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

Summary

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

Abstract

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.

Introduction

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....

Protocol

1. Red beet and spinach cultivation

1. Grow red beet (B. vulgaris cv Moulin Rouge) and spinach (S. oleracea cv Industria) plants in a growth chamber, using 150 µE of light intensity, 65% relative humidity, 12 h light/dark cycle at 23/21 °C, respectively.
2. 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 re.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
0.2-μm Minisart RC4 membrane filters	Sartorius-Stedim	17764
2–mercaptoethanol	Sigma	M3148	Toxic; 4 % to make loading buffer with glycerol, SDS and Tris-HCl
4-Morpholineethanesulfonic acid (MES)	Sigma	M8250	pH 5.5
96-well plate	Sarstedt	833924
Acetic acid	Sigma	27221	Corrosive
Acetonitrile LC-MS grade	Sigma	34967
Acetosyringone	Sigma	D134406	Toxic – 0.1 M stock in DMSO
Agar Bacteriological Grade	Applichem	A0949	15 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Tryptone
Ammonium formate	Sigma	70221
Anti-eGFP antibody	ABCam	ab290
Anti-GAD 65/67 antibody	Sigma	G5163
Anti-LHCB2 antibody	Agrisera	AS01 003
Brilliant Blue R-250	Sigma	B7920
C18 Column	Grace	-	Alltima HP C18 (150 mm x 2.1 mm; 3 μm) Column
C18 Guard Column	Grace	-	Alltima HP C18 (7.5 mm x 2.1 mm; 5 μm) Guard  Column
CalMag Grower	Peter Excel	15-5-15	Fertilizer
Carbenicillin disodium	Duchefa Biochemie	C0109	Toxic
Chemiluminescence imaging system	BioRad	1708370	ChemiDoc Touch Imaging System
Chloroform	Sigma	C2432
Detergent	Sigma	P5927	Polysorbate 20
Fluorescence reader	Perkin-Elmer	1420-011	VICTOR Multilabel Counter
Formic acid LC-MS grade	Sigma	94318
Glycerol	Sigma	G5516	15 % to make loading buffer with Tris-HCl, SDS and 2–mercaptoethanol
GoTaq G2 polymerase	Promega	M7841
HCl	Sigma	H1758	Corrosive
HILIC Column	Grace	-	Ascentis Express HILIC (150 mm x 2.1 mm; particles size 2.7 μm) Column
HILIC Guard Column	Grace	-	Vision HT HILIC (7.5 mm x 2.1 mm; 3 μm) Guard  Column
Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody	Sigma	A6154	Do not freeze/thaw too many times
HPLC Autosampler	Beckman Coulter	-	System Gold 508 Autosampler
HPLC System	Beckman Coulter	-	System Gold 128 Solvent Module HPLC
Isopropanol	Sigma	24137	Flamable
Kanamycin sulfate	Sigma	K4000	Toxic
KCl	Sigma	P9541	2 g/L with NaCl , Na2HPO4 and KH2PO4 to make PBS
KH2PO4	Sigma	P9791	2.4 g/L with NaCl , Na2HPO4 and KCl to make PBS
Loading Buffer
Luminol solution	Ge Healthcare	RPN2232	Prepare the solution using the ECL Prime Western Blotting System commercial kit
Lyophilizator	5Pascal	LIO5P0000DGT
Mass Spectometer	Bruker Daltonics	-	Bruker Esquire 6000; the mass spectrometer was equipped with an ESI source and the analyzer was an ion trap
Methanol	Sigma	32213
MgSO4	Sigma	M7506
Milk-blocking solution	Ristora	-	3 % in PBS
Na2HPO4	Sigma	S7907	Use with NaH2PO4 to make Sodium Phospate buffer
NaCl	Sigma	S3014	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
NaH2PO4	Sigma	S8282	Use with Na2HPO4 to make Sodium Phospate buffer; 14.4 g/L to make PBS
NaOH	Sigma	S8045
Nitrocellulase membrane	Ge Healthcare	10600002
Pepsin from porcine gastric mucosa	Sigma	P7000
Peroxidase substrate ECL	GE Healthcare	RPN2235	Light sensitive material
Pump Vacuum Press	VWR	111400000098
Reagent A	Sigma	B9643	Use 50 parts of this reagent with 1 part of reagent B to prepare BCA working solution
Reagent B	Sigma	B9643	Use 1 part of this reagent with 50 parts of reagent A to prepare BCA working solution
Rifampicin	Duchefa Biochemie	R0146	Toxic – 25 mg/mL stock in DMSO
SDS (Sodium dodecyl sulphate)	Sigma	L3771	Flamable, toxic, corrosive-10 % stock; 3 % to make loading buffer with Tris-HCl, Glycerol and 2–mercaptoethanol
Sodium metabisulphite	Sigma	7681-57-4
Sonicator system	Soltec	090.003.0003	Sonica® 2200 MH; frequency 40 khz
Syringe	Terumo	-
Transparent fixed 300-µL insert glass tubes	Thermo Scientific	11573680
Trizma Base	Sigma	T1503	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–mercaptoethanol
Tryptone	Formedium	TRP03	10 g/L to make LB medium (pH 7.5 with NaOH) with Yeast extract, NaCl and Agar Bacteriological Grade
Vacuum concentrator	Heto	3878 F1-3	Speed-vac System
Water LC-MS grade	Sigma	39253
Yeast extract	Sigma	Y1333	5 g/L to make LB medium (pH 7.5 with NaOH) with Tryptone, NaCl and Agar Bacteriological Grade