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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing coronavirus disease (COVID-19) pandemic which is characterized by respiratory illness and severe pneumonia, and currently accounts for > 2.5 million deaths worldwide. Recently, diverse mutations in the spike protein of SARS-CoV-2 were reported in United Kingdom (Alpha) and South Africa (Beta) strains which raise concerns over the potential increase in binding affinity towards the host cell receptor and diminished host neutralization capabilities. In order to study the effect of mutation in the binding efficiency of SARS-CoV-2 receptor binding domain (RBD) with anti-SARS-CoV/CoV-2 monoclonal antibodies (mAbs), we have produced SARS-CoV-2 RBD and two variants SARS-CoV-2 RBD (Alpha RBD and Beta RBD) in Nicotiana benthamiana by transient expression. Plant-produced SARS-CoV-2 RBD-Fc, Alpha RBD-Fc and Beta RBD-Fc exhibited specific binding to human angiotensin converting enzyme 2 (ACE2) receptor determined by ELISA. Intriguingly, the binding of plant-produced SARS-CoV-2 RBD proteins to plant-produced mAbs CR3022, B38, and H4 was found to be different depending on the variant mutation. In contrary to the plant-produced SARS-CoV-2 RBD-Fc and Alpha RBD-Fc, Beta RBD-Fc variant showed weak binding affinity towards the mAbs. The result suggested that the Beta RBD variant might have acquired partial resistance to neutralizing antibodies compared to other variants. However, further studies with sera from convalescent or vaccinated individuals are required to confirm this finding.

Inductive molecules are critical components for successful bone tissue engineering. Dentin matrix protein-1 (DMP1), a non-collagenous protein in the bone matrix, has been shown to play roles in osteogenic differentiation and phosphate homeostasis. This study aimed to produce recombinant human dentin matrix protein-1 (hDMP1) in Nicotiana benthamiana and investigated the ability of this plant-produced DMP1 to induce osteogenesis in human periodontal ligament stem cells (hPDLSCs). The hDMP1 gene was cloned into the geminiviral vector for transient expression in N. benthamiana. We found that hDMP1 was transiently expressed in N. benthamiana leaves and could be purified by ammonium sulphate precipitation followed by nickel affinity chromatography. The effects of hDMP1 on the induction of cell proliferation and osteogenic differentiation were investigated. The results indicated that plant-produced hDMP1 could induce the cell proliferation of hPDLSCs and increase the expression levels of osteogenic genes, including osterix (OSX), type I collagen (COL1), bone morphogenetic protein-2 (BMP2), and Wnt3a. Moreover, the plant-produced hDMP1 promoted calcium deposition in hPDLSCs as determined by alizarin red S staining. In conclusion, our results indicated that plant-produced hDMP1 could induce osteogenic differentiation in hPDLSCs and could potentially be used as a bone inducer in bone tissue engineering.

SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic. The virus spreads rapidly with a high transmission rate among humans, and hence virus management has been challenging owing to finding specific therapies or vaccinations. Hence, an effective, low-cost vaccine is urgently required. In this study, the immunogenicity of the plant-produced S1 subunit protein of SARS-CoV-2 was examined in order to assess it as a potential candidate for SARS-CoV-2. The SARS-CoV-2 S1-Fc fusion protein was transiently produced in Nicotiana benthamiana. Within four days of infiltration, the SARS-CoV-2 S1-Fc protein was expressed in high quantities, and using protein A affinity column chromatography, plant-produced S1-Fc protein was purified from the crude extracts. The characterization of plant-produced S1-Fc protein was analyzed by SDS-PAGE and Western blotting. Immunogenicity of the purified S1-Fc protein formulated with alum induced both RBD specific antibodies and T cell immune responses in mice. These preliminary results indicated that the plant-produced S1 protein is immunogenic in mice.