Figure 5.

GE-AAV production and ADCC activity validation

(A) 3BNC117 was produced in HEK293T cells by AAV2 transduction at an MOI of 100,000. 72 h after transduction, cells were washed and transferred to serum-free media. After an additional 4 days of culture, supernatants were harvested, clarified, and analyzed for 3BNC117 concentration by protein A/anti-rhesus IgG ELISA. Antibody concentrations were normalized and analyzed for ADCC activity. ADCC levels were compared to 3BNC117 produced in HEK293T cells, as well as FUT8 KO cells as a representation of ADCC in IgG devoid of fucose. Both control antibodies were produced by plasmid transfection. ADCC activity was measured against HIV-1 NL(AD8)-infected target cells in triplicate. Samples were analyzed for statistical significance using a two-tailed paired t test. (B) Genome copies per AAV preparation was calculated by taking the total genome copies divided by the number of preparations performed.