key: cord-0741938-9ju00ryh authors: Santos, K. S.; Oliveira, J. R.; Machado, R. R.; Magawa, J. Y.; Daher, I. P.; Urbanski, A. H.; Schmitz, G. J.; Silva, R. C. V.; Durigon, E. L.; Boscardin, S. B.; Rosa, D. S.; Schechtman, D.; Nakaya, H. I.; Cunha-Neto, E.; Gadermaier, G.; Coelho, V.; Kalil, J. title: Immunodominant B cell epitope in SARS-CoV-2 RBD comprises a B.1.351 and P.1 mutation hotspot: implications for viral spread and antibody escape date: 2021-03-12 journal: nan DOI: 10.1101/2021.03.11.21253399 sha: 5fc2ca54b6535130b999af0cd24f41cbc1321c94 doc_id: 741938 cord_uid: 9ju00ryh Recent SARS-CoV-2 variants pose important concerns due to their higher transmissibility (1) and escape (2) from previous infections or vaccine-induced neutralizing antibodies (nAb). The receptor binding domain (RBD) of the Spike protein is a major nAb target (3), but data on its B cell epitopes are still lacking. Using a peptide microarray, we identified an immunodominant epitope (S415-429) recognized by 68% of sera from 71 convalescent Brazilians infected with the ancestral variant. In contrast with previous studies, we have identified a linear IgG and IgA antibody binding epitope within the RBD. IgG and IgA antibody levels for this epitope positively correlated with nAb titers, suggesting a potential target of antibody neutralizing activity. Interestingly, this immunodominant RBD region harbors the mutation hotspot site K417 present in P.1 (K417T) and B.1.351 (K417N) variants. In silico simulation analyses indicate impaired RBD binding to nAb in both variants and that glycosylation in the B.1.351 417N could further hinder antibody binding as compared to the K417T mutation in P.1. This is in line with published data showing that nAb from either convalescents or anti-CoV-2 vaccinees are less effective towards B.1.351 than for P.1. Our data support the occurrence of immune pressure and selection involving this immunodominant epitope that may have critically contributed to the recent COVID-19 marked rise in Brazil and South Africa, and pinpoint a potential additional immune escape mechanism for SARS-CoV-2. Some rapidly spreading SARS-CoV-2 variants of concern (VOC) have been shown to be less recognized by neutralizing antibodies (nAb) from both Covid-19 convalescents and anti-CoV-2 vaccinees. This has been attributed to loss of neutralizing antibody recognition of key regions in the Spike protein due to single or multiple mutations in RBD (3) , and/or deletions in the Spike N-terminal domain (NTD) (4) . The nAb binding regions have been indirectly indicated by the loss of neutralization efficiency, using SARS-CoV-2 pseudovirus assays with VOC Spike proteins as compared to ancestral virus Spike. However, immunodominant regions target of anti-CoV-2 antibody responses have not, to date, been directly determined. In this study, we have systematically screened SARS-CoV-2 RBD-derived overlapping peptides to identify linear IgG and IgA binding regions and integrate results with antibody neutralizing titers, using sera from Brazilian COVID-19 convalescent subjects from the beginning of the pandemic. All 71 COVID-19 convalescent individuals (52% female, 48% male, median 42 years old) had RT-PCR diagnostic confirmation from March to April 2020. Serum samples were collected 30-50 days after symptom onset at the Hospital das Clínicas da Universidade de São Paulo, Brazil. The 71 study subjects were selected from a larger cohort (with statistical power of 73.4% for 71 samples) and grouped according to high or low neutralization titers. Among participants, 21 individuals had been hospitalized, while 50 presented mild symptoms. The neutralization capacity was determined by a cytopathic effectbased virus neutralization test (VNT) using ancestral SARS-CoV-2 and sera diluted from 1:20 to 1:5120 (5) . According to the EU recommendation for COVID-19 plasma donation (6) , we clustered the subjects in two groups: (i) 52 individuals with high neutralization (≥1:160 titers) and (ii) 19 individuals with low neutralization (<1:160 titers) capacity (Online is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint To identify IgG and IgA binding regions, 91 overlapping peptides (15-mers) spanning the whole RBD sequence of the Spike protein (P1-P91 peptides) were analyzed in a microarray (Online Table 1 ). Using optimized serum dilutions of 1:10, we successfully identified linear anti-CoV-2 RBD antigenic regions, providing a comprehensive profile of IgG and IgA antibody reactivity of individuals after infection with the ancestral SARS-CoV-2 (Fig. 1A) . In contrast to our findings, RBD was previously considered to essentially lack linear antibody binding epitopes, since less than 10% of convalescent sera reacted with RBD peptides (7) . We believe that our optimized assay, using previously titrated sera and shorter peptides of 15mer with overlaps of 13-mer, contributed to our success in identifying linear epitopes. Considering all 91 peptides, IgG antibodies showed higher binding intensities than IgA (p<0.0001), possibly related to higher concentrations of circulating IgG antibodies. Peptides recognized by more than 30% of subjects were selected for in-depth analysis (Fig. 1B) . Of those peptides, seven (P12, P14, P18, P30, P40, P44 and P50) were frequently detected by IgG antibodies, while eight (P6, P12, P14, P18, P30, P44, P66 and P108) by IgA antibodies, including five peptides recognized by both antibody isotypes. Peptide P44 (S415-429, TGKIADYNYKLPDDF) was the topmost recognized peptide, with 68% of cohort presenting IgG and 82% IgA specific antibodies against it. Comparing reactivity between high versus low neutralizers, P44 showed significantly higher IgG (p=0.0047) and IgA (p=0.0176) levels in individuals with higher neutralizing capacity (≥1:160) ( Fig. 2A ). In addition, IgG reactivity to P44 also positively correlated with the neutralizing activity (r= 0.4846, p<0.0001, 95% confidence interval 0.2769 -0.6491) and to a smaller extent, also for IgA (r=0.3103, p=0.0084, 95% confidence interval 0.07602 -0.5121) (Fig. 2B) . These findings point P44 to be a relevant immunodominant RBD region and potential target of IgG and IgA neutralizing activity. In concordance with this interpretation, in silico B cell epitope predictions revealed that peptide P44 lies within an area with the highest epitope probability (Fig. 2C) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint Of note, IgA reactivity to P44 was higher in hospitalized individuals (p=0.0092), but there was no significant difference for IgG (p=0.051) (Fig. 2D ). Although still controversial and mechanistically unclear, high levels of circulating anti-CoV2 IgA have been previously associated with severe COVID-19 (8) . Nevertheless, mucosal IgA, mostly produced in the dimeric form by residing plasma cells, is likely to play a significant role in virus neutralization at the infected mucosal sites (9). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint using median values within each group). Highlighted is the P44 (S415-429) peptide and respective amino acid sequence. High N: subjects displaying serum with high neutralizing activity (≥1:160), Low N: subjects displaying serum with low neutralizing activity (<1:160). MFI: mean fluorescence intensity. B. Selected peptides (P6, P12, P14, P18, P30, P44, P50, P66 and P108) recognized by IgG and IgA of at least 30% of individuals in the cohort (n=71) are represented with their total percentage of recognition and neutralization titers ranges. In contrast to P44, several other peptides located outside the antibody binding region (Fig. 3A ) showed higher IgG binding intensities in individuals with low neutralization titers indicating limited relevance for virus neutralization (Online Fig. 2 ). . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint Figure 2 . A. IgA and IgG binding to P44 peptide. Mean fluorescence intensity (MFI) of high neutralizers compared to low neutralizers (Mann Whitney). B. Correlation of IgG and IgA MFI specific for P44 and virus neutralization titers (Spearman correlation for IgG r= 0.4846, p<0.0001, 95% confidence interval 0.2769 -0.6491, and for IgA r=0.3103, p=0.0084, 95% confidence interval 0.07602 -0.5121)). C. In silico epitope prediction using BepiPred. Highlighted is the region corresponding to P44 showing a high epitope predictive value. D. IgA and IgG binding to P44. Mean fluorescence intensity (MFI) of hospitalized compared to non-hospitalized (Mann Whitney). Structurally, P44 is located in direct proximity of the binding area, facilitating the interaction with the human ACE2 receptor (Fig. 3B ). More importantly, it is also close to the binding sites of several therapeutic antibodies frequently shown to overlap with the ACE2 binding site (10) . The therapeutic neutralizing antibody REGN10933 (11), for example, recognizes the core binding region of the RBD wherein P44 is located (Fig. 3A ). Based on our data, the identified linear epitope P44 seems to be also a relevant antigenic determinant for neutralizing antibodies generated during COVID-19 infection and are sustained in convalescence. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint Notably, P44 comprises the mutation hotspot site K417, which is mutated in both the South African variant B.1.351 (K417N) and the Brazilian P.1 (K417T) (12) . This residue is likely a result of SARS-CoV-2 of adaptive evolution providing stronger receptor binding (13) . It is expected that selective pressure due to an increasing number of people having specific antibodies against this immunodominant region would affect variants' selection, favoring immune escape from an established humoral response. Reports in the literature show intriguing data that the VOC containing K417N mutation (B.1.351) is more effective in escaping antibody neutralization than VOC P.1 that presents K417T mutation (2, 14) . To investigate the potential relevance of K417 mutations for antibody binding, we performed in silico equilibrium molecular dynamics simulations of B.1.351 (mutations: E484K, K417N and N501Y) and P.1 (mutations: E484K, K417T and N501Y) variants interacting with the therapeutic mAb REGN10933, previously shown to present loss of neutralizing activity in a mutated K417N pseudovirus assay (3) (Fig. 4A, B) . In contrast to the ancestral virus RBD, both variants showed loss of their binding regions in the core around residue 484, corroborated by in vitro studies showing that mutation at this site negatively impacts neutralization capacity (3). E484 is located in a molecular loop stabilized by two close disulfide bonds, most likely establishing a conformational antibody binding epitope. This potential conformational recognition requirement could explain the lack of antibody reactivity to the corresponding linear peptides investigated in our work. Additionally, comparing the two variants, the exchange of the positively charged K417 to the neutral asparagine (B.1.351) or threonine (P.1) led to a shift in the helix structure, further increasing the distance of molecular interaction, likely impairing antibody binding. However, the simulation showed only a minor binding difference when comparing these two mutants. Therefore, this K417 asparagine/threonine alteration seems insufficient to explain the . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint markedly more pronounced neutralizing resistance observed for the B.1.351 South African variant compared to P.1, following infection or vaccination with ancestral virus (2). The crystallographic structure (pdb: 7JJI) of the Spike protein shows a potentially functionally relevant molecular feature: all exposed asparagine residues are glycosylated. We therefore suggest that the introduction of asparagine at 417 position in B.1.351 would generate an additional glycosylation site, as also predicted by the NetNGlyc Prediction tool (data not shown). Accordingly, glycans have been shown to increase the infectivity of SARS-CoV-2 (15), while the presence of glycosylation also reduces the interaction of Spike with relevant antibodies (16) . To further explore the interference of glycosylation in binding, we performed a docking simulation and found that glycosylation at 417N would negatively impact antibody binding and could, therefore, together with the other mutations, lead to the observed antibody neutralization immune escape (Fig. 4A, B) . Interestingly, introducing a glycosylation site is a general mechanism of immune escape used by other viruses such as the influenza virus (17) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint 1.4 to 2.5 fold more transmissible (21) (22) (19) than the ancestral virus, and 25-61% more likely to evade previous protective immunity (22) . However, to date, no information on P.1 differential pathogenicity is available, neither on potential mechanisms involved in its putative higher transmissibility. Likewise, B.1.351 has been reported to be more transmissible (23) , and likely to evade the immune response, favoring reinfection. Nevertheless, these critical issues are still controversial and await further investigations. Noteworthy, numerous reports shows that CD4+ and CD8+ T cell responses play important role in resolution of SARS-CoV-2 infection and COVID-19 (24) and by all means, need to be taken into account for the evaluation of immunological protection. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint residue 417 (N-Glyc) or without glycosylation (Non N-Glyc). A. Structure bonds showing Anc presents several binding bridges nearby residue E484 while P.1 and B.1.351 do not. Glycosylated B.1.351 presents several binding bridges between the glycan residue and N417 evidencing a competitive site that hinders antibody binding. B. Surface solvent accessibility evidencing charged regions. In blue, positive charges and in red negative charges. Highlighted is the region of contact between mAb and RBD. There is a perfect binding of two molecules if there is no glycosylation at the residue 417. When a glycosylation site is introduced there is a visible distancing between the proteins suggesting that binding is sterically hindered. 01.20.0009.00. Seventy-one COVID-19 convalescent individuals (52% Female, 48% Male, median age 42 years old) with RT-PCR diagnostic confirmation from March to April 2020 were included in this study. Samples were collected 30-50 days after symptoms onset at the Hospital das Clínicas da Universidade de São Paulo, Brazil. Participants were selected from a larger cohort based on high or low neutralization titers. Neutralization capacity was determined by a is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint cytopathic effect (CPE)-based virus neutralization test (VNT) using SARS-CoV2 and study participants' sera diluted 1:20 to 1:5120 (5) . Two pre-pandemic sera were used as negative controls. According to the EU recommendation for COVID-19 plasma donation (6), we clustered the subjects in two groups: 52 with high neutralization (neutralizing antibody titers ≥1:160) and 19 individuals with low neutralization (neutralizing antibody titers <1:160 titers) capacities (Online Figure 1) . From those selected individuals, 21 individuals had been hospitalized with 9 needing Intensive Unite Care without mechanical ventilation while 50 presented mild symptoms that did not involve hospitalization. The study was approved by CAPPesq (Comissão de Ética Para Análise de Projetos de Pesquisa do HC-FMUSP) and CONEP (Comissão Nacional de Ética em Pesquisa) (CAAE: 30155220.3.0000.0068). All study participants signed informed consents. The mapping of the IgG and IgA-specific epitopes was done by microarray using PEPperMAP® Linear Epitope Mapping from PEPperPRINT (Heidelberg, Germany). The SARS-CoV-2 Spike RBD sequence (S335 to S516) was synthesized as overlapping peptides of 15 amino acid residues in length with 13 overlapping residues, totalizing 91 peptides and printed in duplicate onto glass slides. Each chip produced uses as controls Influenza Hemagglutinin and polio assay control peptide spots. To ensure that the secondary antibodies do not interact with the antigen-derived peptides printed on the arrays, one copy of the array was pre-stained with goat anti-human IgG (H+L) DyLight680 (Invitrogen, USA) secondary antibody or goat anti-human IgA (chain alpha) DyLight800 (Rockland Immunochemicals Inc., USA) diluted 1:2000 in staining buffer (PBS with 10% blocking buffer) on an orbital shaker at room temperature for 45 min. No background fluorescence due to nonspecific binding of the secondary antibody was observed. Subsequently, serum samples from convalescent individuals were serially diluted from . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint 1:1000 to 1:10 in staining buffer. Best dilution of 1:10 was chosen and added to the microarrays for overnight incubation at 4°C. After three washing steps of 1 min each with 200 µL of the standard buffer, the microarrays were incubated with previously titrated secondary antibodies, anti-IgG and anti-IgA at a dilution of 1:2000, on an orbital shaker at room temperature for 45 minutes. Following secondary antibody incubation, three wash steps were performed and the microarrays were dipped in dipping buffer (1 mM TRIS) and centrifuged at 250 g for 5 minutes for drying. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint incubated at 56°C for 30 min for inactivation and a series of dilutions (1:20 to 1:5120) was prepared for the assay. Serum dilutions were mixed at equal volumes with the virus (100 tissue culture infectious doses, 50% endpoint per well) and pre-incubated for virus neutralization for 1 hour at 37°C. The mixtures containing serum and virus were transferred onto the confluent cell monolayer and incubated at 5% CO2 for 3 days at 37°C. After 72 hours, plates were analyzed by light microscopy. Gross CPE was observed on Vero cells, distinguishing the presence/absence of CPE-VNT. To determine neutralizing antibody titers, the highest serum dilution that was able to neutralize virus growth was considered. As further check, plates were fixed and stained for 30 min with amido black (0.1% amido black [w/v] solution with 5.4% acetic acid, 0.7% sodium acetate). As positive control, an internal serum from a RT-qPCR positive individual and a plaque reduction in the neutralization test >640 was used in each assay. Following recommendations of the World Health Organization, all cytopathic effect-based virus neutralization assays were performed in a Biosafety Level 3 laboratory. According to the EU recommendation for COVID-19 plasma donation (9), the study subjects were clustered into two groups resulting in 52 with high neutralization capacity (≥1:160 titers) and 19 individuals with low neutralization (<1:160 titers). Neutralizing antibody titers were transformed in natural logarithm (ln) for normal distribution. Linear B-cell epitopes were predicted using the BepiPred-2.0 web server (http://www.cbs.dtu.dk/services/BepiPred/). BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This method was superior to other available tools for sequence-based epitope prediction, with regard to both epitope data derived from solved 3D structures and a large collection of linear epitopes downloaded from the IEDB database. In this study, we used a threshold value of . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint 0.55 to obtain the maximum accuracy of prediction. The region corresponding to P44 was highlighted in the SARS-CoV-2 RBD protein sequence in the graph. Structural representations of RBD interacting with ACE2 (pdb: 6M0J) and the neutralizing antibody REGN10933 (pdb: 6XDG) were generated using UCSF Chimera is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253399 doi: medRxiv preprint GraphPad Prism 9.0.1 was used for statistical analyses of individual peptide reactivity comparing sera of subjects with high and low neutralization capacity (Mann Whitney) and Spearman for correlation analysis, p values <0.05 were considered statistically significant. One-sided Mann-Whitney test was performed to investigate differences between the mean MFI values of high and low neutralization groups, for each peptide, using the rstatix R package (v0.6.0). To visualize the results, the -log2 (p-values) were plotted in a heatmap using the ggpubr package (0.4.0). The statistical power (1-b) of 50 versus 21 individuals for P44 was calculated. Updated rapid risk assessment from ECDC on the risk related to the spread of new SARS-CoV-2 variants of concern in the EU/EEA -first update Circulating SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. medRxiv Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization. bioRxiv Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape Screening for SARS-CoV-2 antibodies in convalescent plasma in Brazil: Preliminary lessons from a voluntary convalescent donor program processing, storage, distribution and monitored use. (Directorate-general for health and food safety Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population Severe COVID-19 is associated with elevated serum IgA and antiphospholipid IgA-antibodies Strong Clonal Relatedness between Serum and Gut IgA despite Different Plasma Cell Origins Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions Neutralizing Activity of BNT162b2-Elicited Serum Inhibition of SARS-CoV-2 viral entry upon blocking N-and O-glycan elaboration The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity Adsorptive mutation and N-linked glycosylation modulate influenza virus antigenicity and fitness Three-quarters attack rate of SARS-CoV-2 in the Brazilian Amazon during a largely unmitigated epidemic Africa Succeeded Against COVID-19's First Wave, but the Second Wave Brings New Challenges Model-based evaluation of transmissibility and reinfection for the P.1 variant of the SARS-CoV-2. medRxiv Genomics and epidemiology of a novel SARS-CoV-2 lineage in Manaus, Brazil. medRxiv Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv Adaptive immunity to SARS-CoV-2 and COVID-19 We would like to acknowledge all participants enrolled in this study. The authors reported no conflicts of interest. This project was supported by FAPESP project N. 2020/05256-7 and FINEP grant N.