Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Construction and characterization of CD40L-fused multi-epitope trimeric proteins. ( A ) Trimer-40L-TB expresses a trimeric fusion protein containing the extracellular domain of CD40L and TB epitopes. 5B-TB encodes a fusion protein lacking CD40L. 5B-B encodes a fusion protein lacking CD40L and T epitopes. Monomer TB encodes a monomeric vaccine, and 5B serves as a trimerization motif without antigenic epitopes. Numbers indicate the expected molecular weights of proteins in each construct. ( B ) SDS-PAGE and western blotting analysis of protein expression, identified using FMDV-positive serum. ( C ) ELISA detection of the reactivity of five proteins. Equal concentrations of the five proteins were coated, serially diluted, and detected using biotinylated O8 single-domain Ab. Repeat three times for each sample ( n = 3), and data are presented as the mean ± standard deviation. ( D ) SPR analysis of the binding affinity of trimeric and monomeric proteins. O8 single-domain Ab was immobilized on an M5 sensor chip, and four proteins were applied as analytes. Note: B-cell epitopes: VP1: 137–156 (O/BY; O/Panasia; A/GDMM; A/HW); sCD40L: residues 113–261; T-cell epitopes: 2C [157–174], 2C [151–168], 2C [109–126], and 2B [31–48]; AP: RRRWCKPPP; 5B: artificially designed protein self-assembling trimerization motif.

Article Snippet: Plates were then washed, incubated with biotinylated anti-pig IgG (5 μg/mL, Mabtech) for 2 h at room temperature, followed by streptavidin-HRP for 1 h. Spots were developed using TMB substrate (Mabtech), and the reaction was stopped by rinsing with deionized water.

Techniques: Construct, SDS Page, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Binding Assay

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Analysis of Trimer-CD40L-TB vaccine-induced FMDV GH-Loop-specific GC B-cell counts, IgG Abs, neutralizing Abs, and accompanying broad-spectrum protection against FMDV serotypes O and A. Two pigs from each of the Trimer-CD40L-TB, Trimer-TB, and control groups were euthanized at 35 dpv, and lymphoid tissues were collected. ( A ) Isolating single cells was assessed by flow cytometry. Representative dot plots of GH-Loop-specific GC B cells in the Trimer-CD40L-TB, Trimer-TB, and control groups ( n = 2). ( B ) Bar graph analyzing the differences in the number of GH-Loop-specific GC B cells among the three groups ( n = 2/group). ( C ) Fluorescence staining analysis of GC B cells in tissue sections, with BCL-6 (red) and IgG (purple) used to localize B cells in GC. ( D ) Analysis of differences in the number of IgG + BCL-6 + fluorescent cells among the three groups ( n = 2/group). ( E ) PBMCs were isolated from pigs at 35 dpv. After coating with anti-porcine IgG Ab, PBMCs were added and incubated for 24 h, then biotinylated Trimer-B protein was used to detect the number of IgG antibodies secreted by B cells specific to B epitopes ( n = 4/group). ( F ) Serum samples were collected from each group of pigs, and FMDV NAb titers were detected by a neutralization test. FMDV NAb titers among the four groups were compared at 7, 14, 21, 28, and 35 dpv ( n = 4/group). All data assay is presented as the mean ± standard deviation ( n = 2 or 4). All data were assessed by one or two-way ANOVA, ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Plates were then washed, incubated with biotinylated anti-pig IgG (5 μg/mL, Mabtech) for 2 h at room temperature, followed by streptavidin-HRP for 1 h. Spots were developed using TMB substrate (Mabtech), and the reaction was stopped by rinsing with deionized water.

Techniques: Control, Flow Cytometry, Fluorescence, Staining, Isolation, Incubation, Neutralization, Standard Deviation