The full-length S glycoprotein of SARS-CoV-2, strain hCoV-19/USA/CA2/2020, corresponding in sequence to nucleotides 21,563–25,384 from EPI_ISL_406036 in the GISAID database (https://www.gisaid.org/ ), was expressed in N. benthamiana plants using A. tumifaciens transfection, and the downstream purification processes were very similar to those previously described to produce VLPs bearing influenza hemagglutinin proteins. In this system, Spike protein expression is not plasmid-driven per se. Rather, the Agrobacterium vector cuts a defined segment of the plasmid and transfers it to the nucleus of the plant cells. This segment remains episomal for some time before being degraded (hence, transient expression) and drives the expression of Spike protein. For the CoVLP vaccine candidate, the Spike protein was modified with R667G, R668S, and R670S substitutions at the S1/S2 cleavage site to increase stability and K971P and V972P substitutions to stabilize the protein in pre-fusion conformation. The signal peptide was replaced with the protein disulfide isomerase from alfalfa, and the transmembrane (TM) domain and cytoplasmic tail (CT) of S protein were replaced with TM/CT from influenza H5 A/Indonesia/5/2005 to increase VLP assembly and budding. Expression of the Spike protein was driven using the double 35 S promoter and proprietary 5′ and 3′ untranslated regions developed to maximize mRNA stability and protein translation. The TBSV P19 suppressor of gene silencing, used under license from Plant Bioscience Limited, is co-expressed to maximize the transient expression of Spike protein. The self-assembled VLPs bearing Spike protein trimers were isolated from the plant matrix and subsequently purified using a process similar to that described for the influenza vaccine candidates. Briefly, N. benthamiana plants were grown in a controlled greenhouse environment for approximately five weeks before being exposed to the A. tumefaciens transfer vector by vacuum infiltration. After infiltration, plants were placed in a growth chamber under optimal conditions for CoVLP production for up to 6 d. Aerial parts of the plants were then harvested, and the VLPs were released using a proprietary extraction method. The bulk drug substance containing concentrated CoVLPs was then purified using a series of standard industrial filtration and chromatography unit operations steps. The AS03 adjuvant, an oil-in-water emulsion containing DL-α-tocopherol (11.69 mg per dose) and squalene (10.86 mg per dose), was supplied by GlaxoSmithKline.
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