Origin of our idea
After a lot of brainstorming and reflection we ended up choosing the “edible vaccine against SARS-CoV-2” as our project. Selecting a novel, smart and achievable project was not an easy task. It is a peculiar process that needs knowledge and experience but also young and curious minds to think of something new. As a team, at first, we needed time to bond with each other, eventually though we found our way and became a powerful team and friends in the process. Unfortunately, we were left behind schedule and we had to rush to find our perfect project for the iGEM competition. Therefore we started looking for new biological technologies as an inspiration. We worked hard to study a great deal of bibliography to decide if every idea we had was doable. After all that, we were presenting those same ideas to our P.I.s so we could receive their feedback and criticism on them. It was a time-consuming and stressful assignment especially under the pressure of time. In the end we found our final idea. Something fresh, smart, trending and above all something that can really help people, maybe even save some lives. It was a combination of things that helped us come up with this idea. We were very inspired by old iGEM projects, the technology we were looking at from the bibliography at the time, the teamwork and of course our P.I.s . They were always there to supervise, assist us, and indicate to us the right way.
Advantages over Classical Vaccines
The coronavirus, a disease that arrived suddenly and progressed swiftly in just a few months, highlighted the importance of immunizations once more!
Conventional vaccinations have a number of drawbacks. The issue of safety is one of the most serious ones. Ιnjectable vaccinations can stimulate systemic humoral responses, although T cell effector function and mucosal immunity are critical for infectious disease prevention. Secondary effects of parenteral vaccination injection include local inflammation at the inoculation site, fever, and, in rare cases, hypersensitivity. Conventional approaches also make it impossible to generate vaccinations for all diseases. Due to these drawbacks, the idea of alternate vaccine delivery techniques emerged, paving the way for the creation of plant-based vaccines known as edible vaccines. Our team focused on developing edible vaccines as an alternative to injectable vaccinations in order to improve antigen stability and overall immunogenicity. New vaccine formulations that are edible or intradermal have been shown to elicit both a systemic and mucosal immune response.
Oral vaccinations have a number of advantages versus injectable vaccines, including:
The production of edible vaccines
The manufacturing of edible vaccines is done by inserting a transgene into the selected plant cell. Transgene integration can be achieved either by vector (direct gene delivery) or without (indirect gene delivery). Gene delivery is facilitated by an Agrobacterium vector in order for plant cells to synthesize the desired protein in indirect gene delivery.
Agrobacterium is a gram-negative bacterium that affects plants by transferring its genes to the nucleus of the plant. Agrobacterium tumefaciens and Agrobacterium rhizogenes are the two most widely employed Agrobacterium species. The tumor-causing plasmid Ti is carried by Agrobacterium tumefaciens, while the root-inducing plasmid Ri is carried by Agrobacterium rhizogenes. This procedure is straightforward and cost-effective, but it is time-consuming and yields modest results.
When gene transfer via Agrobacterium is not possible, direct gene delivery involves inserting chosen DNA or RNA directly into the plant cell without the use of a vector, commonly by the biobalistic approach known as gene gun or microprojectile bombardment.
In our project, we picked Agrobacterium Tumefaciens for indirect gene administration since it is a current method of gene integration that is extensively utilized in transgenic plants while also being a simple and safe procedure of plant transformation. Agrobacterium transformation is also popular because it allows for the integration of well-defined DNA fragments, has a high rate of transgenic plant development, and is very inexpensive. Finally, most attempts to make edible vaccines, such as the cholera, Norwalk, and hepatitis B vaccines, used this strategy.
Exploiting the mucosal immunity
In addition to all of the advantages that edible vaccines give, they also provide critical protection by inducing both systemic and mucosal immunity, as opposed to standard vaccines that only elicit a mucosal response. The mucosal immune system is divided into two sections that work together. The mucosa-associated lymphoid tissues (MALTs) are inductive sites, which means that immune responses begin there. The effector locations, such as the lamina propria, are the second (LP). Immune responses and antibodies are produced in the effector sites. Our research is focused on the immunological response that Gut-associated lymphoid tissue (GALT) is in charge of.
Focusing on our topic, we will briefly discuss what happens when an immune response occurs following the ingestion of an antigen, in this case the spike S1 protein from SARS-CoV-2. M cells, which are found in the epithelial layer of the mucosal tissue and, more precisely, in the effector site (PPs), take the antigen and offer it to antigen-presenting cells (APCs), such as dendritic cells, at the beginning. M cells, more precisely, 'wake up' dendritic cells in the digestive system lumen, which now begin to transport the antigen (or peptides of it).
Finally, using chemokines, the processed peptides are delivered to naive T-cells, which begin to develop into antigen-specific cells such as Th1, Th2, Th17, and cytotoxic T cells.
The B-cells that have now matured into IgA-B cells migrate from the inductive site (PPs) through the lymph vessels to the lymph nodes and the effector site once the immune response has been initiated (LP). Finally, the Th2-type cytokines lead IgA-B cells to develop into plasma cells (IL-5 and IL-6). The interaction of IgA with the polymeric Ig receptor results in the production of secretory IgA. (SIgA).
The mucosal surfaces of the gut intestines are important routes of entry into the body for the majority of infections due to their large surface area. As a result, novel vaccinations, like the one we propose, are very desirable. Mucosal immunization with an appropriate vaccine delivery method induces both protective mucosal and systemic immune responses, resulting in a second layer of pathogen protection. However, only a low number of mucosal vaccines has been produced and used against human diseases. This is owing to obstacles such as determining the best vaccine formulation, including the adjuvant, to enhance immunogenicity, stability, and delivery. We want to contribute to such a vaccination development through our project: 'vaccinATEd.'
Agrobacterium tumefaciens cells, transformed with a plasmid construct expressing the antigen, will be introduced into Lactuca sativa (lettuce) leaves for transient expression of the synthetic protein. The synthetic protein consists of a signal peptide (SP), three copies of the S1 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) connected with an amino acid linker, a transmembrane (TM) domain, an HA (Human influenza hemagglutinin) tag and a fluorescent protein (mCherry).
The signal peptide mediates the membrane targeting of the protein upon its expression in the plant cells. The three S1 subunits originate from the spike (S) protein) and resemble the wild type version of this subunit, the alpha variant (lineage B.1.1.7) identified in the United Kingdom, and the beta variant (lineage B1.351) identified in South Africa. Linker amino acids between each subunit serve for the subunits to fold in a desired three-dimensional conformation. The transmembrane domain originated from the plant FLS2 immunity receptor protein and it is involved in reception of bacterial components. TM-domain functions in such a way as to incorporate the synthetic protein into plant cells' plasma membranes. The HA tag is a general antibody epitope tag enabling easy purification, detection and future quantification of the synthetic protein. Finally, the fluorescent protein serves for real-time detection of the cellular localisation of the synthetic protein, but also in primary quantifications. After its expression in the plant cells, the protein will be targeted to the plasma membrane. Thus, the S1 subunits will be exposed to the extracellular space and hopefully they will maintain their appropriate conformation. Upon consuming the transformed lettuce leaves by mice (Mus musculus), the S1 subunits will serve as antigens for the induction of mucosal immunity. The goal is to achieve the immunization of the mice against the wild-type SARS-CoV-2 and its selected variants, laying the ground for the development of an edible vaccine.
Not all plants can be used to create proteins due to their unique restrictions, therefore choosing the optimal host plant to express the protein for the intended purpose is critical. During our investigation for the edible vaccine, we learned that lettuce is an ideal plant for our project, as it is currently one of the foods utilized in the creation of edible vaccines. The plant used to contain the vaccine must have a pleasant flavor, be hardy, and have a good nutritional value. One of the most essential characteristics of lettuce is that it is simple to deliver to mice, allowing us to move forward with the laboratory part of our project. Furthermore, lettuce is a fast-growing, easy-to-cultivate plant. As a result, edible vaccines are a more sustainable solution than standard vaccines, which must be brought to the location of administering. Additionally, lettuce leaves can be freeze-dried, allowing us to explore the option of using capsules as a different method of drug administration.
After doing research and a series of tests on various lettuce cultivars, we concluded that romaine-type lettuce is suited for our trials. Its leaves are particularly soft and have a small amount of lateral veins, allowing injection an easier through the procedure.
Do you want to review every step of a vaccine's production?
Check out our engineering section for more information on our project's extensive design and engineering.
- Lamichhane, Aayam, et al. “The Mucosal Immune System for Vaccine Development.” Vaccine, vol. 32, no. 49, Nov. 2014, pp. 6711–6723, 10.1016/j.vaccine.2014.08.089. Accessed 21 July 2020.
- Lycke N. Recent progress in mucosal vaccine development: Potential and limitations. Nature Reviews Immunology. 2012;12(8):592–605.
- Criscuolo E, et al. Alternative methods of vaccine delivery: An overview of edible and intradermal vaccines. Journal of Immunology Research. 2019;2019:13.
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- Karamloo F., König R. SARS-CoV-2 immunogenicity at the crossroads. Allergy. 2020;75:1822–1824. doi: 10.1111/all.14360.
- Van Eerde A., Gottschamel J., Bock R., Hansen K.E.A., Munang Andu H.M., Daniell H., Liu Clarke J. Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development. Plant. Biotechnol. J. 2019;17:1408–1417. doi: 10.1111/pbi.13065.