Cells and Viruses
BHK-21 (CCL-10), A549 (CCL-185), HeLa (CCL-2), 293T (CRL-1573), 143B (CRL-8303), MRC-5 (CCL-171), HEK293/17 (CRL11268), THP-1 (TIB-202), ARPE-19 (CRL-2302) were purchased from the American Type Culture Collection (ATCC) and grown according to ATCC recommendations. CEF were purchased from Charles River (10100795) and grown in minimum essential medium (MEM) with 10% FBS (MEM10). HEK293T/ACE2 were a kind gift of Pamela J. Bjorkman47. We acknowledge Bernard Moss (LVD, NIAID, NIH) for the gift of wtMVA (NIH Clone 1) that was used solely as a reference standard. To produce sMVA and wtMVA virus stocks, CEF were seeded in 30 × 150 mm tissue culture dishes, grown to ~ 70–90% confluency, infected at 0.02 multiplicity of infection (MOI) with sMVA or wtMVA. Two days post infection, ultra-purified virus was prepared by 36% sucrose density ultracentrifugation and virus resuspension in 1 mM Tris-HCl (pH 9)48. Virus stocks were stored at -80ºC. Virus titers were determined on CEF by immunostaining of viral plaques at 16–24 h post infection using polyclonal Vaccinia antibody. FPV stocks were produced following propagation on CEF using FPV strain TROVAC (ATCC VR-2553)35 or HP1.44136, kindly provided by Bernard Moss. FPV titers were evaluated on CEF by virus plaque determination.
Construction of sMVA fragments
The three ~ 60 kbp sMVA fragments (F1-F3; Fig. 1) comprising the complete MVA genome sequence reported by Antoine et al. (NCBI Accession# U94848)4 were constructed as follows: sMVA F1 contained base pairs 191-59743 of the MVA genome sequence; sMVA F2 comprised base pairs 56744–119298 of the MVA sequence; and sMVA F3 included base pairs 116299–177898 of the reported MVA genome sequence4. A CR/HL/CR sequence arrangement composed of 5’-TTT TTT TCT AGA CAC TAA ATA AAT AGT AAG ATT AAA TTA ATT ATA AAA TTA TGT ATA TAA TAT TAA TTA TAA AAT TAT GTA TAT GAT TTA CTA ACT TTA GTT AGA TAA ATT AAT AAT ACA TAA ATT TTA GTA TAT TAA TAT TAT AAA TTA ATA ATA CAT AAA TTT TAG TAT ATT AAT ATT ATA TTT TAA ATA TTT ATT TAG TGT CTA GAA AAA AA-3’ was added in the same orientation to both ends of each of the sMVA fragments, wherein the italicized letters indicate the duplex copy of the MVA terminal HL sequence and the underlined letters indicate the CR sequences. Notably, the CR/HL/CR sequences incorporated at the ITRs of sMVA F1 and F3 were added in identical arrangement as the CR/HL/CR sequences occur at the ITRs at the genomic junctions of putative MVA replication intermediates4. The sMVA fragments were produced and assembled by Genscript using chemical synthesis, combined with a yeast recombination system. All sMVA fragments were cloned into a yeast shuttle vector, termed pCCI-Brick, which contains a mini-F replicon for stable propagation of large DNA fragments as low copy BACs in E. coli. sMVA F1 and F3 were cloned and maintained in EPI300 E. coli (Epicentre), while sMVA F1 was cloned and maintained in DH10B E. coli (Invitrogen).
SARS-CoV-2 S and N antigen sequences were inserted into the sMVA fragments by En passant mutagenesis in GS1783 E. coli cells49,50. Briefly, transfer constructs were generated that consisted of the S or N antigen sequence with upstream mH5 promoter sequence and downstream Vaccinia transcription termination signal (TTTTTAT), and a kanamycin resistance cassette flanked by a 50 bp gene duplication was introduced into the antigen sequences. The transfer constructs were amplified by PCR with primers providing ~ 50 bp extensions for homologous recombination and the resulting PCR products were used to insert the transfer constructs into the sMVA DNA by a first Red-recombination reaction49,50. Primers 5’- AAA AAA TAT ATT ATT TTT ATG TTA TTT TGT TAA AAA TAA TCA TCG AAT ACG AAC TAG TAT AAA AAG GCG CGC C-3’ and 5’-GAA GAT ACC AAA ATA GTA AAG ATT TTG CTA TTC AGT GGA CTG GAT GAT TCA AAA ATT GAA AAT AAA TAC AAA GGT TC-3’ were used to insert the N antigen sequence into the Del2 site. Primers 5’- ATA TGA ATA TGA TTT CAG ATA CTA TAT TTG TTC CTG TAG ATA ATA ACT AAA AAT TTT TAT CTA GTA TAA AAA GGC GCG CC-3’ and 5’-GGA AAA TTT TTC ATC TCT AAA AAA AGA TGT GGT CAT TAG AGT TTG ATT TTT ATA AAA ATT GAA AAT AAA TAC AAA GGT TC-3’ were used to insert the S antigen sequence into the IGR69/70 insertion site primers. Primers 5’- TTG GGG AAA TAT GAA CCT GAC ATG ATT AAG ATT GCT CTT TCG GTG GCT GGT AAA AAA TTG AAA ATA AAT ACA AAG GTT C-3’ and 5’-ACA AAA TTA TGT ATT TTG TTC TAT CAA CTA CCT ATA AAA CTT TCC AAA TAC TAG TAT AAA AAG GCG CGC C-3’ were used to insert the S or N antigen sequence into the Del3 site. Underlined letters indicate the sequences used to produce ~ 50 bp extensions for homologous recombination. The S and N antigen sequences were based on the SARS-CoV-2 reference strain (NCBI Accession# NC_045512) and codon-optimized for Vaccinia10,38. Inserted antigen sequences were verified by PCR, restriction enzyme digestion, and sequencing.
sMVA virus reconstitution
sMVA virus reconstitution from the three sMVA DNA plasmids in BHK cells using FPV as a helper virus was performed as follows8–10. The three sMVA DNA plasmids were isolated from E. coli by alkaline lysis51 and co-transfected into 60–70% confluent BHK cells grown in 6-well plate tissue culture plates using Fugene HD transfection reagent (Roche) according to the manufacturer’s instructions. At 4 hours post transfection, the cells were infected with approximately 0.1-1 MOI of FPV to initiate the sMVA virus reconstitution. The transfected/infected BHK cells were grown for 2 days and then every other day transferred, re-seeded, and grown for additional two days in larger tissue culture formats over a period of 8–12 days until most or all of the cells showed signs of sMVA virus infection. Using this procedure, characteristic MVA viral plaque formation and cytopathic effects (CPEs) indicating sMVA virus reconstitution was usually detected at 4–8 days post transfection/infection. Fully infected BHK cell monolayers were usually visible at 8–12 days post transfection/infection. sMVA virus from infected BHK cell monolayers was prepared by conventional freeze/thaw method and passaged once on BHK cells before producing ultra-purified virus stocks on CEF. sMVA or recombinant sMVA-CoV-2 vectors were reconstituted either with FPV HP1.441 (sMVA hp, sMVA-N/S, sMVA-S/N hp) or TROVAC (sMVA tv1 and tv2, sMVA-S tv, sMVA-N tv, sMVA-N/S tv, sMVA-S/N tv).
Host cell range
sMVA and wtMVA host cell range using various human cell lines (HeLa, 293T, MRC-5, A549, and 143B) BHK cells, and CEF was determined as follows. The cells were seeded in 6-well plate tissue culture format and at 70–90% confluency infected in duplicates with 0.01 MOI of sMVA or wtMVA using MEM2. At 2 hours post infection, the cells were washed twice with PBS and incubated for two days in normal growth medium (as described under cells and viruses). After the incubation period, virus was prepared by conventional freeze/thaw method and the virus titers of each duplicate infection was determined in duplicate on CEF.
To compare the replication kinetics of sMVA and wtMVA, CEF or BHK cells were seeded in 6 well-plate tissue culture format and at 70–90% confluency infected in triplicates at 0.02 MOI with sMVA or wtMVA using MEM2. After 2 hours of incubation, the cells were grown in MEM10. At 24 and 48 hours post infection, virus was prepared by freeze/thaw method and the virus titers of each triplicate infection and the inoculum was determined in duplicate on CEF.
Plaque size analysis
To compare the plaque size of sMVA virus and wtMVA, CEF or BHK cells were seeded in 6-well plate tissue culture format and at 70–90% confluency infected with 0.002 MOI with sMVA or wtMVA using MEM2. After 2 hours of incubation, MEM10 was added and the cells were grown for 16–24 hours. The cell monolayers were stained with Vaccinia virus polyclonal antibody and viral plaques were imaged using Leica DMi8 inverted microscope and measured using LAS X software. The size of 25 viral plaques per sMVA or wtMVA was calculated using the formula 𝐴𝑟𝑒𝑎= 𝜋×𝑎×𝑏, where a and b are the major and minor radius of the ellipse, respectively.
To characterize the viral DNA of the sMVA vectors by PCR, CEF were seeded in 6-well plate tissue culture format and at 70–90% confluency infected at 5 MOI with sMVA or wtMVA. DNA was extracted at 16–24 hours post infection by the DNA Easy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. All PCR reactions were performed with Phusion polymerase (ThermoFischer Scientific). Primers 5’-TCG TGG TGT GCC TGA ATC G-3’ and 5’-AGG TAG CGA CTT CAG GTT TCT T-3’ were used to detect MVA ITR sequences; primers 5’-TAT CCA CCA ATC CGA GAC CA-3’ and 5’-CCT CTG GAC CGC ATA ATC TG-3’ were used to verify the transition from the left ITR into the unique region; primers 5’-AGG TTT GAT CGT TGT CAT TTC TCC-3’ and 5’- AGA GGG ATA TTA AGT CGA TAG CCG-3’ were used to verify the Del2 site with or without inserted N antigen sequence; primers 5’-TGG AAT GCG TTC CTT GTG C-3’ and 5’-CGT TTT TCC CAT TCG ATA CAG-3’ with binding sites flanking the F1/F2 homologous sequences were used to verify the F1/F2 recombination site; primers 5’-TAT AGT CTT TGT GGC ATC CGT TG-3’ and 5’-ACC CAA ACT TTA GTA AGG CCA TG-3’ were used to verify the IGR69/70 insertion site with or without inserted S antigen; primers 5’-ATA AGC GTT GTC AAA GCG GG-3’ and 5’-AGG AAA TAG AAA TTG TTG GTG CG-3’ with binding sites flanking the F2/F3 homologous sequences were used to verify the F2/F3 recombination site; primers 5’-ACA TTG GCG GAC AAT CTA AAA AC-3’ and 5’-ATC ATC GGT GGT TGA TTT AGT AGT G-3’ were used to verify the Del3 insertion site with and without inserted S or N antigen sequences; primers 5’-TAT CCA CCA ATC CGA GAC CA-3’ and 5’-GTC TGT CCG TCT TCT CTA TTG TTT A-3’ were used to verify the transition from the unique region into the right ITR; and primers 5’-TTA ACT CAG TTT CAA TAC GGT GCA G-3 and 5’-TGG GGT TTC TTC TCA GGC TAT C-3’ were used to detect the SopA element of the BAC vector.
Restriction pattern analysis
BHK cells were seeded in 20 × 150 mm tissue culture dishes, grown to ~ 70–90% confluency, and infected at 0.01 MOI with wtMVA, sMVA tv1, or sMVA tv2. Ultra-purified virus was prepared two days post-infection as previously described48. Viral DNA (vDNA) was phenol/chloroform extracted, followed by ethanol precipitation as previously described52. DNA concentration and A260/A280 ratios were determined using NanoVue (GE Healthcare Bio-sciences Corp). 10 µg of vDNA were digested with 3 units of either KpnI or XhoI, followed by visualization on 0.5% EtBr-stained agarose gel that was run at 2.4v/cm, overnight.
Sequencing of sMVA fragments and genome
PacBio Long Read Sequencing analysis confirmed the integrity of the sMVA fragments and sMVA genome, including a single point mutation in a non-coding determining region at 3 base pairs downstream of 021L4 that was found both in sMVA F1 and in reconstituted sMVA. Briefly, 5 ug of fragmented DNAs were converted to SMRTbell libraries using the SMRTbell Template Prep Kit 1.0 (PacBio). The libraries were size-selected (7-kb size cutoff) with BluePippin (Sage Science). The size-selected libraries were loaded to SMRT cells using MagBeads loading and sequenced on a PacBio RSII with 10 hour movie. Read demultiplexing, mapping to the reference sequence (Vaccinia virus strain Ankara U94848.1), and variants calling were performed using the SMRT Link (v22.214.171.124841). The identified variants were visually inspected in SMRT view Genome Browser for confirmation. De novo assembly was done using either canu v1.7.1 or wtdbg2 v2.5. The 5’ start position of the assembled contig was edited by comparing to the U94848.1 reference.
BHK cells infected at 5 MOI were harvested 24-post infection. Proteins were solubilized in PBS with 0.1% Triton X-100, supplemented with protease inhibitor, then reduced and denatured in Laemmli buffer containing DTT and boiled at 95 °C for ~ 10 minutes. Proteins were resolved on a 4–20% Mini Protean TGX gradient gel (BioRad), and transferred onto PVDF membrane. S protein was probed with anti-SARS-CoV-1 S1 subunit rabbit polyclonal antibody (40150-T62-COV2, Sino Biological); N protein was probed with anti-SARS-CoV1 NP rabbit polyclonal antibody (40413-T62, Sino Biological). Vaccinia BR5 protein was probed as a loading control. Anti-rabbit polyclonal antibody conjugated with horseradish peroxidase (Sigma-Aldrich) was used as a secondary antibody and protein bands were visualized with chemiluminescent substrate (ThermoFisher).
HeLa cells were seeded in a 6-well plate (5 × 105/well) and infected the following day with sMVA vaccine candidates at an MOI of 5. Following an incubation of 6 hours, cells were detached with non-enzymatic cell dissociation buffer (13151014, GIBCO). Cells were either incubated directly with primary antibody or fixed and permeabilized prior to antibody addition. Anti-SARS-CoV-1 S1 mouse (40150-R007, Sino Biological) and S2 rabbit (GTX632604, GeneTex) monoclonal antibodies, anti-SARS-CoV-1 N rabbit monoclonal antibody (40143-R001, Sino Biological), and anti-vaccinia rabbit polyclonal antibody (9503 − 2057, Bio Rad) were used in dilution 1:2,000. One hour later anti-mouse or anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (A11001, A21206; Invitrogen) were added to the cells at a dilution of 1:4,000. Live cells were ultimately fixed with 1% paraformaldehyde (PFA).
BHK or HeLa cells were grown on glass coverslips and infected with sMVA or recombinant sMVAs encoding S and/or N proteins at an MOI of 5 for 6 hours at 37°C in a humidified incubator (5% CO2). After infection, cells were fixed for 15 min in 2% PFA and then directly permeabilized by addition of ice cold 1:1 acetone/methanol for 5 min on ice. Cells were blocked for 1 hr with 3% BSA at room temperature, incubated with primary antibody mix (1:500) against the S2 subunit or N for 1 hr at 37°C, and then incubated with Alexa-conjugated secondary antibodies (ThermoFischer) (1:2000) for 1 hr at 37°C, with washing (PBS + 0.1% Tween20) between each step. For detection of cell membranes and nuclei, cells were incubated with Alexa-conjugated wheat germ agglutinin at 5 µg/mL (Thermo Fisher) and DAPI for 10 minutes at room temperature. Coverslips were washed and mounted onto slides with Fluoromount-G (SouthernBiotech). Microscopic analysis was performed using a laser-scanning confocal microscope (Zeiss, LSM700). Images were acquired and processed using Zen software (Zeiss).
The Institutional Animal Care and Use Committee (IACUC) of the Beckman Research Institute of City of Hope (COH) approved protocol 20013 assigned for this study. All study procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. 6 weeks old C57BL/6 (C57BL/6J, 000664) or Balb/c (BALB/cJ, 000651) were purchased from the Jackson laboratories. C57BL/6 Nramp were bred at the City of Hope animal facility. Mice (N = 4–5) were immunized twice in three weeks interval by intraperitoneal route with 5 × 107 PFU (high dose) or 1 × 107 PFU (low dose) of sMVA, wtMVA, or sMVA-CoV2 vectors. To determine immune stimulation by both the S and N antigen when using separate vectors (Figures S9-10), mice were co-immunized via the same immunization schedule and route with half of the high (2.5 × 107 PFU) or low dose (0.5 × 107 PFU) of each of the vaccine vectors. Blood samples for humoral immune analysis were collected by retro-orbital bleeding two weeks post-prime and one-week post booster immunization. Splenocytes for cellular immune analysis were collected at one week post booster immunization and were isolated by standard procedure after animals were humanely euthanized.
Binding antibodies in mice immunized with sMVA, wtMVA, or sMVA-CoV2 vectors were evaluated by ELISA. ELISA plates (3361, Corning) were coated overnight with 1 µg/ml of MVA expressing Venus fluorescent marker9, S (S1 + S2, 40589-V08B1, Sino Biological), RBD (40592-V08H, Sino Biological) or N (40588-V08B, Sino Biological). Plates were blocked with 3% BSA in PBS for 2 hours. Serial dilutions of the mouse sera were prepared in PBS and added to the plates for two hours. After washing the plate, 1:3,000 dilution of HRP-conjugated anti-mouse IgG secondary antibody (W402B, Promega) was added and incubated for one additional hour. Plates were developed using 1-Step Ultra TMB-ELISA (34028, Thermo Fisher) for one to two minutes after which the reaction was stopped with 1M H2SO4. Plates were read at 450 nanometers wave length using FilterMax F3 microplate reader (Molecular Devices). Binding antibodies endpoint titers were calculated as the latest serum dilution to have an absorbance higher than 0.1 absorbance units (OD) or higher than the average OD in mock immunized mice plus 5 times the standard deviation of the OD in the same group at the same dilution. For evaluation of the IgG2a/IgG1 ratio, mouse sera were diluted 1:10,000 in PBS. The assay was performed as described above except for the secondary antibodies (1:2,000. goat Anti-Mouse IgG2a cross absorbed HRP antibody, Southern biotech, 1083-05; Goat anti-Mouse IgG1 cross absorbed HRP antibody, Thermo Fisher, A10551). The IgG2a/IgG1 ratio was calculated by dividing the absorbance read in the well incubated with the IgG2a secondary antibody divided the absorbance for the same sample incubated with the IgG1 antibody.
MVA neutralization assay.
ARPE-19 cells were seeded in 96 well plates (1.5 × 104 cells/well). The following day, serial dilutions of mouse sera were incubated for 2 hours with MVA expressing the fluorescent marker Venus9 (1.5 × 104 PFU/well). The serum-virus mixture was added to the cells in duplicate wells and incubated for 24 hours. After the 24 hours incubation period, the cells were imaged using Leica DMi8 inverted microscope. Pictures from each well were processed using Image-Pro Premier (Media Cybernetics) and the fluorescent area corresponding to the area covered by MVA-Venus infected cells was calculated.
SARS-CoV-2 pseudovirus production
The day before transfection, HEK293T/17 were seeded in a 15 cm dish at a density of 5 × 106 cells in DMEM supplemented with 10% heat inactivated FBS, non-essential amino acids, HEPES, and glutamine53. Next day, cells were transfected with a mix of packaging vector (pALDI-Lenti System, Aldevron), luciferase reporter vector and a plasmid encoding for the wild type SARS-CoV2 Spike protein (Sino Biological) or vesicular stomatitis virus G (VSV-G, Aldevron), using FuGENE6 (Roche) as a transfection reagent : DNA ratio of 3:1, according to manufacturer’s protocol. Sixteen hours post-transfection, the media was replaced and cells were incubated for an additional 24–72 hours. Supernatants were harvested at 24-, 48- and 72 hours, clarified by centrifugation at 1,500 RPM for 5 minutes and filtered using a sterile 0.22 µm pore size filter. Clarified lentiviral particles were concentrated by ultracentrifugation at 20,000 RPM for 2 hours at 4 °C. The pellet was resuspended in DMEM containing 2% heat inactivated-FBS and stored overnight at 4 °C to allow the pellet to completely dissolve. Next day, samples were aliquoted, snap frozen and stored at -80 °C for downstream assays.
SARS-CoV-2 pseudotype neutralization and ADE assay
Levels of p24 antigen in the purified SARS-CoV-2 pseudotype solution was measured by ELISA (Takara). Mouse sera were heat inactivated, pooled and diluted at a linear range of 1:100 to 1:50,000 in complete DMEM. For the neutralization assay, diluted serum samples were pre-incubated overnight at 4 °C with SARS-CoV-2-Spike pseudotyped luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen. HEK293T cells overexpressing ACE-2 receptor were seeded the day before transduction at a density of 2 × 105 cells per well in a 96-well plate in complete DMEM47. Before infection, 5 µg/mL of polybrene was added to each well. Neutralized serum samples were then added to the wells and the cells were incubated for an additional 48 hours at 37 °C and 5% CO2 atmosphere. Following incubation, cells were lysed using 40 µL of Luciferase Cell Culture Lysis 5x Reagent per well (Promega). Luciferase activity was quantified using 100 µL of Luciferase Assay Reagent (Promega) as a substrate. Relative luciferase units (RLU) were measured using a microplate reader (SpectraMax L, Molecular Devices) at a 570 nm wave length. The percent neutralization titer for each dilution was calculated as follows: NT = [1-(mean luminescence with immune sera/mean luminescence without immune sera)] x 100. The titers that gave 90% neutralization (NT90) were calculated by determining the linear slope of the graph plotting NT versus serum dilution by using the next higher and lower NT. In all the experiments RLU of uninfected cells was measured and was always between 50 and 90.
For the ADE assay, THP1 cells were seeded at a confluency of 2 × 106 cells/mL in a 96 well plate and co-incubated for 48 hours with serum samples diluted at 1:5,000 or 1:50,000 in the presence of SARS-CoV-2-Spike pseudotyped or VSV-G luciferase lentiviral vector, normalized to 100 ng/mL of p24 antigen. Following incubation, cells were lysed using 100 µL of ONE-Glo Luciferase Assay System per well (Promega). RLU were measured as above.
SARS-CoV-2 focus reduction neutralization test (FRNT)
FRNT assay was performed as described recently54. Briefly, HeLa-ACE2 cells were seeded in 12 µL complete DMEM at a density of 2 × 103 cells per well. In a dilution plate, pooled mouse serum was diluted in series with a final volume of 12.5 µL. Then 12.5 µL of SARS-CoV-2 was added to the dilution plate at a concentration of 1.2 × 104 pfu/mL.
After 1 h incubation, the media remaining on the 384-well plate was removed and 25 µL of the virus/serum mixture was added to the 384-well plate. The plate was incubated for 20 h after which the plate was fixed for 1 h. Each well was then washed three times with 100 µL of 1xPBS 0.05% tween. 12.5 µL of human polyclonal sera diluted 1:500 in Perm/Wash buffer (BD Biosciences 554723) were added to each well in the plate and incubated at RT for 2 h. Each well was further washed three times and peroxidase goat anti-human Fab (Jackson Scientific) was diluted 1:200 in Perm/Wash buffer, then added to the plate and incubated at RT for 2 h. The plate was then washed three times and 12.5 µL of Perm/Wash buffer was added to the plate then incubated at RT for 5 min. The Perm/Wash buffer was removed and TrueBlue peroxidase substrate was immediately added (Sera Care 5510-0030). Sera were tested in triplicate wells. Normal human plasma was used as negative controls for serum screening.
SARS-CoV-2 convalescent plasma samples
IBC Protocol 20004 approved the use of SARS-CoV-2 convalescent plasma. Anonymized plasma samples of SARS-CoV-2 convalescent individuals (N = 19) were obtained from UCSD. Individuals were confirmed to be infected in the previous three to ten weeks by PCR and lateral flow assay. All individuals were symptomatic with mild to moderate-severe symptoms. Serum samples (DS-626-G and DS-626-N, Seracare) purchased before SARS-CoV-2 pandemic were used as a negative control. SARS-CoV-2-specific binding antibodies in plasma samples were measured as described above. Cross-adsorbed goat anti-human IgG (H + L) secondary antibody (A18811, Invitrogen) was used at a dilution of 1:3,000.
T cell analysis
Spleens were harvested and dissociated using a cell mesh following which blood cells were removed using RBC Lysis Buffer (BioLegend). 2.5 × 106 splenocytes were stimulated with S or N peptide libraries (GenScript, 15mers with 11aa overlap, 1 µg/ml), 0.1% DMSO, or phorbol myristate acetate (PMA)-ionomycin (BD Biosciences) for 1.5 h at 37 °C. Anti-mouse CD28 and CD49d antibodies (1 µg/ml; BioLegend) were added as co-stimulation. Brefeldin A (3 µg/ml; eBioscience) was added, and the cells were incubated for additional 16 h at 37 °C. Cells were fixed using Cytofix buffer (BD Biosciences) and surface staining was performed using fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD3 (Clone 17A2, 555274, BD), BV650 anti-mouse CD8a (Clone 53 − 6.7, 563234, BD). Following cell permeabilization using Cytoperm buffer (BD Biosciences), ICS was performed using allophycocyanin (APC)-conjugated anti-mouse IFN-γ (Clone XMG1.2, 554413, BD), phycoerythrin (PE)-conjugated anti-mouse TNF-α (Clone MP6-XT22, 554419, BD), and PE-CF594 anti-mouse IL-2 (BD Biosciences (Clone JES6-5H4, 562483, BD). In experiments testing double recombinants SARS-CoV2 vectors IL-2 antibody was not included and PE-CF594 anti-mouse IL-4 (clone 11B11, 562450, BD) and BV421 rat anti mouse IL-10 (clone JES5-16E3, 563276, BD) were added. Events were acquired using a BD FACSCelesta flow cytometer (2 × 105 cells/tube). Analysis was performed using FlowJo. Antigen specific T cells were identified by gating on size (FSC vs SSC), doublet negative (FSC-H vs FSC-A), CD3+, CD8+/CD4+. Cytokine positive responses are presented after subtraction of the background response detected in the corresponding unstimulated sample (media added with Brefeldin A one hour after beginning of mock stimulation) of each individual mouse sample.
Splenocytes (1 × 106) from immunized mice were incubated in v-bottom wells in the presence of 2 µg/ml S or N peptide pools, or without stimulus in a volume of 200 µl. 48 hours later, plates were centrifuged 2000 RPM for 10 minutes and cell supernatant was collected and stored at -80˚C. Mouse TNF-alpha (MTA00B), Quantikine ELISA kit (R&D systems) was used according to manufacturer’s recommendations.
Statistical evaluation was pursued using GraphPad Prism (v8.3.0). For evaluation of differences in sMVA and wtMVA plaque area in BHK-21 and CEF cells and differences in sMVA and wtMVA host cell range, one-way ANOVA followed by Tukey’s and Dunnet’s multiple comparison tests were used, respectively. For sMVA and wtMVA growth kinetic analysis, mixed-effects model with the Geisser-Greenhouse correction, followed by Tukey’s multiple comparisons test were applied. For ELISAs, one-way ANOVA and Tukey’s multiple comparison tests were used to calculate differences in endpoint titers and group means between groups. For IgG2a/IgG1 ratio analysis, one-way ANOVA with Dunnett’s multiple comparison test was used to compare the IgG2a/IgG1 ratio measured in each group to a ratio of 1. Pearson correlation analysis was performed to calculate the correlation coefficient r and its significance. For T cell responses analysis, one-way ANOVA followed by Dunnett’s multiple comparisons test with a single pooled variance was used to compare the mean of each group.