High-Yield Expression of a Viral Peptide Vaccine In Transgenic Plants (original) (raw)
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High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts
Plant Biotechnology Journal, 2004
The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV ), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant).
Plant Biotechnology Journal, 2004
The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and GM1-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (∼100 mg for a dose of 500 µg antigen) and would permit encapsulation of freeze-dried material or pill formation.
Recent Progress on Vaccines Produced in Transgenic Plants
Vaccines
The development of vaccines from plants has been going on for over two decades now. Vaccine production in plants requires time and a lot of effort. Despite global efforts in plant-made vaccine development, there are still challenges that hinder the realization of the final objective of manufacturing approved and safe products. Despite delays in the commercialization of plant-made vaccines, there are some human vaccines that are in clinical trials. The novel coronavirus (SARS-CoV-2) and its resultant disease, coronavirus disease 2019 (COVID-19), have reminded the global scientific community of the importance of vaccines. Plant-made vaccines could not be more important in tackling such unexpected pandemics as COVID-19. In this review, we explore current progress in the development of vaccines manufactured in transgenic plants for different human diseases over the past 5 years. However, we first explore the different host species and plant expression systems during recombinant protein ...
Plant Viral Vectors as a Tool for Recombinant Vaccine Production
Over the last few years the development of plant viral vectors for protein expression has made rapid and impressive progress. Plant viruses are versatile vectors for production of proteins since they are easy to manipulate, quick to evaluate and offer the possibility of great production yields. They are particularly powerful expression systems for the production of recombinant proteins and peptides for vaccination, since they are able to produce antigens alone or conjugated to viral capsid. Several viral vectors have been developed for vaccine production, mainly Tobacco mosaic virus (TMV) and Potato virus X (PVX). In this review, we will discuss the advantages of the use of plant viral vectors as expression vectors, the principal vectors that have been developed to date, and the different strategies used for expression, with emphasis on recent research conducted in Argentina.
Transgenic plants for the production of veterinary vaccines
Immunology and Cell Biology, 2005
The expression of antigens in transgenic plants has been increasingly used in the development of experimental vaccines, particularly oriented to the development of edible vaccines. Hence, this technology becomes highly suitable to express immunogenic proteins from pathogens. Foot and mouth disease virus, bovine rotavirus and bovine viral diarrhoea virus are considered to be the most important causative agents of economic loss of cattle production in Argentina, and they are thus optimal candidates for alternative means of immunization. Here, we present a review of our results corresponding to the expression of immunogenic proteins from these three viruses in alfalfa transgenic plants, and we discuss the possibility of using them for the development of plant-based vaccines.
Use of Plant Viruses for Delivery of Vaccine Epitopes
Annals of the New York Academy of Sciences, 1996
Certain plant viruses have the potential to be developed for the synthesis and accumulation of proteins and epitopes that are inducers of protective immunity against human and animal diseases. Such plant viruses should replicate to high levels in infected plant tissues, be readily purified, be stable in viuo and in uitro, and be sufficiently characterized both genetically and structurally to allow for directed molecular modifications. Viruses that have been used specifically to produce vaccine epitopes include tobacco mosaic tobarnovirus (TMV), cowpea mosaic comovirus (CPMV), and johnsongrass mosaic potyvirus (JMV). Discussion in this paper is limited to generic modification of replicating viruses that lead to production and accumulation of virions that display unique sequences on the surface of the virus particle. The earliest report of research to modify a plant virus capsid (coat) protein to carry a foreign epitope described genetic modification to cause the addition of eight amino acids derived from a poliovirus capsid protein to the coat protein of TMV and expression of the modified protein in Escherichia coli. Injecting the assembled protein subunits of TMV to mice caused production of antibody against the poliovirus epitope.' Recently a similar approach was taken with JMV to demonstrate that the coat protein of this potyvirus could assemble to form virus-like particles in E. coli when up to 26 kD of foreign protein sequences were added to, or replaced, the amino terminal sequence of the coat protein.* Recently there have been a number of reports on the construction of cloned cDNAs that represent the full-length genome(s) of viral RNAs from which infectious viral RNAs can be transcribed in uitro. The infectious cloned cDNAs (the term used to describe such constructs) of several plant viruses have been subjected to in uitro mutagenesis so that they encode modified coat proteins during virus replication, including proteins that can serve as immunogenic epitopes. Furthermore, knowledge of the crystal structures of TMV and CPMV makes it possible to predict the impact of foreign sequences on capsid and virion structures. These advances allow the design of strategies to develop these viruses as delivery vehicles for vaccine epitopes.
Vaccine, 2002
The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ß-glucuronidase (ßGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ßGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ßGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.
Vaccine Production in Transgenic Plants for Animal and Human Diseases
2019
Introduction Vaccines are a biological preparation that provides active acquired immunity to a particular disease. They can be produced by using different methods of which the egg-based vaccines, cell-based vaccines, and vaccines produced using investigational-manufacturing (plant, bacterial culture and insect cell) systems are some to mention. The technology of the use of plants as a bioreactor to produce human or animal therapeutic vaccines receives increasing attention. Many of these subunit vaccines have been purified and also administered orally in a non-purified form as a food or feed product and this review mainly focuses on edible vaccines. Antigens from several human and veterinary pathogens have been expressed in transgenic plants, including Norwalk virus, rabies, measles, hepatitis B, anthrax, infectious bursal disease virus, and avian influenza virus. Production of edible subunit-based recombinant vaccine proteins in the form of leaves, seeds or fruit is expected to be c...
Immunotherapy of plant viral diseases
Trends in microbiology, 1995
The stable expression of antibodies in plants is one recent strategy for the unconventional control of plant viruses that is undergoing development. The advantages of this approach are its wide applicability and intrinsic safety; however, to be successful, the 'genetic immunization' of plants requires careful antibody design, efficient expression and targeting to appropriate cell compartments.