Antibody-mediated enhancement aggravates chikungunya virus infection and disease severity

Lum, Fok-Moon; Couderc, Thérèse; Chia, Bing-Shao; Ong, Ruo-Yan; Her, Zhisheng; Chow, Angela; Leo, Yee-Sin; Kam, Yiu-Wing; Rénia, Laurent; Lecuit, Marc; Ng, Lisa F. P.

For detection of CHIKV antigens in primary human cells (B cells, monocytes), staining was done as described previously using a commercially available anti-alphavirus mAb (Santa Cruz; SC-58088) 37, 47 . For whole blood and human MDMs, CHIKV infection was directly quantified by the detection of Zs-Green signal under the FITC channel. For detection of CHIKV antigen in RAW264.7 cells, staining was performed with rabbit anti-CHIKV nsP2 antibodies followed by secondary staining with Alexa Fluor 488-conjugated Goat anti-rabbit IgG (H + L) (Invitrogen). For primary human cells, an additional staining step was performed. Surface markers Pacific-Blue-conjugated mouse anti-human CD45, Qdot-605-conjugated mouse anti-human CD19, PerCP Cy5.5 mouse anti-human CD14 and PE-Cy7-conjugated mouse anti-human BDCA2 (all antibodies were from eBiosciences) were stained following manufacturer's protocol. Human monocytes, B cells and pDCs are defined by the specific surface expression of CD14, CD19 and BDCA2 respectively. Data was acquired using either BD FACS Calibur or BD FACS Canto II (BD Biosciences). Softwares used include BD CellQuest Pro software (for FACSCalibur) and BD FACSDiva software (for FACSCanto II) (BD Biosciences). Gating strategy is shown in Fig. S5 . A total of 30,000-50,000 cells were acquired and results were analyzed with FlowJo (version 10) (Tree Star).