One or two injections of MVA-vectored vaccine shields hACE2 transgenic mice from SARS-CoV-2 upper and lower respiratory tract infection

Significance Vaccines are required to control COVID-19 during the current pandemic and possibly afterward. Recombinant nucleic acids, proteins, and virus vectors that stimulate immune responses to the SARS-CoV-2 S protein have provided protection in experimental animal and human clinical trials, although questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic, and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited SARS-CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.

BioLegend). For surface detection, the cells were stained directly using the same primary and secondary antibodies. The binding of hACE2 to surface expressed S protein on infected HeLa cells was detected by incubating with 100 ng/10 6 cells of biotinylated human ACE2 protein (Cat# 10108-H08H-B, Sino Biological) followed by Alexa Fluor 647-conjugated anti-hACE2 antibody (Cat# FAB9332R, R&D Systems). The stained cells were acquired on a FACSCalibur cytometer using Cell Quest software and analyzed with FlowJo (BD Biosciences).
Detection of S-Binding Antibodies by ELISA. CoV-2 S protein produced in HEK293 cells was obtained from the NIAID Vaccine Research Center or Sino Biological and diluted in cold PBS to a concentration of 1 µg/ml. Diluted S protein (100 µl) was added to each well of a MaxiSorp 96well flat-bottom plate (Thermo Fisher). After incubation for 16-18 h at 4 o C, the wells were washed 3 times with 250 µl of PBS + 0.05% Tween 20 (PBS-T, Accurate Chemical) and plates were blocked with 200 µl PBS-T + 5% Nonfat Dry Milk for 2 h at room temperature. During the blocking phase, a series of eight 4-fold dilutions of each mouse serum sample was prepared in blocking buffer. After blocking, plates were washed 3 times with 250 µl of PBS-T and 100 µl of each 4-fold dilution of serum was added to the appropriate well(s) and incubated for 1 h at room temperature. After incubation with serum, plates were washed 3 times with 250 µl of PBS-T. HRP-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher) was diluted 1:4000 in blocking buffer and 100 µl of the secondary antibody was added to each well for 1 h at room temperature. For detection of antibody isotypes, peroxidase-conjugated isotype-specific antibodies were used (Thermo Fisher). Plates were washed 3 times with 250 µl of PBS-T and then 100 µl of KPL SureBlue TMB 1-component microwell peroxidase substrate (SeraCare) was added to each well. The chemiluminescence reaction was stopped after 10 min by addition of 100 µl of 1N sulfuric acid. Spectrophotometric measurements were made at A450 and A650 using a SpectraMax Plus 384 plate reader with SoftMax Pro analysis software (Molecular Devices). Final endpoint titers (1/n) for each sample were determined as 4-fold above the average OD of those wells not containing primary antibody (OD 0.03-0.04).
Stimulation and Staining of Lymphocytes. Splenocytes from individual mice or pooled from 3-5 mice were suspended at 1.5x10 7 cells/ml in RPMI (Quality Biological) supplemented with 10% heat-inactivated FBS, 10 U/ml penicillin, 10 µg/ml streptomycin, 2 mM L-glutamine, and 2 mM HEPES as previously described (3). Splenocytes (100 µl) were mixed with 100 µl of individual peptide pools in 96-well plates and incubated at 37 o C for 1 h after which brefeldin A (Sigma Aldrich) was added and incubation continued for 4-5 h. Staining of cells was performed at 4 o C. Fc receptors were blocked with anti-CD16/32 (Clone 2.4G2, a gift from Jack Bennink, NIAID) for 30 min. Surface staining was performed with anti-mouse CD3-FITC (Clone17A2; BioLegend), anti-mouse CD4-PE (Clone H129.19; BD Biosciences), and anti-CD8-PerCP-Cy5.5 (BD Biosciences) for 1 h. Cells were then fixed and permeabilized with Cytofix/Cytoperm solution and stained with IFNg-APC (BD Biosciences) for 1 h. Cells were washed with PBS and suspended in PBS containing 2% paraformaldehyde. Approximately 100,000 events were acquired on a FACSCalibur cytometer using Cell Quest software and analyzed with FlowJo (BD Biosciences).
Pseudovirus Neutralization Assay. The CoV-2 lentivirus pseudotype assay was carried out as described by Corbett et al. (4) using cells and plasmids obtained from the NIAID Vaccine Research Center. To determine neutralization titers, serum samples were heat inactivated for 30 min at 56°C and clarified by high speed microcentrifugation. The day before titration, 5,000 293T-hACE2.MF cells were seeded per well in 96-well white walled clear bottom tissue culture plates (Corning) in DMEM supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin with 3 µg/ml of puromycin. For each serum sample, duplicate 4-fold dilution series were prepared in 96-well U-bottom plates (Corning) in DMEM supplemented with 5% heat inactivated FBS with the starting dilution being 1:20 in a final volume of 45 µl per well. The pseudovirus was thawed at 37°C and 45 µl of a dilution previously shown to exhibit a 1000-fold difference in luciferase between uninfected and infected cells was added to all wells except for controls. After 45 min at 37°C, the medium was aspirated and 50 µl sample-virus mixture was added to each well and incubated for 2 h at 37°C. DMEM (150 µl) supplemented with 5% heat inactivated FBS was added per well and plates were incubated for 72 h at 37°C. Medium was removed from the wells and the cells were lysed with 25 µl per well of 1X cell lysis reagent (Promega), shaken at 400 rpm for 15 min at room temperature. Luciferase reagent (50 µl, Promega) was added per well and 90 s later relative luciferase units (RLU) were read on the luminometer (EnSight, Perkin Elmer, 570 nm wavelength, 0.1 mm distance, 0.3 s read). NT50 were calculated using Prism (GraphPad Software) to plot dose-response curves, normalized using the average of the no virus wells as 100% neutralization, and the average of the no serum wells as 0%.

MVA Neutralization Assay.
A semi-automated flow cytometric assay was carried out as previously described (5) except for substitution of MVA expressing GFP for the WR strain of VACV. Briefly, ten 2-fold serial dilutions of heat-inactivated serum from vaccinated mice were prepared in a 96-well plate and 6.25x10 3 PFU of MVA-GFP was added to each well and incubated at 37 o C for 1 h. Approximately 10 5 HeLa suspension cells were added to each well in the presence of 44 µg/ml of cytosine arabinoside. After 18 h at 37 o C, the cells were fixed in 2% paraformaldehyde and acquired with a FACSCalibur cytometer using Cell Quest software and analyzed with FlowJo. The dilution of mouse serum that reduced the percentage of GFPexpressing cells by 50% (IC50) was determined by nonlinear regression using Prism.

CoV-2 Challenge Virus.
CoV-2 USA-WA1/2019 was obtained from BEI resources (Ref# NR-52281) and propagated in a BSL-3 laboratory using Vero E6 cells cultured in DMEM+Glutamax supplemented with 2% heat-inactivated FBS and penicillin, streptomycin, and fungizone by Bernard Lafont of the NIAID SARS Virology Core laboratory. The TCID50 of the clarified culture medium was determined on Vero E6 cells after staining with crystal violet and scored by the Reed-Muench method.
Vaccination and Challenge Experiments. Prior to vaccination, the virus was thawed, sonicated twice for 30 s on ice and diluted to 2 x 10 8 PFU/ml in PBS supplemented with 0.05% bovine serum albumin. In an ABSL-2 laboratory, 50 µl of diluted virus was injected IM into each hind leg of the animal for a total dose of 2 x 10 7 PFU. Unless otherwise stated, baculovirus RBD protein provided by Eugene Valkov (NCI) was diluted to 0.2 mg/ml in PBS containing 0.3 mg/ml of QS-21 adjuvant (Desert King International, San Diego, CA) and 10 µg of RBD was injected IM into the left hind leg. All mice scheduled to be infected with SARS-CoV-2 were transferred to an ABSL-3 laboratory a few days prior to virus challenge. The challenge stock of SARS-CoV-2 USA-WA1/2019 was diluted to 2 x 10 6 TCID50/ml in PBS. Mice were lightly sedated with isoflurane and inoculated IN with 50 µl of SARS-CoV-2. After infection, morbidity/mortality status and weights were assessed and recorded daily for 14 days by the NIAID Comparative Medical Branch.

Determination of CoV-2 in Lungs and Nasal Turbinates. At 2-and 5-days post-infection with
CoV-2, lung and nasal turbinates were removed and placed in 1.5-2 ml of ice-cold Dulbecco's PBS and weights of lungs were recorded. Tissues were homogenized for three 25 s intervals in ice water using a GLH-1 grinder equipped with a disposable probe and aerosol proof cap (Omni International). Homogenates were cleared of debris by centrifugation at 4,000 xg for 10 min and the supernatants were transferred to sterile tubes and stored at -80 o C. Clarified homogenates were thawed and titrated in quadruplicate on Vero E6 cells using 10-fold serial dilutions in 96well microtiter plates. After 72-96 h, the plates were stained with crystal violet and scored using the Reed-Muench method to determine TCID50.
Determination of CoV-2 RNA in Lungs and Nasal Turbinates. Immediately after homogenization of lungs and turbinates, 0.125 ml was transferred to sterile tubes, 0.9 ml Trizol (Thermo Fisher) was added and the mixture frozen. After thawing, RNA was extracted using the Trizol Plus RNA Purification Kit with Phasemaker tubes (Thermo Fisher) following the manufacturer's instructions. Contaminating DNA was removed from the eluted RNA using the Turbo DNA-free kit (Thermo Fisher) and RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). CoV-2 S and N transcripts and 18s rRNA were quantified by ddPCR with specific primers (CoV-2 RNA Leader, Forward -CGA TCT CTT GTA GAT CTG TTC TCT AAA C; CoV-2 S, Reverse -TCT TAG TAC CAT TGG TCC CAG AGA; CoV-2 N, Reverse -GGT CTT CCT TGC CAT GTT GAG T; 18S, Forward -GGC CCT GTA ATT GGA ATG AGT C; 18S, Reverse -CCA AGA TCC AAC TAC GAG CTT) using an automated droplet generator and QX200 Droplet Reader (Bio-Rad). The values for CoV-2 S transcripts were normalized using the 18s RNA in the same sample.
Passive Serum Transfer. Serum for passive transfer was obtained from 20 BALB/c mice that were inoculated IM with rMVA S (WT) and 10 BALB/c mice with parental MVA at 0 and 3 weeks. Two weeks after the boosts, the MVA S and control MVA sera were pooled separately. Four naive K18-hACE2 mice each received 0.4 ml of MVA S serum and three received 0.4 ml of the control MVA serum. The following day, mice were bled to determine levels of SARS-CoV-2 binding and neutralizing antibody. Approximately 4 h later, the mice were challenged IN with 10 5 TCID50 of CoV-2. Mice were observed and weighed over the next two weeks.

Safety and Ethics.
All experiments and procedures involving mice were approved under protocol LVD29E by the NIAID Animal Care and Use Committee according to standards set forth in the NIH guidelines, Animal Welfare Act, and US Federal Law. Euthanasia was carried out using carbon dioxide inhalation in accordance with the American Veterinary Medical