Detection and Validation of Influenza Virus Hemagglutinin Specific, Cross‐Reactive, and Heteroclitic B Cells Induced by a Combination Vaccine Adjuvant

Inherent cross-reactivity for shared epitopes is thought to be critical for vaccines, since this is likely the only way in which a vaccine can be anticipatory, similar to immune responses induced by natural infections. However, measuring antigen-specific and cross-reactive B cells remains technically challenging due to low abundance of these cells and background binding. Here, we have assessed different antigen-labeling methods to optimize the measurement of B cell specificity, cross-reactivity, heterocliticity, and average affinity for antigen by flow cytometry. Antigens covalently labeled with NHS ester amine-reactive dyes gave reliable results compared to other antigen labeling methods: namely conjugation of His-tagged antigen on fluorescent streptavidin nanoparticles, or on soluble streptavidin tetramers, bridged via the biomolecular adapter biotin-trisNTA(Ni). Blocking with unlabeled H1 or H7 clearly outcompeted H1- and/or H7-specific cells, indicating that cross-reactive antibodies possess lower affinities compared to the mono-reactive or the rare heteroclitic antibodies. Overall, these studies suggest that evaluating the antigen specificity and breadth of candidate vaccines, including multi-season or universal vaccines, may be best accomplished using flow cytometric quantification of clonally distributed B lineage cells using antigens covalently derivatized with fluorescent dyes, which is also analogous with the standard flow cytometry approach of quantifying antigen biomarkers on cells using fluorescent monoclonal antibodies (mAbs).