Journal: bioRxiv

Article Title: Rationally designed minimized TbpB confers broad protection against meningococcal infection

doi: 10.1101/2025.10.30.685603

Figure Lengend Snippet: A-C: Protein ELISA measuring reactivity of TbpB, C-lobe, and LCL-immune sera against full-length TbpB, TbpB C-lobe, and TbpB N-lobe, respectively. D: IgG titre elicited by the different immunogens towards heat inactivated Nme M982. A-D: N=10-15 per group; squares represent pre-challenge sera from C57BL/6 animals from the sepsis study presented in ; circles represent terminal serum from hCEACAM1 FvB mice from the colonization study presented in , each symbol represents serum from one animal tested in duplicate. E: Serum bactericidal activity (SBA) titre against iron-starved Nme M982. N=19-25 per group, including terminal hCEACAM1 FvB serum from , plus additional terminal serum from immunized hCEACAM1 FvB from pilot studies. SBA titre (>50% killing) reached for 19/25 (76%) TbpB, 8/19 (42%) C-lobe, 14/22 (64%) LCL and 3/21 (14%) Alum samples. Dotted lines represent lowest dilution tested. Line at median for each group. One-way ANOVA with Tukey’s post-hoc test comparing each group to every other group performed using GraphPad Prism 10.2.0. For D, E: Statistics performed on Log2 transformed data. ns, not significant; **, p<0.01; ***, p<0.001; ****, p<0.0001. F: Meningococcal growth inhibition assay. Iron-starved M982 strain was grown in the presence of hTf and pooled heat-inactivated serum from TbpB, C-lobe and LCL immunized animals from . Change in absorbance (OD600) relative to the initial time point is graphed; error bars depict standard deviation of two technical replicates.

Article Snippet: Assay was set up in 40 μL total volume using ∼500 bacteria suspended in PBS++, 10% baby rabbit complement (Cedarlane, CL3441-S100) and 2-fold serial dilutions of heat inactivated terminal mouse serum.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Transformation Assay, Growth Inhibition Assay, Standard Deviation

Journal: bioRxiv

Article Title: Rationally designed minimized TbpB confers broad protection against meningococcal infection

doi: 10.1101/2025.10.30.685603

Figure Lengend Snippet: A: Phylogenetic tree depicting Neisserial TbpB diversity. The location of M982 TbpB is highlighted, along with 16 additional TbpBs spanning various clusters used for cross-reactivity analysis. B: Heat map depicting IgG cross-reactivity against a broad panel of TbpB protein variants by ELISA. Optical density (OD) readings denoted by the colour gradient. Background noise from no-protein control subtracted. Each row is a different TbpB protein capture, with the phylogenetic cluster indicated to the left. Each column is a different mouse sample, with the immunizing antigens indicated above. Numbered labels starting with A are pre-challenge serum from the immunized C67BL/6 sepsis cohort; B are terminal serum from the immunized hCEACAM1 FvB colonization cohort. C: Grouped bar graph summarizing cross-reactive antibody data from B. Bars represent mean, error bars represent standard error, each circle represents serum from a single mouse (N=8 for immune sera, N=3 for Alum). hTf-HRP signal quantifying properly folded TbpB capture is depicted in the inset, mean ± standard deviation. D: Serum reactivity against representative Nme and Ngo strains grown under iron limitation using inactivated whole bacterial ELISA. TbpB cluster indicated below the x-axis. Background noise from no serum control subtracted. Bars represent mean, error bars represent standard error, each circle represents serum from a single mouse, N=5-8/group. Inset depicts hTf-binding as an indicator of TbpAB receptor expression on bacteria used for coating ELISA plates, mean ± standard deviation. For C and D, two-way ANOVA with Dunnett’s post-hoc test comparing each group to Alum control performed using GraphPad Prism 10.2.0. Only p-values <0.05 shown. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001.

Article Snippet: Assay was set up in 40 μL total volume using ∼500 bacteria suspended in PBS++, 10% baby rabbit complement (Cedarlane, CL3441-S100) and 2-fold serial dilutions of heat inactivated terminal mouse serum.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Standard Deviation, Binding Assay, Expressing, Bacteria