Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals

Immunization with mosaic-8b (nanoparticles presenting 8 SARS-like betacoronavirus [sarbecovirus] receptor-binding domains [RBDs]) elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efﬁcacy, we evaluated the effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, ﬁnding the greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate mapping, in which antibodies from speciﬁc cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b-and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were speciﬁc for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19-vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.


INTRODUCTION
Spillover of animal SARS-like betacoronaviruses (sarbecoviruses) has resulted in two health crises in the past 20 years: the SARS-CoV (SARS-1) epidemic in the early 2000s and the recent COVID-19 pandemic caused by SARS-CoV-2 (SARS-2).6][7][8] For example, in Omicron VOCs, substitutions in the SARS-2 spike protein receptor-binding domains (RBDs), the major target of neutralizing Abs and detectable cross-variant neutralization, 9,10 have reduced the efficacies of vaccines and therapeutic monoclonal Abs (mAbs). 8,11One strategy to increase protective responses to SARS-2 VOCs involves novel variant vaccine boosters. 12Since repeated updating of COVID vaccines is impractical and expensive, a more optimal strategy would be a vaccine that does not require changing to protect against both emerging sarbecoviruses and SARS-2 VOCs.
To develop a vaccine that would protect against unknown sarbecoviruses and new SARS-2 variants, we used an approach involving simultaneous display of eight different sarbecovirus RBDs arranged randomly on protein-based 60-mer nanoparticles (mosaic-8b RBD nanoparticles) (Figure 1A) and evaluated Ab responses against RBDs representing both matched (RBD on the nanoparticle) and mismatched (RBD not on the nanoparticle) viruses. 16We found that mosaic-8b nanoparticles showed enhanced heterologous binding, neutralization, and protection from sarbecovirus challenges compared with homotypic (SARS-2 RBD-only) nanoparticles in animal models. 16,17For example, mosaic-8b immunizations showed protection in K18-hACE2 transgenic mice, a stringent model of coronavirus infection, 18 from both SARS-2 and a mismatched SARS-1 challenge, whereas homotypic SARS-2 immunized mice were protected only from SARS-2 challenge. 17o explain the increased cross-reactivity of Abs elicited by mosaic-8b, we hypothesized that B cells with B cell receptors (BCRs) that can crosslink using both of their antigen-binding Fab arms between adjacent non-identical RBDs to allow recognition of conserved epitopes would be preferentially stimulated to produce cross-reactive Abs, as compared with B cells presenting BCRs that bind to variable epitopes, which could rarely, if ever, crosslink between non-identical RBDs arranged randomly on a nanoparticle 17 (Figure S1A).By contrast, homotypic RBD nanoparticles presenting identical RBDs are predicted to bind BCRs against immunodominant strain-specific epitopes presented on adjacent identical RBDs.Epitope mapping of polyclonal antisera elicited by mosaic-8b versus homotypic SARS-2 RBD nanoparticles using deep mutational scanning (DMS) 19 provided evidence supporting this model: Abs from mosaic-8b antisera primarily targeted more conserved class 4 and class 1/4 RBD epitopes (Figure 1B) (RBD epitope nomenclature from Barnes et al. 20 ) that show less variability in sarbecoviruses and SARS-2 VOCs because they contact other portions of the spike trimer, 17 whereas homotypic antiserum Abs primarily targeted variable class 1 and 2 RBD epitope regions that are more accessible and are not involved in contacts with non-RBD portions of spike. 17These results, combined with animal experiments showing promising cross-reactive binding and neutralization data for elicited polyclonal antisera 16,17 and mAbs, 21 suggested that mosaic-8b RBD nanoparticles repre-sent a promising vaccine strategy to protect against current and future SARS-2 VOCs as well as additional animal sarbecoviruses that could spill over into humans.
The mosaic-8b-immunized animals in our previous studies were naive with respect to SARS-2 exposure.By contrast, the majority of humans receiving a mosaic-8b vaccine would have already been infected with SARS-2, vaccinated, or both.An important question, therefore, is whether mosaic-8b immunization would elicit broadly cross-reactive recognition of conserved sarbecovirus RBD epitopes in animals that were pre-vaccinated with spike-based COVID-19 vaccines, e.g., mRNA-lipid nanoparticle (LNP) vaccines.Specifically, we aimed to understand the role of immune imprinting, also known as original antigenic sin (OAS), as it relates to eliciting breadth in non-naive individuals.Immune imprinting was initially postulated to explain why influenza virus infections with new and distinct viral strains resulted in preferential boosting of Ab responses against epitopes shared with the original strain. 22,23Recently, experiments in Ab fatemapping (Igk Tag ) mice demonstrated that, upon repeated immunization, serum Abs continue to derive from B cells that were initially activated in the primary response. 24Abs induced de novo in subsequent responses were suppressed, consistent with OAS.This reliance on primary cohort B cells (primary addiction) decreased with antigenic distance, resulting in partial alleviation of OAS upon boosting with BA.1 spike mRNA-LNP after single-dose WA1 mRNA priming. 24To investigate basic mechanisms in B cell activation in animals with previous antigenic exposure and to inform future mosaic-8b immunizations in humans with previous SARS-2 exposure, we designed experiments to compare immune responses to RBD nanoparticles and additional COVID-19 immunizations in pre-vaccinated animals.
Here, we evaluated immune responses to mosaic-8b, mosaic-7 (mosaic-8b without a SARS-2 RBD), homotypic SARS-2, and admix-8b (mixture of 8 homotypic nanoparticles) (Figure 1A) in non-human primates (NHPs) and in mice that were previously vaccinated with DNA, mRNA, a self-amplifying replicon RNA (repRNA), or adenovirus-vectored COVID-19 vaccines (Table S1).As previously observed in SARS-2 naive animals, 16,17 we found that mosaic-8b immunization in three independent pre-vaccinated animal models elicited broadly crossreactive binding and neutralizing Ab responses against viral strains that were both matched and mismatched.As also observed in naive animals, we found increased cross-reactivity of Abs elicited by mosaic-8b compared with homotypic SARS-2 RBD nanoparticles in pre-vaccinated animals and also Figure 1.RBDs used to make nanoparticles and for assays (A) Models of mosaic-8b, mosaic-7 (mosaic-8b without SARS-2 RBD), homotypic SARS-2, and admix-8b RBD nanoparticles constructed using coordinates of an RBD (PDB: 7BZ5), SpyCatcher (PDB: 4MLI), and i3-01 nanoparticle (PDB: 7B3Y).(B) Sequence conservation determined using the ConSurf Database 13 of the 16 sarbecovirus RBDs used to make nanoparticles and/or for assays shown on two views of an RBD surface (PDB: 7BZ5).Classes 1, 2, 3, 4, and 1/4 epitopes are outlined in different colors using information from Fab-RBD or Fab-spike trimer structures (C102, PDB: 7K8M; C002, PDB: 7K8T; S309, PDB: 7JX3; CR3022, PDB: 7LOP; and C118, PDB: 7RKV).(C) List of sarbecoviruses from which the RBDs in mosaic-8b and admix-8b were included (matched) or not included (mismatched).Clades were defined as described. 14D) Phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega. 15Viruses with RBDs included in mosaic-8b are highlighted in gray rectangles.The scale bar represents phylogentic distance of 0.05 nucleotide substitutions per site.(E) Amino acid sequence identity matrix of RBDs based on alignments using Clustal Omega. 15ee also Figure S1 and Table S1.demonstrated increased cross-reactivity for mosaic-8b versus admix-8b sera in mice that were pre-vaccinated with a Pfizerlike mRNA-LNP.Molecular fate-mapping 24 studies in mice showed that de novo Abs were predominantly induced in prevaccinated mice only with mosaic-8b boosting and that these Abs were specific for variant RBDs with increased identity to RBDs on mosaic-8b.Results from these studies contribute to increased understanding of basic immune phenomena involving B cell responses under potential OAS conditions.In addition, they support the use of a mosaic-8b RBD-nanoparticle vaccine in humans with previous SARS-2 exposure to protect against future SARS-2 variants and also of critical importance, to prevent another pandemic caused by spillover of an animal sarbecovirus with the potential for human-to-human transmission.
Throughout the paper, we refer to COVID-19 pre-vaccinations in NHPs and mice as ''vaccinations,'' whereas we describe subsequent injections of additional immunogens (either RBD nanoparticles or a genetically encoded immunogen) in the non-naive animals as ''immunizations,'' with the first injection after prevaccination referred to as a prime and subsequent immunizations as boosts.For each immunogen evaluated in a pre-vaccinated animal model, we assessed serum Ab binding to spike antigens (different sarbecovirus RBDs or WA1 spike) by ELISA and using an in vitro neutralization assay.Of potential relevance to results in humans, both binding and neutralizing Ab levels correlate with vaccine efficacy. 28We present binding and neutralization data against sarbecovirus antigens (ELISAs) or pseudoviruses (neutralization assays) for each pre-vaccinated and then immunized cohort in the form of line plots with determinations of statistically significant differences between cohorts evaluated using pairwise comparisons for each viral strain.

Mosaic-8b immunizations in previously vaccinated NHPs elicit cross-reactive Ab responses
To address the immunogenicity of a mosaic-8b immunization in a pre-vaccinated animal model, we immunized 14 NHPs that had previously received nucleic acid-based SARS-2 vaccines.0][31] 8 weeks before immunization, we restratified the animals into three groups (n = 4-5), each of which was similar in mean and range of weights, ages, sex, and Ab neutralization titers against SARS-2 D614G (Figure S2A), without considering vaccine history.The mixed immune history in these groups of pre-vaccinated NHPs is representative of a complex immune history in people who have been vaccinated and/or infected multiple times.The NHPs were immunized at week 0 with two doses (8 weeks apart) of either mosaic-8b or homotypic SARS-2 RBD nanoparticles or with a bivalent RNA replicon vaccine formulated with Lipid InOrganic Nanoparticles (repRNA-LION 31 ) encoding the WA1 and the Omicron BA.1 spike proteins, which mimicked immunogens used in commercial bivalent mRNA-LNP vaccines (Figure 2A).Serum was then sampled every 2 weeks for 22 weeks after the first nanoparticle or bivalent repRNA boost to measure the peak and subsequent contraction of serum binding and neutralizing Ab responses (Figure 2A).
We performed ELISAs against a panel of RBD and spike proteins to assess serum Ab binding titers and pseudovirus assays against available viral strains to determine Ab neutralizing titers (Figures 2, S2B, and S2C).Responses at week 0 (immediately prior to nanoparticle or repRNA-LION immunizations) showed equivalent titers across the three groups (Figure 2A).The mosaic-8b and homotypic SARS-2 nanoparticle-immunized NHPs elicited significantly higher levels of binding (Figures 2B  and S2B) and neutralization (Figures 2C and S2C) than the bivalent repRNA-LION immunization after week 0. Notably, RBDnanoparticle immunizations in pre-immunized NHPs elicited high overall ELISA and neutralization titers against a diverse array of both matched and mismatched sarbecoviruses (Figures 2B, 2C, S2B, and S2C).As generally observed for vaccines, [32][33][34] we found that elicited Ab binding responses contracted after both RBD-nanoparticle and bivalent repRNA immunizations (5-to 8-fold from weeks 2 to 8) and after boosting (10-fold from weeks 10 to 22).Contractions in binding Ab levels largely correlated with drops in neutralizing Ab titers (Figures 2B, 2C, S2B, and S2C).
Mean serum binding titers for all RBD variants evaluated by ELISA were significantly higher at 5 of 6 time points evaluated after priming pre-vaccinated NHPs for mosaic-8b compared with homotypic SARS-2 samples (Figures 2B and S2B).In addition, at weeks 2, 4, and 8 (after only a single nanoparticle immunization), mosaic-8b elicited significantly higher neutralization titers compared with immunization with homotypic SARS-2 (Figures 2C and S2C).At every time point, either mean binding titers, mean neutralization titers, or both were significantly higher for mosaic-8b than for homotypic samples across divergent sarbecoviruses, a required result for potentially broad protection since both Ab binding titer and neutralization correlate with vaccine efficacy in licensed SARS-2 vaccines. 34In summary, critical for its potential use in non-naive humans, mosaic-8b elicited cross-reactive Ab responses even in a background of four prior COVID-19 vaccinations, and mosaic-8b immunizations consistently elicited a broader Ab response than homotypic SARS-2 and bivalent repRNA immunizations in the pre-vaccinated NHP model (Figures 2 and S2).S1).For mouse experiments, we could increase the number of animals per cohort and also add new cohorts (admix-8b and mosaic-7) to comparisons of mosaic-8b, homotypic SARS-2, and mRNA-LNP or viral vector immunizations after pre-vaccinations (Figures 3A, 3D, and 4A).

Mosaic
Pre-vaccinated mice were bled prior to immunizations at day 0 and after immunizations at days 28 and 56.As in the NHP experiments, we performed ELISAs against a panel of RBD and spike proteins and pseudovirus assays against available viral strains (Figures 3, 4, S3, and S4).In the WA1 mRNA-LNP prevaccination experiments, responses at day 0 (after all animals had received the same course of mRNA-LNP vaccines) showed significant differences in binding titers elicited by the pre-vaccinations across the cohorts (Figures S3B and S3E), possibly because of differences in initial vaccine responses or in Ab contraction.Thus, we applied baseline corrections to (A) Left: schematic of vaccination/immunization regimen.NHPs were vaccinated at the indicated weeks prior to RBD nanoparticle or repRNA prime and boost immunizations at weeks 0 and 8. Middle: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega. 15RBDs included in mosaic-8b are highlighted in gray rectangles.Right: geometric mean ELISA binding titers at week 0 (after pre-vaccinations but prior to RBD nanoparticle or repRNA immunizations) against indicated viral antigens.(B) Geometric mean ELISA binding titers at the indicated weeks after immunization with mosaic-8b, homotypic SARS-2, or bivalent WA1/BA.1 repRNA against indicated viral antigens.(C) Geometric mean neutralization titers at the indicated weeks after immunization with mosaic-8b, homotypic SARS-2, or bivalent WA1/BA.1 repRNA against indicated sarbecovirus pseudoviruses.See also Figure S2.
account for different mean responses at day 0 in each of the four groups (see STAR Methods).Neutralization potencies at day 0 were similar for all cohorts (Figures S3C and S3F) and therefore were not baseline-corrected.Baseline corrections were not required for the ChAdOx1 pre-vaccination experiments (Figure S4).mRNA-LNP pre-vaccinated mice In the WA1 Pfizer-like mRNA-LNP pre-vaccinated animals (Figure 3A; Table S1), the increase in Ab binding titers was significantly higher at days 28 and 56 for mosaic-8b than for the other cohorts when evaluated against sarbecovirus RBDs (especially notable for day 56) (Figure 3B).In general, while RBD-nanoparticle immunizations boosted Ab binding against RBDs, immunization with an additional dose of WA1 mRNA-LNP did not appreciably boost Ab responses, especially at day 56 (Figure 3B).When comparing responses to different protein nanoparticles, mosaic-8b immunizations induced significantly higher increases in mean binding titers than either admix-8b or homotypic SARS-2 immunizations.However, mean neutralization titers did not show as pronounced differences between cohorts, although the titers for mosaic-8b were higher than titers for admix-8b or homotypic SARS-2 against some viral strains (e.g., SHC014, WIV1, SARS-1) (Figure 3C).
To evaluate whether a mosaic RBD nanoparticle without a SARS-2 RBD would also overcome potential OAS effects, we compared Ab responses raised by mosaic-7 (includes all mosaic-8b RBDs except for SARS-2; Figure 1A) to mosaic-8b responses (Figures 3D-3F).Mice were pre-vaccinated with two doses of WA1 mRNA-LNP and one dose of WA1/BA.5 bivalent mRNA-LNP and then immunized with either two doses of an RBD nanoparticle (mosaic-8b or mosaic-7) or an additional dose of WA1/BA.5 mRNA-LNP, which was included to mimic a more up-to-date immune history in the human population (Figure 3D).We found consistently greater increases in mean Ab binding titers at days 28 and 56 with respect to day 0 for mosaic-7 compared with mosaic-8b-immunized animals across all RBDs (Figure 3E).Although increases in binding titers with respect to day 0 values were higher for mosaic-7 than for mosaic-8b, differences in mean titers for the corresponding non-baseline-corrected data were only marginal between the two groups at day 28, dropping to no significance at day 56 (Figure S3G), suggesting that immunization by these RBD nanoparticles had elicited a maximum binding anti-RBD Ab response.In addition, there were no neutralization differences for mosaic-8b and mosaic-7 antisera across strains (Figure 3F), as also observed for the non-baseline-corrected mean binding titers.In this pre-vaccination/immunization regimen, both mosaic-8b and mosaic-7 were significantly better in inducing binding Abs than an additional dose of WA1/BA.5 (Figure S3G).

ChAdOx1 pre-vaccinated mice
We also examined the responses to mosaic-8b, homotypic SARS-2, and admix-8b in ChAdOx1 pre-vaccinated animals (Figures 4 and S4; Table S1).In these experiments, we compared RBD-nanoparticle immunization with immunization of an additional dose of ChAdOx1 or of WA1 mRNA-LNP (Figure 4A).Overall Ab binding responses across RBDs were generally higher for mosaic-8b and admix-8b than for homotypic SARS-2, and the three nanoparticle immunizations produced higher binding titers than an additional immunization with WA1 mRNA-LNP or ChAdOx1, as reflected in some cases with significant differences in mean ELISA binding titers (Figure 4B).Neutralization titers against SARS-1 were higher for mosaic-8b and admix-8b than for homotypic SARS-2 at day 56, but differences in mean of mean neutralization titers were not significant across cohorts (Figure 4C).Overall, mosaic-8b and admix-8b prime/boost immunizations in ChAdOx1 pre-vaccinated mice elicited the broadest binding and neutralization responses.
To determine which RBD epitopes were targeted in prevaccinated and then immunized mice, we performed DMS 19 using yeast display libraries derived from WA1 and XBB.1.5RBDs for ChAdOx1 pre-vaccinated mice that were immunized with either mosaic-8b, admix-8b, homotypic SARS-2, or WA1 For experiments shown schematically in (A) and (D), binding responses at day 0 (immediately prior to nanoparticle or other vaccine immunizations) showed significant differences across cohorts in titers elicited by the pre-vaccinations.We therefore applied baseline corrections to account for different mean responses at day 0 prior to immunizations in each of the four groups for data in (B) and each of the three groups for data in (E) (see STAR Methods).Non-baseline-corrected binding data for (E) are shown in Figure S3G.Geometric means of fold change in ED 50 or geometric means of ID 50 values for all animals in each cohort are indicated by symbols connected by thick colored lines.Mean titers against indicated viral antigens or pseudoviruses were compared pairwise across immunization cohorts by Tukey's multiple comparison test with the Geisser-Greenhouse correction (as calculated by GraphPad Prism).Significant differences between cohorts linked by vertical lines are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(A) Left: schematic of vaccination/immunization regimen for (B) and (C).Mice were vaccinated at the indicated days prior to RBD-nanoparticle prime and boost immunizations at days 0 and 28 or mRNA-LNP prime immunization at day 0. Right: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega. 15RBDs included in mosaic-8b are highlighted in gray rectangles.(B) Geometric mean fold change ELISA binding titers at the indicated days after immunization with mosaic-8b, admix-8b, homotypic SARS-2, or WA1 mRNA-LNP against indicated viral antigens.(C) Geometric mean neutralization titers at the indicated weeks after immunization with mosaic-8b, admix-8b, homotypic SARS-2, or WA1 mRNA-LNP against indicated sarbecovirus pseudoviruses.(D) Schematic of vaccination/immunization regimen for (E) and (F).Mice were vaccinated at the indicated days prior to RBD-nanoparticle prime and boost immunizations at days 0 and 28 or mRNA-LNP prime immunization at day 0. (E) Geometric mean fold change ELISA binding titers at the indicated days after immunization with mosaic-8b, mosaic-7, or WA1/BA.5 mRNA-LNP against indicated viral antigens.Compare with Figure S3G (non-baseline-corrected geometric mean ELISA binding titers).(F) Geometric mean neutralization titers at the indicated weeks after immunization with mosaic-8b, mosaic-7, or WA1/BA.5 mRNA-LNP against indicated sarbecovirus pseudoviruses.See also Figure S3.
mRNA-LNP (Figures 4A and 5).To aid in interpreting epitope mapping of polyclonal antisera, we first conducted control experiments by comparing DMS of individual mAbs with known epitopes (classes 1, 2, 3, 4, and 1/4; Figure 1B) to DMS using different mixtures of the same mAbs (Figure S5).As previously shown for mapping of mAb epitopes, 35,36 DMS revealed the expected escape profiles matching the epitopes of the control mAbs (Figure S5).However, when mAbs were mixed in various ratios, DMS signals were obscured, with little or no highlighting of epitopes in the equimolar mix and only partial highlighting when the mixtures contained either more class 3, class 1/4, and class 4 mAbs or more class 1/class 2 mAbs (Figure S5).Thus, a polyclonal serum including anti-RBD Abs that are equally distributed across all classes (i.e., a ''polyclass'' antiserum) will likely display low DMS escape fractions across all RBD residues, whereas a polyclonal serum response that is more focused on particular epitope(s) will show more defined escape peaks.
We next analyzed differences in epitope targeting of Abs elicited by mosaic-8b, admix-8b, homotypic SARS-2, and WA1 mRNA-LNP in ChAdOx1 pre-vaccinated mice (Figure 5), keeping in mind (1) the possibility of inducing polyclass Abs and (2) that DMS results can depend on the particular RBD library used in an experiment.For example, class 1 and class 2 anti-RBD Abs from WA1 mRNA-LNP-vaccinated animals do not bind to XBB.1.5,which would emphasize detection of responses to the more conserved class 3 and class 4 epitopes.
We found that both homotypic SARS-2 and WA1 mRNA-LNP immunizations resulted in relatively defined classes 1, 2, and 3 DMS profiles against the WA1 library (Figures 5A and 5B) and defined class 3 and 4 profiles against the XBB.1.5library (Figures 5C and 5D).By contrast, the mosaic-8b and admix-8b DMS profiles were consistent with polyclass Ab induction (Figures 5A-5D).Although sometimes difficult to discern, the majority of responses against the WA1 library mapped to classes 1, 2, or 3 epitopes (Figure 5B), whereas most responses against the XBB.1.5library mapped to class 3 or class 4 epitopes (Figure 5D).We conclude that immunizing pre-vaccinated ChAdOx1 mice with a mosaic or admix RBD-nanoparticle immunogen induces a more polyclass Ab response than immunization with a homotypic protein or mRNA-LNP immunogen.In addition, we note that serum boosting responses after mosaic-8b and admix-8b immunizations in the pre-vaccinated mice showed only weak signals for the more conserved class 3 and class 4 epitopes (Figures 5A and 5B), similar to results for the mAb mix 3 experiment, in which class 1 and class 2 mAbs were in excess over other mAbs (Figure S5).These results imply a diversified response to RBDs after mosaic or admix immunization, i.e., class 1 and class 2 anti-RBD Abs originally induced by ChAdOx1 vaccination dominated serum responses and subsequently masked DMS signals from class 3 and class 4 Abs induced by mosaic-8b and admix-8b.
Molecular fate mapping of mosaic-8b boosting in previously vaccinated mice reveals efficient recall of cross-reactive Abs and generation of strain-specific de novo responses DMS comparisons in pre-vaccinated mice (Figures 5 and S5) involved analyses of mixtures of de novo and recall Ab responses.Thus, we could not determine whether the improved breadth elicited by mosaic-8b immunization was due to de novo responses or recall of cross-reactive Abs elicited by the primary vaccination series.To address this issue, we used recently developed molecular fate-mapping S1pr2-Cre ERT2 .Igk Tag/Tag (S1pr2-Igk Tag ) mice that enable antigen-specific serum Abs to be traced to their cellular and temporal origins. 24In these mice, the C terminus of the immunoglobulin kappa light chain (Igk) is extended to encode a LoxP-flanked FLAG tag and stop codon followed by a downstream Strep tag.B cells bearing this Igk Tag allele produce Abs that are FLAG-tagged unless they are exposed to Cre recombinase, after which they permanently switch the FLAG tag for a Strep tag. 24In S1pr2-Igk Tag mice, tamoxifen-inducible Cre is expressed in B cells as they undergo affinity maturation in germinal centers (GCs). 37Abs derived from the cohort of B cells engaged in GCs at the time of tamoxifen administration, including the cohort's memory B cell and plasma cell progeny, will be fate-mapped as Strep + .Boost-induced de novo GCs that are mainly formed by new cohorts of naive B cells 38 remain FLAG + in the absence of tamoxifen administration, and therefore de novo Abs derived from these B cells are reverse fate-mapped with FLAG. 24Thus, by using tamoxifen to initiate fate mapping of the B cells and Abs elicited by primary mRNA-LNP vaccinations, the FLAG + Abs induced de novo by RBD-nanoparticle immunizations can be distinguished from the recalled primary-cohort-derived Strep + Abs (Figures 6, S6, and S7).
S1pr2-Igk Tag mice were vaccinated twice with WA1 spike mRNA-LNP and then immunized twice with either mosaic-8b Figure 6.Fate mapping of serum antibodies reveals elicitation of cross-reactive recall responses and strain-specific de novo responses elicited by mosaic-8b nanoparticles in previously vaccinated animals Antigenic distance score is defined as the ratio of % amino acid sequence identity of a strain to WA1 RBD divided by % sequence identity of that strain to the closest non-self RBD relative on the nanoparticle.The primary addiction index 24 is defined as Strep/(FLAG + Strep) 3 100.See Figure 1B for RBD epitope classifications.(A) (Left) Schematic of vaccine regimen in S1pr2-Igk Tag/Tag immunized mice.Mice were vaccinated with an mRNA-LNP vaccine encoding WA1 spike at the indicated days prior to RBD-nanoparticle immunizations at days 0 and 28.After each dose of mRNA-LNP vaccine, mice were treated with tamoxifen to switch epitope tags on the Abs produced by B cells activated by the mRNA-LNP vaccinations from FLAG to Strep, so that Ab responses elicited by RBD-nanoparticle boosting can be separated into recall (Strep + ) and de novo (FLAG + ) responses.(Right) Models of RBD nanoparticles that were used for immunizations at days 0 and 28.(B) Comparison of the FLAG + /de novo (blue) and Strep + /recall (red) anti-RBD titers shown at day 42, 2 weeks after a second immunization with mosaic-8b or homotypic SARS-2 nanoparticles.For each strain, the antigenic distance score is shown above the plots and colored with a gradient from purple (score = 1) to yellow (score = 0).Gray-shaded strains are matched to mosaic-8b.(C) The primary addiction index 24 for each RBD is plotted for day 42 serum after two doses of mosaic-8b or homotypic SARS-2.The antigenic distance score is shown above the plots and colored with a gradient from purple (score = 1) to yellow (score = 0).Gray-shaded strains are matched to mosaic-8b.(D) Correlation of the primary addiction index 24 with the antigenic distance score.The best-fit line, R 2 , and p value were calculated by linear regression using Prism.(E) DMS analysis of Strep + Abs from day 0 (prior to RBD-nanoparticle immunization) serum from n = 3 pre-vaccinated mice using an RBD mutant library derived from WA1. Ab binding sites are shaded red according to degree of Strep + Ab escape on the surface of the WA1 RBD (PDB: 6M0J).Locations of residues with high escape scores are indicated on RBD surfaces with gray indicating no escape and shades of red indicating sites with most escape.Residue numbers are color coded according to their RBD epitope classification.(F) DMS analysis using SARS-1 (antigenic distance score = 0.81) and RmYN02 (antigenic distance score = 0.63) RBD mutant libraries of day 56 serum from n = 4 or n = 3 RBD-nanoparticle immunized mice.Ab binding sites are shaded according to degree of Ab escape, with blue for FLAG/de novo responses and red for Strep/ recall responses on the surface of the WA1 RBD (PDB: 6M0J).Comparisons are made for FLAG/de novo and Strep/recall elicited by mosaic-8b and for Strep/ recall elicited by homotypic SARS-2 (FLAG/de novo responses after homotypic SARS-2 immunization were weak to undetectable).Gray-shaded virus names represent strains that are matched to mosaic-8b.Locations of residues with high escape scores are indicated on RBD surfaces with gray indicating no escape and darker shades indicating sites with most escape.Residue numbers are color coded according to their RBD epitope classification.(G) DMS analysis of the Strep/recall compartment of day 56 serum from n = 5 or n = 6 mosaic-8b or homotypic SARS-2 immunized mice using RBD mutant libraries derived from strains with antigenic distance scores of 1.01 (WA1), 1.01 (XBB.1.5),and 0.94 (PRD008).Ab binding sites are shaded red according to degree of Strep/Recall Ab escape on the surface of the WA1 RBD (PDB: 6M0J).Locations of residues with high escape scores are indicated on RBD surfaces with gray indicating no escape and darker shades indicating sites with most escape.Residue numbers are color coded according to their RBD epitope classification.See also Figure S6.
or homotypic RBD nanoparticles (Figure 6A; Table S1).We analyzed binding of total immunoglobulin G (IgG), Strep + (recall), or FLAG + (de novo) Abs as a function of time after vaccination and immunization (Figure S6), obtaining data for the pre-vaccination time frame that were consistent with published results. 24ased on these results, we focused on day 42 serum (2 weeks after final immunization) for assessment of FLAG/Strep Ab reactivity against a panel of RBDs.While both groups of mice recalled cross-reactive Strep + Abs derived from primary responses, only the mosaic-8b-boosted mice induced de novo FLAG + Abs (Figure 6B).The dominance of primary-derived Abs against most RBDs in both groups (Figure 6C) confirmed the strong OASlike ''primary addiction'' previously observed with homologous mRNA-LNP boosting. 24In mRNA-LNP pre-vaccinated mosaic-8b-immunized mice, de novo Ab reactivity was strongest against RBDs antigenically divergent from the priming WA1 RBD and from RBDs that were matched or antigenically highly similar to those on mosaic-8b: the lower the antigenic distance score (ratio between % amino acid identity to WA1 divided by the % identity to the most closely related non-self RBD on the immunizing RBD nanoparticle), the higher the de novo FLAG Ab response against the tested RBD (Figures 6B and 6C).This resulted in a lower reliance on primary cohort B cells, as evaluated by the primary addiction score (Strep/(FLAG + Strep)*100) (Figure 6C), which correlated with the antigenic distance score (R 2 = 0.82, p < 0.0001; Figure 6D).Thus, we find that RBD-nanoparticle immunizations in pre-vaccinated mice elicited mostly cross-reactive recall responses first induced by mRNA-LNP vaccination, while subsequent immunization with mosaic-8b additionally induced de novo Ab responses against RBDs closely related to those present on the nanoparticle.
To determine the epitope specificity of de novo versus recall Abs in pre-vaccinated mice that were immunized with mosaic-8b, we performed DMS 19 using libraries derived from WA1, XBB.1.5,SARS-1, RmYN02, and PRD-0038 RBDs (Figures 6E-6G and S7).As expected, day 0 responses against the WA1 library revealed a predominant class 1/class 2 response (Figure 6E).We compared FLAG + /de novo and Strep + /recall Ab epitopes at day 56 mapped onto the SARS-1 and RmYN02 RBDs (Figure 6F): for SARS-1, we found that de novo responses to mosaic-8b immunizations after WA1 mRNA-LNP pre-vaccination targeted more variable portions of class 2 and class 3 RBD epitopes, whereas recall responses targeted the more conserved class 4 epitope and conserved portions of the class 3 epitope (Figures 6F and S7A).For RmYN02, de novo responses were mainly class 1, and recall responses were class 3 and class 4 (Figures 6F and S7B).In each individual mouse, de novo FLAG + Abs targeted epitopes that were distinct from those targeted by primary-derived Abs (Figure S7), as observed previously for WA1 priming followed by BA.1 boosting. 24The Strep/recall response elicited by homotypic SARS-2 targeted similar conserved epitopes on SARS-1, RmYN02, XBB.1.5,and PRD-0038 as responses to mosaic-8b (Figures 6F and 6G), as expected since Abs that bind to SARS-1, RmYN02, XBB.1.5,or PRD-0038 are necessarily cross-reactive.Accordingly, when comparing responses against WA1, mosaic-8b elicited a more conserved class 3/class 4 response, whereas homotypic SARS-2 elicited primarily class 1 responses combined with a minority class 4 re-sponses, suggesting that mosaic-8b preferentially boosted cross-reactive recall Abs.These results are consistent with Abmediated feedback in which de novo Ab responses form against available epitopes that are not blocked by pre-existing high affinity Abs 24,39-42 and with a model in which primary vaccinederived Abs shift recall GC B cells away from previously targeted sites, thereby directing de novo Ab responses specifically toward other epitopes. 43

DISCUSSION
Much of the human population has already mounted immune responses to SARS-2, either from vaccination, infection, or both. 44,45Thus, new vaccines, especially pan-sarbecovirus vaccines designed to protect from future zoonotic spillovers and from SARS-2 VOCs, should be evaluated in non-naive animal models.Here, we demonstrate that the advantages of mosaic-8b RBD nanoparticles in eliciting cross-reactive and broad Ab responses in naive animals 16,17 are maintained in experiments in animals previously vaccinated with two to four doses of multiple types of COVID-19 vaccines: DNA or repRNA vaccines in NHPs, a Pfizer-equivalent mRNA-LNP in mice, or adenovirus-vectored AstraZeneca ChAdOx1 in mice.
7][48] We found that RBDnanoparticle immunizations (mosaic-8b, admix-8b, mosaic-7, or homotypic SARS-2) produced higher binding and neutralizing titers than additional booster immunizations with mRNA-LNP, repRNA-LION, or adenovirus-vectored vaccines in pre-vaccinated mice or NHPs.0][51][52][53][54][55][56][57][58][59][60] This result suggests that a single mosaic RBD-nanoparticle immunization could be more broadly protective in non-immunologically naive humans than one dose of a SARS-2 homotypic vaccine.We also note that our pre-vaccination experimental setups mimicked likely vaccination scenarios in humans, e.g., in which mosaic-8b vaccinees would have received one or more COVID-19 vaccination(s) months to year(s) before a mosaic-8b immunization (as in our mouse and NHP experiments in which mosaic-8b was injected 0.4-1.2 years after COVID-19 vaccinations) and may have also previously received different vaccine modalities.In summary, our results in three pre-vaccinated animal models, taken together and individually, support the premise that a mosaic-8b vaccine would elicit broad Ab responses in humans.
In a previous study in naive mice, 17 we used DMS to show that immunizations of mosaic-8b and homotypic SARS-2 elicited different types of Abs: for mosaic-8b, mainly Abs against the more conserved class 3, class 4, and class 1/4 RBD epitopes that exhibit increased cross-reactivity across sarbecoviruses and SARS-2 VOCs, [61][62][63][64][65][66] versus for homotypic SARS-2, mostly class 1 and class 2 epitopes that rapidly evolve and vary between zoonotic and human sarbecoviruses. 35,67,68Here, DMS analyses of recall anti-RBD Abs from molecular fate-mapping studies demonstrated that mosaic-8b immunization boosted class 3 and 4 anti-RBD Abs originally elicited by primary vaccination with mRNA-LNP.Since analogous Abs have been identified in human donors, 61,[68][69][70][71][72][73][74] our DMS analyses of elicited Abs suggest that mosaic-8b vaccination in humans would boost cross-reactive Abs that broadly recognize mismatched zoonotic sarbecoviruses, including more distantly related clade 3 sarbecoviruses that could initiate another pandemic and SARS-2 VOCs.Critical for the efficacy of mosaic-8b vaccination in humans, boosted responses in pre-vaccinated mice mirrored the responses of mosaic-8b in naive mice. 17olecular fate mapping 24 allowed us to separately characterize de novo and recall Ab responses to RBD nanoparticles after mRNA-LNP prime and boosting vaccinations, demonstrating that Abs were derived mostly from B cells that were first engaged by mRNA-LNP vaccination (an example of primary addiction 24 ) and that mosaic-8b, but not homotypic SARS-2, induced substantial de novo Ab responses.In general, the recall Abs were broader and more cross-reactive than the more strain-specific de novo responses.1][42][43] Our results also suggest that immunizing with mosaic-8b after COVID-19 vaccination elicits de novo strainspecific Abs that recognize largely variable epitopes on non-SARS-2 strains that are completely or closely matched to RBDs on mosaic-8b.Indeed, eliciting Abs against the seven non-SARS-2 RBDs on mosaic-8b could provide additional protection from future zoonotic spillovers of sarbecoviruses that are closely related to those represented by RBDs on mosaic-8b.Thus, it could be advantageous to introduce a clade 3 RBD to a mosaic RBD nanoparticle to promote further cross-reactive recognition.This could be achieved by replacing the SARS-2 Beta RBD on mosaic-8b with a clade 3 RBD since we found improved Ab responses for mosaic-7, a mosaic RBD nanoparticle without SARS-2 Beta, compared with mosaic-8b, which includes SARS-2 Beta.Additional RBD compositions on a mosaic RBD nanoparticle could also be explored; for example, recent results show that mosaic-7 COM (computationally chosen from natural sarbecoviruses) outperformed mosaic-8b and mosaic-7 in both naive and pre-vaccinated animals. 75n addition to informing future mosaic RBD vaccine design, our results are relevant to understanding OAS, first described with reference to influenza infections and more recently extended to studies of SARS-CoV-2 and other viruses. 22,23,76,77Our studies showed that OAS did not entirely prevent mosaic-8b's induction of potent and cross-reactive Abs after vaccination with monovalent or bivalent genetically encoded vaccines in three independent pre-vaccinated animal models, reproducing our findings of mosaic-8b advantages over homotypic SARS-2 immunogens in naive animals. 16,17In our pre-vaccinated animals, we observed that RBD-nanoparticle immunizations mainly produced recall Abs but that de novo responses could be elicited by including antigenically distant antigens, consistent with previous molecular fate-mapping studies 24 and the strong correlation (R 2 = 0.82; p < 0.0001) observed in this study between primary addiction and antigenic distance scores.This is relevant to the use of mosaic-8b as a vaccine because it includes RBDs that are both closely (91% sequence identity) and more distantly (62%) related to the WA1 RBD in spike-containing vaccinations; thus, we could use OAS to our advantage to both boost recall responses and to elicit de novo responses.By contrast, when immunizing pre-vaccinated animals with homotypic SARS-2, only recall responses were elicited.Taken together, our results are consistent with the general premise that the immune system evolved to respond to new pathogens.
9][70][71][72][73][74] Thus, mosaic-8b immunization in, e.g., an mRNA-LNP-vaccinated person would prepare that person's humoral immune system for a novel sarbecovirus, akin to the idea of using an influenza vaccine encoding 20 different hemagglutinins to prime humans for the next influenza epidemic. 78Finally, mosaic-8b immunization of the non-naive human population would produce de novo responses to the 7 other RBDs that could then be recalled or boosted after an infection by a zoonotic sarbecovirus in a new spillover or by a novel SARS-2 VOC.Taken together, our results support a model in which OAS influences responses to immunogens but that the immune system can also make new responses.Thus, we advocate using maximally multivalent sarbecovirus next-generation vaccines (i.e., with as much antigenic distance as possible) that do not require updating in a single immunization rather than continuously updating with new monovalent or bivalent vaccines.

Limitations of the study
We performed three independent experiments in pre-vaccinated animal models to compensate for not being able to compare responses to RBD-nanoparticle immunogens in vaccinated versus non-vaccinated humans.We used different types of vaccines encoding SARS-2 spike to pre-vaccinate animals: DNA-based, mRNA-LNP, and an adenovirus-vectored vaccine.However, time and financial limitations prevented evaluating other types of COVID-19 vaccines, e.g., a protein-based vaccine such as Novavax, 48 in animal models.Because we were unable to obtain licensed Pfizer-BioNTech or Moderna mRNA-LNP vaccines for research purposes from the respective companies, we used mRNA-LNP formulations from other sources that may not perform identically to clinically available vaccines.In addition, potential improvements to mosaic-8b (e.g., mosaic-7 or a mosaic-8 with clade 3 RBD instead of SARS-2 RBD) could not be evaluated in the time frame of these experiments, nor could we include more recent SARS-2 VOCs in assays due to limited remaining quantities of immune serum.We note that our assays to compare immune responses included serum Ab binding and neutralization but not protection assessment in a challenge model.We believe this concern is mitigated by our previous demonstration of mosaic-8b protection from matched and mismatched challenges in K18-hACE2 transgenic mice, 17 a stringent model of coronavirus infection, 18 and by the observation that protection has been widely reported for most, if not all, COVID-19 vaccine candidates in less stringent animal challenge models.A K18-hACE2 challenge experiment involving a mismatched sarbecovirus in pre-vaccinated and then immunized animals could be informative with respect to OAS effects but was outside the scope of this study.Finally, it is not possible to find analogous doses for genetically encoded vaccines versus protein-based vaccines; thus, it is hard to interpret differences in elicited Ab quantities and neutralization potencies when comparing the effects of boosting vaccinated animals with an mRNA-LNP or adenovirus-vectored vaccine to boosting with RBD nanoparticles.
Future studies of interest in pre-vaccinated animals immunized with mosaic-8b include isolating mAbs and investigating how the memory B cell compartment is altered.and WA1 RBDs fused to the influenza H3N2 hemagglutinin HA2 stem domain.Vaccines were formulated and administered into different animals through different modalities: n=4 macaques received a self-amplifying replicon RNA vaccine formulated with a cationic nanocarrier (repRNA-LION) and delivered IM as described 31 ; n=5 macaques received the same repRNA vaccine formulated on 1 micron gold particles and delivered by gene gun into the skin; and n = 5 macaques received DNA vaccine adjuvanted with a plasmid expressing IL-12 formulated at a 10:1 vaccine plasmid to adjuvant ratio on 1 micron gold particles and delivered by gene gun into the skin as described. 92ecause we were unable to obtain licensed Pfizer-BioNTech or Moderna mRNA-LNP vaccines for research purposes from the respective companies, Pfizer-like mRNA-LNP formulations for WA1 and BA.5 were purchased from Helix Biotech and used for mRNA-LNP vaccinations (Figures 3 and S3; Table S1).Bivalent mRNA-LNP (Figure 3F-3J and S3F-S3N; Table S1) was prepared by mixing WA1 mRNA-LNP and BA.5 mRNA-LNP in a 1:1 ratio by mRNA mass.Moderna COVID-19 vaccine used as a boost in the ChAdOx1 study (Figures 4, 5; S4, and S5; Table S1) was the gift of Jeffrey Ravetch, Rockefeller University.Research grade ChAdOx1 nCoV-19 was kindly provided by the Viral Vector Core Facility, Oxford University (Figures 4, 5, S4, and S5; Table S1).Nucleoside-modified mRNA-LNP encoding SARS-2 spike protein WA1 strain (GenBank: MN908947.3)used for S1pr2-Igk Tag experiments (Figure 6; Table S1) was generated at the University of Pennsylvania and Acuitas as described. 89munization of NHPs Mosaic-8b or homotypic SARS-2 Beta produced at Caltech as described 17 were diluted with Dulbecco's PBS and mixed 1:1 (v/v) with AddaVax for a final vaccine dose of 25 mg of RBD equivalents in 0.5 mL delivered IM.WA1/BA.1 spike repRNA was produced by HDT Bio as described. 3125 mg WA1 and 25mg BA.1 repRNA were individually co-formulated with citrate buffer, water, sucrose, and LION 31 and then mixed together for a final vaccine dose of 50 mg in 0.5 mL delivered IM.Prior to vaccine delivery, animals were sedated and hair over the injection site was clipped and wiped with ethanol.

STAR+METHODS KEY RESOURCES
Immunization of mice mRNA-LNP and ChAdOx1 pre-vaccinated mouse experiments were conducted using 6-7 week old female BALB/c mice (Charles River Laboratories) with 10-14 animals in each cohort.
Mice in the mRNA-LNP pre-vaccination study were vaccinated IM with 20 mL of WA1 mRNA-LNP at day -192 and day -171 containing 1 mg mRNA diluted in PBS (Figures 3 and S3; Table S1).Mice in groups that received a bivalent mRNA-LNP boost were additionally vaccinated IM with 20 mL of WA1/BA.5 mRNA-LNP (0.5 mg WA1 and 0.5 mg BA.5 mRNA) at day -73 (Figures 3F and S3F).Mice were immunized IM with 5 mg of protein nanoparticle (RBD equivalents) in 100 mL containing 50% v/v AddaVax adjuvant on days 0 and 28 or received an additional dose of 1 mg WA1 mRNA-LNP at day 0.
Mice in the ChAdOx1 study (Figures 4 and S4; Table S1) were vaccinated IM with 1x10 8 IU of ChAdOx1 on days -163 and -135.Mice were immunized IM with 5 mg of protein nanoparticle (RBD equivalents) in 100 mL containing 50% v/v AddaVax adjuvant on days 0 and 28 or received WA1 mRNA-LNP (1 mg mRNA in PBS) or 1x10 8 IU of ChAdOx1 at day 0. Mice in both studies were bled as indicated in Figures 3A and 4A, including day 0 and day 28 via tail veins, and then euthanized at day 56 and bled through cardiac puncture.Blood samples were allowed to clot and serum was harvested and stored at -80 C until use.
For Ab fate-mapping experiments, immune responses were induced in 7-to 12-week-old S1pr2-Igk Tag mice (males and females) by IM immunization of right quadricep muscles with 3 mg WA1 mRNA-LNP (Figures 6, S6, and S7; Table S1).Mice were re-immunized using the same procedure 28 days later.These two primary immune responses were fate-mapped by oral gavage of 200 ml tamoxifen (Sigma) dissolved in corn oil at 50 mg/ml, on days 4, 8, 12 after the first and on days 6 and 12 after the second immunization.The third immunization was performed 28 days later with either mosaic-8b or homotypic nanoparticles (5 mg, adjuvanted 1:1 (v/v) with AddaVax) in the left quadricep muscles.The fourth immunization occurred 28 days after the third (month 3 of the experiment).Blood samples were collected at key time points throughout the experiment via cheek puncture into microtubes prepared with clotting activator serum gel (Sarstedt; catalog #41.1378.005).
Expression and purification of SpyCatcher003-mi3 nanoparticles SpyCatcher003-mi3 nanoparticles 82 were expressed in B. subtilis to avoid endotoxins associated with production in E. coli.Strain CVD175 (B.subtilis 168 DaprE DnprE Dvpr Dbpr DnprB Dmpr Depr DhtrA DwprA spoIIE::kan hag::ery; Ingenza Ltd) was transformed with high-copy plasmid pCVD148 encoding for IPTG-inducible expression and secretion of SpyCatcher003-mi3.The resulting strain CVD326 was cultured in Terrific Broth medium (Merck) supplemented with 10 mg/mL chloramphenicol, 10 mg/mL kanamycin and 1% (v/v) glycerol at 37 C with 250 rpm agitation.When OD 600 reached a value of 1.0, SpyCatcher003-mi3 expression was induced for 20 hours by addition of IPTG to a final concentration of 1 mM.The culture supernatant was harvested by centrifugation at 4424 x g for 45 minutes at 4 C. SpyCatcher003-mi3 particles were then precipitated by addition of ammonium sulfate at a final concentration of 20% (w/v) with incubation for 45 min at room temperature on a stirrer plate.Precipitated particles were pelleted by centrifugation at 4424 3 g for 45 min at 4 C.The obtained pellet was resuspended in TBS (20 mM Tris, 150 mM NaCl, pH 8.0) and filtered through a 0.22 mm syringe filter.Glycerol was added to a final concentration of 10% (v/v), and purified nanoparticles were stored at -80 C until use.

Preparation of RBD SpyCatcher-mi3 nanoparticles
Purified SpyCatcher003-mi3 was incubated with a 2-fold molar excess (total RBD to mi3 subunit) of SpyTagged RBD (an equimolar mixture of eight RBDs for mosaic-8b, seven RBDs (all RBDs in mosaic-8b except for the SARS-2 RBD), or only one RBD for homotypic nanoparticles) overnight at room temperature in TBS.Conjugated RBD-mi3 particles were separated from free RBDs by SEC on a Superose 6 10/300 column (GE Healthcare) equilibrated with PBS (20 mM sodium phosphate pH 7.5, 150 mM NaCl).RBD-mi3 nanoparticle conjugations were assessed via SDS-PAGE and western blot.Concentrations of conjugated mi3 particles are reported based on RBD content determined using a Bio-Rad Protein Assay.RBD-mi3 nanoparticles used for immunization of NHPs, ChAdOx nCoV-19-vaccinated mice, and fate mapping studies in mice were made from Expi293-expressed RBDs as previously described. 16,17RBD-mi3 nanoparticles used to immunize mice previously vaccinated with mRNA-LNP were produced from Pichia-expressed RBDs.Pichia-produced and Expi293-produced RBD-mi3 nanoparticles elicit comparable immune responses in immunized mice as do RBD-mi3 nanoparticles constructed using Bacillus (this study) or E. coli mi3 (previous studies 16,17 ) (A.A.C., J.R.K., H.G., and P.J.B., unpublished results).RBD-mi3 nanoparticles were aliquoted and flash frozen in liquid nitrogen before being stored at -80 C until use.RBD-nanoparticles were characterized by SEC and SDS-PAGE.

Serum ELISAs
Affinity purified His-tagged RBD produced by Expi293 transient transfection was diluted in 0.1 M NaHCO 3 pH 9.8 (20 mL of a 2.5 mg/mL solution) and coated onto Nuncâ MaxiSorpÔ 384-well plates (Sigma) and incubated overnight at 4 o C. Plates were blocked with 3% bovine serum albumin (BSA), 0.1% Tween 20 in TBS (TBS-T) for 1 hr at room temperature and then the blocking was removed by aspiration.1:100 dilutions of NHP or mouse serum were serially diluted by 3.1-fold in TBS-T/3% BSA and then added to the plates for 3 hr at room temperature followed by washing with TBS-T.After incubating for one hour in a 1:100,000 dilution of HRP-conjugated anti-IgG secondary (goat anti-mouse IgG (Abcam; RRID: AB_955439) for mouse serum or mouse-anti-monkey IgG (SouthernBiotech; RRID: AB_2796069) for NHP serum), the plates were washed with TBS-T and SuperSignalÔ ELISA Femto Maximum Sensitivity Substrate (ThermoFisher) was added following manufacturer's instructions.Luminescence was read at 425 nm.We used Graphpad Prism 10.1.0to plot and analyze curves assuming a one-site binding model with a Hill coefficient to obtain midpoint titers (ED 50 values).For the WA1 mRNA-LNP pre-vaccinated mouse experiments (Figures 3 and S3), ED 50 s were normalized by dividing the ED 50 response at day 28 or day 56 by the ED 50 response at day 0 to control for differences in Ab binding response between groups from the pre-vaccination (Figure S3).Mean ED 50 s are reported for ELISA data in Figures 2, 3, and 4 and associated supplementary figures, as previously described. 16,17LAG/Strep ELISAs for molecular fate-mapping experiments were performed side-by-side and with internal standards on each 96-well plate as described. 24Plates were incubated overnight at 4 o C with 2 mg of RBD in PBS, wells for standard curves were coated with 10 mg/ml purified IgY (Gallus Immunotech).Plates were washed with PBS-Tween (PBS + 0.05% Tween20; Sigma) and blocked with 2.5% BSA in PBS for 2 hours at room temperature.Serum samples were diluted 1:100 in PBS and serially diluted in 3-fold steps to determine endpoint titers.Mouse anti-IgY mAb-FLAG or mAb-Strep were also serially titrated in 3-fold dilutions.After 2 hours of incubation, followed by washing, rabbit anti-FLAG-HRP (clone D6W5B, CellSignalingTechnology #86861S, RRID: AB_2800094) or anti-Strep (clone Strep-tag II StrepMAB-Classic, Biorad #MCA2489P, RRID: AB_609796) detection Abs were added for 30 minutes.Dilutions of detection Abs were defined such that FLAG-and Strep-tagged mAb dilution curves overlapped.After washing with PBS-Tween, samples were incubated with 3,3 0 ,5,5 0 -Tetramethylbenzidine substrate (slow kinetic form, Sigma) and the reaction was stopped with 1N HCl.Optical Density (OD) absorbance was measured at 450 nm on a Fisher Scientific accuSkan FC plate reader.To normalize FLAG and Strep endpoint titers, the serum titer dilution was calculated at which each sample passed the threshold OD value of its respective mAb at 20 ng/ml.Titers were calculated by logarithmic interpolation of the dilutions with readings immediately above and immediately below the mAb OD used.Antibody OD and endpoint titer levels were plotted in GraphPad Prism 10.1.0,titer values below 100 were set to 100, the top dilution and therefore the limit of detection (LOD).Median anti-RBD titers are reported in Figure 6 and associated supplementary figures, consistent with analyses of previous molecular fate-mapping experiments. 24rum pseudovirus neutralization assays Lentiviral-based pseudoviruses were prepared as described 73,91 using genes encoding S protein sequences lacking C-terminal residues in the cytoplasmic tail: 21 residue (SARS-2 variants, WIV1, and SHC014) or 19 residue cytoplasmic tail deletions (SARS-CoV, Khosta-2-SARS-1 chimera, BtKY72-SARS-1 chimera).BtKY72 (containing K493Y/T498W substitutions) and Khosta-2 pseudoviruses were made with chimeric spikes in which the RBD from SARS-1 (residues 323-501) was substituted with the RBD from BtKY72 K493Y/T498W (residues 327-503) or Khosta-2 (residues 324-500) as described. 95For neutralization assays, pseudovirus was incubated with three-fold serially diluted sera from immunized NHPs or mice for 1 hour at 37 C, then the serum/virus mixture was added to 293T ACE2 target cells or high-hACE2 HEK-293T cell line expressing hACE2 encoded with a consensus Kozak sequence (kindly provided by Kenneth Matreyek, Case Western Reserve University) for SHC014 assays and incubated for 48 hours at 37 C. Media was removed, cells were lysed with Britelite Plus reagent (Revvity Health Sciences), and luciferase activity was measured as relative luminesce units (RLUs).Relative RLUs were normalized to RLUs from cells infected with pseudotyped virus in the absence of antiserum.Half-maximal inhibitory dilutions (ID 50 values) were derived using 4-parameter nonlinear regression in AntibodyDatabase. 88

DMS
Yeast library sorting to identify mutations that reduce binding to polyclonal serum and monoclonal Abs for epitope mapping.DMS studies used to map epitopes recognized by serum Abs were performed in biological duplicates using independent mutant libraries (WA1, 84 XBB.1.5, 85SARS-1, 86 RmYN02, 86 and PRD-0038, 86 generously provided by Tyler Starr, University of Utah) as described previously. 17,36Serum samples were heat inactivated for 30 min at 56 C before DMS analysis.In order to remove non-RBD specific yeast-binding Abs, heat-inactivated sera were then incubated twice with 50 OD units of AWY101 yeast transformed with an empty vector to remove yeast-binding Abs.Monoclonal Abs for DMS analyses were not heat inactivated or yeast depleted.RBD libraries were induced for RBD expression in galactose-containing synthetic defined medium with casamino acids (6.7g/L Yeast Nitrogen Base, 5.0 g/L Casamino acids, 1.065 g/L MES acid, and 2% w/v galactose + 0.1% w/v dextrose).After inducing for 18 hours, cells were washed 2x and then incubated with serum or monoclonal Abs (dilutions chosen to give sub-saturating binding to RBDs; data posted in https://github.com/bjorkmanlab/Mosaic-8b_prevax_fate_mapping)for 1 hour at RT with gentle agitation after which cells were washed 2x and labeled for 1 hour with secondary Ab: either 1:200 Alexa Fluor-647-conjugated goat anti-mouse-IgG Fc-gamma (Jackson ImmunoResearch 115-605-008, RRID: AB_2338904) for serum mouse Abs, 1:200 Allophycocyanin-AffiniPure Goat Anti-Human IgG/Fcg Fragment Specific (Jackson ImmunoResearch 109-135-098, RRID: AB_2337690) for human monoclonal Abs, Streptavidin-APC (eBioscience 17-4317-82) for Strep tag Abs, or SureLightâ APC Anti-DDDDK tag (Abcam ab72569, RRID: AB_1310127) for Flag tag Abs.
Stained yeast cells were processed on a Sony SH800 cell sorter.Cells were gated to capture RBD mutants that had reduced antibody binding for a relatively high degree of RBD expression (data posted in https://github.com/bjorkmanlab/Mosaic-8b_prevax_fate_mapping).For each sample, cells were collected up until around 5 x 10 6 RBD + cells were processed (which corresponded to around 5 x 10 5 -1 x 10 6 RBD + Ab escaped cells; data posted in https://github.com/bjorkmanlab/Mosaic-8b_prevax_fate_mapping).Antibody-escaped cells were grown overnight in a synthetic defined medium with casamino acids (6.7 g/L Yeast Nitrogen Base, 5.0 g/L Casamino acids, 1.065 g/L MES acid, and 2% w/v dextrose + 100 U/mL penicillin + 100 mg/mL streptomycin) to expand cells prior to plasmid extraction.DNA extraction and Illumina sequencing were carried out as previously described. 96aw sequencing data are available on the NCBI SRA under BioProject PRJNA1067836, BioSample SAMN40463706 (for serum and mAb DMS in Figures 5 and S5) and SAMN39552627 (for molecular fate mapping in Figures 6 and S6).Escape fractions were computed using processing steps described previously 36,96 (https://github.com/jbloomlab/SARS-CoV-2_Bjorkman_pilot)and implemented using a new Swift DMS program (available from authors upon request).Escape scores were calculated with a filter to remove variants with deleterious mutations that escape binding due to poor expression, >1 amino acid mutation, or low sequencing counts as described. 96,97tatic line plot visualizations of escape maps were created using Swift DMS. 96In all cases, the line height indicates the escape score for that amino acid mutation, calculated as previously described. 96In some visualizations, RBD sites are categorized based on epitope region, 20 which we defined as: class 1 (pink) (residues 403, 405, 406, 417, 420, 421, 453, 455-460, 473-478, 486, 487, 489, 503, 504); class 2 (purple) (residues 472, 479, 483-485, 490-495), class 3 (blue) (residues 341, 345, 346, 354-357, 396, 437-452, 466-468, 496, 498-501,462), class 4 (orange) (residues 365-390, 408).For structural visualizations, an RBD surface (PDB: 6M0J) was colored by the site-wise escape metric at each site, with magenta scaled to be the maximum used to scale the y-axis for serum and monoclonal Abs, red scaled to be the maximum for Strep tag Abs, and blue scaled to be the maximum for Flag tag responses, and gray indicating no escape.Residues that exhibited the greatest escape fractions were highlighted with their residue number and colored according to epitope class.

QUANTIFICATION AND STATISTICAL ANALYSES
We used pairwise comparisons, a method to evaluate sets of mean binding or neutralization titers against individual viral strains for different immunization cohorts, to determine whether results from different cohorts were significantly different from each other.Statistically significant titer differences between immunized groups of NHPs and mice for ELISAs and neutralization assays were determined using analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc tests with the Geisser-Greenhouse correction, with pairing by viral strain, of ED 50 s/ID 50 s (converted to log 10 scale) calculated using GraphPad Prism 10.1.0.To address whether our data were normally distributed (a requirement for Tukey's multiple comparison tests), we used quantile-quantile (Q-Q) probability plots to compare actual and predicted binding and neutralization titers.Most of the data were normally distributed.However, two circumstances resulted in deviations from normal distributions: (1) Setting of background binding or neutralization data points to limits of detection (titers of 1:50 or 1:100); and (2) Neutralization or binding titers against the WA1 spike antigen (D614G pseudovirus or WA1 spike) that was matched to the strain in the COVID vaccines used for pre-vaccination, which resulted in skewing the distributions because of high titers and inclusion of non-RBD Ab epitopes.
In Figure 6D, the linear regression of primary addiction index against antigenic distance score was calculated using Prism.
Figure S1.Preparation of RBD-mi3 nanoparticles, related to Figure 1 (A) Hypothesis illustrating potential mechanism for mosaic RBD-nanoparticle induction of cross-reactive Abs.Left: both Fabs of a strain-specific membranebound BCR can bind to a strain-specific epitope (pale yellow triangle) on yellow antigens attached to a homotypic nanoparticle.Middle: strain-specific BCRs can only bind with one Fab to a strain-specific epitope (triangle) on yellow antigen attached to a mosaic nanoparticle.Right: cross-reactive BCRs can bind with both Fabs to a common epitope present on adjacent antigens (green circles) attached to a mosaic particle, but not to strain-specific epitopes (triangles).Left: line plots for DMS results from individual mAbs or the indicated mAb mixtures recognizing different RBD epitopes (epitopes defined in Figure 1B).C102, C118, C144, and S309 are mAbs that were derived from COVID-19 convalescent donors. 63,73CR3022 is an anti-SARS-CoV mAb that binds to SARS-2 RBD. 69pitope assignments for these mAbs were previously described. 93DMS was conducted for the mAb reagents using a WA1 RBD library.The x axis shows the RBD residue number, and the y axis shows the sum of the Ab escape of all mutations at a site (larger numbers indicating more Ab escape).Each line represents one antiserum with heavy lines showing the average across the n = 3 sera in each group.Lines are colored differently for RBD epitopes within different classes 20 (epitopes defined in Figure 1B); gray for residues not assigned to an epitope.Right: the average site-total Ab escape for the indicated mAbs and mAb mixtures (mix 1 = equimolar mixture of all five mAbs; mix 2 = 10-fold more S309, C118, and CR3022 than C102 and C144; mix 3 = 10-fold more C102 and C144 than S309, C118, and CR3022) mapped to the surface of the WA1 RBD (PDB: 6M0J).The locations of individual residues are highlighted on the RBD surfaces in colors corresponding to their epitope.6).Data up to day 0 are from the two independent experimental cohorts shown in Figure 6.Data from one of the experimental cohorts was collected up to day 56.For accurate comparisons, each data point within each plot is from samples analyzed together in one assay.RmYN02 RBD is included on the mosaic-8b nanoparticle.
-8b immunizations outperform genetically encoded vaccines and non-mosaic RBD-nanoparticle immunizations in pre-vaccinated mice To generalize our findings in pre-vaccinated NHPs to a different animal model and to other vaccine modalities, we conducted experiments in mice that had been pre-immunized with genetically encoded vaccines, either an mRNA-LNP formulation equivalent to Pfizer-BioNTech's WA1 spike-encoding BNT162b2 vaccine (Figures 3 and S3; Table S1) or with an adenovirus-vectored vaccine (research-grade ChAdOx1 nCoV-19 produced by the Viral Vector Core Facility, Oxford University) also encoding the WA1 spike (Figures 4 and S4; Table

Figure 2 .
Figure 2. Mosaic-8b immunizations in previously vaccinated NHPs elicit cross-reactive Ab responses Data are shown for ELISA and neutralization analyses for serum samples from weeks 0, 2, 8, and 10. (Data for samples from weeks 4, 12, and 22 are in Figure S2.)Geometric means of the effective dilution at half-maximal binding (ED 50 ) or half-maximal neutralization (ID 50 ) for all animals in each cohort are indicated by symbols connected by thick colored lines; results for individual animals are indicated by dotted colored lines.Mean titers against indicated viral antigens or pseudoviruses were compared pairwise across immunization cohorts by Tukey's multiple comparison test with the Geisser-Greenhouse correction (as calculated by GraphPad Prism).Significant differences between cohorts linked by vertical lines are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(A) Left: schematic of vaccination/immunization regimen.NHPs were vaccinated at the indicated weeks prior to RBD nanoparticle or repRNA prime and boost immunizations at weeks 0 and 8. Middle: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega. 15RBDs included in mosaic-8b are highlighted in gray rectangles.Right: geometric mean ELISA binding titers at week 0 (after pre-vaccinations but prior to RBD nanoparticle or repRNA immunizations) against indicated viral antigens.(B) Geometric mean ELISA binding titers at the indicated weeks after immunization with mosaic-8b, homotypic SARS-2, or bivalent WA1/BA.1 repRNA against indicated viral antigens.(C) Geometric mean neutralization titers at the indicated weeks after immunization with mosaic-8b, homotypic SARS-2, or bivalent WA1/BA.1 repRNA against indicated sarbecovirus pseudoviruses.See also Figure S2.

Figure 3 .
Figure 3. Mosaic-8b immunizations in mice previously immunized with an mRNA-LNP vaccine elicit cross-reactive Ab responses

Figure 4 .
Figure 4. Mosaic-8b immunizations in ChAdOx1-vaccinated mice elicit cross-reactive Ab responses Geometric means of ED 50 or ID 50 values for all animals in each cohort are indicated by symbols connected by thick colored lines.Mean titers against indicated viral antigens or pseudoviruses were compared pairwise across immunization cohorts by Tukey's multiple comparison test with the Geisser-Greenhouse correction (as calculated by GraphPad Prism).Significant differences between cohorts linked by vertical lines are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(A) Left: schematic of vaccination/immunization regimen.Mice were vaccinated at the indicated days prior to RBD-nanoparticle prime and boost immunizations at days 0 and 28 or mRNA-LNP or ChAdOx1 prime immunizations at day 0. Right: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega. 15RBDs included in mosaic-8b are highlighted in gray rectangles.(B) Geometric mean ELISA binding titers at the indicated days after immunization with mosaic-8b, admix-8b, homotypic SARS-2, WA1 mRNA-LNP, or ChAdOx1 against indicated viral antigens.(C) Geometric mean neutralization titers at the indicated weeks after immunization with mosaic-8b, admix-8b, homotypic SARS-2, WA1 mRNA-LNP, or ChAdOx1 against indicated sarbecovirus pseudoviruses.See also Figure S4.

Figure 5 .
Figure 5. Differences in epitope targeting of Abs elicited in ChAdOx1 pre-vaccinated mice by mosaic-8b and admix-8b compared with homotypic SARS-2 and WA1 mRNA-LNP Sera were derived from ChAdOx1 pre-vaccinated mice that were immunized with either two doses of an RBD-nanoparticle immunogen or one dose of WA1 mRNA-LNP.(A) Line plots for DMS results from sera from ChAdOx1-vaccinated mice that were immunized with the indicated immunogens (immunization schedule in Figure4A).DMS was conducted using a WA1 RBD library.The x axis shows the RBD residue number, and the y axis shows the sum of the Ab escape of all mutations at a site (larger numbers indicating more Ab escape).Each line represents one antiserum with heavy lines showing the average across the n = 3 sera in each group.Lines are colored differently for RBD epitopes within different classes20 (color definitions in upper right legend; epitopes defined in Figure1E; gray for residues not assigned to an epitope).(B) The average site-total Ab escape for the WA1 RBD library for mice immunized with the indicated immunogens after ChAdOx1 vaccination mapped to the surface of the WA1 RBD (PDB: 6M0J), with gray indicating no escape, and epitope-specific colors indicating sites with the most escape.(C) Line plots for DMS results from sera from ChAdOx1-vaccinated mice that were immunized with the indicated immunogens (immunization schedule in Fig-ure 4A).DMS was conducted using an XBB.1.5RBD library.The x axis shows the RBD residue number, and the y axis shows the sum of the Ab escape of all mutations at a site (larger numbers indicating more Ab escape).Each line represents one antiserum with heavy lines showing the average across the n = 3 sera in each group.Lines are colored differently for RBD epitopes within different classes20 (color definitions in upper right legend; epitopes defined in Figure1E; gray for residues not assigned to an epitope).(D) The average site-total Ab escape for the XBB.1.5RBD library for mice immunized with the indicated immunogens after ChAdOx1 vaccination mapped to the surface of the WA1 RBD (PDB: 6M0J), with gray indicating no escape, and epitope-specific colors indicating sites with the most escape.See also FigureS5.

Figure S2 .
Figure S2.RBD-nanoparticle immunizations in previously vaccinated NHPs elicit cross-reactive Ab responses, related to Figure 2 Data are shown for ELISA and neutralization analyses for serum samples from weeks 4, 12, and 22. (Data for samples from weeks 0, 2, 8, and 10 are in Figure 2.) Geometric means of ED 50 or ID 50 values for all animals in each cohort are indicated by symbols connected by thick colored lines; results for individual animals are indicated by dotted lines.Mean titers against indicated viral antigens or pseudoviruses were compared pairwise across immunization cohorts by Tukey's multiple comparison test with the Geisser-Greenhouse correction (as calculated by GraphPad Prism).Significant differences between cohorts linked by vertical lines are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(A) Left: stratification of pre-vaccinated NHPs into groups used for immunizations with mosaic-8b, homotypic SARS-2, and WA1/BA.1 repRNA based on weight, age, sex, and neutralization ID 50 values (ns, no significant difference; Mann-Whitney test).Neutralization ID 50 s were derived from samples taken at week 8 in the vaccination/immunization regimen in Figure 2A.Right: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega. 15RBDs included in mosaic-8b are highlighted in gray rectangles.(B) Geometric mean ELISA binding titers at the indicated weeks after immunization with mosaic-8b, homotypic SARS-2, or bivalent WA1/BA.1 repRNA against indicated viral antigens.(C) Geometric mean neutralization titers at the indicated weeks after immunization with mosaic-8b, homotypic SARS-2, or bivalent WA1/BA.1 repRNA against indicated sarbecovirus pseudoviruses.

Figure S3 . 3
Figure S3.Cohorts of mRNA-LNP-vaccinated mice showed significant differences in midpoint ED 50 titers at day 0 (prior to RBD-nanoparticle immunizations), related to Figure3Geometric means of ED50 or ID50 values for all animals in each cohort are indicated by symbols connected by thick colored lines.Mean titers against indicated viral antigens or pseudoviruses were compared pairwise across immunization cohorts by Tukey's multiple comparison test with the Geisser-Greenhouse correction (as calculated by GraphPad Prism).Significant differences between cohorts linked by vertical lines are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(A) Left: schematic of vaccination/immunization regimen for (B) and (C).Mice were vaccinated at the indicated days prior to RBD-nanoparticle prime and boost immunizations at days 0 and 28 or mRNA-LNP prime immunization at day 0. Right: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega.15RBDs included in mosaic-8b are highlighted in gray rectangles.(B) Geometric mean ELISA binding titers of serum from mice assigned to each cohort at day 0 (192 and 171 days after the first and second mRNA-LNP vaccinations but prior to nanoparticle or additional mRNA-LNP immunizations) showing significant differences between cohorts prior to immunizations.(C) Geometric mean neutralization binding titers of serum from mice assigned to each cohort at day 0 (192 and 171 days after the first and second mRNA-LNP vaccinations but prior to nanoparticle or additional mRNA-LNP immunizations).(D) Schematic of vaccination/immunization regimen for (E)-(G).Mice were vaccinated at the indicated days prior to RBD-nanoparticle prime and boost immunizations at days 0 and 28 or mRNA-LNP prime immunization at day 0. (E) Geometric mean ELISA binding titers of serum from mice assigned to each cohort at day 0 (192, 171, and 73 days after the first, second, and third mRNA-LNP vaccinations but prior to nanoparticle or additional mRNA-LNP immunizations) showing significant differences between cohorts prior to immunizations.(F) Geometric mean neutralization binding titers of serum from mice assigned to each cohort at day 0 (192, 171, and 73 days after the first, second, and third mRNA-LNP vaccinations but prior to nanoparticle or additional mRNA-LNP immunizations).(G) Non-baseline-corrected geometric mean ELISA binding titers at the indicated days after immunization with mosaic-8b, mosaic-7, or WA1/BA.5 mRNA-LNP against indicated viral antigens.Compare with Figure3E(baseline-corrected geometric mean ELISA binding titers).

Figure S4 .
Figure S4.Cohorts of ChAdOx1-vaccinated mice showed no significant differences in midpoint ED 50 titers at day 0, related to Figure 4 Geometric means of ED 50 or ID 50 values for all animals in each cohort are indicated by symbols connected by thick colored lines.Mean titers against indicated viral antigens or pseudoviruses were compared pairwise across immunization cohorts by Tukey's multiple comparison test with the Geisser-Greenhouse correction (as calculated by GraphPad Prism).Significant differences between cohorts linked by vertical lines are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(A)Top: schematic of vaccination/immunization regimen.Mice were vaccinated at the indicated days prior to RBD-nanoparticle prime and boost immunizations at days 0 and 28 or mRNA-LNP or ChAdOx1 prime immunizations at day 0. Bottom: phylogenetic tree of selected sarbecoviruses calculated using a Jukes-Cantor generic distance model using Geneious Prime 2023.1.2based on amino acid sequences of RBDs aligned using Clustal Omega.15RBDs included in mosaic-8b are highlighted in gray rectangles.(B) Geometric mean ELISA binding titers of serum from mice assigned to each cohort at day 0 (163 and 134 days after the first and second ChAdOx1 vaccinations but prior to nanoparticle, mRNA-LNP, or additional ChAdOx1 immunizations).Binding titers are represented as mean ED 50 values for serum IgG binding to RBD or spike proteins from the indicated sarbecovirus strains.

Figure S5 .
Figure S5.Comparison of DMS profiles of individual mAbs and mAb mixtures, related to Figure 5

Figure S6 .
Figure S6.Time course of serum from fate-mapping studies reveals differences in Ab responses before RBD-nanoparticle immunization, related to Figure 6 Time course of serum antibody responses.FLAG + /de novo (blue) or Strep + /recall (red) Abs after immunization with RBD nanoparticles.Red arrows indicate tamoxifen (Tmx) treatment in all panels; black arrows indicate vaccination with WA1 mRNA-LNP or the indicated RBD nanoparticles, as denoted in the upper left panel.Mice immunized with mosaic-8b are indicated in green; mice immunized with homotypic SARS-2 Beta are indicated in orange.Individual mice are indicated by thin lines; median responses for groups are shown in thick lines.(A and B) Time course of levels of total IgG (left), FLAG + /de novo Igk (center), or Strep+/recall Igk (right) Ab responses after immunization with mosaic-8b (green) or homotypic SARS-2 Beta (orange) RBD nanoparticles measured against RBDs of (A) SARS-2 WA1 and (B) SARS-2 Beta.

Figure S7 .
Figure S7.Individual animal DMS results, related to Figure 6 DMS analyses shown for individual animals for which we had sufficient sera, and the sera exhibited detectable levels of FLAG + or Strep + Abs.(A) DMS analyses for individual animals (indicated by 4-digit numbers) for compiled results shown in Figure 6F of day 56 serum from RBD-nanoparticle immunized mice using SARS-1 (antigenic distance score = 0.81) RBD mutant library.Ab binding sites are shaded according to degree of Ab escape, with blue for FLAG/de novo responses and red for Strep/recall responses on the surface of the WA1 RBD (PDB: 6M0J).Comparisons are made for FLAG/de novo and Strep/recall elicited by mosaic-8b and for Strep/recall elicited by homotypic SARS-2 (there were weak to no FLAG/de novo responses after homotypic SARS-2 immunization).(B) DMS analyses for individual animals (indicated by 4-digit numbers) for compiled results shown in Figure 6F of day 56 serum from RBD-nanoparticle immunized mice using RmYN02 (antigenic distance score = 0.63) RBD mutant library.Ab binding sites are shaded according to degree of Ab escape, with blue for FLAG/de novo responses and red for Strep/recall responses on the surface of the WA1 RBD (PDB: 6M0J).Comparisons are made for FLAG/de novo and Strep/recall elicited by mosaic-8b and for Strep/recall elicited by homotypic SARS-2 (there were weak to no FLAG/de novo responses after homotypic SARS-2 immunization).RmYN02 RBD is included on the mosaic-8b nanoparticle.