Name: Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes (Video) | JoVE Journal
Uploaded: 2019-03-19T15:00:00.000+00:00
Duration: 10 min 16 s
Description: Learn transient expression in red beet for oral vaccine development against Type 1 diabetes using plant-based systems. This method enables high-yield recombinant protein production for immunological applications.

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5 minutes

This work was supported by the Joint Project &#8220;The use of plants for the production of an autoimmune diabetes edible vaccine (eDIVA)&#8221; (Project ID: 891854) funded by the University of Verona in the framework of the call 2014.

1. Red beet and spinach cultivation
<ol>
	<li>Grow red beet (B. vulgaris cv Moulin Rouge) and spinach (S. oleracea cv Industria) plants in a growth chamber, using 150 &#181;E of light intensity, 65% relative humidity, 12 h light/dark cycle at 23/21 &#176;C, respectively.</li>
	<li>After seed germination, fertilize the plants twice a week with a 1 g/L solution of a commercially available fertilizer (Table of Materials). For agroinfiltration use five-week-old spinach and six-week-old red beet plants.</li>
</ol>
2. Transient expression through the deconstructed plant virus-based technology
<ol>
	<li>Construction of plant expression vectors
	<ol>
		<li>Introduce the vectors - the 5&#8217; module (pICH20111), the 3&#8217; modules (pICH31070.GAD65mut, pICH31070.&#8710;87G65mut and pICH7410.eGFP), prepared as previously described1,2,7, and the integrase module (pICH14011) - in Agrobacterium tumefaciens GV3101 strain using standard techniques. Grow on LB medium containing 50 &#956;g/mL rifampicin and appropriate vector-specific antibiotics (50 &#956;g/mL carbenicillin for pICH20111, pICH14011 and pICH7410.eGFP, 50 &#956;g/mL kanamycin for pICH31070) for 2 days at 28 &#176;C.</li>
		<li>Screen the colonies by colony PCR using the following specific primers for each vector: 5&#8217;-ATCTAAGCTAGGGTACCTCG-3&#8217; and 5&#8217;-ACACCGTAAGTCTATCTCTTC-3&#8217; for both the 3&#8217; modules pICH31070.GAD65mut and pICH31070.&#8710;87G65mut, with an annealing temperature of 55 &#176;C and an elongation time of 110 s, 5&#8217;-TGAAGTTCATCTGCACCAC-3&#8217; and 5&#8217;-ACACCGTAAGTCTATCTCTTC-3&#8217; for the third 3&#8217; module pICH7410.eGFP, with an annealing temperature of 53 &#176;C and an elongation time of 30 s, 5&#8217;-AATGTCGATAGTCTCGTACG-3&#8217; and 5&#8217;-TCCACCTTTAACGAAGTCTG-3&#8217; for the 5&#8217; module, with an annealing temperature of 53 &#176;C and an elongation time of 20 s and 5&#8217;-GGCAACCGTTATGCGAATCC-3&#8217; and 5&#8217;-GATGCGTTCCGCAACGAACT-3&#8217; for the integrase module with an annealing temperature of 57 &#176;C and an elongation time of 45 s.</li>
		<li>Carry out the PCR reaction in a total volume of 20 &#956;L using the following specific reaction cycle: 5 min initial denaturation at 95 &#176;C, 35 cycles of 30 s at 95 &#176;C, 30 s at the annealing temperature and the elongation step at 72 &#176;C (annealing temperature and elongation time are specific for each primer couple) and a final elongation step of 7 min at 72 &#176;C.</li>
	</ol>
	</li>
	<li>Syringe agroinfiltration
	<ol>
		<li>Inoculate the three A. tumefaciens transformants in 50 mL of LB medium containing 50 &#956;g/mL rifampicin and the following appropriate vector-specific antibiotics: 50 &#956;g/mL carbenicillin for A. tumefaciens transformed with pICH20111, pICH14011 or pICH7410.eGFP, or 50 &#956;g/mL kanamycin for A. tumefaciens transformed with pICH31070. Grow by shaking overnight at 28 &#176;C.</li>
		<li>Pellet overnight bacterial cultures by centrifugation at 4,500 x g for 20 min and resuspend them in 100 mL (or two volumes of the initial bacterial culture) of infiltration buffer containing 10 mM 4-morpholineethanesulfonic acid (MES; pH 5.5) and 10 mM MgSO4, without considering the OD600. Incubate the suspensions at room temperature (RT) for 3 h.</li>
		<li>Mix equal volumes of bacterial suspensions containing one of the three modules, GAD65mut, &#8710;87GAD65mut or eGFP 3&#8217; module, with 5&#8217; module and integrase module. Use the suspension mix for syringe infiltration of red beet and spinach leaves.</li>
		<li>Place 5 mL of the suspension in a syringe without the needle. Press the tip of the syringe against the underside of the leaf for both spinach and red beet plants, and meanwhile apply a gentle counterpressure to the other side of the leaf.</li>
		<li>Infiltrate the first three completely expanded leaves starting from the apex for each plant. Label the agroinfiltrated leaves with a paper tag on the leaf stem. Return the plants in a growth chamber under standard conditions. 
		NOTE: For health and safety reasons, wear eye protection and gloves during infiltration process.</li>
		<li>Collect agroinfiltrated leaves from 4 to 14 days post infection (dpi) and freeze them in liquid nitrogen. Store plant tissue at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Vacuum agroinfiltration
	<ol>
		<li>Grow separately the three A. tumefaciens transformants in 50 mL of LB medium containing 50 &#956;g/mL rifampicin and appropriate vector-specific antibiotic by shaking overnight at 28 &#176;C.</li>
		<li>Pellet overnight bacterial cultures by centrifugation at 4,500 x g for 20 min. Resuspend the pellet in 1 L of infiltration buffer to an OD600 of 0.35 and incubate the suspensions at RT for 3 h.</li>
		<li>Add 0.01% v/v of the detergent (polysorbate 20) to each suspension. Mix equal volumes of bacterial suspensions containing one of the three modules, GAD65mut, &#8710;87GAD65mut or eGFP 3&#8217; module, with 5&#8217; module and integrase module.</li>
		<li>Insert one plant (six-week-old red beet plant, see section 1) in the holder. Invert the holder and place on top of a beaker containing the infiltration bath (2 L) to submerge the leaves in the infiltration suspension. 
		NOTE: Ensure that all the leaves are completely dipped in the bacterial suspension. Raise the level with the addition of extra infiltration suspension if required.</li>
		<li>Transfer the infiltration bath with the submerged plant to the infiltration chamber and close it. Turn on the vacuum pump and open the vacuum intake valve on the infiltration chamber.</li>
		<li>Once the pressure in the infiltration chamber has reduced to 90 mbar, keep the vacuum for 3 min. Release the vacuum for 45 s. Once the infiltration chamber has returned to atmospheric pressure, open the chamber and remove the infiltrated plant from the bacterial bath.</li>
		<li>Return the plants in a growth chamber under standard conditions.</li>
		<li>Collect agroinfiltrated leaves at the maximum expression dpi, depending on the recombinant protein, and freeze them in liquid nitrogen. Store plant tissue at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Recombinant protein expression analysis
<ol>
	<li>Total soluble protein (TSP) extraction
	<ol>
		<li>Grind the syringe or vacuum infiltrated red beet and spinach leaves, collected in steps 2.2.6 and 2.3.8, to fine powder in liquid nitrogen using mortar and pestle. Transfer the powder into 15 mL plastic tubes and store the material at -80 &#176;C.</li>
		<li>Add 900 &#181;L of extraction buffer (50 mM sodium phosphate pH 8.0, 20 mM sodium metabisulphite) to 300 mg of leaf powder. 
		NOTE: The selected ratio between plant tissue weight (mg) to buffer volume (&#181;L) is 1:3.</li>
		<li>Homogenize the mixture by vortexing for 1 min, then centrifuge at 30,000 x g for 40 min at 4 &#176;C.</li>
		<li>Collect the supernatant in a clean tube and store it at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Plant eGFP visualization and quantification
	<ol>
		<li>Load 100 &#181;L of each TSP extract obtained from eGFP expressing leaves, in three technical replicates, on a 96-well plate.</li>
		<li>Put the 96-well plate in a fluorescence reader and start the measurement. Use the 485/535 nm filter set required for eGFP fluorescence detection.</li>
		<li>For the absolute quantification, in the same plate prepare a calibration curve loading different quantities (62.5, 125, 500, 750 and 1,000 ng) of a purified eGFP.</li>
	</ol>
	</li>
	<li>Bicinchoninic acid (BCA) assay for TSP quantification
	<ol>
		<li>Mix 50 parts of Reagent A (Table of Materials) with 1 part of Reagent B (Table of Materials). Prepare sufficient volume of fresh BCA working solution for the samples to be assayed and the calibration standards. 
		NOTE: The volume of BCA working solution required for each sample is 1.9 mL. For the standard procedure with 9 standards (including a blank), 17.1 mL of BCA working solution is required.</li>
		<li>Pipette 0.1 mL of each standard (including a blank), and TSP extracts into a labelled tube. 
		NOTE: As calibration standards, prepare a fresh set of bovine serum albumin (BSA) standards in the 10-1,000 &#956;g/mL range, preferably using the same diluent as samples, such as water. The blank consists of 0.1 mL of the diluent used for calibration standard and sample preparation.</li>
		<li>Add 1.9 mL of BCA working solution and mix thoroughly. Cover the tubes and incubate at 37 &#176;C for 30 min.</li>
		<li>Cool the tubes to RT. Measure the absorbance at 562 nm of all the samples within 10 min. 
		NOTE: Even at RT, the color development continues. No significant error will be introduced if the absorbance measurements of all tubes are done within 10 min.</li>
		<li>Subtract the 562 nm absorbance value of the blank from the readings of the standards and the TSP extracts.</li>
		<li>Plot the blank-corrected 562 nm reading for each standard on its concentration. Determine the protein concentration of each TSP extract.</li>
	</ol>
	</li>
	<li>Perform Coomassie gel staining as previously described in Gecchele et al.8.</li>
	<li>Western blot analysis
	<ol>
		<li>Perform the western blot analysis as previously described in Gecchele et al.8.</li>
		<li>After the electrophoretic separation of proteins, transfer them onto a nitrocellulose membrane using standard techniques. Prepare the blocking solution by mixing 4% milk in phosphate-buffered saline (PBS) pH 7.4. Block the membrane with 10 mL of the blocking solution at RT for 1 h.</li>
		<li>Prepare the rabbit primary antibody at 1:10,000 for the anti-GAD65/67 and anti-LHCB2, and at 1:20,000 for the anti-eGFP in 5 mL of blocking solution with 0.1% detergent. Incubate the membrane with the prepared primary antibody solutions overnight at 4 &#176;C or for 4 h at RT with constant agitation.</li>
		<li>Discard the primary antibody and wash the membrane 3 times for 5 min each with blocking solution containing 0.1% detergent.</li>
		<li>Prepare the horseradish peroxidase (HRP)-conjugate anti-rabbit antibody at 1:10,000 in blocking solution with 0.1% detergent. Incubate the membrane for 1.5 h at RT with constant agitation.</li>
		<li>Discard the secondary antibody and wash the membrane 5 times for 5 min each with PBS-T (PBS supplemented with 0.1% detergent).</li>
		<li>Incubate the membrane with a commercially available luminol solution following the manufacturer&#8217;s instructions. Detect the signal using a chemiluminescence imaging system.</li>
	</ol>
	</li>
</ol>
4. Plant material processing
<ol>
	<li>Harvest the vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaves at the expression peak (11 dpi) and freeze them in liquid nitrogen.</li>
	<li>Lyophilize the frozen leaves for 72 h at -50 &#176;C and 0.04 mbar. Store them at -80 &#176;C.</li>
	<li>Grind the leaves to fine powder and store it at RT in a sealed container with silica gel to exclude the moisture.</li>
</ol>
5. Gastric digestion simulation and cell integrity analysis
<ol>
	<li>Gastric digestion simulation
	<ol>
		<li>Weigh 100 mg of grinded freeze-dried red beet leaves and resuspend it in 6 mL of PBS (pH 7.4).</li>
		<li>Adjust the sample pH to 2 with 6 M HCl.</li>
		<li>Add 4 mg/mL pepsin from porcine gastric mucosa in 10 mM HCl to obtain a final pepsin concentration of 1 mg/mL or a ratio of 1:20 to total soluble proteins. Shake the sample at 37 &#176;C for 120 min.</li>
		<li>Adjust the samples to pH 8 with 1 M NaOH to inactivate the pepsin.</li>
		<li>Centrifuge 750 &#181;L aliquots of each sample at 20,000 x g for 20 min at 4 &#176;C. Collect separately the supernatant and resuspend the pellet in one supernatant volume of loading buffer (1.5 M Tris HCl, pH 6.8, 3% SDS, 15% glycerol, and 4% 2-mercaptoethanol). 
		NOTE: For health and safety reasons, wear gloves and work under fume hood for sample preparation.</li>
		<li>Analyze both the supernatant and the resuspended pellet by western blot analysis8.</li>
	</ol>
	</li>
	<li>Cell integrity analysis
	<ol>
		<li>Prepare two samples of 100 mg of grinded freeze-dried red beet leaves and resuspend both in 6 mL of PBS (pH 7.4).</li>
		<li>Adjust the pH of only one sample to 2 with 6 M HCl. Shake both samples at 37 &#176;C for 120 min.</li>
		<li>Centrifuge 750 &#181;L aliquots of each sample at 20,000 x g for 20 min at 4 &#176;C. Collect separately the supernatant and resuspend the pellet in one supernatant volume of loading buffer.</li>
		<li>Analyze both the supernatant and the resuspended pellet by western blot analysis.</li>
	</ol>
	</li>
</ol>
6. Bioburden assay
<ol>
	<li>Weigh 100 mg of freeze-dried red beet leaves. Resuspend the powder in 8 mL of sterile PBS (pH 7.4) and vortex for 1 min.</li>
	<li>Prepare the LB medium without antibiotics or containing (i) 50 &#181;g/mL rifampicin, (ii) 50 &#181;g/mL each of rifampicin and carbenicillin, (iii) 50 &#181;g/mL each of rifampicin and kanamycin, or (iv) 50 &#181;g/mL each of rifampicin, carbenicillin, and kanamycin.</li>
	<li>Plate 1 mL of each freeze-dried leaf homogenate in one of the 5 selective LB media.</li>
	<li>Incubate all the plates for 3 days at 28 &#176;C.</li>
	<li>Count the Agrobacterium colonies grown on each plate.</li>
	<li>Calculate and define the residual bacterial charge as the number of colony forming units (CFU) per mL of the freeze-dried leaf homogenate (CFU/mL).</li>
</ol>
7. Metabolite extraction
<ol>
	<li>Primary metabolite extraction
	<ol>
		<li>Weigh 30 mg of -80 &#176;C stored, vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaf powder in a 2 mL plastic tube.</li>
		<li>Add 750 &#181;L of cold 70/30 (v/v) methanol/chloroform and vortex for 30 s. Incubate at -20 &#176;C for 2 h. 
		NOTE: All the solvents and additives must be LC-MS grade.</li>
		<li>Add 600 &#181;L of cold water and centrifuge at 17,900 x g at 4 &#176;C for 10 min. Collect and transfer the upper hydroalcoholic phase into a new 2 mL tube. Discard the lower chloroformic phase and the interphase.</li>
		<li>Put the samples into a vacuum concentrator for 3 h to evaporate the solvents.</li>
		<li>Dissolve the pellet obtained from step 7.1.4 in 300 &#181;L of 50/50 (v/v) acetonitrile/water and sonicate the samples for 3 min.</li>
		<li>Pass the solutions through 0.2 &#956;m membrane filters and put them into a transparent fixed 300 &#181;L insert glass tubes.</li>
	</ol>
	</li>
	<li>Secondary metabolite extraction
	<ol>
		<li>Weigh 300 mg of -80 &#176;C stored, vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaf powder in a 15 mL plastic tube.</li>
		<li>Add 3 mL of methanol, vortex for 30 s, and sonicate at 40 kHz for 15 min. 
		NOTE: All the solvents and additives must be LC-MS grade.</li>
		<li>Centrifuge the samples at 4,500 x g at 4 &#176;C for 10 min and transfer the supernatants into new 15 mL tubes.</li>
		<li>Dilute 100 &#181;L of extract 1:3 (v/v) with water and pass the solution through a 0.2 &#956;m membrane filter.</li>
		<li>Put the solution into a transparent fixed 300 &#181;L insert glass tube.</li>
	</ol>
	</li>
	<li>Polar lipid extraction
	<ol>
		<li>Weigh 200 mg of -80 &#176;C stored, vacuum agroinfiltrated &#8710;87GAD65mut-expressing red beet leaf powder in a 15 mL plastic tube.</li>
		<li>Add 200 &#181;L of water and then 2 mL of methanol, and then keep on ice for 1 h. 
		NOTE: All the solvents and additives must be LC-MS grade.</li>
		<li>Vortex for 30 s and sonicate at 40 kHz for 15 min.</li>
		<li>Centrifuge the samples at 4,500 x g at 4 &#176;C for 25 min. Collect and transfer the chloroform phase into 2 mL tubes. Discard the upper hydroalcoholic phase and the interphase.</li>
		<li>Put the samples into a vacuum concentrator for 3 h to evaporate the solvent.</li>
		<li>Dissolve the pellet obtained from step 7.3.5 with 600 &#181;L of methanol.</li>
		<li>Dilute 100 &#181;L of extract 1:5 (v/v) with methanol and pass the solution through a 0.2-&#956;m membrane filter.</li>
		<li>Put the solution into a transparent fixed 300 &#181;L insert glass tube.</li>
	</ol>
	</li>
</ol>
8. Liquid chromatography mass spectrometry analysis and data processing
<ol>
	<li>Set up the LC-MS system as recommended by the supplier. 
	NOTE: The LC section consists of an autosampler coupled with HPLC equipped with a C18 guard column (75 x 2.1 mm, particle size 5 &#181;m) in front of a C18 column (150 x 2.1 mm, particle size 3 &#181;m) for the analysis of secondary metabolites and polar lipids, whereas with a HILIC guard column (7.5 x 2.1 mm, 3 &#181;m) in front of an HILIC column (150 x 2.1 mm, particle size 2.7 &#181;m). The MS is an ion trap mass spectrometer provided with either an electrospray ionization (ESI) or an atmospheric pressure chemical ionisation (APCI) sources.</li>
	<li>Prepare the appropriate solvents for the HPLC gradients. For primary metabolite analysis, use 20 mM ammonium formate as solvent A; 95% acetonitrile, 5% water plus 10 mM ammonium formate as solvent B. For secondary metabolite analysis, use water plus 0.05% formic acid (A) and acetonitrile plus 0.05% formic acid (B). For polar lipid analysis, use water plus 0.05% formic acid (A) and 100% acetonitrile (B). 
	NOTE: The solvents and additives must be LC-MS grade.</li>
	<li>Use the gradients reported in Table 1 for metabolite elution. Set the flow rate at 0.2 mL/min. Inject 10 &#181;L of each sample for both secondary metabolites and polar lipids, whereas inject 5 &#181;L for primary metabolites.</li>
	<li>Prepare a quality control (QC) sample by mixing equal portions of different samples to have a representative mixture of each experimental condition. Analyse the QC sample during the experiment to monitor instrument efficiency. Specifically, insert a QC sample analysis after each 10 sample-batch.</li>
	<li>Randomize the samples to avoid instrument-driven effects.</li>
	<li>After 9 analyses, insert a column cleaning method and a blank analysis immediately after. 
	NOTE: Perform a slow gradient between the two solvents with an isocratic elution at high percentage of the strongest solvent. The blank consists of an injection of a pure methanol: water (50/50, v/v) to improve retention time reproducibility in the following analysis.</li>
	<li>Set up the instrument to acquire mass spectra in alternate positive and negative ionization modes using the parameters listed in Table 2. 
	NOTE: Other parameters depend on the specific platform.</li>
	<li>Perform the next data processing as explained in Dal Santo et al.9.</li>
</ol>

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<li>Kamei, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complexation+hydrogels+for+intestinal+delivery+of+interferon+beta+and+calcitonin%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Complexation hydrogels for intestinal delivery of interferon beta and calcitonin.</a> Journal of Controlled Release. 134, 98-102 (2009).</li>
<li>Tuesca, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complexation+hydrogels+for+oral+insulin+delivery%3A+effects+of+polymer+dosing+on+in+vivo+efficacy%5BTitle%5D)+AND+%22Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Complexation hydrogels for oral insulin delivery: effects of polymer dosing on in vivo efficacy.</a> Journal of Pharmaceutical Sciences. 97, 2607-2618 (2008).</li>
<li>Twyman, R. M., Schillberg, S., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+the+yield+of+recombinant+pharmaceutical+proteins+in+plants%5BTitle%5D)+AND+%22Current+Pharmaceutical+Design%22%5BJournal%5D)">Optimizing the yield of recombinant pharmaceutical proteins in plants.</a> Current Pharmaceutical Design. 19, 5486-5494 (2013).</li>
<li>Dhama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant-based+oral+vaccines+for+human+and+animal+pathogens+%E2%80%93+a+new+era+of+prophylaxis%3A+current+and+future+perspectives%5BTitle%5D)+AND+%22Journal+of+Experimental+Biology+and+Agricultural+Sciences%22%5BJournal%5D)">Plant-based oral vaccines for human and animal pathogens – a new era of prophylaxis: current and future perspectives.</a> Journal of Experimental Biology and Agricultural Sciences. 447 (0), 75-93 (2013).</li>
<li>Hefferon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconceptualizing+cancer+immunotherapy+based+on+plant+production+systems%5BTitle%5D)+AND+%22Future+science%22%5BJournal%5D)">Reconceptualizing cancer immunotherapy based on plant production systems.</a> Future science. O3, FSO217 (2017).</li>
</ol>

The authors have nothing to disclose.

In this study we showed preliminary analysis for the design of a candidate oral vaccine for autoimmune diabetes. The target protein for this experiment was a mutated form of the human 65 kDa Glutamate Decarboxylase, which production and functionality are easily detectable and measurable12. Its expression in different edible plant tissues was mediated by the vectors5, which mediate a high level of recombinant protein production in a very short time frame. The selection of th...

Since the plant molecular biology revolution in 1980s, plant-based systems for the production of biopharmaceuticals can be considered as an alternative to traditional systems based on microbial and mammalian cells1. Plants display several advantages over traditional platforms, with scalability, cost-effectiveness and safety being the most relevant2. The recombinant product can be purified from transformed plant tissue and then administered, either parenterally or orally and, moreover, transformed edible plant can be used directly for oral delivery. The oral route simultaneously promotes mucosal and systemic immunity, and it eliminates the need for needles and specialized medical personnel. Furthermore, oral delivery eliminates the complex downstream processing, which normally accounts for 80% of the total manufacturing cost of a recombinant protein3. All those advantages can be translated into savings in production, supplies and labor reducing the costs of each dose, making the drug affordable to most of the global population.
Several strategies, both for stable transformation and transient expression, were developed for the production of recombinant proteins in plants. Among them, a high-yield deconstructed plant virus-based expression system (e.g., magnICON) provides superior performance leading high yields of recombinant proteins over relatively short timescales4. Many examples of transient expression using the plant virus-based expression&#160;system in Nicotiana benthamiana plants are reported, being the gold standard production host. However, this model plant is not regarded as an edible species due to the alkaloids and other toxic metabolites that are accumulated in its leaves.
In this work, we describe the comparison between two edible plant systems, red beet (Beta vulgaris cv Moulin Rouge) and spinach (Spinacea oleracea cv Industria), for the expression of two candidate forms of the 65 kDa isoform of glutamic acid decarboxylase (GAD65), carried out by the plant virus-based vectors5. GAD65 is a major autoantigen associated to Type 1 Diabetes (T1D) and it is currently under investigation in human clinical trials to prevent or delay T1D by inducing tolerance6. The production of GAD65 in plants has been extensively studied in model plant species as Nicotiana tabacum and N. benthamiana4,5,6,7. Here, we describe the use of edible plant species for the production of the molecule in tissues that can be meant for a direct oral delivery. From a technical point of view, we studied and selected the system for plant agroinfiltration and the edible plant platform for GAD65 production by evaluating different parameters: the recombinant protein expression levels, the residual microbial charge in plant tissue meant for oral delivery, the resistance of GAD65 to the gastric digestion, and the bioequivalence of the transformed plants with the wild type.

<table><tbody><tr><td>0.2-&amp;mu;m Minisart RC4 membrane filters</td><td>Sartorius-Stedim</td><td>17764</td><td></td></tr><tr><td>2&amp;ndash;mercaptoethanol</td><td>Sigma</td><td>M3148</td><td>Toxic; 4 % to make loading buffer with glycerol, SDS and Tris-HCl</td></tr><tr><td>4-Morpholineethanesulfonic acid (MES)</td><td>Sigma</td><td>M8250</td><td>pH 5.5</td></tr><tr><td>96-well plate</td><td>Sarstedt</td><td>833924</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>27221</td><td>Corrosive</td></tr><tr><td>Acetonitrile LC-MS grade</td><td>Sigma</td><td>34967</td><td></td></tr><tr><td>Acetosyringone</td><td>Sigma</td><td>D134406</td><td>Toxic &amp;ndash; 0.1 M stock in DMSO</td></tr><tr><td>Agar Bacteriological Grade</td><td>Applichem</td><td>A0949</td><td>15 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Tryptone</td></tr><tr><td>Ammonium formate</td><td>Sigma</td><td>70221</td><td></td></tr><tr><td>Anti-eGFP antibody</td><td>ABCam</td><td>ab290</td><td></td></tr><tr><td>Anti-GAD 65/67 antibody</td><td>Sigma</td><td>G5163</td><td></td></tr><tr><td>Anti-LHCB2 antibody</td><td>Agrisera</td><td>AS01 003</td><td></td></tr><tr><td>Brilliant Blue R-250</td><td>Sigma</td><td>B7920</td><td></td></tr><tr><td>C18 Column</td><td>Grace</td><td>&amp;nbsp;&amp;nbsp; -</td><td>Alltima HP C18 (150 mm x 2.1 mm; 3 &amp;mu;m) Column</td></tr><tr><td>C18 Guard Column</td><td>Grace</td><td>&amp;nbsp;&amp;nbsp; -</td><td>Alltima HP C18 (7.5 mm x 2.1 mm; 5 &amp;mu;m) Guard&amp;nbsp; Column</td></tr><tr><td>CalMag Grower</td><td>Peter Excel</td><td>15-5-15</td><td>Fertilizer</td></tr><tr><td>Carbenicillin disodium</td><td>Duchefa Biochemie</td><td>C0109</td><td>Toxic</td></tr><tr><td>Chemiluminescence imaging system</td><td>BioRad</td><td>1708370</td><td>ChemiDoc Touch Imaging System</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432</td><td></td></tr><tr><td>Detergent</td><td>Sigma</td><td>P5927</td><td>Polysorbate 20</td></tr><tr><td>Fluorescence reader</td><td>Perkin-Elmer</td><td>&amp;nbsp;1420-011</td><td>VICTOR Multilabel Counter</td></tr><tr><td>Formic acid LC-MS grade</td><td>Sigma</td><td>94318</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>15 % to make loading buffer with Tris-HCl, SDS and 2&amp;ndash;mercaptoethanol</td></tr><tr><td>GoTaq G2 polymerase</td><td>Promega</td><td>M7841</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td>Corrosive</td></tr><tr><td>HILIC Column</td><td>Grace</td><td>&amp;nbsp;&amp;nbsp; -</td><td>Ascentis Express HILIC (150 mm x 2.1 mm; particles size 2.7 &amp;mu;m) Column</td></tr><tr><td>HILIC Guard Column</td><td>Grace</td><td>&amp;nbsp;&amp;nbsp; -</td><td>Vision HT HILIC (7.5 mm x 2.1 mm; 3 &amp;mu;m) Guard&amp;nbsp; Column</td></tr><tr><td>Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td><td>Sigma</td><td>A6154</td><td>Do not freeze/thaw too many times</td></tr><tr><td>HPLC Autosampler</td><td>Beckman Coulter</td><td>&amp;nbsp;&amp;nbsp; -</td><td>System Gold 508 Autosampler</td></tr><tr><td>HPLC System</td><td>Beckman Coulter</td><td>&amp;nbsp;&amp;nbsp; -</td><td>System Gold 128 Solvent Module HPLC</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>24137</td><td>Flamable</td></tr><tr><td>Kanamycin sulfate</td><td>Sigma</td><td>K4000</td><td>Toxic</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td>2 g/L with NaCl , Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; to make PBS</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td>2.4 g/L with NaCl , Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and KCl to make PBS</td></tr><tr><td>Loading Buffer</td><td></td><td></td><td></td></tr><tr><td>Luminol solution</td><td>Ge Healthcare</td><td>RPN2232</td><td>Prepare the solution using the ECL Prime Western Blotting System commercial kit</td></tr><tr><td>Lyophilizator</td><td>5Pascal</td><td>LIO5P0000DGT</td><td></td></tr><tr><td>Mass Spectometer</td><td>Bruker Daltonics</td><td>&amp;nbsp; -</td><td>Bruker Esquire 6000; the mass spectrometer was equipped with an ESI source and the analyzer was an ion trap</td></tr><tr><td>Methanol</td><td>Sigma</td><td>32213</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>Milk-blocking solution</td><td>Ristora</td><td>&amp;nbsp;&amp;nbsp; -</td><td>3 % in PBS</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907</td><td>Use with NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; to make Sodium Phospate buffer</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014</td><td>80 g/L with KCl, Na2HPO4 and KH2PO4 to make PBS; 10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, Tryptone and Agar Bacteriological Grade</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S8282</td><td>&amp;nbsp;Use with Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; to make Sodium Phospate buffer; 14.4 g/L to make PBS</td></tr><tr><td>NaOH</td><td>Sigma</td><td>S8045</td><td></td></tr><tr><td>Nitrocellulase membrane</td><td>Ge Healthcare</td><td>10600002</td><td></td></tr><tr><td>Pepsin from porcine gastric mucosa</td><td>Sigma</td><td>P7000</td><td></td></tr><tr><td>Peroxidase substrate ECL</td><td>GE Healthcare</td><td>RPN2235</td><td>Light sensitive material</td></tr><tr><td>Pump Vacuum Press</td><td>VWR</td><td>111400000098</td><td></td></tr><tr><td>Reagent A</td><td>Sigma</td><td>B9643</td><td>Use 50 parts of this reagent with 1 part of reagent B to prepare BCA working solution</td></tr><tr><td>Reagent B</td><td>Sigma</td><td>B9643</td><td>Use 1 part of this reagent with 50 parts of reagent A to prepare BCA working solution</td></tr><tr><td>Rifampicin</td><td>Duchefa Biochemie</td><td>R0146</td><td>Toxic &amp;ndash; 25 mg/mL stock in DMSO</td></tr><tr><td>SDS (Sodium dodecyl sulphate)</td><td>Sigma</td><td>L3771</td><td>Flamable, toxic, corrosive-10 % stock; 3 % to make loading buffer with Tris-HCl, Glycerol and 2&amp;ndash;mercaptoethanol</td></tr><tr><td>Sodium metabisulphite</td><td>Sigma</td><td>7681-57-4</td><td></td></tr><tr><td>Sonicator system</td><td>Soltec</td><td>090.003.0003</td><td>Sonica&amp;reg; 2200 MH; frequency 40 khz</td></tr><tr><td>Syringe</td><td>Terumo</td><td>&amp;nbsp;&amp;nbsp; -</td><td></td></tr><tr><td>Transparent fixed 300-&amp;micro;L insert glass tubes</td><td>Thermo Scientific</td><td>11573680</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td>Adjust pH with 1N HCl to make Tris-HCl buffer, use 1,5M Tris-HCl (pH 6.8) to make loading buffer with SDS, Glycerol and 2&amp;ndash;mercaptoethanol</td></tr><tr><td>Tryptone</td><td>Formedium</td><td>TRP03</td><td>10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Agar Bacteriological Grade</td></tr><tr><td>Vacuum concentrator</td><td>Heto</td><td>3878 F1-3</td><td>Speed-vac System</td></tr><tr><td>Water LC-MS grade</td><td>Sigma</td><td>39253</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma</td><td>Y1333</td><td>5 g/L to make LB medium (pH 7.5 with NaOH) with Tryptone, NaCl and Agar Bacteriological Grade</td></tr></tbody></table>

transient expression in red beet of a biopharmaceutical candidate vaccine for type-1 diabetes

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

In this work, the workflow for the development of an oral vaccine in edible plant tissues is presented. The focus of this work is the expression of a target protein in an edible host plant species and the characterization of the potential oral vaccine.
The first step involved the evaluation of the suitability of the plant virus-based expression technology to produce recombinant proteins in edible plant systems. For this aim, we ...

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

transient-expression-red-beet-biopharmaceutical-candidate-vaccine-for

Research

JoVE Journal

Bioengineering

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Learn transient expression in red beet for oral vaccine development against Type 1 diabetes using plant-based systems. This method enables high-yield recombinant protein production for immunological applications.

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

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