mRNA booster vaccination protects aged mice against the SARS-CoV-2 Omicron variant

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Animals

Feminine, 3-month-old BALB/c mice have been bought from the Jackson Laboratory. Feminine, 11-month-old BALB/c mice have been bought from Taconic Biosciences and used for aged mice experiments. Mice have been housed beneath particular pathogen-free circumstances at Boston Youngsters’s Hospital. All of the procedures have been authorised beneath the Institutional Animal Care and Use Committee (IACUC) and operated beneath the supervision of the Division of Animal Assets at Youngsters’s Hospital (Protocol quantity 19-02-3897 R). On the College of Maryland College of Drugs, mice have been housed in a biosafety degree 3 facility for SARS-CoV-2 infections with all of the procedures authorised beneath the IACUC (Protocol #1120004).

Immunization

Mice have been injected with BNT162b2 SARS-CoV-2 spike mRNA vaccine (Pfizer-BioNTech). BNT162b2 suspension (100 µg of mRNA/mL) was diluted 1:5 in phosphate-buffered saline (PBS) and 50 µL (1 µg) was injected intramuscularly within the caudal thigh. BNT162b2 was obtained as residual overfill volumes in used vials from the Boston Youngsters’s Hospital vaccine clinic, utilizing solely materials that may in any other case be discarded, and was used inside 6 h from the time of reconstitution. Mock therapy mice acquired PBS alone.

SARS-CoV-2 wildtype spike and RBD expression and purification

Full size SARS-CoV-2 Wuhan-Hu-1 spike glycoprotein (M1-Q1208, GenBank MN90894) and RBD constructs (amino acid residues R319-K529, GenBank MN975262.1), each with an HRV3C protease cleavage web site, a TwinStrepTag and an 8XHisTag at C-terminus have been obtained from Barney S. Graham (NIH Vaccine Analysis Middle) and Aaron G. Schmidt (Ragon Institute), respectively. These mammalian expression vectors have been used to transfect Expi293F suspension cells (Thermo Fisher) utilizing polyethylenimine (Polysciences). Cells have been allowed to develop in 37 °C, 8% CO2 for a further 5 days earlier than harvesting for purification. Protein was purified in a PBS buffer (pH 7.4) from filtered supernatants by utilizing both StrepTactin resin (IBA) or Cobalt-TALON resin (Takara). Affinity tags have been cleaved off from eluted protein samples by HRV 3C protease, and tag eliminated proteins have been additional purified by size-exclusion chromatography utilizing a Superose 6 10/300 column (Cytiva) for full size Spike and a Superdex 75 10/300 Improve column (Cytiva) for RBD area in a PBS buffer (pH 7.4).

ELISA

Spike protein-specific antibody concentrations have been quantified in serum samples by ELISA by modification of a beforehand described protocol28. Briefly, high-binding flat-bottom 96-well plates (Corning) have been coated with spike protein (25 ng per nicely) and incubated in a single day at 4 °C. Plates have been washed with 0.05% Tween 20/PBS and blocked with 1% bovine serum albumin (BSA)/PBS for 1 h at room temperature. Serum samples have been serially diluted fourfold from 1:100 as much as 1:1.05 × 108 after which incubated for two h at room temperature. Plates have been washed 3 times and incubated for 1 h at room temperature with horseradish peroxidase (HRP)–conjugated anti-mouse IgG, IgG1, and IgG2a (SouthernBiotech). Plates have been washed 5 instances and developed with tetramethylbenzidine (BD OptEIA Substrate Answer, BD Biosciences) for five min after which stopped with 2 N H2SO4. Optical densities (ODs) have been learn at 450 nm with a SpectraMax iD3 microplate reader (Molecular Units). Endpoint titers have been calculated because the dilution that emitted an OD exceeding a 3x background. An arbitrary worth of fifty was assigned to the samples with OD values beneath the restrict of detection for which it was not doable to interpolate the titer.

hACE2-RBD inhibition assay

The hACE2-RBD inhibition assay used a modification of a beforehand printed protocol29. Briefly, high-binding flat-bottom 96- nicely plates (Corning) have been coated with recombinant hACE2 (100 ng per nicely) (Sigma-Aldrich) in PBS, incubated in a single day at 4 °C, washed 3 times with 0.05% Tween 20 PBS, and blocked with 1% BSA PBS for 1 h at RT. Serum samples have been diluted 1:160, preincubated with 3 ng of wildtype RBD-Fc in 1% BSA PBS for 1 h at RT, after which transferred to the hACE2-coated plate. RBD-Fc with out preincubation with serum samples was added as a optimistic management, and 1% BSA PBS with out serum preincubation was added as a detrimental management. Plates have been then washed 3 times and incubated with HRP-conjugated anti-human IgG Fc (SouthernBiotech) for 1 h at RT. Plates have been washed 5 instances and developed with tetramethylbenzidine (BD OptEIA Substrate Answer, BD Biosciences) for five min after which stopped with 2 N H2SO4. The OD was learn at 450 nm with a SpectraMax iD3 microplate reader (Molecular Units). Proportion inhibition of RBD binding to hACE2 was calculated as: Inhibition (%) = [1 − (Sample OD value − Negative Control OD value)/(Positive Control OD value − Negative Control OD value)] × 100.

Pseudovirus neutralization assay

The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene have been generated in an method much like as described beforehand30,31. Briefly, the packaging plasmid psPAX2 (AIDS Useful resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT of variants have been co-transfected into HEK293T cells by lipofectamine 2000 (ThermoFisher). Pseudoviruses of SARS-CoV-2 variants have been generated by utilizing WA1/2020 pressure (Wuhan/WIV04/2019, GISAID accession ID: EPI_ISL_402124), B.1.617.2 (Delta, GISAID accession ID: EPI_ISL_2020950), or B.1.1.529 (Omicron, GISAID ID: EPI_ISL_7358094.2). The supernatants containing the pseudotype viruses have been collected 48 h post-transfection, which have been purified by centrifugation and filtration with 0.45 µm filter. To find out the neutralization exercise of the serum samples, HEK293T-hACE2 cells have been seeded in 96-well tissue tradition plates at a density of two × 104 cells/nicely in a single day. Three-fold serial dilutions of warmth inactivated serum samples have been ready and combined with 50 µl of pseudovirus. The combination was incubated at 37 °C for 1 h earlier than addition to HEK293T-hACE2 cells. After 48 h, cells have been lysed in Regular-Glo Luciferase Assay (Promega) in line with the producer’s directions. SARS-CoV-2 neutralization titers have been outlined because the pattern dilution at which a 50% discount in relative mild unit (RLU) was noticed relative to the common of the virus management wells.

Splenocyte restimulation, intracellular cytokine staining and move cytometry

Mouse spleens have been mechanically dissociated and filtered by a 70 µm cell strainer. After centrifugation, cells have been handled with 1 mL ammonium-chloride-potassium lysis buffer for two min at RT. Cells have been washed and plated in a 96-well U-bottom plate (2 × 106/nicely) and incubated in a single day in RPMI 1640 supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), streptomycin (100 mg/ml), 2-mercaptoethanol (55 mM), non-essential amino acids (60 mM), HEPES (11 mM), and L-Glutamine (800 mM) (all Gibco). Subsequent day, SARS-CoV-2 wildtype (PM-WCPV-S-1) or Omicron (PM-SARS2-SMUT08-1) spike peptide swimming pools (JPT) have been added at 0.6 nmol/ml within the presence of anti-mouse CD28/49d (1 μg/mL, BD) and brefeldin A (5 μg/ml, BioLegend). After 6 h stimulation, cells have been washed twice and have been handled with Mouse Fc Block (BD) in line with the producer’s directions. Cells have been washed and stained with Aqua Dwell/Useless stain (Life Applied sciences, 1:500) for 15 min at RT. Following two further washes, cells have been incubated with the next Abs for 30 min at 4 °C: anti-mouse CD44 [IM7, PerCP-Cy5.5, BioLegend #103032, 1:160], anti-mouse CD3 [17A2, Brilliant Violet 785, BioLegend #100232, 1:40], anti-mouse CD4 [RM4-5, APC/Fire 750, BioLegend 100568, 1:160] and anti-mouse CD8 [53-6.7, Brilliant UltraViolet 395, BD #563786, 1:80]. Cells have been then mounted and permeabilized by utilizing the BD Cytofix/Cytoperm equipment in line with the producer’s directions, and have been subjected to intracellular staining (30 min at 4 °C) utilizing the next Abs: anti-mouse IFNγ [XMG1.2, Alexa Fluor 488, BioLegend #505813, 1:160], anti-mouse TNF [MP6-XT22, PE Cy7, BioLegend # 506324, 1:160], anti-mouse IL-2 [JES6-5H4, PE, BioLegend # 503808, 1:40]. Lastly, cells have been mounted in 1% paraformaldehyde (Electron Microscopy Sciences) for 20 min at 4 °C and saved in PBS at 4 °C till acquisition. Samples have been analyzed on an LSR II (BD) move cytometer and FlowJo v10.8.1 (FlowJo LLC).

SARS-CoV-2 Omicron problem research

Mice have been anesthetized interperitoneally with 50 μL ketamine (1.3 mg/mouse) and xylazine (0.38 mg/mouse) diluted in PBS. Mice have been then intranasally inoculated with 1 ×105 PFU of SARS-CoV-2 Omicron (BA.1), courtesy of Dr. Mehul Suthar, in 50 μL of PBS divided between nares. Challenged mice have been weighed each day. On day 2 post-infection, the mice have been euthanized, and the lungs have been harvested for histology, evaluation of host responses by qPCR, and dedication of viral titer by plaque assay.

SARS-CoV-2 plaque assay

The day previous to an infection, 2.5e5 VeroTMPRSS2 cells have been seeded per nicely in a 12-well plate in 1 mL of VeroTMPRSS2 media [DMEM (Quality Biological), 10% FBS (Gibco), 1% Penicillin-Streptomycin (Gemini Bio Products), and 1% L-Glutamine (Gibco)]. Tissue samples have been thawed and homogenized with 1 mm beads in a Bead ruptor (Omni Worldwide Inc.) after which spun down at 21,000 g for two min. A 6-point dilution curve was ready by serial diluting 25 μL of pattern 1:10 in 225 μL DMEM. 200 μL of every dilution was then added to the cells and the plates have been rocked each 15 min for 1 h at 37 °C. After 1 h, 2 mL of a semi-solid agarose overlay was added to every nicely [DMEM, 4% FBS, and 0.06% UltraPure agarose (Invitrogen)]. After 72 h at 37 °C and 5% CO2, plates have been mounted in 2% PFA for 20 min, stained with 0.5 mL of 0.05% Crystal Violet and 20% EtOH, and washed twice with H2O previous to counting of plaques. The titer was then calculated. For tissue homogenates, this titer was multiplied by 40 based mostly on the common tissue pattern weight being 25 mg.

Gene expression evaluation by qPCR

RNA was remoted from TRI Reagent samples utilizing phenol-chloroform extraction or column-based extraction techniques (Direct-zol RNA Miniprep, Zymo Analysis) in line with the producer’s protocol. RNA focus and purity (260/280 and 260/230 ratios) have been measured by NanoDrop (Thermo Fisher Scientific). Samples with an A260/A280 ratio of <1.7 have been excluded for additional evaluation. cDNA was ready from purified RNA with RT2 First Strand Equipment, per the producer’s directions (Qiagen). cDNA was quantified by qPCR on a 7300 real-time PCR system (Utilized Biosystems) utilizing pre-designed SYBR Inexperienced Primers (QIAGEN) particular for Ifit2 (PPM05993A), Rsad2 (PPM26539A), and Rpl13a (PPM03694A).

Statistics and reproducibility

Mouse experiments aimed to incorporate in complete 20 mice per group and have been from single experiments. Pattern measurement and age standards have been chosen empirically based mostly on the outcomes of earlier research. Mice have been randomly assigned to totally different therapy teams. No information outliers have been excluded. Statistical analyses have been carried out utilizing Prism v9.0.2 (GraphPad Software program). Two-tailed P values <0.05 have been thought of vital. Usually distributed information have been analyzed by one-way analyses of variance (ANOVAs) corrected for a number of comparisons. Non-normally distributed information have been log-transformed and analyzed by ANOVA or analyzed by Kruskal–Wallis take a look at corrected for a number of comparisons.

Reporting abstract

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

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