Breadth and function of antibody response to acute SARS-CoV-2 infection in humans

Abstract

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

The RNA sensor MDA5 detects SARS-CoV-2 infection

“…Cells were stained with Live/Dead Fixable Aqua Cell Stain (1:200 in PBS, Life Technologies, L34957) combined with FcR block (1:200 in PBS, eBioscience), fixed in 4% formaldehyde (10 min at room temperature), and permeabilised in PBS containing 0.1% Triton-X (20 min at room temperature). Cells were incubated with antibodies against human MxA (clone M143, kind gift from G Kochs) and SARS-CoV-2 N protein (clone EY-2A, kind gift from Alain Townsend 59 ; 1:200, 30 min, 4 °C), and goat anti-mouse AlexaFluor 488 (Life Technologies, A11029) and anti-human AlexaFluor 647 (1:500, 30 min, 4 °C; Life Technologies, A21445), and resuspended in CellFix (1:10 in water; BD, 340181). Cells were analysed by flow cytometry on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and data were analysed using FlowJo software (BD).…”

The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells

“…This control was included in all pp experiments and the luciferase values subtracted from values acquired with the SARS-CoV-2pp. To confirm spike-dependent infection, SARS-CoV-2pp were incubated with the anti-S-mAb FI-3A (1µg/mL) (Hauang et al, 2020) for 30 min prior to infection for all experiments. SARS-CoV-2 VSV pp were generated as previously reported (Hoffmann et al, 2020b) using reagents provided by Stefan Pöhlmann (Infection biology unit, German Primate Center, Göttingen, Germany).…”

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

“…After permeabilisation, cells were blocked in blocking solution (50% Li-Cor Odyssey blocking solution, pretreated with RNASecure for 30 min and supplemented with 2 mM ribonucleoside vanadyl complex and 0.1% Tween-20) for 30 min at room temperature. Then, cells were incubated with J2 primary antibody (Scicons 10010200) at 0.5 µg/ml or human anti-N primary antibody (Ey2B clone 1:2000) (Huang et al, 2020) for 2 h at room temperature. Cells were washed three times in PBS/ 0.1% Tween-20 (PBSTw) for 10 min each at room temperature and incubated with fluorescent secondary antibodies (1:500) diluted in blocking solution for 1 h at room temperature.…”