Plant-based Vaccines Research Articles

Chapter One - Plant-Based Vaccines: Novel and Low-Cost Possible Route for Mediterranean Innovative Vaccination Strategies

A plant bioreactor has enormous capability as a system that supports many biological activities, that is, production of plant bodies, virus-like particles (VLPs), and vaccines. Foreign gene expression is an efficient mechanism for getting protein vaccines against different human viral and nonviral diseases. Plants make it easy to deal with safe, inexpensive, and provide trouble-free storage. The broad spectrum of safe gene promoters is being used to avoid risk assessments. Engineered virus-based vectors have no side effect. The process can be manipulated as follows: (a) retrieve and select gene encoding, use an antigenic protein from GenBank and/or from a viral-genome sequence, (b) design and construct hybrid-virus vectors (viral vector with a gene of interest) eventually flanked by plant-specific genetic regulatory elements for constitutive expression for obtaining chimeric virus, (c) gene transformation and/or transfection, for transient expression, into a plant-host model, that is, tobacco, to get protocols processed positively, and then moving into edible host plants, (d) confirmation of protein expression by bioassay, PCR-associated tests (RT-PCR), Northern and Western blotting analysis, and serological assay (ELISA), (e) expression for adjuvant recombinant protein seeking better antigenicity, (f) extraction and purification of expressed protein for identification and dosing, (g) antigenicity capability evaluated using parental or oral delivery in animal models (mice and/or rabbit immunization), and (h) growing of construct-treated edible crops in protective green houses. Some successful cases of heterologous gene-expressed protein, as edible vaccine, are being discussed, that is, hepatitis C virus (HCV). R9 mimotope, also named hypervariable region 1 (HVR1), was derived from the HVR1 of HCV. It was used as a potential neutralizing epitope of HCV. The mimotope was expressed using cucumber mosaic virus coat protein (CP), alfalfa mosaic virus CP P3/RNA3, and tobacco mosaic virus (TMV) CP-tobacco mild green mosaic virus (TMGMV) CP as expression vectors into tobacco plants. Expressed recombinant protein has not only been confirmed as a therapeutic but also as a diagnostic tool. Herpes simplex virus 2 (HSV-2), HSV-2 gD, and HSV-2 VP16 subunits were transfected into tobacco plants, using TMV CP-TMGMV CP expression vectors.

Plant Bioreactors for the Antigenic Hook-Associated flgK Protein Expression

Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seed-specific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

Plant-based vaccines against viruses

Plant-made or “biofarmed” viral vaccines are some of the earliest products of the technology of plant molecular farming, and remain some of the brightest prospects for the success of this field. Proofs of principle and of efficacy exist for many candidate viral veterinary vaccines; the use of plant-made viral antigens and of monoclonal antibodies for therapy of animal and even human viral disease is also well established. This review explores some of the more prominent recent advances in the biofarming of viral vaccines and therapies, including the recent use of ZMapp for Ebolavirus infection, and explores some possible future applications of the technology.

Algae-based oral recombinant vaccines

Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity.

Norovirus Narita 104 virus-like particles expressed in Nicotiana benthamiana induce serum and mucosal immune responses.

Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.

Expression of the nucleotide sequence for the M2e peptide of avian influenza virus in transgenic tobacco plants

The M2e peptide of the H5N1 A/Chicken/Kurgan/05/2005 avian influenza virus was successfully synthesized in transgenic tobacco plants. The amino-terminal segment of the M2 protein, comprised of 22, 30, or 43 amino acids, including the M2e peptide (M122, M130, and M143 variants, respectively), was translationally fused with the N-terminus of β-glucuronidase in the pBI121 plant expression vector. The nucleotide sequence of the target fragment was synthesized by ligation from synthetic oligonucleotides; its codon composition was adapted for expression in plants. Tobacco plants were successfully transformed with the obtained vectors (pBIM122, pBIM130, and pBIM143, respectively). In the plants transformed with pBIM143, the Ml43-β-glucuronidase fusion protein was not produced, probably due to the presence of the M2 protein transmembrane domain (25–43 aa of M2) in this construct. In the pBIM122- and pBIMl30-transformed plants, the target M2e peptide was expressed as a component of the Ml22-β-glucuronidase and M130-β-glucuronidase fusion proteins, respectively, as was detected by Western blot analysis. These proteins were detected as bands of the expected size without apparent degradation. As a result, the M2e peptide of the H5N1 avian influenza virus was successfully synthesized for the first time in nuclear-transformed transgenic plants. The results obtained in this study will be used for developing a transgenic plant-based edible antiinfluenza vaccine.

Immunogenicity of Brugia malayi Abundant Larval Transcript-2, a potential filarial vaccine candidate expressed in tobacco

Transgenic tobacco plants with Bm ALT-2, a filarial vaccine candidate, were developed. The plant-produced antigen showed immunogenicity on par with the E.coli product. Transgenic tobacco plants were developed using Brugia malayi Abundant Larval Transcript-2 (Bm ALT-2), a major antigen produced from recombinant E.coli found to be experimentally successful as potential vaccine candidate against lymphatic filariasis. Results of experiments on the transformation and expression of the Bm ALT-2 in tobacco plant to produce plant-based vaccine are presented here. We have successfully transformed the tobacco plant with Bm ALT-2 and confirmed that the plants expressed the filarial protein by PCR analysis and Western blotting. The level of expression varied from 50 to 90ng/μg of total soluble protein for ALT-2. Immunization of mice with plant-extracted protein indicated that the plant-produced protein had immunological characteristics similar to the E.coli-produced protein. Antibody titres produced by plant-produced recombinant ALT 2-immunized mice were on par with those immunized with recombinant protein produced by E.coli. Antibody isotype assay showed that plant-produced recombinant ALT-2 induced significant IgG1, whereas E.coli-produced recombinant ALT-2 induced IgG3. This result is a step forward towards the development of a model eukaryotic system for the production of recombinant filarial proteins, which can be utilized to produce therapeutic and diagnostic molecules against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. In addition, this is the first report of the immunogenicity of a plant-derived filarial antigen.

Chloroplast expression of an HIV envelop-derived multiepitope protein: towards a multivalent plant-based vaccine

The development of a vaccine is still a priority in the fight against human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). Since conventional vaccine strategies have failed to provide a highly immunoprotective effect, approaches based on the rational design of vaccines composed of multiple HIV neutralizing epitopes have been proposed as potential vaccines. The aim of this study is to design a multiepitopic protein (Multi-HIV) carrying several neutralizing epitopes from both gp120 and gp41 as an effort to develop a new broad immunization scheme against HIV. This Multi-HIV was initially produced in a recombinant Escherichia coli strain either as a single protein or fused to glutathione-S-transferase. These proteins were purified by immobilized metal ion affinity chromatography and shown to be antigenic by positive reactivity in Western blot analyses using sera from HIV-positive patients for labeling. Since global immunization strategies are often limited by costs, platforms that require minimal processing are the priority in this field. Therefore, we explored the possibility of using transplastomic tobacco plants as an experimental model of a low cost plant-based vaccine against HIV. Transplastomic tobacco plants carrying the multi-HIV gene were developed and verified by PCR analyses. The expected Multi-HIV recombinant protein was localized in the chloroplast as proven first by confocal microscopy and subsequently by Western blot analysis. Tobacco-derived Multi-HIV protein was clearly able to evoke humoral responses in mice when orally administered without adjuvants. This report constitutes an effort to explore a new low-cost candidate that could have future implications on the development of affordable HIV vaccines.

Veterinary Vaccines from Transgenic Plants: Highlights of Two Decades of Research and a Promising Example

In the last two decades the development of efficient plant-based expression strategies and new concepts for the purification of recombinant proteins prompted the application of plant-derived vaccines for veterinary purposes. The expression of recombinant proteins in plants possesses advantages over conventional eukaryotic expression systems and therefore represents a versatile tool for the production of "edible" and "seasonal" vaccines. This review aims to provide an overview about the expression of vaccines using transgenic plants for veterinary medicine with the focus on increasing the amount of the recombinant proteins as well as concepts for their efficient purification. ELPylation of recombinant proteins is one strategy for on one side boosting the amount of the recombinant protein and on the other side simplifying its purification. This up-and-coming tool was applied for the development of effective production and purification strategies for antigens against Avian Flu, a very important animal disease with a strong economic impact. Future perspectives of plant-based veterinary vaccines in the context of purification and economy are also discussed within this article.

Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine “Hokken” by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.

Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against Infectious Bursal Disease Virus

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV.

The Twenty-Year Story of a Plant-Based Vaccine Against Hepatitis B: Stagnation or Promising Prospects?

Hepatitis B persists as a common human disease despite effective vaccines having been employed for almost 30 years. Plants were considered as alternative sources of vaccines, to be mainly orally administered. Despite 20-year attempts, no real anti-HBV plant-based vaccine has been developed. Immunization trials, based on ingestion of raw plant tissue and conjugated with injection or exclusively oral administration of lyophilized tissue, were either impractical or insufficient due to oral tolerance acquisition. Plant-produced purified HBV antigens were highly immunogenic when injected, but their yields were initially insufficient for practical purposes. However, knowledge and technology have progressed, hence new plant-derived anti-HBV vaccines can be proposed today. All HBV antigens can be efficiently produced in stable or transient expression systems. Processing of injection vaccines has been developed and needs only to be successfully completed. Purified antigens can be used for injection in an equivalent manner to the present commercial vaccines. Although oral vaccines require improvement, plant tissue, lyophilized or extracted and converted into tablets, etc., may serve as a boosting vaccine. Preliminary data indicate also that both vaccines can be combined in an effective parenteral-oral immunization procedure. A partial substitution of injection vaccines with oral formulations still offers good prospects for economically viable and efficacious anti-HBV plant-based vaccines.

Is An Oral Plant-based Vaccine against Hepatitis B Virus Possible?

Prevention of hepatitis B, one of the most prevalent human diseases, still requires cheap and commonly available vaccines. Oral vaccines, including plant-based formulations, have been considered as alternatives or supplements for standard injection vaccines, due to the assumed low-cost production and simplified vaccination. Although plant production of HBV antigens is sufficiently efficient, despite almost 20 years of research still no anti-HBV plant-based vaccine has been developed. The basic difficulty has been to elaborate an effective immunisation procedure. Immunisation by parenteral priming and oral boosting with raw plant tissue adjuvanted with the cholera toxin, although effective, seemed to be unfeasible and controversial. Exclusively oral immunisation using lyophilised tissue, despite its appropriate form, appeared also impractical because of too low efficiency. Oral tolerance turned out to be the main barrier for anti-HBV plant-based vaccines. Based on previous results and knowledge on the mucosal immune system, a possible vaccine may consist of two components, parenteral for priming and oral for boosting. Probably the oral constituent could independently serve for further booster vaccinations. Both vaccine components can be produced in plants and used after some processing - purification for the injection constituent and lyophilisation for the oral one. Lyophilised tissue can be converted into tablets, capsules, etc. Previous and recent data show that the injection-oral immunisation regime may be efficient. A combination of parenteral and oral vaccination offers good prospects for a truly efficacious plant-derived anti-HBs vaccine and even a partial substitution of parenteral vaccines by an oral formula may prove to be economically reasonable.

Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

Infection with Human papillomaviruses (HPVs) is linked to cervical cancer, which is one of the most common cancers found among women worldwide. Despite preventive immunization, a therapeutic vaccine, targeting infected individuals, is required. Vaccine strategies for treatment of HPV—induced cervical cancer are based on E7 and E6 oncoproteins. In this work we report the transient expression of chimeric particles containing the E6 oncoprotein from Human papillomavirus type 16 (HPV-16) in plants. We fused a mutagenized coding sequence of the E6 oncoprotein (E6GT) with the coding sequence of Potato virus X coat protein (PVX CP) both with the 5′- and 3′-terminus using linkers of different length (0, 4 or 15 amino acids). The expression in E. coli was performed to assess the characteristics of the recombinant protein prior to plant expression. The yield and immunological reactivity of the expressed proteins were screened with anti-PVX CP and E6 antibodies. The highest yields of chimeric particles were observed in the transgenic N. benthamiana expressing Potato virus A HC-Pro protein and the Tobacco mosaic virus movement protein. When inoculated on host plants, these recombinant viruses were not able to spread systemically. The obtained results revealed the new relations for design of expression cassettes for plant-based vaccine production.

Pulmonary hypertension in dialysis patients: a prevalent, risky but still uncharacterized disorder

1166–1173 48. Van Damme-Lombaerts R, Proesmans W, Van Damme B et al. Heparin plus dipyridamole in childhood hemolytic-uremic syndrome: a prospective, randomized study. J Pediatr 1988; 113: 913–918 49. Loirat C, Beaufils F, Sonsino E et al. Treatment of childhood hemolytic-uremic syndrome with urokinase. Cooperative controlled trial. Arch Fr Pediatr 1984; 41: 15–19 50. Sorrenti LY, Lewy PR. The hemolytic-uremic syndrome: experience at a center in the Midwest. Am J Dis Child 1978; 132: 59–62 51. Perez N, Spizzirri F, Rahman R et al. Steroids in the hemolytic uremic syndrome. Pediatr Nephrol 1998; 12: 101–104 52. Bitzan M, Poole R, Mehran M et al. Safety and pharmacokinetics of chimeric anti-Shiga toxin 1 and anti-Shiga toxin 2 monoclonal antibodies in healthy volunteers. Antimicrob Agents Chemother 2009; 53: 3081–3087 53. Lopez EL, Contrini MM, Glatstein E et al. Safety and pharmacokinetics of urtoxazumab, a humanized monoclonal antibody, against Shiga-like toxin 2 in healthy adults and in pediatric patients infected with Shiga-like toxin-producing Escherichia coli. Antimicrob Agents Chemother 2010; 54: 239–243 54. Dowling TC, Chavaillaz PA, Young DG et al. Phase 1 safety and pharmacokinetic study of chimeric murine-human monoclonal antibody c alpha Stx2 administered intravenously to healthy adult volunteers. Antimicrob Agents Chemother 2005; 49: 1808–1812 55. Mulvey GL, Marcato P, Kitov PI et al. Assessment in mice of the therapeutic potential of tailored, multivalent Shiga toxin carbohydrate ligands. J Infect Dis 2003; 187: 640–649 56. Watanabe M, Matsuoka K, Kita E et al. Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections. J Infect Dis 2004; 189: 360–368 57. Mukhopadhyay S, Linstedt AD. Manganese blocks intracellular trafficking of Shiga toxin and protects against Shiga toxicosis. Science 2012; 335: 332–335 58. Ahmed A, Li J, Shiloach Y et al. Safety and immunogenicity of Escherichia coli O157 O-specific polysaccharide conjugate vaccine in 2–5-year-old children. J Infect Dis 2006; 193: 515–521 59. Wen SX, Teel LD, Judge NA et al. A plant-based oral vaccine to protect against systemic intoxication by Shiga toxin type 2. Proc Natl Acad Sci 2006; 103: 7082–7087 60. Gao X, Cai K, Shi J et al. Immunogenicity of a novel Stx2BStx1B fusion protein in a mice model of Enterohemorrhagic Escherichia coli O157:H7 infection. Vaccine 2009; 27: 2070–2076 61. Gu J, Liu Y, Yu S et al. Enterohemorrhagic Escherichia coli trivalent recombinant vaccine containing EspA, intimin and Stx2 induces strong humoral immune response and confers protection in mice. Microbes Infect 2009; 11: 835–841 62. Bentancor LV, Bilen M, Brando RJ et al. A DNA vaccine encoding the enterohemorragic Escherichia coli Shiga-like toxin 2 A2 and B subunits confers protective immunity to Shiga toxin challenge in the murine model. Clin Vaccine Immunol 2009; 16: 712–718

Automated Production of Plant-Based Vaccines and Pharmaceuticals

A fully automated "factory" was developed that uses tobacco plants to produce large quantities of vaccines and other therapeutic biologics within weeks. This first-of-a-kind factory takes advantage of a plant viral vector technology to produce specific proteins within the leaves of rapidly growing plant biomass. The factory's custom-designed robotic machines plant seeds, nurture the growing plants, introduce a viral vector that directs the plant to produce a target protein, and harvest the biomass once the target protein has accumulated in the plants-all in compliance with Food and Drug Administration (FDA) guidelines (e.g., current Good Manufacturing Practices). The factory was designed to be time, cost, and space efficient. The plants are grown in custom multiplant trays. Robots ride up and down a track, servicing the plants and delivering the trays from the lighted, irrigated growth modules to each processing station as needed. Using preprogrammed robots and processing equipment eliminates the need for human contact, preventing potential contamination of the process and economizing the operation. To quickly produce large quantities of protein-based medicines, we transformed a laboratory-based biological process and scaled it into an industrial process. This enables quick, safe, and cost-effective vaccine production that would be required in case of a pandemic.

Can a plant-based vaccine treat hypertension?

Vaccines against hypertension constitute a viable approach to decrease blood pressure. In particular, two vaccines against hypertension (HP) targeting angiotensin II (AgII) have showed promising results and these are currently on evaluation in clinical trials. In parallel, plant-based vaccines have become a biotechnological application that has been assessed in clinical trials for some cases. This report proposes a hypothesis that involves developing a plant-based vaccine against HP. It is hypothesized that a plant-based vaccine having AgII or its AT1 receptor (ATR1) as targets, constitutes a safe, suitable and efficient therapeutic approach for HP. It is known that a number of carrier proteins can be produced in plants retaining its adjuvanticity. Therefore the production in plants of chimeric proteins where either AgII or ATR1 domains are fused to these carriers would be a promising approach to be investigated. Mucosal immunization using plant-derived AgII/ATR1 chimeric proteins would imply several advantages such as low cost and friendly delivery. However due to the lack of a detailed knowledge on the physiological role of AgII at the gastrointestinal tract, the effects of partially blocking the AgII action must be extensively evaluated. An alternative related to this aspect would be the use of transient expression systems where productivity is sufficiently high to allow the purification of the antigen of interest at convenient yields, so that it can constitute a parenteral vaccine. Proving the concept for a plant-based vaccine against HP may have profound implications on the development of a new HP therapy which offers convenient features such as low cost and easier compliance in comparison to pharmacological treatment.

Production of highly concentrated, heat‐stable hepatitis B surface antigen in maize

Plant-based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low-cost, heat-stable, oral alternative to parenterally administered commercial vaccines. Here, we describe high-level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed.

Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants

During the last decade interferons are regarded as potent candidates for generation of plant-based edible vaccines because of broad spectrum of antiviral activities and adjuvant properties. Establishment and certification of numerous interferon producing plant systems requests development of fast and efficient multiplex PCR protocol for the transgene detection in GM plants. Here we represent a protocol for simultaneous amplification in one assay of fragments of hIFN alpha 2b gene and two control genes, namely virD1 of Agrobacterium tumefaciens and conservative region of plant actin gene.

Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli.

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE–Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE–Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE–Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE–Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer’s patches from immunized mice. These results indicated that oral immunization of plant-produced COE–Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice.