Development of a plant-based platform for the production of SARS-CoV-2 antigen

The coronavirus disease 2019 (COVID-19) pandemic has been caused by an RNA virus called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belonging to the family Coronaviridae. This virus is extremely infectious, and to date, it has claimed more than 4.9 million lives worldwide. Scientists have stated that rapid vaccination of the world’s population is the only effective way to contain the COVID-19 pandemic.

Study: Plant-based production of SARS-CoV-2 antigens for use in a subunit vaccine. Image Credit: Kateryna Kon/ ShutterstockStudy: Plant-based production of SARS-CoV-2 antigens for use in a subunit vaccine. Image Credit: Kateryna Kon/ Shutterstock

Plant-based recombinant protein platforms and COVID-19

The continual emergence of SARS-CoV-2 variants due to mutations has posed a threat to the efficacy of the available vaccines designed based on the original SARS-CoV-2 strain. These variants are more infectious and virulent than the original strain and have been classified as variants of Interest (VOI) or variants of concern (VOC). Therefore, there is an urgent need for new vaccines that are highly effective against SARS-CoV-2 variants.

Scientists have recently focused on the development of plant-based recombinant protein platforms. As the production of recombinant proteins is simple, cost-effective, highly scalable, and easily modifiable according to the circulating viral sequences, these could be a true alternative to conventional protein production. Although viral glycoproteins are extremely challenging to be produced in plants, recently, researchers have achieved this feat. This study is available on the bioRxiv* preprint server.

Development of plant-based SARS-CoV-2 antigens

Researchers have successfully produced plant-expressed wild-type glycosylated SARS-CoV-2 Spike RBD (receptor binding domain) protein. This protein is recognized by anti-RBD antibodies. Interestingly, a high-affinity binding between the plant-based Spike RBD protein and ACE2 (angiotensin-converting enzyme 2) receptor of the host has been reported.

In this study, a codon-optimized RBD sequence corresponding to the original SARS-CoV-2 strain was cloned using the pHREAC vector backbone, which is a plant-specific expression platform. The RBD sequence was flanked on the N-terminal side via endoplasmic reticulum targeting signal peptide obtained from Nicotiana tabacum PR-1a signal peptide and on the C-terminal side by a tandem affinity tag comprised of an 8xHis tag and a Twin-Strep-tag. The dual affinity tags were separated by a glycine-serine linker, flanked by a thrombin cleavage site (LVPRGS) placed directly upstream of the affinity tags, and an ER retention signal (KDEL) was positioned downstream and adjacent to two tandem stop codons.

Based on previous studies on plant-based protein expressions, researchers demonstrated the expression of a trimeric Spike mimetic protein in Nicotiana benthamiana co-expressed in the presence and absence of human calreticulin. Compared to the absence of calreticulin, researchers found co-infiltrating with calreticulin influenced a significant elevation in RBD expression levels. In this study, all RBD samples had shown a single band that moved slightly above the expected molecular weight of 31.3 kDa, and slightly above a mammalian-expressed RBD control.

Initially, the protein was partially purified using the Ni-NTA column, and the fractions were analyzed using immunoblot analysis. These eluted proteins confirmed that the plant-produced RBD protein was glycosylated. Researchers assessed the function and folding of RBD produced in N. benthamiana. The correct folding of the RBD antigen is crucial for its interaction with the human ACE2 receptor. This will ensure the development of antibodies capable of neutralizing SARS-CoV-2 original strain and variants.

The authors of the current study revealed that plant-based RBD preserves proper folding and functionality similar to mammalian-produced RBD. This was determined by studying the binding affinity of the protein to host cell receptor ACE2, recognition by the conformation-dependent monoclonal CR3022 antibody, and polyclonal antibodies from sera of SARS-CoV2- infected individuals.

This study reported that IgM, IgA could easily recognize the plant-based protein and IgG antibodies isolated from naturally infected convalescent and vaccinated individuals. However, compared to mammalian-expressed RBD, plant-produced RBD showed a reduced binding affinity with IgM and IgA antibodies but not with IgG antibodies. Additionally, the current study revealed that the newly developed plant-based recombinant protein closely recapitulates the antigenic and biochemical characteristics of RBD produced in human cells. This is a crucial feature for a plant-based human vaccine antigen.

Advantage of a plant-based nasal vaccine

Most of the available COVID-19 vaccines are based on intramuscular delivery systems. This system requires skilled personnel for operation, a deficiency of which has slowed the vaccination process in some low-income countries. Scientists believe that the development of a subunit nasal spray vaccine could be extremely beneficial. This vaccination system could increase mucosal immunity to SARS-CoV-2 variants and help reduce the rate of vaccine-hesitancy as it would provide needle-phobic individuals with alternative immunization options.


The current study revealed that RBD binding to the ACE2 receptor was efficiently neutralized by antibodies obtained from sera of SARS-CoV-2 recovered and partially and fully vaccinated individuals. Therefore, based on all experiments, the authors suggested that recombinant RBD produced via plants contains relevant biochemical and antigenic features and could be used in a subunit vaccine platform.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Priyom Bose

Written by

Dr. Priyom Bose

Priyom holds a Ph.D. in Plant Biology and Biotechnology from the University of Madras, India. She is an active researcher and an experienced science writer. Priyom has also co-authored several original research articles that have been published in reputed peer-reviewed journals. She is also an avid reader and an amateur photographer.


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