Method details

Codon-optimized coding sequences of hemagglutinin (HA) of A/Puerto Rico/8/1934 (PR8), receptor binding domain (RBD, amino acids 1-14 fused with amino acids 319-541) of SARS-CoV-2 (Wuhan-Hu-1, GenBank: {"type":"entrez-nucleotide","attrs":{"text":"MN908947.3","term_id":"1798172431","term_text":"MN908947.3"}}MN908947.3) and firefly luciferase (Luc) were synthesized and cloned into an mRNA production plasmid as described (Freyn et al., 2020). mRNA production was performed as described (Freyn et al., 2020). Briefly, mRNAs were produced to contain 101 nucleotide-long poly(A) tails. m1Ψ-5′-triphosphate instead of UTP was used to generate modified nucleoside-containing mRNA. Capping of the in vitro transcribed mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog, CleanCap. mRNA was purified by cellulose purification, as described (Baiersdörfer et al., 2019). All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at −20°C. Cellulose-purified m1Ψ-containing RNAs were encapsulated in LNPs using a self-assembly process as previously described wherein an ethanolic lipid mixture of ionizable cationic lipid, phosphatidylcholine, cholesterol and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing mRNA at acidic pH (Maier et al., 2013). The LNP formulation used in this study is proprietary to Acuitas Therapeutics; the proprietary lipid and LNP composition are described in US patent US10,221,127. As the DOTAP-containing LNP could not be easily concentrated after the self-assembly process, the DOTAP-LNP was made using an extrusion process (Mui et al., 2003). The lipids (DOTAP, cholesterol, DSPC, PEG-lipid) were first solubilized in chloroform. The chloroform was removed using a stream of nitrogen gas and the lipid film was put under vacuum for 2 h to remove residual solvent. The lipid film was hydrated in PBS, freeze-thawed 5 times using liquid nitrogen and a 60°C water bath, and then extruded 10 times through 80 nm pore-sized filters. The RNA-loaded and empty particles were characterized and subsequently stored at −80°C at an RNA concentration of 1 μg μl^-1 (in the case of loaded particles) and total lipid concentration of 30 μg μl^-1 (both loaded and empty particles). The mean hydrodynamic diameter of mRNA-LNPs was ∼80 nm with a polydispersity index of 0.02-0.06 and an encapsulation efficiency of ∼95%. Two or three batches from each mRNA-LNP formulations were used in these studies and we did not observe variability in vaccine efficacy.

The PR8 HA recombinant protein and the SARS-CoV-2 RBD protein used for vaccine studies were purchased from Sino Biological.

The PR8 HA and RBD proteins used for generating fluorescently-labeled probes for B cell analyses and PR8 HA ELISA studies were produced as described previously (Amanat et al., 2020; Margine et al., 2013; Stadlbauer et al., 2020). The RBD protein used for ELISA was purchased from GenScript.

Fluorescently-labeled recombinant RBD protein was prepared as previously described (Laczkó et al., 2020; Lederer et al., 2020). Briefly, rRBD was independently conjugated to either PE or AlexaFluor 647 using the Lightning-Link® R-Phycoerythrin (R-PE) and Lightning-Link (R) Rapid Alexa Fluor 647 according to manufacturer’s instructions. To create fluorescently labeled RBD and HA tetramers, recombinant RBD or HA was biotinylated using the EZ-Link Micro Sulfo-NHS-Biotinylation Kit. Streptavidin-conjugated Alexa Fluor 647, Alexa Fluor 488, or Brilliant Violet 421 were then added at a 6:1 molar ratio (biotinylated-protein to streptavidin-conjugate). Specifically, after the volume of fluorochrome needed to achieve a 6:1 molar ratio was determined, the total volume of fluorochrome was split into 10 subaliquots. These subaliquots were then added, on ice, to the biotinylated protein and mixed by pipetting every 10 minutes (for a total of 10 additions).

The hydrodynamic diameter (Z-average size) of LNPs (with and without mRNA) was measured at 25°C by DLS using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd) equipped with a solid-state HeNe 633nm laser at a scattering angle of 173°. LNP samples formulated at 1 mg/ml mRNA (or an equivalent of 1 mg/ml for empty LNP) were diluted 1:500 using sterile phosphate-buffered saline before determination of size/PDI. The Z-average is represented as diameter in nm ± width of distribution.

eLNPs with and without ionizable lipid were vitrified using Vitrobot Mark IV System (FEI/Thermo Scientific), and frozen grids were imaged with 300 kV Titan Krios Cryo-TEM with a Falcon III camera (FEI/Thermo Scientific). Images were analyzed using ImageJ. Between 200 to 300 particles were manually measured to determine the diameter of the LNPs. The size of the LNPs are represented as diameter in nm ± standard deviation.

Vaccines were administered via the intramuscular (IM) route into the gastrocnemius muscle, or the intradermal (ID) route across two sites localized toward the base of the tail using a 3/10cc 29½G insulin syringe (Covidien). Mice were immunized with one of the following combinations: 10 μg recombinant PR8 HA (rHA), 10 μg rHA + 30 μg Luc mRNA-LNP (containing 30 μg mRNA and 900 μg total lipid), 10 μg rHA or rRBD + Addavax according to manufacturer’s instructions (equal volumes of protein and AddaVax™ were mixed), 10 μg rHA or rRBD + eLNP (total lipid content: 900 μg; equivalent to lipid content of 30 μg mRNA-LNP), or 10 μg PR8 HA or RBD mRNA-LNP.

In the IL-6 Ab blockade experiments, mice were given 0.5 mg of anti-IL6 antibody or isotype antibody intraperitoneally (IP) one day prior to immunization and 0.25 mg every other day following immunization.

Mice were immunized IM in the gastrocnemius muscle. Mice were immunized with either 30 μg PR8 HA mRNA-LNP or 30 μg rHA + eLNP (total lipid content equivalent to 30 μg mRNA-containing mRNA-LNP).

Mice were isoflurane-anesthetized and blood was collected through the retro-orbital route. Serum was separated from blood following an incubation period of 30 minutes at room temperature, and samples were centrifuged at 10 000 g for 5 minutes in a non-refrigerated Eppendorf 5424 centrifuge. Separated serum was stored at −20°C.

8-week-old BALB/c mice were immunized ID with 10 μg rHA, 10 μg rHA + Addavax, 10 μg rHA + eLNP (30 μg mRNA-LNP equivalent) or 10 μg HA mRNA-LNP. Animals were terminally bled 9 months post-immunization, and HAI titers were determined. 400 μL of each individual immune sera was injected IP into naive recipient mice. Two hours after serum transfer, recipient mice were put under isoflurane anesthesia, and blood was collected for post transfer HAI titer determination. Then, mice were infected with 5000 focus forming units (FFU) of mouse-adapted A/Puerto Rico/8/1934 influenza virus (see IAV LD in Tapia et al., 2013) diluted in a final volume of 40 μL PBS. Viral solution was applied dropwise to nasal orifice, and was inhaled by serum-transferred mice. Weight loss was followed daily for 14 days. Animals were euthanized when they lost 20% of their starting bodyweight, as per IACUC protocol.

Sera were heat-inactivated (55°C) for 30 minutes, spun down at 11000 rpm for 2 minutes, and diluted 1:20 in PBS, then serially diluted 1:2 in 50 μl in 96-well U-bottom plates (lowest concentration: 1:2560) using a multichannel pipette. Then, four hemagglutinating doses of A/Puerto Rico/8/1934 virus was added in the same volume as diluted sera. Finally, 12.5 μL of turkey erythrocyte solution - washed twice in phosphate buffered saline and diluted to a final concentration of 2% (v/v) - was added, and gently mixed with the sera-virus solution (final volume of 125 μl). Samples were incubated for 45 minutes at room temperature, after which the plates were turned on the side for one minute, then scanned on a regular office scanner. HAI titers were determined as the highest dilution of the sample that inhibited four agglutinating doses of the influenza virus. Inhibition of agglutination was observed as the blood forming a “tear drop” shape.

Lymph nodes were collected at specific time points and snap-frozen in a mixture of isopropanol and dry ice and stored at −80°C until use. Frozen tissues were cut on dry ice, weighed (∼20 mg), and disrupted using the TissueLyzer® II system (QIAGEN). Tissues were disrupted using 5 mm steel beads (QIAGEN) under the following conditions: 2 × 30 Hz, 20 s per cycle. Homogenized tissues were resuspended in 750 μl of M-Per lysis buffer, in the presence of CoMplete protease inhibitor cocktail and incubated on ice for 30 minutes with inversions every 10 minutes. For the IL-6 ELISA, whole lymph nodes were resuspended in lysis buffer (as above) prior to homogenization (as above). Lysates were cleared by centrifugation (2270 g, 30 minutes, 4°C), transferred to new tubes, and stored at −80°C until use. An aliquot was diluted 1:100 in PBS and total protein content of the lysate sample was determined using the Pierce microBCA protein assay kit or measured undiluted using the Pierce BCA protein assay kit for the IL-6 ELISA.

Tissue lysate collected at 4 and 24 hours post administration of test articles were assayed for the induction of a 32 pro-inflammatory cytokine panel (MCYTOMAG-70K; including TNF-α, IL-1β, IL-6, KC, and IFN-γ) using the Luminex® technology. Plates were designed using the Milliplex® assay builder (Millipore-Sigma) and subjected to the manufacturer’s quality control. For each plate, a standard curve was prepared by diluting the Milliplex® Pro Mouse Cytokine Standard 32-Plex in the Milliplex® in standard diluent followed by 4-fold serial dilutions from 1:4 to 1:65536 in the same diluent. Samples were thawed on ice, cleared by centrifugation (10,000 g, 10 minutes, 4°C), diluted 1:2 using the Milliplex® Sample diluent, and a volume of 25 μl transferred to assay plates prefilled with pooled capture Abs. The plates were incubated for 30 minutes under orbital shaking (800 rpm, room temperature), washed as per manufacturer recommendation, incubated with biotinylated detection Abs (30 minutes, 800 rpm, room temperature), washed and revealed post-incubation for 10 minutes with streptavidin-phycoerythrin (800 rpm, room temperature). Data was acquired on a BioPlex2100® system using RP1 PMT setting with a minimum of 50 beads per region analyzed. For each cytokine, a 5-parameter regression algorithm (5-PL) was used to fit the data and interpolate cytokine values in tissue lysate samples. In order to account for inter-plate variability, two samples (PBS and LPS) were used as inter-plate calibrators.

High Bind Stripwell Corning 96 Well Clear Polystyrene Microplates were coated overnight with 1 μg/ml purified recombinant PR8 HA or SARS-CoV-2 RBD. Plates were washed once with wash buffer (0.05% Tween-20 in PBS), and blocked for two hours at room temperature using a solution of heat inactivated, IgG depleted, protease free bovine serum albumin (2% w/v BSA in PBS). After blocking, plates were washed three times, and mouse sera was serially diluted in the blocking solution and incubated for 2 hours at room temperature. Plates were washed three times before the addition of horse radish peroxidase (HRP)-conjugated anti-mouse secondary Ab specific to total IgG or subclasses in blocking buffer (total IgG, IgG2a, IgG2b: 1:10 000, IgG1: 1:15000, IgA: 1:7500). Plates were incubated for 1.5 hours, washed three times before the addition of 100 μl per well of KPL TMB substrate for 8 minutes. The reaction was stopped with 50 μl 2N sulfuric acid, and the absorbance was measured at 450nm using a SpectraMax 190 microplate reader. Endpoint dilution titer was defined as the highest dilution of serum to give a value 0.01 OD greater than the background (no serum) cut-off OD value. All samples were run in technical duplicates.

The Mouse IL-6 Uncoated ELISA kit was used according to the manufacturer’s instructions. In brief, 96-well MaxiSorp plates were coated with IL-6 capture Ab overnight, blocked and washed according to the manufacturer’s instructions. For each plate, an IL-6 standard curve was prepared by dissolving lyophilized IL-6 in lysis buffer at 500 pg/ml and then performing a 2-fold serial dilution to ∼4 pg/ml. Tissue lysate samples (described in “Protein extraction from lymph nodes”) collected at 4, 8, 12, 24, and 48 hours post immunization were thawed on ice and cleared by centrifugation. 100 μl of each sample was added to the plates undiluted and diluted at a 1:1 ratio with ELISA diluent. Plates were incubated on an orbital shaker at room temperature for 2 hours, before detection Ab and HRP were added according to the manufacturer’s instructions. TMB was added to plates for 10 minutes before the addition of a 2N sulfuric acid stop solution. Absorbance was measured at 450 nm using a BioTek Synergy HT plate reader. A third order polynomial regression was used to fit the data and interpolate a standard curve in Prism v9.0.0. IL-6 concentrations were then normalized on a per sample basis by dividing the IL-6 concentration (pg/ml) generated from the ELISA by the total protein content of the lysate as determined by BCA (mg/ml) to yield pg IL-6 per mg of total protein. Finally, the normalized IL-6 concentrations from the undiluted and the diluted (1:1) samples were averaged prior to plotting. All samples were run in technical duplicates.

293T cells plated 24 hours previously at 5 X 10⁶ cells per 10 cm dish were transfected using calcium phosphate with 35 μg of pCG1 SARS-CoV-2 S D614G delta18 expression plasmid encoding a codon optimized SARS-CoV S gene with an 18 residue truncation in the cytoplasmic tail (kindly provided by Stefan Pohlmann) with a single amino acid substitution (D614G) found in recent circulating variants. Twelve hours post transfection the cells were fed with fresh media containing 5mM sodium butyrate to increase expression of the transfected DNA. Thirty hours after transfection, the SARS-CoV-2 spike expressing cells were infected for 2-4 hours with VSV-G pseudotyped VSVΔG-RFP at an MOI of ∼1-3. After infection, the cells were washed twice with media to remove unbound virus. Media containing the VSVΔG-RFP SARS-CoV-2 pseudotypes was harvested 28-30 hours after infection and clarified by centrifugation twice at 6000 g then aliquoted and stored at −80°C until used for Ab neutralization analysis.

Vero E6 cells stably expressing TMPRSS2 were seeded in 100 μL at 2.5x10⁴ cells/well in a 96 well collagen coated plate. The next day, 2-fold serially diluted serum samples were mixed with VSVΔG-RFP SARS-CoV-2 pseudotype virus (50-200 focus forming units/well) and incubated for 1 hour at 37°C. Also included in this mixture to neutralize any potential VSV-G carryover virus was 8G5F11, a mouse anti-VSV Indiana G, at a concentration of 100 ng/ml. The Ab-virus mixture was then used to replace the media on VeroE6 TMPRSS2 cells. 20 hours post infection, the cells were washed and fixed with 4% paraformaldehyde before visualization on an S6 FluoroSpot Analyzer (CTL). Individual infected foci were enumerated and the values compared to control wells without Ab. The focus reduction neutralization titer 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 titers for each sample were measured in two technical replicates performed on separate days.

Draining inguinal and/or popliteal lymph nodes were harvested after immunization, homogenized with a syringe plunger and filtered through a 40 μm cell strainer on ice. All staining steps were carried out at 4°C in FACS buffer (PBS with 2% heat inactivated FBS). Single cell suspensions were Fc blocked with anti-CD16/CD32 monoclonal Ab (mAb) prior to staining. Splenocytes were harvested from spleens by mechanical disruption between the frosted ends of microscope slides and filtered through 70 μm cell strainer. Bone marrow was flushed from femurs and tibia from each mouse using a 23 g X ¾” needle and syringe into RPMI-1640 media supplemented with 10% heat inactivated FBS and filtered through 70 μm cell strainer. Splenocytes and bone marrow cells were then subjected to red blood cell lysis for 5 min in 2 mL ACK buffer on ice and resuspended in 1 mL RPMI-1640 media.

Cells were incubated with biotinylated CXCR5 Ab for 1 hour, washed, followed by incubation with streptavidin-conjugated BV421 for 30 minutes. After washing, cells were incubated with 20 μg/ml I-A(d) Influenza A HA PE-conjugated tetramer (HNTNGVTAACSHE) in RPMI for 2 hours, then washed. This last step was only performed for the analysis of HA-specific Tfh cells. Cells were next incubated for 30 minutes with a cocktail of Fixable Viability dye eFluor780 and all other surface Abs. Cells were washed with FACS buffer, then fixed and permeabilized in FoxP3/Transcription Factor Staining Buffer Set according to manufacturer’s instructions before intranuclear staining with Bcl-6 (and Foxp3 for the Tfr staining) for 30 minutes. All incubations were performed at 4°C. Staining panel details in Tables S3–S5.

Cells were incubated for 30 minutes at 4°C with a cocktail containing Fixable Viability dye eFluor780 and a cocktail of surface Abs. For antigen-specific GC B cells: HA AF488 tetramer and HA AF647 tetramer, or RBD BV421 tetramer were also added during this step. Excess Abs were washed away, and cells were fixed with 1% paraformaldehyde (PFA) for 30 minutes prior to acquisition. Staining panel details in Tables S6 and S7.

All samples were acquired on a 5 laser Cytoflex LX (Beckman Coulter) or a 5 laser Aurora (Cytek), and data analyzed in FlowJo v10.

5 million cells were stained with fixable live dead aqua (Zombie Aqua) for 15 minutes at room temperature, washed with FACS buffer and stained with labeled HA or RBD probes and a cocktail of surface Abs (see below) in BD Brilliant Buffer for 15 minutes at 4°C. Cells were then washed and resuspended in FACS buffer (PBS + 0.2% bovine serum albumin). ∼2 million events per sample were acquired on a LSRII (PR8 HA experiments), or on BD Symphony A3 Lite (SARS-CoV-2 experiments) and analyzed with FlowJo 10.x software. Staining panel details in Table S1.

Prior to sorting, splenic B cells were enriched by negative selection using Miltenyi LD columns, Miltenyi anti-biotin MACS microbeads, and biotin-conjugated CD4, CD8, F4/80 and Ter119 Abs, according to the manufacturer’s protocol. Enriched cells were stained with a cocktail of surface Abs and with recombinant HA conjugated to PE and AF647. IgD⁺ follicular B cells, HA⁺IgD^-IgM^- memory, and HA^-IgD^-IgM^- B cells were sorted using a BD FACS Aria II sorter (see Figure 3D for gating strategy). Staining panel details in Table S2.

mouse spleens were harvested at day 80 post immunization, and single-cell suspension was prepared in RPMI-1640 media following physical tissue homogenization. Genomic DNA was extracted from sorted cells using the QIAGEN Gentra DNA purification kit following the manufacturer’s recommendations. Amplification and sequencing of Ab heavy chain gene rearrangements were performed as described previously (Johnson et al., 2020). Libraries were pooled at equimolar ratios and loaded onto an Illumina MiSeq in the Human Immunology Core Facility at the University of Pennsylvania, and subjected to pair-end sequencing (2x300 bp) using the Illumina MiSeq Reagent kit.

raw sequencing data in FASTQ format were processed with pRESTO version 0.5.10 (Vander Heiden et al., 2014). Paired reads were aligned and sequences were subsequently subjected to quality filtering as described previously (Johnson et al., 2020). IgBLAST was used for gene identification and ImmuneDB v0.29.9 was used for clonal inference (Rosenfeld et al., 2018). After gene identification, sequences were trimmed to IMGT position 20 to remove 5′ primer sequences. Sequences sharing the same VH gene, JH gene, and 85% CDR3 amino-acid similarity were then grouped into clones and clones averaging less than 85% IGHV nucleotide identity were excluded from further analysis to minimize sequencing artifacts.