Special Feature: Plant Derived Vaccines

Immunology and Cell Biology (2005) 83, 271–277; doi:10.1111/j.1440-1711.2005.01336.x

Expression systems and developments in plant-made vaccines

M Manuela Rigano1 and Amanda M Walmsley1

1The Biodesign Institute at Arizona State University, School of Life Sciences, Arizona State University, Tempe, Arizona, USA

Correspondence: Amanda M Walmsley, The Biodesign Institute at Arizona State University, School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA. Email: amanda.walmsley@asu.edu

Received 14 February 2005; Accepted 16 February 2005.

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Abstract

Delivery of vaccines to mucosal surfaces can elicit humoral and cell-mediated responses of the mucosal and systemic immune systems, evoke less pain and discomfort than parenteral delivery, and eliminate needle-associated risks. Transgenic plants are an ideal means by which to produce oral vaccines, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of the stomach, enabling intact antigen to reach the gut associated lymphoid tissue. In the past few years, new techniques (such as chloroplast transformation and food processing) have improved antigen concentration in transgenic plants. In addition, adjuvants and targeting proteins have increased the immunogenicity of mucosally administered plant-made vaccines. These studies have moved plant-made vaccines closer to the development phase.

Keywords:

expression system, oral immunization, plant-made vaccine, targeting protein, transgenic plant

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Introduction

Subunit vaccines that consist of one or more antigenic epitopes or proteins are often preferred to traditional vaccines made of killed or attenuated organisms. Mammalian, yeast and insect cell cultures are used to produce subunit vaccines because of their ability to process recombinant proteins in a manner similar to that of the native organism. However, expensive media and the purification steps needed for recovering recombinant proteins expressed in these organisms increase the cost of producing these subunit vaccines. In addition, most subunit vaccines produced in these systems are heat sensitive and require parenteral delivery. This restricts use of subunit vaccines in the poorly funded health systems of developing countries.

A promising alternative is to transform plants with a gene(s) encoding an immunogenic protein capable of preventing infection by a pathogenic agent. The production of vaccines in transgenic plants overcomes the risk of contamination with mammalian pathogens and can enable oral delivery. These characteristics simplify vaccine delivery and decrease the cost of an immunization program.

In the past 14 years, a range of different plant and vector systems have been used for the production of a long list of antigens that includes viral1, bacterial2, 3, enteric4 and nonenteric5 pathogen antigens as well as autoimmune antigens6, 7. Plant-made vaccines have prevented the onset of disease in animal models and have proven to be safe in human clinical trials (reviewed by Walmsley and Arntzen 20038). However, before the full potential of this technology can be reached, studies need to focus on increased immunogenicity of mucosally administered subunit vaccines and investigate antigen stability. In this article, we investigate issues to be considered before plant-made vaccines can become available for use.

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Oral immunization

Advances in molecular biology, microbiology and immunology have led to novel approaches to develop vaccines against diseases that still plague animals and humans. However, health organizations such as the World Health Organization (WHO) and National Institutes of Health (NIH) are interested in the development of technologies for oral vaccination. The mucosal surfaces are the most important portal of entry for mammalian pathogens, particularly bacteria and viruses. Although traditional parenteral vaccination has been successful with some diseases, it is less effective at providing protection against enteric and respiratory diseases. This is thought to be partly because systemic vaccination is a poor inducer of mucosal immunity, and therefore, organisms can invade the host before the systemic immunity can slow or stop the infection. Mucosal delivery has the added advantage of not needing needle administration, thereby decreasing cost and increasing ease of vaccination. A mucosal immune response is best achieved by direct application of a vaccine to mucosal surfaces. In addition to facilitating a mucosal immune response, mucosal application of a vaccine can also induce humoral, cell-mediated and systemic immune responses. The successful initiation of mucosal immunization has been identified to be dependent on the following criteria9: (i) effective delivery of antigen to the mucosal immune induction site (e.g. the epithelial cells of the Peyer's patches); (ii) enhancing mucosal immune responses using live attenuated vaccine strains; (iii) improved antigen delivery systems or mucosal immune modulators such as bacterial enterotoxins and cytokines; and (iv) choice of a regimen and route of immunization to induce protective responses at the desired mucosal site and preferably systematically as well10. Transgenic plants that express protective antigens are ideal vehicles to orally deliver protective antigens, as the rigid walls of the plant cell protect the antigen from the acidic environment of the stomach.

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Plant production systems

There are many options when choosing an expression system for producing antigens in plants; the decision is complicated by the fact that each system has advantages and disadvantages depending on the desired product. We compare three broad plant production systems – plant cell culture, culture of organized plant tissue and whole plants – using reports of foreign protein expression.

All plant production systems can perform post-translational modification (PTM). The PTM of a recombinant protein is of special importance for its antigenicity. The first option for those antigens not requiring PTM is to translate the protein in the cell cytoplasm. We have found, however, that this option results in low expression levels (perhaps caused by transcript instability or rapid turnover) or causes stunted growth of the resulting plants. The second option that results in no PTM is transformation into the genome of the chloroplast. Should PTM be needed or not effect the antigenicity of the protein of interest: the protein should be targeted to the endoplasmic reticulum (ER) where it will be N-glycosylated. The protein of interest can be retained in the ER by using a KDEL sequence, or allowed to move through the Golgi apparatus and secretory pathway where high mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycan modifications occur11. In our experience, proteins targeted to the ER without a KDEL sequence accumulate between the cell membrane and cell wall (Walmsley et al., unpubl. data, 2004).

Chloroplast transformation is an attractive alternative to nuclear transformation because it produces high transgene expression. It also imposes transgene containment through maternal inheritance, lacks gene silencing, lacks pleiotropic effects, and lacks position effect because of site-specific transgene integration12. However, the technique is far from routine except in algae such as Chlamydomonas reinhardtii13 and in tobacco14. Chloroplasts use prokaryote-like translation, therefore chloroplast transformation should not be used for proteins needing PTM for structural authenticity and biological activity.

Plant cell culture

Plant cells can be cultured as undifferentiated, individual entities in liquid. In the laboratory, 50 mL of cells and media in 250 mL flasks are subcultured weekly, while production on a larger scale may involve either fermentation equipment or growth in large ponds or tanks. Either method involves constant media circulation and replacement. Plant cell cultures are independent of seasonal variation and enable continuous supply of product.

Technically belonging to the Kingdom Protista, algae is an informal term used for photosynthetic eukaryotes other than plants and cyanobacteria15. The green algae are the most diverse of all algae and consist of unicellular and multicellular species. The alga of most interest concerning 'plant'-made vaccines is the genetic workhorse of chloroplast molecular biology, the unicellular Chlamydomonas reinhardtii. Chlamydomonas has several benefits for producing recombinant proteins: (i) the nucleus, chloroplast and mitochondria are easily transformed; (ii) there is a short period between generating initial transformants and scale-up to production volumes; (iii) gametogenesis can be induced (allowing genetic crossing); (iv) it can be grown either heterotrophically or phototrophically; (v) a wide range of promoters are available; (vi) it can produce secreted, glycosylated proteins; and (vii) it can be grown in cultures on scales ranging from a few millilitres to 500 000 L in a contained and cost-effective way16. However, expression of foreign genes transformed into the Chlamydomonas nucleus is problematic. For reasonable protein expression, genes have to be engineered for codon preferences17 and contain introns that act as enhancer-like elements18, 19. It has also been found that transgenic Chlamydomonas strains expressing high levels of foreign genes are quickly silenced16. Although chloroplast transformation is more reliable for high expression, 0.5–1% total soluble protein (TSP)20, 21 it is not capable of producing glycosylated proteins.

Two tobacco lines commonly found in cell culture are BY2 and NT1. The lines, derived from Nicotiana tobaccum, have lost the ability to regenerate. Agrobacterium is routinely used to transform the cells while in liquid form, then transgenic callus is regenerated from a lawn of cells plated on solid medium. The features of transgenic lines are that (i) they can be identified quickly (5–8 weeks depending on the selective agent used) and amplified in liquid culture for larger batches; (ii) harvest of transgenic cells can start 1 month after seeding the culture and can continue weekly thereafter; (iii) on selecting elite lines, gene of interest expression is constant and high (our usual top lines express between 200 and 400 microg/g dry weight, although we have had lines expressing up to 1 mg/g dry weight); (iv) containment is inherent; (v) culture conditions are closed so good laboratory and manufacturing practices (GLP and GMP, respectively) are easily applied; (vi) the lines have low alkaloid content so can be used for oral delivery; and (vii) the media used is inexpensive. However, gene silencing may occur (although the frequency of this is decreased by selecting lines with low transgene copy numbers) and some high expressing lines are slow growing.

Culture of organized plant tissue

More than 116 plants have been induced to produce hairy roots in culture22; therefore, which species to use may rely on the technology on hand, as well as palatability. Hairy roots grow fast without need of an external supply of auxins or light, they are fairly stable in metabolite yield because of genetic stability23, and they may be engineered to secrete the protein of interest into the surrounding medium should oral delivery not be desired24, 25. Production of pharmaceuticals by hairy root cultures originally involved over-production of a pharmaceutical naturally produced by the plant26, 27, 28, 29, 30, 31, but hairy roots have also produced a foreign protein of pharmaceutical interest24, 25, 32. Sharp and Doran (2001)32 directly compared expression of Guy's 13 monoclonal antibody by N. tobaccum cell culture, shoot culture and hairy root culture. Hairy root growth was considerably slower than shoot culture and suspended cells, while antibody in the biomass of root and suspension cultures were similar and higher than that in the shoot cultures. The maximum antibody concentrations as percentages of TSP were 2.4, 1.2 and 6.5 for hairy roots, shoot culture and suspended cells, respectively. Sharp and Doran found that high antibody accumulation was not maintained in the suspended cultures over 12 months of culture maintenance.

Whole plants

Plant-made vaccines can be made from transient or stable transformation of plants. Our discussion focuses on stably transgenic plants except where indicated. Production of plant-made vaccines in whole plants needs simple and inexpensive inputs (sunlight, water and nutrients), it enables unsophisticated and rapid increase of dose production through increasing area put to seed, and low technology harvest and processing for vaccine production. As found for other production systems, vaccine production by whole plants has its disadvantages. Many disadvantages can be eliminated by selecting lines with particular characteristics; therefore, much time is spent characterizing lines before one is chosen for production. The probability of gene silencing is reduced by selection of lines that have low number and simple patterns of transgene insertion, and 'stable' transgenic lines (with regards to variability of antigen expression) can usually be selected after six generations in species such as tomato, maize and potato (using minitubers as the propagation method). All transgenic plants, including those expressing pharmaceuticals, must be contained to prevent accidental release of the transgene into the environment and possible contamination of the food chain. This is discussed in further detail later in this review.

Although the disadvantages of producing plant-made vaccines in whole plants can be negated, it should be made certain that the cost of doing so does not outweigh the benefit. For example, the cost of downstream processing for perishable tissues such as leaves and fruit should not be such that it prohibits use of the vaccine even though these tissues provide stable antigen content. A comparison of the three plant production systems described here can be found in Table 1.


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Perceived problems of plant-made vaccines

Poor immunogenicity of orally delivered subunit vaccines

The broad application of plant-made vaccines is limited by the poor response of the mammalian immune system to non-particulate, subunit vaccines. Co-administration of a plant-made vaccine antigen with a carrier protein can increase the immunogenicity and compensate in part for low antigen recognition. Two bacterial products with great potential to act as carriers for subunit vaccines are the heat labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) and the cholera toxin (CT) of Vibrio cholerae. LT and CT are heterohexameric proteins with similar structure. They abrogate tolerance towards co-administered antigens33, 34 and are recognized as two of the most potent mucosal adjuvants yet identified35. However, their capacity to function as mucosal adjuvants is markedly different35, most likely due to differences in ganglioside affinity36. Studies have proven the B subunit of LT (LTB), but not the B subunit of CT (CTB), to be an effective intranasal adjuvant35, 37. In addition, LTB is more stable that CTB at low pH, and LTB may have a greater capacity to maintain receptor binding activity following trafficking across the nasal epithelium38. The B subunit of LT has been successfully expressed in tobacco, potato tissues and maize, and has been found to be immunogenic in mice feed trials39, 40, 41 and human clinical trials42.

Conjugation with LTB and CTB may greatly facilitate antigen delivery and presentation to the gut-associated lymphoid tissue due to their affinity for the GM1 gangliosides43, 44, 45. Immunomodulatory activities of LTB include elevation of Th1- and Th2-type cytokines, CD8+ and CD4+ T-cell apoptosis, B-cell activation of MHC II, and modulation of antigen presentation by B cells and macrophages35.

CTB and LTB have been extensively studied as carrier proteins within bacterial expression systems46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, and they have been recently used in plant-made vaccines3, 7, 45, 57, 58, 59.

Arakawa et al. (1998)57 fused the 258 base pair (bp) proinsulin to the carboxyl terminus of CTB and expressed the fusion protein in transgenic potato. The CTB/proinsulin fusion protein formed pentamers, and CTB and its insulin counterpart were shown to retain native antigenicity. Non-obese diabetic mice fed transformed potato tuber tissues containing the fusion protein showed a substantial reduction in pancreatic islet inflammation (insulitis), and a delay in the progression of clinical diabetes.

In 2004, Kim et al.45 expressed a fusion protein containing CTB and the HIV-1 envelope glycoprotein gp120 V3 loop in potato tuber tissues. To facilitate CTB pentamerization, a flexible hinge peptide was introduced between the two fusion proteins. The hinge peptide contained two glycine and proline residues and included codons less frequently used in plants to facilitate CTB subunit folding before gp120 translation initiation.

Our group reported the ability of transgenic plants to produce a fusion protein consisting of LTB and a 6 kDa tuberculosis antigen (the early secretory antigenic target [ESAT]-6)3. Both components of the fusion protein were shown to retain native antigenicity and the ability to form pentamers. The gene for the LTB/ESAT-6 fusion protein has been effectively produced in the model plant species Arabidopsis thaliana and in the palatable plant, tomato.

Yu and Langridge (2001)58 expressed a multicomponent CT fusion vaccine in potato. CTB and CTA2 (the component of the A subunit [CTA] that associates CTA to the CTB subunit) were fused to a rotavirus enterotoxin protein (NSP4) and an ETEC fimbrial colonization factor (CFA/I). The two fusion proteins assembled into a cholera holotoxin-like structure that retained GM1 binding affinity. In mice, oral immunization with the plant-made vaccine generated detectable levels of serum and intestinal antibodies against the three antigens. Moreover, elevated levels of IL-2 and IFN-gamma were detected in antigen-challenged spleen cells from the immunized mice. Furthermore, diarrhoea symptoms were reduced in severity in pups of immunized dams following rotavirus challenge.

This work was expanded by Lee et al. (2004)59 who demonstrated that the CFA/I protein in the multicomponent vaccine elicited levels of anti-CFA/I antibodies in serum and in the intestine of immunized mice that specifically recognized ETEC CFA/I fimbrial structures and inhibited ETEC binding to Caco-2 human colon carcinoma cells59. This work suggested that CTB pentamers functioned as a delivery system for the CTA-linked CFA/I antigen to the enterocyte membrane receptor site. The CTB moiety in the CTB/NSP4 fusion protein mediated CFA/I/CTA2 fusion protein binding to the surface of enterocyte and M cells. Furthermore, CTB may also have played a role in enhancement of the immune response to mucosally administered antigens. These findings demonstrated the feasibility of a plant-made multicomponent vaccine for protection against three enteric diseases: ETEC infection, rotavirus infection and cholera.

Dose variability and low antigen expression

Many features combine to make a safe vaccine. The ability to consistently deliver a known dose of small volume is one such feature. Due to pleiotropic and position effects, there is inherent variability in expression of recombinant proteins in transgenic plants. This variability makes it difficult to control the exact dose of antigen delivered by a plant-made vaccine. Food-processing techniques such as batch processing and freeze-drying can standardize the antigen concentration in transgenic plant materials.

Our group batch processed and freeze-dried A. thaliana expressing a fusion protein consisting of LTB and the tuberculosis antigen ESAT-63. Freeze-drying the Arabidopsis material concentrated the antigen sevenfold. Western blot analysis and ELISA showed the LTB/ESAT-6 protein in the freeze-dried Arabidopsis material retained correct conformation and native antigenicity. No loss in antigen activity or content was observed in freeze-dried material kept at –20°C for 4 months or room temperature for 6 months. The material also proved to be immunogenic in small animal trials (Rigano et al., unpubl. data, 2004).

We also conducted a similar study in tomato7. Tomato plants produce large masses of palatable fruit, grow quickly, and are cultivated broadly. Unfortunately, a vaccine expressed in tomato fruit has a short shelf life. Fresh tomato fruit expressing a fusion protein containing LTB and a species-specific immunocontraceptive epitope were pooled and freeze-dried. Freeze-drying tomato fruit concentrated antigen 16-fold and extended shelf life to 5 months (before materials were used). These materials also proved to be immunogenic in animal trials (Walmsley et al., unpubl. data, 2003).

Freeze-drying has also been applied to a potato-made vaccine60. Castañón et al. (2002) lyophilized and powdered transgenic potatoes expressing a rabbit haemorrhagic disease virus (RHDV) vaccine consisting of the VP60 gene. The freeze-dried material was stored for 3 months and then used in animal trials. Protein extracts made from the potato powder was used in rabbits to prime subcutaneously and boost intramuscularly. The immunized rabbits showed specific antibody responses and were protected against challenge with virulent RHDV60.

Antigen content can also be increased by transient expression of antigens using viral vectors. Viral replication amplifies gene transcripts and results in a higher expression of the foreign protein within a short period. However, this system requires individually inoculating plants and may be less attractive for large-scale protein production. We have also increased antigen content by self-pollinating high-expressing tomato lines. Antigen content was increased 20–30-fold in one generation7. A similar tactic has worked in maize where a selection and back-crossing program resulted in a greater than 70-fold increase in antigen concentration over six generations and a 150-fold increase over eight generations61.

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Safety of plant-made vaccines

Plant-made vaccines will be subjected to the same, if not more stringent, quality control and safety standards as vaccines derived from bacterial, yeast, filamentous fungi or mammalian cell systems. Protocols defining GMP and GLP, as established for other vaccines, must be redefined for plant-made vaccines62.

One concern regarding the production of antigens in plants is the safety of the food chain from contamination with pharmaceuticals. This could occur if pollen from transgenic plants out-crossed with food crops, if equipment used to harvest or process plant-made vaccines was used to harvest or process food crop plants without thorough decontamination, and/or if fields used for production of plant-made vaccines were used to grow food crops without a preassigned fallow period or decontamination. Containment is achieved by strict regulations regarding geographical isolation, buffer plants, physical containment in glasshouses, harvest and processing, and transport of the plants and resulting products. Some groups also help contain the transgene by using regulated or localized antigen expression, for example localized to the fruit or grain63 and/or expression triggered by application of ethanol. Expression of antigens in seeds has the proposed advantage of long-term stability for storage. However, seeds are viable organisms and could face tough regulations.

In the USA, the Department of Agriculture (USDA) and the Food and Drug Administration (FDA) currently regulate plant-made pharmaceuticals. The USDA and FDA, respectively, regulate the location and containment of plant-expressing pharmaceuticals and ensure that quality assurance and quality control standards are maintained during production and preparation of the materials. They take a 'no tolerance' approach with regards to contamination of food crops with genetically modified organisms expressing pharmaceuticals. If contamination is found, the entire crop is destroyed. The FDA ensures safety and efficacy of the materials before human administration and determines the relative risk of progressing plant-made pharmaceuticals to licensure through phased clinical trial monitoring8. Alternative ways of containment include using male-sterile traits62 and further developing chloroplast transformation of crop plants to insert transgenes into the genome of the chloroplast. Because plastids in most agronomically important plant species are maternally inherited, the gene would not be present in pollen.

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Conclusion

The plant system used to produce a plant-made vaccine is as important as the antigen itself. The plant production system can influence antigen content, stability and authentic conformation and ease of production scale-up, harvest and processing. The cost of the vaccine is also influenced by the plant-production system, as it has direct impact on the time spent in development and the cost of containment and processing. Future research needs to focus on making plant-made vaccines as safe, consistent and efficient as pharmaceuticals produced by other sources. Much progress has been made. Increase of antigen expression levels has been achieved through chloroplast transformation, plant breeding and use of food-processing techniques. In addition, freeze-drying has provided antigen stability at ambient temperatures, batch consistency and concentrated antigen. The use of carrier proteins and adjuvants has increased the ability of plant-made antigens to be recognized by the mucosal immune system. The research conducted by an increasing number of laboratories is moving us closer to the production of low-cost, safe, orally delivered, plant-made vaccines that will be able to prevent infectious disease in developing and industrial countries.

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