Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.
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.
Novartis Vaccines & Diagnostics Siena, Italy.
Nitya
N
Nair
Novartis Vaccines & Diagnostics Siena, Italy ; Whitehead Institute for Biomedical Research Cambridge, Massachusetts.
Ludovico
L
Buti
Novartis Vaccines & Diagnostics Siena, Italy.
Elisa
E
Faenzi
Novartis Vaccines & Diagnostics Siena, Italy.
Francesca
F
Buricchi
Novartis Vaccines & Diagnostics Siena, Italy.
Sandra
S
Nuti
Novartis Vaccines & Diagnostics Siena, Italy.
Chiara
C
Sammicheli
Novartis Vaccines & Diagnostics Siena, Italy.
Simona
S
Tavarini
Whitehead Institute for Biomedical Research Cambridge, Massachusetts ; University of Rochester, School of Medicine & Dentistry Rochester, New York ; Department of Biology, Massachusetts Institute of Technology Cambridge, Massachusetts.
Maximilian Wl
MW
Popp
Whitehead Institute for Biomedical Research Cambridge, Massachusetts ; Department of Biology, Massachusetts Institute of Technology Cambridge, Massachusetts.
Hidde
H
Ploegh
Novartis Vaccines & Diagnostics Siena, Italy.
Francesco
F
Berti
Novartis Vaccines & Diagnostics Siena, Italy.
Mariagrazia
M
Pizza
Novartis Vaccines & Diagnostics Siena, Italy.
Flora
F
Castellino
Novartis Vaccines & Diagnostics Siena, Italy.
Oretta
O
Finco
Novartis Vaccines & Diagnostics Siena, Italy.
Rino
R
Rappuoli
Novartis Vaccines & Diagnostics Siena, Italy.
Giuseppe
G
Del Giudice
Novartis Vaccines & Diagnostics Siena, Italy.
Grazia
G
Galli
Novartis Vaccines & Diagnostics Siena, Italy.
Monia
M
Bardelli
Antigen-specific memory B cells; Neisseria meningitidis MenB; flow cytometry; sortagging; vaccination