HomeScienceSARS-CoV-2 mRNA-vaccine candidate; COReNAPCIN®, induces robust humoral and cellular immunity in mice...

SARS-CoV-2 mRNA-vaccine candidate; COReNAPCIN®, induces robust humoral and cellular immunity in mice and non-human primates

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Generation of SARS-CoV-2 mRNA vaccine construct

For mRNA production as the active ingredient of COReNAPCIN® vaccine candidate a genetic construct was designed based on the sequence of Wuhan-Hu-1 SARS-CoV-2 Spike glycoprotein (GenBank: MN908947.3). The codon optimized DNA sequence encoding the full length 1273 amino acid sequence of SARS-CoV-2 Spike glycoprotein was mutated to bear two substitutions (L986P and V987P) for encoding the pre-fusion stabilized form of the protein43. This DNA sequence was cloned into ReNAP-IVT1 plasmid in between special 5’-UTR and 3’-UTR-polyA tail, for transcribing the mRNA under the control of a T7 promoter. Upon sequence verification, the target recombinant plasmid DNA was transformed into E.coli-DH10B bacterial cells and single colonies were selected for preparing COReNAPCIN® vaccine master- and working cell banks (COReNAPCIN®-MCB and WCB). After full characterization of master- and working cell banks the WCB was used for the manufacturing step.

COReNAPCIN® mRNA manufacturing

Manufacturing of COReNAPCIN® mRNA starts from one vial COReNAPCIN®-WCB. Fed-batch fermentation was performed using a basic defined medium free of animal-derived components. The bacterial pellet was harvested and disrupted by the chemical lysis method with a novel batch operation methodology. Plasmid DNA purification was performed by two-step column chromatography for purification and well separation of plasmid DNA isoforms followed by Ultrafiltration/Diafiltration (UF/DF). The purified circular plasmid DNA was then incubated with restriction enzyme followed by another round of UF/DF to produce linearized plasmid. The ultrapure linearized plasmid was then used as DNA template for producing 5’-capped and polyA tailed COReNAPCIN® mRNA in an in-vitro transcription (IVT) reaction. In addition to linearized plasmid, the IVT reaction was set to contain 5’-cap analog, the mRNA building blocks; ATP, CTP, GTP and N1-Methylpseudouridine-5’-Triphosphate (me1Ψ-UTP), the enzymes; T7 RNA polymerase and inorganic pyrophosphatase, and the RNase inhibitor in a specialized IVT reaction buffer free of animal-derived components. The produced mRNA was then purified quickly with ion exchange (IEX) chromatography followed by oligo-dT affinity chromatography for removing all major impurities such as free 5’-cap and nucleotide analogues, enzymes, plasmid DNA template, as well as degraded, truncated and double-stranded RNAs. The integrity and purity of in-vitro transcribed COReNAPCIN® mRNA was verified before application in the formulation step.

COReNAPCIN® formulation

Following mRNA purification, mRNA was diluted in citrate buffer (pH 4) to reach the desired concentration. Lipid mixture (lipid mix) solution was prepared (12.5 mM concentration) by dissolving a specialized ionizable lipid, cholesterol, DSPC, and DMG-PEG in absolute ethanol with the molar ratio of 50, 38.5, 10, and 1.5, respectively. mRNA and lipid solutions were mixed within a dedicated microfluidic device at the ratio of 3:1 (aqueous: organic phase). Then buffer exchange (acetate buffer, pH 7.4), and concentration processes of formulated mRNA-LNP were performed using tangential flow filtration (TFF). Finally, the formulated mRNA-LNP (COReNAPCIN®) was filtrated by 0.22 μm filters and stored at −20 °C at the concentration of 0.1 mg/mL.

Cell transfection

Primarily, HEK 293 T cells were seeded into a 12-well cell culture plate, 2 × 105 cells per well in HG-DMEM medium (Biowest). After 24 h of incubation, cells were transfected with either 1 μg mRNA using 2 μL Lipofectamine™ (Invitrogen) or 1 μg of mRNA-LNP. Untransfected cells were used as negative control. Following 24 h of incubation at 37 °C, cells were analyzed for SARS-CoV-2 S protein expression by western blot and flow cytometry.

Western blot

Briefly, equal amounts (20 μg) of total protein extracts from cultured cells were loaded and run on a 10% SDS-PAGE and then transferred to a nitrocellulose membrane. After 2 h blocking in a buffer containing 5% fat-free dried milk and 0.5% Tween-20, the membrane was incubated with anti- SARS-CoV-2 spike polyclonal antibody (Sino biological) or anti-β-actin polyclonal antibody (Abcam) overnight at 4 °C. The membrane was then washed three times and incubated for one hour with HRP-conjugated goat anti-rabbit IgG (Sigma) at room temperature. Finally, the result was visualized by DAB staining. All gels derive from the same experiment and they were processed in parallel (Supplementary Fig. 1).

Flow cytometry

In order to analyze transfected cells for cell surface expression of S protein, 2 × 105 cells of each of transfected and untransfected cells stained with SARS-CoV-2 Spike antibody (SinoBiological) and Anti-human IgG FITC (Sigma–Aldrich®). After staining with primary and secondary antibodies, cells were acquired on BD FACS Lyrics and analyzed by Flowjo V10 (BD Biosciences).

Animal models and immunization

Immunogenicity of COReNAPCIN® vaccine candidate was assessed in mice (BALB/c and C57BL/6) and rhesus macaques. All experiments involving animal, were conducted in accordance with the ethical regulations for the care and use of laboratory animal and approved by Vice-Chancellor in Research Affairs-Tehran University of Medical Sciences with an ethic code of: IR.TUMS.VCR.REC1398.1055. COReNAPCIN® Immunogenicity assessments is summarized in Table S1.

BALB/c and C57BL/6 mice (16–18 g, 4–6 weeks old) were purchased from the Pasteur Institute of Iran. Mice were housed in a standard animal facility with the room temperature of 23 °C, relative humidity of 65% in 12/12-hour light/dark cycles, with free access to water and rodent chow.Total number of 80 BALB/c mice in four groups (15 females and 5 males per group), and 48 C57BL/6 mice in four groups (12 females in each group), were injected intramuscularly (IM) with either phosphate-buffered saline (PBS) or 0.05, 0.5, or 3 µg mRNA-LNP at days 0 and 21. To evaluate the humoral immune response, blood samples were collected from the retro-orbital sinus of mice under anesthesia. The summary of the mice immunogenicity study design, is presented in Fig. 2a. On day 42 post-prime (21 days post-boost), 5 mice from each group (in both BALB/c and C57BL/6 mice) were euthanized, and cellular immune response was assessed by analyzing the splenocytes.

Rhesus macaques were housed individually in cages in a climate-controlled room (temperature of 18–25 °C and humidity 30–70%), with a 12 h light/dark cycles, given chow and fruits in strict accordance to the animal welfare requirements and allowed free access to water. During the challenge study, macaques were housed in the biosafety level 3 (BSL3) facility.

Each group was made of 2 rhesus macaques (1 male and 1 female) injected with either PBS, 30 or 50 µg of mRNA-LNP into the right hind leg (IM) at days 0 and 28. Blood samples were collected and subjected for analyzing humoral immune responses. Cellular immunity was assessed at day 21 post boost injection. The rhesus macaque immunogenicity study design, is illustrated in Fig. 5a.

Rhesus macaques were challenged on day 21 post-boost, under anesthesia, with 2 × 108 PFU of SARS-CoV-2 in 2 ml PBS, which was divided into two equal volumes for administrating intranasal (1 ml, 0.5 ml per nostril) and intratracheal route (1 ml). At days 2, 4, 7, 14, 21, 28 post-challenge, nasal and rectal swabs were collected and analyzed by quantitative Real-time PCR (qRT-PCR) for detection of RdRp and nucleocapsid protein (N) genes of SARS-CoV-2. On day 7 post-challenge, one rhesus from each group was euthanized. After proper fixation of lungs in 10 % buffered formalin, tissue sectioning in 5 µm diameter, and hematoxylin and eosin (H&E) staining, lung sections were analyzed by a veterinary pathologist.

Quantification of SARS-CoV-2 RNA by qRT-PCR

To detect and measure the presence of SARS-CoV-2 viral RNA in nasal turbinates and rectal swab samples of macaques, two SARS-CoV-2 genes, N and RdRp genes, were analyzed by qRT-PCR test, using SENMURV multi-star SARS-CoV-2 qRT-PCR kit (STRC). Nasal and rectal swab specimens were subjected to total RNA extraction using the RNeasy Mini Kit (QIAGEN). The qRT-PCR reaction, was run with the following thermal profile: 20 min in 50 °C, 10 min in 95 °C, and followed by 40 cycles of 95 °C and 55 °C for 10 and 40 s, respectively.

Enzyme-linked immunosorbent assay (ELISA)

SPL Maxibinding ELISA plates (SPL) were coated with 33 ng of SARS-CoV-2 S protein (Acro-Biosystems) or SARS-CoV-2 receptor binding protein (RBD) (Sino Biological) in 100 µl (final concentration of 330 ng/ml) coating buffer (Biolegend) with overnight incubation at 4 °C. After routine wash and block with 2% Bovine Serum Albumin, (BSA) (Sigma–Aldrich), 100 µl of serial diluted mice or macaques serum were added to wells and incubated at room temperature for one hour. After washing, 50 µl of either Anti-Mouse IgG (γ-chain specific), Anti-mouse IgG1, Anti-mouse IgG2a (Sigma–Aldrich, USA) or Anti-monkey IgG in 1:5000 dilutions were added to each well and plates were incubated at room temperature for one hour. After 4 rounds of wash, 100 µl of Tetramethylbenzidine (TMB) (RaziBIOTech) were added to each well, followed by 10 min of incubation. Finally, 1 N HCl was used as a stop reagent and the absorbance was measured by BioTek® 800™ TS at 450 nm and 630 nm. To calculate endpoint titers, the baseline serum samples of 30 mice, were used and calculated as described by Frey et al.44.

Pseudovirus neutralization assay (pVNT)

Spike pseudotyped lentivirus, was produced by the co-transfection of plex307-egfp, pCMV3-spike (Wuhan-D614G, Delta and beta), and pspax2 with Lipofectamine 3000 (Thermo). In order to make a cell-free virus, the virus-containing supernatants were harvested 48 and 72 h post-transfection and were passed through 0.45 um filters to eliminate any cellular debris. Spike pseudotyped lentivirus encodes the EGFP protein that serves as a reporter gene. To perform the pVNT assay, ACE2-positive cells are required. Thus, we stabilized a single-cell ACE2 cell line using a VSVG lentivirus which packages the human ACE2 (hACE2). The hACE2 gene was amplified from the pCMV-hACE2 plasmid (Sino biological) and subcloned into the Plex307 expression vector. We added 50 ul of DMEM-High Glucose/10% FBS/1% P/S sera, which was serially diluted 2-fold from 1/40 up to 1/5120 in a 96-well cell culture plate. 50 ul of spike pseudotyped lentivirus was added to each well, following an hour of incubation at 37 °C. 1.4 × 104 of HEK-293T-hACE2 cells were then added to each well. Fluorescent microscopy was used to count the number of EGFP-positive cells in each well, after 48 to 60 h post-transduction. IC50 was calculated using the percentage of GFP positive cells versus the logarithm (dilution factor) as a non-linear regression curve fit in GraphPad Prism V845.

Convalescent sera from COVID-19 patient aged 18–63 years old (n = 32), collected 14 days after PCR-confirmed diagnosis (pre delta wave) and used as benchmark for pVNT of rhesus macaques. All serum samples collected from symptomatic patient and 9 of patients experienced severe form of COVID-19.

Conventional virus neutralization test (cVNT)

The cVNT assay, was conducted on serum samples of rhesus macaques, collected on day 14 post boost. Briefly, 50 μL of 100 median tissue culture infecting dose per mL (TCID50/mL) of SARS-CoV-2 (Wuhan or Omicron), were mixed with 50 μL of two-fold serial dilutions of heat-inactivated serum (at 56 °C for 30 min). After 60 min incubation at 37 °C, the virus-serum mixture was added to 96 well-plate previously seeded with 1.5 × 104 Vero cells per well (performed in triplicate). One hour post-incubation, the supernatant was removed and cells were washed twice with Dulbecco’s Modified Eagle Medium (DMEM). Then, cells were incubated for 72 h at 37 °C in a 5% CO2 in DMEM with 10% heat-inactivated Fetal bovine serum (FBS). Half-maximal inhibitory concentration (IC50) titer, was measured by microscopy and reported as the highest dilution inhibiting 50% cytopathic effect (CPE) formation.

Surrogate virus neutralization test (sVNT)

We used sVNT (ACE2 inhibition test) ELISA (Pishtaz Teb) (with a detection limit of 40 µg/ml), for measuring the SARS-COV-2 neutralizing ability of serum samples of mice and rhesus macaques collected 40 days post-prime. The assay was performed according to the manufacturer’s instructions; in summary, 50 µl of serum samples, negative and positive controls were added to the wells. Then immediately, 50 µl RBD conjugated HRP, was added and incubated for 30 min at 37 °C. Following the routine wash, 100 µl TMB was added to each well and plates were placed at room temperature for 15 min. Finally, 100 µl 1 N HCL as stop solution was added to each well and the absorbance was measured by BioTek® 800™ TS at 450 nm and 630 nm (as Reference filter).

Intracellular cytokine staining

Mice fresh splenocytes were separated by mechanical homogenization then filtered through a 70 µm cell strainer (Biologics). Red Blood Cells (RBCs) were removed by using RBC lysis buffer, then following wash with PBS, 106 splenocytes were ex vivo stimulated with SARS-CoV-2 peptide pool (JPT PM-WCPV-S-1) at concentration of 2 µg/ml of each peptide in RPMI (Biowest) supplemented with 10% heat inactivated FBS (Gibco). PMA/Ionomycin (BD Pharmingen™) and dimethyl sulfoxide (DMSO) (Sigma–Aldrich) were used as a positive and negative control, respectively. Brefeldin (Biolegend) as Golgi stop was added to each well one-hour post-incubation. Following eight hrs of incubation, the harvested cells, washed with PBS, and stained with Zombie Violet (Biolegend) at 1/200 dilution and incubated at room temperature for 30 min. After washing cells with staining buffer (PBS supplemented with 2% FBS and 0.05% NaN3), anti CD3-Percp, anti CD8-PE, anti CD4-PE, anti CD44-APC (Biolegend) were used for cell surface staining followed by 20 min incubation at room temperature. For intracellular staining, cells were fixed and permeabilized with Cytofast fix/perm (Biolegend) according to the manufacturer instruction, then the anti IFN-γ-FITC (Biolegend) was added to the tubes. After 20 min incubation and washing with permeabilization and staining buffer (Biolegend), cells were acquired on BD FACS Lyrics and analyzed by Flowjo V10 (BD Biosciences).

Enzyme-linked immunosorbent spot assay (ELISpot)

Cryopreserved mice splenocytes were thawed in pre warmed RPMI (Biowest) and rested for 24 h. IFN-γ ELISpot precoated plates (Abcam) were used according to the manufacturer’s instruction. Briefly, the total number of 2 × 105 harvested splenocytes, were stimulated with 2 µg/ml of SARS-CoV-2 peptide pool (JPT product PM-WCPV-S-1, Germany), for 18 h at 37 °C. DMSO (Sigma-Aldrich, USA) and phytohemagglutinin (PHA) (Gibco) were used as negative and positive control, respectively. After several wash steps, 100 µl streptavidin-AP (Abcam) was added to each well, followed by incubation at room temperature for 1 h. Finally, 100 µl BCIP/NBT was added to the each well and after 15 min incubation at room temperature, plate was rinsed in tap water, dried and spots were counted by image processing software.

For quantitative measuring of macaque specific T cell, Monkey IFN-γ ELISpot plates (Mabtech) were used. After thawing and overnight incubation, total number of 3 × 105 NHP PBMC, were added to each well and stimulated with 2 µg/ml of SARS-CoV-2 peptide pools (JPT, PM-WCPV-S-1). DMSO (peptide solvent) and anti CD3 (Mabtech) used for negative and positive control, respectively. The plate then incubated at 37 °C for 18 h followed by wash according to the manufacturer instruction. After incubation with detection antibody (7-B6-ALP) and subsequent washing, 100 µl substrate solution (BCIP/NBT-plus) added to each well. When spots were formed, plate rinsed in tap water, dried and spots were counted by image processing software.

Cytokine analysis

Splenocytes and PBMCs, respectively, from immunized mice and rhesus macaques were collected 21 days post boost. Five-million mice splenocytes or rhesus macaque PBMCs were stimulated for 24 h with SARS-CoV-2 peptide pool (JPT, PM-WCPV-S-1) at final concentration of 2 µg/ml per peptide. Negative control wells received same volume of DMSO with no peptide and PMA/Ionomycin added to positive control wells. Concentrations of IFN-γ and IL-4 in supernatants were analyzed by DuoSet ELISA kit (Biolegend) according to the manufacturer’s instructions.

Statistical analysis

Graph Pad V8 Prism was used to illustrates the figures and analyze the results statistically. Dotted lines indicate assay limits of detection. For comparing values between two groups, t-test was conducted and Group comparisons were made by one-way ANOVA followed by Dunn’s test. A p value less than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was assumed to be statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.


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