Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo. Issue 1 (1st August 2013)

• Record Type: Journal Article
• Authors: 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
• Is Part Of: Immunity, inflammation and disease. Volume 1:Issue 1 (2013)
• Page Start: 3
• Page End: 13
• Publication Date: 2013-08-01
• Subjects: Antigen‐specific memory B cells -- flow cytometry -- Neisseria meningitidis MenB -- sortagging -- vaccination; Immunology -- Periodicals; Immunity -- Periodicals; Inflammation -- Periodicals; 616.079
• DOI: 10.1002/iid3.3
• Languages: English
• ISSNs: 2050-4527

Abstract

Abstract: 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. Abstract : We describe two independent methods to optimize protein antigen labeling with fluorochromes for FACS identification and analysis of rare antigen‐specific memory B cells: conventional amine labeling with stringently controlled reaction parameters, and a novel site‐specific method, named sortagging, where nucleophilic fluorochromes are added in known numbers to LPTEG motifs expressed on the target proteins by using the staphylococcal sortase A. Using the meningococcal serogroup B NadA antigen and a mouse model of vaccination, we show that both methods permit the specific identification of all NadA‐specific memory B cells by FACS directly in ex vivo samples. We demonstrate that both techniques minimize the degree of labeling while maintaining adequate signal to noise ratios for sensitive and identification of rare memory B cells specific for at least two different antigens. This method facilitates accurate control of antigen integrity thus providing the potential for more valid analysis and sorting of antigen‐specific B cell repertoires and a powerful tool for best understanding B cell dynamics following vaccination or infection.