{"doi":"10.1002/cytoa.70000","title":"Detection and Validation of Influenza Virus Hemagglutinin Specific, Cross‐Reactive, and Heteroclitic B Cells Induced by a Combination Vaccine Adjuvant","abstract":"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).","journal":"Cytometry Part A","year":2025,"id":549430,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9565,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":551486,"name":"Jiin Felgner","orcid":null,"position":1,"is_corresponding":false},{"id":832784,"name":"Jenny E. Hernandez‐Davies","orcid":null,"position":2,"is_corresponding":false},{"id":607046,"name":"Johannes S. Gach","orcid":"0000-0002-5043-5710","position":3,"is_corresponding":false},{"id":729305,"name":"Tyler J. Albin","orcid":null,"position":4,"is_corresponding":false},{"id":550812,"name":"D. Huw Davies","orcid":"0000-0002-6763-0109","position":5,"is_corresponding":false},{"id":832378,"name":"Egest J. Pone","orcid":"0000-0001-5755-6788","position":0,"is_corresponding":true}],"reference_count":52,"raw_metadata":null,"created_at":"2026-07-19T02:54:07.823422Z","pmid":"41392330","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}