Journal: NPJ Vaccines

Article Title: Bivalent norovirus mRNA vaccine elicits cellular and humoral responses protecting human enteroids from GII.4 infection

doi: 10.1038/s41541-024-00976-z

Figure Lengend Snippet: Expi293F cells were transfected with mRNA-encoding ( A ) norovirus GI.1 or ( B ) GII.4 VP1 and analyzed for protein expression by western blot as described in Methods. The supernatant was collected, clarified, ultrafiltered, and visualized via NSEM. Representative micrographs are shown from cells transfected with ( C ) GI.1 or ( D ) GII.4, with n = 5 micrograph images per group. E Experimental schematic of the study. Balb/c mice ( n = 10 per group) were immunized on days 0 and 28 with 10 μg of mRNA-LNP vaccine encoding Norwalk1968/GI.1 or CapeTown2012/GII.4 VP1, or empty LNP as control at an equivalent dose. Sera were collected at various time points (4, 8, 14, 22, 26, and 34 weeks post-prime) and screened against ( F ) Norwalk1968/GI.1 and ( G ) CapeTown2012/GII.4 VLPs to assess their ability to block VLP binding to its carbohydrate ligand in a surrogate neutralization assay. The data was analyzed using GraphPad Prism, and the dilution at which 50% of VLP-ligand binding was blocked (ID50) was calculated. Sera that did not block at least 50% of VLP-ligand binding were assigned a titer of 0.5X the lower limit detection (ID50 = 25) and marked below this limit (dashed line). The nAb titer is presented as 50% inhibitory dilution (ID50) values (Mean ± SD), n = 10 per group (5 male and 5 female). The nAb titers were compared with the values from Day 56 for each strain, ∗ p < 0.05 F:(D56 vs D28 p = 0.0135) G:(D56 vs D28 p = <0.0001, D56 vs D150 p = <0.0001, D56 vs D180 p = <0.0001, d56 vs d240 p = <0.0001), one-way ANOVA, Tukey’s post hoc tests. A red square symbol indicates nAb titers in a serum sample collected on Day 14 from a patient infected with the GII.4 norovirus strain. nAb titer values for empty LNP are not shown because they were below the level of detection. Schematic for Fig. 1E created with Biorender.

Article Snippet: Splenocytes were stimulated with 80 μl of a synthetic peptide pool of CapeTown/GII.4 VP1 (150 peptides, each with 15 AA length and 11 AA overlap, a purity level between 80 and 90%, synthesized by GenScript) at a final concentration of 1.5 μg/mL, and 1 μg/mL of CD28 (co-stimulatory signal) for 1 h at 37 °C.

Techniques: Transfection, Expressing, Western Blot, Control, Blocking Assay, Binding Assay, Neutralization, Ligand Binding Assay, Infection

Journal: NPJ Vaccines

Article Title: Bivalent norovirus mRNA vaccine elicits cellular and humoral responses protecting human enteroids from GII.4 infection

doi: 10.1038/s41541-024-00976-z

Figure Lengend Snippet: Balb/c mice ( n = 10 per group) were immunized on days 0 and 28 with 10 μg of mRNA-LNP vaccine encoding Norwalk1968/GI.1 or CapeTown2012/GII.4 VP1, or empty LNP as control at an equivalent dose. A nAb response titers to three genogroup I strains (GI.1, GI.3, and GI.5) and ( B ) against four genogroup II strains (GII.3, GII.4 Sydney 2012, GII.4 New Orleans 2009, and GII.4 Farmington Hills 2002) at day 56 post prime in a surrogate neutralization assay. The nAb titer is presented as 50% inhibitory dilution (ID50) values (Mean ± SD), n = 10 per group (5 male and 5 female). Only the cross-reactivity nAb titer values of GII.4 variants were compared, ∗ p < 0.05, (2012 vs 2009 p = <0.0001, 2012 vs 2002 p = <0.0001) one way-ANOVA, Tukey’s post hoc tests.

Article Snippet: Splenocytes were stimulated with 80 μl of a synthetic peptide pool of CapeTown/GII.4 VP1 (150 peptides, each with 15 AA length and 11 AA overlap, a purity level between 80 and 90%, synthesized by GenScript) at a final concentration of 1.5 μg/mL, and 1 μg/mL of CD28 (co-stimulatory signal) for 1 h at 37 °C.

Techniques: Control, Neutralization

Journal: NPJ Vaccines

Article Title: Bivalent norovirus mRNA vaccine elicits cellular and humoral responses protecting human enteroids from GII.4 infection

doi: 10.1038/s41541-024-00976-z

Figure Lengend Snippet: A Experimental schematic of the study. Balb/c and C57BL/6 mice were i.m. immunized on days 0 and 28 with varying doses (0.25 μg, 0.5 μg, 1 μg, and 3 μg) of each mRNA-LNP vaccine encoding Norwalk1968/GI.1 and CapeTown2012/GII.4 VP1, or empty LNP as control. Sera were collected 4 weeks after the prime and after the boost, and then screened against ( B , D ) Norwalk1968/GI.1 and ( C , E ) Sydney2012/GII.4 VLP to evaluate their capacity to block VLP binding to their carbohydrate ligand in a surrogate neutralization assay. The nAb titer is presented as 50% inhibitory dilution (ID50) values (Mean ± SEM), n = 5. Dashed line = limit of detection. nAb titer values for empty LNP are not shown because they were below the level of detection. Only nAb titer values within Day 56 were compared, ∗ p < 0.05, B:(D56 0.25 μg vs D56 3 μg p = 0.0007), C:(D56 0.25 μg vs D56 3 μg p = 0.0001) ANOVA, Tukey’s post hoc tests. Schematic for Fig. 3A created with Biorender.

Article Snippet: Splenocytes were stimulated with 80 μl of a synthetic peptide pool of CapeTown/GII.4 VP1 (150 peptides, each with 15 AA length and 11 AA overlap, a purity level between 80 and 90%, synthesized by GenScript) at a final concentration of 1.5 μg/mL, and 1 μg/mL of CD28 (co-stimulatory signal) for 1 h at 37 °C.

Techniques: Control, Blocking Assay, Binding Assay, Neutralization

Journal: NPJ Vaccines

Article Title: Bivalent norovirus mRNA vaccine elicits cellular and humoral responses protecting human enteroids from GII.4 infection

doi: 10.1038/s41541-024-00976-z

Figure Lengend Snippet: A Experimental schematic of the study. Human intestinal enteroids were infected for 2 h at 37 °C with GII.4 Sydney virus (stool sample #20942) that was pre-incubated with serial dilution of sera from mice immunized by i.m. with ( B ) 10 μg or ( C ) 0.25 μg of each mRNA-LNP vaccine encoding Norwalk1968/GI.1 and CapeTown2012/GII.4 VP1 for 1 h at room temperature. Following infection, 3D HIE were washed and cultured in BME for 3 days post-infection (see Methods and Materials). Viral titers were measured by RT-qPCR. * p < 0.05 vs control (empty LNP). B , C Each dot in the graph at a given dilution represents an independent infection performed on a separate day, while each set of bars, i.e., dilutions 1:100 to 1:30,000 ( B ), or 1:300 to 1:10,000 ( C ) represents the dilution series from an individual serum sample. Schematic for Fig. 5A created with Biorender.

Article Snippet: Splenocytes were stimulated with 80 μl of a synthetic peptide pool of CapeTown/GII.4 VP1 (150 peptides, each with 15 AA length and 11 AA overlap, a purity level between 80 and 90%, synthesized by GenScript) at a final concentration of 1.5 μg/mL, and 1 μg/mL of CD28 (co-stimulatory signal) for 1 h at 37 °C.

Techniques: Infection, Virus, Incubation, Serial Dilution, Cell Culture, Quantitative RT-PCR, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Krüppel‐Like Factor 15/Interleukin 11 Axis‐Mediated Adventitial Remodeling Depends on Extracellular Signal‐Regulated Kinases 1 and 2 Activation in Angiotensin II–Induced Hypertension

doi: 10.1161/JAHA.120.020554

Figure Lengend Snippet: A, Adventitial fibroblasts (AFs) were incubated with IL‐11 neutralizing antibody 1 hour before Ang II 24‐hour treatment. The protein levels of collagen, type I, α 1 (COL1a1) (1.00‐, 1.19‐, 2.10‐, and 0.95‐fold), α‐smooth muscle actin (ACTA2) (1.00‐, 1.00‐, 1.49‐, and 0.97‐fold), and Krüppel‐like factor 15 (KLF15) (1.00‐, 0.87‐, 0.66‐, and 0.63‐fold) were measured by Western blotting. N=4. The right panel was the quantity of the Western blot. B, AFs were incubated with IL‐11 neutralizing antibody 1 hour before Ang II 24‐hour treatment. The mRNA levels of COL1a1 (1.00‐, 1.09‐, 1.40‐, and 1.52‐fold), ACTA2 (1.00‐, 0.91‐, 1.80‐, and 1.58‐fold), and KLF15 (1.00‐, 0.98‐, 0.37‐, and 0.51‐fold) were measured by quantitative reverse transcription–polymerase chain reaction. N=5. C, AFs were incubated with ribosomal S6 kinase inhibitor (CMK) (10 µmol/L) or ERK1/2 inhibitor (ERKi) (10 µmol/L) 1 hour before recombination mouse IL‐11 ( rmIL‐11) (5 ng/mL) 24‐hour treatment. The protein levels of phosphorylated p90RSK (p‐p90RSK) (1.00‐, 2.10‐, 0.93‐, and 0.74‐fold), p90RSK, phosphorylated ERK1/2 (p‐ERK1/2) (1.00‐, 10.71‐, 10.89‐, and 2.53‐fold), and ERK1/2 were measured by Western blotting. N=4. The right panel was the quantity of the Western blot. D, AFs were incubated with dimethyl sulfoxide, CMK (10 µmol/L), or ERKi (10 µmol/L) 1 hour before PBS or rmIL‐11 (5 ng/mL) 24‐hour treatment. The protein levels of COL1a1 (1.00‐, 1.40‐, 0.68‐, and 0.79‐fold) and ACTA2 (1.00‐, 1.13‐, 0.89‐, and 0.95‐fold) were measured by Western blotting. N=4. The right panel was the quantity of the Western blot. CON indicates control. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Cell extracts were clarified by a 15‐minute centrifugation at 4°C and 13000 g . Equal amounts of samples were separated by SDS/PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore Sigma; IPVH00010), and subjected to immunoblotting analysis of IL‐11 (R&D; MAB218), IL‐11 receptor subunit α (Santa Cruz Biotechnology; sc‐130920), KLF15 (Millipore; ABC471), collagen type 1a1 (Servicebio; GB11022‐1), ACTA2 (Millipore Sigma; A2547), phosphorylated ERK1/2 (Thr202/Tyr204; 4370), ERK1/2 (Cell Signaling Technology; 4695), phosphorylated P90 ribosomal S6 kinase (p90RSK) (Cell Signaling Technology; 11989), and p90RSK (Cell Signaling Technology; 9355) at dilution of 1:1000, and GAPDH (Kangchen, Shanghai, China; KC‐5G5) at dilution of 1:4000.

Techniques: Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction