Production of purified and inactivated SARS-CoV-2
SARS-CoV-2 Bank: SARS-CoV-2/SP02/2020HIAE (GenBank MT126808.1. B.1.1.28) is a clinical isolate from one of the first patients diagnosed with COVID-19 in Brazil, admitted to the Hospital Israelista Albert Einstein (São Paulo, Brazil). The clinical material was tested for SARS-CoV-29 and for 15 viral agents using real-time PCR (influenza A and B, endemic coronavirus—CoV-NL63, -229E, -HKU1 and -OC43, enterovirus, parainfluenza virus 1, 2, 3 and 4; human metapneumovirus, rhinovirus, respiratory syncytial virus and adenovirus)10. No viral agent, except for SARS-CoV-2, was detected in the sample. The virus was isolated in VERO E6 cells, and the characterization of the strain was described by Araujo et al.10. A nucleotide identity comparison between the genomes of the used strain (GenBank MT126808.1. B.1.1.28) and the original Wuhan-Hu-1 genome (NC_045512.2) was also performed. Their whole genome identity is 99.99%, with only four different nucleotides and no changes in the amino acid sequence of the spike protein. Working virus seed stock (WVSS) was prepared by infecting VERO CCL-81.4 cells (ECAAC General Collection 88020401) adapted to VP serum-free medium (Thermo Fisher, MA, USA) at Butantan Institute with an MOI (multiplicity of infection) of 0.1 for 48 h at 37 °C in a 5% CO2 atmosphere. The produced virus was filter-sterilized (0.22 µm), formulated with SPG (7.462 g/L sucrose, 0.0517 g/L KH2PO4, 0.1643 g/L K2HPO4·3 H2O and 0.0907 g/L potassium glutamate) and frozen at − 80 °C. The final WVSS titre was 9.79E + 06 TCID50/mL. All manipulations of the active virus were performed in a BSL3 laboratory.
Virus titration
Determination of the 50% Tissue Culture Infective Dose (TCID50): Virus samples were taken from culture or purification steps and immediately frozen at − 80 °C before analysis. After thawing, samples were serially diluted ten-fold (10–1–10–12) with Dulbecco’s modified Eagle’s medium (DMEM) containing 2.5% foetal bovine serum (FBS) and inoculated in six replicates in 96-well plates in which VERO ATCC CCL-81.4 cells were seeded (2 × 10E + 04 cells/well). After an incubation at 37 °C with 5% CO2 for 72 h, the plates were microscopically inspected for cytopathic effects (CPE) caused by SARS-CoV-2. The monolayers were fixed and stained with a Naphthol Blue Black (Sigma-Aldrich) solution (0.1% naphthol blue w/w with 5.4% acetic acid and 0.7% sodium acetate) and analysed to confirm the results. The viral titre was calculated using the Spearman & Kärber algorithm11 and reported in TCID50/mL.
Antigen production and inactivation
For antigen production, Vero ATCC CCL-81.4 cells were seeded in T 225 cm2 flasks (107 cells/flask) and grown for 48 h before infection. SARS-CoV-2/SP02/2020HIAE was inoculated at an MOI of 0.1 in 5 mL of culture medium and allowed to adsorb for one hour at 37 °C. Serum-free medium was added to cultures to a volume of 100 mL. The cultures were incubated at 37 °C with 5% CO2 for 48 h. Before harvest, flasks were microscopically inspected for cytopathic effects and the absence of contamination, and then the culture medium was transferred to a new flask. Three batches of active SARS-CoV-2 were clarified through 0.22 µm filters (Opticap XL2 Durapore, Merck Millipore) and pooled (6 L) for the following purification process. Clarified material was concentrated by tangential flow filtration (TFF) using a Mini Pellicon system with an Ultracell 300 kDa membrane (Merck Millipore). The concentrated material (2 L) was subjected to two rounds of ultracentrifugation (XPN 90, Beckman Coulter) in sucrose gradients (65–34%) using a zonal Ti15 rotor for 3 h at 4 °C and 25 krpm. Fractions containing the purified virus were pooled after each ultracentrifugation procedure. The final purified material was diluted with 1× PBS to a sucrose concentration of 20%, filter-sterilized (0.22 µm) and frozen at − 80 °C. Frozen aliquots of purified SARS-CoV-2 were subjected to virus inactivation using gamma irradiation at Instituto de Pesquisas Nucleares (IPEN, São Paulo, Brazil) using GammaCell Irradiation unit, (Nordion, Canada). The radiation dose for virus inactivation was previously chosen through a D10 experiment where several doses were used to reduce the virus titer proportionally. The gamma radiation dose necessary to inactivate SARS-CoV-2 (Wuhan strain) was determined as 15 kGy already considering the sterilization assurance level required for animal or pharmaceutical products. The virus titre was quantified by determining the TCID50/mL.
Virus inactivation test
The effectiveness of the SARS-CoV-2 inactivation process was evaluated by detecting cytopathic effects and performing a kinetic analysis of subgenomic viral RNA using RT-qPCR in VERO CCL 81.4 cells after SARS-CoV-2 antigen inoculation. Briefly, aliquots of 0.1 mL of the inactivated virus were incubated with 1E + 05 VERO ATCC CCL-81.4 cells for 1 h at 37 °C, followed by the addition of 1.4 mL of DMEM supplemented with 10% FBS. The plates were incubated for 72 h, and in the absence of cytopathic effects, a 0.1 mL aliquot of the supernatant was transferred to a new culture of VERO ATCC CCL-81.4 cells for the second passage. The procedure was repeated at least one more time. For the kinetic analysis of subgenomic viral RNA, 1 mL of inactivated virus was inoculated in 0.1 mL aliquots in ten wells of a 12-well plate containing VERO ATCC CCL-81.4 cells and incubated for 72 h. The supernatant of all wells was pooled, and aliquots of 0.1 mL were placed in a new well of a 12-well plate for the second passage in VERO cells.
For the analysis of increasing amounts of subgenomic RNA during incubation, which indicates virus replication, samples were collected after 1 h, 24 h and 48 h of incubation by lysing the cell monolayer with lysis buffer from the PureLink™ RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA was extracted, and 100 ng of total RNA were subjected to reverse transcription followed by qPCR using High-Capacity cDNA Reverse Transcription and Fast SYBR™ Green Master Mix kits, respectively (Thermo Fisher Scientific). Quantitative PCR was performed using serial dilutions of a synthetic sequence corresponding to the fragment of amplification as a standard curve (Gene Blocks, IDT). Primers were synthesized based on published methodology12.
Characterization of the antigen
The purity of the inactivated virus samples was evaluated by performing SDS-PAGE using 4–15% Criterion™ TGX™ Precast Midi Protein gels under nonreducing conditions (Bio-Rad). Electrophoresis was performed for 1 h at 100 V, and the gel was stained using a Pierce™ Silver Stain Kit (Thermo Fisher Scientific). In addition, the amount of residual protein from VERO ATCC CCL-81.4 cells was quantified using the Immunoenzymetric Assay of VERO Cell Host Cell Proteins (Cygnus Technology), as recommended by the manufacturer. The endotoxin content in the purified and inactivated virus samples was quantified using the PYROGENT™ Plus Gel Clot LAL Assay (Lonza, USA) according to the manufacturers’ recommendations. Finally, the purity and identity of the antigen were also evaluated through mass spectrometry analysis as described below.
The antigen quality was analysed based on antigen preservation after the inactivation process using Western blotting. For this analysis, 400 µL of the purified and inactivated antigen were separated on SDS-PAGE gels as described above, and proteins were transferred to a nitrocellulose membrane for 1 h at 90 V—150 mA current. The membrane was blocked with the blocking solution for 3 h at room temperature under agitation. Then, the membrane was washed with phosphate buffer containing 0.05% Tween 20 (3 × 5 min), followed by an incubation with rabbit monoclonal [CR3022] anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (Abcam—Ab273074) and rabbit polyclonal anti-SARS-CoV-2N protein antibody, which was kindly provided by Dr Roxane Piazza (Butantan Institute, Brazil), diluted in sample diluent for 1 h. Peroxidase labelled goat anti-rabbit IgG (KPL) was used for labelling, and the membrane was revealed with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Images were acquired with Uvitec Alliance 2.7 equipment.
Immunogenicity analysis of the inactivated virus
The use of mice in the present study was approved by the Institutional Animal Care and Use Committee of the Butantan Institute (n° 9165080620). BALB/c female mice (18–22 g) were obtained from the Center for Animal Breeding of the Butantan Institute. Animals (n = 5) were immunized subcutaneously with purified and inactivated SARS-CoV-2 (corresponding to 1.74 × 10E + 04 TCID50/animal) mixed with Al(OH)3 (0.25 mg/animal) to achieve a final volume of 200 µL. These animals received three booster doses (1.74 × 10E + 04 TCID50/animal) at 7-day intervals. Control animals (n = 5) were inoculated with sterile saline containing Al(OH)3 (0.25 mg/animal). Blood was collected 21 and 35 days after priming for serum collection, and samples were stored at − 20 °C until use. Serum samples were further analysed using ELISA as described below and a cytopathic effect-based virus neutralization test (CPE-VNT, as described below). For CPE-VNT, the sera were previously inactivated at 56 °C for 45 min.
Production of anti-SARS-CoV-2 equine immunoglobulin
Animals
The procedures involving horses were approved by the Institutional Animal Care and Use Committee of the Butantan Institute (n° 6016030720).
Ten healthy horses (Creole race) that had not been previously immunized were selected and trained to be manageable for the work. Clinical conditions and behaviours were evaluated, and those horses that were suitable had blood samples collected for laboratory tests, including a complete blood cell count (red blood cells, white blood cells, platelets, haemoglobin, and haematocrit), chemistry panel (total plasma protein, albumin, gamma globulin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin, calcium, glucose, sodium, potassium, blood urea nitrogen, and creatinine levels), lipid panel (high-density lipoprotein and low-density lipoprotein levels), creatine phosphokinase levels, and coagulation tests (prothrombin time test and activated thromboplastin time test). Blood samples were collected from the jugular vein and analysed in a veterinary clinical analysis laboratory.
Immunization of horses
Horses (n = 10) were subcutaneously injected four times with the inactivated SARS-CoV-2 suspension (corresponding to 4.5 × 10E + 05 TCID50/animal/dose) at 1-week intervals (days 0, 7, 14, and 21). Montanide™ ISA 50 V oil-in-water adjuvant (Seppic; Shangai, China) was included in the first and third doses to enhance the antibody response. Serum samples were collected before (day 0), during (day 14), and after (days 28, 35, and 49) immunization (Supplementary Fig. S1) and stored at − 20 °C until use. Haematological and biochemical parameters and antibody titres against SARS-CoV-2 proteins (determined using ELISA and CPE-VNT) were monitored before and after the immunization schedule.
Horse plasma collection
Plasma was collected by automated plasmapheresis using a Cobe Spectra® (Terumo BCT) system for therapeutic apheresis procedures. Briefly, a double-lumen catheter was inserted in the jugular vein. One lumen allowed whole blood to be drawn from the horse to which ACD anticoagulant was added. Blood components were separated based on the specific gravity of cells using continuous flow centrifugal technology. Plasma was collected in a transient bag and transferred to a customized bag. The other lumen of the catheter allowed the return of uncollected components to the animal with Ringer’s lactate solution. The plasmapheresis procedure was performed three times (Days 28, 35 and 49 after first dose), according to Supplementary Fig. S1. Each plasmapheresis procedure generated one hyperimmune plasma pool; thus, three pools were obtained sequentially. Plasma samples were analysed for bioburden testing and antibody titres against SARS-CoV-2. Samples from animals were investigated for the presence of SARS-CoV-2 and equine viral encephalitis [Eastern Equine Encephalitis (EEE), Western Equine Encephalitis (WEE), Venezuelan Equine Encephalitis] and equine herpesvirus using PCR in an Equine Veterinary Diagnostic Laboratory to ensure a minimal risk of virus contamination in the starting raw material (plasma).
Purification of immunoglobulins from horse plasma
The starting plasma pool was subjected to fractionation by precipitation with ammonium sulphate. The precipitate was centrifuged, and the liquid fraction was discharged. The product was then solubilized, and the pH was adjusted for enzymatic digestion with pepsin to hydrolyse the non-IgG proteins and cleave the molecule into F(ab′)2 fragments, removing the Fc fragment. A second precipitation was performed by adding ammonium sulphate, and the pH was adjusted by adding caprylic acid with stirring. Next, thermocoagulation at 56 °C separated nonspecific thermolabile proteins, whereas the precipitate was eliminated. The supernatant was subjected to diafiltration and ion-exchange chromatography and concentrated through tangential ultrafiltration. Phenol was added as a preservative, and the chloride concentration was adjusted. The F(ab′)2 solution was then subjected to 0.22 µm filtration and maintained in disposable plastic bags. All production steps were performed under GMP (good manufacturing practices) conditions.
Protein profile of the final product (batches 00001, 00002, and 00003)
The protein profiles of the batches of final products (equine anti-SARS-CoV-2 F(ab′)2 immunoglobulin) were analysed using SDS-PAGE (12.5%) under reducing conditions. Samples of each batch were incubated at 37 °C for 30 min, 1 h, 2 h or 3 h and separated on SDS-PAGE gels to evaluate the presence of any proteolytic product. Gels were stained with Coomassie Blue dye (40% methanol, 10% acetic acid and 0.25% Coomassie blue) and destained with 40% methanol containing 10% acetic acid.
Protein identification using LC–MS/MS analysis (liquid chromatography and tandem mass spectrometry)
Samples of the purified inactivated virus (batch 8911/20) and from the equine anti-SARS-CoV-2 F(ab′)2 immunoglobulin (batches 00001, 00002, and 00003) were subjected to the FASP protocol13 using 10 kDa filter units (Merck-Millipore). Tryptic peptides were desalted using homemade stage tips, and the eluted peptides were dried under a vacuum using a centrifugal concentrator (Eppendorf). Samples were dissolved in 20 µL of aqueous buffer containing 0.1% formic acid, and 2 µL of each sample were injected into an Acclaim PepMap100 C18 trap column (Thermo Fisher Scientific) with a 3 μm particle size, 100 Å pore size, 75 μm internal diameter, and 20 mm column length and analysed using an Orbitrap Q-Exactive plus (Thermo Fisher Scientific) mass spectrometer coupled to an Easy nanoLC 1200 (Thermo Fisher Scientific) at a flow rate of 200 nL/min. Buffer A contained 0.1% (v/v) formic acid, and buffer B contained 0.1% (v/v) formic acid in 80% acetonitrile. Peptides were applied with an increasing gradient of acetonitrile over time (0–90% (v/v) acetonitrile in 30 min) to separate the peptides on an analytical Acclaim PepMap column (Thermo Scientific) with a 2 µm particle size, 100 Å pore size, length of 150 mm, and inner diameter of 50 µm. The spray voltage was set to 2.4 kV, and the mass spectrometer was operated in positive, data-dependent mode, in which one full MS scan was acquired in the m/z range of 300–1500 followed by MS/MS acquisition using higher-energy collisional dissociation (HCD) of the seven most intense ions from the MS scan using an isolation window of 2.0 m/z.
LC–MS/MS data analysis
The obtained MS and MS/MS spectra were analysed using PEAKS STUDIO version X, and the searches were performed against a customized database. Briefly, for antigen data analysis, the reference bank used included all SARS-CoV-2 sequences and reviewed Chlorocebus aethiops sequences downloaded from UniProt (total of 162 sequences, downloaded on 10-01-2020). For the analysis of the three batches of equine anti-SARS-CoV-2 F(ab′)2 immunoglobulin, the reference bank was constructed using all anti-SARS-CoV-2 sequences downloaded from GenBank, and reviewed Equus caballus sequences from UniProt (total of 2190 sequences downloaded on 10-09-2020). Both reference databases were concatenated with common contaminants for mass spectrometry experiments (116 sequences), and the decoy sequences were used for false discovery rate control. The search engine was set to detect semitryptic peptides at an FDR (false discovery rate) of 0.01. Methionine oxidation and acetylation of the protein N-termini were set as variable modifications, and carbamidomethylation of cysteine was set as a fixed modification. Features identified as contaminants and proteins without unique peptides were excluded. Spectral counts were used to estimate the protein proportion in each sample.
Anti-SARS-CoV-2 serum potency analyses
ELISA for anti-SARS-CoV-2 IgG detection
ELISA plates (Costar®) were coated with 100 µL of purified and inactivated SARS-CoV-2 (1 × 10E + 03 virus/mL or 2 × 10E + 03 virus/mL for mouse and horse, respectively) and incubated overnight at 4 °C to evaluate the serum antibody titres of the immunized mice and horses. The plates were then blocked with 5% BSA in PBS for 2 h at 37 °C and incubated with serial dilutions of nonimmune or experimental serum samples for 1 h at 37 °C. After incubation, the plates were washed with PBS containing 0.05% Tween 20 and incubated with anti-mouse IgG-HRPO (1:2000, Zymed) or anti-horse IgG-HRPO antibodies (1:5000, Jackson ImmunoResearch Lab) in 0.1% BSA/PBS-Tween 20 0.05% for 1 h at 37 °C. The plates were then washed, and the reactions were developed with TMB substrate (BD OptEIA™) according to the manufacturer’s instructions. Absorbance values were recorded in an ELISA reader (BioTek Elx800 spectrophotometer) at 450 nm. The titre was established as the highest serum dilution in which the measured absorbance was twice as high as that determined for nonimmunized mouse serum (control group) or for preimmune horse plasma.
Cytopathic effect-based virus neutralization test (CPE-VNT)
CPE-VNT was carried out using SARS-CoV-2 wild-type variant B.1.1.28 (SARS-CoV-2/human/BRA/SP02/2020; MT126808.1) to evaluate the plasma neutralizing antibody titres of the immunized mice and horses. The 3 batches of the final product (equine anti-SARS-CoV-2 serum) were also tested against the P.1./Gamma SARS-CoV-2 variant (IMT 87201 strain) and P.2./Zeta variant (hCoV-19/Brazil/RS-00601/2020-LMM-EPI_ISL_779155, which were kindly provided by Dr. Fernando Spilki—Feevale University, RS, BR. The assays were performed using 96-well plates seeded with 2 × 10E + 04 VERO ATCC CCL-81.4 cells per well, 24 to 28 h prior to the experiment. A serial dilution of each serum (1:20 to 1:40,960) in DMEM supplemented with 2.5% FBS was performed, and then 100 TCID50 of the virus (v/v) was added. The virus/serum mixture was incubated at 37 °C for 1 h to allow virus neutralization. Thereafter, the mixture was transferred onto the confluent VERO ATCC CCL-81.4 cell monolayer and incubated for 72 h at 37 °C with 5% CO2. After the incubation period, the plates were microscopically inspected for CPE caused by SARS-CoV-2 and subsequently stained with 0.2% naphthol blue black solution and analysed to confirm the titre. The virus neutralization titre, referred to as VNT100, is described as the highest dilution of serum that neutralizes virus growth. In each assay, a positive serum and a negative serum were included as controls.
ELISA kit cPass™
The cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript, Cat.: L00847) was used according to the manufacturer’s protocol for qualitative direct detection of total neutralizing antibodies to SARS-CoV-2 in equine and hamster sera14. For the three batches of equine anti-SARS-CoV-2, samples were diluted 1:100, 1:1000 and 1:2500 with PBS, pH 7.4. For the hamster serum, samples were diluted 1:10, 1:20 or 1:40 with PBS, pH 7.4. The serum neutralization plate was blocked with 200 μL/well of PBS + BSA 1% and then incubated at 37 ± 2 °C for 1 to 2 h. Samples and controls were added to the wells and incubated at 37 ± 2 °C for 30 min with the anti-SARS-CoV-2 RBD-HRP solution. The same samples and controls were then transferred from serum neutralization plates to capture plates (ELISAs), incubated at 37 ± 2 °C for 15 min and washed four times with a 1 × wash solution (1:20 dilution with ddH2O, 300 µL/well). After the washing step, the TMB substrate solution was added (100 µL/well) and incubated at ambient temperature (25 ± 3 °C) for 20 min in the dark. Finally, the provided stop solution was added (50 µL/well) to interrupt the HRP and TMB reaction. The absorbance of the samples was measured immediately at 450 nm. Competitive ELISA was performed under GLP conditions, and the methodology was previously validated for usage with equine serum.
Elecsys®Anti-SARS-CoV-2 S
The three batches of equine anti-SARS-CoV-2 F(ab′)2 immunoglobulin were diluted in sterile pyrogenic saline solution at a ratio of 2 from 1/20 to 1/20,480. The samples were transferred to collection tubes of the vacutainer type compatible with Cobas and Roche equipment. The analyses were performed in an automated manner using the Elecsys® Anti-SARS-CoV-2 kit (Roche Diagnostics International AG) for the quantitative determination of antibodies (including IgG) against the RBD domain of the SARS-CoV-2 spike protein. The results are reported as U/mL based on the kit’s standard monoclonal antibody curve.
Challenge test in Golden Syrian hamsters
Animals
The procedures described here were approved by the Institutional Animal Care and Use Committee of Butantan Institute (n°9165080620) and the Institute of Biomedical Sciences of the University of São Paulo (n° 6103261120).
Study design
Seventy-two conventional Golden Syrian hamsters (Mesocricetus auratus) (36 males and 36 females, 115–148 g, 10–11 weeks old) were obtained from the Center for Animal Breeding of Butantan Institute. Experiments were performed in a biosafety level 3 animal facility. Hamsters were housed individually and divided into four groups of 18 animals each (9 males and 9 males), normalized based on weight. On Day zero, animals were anaesthetized with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (7 mg/kg), and two groups (G1 and G2) were inoculated intranasally with 50 μL of 1 × 10E + 05TCID50 of SARS-CoV-2 (SARS-CoV-2/SP02/2020HIAE, GenBank MT12680.1, 4th passage in VERO ATC-CCL81.4 cells) in DMEM with 2% FBS, while the remaining two groups (G3 and G4) were inoculated with 50 μL of DMEM containing 2% FBS. On Day 2 p.i. (postinfection), hamsters of G1 and G3 were intraperitoneally injected with 500 µL of equine anti-SARS-CoV-2 F(ab′)2 immunoglobulin (7.2 mg/animal, equivalent to ~ 3365 U/RBD/animal), while hamsters of G2 and G4 received 500 μL of sterile apyrogenic saline solution via intraperitoneal injection. On Days 3, 5 and 7 p.i., subgroups of 6 animals (3 males and 3 females) from each group were euthanized with overdoses of ketamine hydrochloride (600 mg/kg) and xylazine hydrochloride (30 mg/kg) and necropsied. Nasal turbinates, trachea, and fragments of the lungs were separately collected in lysis buffer (Mag MaxCore kit, Thermo Fisher Scientific) (for RNA extraction), in viral transportation medium [VTM − DMEM + 2% FBS + antibiotics/antifungal (Vitrocell − penicillin 10.000U, streptomycin 10 mg, 25 μg de amphotericin B/mL] (for virus titration), and in 10% formalin (for histopathology analysis). All five lobes of the lungs were represented in each sample for RNA extraction and virus isolation and were analysed separately using histopathology. Whole lungs and all tissue fragments were weighed. Samples collected for virus isolation were quickly frozen in liquid nitrogen and stored at − 80 °C until processing. The animals were also bled, and the serum was analysed for the presence of horse antibodies using capture ELISA and the ELISA kit cPass™.
Viral load quantification from tissue samples
The viral load was quantified by determining the TCID50/g of tissue (using samples in VTM) and the number of viral RNA copies/β actin RNA copies/gram of tissue (using samples in lysis buffer). Briefly, all samples of nasal turbinates, trachea, and lungs were thawed and subjected to disruption using 2 mm glass beads in TissueLyser II equipment (Qiagen) at 30 Hz for 2 min twice, followed by centrifugation at 13,000 rpm (Eppendorf 5804R centrifuge) for 30 s. The supernatants were used for viral load quantification. The TCID50 determination was performed as described above.
Total nucleic acids were extracted using a Magmax Core Kit with a MagMAX Express Magnetic Particle Processor (Thermo Fisher Scientific) according to the manufacturer’s instructions. The detection of SARS-CoV-2 RNA was performed based on a previously described protocol9 using a one-step real-time quantitative PCR (qRT-PCR) assay kit (AgPath-ID™ One-Step RT-PCR Reagents, Applied Biosystems Inc.) and an ABI 7500 SDS real-time PCR machine (Applied Biosystems). The duplex reaction was performed using specific primers for SARS-CoV-2 and primers for hamster β actin (ActB Rv 5′ CAC CAT CAC CAG AGT CCA TCA C 3′, ActB F CTG AAC CCC AAA GCC AAC; ActB_P- HEX TGT CCC TGT ATG CCT CTG GTC GTA ZEN/IOWA BLACK) as a housekeeping gene. The number of RNA copies/mL was quantified based on a standard curve obtained by serially diluting a synthetic dsDNA sequence (Gene Blocks, IDT) corresponding to the amplification fragment of the target gene.
Capture ELISA for the detection of horse antibodies in hamster serum samples
Capture ELISAs were performed to identify horse antibodies in hamster serum samples. Briefly, ELISA plates were coated with 100 µL of rabbit anti-horse IgG (500 ng/mL) (Sigma) overnight at 4 °C. The plates were washed with 200 µL of PBS and blocked with 200 µL of BSA 5% in PBS for 2 h at 37 °C. The plates were then washed three times with 200 µL of PBS, and the previously prepared standard curve (anti-SARS-CoV-2 horse F(ab′)2) immunoglobulin serially diluted in PBS, from 262 µg/mL to 0.13 µg/mL) and serum samples (diluted 1:10 in PBS) were added in triplicate. After 1 h of incubation at 37 °C, the plates were washed three times with PBS containing 0.05% Tween 20 and incubated with peroxidase-conjugated anti-horse IgG (diluted 1:5000 in BS with 0.05% Tween 20 and 0.1% BSA) (Jackson ImmunoResearch Lab) for 1 h at 37 °C. The plates were washed three times with PBS containing 0.05% Tween 20, and the reactions were developed for 15 min by an incubation with TMB substrate (BD OptEIA™) according to the manufacturer’s instructions. Absorbance values were recorded using an ELISA reader (BioTek Elx800 spectrophotometer) at 450 nm. The standard curve was linearized by log–log transformation and then subjected to linear regression analysis. The concentration of horse F(ab′)2 in serum samples was estimated by interpolating the samples to the standard curve.
Histopathological examination
Lung samples preserved in 10% formalin were subjected to routine fixation and paraffin embedding, cut and stained with haematoxylin and eosin (HE). Histopathological findings of changes related to SARS-CoV-2 infection were assessed as described in the literature15,16,17. Tissue samples were blindly evaluated by certified veterinary pathologists and examined under a light microscope for the presence of cellular/tissue alterations in the lung, as described in detail in Table S1 (Supplementary Material). Five anatomical structures of the lungs were analysed per animal and identified as the right cranial lobe, right middle lobe, right caudal lobe, accessory lobe and left lung. Histopathological scores were established as very light (I), light (II), moderate (III), and severe (IV). Using this metric, we were able to quantify the degree of bronchitis, oedema, and pneumonia18,19.
Analysis of leukocyte infiltration in the lung tissue
The numbers of neutrophils, monocytes and lymphocytes present in the hamster lung tissue were determined to better analyse the inflammatory process. For this experiment, images of five fields of H&E-stained sections from each animal were captured under a microscope (Nikon Eclipse Ti-S) using a digital camera (DS-Fi1c, Nikon). For leukocyte counting, ImageJ software and the Colour Deconvolution 2 plugin were used to visualize and separate nuclei from the cytoplasm (Fig. S2—Supplementary Material)20. For cell counting, the Cell Counter plugin was used. This analysis facilitated the differential counting of segmented and mononuclear nuclei.
Analysis of cellularity and functional area in the lung tissue
Cellularity and functional lung area represented by the intra-veolar regions in hamster lung tissue were determined conformed21. For this experiment, five H&E stained sections of each lung lobe per animal were captured under a microscope (Nikon Eclipse Ti-S) using a digital camera (DS-Fi1c, Nikon). To measure the areas of interest, ImageJ software and the Color Deconvolution 2 plugin were used to visualize and separate the cytoplasmic nuclei. The images were transformed into 8-bit and treated with threshold and percentage of the measured area.
Immunofluorescence staining of lung tissue
Thin sections of lung tissue were subjected to deparaffinization, rehydration and antigen recovery. Endogenous peroxidases were then blocked, followed by tissue permeabilization using 0.4% Triton X-100 in PBS. Nonspecific binding was blocked, and the thin sections were washed twice with PBS containing 0.05% Tween 20 and incubated with anti-SARS-CoV-2 spike glycoprotein rabbit antibody (Abcam, ab272504, 1:100) for one hour at room temperature. The thin sections were then washed with PBS containing 0.05% Tween 20 twice and incubated with the Alexa Fluor 647-conjugated anti-rabbit antibody (Thermo Fisher Scientific; A21244, 1:100) for 30 min in a dark chamber at room temperature. After washing, anti-horse IgG FITC (Sigma-Aldrich; F-7759, 1:100) was added and incubated for one hour at room temperature. The thin sections were washed, and the slides were mounted using Hoechst (Thermo Fisher Scientific; 62249, 1:1000) and ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific; P36980). The sections were analysed under a confocal microscope (Leica TSC SP8 DSL Hyvolution). The images were assembled and analysed in 3D using Imaris Viewer software.
Statistical analysis
Data are presented as the means ± standard errors and were statistically analysed with GraphPad Prism version 9.1 software for Windows (San Diego, CA, USA). For comparisons, the following tests were used: one-way ANOVA followed by Tukey’s posttest or Student’s t-test followed by the Mann–Whitney test.
Ethics declarations
All methods were carried out in accordance with the relevant guidelines and regulations. All animal experiments comply with the ARRIVE guidelines.
