To effectively induce a robust nAb response against SARS-CoV-2 in vivo, the RBD of the S protein was selected as the immunogen ( Supplementary Data , Figure S1A ). Based on our previous study (39), honeybee melittin (HBM) signal peptide was added to the N-terminal of the RBD region to promote the expression of SARS-CoV-2 RBD protein in secretory forms. Baculovirus expression system and 6×His tags were applied for mass expression and purification of the recombinant RBD protein. As shown in Supplemental Figure 1 , Western blot results suggested that the recombinant RBD protein was represented by a 35 kDa band ( Figure S1C ). Meanwhile, the purity of the recombinant RBD preparation, as determined by SDS‒PAGE followed by thin layer chromatography, was determined to be 90% ( Figure S1D ). The above results show that the RBD protein of SARS-CoV-2 was well prepared for further research.

Three horses (No. 15, No. 16, and No. 17) were immunized with adjuvanted RBD protein purified from Sf9 cells. The results showed that the neutralizing antibody titers of the sera from the three horses all showed a trend of an unremitting increase in the number following a series of immunizations. The neutralizing serum titer was detected with the SARS-CoV-2 Wuhan01 strain in the BSL-3 laboratory. The neutralizing antibody titers of the three horses were 1:34,000, 1:51,200, and 1:25,600 after five immunizations, respectively ( Figure S2B ). In view of the above results, the sera of horses No. 15 and No. 16 after five immunizations were selected for subsequent IgG and F(ab’)₂ purification. The saturated ammonium sulfate precipitation method was applied to purify IgG from equine serum, and pepsin was used to digest the Fc region to obtain purified F(ab’)₂ fragments. Finally, the sample was run through a Protein A column to remove the residual immunoglobulin G, and the purified F(ab’)₂ fragments were collected by running through a Protein-G column. The integrity of IgG and F(ab’)₂ fragments was determined by SDS‒PAGE ( Figures S2C, D ). After purification, the purities of IgG and F(ab’)₂ were determined to be 92.6% ( Figure S1E ) and 93.7%, respectively ( Figure S2F ). These results collectively demonstrated that the purity of IgG and F(ab’)₂ products were obtained from equines.

Neutralizing activity against SARS-CoV-2 is an indicator of functional antibodies that confer a protective immune response. The binding activity to RBD and neutralizing activity of equine IgG and F(ab’)₂ were investigated. The RBD protein produced by the prokaryotic expression system was used to detect the binding ability of purified IgG. The results showed that the ELISA titer of IgG was 1:12,280 ( Figure 1B ). Hyperimmune serum, purified IgG and F(ab’)₂ derived from the No. 15 and No. 16 equines were selected to evaluate whether the purification operation affected the neutralizing ability. As shown in Figure 1A , purified IgG and F(ab’)₂ of equine No. 15 were found to possess similar neutralizing activity levels, with a neutralizing titer of over 50,000, while the titer of equine No. 15 serum products was over 40,000. The neutralizing titer of serum and purified IgG derived from equine No. 16 was over 30,000, but the purified F(ab’)₂ has a slightly decreased neutralizing titer of over 20,000. No significant differences in neutralizing activity between hyperimmune serum and purified equine immunoglobulins were found after purification. These results indicated that the preparation and purification processes do not adversely affect the neutralizing capacities of these antibodies.

To evaluate the broad-spectrum neutralizing activity of equine immunoglobulin, VSV-vectored pseudoviruses of the five SARS-CoV-2 mutants recommended by the WHO were applied. Table S1 shows the source of each variant of VOCs and VOIs and the specific information of mutation sites on the S protein. It is not difficult to find that most variants of VOCs and VOIs shared D614G mutation sites since the D614G mutation occurred in the B.1.1.7 variant. Additionally, the N501Y mutation was identified in most SARS-CoV-2 VOCs except for the B.1.617.2 variant. N501Y and D614G mutations were stable in the Omicron variant (BA.1 and BA.2), which has recently continued to circulate around the world ( Table S1 ). The results of the neutralization assay suggested that IgG or F(ab’)₂ derived from equine No. 15 and No. 16 have similar neutralizing activity against SARS-CoV-2 Wuhan01 ( Figures 2A, B ). The mean neutralizing titer of IgG from the two equines was 1:51,200, while the mean neutralizing titers of F(ab’)₂ were 51,200 and 42,000, respectively. However, the SARS-CoV-2 VOCs defined by the WHO, including Alpha B.1.1,7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron), weakened the neutralizing effect of IgG and F(ab’)₂ to varying degrees. Compared to SARS-CoV-2 Wuhan01, the VOCs weakened the neutralizing titer by 1.5~4.0 folds of IgG and 2.4~8.0 folds of F(ab’)₂ for No.15 equine ( Figure 2A ), 2.4~9.6 folds of IgG and 2.0~13.3 folds of F(ab’)₂ for No.16 equine ( Figure 2C ). VOIs weakened the neutralizing titer by 1.2~3.0 folds of IgG and 1.5~4.8 folds of F(ab’)₂ for No.15 equine ( Figure 2B ), 1.2~3.0 folds of IgG and 1.7~6.7 folds of F(ab’)₂ for No.16 equine ( Figure 2D ). Among the VOCs, regardless of whether they were IgG or F(ab’)₂, Omicron had the most significant neutralization weakening ability, with a mean neutralizing titer of 12,800 for No. 15 ( Figure 3A ) and 4,200 for No. 16 ( Figure 3C ). IgG and F(ab’)₂ products still maintained a strong neutralizing effect. These results indicated that equine polyclonal antibodies against SARS-CoV-2 have potential broad-spectrum neutralizing activity against SARS-CoV-2 variants. For the VOIs, IgG and F(ab’)₂ of No. 15 equine have a neutralizing capability against pseudoviruses of B1,1,42 (Epsilon) and P.2 (Zeta) similar to that of the parental virus SARS-CoV-2 Wuhan01. Meanwhile, other variants of VOIs to a lesser extent weakened the neutralizing ability ( Figure 2C ). In comparison to equine No. 15, the neutralizing ability of equine No. 16 IgG or F(ab’)₂ was slightly weaker than that of equine No. 15 ( Figure 2D ). In the following study, we selected the equine serum product of horse No. 15 to evaluate the protective efficacy in rodent models.

The half-lives of IgG and F(ab’)₂ were assessed in guinea pigs and found to be 5~6 days and 24~48 h, respectively (Table 1). To verify the protective effect of equine antibodies in vivo, purified IgG and F(ab’)₂ were given to wild-type (WT) BALB/c mice by intraperitoneal injection with 250 µg per dose (10 mg/kg) at 1 day post-infection (dpi) or 1 day preinfection with a lethal dose mouse-adapted SARS-CoV-2 ( Figure 3A ). The results indicated that all BALB/c mice injected with IgG or F(ab’)₂ survived after SARS-CoV-2 infection, while the prevention control and treatment control succumbed to the infection at 4~7 dpi and 5~7 dpi, respectively ( Figure 3B ). Throughout the challenge experiment, all mice in control group had obvious weight loss. At the endpoint of the study, the weight of the surviving mice recovered to their original weight, and the weight of the IgG treatment group increased by 1.50 ± 3.04% (mean ± SD). The body weight of the two control groups dropped to 80% of the initial weight within 2~5 dpi ( Figure 3C ). The mice were euthanized if their body weight was reduced by 20% based on the requirements of the Animal Experimentation Committee. The results of temperature changes showed that the body temperature of mice decreased continuously after SARS-CoV-2 infection, and the temperature changes in the control group were 5 ± 1.55°C in the prevention control group and 5 ± 1.14°C in the therapeutic control group. The body temperature of the prevention group and therapeutic group fluctuated within a normal range. The temperature changes were 0.14 ± 1.09°C in the IgG prevention group and 0.44 ± 0.62°C in the F(ab’)₂ prevention group; the temperature changes were 2.28 ± 0.82°C in the IgG treatment group and 1.56 ± 0.67°C in the F(ab’)₂ treatment group ( Figure 3D ).

Hematological analysis revealed that BMA8-infected BALB/c mice in the control group showed a moderate increase in white blood cell (WBC) count ( Figure 4A ), with significant decreases in the percentage of lymphocytes (LYM%) ( Figure 4C ) and monocytes (Mon%) ( Figure 4E ) and a decrease in platelet count (PLT) ( Figure 4D ) at 3~5 dpi in comparison to all treatment groups. A marked increase in the percentage of neutrophils (Neu%) ( Figure 4B ) was also observed in the control group rather than the treatment groups injected with IgG or F(ab’)₂, which is a typical severe characteristic of SARS-CoV-2 infection observed in clinical COVID-19 patients.

To assess the bio-distribution of SARS-CoV-2 infection in the upper and lower respiratory tracts, the turbinates and lungs were harvested at selected times (3 dpi and 5 dpi) after infection, and viral RNA (vRNA) was quantified by RT-qPCR and TCID₅₀. As shown in Figure 5 , there were much higher levels of vRNA loads in the lung and turbinate at 3 and 5 dpi in both the prevention control group and the treatment control group than in all administration groups. The vRNA loads reached 10⁵~10¹¹ copies/g at 3 dpi ( Figure 5A ) and 10⁷~10¹¹ copies/g at 5 dpi ( Figure 5B ) in the prevention control group, while the viral loads reached 10⁸~10¹² copies/g at 3 dpi ( Figure 5C ) and 10⁷~10¹¹ copies/g at 5 dpi ( Figure 5D ) in the treatment control group. Regardless of the prevention group or the treatment group, no significant differences in viral copies between IgG and F(ab’)₂ were found at 3 dpi or 5 dpi. The TCID₅₀ results tended to coincide exactly with the RT-qPCR results, and significantly higher viral titers were confirmed in all control groups ( Figures 5E–H ). Although the treated mice were positive for vRNA in the turbinates and lungs, infectious viruses were not recovered from those tissues from most of the surviving animals at 5 dpi ( Figures 5F, H ). Taken together, the above results illustrated that equine IgG or F(ab’)₂ treatment significantly inhibited viral replication in the upper and lower tracts, which may block shedding of infectious SARS-CoV-2.

The gross pathology and histological assays were evaluated to check the pathological changes in the lungs at 3 dpi. The pathological section of the lung from controlled animals showed that the tissue airway epithelial cells were tightly arranged, and there was no obvious epithelial cell degeneration and necrosis; more necrotic epithelial cells (blue arrows) can be found in individual airway cavities in the local tissues ( Figure 6E ), and the number of airway epithelial cells were reduced; a large range of alveolar cavities were narrowed, and a small amount of alveolar wall capillary congestion (black arrow), and a small amount of neutrophil infiltration was obvious and perivascular edema with a handful of inflammatory cell infiltration (yellow arrow) in the local alveolar cavity ( Figures 6A, B, F ); spleen result prompted that the red pulp and white pulp were clearly demarcated and spotted apoptosis of lymphocytes, nuclear pyknosis and deep staining or fragmentation (black arrows) were shown in the spleen nodules, and the expansion of germinal centers (yellow arrows) could be seen locally ( Figures 6C, G ); scattered neutrals were mostly seen in the red pulp granulocyte infiltration (red arrow) ( Figure 6D ); more brown‒yellow particles (blue arrow) were found in the red pulp ( Figures 6G, H ).

The lungs of the surviving mice showed that the bronchial epithelial structure was intact, the epithelial cells were normal and tightly arranged, and the alveolar structure was clear and not significantly thickened without any obvious inflammation ( Figure 6I ). The splenic tissue capsule was composed of dense connective tissue with uniform thickness and rich in elastic fibers and smooth muscle fibers. The capsule connective tissue extended into the spleen to form trabeculae, with no obvious abnormalities. Hepatic cords were arranged neatly, with no obvious expansion or squeezing of liver sinusoids ( Figure 6J ). The liver lobule was clearly demarcated, and the staining nuclei in the liver cells were blue. The nuclei were large, round, and centered, with little heterochromatin and light staining, and the nucleoli could be clearly seen ( Figure 6K ). The renal tissue was evenly stained, the renal cortex and medulla were clearly demarcated, the glomerulus was normal in shape and structure, and the epithelial cells of renal tubules were closely arranged without obvious inflammation ( Figure 6L ).

To further identify the effectiveness of purified equine immunoglobulin IgG and F(ab’)₂, after combining the observation of pathological sections, we continued to perform a detailed histopathological assessment of the lungs of the mice at 5 dpi after challenge using immunohistochemistry by detecting SARS-CoV-2 nucleocapsid. Regardless of the prevention group or the treatment group, after the application of IgG or F(ab’)₂, the mouse lungs were normal and no virus was detectable ( Figures 6M, N, P, Q ). SARS-CoV-2 nucleocapsid was detected in the lung tissue by immunohistochemistry (IHC) staining, especially in conducting airway epithelia and in the alveoli ( Figures 6O, R ). Based on the above histopathological results, equine immunoglobulin IgG and F(ab’)₂ could effectively clear SARS-CoV-2 in the lungs of mice.

The immunocompetent Syrian golden hamster has also been used to evaluate equine pAb activity against SARS-CoV-2 infection in the upper and lower respiratory tracts (40, 41). We used this animal model to independently assess the inhibitory activity of equine IgG or F(ab’)₂. Weights were followed for 6 days, and then tissues were harvested for virological, histopathology and immunohistochemistry analysis. The treatment strategy is shown in Figure 7A . At 500 μg of equine immunoglobulins, hamsters treated with equine immunoglobulin IgG or F(ab’)₂ and infected with wild-type SARS-CoV-2 Wuhan01 showed a 100% survival rate ( Figure 7B ), protection against weight loss ( Figure 7C ), and reduced viral burden levels in the lungs and nasal swabs compared to the independent control ( Figures 7D, E ). Correspondingly, RT‒qPCR and TCID₅₀ analysis showed reduced SARS-CoV-2 RNA replication in the lungs of hamsters treated with equine immunoglobulin IgG or F(ab’)₂. Although treated animals were positive for vRNA in turbinates and lungs by RT-qPCR ( Figures 7D, E ), infectious viruses were not recovered from most of these animals via TCID₅₀ analysis ( Figures 7F, G ). Overall, when a 500 μg dose of IgG or F(ab’)₂ was administered, equine pAbs established a trend toward protection against weight loss in hamsters infected with SARS-CoV-2 Wuhan01.

For the surviving hamster, the structure of the lung tissue was basically normal, and the alveolar wall in the visual field was uniform without thickening. The alveolar outline was clear with normal size. The alveolar epithelial cell nucleus was round with no necrosis; no inflammatory cell infiltration was found in the lung parenchyma ( Figures S3A, E ). For the control group, the lung tissue structure was severely abnormal ( Figures S3B, F ), and proliferation of alveolar epithelial cells is shown by the yellow arrow ( Figure S3B ). A small amount of inflammatory cell infiltration and hemorrhage was found in the lung parenchyma, as shown by the red arrow ( Figure S3F ). Immunohistochemistry results indicated that SARS-CoV-2 was negative in all treatment groups ( Figures S3C, G ) and was detectable in all control groups ( Figures S3D, H ).

Female BALB/c mice aged 9 months, female hartly guinea pigs aged 6~8 weeks, and female golden hamsters aged 4~6 weeks were purchased from Changchun Yisi Experimental Animal Technology Center. Horses aged 5~6 years were kept in Red Hill Horse Farm and given proper animal welfare. BALB/c mouse-adapted SARS-CoV-2 (BMA8), VSV-vectored pseudoviruses of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) VOCs and VOIs expressing the reporter gene of enhanced green fluorescent protein (eGFP) were generated and stored in the Biosafety Level 3 Laboratory (BSL-3). Our previous study has reported that BMA8 was obtained by passaging the SARS-CoV-2 Wuhan01 in aged mice for 8 generations, which a mouse-adapted virus cause 100% death in aged BALB/c mice. The mouse model was characterized by high viral loads in the upper and lower respiratory tract, accompanied by lymphopenia, and neutrophilia. Q498H (S), A22D (E) and A36V (E) emerged in structural protein, while five mutations were identified in non-structural protein (69). Wild type SARS-CoV-2 Wuhan01 was gifted from Pro.Chenfeng Qin, an isolation derived from a COVID-19 patient of Wuhan (BetaCov/Wuhan/AMMS01/2020). Spodoptera frugiperda Sf9 (ATCC^® CRL-1711™) insect ovary cells were grown in Sf-900™ II serum-free medium (Life Technologies, San Diego, CA, USA) at 27°C at 200 rpm in suspension culture for baculovirus infection and protein expression. Vero E6 cells (ATCC^® CRL-1587™) were cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) (DMEM; GIBCO, Grand Island, NY) containing 10% fetal bovine serum (Gibco, ThermoFisher, MA, USA) with 5% CO₂ for BMA8 propagation and titration.

The complete genome sequence of SARS-CoV-2 was obtained from GenBank (ID: NC_045512.2), and codons in the RBD (aa319~541) were optimized for protein expression in Sf9 cells (Sangon Biotech, Shanghai, CN). The RBD region gene was cloned into the pFastBac1 vector to generate the recombinant plasmid pFastBac1-RBD. pFastBac1-RBD was transformed into E. coli DH10Bac competent cells (Life Technologies, San Diego, CA, USA) to produce recombinant bacmid. According to the manufacturer’s instructions, ExpiFectamine Sf transfection reagent (Gibco, Thermo Fisher, MA, USA) was used to transfect recombinant bacmid into Sf9 cells. The recombinant baculovirus rBV-RBD was harvested 4 days after transfection. To generate recombinant RBD protein, Sf9 cells were infected with rBVs in a volume of 300 mL with a cell density of 1~2×10⁷/mL. At 120 h post-infection, cells and debris were removed from the supernatant by centrifugation at 5,000 rpm for 20 min in a JA-10 rotor (Beckman J-20XP, Fullerton, USA). The cell culture supernatant was harvested and subjected to affinity purification through HisPur™ Ni-NTA Spin Columns (Thermo Fisher, MA, USA) to obtain high-purity recombinant RBD protein.

For western blot analysis, 10 ug high-purity recombinant RBD protein samples separated through SDS-PAGE were transferred onto Polyvinylidene fluoride (PVDF) membranes (Immobilin-p, Millipore, USA). The membranes were then blocked with 5% milk blocking buffer in Tris-buffered saline with Tween 20 (TBST) for 1 hour. A 1,000-fold dilution of the mouse polyclonal antibody (SinoBiological, CN) were used to detect the presence of RBD protein. A 10,000-fold dilution of the HRP-labeled goat anti-mouse IgG was utilized to identify and combine the primary antibody (Abcam, USA). The membranes were visualized using Western Blue stabilized substrate (Promega, USA).

Briefly, recombinant baculoviruses-infected Sf9 cells were fixed with 80% cold acetone. Next, the cells were subjected to IFA analysis by using 1,000-fold dilution of mouse monoclonal antibodies against SARS-CoV-2 RBD (SinoBiological, Peking, CN). After incubation with primary antibody, the cells were thoroughly washed with PBS and blocked with PBST. Then, FITC-labeled goat anti-mouse IgG (Millipore, Bedford, USA) was added with 0.3% Evans blue for 1 h as a secondary antibody. The plate was placed under a fluorescence microscope (ZEISS Axio Vert. A1, Germany) to observe and capture the fluorescence.

Three 5~6-year-old healthy male horses were fed under standard conditions. After emulsifying 1.5 mg of RBD protein in 1 mL PBS with an equal volume of Freund’s complete adjuvant (Thermo Fisher, USA), horses were vaccinated intramuscularly via a subcutaneous multipoint injection route. The subsequent immunization dosage was, respectively given 2.5, 2.5, 3.0, and 4.0 mg of protein in 1 mL PBS mixed with equal volume Freund’s incomplete adjuvant (Thermo Fisher, USA) for each horse via subcutaneous multipoint injection route. Each horse was immunized 5 times in total at 14-day intervals ( Figure S2A ). One week after each immunization, and the serum was harvested for further use.

The RBD protein expressed in E. coli was coated into a 96-well microtiter plate at a final concentration of 1 μg/ml and incubated overnight at 4°C. Subsequently, the plate were blocked with 2% BSA solution for 2 hours at 37°C, 100 μL of serum samples after serial twofold dilutions were added to each well and incubated at 37°C for 2 h. After the cells were thoroughly washed with PBS containing 0.5% Tween-20, 100 μL of 5,000-fold diluted HRP-labeled goat anti-horse antibody was added to each well, and the plates were incubated at 37°C for 1 h. After three washes, 100 μL TMB (3,3’,3,5’-tetramethyl benzidine) was added to each well and incubated for 15 min. Then, 50 μL 0.5 M H₂SO₄ solution was added to the well to stop the reaction in each well. The optical density (OD) was measured at 450 nm. The titer result was defined as the inverse of the maximum dilution of serum.

A 1/2 volume of saturated ammonium sulfate solution was added to an equal volume of serum diluted with saline. The mixture was gently stirred for 30 min at room temperature, centrifuged at 5,000 rpm for 20 min, and 1/3 volume of saturated ammonium sulfate solution was added. The mixture was incubated at room temperature for 30 min and separated at 5,000 rpm for 20 min. The precipitate was dialyzed in saline overnight at 4°C to remove salt.

The pH of equine antiserum was adjusted to 3.3 with 1 mol/L HCl. The equine antiserum was digested at 30°C for 2.5 h with pepsin activated by NaAc solution (pH=3.3) at a final concentration of 10,000 IU/mL. The digestion reaction was terminated by adding 1 mol/L NaOH to the phase solution to adjust the pH to 7.2. Subsquently, the above solution was purified by protein a column and then by protein g column. The purified protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purity of F(ab’)₂ was detected by a thin slice scan.

Immunoaffinity chromatography was used to purify RBD-specific IgG and F(ab’)₂. Sepharose 4B gel was utilized to prepare the RBD immunoaffinity column. The gel was resuspended in the coupling solution in which the RBD protein was dissolved and was placed on an overturning shaker overnight at 4°C. Subsequently, the gel was transferred to an empty purification column. After washing with coupling buffer, the gel was resuspended in 2% sodium borohydride (V/V) and incubated at room temperature for 30 min. After washing again, 0.1 mol/L Tris-HCl (pH=8.0) solution was used to resuspend and block at room temperature for 2 h. After washing, the gel was washed alternately with 0.1 mol/L glycine-HCl (pH=2.7) solution and washing buffer. The residual liquid was removed, and 0.02 mol/L PBS was used for equilibration. Total IgG prepared by the precipitation method or F(ab’)₂ digested by pepsin was mixed and combined with RBD-4B gel overnight at 4°C. RBD-specific equine immunoglobulin IgG and F(ab’)₂ were eluted with 0.1 mol/L glycine-HCl (pH=2.7). Residual glycine-HCl in the eluent was terminated with 0.1 mol/L Tris-HCl (pH=9.0) to prevent antibody inactivation. After purification, RBD-specific equine immunoglobulin IgG and F(ab’)₂ were obtained.

The purified immunoglobulin and F(ab’)₂ fragment prepared were analyzed by SDS-PAGE. Samples were mixed with SDS-PAGE loading buffer and boiled for 10 min. The prepared samples were electrophoresed through a 10% precast SDS‒PAGE gel. Finally, the SDS-PAGE gel was stained with Coomassie Brilliant Blue staining solution and incubated at room temperature for 30 min. A Tanon 3,500 automatic gel image analysis system (Bio-Equip, Pecking, CN) was used to capture images after decolorization.

We measured the cross-neutralizing activity of equine antibodies by wild type SARS-CoV-2 Wuhan01 and pseudovirus-based neutralizing assay. After the inactivated serum samples were diluted from 1:40 with a serial twofold dilution, 100 μL of diluted serum samples were added to a 96-well plate, and 100 TCID₅₀ wild type SARS-CoV-2 or VSV-eGFP-SARS-CoV-2 was added to each well. 1×10⁴ Vero E6 cells were added to each well after the virus-serum mixture was incubated at 37°C for 1 hour. Cytopathic effect (CPE) was detected under a microscope after the cells were cultured at 37°C for 48~72 hours. The neutralizing antibody titers were defined as the inverse of the highest dilution, which could completely inhibit the production of the cytopathic effect (CPE) compared with viral controls. Virus control and cell control were included in each test.

Two groups of 5 inbreed hartly guinea pigs were administered an intraperitoneal (IP) injection of l mL purified equine antisera (neutralization titer 1:20,480) or F(ab’)₂ (neutralization titer 1:20,480). Blood samples were collected at 0, 1, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h post-injection, and sera were used for determination of neutralization titers against wild-type SARS-CoV-2 Wuhan01. The half-life was defined as the time range post-infection in which during which the serum neutralizing antibody titer decreased by 50%.

Mice were randomized into six groups of 13. Each mouse was given 250 µg of antibody at a dose of 10 mg/kg. In the prevention group, BALB/c mice were first intraperitoneally given 250 µg purified equine IgG or F(ab’)₂ and challenged by intranasal administration of 100 TCID₅₀ mouse-adapted SARS-CoV-2 in 50 µL DMEM after 24 h. In the treatment group, BALB/c mice were first challenged with 100 TCID₅₀ mouse-adapted SARS-CoV-2 in 50 µL DMEM and given 250 µg purified equine IgG or F(ab’)₂ via IP injection 24 h post-infection. At 3 dpi and 5 dpi, four mice in each group were sacrificed. Lungs and turbinates were harvested to test the viral RNA loads, histopathology and immunohistochemistry test. The challenge strain of mouse-adapted SARS-CoV-2 named BMA8 was previously described (69). All mice were monitored for clinical signs of disease, survival rate, weight change and temperature changes.

Briefly, viral RNA in each tissue homogenates sample was extracted with an RNeasy Mini kit (QIAGEN, USA) following the manufacturer’s protocol. The viral RNA quantification was performed by RT-qPCR targeting the N gene of SARS-CoV-2. RT-qPCR was performed using One Step PrimeScript RT-PCR Kit (Takara, Dalian, China) with the following primers and probes: NF (5’-GGGGAACTTCTCCTGCTAGAAT-3’); NR (5’-CAGACATTTTGCTCTCAAGCTG-3’); and NP (5’-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3’). The details can be referred to our previous study (69).

The supernatant of tissue homogenate was serially diluted at a tenfold ratio, and 100μL were added into 96-well cell culture plates that had been plated with Vero E6 cells. The plates were incubated at 37°C for 1 h and then washed 3 times with sterile PBS. 100μL of incomplete medium containing 2% FBS and 1% penicillin‒streptomycin was added to each well of 96-well plate. The median tissue culture infective dose (TCID₅₀) was calculated by observing the number of wells with cytopathic effect (CPE) based on the Reed and Muench method after incubating for 72 h.

To determine the complete blood cell counts in mice, Blood was collected through the orbital venous plexus, blood was collected through the orbital venous plexus into anticoagulant tubes containing EDTAK2. Blood cell count was carried out under the automatic blood analyzer (BC-5000vet, Mindray, China), and the operation procedures were carried out according to the instrument operation instructions.

Mice were euthanized at the appointed time. Lungs, spleens, kidneys and livers were collected. Tissue samples from mice were fixed in 10% buffered formalin and embedded in paraffin wax. Lung tissue was stained with hematoxylin and eosin (H&E) for histopathological assessment. For immunohistochemistry (IHC), quenching of paraffin-embedded lung tissues was performed with 3% hydrogen peroxide in methanol for 10 min. The tissue was added with 500-fold dilution of mouse monoclonal anti-SARS-CoV-2 N antibody at 4°C for overnight. After incubation with primary antibody, sections were washed with PBS for 3 times and species-matched secondary antibodies were applied for 2 h at 4°C. The sections were observed under ZEISS Axio Vert. A1 microscope. The details of the histopathology and immunohistochemistry assays can be found in our previous study (69).

Hamsters were randomized into six groups of 9. Each Syrian hamster was given 250 µg of antibody at a dose of 10 mg/kg. In the prevention group, hamsters were administered purified equine IgG or F(ab’)₂ by IP injection and challenged with 1,000 TCID₅₀ wild-type SARS-CoV-2 Wuhan01 by the intranasal route in a final volume of 100 μL after 24 h. In the treatment group, hamsters were first challenged with 1,000 TCID₅₀ SARS-CoV-2 Wuhan01 and given purified equine IgG or F(ab’)₂ by IP injection 24 hours post-infection. All hamsters were monitored for body weight loss. At 3 dpi, four hamsters in each group were euthanized. Lungs and turbinates were harvested to detect the viral loads, histopathology and immunohistochemistry test.

All procedures involving cells and animals were conducted in a biosafety level 3 laboratory (BSL-3) and approved by the animal experimental committee of the Laboratory Animal Center, Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval number: IACUC of AMMS-11-2020-020). Animals are given enough drinking water, adequate food and comfortable living conditions in accordance with animal welfare principles.

Figures were generated using GraphPad Prism 8.0 software. Differences between means were evaluated using one-way ANOVA or two-way ANOVA and were deemed significant at P values of 0.05 or less.