Intratracheal administration of a cocktail of trimerized nanobodies suppresses weight loss and prolongs survival in mice infected with SARS-CoV-2 – Communications Medicine

Production and trimerization of nanobodies

Nanobodies targeting SARS-CoV-2 spike protein were selected as previously described^12.13. N-terminal labeled tandem homotrimer PelB (MKYLLPTAAAGLLLLAAQPAMA) genes of the two-connected nanobody (GGGGS)₄ linkers and 6 x C-terminal His-tag were synthesized and subcloned into the pMES4 vector. The exact amino acid sequences are as follows:

TP17, MKYLLPTAAAGLLLLAAQPAMAQVQLQESGGGLVQAGGSLRLSCAASGRTSSVYNMAWFRQTPGKEREFVAAITGNGGTTLYADSVKGRLTISRGNAKNTVSLQMNVLKPDDTAVYYCAAGGWGKERNYAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCAASGRTSSVYNMAWFRQTPGKEREFVAAITGNGGTTLYADSVKGRLTISRGNAKNTVSLQMNVLKPDDTAVYYCAAGGWGKERNYAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCAASGRTSSVYNMAWFRQTPGKEREFVAAITGNGGTTLYADSVKGRLTISRGNAKNTVSLQMNVLKPDDTAVYYCAAGGWGKERNYAYWGQGTQVTVSSHHHHHH;

TP86, MKYLLPTAAAGLLLLAAQPAMAMAQVQLQESGGGLVQAGGSLRLSCVASGRTFSSLNIVWFRQAPGKERKFVAAINDRNTAYAESVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCHSADVNGGMDYWGKGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCVASGRTFSSLNIVWFRQAPGKERKFVAAINDRNTAYAESVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCHSADVNGGMDYWGKGTQVTVSSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCVASGRTFSSLNIVWFRQAPGKERKFVAAINDRNTAYAESVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCHSADVNGGMDYWGKGTQVTVSSHHHHHH.

These expression vectors were introduced into the lipopolysaccharide-free electrocompetent BL21 (DE3) E.coli according to manufacturer’s protocol (ClearColi: LGC Ltd., Middlesex, UK). Transformed colonies were picked and grown in phosphate buffered broth. When the E.coli the culture broth reached an OD of 0.6 AU, the final concentrations of 1 mM isopropyl-β-D-thiogalactopyranoside were added to the cells, and the cells were continued in culture for several hours. the cultivated E.coli cells were harvested by 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 incubated at 4°C for 45 min, and supernatants were centrifuged (20,000 ×g, 4°C for 10 min) and recovered. 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 the preparation of the SARS-CoV-2 virion.

Virus-like infectivity assay

HIV-1-based SARS-CoV-2 pseudotyped virus was prepared as previously described^12.13. Briefly, LentiX-HEK293T cells were transfected with plasmids encoding full-length C9-tagged SARS-CoV-2 spike variants (D614G, Beta, Gamma, Delta, and Omicron) and the transfer vector of the HIV-1 encoding a reporter luciferase 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 an additional 48 h. The supernatants were then collected, filtered through a 0.45 mm membrane and centrifuged. Pseudoviruses were incubated with nanobodies sequentially diluted four times for 1 h at 37°C. As a control, pseudoviruses were also incubated without nanobodies. Then, the pseudoviruses with and without nanobodies were added on HOS-ACE2-TMPRSS2 cells and cultured for 2 days. 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 tests were performed in triplicate and IC₅₀ values ​​were calculated using GraphPad Prism software. The original data is 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. The delta strain (TKYTK1734) was provided by the Tokyo Metropolitan Institute of Public Health. The Tokyo strain and the Delta strain were infected with VeroE6/TMPRSS2 at an MOI of 0.1, then cultured in DMEM containing 2% FBS at 37°C for 1 day. The Omicron strain was infected with VeroE6/TMPRSS2 at an MOI of 0.1, then cultured in DMEM containing 2% FBS at 37°C for 3 days. Culture media were centrifuged at 1500 ×g for 10 min, then stored at –80°C. To measure viral titer, culture media were serially diluted 10-fold with RPMI1640 containing 2% FBS and PS. Diluted culture media were incubated with VeroE6/TMRPSS2 cells (2 × 10⁴ cells/well) in 96-well plates for 3-5 days, and viral titers of each strain were calculated using the Reed-Muench calculation method.

In vivo infection test using huACE2 transgenic mice

The huACE2 mice were obtained from the laboratory animal resource bank of the National Institute of Biomedical Innovation, Health and Nutrition. To maintain heterozygous huACE2 mice, C57BL/6 mice and heterozygous huACE2 mice were mated. Mouse genotypes were analyzed by PCR for ear DNA using the primer sets 5′-CTTGTGATATGGTGGGGTAGA-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 randomly assigned to two groups (PBS treatment (not= 6) and TP17/86 cocktail-care (not= 6)) to evaluate the protective efficacy of the TP17/86 cocktail. huACE2 Tg mice were inserted into an intubation tube (22G 32 mm, KN-1008-2, Natsume Seisakusho) using an otoscope and an intubation platform under anesthesia (100 μl/mouse , Medetomidine: 20 μg/ml, Midazolam: 600 μg/ml, Butorphanol: 1 mg/ml) then infected by the respiratory tract with the SARS-CoV-2 virus (ancestral and Delta, at a dose of 1 × 10⁴ TCID₅₀ in 25 µl; Omicron at the dose of 1 × 10⁵ TCID₅₀ in 25 μl) using a 100 μl micropipette. The infected mice received an intraperitoneal injection of 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 infected mice were monitored daily for up to 14 days. Mice that were clearly emaciated were euthanized after recording their body weight and were considered dead. The original data is available in Supplementary Data.

Ethics statements

All mouse experiments were performed in accordance with the guidelines of the Scientific Council of Japan for the proper conduct of animal experiments. Protocols were approved by the NIBIOHN Institutional Animal Care and Use Committee (Approval ID: DSR02-24R3). All experiments with SARS-CoV-2 infected huACE2 Tg mice were performed in improved BSL3 containment laboratories at NIBIOHN’s Tsukuba Primate Research Center in accordance with approved standard operating procedures of the BSL3 facility.

Purification of viral RNA and RT-qPCR

HuACE2 Tg mice were randomly assigned to two groups (PBS treatment (not = 5) and TP17/86 cocktail-care ( not= 5)) to assess the viral load of SARS-CoV-2. Viral infection and administration of the TP17/86 cocktail were carried out as above. To measure the viral load of the SARS-CoV-2 Tokyo strain in the lungs, the organs were homogenized in 3 ml of PBS using gentleMACS^MT Dissociator and M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany). Lung RNAs were purified using 250 µl 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 lung RNA. To quantify SARS-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 the THUNDERBIRD Probe qPCR Mix (TOYOBO) and primer/probe sets as follows; 5′-CGATCTCTTGTAGATCTGTTCTC-3′ (forward primer), 5′-ATATTGCAGCAGTTACGCACACA-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 subgenomic RNA copy number, PCR fragments amplified with the same set of primers as RT-qPCR were cloned into the pMD vector and used for RT-qPCR standards. To quantify the number of subgenomic RNA copies in the lung ( there), copy number obtained by RT-PCR (a) was calculated as follows:

The original data is available in Supplementary Data.

statistical analyzes

Statistical analyzes were performed by GraphPad Prism 7.0f (GraphPad Software, La Jolla, CA, USA). The Two-Tailed StudentyouThe test 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.

Summary of reports

Further information on the research design can be found in the summary of Nature portfolio reports linked to this article.