Expression of monkeypox virus-specific antibodies in Nicotiana benthamiana

Abstract

Mpox is a re-emerging viral disease that has raised global public health concerns, highlighting the need for rapid antibody production and diagnostic development platforms. This study aimed to develop and evaluate a plant-based transient expression system for the production of Mpox virus-specific monoclonal antibodies, anti-A35 and anti-B6, in Nicotiana benthamiana, and to investigate their biological activity and diagnostic potential. Recombinant anti-A35 and anti-B6 antibody genes were cloned into a geminiviral plant expression vector and introduced into Agrobacterium tumefaciens for transient expression in N. benthamiana. Recombinant antibodies were extracted and purified using Protein A affinity chromatography. Protein expression and assembly were analyzed by SDS-PAGE and Western blot. Optimization studies evaluated the effects of heavy chain:light chain (HC:LC) ratios and harvest time points on recombinant antibody production. The results demonstrated successful production of recombinant anti-A35 and anti-B6 antibodies in N. benthamiana. SDS-PAGE and Western blot analyses confirmed correct assembly of full-length IgG molecules. The 1:1 HC:LC ratio produced the most balanced expression profile with lower fragmentation. Anti-A35 showed the highest yield at 3 days post infiltration (dpi), whereas anti-B6 reached maximal accumulation at 7 dpi. Both antibodies specifically recognized Mpox-infected Vero cells and retained detectable biological activity against Mpox virus. In addition, integration of the recombinant antibodies into an electrochemical immunosensor platform generated measurable concentration-dependent electrochemical responses upon virus binding. Overall, this study demonstrates the feasibility of transient plant-based expression systems for recombinant anti-Mpox monoclonal antibody production and highlights their potential applications in diagnostic development and future biomedical research.