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[Preprint]. 2024 Dec 9:2024.12.06.625234.
doi: 10.1101/2024.12.06.625234.

Defining a highly conserved B cell epitope in the receptor binding motif of SARS-CoV-2 spike glycoprotein

Sameer Kumar Malladi  1 , Deepika Jaiswal  2 , Baoling Ying  3 , Wafaa B Alsoussi  1 , Tamarand L Darling  3 , Bernadeta Dadonaite  4 , Alesandro Civljak  2 , Stephen C Horvath  1 , Julian Q Zhou  1 , Wooseob Kim  1   5 , Jackson S Turner  1 , Aaron J Schmitz  1 , Fangjie Han  1 , Suzanne M Scheaffer  3 , Christopher W Farnsworth  1 , Raffael Nachbagauer  6 , Biliana Nestorova  6 , Spyros Chalkias  6 , Michael K Klebert  7 , Darin K Edwards  6 , Robert Paris  6 , Benjamin S Strnad  8 , William D Middleton  8 , Jane A O'Halloran  9 , Rachel M Presti  9   10 , Jesse D Bloom  4   11 , Adrianus C M Boon  1   3   12 , Michael S Diamond  1   3   10   12   13 , Goran Bajic  2 , Ali H Ellebedy  1   10   12   13
Affiliations

Defining a highly conserved B cell epitope in the receptor binding motif of SARS-CoV-2 spike glycoprotein

Sameer Kumar Malladi et al. bioRxiv. .

Abstract

SARS-CoV-2 mRNA vaccines induce robust and persistent germinal centre (GC) B cell responses in humans. It remains unclear how the continuous evolution of the virus impacts the breadth of the induced GC B cell response. Using ultrasound-guided fine needle aspiration, we examined draining lymph nodes of nine healthy adults following bivalent booster immunization. We show that 77.8% of the B cell clones in the GC expressed as representative monoclonal antibodies recognized the spike protein, with a third (37.8%) of these targeting the receptor binding domain (RBD). Strikingly, only one RBD-targeting mAb, mAb-52, neutralized all tested SARS-CoV-2 strains, including the recent KP.2 variant. mAb-52 utilizes the IGHV3-66 public clonotype, protects hamsters challenged against the EG.5.1 variant and targets the class I/II RBD epitope, closely mimicking the binding footprint of ACE2. Finally, we show that the remarkable breadth of mAb-52 is due to the somatic hypermutations accumulated within vaccine-induced GC reaction.

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Conflict of interest statement

Competing interests The Ellebedy laboratory received funding from Moderna, Emergent BioSolutions, and AbbVie that is unrelated to the data presented in the current study. A.H.E. has received consulting and speaking fees from InBios International, Fimbrion Therapeutics, RGAX, Mubadala Investment Company, Moderna, Pfizer, GSK, Danaher, Third Rock Ventures, Goldman Sachs and Morgan Stanley and is the founder of ImmuneBio Consulting. JST, WBA, AJS and AHE are recipients of a licensing agreement with Abbvie that is unrelated to the data presented in the current study. M.S.D. is a consultant or advisor for Inbios, Vir Biotechnology, IntegerBio, Akagera Medicines, Moderna, Merck, and GlaxoSmithKline. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Emergent BioSolutions, and IntegerBio. The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., and Nano targeting & Therapy Biopharma Inc. The Boon laboratory has received funding support from AbbVie Inc., for the commercial development of SARS-CoV-2 mAb. RP, BN, SC, DE and RN are employees of and shareholders in Moderna, Inc. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official view of NIAID or NIH. JDB consults for Apriori Bio, Pfizer, Invivyd, and the Vaccine Company. JBD and BD consult for Moderna. JDB and BD are inventors on Fred Hutch licensed patents related to the deep mutational scanning of viral proteins.

Figures

Figure 1.
Figure 1.. mRNA1273.214 bivalent booster vaccinees’ B cell responses.
(A) Nine participants immunized previously with three doses of ancestral WA1 vaccine were enrolled and followed after boosting. All participants’ blood was collected at baseline, week 1, 4, 8 and 17 following boosting. Fine needle aspirate of draining axillary lymph nodes were collected from all participants at week 8 post-boosting. (B) Frequency of bivalent vaccine WA1 and BA.1 S+ antibody responses were probed by ELISpot at week 1 corresponding to peak plasmablast response time-point. (C) All participants’ (n = 9) plasma anti-S IgG titres were measured at week 0 and 4 against WA1 and BA.1 Spike protein. Results are from technical duplicates of one experiment. P values were determined by two-tailed Wilcoxon matched-pairs signed rank test. (D) and (E) Representative flow cytometry plots and frequencies of S-binding germinal centre B cells (BCL6+CD38intIgDloCD19+CD3−) and lymph node plasma cells (CD20loCD38+IgDloCD19+CD3−) of fine needle aspirates from draining axillary lymph nodes at week 8 post boosting. Horizontal lines indicate median in frequency plots.
Figure 2.
Figure 2.. Lymph node fine needle aspirate B cell analysis of vaccinees.
(A) Uniform manifold approximation and projection (UMAP) representing B cell transcriptional clusters from single cell RNA sequencing (scRNA-seq) of lymph node FNAs (left) with spike-specific clones overlaid (right). Each dot represents a cell, coloured by phenotype as defined by transcriptomic profile (left) and S-specificity (right). Total number of cells (left) and number of S+ cells (right) are on the top left corner. GC, GC B cell; LNPC, Lymph node plasma cell; MBC, Memory B cell. (B) Antigen binding landscape of GC B cell and LNPC mAbs (n = 598) derived from distinct B cell clonal lineages from lymph node FNA at week 8 of five participants. (C) Binding of mAbs from S-specific GC B cells and LNPCs at day 57 post boosting to constituent domains of WA1/2020 S protein measured by ELISA. (D) WA1/2020 RBD-binding mAbs binding variants of concern RBD. The number of mAbs binding RBD of WA1/2020; WA1/2020 and BA.1; and WA1/2020, BA.1, and XBB.1.5 are depicted. (E) Neutralizing activity of mAbs cross reacting with WA1/2020, BA.1, and XBB.1.5 as determined with VSV-S (WA1/2020 D614G) chimeric virus assays. Each symbol represents one mAb. Percentages indicate the proportion of mAbs above 90% infection reduction threshold. (F) Neutralizing activity of mAbs in infection against WA1/2020 D614G, BA.1, XBB.1.5, EG.5.1, BA.2.86, HV.1, JN.1 (F) and KP.2 (G) authentic viruses. Each symbol in (F) represents one mAb. Authentic virus neutralization IC50 quantitated in ng/ml and mAbs are considered neutralizing given IC50 < 1000 ng/ml. mAb 52 potently neutralized WA1 D614G, BA.1, XBB.1.5, EG.5.1, BA.2.86, HV.1, JN.1 (F) and KP.2 (G) viral variants. Results are from technical duplicates of one experiment.
Figure 3.
Figure 3.. mAb-52 protects hamsters from EG.5.1 challenge.
(A) EG.5.1 challenge of five-week-old male Syrian golden hamsters. One day prior to challenge (d-1), the hamsters received an intraperitoneal injection with mAb-52 or 1G05 isotype control at 10 mg/kg. The following day (d0), the hamsters were intranasally challenged with 104 PFU of SARS-CoV-2 EG.5.1. Following challenge, the hamsters were monitored daily and their (B) nasal wash, (C) nasal turbinate, and (D) left lungs were harvested on day 3 for measurement of infectious virus by plaque assay and viral RNA by RT-qPCR. The data is from one experiment with 6 hamsters per group/experiment. P values were determined by two-tailed Mann-Whitney test.
Figure 4.
Figure 4.. XBB.1.5 RBD deep mutational scanning escape of mAb-52.
(A) Total escape at each site in the XBB.1.5 RBD as measured by pseudovirus deep mutational scanning library. (B) Escape caused by individual mutations at key sites of escape. XBB.1.5 wild type amino acids are depicted with X and amino acids in grey are absent in the library or highly deleterious for spike function. The complete data for line plot, heat map and analysis code can be accessed at dms-vep.org/SARS-CoV-2_XBB.1.5_RBD_DMS_mAB-52/htmls/mAb_52_mut_icXX.html (C) KD fold change determined by BLI binding of RBD escape mutants binding Fab-52. RBD mutants with KD fold change >10 are considered the footprint of mAb-52. (D) Mutational escape at key residues highlighted in heat map mapped onto surface representation of RBD. The higher intensity depicts higher escape. The epitope can be categorized as class I/II based on previous RBD antibody nomenclature. Results are the average of two independent libraries.
Figure 5.
Figure 5.. Cryo-EM structure of Fab-52 in complex with XBB.1.5 spike.
(A) 2.58 Å cryo-EM density for Fab-52-XBB.1.5 Spike trimer complex; Fab-52 VH (orange red) and VL (yellow) bound to the RBD (light gray). (B) Fab-52 footprint (orange), as defined by buried surface area, depicted on a surface representation of the RBD (light gray) with CDR loops of the Fab-52 VH (orange red) and VL (yellow), ACE2 footprint is outlined as red line track on RBD. (C) RBD epitope residues denoted by arrows in FAb-52:RBD interface. (D) Fab-52 paratope residues denoted by arrows in orange red and yellow. (E) and (F) are two 180° views along the y-axis that show details of the Fab-52:RBD molecular interface with numerous polar interactions.
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