key: cord-0905938-r66vfirw authors: Rees-Spear, C; Muir, L; Griffith, SA; Heaney, J; Aldon, Y; Snitselaar, JL; Thomas, P; Graham, C; Seow, J; Lee, N; Rosa, A; Roustan, C; Houlihan, CF; Sanders, RW; Gupta, R; Cherepanov, P; Stauss, H; Nastouli, E; Doores, KJ; van Gils, MJ; McCoy, LE title: The impact of Spike mutations on SARS-CoV-2 neutralization date: 2021-01-19 journal: bioRxiv DOI: 10.1101/2021.01.15.426849 sha: 03dc4d39b32ae45b7575c4025b8dff4d9f4fb499 doc_id: 905938 cord_uid: r66vfirw Multiple SARS-CoV-2 vaccines have shown protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, Spike. Antibodies from SARS-CoV-2 infection neutralize the virus by focused targeting of Spike and there is limited serum cross-neutralization of the closely-related SARS-CoV. As new SARS-CoV-2 variants are rapidly emerging, exemplified by the B.1.1.7, 501Y.V2 and P.1 lineages, it is critical to understand if antibody responses induced by infection with the original SARS-CoV-2 virus or the current vaccines will remain effective against virus variants. In this study we evaluate neutralization of a series of mutated Spike pseudotypes including a B.1.1.7 Spike pseudotype. The analyses of a panel of Spike-specific monoclonal antibodies revealed that the neutralizing activity of some antibodies was dramatically reduced by Spike mutations. In contrast, polyclonal antibodies in the serum of patients infected in early 2020 remained active against most mutated Spike pseudotypes. The majority of serum samples were equally able to neutralize the B.1.1.7 Spike pseudotype, however potency was reduced in a small number of samples (3 of 36) by 5–10-fold. This work highlights that changes in the SARS-CoV-2 Spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their impact on vaccine efficacy. Serum neutralization activity is a common correlate of protection against viral infection 37 following vaccination or natural infection (Plotkin, 2008) . However, effective protection from 38 viral infection can also require sufficient breadth of serum neutralization rather than potency 39 alone. This is because of the high-levels of variation observed in major antigens across some domain (RBD -found within the S1 subunit of Spike). There is considerable amino acid 65 variation between the two RBDs, despite their conserved binding to ACE2, which explains why 66 the majority of SARS-CoV-induced neutralizing monoclonal antibodies (mAbs) were found not 67 to neutralize SARS-CoV-2, although some cross-binding activity has been observed (Wu et al., 68 2020) and targeted mutations can enable neutralization . Similarly, the 3 majority of COVID-19 sera have either weaker or no neutralizing activity against SARS-CoV, but 70 cross-neutralizing mAbs have been isolated (Brouwer et al., 2020 for a particular mAb but not for other mAbs from within the same binding cluster. This 300 highlights that different antibodies use distinct molecular contacts within shared epitopes, such 301 that a single mutation may not be detrimental to all antibodies within the same binding cluster. Thus, because polyclonal sera contain multiple antibodies that target the major neutralizing 303 sites in subtly different ways, it is less sensitive to Spike mutations. The Spike mutations studied here were designed to identify potential escape variants by 305 mimicking in part the natural variation observed between SARS-CoV and SARS-CoV-2, and are 306 focused mainly on the RBD as the major site of neutralizing antibody activity. Therefore, it was 307 unsurprising that many of the RBD-specific mAbs evaluated here lost neutralization activity 308 against one or more of these mutations. 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Elife, 9 D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization Possible host-adaptation 591 of SARS-CoV-2 due to improved ACE2 receptor binding in mink. Virus evolution A natural 594 mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-595 reactive antibody Neutralization of N501Y mutant SARS-CoV-2 by BNT162b2 vaccine-elicited sera Conformational 600 dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 601 revealed by cryo-EM Key residues of the receptor 603 binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and 604 neutralizing antibodies Immunodominance and Antigenic 607 Variation of Influenza Virus Hemagglutinin: Implications for Design of Universal 608 Indicated mAbs were serially diluted in duplicate and incubated with each 614 mutant SARS-CoV-2 luciferase-encoding pseudotyped virus (as noted in the legend) prior to the 615 addition of HeLa cells expressing ACE-2. After two days, neutralization was measured as the 616 relative reduction in relative light units (RLU). Data are representative of three independent 617 repeats inhibitory concentration (IC50) values were calculated for each mAb against the mutant SARS-619 CoV-2 pseudotyped viruses indicated in the left hand column. IC50 values are color-coded as 620 follows: pale grey >1μg/ml, light grey 0.1-1 μg/ml, medium grey 0.1-0.01 μg/ml and dark grey 621 The previously established binding cluster for each mAb is indicated above each 622 column, and whether or not the mAb binds RBD is also indicated Magnified image shows mutated amino acid side 626 chains at residues of interest. (B) IC50 values for each mAb against SARS-CoV-2 wildtype 627 pseudotyped virus were divided by the IC50 for each mutant pseudotyped virus against the 628 corresponding mAb to generate the fold decrease in neutralization on the Y-axis. The dotted 629 horizontal line indicates a 5-fold drop in neutralization potency. The competitive binding 630 clusters of each mAb Thirty six serum samples were serially titrated and incubated with the mutant SARS-CoV-2 632 luciferase-encoding pseudotyped viruses indicated in the legend prior to the addition of HeLa 633 cells expressing ACE-2. After two days, neutralization was measured as the relative reduction in 634 relative light units (RLU) and 50% inhibitory dilution factors calculated using Graphpad Prism ID50 values for each sera against SARS-CoV-2 wildtype pseudotyped virus were divided by the 636 ID50 for each mutant pseudotyped virus against the corresponding sera to generate the fold 637 decrease in neutralization on the Y-axis. The dotted horizontal line indicates a 5-fold drop in 638 neutralization potency. The 18 serum samples from hospitalized patients are shown in the 639 upper graph labeled "severe illness" and the 18 serum samples from healthcare workers who 640 experience mild/asymptomatic COVID-19 are shown in the lower graph labeled Serum responses following severe COVID-19 have greater polyclonality but less 643 efficient neutralization. (A) Spike S1 subunit semi-quantitative titers measured by ELISA (see 644 Methods) are shown on the Y-axis for 94 serum samples from hospitalized COVID-19 patients 645 and 105 serum samples from healthcare workers who experienced mild COVID-19 disease hospitalized COVID-19 patients and 99 serum samples 648 from healthcare workers who experienced mild COVID-19 disease. Note, some serum samples 649 from the original cohort that had binding titers gave no neutralization titer (6 from healthcare 650 workers, 1 from a hospitalized patient). (C) ID50 values measured by pseudotyped 651 neutralization assay for serum samples from hospitalized COVID-19 patients plotted on the Y-652 axis against the corresponding S1 IgG binding titer for each sample Serum sample groups are color-coded according to the 659 legend. (E) Concentrations of S1-specific serum IgG (pg) at ID50 dilutions were calculated using 660 the IgG titers quantified via the semi-quantitative ELISA and the known ID50 value. Only sera 661 that gave a measurable titer in both semi-quantitative ELISA and pseudotype neutralization 662 assay were included Figure 4: Variant B.1.1.7 SARS-CoV-2 Spike effect on mAb and serum neutralization Data are representative of three independent repeats. The horizontal 670 dotted line on each graph indicates 50% neutralization. (B) IC50 values for each mAb or ID50 671 values for each serum sample against SARS-CoV-2 D614G pseudotyped virus were divided by the 672 IC50 for each mutant pseudotyped virus against the corresponding mAb to generate the fold 673 decrease in neutralization on the Y-axis, as color-coded in the key. The dotted horizontal line 674 indicates a 5-fold drop in neutralization potency. Whether fold decrease in neutralization 675 potency refers to mAbs, 18 serum samples from hospitalized patients or 18 serum samples 676 from healthcare workers who experience mild/asymptomatic COVID-19 is The authors would like to thank James E Voss for the gift of HelaACE2 expressing cells, George 406 Kassiotis, Dan Frampton, Ann-Kathrin Reuschl and Joe Grove for helpful discussion and critical