Adjuvant lipidoid-substituted lipid nanoparticles increase the immunogenicity of SARS-CoV-2 mRNA vaccines

Supplies

TLR7/8 agonist 1 dihydrochloride was bought from Cayman Chemical. 1,2-Epoxydodecane (C12) was obtained from Sigma-Aldrich. Core 200 was personalized from Enamine, and different polyamine cores had been bought 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 had been bought 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 Crimson, LysoTracker Inexperienced, DiO and DiR had been purchased from Invitrogen. Mouse haptoglobin ELISA and mouse IP-10 ELISA had been obtained from Abcam. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine, DMG-PEG and ldl cholesterol had been obtained from Avanti Polar Lipids. DLin-MC3-DMA and SM-102 had been bought from MedChem Categorical. Codon-optimized m1ψ-modified luciferase mRNA and SARS-CoV-2 diproline-modified spike (S2P) mRNA had been produced by in vitro transcription¹⁴. Cy5-tagged luciferase mRNA was produced in home by incorporating Cy5-UTP (TriLink) into the in vitro transcription response.

Synthesis of adjuvant lipidoid

Adjuvant lipidoid C12-TLRa was synthesized by reacting epoxydodecane (C12) with TLR7/8 agonist 1 dihydrochloride utilizing the ring-opening response²⁵. 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 earlier than including 20 mg of C12. The vial was sealed, and the combination 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 specified fraction was collected (yield, 44%). C12-TLRa was characterised by mass spectrometry (calculated MS, 728.12; discovered [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).

Normal technique for the synthesis of polyamine-derived lipidoids

The polyamine cores had been reacted with extra moles of C12 as wanted to saturate the amines^25,33. Taking C12-113 for instance, 113 core (1 equiv.) was combined with C12 (4.8 equiv.) for 48 h at 80 °C in a neat situation. The crude product was used for the preliminary library screening. To purify the top-performing C12-113 lipidoid, the crude product was separated as described above, and the absolutely saturated product was collected and recognized by mass spectrometry (calculated MS, 854.49; discovered [M + 2H]²⁺, 429.13) and used for subsequent experiments.

Structural simulation of agonist–TLR7 interplay

The buildings of TLR7/8 agonist 1 and C12-TLRa had been first optimized by molecular dynamics simulation with the CHARMm drive discipline⁴⁵. The precise TLR7 protein crystal construction was derived from the construction of the TLR7/R848 advanced (PDB ID, 5GMH), eradicating 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 websites between TLR7 dimer and the corresponding agonists had been generated utilizing BIOVIA Discovery Studio 2018.

LNP formulation

LNPs had been formulated by microfluidic mixing³⁰. Briefly, an ethanol section containing lipidoid (with or with out C12-TLRa substitution), phospholipid, ldl cholesterol and DMG-PEG at a chosen molar ratio (Supplementary Desk 1) was combined with an aqueous section (10 mM citrate buffer, pH 3) containing mRNA at a move price ratio of 1:3 and at a lipidoid/RNA weight ratio of 10:1 in a microfluidic chip system. LNPs had been dialysed towards 1× PBS in a 20 kDa molecular weight cut-off cassette for two h, sterilized by way of a 0.22 μm filter and saved at 4 °C. DiO- or DIR-labelled LNPs had been obtained by mixing DiO or DiR (1 mol% of complete lipids) with LNPs earlier than dialysis.

LNP characterization

The hydrodynamic dimension, PDI and zeta potential of LNPs had been measured utilizing a Zetasizer Nano ZS90 (Malvern Devices). The morphology of LNPs was characterised by TEM (JEOL 1010) and cryo-EM (Titan Krios, Thermo Fisher) with a K3 Bioquantum (Gatan). The mRNA encapsulation effectivity and the pOk_a of LNPs had been decided utilizing a modified Quant-iT RiboGreen RNA assay (Invitrogen) and a 6-(p-toluidinyl)naphthalene-2-sulfonic acid assay, respectively^30,33. LNP formulations had been routinely examined by the Limulus amebocyte lysate (LAL) take a look at, and endotoxin ranges had been constantly discovered to be <1 endotoxin unit per ml.

Cell tradition and animal research

The HEK-Blue mTLR7 cell line was kindly offered by J. Shi at Harvard Medical Faculty, who obtained it from InvivoGen (#hkb-mtlr7). These cells had been maintained in response to vendor’s instruction. Murine macrophage DC2.4 cell line was obtained from American Sort Tradition Assortment 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 had been cultured at 37 °C in a humidified incubator of 5% CO₂, and routinely examined for mycoplasma contamination.

BMDCs had been generated from C57BL/6 mice. Briefly, bone marrow cells had been flushed from mouse femurs and tibias, lysed by ammonium–chloride–potassium (ACK) buffer to take away crimson blood cells after which 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 issue (#315-03, PeproTech). On day 6, non-adherent and loosely adherent cells had been collected for research.

MoDCs had been generated from a 38-year-old wholesome male volunteer donor. Monocytes had been remoted from donated apheresis blood utilizing the RosetteSep Human Monocyte Enrichment Cocktail (#15068, Stemcell Applied sciences), and offered by the Human Immunology Core on the College of Pennsylvania. These cells had been induced into MoDCs by culturing in full RPMI medium supplemented with 20 ng ml⁻¹ human IL-4 (#574002, Biolegend) and 20 ng ml⁻¹ human granulocyte-macrophage colony stimulating issue (#572902, Biolegend) for six days. This examine was accredited by the Institutional Assessment Board of the College of Pennsylvania (#705906). Knowledgeable consent was obtained from the donor, who was compensated for this blood donation.

All animal protocols had been accredited by the Institutional Animal Care and Use Committee of the College of Pennsylvania (#806540), and animal procedures had been carried out in accordance with the Pointers for Care and Use of Laboratory Animals on the College of Pennsylvania. C57BL/6 feminine mice (6–8 weeks of age, 18–20 g physique weight) had been bought from Jackson Laboratory.

In vitro mLuc supply

DC cells, BMDCs or MoDCs had been seeded onto a 96-well plate at a density of 10,000 per properly in a single day after which mLuc-loaded LNPs had been used to deal with cells on the indicated doses for twenty-four h. Luciferase expression was evaluated by Luciferase Reporter 1000 Assay System (E4550, Promega), and cell viability was measured utilizing a CellTiter-Glo Luminescent Cell Viability Assay (G7572, Promega) in response to the producer’s protocols. The relative luciferase expression was reported as relative gentle items normalized to cell viability. Free mRNA was used as a management.

TLR7 reporter assay

The TLR7-agonistic exercise of C12-TLRa was examined on HEK-Blue mTLR7 reporter cells utilizing a HEK-Blue Detection Package (#hb-det2, InvivoGen) in response to the producer’s directions. Briefly, HEK-Blue mTLR7 reporter cells had been seeded right into a 96-well plate at a density of 40,000 cells per properly in HEK-Blue Detection medium containing completely different concentrations of C12-TLRa. After incubation for twenty-four h, the absorbance at 650 nm was measured utilizing a plate reader (Infinite M200, Tecan) and information had been normalized to untreated cells. TLR7/8 agonist 1 was used as a constructive management. Equally, the TLR7-agonistic exercise of LNPs was measured.

Mobile uptake

DC2.4 cells had been seeded into 35 mm glass-bottom dishes for twenty-four h after which handled with DiO-labelled C12-113 LNP or DiO-labelled C12-113/TLRa LNP at an mRNA focus of 500 ng ml⁻¹ for two h. Cells had been sequentially stained with LysoTracker Deep Crimson (100 nM) for 30 min and Hoechst 33342 (10 μg ml⁻¹) for five min. Photographs had been taken instantly utilizing a confocal laser scanning microscope (LSM 710, Zeiss).

Evaluation of DC maturation and cytokine manufacturing in vitro

DC2.4 cells or BMDCs had been seeded right into a 12-well plate at a density of 1 × 10⁶ cells per properly in a single day after which handled with SARS-CoV-2 mRNA-loaded LNPs (500 ng ml⁻¹) for twenty-four h. Cell cultures had been collected for ELISA of TNF-α, IL-12p70 and IL-1β. Cells had been collected, blocked with anti-mouse CD16/32 antibody after which stained with APC anti-mouse CD11c antibody, FITC anti-mouse CD80 antibody and PE anti-mouse CD86 antibody for 30 min at 4 °C earlier than being analysed by move cytometry (BD, LSR II). Equally, MoDCs had been handled. Cell cultures had been collected for ELISA of human TNF-α, IL-12p70 and IL-1β. MoDCs had been collected and stained with APC anti-human CD11c antibody, FITC anti-human CD80 antibody and PE anti-human CD86 antibody earlier than evaluation. Antibodies had been used in accordance the producer’s instruction with a typical dilution at 1:100.

Evaluation of DC maturation and cytokine manufacturing in vivo

Two iLNs from every mouse had been harvested at 24 h post-injection of SARS-CoV-2 mRNA-loaded LNPs (5 μg mRNA per mouse) on the tail base and had been gently mechanically disrupted utilizing sterile pestles in 0.1 ml of RPMI full medium in a 1.5 ml tube. The ensuing cell suspensions had been collected, blocked with anti-mouse CD16/32 antibody after which stained with APC anti-mouse CD11c antibody, FITC anti-mouse-CD80 antibody and PE anti-mouse-CD86 antibody earlier than being analysed by move cytometry.

Blood was collected into serum separator tubes (BD #365967) by way of the retro-orbital route at 6 and 24 h post-immunization. Serum was separated from blood following an incubation interval of 30 min at room temperature (r.t.), and samples had been centrifuged at 10,000g for five min. The serum was saved at −20 °C till use. To analyse the intralymphatic cytokine manufacturing, the ensuing cell suspensions from iLNs had been positioned to a 96-well plate at a density of 10,000 cells per 100 μl per properly and cultured for 8 h. Supernatant was collected for ELISA of TNF-α, IL-12p70 and IL-1β along with serum samples. Moreover, 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 had been s.c. injected on the tail base with mLuc-loaded LNPs at a dose of 5 μg mRNA per mouse. At 6 or 24 h post-injection, mice had been intraperitoneally (i.p.) injected with d-luciferin potassium salt (150 mg per kg (physique weight)), and bioluminescence imaging was carried out on an IVIS imaging system (PerkinElmer). To allow concurrent bioluminescence and fluorescence imaging, DiR-labelled, mLuc-loaded LNPs had been s.c. injected into mice. At 24 h post-injection, mice had been i.p. injected with d-luciferin potassium salt, and main organs and iLNs had been collected for bioluminescence and fluorescence imaging.

In vivo immunization

Mice had been s.c. immunized with SARS-CoV-2 mRNA-loaded LNPs at a dose of 1 or 5 μg mRNA per mouse twice utilizing a prime-boost technique at a 3 week interval. Physique weight was recorded twice every week in the course of the experiment. Serum was collected utilizing serum separator tubes as described above, saved at −20 °C and used for ELISA and virus neutralization assay. Two weeks after the enhance vaccination, mice had been anaesthetized and spleens had been collected for move cytometry evaluation.

Willpower of anti-RBD antibody titres utilizing ELISA

Purified SARS-CoV-2 His tagged RBD (1 μg ml⁻¹) (Sino Organic, #40592-V08H) was used to coat Excessive Bind Stripwell Corning 96-well clear polystyrene microplates in a single day. Plates had been washed with wash buffer (0.05% Tween 20/PBS) as soon as, and blocked for two h at r.t. utilizing an answer of heat-inactivated, IgG-depleted, protease-free bovine serum albumin (2% w/v BSA/PBS). Afterwards, plates had been washed 3 times, and mouse sera had been serially diluted within the blocking answer and incubated for two h at r.t. Plates had been washed 3 times earlier than including horseradish-peroxidase-conjugated anti-mouse secondary antibody particular to complete IgG (1:10,000, Abcam #ab97040) or subclasses (IgG1, 1:10,000, Abcam, #ab98693; IgG2c, 10,000, Abcam, #ab98722) in blocking buffer. Plates had been incubated for 1.5 h and washed 3 times earlier than the addition of 100 µl KPL 3,3′,5,5′-tetramethylbenzidine substrate per properly for 8 min. The response was stopped by including 50 µl of two N sulfuric acid, and the absorbance was measured at 450 nm utilizing a SpectraMax 190 microplate reader. RBD-specific IgG endpoint dilution titre was outlined as the best dilution of serum to provide an optical density larger than the cut-off optical density worth decided utilizing the Frey technique⁴⁷.

Pseudovirus neutralization assay

A VSV pseudotype with SARS-CoV-2 S was first produced³⁶. We carried out an antibody neutralization assay utilizing VSVΔG-RFP SARS-CoV-2. Vero E6 cells stably expressing TMPRSS2 had been seeded in 100 μl DMEM at 2.5 × 10⁴ cells per properly in a 96-well collagen-coated plate. After 12 h, twofold serially diluted serum samples had been combined with VSVΔG-RFP SARS-CoV-2 pseudotype virus (50–200 focus-forming items per properly) 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 additionally included on this combination to neutralize any potential VSV-G carryover virus at a focus of 100 ng ml⁻¹. The antibody–virus combination was then used to interchange the media on Vero E6 TMPRSS2 cells. At 20 h post-infection, the cells had been washed and stuck with 4% PFA earlier than visualization on an S6 FluoroSpot Analyzer (CTL). Particular person contaminated foci had been enumerated, and the values had been in contrast with management wells with out antibody. The main target discount neutralization titre 50% (FRNT₅₀) was measured as the best serum dilution at which focus rely was diminished by not less than 50% relative to regulate cells that had been contaminated with pseudotype virus within the absence of mouse serum. FRNT₅₀ titres for every pattern had been measured in two technical replicates carried out on separate days.

Move cytometry evaluation of T and B cells

T cell

Spleens had been collected, processed as single cells, filtered utilizing a 70 µm cell strainer in full RPMI 1640 and centrifuged, and crimson blood cells lysed in ACK lysis buffer to acquire a transparent single-cell suspension. To measure antigen-specific T cells, two million splenocytes had been stimulated with 2.5 µg ml⁻¹ of SARS-CoV-2 RBD peptide swimming pools (#PM-WCPV-S-RBD-1, JPT) in a FACS tube for six h at 37 °C, 5% CO₂ with 2 mg ml⁻¹ anti-CD28 (Tonbo #40-0281-M001) offering co-stimulation. Stimulations proceeded for 1 h earlier than including 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 five h. DMSO served as a detrimental management, and the mix of fifty mg ml⁻¹ phorbol 12-myristate 13-acetate and 1 mg ml⁻¹ ionomycin served as a constructive management. After a complete of 6 h, samples had been washed with PBS, stained with Dwell/Useless Aqua for five min, blocked utilizing anti-mouse CD16/32 antibody for 20 min and stained extracellularly for 30 min utilizing antibodies (Supplementary Figs. 21d and 27d). Cells had been washed in FACS buffer, mounted and permeabilized utilizing the Cytofix/Cytoperm package (BD Biosciences #554714), and stained intracellularly utilizing antibodies for 30 min (Supplementary Figs. 21d and 27d). After intracellular staining, cells had been washed twice and stuck with 300 µl (1% PFA), and samples had been acquired on a BD LSR II geared up with 4 laser strains and 18 photomultiplier tubes. The gating technique, and the antibody checklist and catalogue numbers are offered in Supplementary Figs. 21 and 27.

Reminiscence B cell

Spleens had been collected, processed as single cells, filtered utilizing a 40 µm cell strainer in full RPMI 1640 and centrifuged at 300g for five min; crimson blood cells had been lysed with ACK (1 min), washed twice and counted, and two million cells per pattern had been incubated with anti-mouse CD16/32 antibody for 20 min at 4 °C. Cells had been then washed with FACS buffer (1% BSA/PBS) and stained for 1 h utilizing antibodies (Supplementary Fig. 23d). Following staining, cells had been washed twice and stuck with 300 µl (1% PFA), and samples had been acquired on a BD LSR II geared up with 4 laser strains and 18 photomultiplier tubes. The gating technique, and the antibody checklist, fluorescent RBD probes¹⁴ and catalogue numbers are offered in Supplementary Fig. 23.

ELISpot assay

Bone marrow was flushed from femurs and tibia into FACS buffer and filtered by way of a 63 µm Nitex mesh. Crimson blood cells had been lysed in ACK buffer for five min on ice, and washed twice with FACS buffer. The ensuing cells had been counted utilizing a Beckman Coulter ViCell. MultiScreenHTS IP filter plates, 0.45 µm (Millipore Sigma, MSIPS4W10), had been 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 had been then washed with 200 µl PBS per properly 3 times and blocked at 37 °C in full RPMI for 30 min. Bone marrow cells had been plated in six halving dilutions starting with one million complete bone marrow cells per properly and incubated in a single day in full RPMI. Plates had been then washed with wash buffer (1× PBS + 0.1% Tween 20) 5 instances, and biotinylated anti-IgG detection antibody (goat anti-mouse IgG human ads-BIOT; Southern Biotech, 1030-08) was added at a remaining dilution of three μg ml⁻¹ in 2% BSA/PBS and incubated at r.t. for 1 h. Plates had been as soon as once more washed 5 instances, and streptavidin-alkaline phosphatase (1:20,000 dilution in 2% BSA/PBS) was added previous to incubation at r.t. for 30 min. Plates had been then washed 5 instances with wash buffer, and 50 µl per properly 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium chloride answer (Sigma, #B1911, 100 ml) was added for ~10 min or till spots developed at which period the response was quenched with 100 µl 1 M sodium phosphate monobasic answer. After plates had been rinsed with deionized H₂O and dried in a single day, they had been scanned and counted utilizing an S6 FluoroSpot Analyzer.

Statistics and reproducibility

All information are introduced as imply ± s.d. Pupil’s t-test or one-way evaluation of variance (ANOVA) adopted by Tukey’s take a look at was utilized for comparability between two teams or amongst a number of teams utilizing Graphpad Prism 7.0, respectively. P < 0.05 was thought of to be statistically vital. Every experiment is repeated not less than 3 times independently with related outcomes, and the consultant dataset is introduced.

Reporting abstract

Additional info on analysis design is out there within the Nature Portfolio Reporting Abstract linked to this text.