Chimeric spike design and expression
Sequences of spike proteins of available variants were collected from public database (GISAID). Selected mutations from variants and predicted potential mutations were incorporated into a Hexapro spike sequence ‘backbone’’ and sequences were run through ROBETTA36 and SWISS-MODEL37 for homology structure prediction for trimer formation. DNA constructs were synthetised by ATUM (Menlo Park, USA) and provided with CMC relevant documentation. The furin cleavage-site RRAR was mutated to be non-functional. The transmembrane domain and the C-terminal intracellular tail were removed and replaced by a T4 foldon sequence in trimer designs38. A four amino acid C-terminal ‘C-tag’’ was added to purify chimeras that could not bind commercially available resins39.
Transfection and culturing of CHOExpress® cells (ExcellGene SA, Monthey, Switzerland) was performed as previously described18. After transfections, suspension cells were selected with puromycin, stable pools selected with high secretion and clonal cell lines were obtained by image-assisted cell distribution (f-sight, Cytena GmbH, Freiburg, Germany). The lead clonal cell lines were used for scale-up in an optimised fed-batch process at the 0.2, 10 and 50 L bioreactor scale of operation. The production medium utilised was EX-CELL® Advanced™ CHO Fed-batch medium (Merck, St. Louis, Missouri, United States). Bioreactors were seeded at a density of 5 x 105 cells/mL and maintained at 37 °C for 4 days. Production culture fluids were harvested, clarified using Harvest RC (3MTM), and proteins purified via affinity chromatography. The loading, washing, and elution steps were performed on an NGL COVID-19 resin (Repligen, Waltham, United States) as per the manufacturer’s recommendations. The eluted product was further purified using Capto adhere anion exchange resin (Cytiva, Marlborough, United Sates), followed by tangential flow filtration with a 100 kD cut-off to isolate trimeric spike proteins.
Mouse immunisation
Female C57BL/6 mice (6–8 weeks of age) were purchased from Australian BioResources (Moss Vale, Australia) or Walter and Eliza Hall Institute of Medical Research (Parkville, Australia); K18-hACE2 mice were bread as hemizygous at Centenary Institute, Newtown, Australia. All mice were housed at the Centenary Institute in specific pathogen-free conditions. All mouse experiments were performed according to the National Health and Medical Research Council Australian code for the care and use of animals for scientific purposes, and were approved by the Sydney Local Health District (SLHD) Animal Ethics and Welfare Committee (ethics approval number 2020-009).
Mice were immunised twice, three weeks apart i.m. Each vaccine contained 5, 1 or 0.1 μg of different stabilised full length spike antigen (chimeric, Ancestral, Delta or BA.1) in endotoxin free PBS with 25 μl of Sepivac SWE™ adjuvant (1:1 volume ratio; Seppic, France). For boosting experiments mice received a prime with 0.05 μg of Comirnaty Omicron XBB.1.5 vaccine twice, 2 weeks apart i.m., rested seven weeks before being boosted with chimeric spike in SWETM (1 μg of antigen) or Comirnaty Omicron XBB.1.5 (5 μg; twice, 2 weeks apart.) In some experiments, mice were injected with 3 μg of anti-CD45 biotin (Becton Dickinson, New Jersey, USA) intravenously prior to organ collection.
Pseudovirus neutralisation assays
Replication-deficient SARS-CoV-2 Spike pseudotyped lentivirus particles were generated by co-transfecting a GFP-luciferase, mTAG-BFP2 or LSSmOrange encoding lentivirus vector and a spike expression construct together with lentivirus packaging and helper plasmids into 293 T cells using Fugene HD (Promega, Wisconsin, USA) as previously described40. To determine nAb titres (NT50), pseudovirus particles were incubated with serially diluted plasma samples at 37 °C for 1 h prior to spinoculation (800 x g) of ACE2 over-expressing 293 T-cells. Seventy-two hours post-transduction, cells were fixed and stained with SYTO™ 60 Red Fluorescent Nucleic Acid Stain (Invitrogen) as per the manufacturers instructions, imaged used an Opera Phenix high content screening system (Revvity, Massachusetts, USA) and the percentage of GFP, mTAG-BFP2 or LSSmOrange positive cells was enumerated (Harmony® high-content analysis software, Revvity). NT50 titres were converted to international units (IU)/ml using WHO working standard 21/234. For each pseudovirus variant the fold change in NT50 from ancestral was calculated and used to transform IU/ml for each serum sample. To determine the breath of neutralisation across variants, neutralisation titres for each were plotted on the y-axis, while the x-axis displayed the genetic distance of the RBD for each pseudotyped spike virus relative to ancestral RBD. This generated a unique curve for each mouse plasma sample. AUC was measured for each curve generated using GraphPad Prism. For testing of human sera, samples were collected from patients 5–32 days (Ancestral) or 14–-28 days (Delta) post positive PCR swab in Sydney, Australia during March 2020 or June 2021, respectively, as described41. Ethics approval was granted by the RPA ethics committee, human ethics number X20-0117 and 2020/ETH00770, or the Ethics Committees of the Northern SLHD and the University of New South Wales, NSW Australia (ETH00520). Written or verbal consent was obtained from all patients.
Flow cytometry
Blood was collected from the lateral tail vein and peripheral blood mononuclear cells (PBMCs) were isolated via Histopaque 1083 (Merck) stratification. Spleen and lung samples were processed as described previously42. To assess cytokine secretion by Spike-specific T cells, murine PBMCs, lung, or spleen, cells were stimulated for 4 h with 5 μg/mL of full length Ancestral spike with 1 μg/mL of Spike peptide (amino acid positions aa 538–54643), or for 30 min with 1 μg/mL of Peptivator Spike peptide pool (Miltenyi biotec) and then supplemented with Protein Transport Inhibitor Cocktail (Life Technologies, Carlsbad, USA) for a further 12 or 4 h, respectively. Cells were surface stained with fixable blue dead cell stain (Life Technologies) and marker-specific fluorochrome-labelled antibodies (Supplementary Table 2). Cells were then fixed and permeabilised using the BD Cytofix/CytopermTM kit (Becton Dickinson) according to the manufacturer’s protocol and intracellular staining was performed to detect cytokines. All samples were acquired on an Aurora five-laser spectral cytometer (Cytek Biosciences, Fremont, United States) and assessed using FlowJoTM analysis software v10.10 (Treestar, Oregon, USA).
Mouse SARS-Cov-2 challenge
Male hemizygous K18-hACE2 mice were challenged as described previously40. Briefly, mice were anaesthetised with isoflurane followed by intranasal challenge with 1 × 103 PFU SARS-CoV-2 (Delta strain). Mice were weighed and monitored daily. Mice were euthansed by carbon dioxide (CO2) inhalation. Clinical scores were defined as follows: No clinical score refers to mice that have no visible signs of disease, they may have experienced body weight loss but do not show any phenotypic alterations to body condition or behaviour; Category 1 mice are typically lethargic, with some slight hunched posture and ruffling; Category 2 mice are lethargic and slow to move, with clear hunching, ruffling, signs of laboured breathing, closed or partially closed eyes; Category 3 mice are moribund; requiring immediate euthanasia, typically cannot hold themselves up, cold to the touch, shallow and laboured breathing.
At day 6 post-infection, mice were euthanised and BALF was collected and enumerated using a haemocytometer. Tissue was homogenised using a gentle MACS tissue homogeniser, after which homogenates were centrifuged (300 × g, 7 min) to pellet cells, followed by collection of supernatants for viral quantification by plaque assay. VeroE6 cells (CellBank Australia, Australia) were grown in Dulbecco’s Modified Eagles Medium (Thermo Fisher, Waltham, United States) supplemented with 10% heat-inactivated foetal bovine serum (Sigma-Aldrich, Saint Louis, United States) at 37 °C/5% CO2. Cells were placed into a 24-well plate at 1.5 × 105 cells/well and allowed to adhere overnight. The following day, virus-containing samples (BALF, Lung and brain homogenates) were serially diluted in modified eagles medium (MEM), cell culture supernatants removed from the VeroE6 cells and 250 μL of virus-containing samples was added to cell monolayers. After 1 h, 250 μL of 0.6% agar/MEM solution was gently overlaid onto samples and placed back into the incubator. At 72 h post-infection, each well was fixed with an equal volume of 8% paraformaldehyde solution (4% final solution) for 30 min at RT, followed by several washes with PBS and incubation with 0.025% crystal violet solution for 5 min at RT to reveal viral plaques.
Hamster SARS-CoV-2 challenge
The hamster challenge studies were performed at the vaccine and infectious disease organisation (VIDO, University of Saskatchewan, Canada). The University of Saskatchewan’s University Animal Care Committee and Animal Research Ethics Board approved the animal work as per guidelines of the Canadian Council of Animal Care’s criteria. Male Golden Syrian hamsters (7–8 weeks old) were purchased from Charles River Laboratories (Charles River, Kingston, U.S.A.). Hamsters were immunised i.m. twice three weeks apart in the quadriceps once with a total volume of 100 μl. Each vaccine contained five or 20 μg of chimeric spike antigen in endotoxin free PBS with SWETM, 100 μl of Novavax NVX-CoV2373 (1 μg of the ancestral spike antigen formulated in Matrix-M adjuvant) or Comirnaty Omicron XBB.1.5 (5 μg). Three weeks after the second immunisation, animals were challenged intranasally with 1 × 105 TCID50 of SARS-CoV-2 variant Omicron (XBB.1.5), 1 × 105 TCID50 of SARS-CoV-2 variant Delta (B.1.617.2) or SARS-CoV-1 (Tor-2). Administration of the challenge virus was in both nares with 50 μL/nare. Body weight of each animal was measured daily. Animals were euthanised by CO2 inhalation either at 3 days post-challenge or at 10 days post-challenge. At necropsy, blood, lung tissues and nasal turbinate were collected for assessment of lesions, infectious virus quantification and histopathological examination. Infectious virus was determined by TCID50 analysis. Assays were conducted in 96-well plates using Vero 76 cells (ATCC CRL-1587). TCID50 was determined by microscopic observation of the cytopathic effect on cells.
Statistical analysis
The significance of differences between experimental groups was evaluated by either one-way or ANOVA, with pairwise comparison of multi-grouped data sets achieved using Tukey’s post-hoc test, as indicated in the relevant figure legend. Differences were considered statistically significant when p ≤ 0.05. Statistical analysis was performed using GraphPad Prism Version 10.2.1.
