Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 hijacks neutralizing dimeric IgA for nasal infection and injury in Syrian hamsters 1

doi: 10.1080/22221751.2023.2245921

Figure Lengend Snippet: B8-dIgA1 and B8-dIgA2 enhance SARS-CoV-2 infection via CD209. (A) The effects of B8-dIgA2 on SARS-CoV-2 infection in the MucilAir TM model, consisting of primary human nasal epithelial cells but no DCs. B8-mIgA2 or B8-dIgA2 were pre-incubated at doses of 10, 100, and 1000 ng/ml, respectively, in the apical compartment with or without mucus for 1 hour, before adding 10 4 PFU of SARS-CoV-2 (BetaCoV/France/IDF00372/2020) for 4 hours. The viral RNA loads were measured by RT-PCR in both the apical and basal compartments and are shown in log-transformed units; (B) Representative confocal images (400×) of olfactory epithelium in NT showed the expression of CD209 (DC-SIGN) in green and ACE2 in magenta by immunohistochemical staining of experimental hamsters treated with B8-dIgA2 without (left) or with (middle and right) SARS-CoV-2 infection. Color-coding indicates specific antibodies used for double staining. Infected CD209 + cells are visualized in yellow as indicated by arrows (right); (C) The CD209 or CD299 overexpressed-HEK 293 T cells were pre-treated for 6 hours with 10 ng/ml of B8-dIgA1 or B8-dIgA2 or control dIgA1 or control dIgA2 or PBS, respectively, prior to SARS-CoV-2 infection (MOI: 0.05). Each treatment was performed in triplicate. Two days after infection, SARS-CoV-2 NP expression (green) was quantified by the mean fluorescence intensity (MFI) after anti-NP IF staining. Statistics were generated using student- t tests. * p < 0.05; ** p < 0.01; *** p < 0.001L; (D) The effects of B8 antibodies on cell-cell fusion. 293 T cells co-transfected with SARS-CoV-2 spike and GFP were pre-treated with 100× the IC 90 dose of B8-IgG1, B8-mIgA1, B8-mIgA2, B8-dIgA1, B8-dIgA2 or and IgG isotypic control for 1 hour, respectively. Vero-E6 cells transfected with TMPRSS2 were then added to the treated 293T-spike-GFP cells and co-cultured for 48 hours. Cell-cell fusion was imaged under a fluorescence confocal microscope at the 50× magnification.

Article Snippet: For identification of DC-SIGN expression, we stained the NT slices with rabbit anti-DC-SIGN primary antibody (Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (Life Technologies) according to the manufacturer’s instructions.

Techniques: Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Expressing, Immunohistochemical staining, Staining, Double Staining, Control, Fluorescence, Generated, Transfection, Cell Culture, Microscopy