Cell and viruses

RBD-mFc protein (Cat. 40,592-V05H) were purchased from Sino Biological, Inc. (Beijing, China). The HEK293 cell line, pseudovirus PSV-Luc-Spike (M), and the ACE2-overexpressing 293FT (293FT-ACE2) cell line were provided by Sinocelltech Ltd (Beijing, China). The SARS-CoV-2 isolate Wuhan-Hu-1(GenBank accession no. 622,319), Omicron BA.2.76(EPI_ISL_12810061) and Vero E6 cells were provided by the biosafety level 3 laboratory of the Second Military Medical University (Shanghai, China).

Immunogen RBD-mFc protein expression, purification, and quality control

The target genes encoding the RBD protein of the SARS-CoV-2 spike protein (Arg319-Phe541; GenBank accession no. YP_009724390.1) were amplified by PCR, subcloned into the vector, and the plasmid DNA was extracted and sequenced to confirm the correct insertion sequence. The protein was expressed in the HEK293E cells through transfection with transfection reagent TF1 (Sino Biological Inc.), and purified protein was processed from the medium supernatant after 7 days. The obtained protein samples were monitored with SDS-PAGE.

ELISA determination of purified RBD-mFc protein binding activity with ACE2

ELISA was performed to detect the binding of RBD-mFc protein to ACE2 receptor, as previously described [11]. The recombinant RBD-mFc protein was diluted to 2 µg/mL in phosphate-buffered saline (PBS, pH 7.0) and 100 µL was added into each well in 96-well plates. The plate was covered and incubated at 4℃ overnight, then aspirated and washed once with phosphate-buffered saline with 0.05% Tween 20 (PBST). Next, 300 µL blocking solution (2% bovine serum albumin [BSA] in PBST, pH 7.4) was added to each well, incubated at room temperature for 60 min, then aspirated and washed twice. Human recombinant ACE2 protein (His tag) (Cat. 10,108-H08H, Sino Biological) was diluted to 20 µg/mL in dilution buffer (0.1% BSA in PBST, pH 7.4). A 7-point curve was prepared using 5-fold serial dilutions stating from 20 µg/mL. The dilution buffer was used as the zero standard (0 µg/mL). The sample (100 µL) was transferred to each well and the plate was covered and incubated at room temperature for 60 min before it was aspirated and washed three times. Anti-His tag antibody (HRP) (Cat. 105,327-MM02T-H, Sino Biological, Inc.) was diluted to 0.12 µg/mL in dilution buffer (0.5% BSA in PBST, pH 7.4) and 100 µL was transferred to each well. The plate was covered and incubated at room temperature for 60 min, then 200 µL tetra-methyl benzidine (TMB) was transferred to each well. The plate was covered again and incubated at room temperature for 20 min. H2SO4 (50 µL, 2 M) was added to each well to halt the TMB reaction. The optical density at 450 nm (OD 450 nm) was detected. Data analysis was carried out with GraphPad Prism 8.0.1. The value of EC50 was calculated from the best-fit curves for experiment using Prism EC50 curve-fitting algorithm.

RBD-mFc protein immunization of hens

As the immunogen, pure RBD-mFc protein was combined and emulsified in equal proportions with Freund’s immunological adjuvant. The first immunization used complete Freund’s immune adjuvant while the remaining immunizations used incomplete Freund’s immune adjuvant. Each hen (Ten 5-month-old Kangle chickens, weighing about 1600 g; Eggs laid before immunization were collected as a control.) was injected at multiple sites (i.m.) with 300 µg recombinant spike protein and immunized at 10-day intervals for the first five immunizations and then at 1-month intervals afterwards. We collected the eggs, and the egg supernatant titer was tested after a week off per vaccination. The egg yolks were separated, diluted with ultrapure water at 1:7 volume, 1 M HCl solution was added to adjust the pH to 5.0, and then incubated overnight at 4℃ or stored at -20 °C until used. After 30-min centrifugation at 4120 ×g, the egg supernatant titer was tested by ELISA.

IgY production and purification

We used an improved extraction, namely, lipoprotein was first separated at -20℃, then the antibodies were purified using 2-step ammonium sulfate precipitation. The details are as follows: the yolk was carefully removed from the egg white and rolled on a paper towel to remove any egg white before being diluted eight folds with double-distilled water (ddH2O) (pH 5.0). Next, it was incubated for 2 h at -20℃, thawed at room temperature, and centrifuged at 4120 ×g and 4℃ for 30 min. The supernatant was removed and filtered through a 0.22-µm filter. Ammonium sulfate was added to the supernatant until the concentration was 40%, then the supernatant was placed at 4℃ for 2 h, centrifuged at 4120 ×g for 30 min, and the supernatant was removed. The precipitation was dissolved in PBS (pH 7.4). Then, ammonium sulfate was added to form a final concentration of 35%, and the mixture was placed at 4℃ for 2 h, then centrifuged at 4120 ×g for 30 min. The supernatant was discarded and the precipitation was dissolved in PBS (pH 7.4). The concentration of IgY protein was measured photometrically at 280 nm using UV spectra (NanoDrop One, Thermo Fisher Scientific Inc, USA) and was calculated according to the Lambert-Beer law with an extinction coefficient of 1.36 for IgY. The purity of IgY was monitored with SDS-PAGE.

ELISA testing of IgY binding ability with RBD protein

ELISA was performed to detect the binding of IgY to RBD protein [12]. The SARS-CoV-2 spike RBD-mFc was combined with solid-phase carriers to form the solid-phase conjugate. After washing unbound protein and impurities away, the test product IgYs (3-fold serial dilutions stating from 300 µg/mL) were added to bind with the solid-phase antigen to form the solid-phase immune complex. Next, horseradish peroxidase (HRP)-labeled rabbit anti-chicken IgYs were added. The IgYs on the solid-phase immune complex were combined with an HRP-conjugated antibody. The enzyme catalyzed the substrate that had been added earlier. The OD450 was read by a multi-well spectrophotometer. Data analysis was carried out with GraphPad Prism 8.0.1. The value of EC50 was calculated from the best-fit curves for experiment using Prism EC50 curve-fitting algorithm.

Competition ELISA

We used a competition ELISA to evaluate the ability of the IgY to inhibit binding of RBD protein to the human ACE2 [13]. The solid-phase conjugate consisted of SARS-CoV-2 spike RBD-mFc and the solid-phase carrier. The RBD were incubated overnight at 4℃ in high bind 96 well plate. After removing unbound protein and impurities, a serial dilution of purified the IgY and ACE2 (Fc Tag) (Sino Biological Inc., cat. no.: 10,108-H02H) were added to combine with the solid-phase antigen to form the solid-phase immune complex. After incubated for 1 h at 37 ℃, HRP-labeled goat anti-human IgG (FC) was added, and the ACE2-FC on the solid-phase immune complex was combined with HRP-conjugated antibody to catalyze the solid-phase substrate. The inhibition rate was calculated using colorimetry. Data were analyzed using GraphPad Prism 8.0.1.

Pseudovirus neutralizing assay

The effectiveness of IgY suppression of the SARS-CoV-2 pseudovirus [PSV-Luc-Spike(M)] was measured using luciferase-generated luminescence [14]. 293FT-ACE2 cells were infected with lentivirus expressing SARS-CoV-2 spike protein and luciferase. The light emission was detected by a microporous plate luminometer when the pseudovirus invaded the cells, with the luciferase reacting with the substrate. Lower bioluminescence intensity (relative light unit [RLU]) indicated higher IgY antibody activity against the SARS-CoV-2 pseudovirus. In brief, IgYs were serially diluted, incubated with 100 TCID50/well pseudovirus (1 h at 37℃, in a 5% CO2 incubator), and co-cultured with 3 × 104 293FT-ACE2 cells for 20 h. After incubation, the supernatant was removed and 1×Passive lysis buffer was added at 50 µl/well to lyse the cells. Relative light unit (RLU) of the cell lysate was measured to evaluate luciferase activity. The neutralization percentage was calculated by the formula: Neutralization (%) = (Positive Control RLUs-Sample RLUs) / (Positive Control RLUs-Negative Control RLUs) ×100%. Neutralization titers of the antibodies were presented as 50% maximal inhibitory concentration (EC50) which determined by the Reed-Muench method. Data analysis was carried out with Excel 2016.

Authentic SARS-CoV-2 omicron strain neutralization assay of IgY

We performed an authentic SARS-CoV-2 Omicron BA.2.76 strain virus neutralization assay with anti- SARS-CoV-2 Omicron BA.2.76 strain IgYs in a biosafety level 3 laboratory. The cell culture medium contained a series of anti-SARS-CoV-2 Omicron BA.2.76 strain IgYs from 2-fold serial dilutions starting from 50 µg/mL concentration. After 1-h incubation with the SARS-CoV-2 Omicron BA.2.76 strain virus at 37℃, the IgYs were added to Vero E6 cells, and the cells were cultured for 24 h. Then, the supernatant of the cell culture was discarded and the cells were treated with anhydrous methanol at -20°C for 20 min. The cells were then washed with precooled PBS and incubated with 3% BSA for 2 h at room temperature. Serum from convalescent patients (1:400 dilution) was added as the primary antibody and incubated at 4℃ overnight. The supernatant was discarded and the cells were washed in precooled PBS three times. Then, FITC-labeled anti-human IgG antibody was added for 1-h incubation at room temperature. The supernatant was discarded and the cells were washed with precooled PBS and stained with DAPI (4’, 6-diamidino-2-phenylindole) at room temperature for 10 min. The cells were then washed with precooled PBS once again. The cytopathic phenotype was observed every day and the number of positive cells were counted.

Large-scale purification of IgYs

The egg yolk of immunized hens was separated by egg yolk sieving, then 8-fold distilled water was added to stir and mix to prepare the mixture at pH 5.0–5.2 (adjusted by 1 M HCl) and held for precipitation at 4℃ overnight. The mixture was centrifuged at 8230 ×g for 20 min and the precipitate was discarded. Octanoic acid was slowly added to the collected supernatant to 1% octanoic acid to remove impurities. The mixture was stirred until smooth and the supernatant was collected for ultrafiltration concentration after standing at 4℃ for 2 h and was centrifuged at 4℃ for 30 min at 8230 ×g. Diatomite (3%) was added as a filtration aid, and the filtrate was extracted by vacuum filtration with a 0.45-µm mixed cellulose ester membrane (0.09 MPa). A 50,000-molecule interception ultrafiltration membrane was selected, the material was fed at room temperature, and the filtrate was concentrated by ultrafiltration in single-stage batch operation mode to obtain concentrated IgYs, followed by Pasteur disinfection and 0.22-µm membrane filtration.

Hamster model of SARS-CoV-2 infection

The SARS-CoV-2 strain had been isolated from a patient with laboratory-confirmed COVID-19 by passaging in Vero E6 cells [11]. The virus working stocks were propagated and titrated in Vero E6 cells in the presence of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin at 2 µg/mL. The stocks were stored at -80 °C prior to experimental infections. All experiments involving infectious viruses were performed in the biosafety level 3 facility of the Second Military Medical University.

The hamsters were randomly classified into three groups (n = 6 per group) for the SARS-CoV-2 challenge experiments. Animals were mock-infected in the first group (naïve group). In the second and third groups (control and treatment, respectively), the animals were treated placebo (control group) or the RBD IgY (8.6 mg intraperitoneally and 1.7 mg i.n.) 1 day before challenge, and 1.7 mg i.n. twice daily for five days after challenge. Hamsters in the control and treatment groups were infected i.n. with 8 × 104 TCID50 (median tissue culture infective dose) SARS-CoV-2 diluted in 80 µL Dulbecco’s modified Eagle’s medium as previously described27 [11, 15]. At 4 dpi, the three hamsters in each group were killed and their lungs were obtained for RT-PCR virus load analysis and pathological testing. The weights of the remaining three hamsters in each group were tracked every day for 14 days.

Hematoxylin and eosin (H&E) staining

The hamster lung tissues were preserved in 4% paraformaldehyde and embedded in paraffin. The tissue Sect. (3 μm) were dewaxed, rehydrated, and underwent routine H&E staining. The tissue sections were examined and imaged under a light microscope. The results were scored semi-quantitatively for alveolar septal thickening and inflammatory cell infiltration, alveolar exudate, and hemorrhage based on the H&E-stained scans [14]. Date were presented as Mean ± SD and were analyzed using unpaired t test with GraphPad Prism 8.0.1. p < 0.05 was considered statistically significant.

RNA extraction and RT-qPCR

As previously reported, hamster lung samples were obtained postmortem for viral detection using RT-qPCR [15]. Briefly, total RNA was extracted from the lung tissues using TRIzol (Thermo Fisher Scientific, Shanghai, China). The RNA concentrations and absorbance at 260 nm (A260)/A280 ratio were assessed with a multiplate reader (Synergy 2; BioTek, Shanghai, China). RNA was converted into complementary DNA (cDNA) using a reverse transcription system (Promega, Madison, WI, USA). The cDNA product was used for the following qPCR analysis directly with TB Green Fast qPCR Mix (TaKaRa, Otsu, Japan) and gene-specific primers. Hamster β-actin expression was used for normalization. Additionally, a standard curve was constructed using expression plasmids of the SARS-CoV-2 N gene and hamster β-actin. The SARS-CoV-2 RT-qPCR was quantified as copy numbers. Date were presented as Mean ± SD and were analyzed using unpaired t test with GraphPad Prism 8.0.1. p < 0.05 was considered statistically significant.