Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2

Molecular cloning

The Omicron spike amino acid sequence was derived from two lineage BA.1 Omicron cases identified in Canada on November 23 2021 (GISAID EpiCoV, EPI_ISL_6826713, and EPI_ISL_6826714). Omicron spike cDNA were codon optimized, synthesized as gblocks (IDT) and cloned to mRNA vector with 5′, 3′ untranslated region (UTR) and poly A tail. The furin cleave site (RRAR) was replaced with a GSAS short stretch in the mRNA vector. HexaPro mutations were introduced in the WT sequence (Wuhan-Hu-1, which was used for the current clinical mRNA vaccines) and Omicron variant spike sequence of mRNA vector to improve expression and prefusion state²⁰. The accessory plasmids for pseudovirus assay including pHIVNLGagPol and pCCNanoLuc2AEGFP were from Dr. Bieniasz’ lab⁴⁹. The C-terminal 19 amino acids were deleted in the SARS-CoV-2 spike sequence for the pseudovirus assay. A list of oligos has been provided in supplementary table 1.

Cell Culture

HEK293T (ATCC CRL-3216), HEK293FT (Thermo Fisher Cat. No. R70007), and 293T-hACE2 (gifted from Dr Bieniasz’ lab) cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo fisher) supplemented with 10% Fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco, final concentration penicillin 100 unit/ml, streptomycin 100 µg/ml), which is denoted as complete growth medium. Cells were split every 2 days at a split ratio of 1:4 when the confluency reached over 80%. Vero-E6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 5% heat-inactivated fetal bovine serum (FBS).

In vitro mRNA transcription and vaccine formulation

A Hiscribe™ T7 ARCA mRNA Kit (with tailing) (NEB, Cat # E2060S) was used to in vitro transcribe codon-optimized mRNA encoding HexaPro spikes of SARS-CoV-2 WT and Omicron variant with 50% replacement of uridine by N1-methyl-pseudouridine. The DNA template was linearized before mRNA transcription and contained 5′ UTR, 3′ UTR and 3′polyA tail as flanking sequence of spike open reading frame.

The purified mRNA was generated by following NEB manufacturer’s instructions and kept frozen at −80 °C until further use. The lipid nanoparticles mRNA was assembled using the NanoAssemblr® Ignite™ instrument (Precision Nanosystems) according to manufacturers’ guidance. In brief, lipid mixture composed of 46.3% ALC-0315 (MedChemExpress, HY-138170), 1.6% ALC-0159 (MedChemExpress, HY-138300), 9.4% DSPC (Avanti polar lipids, 850365 P), and 42.7% Cholesterol (Avanti polar lipids, 700100 P), was mixed with prepared mRNA in 25 mM sodium acetate at pH 5.2 on Ignite instrument at a molar ratio of 6:1 (LNP: mRNA)^47,50. The LNP encapsulated mRNA (LNP-mRNA) was buffer exchanged to PBS using 100 kDa Amicon filter (Macrosep Centrifugal Devices 100 K, 89131-992). Sucrose was added as a cryoprotectant. The particle size of mRNA-LNP was determined by DLS device (DynaPro NanoStar, Wyatt, WDPN-06) and TEM described below. The encapsulation rate and mRNA concentration were quantified by Quant-iT™ RiboGreen™ RNA Assay (Thermo Fisher).

Validation of LNP-mRNA mediated spike expression in vitro and receptor-binding capability of expressed Omicron HexaPro spikes

On day 1, HEK293T cells were seeded at 50% confluence in 24-well plate and mixed with 2 µg Omicron LNP-mRNA. After 16 hours, the cells were collected for flow cytometry. The spike expression on cell surface were detected by staining cells with human ACE2–Fc chimera (Sino Biological, 10108-H02HG) in MACS buffer (D-PBS with 2 mM EDTA and 0.5% BSA) for 20 min on ice. Cells were washed twice after the primary stain and incubated with PE–anti-human Fc antibody (Biolegend, Cat. No. 410708, Clone No. M1310G05, 1:100 dilution) in MACS buffer for 20 min on ice. During secondary antibody staining, live/Dead aqua fixable stain (Invitrogen) was used to assess cell viability. Data was collected on BD FACSAria II Cell Sorter (BD) and analyzed using FlowJo software (version 10.7.2, FlowJo LLC).

Negative-stain TEM

Formvar/carbon-coated copper grid (Electron Microscopy Sciences, catalog number FCF400-Cu-50) was glow-discharged and covered with 6 μl of the sample for 1 min before blotting away the sample. The sample was double-stained with 6 μl of 2% (w/v) uranyl formate (Electron Microscopy Sciences, catalog number 22450) for 5 s (first stain) and 1 min (second stain), blotting away after each stain. Images were collected using a JEOL JEM-1400 Plus microscope with an acceleration voltage of 80 kV and a bottom-mount charge-coupled device camera (4k by 3k, Advanced Microscopy Technologies).

Mouse vaccination

All experiments in this vaccine immunogenicity study used 6–8-weeks-old female C57BL/6Ncr (B6) mice purchased from Charles River. The mice-housing condition was maintained at regular ambient room temperature (65–75 °F, or 18–23 °C), 40–60% humidity, and a 14 h:10 h day/night cycle. Each mice cage was individually ventilated with clean food, water, and bedding. Two sets of immunization experiments were performed: vaccination with Omicron LNP-mRNA, and sequential vaccination with WT LNP-mRNA, followed by WT or Omicron LNP mRNA booster. For the Omicron LNP-mRNA vaccination experiment, five mice were immunized with 10 µg Omicron LNP-mRNA on day 0 (prime) and day 14 (boost). Retro-orbital blood was collected prior to vaccine injection on day 0, day 13, and day 21. For WT and Omicron LNP-mRNA sequential vaccination experiment, 18 mice were administered with either 100 µl PBS (3 + 3 mice, two independent experiments) or two-dose 1 µg WT (on day 0 and day 21, 3 + 9 mice, two independent experiments) and 10 µg Omicron LNP-mRNA (over 3.5 months post prime). Retro-orbital blood was collected prior to booster shot or two weeks post booster and 2^nd dose of WT LNP-mRNA.

Institutional approval

This study has received institutional regulatory approval. All recombinant DNA (rDNA) and biosafety work were performed under the guidelines of Yale Environment, Health and Safety (EHS) Committee with approved protocols (Chen 18–45, 20–18, and 20–26). All animal work was performed under the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC) with approved protocols (Chen 2020-20358; Chen 2021-20068; Wilen 2021-20198).

Isolation of plasma and PBMCs from blood

At the defined time points, retro-orbital blood was collected from mice. The isolation of PBMCs and plasma was achieved via centrifugation using SepMate-15 and Lymphoprep gradient medium (StemCell Technologies). 200 µl blood was immediately diluted with 800 µl PBS with 2% FBS. The blood diluent was then added to SepMate-15 tubes with 6 ml Lymphoprep (StemCell Technologies). Centrifugation at 1200 × g for 20 min was used to isolate RBCs, PBMCs and plasma. 250 µl diluted plasma was collected from the surface layer. The remaining solution at the top layer was poured to a new tube to isolate PBMCs, which were washed once with PBS + 2% FBS. The separated plasma was used in ELISA and neutralization assay.

ELISA

In all, 3 µg/ml of spike antigens were coated onto the 384-well ELISA plates (VWR, Cat # 82051-300) overnight at 4 degree. The antigen panel used in the ELISA includes RBDs of SARS RBD (AcroBiosystems, SPD-S52H6), MERS RBD (AcroBiosystems, SPD-M52H6), 2019-nCoV WA-1 (Sino Biological 40592-V08B), Delta variant B.1.617.2 (Sino Biological 40592-V08H90), Beta variant B.1.351 (Sino Biological 40592-V08H85) and Omicron variant B.1.1.529 (Sino Biological 40592-V08H121). Plates were washed with PBST (PBS plus 0.5% Tween 20) three times in the 50TS microplate washer (Fisher Scientific, NC0611021) and blocked with 0.5% BSA in PBST at room temperature for one hour. Plasma was fourfold serially diluted starting at a 1:500 dilution. Diluted plasma samples were added to the plates and incubated at room temperature for one hour, followed by washes with PBST five times. Anti-mouse secondary antibody (Fisher, Cat. No. A-10677) at 1:2500 dilution in blocking buffer was incubated at room temperature for one hour. Plates were washed five times and developed with tetramethylbenzidine substrate (Biolegend, 421101). The reaction was stopped with 1 M phosphoric acid after 20 min at room temperature, and OD at 450 nm was measured by multimode microplate reader (PerkinElmer EnVision 2105, Envision Manager v1.13.3009.1401). The binding response (OD450) was plotted against the dilution factor in log10 scale as the dilution-dependent response curve. The area under curve of the dilution-dependent response (Log10 AUC) was calculated to quantify the potency of the plasma antibody binding to spike antigens. The fold change of antibody titer was estimated using this equation: ratio = 10 ^ (AUC1 − AUC2).

hACE2 and antibody competition ELISA

The 384-well plate was coated with 0.6 µg/ml Omicron RBD at 4 degree overnight before washed with PBST (0.5% Tween-20) three times and blocked with 2% BSA in PBST for 1 h at room temperature. In hACE2 and antibody competition ELISA, 15 µg/ml hACE2 (Sino, 10108-H08H) or 10 µg/ml antibodies including Clone 13 A (Chen lab, in house), CR3022 (Abcam, Cat. No. Ab273073, Clone No. CR3022) and S309 (BioVision, Cat. No. A2266, Clone No. S309) were respectively added to the plate 1 hour prior to subsequent incubation with serially diluted plasma for another hour at room temperature. After coincubation of plasma and hACE2/antibodies, the plate was washed five times with PBST and incubated with anti-mouse secondary antibody with minimal cross reactivity with human IgG (Biolegend, Cat. No. 405306, Clone No. Poly4053, 1:2500 dilution). The plate was washed five times after 1-hour secondary antibody incubation and developed with tetramethylbenzidine substrate (Biolegend, 421101). The reaction was stopped with 1 M phosphoric acid after 20 min at room temperature, and OD at 450 nm was measured by multimode microplate reader (PerkinElmer EnVision 2105). The normalized AUC was calculated by normalizing the value with AUC determined in PBS group.

Omicron, WA-1, and Delta pseudovirus production and characterization

For the neutralization assay, HIV-1 based SARS-CoV-2 WA-1, B.1.617.2 (Delta) variant, and B.1.1.529 (Omicron) variant pseudotyped virions were packaged using a coronavirus spike plasmid, a reporter vector and a HIV-1 structural protein expression plasmid. The reporter vector, pCCNanoLuc2AEGFP, and plasmid expressing HIV-1 structural proteins (pHIVNLGagPol) were gifts from Dr Bieniasz’s lab. The spike plasmid for SARS-CoV-2 WA-1 pseudovirus truncated C-terminal 19 amino acids (denoted as SARS-CoV-2-Δ19) and was from Dr Bieniasz’ lab. Spike plasmids expressing C-terminally truncated SARS-CoV-2 B.1.617.2 variant S protein (Delta variant-Δ19) and SARS-CoV-2 B.1.1.529 variant S protein (Omicron variant-Δ19) were made based on the pSARS-CoV-2-Δ19. All pseudoviruses were produced under the same conditions. Briefly, 293FT cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of corresponding plasmids, in the presence of 198 µl PEI (1 mg/ml, PEI MAX, Polyscience). At 48 h after transfection, the supernatant was filtered through a 0.45-μm filter, and frozen in −80 °C.

To characterize the titer of WA-1, Delta, and Omicron pseudoviruses packaged, 1 × 10⁴ 293T-hACE2 cells were plated in each well of a 96-well plate. In the next day, different volumes of pseudovirus supplemented with culture medium to a total volue of 100 μL were added into 96-well plates with 293T-hACE2. Plates were incubated at 37 °C for 24 h. Then cells were washed with MACS buffer once and the percent of GFP-positive cells were counted by Attune NxT Acoustic Focusing Cytometer (Thermo Fisher, Attune NxT Software v3.1). To normalize pseudovirus titer, 1 × 10⁴ 293T-hACE2 cells were plated in each well of a 96-well plate. In the next day, 50 μL pseudovirus was mixed with 50 μL culture medium to 100 μL. The mixture was incubated for 1 hr in the 37 °C incubator, supplied with 5% CO2, and added into 96-well plates with 293T-hACE2. Plates were incubated at 37 °C for 24 hr. Then cells were washed with MACS buffer once and the percent of GFP-positive cells were counted by Attune NxT Acoustic Focusing Cytometer (Thermo Fisher). Delta pseudovirus and Omicron pseudovirus were diluted accordingly to match the functional titer of WA-1 pseudovirus for neutralization assay of plasma samples.

Pseudovirus neutralization assay

The SARS-CoV-2 pseudovirus assays were performed on 293T-hACE2 cells. One day before infection, 1 × 10⁴ 293T-hACE2 cells were plated in each well of a 96-well plate. In the next day, plasma collected from mice were serially diluted by 5 fold with complete growth medium at a starting dilution of 1:100. 55 μL diluted plasma was mixed with the same volume of SARS-CoV-2 WA-1, Delta variant, or Omicron variant pseudovirus and was incubated for 1 hr in the 37 °C incubator, supplied with 5% CO2. 100 μL of mixtures were added into 96-well plates with 293T-hACE2. Plates were incubated at 37 °C for 24 h. Then cells were washed with MACS buffer once and the percent of GFP-positive cells were counted by Attune NxT Acoustic Focusing Cytometer (Thermo Fisher). The 50% inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism (version 9.3.1, GraphPad Software Inc.). If the fitting value of IC50 is negative (i.e. negative titer), which suggested undetectable neutralization activity, the value was set to baseline (1, 0 in log scale).

Omicron and Delta live virus production and characterization

Full-length SARS-CoV-2 Omicron (BA.1) and Delta (B.1.617.2) isolates were a gift of Carolina Lucas and Akiko Iwasaki, and were isolated and sequenced⁵¹. Remnant nasopharyngeal swap samples selected for virus isolation were diluted in DMEM by 10 fold and then filtered through a 45-µm filter. Tenfold serial dilution of samples was made from 1:50 to 1:19,531,250. The diluted samples were subsequently co-incubated with TMPRSS2-Vero E6 in a 96-well plate and adsorbed for 1 h at 37 °C. Replacement medium was added after adsorption, and cells were incubated at 37 °C for up to 5 days. Supernatants from cells with cytopathic effect were collected, frozen, thawed and subjected to RT–qPCR.

To expand viral stocks, 107 Vero-E6 cells stably overexpressing ACE2 and TMPRSS2 were infected with SARS-CoV-2 at an MOI of ~0.01. The Omicron stock was collected 2 dpi, clarified by centrifugation (450 × g for 10 min), filtered through a 0.45-micron filter, and concentrated tenfold using Amicon Ultra-15 columns. To increase titer, the Delta stock was collected at 1 dpi, clarified, filtered, and used to infect 5 × 10⁷ Vero-E6 cells overexpressing ACE2 and TMPRSS2. At 1 dpi, supernatant was harvested, clarified, filtered, and concentrated as above. Viral stocks were titered by plaque assay in Vero-E6 cells⁵². In brief, 7.5 × 10⁵ and 4 × 10⁵ Vero-E6 cells were seeded in each well of 6-well plates or 12-well plates. The media was replaced the next day with 100 μl of 10-fold serially diluted virus. Gentle rocking was applied to the plates incubated at 37 °C for 1 h. Subsequently, overlay media, DMEM with 2% FBS and 0.6% Avicel RC-581 was added to each well. At 2 dpi for SARS-CoV-2, plates were fixed with 10% formaldehyde for 30 min, stained with crystal violet solution (0.5% crystal violet in 20% ethanol) for 30 min, and then rinsed with deionized water to visualize plaques.

Infectious virus neutralization assay

The complements and other potential neutralizing agents were heat inactivated in mouse plasma prior to infectious virus neutralization assay. Mouse plasma samples were serially diluted, then incubated with SARS-CoV-2 Omicron live virus for 1 h at 37 °C. The Omicron live virus was isolated from nasopharyngeal specimens and sequenced as part of the Yale SARS-CoV-2 Genomic Surveillance Initiative’s weekly surveillance Program in Connecticut⁵³. After coincubation, plasma/virus mixture was added to Vero-E6 cells overexpressing ACE2/TMPRSS2. Cell viability was measured at 3dpi or 5dpi using CellTiter Glo.

Statistics and reproducibility

Standard statistical methods were applied to non-high-throughput data. The statistical methods are described here, in figure legends and/or supplementary Excel tables. Data on dot-bar plots are shown as mean ± s.e.m. with individual data points in plots. Two-way ANOVA with Tukey’s multiple comparisons test and one-way ANOVA with Dunnett’s multiple comparisons test were used to assess statistical significance for grouped and non-grouped datasets respectively. Statistical significance labels: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Non-significant comparisons are not shown, unless otherwise noted as n.s., not significant. Sample number is designated as n from biologically independent samples. Prism (version 9.3.2, GraphPad Software Inc.) and RStudio (version 1.3.959, RStudio software company) were used for these analyses. Additional information can be found in the supplementary excel tables. Most of the data were collected from one independent experiment unless specifically stated otherwise in figure legends. Over 40 TEM micrographs were collected at various magnifications in one independent experiment and a representative micrograph was shown in Fig. 1.

Schematic illustrations

Schematic illustrations were created with Affinity Designer or BioRender.

Replication, randomization, blinding, and reagent validations

Biological or technical replicate samples were randomized where appropriate. In animal experiments, mice were randomized by littermates.

Experiments were not blinded.

Commercial antibodies were validated by the vendors, and re-validated in house as appropriate. Custom antibodies were validated by specific antibody – antigen interaction assays, such as ELISA. Isotype controls were used for antibody validations.

Cell lines were authenticated by original vendors, and re-validated in lab as appropriate.

All cell lines tested negative for mycoplasma.

Reporting summary

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