Team:Crete/Proof Of Concept

Theoretical project expansion Consumption of edible vaccines by mice to test possible immune response.

Our project essentially in its plainest form aims to demonstrate the proper function of an edible vaccine. Given that our goal is to achieve transmembrane expression of our own synthetically constructed protein (SARS-CoV-2 spike S1 by transient expression in lettuce leaves through the agroinfiltration process) we want to induce an immune response in humans and other vertebrates through the gut mucosa. From the beginning we were aware of the necessity of using laboratory animals in order to prove that the edible vaccine actually induces an immune response in vertebrates and specifically in mice. However, as undergraduate students we weren't authorized to handle experiments with mice in the laboratory which made it quite challenging for us!
In the early stages of the experimental design for the manipulation of mice, we had all the proper guidance from our supervisors and other specialised staff. We were given a theoretical training that highlighted the mice's care inside the lab, potential risks and how to handle these animals, following all the universal regulations
Unfortunately, given the existing state of the coronavirus, most laboratories in our country were closed for over 6 months and others could only accept a small number of people, mostly post-graduate students. Therefore, since we could only begin our bench work at the very beginning of July, we didn't get to catch up with the second series of experiments involving mice immunization, because the antigen production would take around two months in order for us to have reliable data. From the beginning of the 2021, our team worked hard towards creating a well-documented experimental procedure for immune response induction and in this section we’d like to share with you our detailed experimental design around laboratory mice and what exactly we’d have done, if we had more time inside the laboratory.

Why was such an experimental procedure necessary?

First and foremost, it must be determined why animals are required to confirm our concept, as well as why mice are the most appropriate species to utilize. Because the efficacy of immunization and specific antigen induction of the adaptive immune system in mice must be determined, there is no other option but to use live animals to study the murine immune system.
The suggested investigations will require live vertebrate animals because the immune system is an intrinsic aspect of the entire animal and cannot be successfully replicated in cell culture. Cell lines are known to contain a variety of chromosomal and chromatin abnormalities, including duplicated chromosomes, altered epigenetic chromatin profiles, and unregulated gene expression patterns. Furthermore, we will choose mice for the proposed project due to their utility as a model system for the study of the human body.

What are the basic steps of this experiment?

First of all, it is crucial that before the oral administration of the vaccine, inbred female C57BL/6 5-week old mice had been acclimating for at least 1 week in cages that were randomly distributed. After this period of time, our wet lab team would feed the mice at days 0, 21, 42, and 63 the lyophilized antigen-containing lettuce tissue whereas for the proper evaluation of the H0 hypothesis a control group of mice has to be used. This small population of mice would be fed regular food. Furthermore, to test if a proper production of antigens occurred our team would be obligated to take blood samples from the mice on days 0,14,35, and 56 by submandibular vein bleed in accordance with the Institutional Animal (FORTH) Care and Use Committee. Finally, on the 84th day of experiments, the mice would be euthanized in accordance with the Institutional (FORTH) IACUC.

How does our team guarantee the least possible stress of the animals during the experiment?

Because of our team's high ethical standards and belief in the importance of compassionate scientific procedures, we believe it is at the very least required to inform the public about euthanasia methods and living conditions. More accurately, we strongly consider that the best method for euthanasia is the use of CO2. This is because quick unconsciousness happens without pain, and as a result, we reduce animal stress to the lowest level feasible. The absence of cardiovascular function is strictly linked to death confirmation. During the tests, all mice would be checked on a regular basis for signs of discomfort, and any that showed overt signs of discomfort (lack of activity or feeding behavior) would be terminated right away. Also, on a daily basis, the expert veterinarian of the hosting institution's mouse facility would perform monitoring of the mice in order to evaluate their health and well-being.

How is your team going to test if antibodies were produced by mice after the consumption of the lyophilized antigen-containing lettuce tissue?

In order to determine the concentration of IgG antibodies against the S1 antigen our wet lab team would have to conduct a quantitative ELISA experiment. More specifically, we would choose a sandwich ELISA, meaning that human anti-spike IgG antibodies are used to coat the wells of the 96 well-plates. After several washes the S1 antigen which is added binds through the receptor binding domain (RBD) to the antigen (IgG) with high affinity and then, again after several washes, serum from the blood that had been collected from the submandibular vein of mice is added. Finally, after washes, a third biotin labeled polyclonal antibody is added, and an enzymatic color reaction occurs after streptavidin-HRP connects with biotin. The amount of color measured is proportional with the IgG antibodies produced by mice.
  • Donini, M.; Marusic, C. Current state-of-the-art in plant-based antibody production systems. Biotechnol. Lett. 2019, 41, 335–346.
  • Choubini, E.; Habibi, M.; Khorshidi, A.; Ghasemi, A.; Karam, M.R.A.; Bouzari, S. A novel multi-peptide subunit vaccine admixed with AddaVax adjuvant produces significant immunogenicity and protection against Proteus mirabilis urinary tract infection in mice model. Mol. Immunol. 2018, 96, 88–97.
  • Sun, S.; He, L.; Zhao, Z.; Gu, H.; Fang, X.; Wang, T.; Yang, X.; Chen, S.; Deng, Y.; Li, J.; et al. Recombinant vaccine containing an RBD-Fc fusion induced protection against SARS-CoV-2 in nonhuman primates and mice. Cell. Mol. Immunol. 2021, 18, 1070–1073.
  • Huang Y, Yang C, Xu X, Xu W, Liu S. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin. 2020;41(9):1141-1149. doi:10.1038/s41401-020-0485-4
  • Maharjan, P. M., et al. Plant-Expressed Receptor Binding Domain of the SARS-CoV-2 Spike Protein Elicits Humoral Immunity in Mic . Vaccines 2021, 9(9), 978;