Materials
The SARS-CoV-2 pseudovirus was obtained from (GENEWIZ, Batch Number: PSEUV02). The SARS-CoV-2 authentic virus was obtained from (Harbin Veterinary Research Institute, Batch Number: SARS-CoV-2/HRB24/human/2020/CHN). The recombinant SARS-CoV-2 S wild-type (Cat.# SPN-C52H9-50ug), SARS-CoV-2 Delta (Cat.# SPN-C52He-50ug), SARS-CoV-2 Lambda (Cat.# SPN-C52Hs-50ug), SARS-CoV-2 Omicron (Cat.# SPN-C52Hz), ACE-2 (Cat.# AC2-H52H8-50ug), biotinylated ACE-2 (ACE-2-b) (Cat.# AC2-H82E6-200ug) were obtained from ACROBiosystems. The mFc-tagged ACE-2 (Cat.# 10,108-H05H) and SARS-CoV-2 S Omicron (Cat.# 40,589-V08H26) was obtained from SinoBiologicals.
Cell transfection, antibody expression, and antibody purification
The Expi293F cells (Thermo Fisher Scientific) were cultured in a humidified chamber at 37 °C, 5% CO2 in Expi293 expression medium (Thermo Fisher Scientific). They were transiently transfected with plasmids encoding antibodies using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific) as instructed by the manufacturer and stated in detail in our previous publication8. Briefly, the Expi293F cells were plated in fresh media at a density of 1.7 × 106 cells/ml and cultured overnight. The next day, DNA and ExpiFectamine reagent were separately mixed in Opti-MEM, and incubated at room temperature for 3 min, and combined. After further incubation for 20 min at room temperature, it was added to cells followed by the addition of enhancers at 17 h after transfection. The cells were harvested 72 h after transfection, the cell supernatant was passed through a 0.45 µm membrane, and the antibody concentration was measured by biolayer interferometry using GatorPrime (Gator Bio) using a Protein A probe. The antibodies were purified by Protein A columns in an AKTA Explorer 100 purification system, dialyzed twice in PBS, and finally passed through a 0.22 µm membrane.
Animal studies
All animal studies were performed in contract with Shanghai Model Organisms Center, Inc.
Pseudovirus infection study
6–8-week-old female Ubc-CreER x Rosa-CAG-LSL-ACE2-IRES-tdTomato mice, weighing ~ 20–30 g, were used for this study. A total of 11 mice were obtained from Shanghai Model Organisms Center, Inc. Animals were housed in a specific pathogen free (SPF) laboratory animal room and provided food and water ad libitum. To induce the expression of huACE-2, the mice were administered with 100 mg/kg tamoxifen by intraperitoneal (I.P.) injection five times from pre-inoculation day 11 to 3, on every other day. Three days after the last tamoxifen dose (day 0), mice were intranasally (I.N.) challenged with 6.05 × 104 transducing units (TU) of SARS-CoV-2-luc pseudovirus expressing luciferase at Day 0 (D0). Mice were then randomly divided into 3 groups. No treatment was provided to the control group of 4 mice (Group 1). The treatment groups were treated with 10 mg/kg of ABS-VIR-001 by I.N. administration at 10 h pre-infection (-10 hpi, Group 2, prophylaxis model) of 3 mice or 2 h post-infection (+ 2 hpi, Group 3, post-exposure treatment model) of 3 mice, respectively. The body weights of mice were monitored daily and bioluminescence (BLI) measurements of SARS-CoV-2-luc were obtained on D3, D4 and D7.
Authentic SARS-CoV-2 infection study
6–8-week-old male NM-KI-200272 CAG-human ACE2-IRES-Luciferase-WPRE-polyA mice, weighing ~ 20–30 g, were used for this study. A total of 22 mice were obtained from Shanghai Model Organisms Center, Inc. Animals were housed in a SPF laboratory animal room in a Biosafety Level 3 Laboratory of Harbin Veterinary Research Institute and provided food and water ad libitum. The mice were I.N. challenged with 1000 PFU of SARS-CoV-2 (SARS-CoV-2/HRB24/human/2020/CHN) at D0. Then, the mice were randomly divided into 4 groups. No treatment was provided to the control group of 5 mice (Group 1). The treatment groups were treated with 25 mg/kg of ABS-VIR-001 by I.N. administration at 10 h pre-infection (-10 hpi, Group 2, prophylaxis model) of 5 mice or 2 h post-infection (+2 hpi, Group 3, post-exposure treatment model) of 6 mice, or with 10 mg/kg of ABS-VIR-001 by intraperitoneal (I.P.) administration 2 h post-infection (+ 2 hpi, Group 4, post-exposure treatment model) of 6 mice. The body weights and survival of mice were monitored daily and after D4 (D3 if the mouse succumbed to infection sooner), the lung was harvested and measured for SARS-CoV-2 titers using rt-PCR.
ABS-VIR-001 safety study
6–8-week-old female M-NSG mice weighing ~ 17–23 g was used for this study. A total of 16 mice were obtained from Shanghai Model Organisms Center, Inc. Animals were housed in a SPF laboratory animal room and provided food and water ad libitum. The mice were randomly divided into 4 groups. PBS was provided I.N. and intravenously (I.V.) to the control group of 4 mice (Group 1). The treatment groups were treated with 25 mg/kg of ABS-VIR-001 by I.N. administration (Group 2) of 4 mice, 10 mg/kg of ABS-VIR-001 by I.N. administration (Group 3) of 4 mice, or 10 mg/kg of ABS-VIR-001 by I.V. administration (Group 4) of 4 mice. The body weights of mice were monitored and after D6, the mice were euthanized, and the blood was analyzed for hematological and metabolic parameters.
Antibody thermostability analysis
The thermostability of ABS-VIR-001 was assessed by heating the antibody to 45º C for up to four weeks. Each week, a sample of the heated ABS-VIR-001 sample was analyzed by differential scanning fluorimetry (DSF) method using the UNcle system (Unchained Labs) v4.01 (https://www.unchainedlabs.com/uncle/). For the DSF assay, the temperature was increased at 1 °C/min from 25 °C to 95 °C to obtain the melting temperature (Tm) of the sample. The data were analyzed and calculated by the UNcle Analysis Software. The binding kinetics of heated ABS-VIR-001 were also analyzed using biolayer interferometry, weekly.
Biolayer interferometry
Biolayer interferometry assays were performed to assess quantitative and qualitative ABS-VIR-001 binding using the GatorPrime system (Gator Bio) v2.7.3.0728 (https://www.gatorbio.com/).
Quantitative ABS-VIR-001 concentration assessment
A glass Protein A probe (Gator Bio) was dipped in purified ABS-VIR-001. Then, the binding signal was analyzed against an in-house Protein A-based standard curve and calculated by the GatorPrime (Gator Bio) software.
Quantitative ABS-VIR-001 binding kinetics
A glass hFC probe (Gator Bio) was initially dipped into Q Buffer (Gator Bio) for about 120 s yielding the baseline signal. Then the probe was loaded with 3–5 µg/ml of ABS-VIR-001 diluted Q Buffer for about 200 s and followed by a Q Buffer wash step of about 120 s. Next, the bound ABS-VIR-001 was associated with 100–3.125 nM of S protein (wild-type or mutants), including a no S protein control (0 nM) for about 180 s followed by a dissociation step of up to 900 s. The association and dissociation curves were graphed and calculated by the GatorPrime (Gator Bio) software to yield binding kinetics values (koff, Kon, and KD).
Qualitative ABS-VIR-001 binding and inhibition
A glass streptavidin probe (Gator Bio) was initially dipped into Q Buffer (Gator Bio) for about 120 s yielding the baseline signal. Then the probe was loaded with 5 µg/ml of ACE-2-b diluted in Q Buffer for about 60–120 s and followed by a Q Buffer wash step of about 120 s. Next, the bound ACE-2-b was associated with a 1:5 ratio or 1:20 ratio S protein (wild-type or mutants) to ABS-VIR-001 pre-mixture, including a no ABS-VIR-001 control (0 nM) for about 180 s followed by a dissociation step of up to 300 s. The association curves were graphed by the GatorPrime (Gator Bio) software to yield qualitative binding and inhibition signals in the absence or presence of ABS-VIR-001.
ELISA
High-binding 96-well plates were coated overnight with 1 µg/ml of SARS-CoV-2 S RBD at 4ºC. ABS-VIR-001 was prepared at 125 µg/ml and serially diluted fivefold and the three VHH-Fcs were prepared for the VHH-cocktail at 75 µg/ml of each and serially diluted fivefold in ELISA assay buffer consisting of 1 × PBS with 1% (w/v) BSA. After washing and blocking the plate, antibody dilutions were added and incubated for 30–45 min at RT with shaking. The plate was washed again and treated with goat anti-human Fc-HRP for an additional 30–45 min at RT with shaking. Finally, the plate was washed and treated with development buffer containing Amplex Red and H2O2 before detecting emitted fluorescent signal from each well on a SpectraMax Gemini XPS plate reader using SoftMaxPro v5.4 to detect binding. In contrast, if blocking was assessed, 0.45 ug/ml of ACE-2-b was added in lieu of anti-human Fc-HRP, followed by a Strep-HRP detection antibody, and emitted fluorescent signals were read using the SpectraMax Gemini XPS plate reader using SoftMaxPro v5.4.
Data analysis
The data was analyzed by the software Prism (GraphPad) v9.2.0 (https://www.graphpad.com/). Any statistical analysis performed are indicated in the figure legends.
Ethics statement
The mouse experiments were approved by the Institutional Animal Care and Use Committee of Harbin Veterinary Research Institute and performed in accordance with institutional guidelines. The studies were performed in compliance with the ARRIVE guidelines.
