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

Abstract

Plasmablast responses and derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients. An average of 13.7% and 13.0% of plasmablast-derived IgG MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. Of thirty-two antibodies specific for the spike glycoprotein, ten recognised the receptor-binding domain (RBD), thirteen were specific for non-RBD epitopes on the S1 subunit, and nine recognised the S2 subunit. A subset of anti-spike antibodies (10 of 32) cross-reacted with other betacoronaviruses tested, five targeted the non-RBD S1, and five targeted the S2 subunit. Of the plasmablast-derived MAbs reacting with nucleocapsid, over half of them (19 of 35) cross-reacted with other betacoronaviruses tested. The cross-reactive plasmablast-derived antibodies harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. We identified 14 of 32 anti-spike MAbs that neutralised SARS-CoV-2 in independent assays at ≤ 133 nM (20 μg/ml) (five of 10 anti-RBD, three of 13 anti-non-RBD S1 subunit, six of nine anti-S2 subunit). Six of 10 anti-RBD MAbs showed evidence of blockade of ACE2 binding to RBD, and five of six of these were neutralising. Non-competing pairs of neutralising antibodies were identified, which offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2

“…Plate out µL of 1:20 serum/plasma in alternate columns 1, 3,5,7,9,11 (add 2.5 µL sample to 47.5 µL PBS). (Huo, Zhao, et al, 2020;ter Meulen et al, 2006) EY-6A 16 (Zhou et al, 2020) VHH-72-Fc Negative (Wrapp et al, 2020) FI-4A 16 (Huang et al, 2020) ACE2-Fc 4 (Huo, Le Bas, et al, 2020) FI-3A 8 (Huang et al, 2020) FI-1C 4 (Huang et al, 2020) H11-H4-Fc 16 (Huo, Le Bas, et al, 2020) FD-5D Negative (Huang et al, 2020) FD-11A 31 (Huang et al, 2020) FN-12A 31 (Huang et al, 2020) FJ-10B 62 (Huang et al, 2020) FM-7B 62 (Huang et al, 2020) EZ-7A Negative (Huang et al, 2020) S309 125 (Pinto et al, 2020) EW-8B non-RBD anti Spike Neg Control (Huang et al, 2020) EW-9C non-RBD anti Spike Neg Control (Huang et al, 2020) FJ-1C anti NTD Neg Control (Huang et al, 2020) Supplementary Figure 2. A) The 232 samples from figure in the main paper were titrated a second time 34 days after the first measurement, as described for figure 4 (Methods). The values for the end point doubling dilution were compared and a correlation coefficient, and slope calculated (Prism v8).…”

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2

“…The addition of the IH4-RBD reagent at between 12.5-800 ng/well (in µL PBS) induced a concentration dependent agglutination of the red cells, detected by the formation of a visible mat or plug of agglutinated cells, and the loss of teardrop formation on tilting the plate (Figure 2A (Huo, Zhao, et al, 2020;Lan et al, 2020;Yan et al, 2020;Zhou et al, 2020). EW-9B and EW-9C are monoclonal antibodies against non-RBD epitopes on the spike protein (Huang et al, 2020). ACE2-Fc has been described (Huang et al, 2020).…”

“…Having established a standard addition of 100 ng/well of the IH4-RBD reagent, we screened a set of twelve human monoclonal antibodies, two divalent nanobodies, and divalent ACE2-Fc, that are known to bind to the RBD (Huang et al, 2020;Huo, Le Bas, et al, 2020;Pinto et al, 2020;Wrapp et al, 2020;Zhou et al, 2020). These reagents bind to at least three independent sites on the RBD, and some are strongly neutralising and capable of profound ACE2 blockade (Huang et al, 2020;Huo, Le Bas, et al, 2020). Twelve of the 15 divalent molecules agglutinated red cells and titrated in the HAT to an end point between 2-125 ng/well, after addition of 100 ng IH4-RBD ( Figure 2B and Supplementary Table 1).…”

“…The RBD of betacoronaviruses folds independently of the rest of the spike protein (Lan et al, 2020;Li, Li, Farzan, & Harrison, 2005;Yan et al, 2020). This useful property provides an Achilles' heel for the virus and allows many potential applications in vaccine design (Dai et al, 2020;Mulligan et al, 2020;Tan et al, 2020;Walls et al, 2020;Yang et al, 2020;Zha et al, 2020), and serology (Amanat et al, 2020;Piccoli et al, 2020; The National SARS-CoV-2 Serology Assay Evaluation Group, 2020), see also www.gov.uk/government/publications/COVID-19-laboratoryevaluations-of-serological-assays). The majority of neutralising antibodies bind to the RBD (Barnes et al, 2020;Piccoli et al, 2020), and the level of antibody to the RBD detected in ELISA correlates with that of neutralising antibodies (Amanat et al, 2020;Piccoli et al, 2020;Robbiani et al, 2020).…”

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2

“…• 100 µL, 20 µL pipettes, Multichannel pipettes (Huo, Zhao, et al, 2020;ter Meulen et al, 2006) EY-6A 16 (Zhou et al, 2020) VHH-72-Fc Negative (Wrapp et al, 2020) FI-4A 16 (Huang et al, 2020) ACE2-Fc 4 (Huo, Le Bas, et al, 2020) FI-3A 8 (Huang et al, 2020) FI-1C 4 (Huang et al, 2020) H11-H4-Fc 16 (Huo, Le Bas, et al, 2020) FD-5D Negative (Huang et al, 2020) FD-11A 31 (Huang et al, 2020) FN-12A 31 (Huang et al, 2020) FJ-10B 62 (Huang et al, 2020) FM-7B 62 (Huang et al, 2020) EZ-7A Negative (Huang et al, 2020) S309 125 (Pinto et al, 2020) EW-8B non-RBD anti Spike Neg Control (Huang et al, 2020) EW-9C non-RBD anti Spike Neg Control (Huang et al, 2020) FJ-1C anti NTD Neg Control (Huang et al, 2020) Supplementary Figure 1 A, B) Three MAbs or Nanobody-Fc reagents of 15 tested specific for RBD failed to cross-link red cells but could be developed with an anti IgG reagent. Antibodies were titrated from 500 ng/well to 1 ng/well in 50 µL, 1:40 red cells were added in 50 µL, followed by 100 ng/well IH4-RBD in 50 µL, and incubated for 1 hour.…”