DNA template design
The DNA template for the Comirnaty vaccine was constructed by cloning the publicly available mRNA vaccine sequence58 into a pUC-based plasmid under the control of a T7 promoter.
For the CPVax-CoV vaccine, the coding sequence of the Spike SARS-CoV-2 protein from Wild wild-type-H1 strain (GISAID59 accession number EPI_ISL_402124) was codon-optimized for human expression. This sequence included the K986P and V987P (2P) stabilizing mutations and the substitution of the RRAR furin cleavage site at residues 682–685 with GSAS. For the CPVax-CoV-XBB vaccine, the DNA template was generated using the XBB.1.5 strain sequence (GISAID accession number EPI_ISL_15851788). The same 2P and RRAR to GSAS modifications were introduced, and the sequence was similarly codon-optimized for human use.
Both CPVax-CoV and CPVax-CoV-XBB, optimized sequences were inserted in-frame immediately downstream of the 5´UTR into a pUC-based plasmid. This plasmid featured the following elements arranged in a 5′ to 3′ orientation: a T7 RNA polymerase promoter, an optimized 5′ UTR from human APOA216, the 3′ UTR from human beta-globin (GenBank: NM_000518), a polyadenylation tail consisting of 100 adenines, and a BspQI restriction enzyme site. The complete 5′ and 3′ UTR sequences are provided in the Supplementary Information (Supplementary Fig. 3).
Gene synthesis, cloning, and plasmid preparations were outsourced to Genscript. The purified plasmids were subsequently used for in vitro transcription.
In vitro transcription and mRNA purification
Each plasmid containing the DNA template sequence of interest was digested with BspQI (ON-124, HONGENE), which cleaved the plasmid immediately after the segment to be transcribed. The linearization reaction was then purified with the Wizard SV Gel and PCR Clean-Up (Promega A7270), in accordance with the manufacturer’s instructions.
The purified linear DNA was subsequently employed for mRNA production by in vitro transcription using T7 RNA polymerase following the manufacturer’s instructions. Briefly, transcription reactions were performed at 37 °C for 3 h using the following materials:
Template linear DNA (50 µg/mL), T7 RNA polymerase (5000 U/mL; HONGENE, ON-004), RNAse inhibitor (1000 U/mL; HONGENE, ON-039), Inorganic Pyrophosphatase (2 U/mL; HONGENE, ON-025), ATP (5 µg /mL; HONGENE, R1331), GTP (5 µg/mL; HONGENE, R2331), CTP (5 µg/mL; HONGENE, R3331), N1-Methylpseudouridine (5 µg/mL, HONGENE, R5-027), CAP AG (4 µg mL, Hongene, ON-134), and RNAse-free double-distilled water.
The generated mRNA transcripts were initially treated using DNaseI incubation (Hongene, ON-109) according to the manufacturer’s instructions.
In order to reduce double-stranded RNA (dsRNA) contaminants, the resulting mRNA was purified through a process involving two sequential chromatography steps, starting with anion exchange chromatography (AEX) followed by affinity chromatography60. Initially, the mRNA sample was purified using the CIMmultus PrimaS Chromatography Column (BIA Separations). IVT mixture was diluted once in sample loading/equilibrate buffer A (20 mM Tris, 20 mM BTP, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH = 8.0) and loaded onto the column. After unbound IVT components eluted in flow-through, a wash 1 with equilibration buffer was necessary, followed by a high-salt wash with 50 mM Tris, 3.0 M guanidine-HCl, 20 mM EDTA, pH 8.0. And after a wash 3 with buffer A until UV returns to baseline, a step elution was performed with buffer C (20 mM Tris, 20 mM BTP, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH = 11.0). Fractions were neutralized immediately after elution. Following, additional purification was carried out through affinity chromatography using a POROS Oligo (dT)25 column (ThermoFisher). Specifically, the buffers employed were as follows: Buffer A, which contained 50 mM disodium phosphate, 0.5 M NaCl, 5 mM EDTA, pH = 7.0, and Buffer B, which contained 50 mM sodium dihydrogen phosphate, 5 mM EDTA, pH = 7.0. The mRNA samples were initially half-diluted in Buffer A 2x. Following this, the column was equilibrated with 100% Buffer A, loaded with mRNA, washed with Buffer B, and ultimately eluted using double-deionized water. To completely remove Buffer B, mRNA was washed with a 30 KDa Amicon® filter and then equilibrated through a one-tenth dilution in citrate buffer 10× with a pH of 6.5.
The concentration of mRNA was determined by measuring the optical density at 260 nm, then adjusted to a final concentration of 1 mg/mL, aliquoted, and stored at −80 °C until needed.
For quality assurance, all mRNAs underwent analysis through automated electrophoresis (2100 Bioanalyzer G2938B, Agilent). Subsequently, the mRNA samples were aliquoted and stored at −80 °C until needed.
Synthesis and characterization of ionizable lipids
All intermediate compounds and ionizable lipids were synthesized following protocols based on the Sequential Thiolactone Amine Acrylate Reaction23. Briefly, following the synthesis of thiolactone derivatives, the ionizable lipid was obtained via a multicomponent one-pot reaction. Thus, the corresponding thiolactone derivatives (0.15 mmol, 1 equiv.), acrylate (0.15 mmol, 1 equiv.) amine (0.15 mmol, 1 equiv.) were dissolved in 300 μL of tetrahydrofuran (THF) at room temperature. After stirring for 2 h, the THF was removed under reduced pressure, and the product was purified using a CombiFlash NextGen 300+ (gradient of elution: from 100% dichloromethane to 50% of an 80/20/1 mixture of DCM/MeOH/NH4OH (aq)). All synthesized ionizable lipids were characterized by high-performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD) and mass spectrometry (ThermoFisher ISQ). Noteworthy, in previous publications, CP-LC-0743 was also referred to as A4B2C1.
Molecular structures are shown in Supplementary Fig. 1. Theoretical and experimental molecular weights (MW):
CP-LC-0729 theoretical [M + H]+ = 740.63, experimental [M + H]+ = 740.85
CP-LC-0867 theoretical [M + H]+ = 684.57, experimental [M + H]+ = 684.74
CP-LC-0431 theoretical [M + H]+ = 766.65, experimental [M + H]+ = 766.85
CP-LC-0474 theoretical [M + H]+ = 768.66, experimental [M + H]+ = 768.76
CP-LC-0743 theoretical [M + H]+ = 766.65, experimental [M + H]+ = 766.81
CP-LC-0729 1H-RMN was obtained using a Bruker 400 MHz NMR spectrometer. 1H NMR (400 MHz, CDCl3) δ: 6.76 (m, 1H); 6.37 (d, J = 7.9 Hz, 1H); 4.60 (dt, J = 7.1 Hz J = 7.1 Hz, 1H); 3.98 (d, J = 5.8 Hz, 2H); 3.36 (dt, J = 5.7 Hz J = 5.7 Hz, 2H); 2.78 (t, J = 6.8 Hz, 2H); 2.59 (m, 4H); 2.48 (t, J = 5.9 Hz, 2H); 2.28 (s, 6H); 2.06 (m, 2H); 1.96 (m, 1H); 1.58 (m, 3H); 1.41 (m, 2H); 1.25 (m, 44H); 0.87 (m, 12H) (full spectrum available in Supplementary Fig. 1).
mRNA encapsulation into LNPs
LNPs formulations were prepared following a previously described method with modifications20. Briefly, the purified mRNAs were initially diluted in 10 mM sodium citrate buffer at pH = 4, reaching a final concentration of 266 µg/mL. Simultaneously, the lipid mixture was prepared in ethanol at the different molar ratios used of 46.6:9.4:42.7:1.6 (Comirnaty), 50:10:38.5:1.5 (CP ionizable lipids formulations in Fig. 1) and 40.7:34.9:23.3:1.2 (CPVax-CoV) for ionizable lipids CP-LC-0729, CP-LC-0867, CP-LC-0431, CP-LC-0743, CP-LC-0474 or ALC-0315 (Merck 586224): helper lipids DSPC (Merck 850365P) or DOPE (Merck 850725P): Cholesterol (Sigma C3045) and DMG-PEG2000 (Cayman 33945-1) or ALC-0159 (Cayman 34336). Aqueous mRNA solution and lipid mixture were combined at a molar N/P ratio of 6:1 (Moderna/Pfizer) or ionizable lipid/RNA weight ratio of 10:1 (CPVax-CoV formulation). Microfluidic technique was used for the synthesis of LNPs, thereby NanoAssemblr® Ignite microfluidic device (Precision Nanosystems) was set at a total flow rate (TFR) of 12 mL/min and a aqueous:ethanol flow rate ratio (FRR) of 3:1. The resulting LNPs were dialyzed (Pur-A-Lyzer™ Midi Dialysis Kit) overnight against Tris buffer containing cryoprotectants. Each resulting LNP solution was then collected and adjusted to a final concentration of mRNA of 100 µg/mL, filtered through a 0.22 mm filter, and stored at −80 °C for further use.
The average size, PDI, and zeta potential of LNPs were determined using a Malvern Zetasizer Advance Lab Blue Label (Malvern Instruments Ltd., UK) with a capillary cell (DTS1070) and diluting the sample (typically 1:100) in KCl 10 mM filtrated solution.
The concentration of mRNA in LNPs was measured using Quant-iT™ RiboGreen™ RNA Assay Kit from Thermo Fisher Scientific following the manufacturer’s protocols. Thus, the % of RNA encapsulated was calculated by comparing the total RNA obtained by the lysis of mRNA-LNPs using 0.5% Triton X-100 and the non-encapsulated RNA obtained when the LNPs are not lysed in the absence of detergent. Fluorescence was quantified in a Fluostar Omega microplate reader (BMG Labtech). Agarose gel electrophoresis was additionally used to determine the encapsulation of mRNA in LNPs. The quantification of encapsulated mRNA was determined by band densitometry using ImageJ software. Samples were loaded in a 1% agarose gel including SYBR-Safe, and the electrophoresis was run at 120 V for 30 min. Gels were visualized with a UV transilluminator iBright™ CL750 imaging system, using adequate exposure times to avoid image saturation.
LNP lyophilization
After adjusting the concentration of mRNA-LNPs to 100 µg/mL, the mRNA-LNPs suspension was aliquoted into glass vials with a volume of 300 µL per vial. Lyophilization was conducted using a Genesis Pilot Freeze Dryer, following a three-stage process: initial freezing, primary drying, and secondary drying. After lyophilization, vials were backfilled with pure nitrogen, capped, and transferred to various temperatures for stability assessments. The lyophilized mRNA-LNPs were stored at either 4 °C or 25 °C for different times and compared with the non-lyophilized solution stored at −80 °C as a control. To reconstitute lyophilized samples, 300 μL of RNase-free water was added to each vial and softly mixed until the solution turned into a homogeneous, slightly white, clear suspension.
Cell lines
HEK293T cells were obtained from the American Type Culture Collection (ATCC, CRL-3216 and CRL-1586). Vero E6 cells were kindly provided by Júlia Vergara from the Centro de Investigación en Sanidad Animal IRTA-CReSA (Barcelona, Spain). HEK293T-ACE2-TMPRSS2 cells were obtained from the National Institute for Biological Standards and Controls (NIBSC, 101008).
Cells were cultured on complete DMEM, which consists of high-glucose Dulbecco’s Modified Eagle’s Medium (Merck, D6429) supplemented with 10% Fetal Bovine Serum (Sigma, F7524), 1% Penicillin-Streptomycin Solution (Gibco, 15140122), and 2 mM Glutamax (Fisher, 35050038). Vero E6 cells were additionally supplemented with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperanzineethanesulfonic acid) (Biowest), and HEK293T-ACE2-TMPRSS2 cultures were additionally supplemented with 1 µg/mL puromycin dihydrochloride (Sigma, P8833).
Animals
All animal experiments were conducted in agreement with European and national directives for the protection of experimental animals, and experimental procedures were approved by the Ethics Committee for Animal Experiments of the University of Zaragoza (PI59/21 and PI36/22) or the University of Navarra (Protocol ref. CEEA026/20). In addition, animal experiments using the SARS-CoV-2 strains were approved by the Biosafety Committee of the University of Zaragoza (Refs: 124/20, 156/21, and 184/22) and the GMO Interministerial Council (A/ES/20/99).
For the immunity studies, BALB/cAnNRj mice were purchased from Janvier Labs. For the viral challenge studies, BALB/cOlaHsd and C57BL/6J K18-hACE2 transgenic mice were purchased from Envigo and Charles River (K18-hACE2 JAX Mice Strain), respectively.
Male and female mice aged 8–10 weeks and weighing 18–28 g were used for all the experiments. All mice underwent an acclimation period lasting 3–7 days to adapt to the experimental conditions upon arrival at the research facilities, and were housed and maintained in specific pathogen–free conditions in the facilities of Centro de Investigaciones Biomédicas de Aragón (Zaragoza, Spain; reference ES 50 297 0012 011) for immunity studies and Centro de Investigación de Encefalopatías y Enfermedades Transmisibles Emergentes (Zaragoza, Spain; reference ES 50 297 0012 009) for challenge studies. Housing conditions were controlled, maintaining a room temperature of 20–24 °C, humidity levels ranging from 50% to 70%, and a light intensity of 60 lux, with a light-dark cycle lasting 12 h. During the studies, all animals were monitored by the animal resources center or laboratory staff daily.
For the procedures requiring anesthesia (SPECT-CT imaging and SARS-CoV-2 challenge), all animals were anesthetized via inhalation using isoflurane (IsoVet) mixed with oxygen at a flow rate of 1 L/min in a rodent-specific anesthesia station. Anesthesia was induced with 5% isoflurane and maintained at 2%. Animals were monitored continuously during anesthesia and observed post-procedure until fully recovered. No anesthesia was used for intramuscular injections, blood collection, and weighing. Mice were humanely euthanized with CO2 asphyxiation followed by cervical dislocation.
Virus strains
The SARS-CoV-2, hCoV-19/Sweden/20-53846/2020, (Lineage B.1.1.7; Alpha variant) was provided by the Public Health Agency of Sweden. The mouse-adapted strain SARS-CoV-2 MA20 was obtained by serial passaging of SARS-CoV-2, hCoV-19/Sweden/20-53846/2020, (Lineage B.1.1.7; Alpha variant) in mice as described in ref. 27. The SARS-CoV-2 hCoV19/USA/CA-Stanford-109_S21/2022 (Lineage XBB; Omicron Variant) (GISAID: EPI_ISL_15509864) was isolated from a human on October 10, 2022, in California and was obtained from BEI resources (NR-58925). All procedures involving infectious viruses, including in vivo experiments, were performed under biosafety level 3 (BSL-3) conditions.
mRNA in vitro transfection
HEK293T cells were seeded in 6-well cell culture plates at a density of 4 × 105 cells/well and incubated overnight. The SARS-CoV-2 Spike-coding mRNAs were transfected using Lipofectamine MessengerMax Transfection Reagent (Invitrogen, 15397974) according to the manufacturer’s protocol. Briefly, a mixture of each mRNA (2.5 µg/well) and Lipofectamine MessengerMAX (5 µL/well) was pre-incubated in OptiMEM media. The mRNA-lipofectamine mixture was added to the corresponding wells in duplicate, resulting in a final mRNA concentration of 2.5 µg/well. The cells were incubated for 24 h at 37 °C and 5% CO2.
For Western Blot, cells were lysed after incubation with 0,1% TritonX and centrifuged at 13,000×g for 10 min. The cell lysates were run under reducing conditions by SDS-PAGE (SurePAGE, 4-12% Genscript, M00653) and transferred to a nitrocellulose PVDF membrane (Bio-Rad). The membranes were blocked with 3% BSA in TBST buffer (Tris-buffered saline + 0.1% Tween 20) and incubated with a rabbit polyclonal SARS-CoV-2 Spike antibody (SinoBiological, 40591-T62) overnight at 4 °C. The membrane was washed and incubated with a goat anti-rabbit Ig, Human ads-HRP (Southern Biotech, 4010-05) as a secondary antibody. Colorimetric detection of the samples was performed with Pierce ECL Western Blotting Substrate (Thermo, 32106), and images were acquired using the iBright CL750 Imaging system (Invitrogen).
For flow cytometry analysis, cells were trypsinized after incubation. Collected cells were incubated with human FcR blocking reagent (Miltenyi, 130-059-901) for 15 min at 4 °C. Cells were washed and incubated with fixation buffer (eBioscience, 88-8824-00) for 30 min at 4 °C, followed by three washing steps with permeabilization buffer (eBioscience, 88-8824-00) and incubation with SARS-CoV-2 Spike S1 Subunit Antibody (R&D Systems, MAB105403) diluted in permeabilization buffer for 1 h at room temperature. Subsequently, cells were washed and incubated with FITC Goat Anti-Mouse IgG/IgM (BD, 555988) for 1 h at room temperature. After final washing steps, labeled cells were resuspended in PBS buffer, and acquisition was performed using a Gallios flow cytometer (Beckman Coulter).
In vivo immunization and blood collection
Mice were immunized with LNPs prepared as described above, receiving an intramuscular injection of 1 µg of the specified mRNA-LNP into the right thigh muscle, diluted in Tris buffer containing 15% sucrose in a final volume of 30 µL, and administered using a 30 G insulin syringe. An equal booster dose was administered on day 21 following the initial immunization. Blood samples were collected from the submandibular vein on days 0, 21, and 42 post-prime. Blood was allowed to clot and then centrifuged at 6500×g and 4 °C for 10 min. The sera were collected and stored at −80 °C for antibody analysis.
Determination of antibody levels in serum
The serum samples from immunized mice were analyzed for RBD-specific IgG antibody titers using an indirect enzyme-linked immunosorbent assay (ELISA). 96-well Nunc MaxiSorp microplates (Thermo Scientific) were coated with 50 ng/well of recombinant RBD (Certest Biotec) diluted in carbonate/bicarbonate buffer pH 9.6 and incubated overnight. The next day, plates were washed with phosphate buffer saline–Tween 20 0.05% (PBST) followed by blocking with 3% BSA (Seqens) in PBST for 1 h. After washing, serially diluted mouse sera were added to the plate and incubated for 1.5 h at 37 °C, followed by washing steps and addition of IgG goat anti-mouse horseradish peroxidase (HRP) antibody (Southern Biotech) at a dilution of 1:10.000. The plates were developed using 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Abcam) and 0.2 M H2SO4 (aq) to stop the reaction. Finally, the absorbance was measured at 450 and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech).
The reciprocal endpoint titer was defined as the highest dilution at which the optical density (OD 450-630 nm) of the sample reached a predetermined cutoff of 0.1 or greater.
Organ single-cell suspensions
Organs were harvested and placed in RPMI 1640 medium (Fisher, 10379144). Spleens and lymph nodes (LNs) were homogenized using a syringe plunger and filtered through a 70 µm cell strainer. The cell strainer was washed with 10 mL of RPMI, and cells were centrifuged at 450×g for 5 min. Red blood cells (RBCs) in spleens were lysed using 1 mL of eBioscience RBC Lysis Buffer (Thermofisher, 00-4300-54) for 1 min. After lysing, the cells were washed with 10 mL of RPMI and centrifuged at 450×g for 5 min.
Cells from tissues were resuspended in 1 mL of RPMI 1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (Sigma F7524), 1% Glutamax (Fisher, 35050038), 1% Penicillin-Streptomycin (Gibco, 15140122), and 0.00035% β-mercaptoethanol (Merck, M3148). The resuspended cells were immediately used for counting, culture, or staining.
ELISPOT assay
Enzyme-linked immunospot (ELISPOT) assay was employed to quantify the frequency of cytokine-secreting splenocytes. ELISpot assays were performed using mouse IFN-γ ELISpot kits (CTL Immunospot) according to the manufacturer’s instructions.
96-well immunospot plates were coated with 60 µL of murine IFN-γ capture solution and incubated at 4 °C overnight. The next day, plates were washed with PBS, and 4 × 105 splenocytes/well were stimulated with 50 µg/mL of a peptide pool covering the immunodominant sequence domains of the S protein (Miltenyi, 130-126-700). Phorbol-12-myristate-13-acetate (PMA) 50 ng/mL (Merck, 524400) and ionomycin 500 ng/mL (Merck, 407953) were used as positive control, while splenocytes without stimulation were utilized as negative controls. Samples were incubated for 20 h at 37 °C and 5% CO2. The following day, plates were washed, and 10 µL of anti-murine IFN-γ detection solution was added. After washing, a tertiary solution containing streptavidin-HRP was used, followed by the addition of a chromogen substrate for enzyme activity detection. Finally, plates were scanned using an ImmunoSpotS6 Ultra-V analyzer (CTL EUROPE GMBH), and spot numbers were assessed using ImmunoSpot 7.0.34.0 Professional Analyzer DC software.
ELISA cytokine assay
The 1 × 106S splenocytesl were seeded in 96-well plates at 1 x106 and stimulated with 50 µg/mL of a peptide pool covering the immunodominant sequence domains of the S protein (Miltenyi, 130-126-700). Phorbol-12-myristate-13-acetate (PMA) 50 ng/mL (Merck, 524400) and ionomycin 500 ng/mL (Merck, 407953) were used as positive controls, while splenocytes without stimulation were utilized as negative controls. Samples were incubated for 20 h at 37 °C and 5% CO2. Supernatants were collected after centrifugation at 1500×g for 5 min, and samples were stored at −80 °C until analysis.
Cytokine quantification was conducted using Mabtech ELISA Flex (HRP) kits following the manufacturer’s instructions. Briefly, Nunc MaxiSorp 96-well plates (Thermo Fisher) were coated with capture antibody and incubated at 4 °C overnight. The next day, plates were washed with PBST and blocked with 0.1% BSA in PBST buffer for 1 h at room temperature. After washing, samples and standards dilutions were added and incubated for 2 h at room temperature, followed by the addition of detection biotinylated antibody and incubation for 1 h. Streptavidin-HRP was added and incubated for 1 h at RT. Finally, the plates were washed, and TMB substrate (Abcam) was added. Reaction was stopped with 0.2 M H2SO4, and the absorbance was measured at 450 nm and 630 nm using a FLUOstar Omega microplate reader (BMG Labtech).
Luminex assay
Lung samples were acquired from a weighed portion of murine lung, previously homogenized in 1 mL of DMEM using a GentleMACS Dissociator (Miltenyi), and then clarified by collecting the supernatant after centrifugation at 450 rpm for 5 min. Before analysis, samples were inactivated by the addition of 0.5% Triton X100 and incubated for 30 min at 4 °C. Protease inhibitor cocktail (Complete, Roche) was added to prevent protein degradation during incubation. Samples were stored at −80 °C until the Luminex assay was conducted.
Cytokine/Chemokine array analysis was carried out using the Luminex Mouse Discovery Assay 11-plex kit (R&D Systems). Samples were centrifuged at 1000×g for 10 min and then diluted 1:2 in calibrator diluent, and the assay was conducted following the manufacturer’s instructions. Measurements were carried out using Luminex LABSCAN 100 and quantified by comparison to a standard curve. Data collection and analysis were performed utilizing LUMINEX 100 IS software.
Flow cytometry analysis of Tfh and GC B cells
Lymph node single cell suspensions were incubated with mouse FcR blocking reagent (Miltenyi, 130-092-575) for 15 min at 4 °C. Cells were washed and incubated with anti-mouse antibodies for surface staining for 15 min at 4 °C. Cells were washed and fixed with 4% paraformaldehyde for 30 min at room temperature and resuspended in PBS buffer. Acquisition was performed using a Gallios flow cytometer (Beckman Coulter).
Tfh cells were defined as CD45R–CD4+, CD44high, CXCR5+, PD-1+, and the following antibodies were used for surface staining: CD45R (B220)-APC-Vio770 (Miltenyi, 130-110-849), CD4-VioBright FITC (Miltenyi, 130-118-692), CD44-PerCP-Vio700 (Miltenyi, 130-128-625), CD185 (CXCR5)-APC (Miltenyi, 130-119-129), and CD279 (PD1)-PE (Miltenyi, 130-111-953). GC B cells were defined as CD19+GL7+CD95+, and the following antibodies were used for surface staining: CD19-APC (Miltenyi, 130-112-036), CD95 (FAS)-PE (Miltenyi, 130-112-036), and GL7-Alexa Fluor 488 eBioscience (Thermofisher, 53-5902-82).
Pseudotyped SARS‑CoV‑2 neutralization assay
The viral particles used in the neutralization assays were synthesized using an HIV-based second-generation lentiviral system, characterized by the utilization of three plasmids. The transfer plasmid (pwGFP) housed the viral particle’s genome (eGFP). The packaging plasmid (EMP0) was used to encode essential viral proteins, including Gag (structural proteins), Pol (polymerase), Rev (gene expression regulator), and Tat (transcriptional activator). The envelope plasmid encoded the full-length S protein of SARS-CoV-2. Two envelope plasmids were employed to pseudotype viral particles with the S protein variants of WT and XBB strain of SARS-CoV-2, respectively.
To produce the viral particles, 2.75 × 106 HEK293T cells were seeded in a T75 flask containing 13 mL of complete DMEM. The following day, the cells were transfected with 12.5 µg each of pwGFP and EMP0 plasmids, along with 2.5 µg of the different envelope plasmids, using FuGENE transfection reagent (Promega) according to the manufacturer’s instructions. After 48 h the viral supernatant was collected and filtered through an Acrodisc® Supor® 0.45 µm filter before being stored at −80 °C.
The capacity of each pseudovirus variant to infect ACE2-TMPRSS2 cells was determined empirically with an infectivity assay. The 2 × 105 cells/mL HEK293T-ACE2-TMPRSS2 cells were infected with an equal volume of serially diluted pseudovirus in DMEM. The cells were seeded in a black 96-well microplate (Thermo Scientific) and incubated at 37 °C and 5% CO2 for 48 h. Following incubation, cells were fixed with 4% paraformaldehyde in PBS for 1 h. The plates were washed twice with PBS and dried. The fluorescent spots were counted using a C.T.L. S6 Ultra-V analyzer with the FluoroSpot-X suite selected. The dilution of the pseudovirus chosen was the one that yielded 500–1000 infected cells per well (Supplementary Fig. 3).
For the neutralization assay, mouse sera were serially diluted in complete DMEM and added to black 96-well microplates, alongside the previously established volume of pseudovirus. The mixture was then incubated at 37 °C and 5% CO2 for 1 h to allow neutralization. After incubation, 2 × 104 HEK293T-ACE2-TMPRSS2 cells were added to each well, followed by 48 h of further incubation. The cells were then fixed as previously described, and the fluorescent spots were counted using a C.T.L. S6 Ultra-V analyzer with the FluoroSpot-X suite selected. The NT50 titer was defined as the reciprocal of the highest dilution at which a 50% or higher reduction in the number of spots compared to the untreated condition was achieved.
In vivo biodistribution
In vivo biodistribution assays were performed using Indium-111 radiolabeled LNPs. Lipid nanoparticle suspensions were incubated with 55 MBq Indium-111 oxine for 15 min at 37 °C. Labeled LNPs were afterwards purified using centrifugal concentrators (Vivaspin 500, 10.000 MWCO PES).
In vivo biodistribution assays were performed on BALB/c mice (n = 3 male, n = 3 female) on the U-SPECT6/E-class (MILabs) single photon emission computed tomography (SPECT) system. Radiolabeled LNPs were administered via intramuscular injection as described above. At 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 168 h post-administration, animals were anesthetized using isoflurane (2% in 100% O2), and SPECT images were acquired. Computer tomography (CT) scan was performed immediately after adjusting voltage and current to 55 kV and 0,33 mA, respectively. After image acquisition, these were reconstructed using indium-111 photopeaks with a window of 20% and a calibration factor to obtain the exact activity information (MBq/mL).
Images were analyzed using PMOD v3.2 software (PMOD Technologies, Switzerland). Signal values were corrected using the specific indium-111 radioactive decay correction factor and then transformed to standardized uptake value (SUV) units using the formula:
SUV = [tisular activity concentration (MBq/cm3)/dose (MBq)] × bodyweight (g).
Quantitative analysis was performed on organs/areas where signal could be correctly detected, setting a volume of interest (VOI) and quantifying over time, calculating the average signal in the VOI (Suvmean). Due to the difficulty in quantifying bone marrow signal, a visual analysis was performed to identify the timing of signal appearance in various bones.
At 168 h, the animals were euthanized, and the following organs/tissues were dissected: lungs, spleen, liver, kidneys, bone, muscle at the injection site, contralateral muscle, presacral lymph nodes, inguinal lymph nodes, and brain. Tissue activity was measured using a gamma counter (Hidex) to determine counts per minute (cpm) for each tissue. To quantitatively assess the amount of radioactivity in each sample, a ratio was calculated using the brain (background) as the reference tissue, and the values were normalized to tissue weight (g).
Serum biochemical analysis
Serum samples were collected as specified above. Biochemical analysis of AST and ALT levels was performed using the Cobas c-311 automatic analyzer (Roche Diagnostics).
SARS-CoV-2 challenge
Mice were anaesthetized with isoflurane (5% for induction and 2.5% for maintenance) and infected intranasally with the corresponding amount of virus to achieve the indicated doses, which was diluted in PBS in a total volume of 40 µL. After virus challenge, mice were periodically weighed and assessed using a clinical scoring system evaluating various parameters, including mouse appearance, level of consciousness, activity, response to stimuli, eye appearance, and frequency and quality of respiration61. Humane endpoints were established on a weight loss threshold of 25% or greater, alongside clinical scoring criteria.
Viral load determination by TCID50 assay
Lungs were harvested, weighed, and homogenized in 1 mL of DMEM using a GentleMACS Dissociator (Miltenyi). Homogenates were centrifuged at 450×g for 5 min, and the supernatant was taken for titration.
Virus titration was determined by TCID50 in Vero E6 cells. Vero E6 cells were seeded in 96-well plates at a density of 104 cells/well and cultured overnight at 37 °C and 5% CO2. Lung homogenates serial 1-log dilutions were prepared in complete DMEM with only 2% FBS and added to the cultured cells. 72 h after cell infection, plates were evaluated for cell death, and TCID50 was calculated using Ramakrishan’s newly proposed method formula62 and normalized to the weight (g) of the lung and the amount of buffer (mL) used for lung homogenization.
Statistical analysis
In experimental studies, GraphPad Prism 10 software was used for representation and statistical analyses.
We used ordinary one-way or two-way ANOVA with Tukey’s multiple comparison post-test to compare experimental groups. Statistical significances are denoted in the Figures by asterisks, as follows: *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, or ****p-value < 0.0001. The absence of asterisks indicates non-statistical significance with a p-value > 0.05. Outliers were identified and removed using Grubbs’ test, applying a significance threshold of p < 0.05.
