key: cord-0693082-pqhzr648
authors: Hoffmann, Markus; Arora, Prerna; Groß, Rüdiger; Seidel, Alina; Hörnich, Bojan; Hahn, Alexander; Krüger, Nadine; Graichen, Luise; Hofmann-Winkler, Heike; Kempf, Amy; Winkler, Martin Sebastian; Schulz, Sebastian; Jäck, Hans-Martin; Jahrsdörfer, Bernd; Schrezenmeier, Hubert; Müller, Martin; Kleger, Alexander; Münch, Jan; Pöhlmann, Stefan
title: SARS-CoV-2 variants B.1.351 and B.1.1.248: Escape from therapeutic antibodies and antibodies induced by infection and vaccination
date: 2021-02-11
journal: bioRxiv
DOI: 10.1101/2021.02.11.430787
sha: ac6f9859ae0756c4accce4d50d83182fae962248
doc_id: 693082
cord_uid: pqhzr648

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of UK, South Africa and Brazil variant into human cells is susceptible to blockade by entry inhibitors. In contrast, entry of the South Africa and Brazil variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment and was less efficiently inhibited by serum/plasma from convalescent or BNT162b2 vaccinated individuals. These results suggest that SARS-CoV-2 may escape antibody responses, which has important implications for efforts to contain the pandemic.

The pandemic spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the 53 causative agent of coronavirus disease 2019 , is ravaging economies and health 54 system worldwide and has caused more than 2.3 million deaths ((WHO), 2020). The 55 identification of antivirals by drug repurposing was so far largely unsuccessful. Remdesivir, an 56 inhibitor of the viral polymerase, is the only antiviral with proven efficacy (Beigel et al., 2020) . 57 However, the clinical benefit reported for Remdesivir treatment is moderate and has been called 58 into question (Consortium et al., 2020; Wang et al., 2020) . Recombinant antibodies, which target 59 the viral spike protein (S) and neutralize infection in cell culture and animal models (Baum et al., 60 2020a; Chen et al., 2020) , have been granted emergency use authorization (EUA) and may 61 provide a valuable treatment option in the absence of other antivirals. In contrast to the moderate 62 success in the area of antivirals, protective mRNA-and vector-based vaccines encoding the 63 SARS-CoV-2 S protein have been approved for human use and are considered key to the 64 containment of COVID-19 (Baden et al., 2021; Polack et al., 2020) . 65 SARS-CoV-2, an enveloped, positive-strand RNA virus that uses its envelope protein 66 spike (S) to enter target cells. Entry depends on S protein binding to the cellular receptor ACE2 67 and S protein priming by the cellular serine protease TMPRSS2 (Hoffmann et al., 2020b; Zhou et 68 al., 2020) and these processes can be disrupted by soluble ACE2 and serine protease inhibitors 69 (Hoffmann et al., 2020b; Monteil et al., 2020; Zhou et al., 2020) . Further, the S protein of SARS- 70 CoV-2 and other coronaviruses is a major determinant of viral cell and species tropism and the 71 main target for the neutralizing antibody response. The genetic information of SARS-CoV-2 has 72 remained relatively stable after the detection of first cases in Wuhan, China, in the winter season 73 of 2019. The only exception was a D614G change in the viral S protein that became dominant 74 early in the pandemic and that has been associated with increased transmissibility (Korber et al., 75 4 2020; Plante et al., 2020; Volz et al., 2021) . In contrast, D614G has only a moderate impact on 76 SARS-CoV-2 neutralization by sera from COVID-19 patients and by sera from vaccinated 77 individuals (Korber et al., 2020; Weissman et al., 2021) . 78 In recent weeks several SARS-CoV-2 variants emerged that seem to exhibit increased 79 transmissibility and that harbor mutations in the S protein. The SARS-CoV-2 variant B.1.1.7 (UK 80 variant), also termed variant of concern (VOC) 202012/01 or 20I/501Y.V1, emerged in the 81 United Kingdom and was associated with a surge of COVID-19 cases (Leung et al., 2021) . 82 Subsequently, spread of the UK variant in other countries was reported (Claro et al., 2021;  83 Galloway et al., 2021) . It harbors nine mutations in the S protein, six of which are located in the 84 surface unit, S1, and three are found in the transmembrane unit, S2 (Fig. 1 ). Exchange N501Y is 85 located in the receptor binding domain (RBD), a domain within S1 that interacts with ACE2, and 86 its presence was linked to increased human-human transmissibility (Leung et al., 2021; (Muik et al., 2021) 

The spike proteins of the SARS-CoV-2 variants mediate robust entry into human cell lines 125 We first investigated whether the S proteins of SARS-CoV-2 WT (Wuhan-1 isolate with D614G 

All S proteins studied were robustly expressed and mediated formation of syncytia in 139 transfected cells ( Fig. 2A) . Entry into all cell lines was readily detectable but the relative entry 140 efficiency varied. Particles bearing the S proteins of the SARS-CoV-2 variants entered 293T 141 (Brazil variant) and 293T-ACE2 (South Africa and Brazil variants) cells with slightly reduced 142 efficiency as compared to particles bearing WT S protein, while the reverse observation was 143 made for Calu-3 cells (UK variant) . For the remaining cell lines, no significant differences in 144 entry efficiency were observed between SARS-CoV S WT and S proteins from SARS-CoV-2 145 7 variants (Fig. 2B) . Collectively, these results indicate that the mutations present in the S proteins 146 of UK, South Africa and Brazil variant are compatible with robust entry into human cells.

The spike proteins of the SARS-CoV-2 variants mediate fusion of human cells 149 The S protein of SARS-CoV-2 drives cell-cell fusion resulting in the formation of syncytia and 150 this process might contribute to viral pathogenesis (Buchrieser et al., 2021) . We employed a cell- (Hoffmann et al., 2020a) . In contrast, the SARS-CoV-2 161 S protein mediated efficient membrane fusion in the absence of TMPRSS2 expression in target 162 cells (Fig. 3A ,B) and this property is known to depend on the multibasic S1/S2 site of this S 163 protein which is absent in SARS-CoV S (Hoffmann et al., 2020a) . Finally, the S proteins of all 164 SARS-CoV-2 variants tested facilitated cell-cell fusion with similar (UK) or slightly reduced 165 (South Africa, Brazil) efficiency as compared to WT S protein (Fig. 3A,B) . We next investigated whether the S proteins of the SARS-CoV-2 variants showed altered 169 stability, which may contribute to the alleged increased transmissibility of the viral variants. For 170 this, we incubated S protein-bearing particles for different time intervals at 33°C, a temperature 171 that is present in the nasal cavity, and subsequently assessed their capacity to enter target cells.

The efficiency of cell entry markedly decreased upon incubation of particles at 33°C for more 173 than 8 h, but no appreciable differences were observed between particles bearing S proteins from 174 SARS-CoV-2 WT or variants (Fig. 4A ).

Although the S proteins of the SARS-CoV-2 variants under study did not differ markedly (Monteil et al., 2020) . Similarly, the clinically proven protease inhibitors 189 Camostat and Nafamostat block TMPRSS2-dependent SARS-CoV-2 cell entry and their potential 190 for COVID-19 treatment is currently being assessed (Hoffmann et al., 2020b; Hoffmann et al., 191 2020c). Finally, the membrane fusion inhibitor EK1 and its optimized lipid-conjugated derivative 192 9 EK1C4 block SARS-CoV-2 entry by preventing conformational rearrangements in S protein 193 required for membrane fusion (Xia et al., 2020) . We asked whether entry driven by the S proteins 194 of UK, South Africa and Brazil variant can be blocked by these inhibitors. All inhibitors were 195 found to be active although entry mediated by the S proteins of the SARS-CoV-2 variants was 196 slightly more sensitive to blockade by sACE2 as compared to WT S protein, at least for certain 197 sACE2 concentrations (Fig. 5) . Conversely, entry driven by the S protein of the Brazil variant 198 was slightly more sensitive to blockade by EK1 and EK1C4 as compared to the other S proteins 

The vaccine BNT162b2 is based on an mRNA that encodes for the viral S protein and is highly 237 protective against COVID-19 (Polack et al., 2020) . While the S protein harbor T-cell epitopes 238 (Grifoni et al., 2020; Peng et al., 2020) , efficient protection is believed to require the induction of 239 neutralizing antibodies. We determined neutralizing activity of sera from 15 donors immunized 240 11 twice with BNT162b2 (Table S1 ). All sera efficiently inhibited entry driven by the WT S protein 241 and inhibition of entry driven by the S protein of the UK variant was only slightly reduced ( Fig.   242 7B,C). In contrast, 12 out of 15 sera showed a markedly reduced inhibition of entry driven by the 243 S proteins of the South Africa and Brazil variant (Fig. 7B,C) , although it should be stated that all 244 sera completely inhibited entry at the lowest dilution tested. In sum, these results suggest that 245 BNT162b2 may offer less robust protection against infection by these variants as compared to Thus, the S proteins of these viruses mediated entry into various cell lines with roughly 285 comparable efficiency and no evidence for increased S protein stability or differences in entry 286 kinetics were obtained. Similarly, the S proteins of all variants were able to mediate fusion of 287 human cells. Moreover, entry driven by all S proteins studied was blocked by sACE2, protease 288 13 inhibitors targeting TMPRSS2 and a membrane fusion inhibitor. However, it should be noted that 289 the S proteins of all variants were slightly more susceptible to blockade by sACE2, suggesting 290 differences in ACE2 engagement between WT and variant S proteins.

Although host-cell interactions underlying viral entry might not differ markedly between 292 SARS-CoV-2 S protein WT and the variants studied here, major differences in susceptibility to Fígure S1. Location of SARS-2-S RBD mutations K417N/T, E484K and N501Y with respect to 530 the binding interface of the REGN-COV2 antibody cocktail (related to Figure 6 ).

The protein models of the SARS-2-S receptor-binding domain (RBD, blue) in complex with 532 antibodies Casirivimab (REGN10933, orange) and Imdevimab (REGN10987, green) were 533 constructed based on the 6XDG template (Hansen et al., 2020) . Residues highlighted in red light blue = S1 subunit with RBD in dark blue, grey = S2 subunit, orange = S1/S2 and S2' 736 cleavage sites, red = mutated amino acid residues. 

The glycoprotein of vesicular stomatitis virus promotes release of virus-like 601 particles from tetherin-positive cells

Syncytia formation by SARS-CoV-2-604 infected cells

Distinct conformational states of SARS-CoV-2 spike protein

Novavax offers first evidence that COVID vaccines 609 protect people against variants

Emerging SARS-CoV-2 Variants

SARS-CoV-2 Neutralizing Antibody LY-CoV555 in 613 Outpatients with Covid-19

Local Transmission of 616 SARS-CoV-2 Lineage B.1.1.7, Brazil

Repurposed Antiviral Drugs for Covid-19 -Interim WHO Solidarity Trial Results

Emergence of SARS-CoV

B.1.1.7 Lineage -United States

Effect of Bamlanivimab as Monotherapy or in 627

Combination With Etesevimab on Viral Load in Patients With Mild to Moderate COVID-19: A 628 Randomized Clinical Trial

Targets of T Cell Responses to

SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Studies in humanized mice and convalescent humans yield a 635 SARS-CoV-2 antibody cocktail

TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 638 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein

A Multibasic Cleavage Site in the 641

Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Differential sensitivity of bat cells 649 to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and 650 influenza viruses

Nafamostat Mesylate Blocks Activation of SARS-CoV-2: New Treatment Option for 653 COVID-19

Functional analysis of 655 potential cleavage sites in the MERS-coronavirus spike protein

Mutations in the Spike Protein of Middle East Respiratory 658

Tracking Changes in SARS-CoV-2 Spike: 662 Evidence that D614G Increases Infectivity of the COVID-19 Virus

Early transmissibility 664 assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2

Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus 672 by BNT162b2 vaccine-elicited human sera

Broad and strong memory CD4(+) and CD8(+) T cells induced 675 by SARS-CoV-2 in UK convalescent individuals following COVID-19

Spike mutation D614G alters SARS-CoV-2 679 fitness

Safety and Efficacy of the BNT162b2 mRNA 682

Covid-19 Vaccine

Specific Immune Memory Persists after Mild COVID-19

Characterization of the sialic acid binding activity of 688 influenza A viruses using soluble variants of the H7 and H9 hemagglutinins

Evaluating the Effects of SARS-CoV-2 Spike 692

Robust neutralizing antibodies to SARS-695

CoV-2 infection persist for months

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-698 controlled, multicentre trial

D614G Spike Mutation Increases SARS 701

CoV-2 Susceptibility to Neutralization

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent

coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate 705 membrane fusion

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and 708 N501Y variants by BNT162b2 vaccine-elicited sera

Quantifying the transmission advantage associated with 711 N501Y substitution of SARS-CoV-2 in the United Kingdom: An early data-driven analysis

A pneumonia outbreak associated with a new coronavirus of probable 715 bat origin

Fígure S1. Location of SARS-2-S RBD mutations K417N/T, E484K and N501Y with respect to 846 the binding interface of the REGN-COV2 antibody cocktail (related to Figure 6 ).

The protein models of the SARS-2-S receptor-binding domain (RBD, blue) in complex with 848 antibodies Casirivimab (REGN10933, orange) and Imdevimab (REGN10987, green) were 849 constructed based on the 6XDG template (Hansen et al., 2020) . Residues highlighted in red