Cloning of expression vectors

The tetanus toxoid fragment C (TTC) DNA sequence (GenBank accession no. FJ917402.1) was synthesized by GeneArt® Gene Synthesis (Life Technologies, Darmstadt, Germany) and included the restriction sites SfiI and NotI. After digestion with these enzymes, the TTC DNA sequence was ligated into the SfiI/NotI sites of pBM1.1 with and without the ETA’ sequence [18] and pMS [19] (Fig. 1). Successful cloning was confirmed by colony polymerase chain reaction and DNA sequencing.

Expression of TTC-ETA’ and TTC in Escherichia coli and protein purification

Escherichia coli BL21 (DE3) cells (Novagen, Darmstadt, Germany) were transformed with the TTC and TTC-ETA’ encoding expression vectors and the corresponding proteins were expressed into the periplasm under osmotic stress in the presence of compatible solutes [20]. The protein was purified from the periplasmic fraction by immobilized metal-ion affinity chromatography (IMAC) using a Nickel-Sepharose (Ni-NTA) Superflow Cartridge (Qiagen, Hilden, Germany) on the ÄKTApurifier system (GE Healthcare Life Sciences, Freiburg, Germany) followed by a size-exclusion chromatography using a Superdex 200 (GE Healthcare). The TTC proteins were eluted into phosphate buffered saline (PBS, pH 7.4) and concentrated using Vivaspin 6 columns (Sartorius, Goettingen, Germany). The proteins were passed through a 0.22-μm sterile filter (Nalgene, Roskilde, Denmark) and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [21]. After gel staining with Coomassie Brilliant Blue, the protein concentration was estimated using AIDA Image Analyzer (Raytest GmbH, Straubenhardt, Germany) against 0.25-3 μg standards of bovine serum albumin (BSA). Unstained gels were blotted onto nitrocellulose membranes and probed with a primary anti-penta-his antibody diluted 1:5000 (Qiagen, Hilden, Germany; Catalogue number: 34660) and an alkaline phosphate (AP)-coupled goat-anti-mouse secondary antibody also diluted 1:5000 (Dianova, Hamburg, Germany; Catalogue number: 115-055-020).

Expression of SNAP-TTC in HEK293T cells

Human embryonic kidney cells (HEK293T, ATCC, Wesel, Germany, CRL-11268) were cultured under standard conditions (RPMI 1640 medium, 10 % v/v fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin, 37 °C, 5 % CO₂) and transfected with the pMS expression vectors using Roti®-Fect (Carl Roth, Karlsruhe, Germany) according to the manufacturer’s instructions. Briefly, 2 μg of DNA was mixed with 2 μl of Roti®-Fect and applied to the cells for 3 h. The cultures were then supplemented with 100 ng/ml Zeocin® (Invitrogen, Carlsbad, USA) for selection. The supernatant was collected from the transfected HEK293T cells and the protein was purified by IMAC as described above. After overnight dialysis against 1 x PBS at 4 °C, the SNAP-TTC protein was passed through a 0.22-μm sterile filter (Nalgene, Roskilde, Denmark). The protein concentration was determined using the AIDA Image Analyzer as described above.

Coupling SNAP-TTC to the fluorescent dye

Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [17]. Briefly, 1 μg SNAP-TTC protein was mixed with 2 nmol BG-647 or BG-488 solution prepared from a 50 nmol stock and incubated for 1 h at room temperature. Samples were taken to determine the coupling efficiency by SDS-PAGE and the fluorescence signal was detected using the CRi Maestro imaging system with appropriate filter sets (Perkin Elmer, Waltham, MA, USA).

Cultivation of hybridoma cells and cell binding analysis by flow cytometry

TTC-reactive 5E4 hybridoma cells (kindly provided by Prof. Dr. M. Shapiro, Rockville, USA) and myelin oligodendrocyte glycoprotein (MOG)-reactive 8.18-C5 hybridoma cells (kindly provided by Prof. C. Linington, Glasgow, UK) were cultured under standard conditions (RPMI 1640 medium, 10 % FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 37 °C, 5 % CO2). The binding of TTC fusion proteins to 5 × 10⁵ TTC-reactive hybridoma cells was evaluated by flow cytometry using a BD FACSVerse flow cytometer (Becton Dickinson, Heidelberg, Germany) and BD FACSuite analysis software. Briefly, the cells were incubated with 100 nM purified TTC fusion proteins for 20 min and then with 1 μg/ml anti-His5 AlexaFluor 488 antibody (Cat. no. 35310, Qiagen, Hilden, Germany) for 20 min, in each case on ice with intermediate washes in 1.8 ml 1 x PBS. Dose-dependent binding of TTC-ETA’ was analyzed on TTC-reactive 5E4 hybridoma cells. MOG-reactive 8.18-C5 hybridoma cells were used as a negative control. Briefly, 2 x 10⁵ target cells were incubated with various concentrations (1–400 nM) of TTC-ETA' followed by incubation with 1 μg/mL Penta-His Alexa Fluor 488 Conjugate antibody (Cat. no. 35310, Qiagen, Hilden, Germany). Cells were washed twice with 1x PBS in each step and finally eluted in FACS buffer. The binding activity of TTC-ETA' was analyzed on a BD FACSVerse flow cytometer (Becton Dickinson) using the BD FACSuite analysis software (Becton Dickinson). Mean values of three independent measurements were normalized and fitted using the GraphPad Prism v5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Hybridoma cell viability assay

Hybridoma 5E4 and 8.18-C5 cells (1 x 10⁴ cells/well) were incubated with serially diluted TTC-ETA’ and TTC proteins in cell culture media (RPMI 1640, 10 % v/v FCS) at 37 °C and 5 % CO2 for 72 h. Cell viability was determined by adding XTT/phenazine methosulfate in 50 μl RPMI medium and incubating as above for 4 h. Absorbance was measured at 450 nm and 630 nm (reference wavelength) on a BioTek ELISA reader (Bad Friedrichshall, Germany). Results are presented as the means of three independent experiments with the corresponding standard deviations. The half maximal inhibitory protein concentration (EC50) was calculated relative to untreated control cells. Raw data were fitted using the GraphPad Prism v5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Isolation of B-lymphocytes

For enzyme-linked immunospot (ELISPOT) assays CD27⁺ memory B cells were isolated from leukocyte filters obtained from tetanus-vaccinated blood donors. The local ethics committee approved this procedure. The donors gave written informed consent and were asked for their status of vaccination. First, total B cells were isolated by negative selection using the RosetteSep™ Human B Cell Enrichment Cocktail (STEMCELL Technologies SARL, Grenoble, France) and subsequent density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare, Munich, Germany). After two washing steps with PBS containing 0.5 % BSA (Sigma-Aldrich, Taufkirchen, Germany) the B cells were immediately labeled with CD27 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalogue number: 130-051-601) and subsequently isolated using LS columns and a MidiMACS Separator according to the manufacturer’s instructions (Miltenyi Biotec). Thereafter, the isolated CD27⁺ memory B cells were incubated at a concentration of 10⁵ cells per ml in RPMI 1640 medium supplemented with 10 % v/v FCS, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Life Technologies GmbH, Darmstadt, Germany) on a 96-well round-bottomed plate (Greiner Bio-One, Frickenhausen, Germany) with 2.5 μg/ml CpG oligodeoxynucleotide 2006 (5’-TCG TCGTTTTGTCGTTTTGTCGTT-3’, TIB MolBiol, Berlin, Germany) and 50 ng/ml interleukin (IL)-21 (Life Technologies GmbH, Darmstadt, Germany). After polyclonal activation with the TLR-9 agonist CpG and IL-21, 10 nM TTC or TTC-ETA was added to the culture medium on day 2. On day 4, activated cells were harvested and ELISPOT assays were carried out to determine the frequencies of IgG, anti-tetanus toxoid (TT) IgG as well as anti-TTC specific IgG antibody secreting cells (ASCs). For flow cytometric analyses CD19 MicroBeads (Miltenyi Biotec; Catalogue number: 130-097-055) instead of CD27 MicroBeads were used for positive selection and peripheral blood mononuclear cells (PBMCs) were isolated from a donor 6 and 14 days after booster vaccination with tetanus toxoid to determine the binding of the TTC fusion protein to ex vivo plasma cells and memory B cells, respectively. Briefly, PBMCs were isolated by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare) and stained for TTC-specific CD27⁺⁺CD38⁺⁺ plasma cells or CD27⁺ memory B cells.

Binding analysis of the TTC fusion protein to B-lymphocytes by flow cytometry

The staining was performed with the following monoclonal antibodies against the human B cell specific surface markers CD19 (Becton Dickinson; Catalogue number: 560911), CD27 (Becton Dickinson; Catalogue number: 337169), CD38 (Becton Dickinson, Catalogue number: 345806) and biotinylated IgD (Becton Dickinson; Catalogue number: 555777) in combination with streptavidin Alexa-Fluor 680 (Life Technologies; Catalogue number: S21378) in combination with the recombinant TTC protein followed by an Alexa Fluor 488-conjugated anti-penta-his antibody (Qiagen; Catalogue number: 35310). For comparison, a FITC-labeled TTC peptide (List Biological Laboratories, Inc., CA, USA) was used. For the intracellular TTC staining saponin was added. In the case of surface staining dead cells were excluded by labeling with 220 nM 4’,6-diamidino-2-phenylindole (DAPI; Life Technologies). Flow cytometry was carried out using a FACSCanto™ with FACS DIVA Software (Becton Dickinson) and the results were analyzed using the FlowJo software (Treestar, Ashland, OR, USA).

Quantitation of TT- and TTC-specific ASCs by ELISPOT after polyclonal activation of CD27⁺ memory B cells

After polyclonal activation with the TLR-9 agonist CpG and IL-21 for 4 d, we carried out ELISPOT assays to determine the frequency of IgG⁺ anti-tetanus toxoid (TT) and anti-TTC specific ASCs. Based on the results of the hybridoma cell viability assay, on day 2 of cultivation, the memory B cells were treated with 10 nM TTC or TTC-ETA’. To determine the frequencies of total ASCs, high-binding 96-well ELISA plates (Greiner Bio-One) were pre-coated overnight at 4 °C with goat anti-human kappa and lambda antibodies (SouthernBiotech, AL, USA; Catalogue numbers: SBA1050-04, SBA1060-04). To determine the frequencies of tetanus-specific ASCs, high-binding 96-well ELISA plates were pre-coated with 20 Lf/ml tetanus toxoid (Statens Serum Institute, Copenhagen, Denmark) or with 20 μg/ml TTC protein. After three washes with 150 μl/well PBS plus 0.5 % BSA, the plates were blocked with 200 μl/well PBS containing 3 % BSA and 5 % FCS for 1 h at 37 °C in a 5 % CO2 atmosphere. The obtained cells were titrated and plated in duplicates in the following 10 dilutions: 5–1,000 cells/well for total IgG and 2,000-100,000 cells/well for TT and TTC IgG ELISPOTs. After incubating for 8 h at 37 °C, the plates were washed six times with 150 μl/well PBS. Spots were visualized using AP-conjugated goat anti-human IgG (SouthernBiotech; Catalogue number: SBA1030-04) for 1 h at 37 °C in a 5 % CO2 atmosphere, followed by adding the 2-amino-2-methyl-1-propanol (AMP) buffer supplemented with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitroblue tetrazolium (NBT). After counting the spots, the frequencies of TT and TTC IgG-secreting ASCs were calculated for each individual.

Statistical analysis

Statistical analyses were based on at least three independent series of experiments in triplicates. The two-tailed Student’s t-test was used for statistical analysis. A P value of less than 0.05 was considered statistically significant. Calculations were performed using GraphPad Prism v5 software.

Expression of TTC fusion proteins

The prokaryotic and eukaryotic expression vectors containing the TTC cell-binding domain were sequenced to verify the correct format of the fusion domains. The pBM expression vectors (Fig. 1a) were then introduced into Escherichia coli strain BL21 (DE3) and the pMS expression vector (Fig. 1b) was used for the transient transfection of HEK293T cells. TTC and TTC-ETA’ proteins were periplasmically expressed in E. coli BL21 (DE3) cells under osmotic stress conditions in the presence of compatible solutes. After purification by IMAC using a Ni-Sepharose Superflow cartridge followed by size exclusion chromatography, we achieved yields of up to 1.1 mg TTC (MW: 55 kDa) and 1.8 mg TTC-ETA’ (MW: 95 kDa) per liter of bacterial culture. The purified fusion proteins were analyzed by SDS-PAGE and western blot using a mouse anti-His6 monoclonal antibody (Additional file 1: Figure S1). The SNAP-TTC fusion protein expressed in transfected HEK293T cells was secreted into the cell culture supernatant and purified by IMAC using a Ni-Sepharose Superflow cartridge as above, resulting in a yield of 6 mg per liter of cell culture supernatant. The protein was analyzed by SDS-PAGE and the corresponding western blot confirmed the expression of SNAP-TTC (data not shown). The SNAP-TTC protein was successfully coupled to the fluorescent dyes BG-488 or BG-647 and detected using the CRi Maestro imaging system (Fig. 2a). The SDS polyacrylamide gel was then stained with Coomassie Brilliant Blue and we confirmed that the protein bands matched the corresponding fluorescence signals (Fig. 2b).

Binding of TTC and TTC-ETA’ to hybridoma cells

TTC-ETA’ selectively targets TTC-reactive hybridoma cells and TTC-reactive memory B cells by binding to the TTC-reactive BCR. This binding activity is necessary for the biological function of the protein and was confirmed by investigating the ability of the fusion protein to bind TTC-reactive 5E4 hybridoma cells, which express the membrane-bound form of the BCR. The quantity of protein bound to the cell surface was determined by flow cytometry using an Alexa Fluor 488-conjugated anti-penta-his antibody. This confirmed that both TTC and TTC-ETA’ bound to the TTC-reactive hybridoma cells, whereas no binding was observed on the control cell line (MOG-specific hybridoma 8.18-C5 cells) as shown in Fig. 3a and b. Both hybridoma cell lines showed similar amounts of B cell receptors on their cell surfaces (approx. 1500 receptors per cell) as determined by flow cytometry (data not shown). Coupling SNAP-TTC to BG-647 did not affect the functionality of the protein, and we were able to detect specific binding of the protein to the surface of TTC-reactive hybridoma cells but not to the control hybridoma cells by flow cytometry (Fig. 3c and d). The TTC-ETA’ fusion protein exhibited a concentration-dependent binding to the TTC-reactive hybridoma cell line 5E4 compared to the control hybridoma cell line 8.18-C5 as shown in Fig. 4a and b.

Viability of hybridoma cells following treatment with TTC fusion proteins

After confirming the binding of the TTC fusion protein to the BCR on the surface of TTC-reactive 5E4 hybridoma cells, we investigated the toxicity of the TTC-ETA’ fusion protein in vitro using an XTT cell viability assay. TTC-ETA’ showed specific and dose-dependent toxicity towards TTC-reactive 5E4 hybridoma cells with a half inhibitory protein concentration (EC₅₀) of 1.3 ± 0.4 nM, but they showed no toxicity towards control hybridoma cells (Fig. 5a). Furthermore, the TTC protein alone showed no toxicity towards either of the hybridoma cell lines (Fig. 5b).

Binding analysis of TTC fusion proteins to B-lymphocytes

Next, we determined the binding capacity of the SNAP-TTC-BG-647 protein to polyclonally activated CD19⁺ B cells isolated from leukocyte filters of vaccinated blood donors by flow cytometry. Figure 6 demonstrates that the frequencies of TTC-specific B cells remain low at day 4 after polyclonal activation (intracellular staining, Fig. 6b) compared to residual signals of the control B cells (surface staining, Fig. 6a). Since the frequencies of TTC-specific B cells were extremely low after polyclonal activation in donors without a recent booster vaccination (Fig. 6b), the binding of the TTC fusion protein was additionally verified on plasma cells and memory B cells isolated at day 6 and day 14 from a blood donor vaccinated recently, respectively. The samples were exposed to the recombinant TTC protein followed by an Alexa Fluor 488-conjugated anti-penta-his secondary antibody. Flow cytometry showed that tetanus-reactive CD27⁺ memory B cells (surface staining, Fig. 7a) as well as tetanus-reactive CD27⁺⁺CD38⁺⁺ plasma cells (intracellular staining, Fig. 7c) were detectable within the peripheral B cell pool shortly after booster vaccination. The frequencies of TTC-reactive memory B cells and plasma cells detected using the recombinant TTC protein was comparable to the use of the commercial FITC-conjugated TTC protein (Fig. 7b and d). TTC bound specifically to TTC-reactive memory B cells and plasma cells within the peripheral B cell pool.

Cytotoxic activity of the recombinant TTC-ETA’ protein on polyclonally activated memory B cells

Having confirmed the targeting of TTC-reactive memory B cells, we next set out to measure the depletion effects of the TTC-ETA’ protein. Therefore, CD27⁺ memory B cells were activated polyclonally to develop into antibody-secreting cells (ASCs) and were treated on day 2 of cultivation with 10 nM of recombinant TTC or TTC-ETA’. Next, ELISPOT analyses were performed to determine the frequency of all IgG-secreting cells as well as the TT-specific and TTC-specific IgG-secreting cells. The ASCs secreted IgG antibodies at comparable frequencies after treatment with TTC or TTC-ETA’ (Fig. 8a). The ELISPOT assays were carried out with commercial tetanus toxoid (TT) as the coating agent. When TTC protein was added to the cultures as the control protein, the ASCs secreted the TT-IgG antibodies at a frequency of 0.7 ± 0.6 % (Fig. 8b). Following the treatment with TTC-ETA’ the frequency of TT-IgG antibodies secreted by the ASCs declined to 0.4 ± 0.3 % (Fig. 8b). When tetanus toxoid fragment C (TTC) was used as the ELISPOT coating agent, we observed a small increase in the frequencies of general IgG antibodies following TTC-ETA’ treatment (Fig. 8c). The depletion effect of the TTC-ETA’ protein was confirmed by lower frequencies of secreted TTC-IgG antibodies decreasing significantly from 0.1 ± 0.12 to 0.02 ± 0.03 % (p = 0.0156) compared to treatment with the control protein TTC (Fig. 7d).