Methods

A Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine

iPS cell culture, irradiation, and cryopreservation

Human iPS cells (Thermo Fisher) were cultured on vitronectin-coated T225cm2 flasks using complete mTesSR Plus medium (StemCell Technologies) supplemented with 1% penicillin/streptomycin, Rock inhibitor (StemCell Technologies) at 1:1000 dilution. For drug selection, G148 was used at 500ug/ml and puromycin at 5ug/ml (Sigma-Aldrich). Cultures were maintained at 37 °C, 5% CO2 in a humidified incubator. Harvesting of engineered UVC was performed using accutase (StemCell Technologies) and cells were counted using a CellDrop cell counter (DeNovix). Cells were irradiated at a total single dose of 10 Gy, before centrifugation at 300 xg for 10 minutes followed by resuspension in 100 μl of CryoStor-CS10 freezing media (StemCell Technologies). The UVC preparations for use in non-human primate studies were analyzed for endotoxin levels (Wickham Laboratories Ltd) and absence of mycoplasma (Mycoplasma Experience Ltd).

CRISPR genetic engineering

CRISPR sgRNAs targeting the human B2M gene, PPP1R12C (AAVS1), and the ROSAβgeo26 locus were designed and validated for indel formation at the selected genomic site. Up to 6 sgRNAs per target gene were tested and the most efficient sgRNA was selected containing 2’-O-methyl and 3’ phosphorothioate modifications to the first three 5’ and the last three 3’ nucleotides (Synthego). 2×106 UVC cells were electroporated using a Neon Nucleofector (Lonza) in Buffer P3 (Lonza) with Cas9 protein (IDT) precomplexed with sgRNA, in a total volume of 100 μl using electroporation program CM138. Gene targeting vectors carrying an expression cassette for expression of human MICA or the SARS-CoV-2 WA1/2020 spike gene, targeting the Rosa26 and AAVS1 locus respectively, were co-electroporated at 4 μg. Indels introduced by CRISPR editing were detected by PCR and Sanger sequence using DNA primers designed to amplify a 600-900 base pair region surrounding the sgRNA target site. A minimum of 24 hours after electroporation, genomic DNA was extracted using the DirectPCR Lysis solution (Viagen Biotech) containing Proteinase K and target regions were amplified by PCR using the GoTaq G2 PCR mastermix (Promega). Correct and unique amplification of the target regions was verified by agarose gel electrophoresis before purifying PCR products using the QIAquick PCR Purification Kit (Qiagen). For analysis by TIDE, PCR amplicons were Sanger sequenced (Eurofins or Genewiz) and paired .ab1 files of control versus edited samples were analyzed using Synthego’s ICE tool (https://ice.synthego.com).

Intracellular spike protein staining

Engineered UVC were harvested and then fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Solution (ThermoFisher). Cells were then stained for intracellular spike protein using an Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (Abcam, ab275759, 1:50) followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (Abcam, ab150077, 1:500). Flow analysis was carried out on a Fortessa flow cytometer (BD Bioscience), and data analyzed, and flow cytometry figures generated using FlowJo 10 software (BD Biosciences).

Flow cytometry analysis of cell surface antigen expression

For flow cytometric analysis of cell surface expression of MHC-I, MICA and SARS-CoV-2 spike protein, cells were harvested from culture plates and washed using PBS with 1% Bovine Serum Albumen (Thermo Scientific) and were then stained with PE anti-human MICA/MICB Antibody (6D4, Biolegend), Alexa Fluor 647 anti-human HLA-A,B,C (W6/32, Biolegend), and anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (Abcam, ab275759, 1:50) followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (Abcam, ab150077, 1:500). Live/Dead Fixable Dead Cell Stains (Invitrogen) were included in all experiments to exclude dead cells. After staining, cells were resuspended in PBS with 2% Human Heat Inactivated AB Serum (Sigma) and 0.1 M EDTA pH 8.0 (Invitrogen) before analysis on a Fortessa flow cytometer (BD Bioscience) and data analyzed using FlowJo 10 software (BD Biosciences).

Western blot

The SARS-CoV-2 spike glycoprotein was detected in UVC lysates by western blotting. Briefly, cells were lysed by RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP40, 1x protease inhibitor cocktail). Samples were spun at 4°C for 10 mins at 12,000 xg and the pellet discarded. Protein content was measured using BCA Assay (ThermoFisher) using a PHERAstar plate reader (BMG Labtech) at 560 nm. LDS Sample Buffer was added to 30 ng of protein sample to make a 1x solution, with 0.5 μl of b-mercaptoethanol per well and heated at 70°C for 10 minutes before separation on a polyacrylamide gel (Bio-Rad Mini-PROTEAN TGX Gel 4-15%) and transferred to a PVDF membrane. Membranes were blocked in blocking buffer (5% non-fat powdered milk in TBST), before incubation with primary antibodies in blocking buffer (Rabbit polyclonal anti-SARS-Cov2, Sino Biological 40591-T62, 1:6000 dilution or Mouse b-actin, Abcam 8226, 1 μg/ml), detected with HRP conjugated secondaries in blocking buffer (Goat anti-Rabbit HRP, Sino Biological SSA003, 0.5 μg/ml or Goat anti-Mouse HRP, Abcam ab205719, 1: 4000 dilution) and visualised using the SuperSignal West Femto kit (ThermoFisher) as per kit instructions.

qPCR measurement of stem cell factors

Total RNA was extracted from UVC cells using the ReliaPrep RNA miniprep (Promega) according to the manufacturer’s instructions (a DNase treatment was included for all samples), and RNA concentration and absorbance ratios were measured using a Nanodrop One Spectrophotometer (ThermoFisher). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (the Applied Biosystems) in a total volume of 20 μl to produce DNA that was subsequently assessed by spectrophotometric analysis and diluted to 100 ng/μl. Individual master mixes with each of the DNA-primer combinations for detection of human SOX2, NANOG, OCT4, DNC, Vimentin, HES5 and GATA6 genes were made for 3 replicates using the Brilliant III Ultra-Fast SYBR green qPCR master mix (Agilent Technologies) and analyzed on a CFX Opus Real-Time PCR system (BioRad) using the following program: 95 °C for 15 minutes for 1 cycle; 95°C for 15 seconds for 40 cycles; 60°C for 30 seconds.

SARS-CoV-2 spike protein ELISA

Cell pellets were harvested and lysed in 20 μl Cell Extraction Buffer (Invitrogen) containing protease inhibitors (Sigma) on ice for 30 minutes, with 3 brief vortexing every 10 minutes. Samples were centrifuged at 13,000 rpm for 10 minutes at 4°C to pellet insoluble contents. S1 Spike protein was detected using a Covid-19 S-protein ELISA kit (Abcam) specific to S1RBD. Samples were diluted to a range determined to be within the working range of the ELISA kit used and the assay procedure was follows as per manufacturer’s instructions. The resulting colorimetric signal was detected at 450 nm using a PHERAstar (BMG LABTECH) plate reader. GraphPad Prism was used to plot a standard curve and interpolate the sample values using a 4-parameter logistic fit.

UVC Proliferation and apoptosis assays

To quantify apoptosis of UVC post-irradiation, cells were stained using a FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend). Proliferation of cells was measured staining of control and Irradiated UVC with either 2 μM Cell Trace Yellow (Abcam) according to kit protocol and analyzing the dilution of the dye at 24-hour periods over 3-days and measuring fluorescence intensity. Flow analysis was carried out on a Fortessa flow cytometer (BD Bioscience), and data analyzed, and flow cytometry figures generated using FlowJo 10 software (BD Biosciences).

CAM cytotoxicity assay

Both MHC-I expressing and MHC-I deficient (B2M knockout) UVC were used as target cells for NK cell cytotoxicity assay. Trypsinized cells were stained with calcein acetoxymethyl ester (CAM, Invitrogen) at a 10 μM concentration for 1 hour at 37°C and then washed to remove excess dye. NK cells highly enriched from normal cynomolgus macaque (Macaca fascicularis) blood samples using a CD3 depletion kit (Miltenyi Biotec), were used as effector cells. NK cell effectors and stained target cells were co-cultured in 96 well round bottom plates at effector: target (E:T) ratios of 1:1 and 5:1. Control wells included – only target cells for spontaneous release of CAM and target cells treated with Triton-X100 for maximum release of CAM. At the end of 4-hour incubation, supernatant was collected for CAM measurement in a fluorescent plate reader at 530 nm. Percent-specific lysis = (test release - spontaneous release)/(maximum release - spontaneous release).

Nucleofection of NKG2D ligands in iPS cells

UVC were cultured in EGM2 (Lonza) media supplemented with 20 ng/ml VEG-F (Peprotech) until 70-90% confluent, in tissue culture flasks pre-coated with sterile 0.1% gelatin in PBS for 1 hour at 37°C. The cells were removed from culture flasks using trypsin, washed, and transfected with plasmid DNA containing either MICA, MICB or ULBP-1 genes after optimizing nucleofection conditions using primary cell 4D nucleofector kit and 4D nucleofector system (Lonza). After 48 hours of culture, transfected cells were stained with aqua dye for live/dead discrimination and corresponding antibodies-MICA/MICB (Clone 6D4, PE, BioLegend) or ULBP-1 (clone 170818, PE, R & D Systems). Stained cells were fixed with 2% paraformaldehyde and acquired on LSRII flow cytometer. Transfection efficiency was calculated as % live cells expressing transfected protein.

NK cell intracellular cytokine staining assay

NK cell effectors were enriched from normal cynomolgus macaque (Macaca fascicularis) blood samples using a CD3 depletion Kit (Miltenyi Biotec). Target and effector cells were plated at E:T ratio of 2:1 in a 96 well round bottom plate. Anti-CD107a antibody (clone H4A3, ECD conjugate, BD Biosciences), brefeldin A and monensin (BD Biosciences) were added to all the samples prior to incubation. After 6 hours of incubation at 37°C, the cells were washed and stained with aqua dye used for live and dead cell discrimination for 20 minutes at room temperature. The cells were then washed and stained for surface markers that included CD3 (SP34.2, BV421, BD Biosciences), CD14 (M5E2, BV650, BD Biosciences), CD16 (3G8, BUV496, BD Biosciences), CD20 (L27, BV570, BD Biosciences), CD56 (NCAM1.2, BV605, BD Biosciences), HLA-DR (G46-6, APC-H7, BD Biosciences) and NKG2A (Z199, PE-Cy7, BD Biosciences) to delineate NK effector cells. Following incubation for 20 minutes, cells were washed and permeabilized using fix & perm reagent (Thermofisher Scientific) as per manufacturer’s recommendation. Intracellular cytokine staining was performed for macrophage inflammatory protein 1β (MIP-1β; D21-1351, FITC, BD Biosciences) interferon-γ (IFN-γ; B27, BUV395, BD Biosciences), tumor necrosis factor-α (TNF-α; Mab11, BV650, BD Biosciences) at 4°C for 15 minutes.

Cells were washed, fixed, and acquired on LSRII flow cytometer. Unstimulated NK cells were used for background subtraction of percent positive cells. NK cells stimulated with leukocyte activation cocktail (BD Biosciences) were used as positive control for the assay.

Animals and study design

Outbred adult male and female rhesus macaques (M. mulatta) and cynomolgus macaques (M. fascicularis), 6–12 years old, were randomly allocated to groups. All macaques were housed at Bioqual. Macaques were treated with irradiated UVC at doses of either 1×107 or 1×108 cells (n = 3-6), and sham controls (n = 3-6). Prior to immunization, the cryopreserved doses of irradiated UVC were thawed at 37°C, then 900 μl of 1xPBS was added to each vial of 100 μl UVC in CryoStore freezing media. Macaques received a prime immunization of 1ml of UVC by the intramuscular route without adjuvant at week 0. At weeks 4 or 6, macaques received a boost immunization of either 1×107 or 1×108 UVC. At week 10 all macaques were challenged with 1.0 × 105 TCID50 (1.2 × 108 RNA copies, 1.1 × 104 PFU) SARS-CoV-2, which was derived from B.1.617.2 (Delta). Viral particle titers were assessed by RT– PCR. Virus was administered as 1 ml by the intranasal route (0.5 ml in each nare) and 1 ml by the intratracheal route. All immunological and virological assays were performed blinded. All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (IACUC).

Subgenomic viral mRNA assay

SARS-CoV-2 E gene sgRNA was assessed by RT–PCR using primers and probes as previously described49, 50, 51, 52. In brief, to generate a standard curve, the SARS-CoV-2 E gene sgRNA was cloned into a pcDNA3.1 expression plasmid; this insert was transcribed using an AmpliCap-Max T7 High Yield Message Maker Kit (Cellscript) to obtain RNA for standards. Before RT–PCR, samples collected from challenged macaques or standards were reverse-transcribed using Superscript III VILO (Invitrogen) according to the manufacturer’s instructions. A Taqman custom gene expression assay (ThermoFisher Scientific) was designed using the sequences targeting the E gene sgRNA. Reactions were carried out on a QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems) according to the manufacturer’s specifications. Standard curves were used to calculate sgRNA in copies per ml or per swab; the quantitative assay sensitivity was 50 copies per ml or per swab.

Serum antibody ELISA

RBD-specific binding antibodies were assessed by ELISA as previously described9,10. In brief, 96-well plates were coated with 1 μg ml-1 SARS-CoV-2 RBD protein (A. Schmidt, MassCPR) in 1× DPBS and incubated at 4 °C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween 20 in 1× DPBS) and blocked with 350 μl casein block per well for 2–3 hour at room temperature. After incubation, block solution was discarded, and plates were blotted dry. Serial dilutions of heat-inactivated serum diluted in casein block were added to wells and plates were incubated for 1 hour at room temperature, before three further washes and a 1-hour incubation with a 1:1,000 dilution of anti-macaque IgG HRP (NIH NHP Reagent Program) at room temperature in the dark. Plates were then washed three times, and 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 μl SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.2. The log10(endpoint titers) are reported.

Pseudovirus neutralization assay

The SARS-CoV-2 pseudovirus expressing a luciferase reporter gene were generated in a similar approach to that previously described9,10,16. In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS-CoV-2 SΔCT were co-transfected into HEK293T cells with calcium phosphate. The supernatants containing the pseudotype viruses were collected 48 hours after transfection; pseudotype viruses were purified by filtration with 0.45-μm filter. To determine the neutralization activity of the antisera from vaccinated macaques, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 × 104 cells per well overnight. Twofold serial dilutions of heat-inactivated serum samples were prepared and mixed with 50 μl of pseudovirus. The mixture was incubated at 37 °C for 1 hour before adding to HEK293T-hACE2 cells. After 48 hours, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in relative light units was observed relative to the average of the virus control wells.

Statistical Analyses

Statistical differences between two sample groups, where appropriate, were analyzed by a standard Student’s two-tailed, non-paired, t-test and between three or more sample groups using two-way or three-way ANOVA using GraphPad Prism 9. Analysis of virological data was performed using two-sided Mann–Whitney tests. Correlations were assessed by two-sided Spearman rank-correlation tests. P values are included in the figures or referred to in the legends where statistical analyses have been carried out. P values of less than 0.05 were considered significant.

Article TitleA Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine

Abstract

COVID-19 has forced rapid clinical translation of novel vaccine technologies, principally mRNA vaccines, that have resulted in meaningful efficacy and adequate safety in response to the global pandemic. Notwithstanding this success, there remains an opportunity for innovation in vaccine technology to address current limitations and meet the challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) rationally designed to mimic the natural physiologic immunity induced post viral infection of host cells. Induced pluripotent stem cells were CRISPR engineered to delete MHC-I expression and simultaneously overexpress a NK Ligand adjuvant to increase rapid cellular apoptosis which was hypothesized to enhance viral antigen presentation in the resulting immune microenvironment leading to a protective immune response. Cells were further engineered to express the parental variant WA1/2020 SARS-CoV-2 spike protein as a representative viral antigen prior to irradiation and cryopreservation. The cellular vaccine was then used to immunize non-human primates in a standard 2-dose, IM injected prime + boost vaccination with 1e8 cells per 1 ml dose resulting in robust neutralizing antibody responses (1e3 nAb titers) with decreasing levels at 6 months duration. Similar titers generated in this established NHP model have translated into protective human neutralizing antibody levels in SARS-Cov-2 vaccinated individuals. Animals vaccinated with WA1/2020 spike antigens were subsequently challenged with 1.0 × 105 TCID50 infectious Delta (B.1.617.2) SARS-CoV-2 in a heterologous challenge which resulted in an approximately 3-log order decrease in viral RNA load in the lungs. These heterologous viral challenge results reflect the ongoing real-world experience of original variant WA1/2020 spike antigen vaccinated populations exposed to rapidly emerging variants like Delta and now Omicron. This cellular vaccine is designed to be a rapidly scalable cell line with a modular poly-antigenic payload to allow for practical, large-scale clinical manufacturing and use in an evolving viral variant environment. Human clinical translation of the UVC is being actively explored for this and potential future pandemics.


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