Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (mu-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of mu-tp and KDEL to the ACE2 protein (ACE2 mu K) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 mu K protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 mu K protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks. In this study, we focused on the efficient, rapid, and scalable expression of ACE2 protein as a preventive and therapeutic agent for the future outbreaks of SARS-CoV-2 and other viral pandemics using Nicotiana benthamiana as a plant transient expression system. To enhance the functionality of the protein, we endeavored to produce oligomeric ACE2 mu K by incorporating the KDEL endoplasmic reticulum retention motif and mu tailpiece (mu-tp) of immunoglobulin M, while excluding the Fc region of the antibody. The plant-derived ACE2 mu K protein exhibited enhanced binding activity against various SARS-CoV-2 strains in comparison to ACE2 protein derived from CHO cells. Moreover, the ACE2 mu K protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. To our knowledge, this work is the first attempt to fuse the mu-tp without the Fc region to enhance the activity of a recombinant protein. The graphical abstract was created with BioRender.com. image