Open AccessOptimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo
Author(s) -
Nair Nitya,
Buti Ludovico,
Faenzi Elisa,
Buricchi Francesca,
Nuti Sandra,
Sammicheli Chiara,
Tavarini Simona,
Popp Maximilian W.L.,
Ploegh Hidde,
Berti Francesco,
Pizza Mariagrazia,
Castellino Flora,
Finco Oretta,
Rappuoli Rino,
Del Giudice Giuseppe,
Galli Grazia,
Bardelli Monia
Publication year - 2013
Publication title -
immunity, inflammation and disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.918
H-Index - 18
ISSN - 2050-4527
DOI - 10.1002/iid3.3
Subject(s) - antigen , memory b cell , neisseria meningitidis , epitope , flow cytometry , ex vivo , biology , b cell , antibody , naive b cell , in vivo , t cell , chemistry , microbiology and biotechnology , virology , immunology , antigen presenting cell , immune system , genetics , bacteria
Antigen‐specific memory B cells generate anamnestic responses and high affinity antibodies upon re‐exposure to pathogens. Attempts to isolate rare antigen‐specific memory B cells for in‐depth functional analysis at the single‐cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen‐specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA‐specific memory B cells with high sensitivity and specificity, comparable to NadA amine‐labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA‐specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.