key: cord-0770058-t675jc0u
authors: Sokal, Aurélien; Barba-Spaeth, Giovanna; Fernández, Ignacio; Broketa, Matteo; Azzaoui, Imane; de La Selle, Andrea; Vandenberghe, Alexis; Fourati, Slim; Roeser, Anais; Meola, Annalisa; Bouvier-Alias, Magali; Crickx, Etienne; Languille, Laetitia; Michel, Marc; Godeau, Bertrand; Gallien, Sébastien; Melica, Giovanna; Nguyen, Yann; Zarrouk, Virginie; Canoui-Poitrine, Florence; Noizat-Pirenne, France; Megret, Jérôme; Pawlotsky, Jean-Michel; Fillatreau, Simon; Bruhns, Pierre; Rey, Felix A.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Chappert, Pascal; Mahévas, Matthieu
title: mRNA vaccination of naive and COVID-19-recovered individuals elicits potent memory B cells that recognize SARS-CoV-2 variants
date: 2021-09-21
journal: Immunity
DOI: 10.1016/j.immuni.2021.09.011
sha: 0b931a3227d54d865faf89f97cff54168a20bf38
doc_id: 770058
cord_uid: t675jc0u

In addition to serum immunoglobulins, memory B cell (MBC) generation against SARS-CoV-2 represents another layer of immune protection, but the quality of MBC responses in naive and COVID-19-recovered individuals after vaccination remains ill-defined. We studied longitudinal cohorts of naive individuals and disease-recovered patients for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of VDJ repertoires, affinity and neutralization against variants of concern (VOCs), using unbiased cultures of 2452 MBCs. Upon boosting, the MBC pool of recovered patients selectively expanded, further matured and harbored potent neutralizers against VOCs. Although naïve individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity towards multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 220 million infections and at least 4.5 million deaths, as of Sept 6, 2021. Vaccination represents the main hope to control the pandemic.

COVID-19 vaccines containing nucleoside-modified mRNA encoding the original Wuhan-Hu-1 SARS-CoV-2 spike glycoprotein (S) developed by Pfizer/BioNTech (BNT162b2) and

Moderna (mRNA-1273) are now being deployed worldwide. They were shown to be safe, highly effective to prevent infection and to control disease severity (Baden et al., 2021; Dagan et al., 2021; Polack et al., 2020) .

The emergence of SARS-CoV-2 variants bearing mutations in key B cell epitopes, however, has raised concerns that viral evolution will erode natural immunity or the protection offered by vaccination. One early mutation in the spike protein (D614G), which shifts the equilibrium between the open and closed protein conformation without modifying antibody neutralization, has become globally dominant (Plante et al., 2021; Weissman et al., 2021; Yurkovetskiy et al., 2020) . Since then, novel variants of concern (VOCs) or of interest (beta) variant, K417T, E484K, N501Y in the P.1 (gamma) variant and L452R, E484Q, or L452R, T478K in the B1.617.1 (kappa) and B1.617.2 (delta) variants, respectively (Cherian et al., 2021; Davies et al., 2021; Greaney et al., 2021a; Tegally et al., 2021) .

Higher infectiousness of the B.1.1.7 variant does not impair neutralizing antibody response (Davies et al., 2021; Garcia-Beltran et al., 2021; Planas et al., 2021a; Supasa et al., 2021) . By contrast, E484K and K417T/N mutations in the B.1.351 and P.1 strains markedly reduced the neutralization potency in COVID-19 recovered or naive vaccinated individuals (Cele et al., 2021; Edara et al., 2021; Greaney et al., 2021a; Hoffmann et al., 2021; Planas et al., 2021a; Wang et al., 2021a; Xie et al., 2021) . Even though infection with VOCs or VOIs remain possible after successful vaccination (Hacisuleyman et al., 2021) , 

In parallel to the rapid antibody secreting cell (ASC) and serum immunoglobulin G (IgG) response, the progressive generation of memory B cells (MBCs) against the SARS-CoV-2 virus represents another layer of immune protection (Dugan et al., 2021; Gaebler et al., 2021; Rodda et al., 2021; Sakharkar et al., 2021; Sokal et al., 2021) . MBCs not only persist after infection but continuously evolve and mature by progressive acquisition of somatic mutations in their variable region genes to improve affinity through an ongoing germinal center response, potentially driven by antigenic persistence Rodda et al., 2021; Sokal et al., 2021) . MBCs further drive the recall response after antigenic rechallenge by differentiating into new antibody-secreting cells (ASCs) displaying the diverse array of high-affinity-antibodies contained in the MBC repertoire. However, a strong convergence of the anti-RBD response across COVID-19 recovered and naive vaccinated individuals shaped by recurrent germline gene families has been described. This could favor viral mutational escape as one single mutation in the RBD can confer a selective advantage by reducing binding and neutralizing activity of antibodies (Garcia-Beltran et al., 2021; Greaney et al., 2021a) .

Upon vaccination, COVID-19 recovered patients showed a striking expansion of RBD-specific MBCs (Goel et al., 2021; Wang et al., 2021b) and elicited a strong serum antibody response including cross-neutralizing antibodies against VOCs (Ebinger et al., 2021; Konstantinidis et al., 2021; Krammer et al., 2021; Manisty et al., 2021; Saadat et al., 2021; Samanovic et al., 2021; Stamatatos et al., 2021; Wang et al., 2021b) . Much less is known about the long-term stability, dynamics and functionality of the MBC repertoire after repeated antigenic stimulation. How the MBC pool will contract or expand its diversity after a new challenge is of major importance in the context of vaccination schemes with repeated homologous or heterologous booster doses and coexistence of multiple VOCs.

Here, we longitudinally characterized the dynamics, clonal evolution, affinity and neutralization capacity of anti-SARS-CoV-2 MBCs after mRNA vaccination in naive and SARS-CoV-2 recovered individuals from the analysis of over two thousand naturally expressed antibodies from single-cell cultured RBD-specific MBCs. We demonstrate that mRNA vaccination selects high-affinity neutralizing clones without compromising the overall MBC pool.

J o u r n a l P r e -p r o o f

individuals.

The B cell immune response elicited by vaccination was analyzed in two cohorts, one of patients previously infected with SARS-CoV-2 (SARS-CoV-2 recovered) and one of virusnaive individuals (SARS-CoV-2 naive). We previously characterized the longitudinal evolution and maturation, up to 6 months after infection, of SARS-CoV-2-responding B cells in a cohort of mild ambulatory (M-CoV) and severe forms of COVID-19 requiring oxygen (S-CoV) (Sokal et al., 2021) . Thirty-four patients from this original cohort were included in this study along with 9 additional COVID-19 patients, for a total of 17 S-CoV and 26 M-CoV ( Figure 1A and Table S1A and S1B). All patients in this first cohort received one dose of Pfizer-BioNTech mRNA (BNT162b2) vaccine between 6 and 12 months post infection (median 309 days post COVID-19 symptom onset, range: 183-362 days, see Table S1C ).

This unique vaccine dose for SARS-CoV-2 recovered patients was referred to as "boost". As a parallel cohort, we recruited 25 healthcare workers with no clinical history of and no serological evidence of previous SARS-CoV-2 infection. SARS-CoV-2 naive individuals in this second cohort received 2 doses of BNT162b2 vaccine as part of the French vaccination campaign. The second vaccine dose, also referred to as "boost", was received in median 28 days after the first dose of vaccine, referred to as "prime". Post-boost blood samples were collected from all vaccinated participants in median 8 days (range 5-22 days) and 2 months after the injection ( Figure 1A and see Table S1C ). Eight SARS-CoV-2 naive donors were additionally sampled after initial priming.

We first measured the pre-and post-boost evolution of IgG serum titers against the WT RBD in both cohorts. Anti-RBD IgG titers remained stable between 6 (M6) and 12 months (M12) post-infection in SARS-CoV-2 recovered patients, with only a mild decrease seen in S-CoV patients ( Figure 1B ). In line with previous reports, a single dose of mRNA vaccine elicited a strong recall response in all patients, with anti-RBD IgG titers increasing on average by 24-fold in S-CoV and 53-fold in M-CoV patients as compared with pre-boost titers ( Figure 1C and S1A, Table S2A ). In SARS-CoV-2 naive individuals, the mRNA vaccine boost also induced a robust anti-RBD IgG response (average of 25-fold-increase)

although titers remained inferior to SARS-CoV-2-recovered patients at all time points (mean 10,870 vs. 75,511 AU/mL in S-CoV and 55,024 in M-CoV, P-value 0.005 and <0.0001, J o u r n a l P r e -p r o o f respectively). Despite contraction of the response in all groups, anti-RBD IgG were at least 10-fold higher than pre-boost/prime titers at 2 months post boost ( Figure 1C and S2A).

Twelve months after infection, all sera from SARS-CoV-2 recovered patients demonstrated neutralization potential in a focus reduction assay against an authentic SARS-CoV-2 virus carrying the dominant D614G amino acid change in the S1 domain of its Spike protein ( Figure 1D -E and Figure S1B -C). This potential was more pronounced in S-CoV than in M-CoV but, as expected, was similarly reduced in both groups against the B.1.351 VOC strain, harboring three mutations in its RBD (N501Y, which increases the affinity for the ACE2 receptor, E484K and K417N, which are implicated in escape from neutralizing antibodies (Harvey et al., 2021) ), along with several mutations in other Spike domains. As previously reported (Goel et al., 2021; Reynolds et al., 2021; Stamatatos et al., 2021; Wang et al., 2021b) , a boost mRNA vaccine strongly enhanced the overall neutralizing potency of virus (IC50 >1/2560), although we cannot exclude differences above the tested dilution range for both VOCs ( Figure 1D -E, Figure S1B , J o u r n a l P r e -p r o o f

Similar to the serum IgG titers, the percentage of RBD-specific and S-specific CD27 + IgD -B cells remained stable between 6 and 12 months in SARS-CoV-2 recovered patients, irrespectively of initial disease severity, thus confirming the generation of germinalcenter-derived long-lived MBCs after natural infection (Figure 2A -B and Figure S2A -B).

RBD-specific MBCs, which represent a large fraction of the neutralizing MBC pool against SARS-CoV-2, substantially expanded after one dose of mRNA vaccine before a modest contraction at 2 months ( Figure 2C and Figure S2C , Table S2 ). In contrast, only low numbers of RBD-specific B cells were detectable in naive individuals after prime with a nonsignificant impact of boost vaccination (Figure 2C) , although RBD-specific ASCs were observed in all donors in the early steps of the post-boost response ( Figure S2D ). The frequency of RBD-specific MBCs persisting 2 months after the boost in naive individuals remained significantly lower than the frequency of MBCs observed in SARS-CoV-2 recovered patients before vaccination ( Figure 2C) , with similar profiles observed for spikespecific MBCs ( Figure S2E ). The overall frequency of spike-specific MBCs in naive individuals at two months after the boost (three months after the prime) appeared even reduced as compared to the three-month post-infection time point in SARS-CoV-2 recovered patients ( Figure S2F ).

Unsupervised analysis of CD19 + IgDswitched B cell populations, using a multiparametric flow panel which we previously used to describe the initial response against SARS-CoV-2 in these patients (Sokal et al., 2021) , showed that RBD-specific cells mostly resided in the CD21 + CD27 + IgD -CD38 int/-CD71 int/resting MBC compartment before vaccination ( Figure 2D -F, Table S2C and D). These cells rapidly switched to a CD27 + CD38 int/+ CD71 + activated B cell phenotype (ABC cluster), in the first 7 days after the boost, together with the emergence of a population of RBD + CD38 high CD27 high ASCs. The persistent expression of the BCR on the surface of these cells harboring a classical ASC phenotype suggests that they were mainly newly generated plasmablasts. These activated subsets progressively contracted and matured as resting MBCs at the latest time point in our study. Atypical MBCs (DN2; IgD -CD27 +/-CD11c + ) and ASC precursors (DN1; IgD -CD27 -)

RBD-specific clusters were also observed, with a small DN2 fraction persisting up to two months post boost, notably in S-CoV patients ( Figure 2F ).

The low numbers of RBD-specific B cells in naive patients precluded a robust unsupervised analysis, and we therefore characterized the phenotype of RBD-specific MBCs which also harbored high frequency of convergent RBD-specific clones with SARS-CoV-2 recovered patients based on V-D-J sequences ( Figure 3D) .

We previously reported a progressive accumulation of somatic mutations in RBDspecific clones up to 6 months after infection in this cohort of SARS-CoV-2 recovered patients (Sokal et al., 2021) . The number of mutations in RBD-specific V H sequences J o u r n a l P r e -p r o o f remained stable between 6 and 12 months. In contrast, V H mutation numbers were increased shortly after the boost and maintained in the MBC pool 2 months after ( Figure 3E ). This evolution in mutation profile could further be confirmed at the individual level for 2 patients with complete follow-up from 3 or 6 months post symptom onset to 2 months after boost ( Figure S3C -D, Table S3A ). This rapid increase in overall mutational load suggests that a fraction of matured, pre-existing MBCs was selectively mobilized upon vaccine response and (Yuan et al., 2021) , clones recognizing the K417 residue were highly enriched for IGHV3-53 and 3-66 genes, whereas clones recognizing the E484K/Q residue were mostly enriched for the IGHV1-2 and 1-69 genes ( Figure S5E , Table S2H ). These results also allowed us to ascribe RBD binding residues of B cells within individual MBC repertoires of naive and recovered patients, confirming the major targeting of the E484 and/or L452 residues within the vaccine activated pool ( Figure 5E ), but also highlighting significant J o u r n a l P r e -p r o o f inter-donor variability in the overall recognition profile of the SARS-CoV-2 RBD by their MBC repertoire.

Finally, to evaluate the cross-neutralization potential of RBD-specific clones Figure   6E ), maintenance of neutralization activity against the VOC was strongly dictated by their J o u r n a l P r e -p r o o f original affinity against the WT RBD. Whereas clones with weak initial affinity against WT RBD were mostly impaired (red dots in the grey sector), 40% of clones with high initial affinity remained potent neutralizers against the VOC (white dots in the grey sector, Figure   6E ). This included monoclonals directed against both the E484 and K417 residues of SARS-CoV-2 RBD ( Figure S6C ). This highlights the key role of affinity maturation in shaping the humoral response and anticipating viral escape.

Overall, these results demonstrate that the MBC pool selected by the WT RBD after mRNA vaccination contains, among its diverse and affinity matured repertoire, a substantial fraction of potent neutralizers against VOCs.

MBCs display a diverse repertoire allowing for an adaptive response upon re-exposure to the pathogen, especially in the case of variants (Purtha et al., 2011; Weisel et al., 2016) .

However, repeated antigenic stimulation, either with vaccinations or viral challenge may be deleterious, reducing the diversity of the overall response in which drifted epitopes are less well targeted (Andrews et al., 2015; Mesin et al., 2020) . Thus, understanding how mRNA vaccination impacts the MBC pool shaped by a previous exposure to SARS-CoV-2 and to determine its capacity to neutralize variants is critical. More generally, to decipher how MBCs from naive vaccinees differ and evolve in comparison with SARS-CoV-2 recovered patients is also of major importance in the pandemic context.

We report here a longitudinal study of SARS-CoV-2 recovered patients, followed over Our study highlights the stability of the overall RBD-specific MBC population up to 12 months after infection with a stable mutation profile, extending observations on memory persistence in COVID-19 (Wang et al., 2021b) . Dynamics of RBD-specific cells after mRNA vaccination in SARS-CoV-2 recovered patients reflect the plasticity of the MBC pool, which promptly and widely activates, proliferates and generates ASCs, and then contracts as resting

MBCs (Goel et al., 2021; Reynolds et al., 2021; Wang et al., 2021b) . This mobilized population contains highly mutated affinity-matured clones that settle, expand and persist for up to 2 months after the boost with a higher frequency and mutational load than before the (Wang et al., 2021b) . This phenomenon occurs independently of the mutational load, high-affinity clones against variants being "randomly" present in the MBC repertoire of SARS-CoV-2 recovered or naive individuals. So, despite the fact that amplitude and quality of the MBC response after mRNA vaccine appears to be lower in naive than in previously infected individuals, highaffinity clones with neutralizing potency against VOC settle in their repertoire, suggesting that their MBC pool could compensate for the time-dependent decay of the initial antibody response.

Correlation of neutralization and affinity shows a complex profile for antibodies expressed by MBCs. Immune escape can affect high-affinity clones against WT RBD, but at the same time, a proportion of clones with high affinity for WT RBD maintain neutralizing potency against B.1.351, contrasting with clones with low or weak affinity that constantly failed to neutralize variants. It indicates that high affinity provides some flexibility for antibodies to cope with mutations affecting their binding target and to conserve their neutralization potency. Consistent with structural analysis (Yuan et al., 2021) , determination of targeted epitopes using binding of different RBD of VOC shows that E484 preferentially J o u r n a l P r e -p r o o f affected the binding affinity of the IGHV1-2/1-69 genes, and K417N and N501Y the one of IGHV3-53/3-66 and IGHV1-2. It underlines that, if particular antibody lineages are affected by RBD mutations, others may retain neutralizing properties Greaney et al., 2021 Greaney et al., , 2021b Muecksch et al., 2021; Scheid et al., 2021; Wang et al., 2021b) .

Altogether, these data describe an immune response maturing with time in SARS-CoV-2 convalescent patients, and resulting in a massive, high-affinity response after vaccination, which, even imprinted by the Wuhan-type RBD, displays an improved recognition of the RBD variants as well. In this immune evolution scheme, the response of naive vaccinees nevertheless lags behind the maturation process that took place during infection. As recently proposed, vaccinated individuals will further improve with time the affinity and diversity of their MBC response and therefore probably also improve their quantitative and qualitative antibody response through the persistence of vaccine-induced germinal centers Turner et al., 2021) . Nonetheless, our observations in SARS-CoV-2 recovered patients suggest that repeated challenges, even using the original spike protein, will help to reduce any persisting differences and allow vaccinated people to respond more efficiently to current SARS-CoV-2 variants by recall of affinity-matured

MBCs.

The relatively small size and the limited follow-up of the cohorts do not allow us to identify characteristics of the patients that would predict response to mRNA vaccine and its persistence. Furthermore, patients with severe form of Covid-19 were not matched for age and comorbidities with those who presented a mild form or with naive individuals, thus differences between these groups must be interpreted with caution. Finally, we focused our study on the RBD domain of the SARS-CoV-2 spike protein as it represents the major known .001, **P < 0.01, *P < 0.05). See also Figure S1 and Table S1 . (***P<0.0001, *** P<0.001, **P < 0.01, *P < 0.05). See also Figure S2 and Table S2 . Figure S3 and Table S2 . Figure S4 and Table S2 . Figure S5 and Table S2 . 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Matthieu Mahévas (matthieu.mahevas@aphp.fr).

No unique materials were generated for this study.

 Single cell culture VDJ sequencing data reported in Figure 3 and Figure S3 are directly included in this study as part of  Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

In total, 43 patients with recovered COVID-19 (17 S-CoV and 26 M-CoV) and 25 naive patients were included in this study and sampled at least one time before boost vaccination were enrolled in the naive group (IRB 2018-A01610-55). Detailed information on the individuals, including gender and health status, can be found in Table S1 .

All vaccinated subjects received the BNT162b2 mRNA vaccine. SARS-CoV-2 recovered patients received only one dose, in line with French guidelines, except 3 who received 2 doses (See Table S1 ). First injection was realized in mean 309 days (± SD 44.6 days) after the infection. Naive patients received two doses at a mean 27.7 days (± SD 1.8 days) interval.

Prior to vaccination, samples were collected from SARS-CoV-2 recovered patients 12 months post symptoms onset (mean ± SD: 329.1 ± 15.8 days after disease onset for S-CoV, and 342.0 ± 8.6 days after disease onset for M-CoV). Samples at 12 months post disease onset were defined as "pre-boost". For patients not sampled before vaccination (n=9/34), sample at 6 months was considered as "pre-boost". For SARS-CoV-2 naive patients, the "prime" timepoint was defined as the sampling between the two doses and was drawn at a mean 20.2 ± 5.9

days after the first injection.

Samples were additionally collected shortly after the boost (mean ±SD: 10 ± 5.3 days for S-CoV; 23 ± 6.1 days for M-CoV and 9 ± 4.0 days for naive), and 2 months after the boost (mean ±SD: 64.7 ± 15.3 days for S-CoV ; 63.2 ± 11.9 days for M-CoV and 63.3 ± 9.0 days for naive). Clinical and biological characteristics of these patients are summarized in Table   S1 . Patients were recruited at the Henri Mondor University Hospital (AP-HP), between March and April 2021. MEMO-COV-2 study (NCT04402892) was approved by the ethical committee Ile-de-France VI (Number: 40-20 HPS), and was performed in accordance with the French law. Written informed consent was obtained from all participants.

The reference D614G strain (hCoV-19/France/GE1973/2020) and the B. 

Vero E6 cells and were used at passage 3 and passage 2 respectively. Single use aliquots stored at -80°C were used for all the assays.

Serum samples were analyzed for anti-S-RBD IgG titers with the SARS-CoV-2 IgG Quant II assay (ARCHITECT®, Abbott Laboratories). The latter assay is an automated chemiluminescence microparticle immunoassay (CMIA) that quantifies anti-RBD IgG, with 50 AU/mL as a positive cut-off and a maximal threshold of quantification of 40,000 AU/mL.

All assays were performed by trained laboratory technicians according to the manufacturer's standard procedures.

The ectodomain from the SARS-CoV-2 Spike (residues 1-1208) was designed as a stabilized construct with six proline mutations (F817P, A892P, A899P, A942P, K986P, V987P), a GSAS substitution at the furin cleavage site (residues 682-685) and a C-terminal Foldon trimerization motif (Hsieh et al, 2020) , followed by Hisx8, Strep and Avi tags. This construct was cloned using its endogenous signal peptide in pcDNA3.1(+). 

Single cell culture was performed as previously described (Crickx et al., 2021) . Single B cells were sorted in 96-well plates containing MS40L lo cells expressing CD40L (kind gift from G.

Kelsoe, Luo et al., 2009) . Cells were co-cultured at 37°C with 5% CO2 during 21 or 25 days carbonate during 1h at 37°C. After plate blocking, cell culture supernatants were added for 1hr, then ELISA were developed using HRP-goat anti-human IgG (1 μg/ml, Immunotech) and

TMB substrate (Eurobio). OD450 and OD620 were measured and Ab-reactivity was calculated after subtraction of blank wells. Supernatants whose ratio of OD450-OD620 over control wells (consisting of supernatant from wells that contained spike-negative MBCs from the same single cell culture assay) was over 3 were considered as positive for WT RBD, B.1.1.7 RBD or B.1.351 RBD ELISA. PBS was used to define background OD450-OD620.

Clones whose culture had proven successful (IgG concentration ≥ 1 µg/mL at day 21-25)

were selected and extracted using the NucleoSpin96 RNA extraction kit (Macherey-Nagel) according to the manufacturer's instructions. A reverse transcription step was then performed J o u r n a l P r e -p r o o f using the SuperScript IV enzyme (ThermoFisher) in a 14 μl final volume (42°C 10 min, 25°C

10 min, 50°C 60 min, 94°C 5 min) with 4 µl of RNA and random hexamers (Thermofisher scientific). A PCR was further performed based on the protocol established by Tiller et al (Tiller et al., 2008) . Briefly, 3.5 μl of cDNA was used as template and amplified in a total volume of 40 μl with a mix of forward L-VH primers ( For specific patients and time points (see Table S1 ), some IgH sequences were obtained directly from single-cell sorting in 4µL lysis buffer containing PBS (Gibco), DTT (ThermoFisher) and RNAsin (Promega). Reverse transcription and a first PCR was performed as described above (50 cycles) before a second 50-cycles PCR using 5'AgeI VH primer mix and Cγ-CH1 3' primer, before sequencing.

Processed FASTA sequences from cultured single-cell V H sequencing were annotated using Igblast v1.16.0 against the human IMGT reference database. Clonal cluster assignment (DefineClones.py) and germline reconstruction (CreateGermlines.py) was performed using the Immcantation/Change-O toolkit (Gupta et al., 2015) on all heavy chain V sequences.

Sequences that had the same V-gene, same J-gene, including ambiguous assignments, and same CDR3 length with maximal length-normalized nucleotide hamming distance of 0.15 were considered as belonging to the same clonal group. Mutation frequencies in V genes were then calculated using the calcObservedMutations() function from the Immcantation/SHazaM 

This high-throughput kinetic screening of supernatants using single antigen concentration has recently been extensively tested and demonstrated excellent correlation with multiple antigen concentration measurements (Lad et al., 2015) . Biolayer interferometry assays were performed using the Octet HTX instrument (ForteBio). Anti-Human Fc Capture (AHC)

biosensors ( Figure S4 ) and initial prediction of key binding residues. mAbs were defined as affected against a given variant RBD if the ratio of calculated KD value against that RBD variant and the WT RBD was superior to two. Key binding residues prediction was simply made based on mutations repartition in the different variants ( Figure 5C) (Yuan et al., 2021; Starr et al., 2021) . Sensors with missing values were manually inspected to resolve binding residues attribution, leaving only four with an unresolved profile.

This analysis was further confirmed by conserved intra-clonal residue binding as illustrated in 

ClinicalTrials.gov Identifier: MEMO-CoV2, NCT04402892. 

Butt, A.A., and National Study Group

Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants

Immune history profoundly affects broadly protective B cell responses to influenza

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Distinct conformational states of SARS-CoV-2 spike protein

Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma

Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra

Antibody Evolution after SARS-CoV-2 mRNA Vaccination

Rituximab-resistant splenic memory B cells and newly engaged naive B cells fuel relapses in patients with immune thrombocytopenia

BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass Vaccination Setting

Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets

Antibody responses to the BNT162b2 mRNA vaccine in individuals previously infected with SARS-CoV-2

Infection-and vaccine-induced antibody binding and neutralization of the B.1.351 SARS-CoV-2 variant

Evolution of antibody immunity to SARS-CoV-2

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

Distinct antibody and memory B cell responses in SARS-CoV-2 naïve and recovered individuals following mRNA vaccination

Comprehensive mapping of mutations in the SARS-CoV-2 receptorbinding domain that affect recognition by polyclonal human plasma antibodies

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data

Vaccine Breakthrough Infections with SARS-CoV-2 Variants

SARS-CoV-2 variants, spike mutations and immune escape

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

Structure-based design of prefusionstabilized SARS-CoV-2 spikes

Human neutralizing antibodies elicited by SARS-CoV-2 infection

Levels of Produced Antibodies after Vaccination with mRNA Vaccine; Effect of Previous Infection with SARS-CoV-2

Antibody Responses in Seropositive Persons after a Single Dose of SARS-CoV-2 mRNA Vaccine

Highthroughput kinetic screening of hybridomas to identify high-affinity antibodies using biolayer interferometry

Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes

Antibody response to first BNT162b2 dose in previously SARS-CoV-2-infected individuals

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

Restricted Clonality and Limited Germinal Center Reentry Characterize Memory B Cell Reactivation by Boosting

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies

Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Spike mutation D614G alters SARS-CoV-2 fitness

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

Prior SARS-CoV-2 infection rescues B and T cell responses to variants after first vaccine dose

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19

Binding and Neutralization Antibody Titers After a Single Vaccine Dose in Health Care Workers Previously Infected With SARS-CoV-2

Prolonged evolution of the human B cell response to SARS-CoV-2 infection

Poor antigen-specific responses to the second BNT162b2 mRNA vaccine dose in SARS-CoV-2-experienced individuals

B cell genomics behind crossneutralization of SARS-CoV-2 variants and SARS-CoV

Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera

Detection of a SARS-CoV-2 variant of concern in South Africa

Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection

A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells

D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19

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

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

Vaccination boosts high-affinity RBD-memory B cells (MBCs) in COVID-19 recovered patients

Boosted MBCs retain their diversity and express potent variant-neutralizing antibodies

To better understand B cell responses to SARS-CoV-2 mRNA vaccination, Sokal et al. analyzed memory B cells from COVID-19-recovered and -naive individuals. In recovered patients, vaccination amplifies a broad repertoire of matured MBCs and generates variant-neutralizing plasma cells. In naïve individuals, vaccination induces an MBC pool containing potent neutralizing clones against all current variants of concern, including beta and delta

SARS-CoV2 Recovered: Severe Covid-19 (n= 17) Mild Covid-19