Seed-specific expression of porcine verotoxigenic Escherichia coli antigens in tobacco plants as a potential model of edible vaccines.

Vaccines can reduce the use of antibiotics by preventing specific infective diseases in pigs. Plant-based edible vaccines are particularly attractive because, upon oral ingestion via feed, they can elicit the local immune system against a foreign disease-causing organism. The aim of this study was to engineer two different independent lines of tobacco plants for the seed-specific expression of immunogenic proteins of VTEC as a model of an edible vaccine. For each antigen, fifty Nicotiana tabacum L. cv Xanthi leaf disks were transformed by agroinfection for the seed-specific expression of the structural parts of the fimbrial subunit FedF of F18 and the B-subunit of Vt2e genes. The synthetic genes, optimized by the codon adaptation index for their expression in tobacco, were inserted into expression cassettes under the control of ß-conglycinin promoter. Regenerated tobacco plants (T0) were characterized by molecular and immunoenzymatic techniques. Our results showed that both FedF and Vt2eB genes were integrated into tobacco genome efficiently (> 80%) and they are also maintained in the second generation (T1). Western blotting analyses carried out on the positive producing lines, showed the tissue-specific expression in seeds and the temporal protein accumulation in the mid-late maturation phases. The enzyme-linked immunosorbent assay showed seed expression levels of 0.09 to 0.29% (from 138 to 444 µg/g of seeds) and 0.21 to 0.43% (from 321 to 658 µg/g of seeds) of total soluble protein for the FedF and Vt2eB antigens, respectively. This study confirmed the seed-specific expression of the selected antigens in plant seeds. The expression level is suitable for seed-based edible vaccination systems, which could represent a cost-effective way to prevent VTEC infection. Our findings encourage further in vivo studies focused on the activation of the local immune response.

Prophylactic and Therapeutic Effects of Oral Immunotherapy on Birch Pollen-Induced Allergic Conjunctivitis in Mice with a Rice-Based Edible Vaccine Expressing a Hypoallergenic Birch Pollen Allergen.

We investigated the prophylactic and therapeutic effects of the oral administration of transgenic rice seeds expressing a hypoallergenic Bet v 1 derivative of allergic birch pollen conjunctivitis in mice. Transgenic rice seed depositing a chimeric molecule called TPC7 (tree pollen chimera 7) created by DNA shuffling of Bet v 1 family sequences from birch, alder and hazel in protein bodies of endosperm was generated. BALB/c mice were sensitized to birch pollen in alum and challenged with pollen in eyedrops. They were fed TPC7 transgenic or non-transgenic (control) rice seeds for 14 d before sensitization (prophylactic protocol) or 17 d after sensitization (therapeutic protocol). The clinical score and number of conjunctival eosinophils were significantly lower in TPC7-fed mice than in the control mice based on both the prophylactic and therapeutic protocols. Serum concentration of allergen-specific IgE did not differ between TPC7-fed and control groups in either protocol. Prophylactic administration of TPC7 downregulated the production of IL-4 and IFN-γ, whereas therapeutic administration of TPC7 upregulated the production of IFN-γ by allergen-stimulated splenocytes. Prophylactic or therapeutic oral administration of transgenic rice expressing TPC7 suppressed birch pollen-induced allergic conjunctivitis in mice. Feeding transgenic rice is a potentially effective approach as an allergen-specific immunotherapy for allergic conjunctivitis.

Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines.

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52-90 µg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection.

A cross talk between the immunization and edible vaccine: Current challenges and future prospects.

INTRODUCTION: It is well known that immune system is highly specific to protect the body against various environmental pathogens. The concept of conventional vaccination has overcome the pandemic situation of several infectious diseases outbreak. AREA COVERED: The recent idea of immunization through oral route (edible vaccine) is vital alternatives over conventional vaccines. Edible vaccines are composed of antigenic protein introduced into the plant cells which induce these altered plants to produce the encoded protein. Edible vaccine has no way of forming infection and safety is assured as it only composed of antigenic protein and is devoid of pathogenic genes. Edible vaccines have significant role in stimulating mucosal immunity as they come in contact with digestive tract lining. They are safe, cost-effective, easy-to-administer and have reduced manufacturing cost hence have a dramatic impact on health care in developing countries. EXPERT OPINION: The edible vaccine might be the solution for the potential hazard associated with the parenteral vaccines. In this review we discuss the detailed study of pros, cons, mechanism of immune stimulation, various outbreaks that might be controlled by edible vaccines with the possible future research and applied application of edible vaccine.

A review on edible vaccines and their prospects.

For a long time, vaccines have been the main mode of defense and protection against several bacterial, viral, and parasitic diseases. However, the process of production and purification makes them expensive and unaffordable to many developing nations. An edible vaccine is when the antigen is expressed in the edible part of the plant. This reduces the cost of production of the vaccine because of ease of culturing. In this article, various types of edible vaccines that include algal and probiotics in addition to plants are discussed. Various diseases against which research has been carried out are also reviewed. This article focused on the conception of edible vaccines highlighting the various ways by which vaccines can be delivered.

A multi-epitope plant-made chimeric protein (LTBentero) targeting common enteric pathogens is immunogenic in mice.

KEY MESSAGE: A plant-based multiepitopic protein (LTBentero) containing epitopes from ETEC, S. typhimurium, and V. parahaemolyticus was produced in plants cells and triggered systemic and intestinal humoral responses in immunized mice. Around 200 million people suffer gastroenteritis daily and more than 2 million people die annually in developing countries due to such pathologies. Vaccination is an alternative to control this global health issue, however new low-cost vaccines are needed to ensure proper vaccine coverage. In this context, plants are attractive hosts for the synthesis and delivery of subunit vaccines. Therefore, in this study a plant-made multiepitopic protein named LTBentero containing epitopes from antigens of enterotoxigenic E. coli, S. typhimurium, and V. parahaemolyticus was produced and found immunogenic in mice. The LTBentero protein was expressed in tobacco plants at up to 5.29 µg g-1 fresh leaf tissue and was deemed immunogenic when administered to BALB/c mice either orally or subcutaneously. The plant-made LTBentero antigen induced specific IgG (systemic) and IgA (mucosal) responses against LTB, ST, and LptD epitopes. In conclusion, multiepitopic LTBentero was functionally produced in plant cells, being capable to trigger systemic and intestinal humoral responses and thus it constitutes a promising oral immunogen candidate in the fight against enteric diseases.

Edible Vaccines: Promises and Challenges.

Vaccines are biological preparations that improve immunity to particular diseases and form an important innovation of 19th century research. It contains a protein that resembles a disease-causing microorganism and is often made from weak or killed forms of the microbe. Vaccines are agents that stimulate the body's immune system to recognize the antigen. Now, a new form of vaccine was introduced which will have the power to mask the risk side of conventional vaccines. This type of vaccine was produced from plants which are genetically modified. In the production of edible vaccines, the gene-encoding bacterial or viral disease-causing agent can be incorporated in plants without losing its immunogenic property. The main mechanism of action of edible vaccines is to activate the systemic and mucosal immunity responses against a foreign disease-causing organism. Edible vaccines can be produced by incorporating transgene in to the selected plant cell. At present edible vaccine are developed for veterinary and human use. But the main challenge faced by edible vaccine is its acceptance by the population so that it is necessary to make aware the society about its use and benefits. When compared to other traditional vaccines, edible vaccines are cost effective, efficient and safe. It promises a better prevention option from diseases.

Expressing the immunodominant projection domain of infectious bursal disease virus fused to the fragment crystallizable of chicken IgY in yellow maize for a prospective edible vaccine.

Control of Infectious bursal disease virus (IBDV) in endemic countries has been based on early immunization of chicks using conventional live or inactivated vaccines that became not fully effectual and have biosafety concerns. This endeavor seeks generating a recombinant chimeric protein merging the projection domain (PD) of IBDV VP2 capsid with the fragment crystallizable (Fc) of avian IgY (FcIgY), in maize as a prospective poultry edible vaccine. The PD sequence was built on the basis of very virulent IBDV isolates circulating in Egypt. After optimization of codon-usage in maize, sequences of PD and FcIgY were effectively expressed in two elites of yellow maize via bombardment transformation in immature embryos. Chimeric protein amount in stable transgenic samples ranged from1.36% to 3.03% of the total soluble protein based on tissue age and maize cultivar. IBDV VP2 coding sequence was amplified from viral RNA, cloned, and expressed in E. coli. A group of Balb/C mice were hyper-immunized with purified recombinant VP2 protein for raising anti- recombinant VP2 antibodies (anti-rVP2 Ab). Proper expression in maize and immunoreactivity of the chimeric protein (PD-FcIgY) to chicken anti- IBDV and anti-rVP2 Ab were confirmed by both direct and indirect double antibody sandwich (DAS)-ELISAs as well as western blotting. Seeds of regenerated transgenic maize will be validated for chickens as edible vaccination in further studies.

A review on edible vaccines and their prospects

For a long time, vaccines have been the main mode of defense and protection against several bacterial, viral, and parasitic diseases. However, the process of production and purification makes them expensive and unaffordable to many developing nations. An edible vaccine is when the antigen is expressed in the edible part of the plant. This reduces the cost of production of the vaccine because of ease of culturing. In this article, various types of edible vaccines that include algal and probiotics in addition to plants are discussed. Various diseases against which research has been carried out are also reviewed. This article focused on the conception of edible vaccines highlighting the various ways by which vaccines can be delivered.

Immunogenicity Evaluation of Recombinant Edible Vaccine Candidate Containing HER2-MUC1 against Breast Cancer.

Human epithelial growth factor receptor2 (Her2) and polymorphic epithelial mucin (MUC1) are tumor-associated antigens that have been extensively investigated in adenocarcinomas. Generally, each of these molecules was used separately for diagnosis of adenocarcinomas and as an injective vaccines in cancer therapy researches, but not in the chimeric form as an edible immunogen. In this study, Her2, MUC1, and a novel fusion structure were expressed in the seeds and hairy roots of transgenic plants appropriately. The mice groups were immunized either by feeding of transgenic seeds or hairy roots. All immunized groups showed a considerable rise in anti-glycoprotein serum IgG and IgA, and IFNÉ£ cytokine. However, the animals received chimeric protein showed significant higher immune responses in comparison to ones received one of these immunogen. The results indicated that the oral immunization of an animal model with transgenic plants could effectively elicit immune responses against two major tumor-associated antigens.

Oral immunization with a plant-derived chimeric protein in mice: Toward the development of a multipotent edible vaccine against E. coli O157: H7 and ETEC.

The most bacterial cause of infectious diseases associated with diarrhea are enterotoxigenic and enterohemorrhagic Escherichia coli (ETEC and EHEC, respectively). These strains use colonization factors for the attachment to the human intestinal mucosa, followed by enterotoxins production that could induce more host damage. The Heat-labile enterotoxin (LT) and colonization factors (CFs) are momentous factors for the pathogenesis of ETEC. Also, Intimin and Shiga like toxin (STX) are the main pathogenic factors expressed by EHEC. Because of mucosal surfaces are the major entry site for these pathogens, oral immunization with providing the protective secretary IgA antibody (sIgA) responses in the mucosa, could prevent the bacterial adherence to the intestine. In this study oral immunogenicity of a synthetic recombinant protein containing StxB, Intimin, CfaB and LtB (SICL) was investigated. For specific expression in canola seeds, the optimized gene was cloned in to plant expression vector containing the Fatty Acid Elongase (FAE) promoter. The evaluation of the expression level in canola seeds was approximately 0.4% of total soluble protein (TSP). Following to oral immunization of mice, serum IgG and fecal IgA antibody responses induced. Caco-2 cell binding assay with ETEC shows that the sera from immunized mice could neutralize the attachment properties of toxigenic E. coli. The reduction of bacterial shedding after the challenge of immunized mice with E. coli O157:H7 was significant. The sera from immunized mice in the rabbit ileal loop experiment exhibited a significant decrease in the fluid accumulation compared to the control. The results indicate efficacy of the recombinant chimeric protein SICL in transgenic canola seed as an effective immunogen, which elicits both systemic and mucosal immune responses as well as protection against EHEC and ETEC adherence and toxicity.

A Preliminary Study of a Lettuce-Based Edible Vaccine Expressing the Cysteine Proteinase of Fasciola hepatica for Fasciolosis Control in Livestock.

Oral vaccination with edible vaccines is one of the most promising approaches in modern vaccinology. Edible vaccines are an alternative to conventional vaccines, which are typically delivered by injection. Here, freeze-dried transgenic lettuce expressing the cysteine proteinase of the trematode Fasciola hepatica (CPFhW) was used to orally vaccinate cattle and sheep against fasciolosis, which is the most important trematode disease due to the parasite's global distribution, wide spectrum of host species and significant economic losses of farmers. In the study, goals such as reducing the intensity of infection, liver damage and F. hepatica fecundity were achieved. Moreover, we demonstrated that the host sex influenced the outcome of infection following vaccination, with female calves and male lambs showing better protection than their counterparts. Since differences occurred following vaccination and infection, different immunization strategies should be considered for different sexes and host species when developing new control methods. The results of the present study highlight the potential of oral vaccination with plant-made and plant-delivered vaccines for F. hepatica infection control.

Induction of PrPSc-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease.

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrPC into PrPSc. Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrPSc-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrPSc-specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.

Do microRNAs play a role in the activity of plant-based vaccines?

INTRODUCTION: An important trend in vaccinology is the development of oral vaccines based on genetically modified plants. Areas covered: Several studies have suggested that dietary microRNAs from plants and other organisms are bioavailable upon oral ingestion exerting biological events in the host such as the modulation of gene expression in several cell types. Since oral plant-based vaccines rely on whole cells as vaccine delivery vehicles, miRNAs could play a role in the immunogenic activity of this type of vaccine. In the present report, this hypothesis is discussed under the light of recent evidence on the immunomodulatory activity exerted by miRNAs using in vitro and in vivo evaluations. Expert commentary: The ways to generate new knowledge and exploit the potential of miRNAs in the development of oral vaccines are discussed.

Immunotherapies for neurodegenerative diseases: current status and potential of plant-made biopharmaceuticals.

INTRODUCTION: Neurodegenerative diseases (NDs) have a serious impact on global health with no effective treatments available to date. Vaccination has been proposed as a therapeutic approach for NDs, and clinical evaluations of some candidates for Alzheimer's disease and multiple sclerosis are ongoing. Moreover, monoclonal antibodies for passive immunotherapy are under evaluation for Alzheimer's, synucleinopathies, and multiple sclerosis. Areas covered: With the consolidation of plant-based systems for the production and oral delivery of biopharmaceuticals, interesting perspectives arise in the fight against NDs. Based on analysis of the current biomedical literature, the role of plant-made biopharmaceuticals and the outlook on how this technology is leading to new therapeutic candidates and potential developments for NDs are presented in this review. Expert commentary: Substantial innovations in the following years are expected as a consequence of applying molecular pharming in the fight against NDs.

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B.

Disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. Every year more than 100 million children are vaccinated with the standard World Health Organization (WHO)-recommended vaccines including hepatitis B (HepB). HepB is the most serious type of liver infection caused by the hepatitis B virus (HBV), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. To date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. Among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. Research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently HepB and influenza. More inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. In this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis B surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed.

Oral rice-based vaccine induces passive and active immunity against enterotoxigenic E. coli-mediated diarrhea in pigs.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in both neonatal and weaned pigs. Because the cholera toxin B subunit (CTB) has a high level of amino acid identity to the ETEC heat-labile toxin (LT) B-subunit (LTB), we selected MucoRice-CTB as a vaccine candidate against ETEC-induced pig diarrhea. When pregnant sows were orally immunized with MucoRice-CTB, increased amounts of antigen-specific IgG and IgA were produced in their sera. CTB-specific IgG was secreted in the colostrum and transferred passively to the sera of suckling piglets. IgA antibodies in the colostrum and milk remained high with a booster dose after farrowing. Additionally, when weaned minipigs were orally immunized with MucoRice-CTB, production of CTB-specific intestinal SIgA, as well as systemic IgG and IgA, was induced. To evaluate the cross-protective effect of MucoRice-CTB against ETEC diarrhea, intestinal loop assay with ETEC was conducted. The fluid volume accumulated in the loops of minipigs immunized with MucoRice-CTB was significantly lower than that in control minipigs, indicating that MucoRice-CTB-induced cross-reactive immunity could protect weaned pigs from diarrhea caused by ETEC. MucoRice-CTB could be a candidate oral vaccine for inducing both passive and active immunity to protect both suckling and weaned piglets from ETEC diarrhea.

Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines.

Chikungunya virus is an emerging pathogen initially found in East Africa and currently spread into the Indian Ocean Islands, many regions of South East Asia, and in the Americas. No licensed vaccines against this eminent pathogen are available and thus intensive research in this field is a priority. This review presents the current scenario on the developments of Chikungunya virus vaccines and identifies the use of genetic engineered plants to develop attractive vaccines. The possible avenues to develop plant-made vaccines with distinct antigenic designs and expression modalities are identified and discussed considering current trends in the field.

The potential of plants for the production and delivery of human papillomavirus vaccines.

The available vaccines against human papillomavirus have some limitations such as low coverage due to their high cost, reduced immune coverage and the lack of therapeutic effects. Recombinant vaccines produced in plants (genetically engineered using stable or transient expression systems) offer the possibility to obtain low cost, efficacious and easy to administer vaccines. The status on the development of plant-based vaccines against human papillomavirus is analyzed and placed in perspective in this review. Some candidates have been characterized at a preclinical level with interesting outcomes. However, there is a need to perform the immunological characterization of several vaccine prototypes, especially through the oral administration route, as well as develop new candidates based on new chimeric designs intended to provide broader immunoprotection and therapeutic activity.