HomeHealthMedicineIntratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of...

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-terminal PelB (MKYLLPTAAAGLLLLAAQPAMA)-tagged tandem homotrimer of the nanobody linked to two (GGGGS)4 linkers and C-terminal 6xHis-tagged 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 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 concentrations of 1 mM isopropyl-βD-thiogalactopyranoside were added to the cells and the cells were further cultured for several hours. The cultured E coli cells were collected by centrifugation (2100xg4°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 hour, 2x 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 1 hour. 45 min, and the supernatants were centrifuged (20,000 xg, 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) with 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 SARS-CoV-2 virions.

Pseudoviral infectivity test

HIV-1 based SARS-CoV-2 spike pseudotyped virus was prepared as previously described12.13. Briefly, LentiX-HEK293T cells were transfected with plasmids encoding the C-terminal C9-tagged full-length SARS-CoV-2 spike variants (D614G, Beta, Gamma, Delta, and Omicron) and HIV-1 transfer vector containing encodes a luciferase reporter with PEI MAX transfection reagent (Polyscience #24765). Cells were incubated for 3.5 hours at 37°C with DNA/PEI mixture and an additional 48 hours with DMEM containing 10% FBS. The supernatants were then collected, filtered through a 0.45 mm membrane and centrifuged. The pseudoviruses were incubated for 1 hour at 37°C with 4-fold serially diluted nanobodies. As a control, pseudoviruses were also incubated without nanobodies. Subsequently, the pseudoviruses with and without nanobodies were added to HOS-ACE2-TMPRSS2 cells and cultured for 2 days. The infected cells were lysed and the 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 is available in Supplementary Data.

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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 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 grown in DMEM with 2% FBS at 37°C for 1 day. The Omicron strain was infected with VeroE6/TMPRSS2 at an MOI of 0.1 and then grown in DMEM with 2% FBS at 37°C for 3 days. The culture media was centrifuged at 1500xg for 10 min, then stored at –80 °C. To measure viral titer, culture media was serially diluted 10-fold with RPMI1640 containing 2% FBS and PS. The diluted culture media were incubated with VeroE6/TMRPSS2 cells (2 x 1064 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 test with 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 on ear DNA using the primer sets 5′-CTGGTGATATGTGGGGTAGA-3′ and 5′-CGCTTCATCTCCCACCACTT-3′. Male and female huACE2 mice were kept in plastic cages with free access to food and water and housed at 25 ± 2°C with a 12 hour light/dark cycle. huACE2 Tg mice were randomly assigned 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 32mm, 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 subsequently infected via the respiratory tract with SARS-CoV-2 virus (ancestral and Delta, at a dose 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 injected intraperitoneally with atipamezole (100 µl/mouse, 20 µg/ml). One day after infection, infected mice received intratracheally 1.2 mg/kg 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 considered dead. Original data is available in Supplementary Data.

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Ethical Statements

All mouse experiments were performed in accordance with the Science Council of Japan’s Guidelines for the Correct Conduct of Animal Experiments. The protocols have been 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 BSL3 enhanced containment laboratories at NIBIOHN’s Tsukuba Primate Research Center, following the approved BSL3 facility standard operating procedures.

Purification of viral RNA and RT-qPCR

huACE2 Tg mice were randomly assigned 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 performed 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 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 of lung RNA. To quantify the subgenomic RNA of SRAS-CoV-2, the RT reaction products were diluted by 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′-CGATTCTGTAGATCTGTTCTC-3′ (forward primer), 5′-ATATTGCAGCAGTACGCACACA-3′ (reverse primer), and FAM-5′-ACACTAGCCATCCTTACTGCGCTTCG-3′-BHQ1 (probe). The qPCR conditions were 95°C for 5 minutes and 45 cycles of 15 seconds at 95°C followed by 60 seconds at 60°C. To examine subgenomic RNA copy number, PCR fragments amplified with the same primer set as RT-qPCR were cloned into pMD vector and used for standards of RT-qPCR. To quantify the number of copies of subgenomic RNA in the lung ( j), copy number obtained from RT-PCR (a) was calculated as follows:

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

Original data is available in Supplementary Data.

static analysis

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

Report summary

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

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