Assessing the impact of mRNA vaccination in chronic inflammatory murine model | npj Vaccines – Nature.com

Preparation of mRNA

The antigen was designed using the DNA sequence encoding the spike protein of the SARS-CoV-2 Omicron variant. The mRNA vaccine plasmid was produced by inserting antigen DNA into multiple cloning sites on the mRNA platform using restriction enzymes (Pac1 and Cla1), as previously described46. After the mRNA vaccine template was linearized using Not1, mRNA was produced using the EZ T7 High Yield In vitro Transcription Kit (Enzynomics, Daejeon, Korea) according to the manufacturers protocol. Capping was performed using SC101 (STPharm, Siheung, Korea), and UTP was replaced with N1-methyl-pseudouridine (Trilink, San Diego, CA, USA). Total mRNA was precipitated using lithium chloride and purified using cellulose, as described previously47.

Lipid nanoparticles (LNPs) were prepared according to a reported protocol48. Briefly, all lipid components were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 (SM-102; distearoylphosphatidylcholine cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), and mRNAs were dissolved in sodium citrate buffer (50mM; pH 4) solution at a charge ratio of N/P=6. LNPs were formulated using NanoAssemblr IgniteTM (Precision Nanosystems, Vancouver, Canada) by mixing aqueous and organic solutions at a ratio of 3:1 and a total flow rate of 10mL/min. The LNP solution was concentrated by ultrafiltration using an Amicon Ultracentrifugal filter (UFC9030, Merck Millipore, Billerica, MA, USA), following the manufacturers instructions.

The size and Zeta potential of the mRNA vaccine were measured using a zetasizer with diluted mRNA vaccine in water (Zetasizer Nano Zs, Malvern Panalytical, Malvern, Worcestershire, UK). The encapsulation efficiency of LNP is determined by Ribogreen assay. Both Triton X-100 treated or untreated mRNA vaccines were incubated with Ribogreen reagent of Quant-it RiboGreen RNA Assay Kit (Thermofisher, Waltham, MA, USA, Cat. R11490). A microplate reader analyzed the fluorescence for the total and free mRNA amount. The following formula was used to obtain the encapsulation efficiency of the mRNA vaccine:

$${rm{Encapsulation; efficiency}}left( % right)=left[left({rm{Total}},{rm{mRNA}}-{rm{free}},{rm{mRNA}}right)/{rm{total}},{rm{mRNA}}right]times 100 %$$

The endotoxin level is determined to be lower than 0.1 EU/ml using the LAL assay kit (Cat. L00350, Genscript, Piscataway, NJ, USA).

Three microliter of mRNA vaccine solution was loaded onto a holey carbon grid (Quantifoil R1.2/1.3, 200 mesh Cu, Structure Probe Inc., USA) treated with a glow discharger at 15mA for 60s to increase the loading efficiency. The grid was blotted for 4s at 15C with 100% humidity using a Vitrobot Mark IV (Thermofisher, USA, SNU CMCI) and immediately plunge-frozen in liquid nitrogen-cooled liquid ethane. The grids were imaged with TEM (JEM-2100F, JEOL, Japan) while maintaining the temperature at ~180C at an acceleration voltage of 200keV. Images were recorded using an ultrascan 1000 electron detector.

Female Balb/c and C57BL6 mice at the age of 68 weeks were obtained from Dae-Han Biolink Co. (Eumseong, Korea) and housed in a controlled environment (inverted 12-h daylight cycle) with free access to food and water. All procedures were complied with the ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of the Samsung Biomedical Research Institute (#2022032201). The Samsung Biomedical Research Institute is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and abides by the Institute of Laboratory Animal Resources guidelines. Mice were fed a normal-fat diet (containing 5% fat) and simultaneously treated with LPS from Escherichia coli (Sigma, St Louis, MO, USA). The surgical procedure for inserting the osmotic pump followed the manufacturers instructions. Briefly, after the hair was shaved, the back of the mouse was incised, and a space was made in the subcutaneous tissue. The device was inserted into the space, and the incision was closed using wound clips (7mm). The mice were implanted with an osmotic pump (Alzet model 1004; DURECT Corp., Cupertino, CA, USA) filled with either Tween-saline (0.9% NaCl and 0.1% Tween 80 in distilled water, Sigma), normal saline control, or LPS diluted in Tween-saline infused at 300g/kg/day for 4 weeks (LPS group)49. For blood collection, mice were anesthetized with isoflurane (Isotroy 100, Troikaa, Gujarat, India), and blood was collected from the facial vein. At the defined endpoint (2 days after the boost), mice were euthanized by CO2 inhalation, and their spleens, lungs, blood, hearts, and muscles were harvested.

The pump-implanted animals were randomly assigned to four groups for administering the intramuscular SARS-CoV-2 mRNA vaccine or normal saline control (groups: saline pump, saline pump + mRNA vaccine, LPS pump, and LPS pump + mRNA vaccine). The mice were immunized intramuscularly with 10g of the Omicron S mRNA vaccine with the sequence encoding the spike protein from the SARS-CoV-2 Omicron variant. The mRNA expression platform has been previously described22. The immunization schedule comprised two injections: an initial prime injection followed by a booster injection, with a 2-week interval between injections. Once the immunization protocol was completed, the mice were euthanized 2 days after immunization, and whole blood samples and tissues were collected.

Splenocytes (1106) isolated from immunized mice were cultured in 96-well plates. Subsequently, the samples were treated with Brefeldin A (Golgi plug, BD Biosciences, Franklin Lakes, NY, USA) and stimulated with antigen peptides of the spike protein from the SARS-CoV-2 Omicron variant (5g/mL) in RPMI medium for 12h at 37C. Then, the splenocytes were treated with anti-mouse CD16/32 (Invitrogen, Waltham, Massachusetts, USA) for 20min at 4C. To stain surface proteins such as CD8, the splenocytes were stained with anti-mouse CD8 fluorescent antibody for 30min at 4C in the dark. Then, the samples were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences), stained with anti-mouse tumor necrosis factor alpha (TNF-) and granzyme B fluorescent antibody for 30min at 4C in the dark. For detecting the levels of M1 and M2 macrophages in the lungs using flow cytometry, lung tissues excised from immunized mice were digested with Hanks Balanced Salt Solution containing 1.5mg/ml collagenase A, 0.1mg/ml Dnase1 (Sigma Aldrich, Burlington, Massachusetts, United States) for 1h at 37C and stained using anti-mouse CD11b, CD11c, and F4/80 with the surface protein staining method used for splenocytes. The samples were then fixed and permeabilized using a Foxp3 Fixation Kit (Invitrogen) according to the manufacturers instructions. The cells were stained with anti-mouse CD206 CD8 fluorescent antibody in the dark for 30min at 4C. The samples were analyzed using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA), and data were analyzed using CytExpert (Beckman Coulter, Brea, CA, USA).

Enzyme-linked immunosorbent assays (ELISAs) were performed to assess antigen-specific total IgG levels in mouse serum. Briefly, a 96-well plate was coated with S protein from the SARS-CoV-2 Omicron variant at a concentration of 100ng/well and incubated overnight at 4C. The wells were then blocked with 100l blocking buffer (1% BSA in PBS) for 1h at room temperature. The serum samples diluted with blocking buffer (at 1:20) were added to the wells and incubated for 2h at room temperature (2022C). After the incubation period, the wells were washed thrice with 200l PBS-T (PBS containing Tween 20). Horseradish peroxidase-conjugated anti-mouse IgG antibodies (from Bethyl Laboratories, Montgomery, TX, USA) were added to the wells and then the plates were incubated at room temperature for 1h. The antibodies were appropriately diluted in blocking buffer (at 1:5000). Following three washes with PBS-T, tetramethylbenzidine substrate was added to the wells, and the plates were incubated for 15min. The reaction was stopped by adding 2N H2SO4. Finally, the optical density was measured at 450nm using a microplate reader (GloMax Explorer, Promega, Seoul, Republic of Korea).

Splenocytes (5105) isolated from immunized mice were cultured in 96-well MultiScreen-IP Filter Plates (Millipore, Burlington, MA, USA) and stimulated with antigen peptides of spike protein from the Wuhan SARS-CoV-2 strain (5g/ml) in RPMI medium for 24h at 37C. The ELISpot assay for detecting IFN- secreted from splenocytes was performed as per the manufacturers instructions (Mab-tech, Stockholm, Sweden).

The epididymal fat, liver, lung, muscle, and pancreatic tissues of osmotic pump-implanted mice were fixed in 10% neutral formalin. After fixation, the samples were embedded in paraffin and stained with hematoxylin and eosin (H&E). The percentage area of myocardial inflammation was determined using computer-assisted analysis. Two different areas of each heart were quantified using ImageJ software1.45s (NIH, MA, USA), as described previously50. To analyze the damaged muscle area, a Motic EasyScan Digital Slide Scanner (Motic Hong Kong Ltd., Hong Kong, China) was used to randomly capture 510 images of the affected regions. These images were then analyzed for the cross-sectional area (CSA) of the centrally nucleated regenerating myofibers using ImageJ software51. The procedures were performed by investigators who were blinded to the identities of the samples. In the SARS-CoV-2 spike immunohistochemistry procedure, Dako Retrieval Solution (pH 6.0, S2369) was utilized for antigen retrieval through incubation of sections. To mitigate nonspecific binding, a blocking step of 1-hour duration was carried out using Dako Protein Block Serum-Free (X0909). Subsequently, the sections were exposed to anti-SARS-CoV/SARS-CoV-2 (COVID-19) spike antibody (1:200; GTX632604, GeneTex) for 24h at 4C. The visualization of antigens was enabled using the Dako Envision Detection system Peroxidase/DAB+ (K5007). Following this, the slides were dehydrated and mounted after counterstaining with hematoxylin. Images of the SARS-CoV-2 spike protein were captured randomly using a Motic Easyscan Digital Slide Scanner (Motic Hong Kong Limited).

Total RNA was extracted from lung tissue using the TRIzol reagent (Invitrogen, Grand Island, NY, USA), and cDNA was synthesized using a cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturers instructions. Real-time PCR was performed using the SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) to compare the mRNA levels of genes associated with inflammation, myogenesis, muscle types, and mitochondria. The primer sets were synthesized by SFC-probe (Cheongju, Korea) for inflammation-related genes and Bioneer (Daejeon, Korea) for the remaining genes. The primer sequences are listed in Supplementary Table 1.

Plasma was isolated from mouse blood and processed according to the manufacturers protocol for the Creatine Kinase Activity Assay Kit (3050 Spruce Street, St. Louis, MO, USA). The creatine kinase activity was determined by measuring absorbance (340nm) using a VICTOR Nivo TM multimode Microplate Reader (PerkinElmer, Waltham, MA, USA) following the standard procedure for assessment.

Statistical analyses were performed using Prism 8 software (GraphPad, San Diego, CA, USA). Data were presented as meanstandard deviation. Significant differences between means were determined using Students t-test, and P values less than 0.05 were considered statistically significant.

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

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Assessing the impact of mRNA vaccination in chronic inflammatory murine model | npj Vaccines - Nature.com