Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice

Nanobody production and trimerization

Nanobodies targeting SARS-CoV-2 spike protein were selected as previously described12,13. Genes of N-terminally PelB (MKYLLPTAAAGLLLLAAQPAMA)-tagged tandem homotrimer of the nanobody connected with two (GGGGS)4 linkers and C-terminally 6×His-tagged were synthesized and subcloned in the pMES4 vector. The exact amino acid sequences are as below:



These expression vectors were introduced in the lipopolysaccharide-free electrocompetent BL21 (DE3) E. coli according to the manufacturer’s protocol (ClearColi: LGC Ltd., Middlesex, UK). The transformed colonies were selected and grown in the phosphate buffered broth. When the E. coli culture broth reached an OD of 0.6 AU, the final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside was added to the cells and the cells were continued to culture for several hours. The cultured E. coli cells were collected with centrifugation (2100 ×g, 4 °C for 10 min) and suspended with the TES buffer containing 200 mM Tris (pH 8.0), 0.5 mM EDTA, and 500 mM sucrose. After incubating the cells at 4 °C for 1 h, 2× volumes of a diluted TES buffer containing 50 mM Tris (pH 8.0), 0.125 mM EDTA, and 125 mM sucrose were added and the cells were further incubated at 4 °C for 45 min, and the supernatants were centrifuged (20,000 ×g, 4 °C for 10 min) and collected. The extracted nanobodies were purified using IMAC (Cytiva) and desalted with a dialysis membrane.

Cell lines

LentiX-HEK293T cells (Takara Bio #Z2180N) were maintained in DMEM (high glucose) (Sigma-Aldrich, #6429) containing 10% fetal bovine serum (FBS, Sigma-Aldrich #172012), and 1% penicillin-streptomycin (PS ) (Sigma-Aldrich, #P4333). HOS cells stably express human ACE2 and TMPRSS2 (HOS-ACE2-TMPRSS2 cells) were prepared as previously described15. VeroE6/TMPRSS2 cells were obtained from the JCRB Cell Bank of NIBIOHN for SARS-CoV-2 virion preparation.

Pseudoviral infectivity assay

HIV-1-based SARS-CoV-2 spike pseudotyped virus was prepared as described previously12,13. In brief, LentiX-HEK293T cells were transfected with plasmids encoding the C-terminally C9-tagged full-length SARS-CoV-2 spike variants (D614G, Beta, Gamma, Delta, and Omicron) and HIV-1 transfer vector encoding a luciferase reporter using PEI MAX transfection reagent (Polyscience #24765). Cells were incubated for 3.5 h at 37 °C with DNA/PEI mixture and with DMEM containing 10% FBS for another 48 h. The supernatants were then collected, filtered through a 0.45-mm membrane, and centrifuged. The pseudoviruses were incubated with four-fold sequentially diluted nanobodies for 1 h at 37 °C. As control, pseudoviruses were also incubated without nanobodies. Then, the pseudoviruses with and without nanobodies were added onto HOS-ACE2-TMPRSS2 cells and cultured for 2 days. The infected cells were lysed, and luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega KK, Osaka, Japan) with a microplate spectrophotometer (ARVO X3: PerkinElmer Japan Co., Ltd., Kanagawa, Japan). All assays were performed in triplicate and IC50 values ​​were calculated using the GraphPad Prism software. Original data are available in Supplementary Data.

Preparation of SARS-CoV-2 virions

Tokyo strain (SARS-CoV-2/UT-NCGM02/Human/2020/Tokyo) and Omicron strain (hCoV-19/Japan/NC928-2N/2021) were provided by the National Center for Global Health and Medicine. Delta strain (TKYTK1734) was provided by Tokyo Metropolitan Institute of Public Health. Tokyo strain and Delta strain were infected with VeroE6/TMPRSS2 at an MOI of 0.1 and then cultured in DMEM containing 2% FBS at 37 °C for 1 day. Omicron strain was infected with VeroE6/TMPRSS2 at an MOI of 0.1 and then cultured in DMEM containing 2% FBS at 37 °C for 3 days. The culture media were centrifuged at 1500 ×g for 10 min, then stored at –80 °C. To measure the viral titer, culture media were diluted serially by a factor of 10 with RPMI1640 containing 2% FBS and PS. The diluted culture media were incubated with VeroE6/TMRPSS2 cells (2 × 104 cells/well) in 96 well plates for 3 to 5 days, and viral titers of each strain were calculated using the Reed-Muench calculation method.

In vivo infection assay using huACE2 transgenic mice

huACE2 mice were obtained from the Laboratory Animal Resource Bank of the National Institute of Biomedical Innovation, Health and Nutrition. To maintain the heterozygous huACE2 mice, C57BL/6 mice and heterozygous huACE2 mice were mated. The genotypes of mice were analyzed by PCR for ear DNA using the primer sets 5′- CTTGGTGATATGTGGGGTAGA -3′ and 5′- CGCTTCATCTCCCACCACTT -3′. Male and female huACE2 mice were maintained in plastic cages with free access to food and water and housed at 25 ± 2 °C with a 12 h light/dark cycle. huACE2 Tg mice were assigned randomly to two groups (PBS-treatment (n= 6) and TP17/86 cocktail-treatment (n= 6)) to assess the protective efficacy of the TP17/86 cocktail. huACE2 Tg mice were inserted intubation tube (22G 32 mm, KN-1008-2, Natsume Seisakusho) using an otoscope and intubation platform under anesthesia (100 μl/mouse, Medetomidine: 20 μg/ml, Midazolam: 600 μg/ml, Butorphanol : 1 mg/ml) and then infected via respiratory tract with SARS-CoV-2 virus (ancestral and Delta, at a dosage of 1 × 104 TCID50 in 25 μl; Omicron at a dosage of 1 × 105 TCID50 in 25 μl) using a 100 μl micropipette. Infected mice were intraperitoneally injected with atipamezole (100 μl/mouse, 20 μg/ml). One day after infection, infected mice received intratracheally 1.2 mg/kg of TP17/86 cocktail (VHH) or PBS (Control) similar to how infection was performed. Body weight and survival of the infected mice were monitored every day for up to 14 days. Mice that were clearly emaciated were euthanized after recording their body weight and were considered dead. Original data are available in Supplementary Data.

Ethics statements

All mice experiments were performed in accordance with the Science Council of Japan’s Guidelines for the Proper Conduct of Animal Experiments. The protocols were approved by the Institutional Animal Care and Use Committee of NIBIOHN (approval ID: DSR02-24R3). All experiments with huACE2 Tg mice infected with SARS-CoV-2 were performed in enhanced BSL3 containment laboratories at the Tsukuba Primate Research Center of the NIBIOHN, following the approved standard operating procedures of the BSL3 facility.

Purification of viral RNA and RT-qPCR

huACE2 Tg mice were assigned randomly to two groups (PBS-treatment (n = 5) and TP17/86 cocktail-treatment ( n= 5)) to assess the viral load of SARS-CoV-2. Viral infection and administration of the TP17/86 cocktail were conducted as above. To measure the viral load of SARS-CoV-2 Tokyo strain in the lung, organs were homogenized in 3 ml of PBS using gentleMACSTM Dissociator and M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany). The lung RNAs were purified using 250 ul of lung lysate by TRIzol LS Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Reverse transcriptase (RT) reactions were performed with ReverTra Ace qPCR RT master mix with gDNA remover (TOYOBO, Osaka, Japan) using 500 ng of lung RNA. To quantify the SRAS-CoV-2 subgenomic RNA, the RT reaction products were diluted with 1/10 and 5 μl of the diluents were subjected to quantitative real-time PCR using THUNDERBIRD Probe qPCR Mix (TOYOBO) and primer/probe sets as follows ; 5′-CGATCTCTTGTAGATCTGTTCTC-3′ (forward primer), 5′-ATATTGCAGCAGTACGCACACA-3′ (reverse primer), and FAM-5′-ACACTAGCCATCCTTACTGCGCTTCG-3′-BHQ1 (probe). The qPCR conditions were 95 °C for 5 min, and 45 cycles of 15 s at 95 °C followed by 60 s at 60 °C. To examine the copy number of subgenomic RNA, PCR fragments amplified the same primer set as RT-qPCR were cloned into pMD vector and used for standards of RT-qPCR. To quantify the copy number of subgenomic RNA in the lung ( y), copy number obtained from RT-PCR (a) was calculated as follows:

$${{y}}=, {{a}}times frac{3000,({{{{{{rm{total}}}}}}},{{{{{{ rm{lung}}}}}}},{{{{{{rm{lysate}}}}}}})}{250,({{{{{{rm{lysate}}}}} }}},{{{{{{rm{for}}}}}}},{{{{{{rm{RNA}}}}}}},{{{{{{ rm{extraction}}}}}}})} times frac{({{{{{{rm{total}}}}}}},{{{{{{rm{RNA}}} }}}},{{{{{{rm{in}}}}}}},0.25,{{{{{{rm{ml}}}}}}},{{{ {{{rm{of}}}}}}},{{{{{{rm{lysate}}}}}}})}{500,({{{{{{rm{RNA }}}}}}},{{{{{{rm{for}}}}}}},{{{{{{rm{RT}}}}}}},{{{ {{{rm{reaction}}}}}}})} \ times frac{100,({{{{{{rm{total}}}}}}},{{{{ {{rm{RT}}}}}}},{{{{{{rm{reaction}}}}}}})}{5,({{{{{{rm{RT} }}}}}},{{{{{{rm{reaction}}}}}}},{{{{{{rm{for}}}}}}},{{{{ {{rm{RT}}}}}}}-{{{{{{rm{qPCR}}}}}}})}$$

Original data are available in Supplementary Data.

Statistical analyses

Statistical analyzes were performed by GraphPad Prism 7.0f (GraphPad Software, La Jolla, CA, USA). The Student’s two-tailedttest was used for body weight and RT-qPCR of subgenomic RNA, and the log-rung test was used for survival rate.p< 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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