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

Among the different strategies for production of recombinant protein in plants, the deconstructed plant-based VERS expression provides superior performance, leading to high yields over a relative short time frame. Replication of this technology for the production of an oral vaccine holds a great potential to become an alternative to conventional vaccines in the near future. Lyophilized red beet leaves transiently transformed accumulates an amount of photo antigens suitable for oral tolerance induction in type one diabetes prevention.

This experimental protocol could be extended to many different edible species after a case-by-case evaluation of recombinant protein accumulation. Demonstrating the procedure will be Edoardo Bertini, Mattia Santoni, Anna Cuccurullo, and Roberta Zampieri. To begin this procedure, grow red beet and spinach plants in a growth chamber as outlined in the text protocol.

After seed germination, fertilize the plants twice a week with a solution of commercially available fertilizer at a concentration of one gram per liter. For agroinfiltration, use five week old spinach and six week old red beet plants. After constructing the plant expression vectors, inoculate the three A.tumefaciens transformants in 50 milliliters of LB medium containing rifampicin at a concentration of 50 micrograms per milliliter and vector-specific antibiotics as outlined in the text protocol.

Grow by shaking overnight at 28 degrees Celsius. The next day, pellet the bacterial cultures by centrifuging at 4500 times G for 20 minutes. Resuspend the pellet in 100 milliliters of infiltration buffer containing 10 millimolar MES and 10 millimolar magnesium sulfate.

Incubate the suspensions at room temperature for three hours. Mix an equal volume of a bacterial suspension containing one of the modules shown here, with the five prime module and the integrase module. Using a syringe without a needle, take out five milliliters of the suspension mix.

Press the tip of the syringe against the underside of the plant leaf while applying gently counter pressure to the other side of the leaf. Infiltrate the first three completely expanded leaves, starting from the apex, for each plant. Label the agroinfiltrated leaves with the paper tag on the leaf stem.

Then return the plants to the growth chamber under standard conditions. On days four to 14 post-infection, collect the agroinfiltrated leaves and freeze them in liquid nitrogen. Store this plant tissue at minus 80 degrees Celsius.

First, separately grow the three A.tumefaciens transformants in 50 milliliters of LB medium containing rifampicin at a concentration of 50 microliters per milliliter, and appropriate vector-specific antibiotics by shaking overnight at 28 degrees Celsius. The next day, pellet the bacterial cultures by centrifuging at 4500 times G for 20 minutes. Resuspend the pellet in 1 liter of infiltration buffer to an OD600 of 0.35, and incubate the suspensions at room temperature for three hours.

Then add 0.01%of the detergent to each suspension. Mix an equal volume of a bacterial suspension containing one of the modules shown here with the five prime module and the integrase module. Insert one six week old red beet plant into the holder.

Invert the holder and place it on top of a beaker containing two liters of an infiltration bath to submerge the leaves in the infiltration suspension. After this, 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.

Once the pressure in the chamber has fallen to 90 millibar, maintain the vacuum for three minutes. Then release the vacuum for 45 seconds. When the chamber has returned to atmospheric pressure, open the chamber and remove the infiltrated plant from the bacterial bath.

Return the plant to the growth chamber. On the day of maximum expression, harvest the infiltrated leaves and freeze them in liquid nitrogen as previously described. First, harvest the vacuum agroinfiltrated delta 87 GAD65mut expressing red beet leaves at the expression peak, and freeze them in liquid nitrogen.

Lyophilize the frozen leaves at minus 50 degrees Celsius and at 0.04 millibar for 72 hours. Then store them at minus 80 degrees Celsius. When ready to proceed, grind the leaves to a fine powder and store at room temperature in a sealed container with silica gel to exclude the moisture.

For the gastric digestion simulation, weigh 100 milligrams of the finely ground freeze dried red beet leaves, and resuspend it in six milliliters of PBS. Use six molar hydrochloric acid to adjust the sample pH to two. Add four milligrams per milliliters pepsin from porcine gastric mucosa, and 10 millimolar hydrochloric acid, to obtain a final pepsin concentration of one milligram per milliliter.

Shake the sample at 37 degrees Celsius for 120 minutes. Next, use sodium hydroxide to adjust the sample pH to eight and inactivate the pepsin. Transfer 750 microliter aliquots of each sample to microcentrifuge tubes, and centrifuge the aliquots at 20, 000 times G and at four degrees Celsius for 20 minutes.

Collect each supernatant separately, and resuspend each pellet in one supernatant volume of loading buffer. Then analyze both the supernatant and the resuspended pellet by Western blot analysis. For the cell integrity analysis, prepare two samples of 100 milligrams of the finely ground freeze-dried red beet leaves, and resuspend both in six milliliters of PBS.

Use six molar hydrochloric acid to adjust the pH of one sample to two. Shake both samples at 37 degrees Celsius for 120 minutes. Then aliquot 750 microliters of each sample to microcentrifuge tubes.

Centrifuge at 20, 000 times G and at 4 degrees Celsius for 20 minutes. Collect each supernatant separately, and resuspend each pellet in one supernatant volume of loading buffer. Then analyze both the supernatant and the resuspended pellet by Western blot analysis.

First, weigh 100 milligrams of the finely ground freeze-dried red beet leaves, and resuspend it in eight milliliters of sterile PBS. Vortex for one minute. Plate one milliliter of each freeze-dried leaf homogenate in one of the five prepared selective LB media.

Incubate the plates at 28 degrees Celsius for three days. After this, count the agrobacterium colonies grown on each plate, and calculate the residual bacterial charge as the number of colony forming units per milliliter of the freeze-dried leaf homogenate. In this work, an oral vaccine is developed in edible plant tissue.

Red beet and spinach plants are manually agroinfiltrated with suspensions of A.tumefaciens carrying EGFP recombinant expression vectors, and the fluorescent protein expression is visualized by Western blot analysis and quantified under UV light. The red beet system is characterized by a higher EGFP expression, reaching approximately 544 micrograms per gram of fresh leaf weight at nine days post-infection, while the spinach only reached a maximum level of approximately 113.4 micrograms per gram of fresh leaf weight on 11 days post-infection. Because of this, the red beet is selected as the expression host for all subsequent experiments.

Plants are then vacuum infiltrated with an A.tumefaciens suspension. After the TSP extraction from agroinfiltration leaves, the samples are analyzed by Western blot, and the recombinant protein is relatively quantified by a densitometry analysis. Finally, the parameters for the development of a potential oral vaccine are evaluated.

As seen here, the target protein is seen to be stable after the lyophilization process. Gastric digestion is simulated by adding the porcine gastric enzyme pepsin to freeze-dried material, either to a final concentration of one milligram per milliliter, or a ratio of one to 20, to TSP. Both digestive treatment conditions result in degradation of the recombinant protein.

The absence of a specific signal in the pellet samples after pepsin digestion suggest that after freeze-drying, the plant cells lost their integrity, and this led to the target protein degradation. The procedure should be carefully adapted to the plant species in respect to vacuum infiltration and to the target recombinant protein in respect to time course analysis. The demonstrating procedure might also be used for the production of biopharmaceutical intended for oral delivery, like vaccine and antibodies.