Rice‐derived <scp>SARS</scp>‐<scp>CoV</scp>‐2 glycoprotein <scp>S1</scp> subunit vaccine elicits humoral and cellular immune responses

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has been spreading and mutating globally despite the expedited approval of many commercial vaccines. Therefore, developing safe, effective and affordable vaccines remains essential to meet the global demand, particularly in developing countries. Transgenic plants have emerged as a promising platform to express recombinant proteins for pharmaceutical and vaccine applications. Two binary vectors, pCAMBIA1300Gt1-S1 and pCAMBIA1300Actin-S1, containing distinct promoters, were constructed and transformed into rice via Agrobacterium. Overall, 56 independent transgenic rice lines were regenerated. Expression analysis revealed that the rice-derived S1 (rS1) protein could be expressed in pGt1::S1 transgenic rice seeds. rS1 protein expression levels reached up to 282 μg/g dry weight, with S1 gene insertion having no effect on grain size and weight. The rS1 protein exhibited a high affinity for human angiotensin-converting enzyme 2 (ACE2) in vitro. Moreover, the immunogenicity of purified rS1 protein co-administered with various adjuvants demonstrated that mice vaccinated with Alum-adjuvant rS1 generated enhanced humoral immune responses with high serum IgG, IgG1 and neutralizing antibody levels. Salmonella Typhimurium flagellin (FliC)-adjuvanted rS1 elicited stronger S1-specific IgG2a levels, promoted splenocyte proliferation and induced mixed Th1/Th2/Th17 cytokine responses. This was evidenced by increased proportions of antigen-specific interferon (IFN)-γ, interleukin-4 (IL-4) and IL-17A-positive CD4⁺ T lymphocytes, suggesting its potential to induce both humoral and cellular immune responses. These findings suggest that rS1 protein offers a promising approach for affordable COVID-19 subunit vaccine production, and this strategy can be universally applied to other viral vaccines.