Safety, immunogenicity and efficacy of an mRNA-based COVID-19 … – Nature.com

Ethics statements

All animal work related to this study was conducted following code of ethics for the care and use of animals as guided by the Public Health Service (PHS) Policy on Human Care and Use of Laboratory Animals, an in compliance with the Housing and handling of the animals following the standards of AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). Studies were approved by institutional animal care and use committees(IACUC) at Charles River Laboratory and Bioqual Inc, ensuring that all experimental procedures were performed in compliance with applicable animal welfare laws and regulations. Animals were housed in suitable facilities with access to food, water, and environmental enrichment. Trained personnel performed the procedures, minimizing the number of animals used and optimizing their welfare. All mice were euthanized at terminal study timepoints by CO2 asphyxiation. Hamsters were euthanized via isoflurane inhalation and terminal bleed followed by bilateral thoracotomy. All methods were in accordance with ARRIVE guidelines.

GLB-COV2-043 mRNA encodes for the full-length wild-type spike (S) protein of SARS-CoV-2 from the original Wuhan strain, Wuhan-Hu-1 (GenBank: QHD43416.1). The mRNA is modified and contains pseudouridine (), instead of uracil (U). It is produced by in vitro transcription and purified to generate the final 2.0mg/mL mRNA, which is stored at-65C until encapsulated in the lipid nanoparticle (LNP). The final formulated mRNA-LNP is manufactured in a multi-step process that involves mixing of aqueous GLB-COV2-043 mRNA and an organic phase of the lipid components (a proprietary cationic lipid, a saturated phospholipid, a PEG-lipid and cholesterol), of the lipid nanoparticles. After the LNPs are formed, the organic phase is removed, a buffer exchange is performed, sucrose is added, and the LNPs are diluted to the mRNA target concentration and aseptically filtered to generate the bulk mRNA-LNP dispersion. The mRNA-LNP is tested for critical attributes and stored at65C before final use. The lipid nanoparticle systems were developed by and licensed from Acuitas.

HEK293FT (Thermo Fisher), were cultured in Dulbeccos Modified Eagles Medium (DMEM, Gibco) supplemented with 10% FBS (Gibco), 1% penicillinstreptomycin (Gibco), 6mM L-glutamine (Gibco), 1mM MEM sodium pyruvate (Gibco), 0.1mM MEM non-essential amino acids (Gibco) at 37C and 5% CO2. HEK293T-hACE2 cells (SBI Biosciences) were cultured in DMEM media containing 10% FBS, 1% PenStrep, and 2g/mL puromycin (Fisher Scientific) at 37C and 5% CO2.

HEK293FT (Thermo Fisher) cells were transfected with GLB-COV2-043 mRNA encoding SARS-CoV-2 full-lengthwild-type (Wuhan-Hu-1) Spike protein using Lipofectamine MessengerMax (Thermo Fisher) following the supplier recommendations. For demonstration of cell surface expression of full-length Spike protein, 24h post transfection, cells were collected and detected with anti-Spike rabbit IgG, which in turn were detected using anti-rabbit-IgG-FITC that showed surface expression of the full-length Spike proteins. For intracellular staining of the cells, DAPI was used. For hACE2 binding, 24h post transfection, cells were collected and resuspended in FACS buffer. Cells were stained with biotinylated 1108M hACE2 (ACRO), in FACS buffer for 1h at room temperature. Thereafter, cells were washed twice with FACS buffer and incubated with PE-Streptavidin (Invitrogen) in FACS buffer for 1h at room temperature. The cells were washed and resuspended in FACS buffer, and the acquisition was performed on a BD Symphony instrument (BD Biosciences) and analyzed using FlowJo v10.8.1 software.

5105 HEK293FT cells/well were plated on a 6 well plate (Corning) and were transiently transfected with 2g GLB-COV2-043 mRNA encoding SARS-CoV-2 full lengthwild-type (Wuhan-Hu-1) Spike protein using Lipofectamine MessengerMax (Thermo Fisher) following the supplier recommendations. 20h post transfection, the cells were lysed using RIPA lysis buffer (Thermo Fisher) with protease inhibitors (Thermo Fisher) and DNAse I (NEB). The cell lysates were collected after centrifugation (4C, 12,000g for 15min), reduced with DTT (Thermo Fisher) and denatured in sample buffer (Thermo Fisher) upon heating at 95C for 5min. Cell extracts were run on a 412% BisTris gel (Invitrogen) and transferred to a nitrocellulose membrane (Bio-Rad) using Trans-Blot Turbo transfer system (Bio-Rad). The membrane was then blocked with Intercept blocking buffer (Licor) for 1h at room temperature and incubated with SARS-CoV-2 Spike RBD or anti-S2 antibodies (Sino Biological) at 4C overnight. The following day, the membrane was washed with 1PBS-0.2%Tween-20 (Fisher) and incubated with a HRPlabeled goat anti-rabbit secondary antibody (Abcam) at room temperature for 1h. The membrane was washed again with PBS-0.2% Tween-20 buffer, and the signal was detected using an Odyssey CLx Infrared Imaging System.

C57BL/6 female mice, 68weeks of age, were used for the studies that were performed at Charles River Laboratory (CRL). The protocol was conducted in compliance with CRL IACUC (Institutional Animal Care and Use Committee). Mice were separated into groups of 8 mice each (n=8), bled on day-1 and immunized on day zero with three different intramuscular (IM) doses of 10g, 1g, or 0.1g of GLB-COV2-043 mRNA encapsulated in LNP. All mice received a second dose after 3 weeks. In one study, mice received a booster dose of 10g of mRNA-LNP, 4months after primary immunization series. Monthly bleeds were performed to evaluate humoral immune responses over time, and the animals were euthanized 4 months after the booster dose, when tissues and spleens were collected for immune cells analysis. At terminal time points, blood and spleens were harvested for immune cell analysis.

Golden Syrian hamsters immunization and challenge studies were performed at BIOQUAL Inc. Handling samples and animals occurred in compliance with the biosafety protocols, and study was performed under an IACUC-approved protocol. 60 Golden Syrian hamsters were distributed into five groups of 12 animals each (6 female, 6 male). Animals were weighed and observed for clinical signs once daily during immunization days and before the livevirus challenge. Hamsters received 3 IM doses, 3weeks apart, of 30g or 3g of GLB-COV2-043 or an added (50:50) mix of GLB-COV2-043 (coding for wild-type Wuhan spike) plus GLB-COV2-076 (coding for Omicron BA.1 spike)mRNA-LNPs. Blood was collected for humoral immune responses analysis on each immunization day: 0, 21 (3weeks), and 42 (6weeks). On day 63 (9weeks), animals were challenged via an intranasal route with 4.8104 TCID50 of SARS-CoV-2 Omicron B.1.1.529 (BA.1 variant). Post-challenge, clinical observations were recorded twice daily (AM/PM), and body weights were recorded once daily until termination. For longitudinal viral load analysis, oral swabs were collected on days 65, 67, 68, 70, and 76 or 77. On days 67, 68, 76, and 77. At euthanasia, all animals were necropsied for lung and nasal turbinate for viral load analysis and histopathology.

The Sprague Dawley (SD) rat repeat dose toxicity study was conducted at CRL. This cGLP study, based on approved protocol and led by an American Board of Toxicology certified toxicologist, complied with all applicable sections of the Final Rules of the Animal Welfare Act regulations (Code of Federal Regulations, Title 9), the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the Office of Laboratory Animal Welfare, and the Guide for the Care and Use of Laboratory Animals from the National Research Council. A total of 30 rats, 15 males and 15 females, were used; 10 of those, from each sex, were used in the main study and 5 of those, from each sex, were used in the recovery phase of the study. The rats were intramuscularly immunized with 80g dose, administered once every two weeks for a total of 3 doses. Seroconversion was determined by quantitative ELISA assessment of anti-S (Wuhan) bindingantibodies. The in-life procedures, observations (e.g., post-dose, food consumption, local irritation) and measurements (e.g., individual body weights) were performed for all main and recovery study animals. Clinical pathology included evaluation of samples for hematology parameters, coagulation parameters, clinical chemistry parameters and alpha-2-macroglobulin (2M) analysis.

ELISA 96-well plates (Thermo Scientific) were coated with 100 L solution of the soluble recombinant Wuhan, Beta, Delta, Gamma, Alpha, or Omicron BA.1 Spike S1 subunit proteins (Sino Biological) in 1PBS (0.5g/mL) and incubated overnight at 4C. The wells were then aspirated and washed 3 times with 200 L per well of 1PBS (Phosphate Buffered Saline). 200 L of blocking solution (1% BSA/1PBS) were added to each of the wells and incubated for 2h at room temperature. During the incubation period, mice serum samples were diluted with diluent solution (1% BSA, 0.05% Tween-20, 1PBS) starting at 1:500 and serially diluted 5-fold an additional 7 times to make a total of 8 dilutions. After the blocking incubation period, the blocking solution was discarded. The diluted mice serum sample solutions were added and incubated at room temperature for 2h. Plates were washed 5 times with 200 L of 0.05% Tween-20/1PBS. 100l of goat anti-mouse IgG H+L antibody conjugated to HRP (SouthernBiotech), diluted 1:5,000 in diluent solution, was added and incubated at room temperature for 1h. Plates were washed 5 times with 200 L of 0.05% Tween-20/1PBS. For detection development, 100l of TMB substrate (Fisher) was added to each well and the reaction was stopped after 15min using 100l of 1M H3PO4 (Sigma Aldrich). For avidity ELISA, one additionalstep was added in which half of the plates were incubated for exactly 12min with 100l of 1.5M KSCN (Sigma Aldrich) per well and the other corresponding plates were incubated with 1PBS under the same conditions. OD450 readings were taken using the Biotek Synergy HTX plate reader and the data was analyzed using GraphPad Prism v.9.4.1. The end-point titers were calculated as the lowest dilution that emitted an optical density (OD) value greater than 4 times the background (secondary antibody alone).

HEK293T-hACE2 cells (SBI Biosciences) were cultured in DMEM media containing 10% FBS, 1% PenStrep, and 2g/mL puromycin (Fisher Scientific) as a selection antibiotic at 37C and 5% CO2. Prior to plating cells, plates were coated with poly-d-lysine (Fisher Scientific CB-40210) at a concentration of 2.5g/well for 1h at room temperature; poly-d-lysine was removed, and plates were washed with Gibco 1PBS (Thermo Fisher Scientific) and dried. Cells were plated with 1.25104 cells/well. The following day, fivefold serial dilutions of sera were made in HEK293T-hACE2 media with a starting dilution of 1:50 in a final volume of 50L. 50L of 1107/mL replication-deficient pseudovirus expressing spike of wild-type SARS-CoV-2 Wuhan or its variants Beta, Delta, Gamma, Alpha and Omicron B.1.1.529 (BA.1) and firefly luciferase within a Moloney Murine Leukemia Virus (MLV) backbone (eEnzyme) were added to each well and incubated together for 1h at 37C and 5% CO2. Following incubation, the serum-pseudovirus mixture was added to the cells and incubated at 37C and 5% CO2 for 48h. Each plate included wells with cell-only, as positive control, and pseudovirus-only, as negative control. Following incubation, media was removed and 50L of Glo Lysis Buffer (Promega) was added to each well, incubated for 10min, and transferred to an opaque white 96-well plate. 50 L of Bright-Glo Luciferase Substrate (Promega) was added to each well and luminescence was determined using the GloMax Navigator (Promega) with an integration time of 1s. Relative luciferase units (RLU) were plotted and normalized in GraphPad Prism v.9.4.1 to the cell-only control as 100% neutralization and the pseudovirus-only control as 0% neutralization. A non-linear regression of log(inhibitor) versus normalized response with a HillSlope less than zero was used to determine the IC50 values.

For the ELISpot assay, we followed the manufacturer protocol for mouse IgG B-cell ELISpot using an ELISpot Flex: Mouse IgG (ALP) kit (Mabtech). Briefly, sterile 96-well ELISpot plates with a PVDF membrane (Mabtech) were pre-treated with 15L of 70% ethanol and washed 5 times with 200L of sterile water per well. SARS-CoV-2 spikeantigens (Sino Biological) were diluted to 5g/mL in sterile 1PBS. Coated plates were wrapped in parafilm to avoid evaporation and incubated overnight at 4C. The following day, plates were washed 5 times with 200 L/well of sterile 1PBS to remove excess antigen and then blocked with 200 L/well of R10 [RPMI 1640 (Fisher Scientific), 10% fetal bovine serum (FBS) (Fisher Scientific), 1% penicillinstreptomycin (Fisher Scientific)] for at least 30min at room temperature. Cells were plated at 2.5105 cells/well in 100L using R10 and incubated in the coated plates for 1624h in a 37C humidified incubator with 5% CO2. The following day, the plates were washed 5 times with 200L/well of 1 PBS. The detection antibody, anti-IgG-biotin, was diluted to 1g/mL in 1PBS-0.5% FBS and 100 L was added to incubate for 2h at room temperature. The 5 times wash step was repeated and 100 L of streptavidinalkaline phosphatase (ALP) diluted 1:1000 in 1PBS-0.5% FBS was added to each well. After incubating for 1h at room temperature, plates were washed and 100 L of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (Mabtech) was added to each well and incubated for about 5min until spots emerged. The plates were washed with tap water extensively to stop color development and dried for 24h. The spots were counted on an ImmunoSpot S6 Ultimate M2 analyzer (CTL), using ImmunoSpot 7.0.37.0 Professional DC program.

Spleens were suspended in 1PBS and homogenized through a 70m strainer. Splenocytes were then incubated with ACK (Ammonium-Chloride-Potassium) lysis buffer and passed through a 40m strainer to prepare a cell suspension. Cells were plated at 5105 cells in 100l with T cell medium [RPMI 1640 (Fisher Scientific), 10% fetal bovine serum (FBS) (Fisher Scientific), 1% pyruvate (Fisher Scientific), 1% non-essential amino acids (NEAA) (Fisher Scientific), 0.4% MEM (minimum essential medium) vitamins (Fisher Scientific), 0.1% -MercaptoethanoL (Fisher Scientific), dimethyl sulfoxide (DMSO, Sigma)]. This was followed by stimulating the cells with an overlapping peptide pool (JPT) diluted 1:200 in medium, or 1of a stimulation cocktail (eBioscience). After the cells have incubated at 37C, 5% CO2 for 2h in the dark, media containing 1% of 10Brefeldin A (BFA) (Sigma) was added to each well. After 4h incubation, FcR blocking antibody (Fisher Scientific), diluted to 1g/ml with 0.1% BFA, was added to the cells and incubated for 15min at 4C in the dark. Cells were than stained for 30min at 4C in the dark with viability and surface markers Zombie Aqua Fixable Viability Kit (Biolegend) or eFluor 780 Fixable Viability Dye (ThermoFisher), dump channel [anti- mouse CD19 (clone 6D5, Biolegend; clone 1D3, TONBO Biosciences), F4/80 (clone BM8, Biolegend; clone BM8.1, TONBO Biosciences), Gr-1 (clone RB6-8C5, Biolegend; clone RB6-8C5, TONBO Biosciences), CD11b (clone M1/70, TONBO Biosciences), CD11c (clone N418, TONBO Biosciences), anti-mouse CD3e (clone 145-2C11 Biolegend), CD62L (clone MEL-14, Biolegend), CD4 (clone RM4-5, Biolegend or TONBO Biosciences), CD44 (clone IM7, TONBO Biosciences), CD8a (clone 536.7, TONBO Biosciences). Subsequently, cells were fixed, permeabilized, then stained with intracellular markers anti-mouse TNF-alpha (clone MP6-XT22, Biolegend), IL-2 (clone JES6-5H4, TONBO Biosciences), IL-4 (clone 11B11, Biolegend), IL-5 (clone TRFK5, Biolegend), and IFN-gamma (clone XMG1.2, TONBO Biosciences). For antigen-specific B cell analysis, splenocytes were thawed using 37C pre-warmed R10 (10% FBS in RPMI 1640) and plated in R10 incubated overnight at 37C and 5% CO2. The following day, cells were plated at 5106 cells in R10 and stained for 30min on ice in the dark with tetramers, composed of his-tagged spike protein (R&D Systems and BPS Biosciences) and labelled anti-His tag antibodies (clone J095G46, Biolegend). Cells were then stained for an additional 15min with viability and surface markers Zombie Aqua Fixable Viability Kit (Biolegend), dump channel [anti- mouse CD4 (clone RM4-4, Biolegend), CD8a (clone QA17A07, Biolegend), Gr-1 (clone RB6-8C5, Biolegend), F4/80 (clone BM8, Biolegend), CD11b (clone M1/70, Biolegend), CD11c (clone N418, Biolegend)], anti- mouse GL7 (clone GL7, Biolegend), IgD (clone 11-26c.2a, Biolegend), B220 (clone RA3-6B2, Biolegend), CD138 (clone 281-2, Biolegend), CD38 (clone 90, Biolegend), CD19 (clone 6D5, Biolegend), IgM (clone 1B4B1, SouthernBiotech) and IgG1 (clone X56, BD Biosciences). The cells were analyzed using a BD FACSymphony A3 flow cytometer and data processed using FlowJo v10.8.1 software.

Snap-frozen hamster tissue samples were homogenized in 2mL ice-cold assay medium (DMEM+10% FBS+Puromycin+P/S) for approximately 20s using a hand-held tissue homogenizer (Omni International). The samples were centrifuged (300g, 4C, 10min) to remove cellular debris. Clear flat-bottom 96-well culture microplates (BD Falcon) were seeded with Vero TMPRSS2 cells at 2.5104 cells per well in growth media (DMEM+10% FBS+Puromycin+P/S) and incubated at 37C, 5% CO2 until 80100% confluent. Growth media was aspirated out and replaced with 180L of diluent media (DMEM+2% FBS+Puromycin+P/S) per well. Next, 20L of processed tissue sample was added to the top row of the plate in quadruplicate, mixed via pipetting, and then serially diluted down the rows by 20L (tenfold dilution). Plates were incubated at 37C, 5% CO2 for 4days. After incubation, the presence of cytopathic effects (CPE) in each well was recorded, and the TCID50 value was calculated using the Read-Muench formula.

Arithmetic or geometric means are represented by symbols or the heights of bars, and error bars represent the corresponding SEM (Standard Error of the Mean). Dotted lines indicate assay's lower limits of quantification (LLOQ). Two-sided MannWhitney U-tests were used to compare two experimental groups and two-sided Wilcoxon signed-rank tests to compare the same animals at different time points. To compare more than two experimental groups, Kruskal Wallis ANOVA with Dunns multiple comparisons tests were applied. For pseudovirus neutralization assay, a nonlinear regression of log(inhibitor) versus normalized response with a HillSlope less than zero was used to determine the IC50values. Statistical analyses were done using GraphPad Prism v.9.4.1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.000.

Go here to read the rest:

Safety, immunogenicity and efficacy of an mRNA-based COVID-19 ... - Nature.com