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Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.

Nair N, Buti L, Faenzi E, Buricchi F, Nuti S, Sammicheli C, Tavarini S, Popp MW, Ploegh H, Berti F, Pizza M, Castellino F, Finco O, Rappuoli R, Del Giudice G, Galli G, Bardelli M - Immun Inflamm Dis (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines & Diagnostics Siena, Italy.

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.

No MeSH data available.


Single antigen staining is sufficient to identify NadA-specific switched memory B cells. (A) Representative staining of NadA-specific memory B cells identified by single or dual antigen staining strategies with amine-labeled or sortagged NadA-Alexa fluorochrome conjugates. Gating of NadA-specific memory B cells was based on staining in naïve mice (overlay in light gray) and HSA specificity controls. (B) For each staining strategy, the percentage of NadA-specific memory B cells among total switched memory B cells is shown in immune (top) and naïve (bottom) mice after subtracting background HSA staining. Results are from three independent experiments performed with splenocytes pooled from four mice per group. Box plots depict median values and ranges; the box-crossing lines depict the means.
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fig03: Single antigen staining is sufficient to identify NadA-specific switched memory B cells. (A) Representative staining of NadA-specific memory B cells identified by single or dual antigen staining strategies with amine-labeled or sortagged NadA-Alexa fluorochrome conjugates. Gating of NadA-specific memory B cells was based on staining in naïve mice (overlay in light gray) and HSA specificity controls. (B) For each staining strategy, the percentage of NadA-specific memory B cells among total switched memory B cells is shown in immune (top) and naïve (bottom) mice after subtracting background HSA staining. Results are from three independent experiments performed with splenocytes pooled from four mice per group. Box plots depict median values and ranges; the box-crossing lines depict the means.

Mentions: We compared the frequencies of NadA-specific MBC identified by a single fluorescent bait (amine-labeled or sortagged) to those identified by combining two baits that were amine-labeled with different fluorochromes. The results from three independent experiments showed that the frequencies of switched B cells binding to one or two baits were comparable (Fig. 3A). The mean frequency of NadA-specific memory B cells identified by dual antigen staining did not differ from those observed in samples stained with a single amine-labeled or sortagged NadA bait (P-values for comparison across all groups and between each pair of samples was always >0.9 by the Tukey–Kramer test) (Fig. 3B). In addition, to verify whether the signal to noise ratios were higher in samples stained with two NadA baits than in those stained with a single NadA we compared the ratios of mean fluorescence intensities measured for each bait in the negative and positive gates using the one-tailed Wilcoxon test. The results of this analysis showed that differences in signal to noise ratios in each fluorescence channel were not statistically significant (data not shown).


Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo.

Nair N, Buti L, Faenzi E, Buricchi F, Nuti S, Sammicheli C, Tavarini S, Popp MW, Ploegh H, Berti F, Pizza M, Castellino F, Finco O, Rappuoli R, Del Giudice G, Galli G, Bardelli M - Immun Inflamm Dis (2013)

Single antigen staining is sufficient to identify NadA-specific switched memory B cells. (A) Representative staining of NadA-specific memory B cells identified by single or dual antigen staining strategies with amine-labeled or sortagged NadA-Alexa fluorochrome conjugates. Gating of NadA-specific memory B cells was based on staining in naïve mice (overlay in light gray) and HSA specificity controls. (B) For each staining strategy, the percentage of NadA-specific memory B cells among total switched memory B cells is shown in immune (top) and naïve (bottom) mice after subtracting background HSA staining. Results are from three independent experiments performed with splenocytes pooled from four mice per group. Box plots depict median values and ranges; the box-crossing lines depict the means.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4217542&req=5

fig03: Single antigen staining is sufficient to identify NadA-specific switched memory B cells. (A) Representative staining of NadA-specific memory B cells identified by single or dual antigen staining strategies with amine-labeled or sortagged NadA-Alexa fluorochrome conjugates. Gating of NadA-specific memory B cells was based on staining in naïve mice (overlay in light gray) and HSA specificity controls. (B) For each staining strategy, the percentage of NadA-specific memory B cells among total switched memory B cells is shown in immune (top) and naïve (bottom) mice after subtracting background HSA staining. Results are from three independent experiments performed with splenocytes pooled from four mice per group. Box plots depict median values and ranges; the box-crossing lines depict the means.
Mentions: We compared the frequencies of NadA-specific MBC identified by a single fluorescent bait (amine-labeled or sortagged) to those identified by combining two baits that were amine-labeled with different fluorochromes. The results from three independent experiments showed that the frequencies of switched B cells binding to one or two baits were comparable (Fig. 3A). The mean frequency of NadA-specific memory B cells identified by dual antigen staining did not differ from those observed in samples stained with a single amine-labeled or sortagged NadA bait (P-values for comparison across all groups and between each pair of samples was always >0.9 by the Tukey–Kramer test) (Fig. 3B). In addition, to verify whether the signal to noise ratios were higher in samples stained with two NadA baits than in those stained with a single NadA we compared the ratios of mean fluorescence intensities measured for each bait in the negative and positive gates using the one-tailed Wilcoxon test. The results of this analysis showed that differences in signal to noise ratios in each fluorescence channel were not statistically significant (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines & Diagnostics Siena, Italy.

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.

No MeSH data available.