrical stunning followed by exsanguination. Necropsies were conducted and samples from blood, feces, bile and tissues were aliquoted for RNA extraction and stored at − 80 °C. Infection, treatment and sample analyses were performed by the same examiners. Therefore, no additional blinding measures could be taken.

Statistical analysis

The statistical analysis of the animal test data was performed using the R statistical software (R Core Team [2023]. _R: A Language and Environment for Statistical Computing_. R Foundation for Statistical Computing, Vienna, Austria). Data were evaluated for normality using Shapiro-Wilk test, qq-plot and histogram. Differences between treatment groups were examined using a Welch two-sample t-test and the Wilcoxon Rank Sum Test. In the context of the t-test, the null hypothesis (H0) proposed that the mean of the antibody treatment group exceeded that of the infected control group, while the alternative hypothesis (H1) suggested the contrary: H0 = µ_treatment ≥ µ_{infected control}; H1 = µ_treatment < µ_{infected control}. The code and data used for statistical analysis are available in the Supplemental Data 3 and the Supplemental Table 5.