Rapid and Scalable Production of Functional Anti-Coronavirus Monoclonal Antibody CR3022 in Plants


 Severe acute respiratory syndrome coronavirus-2 is responsible for an ongoing global outbreak of coronavirus disease (COVID-19) and represents a significant public health threat. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of diagnostic reagents/biopharmaceuticals for COVID-19. We explored the possibility of producing an anti-SARS-CoV monoclonal antibody (mAb) CR3022 and the receptor binding domain (RBD) of SARS-CoV-2 in Nicotiana benthamiana. Both RBD and the mAb were transiently expressed with the expression of 8μg/g and 130μg/g leaf fresh weight respectively. The plant-purified mAb binds to SARS-CoV-2, but fails to neutralize it in vitro. This is the first report showing the functional characterization of an anti- SARS-CoV mAb CR3022 in plants. Overall these findings showed that plants are a promising platform to produce anti-SARS-CoV mAb to use as a research reagent or a biotherapeutic in a cost-effective manner, which is especially important to developing economies during epidemics.

Introduction has attracted the attention of the whole world. Tens of thousands of infected cases have been reported and death toll is escalating daily. The continued spread of SARS-CoV-2 in many countries demands the development of cost-effective rapid diagnostic assays and therapeutics for COVID-19.
The receptor binding domain (RBD) located within the spike region of SARS-CoV mediates virus entry into the host cell by interacting with host receptor angiotensin converting enzyme 2 (ACE2) 30. The new virus SARS-CoV-2 is genetically related to SARS-CoV which also utilizes the ACE2 receptor on human cells for its cell attachment and entry 31. The RBD region located in the spike glycoprotein is essential for membrane fusion and is regarded as a major target of the host antibody response. As a result, antibodies targeting the RBD region have been extensively explored as potential coronavirus therapeutic candidates, and these may additionally be utilized for the development of SARS-CoV-2 diagnostics 3, 32.
In the current study, we used a plant expression system for the production of the RBD antigen of SARS-CoV-2 and a mAb CR3022 that is specific to the RBD of SARS-CoV. We used a geminiviral replicon vector derived from the bean yellow dwarf virus 33 for the production of both RBD and mAb CR3022. Our results showed that both RBD antigen of SARS-CoV and mAb CR3022 could be produced rapidly in a large scale in N. benthamiana within a time frame of less than 2 weeks after the gene construct delivery 34. Recent developments in plant expression strategies using viral vectors and transient expression has increased protein yield, significantly reduced the upstream production cost, simplified the downstream processing of plant recombinant proteins which improves the commercial viability of the system 11, 35, 36. Our results indicated that RBD of SARS-CoV-2 was expressed in N.
benthamiana plant as a soluble protein rapidly and accumulated to 8 μg/g leaf fresh weight, whereas mAb CR3022 accumulated at high levels at 130 μg/g leaf fresh weight in N. benthamiana leaves. The Altogether, our results convincingly demonstrate the practicability of using a plant expression system for the rapid and large-scale production of antibodies with diagnostic or therapeutic potential. In particular, this methodology is scalable to a commercial basis without a high capital investment, and is therefore suitable for use in developing economies. Furthermore, this study proved the robustness of plant transient expression system for the production of anti-SARS-CoV mAb CR3022 with high yield and low cost which can likely improve the affordability of mAb-based diagnosis in the developing world.

Conclusion
In summary, we have demonstrated the rapid production of mAb CR3022 in Nicotiana benthamiana.
The expressed antibody was purified and analyzed for antigen-binding and SARS-CoV-2 virus neutralization activity in vitro. The results showed that plant-produced CR3022 mAb could bind to recombinant RBD protein of SARS-CoV-2, but that it did not neutralize the virus in vitro. Our study indicated that plant transient expression systems can greatly reduce the production cost and could be adapted for the low cost, rapid large-scale production of recombinant antibodies that could be used as detection/diagnostic reagents to detect COVID infection.

Construction of expression vectors of mAb CR3022 and RBD
The Institutional Review Board of Chulalongkorn University approved the present study. The coding gene fragments of variable heavy chain (VH) and variable light chain (VL) regions of mAb CR3022 (Accession Nos.: DQ168569.1 and DQ168570.1) were codon optimized for expression in N.
benthamiana and commercially synthesized (Genewiz, Suzhou, China). The VH and VL were fused with human IgG1 CH and CL regions respectively. The resulting full length coding sequences of CR3022 HC and LC were cloned into geminiviral vector (pBY2e) as described previously 10 by a three fragment ligation: the backbone from pBY2e was obtained from XbaI-SacI digestion; VH and CH were obtained by XbaI-NheI and NheI-SacI digestion, respectively while VL and CL were obtained by XbaI-AflII and AflII-SacI digestion, respectively to create the expression cassette pBY2e-CR3022-HC and pBY2e-CR3022-LC.

Transient expression of SARS-CoV-2-RBD and mAb CR3022 in N. benthamiana leaves
The expression vectors were transformed into Agrobacterium tumefaciens strain GV3101 via electroporation, and the resulting strains were confirmed by PCR. Wild type N. benthamiana plants were grown in a green house with a suitable light/dark cycle at 28°C for 6-8 weeks. For mAb CR3022 expression, recombinant Agrobacterium containing pBY2e-CR3022-HC and pBY2e-CR3022-LC were pelleted and resuspended in infiltration buffer to an OD600 of 0.4 and mixed at a 1:1 ratio prior to vacuum infiltration. Similarly, an Agrobacterium strain harboring pBY2e -SARS-CoV-2-RBD was delivered into tobacco leaves by agroinfiltration.

Extraction and purification of recombinant monoclonal antibody and RBDs from plant leaves
Briefly, agroinfiltrated leaves were harvested at 3 days post infiltration (d.p.i) and proteins extracted in extraction buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4) at pH 7.4 using a previously developed method with some modifications 10 for CR3022 mAb. The crude leaf extract was with FITC (Santa Cruz Biotechnology, Inc.). After incubation at 37oC for 1hr, the plate was washed three times and DNA staining dye, Hoechst33342 was added. The plate was then subjected to automated image acquisition and analysis using Operetta (PerkinElmer). All sera were heated inactivated in 56oC for 30 mins before use.

Conflict of Interest
The authors declare no conflict of interest.

Supplementary Files
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