Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Nair, Nitya, Ludovico Buti, Elisa Faenzi, Francesca Buricchi, Sandra Nuti, Chiara Sammicheli, Simona Tavarini, et al. “ Optimized Fluorescent Labeling to Identify Memory B Cells Specific for Neisseria Meningitidis Serogroup B Vaccine Antigens Ex Vivo .” Immun. Inflamm. Dis. 1, no. 1 (October 2013): 3–13. As Published http://dx.doi.org/10.1002/iid3.3 Publisher Wiley Blackwell Version Final published version Citable link http://hdl.handle.net/1721.1/92523 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/3.0/ ORIGINAL RESEARCH Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo Nitya Nair1†, Ludovico Buti1,2‡, Elisa Faenzi1, Francesca Buricchi1, Sandra Nuti1, Chiara Sammicheli1, Simona Tavarini1, Maximilian W.L. Popp2,3,4, Hidde Ploegh2,4, Francesco Berti1, Mariagrazia Pizza1, Flora Castellino1, Oretta Finco1, Rino Rappuoli1, Giuseppe Del Giudice1, Grazia Galli1, & Monia Bardelli1 1Novartis Vaccines & Diagnostics, Siena, Italy 2Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 3University of Rochester, School of Medicine & Dentistry, Rochester, New York 4Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts Keywords Abstract Antigen-specific memory B cells, flow cytometry, Neisseria meningitidis MenB, Antigen-specific memory B cells generate anamnestic responses and high affinity sortagging, vaccination 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 Correspondence been hindered by the lack of tools with adequate sensitivity. We applied two Nitya Nair, Department of Microbiology & independent methods of protein labeling to sensitive and specific ex vivo Immunology, identification of antigen-specific memory B cells by flow cytometry: stringently Stanford University School of Medicine and Veterans Administration Palo Alto Health Care controlled amine labeling, and sortagging, a novel method whereby a single System, Palo Alto, CA 94307-1207. nucleophilic fluorochrome molecule is added onto an LPETG motif carried by Tel: þ1 650 493 5000, Extn. 63125; the target protein. We show that sortagged NadA, a major antigen in the Fax: 650 852 3259 meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with E-mail: [email protected] high sensitivity and specificity, comparable to NadA amine-labeled under stringent Monia Bardelli, Research Center, Novartis reaction parameters in a mouse model of vaccination. We distinguish NadA- Vaccines and Diagnostics srl 53100, Siena, specific switched MBC induced by vaccination from the background signal Italy. Tel: þþ39 0577 245 152. contributed by splenic transitional and marginal zone B cells. In conclusion, we E-mail: [email protected] demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as Funding information conventional chemically labeled antigens in detecting rare MBC, and minimize No funding information provided. the possibility of disrupting conformational B cell epitopes. Received: 24 April 2013; Revised: 10 June 2013; Accepted: 12 June 2013 Immunity, Inflammation and Disease Introduction 2013; 1(1): 3–13 Antigen-specific memory B cells generate anamnestic doi: 10.1002/iid3.3 responses and high affinity antibodies upon re-exposure to bacterial and viral pathogens. The mechanisms through †Present address: Department of Microbiology which memory B cells are involved in the generation and & Immunology, Stanford University School of Medicine and Veterans Administration Palo maintenance of long-term serologic memory remain unclear Alto Health Care System, Palo Alto, CA. since protective antibody titers do not necessarily correlate with the number of memory B cells induced by infection ‡Present address: Ludwig Institute for Cancer and/or vaccination [1–4]. It is likely that both the quality and Research, Nuffield Department of Clinical the size of the memory B cell pool are important Medicine, University of Oxford, Headington, determinants of the overall protective response to infection Oxford, UK. and/or vaccination. © 2013 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd. 3 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Optimized fluorescent labeling to identify Neisseria meningitidis serogroup B vaccine antigens ex vivo N. Nair et al. Qualitative assessments of memory B cells ex vivo have cells by FACS in a mouse model of vaccination. Sortagged been challenging due to their low frequency in peripheral NadA performed as well as amine-labeled NadA prepared blood [1, 5]. As a consequence most studies have relied on using controlled reaction parameters, in terms of sensitivity expansion and conversion of memory B cells into antibody and specificity. Single antigen staining using either detection secreting cells by in vitro polyclonal stimulation with TLR reagent was sufficient to thoroughly identify NadA-specific ligands (CpG-2006, R848) and cytokines (IL-2, IL-10 or memory B cells among the total memory B cell population. IL-6) for subsequent analysis by ELISpot or serial limiting In addition we distinguished the NadA-specific switched dilution assay [1, 2, 6]. An alternative strategy has been to use memory B cells induced by vaccination, from the back- fluorescently labeled proteins to identify antigen-specific ground binding reactivity contributed by transitional and MBC from mice and humans for qualitative analysis by flow marginal zone splenic B cells with low affinity IgM receptors. cytometry [5, 7]. However low signal to noise ratio is often We provide proof of concept that sortagged NadA can be observed due to low memory B cell frequencies and high used in dual staining with other antigens to identify memory background due to the fluorochrome itself [8, 9]. B cells with distinct antigenic specificities. Fluorescent Previous work has shown that dual antigen staining, in sortagged protein antigens therefore are improved alter- which tetanus (TT) or diphtheria (DT) toxoid were natives to conventional chemically labeled baits due to the labeled with different fluorochromes, increased specificity added benefits of quantitative site-specific labeling that and maintained sensitivity in the identification of TT- and DT- permit better epitope preservation. specific memory B cells as a double positive population by flow cytometry [5]. Dual antigen staining requires labeled antigens with equivalent affinities for the B cell receptor (BCR) to Results facilitate unbiased detection of memory B cell populations [8]. Sortagging and amine-labeling of NadA with However most conventional labeling methods involve chemi- fluorochromes cal attachment of fluorochrome molecules to accessible amine groups on the protein of interest [5, 7], during which the According to the proposed three-dimensional model of positions and numbers of labeled amines, cannot be easily NadA [14], sortagging inserted the Alexa fluorochrome to controlled. Furthermore, amine labeling may interfere with the C-terminus of the coiled-coil-rich stalk region of the protein folding and disrupt conformational B cell epitopes at protein (Fig. 1A). Conversely chemical labeling tagged the random, therefore skewing the selection of antigen-specific solvent accessible amine groups at random (Fig. 1B). memory B cells for downstream analysis. NadA equipped with the LPETG sortagging recognition We describe two independent methods to fluorescently motif followed by a C-terminal HA and His(6) affinity handle label protein antigens: conventional amine labeling with were expressed and purified from Escherichia coli. Sortase A stringently controlled reaction parameters, and sortagging, a (srtA) containing an N-terminal His(6) tag was produced as novel site-specific labeling method mediated by staphylo- previously described [10]. The presence of the His tag on coccal sortase A, in which a known number of nucleophilic SrtA and NadA allowed to remove both SrtA and unreacted fluorochrome molecules are added to LPTEG motifs NadA after completion of the reaction. Overnight incuba- expressed on the target proteins [10]. In both methods the tion of NadA with SrtA resulted in the formation of an acyl- degree of labeling is minimized. enzyme intermediate (Fig. 1C, lane two). Addition of the gly- As a model antigen we used Neisseria adhesin A (NadA), a probe (respectively 3gly-488 or 3gly-647) drove the reaction major protein present in a multicomponent meningococcal to the transpeptidation product, and resulted in the site- serogroup B vaccine in advanced stage of development, and a specific labeling of NadA with A488 or A647 (Fig. 1C, lower virulence factor involved in meningococcus invasion and insert and Fig. S1A). The overnight reactions (In) were adhesion to epithelial cells [11, 12]. NadA is an oligomeric loaded on a Ni-Nta column and SrtA and unreacted material coiled-coil adhesin with a trimeric structure and binding of were separated from labeled