Citation
- Authors: Jahanbani S. et al.
- Year: 2022
- Journal: Clin Immunol
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
Heavy chain sequences were used to produce monoclonal antibodies as previous described. In short, the mAb's variable sequences of heavy chain and light chain regions were cloned into an in-house expression vector VRC01. Expi293 cells (2.5 × 106 cells/ml) were transfected with Lyme mAb's cognate heavy and light chains at a final transfection concentration of 0.5μg/mL. FectoPro Transfection Reagent (VWR) was added to the plasmids at a final transfection concentration of 1.3uL/mL. Media was harvested after 7 days and antibody purification was performed with IgG elution buffer (Pierce) by AmMag Protein magnetic beads (Genscript). Antibody concentrations were measured with a nanodrop.
Abstract
Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.