DNA vaccine
The study vaccine, pNTC-Spike, contains a DNA plasmid encoding an unmodified SARS-CoV-2 spike protein derived from the Wuhan-Hu-1 strain (MN908947). The human codon optimized SARS-CoV-2 spike sequence was synthesized by GeneArt (Thermo Fisher Scientific, Germany) and subcloned using EcoRI and XhoI into the NTC8685-eRNA41H vector backbone (Nature Technology Corporation, Lincoln, NE, USA). pNTC-Spike was produced by Nature Technology Corporation using an antibiotic-free selection procedure in NTC4862 E. coli cells (DH5α attλ::P5/6 6/6-RNA-IN-SacV, Cmr)12 at 10 mg/mL in phosphate buffered saline (PBS). The plasmid preparation contained <2.0 EU/mg endotoxin, as determined by a Limulus Amoebocyte Lysate (LAL) test using the Endosafe nexgen-PTS LAL assay (Charles River, Wilmington, MA, USA). The construct was sequenced and tested for expression prior to use.
Generation of antibodies specific for SARS-CoV-2 spike protein
Handling of laboratory animals for the production of monoclonal and polyclonal antibodies complied with the regulations of the German Animal Welfare Act and European legislation for the protection of animals used for scientific purposes (Directive 2010/63/EU). Immunizations of mice to generate monoclonal antibodies S1-1047 (IgG1) and S2-1254 (IgG1) received ethical approval by the State Office for Health and Social Affairs in Berlin (LAGeSo Berlin, Germany) under the registration number H129/19 (approval date 03/07/2019). NMRI mice (Charles River, Sulzfeld, Germany) were immunized three times with intervals of 3 weeks with 30 µg of recombinant SARS-CoV-2 spike domains S1 or S2, respectively (S1 = Cat. # REC31806, S2 = Cat. # REC31807, The Native Antigen Company, Oxford, UK) in Gerbu Adjuvans MM (GERBU Biotechnik GmbH, Heidelberg, Germany) according to the manufacturer´s instructions and finally boosted with 15 µg of the antigens in PBS at the last 3 days prior to fusion. Hybridoma cells were generated by the fusion of splenocytes from immunized mice with myeloma cells (P3-X63-Ag8.653, American Type Culture Collection)51. Cells were fused at 37 °C at a ratio of 4: 1 in polyethylene glycol 1500 (PEG, Roche Diagnostics, Mannheim, Germany) by slowly adding PEG (1 mL per 100 × 106 splenocytes) to the pelleted cells, slow addition of RPMI 1640 (4 mL per 100 × 106 splenocytes) and final addition of a larger volume of RPMI 1640 (10 mL per 100 × 106 splenocytes). Cells were plated in a density of 20,000 splenocytes together with 20,000 BALB/c thymocytes as feeder cells in a volume of 200 µL per well of 96 well cell culture plates in RPMI 1640 media supplemented with 20% fetal calf serum, 50 µM 2-mercaptoethanol, 50 U/mL recombinant murine IL-6, 1% glutamine, 5.7 µM azaserine and 100 µM hypoxanthine. Starting at day 10 after fusion, antibodies from hybridoma supernatants underwent a stringent screening procedure employing e.g. ELISA and surface plasmon resonance spectroscopy to identify hybridoma clones with superior specificity, affinity, and broad applicability in different assays; selected clones were subcloned twice to ensure clonality. A rabbit polyclonal antibody (KSpike) was generated by subcutaneous immunization of a New Zealand rabbit with 25 µg of recombinant SARS-CoV-2 spike S1S2 protein (Cat. # 40589-V08B1, Sino biological, Bejing, China) for two times with an interval of 4 weeks. The IgG fractions were affinity purified from hybridoma culture supernatants or rabbit serum using Protein A or G columns, respectively, on an ÄKTA LC-instrument (ÄKTA, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The monoclonal antibodies S1-1047 and S2-1254 showed high specificity for their respective target domain in the spike protein of SARS-CoV-2 as shown by indirect ELISA using the rabbit pAb KSpike as control reagent (Supplementary Fig. 1).
Western blot
The day before transfection, 1.2 × 105 Vero E6 cells were seeded per well in a six well tissue culture plate with glass coverslips and incubated overnight at 37° C, 5% CO2. Vero E6 cells were transfected with 2 µg of pNTC-Spike using Fugene HD Transfection Reagent (Cat. # E2311, Promega, Madison, WI, USA). Cells were harvested 48 h post-transfection in lysis buffer (0.125 M NaCl, 20 mM Tris [pH 8.0], 0.5% Igepal). The protein content was determined by the Pierce™ BCA Protein Assay Kit (Cat. # 23227, Thermo Fisher, Waltham, MA, USA), and 25 µg cell lysate was separated on a Novex 10% Tris-Glycine Mini Gel (Cat. # XP00105BOX, Thermo Fisher) with the PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (Cat. # 26616, Thermo Fisher). SARS-CoV-2 spike protein expression was detected with a 1:5000 dilution of anti-SARS-CoV-2 S1 and anti-SARS-CoV-2 S2 mouse monoclonal antibodies (S1-1047 and S2-1254, respectively) and visualized with a 1:5000 dilution of an HRP conjugated rabbit anti-mouse IgG polyclonal antibody (Cat. # P0161, Agilent, Santa Clara, CA, USA). A mouse monoclonal anti-β-actin (1:5000 dilution; Cat. # A1978, Sigma-Aldrich, Germany) was used to verify equal loading. As positive controls, a lysate preparation from Vero E6 cells infected with a SARS-CoV-2 clinical isolate and a commercial SARS-CoV-2 spike expressing plasmid (Cat. # VG40589-UT, Sino Biological, China) were used. The white light and chemoluminescence channel overlay of uncropped blots with molecular weight marker are presented in Supplementary Fig. 2.
Immunofluorescence assay and microscopy
Vero E6 cells were seeded on coverslips in six-well plates and transfected with 2 µg of pNTC-Spike using Fugene HD Transfection Reagent (Cat. # E2311, Promega, Madison, WI, USA) or Polyfect Transfection reagent (Cat. # 301105, Qiagen GmbH, Hilden, Germany). Forty-eight hours post-transfection, cells were fixed at room temperature for 10 min in 4% paraformaldehyde (Cat. # HT501128-4L, Sigma-Aldrich), permeabilized at −20 °C for 10 min in 100% methanol. Cells were washed three times with Dulbecco’s phosphate buffered saline (DPBS) and blocked for 1 h at room temperature in blocking buffer (5% BSA in DPBS). The blocking buffer was aspirated, and cells were incubated for 2 h at 4 °C with a 1:1000 dilution of primary antibody in blocking buffer (S1-1047 or S2-1254). Cells were washed three times with DPBS and incubated for 1 h at room temperature in the dark with an Alexa Fluor 488 conjugated goat anti-mouse IgG (H + L) Trial Superclonal™ antibody (1:1000 dilution, Cat. # A28175, Thermo Fisher) in blocking buffer. Coverslips were washed three times with DPBS, dried on a paper towel, mounted with VECTASHIELD medium containing 4, 6-diamino-2-phenylindole hydrochloride (DAPI) (Cat. # H-1200-10, VECTOR Laboratories, Burlingame, CA, USA), and sealed with nail polish. Immunostained cells were observed with an Olympus BX61 fluorescence microscope, using the cellSens Entry software version 1.11 for image capture. Imaging software Image J, version 1.53j (NIH, USA) was used to merge images and visualize the results.
Animals and study design
Mice
Eight week old female CB6F1 mice (Envigo, Netherlands), offspring of a cross between BALB/c and C57BL/6 mice, were randomly assigned to receive either 10 µg pNTC-Spike (N = 5), 50 µg pNTC-Spike (N = 5) or 50 µg of a vector control (N = 5). The unadjuvanted vaccine doses were prepared in PBS in a final volume of 50 µl and administered in two 25 µL injections per immunization. The mice were immunized via the intradermal route with needle injection at the base of the tail at weeks 0, 2, and 4.
Rabbits
Nine to ten week old female New Zealand white rabbits (Charles River, France) were immunized with 125 µg pNTC-Spike in PBS without adjuvant at weeks 0, 2, and 4. Three administration routes were evaluated in independent studies in the following order: intradermal route using the Tropis ID Needle-free Injection System (PharmaJet, Inc.) (N = 4); intradermal route using needle-and-syringe (N = 3); intramuscular route in a single dose of 500 µL using the Stratis Needle-free Injection System (PharmaJet, Inc.) (N = 5). In the latter study, vaccine safety was observed.
Nonhuman primates
Eight male and female adult rhesus macaques (Macaca mulatta), 2–8 years old (mean: 4 years), were randomly divided into two groups: pNTC-Spike vaccinates (N = 6) and sham controls (N = 2). Animals received three immunizations of 2 mg DNA each at weeks 0, 2, and 4. The unadjuvanted vaccine was administered via the intradermal route using the Tropis ID needle-free injection system (PharmaJet, Inc.) with four 100 μL doses per immunization, equally distributed over the left and right scapula region. The interval between last immunization to viral challenge varies for the different SARS-CoV-2 vaccine candidates (adenoviral vector, mRNA, live attenuated, protein and DNA) evaluated in nonhuman primates (median: 25 days; range: 14–77 days)39. To enable comparison with the latter, we selected an interval of 28 days between the last immunization and challenge. At week 8, all eight animals were challenged with 1.0 × 105 TCID50 (1.2 × 108 RNA copies, 1.1 × 104 PFU) SARS-CoV-2 (strain nCoV-WAI-2020; MN985325.1; BEI Resources, Manassas, VA, USA). In similar studies conducted at the same facility, BIOQUAL, Inc., the 1.0 × 105 TCID50 challenge dose consistently resulted in infection of SARS-CoV-2 immune naïve rhesus macaques (N = 28, total) with detectable viral loads by 2 days post-challenge in BAL and nasal swabs.23,26,27,28 The challenge stock was propagated at BIOQUAL, Inc. in Vero E6 cells from a seed stock obtained by Kenneth Plante, World Reference Center for Emerging Viruses and Arboviruses, UTMB, Galveston, TX (lot no. TVP 23156). The stock was deep sequenced (SRA accession no. SRR12749718) by Shelby O’Connor’s laboratory, Univ. Wisconsin-Madison, WI. Sequencing confirmed the expected sequence identity. The virus was diluted in PBS and administered as 1 mL by the intranasal (IN) route and 1 mL by the intratracheal (IT) route.
The mice and rabbits were housed in a pathogen-free and climate-controlled animal facility at Statens Serum Institut, Denmark. All cages were provided with bedding material and environmental enrichment. Animals had access to water and a standard pelleted diet ad libitum. Animal husbandry and procedures comply with the Danish legislation, which is based on the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. The experiments received ethical approval by The Animal Experimentation Council, the National Competent Authority within this field (approval number 2017-15-0201-01322), and were supervised by the laboratory animal veterinarians at Statens Serum Institut. The nonhuman primates were housed at BIOQUAL Inc. (Rockville, MD) and associated animal studies conducted in compliance with relevant local, state, and federal regulations and received ethical approval by the Institutional Animal Care and Use Committee (IACUC). The assays used to quantitate vaccine-induced immune responses in the small animals and rhesus macaques were performed at Statens Serum Institut and BIOQUAL, Inc., respectively, and were depended on the assays available at each institute.
Mouse and rabbit antibody enzyme-linked immunosorbent assay (ELISA)
Spike- and RBD-specific binding immunoglobulin G (IgG) titers were determined by standard ELISA. In brief, Nunc™ MaxiSorp™ plates were coated with 100 µL of 4 µg/mL recombinant SARS-CoV-2 spike S1S2 protein (Cat. # 40589-V08B1; Sino biological) or 4 µg/mL SARS-CoV-2 spike RBD protein (Cat. # 40592-V08B; Sino biological) at 4 °C overnight. In consecutive order, with wash steps in between, plates were incubated at room temperature on an orbital shaker with 150 µL of blocking buffer (SSI Dilution Buffer [Cat. # 1322, SSI Diagnostica], 2% skim milk), 100 µL of 5-fold serial dilutions of a mouse or rabbit sera (1:20 to 1:1562500), 100 µL of horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody (1:10000 dilution) or mouse-anti-rabbit IgG antibody (1:2000 dilution) (Cat. # A4416 and A1949, respectively; Sigma-Aldrich), and 3,3′, 5,5′-Tetramethylbenzidine (TMB) One Substrate (Cat. # 4380, KemEnTec, Denmark). TMB reaction was stopped with H2SO4, and absorbance read at 450 nm using 620 nm as a reference on a FLUOstar Microplate Reader (BMG LABTECH, Germany). Each wash step comprised three washes with 250 µL wash buffer (PBS with 0.05% Tween20) for 1 min.
Spike ectodomain-specific antibody ELISA
White Nunc™ MaxiSorp™ microtiter plates were coated with 100 µL of 1 µg/mL recombinant SARS-CoV-2 Spike His-tag protein (Cat. # 10549-CV-100; R&D Systems) at 4 °C overnight. In consecutive order, with wash steps in between, plates were incubated with 150 µL of blocking buffer (Dilution Buffer pH 7.2 [Cat. # 1322, SSI Diagnostica], 2% bovine serum albumin, and 0.1% Tween20), 100 µL of 5-fold serial dilutions of rabbit sera (1:20 to 1:1562500), 100 µL of HRP conjugated mouse-anti-rabbit IgG antibody (1:2000 dilution) (Cat. # A1949 Sigma-Aldrich), and BM Chemiluminescent Substrate (Cat. # 11582950001, Sigma-Aldrich). Luminescence was read on a FLUOstar Microplate Reader (BMG LABTECH, Germany). Each wash step comprised three washes with 250 µL wash buffer (PBS with 0.05% Tween20) for 30 s. ELISA end-point titers were calculated from a four-parameter logistic regression curve in GraphPad Prism 8.3.0, using the reciprocal serum dilution that yielded an absorbance above a positive cut-off value calculated for each group of animals based on absorbances measured on day 0 at a serum dilution of 1:20 that is (mean absorbance for all animals) + (3 × standard deviation of the absorbance measured for all animals).
Virus microneutralization test
A 2-fold serial dilution of heat-inactivated serum/plasma samples were mixed with 300 × TCID50 SARS-CoV-2 virus, as determined from a virus titration 96 h post-inoculation. The solution was incubated for 1 h at 37 °C, 5% CO2, and added to Vero E6 cells (kindly provided by Bjoern Meyer, Institut Pasteur, Paris, France) in a 96 well tissue culture plate seeded with 104 cells per well the day prior. The inoculated cells were incubated at 37 °C, 5% CO2 for 24 h, and the inhibition of virus infection in the presence of serum measured in a standard ELISA targeting the SARS-CoV-2 nucleocapsid protein. The culture medium was removed from the infected Vero E6 cell monolayers and the cells washed twice with 100 μL PBS. The cells were fixed with cold 80% (v/v) acetone in PBS for 10 min. Following three wash steps with wash buffer (PBS containing 1% (v/v) Triton-X100) for 30 s, a 100 μL of a SARS-CoV-2 nucleocapsid protein monoclonal antibody was added and incubated for 5 min on an orbital shaker (300 rpm) at room temperature and subsequently for 1 h at 37°C. For testing rabbit sera, the mouse monoclonal antibody clone 7E1B was used (1:4000 dilution; Cat. # BSM-41414M, Bioss, Woburn, Massachusetts, USA); for testing mouse sera, a rabbit monoclonal antibody was used (1:2500; Cat. # 40143-R019, Sino Biological, China). The plates were washed and incubated with 100 μL of either a 1:6000 diluted mouse-anti-rabbit IgG HRP conjugate antibody (Cat. # A1949, Sigma-Aldrich) or a 1:10000 diluted goat anti-mouse IgG (H + L) cross-adsorbed HRP conjugate antibody (Cat. # A16078; Invitrogen, Waltham, Massachusetts, USA) for 5 min on an orbital shaker (300 rpm) at room temperature and subsequently for 1 h at 37 °C. The plates were washed five times with wash buffer for 30 s, followed by three washes with deionized water. A 100 μL TMB One Substrate (Cat. # 4380, KemEnTec, Denmark) was added and incubated for 15 min. The reaction was stopped with H2SO4, and absorbance read at 450 nm using 620 nm as a reference on a FLUOstar Microplate Reader (BMG LABTECH, Germany).
Included on each microneutralization plate were quadruplicate wells containing cells with 300 × TCID50 SARS-CoV-2 virus without serum (virus control) and quadruplicate wells containing cells with virus diluent only (cell control). The neutralization antibody titer was determined for each serum sample as the interpolation of a four-parameter logistic regression curve with the 50% virus level cut-off calculated for each assay plate: [(mean OD of virus control wells) + (mean OD of cell control wells)]/2. The reciprocal serum dilution corresponding to that well is reported as the 50% neutralization antibody titer for that sample. The microneutralization assay is validated and has comparable performance to other live virus neutralization assays established at different European laboratories (laboratory 4 in ref. 52). Virus neutralization was measured for the following SARS-CoV-2 viruses: early pandemic (lineage B.1) strain SARS-CoV-2/Hu/Denmark/SSI-H1; Alpha variant (lineage B.1.1.7) strain SARS-CoV-2/Hu/Denmark/SSI-H14; Beta variant (lineage B.1.351) strain hCoV-19/Netherlands/NoordHolland_10159/2021 (Cat. # 014V-04058, European Virus Archive—Global, Marseille, France); and the Delta variant (lineage B.1.617.2) strain SARS-CoV-2/Hu/Denmark/SSI-H11. Strains from Denmark were isolated at Statens Serum Institut from clinical samples on Vero E6 cells. All virus stocks were deep sequenced to confirm identity, confirm the absence of cell culture-derived mutations, and the presence of lineage-specific mutations in the spike protein. The comparison between strains was done on the same day using a single dilution for each serum sample.
Mouse and rabbit splenocyte isolation, restimulation, and cytokine quantification
Following excision, the spleens were submerged in RPMI medium and processed aseptically within an hour. The spleens were homogenized through a 70 µm cell strainer using a syringe plunger. The single-cell suspensions were washed twice with cold PBS followed by lysis of red blood cells using Red Blood Cell Lysis buffer (Cat. # R7757, Sigma-Aldrich). The splenocytes were resuspended in culture medium (RPMI containing 10 % fetal bovine serum, 1 % Penicillin and Streptomycin, 1 mM sodium pyruvate, 10 mM HEPES and 50 µM 2-Mercaptoethanol). A total of 8 × 105 splenocytes were stimulated in duplicate with 2 µg/mL SARS-CoV-2 spike S1S2 protein (Cat. # 40589-V08B1; Sino biological), 2 µg/mL SARS-CoV-2 spike RBD protein (Cat. # 40592-V08B; Sino biological), 5 µg/mL concanavalin A (Cat. # C0412, Sigma-Aldrich) as a positive control, or culture media as mock stimulated control. The re-stimulated splenocytes were kept under standard tissue culture conditions (37˚C with 5% CO2) for 48 h. The level of secreted interferon-γ (IFN-γ), interleukin 5 (IL-5), or interleukin 17 (IL-17) in clarified cell culture supernatants were determined by cytokine ELISA (Cat. # 3321-1H-6, 3391-1H-6, 3521-1H-6, 3110-1H-6; MABTECH AB, Sweden) according to the manufacturer’s instructions. For the rabbit IFN-γ ELISpot (Cat. # 3110-4HPW-10; MABTECH AB, Sweden), a total of 1 × 106 splenocytes were stimulated for 18 h using the aforementioned antigen stimulations and tissue culture conditions. The secretion of IFN-γ was determined according to the manufacturer’s instructions and spots were counted using CTL Immunospot® analyzer and ImmunSpot® Software (version 7.0.22.1).
Nonhuman primate antibody enzyme-linked immunosorbent assay (ELISA)
SARS-CoV-2 spike protein-specific IgG in serum was quantified in duplicate by ELISA. In brief, Nunc™ MaxiSorp™ microtiter plates were coated with 50 µL of 2 μg/mL recombinant SARS-CoV-2 spike S1S2 protein (Cat. # 40589-V08B1, Sino Biological) in PBS and incubated overnight at 4°C. Plates were washed five times with wash buffer (0.05% Tween20 in PBS) and blocked with 100 μL 1% bovine serum albumin in PBS for 2 h at room temperature. The block solution was discarded and 100 μL of serial 4-fold dilutions of serum starting at a 1:20 dilution were added to the wells, followed by a 1 h incubation at room temperature. Plates were washed three times with wash buffer and incubated for 1 h at room temperature with 50 µL a 1:10000 dilution of goat anti-monkey IgG (H + L) (Cat. # PA1-84631, Invitrogen). Plates were washed five times with wash buffer and once with PBS, followed by the addition of 100 μL of SureBlue TMB 1-Component Microwell Peroxidase Substrate (Cat. # 5120-0075, SeraCare, Milford, MA, USA). The reaction was stopped after 10 min with the addition of 100 μL TMB Stop solution per well. The absorbance was measured at 450 nm using 620 nm as a reference. ELISA end-point titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.300.
Plaque reduction neutralization test (PRNT)
The PRNT was performed in six-well tissue culture plates seeded with 1.75 × 105 Vero E6 cells (Cat. # CRL-1586, ATCC, Manassas, VA, USA) per well the day before. Serum samples were heat-inactivated at 56 °C for 30 min and tested in duplicate in a three-fold serial dilution ranging from 1:20 to 1:4860. Each serum dilution was pre-incubated with 30 PFU SARS-CoV-2 for 1 h at 37 °C before addition to the Vero76 monolayers. After an incubation of 1 h at 37 °C, the supernatants containing the serum/virus mixture were removed and the monolayer washed once with PBS before overlaying with a semi-solid culture medium. Following a 3 day incubation at 37 °C with 5% CO2, the cells were fixed and stained with crystal violet. The reciprocal of the serum dilutions causing plaque reductions of 90% (PRNT90) and 50% (PRNT50) were recorded as titers. Virus neutralization was measured for the early pandemic SARS-CoV-2 strain nCoV-WAI-2020 (Cat. # NR-52281, BEI Resources, Manassas, VA, USA) and the Beta variant (lineage B.1.351) strain 2019-nCoV/South Africa/KRISP-K005325/2020 (Cat. # NR-54009, BEI Resources). All virus stocks were deep sequenced to confirm identity.
Subgenomic SARS-CoV-2 RNA assay
Replicating the SARS-CoV-2 virus was detected and measured using a real-time RT-PCR assay targeting viral replication intermediates not packaged into virions24. RNA was extracted using the QIAamp Viral RNA Mini Kit (Cat. # 52904, Qiagen, Hilden, Germany). The SARS-CoV-2 E gene subgenomic messenger RNA (sgmRNA) was detected using a leader-specific primer (SG-F: 5′-CGATCTTGTAGATCTGTTCCTCAAACGAAC-3′) located upstream of SARS-CoV-2 ORF1a and a reverse primer (SG-R: 5′-ATATTGCAGCAGTACGCACACACA-3′) and probe (FAM-5′-ACACTAGCCATCCTTACTGCGCTTCG-3′-BHQ) specific to the E gene. PCR amplification was performed using the SensiFAST™ Probe Lo-ROX One-Step Kit (Cat. # BIO-78005, Meridian Bioscience, Cincinnati, OH, USA) on an Applied Biosystems 7500 Real-Time PCR instrument with the following program: 48 °C for 30 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and 1 min at 55 °C. Subgenomic RNA copies were calculated from a standard curve representing serially diluted plasmid DNA containing the target sequence.
Statistical analyses
Each measurement represents a single animal. Antibody positive and negative responses were determined using a 99.9% confidence interval cut-off value calculated for each serum dilution from the serum controls included in the assays53. Variation in paired continuous variables between multiple time points were compared using the non-parametric Friedman test with Dunn’s correction for multiple comparisons. For the latter, adjusted p-values are reported. The Wilcoxon rank sum test was performed where data for only two time points were available. All statistical tests were two-tailed. Statistical analyses and graphing were done with GraphPad PRISM version 8.3.0. (GraphPad Software Inc., San Diego, CA).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
