Journal: iScience

Article Title: BA.1 breakthrough infection elicits distinct antibody and memory B cell responses in vaccinated-only versus hybrid immunity individuals

doi: 10.1016/j.isci.2025.111962

Figure Lengend Snippet: Prior hybrid immunity status prevents the decline of Fc receptor-dependent anti-Spike IgG functions post breakthrough infection (A) Serum samples were collected from individuals with established hybrid (Hu-1/2RNA/BA.1, purple, n = 15) or vaccination-induced (3RNA/BA.1, green, n = 15) immunity 3 months post BA.1 breakthrough infection. (B) IgG levels specific to the Wuhan Hu-1 (Hu-1) RBD were assessed and concentrations were expressed in binding antibody unit (BAU)/mL. Each serum sample was evaluated as a single measurement. The data are represented using a log 2 scale. The dotted line represents the positivity threshold (≥20.33 BAU/mL). (C) Purified total IgGs from each individual were assayed for Hu-1 RBD-specific IgG off-rate measurement using biolayer interferometry. K off values are expressed as s −1 and each sample was assayed as a single measurement. (D) Subtyping of Hu-1 and BA.1 S-specific IgG was carried out on serum samples. The data are expressed using a log 2 scale. Each sample was assayed as a single measurement. (E) Antibody neutralization capacity was evaluated against the 19A, BA.1, BA.4, BA.5 and XBB.1.5 live isolates. The data are represented using a log 2 scale. Serum samples were tested as technical duplicates. The dotted line represents the positivity threshold (PRNT 50 ≥ 20). (F) The induction of the CD16 pathway was used as a surrogate of ADCC using the ADCC reporter assay. 293T cells stably expressing D614G, BA.1 and BA.4/5 S proteins, or a control plasmid, were used as target cells. The fold change of CD16 activation was calculated in each condition, and the control condition (cells transduced with an empty vector) was subtracted to account for interindividual variations of the background. The y axis indicates the calculated ADCC induction, with a value of zero indicating no induction. Each sample was assayed as a single measurement. Data for all parameters are expressed as box-and-whiskers plots according to the Tukey method with the line inside the box indicating the median, the upper and lower line of the box indicating the interquartile range [IQR]. Multiple regressions (ANCOVA) were realized for each parameter, adjusted on covariate “age,” to compare the results obtained between the two groups. Exact p values are indicated for each parameter. MFI, mean fluorescence intensity.

Article Snippet: For IgG subtyping, mouse anti-human IgG1, IgG2, IgG3, IgG4 antibodies conjugated with phycoerythrin (PE, Southern Biotech) were added at a final concentration of 1.3 μg/mL and the mixture was incubated for 2h at room temperature on a plate shaker.

Techniques: Infection, Binding Assay, Purification, Neutralization, Reporter Assay, Stable Transfection, Expressing, Control, Plasmid Preparation, Activation Assay, Transduction, Fluorescence

Journal: iScience

Article Title: BA.1 breakthrough infection elicits distinct antibody and memory B cell responses in vaccinated-only versus hybrid immunity individuals

doi: 10.1016/j.isci.2025.111962

Figure Lengend Snippet: BA.1 breakthrough infection in individuals with prior hybrid immunity recalls anti-nucleocapsid serological immunity Serum samples were collected from individuals with established hybrid- (Hu-1/2RNA/BA.1, purple, n = 15) or vaccination-induced (3RNA/BA.1, green, n = 15) immunity 3 months post BA.1 breakthrough infection. (A) Anti-N total IgG levels were quantified and expressed as an index. Each sample was assayed as a single measurement. The dotted line represents the positivity threshold (index ≥ 1.4) according to manufacturer’s instruction. (B) Subtyping of N-specific IgG was carried out on serum samples. The data are expressed using a log 2 scale. Each sample was assayed as a single measurement. (C) Antibodies specific to the N protein were assessed for their ability to bind the FcγRIIa and FcγRIIIa receptors. Each sample was assayed as a single measurement. Data for all parameters are expressed as box-and-whiskers plots according to the Tukey method with the line inside the box indicating the median, the upper and lower line of the box indicating the interquartile range [IQR]. Multiple regressions (ANCOVA) were realized for each parameter, adjusted on covariate “age,” to compare the results obtained between the two groups. Exact p -values are indicated for each parameter. MFI: mean fluorescence intensity.

Article Snippet: For IgG subtyping, mouse anti-human IgG1, IgG2, IgG3, IgG4 antibodies conjugated with phycoerythrin (PE, Southern Biotech) were added at a final concentration of 1.3 μg/mL and the mixture was incubated for 2h at room temperature on a plate shaker.

Techniques: Infection, Fluorescence

Journal: iScience

Article Title: BA.1 breakthrough infection elicits distinct antibody and memory B cell responses in vaccinated-only versus hybrid immunity individuals

doi: 10.1016/j.isci.2025.111962

Figure Lengend Snippet:

Article Snippet: For IgG subtyping, mouse anti-human IgG1, IgG2, IgG3, IgG4 antibodies conjugated with phycoerythrin (PE, Southern Biotech) were added at a final concentration of 1.3 μg/mL and the mixture was incubated for 2h at room temperature on a plate shaker.

Techniques: Recombinant, Bioassay, Luciferase, Software, Imaging, Staining, Blocking Assay