Journal: Journal of Medical Virology

Article Title: Structural Adaptability of IgA and IgM Supports Broad SARS‐CoV‐2 Variant Neutralization

doi: 10.1002/jmv.70790

Figure Lengend Snippet: Comparative Enzyme‐linked immunosorbent assay (ELISA)‐based binding analysis of IgG, IgA, and IgM to SARS‐CoV‐2 spike antigens. The binding of mAbs sharing the same Fab domain was measured by ELISA under the following coating conditions: (a) ELISA plates coated with SARS‐CoV‐2 Wuhan strain S1 subunit. (b) ELISA plates coated with SARS‐CoV‐2 Wuhan strain receptor‐binding domain (RBD). Antibody binding was detected using HRP‐conjugated anti‐human IgG, IgA, or IgM secondary antibodies. Symbols: IgG (●), IgA (▲), IgM (■). Data are presented as mean ± SEM from three independent experiments. Statistical significance was assessed using one‐way ANOVA followed by Tukey's post hoc test. Reported p‐values are indicated in each graph.

Article Snippet: Additionally, SARS‐CoV‐2 (2019‐nCoV) Spike RBD Antibody Titer Assay Kit (KIT 002) and SARS‐CoV‐2 (2019‐nCoV) Spike S1 Antibody Titer Assay Kit (KIT 003) were obtained from Sino Biological through Osaka Yaken Co. Ltd, Japan.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Journal of Medical Virology

Article Title: Structural Adaptability of IgA and IgM Supports Broad SARS‐CoV‐2 Variant Neutralization

doi: 10.1002/jmv.70790

Figure Lengend Snippet: AlphaFold2 3D modeling of the SARS‐CoV‐2 Wuhan spike trimer. (a) Top view and (b) side view. Each monomer is represented in purple, orange, and green, while the SASFS peptide is highlighted in red.

Article Snippet: Additionally, SARS‐CoV‐2 (2019‐nCoV) Spike RBD Antibody Titer Assay Kit (KIT 002) and SARS‐CoV‐2 (2019‐nCoV) Spike S1 Antibody Titer Assay Kit (KIT 003) were obtained from Sino Biological through Osaka Yaken Co. Ltd, Japan.

Techniques:

Journal: Journal of Medical Virology

Article Title: Structural Adaptability of IgA and IgM Supports Broad SARS‐CoV‐2 Variant Neutralization

doi: 10.1002/jmv.70790

Figure Lengend Snippet: AlphaFold2 3D modeling of the RBD monomer structures from SARS‐CoV‐2 Wuhan, Omicron BA.1, and Omicron BA.5 variants. The SASFS peptide (residues 371‐375) is highlighted in red in all models. (a) RBD of Wuhan strain (orange), showing antibody binding regions. (b) RBD of Omicron BA.1 strain (Turquoise), showing antibody binding regions. (c) RBD of Omicron BA.5 strain (magenta), showing antibody binding regions. (d) Structural alignment of the Wuhan RBD (orange) with BA.1 RBD (Turquoise). (e) Structural alignment of the Wuhan RBD (orange) with BA.5 RBD (magenta).

Article Snippet: Additionally, SARS‐CoV‐2 (2019‐nCoV) Spike RBD Antibody Titer Assay Kit (KIT 002) and SARS‐CoV‐2 (2019‐nCoV) Spike S1 Antibody Titer Assay Kit (KIT 003) were obtained from Sino Biological through Osaka Yaken Co. Ltd, Japan.

Techniques: Binding Assay

Journal: Journal of Medical Virology

Article Title: Structural Adaptability of IgA and IgM Supports Broad SARS‐CoV‐2 Variant Neutralization

doi: 10.1002/jmv.70790

Figure Lengend Snippet: NS‐EM images of SARS‐CoV‐2 virions. Panels (a–i) show representative virions with spike proteins on the virion distributed over the particle surface. SARS‐CoV‐2 Wuhan strain was fixed with 2.5% glutaraldehyde in cacodylate buffer for 15 min at room temperature, following established protocols for viral inactivation and electron microscopy specimen preparation recommended by WHO BSL‐3 guidelines, CDC TEM preparation manuals, and standard electron microscopy references (Bozzola & Russell). To concentrate viruses, we employed ultracentrifugation . A 5 µL aliquot of each sample was placed on carbon‐covered grids for 1 min, followed by removal of excess liquid via capillary action. Immediately, 10 µL of 2% phosphotungstic acid (PTA) was added for washing, and excess solution was removed via capillary action. Grids were subsequently stained with 10 µL of 2% PTA for 1 min, and excess stain was removed. Finally, air‐dried grids were imaged at 120 keV using a JEOL 1200EX or FEI CM120 transmission electron microscope at 30 K or 50 K magnification. Scale bars: The black bar in panel d represents 20 nm, while scale bars in all other panels correspond to 50 nm.

Article Snippet: Additionally, SARS‐CoV‐2 (2019‐nCoV) Spike RBD Antibody Titer Assay Kit (KIT 002) and SARS‐CoV‐2 (2019‐nCoV) Spike S1 Antibody Titer Assay Kit (KIT 003) were obtained from Sino Biological through Osaka Yaken Co. Ltd, Japan.

Techniques: Electron Microscopy, Staining, Transmission Assay, Microscopy

Journal: Journal of Medical Virology

Article Title: Structural Adaptability of IgA and IgM Supports Broad SARS‐CoV‐2 Variant Neutralization

doi: 10.1002/jmv.70790

Figure Lengend Snippet: Each Ab bound to spike trimer protein at different concentrations coating ELISA. Each mAb s having the same Fab domain was analyzed by anti‐SARS‐CoV‐2 full spike ELISA assay. Plates were developed with HRP conjugated anti‐human IgG/IgA and IgM antibodies. Data represent mean ± SD of triplicate wells. Full Spike Protein Coating Concentrations: (a) 1000 ng/mL, (b) 500 ng/mL, (c) 250 ng/mL, (d) 125 ng/mL, (e) 62,25 ng/mL, (f) 31,125 ng/mL, (g) 15,625 ng/mL, (h) 7,8 ng/mL.

Article Snippet: Additionally, SARS‐CoV‐2 (2019‐nCoV) Spike RBD Antibody Titer Assay Kit (KIT 002) and SARS‐CoV‐2 (2019‐nCoV) Spike S1 Antibody Titer Assay Kit (KIT 003) were obtained from Sino Biological through Osaka Yaken Co. Ltd, Japan.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: a Design and structure of the mRNA constructs encoding the SARS-CoV-2 RBD. The RBD-specific sequence (amino acids 327–528 of the wild-type SARS-CoV-2 S glycoprotein sequence) was fused to the sequence encoding the trimerization domain of the T4 fibritin (foldon). In BNT162b3, the N-terminal sequence (which includes the S SP) was prolonged and the CT was devoid of an ER retention motif the C-terminal 19 amino acids (CTΔ19). b Detection of total (intracellular and surface) and cell surface expression of the encoded RBDs in HEK293T cells by SARS-CoV-2 S1-specific antibody staining and flow cytometry. HEK293T cells were transfected with mRNA formulated with a transfection reagent (hatched bars) or with LNPs (full bars). Bars indicate arithmetic group means + SD. c Representative confocal images of the cellular localization of the RBD in HEK293T cells transfected with BNT162b1 or BNT162b3 mRNA formulated with a transfection reagent. Non-transfected cells were used as a control. aa amino acids, CT cytoplasmic tail, ER endoplasmic reticulum, LNP lipid nanoparticle, RBD receptor-binding domain, S spike; SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, SD standard deviation, SP signal peptide, TM transmembrane domain, UTR untranslated region.

Article Snippet: Polyclonal mouse IgG (Southern Biotech, cat# 0107-01), rabbit monoclonal SARS-CoV-2 S1 IgG (SinoBiological, cat# 40150-R007), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma-Aldrich, cat# A0545, 1:5,000 dilution), HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research, cat# 115-035-071, 1:7,500 dilution), goat anti-rabbit IgG (Jackson ImmunoResearch, cat# 1111-545-003), anti-vesicular stomatitis virus glycoprotein (VSV G) IgG2aΚ (clone 8G5F11, Kerafast, cat# EB0010).

Techniques: Construct, Sequencing, Expressing, Staining, Flow Cytometry, Transfection, Control, Binding Assay, Standard Deviation

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: Female BALB/c mice ( n = 5–8) received a single i.m. dose of 1 µg BNT162b1 or 0.2 µg or 1 µg BNT162b3 or buffer control. Serum samples obtained after vaccination for a follow-up period of 28 days were analyzed for a concentration of RBD-binding IgG, and for b pVNT 50 (log 10 ) against the wild-type SARS-CoV-2 lineage. Horizontal gray line marks the LLOD. c IFN-γ secretion by splenocytes from Day 28 pulsed with RBD overlapping peptide pool, by ELISpot. d Secretion of cytokines by splenocytes from Day 28 pulsed with S1 overlapping peptide pool, determined on culture supernatants by bead-based multiplex analysis. P values, α = 0.05, from two- ( a , b ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( c [BNT162b3], d ), or unpaired t -test ( c , BNT162b1). Symbols represent individual animals; bar heights indicate group arithmetic means ( c , d ) or geometric means ( a , b written above bars). ELISpot enzyme-linked immunosorbent spot, GM-CSF granulocyte-macrophage colony-stimulating factor, IFN-γ interferon gamma, IgG immunoglobulin G, IL interleukin, i.m. intramuscular, pVNT 50 pseudovirus-based VSV-SARS-CoV-2 50% neutralization titers, RBD receptor-binding domain, S spike, S1 N-terminal furin cleavage fragment of S, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, T H T helper, TNF tumor necrosis factor, VSV vesicular stomatitis virus.

Article Snippet: Polyclonal mouse IgG (Southern Biotech, cat# 0107-01), rabbit monoclonal SARS-CoV-2 S1 IgG (SinoBiological, cat# 40150-R007), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma-Aldrich, cat# A0545, 1:5,000 dilution), HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research, cat# 115-035-071, 1:7,500 dilution), goat anti-rabbit IgG (Jackson ImmunoResearch, cat# 1111-545-003), anti-vesicular stomatitis virus glycoprotein (VSV G) IgG2aΚ (clone 8G5F11, Kerafast, cat# EB0010).

Techniques: Control, Concentration Assay, Binding Assay, Enzyme-linked Immunospot, Multiplex Assay, ELISpot Assay, Neutralization, Virus

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: Female BALB/c mice ( n = 12) received two i.m. doses (Days 0 and 92) of 0.2 µg or 5 µg of BNT162b3 or 5 µg control RNA-LNP (C). Serum samples obtained for a follow-up period of 127 days were analyzed for a serum concentration of RBD-binding IgG, and for b pVNT 50 (log 10 ) against the wild-type SARS-CoV-2 lineage. Horizontal gray lines mark the LLODs. c IFN-γ secretion by splenocytes from Day 127 (Day 35 PD2) pulsed with RBD overlapping peptide pool, by ELISpot. P values, α = 0.05, from one-way ANOVA followed by Tukey’s multiple comparisons test ( c ). Symbols represent individual animals; bar heights indicate geometric ( a , b written above bars) or arithmetic means ( c ). ELISpot enzyme-linked immunosorbent spot, IFN-γ interferon gamma, i.m. intramuscular, LLOD lower limit of detection, RNA-LNP ribonucleic acid lipid nanoparticle, PD post dose, pVNT 50 pseudovirus-based VSV-SARS-CoV-2 50% neutralization titers, RBD receptor-binding domain, S spike, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, VSV vesicular stomatitis virus.

Article Snippet: Polyclonal mouse IgG (Southern Biotech, cat# 0107-01), rabbit monoclonal SARS-CoV-2 S1 IgG (SinoBiological, cat# 40150-R007), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma-Aldrich, cat# A0545, 1:5,000 dilution), HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research, cat# 115-035-071, 1:7,500 dilution), goat anti-rabbit IgG (Jackson ImmunoResearch, cat# 1111-545-003), anti-vesicular stomatitis virus glycoprotein (VSV G) IgG2aΚ (clone 8G5F11, Kerafast, cat# EB0010).

Techniques: Control, Concentration Assay, Binding Assay, Enzyme-linked Immunospot, ELISpot Assay, Neutralization, Virus

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: Male macaques (2–4 years old, n = 6) received two i.m. doses of 30 μg of BNT162b1 or BNT162b3 (Day 0 and 21; arrowhead below the x -axes). a RBD-binding IgG concentration (LLOD = 1.1505 U mL −1 ). b VNT 50 (LLOD = 20) against the wild-type SARS-CoV-2 lineage. The blue triangles in a and b mark the mean values for previously published BNT162b1 data . Horizontal gray lines mark the LLOD. Values below the LLOD were set to 1/2 the LLOD. c–f PBMCs collected on Days 0, 14, and 28 or 42 after Dose 1 were pulsed ex vivo with a full-length S overlapping peptide pool. c , IFN-γ and IL-4 ELISpot. d Frequency of S-specific CD4 + T cells expressing IFN-γ, IL-4, IL-21, any T H 1 cytokine (IFN-γ, IL-2 or TNF-α) and all three T H 1 cytokines by flow cytometry. e Frequency of S-specific CD4 + T cells and cTfh cells expressing IL-21, and any T H 1 cytokine (IFN-γ, IL-2 or TNF-α) by flow cytometry. f Frequency of S-specific CD8 + T cells expressing IFN-γ by flow cytometry. Each symbol represents one macaque. Heights of bars indicate the geometric ( a , b ) or arithmetic ( c – f ) means for each group, and values are written above the bars. cTfh circulating T follicular helper, IFN-γ interferon gamma, IL interleukin, i.m. intramuscular, LLOD lower limit of detection, NT not tested, NA not available, PBMC peripheral blood mononuclear cell, RBD receptor-binding domain, RNA-LNP ribonucleic acid lipid nanoparticle, S spike, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, T H T helper, TNF-α tumor necrosis factor alpha, VNT 50 SARS-CoV-2 virus 50% neutralization titers.

Article Snippet: Polyclonal mouse IgG (Southern Biotech, cat# 0107-01), rabbit monoclonal SARS-CoV-2 S1 IgG (SinoBiological, cat# 40150-R007), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma-Aldrich, cat# A0545, 1:5,000 dilution), HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research, cat# 115-035-071, 1:7,500 dilution), goat anti-rabbit IgG (Jackson ImmunoResearch, cat# 1111-545-003), anti-vesicular stomatitis virus glycoprotein (VSV G) IgG2aΚ (clone 8G5F11, Kerafast, cat# EB0010).

Techniques: Binding Assay, Concentration Assay, Ex Vivo, Enzyme-linked Immunospot, Expressing, Flow Cytometry, Virus, Neutralization

Journal: NPJ Vaccines

Article Title: Strong and early immune responses against SARS-CoV-2 in mice and rhesus macaques after BNT162b3 vaccination

doi: 10.1038/s41541-025-01365-w

Figure Lengend Snippet: a Female BALB/c mice ( n = 12) received two i.m. doses (on Days 0 and 92) of 0.2 µg BNT162b3. Sera obtained on Day 91 PD1 and Days 7 and 35 PD2 were analyzed for pVNT 50 against the wild-type lineage and variants. b Male rhesus macaques (2–4 years old, n = 6) received two i.m. doses (on Days 0 and 21) of 30 µg BNT162b3 RNA-LNP, and serum was collected on Day 7 PD2. Graphs show log 10 of pVNT 50 against the wild-type SARS-CoV-2 lineage and variants. Symbols represent individual values; black lines indicate geometric means. Horizontal gray lines mark the LLODs. P values, α = 0.05, from two- ( a ) or one-way ( b ) ANOVA followed by Dunnett’s multiple comparisons test. i.m. intramuscular, LLOD lower limit of detection, PD post dose, pVNT 50 pseudovirus-based VSV-SARS-CoV-2 50% neutralization titers, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, VSV vesicular stomatitis virus.

Article Snippet: Polyclonal mouse IgG (Southern Biotech, cat# 0107-01), rabbit monoclonal SARS-CoV-2 S1 IgG (SinoBiological, cat# 40150-R007), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma-Aldrich, cat# A0545, 1:5,000 dilution), HRP-conjugated goat anti-mouse IgG (Jackson Immuno Research, cat# 115-035-071, 1:7,500 dilution), goat anti-rabbit IgG (Jackson ImmunoResearch, cat# 1111-545-003), anti-vesicular stomatitis virus glycoprotein (VSV G) IgG2aΚ (clone 8G5F11, Kerafast, cat# EB0010).

Techniques: Neutralization, Virus