Plant-Produced Anti-Enterovirus 71 (EV71) Monoclonal Antibody Efficiently Protects Mice Against EV71 Infection

Abstract

Enterovirus 71 (EV71) is the main causative agent of severe hand-foot-mouth disease. EV71 affects countries mainly in the Asia-Pacific region, which makes it unattractive for pharmaceutical companies to develop drugs or vaccine to combat EV71 infection. However, development of these drugs and vaccines is vital to protect younger generations. This study aims to develop a specific monoclonal antibody (mAb) to EV71 using a plant platform, which is a cost-effective and scalable production technology. A previous report showed that D5, a murine anti-EV71 mAb, binds to VP1 protein of EV71, potently neutralizes EV71 in vitro, and effectively protects mice against EV71 infection. Herein, plant-produced chimeric D5 (cD5) mAb, variable regions of murine D5 antibody linked with constant regions of human IgG1, was transiently expressed in Nicotiana benthamiana using geminiviral vectors. The antibody was expressed at high levels within six days of infiltration. Plant-produced cD5 retained its in vitro high-affinity binding and neutralizing activity against EV71. Furthermore, a single dose (10 µg/g body weight) of plant-produced cD5 mAb offered 100% protection against infection in mice after a lethal EV71 challenge. Therefore, our results showed that plant-produced anti-EV71 mAb is an effective, safe, and affordable therapeutic option against EV71 infection.2 of 10 vaccines [16,17]. For EV71 post-exposure treatment in patients with severe infection, EV71-specific intravenous immunoglobulins are used [18]. However, there are disadvantages to using pooled human sera, such as the risk of transmitting human pathogens, availability of donors, and batch-to-batch variability [19]. Therefore, neutralizing monoclonal antibodies (mAbs) are considered a safe alternative for passive immunization against EV71.Over the years, several EV71-neutralizing antibodies have been reported [20][21][22][23][24][25][26]. Among these mAbs, D5 is one of the most promising mAb for development as prophylactic and therapeutic for EV71 infection. D5 mAb binds specifically to SP70 peptide on VP1 GH loop of EV71 and can potently neutralize EV71 infection by blocking viral attachment and internalization and by stabilizing the virus through a bivalent binding mode [26,27]. However, the antibody production in mammalian cell culture has limitations with high production costs and scalability.Recently, plants are being used as an alternative mAb production platform with unique advantages such as low production cost, speed of production, and scalability [28]. This platform offers a solution for developing countries to develop affordable medicines in the region. Previous studies demonstrated the efficacy of plant-produced mAbs for viral infections, such as West Nile virus [29], Ebola virus [30], Rabies virus [31], and others. The goal of this study is to investigate the protective effect of plant-produced EV71 specific mAb against EV71 challenge in mice.In this study, chimeric D5 (cD5) mAb was constructed and produced transiently in Nicotiana benthamiana leaves. After...

Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

“…The V H and V L chains were fused with human IgG1 C H and C L regions respectively. The resulting full length coding sequences of CR3022 HC and LC were cloned into a geminiviral vector (pBY2e) as described previously 10 by a three fragment ligation: the backbone from pBY2e was obtained from Xba I- Sac I digestion; V H and C H were obtained by Xba I- Nhe I and Nhe I- Sac I digestion, respectively while V L and C L were obtained by Xba I- Afl II and Afl II- Sac I digestion, respectively to create the expression cassettes pBY2e-CR3022 HC and pBY2e-CR3022 LC.…”

“…Over the past two decades, biopharmaceuticals have been produced in a number of different expression systems such as yeast, mammalian cells, and plants, but currently most of the commercially available recombinant vaccines or biopharmaceuticals are produced in mammalian or microbial cell cultures. One of the major issues with the biopharmaceuticals produced in mammalian system is the requirement of initial capital investment and the high production costs associated with it 9 , 10 . While each expression system has its own advantages, they have all proven to possess distinct shortcomings, and the limitations of each expression system led to development of alternative production platforms that could significantly reduce the production costs.…”

Monoclonal Antibodies B38 and H4 Produced in Nicotiana benthamiana Neutralize SARS-CoV-2 in vitro

“…To purify anti-SARS-CoV-2 mAb (B38 and H4) from plants, the infiltrated plant leaves were harvested 4 days post infiltration (dpi) and purified by protein A column chromatography as described previously (Rattanapisit et al, 2019a). Briefly, the harvested leaves were homogenized with extraction buffer 1 × phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 ) at a ratio of 1 g leaf tissue to 2 ml buffer, and the plant crude extracts were centrifuged at 26,000 g for 40 min at 4 • C. After centrifugation, supernatant was collected and filtered through 0.45-µm membrane filter.…”

Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 inNicotiana benthamianaas vaccine candidates against COVID-19

“…Plant transient expression system has a number of advantages over stable expression. Using a transient plant expression system, the production of glycosylated form of RBD SARS-CoV-2 in Nicotiana benthamiana plant has been reported recently 29 . However, the expression level of RBD in N. benthamina plant was very low, 8 μg/g, which is unsatisfactory to be economical for commercialization.…”