Fertile hydatids were collected from the livers and lungs of naturally infected cows during the routine work of local abattoirs in Montevideo, Uruguay. The hydatid content was aspirated under aseptic conditions to separate the fluid and protoscoleces. HF was preserved by the addition of 5 mM EDTA and 20 µM 3,5-di-tert-butyl-4-hydroxytoluene (BHT) at -20°C until use. Protoscoleces were used for hydatid genotyping, performed by amplification and sequencing of a fragment of the mitochondrial cytochrome c oxidase subunit 1 (COX1) (Cucher et al., 2011). The sequencing reactions were carried out at Macrogen (Korea), finding that parasite material belonged to E. granulosus s.s. (G1 genotype) and E. ortleppi (G5 genotype). Because of limitations in the availability of fertile hydatids and the minimal HF volume needed for obtaining enough amounts for studies, HF samples were pooled, obtaining pools with a G1/G5 ratio between 100/0 and 78/22 (expressed in volume).

An adult (4 years-old) female llama (Lama glama) from the Montevideo municipal Zoo (Parque Lecocq, Montevideo, Uruguay) was immunised by subcutaneous administration (priming and booster) of 400 µg (protein) of EgAgB emulsified in incomplete Freund´s adjuvant (Sigma-Aldrich, USA). For this purpose, the EgAgB-enriched fraction called Ld_f, with a purity around 98%, was prepared from fertile HF using the methodology based on Q-Sepharose (GE Healthcare, Life Sciences, Sweden) ion exchange chromatography followed by ultracentrifugation on a KBr gradient (Folle et al., 2017). Twenty-one days after the last booster 150 mL of blood was collected in double blood collection bags containing sodium citrate as anticoagulant. All animal manipulation was performed by veterinarians of the Zoo under strict animal welfare guidelines of the Ethical Committee of the zoo.

A phage display library of VHHs/VHs (the heavy chain variable domain of conventional antibodies) was constructed as previously described (Rossotti et al., 2015). Briefly, peripheral blood mononuclear cells were obtained from collected blood (150 mL) by centrifugation on Histopaque-1077 (Sigma-Aldrich) gradients according to the manufacturer’s recommendations. Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) from about 10⁷ cells, and quantified by measuring the absorbance at 260 nm in a Nano Drop (Thermo Fisher Scientific, Fremont, CA) and reverse transcribed using superscript III first-strand synthesis system for RT-PCR (Invitrogen) with random hexamers. The genes encoding both VHH and VH were then PCR-amplified using the obtained cDNA as template and the previously described set of forward and reverse primers (Rossotti et al., 2015). The amplified DNA was digested taking advantage of the SfiI sites introduced with the primers and cloned in the phagemid pComb3X vector (generously donated by Professor Barbas, The Scripps Research Institute, La Jolla, USA). This vector encodes an in-frame 6xHis tag downstream of the cloning site, followed by the encoding sequence of a hemagglutinin epitope (HA), a stop amber codon, and a truncated version of the pIII coat protein of M13. The expression of the inserted gene is under the control of the lacZ promoter, and after induction with 1mM isopropyl-β-d-thiogalactopyranoside (IPTG, Sigma-Aldrich) an important proportion of the recombinant protein is secreted to the media as VHH-6xHis-HA recombinant protein. The ligated vector was electroporated into highly competent ER2738 Escherichia coli cells (Lucigen Corporation, WI). The cells were cultured and the phagemid-borne phage library displaying the VHH/VH repertoire was rescued by superinfection with the M13KO7 helper phage (Pharmacia Biotech, Uppsala, Sweden) and cultured overnight (ON) in the presence of 40 μg/mL of kanamycin. The phages were precipitated with 0.2 volumes of 20% polyethylene glycol 8000, 2.5 M NaCl, on ice during 1 hour, centrifuged and afterwards the collected phages were resuspended in phosphate-buffered saline (PBS) to a final titer of 10¹² cfu/mL.

Microtiter high binding ELISA plates (Maxisorp, Nunc, USA) were coated ON at 4°C with 100 μL/well of 10 μg/mL EgAgB (Ld_f) in PBS. After coating, wells were blocked with 300 μL/well of 3% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. The antibody library (10¹² transducing units) was then mixed with PBS containing 0.05% Tween 20 and 3% BSA, and dispensed into the plates. After incubating for 1 hour at 4°C, plates were washed extensively (8 times) with PBS containing 0.05% Tween 20 (PBS-T), after which the bound phages were eluted by incubation at 37°C with 100 μL/well of 10 mg/ml trypsin (Sigma-Aldrich). The eluted phages (output) were immediately neutralised by adding 100 μL/well of 100 mg/ml trypsin inhibitor (Sigma-Aldrich). The obtained phages were amplified and used for one additional round of panning, but employing ELISA plates coated with 100 μL/well of 0.2 μg/mL EgAgB. After the second round, 10-fold serial dilutions of the phage output (100 μL) were used to infect a fixed amount (100 μL) of ER2738 E. coli cells (0.5 x 10⁸ cells/mL). After infection, cells were plated on ampicillin plates and colony counting allowed assessing the number of phage particles obtained.

For VHHs/VHs expression, the coding sequences from the second output present in pCOM3X vector were cloned en masse into the pINQ-H6HA vector, a modified version of pET28a+ (Novagen) which includes an N-terminal OmpA leader peptide sequence for directing the VHHs/VHs to the periplasmic space and C-terminal HA epitope coding sequence. This vector allows the highly efficient expression of genes under a T7 RNA polymerase promoter when incorporated into inducible T7 RNA polymerase expressing cells, such as E. coli BL21 strain. To that end, plasmid DNA from cells infected with phages from the second output was prepared by performing a miniprep (QIAGEN). The plasmids containing the pool of VHHs/VHs genes were PCR amplified with primers Fw: 5´-GTTACTCGCGGCCCAGGCGGCCATG-3´and Rv: 5´-CCACGATTCTGGCCGGCC TGGCCTGAG-3´, annealing outside SfiI restriction sites. The PCR product was electrophoresed in an agarose gel, gel extracted (QIAGEN), and digested with SfiI restriction enzyme. The digested and purified PCR product was ligated into pINQ-H6HA vector (previously digested with SfiI and purified) with T4 DNA Ligase (Thermo Scientific), and the ligation mix was electroporated into BL21 E. coli electrocompetent cells. The transformants were plated on LB agar plates containing 40 µg/mL kanamycin and 4% glucose and incubated ON at 37°C. The next day, 10 individual clones were tested to check that anti-EgAgB VHHs/VHs were successfully expressed from the pINQ-H6HA vector. For this, the same methodology described above for the screening of positive clones was used except that induction of VHHs/VHs expression was performed at an OD₆₀₀ equals to 0.5, with 3 μM IPTG (final concentration). After induction, cultures were incubated for 4 hours at 37°C, with shaking (250 rpm) to allow VHHs expression. One mL of culture was centrifuged, the supernatant was discarded, and the cells in the pellet were resuspended in PBS (500 μL), followed by lysis by three cycles of freeze/thaw with sonication. After centrifuging at 17,000 x g, 100 µL of the supernatant were transferred to wells of an ELISA plate coated with the EgAgB fraction, Ld_f, (0.2 μg/well) and blocked with BSA (1%). As a control, samples were also transferred to BSA-coated wells. VHH binding was detected by incubation for 1 hour at room temperature (RT) with anti-HA-peroxidase (Roche, 1/3,000 dilution) and the peroxidase activity developed using TMB/H₂O₂ substrate and analysed spectrophotometrically at 450 nm.

Plasmids from EgAgB positive clones expressed from pINQ-HAH6 were isolated by performing minipreps (QIAGEN). Their sequences were obtained by Sanger sequencing by Macrogen (Korea) and manually aligned.

EgAgB positive VHHs/VHs clones (n=9) were evaluated by ELISA for their binding to purified delipidated recombinant EgAgB subunits, named drEgAgB8/1, drEgAgB8/2 and drEgAgB8/3 (E. coli-expressed subunits (Silva-Álvarez et al., 2015b), generously donated by Dr Valeria Silva-Álvarez, Área Inmunología, Facultad de Química, UdelaR). For this purpose, ELISA plates were coated with each of the purified drEgAgB8 subunits at 0.5 μg/well and blocked as mentioned above. Then, the supernatant of cultures expressing anti-EgAgB VHHs/VHs was incubated at 1:5 dilution (1 hour, RT with agitation) and VHH binding was developed as previously described.

The expression of anti-EgAgB VHHs/VHs clones showing the highest binding ability to rEgAgB8/1 (named clones 1, 3 and 7) was analysed in 10 mL LB cultures as described above. After allowing expression, 1 mL of the respective cultures was centrifuged at 5,000 x g for 10 min. The supernatant was discarded and the pellet was resuspended in 100 μL of B-PER reagent (Thermo Fisher Scientific), vortexed, and incubated at RT for 15 min to allow E. coli membrane disruption. After adding PBS (200 μL), the soluble protein fraction was separated by centrifugation for 10 min at 5,000 x g. The remaining pellet was extracted with 8 M urea (30 μL) during 15 min at RT. After PBS addition (270 μL), the supernatant corresponding to the insoluble fraction was separated by centrifugation for 5 min at 17,000 x g. Twenty μL of both soluble and insoluble fractions were loaded on 15% SDS-PAGE gels, run, and stained with Coomassie Blue as detailed below.

The anti-EgAgB VHH clone 1 (for simplicity anti-EgAgB clone 1) was expressed in 500 mL of liquid LB medium by IPTG induction as described above. After 4 hours induction, the culture was centrifuged at 6,000 x g at 4°C for 15 min. The supernatant was discarded and the cellular pellet was washed using LB medium (25 mL), centrifuged as mentioned above and suspended in PBS (10 mL). The cells were then lysed with 0.2 M Tris-HCl pH 8.0 containing 0.5 mM EDTA and 0.5 M sucrose. The lysed cells were then centrifuged at 17,000 x g at 4°C for 20 min and the supernatant was filtered through 0.45 μM pore size filters. VHH purification was performed on Ni-NTA columns in the ÄKTA purification system (General Electric Healthcare, Uppsala, Sweden) according to the manufacturer’s instructions. The purified anti-EgAgB clone 1 was covalently bound to CNBr-Sepharose (GE Healthcare) using a ratio of 10 mg protein/mL matrix and following the protocol recommended by the manufacturer. The efficiency of coupling was around 99% and was estimated by measuring the absorbance at 280 nm of the liquid phase after conjugation. To avoid any possible contamination between native and recombinant EgAgB samples, the obtained anti-EgAgB clone 1-Sepharose matrix was divided into two immunoaffinity columns (1.5 mL each).

EgAgB was prepared from pooled HF taking advantage of the previously described methodology based on anion exchange chromatography followed by ultracentrifugation in a KBr density gradient (Folle et al., 2017), and adding a final step of affinity chromatography on anti-EgAgB clone 1-Sepharose. All buffers were prepared using pyrogen-free water (ICU-VITA, Uruguay). Briefly, HF from hydatids was firstly clarified by centrifugation (10,000 x g for 20 min, 4°C) and filtration through 0.45 µm filter membranes (Millipore). For the whole work, we prepared EgAgB batches (biological replicates, n=5) using between 7 and 20 hydatids each. The clarified HF was then fractionated by anion exchange chromatography on Q-Sepharose equilibrated in 20 mM phosphate buffer, pH 7.4 containing 200 mM NaCl, 5 mM EDTA and 20 µM BHT. The retained fraction containing EgAgB (QS_f), was eluted by changing ionic strength to 500 mM NaCl in a single step. After concentration (~10-fold) and equilibration in PBS containing 5 mM EDTA and 20 µM BHT (PBS_EB) for preventing lipid oxidation, QS_f was ultracentrifuged in a KBr gradient (4 hours at 332,000 x g), obtaining Ld_f, a yellowish-brown band of lower density enriched in EgAgB. Ld_f was equilibrated in PBS_EB and applied on the anti-EgAgB clone 1-Sepharose, previously equilibrated in PBS_EB. After washing with PBS_EB and 0,1M glycine buffers at pH 5 and 4 (2 column volumes each), EgAgB was eluted using 0.1 M glycine pH 2.5. Using a PD-10 desalting column (GE Healthcare, USA) the eluted fraction was equilibrated in PBS_EB plus antibiotic/antimycotic (100 U/mL penicillin, 0.1 mg/m streptomycin and 250 ng/mL amphotericin B, PBS_EBAb) to prevent further contamination. Immunopurified nEgAgB was maintained (for a maximum of 2 months) at 4°C under a N₂ atmosphere. Analysis of the endotoxin level was carried out using a chromogenic Limulus amebocyte lysate assay (LAL assay with a cut-off of 0.03 UE/mL, Beltran Zunino, Montevideo).

The EGR_06805 sequence corresponding to EgAgB8/1 subunit (https://parasite.wormbase.org) was optimised for expression in D. melanogaster using GenScript software. Afterwards, we designed and acquired from GenScript a construct (EgAgB8/1 construct, Supplementary Figure S2 ), containing the optimised EGR_06805 sequence flanked by the sequences to be used as forward and reverse primers for hybridization with the expression vector. Then, we cloned the optimised EGR_06805 sequence, by restriction free (RF)-cloning, in a modified version of the pMT/BiP/V5-His vector, called pDroEx (Ortega et al., 2018). This vector included two tandem sequences for Streptavidin binding (Strep-Tag) and two DNA fragments, flanking the insert gene, to be used for hybridization with generic forward and reverse primers during RF-cloning (Van Den Ent and Löwe, 2006). For Rf-cloning, we used DNA polymerase Phusion, 0.5 µM of generic primers (first PCR) or 100 ng of the EgAgB8/1 construction (second PCR), 20 ng vector and the following conditions: a denaturing step at 98°C for 30 s, 30 amplification cycles (98°C for 10 s, 65°C for 30 s and 72°C for 1 min), and a final extension step at 72°C for 5 min. PCR products were then treated with 20 U of DpnI (Thermo) for 90 min at 37°C followed by 20 min at 80°C to selectively degrade the methylated parental vector. The digestion products were then transformed in competent E. coli XL1 and EgAgB8/1 presence was confirmed by colony-PCR. Positive clones were expanded in LB cultures supplemented with ampicillin (100 µg/ml) and incubated ON at 37°C. Plasmids (pDroEx-EgAgB8/1) were purified using the QIAfilter Plasmid Midi Kit (Qiagen, Germany) and sequenced (Macrogen, Korea) for verification ( Supplementary Figure S2 ).

A stably transformed D. melanogaster S2 cell line secreting EgAgB8/1 was generated. To that end, stable S2 cells lines were cultured in Schneider´s Drosophila media (Gibco, Thermo Fisher Scientific) containing 10% Foetal Bovine Serum (FBS, Gibco) at 28°C. Cultured S2 cells (1.0 - 2.5 x 10⁶ cells/ml) were transfected with 2 µg of pDroEx-EgAgB8/1 and 0.1 µg of pCoPURO (a puromycin resistant plasmid, Invitrogen) using Effectene Transfection Reagent (Qiagen) and following the manufacturer’s recommendations. The presence of pDroEx-EgAgB8/1 in puromycin-resistant cells was controlled by PCR using the generic primers. During subculture of transfected S2 cells, Schneider´s Drosophila medium was progressively substituted by Xpress medium (Lonza, Switzerland) without FBS. When transfected S2 cell culture reached 7 x 10⁶ cell/mL, EgAgB8/1 expression was induced with 5 µM CdCl₂, and the Xpress medium containing CdCl₂ was added to replenish nutrients at 3 and 5 days-post-induction (dpi). At the end point, cell suspension was centrifuged and the supernatant was adjusted at pH 8.0 and kept ON at 4°C. Afterwards, the supernatant was clarified by centrifugation (6,000 x g, 20 min) followed by filtration at 0.22 µm, and fractionated on a Strep-Tactin XT-agarose column (5 mL, IBA, Life Sciences) using 100 mM Tris pH 8.0, containing 150 mM NaCl, 5 mM EDTA, 20 μM BHT and antibiotic/antimycotic as equilibration buffer and adding 25 mM biotin for elution. The eluted fraction was then applied on the anti-EgAgB clone 1-Sepharose column, previously equilibrated in PBS_EBAb. After washing, rEgAgB8/1 was eluted using 0.1 M glycine pH 2.5, and afterwards equilibrated and maintained in PBS_EBAb as described for immunopurified EgAgB. Induction time for rEgAgB8/1 production was evaluated at 4, 7 and 12 dpi in terms of yield and particle size. Analysis of endotoxin level was carried out by LAL assay (Beltran Zunino, Montevideo).

For SDS-PAGE analysis, all samples (HF fractions, nEgAgB and rEgAgB8/1) were resolved on 15% polyacrylamide gels under reducing conditions (40 µM DTT) according to Laemmli (Laemmli, 1970). Gels were stained with Colloidal Coomassie G-250 (Sigma-Aldrich) or transferred onto PVDF membranes (Millipore-Merck, USA) using a Mini Protean Blot Module (Bio-Rad) following the manufacturer’s instructions. Blots were blocked 1 hour at RT in PBS 5% non-fat dry milk, and then incubated with mAb EB7 (diluted hybridoma supernatant) for 1 hour at RT. They were washed with PBS 0.05% Tween-20 and incubated for 1 hour at RT with anti-mouse IgG peroxidase conjugate (diluted 1:4,000; Thermo Scientific). After washing, blots were developed using ECL Supersignal West Pico chemiluminescent substrate (Thermo Scientific) in the G-Box F3 image capture equipment (Syngene).

Both nEgAgB and rEgAgB8/1 were analysed by native gradient gel acrylamide electrophoresis (GGE) using 4-20% gradient gels and the Mini-PROTEAN TGX (Biorad, Life Science, Mexico) and following the instructions of the fabricant. For size comparison the human plasma lipoproteins LDL (density range from 1,019 g/mL - 1,063 g/mL), HDL₂ (density range from 1,063 g/mL- 1,125 g/mL) and HDL₃ (density range from 1,125 g/mL- 1,210 g/mL), purified by sequential ultracentrifugation (Havel et al., 1955) were run in parallel. Gels were stained with Coomassie Brilliant Blue or Sudan Black for visualisation of protein and lipid components, respectively. The relative mobility of all samples was estimated using a ChemiDoc 2,000 densitometer and the software Quantity One (Bio-Rad Laboratories SA, Life Science Group).

Lipids were analysed by HPLC-MS using a ZICpHILIC column (150 x 4.6 mm, 5 µm particle size) supplied by Hichrom Ltd. (Reading, UK) with a mobile phase consisting of 20 mM ammonium carbonate in HPLC-grade water (solvent A) and acetonitrile (solvent B), at a flow rate of 0.3 mL/min. The elution gradient was an A:B ratio of 20:80 (v:v) at 0 min, 80:20 (v:v) at 30 min, 92:8 (v:v) at 30 min 92:8 (v:v) at 35 min, 20:80 (v:v) at 36 min, and 20:80 (v:v) at 45 min. An ACE C4 column (Hichrom Ltd) was used to estimate the unsaturated FAs. The mobile phase for the elution of the ACE C4 column consisted of 1 mM acetic acid in water (A) and 1 mM acetic acid in acetonitrile (B) at a flow rate of 0.4 mL/min. The elution gradient was 40% B (0 min), 100% B (30-36 min) and 40% B (37-41 min). The HPLC-MS system consisted of a Dionex 3,000 HPLC pump interfaced with Orbitrap Exactive mass spectrometer used in positive/negative switching mode. The instrument was calibrated according to the manufacturer’s instructions and operated at 50,000 resolution. The needle voltage was 4.5 kV in positive mode and 4 kV in negative ion mode, the heated capillary temperature was 320°C and the sheath and auxiliary gases 50 and 17 arbitrary units respectively. The data was acquired using Xcalibur 2.0 software (Thermo Fisher Scientific) and the data sets were extracted and aligned by using m/z Mine 2.14.2 and the data was searched against an in-house database of metabolites. All identified lipids were within 5 ppm of their exact masses. Further processing of the data was carried out by using Metaboanalyst 4.0. For a graphical representation of phospholipid and FA content, heatmaps were built using Graphpad Prism 9.4.1 software.

The hydrodynamic radius (R_H) and the polydispersity index (PDI) of EgAgB particles were measured by dynamic light scattering (DLS), using the Zetasizer NanoS (Malvern Panalytical, United Kingdom). Samples (70 µL, 1 mg/mL in PBS_EB) were placed in disposable cuvettes (UVette, Eppendorf) and pre-incubated at 25°C before measurement. Data from triplicate measurements were averaged and analysed with Zetasizer Software v7.13 (Malvern Panalytical) to obtain size distribution of the samples (weighted by volume as assuming sphericity and homogeneity of particles). Reported R_H values were calculated as the mean value for the main peak.

BALB/c mice (female, 8- to 12-week-old) were acquired from DILAVE (Dirección de Laboratorios Veterinarios, Ministerio de Ganadería, Agricultura y Pesca, Uruguay) or Institut Pasteur Montevideo (IP-Montevideo, Uruguay). Animal manipulation and husbandry were done in accordance with the ethical committee guidelines of the Honorary Commission of Animal Experimentation (CHEA) from UdelaR.

The human monocyte-like cell line THP-1 (American Type Culture Collection, USA) was maintained in RPMI medium (RPMI 1640 culture medium containing 10 mM HEPES, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/mL penicillin, 0.1 mg/m streptomycin and 250 ng/mL amphotericin B) plus 10% (v/v) FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% (v/v) CO₂ and sub-cultured every 3-4 days to maintain cell density between 0.2 and 1.0×10⁶ cells/mL Macrophage differentiation was induced by stimulation of THP-1 cells (0.2x10⁶/well in 96-well tissue culture plates) with PMA (50 ng/mL, Sigma-Aldrich) in RPMI supplemented with 5% FBS for 48 hours. Afterwards, cells were cultured in a medium containing 5% FBS for additional 24 hours.

Murine bone marrow-derived macrophages (BMDM) were prepared following a procedure approved by CHEA (protocol N° 538, Exp. No. 101900-000999-17). To that end, bone marrow precursors from Balb/C mice (IP-Montevideo) were differentiated in the presence of conditioned medium from the L929 cell line, containing M-CSF, for 7 days as described in (Mylonas et al., 2013). Cells were then plated (0.5x10⁶ cell/well in 48-well culture plates) using DMEM containing FBS 1% (v/v), and cultured ON at 37°C in a humidified atmosphere with 5% (v/v) CO₂.

THP-1 macrophages or BMDM were exposed to nEgAgB or rEgAgB8/1 (at 1 or 10 µg/mL in analytical triplicates) in the presence or absence of LPS (10 ng/mL, E. coli O127:B8, Sigma-Aldrich), using PBS_EBAb (vehicle) as control. Cell viability and responses (cytokines and nitrite in supernatants or expression of cell surface molecules) were assessed 24 hours post-stimulation using the methods described below.

Animal manipulation and procedures (protocol N° 542, Exp. N° 101900-000972-17) were approved by CHEA. Balb/C mice (DILAVE) were randomly distributed in four groups (n=5 each) and intraperitoneally (i.p.) injected as follows: Vehicle group: PBS_EBAb (EgAgB vehicle) followed by sterile saline solution (SS, as LPS vehicle); EgAgB groups: 50 μg of nEgAgB or rEgAgB8/1 in PBS_EBAb followed by SS; LPS group: PBS_EBAb followed by 15 μg of LPS in SS (E. coli O127:B8, Sigma-Aldrich); EgAgB+LPS groups: 50 μg of nEgAgB or rEgAgB8/1 in PBS_EBAb followed by 15 μg of LPS in SS. To avoid putative interactions before administration, EgAgB was injected 10 min before LPS. After 4 or 24 hours-post-injection (hpi), mice were euthanized and peritoneal cells were collected by lavage with RPMI containing 0.2% FBS and 2 mM EDTA and analysed by flow cytometry as described below. In addition, peritoneal content was retrieved by peritoneal lavage with 1 mL of 0.2% (v/v) FBS in RPMI and maintained at -80°C until cytokine analysis by ELISA.

Cell viability was assessed on the basis of the mitochondrial reductase activity, which convert the water-soluble yellow dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Bio Basic Inc., Canada) to the insoluble, chromogenic, formazan (Kumar et al., 2018). Briefly, macrophage cultures were cultured in the presence of MTT (200 μg/mL in PBS containing 0.1% glucose) for 3 hours at 37°C and 5% CO₂. After washing with PBS, DMSO was added and the produced formazan was determined by measuring the absorbance at 560 nm.

Cytokine responses. IL-1β, IL-6, IL-12p40, IL-10 and interferon-β (IFN-β) were measured in culture supernatants by capture enzyme-linked immunosorbent assay (ELISA) employing OptEIA kits (BD Biosciences), according to the manufacturer’s instructions.

Nitric oxide (NO·) response. Nitrite levels were determined by the colorimetric Griess assay as an indicator of NO· generation. Briefly, macrophage culture supernatants were transferred (50 μL/well) to 96-well flat-bottom plates, and then 50 μL of sulfanilamide (Sigma, 1% wt/vol in 2.5% H₃PO₄) and 50 μL of naphthylethylenediamine dihydrochloride (Sigma, 0.1% wt/vol in 2.5% H₃PO₄) were added to the supernatants. After 5 min incubation, the absorbance at 540 nm (A₅₄₀) was measured and converted to nitrite concentration based on a NaNO₂ standard curve.

Cellular characterization by flow cytometry. Cells (between 0.4-0.5x10⁶) were incubated at RT for 10 min with Live/dead-Aqua viability probe (1:500 (v:v) dilution, Thermo Fisher Scientific) and then for 20 min at RT with 15% normal rat serum in buffer PBS containing 0.1% (w/v) BSA, 2 mM EDTA, pH 7.1 (FACS). Then, cells were incubated on ice for 30 min with antibodies to surface molecules associated with cell activation CD86, CD40, and MHC-II. In the case of peritoneal cells, antibodies to the phenotypic markers F4/80, CD19, and Ly6C were also included ( Supplementary Table S1 ). Afterwards, cells were washed with FACS buffer. Data were acquired on Facs Canto II cytometer and analysed using the FlowJo package. The gating strategy is shown in Supplementary Figure 3 . Resident large peritoneal macrophages (LPM) were defined as CD19⁻F4/80⁺⁺Ly6C^- while small peritoneal macrophages (SPM) as CD19⁻F4/80^+/-SSC^lowLy6C^-MHC-II⁺⁺cells. Infiltrating monocytes were identified as CD19⁻F4/80^+/-SSC^lowLy6C⁺⁺MHC-II^-. When quantifying cell surface molecule expression, the fluorescence intensity (FI) corresponding to the FMO (fluorescence minus one) control was subtracted. In addition, for comparison between mice groups, FI values were normalised to the control (vehicle treated mice).

The number of independent experiments and internal repetitions used for statistical analysis and summarised in the graphs shown is given in each figure legend. For graphical presentation purposes, some data were normalised to the control group or to the LPS response (as indicated); however, statistical analyses were always carried out on the crude data. Significances are indicated by asterisks in figures and are explained in the figure legends. In the case of in vitro studies, multiple comparison was performed by two-way ANOVA with Tukey’s post hoc test, using Graphpad Prism 9.4.1. In the case of in vivo studies, data were analysed by a nonparametric method, since they did not reach normality and homogeneity of variances. Specifically, the Mack Skillings exact test for a two-way layout was applied (Hollander et al., 2015). When the MackSkillings test resulted in a statistic with a p-value of less than 0.05, the test was followed by the post hoc multiple-comparison test described by Conover (Conover, 1999) and the Benjamini and Hochberg correction (McDonald, 2009). Throughout the paper, the symbols *, **, and *** represent p-values of less than 0.05, 0.01, and 0.001, respectively.

A VHHs/VHs phage display library, cloned in the pCOMB3X phagemid vector, was prepared using RNA extracted from peripheral mononuclear cells of a llama immunised with EgAgB (Ld_f). After performing two rounds of panning, screening of EgAgB-specific clones was performed by ELISA using Ld_f for coating. All (10) individual clones expressing VHHs/VHs in the second output of the panning were positive for Ld_f-coated wells, and none of them showed reactivity against BSA-coated wells, indicating they were specific for Ld_f ( Supplementary Figure S4 ). The full coding sequences for VHHs/VHs genes of the second output were then transferred en masse from the pCOMB3x to the pINQ-HAH6 vector, to improve the VHH/VH expression efficacy. The transference of the VHHs/VHs genes was evaluated by performing the same ELISA previously done for the screening, finding that nine out of ten original clones were positive for Ld_f ( Supplementary Figure S5 ). Primary sequencing of the nine Ld_f positive clones allowed identifying five distinct sequences ( Supplementary Figure S6 ) that exhibited a VHH-specific amino acid pattern in framework 2, thereby confirming that all isolated clones were VHHs. The subsequent characterization of their binding specificity was carried out by ELISA using the delipidated EgAgB8 subunits expressed in E. coli (drEgAgB8/1-3), showing that five out of nine positive clones recognized drEgAgB8/1 and none of them were positive for drEgAgB8/2 nor drEgAgB8/3 ( Figure 1A ). The predominance of VHH positive clones specific for EgAgB8/1 agrees with its higher abundance in the nEgAgB present in bovine HF compared to the EgAgB8/2-5 subunits (Folle et al., 2017). Since anti-EgAgB VHHs were developed to allow affinity purification of nEgAgB and its recombinant form, we chose the EgAgB-specific clone that produced higher amounts of soluble VHH as selection criteria. Clones 1, 3 and 7, showing the highest signals for EgAg8/1 by ELISA ( Figure 1A ), were grown and the VHH expression was induced with IPTG. According to SDS-PAGE of soluble and insoluble fractions obtained from cell lysates, clone 1 yielded the highest amount of soluble VHH ( Figure 1B ). This clone was further expressed, purified and immobilised on CNBr-Sepharose for immunoaffinity chromatography.

The native form of EgAgB was purified to homogeneity by immunoaffinity of Ld_f using anti-EgAgB clone1-Sepharose. The unbound proteins were washed with a high salt concentration (1M) and by decreasing pH, and as expected, the nEgAgB was eluted at pH 2.5 as a yellowish fraction. SDS-PAGE and Western blot analysis showed that the fraction eluted at pH 2.5 contained high-purity nEgAgB ( Figures 2A, B , respectively), exhibiting the typical pattern with regularly spaced bands in both analyses. This pattern was almost identical between Ld_f and nEgAgB, indicating no detectable differences in protein composition. However, analysis by the LAL assay revealed higher endotoxin levels in Ld_f (≥ 0.9 ng/µg protein) than nEgAgB (≤ 0.02 ng/µg protein), allowing to test nEgAgB but not Ld_f at 10 µg/mL concentrations in studies for examining macrophage activation. These results were reproduced in six nEgAgB batches derived from different HF pools, suggesting that immunoaffinity contributes to obtaining high-quality nEgAgB preparations suitable for macrophage studies. Only 2.6 mg (protein) of Ld_f, containing approximately 0.0114 micromoles of nEgAgB, saturated the anti-EgAgB clone 1-Sepharose column; this amount was low compared to the estimated 0.75 micromoles (15 mg) of anti-EgAgB VHH immobilised in the Sepharose matrix. Assuming a 1:1 binding ratio, the recovered nEgAgB represented only 1.5% of the theoretical yield, estimated if this VHH would be in a solution.

Expression of rEgAgB8/1 in the S2 cell line was carried out by transfection with the pDroEx-EgAgB8/1 expression vector (containing the sequence EGR_06805) and pCoPURO to select transfected cells using puromycin. The rEgAgB8/1 was detected in cell culture supernatants at 4 dpi by SDS-PAGE and Western blot, and purified using Strep-Tactin XT-agarose column after 4, 7 or 12 dpi (see Supplementary Figures S7A, B ) followed by immunoaffinity with the anti-EgAgB clone1-Sepharose using the same procedure as that for purifying nEgAgB. The SDS-PAGE and Western blot analyses showed that rEgAgB8/1 was pure and had a lower degree of oligomerisation than nEgAgB ( Figures 2C, D ). Using a 50 mL culture, lower amounts of rEgAgB8/1 were obtained at 4 dpi (48 µg) compared to 7 and 12 dpi (117 and 129 µg, respectively). Transfected S2 cells showed a high viability within the first week of cell culture, but it decreased progressively from one week on. These results, together with the characterisation of rEgAgB described below, led us to select 7 dpi as the optimal induction time for expression. At this condition, rEgAgB8/1 preparation showed a modest but reproducible yield in six independent batches (about 3 mg per litre of culture medium). Similarly to nEgAgB, the endotoxin levels detected in rEgAgB8/1 preparations (≤ 0.02 ng/µg protein) enable its use at up to 10 µg/mL in macrophage functional studies.

The analysis by DLS of immunopurified nEgAgB particles showed a multimodal size distribution (weighted by intensity) corresponding to a principal peak (weighted by volume) with an average hydrodynamic radius (R_H ± SD) of 5.6 ± 0.4 ( Figure 3A ) and overall PDI values ranging between 0.236 to 0.419, indicating moderate to broad overall polydispersity. The rEgAgB8/1 preparations obtained at 4, 7 and 12 dpi were also polydisperse (PDI between 0.256 to 0.443) and had a monomodal size distribution with R_H ± SD of 8.3 ± 0.5, 7.8 ± 0.3 and 13.8 ± 0.7, respectively ( Supplementary Figure 7C ). This revealed that after 7 dpi the particle size of rEgAgB8/1 was about 2-fold larger than nEgAgB. In this scenario, we only used for biochemical characterization and functional assays rEgAgB prepared from cell culture supernatants obtained at 7 dpi.

Protein and lipid analysis by native (non-denaturing) gel electrophoresis showed that both nEgAgB and rEgAgB8/1 displayed characteristics of complex lipoproteins, with a principal component of 7 and 12 nm in diameter, respectively ( Figures 3B, C ). The sizes of the nEgAgB and rEgAgB8/1 were similar to those shown by the plasma fraction of human HDL₃ and HDL₂, respectively. These results suggest that the rEgAgB8/1 subunit could also undergo oligomerization during lipid acquisition. Analysis by HPLC-MS of the lipid composition was focused on the phospholipid species and total FAs, which were expected to be in tight association with EgAgB subunits at the lipoprotein surface, conditioning the interactions established with putative cell receptors (Obal et al., 2012). Both EgAgB preparations revealed differences in their 50 most abundant FAs and phospholipids bound to the protein moiety. In agreement with previous studies, C16:0, C18:1, C22:5, C20:4, and C18:2 were the predominant FAs detected in nEgAgB (Obal et al., 2012), while C16:0 showed a high abundance in rEgAgB8/1 followed by C18:1 and C16:1 and C14:0 ( Figure 4 ). In addition, nEgAgB contained a wider variety of phospholipids than rEgAgB8/1, with phosphatidylcholine species (with different FAs occupying positions 1 and 2 in the glycerol) being predominant in both ( Figure 4 ).

Immunopurified EgAgB preparations did not alter macrophage viability at the assayed concentrations (up to 20 µg/mL, Supplementary Figure S8 ). At 10 µg/mL, both nEgAgB and rEgAgB8/1 did not induce per se the secretion of the pro-inflammatory cytokines IL-1β and IL-6 on THP-1 macrophages ( Figures 5A, B , respectively), but at 20 µg/mL triggered the production of low levels of IL-1β ( Supplementary Figures S9A, B , respectively). These results contrast with Ld_f effects on THP-1 macrophages ( Supplementary Figures S9A, B ) and agree with previous results achieved with nEgAgB immunopurified using the EB7 monoclonal antibody [9]. Similar results were obtained using BMDM, suggesting that concentrations up to 10 µg/mL of both nEgAgB and rEgAgB8/1 did not induce per se significant pro-inflammatory effects and thus, were adequate for examining immunomodulation properties. On the other hand, 1 µg/mL of nEgAgB was high enough to inhibit the cytokine secretion induced by LPS in cultured THP-1 macrophages and BMDM. We observed a dose-dependent reduction of IL-1β, IL-6 and IFN-β secretion in activated THP-1 macrophages ( Figures 5A-C ) and of IL-6 and IL-12p40 in activated BMDM ( Figures 5D, E ). It is worth mentioning that in the assayed conditions, IL-1β and IFN-β secretion by BMDM were undetectable in control and LPS-stimulated macrophages. rEgAgB8/1 behaved similarly to nEgAgB, interfering with LPS-mediated responses in THP-1 macrophages and BMDM ( Figures 5A-E ). However, rEgAgB8/1 appeared to exhibit a lower ability to inhibit IL-1β secretion than the native lipoprotein ( Figure 5A ) since inhibition was achieved using 10 but not 1 µg/mL. The EgAgB effects on LPS-induced ·NO response were analysed only in BMDM because nitrite levels were not detectable in LPS-stimulated THP-1 macrophages. Results showed that nEgAgB and rEgAgB8/1 did not induce ·NO response per se and caused a decrease in LPS-induced ·NO generation, even when tested at 1 µg/mL ( Figure 5F ). Finally, EgAgB’s ability to modulate the LPS-induced expression of cell surface MHC-II and the costimulators CD40 and CD86 in BMDM was examined by flow cytometry ( Figure 6 ). The basal cell surface expression of these markers on control macrophages (vehicle-treated) was not affected by nEgAgB and rEgAgB8/1, showing at best a marginal elevation. In contrast, LPS caused a consistent, although modest, significant increase in the relative abundance of cell surface MHC-II (1.8-fold), CD86 (3.4-fold) and CD40 (1.7-fold). Neither nEgAgB nor rEgAgB8/1 controlled the LPS-induced upregulation of these macrophage markers at the cell surface.

The EgAgB effects on monocyte and macrophage activation were examined in vivo using an inflammatory model based on the injection of LPS in the mouse peritoneal cavity. In this model, 15 µg of LPS generated a moderate response, characterised by the presence in the peritoneal lavage of IL-6, IL-12p40 and variable amounts of IL-10 (undetectable in some experiments) at 4 hpi. None of these cytokines was detected at 24 hpi. Following our in vitro observations, nEgAgB modulated the LPS-induced cytokine responses by significantly decreasing IL-6, though marginally, also IL-12p40 ( Figures 7A, B , respectively), and by increasing IL-10 levels ( Figure 7C ). rEgAgB8/1 caused similar modulatory effects despite these trends only reaching statistical significance for IL-12p40 reduction ( Figures 7D-F ).

On the other hand, LPS did not modify the total number of peritoneal cells at 4 hpi but caused a slight reduction in this number at 24 hpi ( Supplementary Figure S10 ); this is likely due to a significant macrophage migration to the omentum (Cassado et al., 2011) that appears not to be compensated by the recruitment of inflammatory blood cells at this time point. LPS activation of resident and self-renewing peritoneal macrophages, referred to as LPM (CD19⁻F4/80⁺⁺Ly6C^-), was null in terms of the cell surface expression of co-stimulators CD86 and CD40 at 4 hpi. However, at 24 hpi LPS caused upregulation of cell surface MHC-II and CD86, and a modest increase in CD40 compared to the vehicle ( Figure 8 , Supplementary Figure S11 ). The presence of nEgAgB only partially reduced the LPS-induced increase of cell surface MHC-II in LPM ( Figure 8A ); however, this effect was not reproduced by rEgAgB8/1 ( Figure 8D ). On the other hand, at 4 hpi LPS caused increments of cell surface CD86 and CD40 in the population of small resident peritoneal macrophages derived from blood monocytes, known as SPM (CD19⁻F4/80^+/-SSC^lowLy6C^-MHC-II⁺⁺, Supplementary Figure S11 ). These increments were inhibited in the presence of nEgAgB ( Figures 9A, B ). rEbAgB8/1 mimicked the nEgAgB modulatory effect on CD86 but not on CD40 ( Figures 9C, D ). Similarly, at 24 hpi, LPS promoted cell surface CD86 and CD40 increases while diminished MHC-II in SPM ( Figure 10 ). In this case, nEgAgB, but not rEgAgB8/1, inhibited partially LPS-induced increases in CD86 and CD40. LPS-recruited monocytes (CD19⁻F4/80^+/-SSC^lowLy6C⁺⁺MHC-II^-) were not detected until 24 hpi. In the presence of EgAgB preparations, trends to lower expression of CD86 and CD40 in recruited monocytes were observed, but the low number (between 100 and 550) of recruited cells makes the comparison unreliable ( Supplementary Figure S12 ). The modulatory effects of EgAgB (both native and recombinant) on LPM and SPM activation by LPS were mostly partial in agreement with the fact that even in the presence of EgAgB, LPS caused some increases in MHC-II and/or cell surface costimulators CD86 and CD40 receptors (indicated as statistically significant differences between EgAgB and EgAgB plus LPS groups, Figures 8 - 10 ). nEgAgB and rEgAgB8/1 caused per se a variable activation of LPM and SPM, according to CD86 and/or CD40 increases ( Figures 8 - 10 ), which appear to be of lower magnitude than those caused by LPS, except for the CD40 induction by rEgAgB8/1 in SPM at 24 hpi.