A Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine
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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 × 10 5 TCID 50 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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SciScore for 10.1101/2021.12.28.474336: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: 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)
IACUC: 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)Sex as a biological variable 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. Randomization Animals and study design: Outbred adult male and female rhesus macaques (M. … SciScore for 10.1101/2021.12.28.474336: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: 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)
IACUC: 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)Sex as a biological variable 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. Randomization 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. Blinding All immunological and virological assays were performed blinded. Power Analysis not detected. Cell Line Authentication Contamination: 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). Table 2: Resources
Antibodies Sentences Resources 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). ab275759suggested: (Abcam Cat# ab275759, RRID:AB_2892127)Anti-Rabbit IgGsuggested: (Abcam Cat# ab150077, RRID:AB_2630356)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) SARS-CoV-2 spike proteinsuggested: Noneanti-human MICA/MICBsuggested: None6D4suggested: NoneAlexa 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). anti-human HLA-Asuggested: NoneW6/32suggested: (Bio-Rad Cat# MCA81A488, RRID:AB_324712)anti-SARS-CoV-2 Spike Glycoprotein S1suggested: (Abcam Cat# ab275759, RRID:AB_2892127)ab150077suggested: (Abcam Cat# ab150077, RRID:AB_2630356)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. anti-SARS-Cov2suggested: Noneb-actin , Abcam 8226 , 1suggested: Noneanti-Rabbitsuggested: (Sino Biological Cat# SSA003, RRID:AB_2814815)anti-Mouse HRPsuggested: (Abcam Cat# ab205719, RRID:AB_2755049)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). antibodies-MICA/MICBsuggested: NoneAnti-CD107a antibody (clone H4A3, ECD conjugate, BD Biosciences) Anti-CD107asuggested: NoneExperimental Models: Cell Lines Sentences Resources , 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. HEK293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)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. HEK293T-hACE2suggested: RRID:CVCL_A7UK)Recombinant DNA Sentences Resources 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. pcDNA3.1suggested: RRID:Addgene_79663)In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program) psPAX2suggested: RRID:Addgene_12260), 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. pLenti-CMVsuggested: NonepcDNA3.1-SARS-CoV-2suggested: NoneSoftware and Algorithms Sentences Resources For analysis by TIDE, PCR amplicons were Sanger sequenced (Eurofins or Genewiz) and paired . Genewizsuggested: (GENEWIZ, RRID:SCR_003177)Intracellular spike protein staining: Engineered UVC were harvested and then fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Solution (ThermoFisher). BD Cytofix/Cytoperm Fixation/Permeabilization Solutionsuggested: NoneFlow 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) FlowJosuggested: (FlowJo, RRID:SCR_008520)GraphPad Prism was used to plot a standard curve and interpolate the sample values using a 4-parameter logistic fit. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)In brief, the packaging construct psPAX2 (AIDS Resource and Reagent Program) AIDS Resource and Reagent Programsuggested: NoneStatistical 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. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:A limitation of the WA1/2020 UVC in a Delta heterologous challenge was the absence of any meaningful protection in nasal swabs. This discordance between vaccine variant, versus virus variant, is the principal challenge facing current vaccinated populations and might suggest the reason for ongoing infectious spread but more limited morbidity and mortality against these emerging variants29, 31. As regards duration of protection, the theoretical hyper-immunity postulated by creating a self-adjuvanting, hyper-immune UVC established robust initial nAb titers. When these animals were rechallenged 6-month later, the nAb response stayed robust at the higher, and now established for clinical use, 1e8 UVC dose. Moreover, the persistent nAb response at 6-months remained robust for SARS-CoV-2 WA1/2020, Beta and Delta variants. As regards intrinsic safety, the UVC undergoes lethal irradiation during manufacture and rapid apoptosis in the immune microenvironment upon vaccination. This is the principal mechanism of efficacy of the UVC, and fortunately its most redeeming safety feature, by virtue of the impossibility of in vivo persistence and teratogenicity of the cellular antigen carrier. The irradiation-induced apoptosis is further enhanced by CRISPR genetic engineering to remove MHC-I expression and introduce cell surface expression of the NKG2D ligand MICA, making the UVC potent targets for host NK cells. Recruited NK cells will likely recognize the UVC as virally infected cell through ...
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