The SARS-CoV-2 mRNA-1273 vaccine elicits more RBD-focused neutralization, but with broader antibody binding within the RBD

The emergence of SARS-CoV-2 variants with mutations in key antibody epitopes has raised concerns that antigenic evolution will erode immunity. The susceptibility of immunity to viral evolution is shaped in part by the breadth of epitopes targeted. Here we compare the specificity of antibodies elicited by the mRNA-1273 vaccine versus natural infection. The neutralizing activity of vaccine-elicited antibodies is even more focused on the spike receptor-binding domain (RBD) than for infection-elicited antibodies. However, within the RBD, binding of vaccine-elicited antibodies is more broadly distributed across epitopes than for infection-elicited antibodies. This greater binding breadth means single RBD mutations have less impact on neutralization by vaccine sera than convalescent sera. Therefore, antibody immunity acquired by different means may have differing susceptibility to erosion by viral evolution.


INTRODUCTION
Mitigation of the SARS-CoV-2 pandemic will depend on population immunity acquired via infection or vaccination. Unfortunately, humans are repeatedly re-infected with the endemic "common-cold" coronaviruses (1), at least in part because these viruses evolve to escape neutralizing antibody immunity elicited by prior infection (2). SARS-CoV-2 is already undergoing similar antigenic evolution, with the recent emergence of new viral lineages with reduced neutralization by antibodies elicited by infection and vaccination (3)(4)(5)(6)(7)(8). Preliminary results suggest that immunity still provides substantial protection against infection and severe disease (9, 10) caused by these new viral lineages-but if SARS-CoV-2 is similar to other human coronaviruses, then at minimum the protection against reinfection will eventually be eroded by viral evolution.
To determine the contribution of RBD-binding antibodies to neutralization, we measured the neutralizing activity of vaccine sera before and after depleting RBD-binding antibodies, using spike-pseudotyped lentiviral particles. For 13 of 14 vaccinated individuals, >90% of the neutralizing activity at both time points was due to RBD-binding antibodies (Fig. 1C,D, Table S1). For 17 of 28 vaccine sera, depletion of RBD-binding antibodies reduced the neutralization titer (reciprocal IC50) from >1000 to <25 (Fig. 1C,D, S1C,D). The percent neutralizing activity due to RBD-binding antibodies was higher for vaccine sera than for convalescent plasmas collected at similar time points (15) (Fig.   1C,D), although the difference was only statistically significant at the day 15-60 time point. These assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (6,20,21). Nonetheless, because the same assay was used for vaccine and convalescent samples, we conclude that the neutralizing activity of the antibody response elicited by the mRNA-1273 vaccine is more focused on the RBD than for infection-elicited antibodies.

Complete mapping of RBD mutations that reduce binding by vaccine-elicited sera at 119 days post-vaccination
We used deep mutational scanning (15,22) to map all mutations to yeast-displayed RBD that reduced vaccine serum antibody binding. Briefly, we incubated duplicate libraries of yeast expressing RBD mutants with each serum, and used fluorescence-activated cell sorting (FACS) to enrich cells expressing RBD mutants with reduced serum binding (Fig. S2, S3, Table S2). These libraries contained 3,804 of the 3,819 possible single amino-acid mutations to the Wuhan-Hu-1 RBD, 2,034 of which are tolerated for proper folding and at least modest ACE2 binding (23). We used deep sequencing to quantify the "escape fraction" for each of the 2,034 tolerated RBD mutations against each serum. These escape fractions range from 0 (no cells with the mutation in the serum-escape bin) to 1 (all cells with the mutation in the serum-escape bin). We represent the escape maps as logo plots, where the height of each le er is proportional to its escape fraction (Fig. 2, Fig. S5, S6).
The escape maps for sera collected at day 119 from individuals who received the 250 µg vaccine dose fell into four qualitative categories (Fig. 2). For 5 of 14 individuals, escape from antibody binding was focused on RBD sites 456 and 484 (Fig. 2, S5). These two sites are on the receptor-binding ridge in the "class 1" and "class 2" RBD epitopes, respectively (24) (Fig. 2A). Two more individuals also had escape maps that were focused on sites 456 and 484, but with a very low overall magnitude of escape ( Fig. 2, S5). For 2 of 14 individuals, serum binding was most affected by mutations in the "class 4" epitope located in the core RBD, including sites 383 to 386 (Fig. 2, S5). The escape maps for the remaining 5 individuals were "flat," meaning that no single mutation had a large effect on serum binding, suggestive of broad binding to multiple RBD epitopes (Fig. 2, S5).
To determine if the vaccine dose affected the RBD binding specificity of the polyclonal antibody response, we mapped binding escape from the day 119 sera from 8 individuals vaccinated with 100 µg rather than 250 ug doses. The escape maps of the 100 µg cohort resembled those of the 250 µg cohort and fell into the 456/484-targeting, core-targeting, or "flat" categories ( Fig. S6). Although the sample sizes are small, and a higher fraction of the 100 µg dose escape maps were "flat" than for the 250 µg cohort (4/8 versus 5/14, respectively), this suggests 100 and 250 ug doses elicit antibody responses similar in the breadth of their RBD binding specificity.
Binding escape maps become more focused from 36

days to 119 days post-vaccination
To examine longitudinal changes in binding specificity of vaccine-elicited serum antibodies to the RBD, we also determined binding-escape maps for sera collected at day 36 post-vaccination from five individuals who received the 250 µg dose (Fig. 3). All of these day-36 sera had relatively "flat" escape maps, meaning that no single mutation had a large effect on serum binding. But by day 119, the escape maps for most individuals were more focused on specific sites in the RBD. Specifically, for four of five individuals, the escape maps became focused on RBD sites 456 and 484 (Fig. 3). For one of these individuals, the focusing on sites 456 and 484 was accompanied by increased focusing on the class 4 epitope, including sites 383-386. Only one individual had a day-119 escape map as flat as the day-36 escape map. These results suggest that as the vaccine-induced RBD-binding antibody response matures over time, it becomes more focused on specific sites in the RBD.

RBD binding by vaccine-elicited sera is broader than for convalescent plasmas
To elucidate differences in the specificity of the RBD-binding antibody response elicited by vaccination versus infection, we compared the vaccine-sera escape maps to ones that we previously determined for convalescent plasmas (15,16). At both 15-60 and 100-150 day time points, the convalescent escape maps were more focused on specific RBD sites than the vaccine escape maps (Fig. 4A). The difference was especially striking at the early time point, where the day 36 vaccine samples all had flat escape maps, whereas the convalescent samples often had escape maps indicating that antibody binding was strongly affected by mutations at specific RBD sites such as 456 and 484 (Fig. 4A). The difference between the vaccine and convalescent samples was less striking at the later time point, but the convalescent maps were still more focused than the vaccine maps. There were also some differences in the RBD sites where mutations affected binding for the vaccine versus convalescent samples. While most samples of both types were affected by mutations at sites 456 and 484, the convalescent samples tended to also be affected by mutations to the 443-450 loop in the class 3 epitope, whereas mutations in the class 4 epitope spanning sites 383-386 sometimes had a more pronounced effect on the vaccine samples (Fig. 4A, 2, S5).
To visualize relationships between vaccine-and infection-elicited antibody responses, we used multidimensional scaling to create a two-dimensional projection of the escape maps for the vaccine sera, convalescent plasmas (15,16), and previously characterized monoclonal antibodies (16,22,(25)(26)(27) ( Fig. 4B). In this projection, antibodies or sera/plasmas with similar binding-escape mutations are located close together, while those affected by distinct mutations are far apart. As previously reported (16), convalescent plasmas clustered closest to class 2 antibodies (Fig. 4B), which are generally most affected by mutations to site 484. The vaccine sera, on the other hand, were more centrally located in the middle of the antibodies of all four classes, reflecting their fla er binding-escape maps that were less dominated by mutations that escape any single antibody class (Fig. 4B).
To examine sites of binding-escape mutations in the context of the RBD's structure, we projected the total escape at each site averaged across all vaccine or convalescent samples at each time point onto the surface of the RBD (Fig. 4C). The sites where mutations affected binding of vaccine sera were broadly distributed across the RBD surface (Fig. 4C), whereas convalescent plasmas were most affected by mutations at just a few key regions (sites 456 and 484, and to a lesser degree the 443-450 loop) (Fig.   4C). However, as noted above, binding escape from the vaccine sera was somewhat more focused at day 119 relative to day 36, including at sites 456, 484, and 383-386.

Single RBD mutations have less impact on vaccine-elicited than infection-elicited neutralizing activity
We tested key RBD mutations in spike-pseudotyped lentiviral neutralization assays against a subset of vaccine and convalescent sera. We used the binding-escape maps to choose six representative samples each from the day 100-150 vaccine and convalescent sera for which >90% of the neutralizing activity was due to RBD-binding antibodies ( Fig. 1, S1, (15)). The escape maps for the vaccine and convalescent samples chosen for these assays are summarized in Fig. 5A and detailed in Fig. 2 and Fig. S7.
For many convalescent sera, single RBD mutations reduced neutralization by approximately the same amount as removing all RBD-binding antibodies (Fig. 5B, S8, S9). However, no single RBD mutation we tested had a comparably large effect on vaccine sera (Fig. 5B). This result is consistent with the binding-escape maps, which generally indicate that vaccine sera have a broader RBD-binding specificity than convalescent sera.
The mutations that most impacted neutralization also differed between vaccine and convalescent sera (Fig. 5B). For convalescent sera, the largest reduction in neutralization was consistently caused by mutations to site E484 in the class 2 epitope (16,22), including the E484K mutation present in multiple emerging viral lineages (28,31,33). But for vaccine sera, E484K generally caused a more moderate decrease in neutralization. For some vaccine sera, another mutation at site E484 (E484P) caused a larger loss of neutralization, but E484P is not found in any sequenced isolates and reduces both ACE2 binding affinity (23) and viral entry titers (Fig. S8D). The F456A mutation to the class 1 epitope often reduced neutralization by vaccine sera, although it had li le effect on convalescent sera; this mutation is also not observed in natural sequences and reduces viral entry titers ( Fig. S8D). Mutations to the class 3 epitope (G446V, L452R) modestly reduced neutralization by some vaccine and convalescent sera (Fig. 5B). However, P384R in the less-neutralizing core RBD class 4 epitope (17,18,36,37) and K417N in the class 1 epitope had li le effect on neutralization by any sera, consistent with previous reports (5)(6)(7)38). Importantly, while single mutations sometimes caused large 5 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. decreases in neutralization by convalescent sera, in no case did they reduce neutralization by vaccine sera >10-fold or to a titer <100 (Fig. 5B, S8).
The fact that single mutations ablate the anti-RBD neutralizing activity of some convalescent sera but only modestly erode the activity of vaccine sera suggests that the vaccine elicits neutralizing antibodies with a greater number of RBD specificities. To test this idea, we performed neutralization assays with a triple mutant (K417N-G446V-E484K) containing a mutation in each of the class 1, 2, and 3 epitopes. For convalescent sera, the E484K mutation alone often caused a decrease in neutralization comparable to the triple mutant (Fig. 5C,D, S8), consistent with the convalescent escape maps showing a strong focus on site E484. In contrast, for vaccine sera, the triple mutant always reduced neutralization more than any of its constituent single mutations (Fig. 5C,D, S8). Moreover, for only one of the six vaccinated individuals did the triple mutant decrease neutralization to the same extent as removing all RBD-binding antibodies (Fig. 5B), indicating that the vaccine usually induces some neutralizing antibodies not escaped by mutations to sites K417, G446, and E484. These results are consistent with the escape maps indicating that the vaccine sera often have a broader RBD-binding specificity. Note that in some cases infection also elicited very broad anti-RBD neutralizing activity: for instance, serum from the convalescent individual with the broadest escape map (subject G, day 94) was substantially more affected by the triple mutant than any of its constituent single mutants (Fig. 5B, S7, S8).

DISCUSSION
We have shown clear differences in the specificity of polyclonal serum antibodies acquired by infection versus vaccination with Moderna mRNA-1273. The neutralizing activity of vaccine sera is even more focused on the RBD than for convalescent sera, with the majority of vaccine sera losing all detectable neutralization at a 1:25 cutoff after depletion of RBD-directed antibodies. This fact is surprising, since the mRNA-1273 vaccine encodes the full spike ectodomain (11), and one conjectured benefit of full-spike versus RBD-only vaccines was elicitation of neutralizing antibodies targeting non-RBD subdomains.
At first glance, the RBD focusing of the vaccine sera neutralization might seem like a "bad" thing, but the rest of our results suggest that this may not be the case. Our comprehensive maps of how RBD mutations reduce serum antibody binding show that vaccine-induced antibodies are usually less affected than infection-elicited antibodies by any single RBD mutation. While infection-elicited RBD antibodies are often strongly focused on an epitope including site E484, vaccine-elicited antibodies bind more broadly across the RBD including to the more conserved "core". This broader binding makes neutralization by vaccine sera more resistant to RBD mutations. For instance, RBD-directed neutralization by convalescent sera was greatly reduced or even eliminated by a combination of key mutations at the three major epitopes in the RBD's receptor-binding motif, but all vaccine sera that we tested retained substantial neutralization against this triple mutant. This result implies that either vaccination induces an antibody response more broadly distributed across the RBD surface, or that the individual antibodies elicited by vaccination are more robust to these mutations (39,40).
CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint Our results do not explain why the vaccine neutralizing response is so RBD-directed, but we note two possibilities. First, the vaccine encodes a stabilized S-2P spike, which could present some epitopes in slightly different conformations. Second, the vaccine is delivered by an mRNA-lipid nanoparticle, which may lead to different kinetics of antigen presentation than viral infection (41,42).
Indeed, another recent study suggests that mRNA vaccination elicits a different distribution of isotypes and fewer antibodies that cross-react to common-cold coronaviruses compared to infection (43). A caveat is that some differences could also be because the vaccinated individuals were relatively young (18-55 years) and healthy, whereas the convalescent individuals were older (23-76 years, median 56) with a range of comorbidities (13).
More generally, our findings suggest that it is important to differentiate antibody immunity acquired by different means when assessing the impact of viral evolution. Significant effort is being expended to identify emerging antigenic variants of SARS-CoV-2 and determine which ones might evade immunity (3,7,8,33). Our findings suggest that the results could vary depending on the source of immunity. Furthermore, carefully characterizing and comparing the specificity of antibody immunity elicited by additional vaccine modalities could provide a basis for determining whether some will be more resistant to viral evolution.

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Study design and SARS-CoV-2 vaccine sera and convalescent plasmas
De-identified post-vaccination sera were obtained as secondary research samples from the National Institutes of Allergy and Infectious Diseases-sponsored mRNA-1273 phase 1 clinical trial (NCT04283461) (12). We obtained samples from 14 individuals who received two 250 µg doses of the mRNA-1273 vaccine, and 8 individuals who received two 100 µg doses. All individuals were between ages 18 and 55 years old. The samples were collected under the human subject approvals described in (12). Due to the de-identified nature of the samples, the work described in this paper was deemed non-human subjects research by the Fred Hutchinson Cancer Research Center Institutional Review Board.
Previously reported results from samples from two cohorts of SARS-CoV-2 convalescent individuals are reanalyzed here (15,16). One cohort of convalescent plasma samples were previously described (13,15) and collected as part of a prospective longitudinal cohort study of individuals with SARS-CoV-2 infection in Sea le, WA February-July 2020. The plasmas from 17 individuals were examined here (8/17 female; age range 23-76 years, mean 51.6 years, median 56 years). All data from this cohort (i.e., the neutralization and RBD-and spike-binding activity of plasmas pre-and post-depletion of RBD-binding antibodies in Fig. 1 and RBD-binding escape maps in Fig. 4, S6B, and S7) were previously reported (15) with the exception of neutralization assays in All data from the second cohort of plasma samples (n=5) (i.e., the aggregated escape maps in Fig. 4) were previously reported (16) and are simply reanalyzed here. The plasmas were originally collected 21-35 days post-symptom onset as part of a prospective longitudinal cohort study of SARS-CoV-2 convalescent individuals in New York, NY, under the human subject approvals described in (14).

RBD deep mutational scanning library
The yeast-display RBD mutant libraries are previously described (22,23). Briefly, duplicate mutant libraries were constructed in the spike receptor binding domain (RBD) from SARS-CoV-2 (isolate Wuhan-Hu-1, Genbank accession number MN908947, residues N331-T531) and contain 3,804 of the 3,819 possible amino-acid mutations, with >95% present as single mutants. Each RBD variant was linked to a unique 16-nucleotide barcode sequence to facilitate downstream sequencing. As previously described, libraries were sorted for RBD expression and ACE2 binding to eliminate RBD variants that are completely misfolded or non-functional (i.e., lacking modest ACE2 binding affinity) (22).

FACS sorting of yeast libraries to select mutants with reduced binding by polyclonal post-vaccination sera
Serum mapping experiments were performed in biological duplicate using the independent mutant RBD libraries, similarly to as previously described for monoclonal antibodies (22) and exactly as previously described for polyclonal plasmas (44). Briefly, mutant yeast libraries induced to express RBD were washed and incubated with serum at a range of dilutions for 1 h at room temperature with gentle agitation. For each serum, we chose a sub-saturating dilution such that the amount of fluorescent signal due to serum antibody binding to RBD was approximately equal across samples. The exact dilution used for each serum is given in Supplementary Table 2. 8 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. After the serum incubations, the libraries were secondarily labeled with 1:100 FITC-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa-647-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064) to label for bound serum antibodies. A flow cytometric selection gate was drawn to capture 3-6% of the RBD mutants with the lowest amount of serum binding for their degree of RBD expression (Fig. S2, S3). We also measured what fraction of cells expressing unmutated RBD fell into this gate when stained with 1x and 0.1x the concentration of serum. For each sample, approximately 10 million RBD+ cells (range 7.3e6 to 1.4e7 cells) were processed on the cytometer, with between 3e5 and 6e5 plasma-escaped cells collected per sample (see percentages in Table S2). Antibody-escaped cells were grown overnight in SD-CAA (6.7g/L Yeast Nitrogen Base, 5.0g/L Casamino acids, 1.065 g/L MES acid, and 2% w/v dextrose) to expand cells prior to plasmid extraction. Miniprep II) as previously described (22). The 16-nucleotide barcode sequences identifying each RBD variant were amplified by PCR and prepared for Illumina sequencing as described in (23). Barcodes were sequenced on an Illumina HiSeq 2500 with 50 bp single-end reads. To minimize noise from inadequate sequencing coverage, we ensured that each antibody-escape sample had at least 2.5x as many post-filtering sequencing counts as FACS-selected cells, and reference populations had at least 2.5e7 post-filtering sequencing counts.

Analysis of deep sequencing data to compute each mutation's escape fraction
Escape fractions were computed as described in (22), with minor modifications as noted below. We used the dms_variants package (h ps://jbloomlab.github.io/dms_variants/, version 0.8.5) to process Illumina sequences into counts of each barcoded RBD variant in each pre-sort and antibody-escape population using the barcode/RBD look-up table from (23).
For each serum selection, we computed the "escape fraction" for each barcoded variant using the deep sequencing counts for each variant in the original and serum-escape populations and the total fraction of the library that escaped antibody binding via the formula provided in (22). These escape fractions represent the estimated fraction of cells expressing that specific variant that falls in the escape bin, such that a value of 0 means the variant is always bound by serum and a value of 1 means that it always escapes serum binding. We then applied a computational filter to remove variants with low sequencing counts or highly deleterious mutations that might cause antibody escape simply by leading to poor expression of properly folded RBD on the yeast cell surface (22,23). Specifically, we removed variants that had (or contained mutations with) ACE2 binding scores < −2.35 or expression scores < −1, using the variant-and mutation-level deep mutational scanning scores from (23).
Note that these filtering criteria are slightly more stringent than those used in (22) but are identical to those used in (15,16,25). We next deconvolved variant-level escape scores into escape fraction estimates for single mutations using global epistasis models (45) implemented in the dms_variants package, as detailed at (h ps://jbloomlab.github.io/dms_variants/dms_variants.globalepistasis.html) and described in (22). The reported scores throughout the paper are the average across the libraries; these scores are also in Supplementary Table 3.
Correlations in final single-mutant escape scores are shown in Fig. S4.
For plo ing and analyses that required identifying RBD sites of "strong escape" (e.g., choosing which sites to show in logo plots in Fig. 2, 3, S5, S6, S7), we considered a site to mediate strong escape if the total escape (sum of mutation-level escape fractions) for that site exceeded the median across sites by >5-fold, and was at least 9 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. 5% of the maximum for any site. Full documentation of the computational analysis is at h ps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Moderna.

Generation of pseudotyped lentiviral particles
We used spike-pseudotyped lentiviral particles that were generated essentially as described in (46), using a codon-optimized SARS-CoV-2 spike from Wuhan-Hu-1 that contains a 21-amino-acid deletion at the end of the cytoplasmic tail (13) and the D614G mutation that is now predominant in human SARS-CoV-2 (47). The plasmid encoding this spike, HDM_Spikedelta21_D614G, is available from Addgene (#158762) and BEI (NR-53765), and the full sequence is at (h ps://www.addgene.org/158762). Point mutations were introduced into the RBD of this plasmid via site-directed mutagenesis. Therefore, all mutations tested in this paper are in the G614 background, and are compared to a "wildtype" spike with G614.

Titering of pseudotyped lentiviral particles
Titers of spike-pseudotyped lentiviral particles were determined as described in (46)  Fraction infectivity of each serum antibody-containing well was calculated relative to a "no-serum" well inoculated with the same initial viral supernatant (containing wildtype or mutant RBD) in the same row of the plate. We used the neutcurve package (h ps://jbloomlab.github.io/neutcurve version 0.5.2) to calculate the inhibitory concentration 50% (IC50) and the neutralization titer 50% (NT50), which is simply 1/IC50, of each

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint Depletion of RBD-binding antibodies from polyclonal sera Two rounds of sequential depletion of RBD-binding antibodies were performed for vaccine-elicited sera. Magnetic beads conjugated to the SARS-CoV-2 RBD (AcroBiosystems, MBS-K002) were prepared according to the manufacturer's protocol. Beads were resuspended in ultrapure water at 1 mg beads/mL and a magnet was used to wash the beads 3 times in PBS with 0.05% BSA. Beads were then resuspended in PBS with 0.05% BSA at 1 mg beads per mL. Beads (manufacturer-reported binding capacity of 10-40 µg/mL anti-RBD antibodies) were incubated with human sera at a 3:1 ratio beads:serum (150 µL beads + 50 µL serum), rotating overnight at 4°C. A magnet (MagnaRack™ Magnetic Separation Rack, ThermoFisher CS15000) was used to separate antibodies that bind RBD from the supernatant, and the supernatant (the post-RBD antibody depletion sample) was removed. A mock depletion (pre-depletion sample) was performed by adding 150 µL of PBS + 0.05% BSA and incubating rotating overnight at 4°C. A second round of depletion was then performed to ensure full depletion of RBD-binding antibodies. For the neutralization assays on these sera depleted of RBD-binding antibodies shown in Fig. S1D; the reported serum dilution is corrected for the dilution incurred by the depletion process.

Measurement of serum binding to RBD or spike by ELISA
The IgG ELISAs for spike protein and RBD were conducted as previously described (48). Briefly, ELISA plates were coated with recombinant spike and RBD antigens described in (48)  100-110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 µg/mL. Both antibodies were recombinantly produced by Genscript. The rREGN10987 is that used in (25) and the variable domain heavy and light chain sequences for r4A8 were obtained from Genbank GI 1864383732 and 1864383733 (21) and produced on a human IgG1 and IgK background, respectively. Pre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates. Secondary labeling was performed with goat anti-human IgG-Fc horseradish peroxidase (HRP) (1:3000, Bethyl Labs, A80-104P). Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction. OD450 was read on a Tecan infinite M1000Pro plate reader. The area under the curve (AUC) was calculated as the area under the titration curve with the serial dilutions on a log-scale.

Data visualization
The static logo plot visualizations of the escape maps in the paper figures were created using the dmslogo package (h ps://jbloomlab.github.io/dmslogo , version 0.6.2) and in all cases the height of each le er indicates the escape fraction for that amino-acid mutation calculated as described above. For each sample, the y-axis is scaled to be the greatest of (a) the maximum site-wise escape metric observed for that sample, (b) 20x the median site-wise escape fraction observed across all sites for that serum, or (c) an absolute value of 1.0 (to appropriately scale samples that are not "noisy" but for which no mutation has a strong effect on antibody binding). Sites K417, L452, S477, E484, and N501 have been added to logo plots due to their frequencies among circulating viruses. The code that generates these logo plot visualizations is available at h ps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Moderna/blob/main/results/summary/escape_profiles.md.
In many of the visualizations (e.g., Fig. 2, 3, 4, S5, S6, S7), the RBD sites are categorized by epitope region (24) and colored accordingly. We define the class 1 epitope as residues 403+405+406+417+420+421+453+455-460+473-476+486+487+489+504, the class 2 epitope as residues 11 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint 472+483-485+490-494, the class 3 epitope to be residues 345+346+437-452+496+498-501, and the class 4 epitope as residues 365-372+382-386. For the static structural visualizations in the paper figures, the RBD surface (PDB 6M0J, (50)) was colored by the site-wise escape metric at each site, with white indicating no escape and red scaled to be the same maximum used to scale the y-axis in the logo plot escape maps, determined as described above. We created interactive structure-based visualizations of the escape maps using dms-view (51) that are available at h ps://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_Moderna/. The logo plots in these escape maps can be colored according to the deep mutational scanning measurements of how mutations affect ACE2 binding or RBD expression as described above.
For the composite line plots shown in Fig. 4, the convalescent (day 15-60) group includes two independent cohorts of individuals, one recruited in New York, NY (n=5) (14), and another recruited in Sea le, WA (n=11) (13). The convalescent (day 100-150) group is from the longitudinal cohort recruited in Sea le, WA (n=11). The escape maps for convalescent individuals were previously reported in (15,16). The mRNA-1273 (day 119) group includes individuals who were vaccinated with either the 100 or 250 µg vaccine dose (n=8 and n=14, respectively). The y-axis maximum is scaled to 1.1 times the maximum group mean site-total escape among all groups, so outlier points exceeding this value are not shown.

Quantification and Statistical Methods
In Fig. 1D, the percent of neutralizing activity of vaccine-elicited sera and convalescent plasma due to RBD-binding antibodies is shown as a Tukey boxplot (middle line=median, box limits=interquartile range) with individual measurements overlaid as points. P-values are from a log-rank test accounting for censoring, p=1.0 ✕ 10 -6 for convalescent vs. vaccine (day 15-60), and p=0.15 for convalescent vs. vaccine (day 100-150).

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint          Table S1. Serum neutralization titers pre-and post-depletion of RBD-binding antibodies.

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Resource Availability
Further information and requests for reagents and resources should be directed to and will be fulfilled by Jesse Bloom (jbloom@fredhutch.org).

Data and Code Availability:
We provide data and code in the following ways: • The complete code for the full computational data analysis pipeline of the mapping experiments is available on GitHub at h ps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Moderna.
• The escape fraction measured for each mutation in Supplementary Table 3

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made occluded in the "down" conformation exposed in the "up" and "down" conformations  T385  K386  K417  G446  G447  N448  Y449  N450  L452  L455  F456  I472  Y473  A475  S477  E484  G485  F486  N487  Y489  F490  Q493  S494  G496  N501  0 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint 24 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. surface after averaging across all sera/plasmas in each group. The RBD surface coloring is scaled from white to red, with white indicating no escape, and red indicating the site with the greatest escape. The color scaling spans the full range of white to red for each serum/plasma group, so the quantitative scale is not comparable across groups. Escape maps for monoclonal antibodies previously described in (16,22,(25)(26)(27), and convalescent plasmas in (15,16).

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint Fig. S1. Raw ELISA and neutralization curves of mRNA-1273 serum samples before and after depletion of RBD-binding antibodies (A) Effect of RBD antibody depletion on binding to RBD and spike by "synthetic sera'' comprised of pre-pandemic pooled serum with the NTD-targeting antibody r4A8 (21) or RBD-targeting antibody rREGN10987 (49). Antibodies were added to pre-pandemic serum at 50 µg/mL. The x-axis indicates the dilution factor of the serum+antibody mix, and the y-axis is the ELISA reading at each dilution. These controls were previously used in (15), and demonstrate that the depletions effectively remove RBD-targeting antibodies but not antibodies targeting other epitopes such as the NTD.  quantify the frequency of each mutation in the initial and "antibody escape" cell populations. We quantified the effect of each mutation as the "escape fraction," which represents the fraction of cells expressing RBD with that mutation that fell in the "antibody escape" FACS bin. Escape fractions are represented in logo plots, with the height of each le er proportional to the effect of that amino-acid mutation on antibody binding. The site-level escape metric is the sum of the escape fractions of all mutations at a site. Experimental and computational filtering was used to remove RBD mutants that were misfolded or unable to bind the ACE2 receptor (see Methods).

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ;

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  The projection includes the escape maps of 22 monoclonal antibodies (escape maps first described in (16,22,(25)(26)(27) of the 4 major structural classes to orient the plot. Antibodies are colored according to epitope, as in

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint The site-wise antibody-binding escape for each of the vaccine and convalescent samples tested in neutralization assays in panels B and C and Fig. 5. (B) The effects of RBD mutations on neutralization of G614 spike-pseudotyped lentiviral particles with the indicated mutations, shown as the inhibitory concentration 50% (IC50). Naturally-occurring mutations are colored in white, and non-naturally-occurring mutations in gray. (C) The fold-change in IC50 compared to wild type spike, grouped by vaccine or convalescent sera (as in Fig. 5C, but shown here for all RBD mutants). Dashed line indicates no change in neutralization relative to wild type spike. Horizontal bars represent the group median fold-change IC50. In (B) and (C), each point represents the IC50 from one individual calculated from technical duplicates. The highest two points for E484K and K417N-G446V-E484K, and the highest 4 points for "all RBD antibodies depleted" are at the limit of detection. (D) Spike-pseudotyped lentiviral particle entry titers for RBD mutants tested in neutralization assays, calculated as the mean relative luciferase units per µL from 16 technical replicates. Mutations that are observed in at least one SARS-CoV-2 sequence in GISAID are colored in white, and non-naturally-occurring mutations in gray. All spike sequences contained G614, which fixed in circulating sequences in 2020 (47). All full neutralization curves are in Fig. S9 and raw IC50 and NT50 values are at h ps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Moderna/blob/main/experimental_data/results/mutant_ne uts_results/fitparams.csv.

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint x-axis is the serum dilution, and the y-axis is the fraction of viral infectivity remaining at that dilution. The neutralization curves were fit and plo ed using neutcurve (h ps://jbloomlab.github.io/neutcurve/ , version 0.5.2).
IC50s were calculated by fi ing 2-parameter Hill curves with the baselines fixed at one and zero. These IC50s were used to determine the fold-change values in Fig. 5 and S8. In each plot, mutants are shown with the wildtype tested on the same date. Error bars represent the standard error of n=2 replicates. For readability, no more than 6 curves are shown per plot. In Fig. 5D, the wildtype curve from the first assay date is shown.

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint Supplementary Files: Table S1. Serum neutralization titers pre-and post-depletion of RBD-binding antibodies. This file contains the serum neutralization titers from vaccinated individuals before (NT50_pre) and after (NT50_post) depletion of RBD-binding antibodies. The table is available online at h ps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Moderna/blob/main/experimental_data/results/rbd_absor ptions/TableS1.csv. Table S2. Information on FACS sorting to select cells expressing RBD mutants with reduced binding by sera from vaccinated individuals. The file gives the number of antibody-escaped cells collected per selection for each replicate library and the percent of RBD+ cells in the antibody-escape gate for each selection, and the exact dilution used for each serum selection. The file is also available on GitHub at h ps://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_Moderna/blob/main/data/TableS2_FACSinfo.csv.

Table S3. Measurements of effects of all amino-acid mutations to the RBD on serum binding.
The file gives the "escape fraction" for each mutation, as well as the total escape fraction at each site and the maximum escape fraction for any mutation at the site. This file includes escape fractions for sera from individuals vaccinated with mRNA-1273 as well as the previously reported escape fractions for convalescent plasma (15,16).

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 14, 2021. ; https://doi.org/10.1101/2021.04.14.439844 doi: bioRxiv preprint