Vaccines represent one of the most successful medical advances of all time, particularly since their introduction as cost-effective measures for preventing acute infectious diseases. Epidemiologically targeted implementation of vaccination programs has diminished morbidity and mortality from infectious diseases that previously were scourges and posed severe economic burdens (such as measles, polio, diphtheria, invasive Haemophilus influenzae type b and pneumococcal infections). Global programs have eradicated smallpox and reduced poliomyelitis transmission such that eradication is feasible. Advances in biotechnology and the understanding of the inductive and effector components of immune responses have provided a new opportunity for vaccine development and implementation. Nowadays, other illnesses, particularly chronic infections and cancers, are becoming the new target of vaccine developments, with the possibility of either treating established diseases (therapeutic vaccines) or preventing them (prophylactic vaccines). Most of the new advances are based on innovative preparation of the antigens and the introduction of new adjuvants. For over a century, conventional vaccines have been based on native pathogens (inactivated or attenuated) or their inactivated toxins (toxoid) along with a single adjuvant (alum salt) to induce an effective humoral response (neutralizing antibodies). The new vaccination strategies are mainly considering antigenic complex structures (including virus-like particles [VLPs] and virosomes) where antigens or individual antigenic epitopes are presented on the surface or within the context of complex structures along with peculiar adjuvants, selected for their property to stimulate T-helper1 (Th1) versus T-helper2 (Th2) polarization of the antigen-presenting cells (APCs). Such new vaccine strategies are also showing promising results against chronic diseases and cancers, invigorating the whole field of vaccinology.
Plants as bioreactors
The application of recombinant technology to plants has also brought about major advances in plant biology, allowing production of genetically modified (GM) plants optimized and/or resistant to pathogens/pesticides. Nevertheless, the introduction of GM plants has been followed by major political arguments about the development and use of GM crops. New technologies, however, have also enabled the development of noncrop plants (i.e., tobacco) to produce pharmaceutical molecules. This approach has major advantages, particularly for the production of pharmaceutical-grade proteins (including glycosylated protein), in comparison to other prokaryotic and eukaryotic expression systems in terms of speed, costs and safety. Plants can be efficiently used as bioreactors in transient systems driven by well-contained infectious vectors, or in stable transgenic systems based on nuclear or plastidial transformation. The latter, because of the expression by way of the plastidial genome, is well contained and does not need particular field limitations.
Plant-based vaccines
A special application of plants as bioreactors is their use to express antigenic molecules to be administered as vaccines. In this special issue on plant-based vaccines, we are reviewing the techniques employed and goals achieved in this field from different angles. Plants are not only used to produce immunogens (from single peptides to complex structures, such as VLPs), but also adjuvants, microbicides and monoclonal antibodies. Plant expression systems cover the whole range of tools needed to produce molecules for an effective preventive as well as therapeutic immunotherapy.
This special issue on plant vaccines includes 13 manuscripts spanning most of the issues involved in plant-based vaccine production, including intellectual property issues. Furthermore, besides the detailed description of antigen expression in different plant systems, adjuvant production as well as plant glycosylation of proteins are analyzed. The short summaries included in the following paragraphs highlight key issues and major achievements discussed in each section. The first six articles deal with general aspects of the plant systems used for the production of pharmaceuticals; the latter seven articles describe specific vaccine approaches pursued in plants.
The issues opens up with an article focused on the legal, economical and social key issues regarding the use of plants as biofactories Citation[1]. Patents associated with recombinant pharmaceutical production in transgenic plants are described and the effect of patenting on innovation in the field and potential dissemination of the technology to the developing world are assessed. It is to be expected that successful results and commercialization of plant-made pharmaceuticals will be accompanied by a corresponding increase in patent applications. It remains to be seen whether the net effect of these activities will be to promote the development of the field Citation[1].
Potential advantages of producing immunogens in plant systems over conventional production systems have been postulated and can be grouped into three major ‘areas’:
• Oral administration of a plant-derived vaccine in the context of a heterologous primer/booster strategy, considering that oral boosting in systemically vaccinated individuals bypasses the issue of inducing oral tolerance;
• The introduction of regulated pharmaceutical products prepared by minimal downstream processing, considering that this would represent a major revolution in pharmaceutical activities whose downstream processing contributes up to 80% of manufacturing costs;
• Enabling the participation of less developed countries in pharmaceutical production, with an emphasis on addressing local health issues Citation[2].
Subunit vaccines need to be glycosylated for the efficient stimulation of the innate immune response. Plants have demonstrated the ability to yield highly homogenous glycan profiles on recombinant proteins after glycoengineering. Highly homogeneously glycosylated antibodies (with ~80% galactosylated structures) can be isolated from glycoengineered Nicotiana benthamiana, comparing favorably with the Chinese hamster ovary cell line production of the same antibody and suggesting that plants are excellent production hosts for glycosylated subunit vaccines yielding homogeneous products Citation[3].
Recombinant antigen production in plants is a safe and economically sound strategy for vaccine development, particularly for oral/mucosal vaccination, but subunit vaccines usually suffer from weak immunogenicity and require adjuvants that escort the antigens, target them to relevant sites and/or activate APCs for elicitation of protective immunity. Genetic fusions of antigens with bacterial adjuvants (e.g., the B subunit of the cholera toxin) and the inclusion of plant defensive molecules, such as lectins and saponins, can be successfully used to induce protective immunity of plant-made vaccines Citation[4].
Viral vectors have been designed on the basis of DNA- and RNA-containing plant virus genomes, and they are used both as single- and multicomponent systems in different combinations, depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, safety, low cost and high yield of pharmaceutical proteins. Among the several available vectors (tobamoviruses, potexviruses, potyviruses, Bromoviridae, Comoviridae and Geminiviridae) special attention is given to illustrate the recently developed MagnICON® expression system (Icon Genetics GmbH, Halle, Germany) Citation[5].
Contained plant systems for the production of biopharmaceuticals represent an alternative, combining the merits of whole-plant with microbial and mammalian cell culture expression systems. In vitro contained production systems, including plant cell suspension cultures or hairy root cultures, novel plants that are grown in contained conditions and microalgae, show intrinsic benefits such as control over growth conditions and avoidance of political resistance to release of genetically modified field crops Citation[6].
Production of recombinant subunit vaccines from genes incorporated into the plastid genome of plants shows some significant benefits: high expression levels due to a large transgene copy number and the absence of gene silencing; biocontainment as a consequence of maternal inheritance of plastids and no transgene presence in the pollen; and expression of multiple transgenes in prokaryotic-like operons. Technology of plastid transformation in Chlamydomonas reinhardtii (a unicellular algae) and Nicotiana tabacum (tobacco; a flowering plant species) is described along with specific vectors Citation[7].
Human papillomavirus and other papillomavirus L1 VLPs can be expressed in plants by several routes and at high yields. These VLPs are ‘biosimilars’ in terms of their morphology, immunogenicity and even efficacy in animal models, to the commercial VLP-based prophylactic vaccines produced in yeast and insect cells. Moreover, plant-made preparations (with or without adjuvants) retain their immunogenicity following lyophilization and oral administration, key issues for development of further innovative approaches: production in plants of L2 protein alone or combinations with L1 in chimeric proteins, second-generation prophylactic vaccine candidates, as well as E6/E7-based therapeutic vaccines Citation[8].
Production in plants of anti-HIV pharmaceuticals also bears some promise. The high flexibility of the plant system offers a wide range of possible products: immunogens spanning from soluble antigens to assembled multicomponent structures (VLPs) with immunogenicity equivalent to that achieved with molecules produced in other eukaryotic expression systems; and highly neutralizing monoclonal antibodies to be preferentially used along with topical microbicides for vaginal and rectal prevention of HIV transmission. Furthermore, natural plant metabolites can contribute to the mucosal efficacy of oral vaccines as well as to the barrier protection of local microbicides Citation[9].
Transgenic plants can be used as bioreactors for the production of mucosally delivered antigens against diseases such as HIV, hepatitis B virus (HBV) and TB. Several issues regarding the oral delivery of subunit vaccines, including degradation of the antigens by digestive enzymes, inefficient transport from the gut lumen to the gut-associated lymphoid tissue and induction of systemic immune tolerance, are some of the issues deserving attention. Different strategies can improve the effectiveness of mucosal vaccines: the size and structure of the vaccine molecules; co-delivery of immunogens with adjuvants or targeting proteins; and the use of natural adjuvants (e.g., tomatine) Citation[10].
HBV vaccines produced in transgenic or transplastomic plants are highly efficient and represent a valid alternative to the yeast recombinant vaccine. In addition, the plant-based mucosal HBV vaccines can be a useful supplement to the currently used recombinant injection vaccines as an additional mucosal vaccine, expanding the range of protective responses in vaccinees. Moreover, a plant-derived oral vaccine is appropriate for the simple and safe revaccination of humans. Immunogenicity of VLPs based on hepatitis B surface antigen (HBsAg) can be further enhanced by production in plants, whose secondary metabolites can play the role of mucosal adjuvants Citation[11].
Plants can be used as bioreactors but also as delivery systems for pharmaceuticals, in particular for the oral delivery of autoantigens and the induction of tolerance. Recent progress in the identification of the major autoantigens associated with autoimmune diseases has paved the way to treatment by exploiting these molecules to prevent the onset of diseases by restoring broken tolerance in patients. The efficacy of treatment with autoantigens in preventing autoimmune diseases is strictly related to the dose and mode of administration, including oral or nasal administrations. Tolerance induction could be augmented by coadministration or fusion with adjuvants or immunomodulators (e.g., IL-10) Citation[12].
Several plant-made vaccines for veterinary purposes are in progress since the regulatory landscape still enables delivery of either crude extracts or minimally processed plant materials to animals for medicinal purposes. Current research directions are mainly pursued with four diseases that are considered significant constraints to international trade in animals (e.g., avian influenza, Newcastle disease, foot-and-mouth disease and enterotoxigenic Escherichia coli). Identification of appropriate plant production platforms with regards to plant species and transformation methodologies represents a key issue Citation[13].
In conclusion, the enormous progress made in plant genomics are opening the possibility of using plants as bioreactors for the production of pharmaceutical products. This new possibility constitutes a major scientific achievement. Examples of this scientific breakthrough are the recent production in tobacco of a H1N1 2009 vaccine based on the hemagglutinin (HA) protein and the initiation of clinical trials with a recombinant, plant-derived, idiotype vaccine to treat B-cell lymphomas Citation[14]. The plant-based production processes are able to compete with conventional methods (i.e., influenza vaccine production in eggs or other recombinant idiotype expression systems in the case of the non-Hodgkin’s lymphoma vaccine), breaking the limits of current standard production technologies and reaching new frontiers for the plant-based production of pharmaceutical-grade proteins.
Financial & competing interests disclosure
John-Edward Butler-Ransohoff is Global Project Leader of Plant Made Pharmaceuticals at Bayer Innovation GmbH. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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