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Cross-reactive SARS-CoV-2 epitope targeted across donors informs immunogen design

Summary The emergence of the antigenically distinct and highly transmissible Omicron variant highlights the possibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape due to viral evolution. This continued evolution, along with the possible introduction of new sarbecoviruses from zoonotic reservoirs, may evade host immunity elicited by current SARS-CoV-2 vaccines. Identifying cross-reactive antibodies and defining their epitope(s) can provide templates for rational immunogen design strategies for next-generation vaccines. Here, we characterize the receptor-binding-domain-directed, cross-reactive humoral repertoire across 10 human vaccinated donors. We identify cross-reactive antibodies from diverse gene rearrangements targeting two conserved receptor-binding domain epitopes. An engineered immunogen enriches antibody responses to one of these conserved epitopes in mice with pre-existing SARS-CoV-2 immunity; elicited responses neutralize SARS-CoV-2, variants, and related sarbecoviruses. These data show how immune focusing to a conserved epitope targeted by human cross-reactive antibodies may guide pan-sarbecovirus vaccine development, providing a template for identifying such epitopes and translating to immunogen design.


In brief
Hauser et al. characterize human antibodies elicited by SARS-CoV-2 vaccination or infection that bind broadly to different sarbecovirus receptorbinding domains. Broadly crossneutralizing antibodies target two conserved epitopes. Structure-guided immunogen design focuses murine antibody responses to one such epitope in the context of pre-existing immunity, conferring improved Omicron neutralization.

INTRODUCTION
As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve because of population immunity, antigenically distinct variants such as Omicron may emerge. 1 This antigenic difference increases the likelihood that there will be re-infections or breakthrough infections following vaccination. [1][2][3][4][5][6][7][8] Furthermore, surveillance efforts have identified related sarbecoviruses in animal reservoirs that use the human ACE2 receptor for viral entry, suggesting possible future pandemics. [9][10][11][12] Consequently, there is a need for cross-reactive therapeutic antibodies or next-generation vaccines that protect against SARS-CoV-2 variants and novel sarbecoviruses with pandemic potential. While many SARS-CoV-2 therapeutic monoclonal antibodies developed early in the pandemic had reduced potency against Omicron, boosting with mRNA vaccines appeared to improve serum neutralization against this and other variants. 4,[13][14][15] Identifying infection-or vaccine-elicited cross-reactive antibodies can inform immunogen design efforts that focus responses to conserved epitopes.
Here, we analyzed 10 donors from a previous cohort that had serum neutralization against SARS-CoV-2 variant pseudovi-ruses 4 to characterize cross-reactive memory B cells to additional variants and pre-emergent sarbecoviruses (Table S1). All donors had at least three documented exposures to SARS-CoV-2: 5 received two SARS-CoV-2 vaccines and had at least one SARS-CoV-2 infection, and 5 had three mRNA vaccinations and no known prior SARS-CoV-2 infection. Serum from each donor neutralized wild-type SARS-CoV-2, Delta, Omicron, and SARS-CoV-1 pseudoviruses 4 (Table S1). Neutralization was significantly reduced against Omicron and SARS-CoV-1 relative to wild-type SARS-CoV-2; neutralization of SARS-CoV-1 was also significantly reduced relative to Delta.
Mapping cross-reactive antibody epitopes We next selected 29 paired heavy and light chains that reflected the genetic diversity of the 228 sequenced B cell receptors (BCRs) for recombinant expression as immunoglobulin G (IgG) for further biochemical characterization. Recombinant antibodies were first tested in an ELISA for binding to wild-type SARS-CoV-2, Omicron, SARS-CoV-1, and SHC014 RBDs (Figure 2A). Twenty antibodies had high-affinity binding (K D < 1 mM) to all four RBDs, ranging from <0.1 to 200 nM affinity; 4 antibodies bound all RBDs except Omicron, and 5 bound to two or fewer RBDs.
We down-selected 19 antibodies from 5 donors (Table S2) based on expression levels for epitope mapping by cross-competition ELISA using the wild-type SARS-CoV-2 RBD ( Figure 2B). We included control antibodies with structurally defined epitopes: B38 (class 1), P2B-2F6 (class 2), S309 (class 3), CR3022 (class 4), and ADI-55688 (precursor to broadly neutralizing therapeutic antibody ADG-2). 16,[20][21][22][23][24] While several antibodies competed with ACE2 receptor-binding motif (RBM)-directed antibodies B38, P2B-2F6, or ADI-55688, most cross-reactive antibodies mapped to two distinct epitopes. One class competed with CR3022 (class 4), while the other class showed no competition with any control antibodies in ELISA. To define the targeted epitope, we chose  representative antibody D2G2 and assayed for competition using biolayer interferometry (BLI) with an additional panel of structurally characterized Fabs: two from mice that represent distinct RBD epitopes, Ab20 and Ab16, 25 and four from human donors targeting minimally overlapping RBM and non-RBM epitopes on the RBD, G32A4, C98C7, G32R7, and G32Q4 26 ( Figure 2C). Only G32R7 competed with D2G2, indicating that the other class of broadly cross-reactive antibodies targets a conserved class 3 epitope that minimally competes with S309, strongly suggesting that the epitope is across that face of the RBD 20,26-28 ( Figure 2D). Of the 11 class 3-directed antibodies characterized, 8 used IGHJ4; only 2 of the 8 class 4-directed antibodies used IGHJ4 (Table S2). There were no other clear patterns of antibody sequence features associated with either of the conserved epitopes, suggesting multiple evolutionary pathways that converge on these conserved epitopes. Broadly reactive antibodies have been previously shown to target both class 3 and 4 epitopes; 27,29 the lack of class 1 and 2 antibodies characterized here indicates that broadly reactive antibodies targeting these epitopes are less represented in the human memory repertoire. 30,31

Cross-reactive antibody neutralization
We next performed pseudovirus neutralization assays on the 19 antibodies down-selected for cross-competition against wildtype SARS-CoV-2, SARS-CoV-1, WIV1, RaTG13, SHC014, and the SARS-CoV-2 variants Delta and Omicron. Representative antibodies targeting the two conserved epitopes based on competition were broadly neutralizing with IC50s ranging from $1 to 200 mg/mL in a pattern that roughly correlated with antibody affinity ( Figures 3A and 3B). Seven antibodies targeting the class 4 conserved epitope and 3 antibodies targeting the class 3 conserved epitope neutralized at least 4 of the 7 pseudoviruses tested (though none of the class 3 antibodies neutralized RaTG13). These data indicate that cross-reactive antibodies elicited by vaccination or infection that target both sites are broadly neutralizing antibodies. We also tested antibodies against authentic wild-type SARS-CoV-2 and Omicron and found preserved neutralization ( Figure S3).

An engineered immunogen for epitope targeting
Based on these observations across human donors, we used a structure-guided approach to design an immunogen to focus antibody responses to the class 4 epitope. Using glycan engineering, we developed a hyperglycosylated immunogen that selectively exposed this epitope while occluding all other epitopes ( Figures 4A, S4A, and S4B); this builds upon our previous hyperglycosylated immunogens to direct immune responses to conserved epitopes. 25 This class 4-focusing immunogen has 11 engineered putative N-linked glycosylation sites; 9 are novel and two are native to SARS-CoV-2. We made trimeric versions of this immunogen using a non-immunogenic, hyperglycosylated GCN4 trimerization tag to increase valency and for subsequent testing in vivo 25 ( Figures S4C-S4F). Two murine cohorts were primed with the SARS-CoV-2 spike to approximate pre-existing vaccine or infection-elicited immunity to SARS-CoV-2 followed by boosting with trimeric wild-type SARS-CoV-2 RBD (''WT cohort'') or the hyperglycosylated immunogen (''HG cohort'') ( Figure 4B).

Assessing immunogenicity and focusing
We assayed sera binding 14 days after the second boost against coronavirus RBDs by ELISA. Both cohorts had similar reactivity to all sarbecovirus RBDs tested, but the WT cohort showed a significant drop off in reactivity against the Omicron RBD versus the SARS-CoV-2 RBD, while the HG cohort did not ( Figures  S4G-S4H). To assess immune focusing to the class 4 epitope, we assayed competition of the murine class 4 antibody Ab16, which competes with CR3022 but has broad neutralization activity across SARS-CoV-2 variants and other sarbecoviruses. 25 Ab16 competes with CR3022 and the class 4 donor antibodies ( Figures 4C and S4F), and the Ab16 footprint was previously structurally characterized; 25 Ab16 was also used to confirm the conformational integrity of the HG immunogen ( Figure S4A). The HG cohort had significant increase in serum competition with Ab16, with a mean serum competition of $2.53 relative to WT ( Figure 4D); this indicates that boosting with the HG immunogen immune focuses to the class 4 epitope.

Immune focusing improves cross-neutralization
We assessed serum pseudovirus neutralization of WT SARS-CoV-2, Delta and Omicron variants, and related sarbecoviruses RaTG13, SARS-CoV-1, WIV1, and SHC014. Sera from all cohorts had broad  Figure 2A for four sarbecoviruses (related to Figure S3). neutralization with NT50s ranging from $10 3 -10 against WT SARS-CoV-2 to $10 2 -10 3 against SARS-CoV-1 and WIV1 ( Figure 4E). Neutralization of WT SARS-CoV-2 and the closely related Delta variant was significantly lower in the HG cohort; however, neutralization of Omicron was significantly improved in the HG cohort ( Figure 4E). Neutralization of related sarbecoviruses RaTG13, SARS-CoV-1, WIV1, and SHC014 showed no significant differences between the WT and HG cohorts ( Figure 4E). This is consistent with the fact that there is no significant decrease in the HG cohort between neutralization of WT SARS-CoV-2 and all other coronaviruses tested, while there are significant decreases in the WT cohort ( Figures S4I-S4J). Because serum antibody responses from the HG cohort significantly competed with Ab16 (class 4 epitope), relative to the WT cohort ( Figure 4D), this suggests that the observed immune focusing elicited by this first-generation immunogen may also neutralize future antigenically distinct SARS-CoV-2 variants. While these data demonstrate a proof-of-principle that focusing SARS-CoV-2 serum antibody responses to a broadly neutralizing epitope can improve breadth, the lack of improved serum neutralization across other sarbecoviruses (Figures S4I-S4J) indicates that further iterative design cycles are necessary.

DISCUSSION
A continued understanding of the nature of the broadly crossreactive repertoire elicited by SARS-CoV-2 infection and vacci-nation is necessary given that additional SARS-CoV-2 variants and other related sarbecoviruses will emerge. Characterizing human cross-reactive antibodies may help identify candidate therapeutic monoclonal antibodies that limit the likelihood of viral escape. Currently available monoclonal antibodies were largely selected based on potent WT SARS-CoV-2 neutralization, but Omicron evades neutralization by many of them, particularly those that target epitopes in the RBM (class 1 and 2 epitopes). [13][14][15] Emerging SARS-CoV-2 variants or other sarbecoviruses may still escape even broadly cross-reactive antibodies, but identifying multiple antibodies with distinct, non-overlapping epitopes may help maintain overall therapeutic efficacy.
Eliciting broadly cross-reactive antibody responses via vaccination, in combination with developing broadly protective therapeutic antibodies, may help reduce the likelihood that ongoing SARS-CoV-2 evolution and potential emerging sarbecoviruses will escape both existing protective immunity and available treatments. Indeed, broadly cross-reactive antibody responses have already been identified in patients vaccinated against SARS-CoV-2 following prior SARS-CoV-1 infection, indicating that it should be possible to elicit these responses in humans. 32,33 Additionally, while boosting with WT SARS-CoV-2 protects against Omicron, protection may vary as other variants evolve; this underscores the need for a pan-sarbecovirus vaccine. 2  (E) Day 35 serum assayed against wild-type SARS-CoV-2, SARS-CoV-2 variants, and related sarbecoviruses for neutralization. Pseudovirus neutralization assays were performed with two technical replicates, and IC50s were determined via non-linear curve fits. For (D) and (E), bars represent mean ± SEM. Statistical significance determined using the Mann-Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant) (related to Figure S4 Efforts to focus the immune response to protective epitopes have extended to several different viruses, including influenza, HIV, respiratory syncytial virus (RSV), and SARS-CoV-2. 25,[36][37][38] Here, we used an immunogen design strategy to re-direct preexisting antibody responses toward conserved epitopes by engineering putative N-linked glycosylation sites to ''mask'' undesirable epitopes. 25,38 Immune re-focusing is particularly important in the context of immune imprinting to influence a memory-recall response that might otherwise be biased toward less conserved epitopes; such re-focusing can improve serum neutralization and protection. These immune imprinting and biased antibody responses were observed for SARS-CoV-2 39-41 and parallel observations from influenza. [42][43][44] Consequently, how to focus humoral immune responses toward broadly protective epitopes must be evaluated in context of pre-existing immunity. Our results show that human subjects generate cross-reactive antibodies targeting conserved epitopes and that these antibodies are genetically diverse. In mice, selectively boosting responses to one of these conserved epitopes using an engineered immunogen improved Omicron variant neutralization in the context of pre-existing immunity. Further experiments will be required to evaluate the impact of this immune focusing on protection, but these data provide proof of principle for an approach to pan-sarbecovirus vaccine design by demonstrating that prior knowledge of the exact variant or emerging sarbecovirus may not be required to elicit a broadly neutralizing antibody response.

LIMITATIONS OF THE STUDY
This study aims to provide proof-of-principle that focusing SARS-CoV-2 serum antibody responses to a conserved epitope in the context of SARS-CoV-2 spike imprinting can improve breadth. Further experiments, including in vivo protection studies, is necessary to establish whether this change is clinically meaningful. Additionally, RBD-directed antibodies represent only one possible class of broadly neutralizing antibodies. Antibodies targeting other conserved regions on the N-terminal domain or elsewhere on the spike glycoprotein could also confer protection, but these antibodies were not isolated in our sorting strategy. However, we note that most potently neutralizing monoclonal antibodies are RBD directed, making additional RBD-directed antibodies promising candidates to achieve broad neutralization. 16,[45][46][47] STAR+METHODS Detailed methods are provided in the online version of this paper and include the following:

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Aaron G. Schmidt (aschmidt@crystal.harvard.edu).

Materials availability
All unique/stable reagents generated in this study will be made available on request, but we may require a payment and/or a completed materials transfer agreement if there is potential for commercial application. For non-commercial use, all unique/stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.
Data and code availability d B cell receptor sequences have been deposited in Genbank. Accession numbers are listed in Table S4. B cell receptor sequences are also available from Mendeley Data at https://doi.org/10.17632/4wmr6xb29g.1. d This paper does not report original code. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Human donor samples Human peripheral blood mononuclear cells were isolated for cell sorting from samples previously published 4 ; samples were obtained with approval from the Partners Institutional Review Board (protocol 2020P002274). All measurements relating to donor samples were obtained from separate samples, rather than repeated measurements of the same samples. Report ll OPEN ACCESS Assay buffers described previously 57 were used to perform cell lysis, and luciferase expression was then measured with a Spectramax L luminometer (Molecular Devices). Background luminescence from cells-only sample wells was calculated and subtracted from each sample well. Neutralization percentage at a given serum or monoclonal IgG concentration was then calculated by dividing by the luminescence of wells containing only virus and cells. GraphPad Prism (version 9) was used to fit nonlinear regressions were to the data and used to calculate IC50 values for all serum samples with neutralization values greater than or equal to 80% at maximum concentration. NT50 values were then calculated by taking the reciprocal of the IC50 values.

Live virus neutralization assay
The live virus neutralization assays were carried out in the Biosafety Level 3 laboratory of the Ragon Institute of MGH, MIT, and Harvard.
The US-WA1/2020 ancestral variant of SARS-CoV-2 was obtained from BEI Resources (Cat # NR-52281). The Omicron BA.1 variant isolate was obtained from the MassCPR variant repository. In brief, the variant had previously been isolated in the Ragon Institute BSL3 by rescue on Vero-E6 cells (ATCC) from primary clinical specimens, and it was then contributed to the MassCPR variant repository. The sequence of all viral stocks was confirmed by whole genome sequencing to ensure that no additional mutation arose during virus expansion. In-house prepared viral stocks (US-WA1/2020 Passage 3 and omicron BA.1 Passage 1) were diluted in D+ media to 32,000 pfu/mL to achieve a multiplicity of infection of 0.01 when added to the cells in the 96-well plates.
The antibody stocks (1 mg/mL) were diluted in D+ media (DMEM supplemented with HEPES, 1X Penicillin 100 IU/mL/Streptomycin 100 mg/mL and 1X Glutamine) to 2x desired final concentration by 3-fold serial dilutions. Equal volumes of viral stock and antibody dilution solutions were mixed and incubated for 1 hour at 37 C at 5% CO 2 . After the incubation, for each condition, 25 mL of virusantibody solutions (or no antibody control) were added to triplicate wells in 96-well plates. The plates were then spinfected at 2,000 x g for 30 minutes at 37C followed by a 48-hour incubation at 37 C at 5% CO 2 .
After 48 hours, the cell culture supernatant was discarded, the cells were washed with PBS (Corning) then harvested using TrypLE (Life Technologies) and D10+ media before being transferred to a V-bottom 96-well plate for flow cytometry staining. Cells were washed in PBS, then stained with live/dead fixable blue stain (Thermo Fisher) for 30 minutes at 4 C. Cells were then washed in flow cytometry buffer (2% FBS in PBS) and fixed using 4% paraformaldehyde (Santa Cruz) for 30 minutes at 4 C. The fixed cells were removed from the BSL3 laboratory and prepared for intracellular staining using Perm/Wash buffer (BD Biosciences). The permeabilized cells were stained with mouse anti-SARS-CoV-2 Nucleocapsid antibody (Biolegend) for 30 minutes at 4 C, then washed and stained with secondary antibody labelled with PE-Cy7 (Biolegend) for 30 minutes at 4 C. Finally, the cells were washed and resuspended in flow cytometry buffer. Flow cytometry was performed on a BD Symphony (BD Biosciences).

QUANTIFICATION AND STATISTICAL ANALYSIS
All FCS files generated from flow cytometry were analyzed using FlowJo software (version 10). GraphPad Prism (version 9) was used to fit asymmetric 4PL curves to the neutralization data which were used to calculate IC50 and NT50 values. GraphPad Prism was also used to perform other statistical analyses: the Kruskal-Wallis non-parametric ANOVA with post hoc analysis using Dunn's test for multiple comparisons was used to compare multiple populations, and the Mann-Whitney U test was used to compare two populations without consideration of paired samples. A p value < 0.05 was considered to be statistically significant.