DOI: 10.5061/dryad.qjq2bvqk9
title: Data for: Immunogenicity of SARS-CoV-2 spike antigens derived from Beta & Delta variants of concern

description: Using our strongly immunogenic SmT1 SARS-CoV-2 spike antigen platform, we
 developed novel antigens based on the Beta & Delta variants of
 concern.  These antigens elicited higher neutralizing antibody
 activity to the corresponding variant than comparable vaccine formulations
 based on the original reference strain, while a multivalent vaccine
 generated cross-neutralizing activity to all three variants. This suggests
 that while current vaccines may be effective at reducing severe disease to
 existing variants of concern, variant-specific antigens, whether in a
 mono- or multivalent vaccine, may be required to induce optimal immune
 responses and reduce infection against arising variants.
description: <strong>Antigens</strong> SmT1v3-R, -B and –D constructs are based on SARS-CoV-2 spike trimers described previously<sup>22,23</sup> but with C-terminal FLAG/His affinity tags removed. Briefly, the SARS-CoV-2 reference strain spike ectodomain sequence (amino acids 1-1208 derived from Genbank accession number MN908947) was codon-optimized for Chinese Hamster Ovary (CHO) cells and synthesized by GenScript.  Within the construct, the spike glycoprotein was preceded by its natural N-terminal signal peptide and fused at the C-terminus to human resistin (accession number NP_001180303.1, amino acids 23-108). Mutations were added to stabilize the generated spike protein as previously described; amino acids 682-685 (RRAR) and 986-987 (KV) were replaced with GGAS and PP, respectively<sup>24,25</sup>. Constructs were then cloned into the pTT241 plasmid. Expression constructs for VOC spike variants were prepared by re-synthesizing and replacing restriction fragments encompassing mutations present in the Beta (SmT1v3-B) (D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V) and Delta (SmT1v3-D) (T19R, G142D, E156-, F157-, R158G, L452R, T478K, D614G, P681R, D950N) variants, while maintaining the codon-optimized sequences of the remaining amino acids used for SmT1v3-R expression. Stably transfected pools were established by MSX selection using the CHO<sup>2353</sup>™ cell line and used for 10-day fed-batch productions with cumate induction as described<sup>23</sup>.  Spike proteins were purified using a proprietary multi-step non-affinity-based process and formulated in Dulbecco’s Phosphate Buffered Saline (DPBS; Hyclone, Logan, Utah, USA) adjusted to pH 7.8 at protein concentrations of 1.1-1.4 mg/ml. Purified proteins analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical size-exclusion ultra-high performance liquid chromatography (SEC-UPLC). SEC-UPLC was run on an Acquity H-Class Bio UPLC system (Wyatt Technology, Santa Barbara, CA, USA) in phosphate-buffered saline (PBS) + 0.02% Tween-20 on a 4.6 × 300 mm Acquity BEH450 column (2.5 μm bead size; Waters Limited, Mississauga, ON, Canada) coupled to a miniDAWN Multi-Angle Light Scattering (MALS) detector and Optilab T-rEX refractometer (Wyatt). The identity and purity of the antigens was also confirmed by mass spectrometry.  Absence of endotoxin contamination was verified using Endosafe cartridge-based Limulus amebocyte lysate tests (Charles River Laboratories, Charleston, SC, USA).  <strong>Immunization & sample collection </strong> Female C57BL/6 mice (6-8 weeks old) were obtained from Charles River Laboratories (Saint-Constant, Canada). Animals were maintained at the small animal facility of the National Research Council Canada (NRC) in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were approved by our Institutional Review Board (NRC Human Health Therapeutics Animal Care Committee) and covered under animal use protocol 2020.10.  All experiments were carried out in accordance with the ARRIVE guidelines. Mouse experiments (n=10 per group) consisted of 2 separate equal-sized cohorts, where animals were treated identically but had procedures conducted on different days. Data from both cohorts was combined and included for analysis. Seven out of a total of 70 mice receiving AddaS03-adjuvanted formulations had to be excluded due to high local reactogenicity at the site of injection. As such, the AddaS03 groups had 9-10 mice per group included in the final analysis, except for one group (1 µg SmT1v3-R) where results from 7 mice were included. Antigen and adjuvant vaccine components were admixed and diluted in phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) prior to administration in a final volume of 50 µL per dose. Sulfated lactosyl archaeol (SLA) archaeosomes are proprietary NRC adjuvants that were prepared as previously described<sup>26</sup>. Levels of endotoxin in the SLA archaeosomes were verified by the Endosafe® cartridge-based Limulus amebocyte lysate test (Charles River Laboratories) and confirmed to be &lt;0.1 EU per mg.  AddaS03 (Invivogen, San Diego, CA, USA) was prepared as per manufacturer’s instructions.  Animals were immunized by intramuscular (i.m.) injection (50 µL) into the left tibialis anterior (T.A.) muscle on Days 0 and 21 with various vaccine formulations as described above.  On Day 28, mice were anesthetized with isoflurane and then euthanized by cervical dislocation prior to collection of spleens for measurement of cellular immune responses by IFN-γ ELISpot. Mice were bled via the submandibular vein on Days 20 and 28 with recovered serum used for quantification of antigen-specific IgG antibody levels and neutralization assays. Samples were simultaneously collected from 10 naïve animals for the assessment of background immune responses. Each of the samples from the individual mice was tested separately in the various readouts. <strong>Anti-Spike IgG ELISA </strong> Anti-spike total IgG titers in serum were measured by indirect ELISA with SmT1-R, -B or -D as previously described<sup>10</sup>. Briefly, 96–well high-binding ELISA plates (Thermo Fisher Scientific) were coated with 0.3 µg/mL SmT1 protein diluted in PBS.  Serum samples were serially diluted 3.162-fold and added to the plates to allow for binding of antibodies to the protein. Bound IgG was detected with goat anti-mouse IgG -HRP (1:4,000, Southern Biotech, Birmingham, AL, USA) prior to the addition of the substrate o-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich). Bound IgG Abs were detected spectrophotometrically at 450 nm.  Titers for IgG in serum were defined as the dilution that resulted in an absorbance value (OD<sub>450</sub>) of 0.2 and were calculated using XLfit software (ID Business Solutions, Guildford, UK).  Samples that did not reach the target OD were assigned the value of the lowest tested dilution (i.e. 100) for analysis purposes. No detectable titers were measured in serum samples from naïve control animals.  <strong>IFN- γ ELISpot</strong> IFN- γ ELISpot was also conducted as previously described<sup>10</sup>. The levels of spike glycoprotein-specific T cells were quantified by ELISpot using a mouse IFN-γ kit (Mabtech Inc., Cincinnati, OH, USA). A spike peptide library (JPT Peptide Technologies GmbH) based on the reference strain sequence and consisting of 315 peptides (15mers overlapping by 11 amino acids with the last peptide consisting of a 17mer) was used to stimulate splenocytes isolated from each of the mice. The library was split into 3 subpools and used to separately stimulate 4x10<sup>5</sup> cells in duplicate at a final concentration of 2 µg/mL per peptide. Cells were also incubated without any stimulants to measure background responses.  Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD, Beachwood, OH, USA). For each animal, values obtained with media alone were subtracted from those obtained with each of the spike peptide pools and then combined to yield an overall number of antigen-specific IFN-γ<sup>+</sup> SFC/10<sup>6</sup> splenocytes per animal.   <strong>Cell-based SARS CoV-2 Spike-ACE2 Binding Assay</strong> The ability serum to neutralize the binding of labeled SARS-CoV-2 spike trimers (SmT1) to Vero E6 cells was measured as previously described<sup>10</sup>. Indicated dilutions of mouse serum were mixed with 250 ng of biotinylated spike and 1x10<sup>5</sup> Vero E6 cells (ATCC® CRL-1586™). The amount of bound spike was quantified using a Streptavidin-phycoerythrin conjugate prior to acquisition on an LSR Fortessa (Becton Dickinson). For analysis purposes, samples with calculated values ≤ 0 were assigned a value of 0. The levels of neutralization were normalized to the World Health Organization human standard reference material (20/136 from NIBSC, South Mimms, UK) to obtain the anti-SARS-CoV-2 activity (IU/mL) in undiluted mouse serum. <strong>Pseudovirus Neutralization Assay</strong> Pseudovirus neutralization assay was performed in 384-well plate format adapted from previously described protocol and modification<sup>27,28</sup>. Briefly, 4-fold serial dilutions of the serum samples were incubated with diluted virus at a 2:1 ratio for 1 hour at 37°C before addition to HEK293-ACE2/TMPRSS2 cells obtained from BEI Resources repository of ATCC and the NIH (NR-55293). Infectivity was then measured by luminescence readout per well. Bright-Glo luciferase reagent (Promega, E2620) was added to wells for 2 min before reading with a PerkinElmer Envision instrument. Neutralization Titer 50 (NT<sub>50</sub>) were calculated with nonlinear regression (log[inhibitor] versus normalized response – variable slope) with the 100% and 0% constraint. Pseudotyped lentiviral particles were produced expressing the SARS-CoV-2 variant spikes under CMV promotor and were packaged onto lentiviral vectors obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Wuhan-Hu-1 (GenBank # NC_045512) Spike-Pseudotyped Lentiviral Kit, NR-52948.  pcDNA3.3-SARS2-B.1.617.2 expressing the SARS-CoV-2 B.1.617.2 (Delta variant) (Addgene plasmid # 172320) and pcDNA3.3_CoV2_501V2  expressing SARS-CoV-2, B.1.351 (Beta variant) (Addgene plasmid # 170449) spike proteins were gifts from David Nemazee. <strong>Statistical analysis</strong> Data were analyzed using GraphPad Prism® version 8 (GraphPad Software). Statistical significance of the difference between groups was calculated by one-way analysis of variance (ANOVA) followed by post-hoc analysis using Tukey's (comparison across all groups) multiple comparison test. Data was log-transformed (except for data from Spike-ACE2 binding assay) prior to statistical analysis. For all analyses, differences were considered to be not significant with p &gt; 0.05. Significance was indicated in the graphs as follows: *p &lt; 0.05, **p &lt; 0.01, ***p&lt;0.001 and ****: p&lt;0.0001. To correlate data sets, R<sup>2</sup> values were determined by fitting the points to a regression line and p-values (two-tailed) were determined using a Spearman correlation. <strong>References</strong> 10.       Akache, B. <em>et al.</em> Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant. <em>Sci. Rep. </em><strong>11</strong>, 21849 (2021). 22.       Stuible, M. <em>et al. </em>Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells. <em>J. Biotechnol.</em> <strong>281</strong>, 39–47 (2018). 23.       Isho, B. <em>et al. </em>Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. <em>Sci. Immunol.</em> <strong>5</strong>, (2020). 24.       Wrapp, D. <em>et al.</em> Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. <em>Science</em> <strong>367</strong>, 1260–1263 (2020). 25.       Pallesen, J.<em> et al.</em> Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. <em>Proc. Natl. Acad. Sci. U. S. A.</em> <strong>114</strong>, E7348–E7357 (2017). 26.       Jia, Y. <em>et al.</em> A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations.<em> Int. J. Pharm.</em> <strong>561</strong>, 187–196 (2019). 27.       Crawford, K. H. D. <em>et al. </em>Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. <em>Viruses</em> <strong>12</strong>, E513 (2020). 28.       Sulea, T. <em>et al.</em> Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody. <em>PLoS ONE</em> <strong>17</strong>, e0266250 (2022).