key: cord-0005063-77lqxgmh authors: Wu, H.; Singh, N.K.; Locy, R.D.; Scissum-Gunn, K.; Giambrone, J.J. title: Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana date: 2004 journal: Biotechnol Lett DOI: 10.1023/b:bile.0000025878.30350.d5 sha: 6e7500915d35939c729504f67c2382c785f77759 doc_id: 5063 cord_uid: 77lqxgmh VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciensby electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens. Infectious bursal disease virus (IBDV) causes infectious bursal disease (IBD), an important disease of commercial chicken flocks worldwide. Control of IBD currently employs biosecurity, sanitation, and vaccination. Commercial vaccines are not totally effective and less attenuated products can cause IBD (Jackwood et al. 1987 , Snyder et al. 1992 . IBDV is a member of the Birnaviridae family, which is characterized by a bisegmented doublestranded RNA genome. The smaller genome, segment B (2.8 kb), encodes VP1, a 90-kDa multi-functional protein with polymerase and capping enzyme activities (Azad et al. 1985 , Spies et al. 1987 . The larger genome, segment A (3.2 kb), encodes a polyprotein that is cleaved by auto proteolysis to form mature viral proteins VP2, VP3, and VP4. VP2 is the major hostprotective antigen with antigenic regions responsible for induction of neutralizing antibodies (Azad et al. 1987 , Jagadish et al. 1998 . Since the introduction of edible plant-based vaccines by Mason et al. (1992) , several laboratories have used transgenic plants for expression of viral and bacterial antigens , Daniell et al. 2001 , Gomez et al. 1998 , Haq et al. 1995 , Kong et al. 2001 , Lauterslager et al. 2001 , Mason et al. 1992 , 1996 , McGarvey et al. 1995 , Ritchter et al. 2000 , Tacket et al. 1998 , Wigdorovitz et al. 1999 . Although oral vaccination can protect against infectious agents entering the body via mucosal surfaces of the host, the mechanisms of ensuing mucosal immunogenicity are not well understood (Shalaby et al. 1995) . Despite enormous potential, exploitation of plants as oral vaccines has been hampered by low immunogenicity, induction of immunotolerance, proteolytic degradation of antigens during passage through the gastrointestinal tract, and exposure to acidic conditions in the stomach (Lauterslager et al. 2001 ). Here Fig. 1 . T-DNA region of rpE-VP2 construct. VP2 from TA vector with XhoI and XbaI was cloned in the plant-binary vector, pE1857 under control of super promoter. The resulting plasmid, rpE-VP2 has a Bar gene cassette as plant selection marker and confers resistance to bialaphos. LB and RB indicate the left and right borders of the T-DNA region. AOCS represents the octopine synthetase promoter. TL represents translational leader sequence. ags-ter represents the agropine synthase terminator region. we demonstrate high level expression of immunologically active VP2 protein in transgenic Arabidopsis thaliana. The infectious bursal disease virus (IBDV) antigenic variant E strain was propagated in 5-week-old specific pathogen free chickens. Birds were kept in Plexiglass isolation units maintained with filtered air under negative pressure. Birds were given a corn-soybean diet and water ad libitum. Care and handling of chicken was according to the Auburn University's Institutional Care and Use Committee. Bursae of Fabricius (the target organ for IBDV infection) were taken from infected birds at 3 days' post-infection. They were dissected and homogenized in TNE buffer (10 mM Tris/HCl, 100 mM NaCl, 10 mM EDTA, pH 8) at a ratio of 1 g bursa to 10 ml TNE buffer. After freezing and thawing three times, homogenates were centrifuged at 17 000 g for 15 min at 4 • C and the supernatant collected for viral RNA extraction with a Trizol RNA extraction kit (Gibco). The VP2 cDNA was prepared from RNA using an RT-PCR preamplification system (Gibco). Primers flanking the VP2 sequence were designed according to information in the GenBank. Primers were designed with XhoI site in Vh-1 and XbaI site in Vb-2, respectively, for directional cloning of amplified sequences: primer Vh-1 GGCCTCGAGAATGGTTAGTAGAGATCAGACA; primer Vb-2 GGCTCTAGATACACCTTCCCCAATT GCAT. The plant expression vector pE1857 (Min et al. 1995) was obtained from a patent transfer agreement from S. Gelvin (personal communication). Briefly, the vector was derived from kanamycin-resistant pGPTV containing the patented super-promoter, TEV translational leader, polylinker derived from pBluescript, and ags terminator in pUC119. The VP2 DNA amplicon was placed under control of super promoter vector between the restriction enzyme sites for XhoI and XbaI. The resulting recombinant construct had bar gene cassette for bialophos (Phosphoinothricin) selection in plants, and was designated as rpE-VP2 ( Figure 1 ). The rpE-VP2 expression cassette and pE1857 control vector were introduced into Agrobacterium tumefaciens strain C58C1 by electroporation and used for transformation of Ar. thaliana by vacuum infiltration (Betchtold et al. 1993) . Seeds were harvested from the self-pollinated primary transformants and used to generate plants for screening as described below. Plants resistant to bialaphos were selected. Seedlings germinating in Promix potting medium were sprayed daily for 3 d with a solution of 50 mg bialaphos l −1 (Sigma). After 5 d, the procedure was repeated for an additional 3 d. Seeds from surviving plants were harvested and bialaphos selection performed for 3 additional generations to obtain homozygous transgenic lines. VP2 in transgenic plants was demonstrated by polymerase chain reaction (PCR) and Southern blotting. Total DNA from leaves was isolated using plant DNAzol reagent (Gibco). The VP2 DNA was amplified with Vh-1 and Vb-2 primers and a 1.5 kb fragment was obtained. This fragment was labeled with 32 P-dCTP for use as a probe in Southern hybridization (Sambrook et al. 1989) . Total DNA from transgenic and control plants was digested with EcoRIV, sep-arated by electrophoresis on 1% agarose gels, and transferred to a nylon membrane. The membrane was hybridized to the VP2 probe at 42 • C for 4 h in presence of 6 SSC and 50% (v/v) formamide. Blots were washed three times with 0.1 SSC and 0.5% SDS at 37 • C for 10 min each, and exposed to an autoradiography film at −80 • C for 24 h. Total RNA from transgenic and control A. thaliana plants were obtained from ∼1 g of leaves by an RNA isolation system (Omega Bio-tek, Inc.). A preamplification kit (Gibco) was utilized in RT-PCR to amplify VP2 DNA with the gene-specific Vh-1 and Vb-2 primers. Leaves were ground to a powder in liquid N 2 and added to 1 ml extraction buffer containing 10 mM 2-Nmorpholino ethanesulfonic acid, pH 6, 10 mM NaCl, 5 mM EDTA, 0.6% Triton X-100, 0.25 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 10 mM DTT, 1 mM phenylmethyl sulfonyl fluoride. The tissue homogenate was centrifuged twice at 12 000 g for 15 min at 4 • C to remove insoluble debris, and resulting supernatant used for VP2 protein analysis. Total soluble protein (TSP) concentration was determined using the Bradford (1976) protein assay. The concentration of VP2 protein in plants was determined by antigen-capture AC-ELISA (Corley et al. 2001) . Plates were coated at 4 • C overnight with polyclonal anti-IBDV-chicken serum diluted at 1:1000 in PBS buffer pH 8; washed 3 times with PBST (phosphatebuffered saline with 0.05% Tween 20) buffer; and blocked with PBST buffer containing 5% skim milk at 37 • C for 3 h. Plates were washed 3 times with PBST and then dried and stored at 4 • C. Since VP2 is about 20% of the IBDV genome, and approximately the same proportion of the viral particle (Lee et al. 2003) , the expression level of VP2 could be estimated from TSP measurements. Purified IBDV, 100 ml, was used as a standard to determine the concentration of VP2 in TSP by Bradford assay. VP2 concentration was estimated by TSP/5. VP2 antigen levels were estimated by AC-ELISA at OD 405 . Both control and transgenic plant extracts were analyzed similarly. The concentration of VP2 in transgenic plants was determined as % TSP and calculated as follows: where OD 405 (leaf extract) represents total soluble protein in leaf extracts detected by AC-ELISA, OD 405 (IBDV) represents total soluble protein in purified IBDV detected by AC-ELISA, and [VP2-IBDV] represents VP2 protein in purified IBDV calculated from purified IBDV concentration divided by five. Purified IBDV were subjected to SDS-PAGE followed by electro-blotting on to nitrocellulose membranes using Semi-dry Trans Blotter (Bio-Rad) according to manufacturer's instruction. Membranes were blocked with 3% (v/v) skim milk. After blotting, different lanes on nitrocellulose were separated. Each strip of nitrocellulose was independently probed with chicken serum orally immunized with leaf extracts from different transgenic lines and serum collected from chicken fed with untransformed plants and plants transformed with vector as negative controls. Monoclonal antibody against VP2 was used as positive control in preliminary experiment. The VP2 antibody produced in chicken in response to oral immunization and forming a complex with VP2 protein on the nitrocellulose was detected by horseradish peroxidase (HRP)-conjugated anti-chicken immunoglobulin G at a 1:1000 dilution following protocol of Jackson Immuno Research Laboratories. Full-length VP2 cDNA was amplified from viral RNA by RT-PCR using primers that placed an XhoI restric- Fig. 3 . Southern blot analysis of VP2 in transgenic plants. Total DNA from leaves of control and transgenic plants were digested with EcoRIV, electrophoresed, transferred to nylon membrane, and hybridized to 32 P labeled VP2 probe. Hybridization signals with VP2 probe in transgenic Ar. thaliana lines V-2, V-3, V-4 (lanes 1, 6, and 9, respectively) are shown in the autoradiogram. DNA from plants transformed with pE1857 (lanes 2, 3, 4, and 5), and control plants (lanes 7 and 8) did not show hybridization with VP2 probe. tion site at the 5 end and XbaI site at the 3 end. The PCR product was ligated into the cloning site of the pGEM T-easy vector (TA vector), and the sequence of the cloned fragment verified. The VP2 DNA was retrieved from the TA vector with the XhoI and XbaI restriction enzymes, and cloned in the plant expression vector pE1857. The recombinant expression vector, rpE-VP2 was used to transform Ar. thaliana. Forty transgenic plants were selected after initial seedling screening with bialaphos. Three selected transgenic lines, V-2, V-3 and V-4 were used in this study and demonstrated stable integration of the VP2 by PCR amplification of total DNA from transgenic lines as template (Figure 2 ). Control plants transformed with pE1857 vector or untransformed plants did not show VP2 DNA. Transgenic plants showing VP2 amplification products also showed VP2 positive hybridization signals on Southern blot (Figure 3 ). The VP2 sequence was integrated into high molecular weight chromosomal DNA. More than one copy of the VP2 transgene was present in the transgenic Ar. thaliana lines, V-3 and V-4. Transgenic plants, expressing VP2 protein showed about 10-20% reduced growth compared to control plants or plants transformed with the pE1857 (data not shown). VP2-specific amplified DNA product was obtained after RT-PCR of RNA from three selected transgenic plants. Non-transformed plants and transgenic plants transformed with control vector showed no VP2 DNA (Figure 4) . Our effort to detect VP2 directly from plant extracts resulted in positive signals with smear across the lanes. Other groups working with VP2 protein of IBDV have experienced similar difficulties (Jagadish et al. 1998 ). It has been suggested that hydrophobic nature of VP2 proteins may be responsible for the conformation dependent distortion on SDS-PAGE. We used a modification of Western blot procedure with IBDV proteins for electrophoresis, and serum proteins from chickens fed with leaf extracts of transgenic plants to detect antibodies in chicken serum against VP2. A 45 kDa band was recognized in lanes where monoclonal anti-IBDV and serum from chicken fed with transgenic plant extracts was used for detection of cross reaction with VP2 protein from IBDV Three transgenic Ar. thaliana lines with high VP2 expression level were designated as V-2, V-3 and V-4, respectively. The ratio of VP2 to total soluble protein ranged from 0.5% to 4.8%. ( Figure 5 ). Chickens fed with the extracts of untransformed plants and plants transformed with control vector did not cross-react with IBDV proteins of blotted nitrocellulose. Antigen capture ELISA was used to determine VP2 expression level in transgenic Ar. thaliana. The level of VP2 ranged from 0.5%-4.8% of TSP in different lines shown in Table 1 . Transgenic line V-3 had the highest level of VP2 expression. The higher level of protein expression in V-3 was consistent with the presence of a higher copy number of the VP2 transgene detected by Southern blot. This is the highest level of recombinant antigenic protein expression in plants, thus far. Other instances where recombinant antigens have been expressed in plants showed lower levels of expression, e.g. B subunits of Escherichia coli enterotoxin (0.01% TSP) (Azad et al. 1985) , hepatitis B surface antigen (0.01% TSP) (Manson et al. 1992) , and the gastroenteritis virus gS gene (0.06% TSP) (Gomez et al. 1998) . VP2 has been expressed in heterologous systems as recombinant protein in E. coli (Jagadish et al. 1998) , yeast (Macreadie et al. 1990 ), bacculovirus (Dybing et al. 1997) , fowl poxvirus (Bayliss et al. 1991) , herpesvirus of turkey (Tsukamoto et al. 1999) , and fowl adenovirus (Sheppard et al. 1998 ). These reports do not provide the concentration of VP2 protein in the experimental systems. Production of recombinant antigenic proteins in plants offer unique advantages over other model expression system. For example, it is economical to produce large quantities of protein in transgenic plants than by traditional industrial fermentation or bioreactor systems. Large scale harvesting and processing technology for plant proteins is readily available. Purification of the recombinant protein is not necessary when the plant tissue is used as animal feed. Plant cells can target proteins into intracellular compartments that are more stable and risks from contamination with pathogens are minimized. VP2 expression in transgenic plants is an interesting model for development of edible vaccines for the control of viral diseases in poultry. Antigenic proteins of some pathogens, such as the gS gene of TGEV (Gomez et al. 1998 ) and VP1 of FMDV , are naturally resistant to degradation in gut when incorporated into the viral particle. Natural bioencapsulation of hepatitis B surface antigen expressed in plants provided protection from degradation in the digestive tract near an immune effector's site in the gut (Kong et al. 2001) . VP2 protein is hydrophobic, and its antigenicity may be conformationdependent (Kibenge et al. 1990 ). VP2 has not been investigated with respect to its resistant to gut degradation. However, our result shows recognition of IBDV by the serum of chickens fed with plant extracts expressing VP2 suggests that the recombinant VP2 produced in plants had the capacity to invoke immune response in chicken. This supported our view that VP2 protein is resistant to degradation in chicken gut and can elicit immune response against IBDV. Transgenic plants offer a novel and safe system for vaccine production. Future demonstration of the efficacy of VP2 antigen produced in transgenic plants in the prevention of IBD will strengthen the concept of edible vaccine production for control of major pathogens of poultry and livestock. The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectiouous bursal disease virus Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus A recombinant fowl poxvirus that expresses the VP2 antigen of infectious bursal disease virus induces protection against mortality caused by the virus In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants Detection of infectious bursal disease vaccine viruses in lymphoid tissues after in ovo vaccination of specific pathogen free embryos Expression of MD infectious bursal disease viral proteins in baculovirus Medical molecular farming -production of antibodies, biopharmaceuticals and edible vaccines in plants Expression of immunogenic glycoprotein S polypeptides from transmissible gastroenteritis coronavirus in transgenic plants Oral immunization with a recombinant bacterial antigen produced in transgenic plants Antigenic diversity of infectious bursal disease viruses Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide Nucleotide sequence analysis of genome segment A of infectious bursal disease virus Oral immunization with hepatitis B surface antigen expressed in transgenic plants Oral immunization of naïve and primed animals with transgenic potato tubers expressing LT-B Purification, crystallization and preliminary X-ray analysis of immunogenic virus-like particles formed by infectious bursal disease virus (IBDV) structural protein VP2 Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT) Expression of hepatitis B surface antigen in transgenic plants Expression of rabies virus glycoprotein in transgenic tomatoes Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes Production of hepatitis B surface antigen in transgenic plants for oral immunization Molecular Cloning: A Laboratory Manual Development of oral vaccines to stimulate mucosal and systemic immunity: barriers and novel strategies Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease Naturally occurringneutralizing monoclonal antibody escape variants define the epidemiology of infectious bursal disease viruses in the United States Properties of RNA polymerase activity associated with infectious bursal disease virus and characterization of its reaction products Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato Protection of chicken against very virulent infectious bursal disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2 Induction of a protective antibody response to footand-mouth disease virus in mice following oral or potential immunization with alfalfa transgenic plants expressing the oral structural protein VP1 This research was supported by a USDA-IFAFS grant (no. 00-52100-9705). We are grateful to Teresa Dormitorio and Chia-chen Weng for technical assistance.