Journal: NPJ Vaccines

Article Title: SARS-CoV-2 spike-ferritin-nanoparticle adjuvanted with ALFQ induces long-lived plasma cells and cross-neutralizing antibodies

doi: 10.1038/s41541-023-00638-6

Figure Lengend Snippet: Spleens from naive, or vaccinated SpFN + ALFQ or SpFN + AH mice ( n = 5 in each group) were collected at week 6 (day 42, Fig. 5d). ELISpot and flow cytometry analysis were performed. a Percentage of plasmablasts (CD3 - CD45R + CD138 + ) in all the three groups. b ELISpot images of the triplicate wells showing S-2P protein-specific- IgG (blue spots) and IgA (red spots) for each group. c Number of S-2P protein-specific IgG and IgA spot-forming units per million splenocytes are presented as a bar graph (Mean ± SEM). Each dot in the bar graph represents the average of triplicate wells for each mouse in each group. The responses were considered positive when the spot count exceeded the Mean ± 3 SD of the negative control wells. d A separate group of mice ( n = 6 in each group) were vaccinated three times with SpFN + ALFQ or SpFN + AH. Bone marrows were collected at week 21 and the percentage of e plasma cells (CD3 - CD45R - CD138 + ) and f intracellular S-2P protein-specific plasma cells were analyzed. The data are represented as bar graphs (Mean ± SD) and each dot represents the data from an individual spleen or bone marrow. The right panels show the representative contour or dot plots for each subset. The flow panel and the gating strategy for the respective subsets are shown in Supplementary Table and Supplementary Fig. , respectively. The statistical differences between the two vaccinated groups were evaluated by Mann–Whitney U -test with P ≤ 0.05 considered as significant.

Article Snippet: ; 1:1000) for four days and kept in a CO 2 incubator at 37 °C were added to the S-2P and RBD-coated wells plates (100,000 cells/well for S2P and 400,000 cells/well for RBD wells), the plates were then placed in the CO 2 incubator at 37 °C or 8 h. The plates were washed twice with PBS containing 0.05% Tween20 (PBS-T) then, anti-mouse IgG/IgA detection solution (C.T.L.) was added, and incubated for 2 h at RT.

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Negative Control, MANN-WHITNEY

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: ( A ) Blood was collected at day 0, day 14, and day 30 from AID-Cre-EYFP mice s.c. injected with aP or wP vaccines or with alum (ctr, control mice). IgM, IgG1, and IgG2b serum Ab titers against pooled proteins of the aP vaccine (PT, PRN, FHA, Fim2,3) ( B ) and IgM, IgG1, IgG2b, IgG2c, and IgG3 serum Ab titers against sonicated Bp ( C ) were detected by ELISA from serum of vaccinated and control mice. Ab titers are arbitrary values and each point in the graphs represents individual mouse data. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions at each time point and the different time points between the same conditions. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Injection, Vaccines, Control, Sonication, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: ( A ) AID-Cre-EYFP mice primed either with aP or wP vaccines or controls injected with alum (ctr) received 2 doses of tamoxifen at days 7 and 10 after prime vaccination. Mice were analyzed at days 14 and 30. ( B ) B220 + EYFP + live cells from dLNs of mice primed with aP and wP vaccines were distinguished into GC (GL7 + ) and memory (GL7 – ) B cells, by flow cytometry. ( C ) Total EYFP + GC and EYFP + memory B cell counts in the 2 dLNs are shown in the graphs. ( D ) A representative flow cytometry profile of heavy chain isotype distribution among EYFP + GL7 + B cells is shown for the aP and wP conditions at day 30 after prime. ( E ) IgM/IgD, IgG1, IgG2, and IgA distribution in the EYFP + GC and memory subsets from mice analyzed at day 30 after prime are shown in the plots. Each point represents an individual mouse analyzed at day 14 or day 30 after prime vaccination. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions at each time point or each Ab isotype. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Vaccines, Injection, Flow Cytometry

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: AID-Cre-EYFP mice primed with aP or wP vaccines or controls injected with alum (ctr) received 2 doses of tamoxifen at days 7 and 10 after prime vaccination. BM cells were collected 30 days after prime vaccination. ( A ) Representative flow cytometry profile of intracellular staining for EYFP + PCs in BM. B220 – EYFP + live cells were gated into CD138 + cells before determination of IgM, IgG1, and IgG2 distribution among PCs (a similar independent analysis was performed for IgA at the place of IgG2, not shown here). Total EYFP + PC cell numbers ( B ) or EYFP + PC isotype distribution ( C ) are represented in the plots. Numbers of IgG1 + ASCs against pooled proteins (PT, PRN, FHA, Fim2,3) ( D ) and numbers of IgG1 + and IgG2 + ASCs against sonicated Bp ( E ) were determined by ELISPOT from total BM cells. Representative spot images for each condition are shown at the left of each panel. Each point in the graphs represents an individual mouse. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Vaccines, Injection, Flow Cytometry, Staining, Sonication, Enzyme-linked Immunospot

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: ( A ) AID-Cre-EYFP mice were primed and boosted (day 30) with homologous and heterologous combinations of the aP and wP vaccines or injected with alum (ctr). Three doses of tamoxifen were administrated at days 7, 10, and 31. Mice were analyzed 50 days after boost injection. Cell numbers of EYFP + GCs ( B ) and cell numbers and isotype distribution of total GC B cells ( C ) or EYFP + memory B cells ( D ) were assessed by flow cytometry in dLNs and reported in the graphs. ( E ) Cell numbers and isotype distribution of EYFP + memory B cells were assessed by flow cytometry in spleen. Numbers of IgG1 + ASCs against pooled proteins (PT, PRN, FHA, Fim2,3) ( F ) or numbers of IgG1 + and IgG2 + ASCs against sonicated Bp ( G ) were determined by a memory B cell ELISPOT assay performed 5 days after in vitro activation of splenocytes in the presence of IL-2 and R848. Each point in the graphs represents an individual mouse. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Vaccines, Injection, Flow Cytometry, Sonication, Enzyme-linked Immunospot, In Vitro, Activation Assay

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: ( A ) AID-Cre-Tomato mice, primed and boosted (day 30) with homologous and heterologous aP and wP vaccine combinations, received tamoxifen at days 7, 10, and 31. Analysis was performed 50 days after boost. ( B ) Representative flow cytometry analysis of d-Tomato + cells selected from B220 + CD45.2 – resident lung B cells. d-Tomato + GL7 – cells were further analyzed for the expression of IgM and IgG1 membrane Ab isotypes. ( C ) Numbers of total d-Tomato + resident memory cells (graph on the left) and their isotype subclasses (graph on the right) are shown. ( D ) dLN, splenic, and lung total d-Tomato + memory B cells belonging to the wP:wP condition were analyzed for the expression of CD73, CD80, and PD-L2 markers. A representative flow cytometry plot is shown. Geometric MFI relative to each membrane marker is indicated in the graphs for all organs. Each point in the graphs ( C and D ) depicts an individual mouse. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Flow Cytometry, Expressing, Membrane, Marker

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: AID-Cre-EYFP mice were primed and boosted (day 30) with homologous and heterologous combinations of the aP and wP vaccines or injected with alum (ctr). Three doses of tamoxifen were administrated at days 7, 10, and 31. Mice were analyzed 50 days after boost injection. ( A ) Cell numbers and isotype distribution of EYFP + PCs assessed by flow cytometry in BM are shown in the graphs. ( B ) Numbers of IgG1 + or IgG2 + ASCs against sonicated Bp were determined by ELISPOT performed on total BM cells. Each point in the graphs represents an individual mouse. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Vaccines, Injection, Flow Cytometry, Sonication, Enzyme-linked Immunospot

Journal: JCI Insight

Article Title: The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings

doi: 10.1172/jci.insight.157034

Figure Lengend Snippet: AID-Cre-EYFP mice were primed and boosted (day 30) with homologous and heterologous combinations of the aP and wP vaccines or injected with alum (ctr). Blood was collected at 5, 25, 40, and 50 days after boost injection. IgG1 and IgG2b Ab titers against pooled proteins (PT, PRN, FHA, Fim2,3) ( A ) or IgG1, IgG2b, IgG2c, and IgG3 Ab titers against sonicated Bp ( B ) were detected by ELISA from serum of vaccinated and control mice. Ab titers are arbitrary values and each point in the graphs represents data from an individual mouse. At least 2 independent experiments were performed for each analysis. Means (±SEM) are shown. Kruskal-Wallis analysis with uncorrected Dunn’s test was performed to compare the different conditions at each time point and the different time points between the same condition. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Goat anti-mouse IgG1, IgG2c, IgG2b, IgG3, and IgA detection Abs (RD-Biotech) were Sulfo-Tag–labeled according to MSD GOLD SULFO-TAG NHS-Ester kit instructions and used for detection.

Techniques: Vaccines, Injection, Sonication, Enzyme-linked Immunosorbent Assay, Control