Ethics statement

All experimental animal procedures described herein were approved by the University of Louisville's Institutional Animal Care and Use Committee (Protocol number: 12006). The maintenance and care of experimental animals complied with the University of Louisville Animal Care and Use Program's guidelines for the humane use of laboratory animals, which adhere to the National Research Council Guide for the Care and Use of Laboratory Animals, the NIH Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the USDA Animal Welfare Regulations promulgated by the Animal Welfare Act.

Construction of transgenic N. benthamiana expressing CTB

Transgenic N. benthamiana plants were created as previously described [30], using Agrobacterium tumefaciens LBA4404 harboring a pGPTV-kan vector containing the plant-expression-optimized synthetic V. cholerae CTB coding sequence (GenBank accession no. AY475128) with an 18-nucleotide extension (TCCGAGAAGGATGAACTC) at the 3′ end that encodes the C-terminal SEKDEL sequence for ER retention [25], [30].

Analysis of N-glycans attached to transgenic N. benthamiana-expressed CTB

Transgenic Nicotiana-expressed CTB and the Escherichia coli-produced counterpart (see below) were purified as described below and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to a poly(vinylidene difluoride) membrane. Blots were probed with goat anti-CTB antiserum (List Biological Laboratories) followed by horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibodies (Sigma-Aldrich), with 1.5 µg/ml concanavalin A (ConA)-HRP conjugate (Sigma-Aldrich), or with rabbit anti-peroxidase (Sigma-Aldrich) followed by HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology) antibodies. Antibodies were detected using chemiluminescence (ECL Prime; GE Healthcare) and images were acquired on a Kodak Image Station 4000R Pro. Glycan profiling was performed as previously described [30]. The glycans were released from CTB by hydrazinolysis, which were pyridylaminated and separated by reversed phase high-performance liquid chromatography (RP-HPLC) and size-fractionation (SF)-HPLC. The glycan structures were determined by mass spectrometry (MS) and/or by comparing their elution profiles in RP-HPLC with commercial 2-aminopyridine (PA)-labeled standards of known isomeric configurations.

Vector construction

A “deconstructed” tobamovirus replicon system [37], [38] (magnICON; Icon Genetics GmbH) was used to express CTB in N. benthamiana. The CTB-SEKDEL coding sequence used for transgenic plant construction was sub-cloned into the magnICON vector pICH11599 to generate pNM47. A standard PCR method was used to remove the V. cholerae secretory signal from the original CTB gene, using pNM47 as a template. The resulting PCR product was sub-cloned into pIHC11599 to generate pNM134. Site directed mutagenesis was performed according to the manufacturer's instructions (Quikchange II Site-Directed Mutagenesis Kit; Agilent Technologies) using pNM134 as the template and primers that mutated the nucleotide A at position 74 (GenBank accession no. AY475128) to a G creating pNM156 (for Asn4→Ser CTB). For expression of pCTB with different secretory signal peptides, pNM156 was used as a template for PCR. An oligonucleotide corresponding to an appropriate secretory signal sequence (Table S1; N. benthamiana codon optimized) flanking the 5′ coding region of the CTB gene and an oligonucleotide corresponding to the 3′ region of the CTB gene+SEKDEL were used to amplify the secretory signal+CTB+SEKDEL-coding sequence. The resulting PCR products were sub-cloned into pIHC11599 to generate pNM226, 227, 228, 229, 230, 231, 232, and 257, respectively. For expression of pCTB with secretory signals other than the above mentioned sequences, the 5′ provectors pICH20155, pICH20188, pICH20388, and pICH20999 (Table S1) were used.

Viral vector-based overexpression of CTB in N. benthamiana

Plant expression of the pCTB was performed using the magnICON system. For expression of pCTB with a secretory signal attached to the 5′ end of CTB, the plasmids pNM226, 227, 228, 229, 230, 231, 232, and 257 were used with pICH20111 and pICH14011. For CTB with the other secretory signals the appropriate 5′ provector (see above; Table S1) was used with pNM156 and pICH14011. The vectors were delivered into N. benthamiana leaves using the Agrobacterium vacuum infiltration method [39]. After 4–6 days, leaf material was homogenized by a Waring blender in extraction buffer (20 mM Tris-Cl, pH 5.0, 500 mM NaCl, 20 mM ascorbic acid, 10 mM sodium metabisulfite) and the extract was filtered through cheese cloth and miracloth. The extract was warmed to 50°C for 25 min and centrifuged at 22,100×g for 15 min at 4°C followed by filtration through a 0.22 µm filter. The clarified extract was analyzed for CTB expression by SDS-PAGE and GM1-ganglioside-capture enzyme-linked immunosorbent assay (GM1-ELISA) as described previously [25], [30], [40], [41].

Expression of CTB in E. coli (eCTB)

The V. cholerae CTB gene (GenBank accession no. AAC34728) was cloned into pET-22b(+) (Novagen). This plasmid was transformed into electrocompetent BL21(DE3) cells. The bacterial cells were grown to an OD600 of ≈0.6. IPTG was added to a final concentration of 400 mM and the cells were cultured for 4 h. The supernatant containing CTB was separated from the cells by centrifugation at 22,100×g for 15 min at 4°C, and used for purification.

Purification of CTB

For plant-produced CTB, clarified leaf extracts were adjusted to pH 8.0 with a Tris, pH 9.0 buffer. Leaf extracts and E. coli culture supernatants were filtered (0.22 µm) and CTB proteins were purified using the following procedure. Chromatography was performed using an AKTA Purifier (GE Healthcare). Talon Superflow Metal Affinity Resin (Clontech), packed in an XK-26 column (GE Healthcare) to a 50 ml bed volume, was equilibrated with 10 column volumes (CV) of buffer A (20 mM Tris-Cl, pH 8.0, 500 mM NaCl). Samples were loaded at 2.5 ml/min. The column was washed with 8 CV of buffer A. The CTB was eluted with a step gradient using 100% buffer B (Buffer A+150 mM imidazole) and collected by monitoring absorbance at 280 nm. SDS-PAGE was employed to assess the collected fractions for the presence of CTB. The CTB-containing fractions were further purified using a Bio-Rad CHT Hydroxyapatite Fast Flow 5 ml pre-packed column. The column was equilibrated with 10 CV of CHT buffer A (10 mM Tris-Cl, pH 8.0, 5 mM sodium phosphate) and the samples were loaded at a flow rate of 2.5 ml/min followed by a 10 CV wash with buffer A. Proteins were eluted using a gradient from 0 to 100% CHT buffer B (10 mM Tris-Cl, pH 8.0, 500 mM sodium phosphate) over 20 CV. Five ml fractions were collected and the CTB-containing fractions, after verification by SDS-PAGE, were combined, and endotoxin contaminants were removed using a Triton X-114 phase separation method [42]. Then, CTB was ultrafiltrated and diafiltrated into sterile Dulbecco's PBS (DPBS) (Gibco) using Amicon Ultra-15 3000 MWCO centrifugal devices (Millipore) according to the manufacturer's instructions. Endotoxin levels were checked with a Charles River PTS Endotoxin test system (Charles River). The concentrations of purified e- and pCTB were determined using theoretical extinction coefficients at 280 nm of 0.8181 (mg/ml)⁻¹ cm⁻¹ and 0.7660 (mg/ml)⁻¹ cm⁻¹, respectively. The molecular mass of purified pCTB was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS at Columbia University Medical Center Protein Core Facility. Under the conditions used, there is a matrix artifact at +202 Da next to the MH+ peak. An internal standard of myoglobin was added as an internal calibrant.

SF-HPLC

The chromatography was performed on a Beckman Coulter System Gold HPLC. An aliquot (17 µl) of pCTB at 1 mg/ml was applied, at 1.0 ml/min, to an SEC column (YMC-Pack Diol-200, 500×8.0 mm I.D., S – 5 µm, 20 nm) equilibrated with 100 mM sodium phosphate, pH 7.0, 200 mM NaCl. After injection, 100 mM sodium phosphate, pH 7.0, 200 mM NaCl was applied to the column at a flow rate of 1.0 ml/min for 35 min. Before and after analysis, an aliquot (17 µl) of Gel Filtration Standard (Bio-rad) was applied to the column to confirm the integrity of the SEC results. CTB elution was monitored by absorbance at 280 nm.

Biochemical analysis

Competitive GM1-ELISA and hemagglutination assay were performed as described previously [25], [30], [40], [41] except for the competitive ELISA; HRP-CTB (Molecular Probes), at the concentration of 2 µg/ml, was used as a competitor. Fifty percent inhibitory concentrations (IC₅₀) were determined by the GraphPad Prism 5.0 (GraphPad Software). The binding affinity (K_d) of GM1 ganglioside to CTB was measured using a Biacore ×100 2.0 instrument at ambient temperature. Briefly, mouse monoclonal antibodies to β subunit Cholera Toxin (Abcam) (mCTB; 25 µg/ml), were immobilized on a CM5 sensor chip (Biacore) in 10 mM sodium acetate pH 5.0 with a flow rate of 5 µl/min and a 10,000 resonance units (RU) target. A reference flow cell was immobilized with mCTB to correct response contributions such as bulk shifts that occur equally in the sample and reference flow cells. CTB was captured on the mCTB chip with a 200 RU target. Serial dilutions of GM1-ganglioside were made in running buffer (HPS-EP, GE Healthcare) and injected, at a flow rate of 5 µl/min, for a contact time of 60 s and a dissociation time of 600 s. A blank cycle (running buffer) was performed and all sample injections were blank subtracted to correct the sensorgrams for drifts and other disturbances that affect the reference subtracted curve. Between sample injections the system was washed with running buffer and the immobilized surface was regenerated with 10 mM glycine-HCl pH 2.5, 0.05% p20 (10% aqueous solution of polysorbate 20, GE Healthcare) for a contact time of 30 s. A replicate of a non-zero concentration of GM1-ganglioside and the blank were injected in each experiment for double referencing thus verifying the reliability of immobilized chip throughout the experiment. The data were analyzed using the 1∶1 binding kinetics analysis in the Biacore ×100 2.0 evaluation software.

Fluorescence-based thermal shift assay (TSA)

The melting temperatures (T_m) of e- and pCTB were determined by the TSA performed on a Bio-Rad iQ5 multicolor real-time PCR system. Each CTB sample, at a final concentration of 25 µM in PBS, was mixed with a final concentration of 5×Sypro orange (Molecular Probes #S-6650) for a total volume of 20 µl/well in a 96 well plate (USA scientific). Blank controls were set up for protein alone. Samples and blanks were analyzed in triplicate. The plate was heated from 20°C to 95°C in 0.2°C increments at intervals of 15 s. Data were plotted using the GraphPad software. For the acid stability, both, e- and pCTB were diluted to 1.0×10⁻⁴ M in the appropriate buffer and pH was checked in each sample. The buffers consisted of 100 mM sodium citrate (pH 3.0), 150 mM sodium chloride; 100 mM sodium citrate (pH 4.0), 150 mM sodium chloride; 100 mM sodium citrate (pH 5.0), 150 mM sodium chloride; 100 mM sodium phosphate (pH 6.0), and 150 mM sodium chloride; PBS (pH 7.4).

Immunofluorescence and flow cytometry

For immunofluorescence studies, mouse monocyte Raw 264.7 (American Type Culture Collection [ATCC]) cells were cultured in chamber slides at 100,000 cells/cm² in the presence of 10 µg/ml eCTB, pCTB, commercial CTB (Sigma Aldrich), or DPBS for 48 h in a humidified environment with 5% CO₂ at 37°C. Analyses were performed in duplicates. After removal of cell culture supernatants, cells were washed and fixed with 1% paraformaldehyde (PFA) in DPBS plus Ca²⁺ and Mg²⁺ (DPBSA). Cells were permeablized with 0.5% tween-20 in DPBSA for 10 min at RT. After 2 h blocking at RT in 5% normal rabbit serum (Jackson ImmunoResearch), the cells were incubated in goat anti-CTB (List Biological) for 2 hours at RT. Finally, cells were washed and incubated with Rabbit anti-goat IgG Cy3 (Jackson ImmunoResearch) for 1 h at RT in the dark; followed by a 5 min staining with 1 µg/ml 4′,6′-diamidino-2-phenylindole (DAPI; Molecular Probes) in the dark. Images were acquired at 40× magnification on a Zeiss Observer.Z1 and processed with Axiovision AxioVs40 V4.6.30 software. Flow cytometry was used to assess the binding of e- and pCTB molecules to RAW264.7, according to a well-established procedure [43]. Briefly, 5×10⁵ cells were seeded in a 24 well plate in the presence of 10 µg/ml e- or pCTB and incubated for 24 h in a humidified environment with 5% CO₂ at 37°C. Next, cells were washed and blocked with purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Biosciences) on ice. Then cells were exposed to goat anti-CTB antibodies (List Biological Laboratories) for 1 h on ice and washed before incubation with Cy3-conjugated AffiniPure rabbit anti-goat IgG (Jackson ImmunoResearch) for 20 min on ice. Finally, cells were washed and analyzed with a FACSCalibur (Becton Dickinson), counting 10,000 cells per sample. Data were acquired and analyzed using CellQuest Pro from BD and PBS was used as a negative control.

Animal housing

Mice were acclimated for approximately one week prior to the initiation of the studies. 8 week-old female C57BL/6J mice were purchased from The Jackson Laboratory in Bar Harbor (Maine, USA). Four animals were housed in each filtertop microisolator cage in a temperature- and humidity-controlled room with alternating light/dark cycles of 12 h, with access to Laboratory Autoclavable Rodent Diet 5010 (LabDiet) and water ad libitum.

Mouse immunization and sample collections

Mice were randomly divided into groups of 4, and eCTB or pCTB (10 or 30 µg), or PBS vehicle control in a volume of 100 µl was administered orally on days 1 and 15 after neutralization of stomach acids using 200 µl of 30 mg/ml sodium bicarbonate solution. No adjuvant was used for immunization. Blood samples were collected from the submandibular vein in BD Microtainer serum separator tubes (Becton Dickinson) and spun at 6000×g for 5 min to obtain the serum samples. Dry fecal pellets were collected at multiple time points during the experiment as indicated in the results. Fecal samples were prepared as described previously [44] with some modifications. Typically, 100 mg of fecal pellets were homogenized in 1 ml cold 0.05% sodium azide in PBS and the homogenates clarified twice by centrifugation at 10,000×g at 4°C for 10 min. In the Chinese hamster ovary (CHO) cell elongation assay, fecal extract preparation buffers did not include sodium azide and the samples were precipitated with 80% ammonium sulfate, dialyzed against PBS, and filtered through a 0.45 µm Supor membrane (Life Sciences).

Quantification of mucosal and systemic Igs

Total and anti-CTB specific IgA and IgG levels were determined in fecal extracts and serum samples, respectively, using ELISA as previously described [25], [44]. Briefly, MaxiSorp ELISA plates (Nalgene Nunc International) were coated overnight with 1 µg/ml purified eCTB. After blocking, 50 µl of fecal or serum samples in appropriate dilutions were added to the plates. Fecal IgA bound to CTB were detected using goat anti-mouse IgA antibodies conjugated with HRP (SouthernBiotech) and a SureBlue TMB 1-Component Microwell Peroxidase Substrate (KPL, Gaithersburg). Absorbance was measured at 450 nm with a background reading at 570 nm in a Synergy HT plate reader (BioTek) after the reaction was stopped. Serum IgG were quantified similarly using HRP-conjugated anti-mouse IgG antibodies (SouthernBiotech). Dilutions of purified mouse IgA or IgG standards were used to calibrate the ELISA. The titer was defined as the greatest dilution factor of the sample with positive OD₄₅₀ reading after subtracting an average background value.

Lamina propria lymphocyte (LPL) isolation

Lymphocytes from mouse intestinal lamina propria were isolated according to a well-established protocol which involves the removal of Peyer's patches and a series of collagenase digestions of tissue to produce a single-cell suspension [45]. LPLs from the same group of animals were pooled for subsequent analysis.

B-cell enzyme-linked immunospot (ELISPOT) assay

B-cell ELISPOT method was carried out using standard procedures [46], [47]. Briefly, MultiScreen-Filter plates (Millipore) were coated with e- or pCTB (antigen specific) in PBS and blocked with complete RPMI medium at room temperature. After washing, dilutions starting at 10⁷cells/ml LPLs were plated and incubated for 4 h at 37°C in a humid environment with 5% CO₂. Plates were then washed thoroughly and incubated with goat anti-mouse IgA HRP conjugate (SouthernBiotech) overnight at 4°C. Finally, the plates were washed and developed at RT for 10–60 min using freshly prepared amino ethyl carbazole (AEC) substrate solution and the reaction was stopped with distilled water. Spots were counted in each well, using an ImmunoSpot Reader (Cellular Technology, Ltd.) of air dried plates and results expressed as the average value of triplicate wells normalized to the counted number of spots per 10⁶ cells after background correction. For total spot count, the plates were submitted to the same procedure with the notable exception that the wells were coated with rat anti-mouse IgA instead of CTB.

Functional analyses of anti-CTB antibodies

The ability of mouse anti-CTB antibodies to bind CT holotoxin was assessed in GM1-ELISA [44]. MaxiSorp ELISA plates were coated overnight with 2 µg/ml monosialogangliosides GM1 (Sigma-Aldrich) at 4°C. Samples consisting of 100 ng/ml CT were pre-incubated for 1 h at 37°C with equal volumes of protease free fecal extracts or 10-times-diluted serum samples from immunized mice. Fifty µl of each sample was added to the plates followed by 1 h incubation at 37°C. Following a wash step, the plates were incubated with primary anti-CT antibodies (Sigma-Aldrich) for 1 h at 37°C. HRP-conjugated goat anti-rabbit secondary antibodies and the TMB substrate were used for detection as described for antibody titer analysis. The results were expressed as percentages of an average OD value signal obtained from samples treated with sera or fecal samples from PBS immunized animals.

CHO cell elongation assay

CHO AA8 cells [48], [49] from ATCC were cultured according to the manufacturer's instructions. Cell elongation assays were carried out as previously described [50], with some modifications. In brief 2,500 CHO AA8 were seeded per well in a 24-well cluster overnight. Ten times diluted fecal or serum samples were added to equal volumes of CT at a final concentration of 10 ng/ml and incubated in a water bath at 37°C for 1 h. Next, the cells were washed and 50 µl of the pre-incubated samples (Antibodies/CT) were added to 450 µl culture medium and used to treat CHO cells overnight. After staining the cells with Crystal Violet, cell length was measured using a Nikon Eclipse TE300 Microscope and the Metamorph software.

Statistical analyses

Group means, standard deviations (SD), and standard errors of the mean (SEM) were derived from the values obtained in at least 3 independent replicates. Statistical significance was analyzed by a one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test or student's t-test unless otherwise stated, using the GraphPad Prism 5.0 software. Differences were considered statistically significant if P<0.05.

Recombinant CTB expressed in transgenic N. benthamiana is N-glycosylated

Upon obtaining kanamycin-resistant transformants, CTB-expressing lines were screened by GM1-ELISA. The CTB expression level of the selected line was up to 0.5 mg of the protein per kg of leaf material (data not shown).

To characterize the transgenic Nicotiana-expressed recombinant CTB in detail, we purified the protein from leaves using immobilized metal affinity chromatography (IMAC) by taking advantage of CTB's natural chelating activity [51], followed by an additional chromatography step using a hydroxyapatite resin. SDS-PAGE analysis of the purified protein under heat-denaturing conditions revealed two distinct bands; a major band at ∼14.5 kDa and a minor one at ∼12.5 kDa. Non-heat-denaturing conditions showed a single band at 60–70 kDa (Fig. 1A, B). The molecular size of the mature CTB monomer deduced from its primary sequence was 12.3 kDa, corresponding well to the size of the lower band under the heat-denatured conditions. Immunoblot analysis using anti-CTB Abs showed that both bands were indeed CTB-related proteins (Fig. 1A). eCTB (devoid of the C-terminal SEKDEL sequence, with a predicted molecular size of 11.6 kDa) migrated slightly faster than the lower band of plant-expressed protein, suggesting that partial degradation did not give rise to the two sub-species of plant-expressed CTB. Based on previous findings on the possible N-glycosylation of CTB, we postulated that the heterogeneity might be attributed to the inefficient recognition of nascent CTB polypeptides by the oligosaccharyltransferase complex in the ER [31], [32]. Accordingly, lectin- and immuno-blot analysis using ConA and anti-peroxidase Abs (the latter recognizes Xyl- and Fuc-containing plant glycans; for example, see [52]) were performed. The results clearly showed that the upper band, but not the lower one, is modified with plant N-glycans (Fig. 1A). The cause of such inconsistent glycosylation is unclear, but not uncommon for recombinantly produced glycoproteins [53], [54].

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Figure 1. Transgenic N. benthamiana-expressed CTB is N-glycosylated.

A, denaturing SDS-PAGE (Coomassie Brilliant Blue Stained, CBB) and an anti-CTB immunoblot showed that transgenic plant-expressed CTB has two monomer species, whereas the E. coli-produced counterpart has only one. The lectin concanavalin A (ConA) and anti-peroxidase antibodies (Anti-HRP) both bound to the upper band of the plant-expressed CTB, but not to the lower band or the eCTB, demonstrating the presence (in the upper band) and absence (lower band) of plant N-glycans in the plant-expressed B subunit (Lane 1 – CTB purified from transgenic Nicotiana benthamiana; 2 – eCTB) B, non-heat denaturing SDS-PAGE showed that transgenic Nicotiana-expressed CTB and the E. coli-made counterpart were assembled into homo-pentamers. The lane numbers are the same as in Fig. 1A. C, analysis of N-glycan structure showing the presence of plant-specific α(1, 3)-fucose/β(1,2)-xylose-containing glycans. The chromatogram shows RP-HPLC separation of 2-aminopyridine (PA)-labeled glycans isolated from Nicotiana-expressed CTB, with representative glycan structures depicted at corresponding PA-glycan fractions. A horizontal line represents the elution position for PA-labeled glycans. Symbols: circle (○), mannose; square (□), N-acetylglucosamine; diamond (◊), fucose; and triangle (Δ), xylose. D, a crystal structure image showing a CTB monomer complexed with GM1-ganglioside (side view; PBD ID: 3CHB). Dot lines in green represent hydrogen bonds involved in binding to GM1-ganglioside, which is shown in yellow. Asn4, which is glycosylated upon expression in eukaryotic cells, is highlighted in red. It is evident that the Asn4 side chain does not interact with GM1-ganglioside. Image was created in Accelrys Discovery Studio Visualizer 2.5.

https://doi.org/10.1371/journal.pntd.0002046.g001

To further dissect the N-glycosylation of Nicotiana-expressed CTB, we analyzed its glycan composition and structure using a combination of comparative HPLC and MS. Nine distinctive fractions of PA derivatives were identified (Fractions 1–9; Fig. 1C), each of which was subsequently subjected to SF-HPLC to separate further (Fig. S1). Table 1 depicts the composition of N-glycans attached to transgenic Nicotiana-expressed CTB. In spite of the ER retention signal attached to the C-terminus of CTB, which would theoretically provide mannose-rich Man5-9GlcNAc2 oligosaccharides, the majority (>80%) showed more processed glycoforms containing a plant-specific β(1,2)-linked xylose and/or an α(1,3)-linked fucose.

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Table 1. N-glycan composition of CTB expressed in transgenic N. benthamiana.

https://doi.org/10.1371/journal.pntd.0002046.t001

High-level expression of a non-glycosylated CTB variant, pCTB, pentamer was obtained by virus vector-based expression

To eliminate N-glycosylation, we performed site-directed mutagenesis to mutate the AAC codon (Asn4) (amino acid numbering based amino acids 22–126 of GenBank accession no. AY475128 without the secretory signal)→AGC (Ser). Serine was chosen because the closely related E. coli heat-labile enterotoxin B subunit has Ser at the corresponding position (for example, see GenBank accession no. AAC60441), and when examining the structure of CTB, Asn4 is not involved in GM1 binding nor does the Asn side chain stabilize the homo-pentamer structure (Fig. 1D). Thus, we anticipated that such a mutation will not affect CTB's structure or function.

Making stable transgenic lines is time consuming and generally yields low accumulation of foreign proteins (∼1% of total soluble proteins) [42]. Hence, we chose a deconstructed tobamovirus-based vector system [55], [56] to overexpress various pCTB variants in N. benthamiana. GM1-ELISA on crude leaf extracts revealed that the aglycosylated B subunit retained a pentameric form although expression was low, i.e., less than 0.1 g/kg (Fig. 2A, column 1). We hypothesized that changing the secretory signal may lead to an increase in pCTB accumulation. Thus, the original V. cholerae secretory signal (corresponding to amino acids 1 to 21; GenBank accession no. AY475128) was replaced with various other signals of plant origin. GM1-ELISA showed that a secretory signal derived from rice-α amylase provided the highest accumulation of pCTB at 0.5–1.5 g/kg of leaf material (Fig. 2A and C), representing ∼40-fold increase from the V. cholerae secretory signal construct. An extraction profile showed that soluble pCTB pentamers were extracted over a wide pH range, pH 3–8. (Fig. 2B). The pH 5 with salt extract was chosen given that the condition provided the best yields of GM1-binding CTB (Fig. 2C) while removing majority of host-derived proteins including ribulose-1,5-bisphosphate carboxylase oxygenase (Fig. 2B), which will aide in purification. High expression is not unusual for the vector used here; however, our expression level is among the highest for plant-based recombinant protein expression reported to date [42]. Importantly, pCTB is not N-glycosylated, as evident from a single band at 12.5 kDa corresponding to the CTB subunit monomer without a glycan (Fig. 2B, 3A), MS analysis (see below), and ConA blot (data not shown). Collectively, these results indicated that N-glycosylation is not necessary for the overexpression of GM1-ganglioside-binding CTB pentamers.

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Figure 2. Tobamoviral vector-based overexpression of the aglycosylated variant pCTB in N. benthamiana.

A, quantification of pCTB accumulation in leaf extracts with twelve different secretory signals. Lane 1- V. cholerae CTB; 2 - Rice-α amylase; 3 - N. plumbagenifolia calreticulin; 4 - Apple pectinase; 5 - Barley-α amylase; 6 - H. Vulgare chitinase; 7 - S. Tuberosum Glucan endo-1,3-beta-D-glucosidase; 8 - A. Thaliana Auxin-binding protein 1; 9 - P. Atrosepticum Pel B; 10 - P. vulgaris endopolygalacturonase-inhibiting protein; 11 - Tobacco PR1a; and 12 - Rice glutelin. GM1-ELISA was employed to quantify the amounts of receptor-binding pCTB. Data are expressed as mean ± SEM of experimental triplicate. The levels were normalized to an average accumulation level obtained with V. cholerae CTB signal peptide (0.016 g/kg). A representation of two separate experiments is shown. B, SDS-PAGE analysis of crude extracts of N. benthamiana leaves expressing pCTB with rice-α amylase signal peptide. Five days post vector inoculation (dpi), leaf proteins expressing pCTB were extracted with different pH buffers without or with 0.5 M sodium chloride salt (S). The arrow indicates the position of pCTB at ∼12 kDa. Control leaf extract are labeled NI. Extraction at pH 5 effectively removed ribulose-1,5-bisphosphate carboxylase oxygenase for simple and efficient pCTB purification based on chromatography. C, quantification of pCTB expressed with rice-α amylase signal peptide in leaf extracts (5 dpi). Data, expressed in gram of pCTB per kg of leaf material, were obtained by GM1-ELISA and plotted as mean ± SEM of three separate production runs. Column numbers 1–12 correspond to NI pH 2 with salt, pCTB pH 2 with salt, pCTB pH 3 with salt, pCTB pH 4 with salt, pCTB pH 5 with salt, pCTB pH 5, pCTB pH 6 with salt, pCTB pH 6, NI pH 7 with salt, pCTB pH 7 with salt, pCTB pH 7, pCTB pH 8 with salt, pCTB pH 8, respectively.

https://doi.org/10.1371/journal.pntd.0002046.g002

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Figure 3. Purity of pCTB.

A, SDS-PAGE analysis. eCTB (lanes 1 and 3) and pCTB (lane 2 and 4) were purified by IMAC and a hydroxyapatite resin as described in Experimental Procedures. A total of 10 µg of purified protein was loaded in each well. Both CTB proteins appear as monomers under heat denaturing conditions (lanes 1 and 2), whereas mostly as pentamers with a trace amount of decamers under non-heat-denaturing conditions (lanes 3 and 4). B, SF-HPLC analysis of pCTB. The large peak corresponds to the CTB pentamer purified to >95% homogeneity. The minor peak is likely a decamer form (see text for details). C, MALDI-TOF-MS analysis of pCTB. The ions (peaks) in the spectrum of singly charged species ([M+H]⁺; magnified in the right panel) contain the mass-to-charge-ratios (m/z) of 12280.53, 12167.10, 12037.73, and 11923.19. These correspond well to the theoretical m/z values of full-length pCTB (12281.98), and variants lacking the C-terminal Leu (12168.78), Glu-Leu (12039.66), and Asp-Glu-Leu (11924.58), respectively.

https://doi.org/10.1371/journal.pntd.0002046.g003

pCTB retains nanomolar GM1-ganglioside binding affinity and physicochemical stability

The observation that pentameric pCTB was expressed at a remarkably high level prompted us to purify the plant-derived aglycosylated B subunit for additional feasibility assessment. Specifically, it was essential to demonstrate that pCTB is comparable to the native bacterial counterpart as the former has Asn4→Ser mutation and a C-terminal ER retention signal. Extraction with a simple aqueous buffer at pH 5 (Fig. 2B and C) followed by two-step chromatography based on IMAC and hydroxyapatite yielded a highly purified protein, as demonstrated by SDS-PAGE analysis and SF-HPLC. The denaturing SDS-PAGE (Fig. 3A) showed each CTB in its monomeric form whereas the non-heat-denaturing SDS-PAGE (Fig. 3A) illustrated that both CTBs retain pentamer formation. Both, e- and pCTB had a single band and showed >95% purity (based on a densitometric analysis). On the SF-HPLC chromatogram (Fig. 3B), pCTB exhibited one large peak and a small peak at a shorter retention time. Because CTB subunits can form a decameric structure [20], the small peak may represent the decameric form of pCTB. The elution time of the large peak roughly corresponded to the size of a pentameric form (50–60 kDa) and therefore the purity of the pentameric form with trace amounts of the decamer was estimated to be >95% for pCTB. MALDI-TOF-MS (Fig. 3C) was performed to determine the actual mass of pCTB. The predicted molecular mass of pCTB was 12281.98 Da. The spectrogram demonstrated that pCTB had a defined peak at 12280.53 Da, thus corresponding well to the expected molecular mass (<0.02% deviation). The approximately 2 Da difference was likely caused by a loss of 2 hydrogen atoms due to the formation of a disulfide bond between the two cysteines, presumably the intramolecular disulfide bond between Cys-9 and Cys-86, which is essential for proper protein folding and GM1-binding activity [7], [57], [58]. The peaks at 12167.10, 12037.73, and 11923.71 Da corresponded to pCTB variants lacking the C-terminal Leu, Glu-Leu, and Asp-Glu-Leu, respectively. The truncation of the ER retention signal did not affect the functionality of the protein (see below).

To test whether pCTB retains high affinity for GM1-ganglioside, a competitive GM1-ELISA was performed (Fig. 4A). There was no significant difference among the apparent affinities of commercial and the two recombinant CTBs to the receptor; commercial CTB (Sigma-Aldrich), pCTB, and eCTB showed 50% inhibitory concentrations (IC₅₀) of 2.44, 2.85 and 4.51 nM, respectively. A haemagglutination assay with GM1-ganglioside-coated red blood cells [41] also revealed that both p- and eCTB displayed a similar haemagglutination effect in nanomolar range (Fig. S3). Lastly, surface plasmon resonance was performed to determine the dissociation constant, K_d (Fig. 4B). Employing the Biacore ×100 1∶1 binding kinetic analysis, it was found that commercial CTB, pCTB, and eCTB had K_d values of 51.40±5.69, 52.58±5.68, and 53.20±1.25 nM, respectively. There was no statistically significant difference between the three K_d values.

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Figure 4. Characterization of pCTB's GM1-ganglioside-binding activity.

A, binding affinity determination based on competitive GM1-ELISA using HRP-labeled CTB. The assay was performed in triplicate. The 50% inhibitory concentrations (IC₅₀) of commercial, p-, and eCTB were determined by non-linear regression analysis (GraphPad Prism 5.0) to be 2.44, 2.85, and 4.51 nM, respectively. B, SPR. Each CTB protein was immobilized on a sensor chip via a CTB-specific monoclonal antibody, and varying concentrations of GM1-ganglioside were used as analytes. For each protein the assay was performed in triplicate. A representative sensorgram obtained with pCTB is shown. The colored curves represent the concentration of GM1-ganglioside (10, 3.33, 1.11, 0.37, and 0.123 µg/ml from top to bottom), and the black lines are the 1∶1 binding kinetics fit. C, analysis of pCTB's binding to RAW264.7 by immunofluorescence. After 48 h incubation, binding of (a) a PBS control, (b) commercial CTB, (c) pCTB, and (d) eCTB to the cells was detected by anti-CTB primary and Cy3-conjugated secondary antibodies on a Zeiss Observer.Z1 microscope (40×magnification) with the Axiovision AxioVs40 V4.6.30 software. The red signals correspond to the presence of CTB proteins. Nuclei were counterstained with DAPI. No significant difference was noted in cell-surface binding or cell morphology among the three proteins. Scale bar, 20 µm. D, a representative flow cytogram showing RAW264.7 cells bound by e- and pCTB, as detected by Cy3-conjugated secondary antibodies. PBS, eCTB, and pCTB histograms are blue, purple, and green, respectively, demonstrating the comparable binding of the bacterial and plant-made aglycosylated B subunits to the cells.

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We also assessed pCTB's capacity to bind to cell-surface GM1-ganglioside. The immunocytochemistry in Fig. 4C demonstrated that commercial CTB, pCTB, and eCTB all bind to the surface of RAW 264.7 cells in a similar manner. No morphological or growth-pattern difference was noted between cells treated with the B subunits from the three different sources. Cell-surface binding was further confirmed by flow cytometry, where a clear and similar shift was observed for cells treated with e- and pCTB as opposed to control PBS treated cells which did not show any fluorescence (Fig. 4D).

In order to discern potential structural instability associated with mutations introduced to create pCTB, we utilized TSA [59] to determine the T_m of the CTB variants. The bacterial and plant-produced proteins showed T_m of 75.5±0.12°C and 72.9±0.31°C, respectively (Fig. 5A) which is statistically different at P<0.05 as determined by the unpaired t-test. Albeit with the slight decrease of T_m, the results indicate that the amino acid modifications placed on pCTB did not significantly affect the thermal stability of the B subunit. The acid stability of both CTBs in different pH buffers was also examined by TSA. As shown in Table 2, both e- and pCTB had similar melting temperatures at the various pH conditions studied. To corroborate these results, we performed GM1-ELISA using the proteins exposed to varying pH conditions (Fig. 5B). The results again showed that there was no significant difference in the pH stability between e- and pCTBs. Taken together, the above data demonstrated that pCTB holds physicochemical stability comparable to the original protein.

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Figure 5. Thermal and pH stability analysis of pCTB.

A, the melting temperature (T_m) determination using TSA. eCTB (dashed line) and pCTB (solid line) were analyzed in triplicate representing three individual lines per protein in the graph. T_m of e- and pCTB were 75.5°C and 72.9°C, as determined by the vertex of the first derivative of the relative fluorescence unit (RFU) values. B, pH stability analysis by GM1-ELISA. e- and pCTB were diluted to 50 ng/ml in various pH buffers (see methods) and incubated for 5 min, and GM1-bound CTB proteins were quantified by GM1-ELISA. The results were normalized to the average value of the corresponding CTB protein at pH 7.4 and expressed as % binding. Data represent the mean ± SEM (n = 6) ns: values not significantly different at P>0.05 determined by unpaired Student t-test.

https://doi.org/10.1371/journal.pntd.0002046.g005

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Table 2. pH stability of e- and pCTB determined by TSA^a.

https://doi.org/10.1371/journal.pntd.0002046.t002

pCTB is orally active to induce robust antibody responses

The results shown above have demonstrated the integrity of pCTB at the molecular level. We next shifted our investigation to examining the in vivo activity of pCTB. Mice were orally immunized with 3, 10, or 30 µg of e- or pCTB followed by a boost 2 weeks after the initial administration. Serum samples from animals immunized with 30 µg CTB per dose showed similar anti-CTB specific IgG titers, of about 100,000 on average, for both bacterial and plant-manufactured B subunits (Fig. 6A). Serum samples from PBS treated animals did not show any reaction above the background (Fig. 6A). Likewise, fecal extracts obtained from mice immunized with 10 µg pCTB and 30 µg of either pCTB or eCTB showed comparable anti-CTB specific IgA titers above 200 (Fig. 6B). Mice treated with 10 µg eCTB showed slightly lower levels of anti-CTB specific IgA in fecal extracts compared with the other groups mentioned above although the difference was not statistically significant (Fig. 6B). Meanwhile, a weak mucosal immune reaction was obtained from the animals immunized with 3 µg of CTB regardless of the origin, plant-made or bacteria-produced, as quantified by fecal IgA and serum IgG titers (data not shown).

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Figure 6. Oral immunogenicity of N. benthamiana-produced pCTB.

A and B, anti-CTB antibody titers. C57bl/6 mice were orally immunized with PBS, eCTB, or pCTB (10 or 30 µg) and again with the same antigens 2 weeks later. Endpoint titers of serum anti-CTB IgG (A) and fecal anti-CTB S-IgA (B) were analyzed 2 weeks after the second immunization. Horizontal bars show mean titers, whereas symbols represent titers of individual mice (for serum IgG) or triplicate analysis (for fecal IgA; fecal samples of each group were pooled for analysis). There was no significant difference between titers induced by the bacterial and plant-made aglycosylated B subunits (ANOVA with Bonferroni's multiple comparison test). C, representation of the percentage of anti-CTB IgA antibody secreting cells (ASCs) relative to the total IgA ASCs isolated from lamina propria, as determined by B-cell ELISPOT assay. Analysis was done 2 weeks after the second immunization. Data shown are mean ± SEM of triplicate analysis. No significant difference was found among e- and pCTB-immunized groups (ANOVA with Bonferroni's multiple comparison test). D, time-course assessment of CTB-specific IgA levels in fecal extracts. Fecal samples were collected 2 to 24 weeks after the second immunization from mice immunized with PBS, 30 µg eCTB, or 10 or 30 µg pCTB. Anti-CTB IgA levels induced by both bacterial and plant-made aglycosylated B subunits were sustained over 6 months. E, fecal anti-CTB IgA endpoint titers at termination (6 months after the second immunization). Horizontal bars show mean titers. Symbols represent titers of individual mice (fecal samples were not pooled). No significant difference was found among e- and pCTB-immunized groups (ANOVA with Bonferroni's multiple comparison test).

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To dissect the CTB-induced immune response at the cellular level, the number of anti-CTB IgA secreting cells isolated from the lamina propria of small intestine were determined in an ELISPOT assay. The total spots representing all cells secreting IgA in the population was similar across groups, including PBS and CTB-immunized animals. As expected, no anti-CTB specific antibody producing cells were detected from PBS treated animals (Fig. 6C). The numbers of anti-CTB IgA-producing cells were comparable between animals that received the same dose of CTB regardless of the system used to produce the protein, whether bacterial or plant-made. There was a trend of dose dependent increase in the number of specific spots, whereby ∼4% of total IgA-secreting cells were CTB-specific with 30 µg dose. At 3 µg dose, both p- and eCTB induced ∼1% of CTB-specific IgA-secreting cells (data not shown).

We also assessed the duration of the anti-CTB specific IgA response in fecal samples after immunization. As shown in Fig. 6D, anti-CTB IgA levels remained high throughout the 6 months that the experiment lasted. At termination the animals treated with 30 µg pCTB or eCTB showed similar titers for anti-CTB specific IgA, relatively higher but not significant compared with the mice that were administered 10 µg of pCTB (Fig. 6E).

pCTB-induced antibodies effectively neutralized cholera holotoxin (CT)

To evaluate the efficacy of antibodies raised against p- and eCTB, we exploited CT holotoxin's ability to bind to GM1-ganglioside. As seen in Fig. 7A, incubation of CT with fecal samples from CTB-immunized mice resulted in significantly less signal compared to samples collected from PBS immunized control group, with inhibition rates between 20–60%, indicating the neutralization of the holotoxin by e- and pCTB-induced intestinal antibodies. The results suggested that the plant-made aglycosylated B subunit has similar if not better efficacy in comparison with the bacteria-derived original protein. Likewise, serum samples collected from bacterial or plant-produced CTB-vaccinated mice prevented the binding of CT to GM1-ganglioside in a dose dependent manner, with both e- and pCTB showing comparable efficacy strengths (Fig. 7B).

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Figure 7. Inhibition of CT holotoxin's GM1-ganglioside binding by pCTB-induced antibodies.

A and B, relative amounts of GM1-bound holotoxin in the presence of (A) fecal and (B) serum Igs. CT holotoxin (100 ng/ml) was pre-incubated with undiluted fecal extracts (A) and 10× diluted sera (B) obtained from animals orally immunized with PBS, 30 µg eCTB, or 10 or 30 µg pCTB and analyzed in a GM1-ELISA. The results are shown as % of the average amount of CT detected with the PBS control group. Data shown are mean ± SEM of triplicate. Both fecal and serum Igs were prepared from animals 2 weeks after the second vaccination. For fecal Igs, all groups showed significant inhibition. For serum Igs, groups that were immunized with 30 µg pCTB and 30 µg eCTB showed significant inhibition: **P<0.01; ***P<0.001 (ANOVA with Bonferroni's multiple comparison test).

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The CHO cell elongation assay [50] was also employed to assess the inhibitory potential of p- and eCTB-induced antibodies. As shown in Fig. 8A, fecal samples from p- and eCTB-treated animals inhibited the CHO cell elongation in a significant manner compared with the extracts obtained from the PBS control group. However, the inhibition was not complete (Fig. 8A, D, E, F, and H), suggesting the partial efficacy at this dilution (10×) of fecal samples. There was no statistically significant difference across groups, e- or pCTB (Fig. 8A, D–F, and data not shown). Higher concentrations of fecal extract samples could not be analyzed due to cell toxicity. By contrast, all serum samples from animals immunized with eCTB, 30 µg, or pCTB, 10 or 30 µg completely abolished the CHO elongation effect of CT (Fig. 8B, H–K). Such high efficacy of serum samples is likely attributed to the higher concentrations of serum Igs than those in fecal samples. As expected fecal extracts and serum samples obtained from PBS immunized mice did not inhibit cell elongation in CHO culture since treatment with these samples lead to a similar cell length compared to the cells incubated in presence of CT only (Fig. 8C, G, and L). Collectively, above results provided compelling evidence that plant-made aglycosylated pCTB can induce holotoxin-neutralizing antibodies upon oral immunization, and this effect is comparable to that of the bacterially produced original B subunit.

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Figure 8. Neutralization of CT holotoxin by pCTB-induced antibodies in the CHO cell elongation assay.

A and B, quantitation of the CHO cell elongation by CT holotoxin pre-treatment with 10× diluted (A) fecal and (B) serum Ig samples. The holotoxin (20 ng/ml) was pre-incubated with 10× diluted fecal or serum samples obtained 2 weeks after the second vaccination from animals immunized with PBS, 10 or 30 µg pCTB, or 30 µg eCTB. Cell length was measured after overnight incubation and crystal violet staining. See Experimental Procedure for the detailed method. Data are shown as mean ± SEM of triplicate. For both fecal and serum Igs, all immunized groups showed significant neutralization of CT-induced cell elongation compared to the PBS control group: ***P<0.001 (ANOVA with Bonferroni's multiple comparison test). C, cells treated with CT. D–G, cells treated with CT and fecal samples from animals immunized with 10 µg pCTB, 30 µg eCTB, 30 µg pCTB, and PBS, respectively. H, untreated control cells. I–L, cells treated with CT and serum samples from animals immunized with 10 µg pCTB, 30 µg eCTB, 30 µg pCTB, and PBS, respectively. Scale bar, 100 µm.

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