- Authors: Derking R. et al.
- Year: 2021
- Journal: Cell Rep 35 108933
- Applications: in vitro / DNA / FectoCHO Expression System, FectoPRO
- Cell type: ExpiCHO-S
Description: Chinese hamster ovary cells
ExpiCHO-S cells were maintained at a density of 1-2x10^6 cells per mL at 37 degrees with 8% CO2 and 135rpm shaking. The WT and NxT T35S T158S encoding plasmids containing a C-terminal D7324-tag were transiently co-transfected with a Furin-encoding plasmid (4:1) using FectoPRO transfection reagent as described by the manufacturer.
Artificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other's occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens.