Vaccination boosts naturally enhanced neutralizing breadth to SARS-CoV-2 one year after infection

Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve individuals2,5–8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants. Should memory responses evolve in a similar manner in vaccinated individuals, additional appropriately timed boosting with available vaccines could cover most circulating variants of concern.

newly arising clones at the 12-month time point in vaccinated individuals (Extended Data Fig  138   5c). Moreover, phylogenetic analysis revealed that sequences found at 6 and 12 months were 139 intermingled and similarly distant from their unmutated common ancestors (Extended Data Fig  140   6). We conclude that clonal re-expansion of memory cells in response to vaccination is not 141 associated with additional accumulation of large numbers of somatic mutations as might be 142 expected if the clones were re-entering and proliferating in germinal centers. 143 144

Neutralizing Activity of Monoclonal Antibodies 145
To determine whether the antibodies obtained from memory B cells 12 months after infection 146 bind to RBD we performed ELISAs (Fig.3a). 174 antibodies were tested by ELISA including: 1. 147 53 that were randomly selected from those that appeared only once and only after 1 year; 2. 91 148 that appeared as expanded clones or singlets at more than one time point; 3. 30 representatives of 149

Epitopes and Breadth of Neutralization 173
To determine whether the loss of non-neutralizing antibodies over time was due to preferential 174 loss of antibodies targeting specific epitopes, we performed BLI experiments in which a 175 preformed antibody-RBD complex was exposed to a second monoclonal targeting one of 3 176 classes of structurally defined epitopes 3,20 (see schematic in Fig. 4a). We assayed 60 randomly 177 selected antibodies with comparable neutralizing activity from the 1.3-and 12-month time 178 points. The 60 antibodies were evenly distributed between the 2 time points and between 179 neutralizers and non-neutralizers (Fig. 4). Antibody affinities for RBD were similar among 180 neutralizers and non-neutralizers obtained at the same time point (Fig. 4b, Extended Data Figure  181 8). Although the differences were small, both neutralizers and non-neutralizers showed increased 182 affinity over time (Fig. 4b, Extended Data Fig. 8). In competition experiments, all but 2 of the 183 30 non-neutralizing antibodies failed to inhibit binding of the class 1 (C105), 2 (C121 and C144) 184 or 3 (C135) antibodies tested and therefore must bind to epitopes that do not overlap with the 185 epitopes of these classes of antibodies (Fig. 4c, and Extended Data Fig. 9). In contrast, all but 2 186 of the 30 neutralizers blocked class 1, or 2 antibodies whose target epitopes are structural 187 components of the RBD that interact with its cellular receptor, the angiotensin-converting 188 enzyme 2 20,21 (ACE2) (Fig. 4c and Extended Data Fig. 9). In addition, whereas 9 of the 15 189 neutralizing antibodies obtained after 1.3 months blocked both class 1 and 2 antibodies, only 1 of 190 the 15 obtained after 12 months did so. In contrast to the earlier time point, 13 of 15 neutralizing 191 antibodies obtained after 12 months only interfered with C121, a class 2 antibody 3,20 ( Fig. 4c and  192 Extended Data Fig. 9). We conclude that neutralizing antibodies are retained and non-193 neutralizing antibodies targeting RBD surfaces that do not interact with ACE2 are removed from 194 the repertoire over time. 195 196 To determine whether there was an increase in neutralization breadth over time, the neutralizing 197 activity of the 60 antibodies was assayed against a panel of RBD mutants covering residues 198 associated with circulating variants of concern: R346S, K417N, N440K, A475V, E484K and 199 N501Y ( Fig. 4d  The increase in breadth and overall potency of memory B cell antibodies could be due to shifts in 204 the repertoire, clonal evolution, or both. To determine whether changes in specific clones are 205 associated with increases in affinity and breadth, we measured the relative affinity and 206 neutralizing breadth of pairs of antibodies expressed by expanded clones of B cells that were 207 maintained in the repertoire over the entire observation period 3,4 . SARS-CoV-2 neutralizing 208 activity was not significantly correlated with affinity at either time point considered 209 independently (Fig. 5a). However, there was a significant increase in overall affinity over time 210 including in the 4 pairs of antibodies with no measurable neutralizing activity (Fig 5b and  211 Supplementary  (Fig. 5c). In contrast, 10 of the 15 antibodies obtained from the same clones after 219 12 months neutralized all variants tested with IC50s as low as 1 ng/ml against the triple mutant 220 convalescents against variants of concern 15,32,33 . 242

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Less is known about selection and maintenance of the memory B cell compartment. SARS-CoV-244 2 infection produces a memory compartment that continues to evolve over 12 months after 245 infection with accumulation of somatic mutations, emergence of new clones, and increasing 246 affinity all of which is consistent with long-term persistence of germinal centers. The increase in 247 activity against SARS-CoV-2 mutants parallels the increase in affinity and is consistent with the 248 finding that increasing the apparent affinity of anti-SARS-2 antibodies by dimerization or by 249 creating bi-specific antibodies also increases resistance to RBD mutations 34-37 . 250 structures over long periods of time 26 . In addition, SARS-CoV-2 protein and nucleic acid has 253 been reported in the gut for at least 2 months after infection 4 . Irrespective of the source of 254 antigen, antibody evolution favors epitopes overlapping with the ACE2 binding site on the RBD, 255 possibly because these are epitopes that are preferentially exposed on trimeric spike protein or 256 virus particles. neutralizing activity against SARS-CoV-2 variants of concern. All experiments were performed at 295  The E484K and K417N/E484K/N501Y (KEN) substitution, as well as the deletions/substitutions 453 corresponding to variants of concern were incorporated into a spike protein that also includes the 454 R683G substitution, which disrupts the furin cleaveage site and increases particle infectivity. 455 Neutralizing activity against mutant pseudoviruses were compared to a wildtype SARS-CoV-2 456 spike sequence (NC_045512), carrying R683G where appropriate. 457 SARS-CoV-2 pseudotyped particles were generated as previously described 3,10 . Briefly, 293T 458 cells were transfected with pNL4-3DEnv-nanoluc and pSARS-CoV-2-SΔ19, particles were 459 harvested 48 hpt, filtered and stored at -80°C. Antibodies were identified and sequenced as described previously 3 . In brief, RNA from single 508 cells was reverse-transcribed (SuperScript III Reverse Transcriptase, Invitrogen, 18080-044) and 509 the cDNA stored at −20 °C or used for subsequent amplification of the variable IGH, IGL and 510 IGK genes by nested PCR and Sanger sequencing. Sequence analysis was performed using 511 MacVector. Amplicons from the first PCR reaction were used as templates for sequence-and 512 ligation-independent cloning into antibody expression vectors. Recombinant monoclonal 513 antibodies were produced and purified as previously described 3 . 514 515

Biolayer interferometry 516
Biolayer interferometry assays were performed as previously described 3 . Briefly, we used the 517 Octet Red instrument (ForteBio) at 30 °C with shaking at 1,000 r.p.m. Epitope-binding assays 518 were performed with protein A biosensor (ForteBio 18-5010), following the manufacturer's clonotypes were assigned based on their V and J genes using in-house R and Perl scripts (Fig.  540 2d). All scripts and the data used to process antibody sequences are publicly available on GitHub 541 (https://github.com/stratust/igpipeline). 542

543
The frequency distributions of human V genes in anti-SARS-CoV-2 antibodies from this study 544 was compared to 131,284,220 IgH and IgL sequences generated by 45 and downloaded from 545 cAb-Rep 46 , a database of human shared BCR clonotypes available at https://cab-546 rep.c2b2.columbia.edu/. Based on the 91 distinct V genes that make up the 6902 analyzed 547 sequences from Ig repertoire of the 10 participants present in this study, we selected the IgH and 548 IgL sequences from the database that are partially coded by the same V genes and counted them 549 according to the constant region. The frequencies shown in (Extended data Fig. 4) are relative to 550 the source and isotype analyzed. We used the two-sided binomial test to check whether the 551 number of sequences belonging to a specific IgHV or IgLV gene in the repertoire is different