key: cord-0900699-9f7k0q1h
authors: McCallum, Matthew; Bassi, Jessica; Marco, Anna De; Chen, Alex; Walls, Alexandra C.; Iulio, Julia Di; Tortorici, M. Alejandra; Navarro, Mary-Jane; Silacci-Fregni, Chiara; Saliba, Christian; Agostini, Maria; Pinto, Dora; Culap, Katja; Bianchi, Siro; Jaconi, Stefano; Cameroni, Elisabetta; Bowen, John E.; Tilles, Sasha W; Pizzuto, Matteo Samuele; Guastalla, Sonja Bernasconi; Bona, Giovanni; Pellanda, Alessandra Franzetti; Garzoni, Christian; Van Voorhis, Wesley C.; Rosen, Laura E.; Snell, Gyorgy; Telenti, Amalio; Virgin, Herbert W.; Piccoli, Luca; Corti, Davide; Veesler, David
title: SARS-CoV-2 immune evasion by variant B.1.427/B.1.429
date: 2021-04-01
journal: bioRxiv
DOI: 10.1101/2021.03.31.437925
sha: f3e656bfe984363f7761519206c6e7d92a84a052
doc_id: 900699
cord_uid: 9f7k0q1h

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.

S 2 X 1 2 8 S 2 X 1 9 2 S 2 X 2 5 9 S 2 X 6 1 5 S 2 H 7 0 S 2 N 1 2 S 2 N 2 2 S 2 X 6 0 8 S 2 X 6 0 9 S 2 X 3 0 S 2 X 3 0 5 S 2 D 1 0 6 S 2 X 6 1 9 S 2 X 5 8 S 2 H 

We previously showed that disruption of the C15/C136 disulfide bond that connects 277 the N-terminus to the rest of the NTD, through mutation of either residue or alteration of the 278 signal peptide cleavage site, abrogates the neutralizing activity of mAbs targeting the NTD 279 antigenic supersite (site i)(12). As the S13I substitution resides in the signal peptide and is 

predicted to shift the signal peptide cleavage site from S13-Q14 to C15-V16, we hypothesized 281 that this substitution indirectly affects the integrity of NTD antigenic site i, which comprises the 282 N-terminus. Mass spectrometry analysis of the S13I and S13I/W152C NTD variants confirmed 283 that signal peptide cleavage occurs immediately after residue C15 ( Figure. 4B-D) . As a result,

C136, which would otherwise be disulfide linked to C15, is cysteinylated in the S13I NTD (Fig.

4C and Supplemental Fig. 5) . Likewise, the W152C mutation, which also introduces a free 286 cysteine, was also found to be cysteinylated in the W152C NTD (Fig. 4E) . Notably, dampening 287 of NTD-specific neutralizing mAb binding is even stronger for the S13I mutant than for the 288 S12P mutant (Fig. 4A) . Conversely, we did not observe any effect on mAb binding of the S12F 289 substitution, which has also been detected in clinical isolates, in agreement with the fact that 290 it did not affect the native signal peptide cleavage site (i.e. it occurs at the native S13-Q14 291 position), as observed by mass spectrometry (Fig. 4F) . While the S13I and W152C NTD 292 variants were respectively cysteinylated at positions C136 and W152C, due to the presence 293 of unpaired cysteines, the double mutant S13I/W152C was not cysteinylated, suggesting that 294 C136 and W152C had formed a disulfide bond with each other (Fig. 4C-E) . Tandem 

Collectively, these findings demonstrate that the S13I and W152C mutations found in with the E484K RBD mutation in the B.1.427 variant . Alternatively, the S13I mutation could 395 emerge in any of these variants. We note that the S13I mutation was also detected in the

We thank Hideki Tani (University of Toyama) for providing the reagents necessary for 404

preparing VSV pseudotyped viruses. This study was supported by the National 405

Institute 

HEK203-hACE2 cells were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep with 538 8% CO2 in a 37°C incubator (ThermoFisher). One day or more prior to infection, 40 μL of poly-539 lysine (Sigma) was placed into 96-well plates and incubated with rotation for 5 min. Poly-lysine 540 was removed, plates were dried for 5 min then washed 1 × with water prior to plating cells.

The following day, cells were checked to be at 80% confluence. In a half-area 96-well plate a 

Intact mass spectrometry analysis of purified NTD constructs

The purpose of intact MS was to verify the n-terminal sequence on the constructs. N-linked 

Non-reducing Peptide Mapping mass spectrometry analysis of purified NTD constructs

The purpose of peptide mapping was to verify the disulfide linkage between C136 and C152 621 on S13I/W152C variant. Combo protease with Glu-C and trypsin was used for protein 622 digestion without adding reducing reagent. 50 μg of deglycosyated protein was denatured (6M 623 guanidine hydrochloride), alkylated (Iodoacetamide), and buffer exchanged (Zeba spin 624 desalting column) before digestion. 10 µg of digested peptide was analyzed on the LC-MS 625 system (Agilent AdvanceBio peptide mapping column and Thermo Q Exactive Plus Orbitrap 

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N-terminal 684 domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

Ultrapotent human 693 antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

A neutralizing human antibody binds to the N-terminal domain of the 721 Spike protein of SARS-CoV-2

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UK) consortium, Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies

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REGN-COV2 862 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Multiple SARS-CoV-2 variants escape neutralization 872 by vaccine-induced humoral immunity

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SARS-CoV-2 variant

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The Impact of Mutations in

Prolonged infectious SARS-CoV-2 shedding from an 910 asymptomatic immunocompromised cancer patient

Persistence and Evolution of 917 SARS-CoV-2 in an Immunocompromised Host

Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive 921 antibody escape

Escape from neutralizing 929 antibodies by SARS-CoV-2 spike protein variants

A novel SARS-CoV-2 variant of concern, B.1.526

A 947 system for functional analysis of Ebola virus glycoprotein

A haploid genetic screen identifies heparan sulfate proteoglycans 951 supporting Rift Valley fever virus infection

Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2

Spike Protein for Neutralization Assays

Elicitation of broadly protective sarbecovirus immunity by 963 receptor-binding domain nanoparticle vaccines. bioRxivorg (2021)

Human immunodeficiency virus-1 viral load is elevated in individuals with reverse-967 transcriptase mutation M184V/I during virological failure of first-line antiretroviral therapy 968 and is associated with compensatory mutation L74I

Characterization of spike glycoprotein 972 of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV