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

Abstract

Current COVID-19 vaccines are largely limited in their ability to induce broad, durable immunity against emerging viral variants. Design and development of improved vaccines utilizing existing platforms requires an in-depth understanding of the antigenic and immunogenic properties of available vaccines. Here we examined the antigenicity of two of the original COVID-19 vaccines, mRNA-1273 and NVX-CoV2373, by electron microscopy-based polyclonal epitope mapping (EMPEM) of serum from immunized non-human primates (NHPs) and clinical trial donors. Both vaccines induce diverse polyclonal antibody (pAb) responses to the N-terminal domain (NTD) in addition to the receptor-binding domain (RBD) of the Spike protein, with the NTD supersite being an immunodominant epitope. High-resolution cryo-EMPEM studies revealed extensive pAb responses to and around the supersite with unique angles of approach and engagement. NTD supersite pAbs were 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.

Figure 1.. NHP serum pAb responses to NVX-CoV2373 and mRNA-1273 and reactivity to variants

(A) Composite maps of Wuhan Spike-Fab complexes and (B) their corresponding epitope plots from ns-EMPEM analysis of serum pAbs from NHPs prime-boosted with either NVX-CoV2373 or mRNA-1273 vaccines. (C) and (D) represent the composite maps of Spike-Fab complexes and their corresponding epitope plots 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.

Figure 2.. NHP serum pAb responses to homologous or heterologous prime-boost immunizations with NVX-CoV2373 and rS-Beta

(A) and (B) represent the composite maps of Spike-Fab complexes and their corresponding epitope plots 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.

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.

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.

Figure 5.. EMPEM analysis of pAbs in clinical trial donors that received either NVX-CoV2373 or mRNA-1273 vaccine series.

Composite maps of Wuhan Spike-Fab complexes and their corresponding epitope plots from ns-EMPEM analysis of pAbs derived from sera collected at (A) three time points (Days 21, 49, and 105) from five donors who received NVX-CoV2373 vaccine and at (B) two time points (Days 29 and 43) from four donors who received mRNA-1273 vaccine. (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.

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.

Figure 7.. Conformational flexibility of NTD.

(A) NTD conformations upon antibody binding captured by MD simulations and (B) ribbon representation overlay of NTDs from published atomic models displaying the plasticity of loops N1 (residues 14–26), N2 (residues 67–79), N3 (residues 141–156), and N5 (residues 246–260).