Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Use of the wound-inducible NtQPT2 promoter from Nicotiana tabacum for production of a plant-made vaccine

The wound-inducible quinolinate phosphoribosyl transferase promoter from Nicotiana tabacum (NtQPT2) was assessed for its capacity to produce B-subunit of the heat-labile toxin (LTB) from enterotoxigenic Escherichia coli in transgenic plant tissues. Comparisons were made with the widely used and constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter. The NtQPT2 promoter produced somewhat lower average concentrations of LTB protein per unit weight of hairy root tissue but allowed better growth thereby producing similar or higher overall average yields of LTB per culture batch. Transgenic tobacco plants containing the NtQPT2-LTB construct contained LTB protein in roots but not leaves. Moreover, wounding NtQPT2-LTB transgenic plants, by removal of apices, resulted in an approximate 500% increase in LTB levels in roots when analysed several days later. CaMV35S-LTB transgenic plants contained LTB protein in leaves and roots but wounding made no difference to their LTB content.

Plant-produced vaccines: promise and reality

Plant-produced vaccines are a much-hyped development of the past two decades, whose time to embrace reality may have finally come. Vaccines have been developed against viral, bacterial, parasite and allergenic antigens, for humans and for animals; a wide variety of plants have been used for stable transgenic expression as well as for transient expression via Agrobacterium tumefaciens and plant viral vectors. A great many products have shown significant immunogenicity; several have shown efficacy in target animals or in animal models. The realised potential of plant-produced vaccines is discussed, together with future prospects for production and registration. © 2008 Elsevier Ltd. All rights reserved.

Intellectual Property Protection For Plant Derived Vaccine Technology: Here They Come Are We Ready or Not ?

Il existe une nouvelle catégorie de technologie, les vaccins dérivés de plantes («VDPs»), qui englobe des produits qui ont un grand potentiel pour l’amélioration de la santé à l’échelle globale. Bien qu’ils ne soient pas encore disponibles pour le public, le développement des VDPs a progressé de façon telle qu’ils devraient être prêts à être mis en marché et distribués sous peu. Ainsi, c’est le moment idéal pour lancer un débat sur la meilleure façon de protéger cette nouvelle catégorie de technologie. Vu leur nature, les VDPs ne se qualifient pas parfaitement pour aucune forme de protection de propriété intellectuelle. En effet, un VDP est à la fois une variété de plante, une biotechnologie, un médicament et un produit qui vise spécifiquement les besoins de pays en voie de développement. Chacune de ces caractéristiques soulève ses propres problématiques en ce qui a trait à la propriété intellectuelle. C’est pourquoi il appert difficile d’identifier la forme de protection la plus adéquate et appropriée pour les VDPs. Cet article traite de la nature d’un VDP, des différentes catégories dans lesquelles il pourrait être classé, des différents types de systèmes de protection de propriété intellectuelle auxquels il pourrait être éligible ainsi que des problèmes qui pourraient être soulevés par tous ces éléments. Ces discussions visent à mettre l’accent sur le fait que nous avons affaire à une toute nouvelle catégorie d’innovation technologique. L’auteure est donc d’avis qu’une approche proactive est nécessaire pour discuter d’un système de protection de propriété intellectuelle en relation avec les VDPs. En ce moment, c’est l’inventeur qui choisi comment il protègera son invention. Les moyens employés par ce dernier pourraient être subséquemment modifiés ou annulés par une décision judiciaire mais comme plusieurs autres inventeurs d’une même catégorie de technologie auront probablement déjà adopté une stratégie de protection similaire, ce type de mesures judiciaire, très tard dans le processus, pourra avoir des résultats néfastes sur les détenteurs de droits. Le développement de lignes directrices d’entrée de jeu, avec l’aide d’un panel d’experts de préférence, peut contribuer à éviter les situations de confusion qui ont déjà été vécues avec l’application d’autres nouvelles technologies et qui devraient servir de leçon pour l’encadrement des VDPs.

Human papillomavirus vaccines in plants

Human papillomaviruses are the etiological agents of cervical cancer, one of the two most prevalent cancers in women in developing countries. Currently available prophylactic vaccines are based on the L1 major capsid protein, which forms virus-like particles when expressed in yeast and insect cell lines. Despite their recognized efficacy, there are significant shortcomings: the vaccines are expensive, include only two oncogenic virus types, are delivered via intramuscular injection and require a cold chain. Plant expression systems may provide ways of overcoming some of these problems, in particular the expense. In this article, we report recent promising advances in the production of prophylactic and therapeutic vaccines against human papillomavirus by expression of the relevant antigens in plants, and discuss future prospects for the use of such vaccines. © 2010 Expert Reviews Ltd.

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.

Plant-made vaccines for humans and animals

Summary: The concept of using plants to produce high-value pharmaceuticals such as vaccines is 20 years old this year and is only now on the brink of realisation as an established technology. The original reliance on transgenic plants has largely given way to transient expression; proofs of concept for human and animal vaccines and of efficacy for animal vaccines have been established; several plant-produced vaccines have been through Phase I clinical trials in humans and more are scheduled; regulatory requirements are more clear than ever, and more facilities exist for manufacture of clinic-grade materials. The original concept of cheap edible vaccines has given way to a realisation that formulated products are required, which may well be injectable. The technology has proven its worth as a means of cheap, easily scalable production of materials: it now needs to find its niche in competition with established technologies. The realised achievements in the field as well as promising new developments will be reviewed, such as rapid-response vaccines for emerging viruses with pandemic potential and bioterror agents. © 2010 The Author. Journal compilation © 2010 Blackwell Publishing Ltd.

Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector

A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 μg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector. © 2006 Elsevier B.V. All rights reserved.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

The release and induced immune responses of a plant-made and delivered antigen in the mouse gut

This study investigated the site of release of a model vaccine antigen from plant cells and the corresponding induced immune response. Three plant tissues (leaf, fruit and hairy root) and two formulations (aqueous and lipid) were compared in two mouse trials. A developed technique that enabled detection of antigen release by plant cells determined that antigen release occurred at early sites of the gastrointestinal tract when delivered in leaf material and at later sites when delivered in hairy roots. Lipid formulations delayed antigen release from all plant materials tested. While encapsulation in the plant cell provided some protection of the antigen in the gastrointestinal tract and influenced antigen release, formulation medium was also an important consideration with regard to vaccine delivery and immunogenicity. Systemic immune responses induced from the orally delivered vaccine benefited from late release of antigen in the mouse gastrointestinal tract. The influences to the mucosal immune response induced by these vaccines were too complex to be determined by studies performed here with no clear trend regarding plant tissue site of release or formulation medium. Expression and delivery of the model antigen in plant material prepared in an aqueous formulation provided the optimal systemic and mucosal, antigen-specific immune responses.

Riding the tide of biopharming in Africa: Considerations for risk assessment

In the past few years, plant biotechnology has gone beyond traditional agricultural production of food, feed and fibre, and moved to address more complex contemporary health, social and industrial challenges. The new era involves production of novel pharmaceutical products, speciality and fine chemicals, phytoremediation and production of renewable energy resources to replace non-renewable fossil fuels. Plants have been shown to provide a genuine and low-cost alternative production system for high-value products. Currently, the principal plant-made products include antibodies, feed additives, vaccine antigens and hormones for human and animal health, and industrial proteins. Despite the unique advantages of scalability, cost and product safety, issues of politics, environmental impact, regulation and socioeconomics still limit the adoption of biopharmaceuticals, especially in the developing world. Plant-based production systems have further complicated biosafety, gene flow and environmental impact assessments with generally genetically modified plants, topics that are already partially understood. This article provides a background to biopharming, highlighting basic considerations for risk assessment and regulation in developing countries, with an emphasis on plant-based vaccine production in South Africa.

Biopharming the SimpliRED™ HIV diagnostic reagent in barley, potato and tobacco

This is the first report of an antibody-fusion protein expressed in transgenic plants for direct use in a medical diagnostic assay. By the use of gene constructs with appropriate promoters, high level expression of an anti-glycophorin single-chain antibody fused to an epitope of the HIV virus was obtained in the leaves and stems of tobacco, tubers of potato and seed of barley. This fusion protein replaces the SimpliRED™ diagnostic reagent, used for detecting the presence of HIV-1 antibodies in human blood. The reagent is expensive and laborious to produce by conventional means since chemical modifications to a monoclonal antibody are required. The plant-produced fusion protein was fully functional (by ELISA) in crude extracts and, for tobacco at least, could be used without further purification in the HIV agglutination assay. All three crop species produced sufficient reagent levels to be superior bioreactors to bacteria or mice, however barley grain was the most attractive bioreactor as it expressed the highest level (150 μg of reagent g-1), is inexpensive to produce and harvest, poses a minuscule gene flow problem in the field, and the activity of the reagent is largely undiminished in stored grain. This work suggests that barley seed will be an ideal factory for the production of antibodies, diagnostic immunoreagents, vaccines and other pharmaceutical proteins.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Drinking from arboreal water sources by mantled howling monkeys (Alouatta palliata Gray).

Despite occasional trips to the ground and feeding in trees whose canopies touched the river, mantled howling monkeys were never seen to drink from any ground water. Drinking from arboreal cisterns was observed, but only during the wet season (meteorologically the less stressful season but phenologically the more stressful season). The lack of sufficient new leaves during the wet season forced the howlers to ingest more mature leaves which contained significantly less water. To compensate for the lowered amount of water in their food, the monkeys utilized arboreal water cisterns. The cisterns dried up during the dry season, but the howlers maintained their water balance by altering their time of actiivity and selecting a diet comprised largely of succulent new leaves. The effect of plant-produced secondary compounds on drinking also was discussed.

Soil management in relation to sustainable agriculture and ecosystem services

Requirements for research, practices and policies affecting soil management in relation to global food security are reviewed. Managing soil organic carbon (C) is central because soil organic matter influences numerous soil properties relevant to ecosystem functioning and crop growth. Even small changes in total C content can have disproportionately large impacts on key soil physical properties. Practices to encourage maintenance of soil C are important for ensuring sustainability of all soil functions. Soil is a major store of C within the biosphere – increases or decreases in this large stock can either mitigate or worsen climate change. Deforestation, conversion of grasslands to arable cropping and drainage of wetlands all cause emission of C; policies and international action to minimise these changes are urgently required. Sequestration of C in soil can contribute to climate change mitigation but the real impact of different options is often misunderstood. Some changes in management that are beneficial for soil C, increase emissions of nitrous oxide (a powerful greenhouse gas) thus cancelling the benefit. Research on soil physical processes and their interactions with roots can lead to improved and novel practices to improve crop access to water and nutrients. Increased understanding of root function has implications for selection and breeding of crops to maximise capture of water and nutrients. Roots are also a means of delivering natural plant-produced chemicals into soil with potentially beneficial impacts. These include biocontrol of soil-borne pests and diseases and inhibition of the nitrification process in soil (conversion of ammonium to nitrate) with possible benefits for improved nitrogen use efficiency and decreased nitrous oxide emission. The application of molecular methods to studies of soil organisms, and their interactions with roots, is providing new understanding of soil ecology and the basis for novel practical applications. Policy makers and those concerned with development of management approaches need to keep a watching brief on emerging possibilities from this fast-moving area of science. Nutrient management is a key challenge for global food production: there is an urgent need to increase nutrient availability to crops grown by smallholder farmers in developing countries. Many changes in practices including inter-cropping, inclusion of nitrogen-fixing crops, agroforestry and improved recycling have been clearly demonstrated to be beneficial: facilitating policies and practical strategies are needed to make these widely available, taking account of local economic and social conditions. In the longer term fertilizers will be essential for food security: policies and actions are needed to make these available and affordable to small farmers. In developed regions, and those developing rapidly such as China, strategies and policies to manage more precisely the necessarily large flows of nutrients in ways that minimise environmental damage are essential. A specific issue is to minimise emissions of nitrous oxide whilst ensuring sufficient nitrogen is available for adequate food production. Application of known strategies (through either regulation or education), technological developments, and continued research to improve understanding of basic processes will all play a part. Decreasing soil erosion is essential, both to maintain the soil resource and to minimise downstream damage such as sedimentation of rivers with adverse impacts on fisheries. Practical strategies are well known but often have financial implications for farmers. Examples of systems for paying one group of land users for ecosystem services affecting others exist in several parts of the world and serve as a model.