Structural and functional characterization of an anti‐West Nile virus monoclonal antibody and its single‐chain variant produced in glycoengineered plants

Abstract

Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ΔXF plants, a glycosylation mutant lacking plant specific N-glycan residues. Glycan analysis revealed that ΔXF plant-derived pE16scFv-CH (ΔXFpE16scFv-CH) and pE16 (ΔXFpE16) both displayed a mammalian glycosylation profile. ΔXFpE16 and ΔXFpE16scFv-CH demonstrated equivalent antigen binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared to the parent mammalian cell-produced E16 (mE16). A single dose of ΔXFpE16 or ΔXFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory, or neoplastic diseases.

Purification of monoclonal antibody against Ebola GP1 protein expressed in Nicotiana benthamiana

“…Specifically, 96-well ELISA plates were coated with 50 μL of 1:2000-diluted irradiated EBV (a gift from Dr. W. Pratt, USAMRIID) at 37 °C for 1 h and blocked with TBST containing 5% skim milk at room temperature for 1 h. After washing with TBST, the plates were incubated with various concentrations of N. benthamiana -produced 6D8 mAb or a N. benthamiana -produced anti-West Nile virus (WNV) mAb (hE16) as a negative control [20] at room temperature for 1 h. Subsequently, the plates were incubated with HRP-conjugated goat anti-human IgG at 37 °C for 1 h and developed with TMB substrate (KPL Inc., Gaithersburg, MD). The OD 450nm was measured with a Bio-Tek Power Wave microplate reader (BioTek, Winooski, VT).…”

Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds

“…However, this issue has recently been bypassed using genetically modified host to produce recombinant proteins that possess a more humanized N-glycan structure without xylose and fucose (Cox et al 2006;Koprivova et al 2004;Loos et al 2011b). Therefore, it is feasible to produce recombinant pharmaceuticals at lower cost, higher glycoform homogeneity, and better bioactivity by combining glycoengineering technologies with different expression strategies in plants (Gasdaska et al 2012;Lai et al 2014;Loos et al 2011a;Zeitlin et al 2011).…”

Risk factors for West Nile virus infection and disease in populations and individuals

“…Both passive and active immunization strategies directed against the envelope (E) proteins of WNV can protect mice from lethal infection (110-113). Single chain antibody (112, 114), targeted nanoparticles (115), attenuated virus vaccines (116), and plant-derived viral proteins have shown efficacy in animal models (117). Indeed, in equine models vaccination was both protective and cost-effective (118, 119).…”