cell
Madin-Darby canine kidney (MDCK) cells were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum (NCS) and 1% penicillin/streptomycin. Human fetal kidney 293 T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Lenti-X 293 T cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. MDCK-SIAT1 cells were maintained in DMEM supplemented with 1 mg/ml G418 geneticin, 10% FBS, and 1% penicillin/streptomycin.
virus
H5N1 viruses (A/Chicken/Ghana/AVL-76321VIR7050-39/2021 (H5N1; clade 2.3.4.4b) and A/India/SARI-4571/2021 (H5N1; clade 2.3.2.1a)) were generated. Genetics using a synthetic gene fragment cloned into pHH21 plasmid based on the sequence of GISAID 11. Contains 0.3% bovine serum albumin (BSA) and 0.5 μg/mL Np-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) were grown in MDCK cells in MEM. -Trypsinization. Experiments with highly pathogenic avian influenza viruses were conducted in a enhanced biosafety level 3 (BSL3) containment laboratory at the University of Tokyo, Japan, and have been approved for such use by Japan’s Ministry of Agriculture, Forestry and Fisheries. .
pseudotyped virus
Pseudotyped virus with HA and NA proteins from strain A/chicken/Ghana/AVL-76321VIR7050-39/2021 was transfected with Lenti-X 293 T by co-transfecting four plasmids, pCAG-H5HA and pCAG-N1NA. produced in cells. Coding sequences of H5-HA and N1-NA proteins, respectively. Lentiviral backbone plasmid pCI-neo synHIVgp-RRE carrying the gag and env genes of HIV. The pGreenFire Transcriptional Reporter Lentivector expresses green fluorescent protein (GFP) and firefly luciferase reporters. Supernatants containing pseudotyped H5N1 viruses were collected 48 h after transfection and filtered. TCID50 values of pseudotyped viruses were determined by measuring luciferase reporter activity in MDCK-SIAT1 cells.
Preparing the DS8390
In vitro T7 RNA polymerase-mediated transcription was used to synthesize mRNA from linearized DNA templates. This template, A/Chicken/Ghana/AVL-76321VIR7050-39/2021 (H5N1; clade 2.3.4.4b), was flanked by 5′ and 3′ untranslated regions and a poly-A tail. HA messenger RNA was purified and encapsulated into lipid nanoparticles (LNPs) composed of ionized lipids, phospholipids, cholesterol, and PEG lipids.
animal experiment
Experiments with mice were performed in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This protocol was approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number: PA20-06). Virus inoculation was performed under anesthesia with isoflurane, and animals were humanely euthanized by cervical dislocation under deep anesthesia with isoflurane to minimize suffering after viral infection.
Immunization of mice
BALB/c mice (7-week-old female, obtained from Japan SLC Co., Ltd.) were anesthetized with isoflurane and mock-immunized intramuscularly with vehicle buffer (10 mM histidine, 300 mM sucrose (pH 7.0)) or immunized with 1 or For immunogenicity analysis and virus challenge studies, 10 μg of DS8390 in 20 μL twice with a 2-week interval between immunizations.
Immunogenicity analysis of immunized mice
For analysis of humoral immunity and T cell responses, immunized mice were anesthetized with isoflurane 2 weeks after the second immunization with DS8390, and blood was collected by cardiac puncture under deep anesthesia. After euthanasia by cervical dislocation, the spleen was harvested and a single cell suspension was prepared.
Microneutralization assay
Virus-neutralizing antibody titers against H5 influenza virus were evaluated in serum samples. Serum samples were treated with receptor-destroying enzyme (RDE; Denka seiken) for 20 h at 37 °C, inactivated at 56 °C for 1 h, and diluted 1:10 in phosphate-buffered saline (PBS). did. Two-fold serial dilutions of serum were prepared in MEM, and each dilution was incubated with the same volume of virus dilution (100 TCID50/50 μL) in MEM containing 1 μg/mL TPCK-trypsin for 1 h at room temperature. did. The serum/virus mixture was added to 100% confluent MDCK cells seeded in 96-well plates the previous day. Cells were incubated at 37 °C for 3 days, and cytopathic effects (CPE) were evaluated visually and microscopically. Virus neutralizing titer was determined as the reciprocal of the highest serum dilution that completely prevented CPE. Each sample was analyzed for geometric mean titer in duplicate.
Pseudotype virus neutralization assay
The serum neutralizing activity against pseudotyped viruses with HA and NA proteins derived from H5 influenza virus was evaluated. Serum was treated with receptor-destroying enzyme (RDE; Denka seiken) for 18–20 h at 37 °C, inactivated at 56 °C for 30 min, and diluted 1:10 in OPTI/MEM. Two-fold serial dilutions of serum were prepared in OPTI/MEM, and each dilution was incubated with an equal volume of virus dilution (100 TCID50/50 μL) in OPTI/MEM for 30 min at 37 °C. The serum/virus mixture was added to 100% confluent MDCK-SIAT1 cells seeded in 96-well plates the previous day. Cells were incubated at 37°C for 2 days, after which luciferase activity was measured using the Bright-Glo luciferase detection system (Promega). Luminescent signals were measured using a plate reader (BMG Labtech). The cutoff value for determining the absence of virus infection was established based on the value of the negative control well, and the virus neutralization titer was determined as the reciprocal of the highest serum dilution that completely blocks infection. Each sample was analyzed for geometric mean titer in duplicate.
Recombinant protein expression and purification
To construct an expression plasmid for soluble recombinant HA (rHA) of A/Chicken/Ghana/AVL-76321VIR7050-39/2021 or A/India/SARI-4571/2021, the HA signal peptide and extracellular domain ( amino acid residues HA1- 1-HA2-176), stabilizing mutations forming disulfide bonds (HA1-M30C and HA2-K51C) and detoxified cleavage sites (KRRKR/G replaced by A/G; slash indicates the cleavage site) followed by T4. The foldon trimerization domain and C-terminal hexahistidine tag were cloned into the pCAGGS plasmid. Proteins were expressed in Expi293F cells (Thermo Fisher Scientific) and purified using TALON metal affinity resin (TaKaRa Clontech).
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed using rHA in mouse serum. ELISA plates were coated with 50 μl of antigen protein at a concentration of 2 μg/ml in PBS overnight at 4°C. After blocking with PBS containing 1% BSA, plates were incubated three times with heat-inactivated (30 min at 56 °C) serum serially diluted 5-fold in PBS containing 0.5% BSA and 0.05% Tween 20 (PBS -BT)). After incubation for 1 h at room temperature, plates were washed four times with PBS containing 0.1% Tween 20 (PBS-T) and treated with anti-mouse IgG (H + L) secondary antibody conjugated with horseradish peroxidase (1: PBS- 1:20,000 dilution in BT) for 1 hour at room temperature. Plates were then washed four times with PBS-T and developed with 1-Step Ultra TMB-ELISA substrate solution (Thermo Scientific). After 10 minutes of incubation, the reaction was stopped by adding 1N sulfuric acid. Absorbance was immediately measured at a wavelength of 450 nm.
Enzyme-linked immunospot (ELISpot) assay
Multiscreen 96-well plates with PVDF membranes (Millipore) were coated with anti-mouse IFN-γ capture antibody overnight at 4 °C and blocked for 2 h in RPMI 1640 medium containing 10% FCS. To prepare single cell suspensions from the spleen, the spleen of each mouse was individually cut into 2–3 mm pieces, incubated with collagenase D in Hank’s Balanced Salt Solution (HBSS) for 30 min at 37 °C, and incubated for 30 min at 37 °C. Gently triturated over balanced salts at 100°C. -μm cell strainer (BD Falcon). Dissociated cells were centrifuged, treated with 1X RBC lysis buffer (eBioscience) for 2 min, and washed with HBSS. Cells were then resuspended in RPMI 1640 containing 10% FCS, 1X MEM nonessential amino acid solution (Gibco), 1 mM sodium pyruvate (Gibco), and 0.05 mM 2-mercaptoethanol. Viable cell numbers were determined using trypan blue staining. Next, 2 × 105 live cells/well from each animal were seeded into precoated assay plates with 3 μg of homologous Ch/Ghana rHA or heterologous India/S4571 rHA protein in 120 μL of RPMI medium and incubated at 37 °C. Cultured in C. 3 days. Plates were then washed with water and processed to stain spots using the Mouse IFN-gamma ELISpot kit (BD) according to the manufacturer’s instructions. Stained spots were counted using an ImmunoSpot analyzer (CTL).
Viral challenge of immunized mice
For virus challenge studies, blood was collected from immunized animals via the submandibular vein under isoflurane anesthesia two weeks after the second immunization. Three weeks after the second immunization, mice were given a median mouse lethal dose (MLD50) of 10 A/Chicken/Ghana/AVL-76321VIR7050-39/2021 (H5N1; clade 2.3.4.4b) or A/India intranasally. I was infected internally. /SARI-4571/2021 (H5N1; clade 2.3.2.1a). Body weight and survival were monitored daily for 14 days. Mice with >25% weight loss or neurological symptoms were euthanized by cervical dislocation under deep anesthesia with isoflurane.
