Advances and challenges in the development and production of effective plant-based influenza vaccines.

Influenza infections continue to present a major threat to public health. Traditional modes of influenza vaccine manufacturing are failing to satisfy the global demand because of limited scalability and long production timelines. In contrast, subunit vaccines (SUVs) can be produced in heterologous expression systems in shorter times and at higher quantities. Plants are emerging as a promising platform for SUV production due to time efficiency, scalability, lack of harbored mammalian pathogens and possession of the machinery for eukaryotic post-translational protein modifications. So far, several organizations have utilized plant-based transient expression systems to produce SUVs against influenza, including vaccines based on virus-like particles. Plant-produced influenza SUV candidates have been extensively evaluated in animal models and some have shown safety and immunogenicity in clinical trials. Here, the authors review ongoing efforts and challenges to producing influenza SUV candidates in plants and discuss the likelihood of bringing these products to the market.

Plant-made vaccine antigens and biopharmaceuticals.

Plant cells are ideal bioreactors for the production and oral delivery of vaccines and biopharmaceuticals, eliminating the need for expensive fermentation, purification, cold storage, transportation and sterile delivery. Plant-made vaccines have been developed for two decades but none has advanced beyond Phase I. However, two plant-made biopharmaceuticals are now advancing through Phase II and Phase III human clinical trials. In this review, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear and chloroplast or transient viral) and their current limitations or challenges. We provide suggestions for advancing this valuable concept for clinical applications and conclude that greater research emphasis is needed on large-scale production, purification, functional characterization, oral delivery and preclinical evaluation.

Mucosal immunization using recombinant plant-based oral vaccines.

The induction of mucosal immunity is very important in conferring protection against pathogens that typically invade via mucosal surfaces. Delivery of a vaccine to a mucosal surface optimizes the induction of mucosal immunity. The apparent linked nature of the mucosal immune system allows delivery to any mucosal surface to potentially induce immunity at others. Oral administration is a very straightforward and inexpensive approach to deliver a vaccine to the mucosal lining of the gut. However, vaccines administered by this route are subject to proteolysis in the gastrointestinal tract. Thus, dose levels for protein subunit vaccines are likely to be very high and the antigen may need to be protected from proteolysis for oral delivery to be efficacious. Expression of candidate vaccine antigens in edible recombinant plant material offers an inexpensive means to deliver large doses of vaccines in encapsulated forms. Certain plant tissues can also stably store antigens for extensive periods of time at ambient temperatures, obviating the need for a cold-chain during vaccine storage and distribution, and so further limiting costs. Antigens can be expressed from transgenes stably incorporated into a host plant's nuclear or plastid genome, or from engineered plant viruses infected into plant tissues. Molecular approaches can serve to boost expression levels and target the expressed protein for appropriate post-translational modification. There is a wide range of options for processing plant tissues to allow for oral delivery of a palatable product. Alternatively, the expressed antigen can be enriched or purified prior to formulation in a tablet or capsule for oral delivery. Fusions to carrier molecules can stabilize the expressed antigen, aid in antigen enrichment or purification strategies, and facilitate delivery to effector sites in the gastrointestinal tract. Many antigens have been expressed in plants. In a few cases, vaccine candidates have entered into early phase clinical trials, and in the case of farmed animal vaccines into relevant animal trials.

Plant-based vaccines.

Plant systems are reviewed with regard to their ability to express and produce subunit vaccines. Examples of different types of expression systems producing a variety of vaccine candidates are illustrated. Many of these subunit vaccines have been purified and shown to elicit an immune response when injected into animal models. This review also includes vaccines that have been administered orally in a non-purified form as a food or feed product. Cases are highlighted which demonstrate that orally delivered plant-based vaccines can elicit immune responses and in some case studies, confer protection. Examples are used to illustrate some of the inherent advantages of a plant-based system, such as cost, ease of scale-up and convenience of delivery. Also, some of the key steps are identified that will be necessary to bring these new vaccines to the market.

Delivery of subunit vaccines in maize seed.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.