Table 3.

Summary of the different expression approaches for producing plant-based vaccines and their function as MERS/SARS-CoV-1 vaccines.

Method	Features	Limitations	Target/Plant Species	The Protein Used/Route of Inoculation	Experimental Phase	Dose	Degree and Type of Protection Generated	Functions	Reference
Stable nuclear transformation	Seed bank possible; Inheritable antigen production; Many methods are available for different crops	Random insertion; Possibility of horizontal gene transfer; position effects and gene silencing; transformation is tedious	Full and truncated S protein/tomato and tobacco	Purified Protein/In saline and oral immunization	Pre-clinical	500 mg of dry tomato fruit, 50 mg of dry tobacco root, 2-week intervals, after a 4-week booster dose of 1 μg of commercially obtained S peptide without adjuvant.	Significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein.	Expression of SARS-CoV S protein (S1) in tomato and tobacco plants and after oral ingestion of tomato fruits, mice display elevated SARS-CoV-specific IgA levels.	[73]
Transient nuclear transformation	High and rapid protein production; Industrial scale production	The seed bank is impossible; requires purification of the antigen;	Partial spike protein of SARS-CoV; recombinant nucleocapsid (rN)and the membrane protein (M)/tobacco	Purified Protein/Intraperitoneally	Pre-clinical	2–4 μg rN protein	Vaccination of BALB/c mice with tobacco-expressed rN protein successfully led to a specific B-cell response.	Produced S1 proteins in chloroplast- and nuclear-transformed plants display potential in safe oral recombinant subunit vaccine. The expression of IL-10 and IFN-γ was up-regulated during the vaccination of rN protein, while IL-4 and IL-2 expression were not.	[74,75,76]
Transplastomic technologies	Multigene expression Highly productive; Better biosafety; site-specific insertion via recombination; Unaffected by silencing or position effects	Lacks complex post-translational modifications; Limited protocols available for limited species; generation of lines are tedious	N-terminal fragment of SARS-CoV S spike protein (S1)/Tomato and tobacco	Purified Protein/In saline and oral immunization	Pre-clinical	500 mg of dry tomato fruit, 50 mg of dry tobacco root, 2-week intervals	The mice parenterally primed with plant-derived antigen developed an immune response after booster immunization.	Sera of mice display the SARS-CoV-specific IgG.	[73,77]