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Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

['Marta Alenquer', 'Cell Biology Of Viral Infection Lab', 'Instituto Gulbenkian De Ciência', 'Oeiras', 'Filipe Ferreira', 'Diana Lousa', 'Itqb Nova', 'Instituto De Tecnologia Química E Biológica António Xavier', 'Universidade Nova De Lisboa', 'Mariana Valério']

Date: None

Abstract Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

Author summary For a SARS-CoV-2 virion to enter a cell, the spike protein displayed at its surface must be recognized by the host ACE2 receptor. Serum neutralizing antibodies, shown to develop upon natural SARS-CoV-2 infection or vaccination, bind spike protein preventing the recognition by ACE2 and, consequently, infection. However, SARS-CoV-2 virus is constantly evolving, and can acquire mutations in spike that render this protein resistant to neutralizing antibodies and make vaccines ineffective. In this paper, we tested how single and a combination of mutations naturally occurring in spike, including in variants of concern, would synergize to affect antibody neutralizing capacity. We then integrated these findings with in silico analyses of amino acids binding to ACE2 and antibodies, and distributed them in a grid as amino acids important for binding to ACE2 or antibodies, or both. We found that changes in amino such as E484 and S494, which frequently interact with antibodies but not with ACE2, promptly evolve immune escape mutants, elicited by infection or vaccination, if the mutation severely alters the binding specificity of the antibody. Our work also suggests that the combination of these mutations with others promoting ACE2 binding, such as N501Y, increases their ability to escape neutralizing-antibody responses.

Citation: Alenquer M, Ferreira F, Lousa D, Valério M, Medina-Lopes M, Bergman M-L, et al. (2021) Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies. PLoS Pathog 17(8): e1009772. https://doi.org/10.1371/journal.ppat.1009772 Editor: Michael S. Diamond, Washington University School of Medicine, UNITED STATES Received: May 20, 2021; Accepted: June 30, 2021; Published: August 5, 2021 Copyright: © 2021 Alenquer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This project is supported by the grants PTDC/CCI-BIO/28200/2017 awarded to DF; FCT RESEARCH4COVID 19 (Refs 580 and 644) awarded by the Fundação para a Ciência e a Tecnologia (FCT, Portugal, https://www.fct.pt/index.phtml.en) to MJA and also by COVID-19 emergency funds 2020 from Fundação Calouste Gulbenkian - Instituto Gulbenkian de Ciência (FCG-IGC, Portugal, https://gulbenkian.pt/ciencia/) to MJA. The work of JD benefited from COVID-19 emergency funds 2020 from Fundação Gulbenkian de Ciência and from Câmara Municipal de Oeiras (https://www.oeiras.pt). The work of HS was supported by ESCMID (https://www.escmid.org) and by Gilead Génese (PGG/009/2017, https://gileadgenese.pt) grants. MJA is funded by FCT 2020.02373.CEECIND; FF is supported by the FCT grant PTDC/BIA-CEL/32211/2017; JG and HS are supported by Fundação para a Ciência e Tecnologia (FCT) through PD/BD/128343/2017 and CEECIND/01049/2020. Competing interests: The authors have declared that no competing interests exist.

Introduction Severe acute respiratory syndrome coronavirus–2 (SARS-CoV-2) is the virus responsible for the pandemic of coronavirus disease 2019 (COVID-19) [1] that has caused more than 164 million infections and provoked the death of over 3 million people (as of April 18, 2021). A pertinent question is whether viral evolution will permit escaping immunity developed by natural infection or by vaccination. The answer to this multi-layered and complex question depends on the type, duration and heterogeneity of the selective pressures imposed by the host and by the environment to the virus, but also on the rate and phenotypic impact of the mutations the virus acquires. The central question is whether the virus can select mutations that escape host immunity while remaining efficient to replicate in the host [2–4]. Identifying immune evasion signatures in the virus is critical for preparedness of future interventions to control COVID-19. SARS-CoV-2 is an enveloped virus, characterized by displaying spike proteins at the surface [5]. Spike is critical for viral entry [6] and is the primary target of vaccines and therapeutic strategies, as this protein is the immunodominant target for antibodies [7–11]. Spike binds to ACE2 displayed at the host cell surface and, upon activation by proteolytic cleavage, mediates membrane fusion of viral and host membranes to deliver virion contents inside the cell. Proteolytic cleavage happens at two sites in spike (S1/S2 and S2’) in a process that starts during viral egress with cleavage of S1/S2 site (by furin) and terminates with cleavage of the S2’ site either at the cell surface by TMPRSS2 or inside lysosomes by, for example, cathepsin L [6]. Interestingly, the spike protein oscillates between distinct conformations to recognize and bind different host factors [12,13]. In the prefusion state, the receptor binding domain (RBD) alternates between open (‘up’) and closed (‘down’) conformations [5,14]. For binding to the ACE2 receptor, the RBD is transiently exposed in the ‘up’ conformation. However, most potent neutralizing antibodies elicited by natural infection [9,11,15] and by vaccination [7,10] bind spike in the closed (‘down’) conformation. Understanding how each amino acid of spike dynamically interacts with either antibodies or ACE2 receptor may reveal the residues that are more prone to suffer mutations driving immunological escape without affecting viral entry. Viral mutations may affect host-pathogen interactions in many ways: affect viral spread, impact virulence, escape natural or vaccine-induced immunity, evade therapies or detection by diagnostic tests, and change host species range [16,17]. Therefore, it is critical to survey circulating variants and assess their impact in the progression of SARS-CoV-2 dynamics in the population, in real time. Being a novel virus circulating in the human population, SARS-CoV-2 evolution displayed a mutational pattern of mostly neutral random genetic drift until December 2020. In fact, the D614G mutation in the spike protein was amongst the few epidemiologically significant variants, resulting in increased transmissibility without affecting the severity of the disease [18,19]. However, from the end of 2020, three divergent SARS-CoV-2 lineages evolved into fast-spreading variants that became known as variants of concern: B.1.1.7 [United Kingdom (UK)] [20], B.1.351 [South Africa (SA)] [21] and P.1 (Brazil) [22]. More recently, other lineages were added to this list: B.1.427, B.1.429 [California, United States of America (USA)], B.1.617 or B.1.617.1–3 (India) [20,23,24], as well as several lineages of interest that carry the mutation E484K, including P.2 [25] and B.1.1.7 containing E484K. These variants present alterations in the spike protein that change its properties: increase transmissibility and virulence [26,27] and/or escape immunity developed by natural infection [28,29], therapies [30–32], vaccination [33], detection [34]; and change the host species range [35]. Mutations in the RBD of spike may severely affect viral replication and host immune response since, as explained above, this region is responsible for binding to ACE2 and is immunodominant. Three mutations—D614G, N501Y and L452R - are associated with an increased ACE2 binding affinity in humans and increased viral transmission [18,19,26,36]. Y453F was associated with a mink-to-human adaptation in cluster 5 [37]. L452R and N439K are associated with a modest reduction in antibody-dependent neutralization by immune sera, whilst variants containing E484K display a reduction that is moderate to substantial [38]. Variants, however, contain several mutations combined and some will have synergetic effects. Interestingly, some mutations are convergent whilst others are unique in lineages [39]. For example, in B.1.351 and P.1 lineages, mutations in amino acid residues at positions 18 (L18F) and at position 417 (S417N, or S417T in some P.1 cases) were observed. A two amino acid deletion in positions 69 and 70 of spike is observed in cluster 5 and lineages B.1.1.7, B.1.525 and B.1.258 [40]. The appearance of shared mutations in distinct and rapidly spreading SARS-CoV-2 lineages suggests that these mutations, either alone or in combination, may provide fitness advantage, as recently observed with the acquisition of S417N by B.1.617.2 lineage. In principle, mutations in different domains of spike may affect its function and result in conformational rearrangements of the RBD. Therefore, it is important to understand how synergetic interactions in spike collectively change the RBD. Serum neutralizing antibodies were shown to develop upon natural SARS-CoV-2 infection [41] or vaccination [42,43], and last at least several months [44–46]. The alarm caused by the emergence of SARS-CoV-2 variants with the potential to escape host immunity acquired through infection or vaccination has been confirmed by a series of reports for lineages B.1.351 and P.1. [28,29,33,47,48]. These raise concerns on whether a reduction in vaccine efficacy could result in re-infections and delay the reduction in mortality caused by circulating SARS-CoV-2. There is, therefore, the urgent need to develop mutation-tolerant vaccines (and biopharmaceuticals) targeting the spike protein. In this sense, it is critical to determine what makes mutations well tolerated for the viral lifecycle whilst efficiently escaping immunity. In this work, we engineered spike-pseudotyped viruses and analyzed individual and combined mutations that convergently appeared in different lineages, over time and across several geographic locations, to determine their synergetic effects on neutralizing-antibody responses. We then used available structures of the complexes spike-antibodies and spike-ACE2 to determine the distance between each amino acid residue in the complex and the frequency of interactions of each amino acid residue of the RBD with either ACE2 or antibodies. We found a moderate reduction in neutralizing potency of sera against SARS-CoV-2 spike-pseudotyped lentivirus containing single mutations at positions 484 (E484K) and 494 (S494P). Interestingly, the reduction became substantial with the addition of synergetic mutations K417N/N501Y to E484K, or E484K/N501Y to S494P. In addition, we show that the amino acid residues at positions 484 and 494 frequently engage in binding to antibodies but not in binding to the receptor ACE2. Our work suggests that the amino acid residues at the RBD that are more dispensable for binding to ACE2 can more promptly evolve immune escape mutants if the amino acid residue substitution severely alters the binding specificity of the antibody. It also suggests that the combination of these mutations with others that promote ACE2 binding, such as N501Y, increases their ability to escape neutralizing-antibody responses.

Discussion It is critical to understand SARS-CoV-2 signatures that allow escaping neutralizing antibodies, which could pose challenges to vaccination and render therapies ineffective. We developed spike-pseudotyped lentiviral particles for high-throughput quantitation that express GFP upon entering cells, contributing to the toolkit of neutralizing assay methodologies [73–75]. Our method of reading fluorescent signals in 96 wells plates using the GloMax Explorer System is suitable to assess the neutralization activity of sera/plasma from individuals infected naturally or vaccinated, and to screen for antiviral drugs that block viral entry, such as therapeutic antibodies, in biosafety level 2 settings, which greatly facilitates the procedure and broadens its usage. It can be easily adapted to include single and multiple mutations, as observed in the present work for sera neutralizing activity (Fig 2). Our results with BNT162b2-elicited plasma (Fig 3) agree with previous publications showing resistance to neutralization by B.1.351 and P.1 lineages [28,29,33,47,76] and validate our neutralization assay. The fact that only mutations containing the E484K substitution promote immune escape, although this effect increases with synergic mutations that improve viral binding to ACE2 (K417N and N501Y), may be a consequence of lack in immunological selective pressure, a recognized driver of evolution [58,77–79], as a large proportion of the population remains susceptible to SARS-CoV-2 infection. However, the emergence of this escape mutant suggests that the continued circulation of the virus may, in the future, impose further immunological constraints and result in viral evolution, as seen for influenza A virus [58,80]. How changes in SARS-CoV-2 will impact the circulation of the virus is not known, and the future will also elucidate whether vaccination will shape evolution of SARS-CoV-2 and permit reinfections. At the moment, reinfections are considered rare events [81,82], but reports that SARS-CoV-2 escape mutants were shown to drive resurgence of cases upon natural infection [23,83] are indicative that viral dynamics in the population may change [26,84]. Therefore, the development of methods able to predict hotspots of immune evasion are in demand. The surveillance of circulating strains across time, the evaluation of the type and duration of host immune responses upon natural infection and vaccination, and the identification of antibodies that efficiently control SARS-CoV-2 are essential measures to understand and control SARS-CoV-2 infection and host response [85–87]. We argue that the structural information on complexes between antibodies, or ACE2, and spike variants may be integrated with mutational maps and their phenotypic characterization [2–4] to predict hotspots of immune evasion. In this work, we analyzed by neutralization assays how a full-length spike carrying single or multiple mutations dispersed throughout the protein affected the conformation of the RBD. With this assay, we probed how antibodies from infected patients or vaccinated individuals block viral entry. We used structural information on the protein complexes spike-antibodies and spike-ACE2 to define the non-overlapping frequency of interactions using a cut-off value of 4.5 Å distance to define a contact. Given that only a few amino acid residues are involved in protein-protein interfaces [88], including in antibody-antigen recognition, we aimed at identifying which amino acid residues may evolve antibody escape mutants without affecting viral entry. With this approach, we identified two amino acid residues at the RBD—positions 484 and 494 (Fig 4E)—that frequently engage in interactions with antibodies but not with ACE2. We observed that E484 is relevant for antibody binding (Fig 2), in agreement with our and previous experimental results, and with the occurrence of this mutation in two variants that are becoming highly prevalent and were shown drive resurgence of infection in sites with high levels of seroconversion [2,33,57,71,72,83]. Additionally, we found that mutation of residue 494 may be problematic, either alone or combined with synergetic mutations, as it reduces neutralization competency of convalescent sera, and thus facilitates antibody escape without substantially altering the affinity for the ACE2 binding (Figs 4E, 4G and S8). The mutation S494P has been found in nature with a prevalence of 0.81% of all sequenced genomes (March 2021, S1 Fig) and has already been observed in combination with mutation E484K (reported on the 22nd of October 2020). Of note, current prevalence of B.1.351 and of P.1 is 1.9% and 0.47%, respectively, being in the same range as S494P. We posit that the prevalence of mutations in amino acids 484, 494 and 501 will increase with viral circulation and/or spike seroconversion and should be surveilled worldwide. We observed a similar pattern relative to the substitution L452R/Q in India, in which the E484Q mutation was acquired posteriorly. At the moment, however, with the majority of the population still susceptible to viral infection, mutations that increase viral transmission, such as N501Y [84] have a selective advantage. In the future, mutations escaping host antibodies whilst increasing binding to ACE2 may become prevent. Our approach has some caveats. Regarding the neutralization assays, and despite spike being the immunodominant protein targeted by antibodies [7–11], other viral proteins may contribute, even if in a small proportion, to neutralization activity in vivo, which we are unable to detect using spike-pseudotyped particles. In addition, the sera/plasma we used were obtained at a fixed time interval. Future experiments should repeat the analysis using sera/plasma of people infected with known genotypes, or from different geographical areas and obtained at different times along the epidemic dynamics. Finally, we inspected a handful of mutations and not a comprehensive mutational map across spike [2,4]. However, we traced several mutations across spike in single and multiple combinations, and thus, we conclude that our analysis is relevant to inform on mutation-driven escape viruses to antibodies. In addition, we only investigated antibody-neutralization responses but other mutations in spike may contribute to overall antibody escape, and hence regulate the host response to infection, which we are unable to detect by our assay. The analysis of frequency of interactions between spike-antibodies or ACE2 has some issues associated as well. The pool of complexes spike protein/RBD and antibodies analyzed here is not necessarily representative of the universe of antibodies found in COVID-19 patients, since it is limited by the availability of structural information and is biased to monoclonal antibodies (S4 Table). All the antibodies analyzed are neutralizing antibodies, which is not the case in nature [54]. Additionally, many structural studies have focused on the interaction with the RBD, while other regions have not been so exhaustively analyzed, which creates a bias in terms of the number of RBD binding antibodies with known structure [50,65,66]. Limitations of the methods used, such as crystallographic contacts, can also influence the results. Nevertheless, its combination with data from the molecular simulation and from experiments helps to predict and explain the emergence of new mutations. In conclusion, our analysis of the spike protein-antibody contacts revealed that (within the available data set) the receptor binding motif, in the receptor binding domain, is an important region for antibody binding, which makes sense, since antibody binding to this region can prevent the interaction with ACE2 and ultimately impair viral infection. Our results are in very good agreement with experimental data on mutation-driven escape to polyclonal antibodies and with the emergence of natural variants, which were reported to re-circulate in previously exposed people [83]. Thus, this analysis represents a simple strategy to predict mutation hotspots that should be kept under surveillance. The predictions will become even better once the pool and diversity of available structures increases.

Materials and methods Ethics statement This study was approved by the Ethics committees of Centro Hospitalar Lisboa Ocidental and Centro Hospitalar Lisboa Central, in compliance with the Declaration of Helsinki, and follows international and national guidelines for health data protection. All participants provided informed written consent to take part in the study. Global variants frequencies A variant surveillance report was retrieved from GISAID on March 26th, 2021. A total of 864131 submissions were used to establish the world frequency of each variant using specific amino acid mutations (e.g., S_D614G, etc.) by using command line search text tools. Counts were then divided by week and year, using available metadata for collection date and just variants over indicated prevalence are reported and plotted per graph. Cells and plasmids Human hepatocellular carcinoma HuH-7 cells (a kind gift from Dr Colin Adrain, Instituto Gulbenkian de Ciência, Portugal), Human Embryonic Kidney 293T (provided by Prof Paul Digard, Roslin Institute, UK) and 293ET cells (from Dr Colin Adrain) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 21969035) supplemented with 10% fetal bovine serum (FBS, Gibco, 10500064), 1% penicillin/streptomycin solution (Biowest, L0022) and 2mM L-glutamine (ThermoFisher, 25030024), at 37°C and 5% CO 2 atmosphere. Plasmids used in this study were obtained as follows: pLEX.MSC was purchased from Thermo Scientific; psPAX2 and pVSV.G were a kind gift from Dr Luís Moita (Instituto Gulbenkian de Ciência, Portugal); pEGFP-N1 was provided by Dr Colin Crump (University of Cambridge, UK); and pCAGGS containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Gene (pCAGGS-SARS-CoV-2-S, NR-5231) was obtained through BEI Resources. Cloning and spike protein mutations To facilitate incorporation of spike protein into lentiviral pseudovirions, the C-terminal 19 amino acids encompassing the ER-retention domain of the cytoplasmic tail of spike were deleted. This construct, named SARS-CoV-2 S trunc , was generated by PCR amplification of the C-terminal region of S without amino acids 1255–1273 (a premature STOP codon was added in the reverse primer), using pCAGGS-SARS-CoV-2-S as template (primers in S7 Table). The amplified sequence was cloned back into pCAGGS-SARS-CoV-2-S, between AgeI and SacI restriction sites, generating the expression vector pCAGGS-SARS-CoV-2-S trunc . Mutations were introduced in the spike protein by site-directed mutagenesis, with the Quickchange Multi Site-Directed Mutagenesis kit (Agilent) following the manufacturer’s instructions (primers in S7 Table). Lentiviral reporter plasmid pLEX-GFP was produced by PCR amplifying GFP from pEGFP-N1 (primers in S7 Table) and cloning the insert into BamHI–XhoI restriction sites in the multi-cloning site of pLEX.MCS vector. For lentiviral plasmid pLEX-ACE2 production, human ACE2 coding sequence was amplified from Huh7 cDNA and cloned into pLEX.MCS, using XhoI and MluI restriction sites (primers in S7 Table). Production of 293T cells stably expressing human ACE2 receptor To produce VSV-G pseudotyped lentiviruses encoding the human ACE2, 293ET cells were transfected with pVSV-G, psPAX2 and pLEX-ACE2 using jetPRIME (Polyplus), according to manufacturer’s instructions. Lentiviral particles in the supernatant were collected after 3 days and were used to transduce 293T cells. Three days after transduction, puromycin (Merck, 540411) was added to the medium, to a final concentration of 2.5μg/ml, to select for infected cells. Puromycin selection was maintained until all cells in the control plate died and then reduced to half. The 293T-ACE2 cell line was passaged six times before use and kept in culture medium supplemented with 1.25 μg/ml puromycin. ACE2 expression was evaluated by flow cytometry (S2 Fig). Production and titration of spike pseudotyped lentiviral particles To generate spike pseudotyped lentiviral particles, 3x106 293ET cells were co-transfected with 8.89μg pLex-GFP reporter, 6.67μg psPAX2, and 4.44μg pCAGGS-SARS-CoV-2-S WT or mutants (or pVSV.G, as a control), using jetPRIME according to manufacturer’s instructions. The virus-containing supernatant was collected after 3 days, concentrated 10 to 20-fold using Lenti-XTM Concentrator (Takara, 631231), aliquoted and stored at -80°C. Pseudovirus stocks were titrated by serial dilution and transduction of 293T-ACE2 cells. At 24h post transduction, the percentage of GFP positive cells was determined by flow cytometry, and the number of transduction units per mL was calculated. Flow cytometry Flow cytometry was performed as in [89]. In brief, HEK 293T and 293T-ACE2 cells were prepared for flow cytometry analysis by detaching from the wells with trypsin, followed by fixation with 4% formaldehyde). For analysis of ACE2 expression, cells were stained with a primary antibody against ACE2 (4μg/ml, R&D Systems, catalog no. AF933) followed by a secondary antibody labelled with Alexa Fluor 568 (1:1000; Life Technologies). Analysis of cell populations was performed in a Becton Dickinson (BD) LSR Fortessa X-20 equipped with FACS Diva and FlowJo (BD, Franklin Lakes, NJ) software’s. Human convalescent sera and post-vaccination plasma/serum Venous blood was collected by standard phlebotomy from health care providers who contracted COVID-19 during spring/summer 2020, as tested by RT-PCR on nasopharyngeal swabs. Serum was prepared using standard methodology and stored at -20°C. During this period, the variant B.1.1, encoding the WT spike protein, was prevalent in Portugal. Peripheral blood from health care workers was collected by venipuncture into EDTA tubes at day 12 and day 33 post-immunization with Pfizer BTN162b2 mRNA vaccine and immediately processed. Alternatively, post-vaccination serum was collected at day 22 and day 51 post-immunization with Pfizer BTN162b2 mRNA vaccine. Peripheral blood was diluted in PBS 1x (VWR), layered on top of biocoll (Biowest) and centrifuged at 1200g for 30 min without break. Plasma was collected to cryotubes and stored at -80°C ultra-low freezer until subsequent analysis. ELISA assay Direct ELISA was used to quantify IgG anti-full-length Spike in convalescent sera. The antigen was produced as described in [90]. The assay was adapted from [91] and semi-automized to measure IgG in a 384-well format, according to a protocol to be detailed elsewhere. For titer estimation, sera were serially diluted 3-fold starting in a 1:50 dilution and cut off was defined by pre-pandemic sera (mean + 2 standard deviation). ELISA assay on post-vaccination plasma and serum was performed based on the protocol [92] and modified as described in Gonçalves et al. [93]. Briefly, 96 well plates (Nunc) were coated with 50 μl of trimeric spike protein at 0.5 μg/mL and incubated overnight at 4°C. On the following day, plates were washed three times with 0.1% PBS/Tween20 (PBST) using an automatic plate washer (ThermoScientific). Plates were blocked with 3% bovine serum albumin (BSA) diluted in 0.05% PBS/T and incubated 1h at room temperature. Samples were diluted using 3-fold dilution series starting at 1:50 and ending at 1:10,9350 in 1% BSA-PBST/T and incubated 1h at room temperature. Plates were washed three times as previously and goat anti-human IgG-HRP secondary antibodies (Abcam, ab97215) were added at 1:25,000 and incubated 30 min at room temperature. Plates were washed three times and incubated ~7min with 50 μl of TMB substrate (BioLegend). The reaction was stopped with 25μl of 1M phosphoric acid (Sigma) and read at 450nm on a plate reader (BioTek). Each plate contained 6 calibrator samples from two high-, two medium-, and two low- antibody producer from adult individuals collected at Hospital Fernando Fonseca that were confirmed positive for SARS-CoV-2 by RT-PCR from nasopharyngeal and/or oropharyngeal swabs in a laboratory certified by the Portuguese National Health Authorities [93]. As negative control, we used pre-pandemic plasma samples obtained from healthy donors collected prior July 2019. The endpoint titer was defined as the last dilution before the absorbance dropped below OD 450 of 0.15. Neutralization assay Heat-inactivated serum and plasma samples were four-fold serially diluted over 7 dilutions, beginning with a 1:30 initial dilution. Dilutions were then incubated with spike pseudotyped lentiviral particles for 1h at 37°C. The mix was added to a pre-seeded 96 well plate of 293T-ACE2 cells, with a final MOI of 0.2. At 48h post-transduction, the fluorescent signal was measured using the GloMax Explorer System (Promega). The relative fluorescence units were normalized to those derived from the virus control wells (cells infected in the absence of plasma or serum), after subtraction of the background in the control groups with cells only. The half-maximal neutralization titer (NT 50 ), defined as the reciprocal of the dilution at which infection was decreased by 50%, was determined using four-parameter nonlinear regression (least squares regression without weighting; constraints: bottom = 0) (GraphPad Prism 9). To assess the specificity of our assay, an anti-spike antibody neutralization assay and an RBD competition assay were performed in SARS-CoV-2 spike pseudotyped and vesicular stomatitis virus (VSV) G pseudotyped lentiviral particles, in parallel (S3 Fig). The anti-spike antibody neutralization assay was performed as above, starting with a concentration of 50μg/ml (Thermo Fisher Scientific, PA5-81795) and three-fold serially diluting over 7 dilutions. For the RBD competition assay, 293T-ACE2 cells were pre-incubated with three-fold serial dilutions of SARS-CoV-2 spike’s receptor binding domain, starting with a concentration of 400μg/ml. After 1h incubation at 37°C, supernatant was aspirated and pseudoviruses were added at an MOI of 0.2. The half maximal inhibitory concentration (IC 50 ) was determined using four-parameter nonlinear regression (least squares regression without weighting; constraints: bottom = 0) (GraphPad Prism 9). Spike protein-antibody contact analysis To determine which residues of the SARS-CoV-2 spike protein contribute the most for the binding of antibodies, we studied 57 PDB structures containing the spike protein (or only the RBD region) bound to antibodies (S4 Table). These complexes were chosen based on their availability in the PDB repositorium [94] and are all neutralizing antibodies. We first used the MDAnalysis library [95,96] to identify the residues of the antibody and the spike protein located in the interface between the two proteins. This was done for all PDB structures using in-house Python scripts. To determine the relevance of each spike protein/RBD residue in the binding, a distance cut-off value of 4.5 Å was applied as a criterion (i.e., a contact is observed when two residues have a minimum distance lower than 4.5 Å). Finally, the numerical python library (NumPy [97]) was used to calculate the frequency of contact for each spike protein residue with an antibody residue, from the sample of PDB structures analyzed. MD simulation of the RBD-ACE2 complex Molecular dynamics (MD) simulations of the RBD bound to the ACE2 protein were performed with the GROMACS 2020.3 package [98], using the Amber14sb [99] force field, starting from the 6m0j structure [49], in a truncated dodecahedron box filled with water molecules (minimum of 1.2 nm between protein and box walls). The TIP3P water model [100] was used and the total charge of the system (-23, including the constitutive Zn- and Cl- ions bound to ACE2) was neutralized with 23 Na+ ions. Additional Na+ and Cl+ ions were added to the solution to reach an ionic strength of 0.1M. The system was energy-minimized using the steepest descent method for a maximum of 50000 steps using position restraints on the heteroatom positions by restraining them to the crystallographic coordinates using a force constant of 1000 kJ/mol in the X, Y and Z positions. Before performing the production runs, an initialization process was carried out in 5 stages of 100 ps each. Initially, all heavy-atoms were restrained using a force constant of 1000 kJ/mol/nm, and at the final stage only the only C-α atoms were position-restrained using the same force constant. In the first stage, the Berendsen temperature algorithm [101] was used to initialize and maintain the simulation at 300 K, using a temperature coupling constant of 0.01 ps, without pressure control. The second stage continues to use the Berendsen temperature algorithm but with a force constant of 0.1 ps. The third stage kept the same temperature control settings, but introduced pressure coupling with the Berendsen pressure algorithm [101] with a pressure coupling constant of 5.0 ps and applied isotropically. The fourth stage changed the temperature algorithm to V-rescale [102], with a temperature coupling constant of 0.1 ps, and the pressure algorithm to Parrinello-Rahman [103] with a pressure coupling constant of 5.0 ps. The fifth stage is equal to the fourth stage, but position restraints are only applied to C-α atoms. During the simulation, the equations of motion were integrated using a timestep of 2 fs. The temperature was maintained at 300 K, using the V-rescale [102] algorithm with a time constant of 0.1 ps, and the pressure was maintained at 1 bar using the Parrinello−Rahman [103] pressure coupling algorithm, with a time constant of 5 ps; pressure coupling was applied isotropically. Long-range electrostatic interactions were treated with the PME [104,105] method, using a grid spacing of 0.12 nm, with a cubic interpolation. The neighbor list was updated every twenty steps and the cutoff scheme used was Verlet with 0.8nm as the real space cut-off radius. All bonds were constrained using the LINCS algorithm [106]. The system was simulated for 8 μs in 5 replicates (to a total of 40 μs). RBD-ACE2 contacts frequency To determine the residues that contribute the most for the interaction between RBD and ACE2, and the persistence of these interactions, we performed a contact analysis throughout the simulation. We started by eliminating the first equilibration μs of all replicates. The MDAnalysis library [95,96] was then used to pinpoint the residues of the RBD that are in contact with the ACE2 protein. A distance cut-off value of 4.5 Å was applied as a criterion (i.e., a contact is observed when two residues have a minimum distance lower than 4.5 Å). Finally, we determined the percentage of time for which a given RBD residue is at less than 4.5 Å of ACE2. Homology-based models of spike protein variants Homology-based models of all the variants analyzed in this study were generated using the software Modeller [107], version 9.23, using the structure of the WT enzyme (PDB code: 6XR8) [12] as a template. The protocol used only optimizes the atoms belonging to the mutated residues and the residues that are located within a 5 Å radius from these residues, maintaining the remaining atoms fixed with the coordinates found in the template structure. The optimization parameters were set to the software default values. The final model corresponds to the one with the lowest value of the DOPE function out of 20 generated structures.

Acknowledgments The authors acknowledge Sean Whelan (Washington University School of Medicine St Louis) for providing reagents, João F. Viana and his team for organizing sera collection at Centro Hospitalar Lisboa Ocidental in Portugal, and the IGC COVID-19 team (Patrícia C. Borges, Christian Diwo, Paula Matoso, Vanessa Malheiro, Lígia A. Gonçalves, Nádia Duarte, Ana Brennand, Lindsay Kosack and Onome Akpogheneta) for processing and ranking samples, for this paper and during early settings. We are grateful to the Unit of Genomics of the Instituto Gulbenkian de Ciência for technical support, sample processing and data collection. The authors thank Isabel Gordo and Mónica Dias (Instituto Gulbenkian de Ciência) for helpful discussions and Rute Castro and Paula M. Alves (Instituto de Biologia Experimental e Tecnológica, Universidade Nova de Lisboa, Portugal) for providing the antigen used in the ELISA and in the RBD competition assay.

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009772

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