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.

Virus stocks. CHIKV-SGP11 was isolated from an outbreak in Singapore in 2008 and prepared in Vero-E6 cells as described 37 . This was used for all in vitro experiments involving isolated primary human monocytes, B cells and the RAW264.7 cells. For the whole blood and MDMs (together with their corresponding precursor monocytes), a full-length CHIKV infectious clone based on the CHIKV LR2006-OPY1 69 expressing the Zs-Green protein was used. CHIKV-SGP11 was further propagated in C6/36 cells and purified by ultra-centrifugation 44 before being used in infection studies in 3 weeks old mice. In infection studies in 11 days old mice, CHIKV-21 isolate obtained from a patient during the 2005-2006 outbreak of CHIKV infection in La Réunion was used 49 . In vitro FcγR expression assay. Cells were first stained with 10 μl of Human FcγR blocking reagent (Miltenyi Biotec), before staining with the following anti-FcγR antibodies and respective fluorophore isotype controls following manufacturer's recommendations: FITC-conjugated mouse anti-human CD64 (eBioscience), APC-conjugated mouse anti-human CD32 (eBioscience), PE-conjugated mouse anti-human CD16 (eBioscience), mouse IgG1κ isotype control FITC (eBioscience), mouse IgG1κ isotype control APC (eBioscience) and mouse IgG1κ isotype control PE (eBioscience). For RAW264.7 cells, staining was performed with FITC-conjugated rat anti-mouse FcγRI and anti-mouse FcγRII antibodies (R&D). Data were acquired with BD FACS Calibur (BD Biosciences) using BD CellQuest Pro software (BD Biosciences). Results were analyzed with FlowJo (version 10) software (Tree Star). In vitro CHIKV infection assay. CHIKV (moi 10) was first incubated with diluted heat-inactivated (56 °C for 30 min) CHIKV-specific patient plasma or animal sera in serum-free IMDM (Hyclone) or DMEM (Gibco) respectively, for 2 h on a shaking heat-block (37 °C; 350 rpm) before being used to infect the respective primary cells or cells lines (2 × 10 6 cells per infection) for 1.5 h in a 37 °C incubator, with atmosphere of 5% (v/v) CO 2 . Virus overlay was removed and cells were washed once with appropriate serum-free medium before they were re-suspended in appropriate complete medium. Cells were further incubated at 37 °C, with atmosphere of 5% (v/v) CO 2, , before being harvested at indicated time points. During harvesting, 140 μl of infected cell suspension was aliquoted for viral RNA extraction. For gene expression studies, an aliquot of cell suspension was spun down and the resultant cell pellet was dried before being stored in −80 °C for downstream total RNA extraction. Remaining cells were collected by centrifugation and fixed with FACS lysing buffer (BD Biosciences) and stored for downstream staining procedures. Mock and non-enhanced control infections were performed by incubating cells with serum-free DMEM (Gibco) and viruses respectively. For ex vivo infection in human whole blood, Zs-Green tagged CHIKV variant (2 × 10 7 PFU) was first incubated with diluted heat-inactivated CHIKV-specific patient plasma in serum free IMDM (Hyclone) for 2 h on a shaking heat-block (37 °C; 350 rpm) before being added to 1 ml of fresh citrate whole blood. 1 ml of whole blood typically contains between 1.5-2 × 10 6 leukocytes. Infection was incubated in 37 °C, with atmosphere of 5% (v/v) CO 2 until being harvested at the indicated time points. During harvesting, the mixture was spun down and 140 μl of plasma was obtained for viral RNA extraction. Red blood cells were