Development of anti-dengue virus D8 neutralizing monoclonal antibody production in Nicotiana benthamiana

• Plant-produced anti-dengue D8 mAb in ΔXF N. benthamiana neutralized all four dengue virus serotypes. • The D8 mAb showed strong NS1 binding and neutralization potency comparable to mammalian-derived HuMAb. • Glycosylation profiling revealed predominant GnGn and GnM3 forms, lacking plant-specific glycans. • FRNT assay showed highest activity against DENV4, with FRNT₅₀ < 1 μg/mL. Dengue is a life-threatening mosquito-borne viral disease ranging from mild symptoms to severe hemorrhagic fever. In this study, a recombinant anti-dengue D8 monoclonal antibody was produced in Nicotiana benthamiana and characterized for protein integrity and glycosylation by LC-MS. The plant-derived antibody lacked plant-specific β1,2-xylose and core α1,3-fucose residues, displaying a mammalian-like glycan profile. Functional evaluation showed potent cross-neutralizing activity against all four dengue virus serotypes (DENV1–DENV4), with the strongest activity against DENV4 (FRNT50 < 1 µg/mL), followed by DENV2 (5.82 µg/mL), DENV3 (9.49 µg/mL), and DENV1 (28.68 µg/mL), comparable to the mammalian-produced counterpart. The antibody also bound strongly to NS1 proteins of all serotypes, especially DENV2, and demonstrated higher reactivity than mammalian-derived anti-NS1 antibodies. Collectively, our results provide proof-of-concept that a glycoengineered plant platform can generate a functional D8 antibody with mammalian-like glycosylation, robust NS1 binding, and cross-neutralizing activity against DENV1–4, prompting further evaluation in Fc-dependent assays and in vivo models.