Production Of Edible Vaccines Research Articles

Agrobacterium tumefaciens: Biology and application in genetic engineering

Agrobacterium tumefaciens is a rod-shaped soil bacterium renowned for its unique ability to transfer tumour-inducing plasmid (Ti plasmid) segments to plant cells. This mechanism has been extensively exploited in plant genetic engineering. This review delves into the intricate biological interactions between A. tumefaciens and plant cells, including the critical steps of bacterial attachment, activation of virulence (Vir) genes, generation and transport of the T-complex, and integration of T-DNA into the plant chromosome. Furthermore, the review examines the engineering of A. tumefaciens as a transformation tool, focusing on the modifications of Ti plasmids to create binary and co-integrative vector systems, which have significantly improved the efficiency and versatility of transformation protocols. The paper also highlights applications of Agrobacterium-mediated transformation in the production of edible vaccines. By providing a detailed examination of the biological, technical, and practical aspects of Agrobacterium-mediated transformation, this review aims to offer insights into optimizing this technique for various plant biotechnology applications. Ultimately, understanding and improving Agrobacterium-mediated transformation is crucial for advancing plant biotechnology.

Suitability of different plant species for experimental agroinfection with Plum pox virus-based expression vector for potential production of edible vaccines.

Nine herbaceous plant species were tested for susceptibility to Plum pox virus (PPV) by Agrobacterium-mediated delivery of its infectious cDNA clone. Two of them became infected, namely spinach (local infection) and oilseed poppy (systemic infection). As acontrol, PPV infection was successfully established in plum seedlings following agroinfiltration, thus providing the first report of agroinfection in Prunus species. According to our results, oilseed poppy can be considered as acandidate host for the production of edible vaccines by aPPV-derived expression vector. Keywords: agroinfiltration; virus host; poppy; spinach.

Plastome engineering in vegetable crops: current status and future prospects.

Plastome (plastid genome) engineering has grown up and got smarter for the transgene expression. Plastid transformation has profound benefits over nuclear transformation, includes a higher level of transgene expression, integration via homologous recombination, transgene containment, lack of gene silencing, and position effect. Substantial and fruitful progress has been achieved in plastome engineering of vegetable crops through the use of improved regeneration/selection procedures, plastid transformation vectors with efficient promoters, and 3/, 5/regulatory sequences. Plastid transformation technology developed for vegetable crops being used as a platform for the production of industrially important proteins and some of the genes of agronomic importance has been stably integrated and expressed in plastome. Although great progress has been accomplished in the plastid transformation of vegetable crops, still it is restricted to few species because of the unavailability of whole plastome sequencing. In this review, the author focus on the technology, progress, and advancements in plastid transformation of vegetable plants such as lettuce, tomato, potato, cabbage, cauliflower, eggplant, carrot, soybean, and bitter melon are reviewed. The conclusions, future prospects, and expansion of plastid transformation technology to other vegetable crops for genetic improvement and production of edible vaccines are proposed.

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.

Role of oral vaccines as an edible tool to prevent infectious diseases.

Plants have been as medicinal mediators for centuries. Recent trends in agro-biotechnology however, improved the therapeutic roles of plants to a significant level and introduced plant-based oral vaccine which can arouse an immune response in consumer. Although conventional vaccines against infectious diseases have been administrated for years the discovery of plant-based oral vaccines can potentially replace them completely in the future. The probable limitations in conventional vaccines are found to be overcome by plant-based oral vaccines. Humans and animals will no longer be dependent upon local or systemic administration of vaccines but they will just receive the vaccines as a routine food. For the purpose, gene of interest is introduced into plant through transformation, and expression of specific antigen is obtained in plant products which are then consumed by humans or animals. Therefore, plants can serve as bioreactors or bio-factories for production of edible vaccines. A detailed overview about edible vaccines, methods for edible vaccine production, candidate bioreactors and future perspectives of edible vaccines has been summarized in current article. The future of vaccination seems to be present within plant-based vaccination system. Keywords: vaccine; edible vaccine; infectious diseases; antigen; edible crops; oral immunization.

A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana

A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.

Escherichia coli expression of NDV fusion protein gene and determination of its antigenic epitopes

AbstractRecurrent outbreaks of Newcastle disease have questioned the usage of existing vaccines that whether they are still adequate to protect clinical diseases and inhibit virus transmission in poultry. Advancement in molecular biology has led to the production of recombinant vaccines in recent years, which can be a more useful strategy to control infections of Newcastle disease virus (NDV). Studies indicate that the pathogenic nature of NDV is mediated by its membrane associated fusion (F) protein. Here we report the cloning of the full-length F gene-pET30a and its expression in Escherichia coli BL21 DE3 cells through isopropyl β-D-1-thiogalactopyranoside induction. Transferring the protein on nitrocellulose membrane in Western blotting confirmed its specificity with histidine-tagged antibody reaction at the proper size of 67 kDa. Protein purification with nickel charged sepharose column affinity chromatography resulted in a single band of 67 kDa purified His-tag F protein on SDS-PAGE. Analysis of its immunogenicity through bioinformatics tools revealed that more than 70% of its sequence is antigenically active comprising 24 linear immunogenic peptides predicted by the Linear epitope prediction tool and 9 immunogenic peptides predicted by ElliPro. This is a key achievement of the study, which may lead towards recombinant vaccine production in future. In conclusion, our findings suggest that rather than employing live viral vaccines, using a purified immunogenic recombinant F protein as a vaccine or cloning the same gene in a suitable plant vector for production of edible vaccine will provide better protection against the NDV into chicken.

Food as a Vaccine

As the science and technology advances, a leap in medical field occurred and all the painful vaccination procedures replaced with modernized techniques like edible vaccines, a non-invasive technique. Vaccine contains weak pathogens of bacteria or virus or a killed bacterial suspension or toxins produced by bacterial strains, when taken inside human body, our immune system finds out the foreign particle and it produces a group of antibodies to eradicate it and making a memory of it. Edible vaccines are genetically modified crop, in which vaccines are produced in the plant parts when antigenic proteins are transgenically introduced into edible crop and work same as that of original vaccines. Present review is focused on several methods used in production of edible vaccines and the types of food used for production of edible vaccines. And the challenges we are still facing in development which needs to be focused more by the future generations.

Isolation of OmpA gene from Salmonella typhimurium and transformation into alfalfa in order to develop an edible plant based vaccine

The outer membrane protein A (OmpA) of Salmonella typhimurium may contribute to immunity and virulence in livestock animals. Introduction of this gene in forage crops like alfalfa may be an alternative and effective way to produce animal edible vaccine. In the present study, the OmpA gene was obtained after polymerase chain reaction (PCR) amplification and sequencing. We successfully identified the complete ORF encoding this protein. In order to express OmpA protein in alfalfa, the gene was inserted into a plant expression vector PBI121.The recombinant OmpA was expressed in Escherichia coli TG1. The new construct was used to transform the Agrobacterium tumefaciens Strain LBA4404 before plant transformation. Transgenic alfalfa plants were then developed by introducing OmpA gene in the plant genome under the control of Camv35s promoter and for the first time we expressed this protein in alfalfa. Releasing this one new transgenic variety may be a considerable progress towards release varieties which enables the production of edible vaccine. Key words : Outer membrane protein A (OmpA), sub cloning, plantibodies, transgenic alfalfa, bioreactor, edible vaccine.

Engineering the Chloroplast Targeted Malarial Vaccine Antigens in Chlamydomonas Starch Granules

BackgroundMalaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii.Methods and FindingsWe describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species.ConclusionThis novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.

Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.).

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB-sCOE), and the sCTB-sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB-sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB-COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB-COE fusion protein to its receptor, G(M1)-ganglioside, in transgenic plants was confirmed via G(M1)-ELISA with anti-cholera toxin and anti-COE antibody. Based on G(M1)-ELISA, the expression level of CTB-COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB-COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.

어류 경구백신 현황과 전망

As the capture fishing industry has declined, the aquaculture industry has become an important source of seafood. With this tendency all fish farming will be performed by large-scale farms where the fish are cultivated in much high density and as a result the incidence of infectious diseases increases. Therefore, vaccination has become an increasingly important part of aquaculture as a cost effective method of controlling various diseases. The early fish vaccines were the formalin inactivated bacteria or virus cultures, which were administered by either immersion or injection. Recombinant DNA biotechnology allowed us to develop orally administrated DNA and recombinant vaccines. In terms of the manufacturing process and cost, Lemna and Spirodela is the most efficient and reliable plant expression system for the production of edible vaccine.

Plant biotechnology for deeper understanding, wider use and further development of agricultural and horticultural crops

Plants bind solar energy to organic matter via photosynthesis and assimilation of carbon dioxide from the atmosphere and comprise the major source of nutrition and bioenergy. Plant biotechnology contributes to solution of important constraints in food and feed production and creates new technologies and applications for the sustainable use of plant resources. Genome-wide approaches such as massive parallel sequencing and microarrays to study gene expression, molecular markers for selection of important traits in breeding, characterization of genetic diversity with the aforementioned approaches, and somatic hybridization and genetic transformation are important tools in plant biotechnology. In this paper, studies carried out on enhanced resistance to viruses and tolerance of cold stress in potato, genetic modification of flower pigmentation and morphology in gerbera, production of edible vaccines in transgenic barley seeds, and expression of heterologous proteins for pharmaceutical purposes from vector viruses were chosen to exemplify the general utility of biotechnological approaches and also how plant biotechnology research has developed on cultivated plants at University of Helsinki. The studies reveal cellular and genetic mechanisms and provide scientific information that can be used for widening the uses of crop plants. They can also be used to detect any putative risks associated with the use of the biotechnological application in agriculture and horticulture and to develop practises which reduce any inadvertent negative consequences that plant production may have to the environment.;

Modern Biotechnology as an Integral Supplement to Conventional Plant Breeding: The Prospects and Challenges

The art of plant breeding was developed long before the laws of genetics became known. The advent of the principles of genetics at the turn of the last century catalyzed the growth of breeding, making it a science‐based technology that has been instrumental in substantial improvements in crop plants. Largely through exploitation of hybrid vigor, grain yields of several cereal crops were substantially increased. Intervarietal and interspecific hybridizations, coupled with appropriate cytogenetic manipulations, proved useful in moving genes for resistance to diseases and insect pests from suitable alien donors into crop cultivars. Plant improvement has been further accelerated by biotechnological tools of gene transfer, to engineer new traits into plants that are very difficult to introduce by traditional breeding. The successful deployment of transgenic approaches to combat insect pests and diseases of important crops like rice (Oryza sativa L.), wheat (Triticum aestivum L.), maize (Zea mays L.), barley (Hordeum vulgare L.), and cotton (Gossypium hirsutum L.) is a remarkable accomplishment. Biofortification of crops constitutes another exciting development in tackling global hunger and malnutrition. Golden Rice, genetically enriched with vitamin A and iron, has, for example, the real potential of saving millions of lives. Yet another exciting application of transgenic technology is in the production of edible vaccines against deadly diseases. How these novel approaches to gene transfer can effectively supplement the conventional breeding programs is described. The current resistance to acceptance of this novel technology should be assessed and overcome so that its full potential in crop improvement can be realized.

Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit.

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.