The COVID-19 pandemic demonstrated the promise and power of vaccination to protect society against infectious diseases. However, factors such as shortage of healthcare workers, ineffective supply chains, and lack of funding to acquire vaccines have also led to a drop in overall vaccination coverage for children, particularly in developing nations.

UNICEF’S State of the World’s Children 2023 report estimated that one in five children are not vaccinated against life-threatening diseases such as cholera, polio and measles, with vast inequalities across urban and rural areas.

While most of us are now familiar with mRNA-based vaccines, the majority of commercially available vaccines still rely on using weakened/inactivated viruses and proteins. These conventional vaccine materials are typically produced using genetically modified cell-based systems including bacteria, fungi, yeast, insect, and mammalian cells.

However, they suffer from limitations including high production costs due to the need for dedicated, sterile facilities to culture cells, and safety concerns, e.g., human cells may carry harmful prions and bacterial cells produce endotoxin after lysis. Importantly, due to the lack of or difference in post-translational modifications, proteins produced by bacterial and insect cells can behave differently (reduced stability and immunogenicity) than expected.

Another big disadvantage as highlighted during the COVID-19 pandemic is that cold chain supply (2–8 oC) is needed for vaccine storage and transport, which can limit accessibility to low- and middle-income nations. For instance, the World Health Organization estimated that in 2011, 2.8 million SARS-CoV-2 vaccine doses were lost due to supply chain disruptions in five countries it surveyed.

Pros and cons of plant systems

Plant molecular farming which refers to the use of plant cells or whole plants as expression platforms is an alternative method to produce vaccines that is purported to be useful for resource-scarce regions and to target niche and orphan vaccines. This concept was introduced in the 1980s to produce human growth hormone in transgenic tobacco and sunflowers. Since then, it has been used to produce seasonal influenza vaccines and for Elelyso for Gaucher disease in the U.S.

BioApp, a company based in South Korea, is also using plants to generate animal vaccines against swine fever. There are several other companies using plant systems to produce viruses, virus-like particles which are subunit vaccines that can self-assemble into non-infectious and non-replicating viruses as vaccines or therapeutics delivery.

Recombinant protein vaccines can be produced in stable transgenic plants or as transient expressions in plants using engineered plant viruses or bacteria in four general steps.

The first step is to choose a plant host. To prevent human and wildlife exposure to modified edible plants, plant molecular farming efforts are concentrated on non-edible tobacco plants such as Nicotiana Benthamiana. Next is to choose a vector to deliver DNA plasmids for transformation, and Agrobacterium tumefaciens is the most popular choice. The third step is to transfect the plants by mechanical inoculation, agrofiltration, or vacuum infiltration. Finally, the proteins are purified by digesting plant leaves, and separating the target proteins by conventional chromatography methods and/or molecular tags.

working with a plant
Plants are safer than bacterial and mammalian systems due to lower possibility of harmful contaminations such as prions and endotoxins. Additionally, plants can be transported more easily compared to cell systems. [Nicolas/Getty Images]

Using plants to produce vaccines has distinct advantages over mammalian systems in terms of biomass generation. It is estimated that the final costs of producing biologics in plants is significantly lower than that of using a conventional mammalian cell system. Plants also share more similar post-translational modifications to mammalian cells compared to insect and bacterial cells and can be further manipulated to improve similarity.

Furthermore, plants are safer than bacterial and mammalian systems due to lower possibility of harmful contaminations such as prions and endotoxins. Additionally, plants can be transported more easily compared to cell systems, making them suitable for onsite extraction and purification when cold chain supplies are not readily available.

Nonetheless, plant-based systems have disadvantages too. A huge limitation is the lower protein yield when compared to bacterial and mammalian cell culture. To better overcome this, Hamel et al. recently found that Agrobacterium-mediated gene expression of foreign proteins can shut down chloroplast gene expression and cause oxidative stress responses to reduce protein yields.

Interestingly, the team showed that the reduction of oxidative stress damage through exogenous application of ascorbic acid can improve plant biomass production to improve yield of viral-like particles.

Another limitation of using plant systems is that protein expression can vary between plant generations and even within leaves. Environmental variability may also influence harvest yield although this issue can be mitigated with controlled environmental facilities.

Finally, plant cells and tissues produce more debris and contaminants than other expression systems which can interfere with protein purification. In fact, this is a key reason that plant-made proteins routinely failed to transition into the clinics. Regardless, with greater innovations in plant genetic engineering and purification techniques, plant-based systems are expected to make increasing contributions to vaccine production for use in animals and humans.

Besides this, Inhwan Hwang, a plant biologist at Pohang University of Science and Technology, adds that “there is also a lack of facilities to produce GMP-grade green biologics from plants for preclinical and clinical trials. Additionally, there is a lack of specialized manpower trained to manufacture GMP-grade green biologics.”

Using plants to produce animal vaccines

Park et al. generated transgenic Nicotiana Benthamiana plants to produce E2 fusion proteins for use as a recombinant vaccine against classical swine fever virus which causes significant damage to the swine industry. The team also adopted a novel protein purification method combining cellulose-binding domain-cellulose-based affinity purification and size exclusion gel filtration chromatography.

plant farming
Plant molecular farming can play a role in green biologics and improve vaccine access. In particular, it can be used to produce vaccines at small scale for personalized treatment and at low cost where resources are limited to set up sterile cell factory facilities, yet where vaccines are most needed. Plant contaminants are also comparatively more tolerable than bacterial endotoxins. [Kilito Chan/Getty Images]

When tested in piglets, a single dose vaccination conferred full virus protection for up to 11 days. Song and co-workers also generated recombinant hemagglutinin proteins as vaccines against the Asian influenza virus. The ectodomains of hemagglutinin of influenza viruses were first expressed in plants as trimers to mimic their native forms. The trimer was then directly bound to inactivated Lactococcus which was used as an antigen-carrying bacteria to generate strong immune response in mice and chickens without adjuvants.

Similar strategies have also been adopted by the fish farming industry to exploit plants to produce transgenic leaves and seeds which are further processed as edible feed pellet vaccines or as purified antigens encapsulated into feed pellets.

While plant molecular farming is unlikely to replace large volume cell culture to produce blockbuster vaccines and biotherapeutics, it can be useful to produce small batch of antigens that are personalized for diseases. For instance, Tusé and colleagues described a plant-made cancer vaccine for treatment of B cell follicular lymphoma in a clinical Phase I study.

Another example is the use of plants to produce virus nanoparticles as a vaccine against the cowpea mosaic virus which was found to show excellent efficacy in preclinical animal models of melanoma, glioma, and breast cancer among others. The virus nanoparticles which were injected directly into the tumors were found to stimulate innate immune cells, resulting in systemic antitumor immunity. Song et al. also made use of Nicotiana Benthamiana to produce recombinant spike proteins of the SARS-CoV-2 virus. The team found that intramuscular injection of the plant-derived vaccine elicited humoral and cellular immune responses in mice, including antibodies that were able to cross-neutralize Alpha, Beta, Delta, and Omicron variants. The survival rates of the vaccinated mice were up to 80% even with lethal SARS-CoV-2 virus challenge.

Eun-ju Sohn, CEO of BioApp, says that “the primary challenge in the PMF (Plant-Made Pharmaceuticals) industry is the lack of successful case studies demonstrating the real-world applicability of PMF technologies. This scarcity of success stories has made it difficult to attract funding, subsequently slowing the industry’s development.

To date, only a few PMF-based products have received formal regulatory approval. With a rising interest in the alternative meat industry, many startups are now focusing on producing animal proteins in plants, moving beyond the constraints of GMOs.

The prospect of consuming pork protein with soy dishes is near, and next could be edible vaccines and therapeutics. Although there are many excellent treatments for conditions like arthritis, it’s not feasible to consume hamster ovary-derived cells or E. coli if they are produced those type of cells.

Plants, however, offer a solution, promising a future where treatment and prevention can be deliciously consumed at home daily happily.”

Conclusions

When Canadian biotechnology company Medicago, which pioneered the use of transient expression in tobacco plants to produce vaccines, ended its operations in early 2023, there were concerns that this could have an adverse impact on future prospects of plant molecular farming. However, the ability of Medicago to license a virus-like particle vaccine (Covifenz) against the SARS-CoV-2 virus in less than two years has helped to validate the promise and derisk this novel technology.

Plant molecular farming can play a role in green biologics and improve vaccine access. In particular, it can be used to produce vaccines at small scale for personalized treatment and at low cost where resources are limited to set up sterile cell factory facilities, yet where vaccines are most needed. Plant contaminants are also comparatively more tolerable than bacterial endotoxins.

Genetically transformed plants can enable onsite isolation and purification of proteins to reduce cold chain requirements. In the future, with greater efforts to improve plant transfection, and protein purification and yield, plant molecular farming will only become more compelling as a method to improve vaccine production and access.

Andy Tay is a freelance writer based in Singapore.

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