Ongoing evolution of SARS-CoV-2 drives escape from mRNA vaccine-induced humoral immunity

Figures

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  Figure 1. SARS-CoV-2 variants of concern harbor spike protein mutations that are enriched in the RBD (A) Scaled stacked area chart of variants emerging during the SARS-CoV-2 pandemic. Arrows denote when each vaccine was made available. Adapted from nextstrain.org. Data as of February 28, 2024. (B) Crystal structures of pre-fusion stabilized SARS-CoV-2 spike trimer (PDB ID 6XR8,59https://doi.org/10.2210/pdb6XR8/pdb) highlighting mutations found in each strain mapped onto their locations on the wild-type spike trimer. From left to right WT, Delta (B.1.617.2), Omicron (BA.1), BA.5, and JN.1 strains. Mutations highlighted for each strain are from corresponding Table S1. (C) Crystal structure of pre-fusion stabilized SARS-CoV-2 spike trimer (PDB ID 6XR8,59https://doi.org/10.2210/pdb6XR8/pdb) with differences with the closely related RaTG13 coronavirus highlighted in red (front and top view) on the WT spike trimer. (D) Crystal structure of pre-fusion stabilized SARS-CoV-2 spike trimer (PDB ID 6XR8,59https://doi.org/10.2210/pdb6XR8/pdb) with all 164 mutations found in variants of concern and variants of interest up to and including JN.1, mapped onto the WT spike, and colored by the number of…

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  Figure 2. Comprehensive neutralization assays with primary vaccine sera revealed regions of vulnerability within the natural mutation landscape (A) Schematic illustrating the time period from which variants were selected and the vaccinee sera to be tested below. (B) Titers (n = 2 per sample per pseudovirus) that achieve 50% of pseudovirus neutralization (pNT50) are plotted for mutants with significant escape primary vaccination regimen (2 doses of Pfizer) found in SARS-CoV-2 through Delta (1.617.2). The solid black line represents geometric mean pNT50 for reference. The following abbreviations are used: NTD, N-terminal domain; RBD, receptor binding domain; S1, S1 subunit; S2, S2 subunit. (C) Spike structure (PDB ID: 6XR8,59https://doi.org/10.2210/pdb6XR8/pdb) with mutations critical for escape from primary vaccine sera mapped onto the spike. The geometric mean pNT50 value for mutations relative to WT was log transformed into a B factor on a scale of −0.9 to 0.9. (D) Individual pseudovirus mutants that escaped primary vaccinees were measured for ACE2 binding. These values were then normalized to WT binding and log transformed. Binding was done for each mutant in technical triplicates (n =…

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  Figure 3. Combinations of individual mutations yield neutralization resistance and variable ACE2 binding (A and B) Primary series vaccinee sera (average of n = 2) was tested via pseudovirus-based neutralization assays against combinations of RBD mutations found in the (A) Beta variant or (B) Delta strain. Each variant was compared to the wild type using a one-way ANOVA with Friedman’s test and Dunn’s multiple comparisons test. (∗ = p < 0.0332, ∗∗ = p < 0.0021, ∗∗∗ = p < 0.0002, ∗∗∗∗ = p < 0.0001). (C and D) Single, double, or triple RBD mutants for the Beta (C) and Delta (D) variants were measured for ACE2 binding. Mutants were co-transfected with a zsgreen plasmid to be expressed on the surface of cells, and ACE2 binding was measured using a fluorescently labeled recombinant ACE2 protein using median fluorescence intensity of ACE2 Alexa 647, spike-positive (green) cells. Experiments were performed in technical triplicates (n = 3). Data are represented as mean ± SD. These values were then normalized to WT binding and log transformed. ACE2 binding was compared to WT using a one-way ANOVA with Dunn’s multiple comparisons test. (∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001). ∗,…

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  Figure 4. mRNA boosters significantly enhance neutralization of SARS-CoV-2 mutants (A) (Top) schematic illustrating the time period from which variants were selected and the vaccinee sera to be tested below. (B) (Bottom) primary or boosted pNT50 (n = 2) for donors across each variant was normalized to geometric mean titer across all donors for WT and log transformed. Length of each vertical line illustrates pNT50 change. (C) Titers (n = 2 per sample per pseudovirus) that achieve 50% of pseudovirus neutralization (pNT50) are plotted for mutants with significant escape boosted vaccination regimen found in SARS-CoV-2 through Delta (1.617.2). The solid black line represents geometric mean pNT50 for reference. The following abbreviations are used: NTD, N-terminal domain; RBD, receptor binding domain; S1, S1 subunit; S2, S2 subunit. (D) Log-transformed pNT50 geometric mean values (average of n = 2) for each variant relative to WT for primary series were plotted against matching boosted values. Linear regression (R2 = 0.5109; slope = −0.098; p < 0.001). Mutations that were escaped for primary vaccinees only or primary and boosted vaccinees are indicated. Labeled mutations are those that had the…

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  Figure 5. Individual Omicron variants reveal sites of resistance that are observed in more recent strains to significantly escape from boosted sera (A) Schematic illustrating the time period from which variants were selected and the vaccinee sera to be tested below. (B) pNT50 are plotted for all individual spike mutations occurring through XBB.1.16 variant with significant escape from 22 COVID naive boosted donors (n = 2 per sample per pseudovirus). The solid black line indicates the geometric mean of the titers against each pseudovirus. The following abbreviations are used: NTD, N-terminal domain; RBD, receptor binding domain; S1, S1 subunit; S2, S2 subunit. (C) Individual pseudovirus mutants that escaped boosted vaccinees were measured for ACE2 binding. These values were then normalized to WT binding and log transformed. All experiments were performed in technical triplicates (n = 3). ∗, significantly worse ACE2 binding. Data are represented as mean ± SD (∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001). (D) Neutralization titer relative to WT (average of n = 2) for boosted vaccinees was plotted against ACE2 binding relative to WT (average of n = 3). Both values are log…

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  Figure 6. Updated COVID boosters improve neutralization against dominant variants throughout the pandemic (A) Schematic illustrating the variants and vaccinee sera to be tested below. (B) Geometric mean pNT50 values of longitudinal serum samples (average of n = 2) over the course of CDC-recommended vaccination schedule against each variant. Strains are plotted in the order in time that they first appear in GISAID. (C) pNT50 of individual serum samples (average of n = 2) were normalized to geometric mean titer across all donors for WT and log transformed. Longitudinal samples from donors after WT, bivalent, or XBB.1.5 boosters are plotted. Length of each vertical line illustrates pNT50 change. (D) Log-transformed pNT50 geometric mean values (average of n = 2) relative to WT for donor sera against variants of concern through JN.1 plotted against the date of first submission to GISAID. Solid lines represent linear regressions for WT booster (R2 = 0.63, slope = −0.298, p = 0.002), bivalent booster (R2 = 0.57, slope = −0.24, p = 0.0018), and XBB.1.5 booster titers (R2 = 0.31, slope = −0.133, p = 0.0379). (E) One-way hierarchical clustering of pNT50 values of sera obtained from boosted donors…

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  Figure 7. SARS-CoV-2 variants of concern have accumulated spike mutations increasing their infectivity and ACE2 binding over time (A) Individual pseudovirus mutants were measured for ACE2 binding. All experiments were done in technical triplicates (n = 3). These values were then normalized to WT binding and compared to SARS-CoV-2 using a one-way ANOVA with Holm-Sidak correction for multiple comparisons. (∗ = p < 0.033, ∗∗ = p < 0.0021, ∗∗∗ = p < 0.0002, ∗∗∗∗ = p < 0.0001). Data are represented as mean ± SD. (B) ACE2 binding values (average of n = 3) plotted for each variant relative to the time it was first submitted to the GISAID database. A simple linear regression was performed with R2 = 0.53, p = 0.0047, slope = 0.707. (C) Pseudovirus infectivity (defined as infectious units per genome copy) was normalized to WT infectivity for each variant spike across technical triplicates (n = 3). Pseudovirus infectivity relative to WT was measured for major SARS-CoV-2 variants by calculating fold change in slope of linear regressions fit across known dilutions. Bars and error bars depict mean and standard error of the mean. Each pseudovirus was compared to SARS-CoV-2 wild type using a one-way ANOVA…