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Clinical Trial
. 2025 Jul 22;44(7):115986.
doi: 10.1016/j.celrep.2025.115986. Epub 2025 Jul 8.

Structural serology of polyclonal antibody responses to mRNA-1273 and NVX-CoV2373 COVID-19 vaccines

Sandhya Bangaru  1 , Abigail M Jackson  1 , Jeffrey Copps  1 , Monica L Fernández-Quintero  1 , Jonathan L Torres  1 , Sara T Richey  1 , Bartek Nogal  1 , Leigh M Sewall  1 , Alba Torrents de la Peña  1 , Asma Rehman  2 , Mimi Guebre-Xabier  2 , Bethany Girard  3 , Rituparna Das  3 , Kizzmekia S Corbett-Helaire  4 , Robert A Seder  4 , Barney S Graham  4 , Darin K Edwards  3 , Nita Patel  2 , Gale Smith  2 , Andrew B Ward  5
Affiliations
Clinical Trial

Structural serology of polyclonal antibody responses to mRNA-1273 and NVX-CoV2373 COVID-19 vaccines

Sandhya Bangaru et al. Cell Rep. .

Abstract

Design and development of improved COVID-19 vaccines that can induce broad, durable immunity against emerging variants require an in-depth understanding of the antigenic and immunogenic properties of vaccines utilizing existing platforms. Here, we examine the antigenicity of two original COVID-19 vaccines by performing secondary analyses of the clinical trials for mRNA-1273 (this study was registered at ClinicalTrials.gov NCT04283461) and NVX-CoV2373 (this study was registered at ClinicalTrials.gov NCT04368988) using electron microscopy-based polyclonal epitope mapping (EMPEM). Both vaccines induce diverse polyclonal antibody (pAb) responses to the N-terminal domain (NTD) in addition to the receptor-binding domain of the Spike protein, with the NTD supersite being an immunodominant epitope. High-resolution cryo-EMPEM studies reveal extensive pAb responses to and around the supersite, with unique angles of approach and engagement. NTD supersite pAbs are also the most susceptible to variant mutations compared to other specificities, indicating that ongoing Spike ectodomain-based vaccine design strategies should consider immuno-masking this site to prevent induction of these strain-specific responses.

Keywords: COVID-19 vaccines; CP: Immunology; CP: Microbiology; EMPEM; NTD supersite; NVX-CoV2373; SARS-CoV-2; Spike; antibody response; mRNA-1273; polyclonal antibodies.

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Conflict of interest statement

Declaration of interests K.S.C.-H., B.S.G., and A.B.W. are inventors on US Patent No. 10/960,070 B2, entitled “Prefusion Coronavirus Spike Proteins and Their Use.” K.S.C.-H. and B.S.G. are inventors on US Patent Application No. 202117798021, entitled “SARS-CoV-2 Vaccine.” A.B.W. is an inventor on US Patent No. 11217328, entitled “Epitope Mapping Method.” B.G., R.D. and D.K.E. are employees of Moderna, Inc. and may hold stock/stock options in the company. A.R., M.G.-X., N.P., and G.S. are employees of Novavax, Inc. and hold stock or stock options.

Figures

Figure 1.
Figure 1.. NHP serum pAb responses to NVX-CoV2373 and mRNA-1273 and reactivity to variants.
(A and B) Composite maps of Wuhan Spike-Fab complexes (A) and their corresponding epitope plots (B) from ns-EMPEM analysis of serum pAbs from NHPs prime-boosted with either NVX-CoV2373 or mRNA-1273 vaccines. (C and D) Composite maps of Spike-Fab complexes (C) and their corresponding epitope plots (D) of pAbs from two NHPs from each vaccine group in (A) complexed with Wuhan (ancestral) or variant Spikes. On composite maps, CoV Spikes are colored grey and the Fabs are colored based on epitope specificities as indicated on the Y-axis of epitope plots, and similar specificities are represented by different shades of the indicated color. The open circles on epitope plots correspond to Fab specificities identified by ns-EMPEM 2D class averages but could not be reconstructed in 3D. See also Figure S1 and Data S1.
Figure 2.
Figure 2.. NHP serum pAb responses to homologous or heterologous prime-boost immunizations with NVX-CoV2373 and rS-Beta.
(A and B) Composite maps of Spike-Fab complexes (A) and their corresponding epitope plots (B) from ns-EMPEM analysis of serum pAbs pooled from four NHPs from each vaccine group complexed with either Wuhan or variant Spikes. CoV Spikes are colored grey and the Fabs are colored based on epitope specificities as indicated on the Y-axis of epitope plots, and similar specificities are represented by different shades of the indicated color. The open circles on epitope plots correspond to Fab specificities identified by ns-EMPEM 2D class averages but could not be reconstructed in 3D. See also Figure S1 and Data S1.
Figure 3.
Figure 3.. Cryo-EMPEM analysis of polyclonal Fabs from NHPs prime-boosted with NVX-CoV2373.
(A) Seven cryo-EMPEM reconstructions of Wuhan Spike complexed with distinct polyclonal Fabs derived from pooled sera from NHPs immunized with NVX-CoV2373, prime and boost. The composite map representing all the identified Fab specificities is shown in the center. The supersite antibodies are colored in dark blue, lateral antibodies in light blue and NTD-down antibodies in turquoise. (B) Zoomed-in views of Fab-NTD (ribbon representation of docked atomic model) complexes from (A) demonstrating the specific NTD loops being engaged by various pAb specificities. The N1, N2, N3, N4, and N5 loops are colored yellow, pink, orange, green, and blue, respectively. PDBs for docked Spike NTD models, selected based on the closest loop conformations for each map, for NVX-NHPWu1–7 are 7l2f, 7c2l, 7mxp, 7jji, 7sjo, 7mxp, and 7mxp, respectively. See also Figures S2, S3 and S4, and Tables S1 and S2.
Figure 4.
Figure 4.. Cryo-EMPEM analysis of polyclonal Fabs from NHP that received heterologous prime-boost immunizations with NVX-CoV2373 and rS-Beta.
(A) Eight distinct cryo-EMPEM reconstructions of Wuhan Spike complexed with polyclonal Fabs derived from an NHP that was primed with NVX-CoV2373 and boosted with rS-Beta. The composite map representing all the identified Fab specificities is shown in the center. The supersite antibodies are colored in dark blue, lateral antibodies in light blue and NTD-down antibodies in turquoise. (B) Zoomed-in views of Fab-NTD (ribbon representation of docked atomic model) complexes from (A) demonstrating the specific NTD loops being engaged by various pAb specificities. The N1, N2, N3, N4, and N5 loops are colored yellow, pink, orange, green, and blue, respectively. PDBs for docked Spike NTD models, selected based on the closest loop conformations for each map, for NVX-NHPSA1–8 are 7mxp, 7l2c, 7jji, 7sj0, 7mxp, 7mxp, 7l2d, and 7mxp, respectively. See also Figures S3 and S5, and Tables S1 and S3.
Figure 5.
Figure 5.. EMPEM analysis of pAbs in clinical trial donors that received either NVX-CoV2373 or mRNA-1273 vaccine series.
(A and B) Composite maps of Wuhan Spike-Fab complexes and their corresponding epitope plots from ns-EMPEM analysis of pAbs derived from sera collected at three time points (Days 21, 49, and 105) from five donors who received NVX-CoV2373 vaccine (A) and at two time points (Days 29 and 43) from four donors who received mRNA-1273 vaccine (B). (C) Ns-EMPEM analysis of pooled serum pAbs from donors prime-boosted with either NVX-CoV2373 (Day 49) or mRNA-1273 (Day 43) in complex with Wuhan- or variant-derived Spikes. On composite maps, CoV Spikes are colored grey and the Fabs are colored based on epitope specificities as indicated on the Y-axis of epitope plots, and similar specificities are represented by different shades of the indicated color. The open circles on epitope plots correspond to Fab specificities identified by ns-EMPEM 2D class averages but could not be reconstructed in 3D. See also Figure S6 and Data S1.
Figure 6.
Figure 6.. Cryo-EMPEM analysis of polyclonal Fabs from donors vaccinated with mRNA-1273.
(A) Eight cryo-EMPEM reconstructions of Wuhan Spike complexed with distinct polyclonal Fabs, two RBD and six NTD, derived from pooled sera from mRNA-1273 vaccinees. The composite map representing all the identified Fab specificities is shown in the center. The RBM Fabs are colored in red, NTD supersite Fabs in dark blue and NTD-down Fab in turquoise. (B) Zoomed-in views of Fab-RBD and Fab-NTD (ribbon representation of docked atomic model) complexes from (A), demonstrating the epitope specificities of various Fabs. The ModWu-RBD1 map, docked with the atomic model of the P4A1-RBD complex (7cjf), shows binding to RBM class 1 epitope that overlaps with ACE2 RBS. For ModWu-NTD1–6 maps, the NTD loops N1, N2, N3, N4, and N5 are colored yellow, pink, orange, green, and blue, respectively. PDBs for docked NTD models, selected based on the closest loop conformations for each map, for ModWu-NTD1–6 are 7jji, 7jji, 7mxp, 7l2e, 8swh, and 7mxp, respectively. (C) Surface representation of the NTD displaying residues in the epitope–paratope interface, identified by MD simulations. (D) Two cryo-EMPEM reconstructions of Delta Spike complexed with distinct polyclonal Fabs, one RBD and one NTD, derived from pooled sera from mRNA-1273 vaccinees used in (A). Bottom panel in (D) is the overlay of Fab-delta Spike complexes, ModDe-RBD1 and ModDe-NTD1 (solid color), with Fab-Wuhan Spike complexes (transparent surface), ModWu-RBD1 and ModWu-NTD6, respectively. See also Figures S4, S7, S8 and S9, and Tables S1, S4 and S5.
Figure 7.
Figure 7.. Conformational flexibility of NTD.
(A and B) NTD conformations upon antibody binding captured by MD simulations (A) and ribbon representation overlay of NTDs from published atomic models (B) displaying the plasticity of loops N1 (residues 14–26), N2 (residues 67–79), N3 (residues 141–156), and N5 (residues 246–260).
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