The peptides (N3-Lys)-GSAKFVAAWTLKAAA (peptide 1), corresponding to pan HLA DR-binding epitope PADRE, designed to stimulate T helper cells in genetically diverse populations), (N3-Lys)-FKEELDKYFKNH (peptide 2), corresponding to residues c of the full spike located right before heptad repeat 2 and is essential for the cell membrane fusion, (N3-Lys)-TESNKKFLPFQQ (peptide 3), corresponding to residues 553-564 of the full spike located on the S1, right after the RBD) have been synthesized by Genscript (Piscataway NJ, USA). DNA oligonucleotides (ssDNA) were synthesized using MerMade 12 DNA synthesizer with the help of the standard phosphoramidite chemistry with details provided in the supplement. The following set of oligonucleotides were synthesized: Strand 1 DBCO-TATACAGCCTACTCACTATA; Strand 2 DBCO-TATACTGAGCTAGTCGTATA; Strand 3 DBCO-TATACCTTCATCCTTATATA and a complement strand (SH)-A9TATAGTGAGTAGG
CTGTATATATACGACTAGCTCAGTATATATATAAGGATGAAGGTATA. Citrate-coated gold nanoparticles 15 nm in diameter and at OD 50 (Luna Nanotech, Markham, ON, Canada) were used for formulation.
Each peptide and DNA strand were dissolved in 6 M guanidinium hydrochloride in the presence of sodium phosphate buffer (50 mM, pH 7.0) at the final concentrations of 200 µM ssDNA and 400 µM peptides. The reaction was carried out for 24 h at 4°C, followed by dialysis to remove excess peptides. Reaction completion was verified by the disappearance of 308 nm absorbance shoulder on UV-Vis, corresponding to the unreacted DBCO.
Assembly of the antigen.
Peptide-coupled DNA strands (1-3) were combined in isotonic 0.5 X PBS pH 7.4 at a concentration of 150 µM and 140 µM of the compliment. The mixture was heated to 95°C followed by gradual cooling to 4°C (2 h). The annealed product was diluted to 1.2 OD at 260 nm in isotonic PBS, and the assembly was verified by the melting experiments using UV-Vis spectrophotometer Varian Cary 50 Bio with a temperature range of 10-85°C, data interval 0.1°C, temperature ramp rate 2°C/minute, signal averaging time 0.1. The melting temperature was compared to the theoretically melting temperature predicted by OligoAnalyzer™ from Integrated DNA Technologies (IDTDNA.com).
Vaccine candidates were formulated by freezing-based (FR), pH-assisted (PA), salt concentration-based (SC) conjugations, and inulin co-crystallization (IN). In the FR method, the annealing product was diluted in water 4-fold (<20mM Na+) and added to the GNP stock solution (136 nM) at a molar ratio of 150:1. The mixture was placed in a -20°C freezer for 1 h. Then, the product was allowed to thaw, and 10 x PBS was added to adjust the tonicity of the final product to isotonic. The solution was diluted to 50 nM (as GNP) in PBS, aliquoted at 100 µl/dose, and frozen for use as an immunogen.
In the PA method, the annealing product was combined with GNP stock solution at a molar ratio of 50:1 and incubated for 5 min. Then, pH was adjusted to 3.0 with 1M citric acid, incubated for 3 min, and neutralized with NaOH to pH 6.5. Heat annealing was performed as described previously. The conjugate was dialyzed against isotonic PBS, diluted, and aliquoted as in the FR method.
In the SC method, the initial procedure was identical to the FR method. Instead of freezing, saturated NaCl (6.15 M) was gradually added to a 1 M final concentration (48 h). The conjugate was dialyzed against isotonic PBS, diluted, and aliquoted as in the FR method.
For IN co-crystallization conjugation, inulin microcrystals (epsilon form) were prepared as described in 64. The microcrystals were washed five times via centrifugation and resuspension in 45°C water. After the final centrifugation, the wet pellet was resuspended in the annealed product at final proportions of 300 mg of wet pellet and 5 moles of annealed product per 1 ml in isotonic PBS. The mixture was then heated up to 55°C and allowed to cool down slowly. The resulting microcrystals were aliquoted at 100 µl/dose and stored at 4°C until further use.
Agarose gel electrophoresis.
Agarose (2%) (Sigma-Aldrich, Inc. St. Louis, MO) gel was prepared in sodium borate buffer (10 mM, pH 8.0) and used for electrophoresis of unconjugated and conjugated GNP prepared by the FR, PA, and SC methods. The nanoparticles were loaded directly in buffer without loading dye at 10 µl/well, and electrophoresis was carried out at 15 V/cm (300 V).
Cells and viral titer determination.
Vero E6 (ATCC® CRL-1586™) and Vero-STAT1 knockout cells (ATCC® CCL-81-VHG™) were cultured in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and 10 mM HEPES. Calu-3 cell (ATCC 184HTB-55) were cultured in Eagle's 188 Minimum Essential Medium (EMEM) (ATCC 30-2003) containing 10% FBS. SARS-CoV-2 isolates USA-WI1/2020 (BEI; cat# NR-52384), USA-WA1/2020 (BEI; cat# NR- NR-52281) were passaged in Vero-STAT1 knockout cells, whereas hCoV-19/USA/PHC658/2021 (Delta Variant) (BEI; cat# NR-55672) was passaged in Calu-3 cells. The viral titer was determined using the plaque assay as described previously 65. In brief, Vero E6 cells (2.510 ^5) were seeded in 6-well plates and incubated for 24 h. After 24 h, cells were washed with sterile 1X PBS, and the virus stock was ten-fold serially diluted in serum-free OptiMEM media and then added to the cells in duplicate. The plates were incubated at 37°C for 1 h with slight shaking every 15 minutes. Then, 2 ml of 0.5% agarose in minimal essential media (MEM) containing 5% FBS and antibiotics were added to each well and incubated at 37°C for 72 h. The cells were fixed with 4% paraformaldehyde overnight, followed by removing the overlay and staining with 0.2% crystal violet to visualize plaque-forming units (PFU). All assays were performed in a BSL-3 laboratory setting.
BALB/c mice (males, 6 to 8 weeks old) were purchased from Jackson Laboratories and housed in micro isolator cages and maintained at 12-h light-dark cycle at 22.2°C, and 30–40% humidity. Mice were given feed and sterile water daily, and they were acclimated to the environment for approximately 1-2 weeks to determine that they were healthy and suitable for experiments. Four groups of mice (n=5 each group) were immunized subcutaneously with GNP-conjugated vaccines (100 µl) by different methods, FR, PA, SC, and IN. The immunogens were diluted in LPS free water and mixed with 25 µg ODN 1826 Class B CpG oligonucleotide (a murine TLR9 ligand) as an adjuvant. Another group of mice immunized with only 25 µg ODN 1826 Class B CpG oligonucleotide serves as the control. Baseline bloods were collected from the submaxillary vein on day 0 (prior to immunization). Blood samples were collected every 14 days with boosts of the same doses were given. All mice were immunized with 2 boosts and finally necropsied at day 61. Blood samples and spleens were collected at necropsy for flow cytometry. All the animal procedures, including housing, are approved by the University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee (IACUC) protocol # 21-020-04-EP. and are conducted according to the National Institutes of Health guidelines. Furthermore, this study is reported in accordance to ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines).
Enzyme-Linked Immunosorbent Assay (ELISA).
Ninety-six-well high binding plates were separately coated with peptide P1, P2 (100 ng/well), whole S-protein, and spike-RBD protein (0.1mg/well) 1X PBS and incubated 4°C overnight. The next day, the plates were washed three times and blocked with 1% BSA in 1X PBS containing 0.1% Tween 20 (PBST) for 1 h at 37°C. Sera samples were diluted starting from 1:40 (please check this) and five-fold serially in 1% BSA containing PBST were added to the plates and incubated at 37°C for 1 h. The plates were washed three times with PBST and HRP-conjugated goat anti-mice IgG Human ads-HRP (SouthernBiotech1:4,000 dilution) (cat#103005) was added and incubated for 1 h at room temperature. Plates were washed five times with PBST before the addition of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reaction was stopped after 5 min by the addition of 0.16 M sulfuric acid to stop the reaction. The OD at 450 nm was measured with a Bio-Rad microplate reader. A graph was plotted as Absorbance at 450 nm vs dilutions.
Immunofluorescence based Neutralization assay.
Neutralization assays were performed against USA_WI/2020, USA-WA1/2020 and hCoV-19/USA/PHC658/2021 strains. Sera samples were serially diluted (5-fold) in serum-free Dulbecco’s modified Eagle’s medium (DMEM) in triplicate wells and incubated with 20000 focus-forming units of SARS-CoV-2 virus at 37°C for 1 h. The serum-virus mixture was added to Vero E6 cell (C1008, ATCC, no. CRL-1586) monolayers seeded in 96 healthy blackout plates and incubated at 37°C for 1 h. The inoculum was removed and replaced with complete DMEM and incubated at 37°C for 24 h. After 24 h, DMEM was removed, cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Following fixation, plates were washed thrice with 1X PBS and permeabilized with 50 µl/well of 0.1% Triton X-100 (Fisher BP151-100) in PBS for 10 min. After permeabilization, cells were washed thrice with 1XPBS and blocked with 3% BSA for 30 mins. Then, cells were stained with 50 µl/well of primary antibody, anti-SARS-CoV-2 spike (rabbit mAb, Sino Biologicals MA14AP0204) at 1:1000 diluted in 3% BSA-PBS and incubated overnight at 4°C on a shaker. The next day, cells were washed three times with 1X PBS and stained with a secondary antibody (Alexa Fluor 488 Goat anti-rabbit) at 1:2000 dilution in 3% BSA-PBS. Finally, cells were incubated at room temperature in a shaker for 1 h, washed three times with 1X PBS, and the nuclei were stained using Hoechst 33342 (Invitrogen H3570) and Cell Mask (Invitrogen C10046) at 1:20000 diluted in 1X PBS. The plate was shaken for 15 mins after proper sealing with aluminum foil/sealer and taken for reading using Operetta Imager. Percentage neutralization was calculated based on the difference in fluorescent intensity.
Analysis of B cells and CD4+/CD8+ memory phenotypes by flow cytometry.
Blood samples and spleen tissues were collected from immunized mice at necropsy. Single-cell suspension from spleen tissues was prepared, and RBCs were lysed using RBC lysis buffer. Cells (5x106) were washed and suspended in staining buffer, and Fc block was performed with purified anti-mouse CD16/32. Dead cells were discriminated by adding Zombie Aqua fixable viability dye. Fluorochrome-labeled mouse-specific antibodies were diluted with staining buffer into cocktails and added to cells at 100 µl per sample. Cells were incubated for 30 min at room temperature and washed twice with staining buffer. Then, cells were fixed using 2% PFA and acquired using the Becton Dickson Fortessa X450 flow cytometer. Fluorescence minus one (FMO) control was performed in parallel, and subsequent gating was determined by FMO. All data were analyzed using FlowJo software
Bars and error bars represent mean G standard error. Data were analyzed using GraphPad Prism (v6.07).