Journal: bioRxiv

Article Title: Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad and durable systemic and mucosal immune responses

doi: 10.1101/2023.02.21.529344

Figure Lengend Snippet: 5-6 weeks old Balb/c mice (n=5 per group) were sc. immunized once with 2 µg of Clec9A-RBD adjuvanted with 50 µg Poly I:C. (A) Percentage of AIM + TFH cells (CD3 + CD4 + CXCR5 + PD-1 + ) in response to re-stimulation with ancestral (left panel) and variant (right panel) SARS-CoV-2 RBD peptides in spleens harvested at 7 days, 2 weeks, and 6-12 months (n=2 at 6 and 9 months old each, and n=1 at 12 months old) post-immunization. (B) Immune detection of GC B cells in spleen sections from immunized and naive mice harvested at 7 days and 6-12 months post-immunization. GC B cells were defined as B220 + PNA + (circled in yellow). DAPI stained cell nuclei. Representative overlay images are shown (scale bar: 2,000 µm). (C) Percentage of spleen RBD-specific GC B cells (B220 + IgD - GL-7 + CD95 + ) measured at 7 days, 2 weeks, 1 month and 6-12 months post-immunization by flow cytometry. (D) Percentage of spleen RBD-specific memory B cells (B220 + IgD - CD27 + ), plasmablasts (B220 + IgD - CD138 + TACI + ) and plasma cells (B220 - IgD - CD138 + TACI + ) measured at 1 month and 6-12 months post-immunization by flow cytometry. (E) Frequency of bone marrow antibody secreting cells present reactive to ancestral (left panel) and variant (right panel) SARS-CoV-2 RBD determined by B cell ELISPOT. SFU = spot forming unit. (A, C-E) Symbols represent individual animals and bars represent the median. Statistical analysis: (A, C-E) Mann Whitney test or Kruskal Walis test with Dunnett’s correction for multiple comparisons. *p < 0.05 or **p<0.01.

Article Snippet: Spots were enumerated using the Mabtech IRIS ™ ELISPOT and FluoroSPOT reader (Mabtech, Nacka Strand, Sweden).

Techniques: Variant Assay, Staining, Flow Cytometry, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad and durable systemic and mucosal immune responses

doi: 10.1101/2023.02.21.529344

Figure Lengend Snippet: (A) Frequency of IFN-γ + splenocytes determined by T cell ELISPOT upon re-stimulation with ancestral SARS-CoV-2 RBD peptides 7 days after single dose immunization with 2 µg Clec9A-RBD or 0.53 µg rRBD, both adjuvanted with 50 µg Poly I:C. (B) Overview of the immunological assays performed at day 7 post-Clec9A-RBD immunization to analyze the RBD-specific cellular responses. (C) Frequency of IFN-γ + and IL-5 + splenocytes determined by IFN-γ/IL-5 FluoroSPOT upon re-stimulation with ancestral (left panel) and variant (right panel) SARS-CoV-2 RBD peptides. (D) Frequency of splenocytes producing IFN-γ, IL-2, and/or TNF-α determined by IFN-γ/IL-2/TNF-α FluoroSPOT upon re-stimulation with ancestral (left panel) and variant (right) SARS-CoV-2 RBD peptides. (A, C, D) SFU = spot forming unit. (E) Percentage of spleen CD4 + and CD8 + T cells expressing IFN-γ, IL-2, and/or TNF-α determined by flow cytometry upon re-stimulation with ancestral (left panel) and variant (right) SARS-CoV-2 RBD peptides. (A) Symbols represent individual animals and bars represent the mean (C-E) ± standard deviation (SD). Statistical analysis: (A, C-E) unpaired two tailed t test or One-Way ANOVA with Tukey’s correction for multiple comparisons. *p < 0.05 or ***p < 0.001.

Article Snippet: Spots were enumerated using the Mabtech IRIS ™ ELISPOT and FluoroSPOT reader (Mabtech, Nacka Strand, Sweden).

Techniques: Enzyme-linked Immunospot, Variant Assay, Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test