The experimental objective was to characterize and evaluate the potential of SCVs as vaccines against SARS-CoV-2 in vitro and in vivo. For the evaluation process, cell cultures (described in detail in Cell lines) and animal models (described in detail in Animals) were used as study subjects. All experiments were controlled laboratory experiments. Animals’ conditions were examined daily and verification of vaccine/challenge virus included RT-qPCR-based techniques as well as Histological and Immunohistochemical procedures. The sample sizes and data collection endpoints were chosen based on previous experiments and literature surveys. No data was excluded and all outlier values are displayed. Experiments were performed at least in replicates and measurements were done in technical replicates or triplicates.
Human samples
Human blood samples for peripheral blood mononuclear cell (PBMC) isolation and neutralization assays were collected from SARS-CoV-2 vaccinated or/and infected (within 4 months) donors, aged 29–66 (median 32), who gave their informed consent. All donors were vaccinated with mRNA vaccines (Moderna or Pfizer/BioNTech) and 5 out of 6 had reported infections with SARS-CoV-2.
Animals
Specific pathogen-free male Syrian hamsters (Mesocricetus auratus) (Janvier labs, RjHan:AURA) were kept at 20 to 22 °C and a relative humidity of 45 ± 10% on a 12-h light/dark cycle, fed with commercial rodent chow (Ssniff, Soest, Germany), and provided with water ad libitum. The age of the animals at prime immunization is 5 weeks for ΔEG and 6 weeks for ΔEG68. Generally, hamsters underwent a daily physical examination and bodyweight routine. Hamsters were euthanized in deep anesthesia using isoflurane by severing the spinal cord in the area of the occiput and blood withdrawal from the cervical veins.
Cell lines
African green monkey kidney cells (Vero E6) were kindly provided by V. Thiel, Bern, Switzerland, or obtained from the Collection of Cell Lines in Veterinary Medicine CCLV-RIE 0929. Adenocarcinomic human alveolar basal epithelial cells expressing ACE2 and TMPRSS2 (A549-AT) were obtained from NIBSC (A549-ACE-2 Clone 8-TMPRSS2; product number 101006). The THP-1 myelomonocytic leukemia cell line was obtained from the American Type Culture Collection. HEK293T cells were kindly provided by D. D. Pinschewer, University of Basel, Switzerland.
All cells were maintained in DMEM high glucose with 10% fetal bovine serum (FBS) + 1% Penicillin/Streptomycin for general propagation or with 2% FBS + 1% Penicillin/Streptomycin for viral infection experiments. During the initial viral rescue, the JAK-I inhibitor Pyridone 6 (CAS 457081-03-7) was added to a final concentration of 2 µM as well as the NFκB inhibitor QNZ (CAS 545380-34-5) at 20 nM. HEK293T-indE received in addition Doxycycline (Merck, D5207) to a final concentration of 2 µg/mL for induction.
Cell line generation
HEK293T-E cells were generated by transfecting HEK293T with 2 µg plasmid DNA containing the SARS-CoV-2 E gene under CMV promoter control in a pcDNA3.1 background containing a Hygromycin resistance gene. After transfection cells were put in DMEM containing 250 µg/mL of Hygromycin. The selection was kept for two weeks and clones were generated by limiting dilution before E expression was tested by RT-qPCR. The clone that showed the highest RNA expression levels was kept for downstream application.
HEK293T-indE (HEK293T-E Tet:E-IRES-ORF6) cells are a derivative of HEK293T-E with a second-generation lentiviral vector generated with the pCW57-E-IRES-ORF6 (Addgene plasmid #80921) as a transfer vector. The vector codes for SARS-COV-2 E and ORF6 under a Tetracycline inducible promoter. After infection, cells were selected in DMEM containing 20 µg/mL of Blasticidin for two weeks. Cells were analyzed by RT-qPCR for E and ORF6 induction following doxycycline treatment (Supplementary Fig. 1c).
HEK293T-ACE2 cells were obtained by infecting the cells with a 2nd generation lentiviral vector with pHR-PGK_hACE2 (Addgene plasmid #161612) as a transfer vector. Cells were sorted for surface expression of ACE2 stained by Mouse anti-human ACE2 (R&D #FAB9332G).
Vero-E2T cells were generated by transfecting Vero E6 cells with 2 µg of an equimolar plasmid mixture containing the SARS-COV-2 E/ORF6/ORF7a/ORF8 genes in individual plasmids all under the CMV promoter in a pcDNA3.1 background containing a Hygromycin resistance gene. After transfection cells were cultivated in DMEM containing 250 µg/mL of Hygromycin. Human TMPRSS2 expression in Vero-E2T and Vero E6 cells (Vero E6-TMPRSS2) was achieved by infecting the cells with a 2nd generation lentiviral vector pLEX307-TMPRSS2-blast (Addgene plasmid #158458) as a transfer vector. After infection cells were selected in DMEM containing 20 µg/mL of Blasticidin for two weeks and analyzed by RT-qPCR for transgene expression (Supplementary Fig. 1b).
Plasmids and lentiviruses
The genes of interest from the Wuhan strain (B.1) were inserted into the pcDNA3.1 backbone under the control of the CMV promoter for expression. The all-in-E plasmid contains the SARS-CoV-2 genes E and ORF6 under the control of an ELF1α promoter or an IRES sequence, respectively, followed by ACE2 and TMPRSS2 under PGK promoter control separated by a P2A cleavage site in a pcDNA3.1 background. The integrity of all plasmids was verified by Sanger sequencing.
The plasmids required for the generation of second-generation lentiviruses were obtained from Addgene. Lentiviruses were generated by transfecting HEK-293T cells with pCMVR8.74 (RRID:Addgene_22036), pMD2.G (RRID:Addgene_12259), and pLEX307-TMPRSS2-blast (RRID:Addgene_158458) plasmids. The culture medium was changed 5 h after transfection, supernatant was collected 24 h later and filtered through a 0.22 µm filter to remove cellular debris.
Viral genome reconstitution procedures
Virus recovery was achieved as described in ref. 27. In brief PCR fragments (fr A-D) spanning the whole SARS-CoV-2 genome were amplified using the high-fidelity proofreading enzyme Q5® High-Fidelity DNA Polymerase (NEB, M0491L) in a 25 µL reaction volume using respective primers (Supplementary Fig. 1a and Supplementary Table 1). Fragment A contains the heterologous CMV promoter upstream of the 5′ UTR and fragment D contains the poly(A) tail, HDV ribozyme, and SV40 termination signal downstream of the 3′ UTR (Fig. 1a).
Cycling conditions were used as recommended by the manufacturer. Fragments were obtained using the following primer combinations: frA: CMV for + frA-frB rev; frB: frB-frA for + frB-frC rev; frC: frC-frB for + frC-frD rev; frD: frD-frC for + SV40 rev. DNA oligonucleotides used are listed in Supplementary Table 1.
12–30 reactions were pooled and purified by PCR column purification using QIAquick PCR purification kit (Qiagen, 28104). DNA concentration was measured by Nanodrop 1000 (Thermo Fisher) or Quantus (Promega, QuantiFluor® ONE dsDNA System, E4871). DNA was further purified by ethanol precipitation and the final concentration was adjusted to 1 µg/µL in nuclease-free water.
Equimolar ratios of frA, frB, frC, or frD (full-length or harboring gene deletions (∆frD)) and all-in-E plasmid were transfected into HEK293T-indE using jetPRIME® (Polyplus, cat. 101000001) as recommended by the manufacturer. 4–24 h post-transfection, the medium was changed to DMEM 2% FBS with the addition of JAK-I inhibitor Pyridone 6 (CAS 457081-03-7) to a final concentration of 2 µM as well as the NFκB inhibitor QNZ (CAS 545380-34-5) at 20 nM and 2 µg/mL Doxycycline and Vero-E2T were added for co-incubation. Every 3–4 days, the medium was exchanged. Screen for virus progeny production was done with SARS-CoV-2 antigen rapid test (Roche, 9901-NCOV-01G) or by cytopathic effect (CPE) in E2T and confirmed by RT-qPCR and FFU (focus forming unit) quantification.
Virus propagation for viral stocks
For wild-type controls, clinical isolates Muc-1/BavPat1 (a Wuhan-1-type virus isolate, provided by G. Kochs, University of Freiburg, Germany and by Bundeswehr Institute of Microbiology, Munich, Germany (SARS-CoV-2 Germany/BavPat1/2020, GISAID accession EPI_ISL_406862)), Omicron XBB.1.5 (isolated from nasopharyngeal aspirates of human donors, who had given their informed consent), synthetic SARS-CoV-2 (Wuhan-1, GenBank No. MT10878443) or rCoV2 (recombinant Wuhan-1-type virus produced by genome reconstitution27), were propagated in Vero E6 cells until CPE was observed.
For deletion mutants, viral particles produced by HEK293T-indE were further amplified in Vero-E2T cells, with additional trans-complementation of the all-in-E plasmid. Viral propagation was observed and monitored by CPE and antigen rapid tests27 and confirmed by RT-qPCR and FFA.
Final viral stocks were harvested, filtered by 0.2 µm filters to remove cells, and frozen in small aliquots. For each viral stock, the viral titer was determined by RT-qPCR and FFA or titration by plaque-forming assay.
Standard plaque-forming assay
Wild-type viral titers were determined by counting plaque-forming units (PFU) after incubation on susceptible cells. Vero E6 cells were seeded at a density of \(4*10^6\) cells/96-well flat bottom plate in DMEM 2% FBS and incubated overnight at 37 °C and 5% CO2. Virus was added 1:10 onto the cell monolayer in duplicates or triplicates and serially diluted 1:2 or 1:3. Plates were incubated for 2 days at 34 °C, 5% CO2 until plaque formation was visible. For virus inactivation, 80 µl of formaldehyde (15% w/v in PBS) (Merck, F8775) was added for 10 min to the cultures. After this period, fixative and culture medium were aspirated, and crystal violet (0.1% w/v) was added to each well and incubated for 5 min. Subsequently, the fixed and stained plates were gently rinsed several times with tap water and dried before analysis on a CTL ImmunoSpot® analyser.
RNA extraction for viral quantification and sequencing of viral stocks
Viral RNA was extracted using the automated Promega Maxwell RSC system (Promega, AS4500) using either the Maxwell® RSC Viral Total Nucleic Acid Purification Kit (Promega, AS1330) or the Maxwell® RSC miRNA from the Tissue and Plasma or Serum Kit (Promega, AS1680).
Sanger sequencing
The region of interest was amplified using SuperScript™ IV One-Step RT-PCR System (Thermo Fisher, 12594100) with either ‘D2 for’ or ‘26847 for’ and ‘29046N rev’ (for primers see Supplementary Table 1). The integrity of the PCR product was checked on agarose gel and subsequently sent for Sanger sequencing to evaluate genome regions affected by deletions/mutations (Microsynth, Switzerland).
Next-generation sequencing (NGS)
Viral RNA was converted to cDNA using a cDNA Synthesis kit (biotechrabbit). cDNA was NGS sequenced using EasySeq SARS-CoV-2 WGS Library Prep Kit (NimaGen, SKU: RC-COV096) on an Illumina NextSeq 2000 system with a P1 flow cell (300 cycles). All NGS sequencing and raw data analysis was done by Seq-IT GmbH & Co. KG.
RT-qPCR quantification of viral and intracellular RNA
For the detection of SARS-CoV-2 RNA, a primer and TaqMan probe set for ORF-1b (Supplementary Table 1) was used as described44. For the detection of SARS-CoV-2 E and TMPRSS2, an in-house primer/probe set was used (Supplementary Table 1). For the normalization of mRNA expression, GAPDH was used (Supplementary Table 1). For RT-qPCR, Luna® Universal Probe One-Step RT-qPCR Kit (E3006E) was used according to manufacturer´s protocol. In brief, Master Mix was set up: for one reaction 1 µL of each primer, 0.5 µL Probe, 10 µL of Luna Universal Probe One-Step Reaction Mix (2X), 1 µL of Luna WarmStart RT Enzyme Mix (20X) were mixed and brought to 15 µL with nuclease-free water. 15 µL of Master Mix were mixed with 5 µL RNA and amplified on ABI7500 fast cycler (ThermoFisher) using the following cycling conditions: 10 min 55 °C, 1 min 95 °C denaturation, followed by 45 cycles for 10 s at 95 °C and 30 s at 58 °C.
In vitro passaging for in vitro safety experiments
For viral passaging experiments, Vero E6 cells were infected with an MOI of 1 (based on FFU) for 3–4 h with wild-type virus or respective deletion candidates. The cells were then washed and fresh 2% DMEM medium was added. Every second day, supernatant (SN) was passaged on freshly seeded Vero E6 (50% confluency). SNs for passage 1 (p1) and p2 were diluted 1:10, for all subsequent passages, SN was diluted 1:100. All collected passages p1 to p10 were subsequently passaged on Vero-E2T. On day 3 and day 6 post-infection SN was sampled for RT-qPCR and images of cell cultures were taken with a Leica DM IL LED inverted microscope. All conditions were treated equally.
Biochemical procedures
For validation and comparison of vaccine candidate viruses, Vero E6-TMPRSS2 cells were infected with virus variants at an MOI of 0.1. 24 h after infection, cells were washed twice with PBS before lysis in cold 140 mM NaCl, 50 mM Tris-HCL, 1% Triton-X100, 0,1% SDS, 0,1% sodium deoxycholate supplemented with protease and phosphatase inhibitors (ThermoFisher, 1861281). Lysates were centrifuged for 10 min, 16,000 × g at 4°C, and supernatants analyzed by Immunoblot. Signals were acquired using an image analyzer (Odyssey CLx, Licor).
Flow cytometry analysis
Transfection
Cells were transfected using JetPrime (Polyplus, 101000001) transfection reagent according to the manufacturer’s protocol. Five hours after transfection, the culture medium was replaced. In the case of THP-1 cells, only ¼ of the recommended amount of DNA and reagents were used to avoid toxicity.
Infection
For cytometry experiments, all infections were conducted in DMEM supplemented with 2% FBS using a MOI of 0.1 based on FFU data.
Cell isolation and monocyte differentiation
PBMCs were isolated using Ficoll-Paque density gradient centrifugation. For each donor, 25 million PBMCs were initially obtained and cryopreserved. Subsequently, 2 million PBMCs of each donor were seeded per well of a 12-well culture plate in RPMI medium. The plate was then incubated at 37 °C for 1 h, after which non-adherent cells were collected and frozen, while the adherent cells underwent three consecutive rinses with room temperature PBS. Subsequently, the adherent cells were resuspended in RPMI medium containing 10% FBS and maintained in culture for 7 days before the experiment to induce monocyte-to-macrophage differentiation. No cytokine was added, and the media was changed every second day.
Cellular immune activation
In 12-well culture plates, 100,000 A549-ACE2-TMPRSS2 (A549-AT) cells were seeded and cultured in DMEM containing either 10,000 FFU of rCoV2, E**fs, ΔEG68, or no virus (control). Following a 24-h incubation period, the supernatant was harvested, centrifuged at 1000 × g to eliminate cellular debris, and subsequently diluted at a 1:3 ratio. This conditioned supernatant was subjected to the monocyte-derived macrophages (at day 7) from each donor. Following a 24-h incubation period, 2 million non-adherent PBMCs from the respective donor, along with 10,000 FFU of the relevant virus or control, were introduced into the wells. This was accompanied by the addition of Monensin at a final concentration of 2 µg/mL and the anti-CD107a antibody, diluted at a 1:200 ratio, to the culture medium. After 20 h of incubation and 4 h before fixation, Brefeldin A at a final concentration of 5 µg/mL was added to the medium.
Staining and fixation
For primary Lymphocytes, after a 24-h incubation and 4 h post Brefeldin A administration, non-adherent PBMCs were collected, washed in PBS, stained with Zombie UV® Fixable Dead Cell Stain (Biolegend) and subjected to a 20-min incubation in a blocking buffer comprising 50% FBS, Brefeldin A, Monensin, and FcR Blocking Reagent (diluted at 1:200). The cells were subsequently incubated for 30 min to label cell surface proteins in a staining buffer containing an antibody mix. Following this, the cells were fixed and permeabilized using the eBioscience™ Transcription Factor Staining Buffer Set, following the manufacturer’s recommendations for intracellular target analysis. Finally, intracellular staining was performed using an anti-IFNγ antibody for 30 min at room temperature. For transfection, cells were washed in PBS and stained with Zombie UV® Fixable Dead Cell Stain (Biolegend), rinsed once with PBS, and blocked in blocking buffer (PBS with 50% FBS), FcR Blocking Reagent 1:150 (Miltenyi Biotec) for 30 min at room temperature, followed by incubation with antibodies against cell-surface molecules in staining buffer (PBS with 15% FBS, FcR Blocking Reagent 1:1000) for 30 min at room temperature. Data were acquired on an Aurora (Cytek, Amsterdam, Netherlands) equipped with 5 lasers (355, 405, 488, 561, and 640 nm) and 60 channels (full spectrum cytometry), unmixed with SpectroFlo®, and analyzed with FlowJo 10.9.0 (TreeStar). The gating strategy is shown in Supplementary Information.
Immunocytochemistry
For detection of infectious vaccine viral particles (focus forming assay (FFA)), protein expression analysis, and surface labeling, Vero E6-TMPRSS2 cells grown on coverslips in 24-well plates were infected with virus variants in 500 µL DMEM medium supplemented with 2% FBS and 1% Penicillin/Streptomycin and incubated overnight. Cells were fixed with 4% PFA in PBS for 10 min at room temperature, washed, and subsequently stained. For FFU determination and protein expression analysis, cells were blocked with 10% Normal Donkey Serum (Jackson ImmunoResearch, 017-000-121) and 0.1% Triton X-100 at room temperature for 60 min followed by incubation with primary antibodies for 60 min at room temperature or overnight at 4 °C in 1% Normal Donkey Serum, 1% BSA and 0.3% Triton X-100 in PBS. Cells were washed three times for 10 min with 0.1% BSA/PBS and incubated with fluorophore-coupled secondary antibodies for 60 min at room temperature in 1% Normal Donkey Serum, 1% BSA and 0.3% Triton X-100 in PBS. Cells were washed once with 0.1% BSA/PBS and washed three times with PBS before mounting on microscope slides using Fluoromount-G (SouthernBiotech, 0100-01). For surface labeling, cells were blocked with 5% milk powder in PBS at room temperature for 1 h and incubated with primary antibodies in 1% BSA/PBS overnight at 4 °C. After 3 washes with PBS, fluorophore-coupled secondary antibodies in 1% BSA/PBS were applied for 60 min at room temperature and washed three times with PBS before mounting on microscope slides. Phalloidin-iFluor488 or -iFluor555 was co-applied with secondary antibodies to label F-actin (Abcam, ab176753 and ab176756 resp.). Hoechst 33342 dye (Merck, B2261) was co-applied during washing at a final concentration of 0.5 µg/mL for nuclear staining.
Images for FFU quantification were acquired on a bright-field microscope (Nikon Ti2 equipped with a Photometrics 95B camera, Nikon NIS AR software), using a 20x Plan-Apochromat objective (numerical aperture 0.75) and were then processed in Fiji and Omero. For quantification of infected foci, images were analyzed with QuPath45. Images for protein expression and surface labeling were acquired on an inverted spinning-disk confocal microscope (Nikon Ti2 equipped with a Photometrics Kinetix 25 mm back-illuminated sCMOS, Nikon NIS AR software), using 40x and 100x Plan-Apochromat objectives (numerical aperture 0.95 and 1.45, respectively) and were then processed in Fiji and Omero.
Electron microscopy
Viral particles were fixed in 1% glutaraldehyde (Thermo Scientific, 233281000). A 4 µL aliquot of sample was adsorbed onto holey carbon-coated grid (Lacey, Tedpella, USA), blotted with Whatman 1 filter paper and vitrified into liquid ethane at –180 °C using a Leica GP2 plunger (Leica microsystems, Austria). Frozen grids were transferred onto a Talos 200C Electron microscope (FEI, USA) using a Gatan 626 cryo-holder (GATAN, USA). Electron micrographs were recorded at an accelerating voltage of 200 kV using a low-dose system (40 e-/Å2) and keeping the sample at –175 °C. Defocus values were –2 to 3 µm. Micrographs were recorded on 4K × 4K Ceta CMOS camera.
Animal immunization and analysis
ΔEG immunization
Eight hamsters were intranasally inoculated with 50 µL of ΔEG virus stock per nostril (\(3.5*10^2\) FFU, Supplementary Fig. 3a, b) at day 0 and boosted with the same dose at day 21. Four hamsters were inoculated with 100 µL of supernatant from uninfected cells and therefore served as sham vaccinated controls. The three direct contact animals were co-housed with ΔEG immunized animals but were separated for 24 h just prior to immunizations and challenge, respectively. Nasal wash samples were taken on days –2, 3, 7, 24, 28, 36, 37, 39, 43, and 47 post immunization (dpim), by applying 200 µL of PBS into each nostril and collecting the reflux under short isoflurane inhalation anesthesia. Serum samples were taken by puncturing the V. saphena at 19 and 33 dpim for serological evaluation. At 35 dpim eight ΔEG immunized animals and four sham vaccinated control animals (intranasally inoculated with filtered medium of non-infected cells) were challenged by intranasal inoculation using 102.5 TCID50/animal of SARS-CoV-2 virus (Wuhan-1, GenBank No. MT10878443) in a 70 µL volume (calculated from back-titration). Five days post challenge (dpc), five ΔEG immunized hamsters and the sham vaccinated control hamsters were sacrificed following the ethical protocol approval, and sera as well as organ samples from the upper and lower respiratory tract were collected during necropsy. At 14 dpc, three ΔEG immunized hamsters and three contact animals were euthanized and serum samples as well as organ samples from upper and lower respiratory tracts were collected during necropsy.
ΔEG68 immunization
Twelve hamsters were intranasal inoculated with 100 µL of ΔEG68 virus stock (\(2.4*10^4\) FFU, Supplementary Fig. 3a, b) at day 0 and boosted with the same dose at day 21. Six direct contact animals were co-housed with ΔEG68 immunized animals but were separated for 24 h before immunizations and challenge infection, respectively. Nasal washing samples were taken at dpim –2, 3, 7, 24, 28, 36, 37, 38, 41 (dpc1), 42 (dpc2), 44 (dpc4) and 48 (dpc8) by applying 200 µL of PBS in each nostril and collecting the reflux under short isoflurane inhalation anesthesia. Serum samples were taken by puncturing the V. saphena at 19 and 33 dpim for serological evaluation. At 35 dpim the ΔEG68 immunized animals were inoculated using a miscalculated low dosage of SARS-CoV-2 virus (Wuhan-1, GenBank No. MT10878443) with less than 1 TCID50/animal. The viral genome copies in this highly over-diluted inoculum were determined by RT-qPCR (RNA-dependent RNA polymerase (IP4) as the target46) with a Ct-value of 35.64, representing 1089 genome copies / mL. With this sample, we were unable to initiate a productive infection even when 70 µL of pure inoculum was applied to Vero E6 cells (0.32 cm2, n = 7). In addition, all nasal wash samples taken from the animals on the first three days after inoculation were negative by RT-qPCR (Supplementary Table 2). Therefore, the challenge infection was repeated with the same animals at 41 dpim applying 70 µL with 102.3 TCID50/animal (Wuhan-1, GenBank No. MT10878443), calculated from a back-titration. Five days post challenge infection, six ΔEG68 immunized hamsters were euthanized and serum samples as well as organ samples from the upper and lower respiratory tract were collected during necropsy. 14 dpc six ΔEG immunized hamsters and their respective six matching contact animals were euthanized and serum samples as well as organ samples from the upper and lower respiratory tract were collected during necropsy.
RNA analysis of hamster samples
RNA from nasal washings and organ samples was extracted using the NucleoMag® VET Kit (Macherey-Nagel, Düren, Germany) in combination with a Biosprint 96 platform (Qiagen, Hilden, Germany). Viral RNA genomes were detected and quantified by real-time RT-qPCR on a BioRad real-time CFX96 detection system (BioRad, Hercules, USA). The target sequence for amplification was viral RNA-dependent RNA polymerase (IP4)34,46. Genome copies per mL sample were calculated based on a quantified standard RNA, where absolute quantification was done by the QX200 Droplet Digital PCR System in combination with the 1-Step RT-ddPCR Advanced Kit for Probes (BioRad, Hercules, USA). The detection limit was calculated to be 1000 copies per reaction.
RBD-specific SARS-CoV-2 antibodies in serum
Serum samples were analyzed using an indirect multispecies ELISA against SARS-CoV-2 RBD47. Briefly, RBD-coated plates or those treated with coating buffer-only were blocked with 5% skim milk in phosphate‐buffered saline, pH 7.5. Serum samples were incubated on the coated and uncoated wells for 1 h at room temperature. Using a multi‐species conjugate (SBVMILK; obtained from ID Screen® Schmallenberg virus Milk Indirect ELISA; IDvet) diluted 1/80 for 1 h at room temperature detection was performed after the addition of tetramethylbenzidine (TMB) substrate (IDEXX) at a wavelength of 450 nm. After each step, the plates were washed three times with Tris‐buffered saline with Tween 20. For readout, absorbances were calculated by subtracting the optical density (OD) measured on the uncoated wells from the values obtained from the protein‐coated wells for each respective sample. Reproducibility was confirmed and normalization was achieved by reference to negative and positive sera samples.
Cytokine measurement
IFN and IL-10 were measured in homogenized hamster organs by ELISA. Organ samples of about 0,1 cm3 size from hamsters were homogenized in a 1 mL mixture composed of equal volumes of Hank’s balanced salts MEM and Earle’s balanced salts MEM (containing 2 mM L-glutamine, 850 mg/L NaHCO3, 120 mg/L sodium pyruvate, and 1% Penicillin/Streptomycin) at 300 Hz for 2 min using a Tissuelyser II (Qiagen) and were then centrifuged to clarify the supernatant. 50 µL of this homogenate was then used as a sample according to the manufacturer’s instruction with the Hamster IFNγ (Assaygenie #HMFI0010) and Hamster IL-10 ELISA Kit (Assaygenie #HMFI0003) for IFNγ and IL-10 respectively.
SARS-CoV-2–specific IgA from organ homogenates
SARS-CoV-2 specific IgA was detected in the supernatant of homogenates from conchae and lung tissue by ELISA. 96-well, flat bottom ELISA plates (Nunc™ MaxiSorp™) were coated with 100 µL of 1,5 µg/mL recombinant SARS-CoV-2 spike protein (S1+S2 ECD, His tag; Sino Biological) in PBS overnight at 4 °C. The following day, plates were washed three times with PBS supplemented with 0,05% Tween 20 (PBS-T) and incubated with 5% skim milk in PBS (blocking buffer) for one hour at room temperature to block unspecific binding. Organ homogenates were centrifuged at 4000 × g for 5 min. Supernatants were diluted at 1:30 in blocking buffer before adding 50 µL of the diluted samples to the plates. Samples were incubated in the plates for two hours at room temperature. The plates were washed three times before adding 50 µL of 1:50 diluted biotinylated anti-hamster IgA detection antibody (Brookwood Biomedical). Following 2 h incubation, plates were washed three times and 50 µL High-Sensitivity NeutrAvidin HRP conjugate was added for 30 min at room temperature. The plates were washed three times and 50 µL 1-Step Ultra TMB ELISA Substrate Solution (ThermoFisher) was added. After five minutes, the reaction was stopped by adding equal volume of 2 M sulfuric acid. The plates were read for absorbance at 450 nm and 570 nm on a Tecan Infinite M200 Pro Microplate reader. Extinction at 570 nm was subtracted as background.
Surrogate virus neutralization test (sVNT)
To evaluate specifically the presence of virus-neutralizing antibodies in the supernatant of lung and conchae nasalis homogenates the cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GeneScript Ref: L00847) was used following the kit instructions. In short, homogenized organ samples and controls were diluted 1:10 in Sample Dilution Buffer and mixed 1:1 with 1:1000 diluted RBD coupled horse-reddish peroxidase and incubated at 37 °C for 30 min. 100 µl of the mixture was added to a respective well of the ACE2-coated assay plate and incubated for 15 min at 37 °C. After that the plate was washed and 100 µl TMB Solution was added to the well and incubated for another 15 min in darkness. Finally, 50 µl Stop solution was added and optical density (OD) was measured at 450 nm wavelength. Percentual signal inhibition was calculated by setting the OD450 value in relation to the negative control.
Neutralization assay
To evaluate specifically the presence of virus-neutralizing antibodies in serum samples a live virus neutralization test was performed. Sera were pre-diluted (starting dilution from 1/16 to 1/512) with Dulbecco’s modified Eagle’s medium (DMEM) in a 96-well deep well master plate. 100 µL of this pre-dilution was transferred into a 96-well plate. A log2 dilution was conducted by passaging 50 µL of the serum dilution in 50 µL DMEM, leaving 50 µL of sera dilution in each well. Subsequently, 50 µL of SARS-CoV-2 (BavPat1) virus dilution (100 TCID50/well) was added to each well and incubated for 1 h at 37 °C. Lastly, 100 µL of trypsinized Vero E6 cells (cells of one confluent T-175 flask per 100 mL) in DMEM with 1% Penicillin/Streptomycin supplementation were added to each well. After 72 h incubation at 37 °C, the cells were evaluated by light microscopy for a specific CPE. A serum dilution was counted as neutralizing in the case no specific CPE was visible and is given as neutralizing dose 100 (ND100). The virus titer was confirmed by virus titration; positive and negative serum samples were included. Tests were performed in 3 technical replicates and average values were used to calculate the 100% neutralizing dose with the Kerber formula: (-log2) = a/b + c ((a) cell culture wells without virus replication, (b) number of cell culture wells per sera dilution, (c) -log2 of pre-dilution of the sera sample).
Pathology
For histopathology, the left lung lobe was processed as described48. The left lung lobe was carefully removed, immersion-fixed in 10% neutral-buffered formalin, paraffin-embedded, and 2- to 3-µm sections were stained with hematoxylin and eosin (HE). Consecutive sections were processed for immunohistochemistry (IHC) used according to standardized procedures of avidin-biotin-peroxidase complex (ABC)-method49. Briefly, endogenous peroxidase was quenched on dewaxed lung slides with 3% hydrogen peroxide in distilled water for 10 min at room temperature. Antigen heat retrieval was performed in 10 mM citrate buffer (pH 6) for 20 min in a pressure cooker. Nonspecific antibody binding was blocked for 30 min at room temperature with goat normal serum, diluted in PBS (1:2). A primary anti-SARS-CoV nucleocapsid protein antibody was applied overnight at 4 °C (1:3000), the secondary biotinylated goat anti-mouse antibody was applied for 30 min at room temperature (Vector Laboratories, Burlingame, CA, USA, 1:200). Color was developed by incubation with ABC solution (Vectastain Elite ABC Kit; Vector Laboratories), followed by exposure to 3-amino-9-ethylcarbazole substrate (AEC, Dako, Carpinteria, CA, USA). The sections were counterstained with Mayer’s hematoxylin. As a negative control, consecutive sections were labeled with an irrelevant antibody (M protein of Influenza A virus, ATCC clone HB-64). An archived control slide from a SARS-CoV-2-infected Syrian hamster was included in each run. All slides were scanned using a Hamamatsu S60 scanner and evaluated using the NDPview.2 plus software (Version 2.8.24, Hamamatsu Photonics, K.K. Japan) by a trained (TB) and reviewed by a board-certified pathologist (AB), blind to treatment. The lung was evaluated using a 500 × 500 µm grid, and the extent of pneumonia-associated consolidation was recorded as the percentage of affected lung fields. We examined for the presence of SARS-CoV-2-characteristic lesions as given in Supplementary Table 3. Following IHC the distribution of virus antigen was graded on an ordinal scale with scores 0 = no antigen, 1 = focal, affected cells/tissue <5% or up to 3 foci per tissue; 2 = multifocal, 6%–40% affected; 3 = coalescing, 41%–80% affected; 4 = diffuse, >80% affected. The target cell was identified based on morphology.
Antibodies
The following antibodies were used in this study: mouse monoclonal anti-β-actin (Cell Signaling Technology; 3700; RRID: AB_2242334; LOT# 20), rabbit polyclonal anti-SARS-CoV-2 nsp2 (GeneTex; GTX135717; RRID: AB_2909866; LOT# B318853), rabbit polyclonal anti-SARS-CoV Nucleocapsid protein (Rockland; 200-401-A50; RRID:AB_828403), mouse monoclonal anti-SARS-CoV-2 Nucleocapsid protein (4F3C4, gift from S. Reiche50), sheep polyclonal anti-SARS-CoV-2 ORF3a51, rat monoclonal anti-SARS-CoV-2 ORF6 (8B10, gift from Y. Miyamoto52), rabbit polyclonal anti-SARS-CoV-2 ORF8 (Novus Biologicals; NBP3-07972; LOT# 25966-2102), mouse monoclonal anti-SARS-CoV-2 Spike protein (4B5C1, gift from S. Reiche).
Fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch (Cy3 donkey anti-rat #712-165-153, Cy3 donkey anti-mouse #715-165-151, Cy5 donkey anti-rabbit #711-175-152, Cy5 donkey anti-mouse #715-175-511), Li-Cor (IRDye 680RD donkey anti-mouse #926-68072, IRDye 680RD goat anti-rabbit #926-68071, IRDye 680RD goat anti-rat #926-68076) and Invitrogen (Alexa Fluor 647 donkey anti-mouse #A31571, Alexa Fluor 680 donkey anti-sheep #A21102).
Flow cytometry antibodies (all anti-human) were from Miltenyi REAfinity™ (VioBlue™ anti-CD44 #130-113-344, VioGreen™ anti-HLA-ABC #130-120-436, PerCP-Vio-700 anti-CD59 #130-128-812, PE-Vio®770 anti-CD275 (B7-H2) #130-116-805, APC anti-CD70 #130-130-100, VioBright V600 anti-IFNγ #130-131-167, Per-CP anti-HLA-DR #130-113-96, PE-Vio770 anti-CD107a #130-111-622, APC anti-CD137 #130-110-764), Biolegend (Brilliant Violet 711 anti-CD80 #305236, Alexa Fluor® 700 anti-HLA-DR #307626, Brilliant Violet 421 anti-CD279 #329920, Brilliant Violet 510 anti-CD3 #300448, Brilliant Violet 650 anti-CD4 #300536, Brilliant Violet 785 anti-CD69 #310931, Alexa Fluor® 700 anti-CD8a #301028, APC/Cyanine7 anti-CD19 #302218) and R&D (mouse monoclonal anti-hACE2 #FAB9332G).
Neutralization assay human samples
Vero E6 cells were seeded in 96-well flat bottom plates, 3.5 × 106 cells / plate in a final volume of 100 µL DMEM complemented with 2% FBS, 1% Penicillin/Streptomycin. Cells were incubated at 37 °C, 5% CO2 overnight to reach confluency. Patient sera were serially diluted 1:2 in a 96-well round bottom plate, starting with a 1:20 dilution. Virus was added to the diluted sera at a final MOI of 0.002 per well and incubated for 1 h at 34 °C, 5% CO2. Pre-incubated sera/virus was added to the cells and incubated for 2 days at 34 °C, 5% CO2. For virus inactivation, 20 µL of formaldehyde (18% w/v in PBS) (Cat# F8775, Sigma-Aldrich) was added for 30 min to the cultures. Fixative and culture medium were aspirated, and crystal violet (0.5% w/v) was added to each well for 5 min. Fixed and stained plates were gently rinsed several times under tap water and dried before analysis on a CTL ImmunoSpot® analyzer.
Statistics
Data were analyzed using GraphPad Prism 9 and 10 software. No statistical methods were used to pre-determine sample sizes. Acquisition and analysis of lung pathology were done by an investigator blinded to the condition. Appropriate statistical tests were chosen based on sample size and are indicated in individual experiments.
Study approval
All experiments involving human donors were approved by Ethikkommission Nordwest- und Zentralschweiz (#2022-00303). All procedures involving animals were in accordance with relevant guidelines and regulations, evaluated by the responsible ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg–Western Pomerania (LALLF M-V) and gained governmental approval under the registration numbers LVL MV TSD/ 7221.3-1-041/20 and 7221.3-1-001/22. All work including infectious SARS-CoV-2 viruses and their recombinant variants was conducted in a biosafety level 3 facility at the Department of Biomedicine within the University of Basel (approved by the Swiss Federal Office of Public Health (BAG) #A202850/3).
Materials & correspondence
This study has generated plasmids, which will be deposited to Addgene. Generated cell lines will be made available upon request. Recombinant viruses will be available through EVA-GLOBAL. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Thomas Klimkait (thomas.klimkait@unibas.ch) or by Fabian Otte (fabian.otte@unibas.ch).
