Bivalent SARS-CoV-2 mRNA vaccines improve breadth of neutralization and defend in opposition to the BA.5 Omicron variant in mice

Cells

African inexperienced monkey Vero-TMPRSS2 (ref. 44) and Vero-hACE2-TMPRRS2 (ref. 45) cells had been cultured at 37 °C in DMEM supplemented with 10% FBS, 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids and 100 U ml−1 of penicillin–streptomycin. Vero-TMPRSS2 cells had been supplemented with 5 µg ml−1 of blasticidin. Vero-hACE2-TMPRSS2 cells had been supplemented with 10 µg ml−1 of puromycin. BHK-21/WI-2 cells and HEK293T/17 cells had been obtained from the American Sort Tradition Assortment (ATCC) and cultured in DMEM supplemented with 10% FBS. All cells routinely examined detrimental for mycoplasma utilizing a polymerase chain response (PCR)-based assay.

Viruses

The WA1/2020 D614G and B.1.617.2 strains had been described previously20,46. The BA.1 isolate (hCoV-19/USA/WI-WSLH-221686/2021) was obtained from a person in Wisconsin as a mid-turbinate nasal swab22. The BA.5 isolate was remoted in California (hCoV-19/USA/CA-Stanford-79_S31/2022) and was a present from M. Suthar (Emory College). All viruses had been passaged as soon as on Vero-TMPRSS2 cells and subjected to next-generation sequencing45 to substantiate the introduction and stability of substitutions. All virus experiments had been carried out in an authorised biosafety stage 3 (BSL-3) facility at Washington College Faculty of Drugs.

Mice

Animal research had been carried out in accordance with the suggestions within the Information for the Care and Use of Laboratory Animals of the Nationwide Institutes of Well being. For research (K18-hACE2 mice) at Washington College Faculty of Drugs, the protocols had been authorised by the Institutional Animal Care and Use Committee at Washington College Faculty of Drugs (assurance no. A3381–01). Virus inoculations had been carried out underneath anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts had been made to reduce animal struggling. For research with BALB/c mice, animal experiments had been carried out in compliance with approval from the Animal Care and Use Committee of Moderna, Inc. Pattern measurement for animal experiments was decided on the premise of standards set by the institutional Animal Care and Use Committee. Experiments had been neither randomized nor blinded.

Heterozygous K18-hACE2 C57BL/6J mice (pressure: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J, cat. no. 34860) had been obtained from The Jackson Laboratory. BALB/c mice (pressure: BALB/cAnNCrl, cat. no. 028) had been obtained from Charles River Laboratories. Animals had been housed in teams of 4–5, fed normal chow diets, subjected to a photoperiod of 12 hours on, 12 hours off darkish/gentle cycle and saved at an ambient animal room temperature of 70° ± 2° F with a room humidity of fifty% ± 5%.

mRNA vaccine and LNP manufacturing course of

A sequence-optimized mRNA encoding prefusion-stabilized Wuhan-Hu-1 (mRNA-1273), BA.1 (mRNA-1273.529, the Omicron gene element in mRNA-1273.214) and BA.5 (mRNA-1273.045, the Omicron gene element in mRNA-1273.222) was designed. The genes of SARS-CoV-2 S2P proteins had been synthesized in vitro utilizing an optimized T7 RNA polymerase-mediated transcription response with full alternative of uridine by N1m-pseudouridine47. Along with the two-proline substitution, the BA.1 spike gene within the mRNA-1273.529 and mRNA-1273.214 vaccines encoded the next substitutions: A67V, Δ69–70, T95I, G142D, Δ143–145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F. The BA.5 gene in mRNA-1273.045 and mRNA-1273.222 vaccines encoded the next substitutions: T19I, Δ24–26, A27S, Δ69–70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K.

A non-translating management mRNA was synthesized and formulated into LNPs as beforehand described48. The response included a DNA template containing the immunogen open studying body flanked by 5′ untranslated area (UTR) and three′ UTR sequences and was terminated by an encoded polyA tail. After RNA transcription, the cap-1 construction was added utilizing the vaccinia virus capping enzyme and a pair of′-O-methyltransferase (New England Biolabs). The mRNA was purified by oligo-dT affinity purification, buffer-exchanged by tangential movement filtration into sodium acetate, pH 5.0, sterile filtered and saved frozen at –20 °C till additional use.

The mRNA was encapsulated in an LNP by means of a modified ethanol-drop nanoprecipitation course of described previously49. Ionizable, structural, helper and polyethylene glycol lipids had been briefly combined with mRNA in an acetate buffer, pH 5.0, at a ratio of two.5:1 (lipid:mRNA). The combination was neutralized with Tris-HCl, pH 7.5; sucrose was added as a cryoprotectant; and the ultimate resolution was sterile-filtered. Vials had been crammed with formulated LNP and saved frozen at –20 °C till additional use. The vaccine product underwent analytical characterization, which included the dedication of particle measurement and polydispersity, encapsulation, mRNA purity, double-stranded RNA content material, osmolality, pH, endotoxin and bioburden, and the fabric was deemed acceptable for in vivo examine. The preclinical supplies used on this examine had been: (1) monovalent mRNA-1273 vaccine that accommodates a single mRNA encoding the SARS-CoV-2 S2P antigen; (2) monovalent mRNA-1273.529 vaccine that accommodates a single mRNA encoding the SARS-CoV-2 S2P antigen for BA.1; (3) monovalent mRNA-1273.045 vaccine that accommodates a single mRNA encoding the SARS-CoV-2 S2P antigen of the BA.4/BA.5 subvariants of Omicron; (4) research-grade bivalent mRNA-1273.214 vaccine, which is a 1:1 benchside mixture of individually formulated mRNA-1273 and mRNA-1273.529 vaccines; (5) research-grade bivalent mRNA-1273.222 vaccine, which is a 1:1 benchside mixture of individually formulated mRNA-1273 and mRNA-1273.045 vaccines; (6) clinically consultant bivalent mRNA-1273.214 vaccine, which is a 1:1 combine within the vial of individually formulated mRNA-1273 and mRNA-1273.529; and (7) clinically consultant bivalent mRNA-1273.222 vaccine, which is a 1:1 combine within the vial of individually formulated mRNA-1273 and mRNA-1273.045. All mRNAs are formulated into a combination of 4 lipids: SM-102, ldl cholesterol, DSPC and PEG2000-DMG.

Clinically consultant materials is made within the course of growth laboratory utilizing a proportionally scaled-down model of the industrial manufacturing course of, whereas the research-grade supplies are ready in an automatic high-throughput, small-scale course of. The principle distinction between the 2, past scale, is when the vaccine parts are combined and the sourcing of uncooked supplies. Within the case of analysis grade, the supplies are individually ready and vialed after which combined 1:1 instantly earlier than injection. For the clinically consultant materials, every mRNA is individually formulated into LNPs after which combined, earlier than vialing in order that each mRNA/LNPs are current within the vial. The preclinical course of entails encapsulating capped, full-length mRNA in LNP, though the precise sequence of unit operations and set factors have been tailored to maximise batch success. Thus, there are batch-to-batch variations within the research-grade materials. Instance Certificates of Analyses for each kinds of supplies will be provided upon request. All vaccines had been ready with the identical technique because the Good Manufacturing Follow for scientific vaccines. Recombinant soluble S and RBD proteins from Wuhan-1, BA.1 and BA.4/5 SARS-CoV-2 strains had been expressed as described50,51.

Recombinant spike and RBD proteins

Recombinant proteins had been produced in Expi293F cells (Thermo Fisher Scientific) by transfection of DNA utilizing the ExpiFectamine 293 Transfection Package (Thermo Fisher Scientific). Supernatants had been harvested at 3 dpi, and recombinant proteins had been purified utilizing Ni-NTA agarose (Thermo Fisher Scientific) after which buffer exchanged into PBS and concentrated utilizing Amicon Ultracel centrifugal filters (EMD Millipore). SARS-CoV-2 B.1.617.2 RBD protein was bought from Sino Organic (40592-V08H90).

ELISA

Assays had been carried out in 96-well microtiter plates (Thermo Fisher Scientific) coated with 100 μl of recombinant Wuhan-1, BA.1 or BA.4/BA.5 spike proteins. Plates had been incubated at 4 °C in a single day after which blocked for 1 hour at 37 °C utilizing SuperBlock (Thermo Fisher Scientific, 37516) after which washed 4 occasions with PBS 0.05% + Tween 20 (PBST). Serum samples had been serially diluted in 5% BSA in TBS (Boston BioProducts, IBB-187), added to plates, incubated for 1 hour at 37 °C after which washed 4 occasions with PBST. Goat anti-mouse IgG-HRP (SouthernBiotech, 1030-05) was diluted in 5% BSA in TBS earlier than including to the wells and incubating for 1 hour at 37 °C. Plates had been washed 4 occasions with PBST earlier than the addition of TMB substrate (Thermo Fisher Scientific, 34029). Reactions had been stopped by the addition of TMB cease resolution (Invitrogen, SS04). The optical density (OD) measurements had been taken at 450 nm, and titers had been decided utilizing a four-parameter logistic curve slot in GraphPad Prism model 9.

Focus discount neutralization check with genuine SARS-CoV-2 strains

Serial dilutions of sera had been incubated with 102 focus-forming models (FFU) of WA1/2020 D614G, B.1.617.2, BA.1 or BA.5 for 1 hour at 37 °C. Antibody–virus complexes had been added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37 °C for 1 hour. Subsequently, cells had been overlaid with 1% (w/v) methylcellulose in MEM. Plates had been harvested 30 hours (WA1/2020 D614G and B.1.617.2) or 70 hours (BA.1 and BA.5) later by eradicating overlays and stuck with 4% paraformaldehyde (PFA) in PBS for 20 minutes at room temperature. Plates had been washed and sequentially incubated with a pool (SARS2–02, −08, −09, −10, −11, −13, −14, −17, −20, −26, −27, −28, −31, −38, −41, −42, −44, −49, −57, −62, −64, −65, −67 and −71 (ref. 52)) of anti-S murine antibodies (together with cross-reactive monoclonal antibodies to SARS-CoV) and HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, A8924, RRID: AB_258426) in PBS supplemented with 0.1% saponin and 0.1% BSA. SARS-CoV-2-infected cell foci had been visualized utilizing TrueBlue Peroxidase Substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Mobile Applied sciences).

VSV pseudovirus neutralization assay

Codon-optimized full-length spike genes (Wuhan-1 with D614G, BA.2.75, BA.1 and BA.5) had been cloned right into a pCAGGS vector. Spike genes contained the next mutations: (a) BA.2.75: T19I, Δ24–26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K; (b) BA.1: A67V, Δ69–70, T95I, G142D/ΔVYY143–145, ΔN211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F; and (c) BA.4/5: T19I, Δ24–26, A27S, Δ69–70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K. To generate VSVΔG-based SARS-CoV-2 pseudovirus, BHK-21/WI-2 cells had been transfected with the spike expression plasmid and contaminated by VSVΔG-firefly-luciferase as beforehand described53. Vero E6 cells had been used as goal cells for the neutralization assay and maintained in DMEM supplemented with 10% FBS. To carry out neutralization assay, mouse serum samples had been heat-inactivated for 45 minutes at 56 °C, and serial dilutions had been made in DMEM supplemented with 10% FBS. The diluted serum samples or tradition medium (serving as virus-only management) had been combined with VSVΔG-based SARS-CoV-2 pseudovirus and incubated at 37 °C for 45 minutes. The inoculum virus or virus–serum combine was subsequently used to contaminate Vero E6 cells (ATCC, CRL-1586) for 18 hours at 37 °C. At 18 hours after an infection, an equal quantity of One-Glo reagent (Promega, E6120) was added to tradition medium for readout utilizing a BMG PHERastar-FSX plate reader. The share of neutralization was calculated based mostly on relative gentle models (RLUs) of the virus management and subsequently analyzed utilizing four-parameter logistic curve (GraphPad Prism 8.0).

Lentivirus-based pseudovirus neutralization assay

Neutralization of SARS-CoV-2 additionally was measured in a single-round-of-infection assay with lentivirus-based pseudovirus assay as beforehand described54. To supply SARS-CoV-2 pseudoviruses, an expression plasmid bearing codon-optimized SARS-CoV-2 full-length spike plasmid was co-transfected into HEK293T/17 cells (ATCC, CRL-11268) with packaging plasmid pCMVDR8.2, luciferase reporter plasmid pHR′CMV-Luc and a TMPRSS2 plasmid. Mutant spike plasmids had been produced by GenScript. Pseudoviruses had been combined with eight serial four-fold dilutions of sera or antibodies in duplicate after which added to monolayers of 293T-hACE2 cells in duplicate. Three days after an infection, cells had been lysed; luciferase was activated with the Luciferase Assay System (Promega); and RLUs had been measured at 570 nm on a SpectraMax L luminometer (Molecular Units). After subtraction of background RLU (uninfected cells), % neutralization was calculated as 100 × ((virus-only management) − (virus + antibody)) / (virus-only management). Dose–response curves had been generated with a five-parameter non-linear perform, and titers had been reported because the serum dilution or antibody focus required to realize ID 50 neutralization. The enter dilution of serum was 1:50; thus, 20 was the decrease LOD. Samples that didn’t neutralize on the LOD at 50% had been plotted at 25, and that worth was used for geometric imply calculations. Every assay included duplicates. As well as, the reported values had been the geometric imply of two impartial assays.

Anti-RBD depletion assay

Hexahistidine-tagged Wuhan-1, BA.1 and BA.4/5 RBD proteins had been produced recombinantly in Escherichia coli and purified as described previously12. After buffer-exchange into PBS or TBS, RBD proteins (15 μg) or no protein (detrimental management) had been incubated with 0.5 mg of Ni-NTA Magnetic Beads (HisPur, 88832) in a single day at 4 °C. After unbound proteins had been washed away with 50 mM Tris (pH 8.0) supplemented with 200 mM NaCl, 50 mM imidazole and 0.05% Tween 20, beads had been combined with diluted serum (1/150 in 50 mM Tris (pH 8.0) supplemented with 200 mM NaCl, 30 mM imidazole and 0.05% Tween 20) obtained from K18-hACE2 mice 1 month after boosting with mRNA-1273, mRNA-1273.214 or mRNA-1273.222 vaccines and incubated with agitation at room temperature for 4 hours. Subsequently, beads had been eliminated with magnetic separation on a KingFisher Flex robotic (Thermo Fisher Scientific), and serial dilutions of the supernatant (in 2% BSA in PBST) had been added to 96-well microtiter plates coated with Wuhan-1 RBD, BA.1 or BA.4/5 RBD (0.1–0.3 μg per properly) for 1 hour at 37 °C. After washing 4 occasions with PBST, goat anti-mouse IgG-HRP (Sigma-Aldrich, A5278) was diluted in 2% BSA in PBST earlier than including to the wells and incubating for 1 hour at 37 °C. Plates had been washed 4 occasions with PBST earlier than the addition of TMB substrate. Reactions had been stopped by the addition of TMB cease resolution. The OD measurements had been taken at 450 nm, and space underneath the curve (AUC) evaluation was carried out utilizing GraphPad Prism 9.4.1.

Mouse experiments

K18hACE2 transgenic mice

Seven-week-old feminine K18-hACE2 mice had been immunized 3 weeks aside with 0.25 µg of mRNA vaccines (management or mRNA-1273) in 50 µl of PBS through intramuscular injection within the hind leg. Animals had been bled 31 weeks after the second vaccine dose for immunogenicity evaluation after which boosted with PBS (no vaccine) or 0.25 µg of mRNA-1273, mRNA-1273.214 or mRNA-1273.222 vaccines. 4 weeks later, K18-hACE2 mice had been challenged with 104 FFU of BA.5 by the intranasal route. Animals had been euthanized at 4 dpi, and tissues had been harvested for virological analyses.

BALB/c mice

Six-to-eight-week-old feminine BALB/c mice had been immunized 3 weeks aside with 1 µg of mRNA vaccines (mRNA-1273, mRNA-1273.529, mRNA-1273.045, mRNA-1273.214 or mRNA-1273.222) or PBS (in 50 µl) through intramuscular injection within the quadriceps muscle of the hind leg underneath isoflurane anesthesia. Blood was sampled 3 weeks after the primary immunization and a pair of weeks after the second immunization, and anti-spike and neutralizing antibody ranges had been measured by ELISA and VSV-based or lentivirus-based pseudovirus neutralization assays.

Measurement of viral burden

Tissues had been weighed and homogenized with zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1 ml of DMEM medium supplemented with 2% heat-inactivated FBS. Tissue homogenates had been clarified by centrifugation at 10,000g for five minutes and saved at −80 °C.

Viral RNA measurement

RNA was extracted utilizing the MagMAX mirVana Whole RNA Isolation Package (Thermo Fisher Scientific) on the KingFisher Flex robotic (Thermo Fisher Scientific). RNA was reverse transcribed and amplified utilizing the TaqMan RNA-to-CT 1-Step Package (Thermo Fisher Scientific). Reverse transcription was carried out at 48 °C for quarter-hour, adopted by 2 minutes at 95 °C. Amplification was completed over 50 cycles as follows: 95 °C for 15 seconds and 60 °C for 1 minute. Copies of SARS-CoV-2 N gene RNA in samples had been decided utilizing a broadcast assay55. The next primers and probe sequence had been used: SARS-CoV-2 N Ahead: 5′-ATGCTGCAATCGTGCTACAA-3′; SARS-CoV-2 N Reverse: 5′-GACTGCCGCCTCTGCTC-3′; and SARS-CoV-2 N Probe: 5′-/56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/−3′.

Viral plaque assay

Vero-TMPRSS2-hACE2 cells had been seeded at a density of 1 × 105 cells per properly in 24-well tissue tradition plates. The subsequent day, medium was eliminated and changed with 200 μl of clarified lung homogenate that was diluted serially in DMEM supplemented with 2% FBS. One hour later, 1 ml of methylcellulose overlay was added. Plates had been incubated for 96 hours after which fastened with 4% PFA (remaining focus) in PBS for 20 minutes. Plates had been stained with 0.05% (w/v) crystal violet in 20% methanol and washed twice with distilled, deionized water.

Cytokine and chemokine protein measurements

Lung homogenates had been incubated with Triton X-100 (1% remaining focus) for 1 hour at room temperature to inactivate SARS-CoV-2. Homogenates had been analyzed for cytokines and chemokines by Eve Applied sciences Company utilizing their Mouse Cytokine Array/Chemokine Array 31-Plex (MD31) platform.

Lung histology

Lungs of euthanized mice had been inflated with ∼2 ml of 10% impartial buffered formalin utilizing a 3-ml syringe and catheter inserted into the trachea and saved in fixative for 7 days. Tissues had been embedded in paraffin, and sections had been stained with hematoxylin and eosin. Photographs had been captured utilizing the NanoZoomer (Hamamatsu) on the Alafi Neuroimaging Core at Washington College.

Supplies availability

All requests for assets and reagents needs to be directed to the corresponding authors. This contains viruses, vaccines and primer-probe units. All reagents might be made obtainable upon cheap request after completion of a supplies switch settlement (MTA). All mRNA vaccines will be obtained underneath an MTA with Moderna (contact: Darin Edwards, [email protected]).

Statistical evaluation

No statistical strategies had been used to predetermine pattern sizes, however our pattern sizes are just like these reported in earlier publications12,20. Significance was assigned when P values had been lower than 0.05 utilizing GraphPad Prism model 9.3. Checks, variety of animals, imply or median values and statistical comparability teams are indicated within the determine legends. Modifications in infectious virus titer, viral RNA ranges or serum antibody responses had been in comparison with mRNA-control immunized and/or PBS-boosted animals and had been analyzed by one-way Kruskal–Wallis or one-way ANOVAwith a number of comparisons assessments or two-tailed Wilcoxon signed-rank check relying on the kind of outcomes, variety of comparisons and distribution of the info. No animals or information factors had been excluded from the analyses.

Reporting abstract

Additional data on analysis design is obtainable within the Nature Portfolio Reporting Abstract linked to this text.