VeroE6/TMPRSS2 (JCRB 1819) cells22,23 were propagated in the presence of 1 mg/ml geneticin (G418; Invivogen) and 5 μg/ml plasmocin prophylactic (Invivogen) in Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Calf Serum (FCS) and antibiotics, and maintained at 37 °C with 5% CO2. Chinese hamster ovary (CHO) cells were maintained in DMEM containing 10% FCS and antibiotics at 37 °C with 5% CO2. Expi293 cells (Thermo Fisher Scientific) were maintained in Expi293 expression medium (Thermo Fisher Scientific) at 37 °C under 8% CO2. The cells were regularly tested for mycoplasma contamination by using PCR, and confirmed to be mycoplasma-free.
hCoV-19/Japan/NC928-2N/2021 (Omicron; NC928)10, SARS-CoV-2/UT- NC002-1T/Human/2020/Tokyo (NCGM02)7, and SARS-CoV-2/UT-HP095-1N/Human/2020/Tokyo (D614G; HP095)23 were propagated in VeroE6/TMPRSS2 cells in VP-SFM (Thermo Fisher Scientific). All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan.
Amino acid sequences for the variable region of the heavy and light chains of the following human monoclonal antibodies against the S protein were used for gene synthesis: clones tixagevimab (COV2-2196/AZD8895; GenBank accession numbers QLI33947 and QLI33948), casirivimab (REGN10933; PDB accession numbers 6XDG_B and 6XDG_D), cilgavimab (COV2-2130/AZD1061; GenBank accession numbers QKY76296 and QKY75909), imdevimab (REGN10987; PDB accession numbers 6XDG_A and 6XDG_A), and S309 (PDB accession numbers 6WS6_A and 6WS6_F). An artificial signal sequence and the constant gamma heavy (IgG1, UniProtKB/Swiss-Prot accession number P01857) and kappa (UniProtKB/Swiss-Prot accession number P01834) or lambda (UniProtKB/Swiss-Prot accession number P0DOY2) light chain coding sequences were added before and after each variable region. Codon usage was optimized for expression in CHO cells. The synthesized genes were cloned into a plasmid for protein expression and transfected into CHO cells. Cell culture media were harvested after incubation for 10–14 days at 37 °C. A human monoclonal antibody (1430E3/9) against the hemagglutinin of influenza B virus24 was previously cloned into the expression vector Mammalian Power Express System (TOYOBO) and was transiently expressed by Expi293 cells. Monoclonal antibodies were purified by using MabSelect SuRe LX (Cytiva) or a protein A column. Purity was confirmed by SDS-PAGE and/or HPLC before use. The reactivities of these antibodies against SARS-CoV-2, including the Alpha, Beta, Delta, Gamma, and Omicron variants, have been tested previously10.
Molnupiravir (EIDD-2801) was purchased from MedChemExpress. S-217622 was kindly provided by Shionogi Co., Ltd.. All compounds were dissolved in 0.5% methylcellulose prior to use in in vivo experiments.
Evaluation of therapeutic efficacy of mAbs and antiviral compounds in Syrian hamsters.
Five- to six-week-old male Syrian hamsters (Japan SLC Inc., Shizuoka, Japan) were used in this study. For the evaluation of mAb efficacy in hamsters, under isoflurane anesthesia, five hamsters per group were inoculated intranasally with 103 PFU (in 30 μl) of HP095 or NC928. On Day 1 post-infection, the hamsters were injected intraperitoneally with 1 ml of a mAb preparation (5 mg/kg). The animals were euthanized on Day 4 post-infection, and the virus titers in the nasal turbinates and lungs were determined by use of plaque assays on VeroE6/TMPRSS2 cells.
For the evaluation of antiviral compound efficacy in hamsters, under isoflurane anesthesia, four hamsters per group were inoculated intranasally with 103 PFU (in 30 μl) of Omicron (NC928). At 24 h after inoculation, hamsters were treated with the following antiviral compounds: (1) molnupiravir, 500 mg/kg (in 1 ml) administered orally twice daily; (2) S-217622, 60/kg (in 1 ml) administered orally twice daily; or (3) methylcellulose (1 ml) as a control for oral treatment. The animals were euthanized on Day 4 post-infection, and the virus titers in the nasal turbinates and lungs were determined by use of plaque assays on VeroE6/TMPRSS2 cells.
All experiments with hamsters were performed in accordance with the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments and the guidelines set by the Institutional Animal Care. The protocols were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number PA19-75).
Enzyme-Linked Immunosorbent Assay (ELISA).
ELISAs were performed as previously reported25. Briefly, ninety-six-well Maxisorp microplates (Nunc) were incubated with the recombinant receptor-binding domain (RBD) of the S protein (50 μl/well at 2 μg/ml), or with PBS at 4 °C overnight and were then incubated with 5% skim milk in PBS containing 0.05% Tween-20 (PBS-T) for 1 h at room temperature. The microplates were reacted for 1 h at room temperature with hamster serum samples that were initially diluted 40-fold in PBS-T containing 5% skim milk and subsequently serially 2-fold diluted, followed by peroxidase-conjugated goat anti-human IgG, Fcγ Fragment specific antibody (Jackson Immuno-Research) for 1 h at room temperature. Then, 1-Step Ultra TMB-Blotting Solution (Thermo fisher scientific) was added to each well and incubated for 3 min at room temperature. The reaction was stopped by the addition of 2 M H2SO4 and the optical density at 450 nm (OD450) was immediately measured. The average OD450 values of two PBS-wells were subtracted from the average OD450 values of the two RBD-wells for background correction. A subtracted OD450 value of 0.1 or more was regarded as positive; the minimum dilution to give a positive result was used as the ELISA titer.
Determination of half-maximal effective concentration (EC50) values.
VeroE6/TMPRSS2 cells were seeded in 96-well plates one day prior to infection, and were incubated at a multiplicity of infection (MOI) of 0.01 with SARS-CoV-2 at 37 °C for 1 h. The inocula were then replaced with MEM containing 5% FCS and serially diluted S-217622, and the cells were incubated at 37 °C with 5% CO2 for 2–3 days to observe cytopathic effects (CPE). The 50% effective concentration (EC50) was determined by using the Spearman–Karber formula26 based on the appearance of visually detectable CPE in quadruplicate experiments.
GraphPad Prism software was used to analyze all the data. We compared virus titers in hamster organs with the control by using a one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test or the Kruskal-Wallis test followed by Dunn's test with multiple comparisons. Differences between groups were considered significant for P values < 0.05.
All data supporting the findings of this study are available within the paper and from the corresponding author upon request. There are no restrictions to obtaining access to the primary data.
No code was used in the course of the data acquisition or analysis.
All reagents described in this paper are available through Material Transfer Agreements.