Recombinant CoV proteins and antibodies
SARS-CoV-2 spike proteins were purchased from ACRObiosystems Inc. SARS-CoV-2 (Cov-19) S protein, His Tag, super stable trimer (MALS & NS-EM verified) (Cat. #SPN-C52H9) was used in early experiments to analyze IgG and IgM response as well as for Activated Immune Cell Marker assays (AIM). Subsequently, with the emergence of new variants of concern and increased availability of recombinant proteins, the following were purchased: SARS-CoV-2 UK Alpha S1 protein (HV69-70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), His Tag (Cat. #SPN-C52H6); SARS-CoV-2 S UK Alpha protein RBD (N501Y), His Tag (Cat. #SPD-C52Hn); SARS-CoV-2 SA Beta S protein (L18F, D80A, D215G, 242-244del, R246I, K417N, E484K, N501Y, D614G, A701V) trimer 50ug #SPN-C52Hk; SARS-CoV-2 SA Beta S protein RBD (K417N, E484K, N501Y), His Tag (MALS verified) (Cat. #SPD-C52Hp); SARS-CoV-2 Brazil Gamma S1 protein (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V11 (Cat. #SPN-C52Hg); SARS-CoV-2 India Delta spike S1 (T95I, G142D, E154K, L452R, E484Q, D614G, P681R), His Tag (Cat. # S1N-C52Ht); SARS-CoV-2 Omicron spike protein HRP (RBD, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, His Tag)-HRP (Cat# Z03730).
SARS-CoV-2 (COVID-19) spike antibody against the S1 and S2 subunits, were purchased from Genetex (Cat. #GTX135356 and #GTX632604) for western blot confirmation of S protein within EDVTM. SARS-CoV-2 (2019-nCoV) spike RBD rabbit PAb, antigen affinity purified (Cat. #40592-T62, Sino Biological) was used for quantitation of the S protein within EDVs using ELISA.
JAWSII mouse bone marrow derived dendritic cells (ATCC® CRL-11904™) were grown in α-minimum essential medium (MEM; #M7145; Sigma) with ribonucleosides and deoxyribonucleosides (4 mM L-glut, 1 mM Sodium Pyruvate, 5 ng/ml GMCSF and 20% FBS) at 37°C, 5% CO2.
Generation of plasmid expressing SARS-CoV-2 S protein under bacterial promoter
An expression cassette was generated by placing the coding nucleotide sequence for SARS-CoV-2 (Covid-19) spike protein (Wuhan sequence; GenBank MN908947.3) on the 3′-end of a modified β-lactamase promoter, which has been previously used for expression in Salmonella typhimurium strains 49. The expression cassette was then inserted between the KpnI 5′ and SalI 3′ sites of the M13 multiple cloning site of pUC57-Kan backbone plasmid to create pLac-CoV2. The sequence was optimised for S. typhimurium codon usage before manufacturing by Genscript services. A negative control plasmid, pLac-control, was created as above by removing the CoV2 sequence from the pLac-CoV2 (Fig. 1).
Cloning of pLac-CoV2 and pLac into Salmonella Typhimurium EDV producing strain and the assessment of plasmid and S protein within EDVs
PLac-Cov2 and pLac-CoV2-control were electroporated into a chemically competent S. typhimurium intermediate strain, which lacks a plasmid restriction mechanism, using a Gene Pulser Xcell™ (Bio-Rad, Hercules CA) with settings 200 ohm, 25 Hz and 2.5 mV. Transformants were recovered in TSB medium for 1.5 h at 37°C before plating on TSB agar plates containing 75 µg/mL kanamycin (#K4000, Sigma-Aldrich, St. Louis, Missouri). Isolates were picked into TSB broth with 75 µg/mL kanamycin and plasmid DNA was extracted using the Qiagen miniprep kit as per manufacturer’s instructions (#27104, Qiagen, Hilden, Germany). Subsequently, the extracted plasmid DNA from the 4004 strain was electroporated as above into the EnGeneIC Pty. Ltd. EDVTM producing S. typhimurium strain (ENSm001). Clones containing pLac-CoV2 produce the encoded Covid-19 spike protein, which along with the plasmid DNA, becomes incorporated into EDVs during cell division to produce EDV-COVID. The EDVs containing pLac (EDV-CONTROL) were created in the same way to be used as a negative control.
To determine the plasmid content of EDV-COVID and EDV-CONTROL, plasmids were extracted from 2x109 EDVs using a Qiaprep Spin miniprep kit (Qiagen) following the manufacturer’s instructions. Empty EDV were processed in the same manner as a control. The quantity of DNA plasmids was then measured by absorption at OD260nm using a biophotometer (Eppendorf). The copy number of the plasmids were calculated using:
Proteins from 2 x 1010 EDV-COVID were extracted using 100 µL B-PER™ (Bacterial Protein Extraction Reagent; ThermoFisher) supplemented with 10% (v/v) lysozyme (Sigma-Aldrich) and 1% (v/v) DNase I (Cat. #EN0521, Qiagen). The extracted samples were then centrifuged at 12000 g for 10 min and the supernatant was collected. The pellet was also collected and resuspended in 100 µL PBS. 23 µL of the supernatant and pellet protein samples were co-incubated with 5 µL of loading buffer and 2 µL DTT (Sigma-Aldrich) at 80oC for 20 min before the entire content of each sample was loaded onto a NuPAGE 4-12% Bis-Tris Mini Protein Gel (Cat. #NP0322BOX, ThermoFisher) and run at 190 V for ~80 min. The gel was then transferred using the iBlot 2 system (ThermoFisher) after which the membrane was blocked using SuperBlock™ blocking buffer (Cat. #37515, ThermoFisher) and subsequently stained with 1:1000 Rabbit poly-clonal anti-SARS-CoV-2 spike (S1) antibody (Genetex) or 1:1000 mouse mono-clonal anti-SARS-CoV-2 spike (S2) antibody (Genetex) and incubated overnight at 4oC. The membrane was then washed with PBST and incubated with HRP conjugated anti-rabbit (1:5000) (Abcam) or anti-mouse (1:5000) (ThermoFisher) IgG secondary antibody for 1 h at RT. The blot was developed using Lumi-Light Western Blot substrate (Cat. #12015200001, Roche) and visualized using a Chemidoc MP (Bio-Rad).
EDV S protein estimation by ELISA
4 x 109 EDV-COVID particles were pelleted by centrifugation at 13000 g for 8 min. 100 µL of B-Per™ Bacteria lysis agent supplemented with 100 µg/reaction of lysozyme (Cat. #L6876, Sigma) and 5U/reaction rDNase I (Cat. #740963, Macherey-Nagel) was added to each sample and incubated on a vortex shaker for 2 h at 600 rpm at RT. The samples were then mixed with 1:5 Dithiothreitol (Cat. # 20290 ThermoFisher) and placed on an 80oC heat block (Eppendorf) at 600 rpm agitation for a further 20 min. Protein quantity was assayed using the DC Protein Assay kit (Cat. #5000111, Bio-rad) following the manufacturer’s specifications.
Standards were generated through serial dilution of the spike protein (ACRObiosystems) to achieve the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.3 pg/mL. EDV-COVID S protein samples were diluted 1:1000 in PBS. Standards and EDV spike protein samples, were then coated on the ELISA plate, sealed, and incubated O/N at 4oC. The plates were then washed 3 times with 300 µL PBST using a plate washer. 200 µL protein free blocking buffer (Cat. # 786-665; Astral Scientific) was added to the plate which was sealed and incubated at RT for 1 h.
Spike RBD Rabbit PAb detection antibody (Sino Biological) was diluted 1:10000 in 10 mL PBST and 100 µL per well was added and incubated for 1 h at RT. The plate was washed in PBST as above before addition of 100 µL sheep anti-rabbit IgG (H+L)-peroxidase (Cat. #SAB3700920; Merck, 1:10000) in 10 mL PBST. Sealed plates were incubated for 30 min at RT in the dark. The plate was washed again as above and 100 µL of TMB solution (Cat #34022; ThermoFisher) was added per well. The reaction was stopped by adding 50 µL of 2 M H2SO4 per well within minutes of TMB addition. The samples were analyzed at OD450nm using a µQuant plate reader (Bio-TEK Instruments, Inc) and KC junior software.
Alpha-galactosylceramide loading into EDV-COVID
EDV-COVID nanoparticles carrying the S protein were purified in large batches through bio-fermentation of the parent bacteria S. typhimurium, followed by tangential flow filtration (TFF) to purify the EDV-COVID particles from the parent as previously described 9. EDV-COVID particles were then buffer exchanged from media into PBS pH 7.4 (Dulbecco’s; ThermoFisher) complemented with 0.5% tyloxapol (Cat. #T8761, Sigma-Aldrich) prior to loading with αGC based on a protocol described in Singh et al (2014) 50.
Alpha-galactosylceramide glycolipid adjuvant (αGC; Advanced Molecular Technologies, Melbourne) stocks were formulated in 100% DMSO (Sigma). Stock αGC was added to EDV-COVID solutions in PBS at a final concentration of 10 µM (8.58 µg/mL equivalent). Co-incubation of EDV-COVID particles and αGC was performed at 37°C with mixing overnight. Unloaded αGC was removed by washing the particles in PBS pH 7.4 (Dulbecco’s; ThermoFisher) through a 0.2 µm TFF system. EDV-COVID-αGC particles were then concentrated in PBS pH 7.4 followed by buffer exchange to 200 mM Trehalose (Cat. #T9531, Sigma) ready for vial filling and freeze-drying.
EDV-COVID-αGC batch vials underwent quality control testing including particle count, uniformity, sterility, S protein concentration, plasmid copy number and αGC concentration per 109 EDV particles, prior to use in animal experiments. Activity of loaded αGC through dendritic cell (DC) uptake and presentation through the CD1d T cell receptor was carried out as described below.
αGC uptake and presentation by murine DCs
JAWSII cells (ATCC) were treated with EDV-COVID-αGC in a 96-well Perfecta3D hanging drop plate (Cat. #HDP1385, Sigma-Aldrich) at 1x109 EDV-COVID-αGC per cell. JAWSII cells treated with 2 µg/mL αGC (Advanced Molecular Technologies) served as a positive control. The cultures were then incubated for 24 h at 37oC with 5% CO2 and cells were collected and stained with a PE conjugated CD1d: αGC complex antibody (Cat. # 12-2019-82, ThermoFisher, 1:2000) and analyzed using a Gallios flow cytometer (Beckman). The results were analyzed using Kaluza Analysis software (V.2.1, Beckman).
Detection of spike protein and CD1d associated αGC in murine DCs following EDV-COVID-αGC co-incubation
JAWSII cells (ATCC) were seeded onto a 96 well hanging drop plate (Sigma-Aldrich) at 5 x 104 cells/well. EDV only, EDV-αGC, EDV-CONTROL, EDV-COVID and EDV-COVID-αGC were co-incubated with the cells at 1x109 EDVs per well. Untreated JAWSII cells were used as controls. The samples were cultured at 37oC with 5% CO2 for 48 h before collected and co-stained with PE anti-mouse αGC:CD1d complex antibody (ThermoFisher, 1:2000) and SARS-CoV-2 S1 protein polyclonal primary antibody (Genetex, 1:2000) at room temperature for 30 min in the dark. The samples were then stained with Alexa Fluor 647 goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (ThermoFisher, 1:1000) at 4oC for a further 20 min and analysed using a Gallios flow cytometer (Beckman Coulter). Mouse IgG2a and rabbit IgG (Biolegend) were used as isotype controls. DAPI was used to differentiate live/dead cells and single stained samples were used to generate compensation. The samples were analysed using the Kaluza analysis software (V2.1, Beckman Coulter).
Extraction of αGC from EDV-COVID-αGC for quantitation
An αGC extraction method was adapted from previous similar studies 51, 52. The necessary number of EDV vials were taken to achieve a total of 4 x 1010 EDVs per sample for extraction of αGC. An EDV only sample was used as a negative control.
All lyophilized vials were resuspended in 400 µL of PBS (Dulbecco’s Ca2+ Mg2+ free, ThermoFisher). Each sample was aliquoted to give ~2 x 1010 EDVs per sample in Eppendorf tubes (i.e., two tubes per sample) and all samples were centrifuged @ 13200 rpm for 7.5 min. The supernatant was removed from each sample and the EDV pellets were resuspended in 800 µL PBS for each 2 x 1010 and centrifuged again as above. The supernatant was removed once again, and all samples were resuspended in 500 µL of UltraPure™ H2O (Cat. # 10977015, ThermoFisher).
For α-GC extraction, each 500 µL sample was transferred to a conical bottom 2 mL microtube (Axygen). One stainless steel bead (5 mm) was added to each sample and samples were then homogenized using agitation on the Qiagen TissueLyser II homogeniser (Qiagen). Homogenisation was carried out in two rounds of 2 min agitations at 25 Hz with a brief stoppage between sets. Lysates were then removed to fresh tubes combining 500 µL aliquots from each sample to give a 1 mL sample (leaving the bead behind).
Each 1mL sample was then extracted for lipids by adding 1 mL of chloroform/methanol (2:1 CHCl3: MeOH ratio), shaking vigorously by hand and incubating at 37°C for 15 min with sonication every 5 min for 1 min. Following 15 min, samples were centrifuged at 2000 g for 10 min in a benchtop micro centrifuge. The organic layer (bottom) was removed to a fresh tube. The samples were then dried before analysis.
Quantitative LC-MS/MS Analysis of αGC
The standard, α-galactosylceramide (αGC), and the internal standard (IS), D-galactosyl-ß-1,1' N-palmitoyl-D-erythro-sphingosine were dissolved in DMSO to 1 mM or 2 mM with heating at 60-80°C for 5 min if necessary to dissolve. Prior to data acquisition the standard stock solution was used to prepare stock dilutions in MEOH:H20 (95:5). A working IS dilution was prepared with final concentration of 200 ng/mL.
Standards were prepared by using five calibration points of αGC (STD) (62.5, 125, 250, 500, and 1 000 ng/mL) spiked with 200 ng/mL IS 52. The standard: IS area ratios were used as calibration curve (CC) points or linearity against which the unknown samples were quantified. The samples were dried and reconstituted in 1ml of working IS dilution.
Samples were acquired along with the freshly prepared CC standards on a TSQ Altis (ThermoFisher) triple quadruple mass spectrometer (MS) interfaced with Vanquish (ThermoFisher) UHPLC (Ultra High-Performance Liquid Chromatography) (LC). The LC-MS instrument method employed for data acquisition was optimised as per Sartorius et. al. (2017) 52. Xcalibur and TraceFinder software were used for data acquisition and analysis respectively (ThermoFisher).
The chromatographic analysis (LC) was performed on an Acquity BEH Phenyl column (Waters, 100 × 2.1 mm, 1.7 μm), eluted with a short gradient program from 95:5 MeOH/H2O to 100% MeOH in 1 min followed by an isocratic elution at 100% MeOH for 4 min. Flow rate was set at 0.4 mL/min and column temperature at 40°C. αGC eluted at a RT of 1.53 min, IS at 1.07 min. Two MRM transitions were monitored for both STD and IS for quantitative purposes and to confirm analytical identification. The most intense transitions for each compound (i.e., m/z 856.7 > 178.9 for STD and m/z 698.5 > 89.2 for IS) were used as analytical responses.
Female BALB/C mice, 6-8 weeks old were obtained from the Animal Resource Centre in Western Australia. The mice were acclimatized for one week before the experiments commenced. All experiments were subject to assessment and approval by the EnGeneIC Animal Ethics Committee according to the “Australian code for the use and care of animals for scientific purposes”. Treatment groups (n = 4-10 depending on experiment) included EDV-COVID-αGC as well as control groups consisting of saline, EDV, EDV-αGC, EDV-CONTROL (Control Plasmid) and EDV-COVID. Initial experiments involved a 2 x 109 i.m. particle dose into a single flank at day 0, followed by a booster of 1 x 109 at day 21. Subsequent experiments applied a higher i.m. only dose of 3 x 109 particles split into 1.5 x 109 per back flank due to limitations of particle volume/concentration acceptable per flank, with a boost of the same dose and mode of delivery at day 21. Depending on the experiment, serum and tissues were collected at 8 h, day 7, day 21 and day 28 post-initial injection. Blood was collected via heart puncture immediately following euthanasia, or tail bleeding for ongoing analysis. Other tissues harvested include spleen, lymph nodes, and bone marrow from the femur.
Isolation of Serum
Whole blood samples in SST vacutainers (Cat. #455092, VACUETTE®) were allowed to clot at RT for 1 h. After centrifugation for 10 min at 800 g the serum layer was aliquoted and stored at -80°C for SARS-CoV-2 specific antibody detection by ELISA and neutralizing antibody assays.
Tissue suspensions were isolated from dissected spleens of treated BALB/C mice using a Dounce homogenizer and resuspended in RPMI-1640 medium (Cat. #R8758, Sigma-Aldrich,). The homogenized tissue was then filtered through sterile 70 µm MACS SmartStrainers (Cat. #130-110-916, Miltenyi Biotec) and incubated with Red Cell Lysing Buffer Hybri-Max™ (Cat. #R7757, Sigma-Aldrich) as recommended by the manufacturer. Cells were then resuspended in 2.5 mL of autoMACS running buffer (Cat. #130-091-221, Miltenyi Biotec) and passed through a 70 µm MACS SmartStrainer to obtain a single-cell suspension.
IFNγ, TNFα, IL-6, IFNα, IL-12p40, IL-10, IL-2 and IL-4 from mouse sera were measured using DuoSet® ELISA kits from R&D Systems according to manufacturer’s instructions (Table S2). Serum levels of IL-21 was analyzed using a LEGEND MAX Mouse IL-21 ELISA kit (Biolegend) following the manufacturer’s instructions. Cytokine concentration was determined by calculating absorbance of the samples against standard curves constructed within the same assay using purified material.
S-protein RBD and S1 IgG/IgM serum titer ELISA
For analysis of anti-RBD specific IgG and IgM antibodies, 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated at 4°C with 50 µL per well of a 2 µg/mL solution of RBD or S1 protein of the corresponding variant being tested (ACRObiosystems) suspended in PBS (GIBCO). On the following day, the coating protein solution was removed and 100 µL of 3% skim milk blocking solution in PBS/0.1% Tween 20 (PBST) or protein free blocking solution (G-Biosciences) was added and incubated at RT for 1 h. Serial dilutions of mouse serum were prepared in 1% skim milk/PBST or protein free blocking solution. The blocking solution was removed and 100 µL of each serum sample was added to the plates and incubated for 2 h at RT. Following incubation, the wells were washed three times with 250 µL of 0.1% PBST, before adding 100 µL of goat anti-mouse IgG (H+L) or IgM (Heavy)–horseradish peroxidase (HRP) conjugated secondary antibody (Cat # 31430, #62-6820; ThermoFisher, 1:3000) prepared in 0.1% PBST. The samples were incubated at RT for 1 h and washed three times with 0.1% PBST. Once completely dry, the samples were visualized by incubating with TMB. The reactions were then terminated, and the samples were read at OD490nm using a KC Junior plate reader (BioTek Instruments).
Antibody titer was determined using ELISA by generating 1:3 serial dilution of the treated mouse serum samples and is expressed as the inverse of the highest dilution with a positive result.
B cell extraction from murine bone marrow
0.5 mL microfuge tubes were punctured at the base with a 21-gauge needle and placed inside a 2 mL tube. The isolated murine tibia and femur were placed in the 1 mL tubes with the cut side of the bone at the bottom. Bone marrow cells were extracted from the tibia and femur via 30 s centrifugation at ≥10000 g. Pelleted cells were resuspended in 1 mL RPMI-1640 medium (Cat. #R8758, Sigma-Aldrich) and incubated with Hybri-Max™ Red Cell Lysing Buffer (Cat. #R7757, Sigma-Aldrich) for 5 min. The lysis buffer was neutralized with 15 mL of RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) (Cat. #AUFBS, Interpath) and centrifuged at 300 g for10 min. Cells were resuspended in a final volume of 10 mL of RPMI-1640 medium for final counting. B cells were isolated using the Pan B Cell Isolation Kit (Cat. #130-095-813, Miltenyi Biotec) as per manufacturers’ instructions.
B cell stimulation and ELISA
ELISA micro plates were coated with 2 µg/mL SARS-CoV-2 spike protein trimer (ACRObiosystems) and incubated overnight at 4°C. Microplates were washed 3x with phosphate-buffered saline (PBS) and blocked with 200 µl/well of Protein-Free Blocking Buffer PBST (Cat. #786-665, G-Biosciences) for 2 h at RT.
Mouse splenocytes were isolated from treated mice and 1x105 cells were seeded into each well in 200 µL AIMV media and incubated at 37°C for 48 h.
At the end of the incubation period, the cells were removed from each well and each microplate was washed 5x with 200 µL/well of 0.05% Tween 20 in PBST. The samples were then incubated in 100 µL/well of 1:5000 mouse IgG-HRP in PBST for 2 h at RT in the dark before washing 3x in 250 µL PBST. The presence of Covid specific IgG was detected by adding 100 µL/well of TMB Substrate System and allowed to incubate up to 20 min or until color solution formed. Enzyme reaction was stopped by adding 50 µL/well of 2N H2SO4 Stop Solution. The samples were analyzed using a CLARIOstar microplate reader (BMG LABTECH) at OD450nm with OD540nm as the reference wavelength and analyzed using the MARS software.
Activation-Induced Markers (AIM) Assay
Isolated spleen cells were seeded at 1 x 106 cells/200 µL/well in AIMV (Life Technologies) serum free media in a 96-well U-bottom plate. Cells were stimulated with 1 µg/mL SARS-CoV-2 trimer (ACRObiosystems) for 24 h at 37oC, 5% CO2. 1 µg/mL DMSO was used as a negative control and 10 µg/mL PHA (Cat. # L2769-2MG; Sigma) as a positive control. After 24 h of stimulation, samples were collected in 1.5 mL microfuge tubes by pipetting up and down to collect the cells and centrifuged at 300 g for 10 min. The supernatant was collected and frozen for processing for IFNγ by ELISA (DuoSet, R&D Systems).
For T-cell activation staining the cell pellets from above were washed twice in 500 µL FACS buffer, centrifuging as above. Final cell pellets were resuspended in 500 µL FACS buffer and stained with the appropriate antibody (included in the kit) for 30 min at RT in the dark (Table S3). Cells were then centrifuged at 300 g for 5 min and washed twice with 500 µL FACS buffer. Cells were then fixed in 1% paraformaldehyde for 10 min at 4°C and after that centrifuged at 300 g for 5 min again. Final resuspension was in 300 µL of FACS buffer before analyzing on a Gallios flow cytometer (Beckman). Single stain samples and mouse IgG isotype controls were used to create compensation for the staining.
Th1/Th2 phenotyping was carried using the Mouse Th1/Th2/Th17 phenotyping kit (Cat. #560758, BD). Firstly, as per AIM assay, isolated spleen cells were seeded at 1 x 106 cells/200 µL/ well in AIMV (Life Technologies) serum free media in a 96-well U-bottom plate. Cells were stimulated with 1 µg/mL SARS-CoV-2 trimer (ACRObiosystems) for 24 h at 37oC, 5% CO2. 1 µg/mL DMSO was used as a negative control. After 24 h of stimulation, 1 μL of BD GolgiStop™ (protein transport inhibitor, Cat. #51-2092KZ; BD) per 200 µL /well of cell culture was added, mixed thoroughly, and incubated for a further 2 h at 37oC. Cells were then centrifuged at 250 g for 10 min and washed 2 times with stain buffer (FBS) (Cat# 554656; BD). The cells were counted and approximately 1 million cells were transferred to each flow test tube for immunofluorescent staining as per manufacturer’s instructions. Cells were protected from light throughout the staining procedure. Firstly, cells were fixed by spinning at 250 g for 10 min at RT and thoroughly resuspending in 1 mL of cold BD Cytofix™ buffer (provided in the kit or Cat# 554655; BD) and incubated for 10-20 min at RT. Following fixation cells were pelleted at 250 g for 10 min at RT and washed twice at RT in stain buffer (FBS). The stain buffer was removed by spinning and the cell pellet was resuspended in 1X BD Perm/Wash™ buffer (Cat# 554723, BD) diluted in distilled water, and incubated at RT for 15 min. Cells were spun down at 250 g for 10 min at RT and the supernatant removed. For staining, the fixed/permeabilized cells were thoroughly resuspended in 50 μL of BD Perm/Wash™ buffer incubated with 20 µL/tube of cocktail included in the kit (Mouse CD4 PerCP-Cy5.5 (clone: RM4-5), Mouse IL-17A PE (clone: TC11-18H10.1), Mouse IFN-GMA FITC (clone: XMG1.2), Mouse IL-4 APC (clone: 11B11) or appropriate negative control. Samples were incubated at RT for 30 min in the dark before proceeding to FACs analysis on a Gallios flow cytometer (Beckman). Compensation was performed manually for each channel using single stained controls.
SARS-Cov-2 Surrogate Virus Neutralization Test (Mouse and Human samples)
Assessment of neutralizing antibodies was carried out using the FDA approved “cPASS SARS-Cov-2 Surrogate Virus Neutralization Test Kit” (Cat. # L00847-A; Genscript) 14. The kit is a blocking ELISA detection tool mimicking the virus neutralization process, suitable for use with serum from mice and other species. The capture plate is precoated with hACE2 protein. The necessary of hACE2 coated plate strips were placed on the plate and the remainder stored at 2-8 °C. HRP-RBD (Wuhan, Genscript) was diluted 1:1000 in HRP dilution buffer provided to a total of 10 mL as per protocol. Mouse and human serum samples, PBMC supernatant and positive and negative controls were diluted 1:10 (10 µL + 90 µL sample dilution buffer) and pre-incubated with HRP-RBD in a 1:1 ratio (60 µL + 60 µL) to allow binding of neutralizing Abs with HRP-RBD. Mixes were incubated at 37°C for 30 min. 100 µL of samples or controls were added to the appropriate wells. The plate was covered with plate sealer and incubated at 37°C for 15 min. The sealer was then removed and the plate washed 4 times with 260 µL of 1X wash solution. The plate was pat dried after washing. 100 µL of TMB solution was then added to each well and the plate incubated in the dark at RT for up to 15 min. 50 µL of stop solution was added to terminate the reactions. Absorbance was analyzed at OD450nm immediately using a CLARIOstar microplate reader. HACE2 receptor binding inhibition was calculated using the formula provided by the manufacturer (% inhibition=1-(OD value of sample/OD value of negative control) x 100%. As per spec sheet a positive value was interpreted as > 30% and a negative as < 30%.
For the assessment of neutralizing antibodies against variant SARS-CoV-2 strains the following HRP-RBD proteins were purchased from Genscript for substitution into the cPASS kit: SARS-CoV-2 Alpha spike protein (RBD, E484K, K417N, N501Y, Avi & His tag)-HRP (Cat. #Z03596), SARS-CoV-2 Beta spike protein (RBD, N501Y, Avi & His tag)-HRP (Cat. #Z03595), SARS-CoV-2 Gamma spike protein (RBD, E484K, K417T, N501Y, Avi & His Tag)-HRP (Cat. #Z03601), SARS-CoV-2 Delta spike protein (RBD, L452R, T478K, Avi & His Tag)-HRP (Cat. #Z03614), SARS-CoV-2 Omicron spike protein HRP (RBD, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, His Tag)-HRP (Cat# Z03730).
Neutralizing titer analysis
Serum samples were diluted in 1:1, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640, and analyzed using the FDA approved “cPASS SARS-Cov-2 Surrogate Virus Neutralization Test Kit” against the wildtype SARS-CoV-2 virus as described previously. The neutralizing titer was determined as the final serum dilution from which resulted in a RBD to hACE2 binding inhibition of greater or equal than 30%.
Student’s T-tests and Ordinary one-way ANOVA with Brown-Forsythe test and Bartlett’s test were conducted using Prism 8 (GraphPad). A p value of <0.05 is statistically significant.
Pancreatic Cancer Clinical Trial Data
All participants in clinical trials conducted at Frankston Private Hospital and Sydney Adventist Hospital signed a patient informed consent form prior to commencement of treatment. Ethics approval granted by Bellberry Limited Human Research Ethics Committee D (00444). Clinical Data presented in this publication is from Clinical Trial ACTRN12619000385145, a Phase I/IIa study of EGFR-Targeted EDVTM carrying the cytotoxic drug PNU-159682 (E-EDV-682) with concurrent immunomodulatory adjuvant non-targeted EDVs carrying an immunomodulator (EDV-αGC) in (i) Cohort 1, subjects with advanced pancreatic cancer solid tumors who have no curative treatment options and (ii) Cohort 2, with other EGFR-expressing solid tumors who failed first or second line therapies or where standard therapies are not appropriate. The results were presented as mean of all 13 patients’ values + SEM.
Individuals provided informed consent and were enrolled in the study (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382580&isReview=true) with approval from St. Vincent’s Hospital Melbourne Human Research Ethics Committee. All participants were otherwise healthy and did not report any history of chronic health conditions. Subjects were identified as SARS-CoV-2 naive via PCR test and naïve for prior COVID-19 vaccines. All subjects received a dose of 8 x 109 EDV-COVID-αGC with an equal booster dose at day 21. Samples were collected at 4 time points: pre-vaccine baseline (time point 1), day 21 before the booster vaccination (time point 2), and day 28 one week post-boost (time point 4). Subjects are also scheduled to return for a 3 month and 6 month time point. Each study visit included collection of 20 mL of peripheral blood. The study began in September 2021 and at time of submission there are a number of volunteers that have come forward to be part of the study. Full data (all time points) at this time is available for four volunteers.
FACS analysis of T cells and B cells in Human Samples
T cell analysis was conducted using DuraClone IM T cell subsets tube (Cat # B53328, Beckman Coulter). 1 x 106 purified PBMCs were added to the tubes directly in 100µL and incubated at RT for 30 min in the dark. The samples were then pelleted at 300g for 5min and washed once in 3mL of PBS. The final samples were resuspended in 500µL of PBS with 0.1% formaldehyde. The compensation for the assay was generated using the Compensation Kit provided in the IM DuraClone T cell subset tube using purified PBMCs.
B cell analysis was conducted using DuraClone IM B cells tube (Cat # B53318, Beckman Coulter). 1 x 106 purified PBMCs were added to the tubes directly in 100µL and incubated at RT for 30 min in the dark. The samples were then pelleted at 300g for 5min and washed once in 3mL of PBS. The final samples were resuspended in 500µL of PBS with 0.1% formaldehyde. The compensation for the assay was generated using the Compensation Kit provided in the IM DuraClone B cell subset tube using purified PBMCs.
Samples were processed using a Gallios flow cytometer (Beckman) and the results were analyzed using the Kaluza Analysis software (ver 2.1, Beckman).
Activation-Induced Markers (AIM) Assay in Human samples
Volunteer PBMCs were seeded at 1 x 106 cells/200 µL/well in AIMV (Life Technologies) serum free media in a 96-well U-bottom plate. Cells were stimulated with 2 µg/mL SARS-CoV-2 trimer (ACRObiosystems) for 24 h at 37oC, 5% CO2. 2 µg/mL DMSO was used as a negative control and PHA (Cat. # 00-49-03; eBiosciences) as a positive control. After 24 h of stimulation, samples were collected in 1.5 mL microfuge tubes by pipetting up and down to collect the cells and centrifuged at 300 g for 10 min. The supernatant was collected and frozen for processing for IFNγ by ELISA (DuoSet, R&D Systems) and for SARS-CoV-2 wildtype surrogate virus neutralization test using the cPASS kit (Genscript). The negative controls of the samples were also used for IL-21 analysis using IL-21 Human ELISA kit (Cat #: BMS2043, ThermoFisher) following the manufacturer’s instructions.