Materials

TLR7/8 agonist 1 dihydrochloride was purchased from Cayman Chemical. 1,2-Epoxydodecane (C12) was obtained from Sigma-Aldrich. Core 200 was customized from Enamine, and other polyamine cores were purchased from Sigma-Aldrich and TCI. Anti-mouse CD16/32 antibody, APC anti-mouse CD11c antibody, FITC anti-mouse CD80 antibody, PE anti-mouse CD86 antibody, APC anti-human CD11c antibody, FITC anti-human CD80 antibody and PE anti-human CD86 antibody were purchased from Biolegend. Mouse IL-1β uncoated ELISA, mouse IL-12p70 uncoated ELISA, mouse TNF-α uncoated ELISA, mouse MCP-1 uncoated ELISA, human IL-1β uncoated ELISA, human IL-12p70 uncoated ELISA, human TNF-α uncoated ELISA, LysoTracker Deep Red, LysoTracker Green, DiO and DiR were bought from Invitrogen. Mouse haptoglobin ELISA and mouse IP-10 ELISA were obtained from Abcam. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, DMG-PEG and cholesterol were obtained from Avanti Polar Lipids. DLin-MC3-DMA and SM-102 were purchased from MedChem Express. Codon-optimized m1ψ-modified luciferase mRNA and SARS-CoV-2 diproline-modified spike (S2P) mRNA were produced by in vitro transcription¹⁴. Cy5-tagged luciferase mRNA was produced in house by incorporating Cy5-UTP (TriLink) into the in vitro transcription reaction.

Synthesis of adjuvant lipidoid

Adjuvant lipidoid C12-TLRa was synthesized by reacting epoxydodecane (C12) with TLR7/8 agonist 1 dihydrochloride using the ring-opening reaction²⁵. Briefly, 10 mg of TLR7/8 agonist 1 dihydrochloride was dissolved in 0.8 ml of ethanol in a glass vial with a magnetic stir bar. Then, 8 μl of triethylamine was added to neutralize the hydrochloride before adding 20 mg of C12. The vial was sealed, and the mixture was stirred for 48 h at 80 °C. The crude product was purified by a CombiFlash NextGen 300+ chromatography system (Teledyne ISCO) with gradient elution from CH₂Cl₂ to 75:22:3 CH₂Cl₂/MeOH/NH₄OH (aq.). The desired fraction was collected (yield, 44%). C12-TLRa was characterized by mass spectrometry (calculated MS, 728.12; found [M + 2H]²⁺, 365.25) and NMR spectroscopy. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 7.78 (d, J = 8.3 Hz, 1H), 7.57 (doublet of doublets, J = 8.4, 1.3 Hz, 1H), 7.35–7.29 (m, 1H), 7.27 (d, J = 7.9 Hz, 2H), 7.05–7.00 (m, 1H), 6.98 (d, J = 8.0 Hz, 2H), 5.84 (s, 2H), 3.61–3.47 (m, 2H), 3.44 (s, 2H), 2.90 (t, J = 7.7 Hz, 2H), 2.68 (q, J = 1.9 Hz, 2H), 2.34 (t, J = 2.8 Hz, 2H), 1.69 (quintet, J = 7.6 Hz, 2H), 1.37 (doublet of triplets, J = 14.9, 7.5 Hz, 2H), 1.22 (s, 36H), 0.90–0.81 (m, 9H).

General method for the synthesis of polyamine-derived lipidoids

The polyamine cores were reacted with excess moles of C12 as needed to saturate the amines^25,33. Taking C12-113 as an example, 113 core (1 equiv.) was mixed with C12 (4.8 equiv.) for 48 h at 80 °C in a neat condition. The crude product was used for the initial library screening. To purify the top-performing C12-113 lipidoid, the crude product was separated as described above, and the fully saturated product was collected and identified by mass spectrometry (calculated MS, 854.49; found [M + 2H]²⁺, 429.13) and used for subsequent experiments.

Structural simulation of agonist–TLR7 interaction

The structures of TLR7/8 agonist 1 and C12-TLRa were first optimized by molecular dynamics simulation with the CHARMm force field⁴⁵. The exact TLR7 protein crystal structure was derived from the structure of the TLR7/R848 complex (PDB ID, 5GMH), removing any ligands or solvent molecules²⁶. Structural simulation between TLR7 dimer and agonists was carried out by CDocker docking simulation⁴⁶ and in situ structural superimposition. Potential non-covalent interactions, binding pockets and overviews of the binding sites between TLR7 dimer and the corresponding agonists were generated using BIOVIA Discovery Studio 2018.

LNP formulation

LNPs were formulated by microfluidic mixing³⁰. Briefly, an ethanol phase containing lipidoid (with or without C12-TLRa substitution), phospholipid, cholesterol and DMG-PEG at a designated molar ratio (Supplementary Table 1) was mixed with an aqueous phase (10 mM citrate buffer, pH 3) containing mRNA at a flow rate ratio of 1:3 and at a lipidoid/RNA weight ratio of 10:1 in a microfluidic chip device. LNPs were dialysed against 1× PBS in a 20 kDa molecular weight cut-off cassette for 2 h, sterilized through a 0.22 μm filter and stored at 4 °C. DiO- or DIR-labelled LNPs were obtained by mixing DiO or DiR (1 mol% of total lipids) with LNPs before dialysis.

LNP characterization

The hydrodynamic size, PDI and zeta potential of LNPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments). The morphology of LNPs was characterized by TEM (JEOL 1010) and cryo-EM (Titan Krios, Thermo Fisher) with a K3 Bioquantum (Gatan). The mRNA encapsulation efficiency and the pK_a of LNPs were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen) and a 6-(p-toluidinyl)naphthalene-2-sulfonic acid assay, respectively^30,33. LNP formulations were routinely examined by the Limulus amebocyte lysate (LAL) test, and endotoxin levels were consistently found to be <1 endotoxin unit per ml.

Cell culture and animal studies

The HEK-Blue mTLR7 cell line was kindly provided by J. Shi at Harvard Medical School, who obtained it from InvivoGen (#hkb-mtlr7). These cells were maintained according to vendor’s instruction. Murine macrophage DC2.4 cell line was obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. All cells were cultured at 37 °C in a humidified incubator of 5% CO₂, and routinely tested for mycoplasma contamination.

BMDCs were generated from C57BL/6 mice. Briefly, bone marrow cells were flushed from mouse femurs and tibias, lysed by ammonium–chloride–potassium (ACK) buffer to remove red blood cells and then cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin, 1% HEPES, 0.1 mM β-mercaptoethanol, 20 ng ml⁻¹ murine IL-4 (#214-14, PeproTech) and 20 ng ml⁻¹ murine granulocyte-macrophage colony stimulating factor (#315-03, PeproTech). On day 6, non-adherent and loosely adherent cells were collected for studies.

MoDCs were generated from a 38-year-old healthy male volunteer donor. Monocytes were isolated from donated apheresis blood using the RosetteSep Human Monocyte Enrichment Cocktail (#15068, Stemcell Technologies), and provided by the Human Immunology Core at the University of Pennsylvania. These cells were induced into MoDCs by culturing in complete RPMI medium supplemented with 20 ng ml⁻¹ human IL-4 (#574002, Biolegend) and 20 ng ml⁻¹ human granulocyte-macrophage colony stimulating factor (#572902, Biolegend) for 6 days. This study was approved by the Institutional Review Board of the University of Pennsylvania (#705906). Informed consent was obtained from the donor, who was compensated for this blood donation.

All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (#806540), and animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals at the University of Pennsylvania. C57BL/6 female mice (6–8 weeks of age, 18–20 g body weight) were purchased from Jackson Laboratory.

In vitro mLuc delivery

DC cells, BMDCs or MoDCs were seeded onto a 96-well plate at a density of 10,000 per well overnight and then mLuc-loaded LNPs were used to treat cells at the indicated doses for 24 h. Luciferase expression was evaluated by Luciferase Reporter 1000 Assay System (E4550, Promega), and cell viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay (G7572, Promega) according to the manufacturer’s protocols. The relative luciferase expression was reported as relative light units normalized to cell viability. Free mRNA was used as a control.

TLR7 reporter assay

The TLR7-agonistic activity of C12-TLRa was tested on HEK-Blue mTLR7 reporter cells using a HEK-Blue Detection Kit (#hb-det2, InvivoGen) according to the manufacturer’s instructions. Briefly, HEK-Blue mTLR7 reporter cells were seeded into a 96-well plate at a density of 40,000 cells per well in HEK-Blue Detection medium containing different concentrations of C12-TLRa. After incubation for 24 h, the absorbance at 650 nm was measured using a plate reader (Infinite M200, Tecan) and data were normalized to untreated cells. TLR7/8 agonist 1 was used as a positive control. Similarly, the TLR7-agonistic activity of LNPs was measured.

Cellular uptake

DC2.4 cells were seeded into 35 mm glass-bottom dishes for 24 h and then treated with DiO-labelled C12-113 LNP or DiO-labelled C12-113/TLRa LNP at an mRNA concentration of 500 ng ml⁻¹ for 2 h. Cells were sequentially stained with LysoTracker Deep Red (100 nM) for 30 min and Hoechst 33342 (10 μg ml⁻¹) for 5 min. Images were taken immediately using a confocal laser scanning microscope (LSM 710, Zeiss).

Analysis of DC maturation and cytokine production in vitro

DC2.4 cells or BMDCs were seeded into a 12-well plate at a density of 1 × 10⁶ cells per well overnight and then treated with SARS-CoV-2 mRNA-loaded LNPs (500 ng ml⁻¹) for 24 h. Cell cultures were collected for ELISA of TNF-α, IL-12p70 and IL-1β. Cells were collected, blocked with anti-mouse CD16/32 antibody and then stained with APC anti-mouse CD11c antibody, FITC anti-mouse CD80 antibody and PE anti-mouse CD86 antibody for 30 min at 4 °C before being analysed by flow cytometry (BD, LSR II). Similarly, MoDCs were treated. Cell cultures were collected for ELISA of human TNF-α, IL-12p70 and IL-1β. MoDCs were collected and stained with APC anti-human CD11c antibody, FITC anti-human CD80 antibody and PE anti-human CD86 antibody before analysis. Antibodies were used according the manufacturer’s instruction with a typical dilution at 1:100.

Analysis of DC maturation and cytokine production in vivo

Two iLNs from each mouse were harvested at 24 h post-injection of SARS-CoV-2 mRNA-loaded LNPs (5 μg mRNA per mouse) at the tail base and were gently mechanically disrupted using sterile pestles in 0.1 ml of RPMI complete medium in a 1.5 ml tube. The resulting cell suspensions were collected, blocked with anti-mouse CD16/32 antibody and then stained with APC anti-mouse CD11c antibody, FITC anti-mouse-CD80 antibody and PE anti-mouse-CD86 antibody before being analysed by flow cytometry.

Blood was collected into serum separator tubes (BD #365967) through the retro-orbital route at 6 and 24 h post-immunization. Serum was separated from blood following an incubation period of 30 min at room temperature (r.t.), and samples were centrifuged at 10,000g for 5 min. The serum was stored at −20 °C until use. To analyse the intralymphatic cytokine production, the resulting cell suspensions from iLNs were placed to a 96-well plate at a density of 10,000 cells per 100 μl per well and cultured for 8 h. Supernatant was collected for ELISA of TNF-α, IL-12p70 and IL-1β together with serum samples. Additionally, serum at 6, 24 and 48 h post-immunization was collected for ELISA of haptoglobin, IP-10 and MCP-1.

Distribution and transfection of LNPs in vivo

Mice were s.c. injected at the tail base with mLuc-loaded LNPs at a dose of 5 μg mRNA per mouse. At 6 or 24 h post-injection, mice were intraperitoneally (i.p.) injected with d-luciferin potassium salt (150 mg per kg (body weight)), and bioluminescence imaging was performed on an IVIS imaging system (PerkinElmer). To enable concurrent bioluminescence and fluorescence imaging, DiR-labelled, mLuc-loaded LNPs were s.c. injected into mice. At 24 h post-injection, mice were i.p. injected with d-luciferin potassium salt, and major organs and iLNs were collected for bioluminescence and fluorescence imaging.

In vivo immunization

Mice were s.c. immunized with SARS-CoV-2 mRNA-loaded LNPs at a dose of 1 or 5 μg mRNA per mouse twice using a prime-boost strategy at a 3 week interval. Body weight was recorded twice a week during the experiment. Serum was collected using serum separator tubes as described above, stored at −20 °C and used for ELISA and virus neutralization assay. Two weeks after the boost vaccination, mice were anaesthetized and spleens were collected for flow cytometry analysis.

Determination of anti-RBD antibody titres using ELISA

Purified SARS-CoV-2 His tagged RBD (1 μg ml⁻¹) (Sino Biological, #40592-V08H) was used to coat High Bind Stripwell Corning 96-well clear polystyrene microplates overnight. Plates were washed with wash buffer (0.05% Tween 20/PBS) once, and blocked for 2 h at r.t. using a solution of heat-inactivated, IgG-depleted, protease-free bovine serum albumin (2% w/v BSA/PBS). Afterwards, plates were washed three times, and mouse sera were serially diluted in the blocking solution and incubated for 2 h at r.t. Plates were washed three times before adding horseradish-peroxidase-conjugated anti-mouse secondary antibody specific to total IgG (1:10,000, Abcam #ab97040) or subclasses (IgG1, 1:10,000, Abcam, #ab98693; IgG2c, 10,000, Abcam, #ab98722) in blocking buffer. Plates were incubated for 1.5 h and washed three times before the addition of 100 µl KPL 3,3′,5,5′-tetramethylbenzidine substrate per well for 8 min. The reaction was stopped by adding 50 µl of 2 N sulfuric acid, and the absorbance was measured at 450 nm using a SpectraMax 190 microplate reader. RBD-specific IgG endpoint dilution titre was defined as the highest dilution of serum to give an optical density greater than the cut-off optical density value determined using the Frey method⁴⁷.

Pseudovirus neutralization assay

A VSV pseudotype with SARS-CoV-2 S was first produced³⁶. We performed an antibody neutralization assay using VSVΔG-RFP SARS-CoV-2. Vero E6 cells stably expressing TMPRSS2 were seeded in 100 μl DMEM at 2.5 × 10⁴ cells per well in a 96-well collagen-coated plate. After 12 h, twofold serially diluted serum samples were mixed with VSVΔG-RFP SARS-CoV-2 pseudotype virus (50–200 focus-forming units per well) encoding the spike of D614G, Beta or Delta variant and incubated for 1 h at 37 °C. A mouse anti-VSV Indiana G, 8G5F11 (#Ab01401-2.0, Absolute Antibody), was also included in this mixture to neutralize any potential VSV-G carryover virus at a concentration of 100 ng ml⁻¹. The antibody–virus mixture was then used to replace the media on Vero E6 TMPRSS2 cells. At 20 h post-infection, the cells were washed and fixed with 4% PFA before visualization on an S6 FluoroSpot Analyzer (CTL). Individual infected foci were enumerated, and the values were compared with control wells without antibody. The focus reduction neutralization titre 50% (FRNT₅₀) was measured as the greatest serum dilution at which focus count was reduced by at least 50% relative to control cells that were infected with pseudotype virus in the absence of mouse serum. FRNT₅₀ titres for each sample were measured in two technical replicates performed on separate days.

Flow cytometry analysis of T and B cells

T cell

Spleens were collected, processed as single cells, filtered using a 70 µm cell strainer in complete RPMI 1640 and centrifuged, and red blood cells lysed in ACK lysis buffer to obtain a clear single-cell suspension. To measure antigen-specific T cells, two million splenocytes were stimulated with 2.5 µg ml⁻¹ of SARS-CoV-2 RBD peptide pools (#PM-WCPV-S-RBD-1, JPT) in a FACS tube for 6 h at 37 °C, 5% CO₂ with 2 mg ml⁻¹ anti-CD28 (Tonbo #40-0281-M001) providing co-stimulation. Stimulations proceeded for 1 h before adding 5 mg ml⁻¹ brefeldin A (Biolegend #420601), 2 mM monensin (Biolegend #420701) and 5 mg ml⁻¹ anti-CD107a Alexa Fluor 647 (Biolegend #121610) for 5 h. DMSO served as a negative control, and the combination of 50 mg ml⁻¹ phorbol 12-myristate 13-acetate and 1 mg ml⁻¹ ionomycin served as a positive control. After a total of 6 h, samples were washed with PBS, stained with Live/Dead Aqua for 5 min, blocked using anti-mouse CD16/32 antibody for 20 min and stained extracellularly for 30 min using antibodies (Supplementary Figs. 21d and 27d). Cells were washed in FACS buffer, fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences #554714), and stained intracellularly using antibodies for 30 min (Supplementary Figs. 21d and 27d). After intracellular staining, cells were washed twice and fixed with 300 µl (1% PFA), and samples were acquired on a BD LSR II equipped with four laser lines and 18 photomultiplier tubes. The gating strategy, and the antibody list and catalogue numbers are provided in Supplementary Figs. 21 and 27.

Memory B cell

Spleens were collected, processed as single cells, filtered using a 40 µm cell strainer in complete RPMI 1640 and centrifuged at 300g for 5 min; red blood cells were lysed with ACK (1 min), washed twice and counted, and two million cells per sample were incubated with anti-mouse CD16/32 antibody for 20 min at 4 °C. Cells were then washed with FACS buffer (1% BSA/PBS) and stained for 1 h using antibodies (Supplementary Fig. 23d). Following staining, cells were washed twice and fixed with 300 µl (1% PFA), and samples were acquired on a BD LSR II equipped with four laser lines and 18 photomultiplier tubes. The gating strategy, and the antibody list, fluorescent RBD probes¹⁴ and catalogue numbers are provided in Supplementary Fig. 23.

ELISpot assay

Bone marrow was flushed from femurs and tibia into FACS buffer and filtered through a 63 µm Nitex mesh. Red blood cells were lysed in ACK buffer for 5 min on ice, and washed twice with FACS buffer. The resulting cells were counted using a Beckman Coulter ViCell. MultiScreenHTS IP filter plates, 0.45 µm (Millipore Sigma, MSIPS4W10), were coated with RBD protein antigen at 10 μg ml⁻¹ in sodium carbonate/sodium bicarbonate buffer pH 9.6 (35 mM NaHCO₃ and 15 mM Na₂CO₃) for 1 h at 37 °C. Plates were then washed with 200 µl PBS per well three times and blocked at 37 °C in complete RPMI for 30 min. Bone marrow cells were plated in six halving dilutions beginning with one million total bone marrow cells per well and incubated overnight in complete RPMI. Plates were then washed with wash buffer (1× PBS + 0.1% Tween 20) five times, and biotinylated anti-IgG detection antibody (goat anti-mouse IgG human ads-BIOT; Southern Biotech, 1030-08) was added at a final dilution of 3 μg ml⁻¹ in 2% BSA/PBS and incubated at r.t. for 1 h. Plates were once again washed five times, and streptavidin-alkaline phosphatase (1:20,000 dilution in 2% BSA/PBS) was added prior to incubation at r.t. for 30 min. Plates were then washed five times with wash buffer, and 50 µl per well 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium chloride solution (Sigma, #B1911, 100 ml) was added for ~10 min or until spots developed at which time the reaction was quenched with 100 µl 1 M sodium phosphate monobasic solution. After plates were rinsed with deionized H₂O and dried overnight, they were scanned and counted using an S6 FluoroSpot Analyzer.

Statistics and reproducibility

All data are presented as mean ± s.d. Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test was applied for comparison between two groups or among multiple groups using Graphpad Prism 7.0, respectively. P < 0.05 was considered to be statistically significant. Each experiment is repeated at least three times independently with similar results, and the representative dataset is presented.

Reporting summary

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

• SEO Powered Content & PR Distribution. Get Amplified Today.
• PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
• PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
• PlatoESG. Automotive / EVs, Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
• BlockOffsets. Modernizing Environmental Offset Ownership. Access Here.
• Source: https://www.nature.com/articles/s41565-023-01404-4