Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines

Abstract

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination^1,2. The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

Extended Data Figure 1.. Multiplexed spike-binding detection assay.

a, Gating strategy for the multiplexed spike-binding assay. After gating on singlets, beads pre-bound with the spike protein of different SARS-CoV-2 strains were separated based on their intensity of SPHERO^™ yellow fluorophore. b, Representative flow cytometry plots of mAb CV3-25 binding to Wuhan-1, B.1.351, BA.1, and BA.5 spike-charged detection beads at the indicated antibody concentrations. c, A CV3-25 standard curve of binding to Wuhan-1, B.1.351, BA.1, and BA.5 spike-charged detection beads (pooled from two experiments, error bars denote standard deviations [SD]). d, Literature-based SARS-CoV-2 spike binding characteristics and escape mutations of the indicated mAbs to Wuhan-1, B.1.351, BA.1, and BA.5 spike proteins. CHK-265 (anti-CHIKV E2) was included as a negative control. +, positive binding; -, negative binding; amino acid substitutions indicate the mutations in the corresponding SARS-CoV-2 strain that results in loss of mAb binding. e, Representative flow cytometry plots of the indicated mAbs to Wuhan-1, B.1.351, BA.1, and BA.5 spike-charged detection beads (three experiments). f, Pearson’s correlation between the spike-binding and ELISA endpoint titers of the mAbs evaluated in d-e. R² and p values are indicated. g, Antibody binding (top panels) and neutralizing activity (bottom panels) of naïve mouse sera and pre-pandemic human sera to Wuhan-1, BA.1, BA.5, and XBB.1.5 spike proteins and chimeric VSV pseudoviruses (n = 4; one experiment). Positive controls of serum collected from a mouse immunized with two doses of mRNA-1273 followed by two doses of mRNA-1273.529 (encodes BA.1 spike protein) and serum collected from a SARS-CoV-2 non-infected clinical trial participant immunized with three doses of mRNA-1273 and boosted with mRNA-1273.222 (Wuhan-1/BA.5 bivalent vaccine) are included (bars indicate mean values; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD for visualization). −, negative SARS-CoV-2 exposure history; +, positive SARS-CoV-2 exposure history.

Extended Data Figure 2.. Antibody depletion assay.

a, Schematic of the anti-spike antibody depletion assay. Dp, depletion. b-c, Wuhan-1 spike-binding and antibody titers obtained after interpolating with CV3-25 standard curves of mAbs SARS2-38 and 2B04 mixed at the indicated ratios and pre-cleared with empty depletion beads (no Dp), B.1.351 spike-loaded beads (B.1.351 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) (numbers at the top in b indicate the derived fraction of Wuhan-1 spike-specific antibody; three experiments; mean values ± SDs are shown, and dotted lines show the LOD). d, Antibody binding of 3-week post-boost sera harvested from mice primed with 0.25 μg and boosted one month later with 1 μg of mRNA-1273 (1273/1273) or mRNA-1273.351 (351/351) (n = 10, two experiments) to Wuhan-1 and B.1.351 spike proteins. Sera were pre-cleared with empty beads, B.1.351 spike-loaded beads, or Wuhan-1 spike-loaded beads (connecting lines represent sera from the same mouse; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD for visualization). e, Percentages of Wuhan-1 (left) and B.1.351 (right) spike-specific antibodies, derived from d. The fraction of Wuhan-1 spike-specific IgG is calculated by dividing the Wuhan-1 spike-binding titer of B.1.351 spike depleted sera (blue circles in d left panel) by that without depletion (dark grey circles in d left panel) from the same individual (B.1.351 Dp/no Dp) (type-specific titers at the LOD in d are designated 0% type-specific response in e; bars and numbers at the top indicate median values). e, Two-sided Mann-Whitney test with Bonferroni correction (p = 0.000011).

Extended Data Figure 3.. Effect of heterologous vaccine boosting on type-specific serum antibody binding and neutralizing responses.

Sera were collected 26 days post-vaccination from mice immunized with a single dose of mRNA-1273 (1273) or mRNA-1273.529 (529) (n = 10, two experiments), or 21 days after the last vaccine dose from mice vaccinated with the one prime/one boost (1273/529, n = 10), two primes/one boost (1273/1273/529, n = 9), or two primes/two boosts (1273/1273/529/529, n = 12; also described in Fig. 1) regimens. All vaccines were given at 0.25 μg mRNA per dose. 1273, mRNA-1273; 529, mRNA-1273.529. a, Antibody binding of sera pre-cleared with empty beads (no Dp), BA.1 spike-loaded beads (BA.1 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) to Wuhan-1 (left) and BA.1 (right) spike proteins (connecting lines represent sera from the same mouse; LOD for the single dose and the one prime/one boost cohorts are 1,600 ng/mL equivalents (eq, derived from a mAb standard curve); LOD (dotted lines) for the two primes/one boost and two primes/two boosts cohorts are 3,200 ng/mL eq; values at the LOD are plotted slightly below the LOD for visualization). b, Percentages of Wuhan-1 (left) and BA.1 (right) spike-specific antibodies (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive IgG in each group. c, Neutralizing activity of pre-cleared sera against VSV pseudoviruses displaying Wuhan-1 (left) or BA.1 (right) spike proteins (numbers above data points indicate the GMT; fractions in parentheses at the top indicate the numbers of mice with detectable neutralization by type-specific antibodies; LOD for the single dose and the one prime/one boost cohorts are 1/25 serum dilution; LOD (dotted lines) for the two primes/one boost and two primes/two boosts cohorts are 1/50 serum dilution). Pie charts illustrate the fraction of neutralization mediated by type-specific and cross-reactive antibodies in each group. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. b, Kruskal-Wallis ANOVA with Dunn’s post-test.

Extended Data Figure 4.. Persistent effects of immune imprinting in the serum of mice receiving heterologous mRNA vaccines.

a, Scheme of immunization regimen and blood draw. Mice were immunized one month apart and boosted with a third dose of mRNA vaccine three months later (n = 10, two experiments, also in Fig. 1e). Sera were collected 15 weeks after the last dose. 1273, mRNA-1273; 529, mRNA-1273.529. b, Antibody binding of sera pre-cleared with empty beads (no Dp), BA.1 spike-loaded beads (BA.1 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) to Wuhan-1 (left) and BA.1 (right) spike proteins (connecting lines represent sera from the same mouse; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD). c, Percentages of Wuhan-1 (left) and BA.1 (right) spike-specific antibodies (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive IgG in each group. d, e, Paired analysis of 3- and 15-week post-boost undepleted sera (d, 15-week data also shown in b "no Dp") and type-specific (e, 15w data also shown in b "BA.1 Dp” or “Wuhan-1 Dp”) antibody reactivity against Wuhan-1 (left) and BA.1 (right) spike proteins (numbers on the top indicate GMT fold reduction). c, Kruskal-Wallis ANOVA with Dunn’s post-test.

Extended Data Figure 5.. Absence of B.1.351 spike-specific antibodies in humans receiving B.1.351-matched booster as a third-dose vaccine.

a, Scheme of immunizations and blood draw. Participants received two doses of 100 μg of mRNA-1273 one month apart and were boosted with 50 μg of the indicated mRNA vaccine at least six months later. Sera were collected one month after the third dose (n = 5). 1273, mRNA-1273; 351, mRNA-1273.351; 211, mRNA-1273.211 (Wuhan-1/B.1.351 bivalent vaccine). b, Antibody binding to Wuhan-1 (top) and B.1.351 (bottom) spike proteins of sera pre-cleared with empty beads (no Dp), B.1.351 spike-loaded beads (B.1.351 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) (connecting lines represent sera from the same individual; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD). c, Percentages of Wuhan-1 (top) and B.1.351 (bottom) spike-specific antibodies (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive IgG in each group. d, Neutralizing activity of pre-cleared sera against Wuhan-1 (top) and B.1.351 (bottom) pseudoviruses (numbers above data points indicate the GMT; fractions in parentheses at the top indicate the numbers of individuals with detectable neutralization by type-specific antibodies). Pie charts illustrate the fraction of neutralization mediated by type-specific and cross-reactive antibodies. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. c, Kruskal-Wallis ANOVA with Dunn’s post-test.

Extended Data Figure 6.. Imprinting effects on MBCs after boosting with bivalent mRNA-1273.214 vaccine.

a, Scheme of immunizations and blood draws. b, Gating scheme for identification of spike-positive MBCs in blood. c-d, Study participants (n = 20) who previously received three doses of mRNA-1273 were boosted with mRNA-1273.214 (Wuhan-1/BA.1 bivalent vaccine), and peripheral blood mononuclear cells (PBMCs) were obtained on days 0 (pre-boost), 28 (post-boost), and 90 (post-boost). c, Representative flow cytometry dot plots showing MBC specificities for pairs of fluorescently labeled WA-1/2020 and BA.1 spike proteins. Quadrant frequencies represent percentage of total MBCs. d, Fractions of spike-binding MBCs with indicated specificities (WA-1⁺BA.1⁺, left; WA-1⁺BA.1⁻, middle; or WA-1⁻BA.1⁺, right) at days 0, 28, and 90 (numbers at the top denote median values). d, Kruskal-Wallis ANOVA with Dunn’s post-test.

Extended Data Figure 7.. Humans with a SARS-CoV-2 infection history administered an XBB.1.5 monovalent vaccine after a previous BA.5-matched booster develop cross-neutralizing antibodies in serum.

a, Scheme of immunizations and blood draws, also described in Fig. 4. Clinical trial participants received a primary two-dose mRNA-1273 series, a third dose of mRNA-1273, a fourth dose of mRNA-1273.222 (Wuhan-1/BA.5 bivalent vaccine), and a fifth dose of mRNA-1273.815 (XBB.1.5 monovalent vaccine) (b-d post-boost, n = 28; b-d pre-boost and e-f, n = 15). Sera were collected immediately before and one month after the fifth dose. Subjects with a history of SARS-CoV-2 infection (anti-N antibody positive before the mRNA-1273.815 vaccination) were selected for analysis. 1273, mRNA-1273; 222, mRNA-1273.222; 815, mRNA-1273.815. b, Paired analysis of pre- and post-boost serum antibody reactivity against Wuhan-1 and XBB.1.5 spike proteins. c, Antibody binding to Wuhan-1 and XBB.1.5 spike proteins of pre- and post-boost sera pre-cleared with empty beads (no Dp), XBB.1.5 spike-loaded beads (XBB.1.5 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) (connecting lines represent sera from the same individual; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD). d, Percentages of Wuhan-1 and XBB.1.5 spike-specific antibodies before and after the fifth immunization (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive antibodies. e, Paired analysis of pre- and post-boost serum neutralizing activity against Wuhan-1 and XBB.1.5 pseudoviruses. f, Neutralizing activity of pre-cleared sera against Wuhan-1 and XBB.1.5 pseudoviruses (numbers above data points indicate the GMT; fractions in parentheses at the top indicate the numbers of individuals with detectable neutralization by type-specific antibodies). Pie charts illustrate the fraction of neutralization mediated by type-specific and cross-reactive antibodies. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. All statistical analysis was performed using a two-sided Wilcoxon signed-rank test.

Extended Data Figure 8.. XBB.1.5 monovalent booster increases serum binding to multiple neutralizing epitopes on the XBB.1.5 spike protein.

a, Schematic of the mAb competition assay (competing serum antibody, red; non-competing serum antibody, salmon; fluorescence-labeled mAb, blue). b, Percent binding of the indicated mAbs competed by serially diluted pre- and post-boost serum of XBB.1.5 monovalent vaccine boosted individuals described in Fig. 4 to XBB.1.5 spike protein. Two naïve sera (black curves, no SARS-CoV-2 infection or vaccination history) were included as control. c, Competition titer of pre- and post-boost sera that inhibits 50% binding of the indicated mAbs (numbers on the top indicate GMT fold increases), derived from b.

Extended Data Figure 9.. Spike protein sequence alignment.

Multiple sequence alignment of SARS-CoV-1 (P59594, UNIPROT), Pang/GD (EPI_ISL_410721, GISAID), XBB.1.5 (EPI_ISL_16713058, GISAID), and Wuhan-1 (EPI_ISL_402124, GISAID) spike proteins. The Wuhan-1 spike is shown in the last row with relative variant sequence changes indicated. The color ribbons beneath the sequence correspond to the specific regions of the spike protein (NTD, green; RBD, blue; S2, pink).

Extended Data Figure 10.. Depletion of Wuhan-1 NTD, RBD, S2, and spike-reactive antibodies from serum.

Antibody binding to Wuhan-1 NTD, RBD, S2, or spike proteins of pre- and post-boost sera of 1273/1273/1273/222/815-vaccinated individuals without (a) or with (b) a history of SARS-CoV-2 infection after pre-clearing with empty beads (no Dp), Wuhan-1 N-terminal domain protein-loaded beads (NTD Dp), RBD-loaded beads (RBD Dp), S2 protein-loaded beads (S2 Dp), a 1:1 mixture of RBD-loaded and S2-loaded beads (RBD & S2 Dp), or spike-loaded beads (spike Dp) (dotted lines show the LOD; values at the LOD are plotted slightly below the LOD; individuals that became infected (anti-N antibody negative before the fourth dose and positive before the fifth dose) are indicated with red borders; all other individuals in b are anti-N positive before the fourth dose). Binding to Wuhan-1 NTD, RBD, and S2 was interpolated from SARS2-57, SARS2-38, and CV3-25 standard curves, respectively. c, Serum neutralizing activity of infection-naïve post-boost sera pre-cleared with empty beads (no Dp), Wuhan-1 RBD-loaded beads (RBD Dp), or a mixture of Wuhan-1 RBD-loaded and S2-loaded beads (RBD & S2 Dp) against SARS-CoV-1 pseudovirus (data also shown in Fig. 5e, third panel) (connecting lines represent sera from the same individual). Statistical analysis: two-sided Wilcoxon signed-rank test.

Extended Data Figure 11.. Neutralizing activity of vaccine-derived serum against Omicron BA.2.86 and MERS-CoV.

As described in Fig. 5, 1273/1273/1273 post-boost sera (first left panels, described in Extended Data Fig. 5), 1273/1273/1273/222 post-boost sera (second left panels, described in Fig. 2), or pre- and post-boost sera relative to immunization with mRNA-1273.815 (XBB.1.5 monovalent vaccine) of SARS-CoV-2 non-infected (third left panels, described in Fig. 4) and infected (right panels, described in Extended Data Fig. 7) individuals were pre-cleared with Wuhan-1 NTD, RBD, S2, RBD together with S2 (RBD & S2), or spike protein. Pre-cleared sera were tested for neutralizing activity against pseudoviruses expressing BA.2.86 (a) or MERS-CoV (b) spike proteins (bars and numbers at the top are GMTs; dotted lines show the LOD; values at the LOD are plotted slightly below; individuals that became infected (anti-N antibody negative before the fourth vaccine dose and positive before the fifth vaccine dose) are indicated with red borders in the right panels).

Figure 1.. Serum antibody responses after BA.1-matched vaccine boosters in mice.

a, e, i, Schemes of the one prime/one boost (a), two primes/one boost (e), and two primes/two boosts (i) immunization regimens and blood collections. a, Female C57BL/6J mice were immunized twice over a 4-week interval with the indicated mRNA vaccines in b-d (n = 10, two experiments). e, Mice were immunized one month apart and boosted with a third dose of mRNA vaccine three months later (n = 10, two experiments; data in f-h). i, Mice were vaccinated one month apart, boosted with a third dose three months later, and received a fourth dose ten months later (1273/1273/1273/1273 group, n = 10; 529/529/529/529 group, n = 14; 1273/1273/529/529 group, n = 12; two experiments; data in j-l). Sera were collected 3 weeks after the last dose. b, f, j, Antibody binding of sera pre-cleared with empty beads (no Dp), BA.1 spike-loaded beads (BA.1 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) to Wuhan-1 (left) and BA.1 (right) spike proteins (connecting lines represent sera from the same mouse; dotted lines show the limit of detection, LOD; values at the LOD are plotted slightly below). c, g, k, Percentages of Wuhan-1 (left) and BA.1 (right) spike-specific antibodies (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive IgG in each group. d, h, l, Neutralizing activity of pre-cleared sera against VSV pseudoviruses displaying Wuhan-1 (left) or BA.1 (right) spike proteins (numbers above data are geometric mean titers (GMT); fractions indicate the numbers of mice with detectable neutralization by type-specific antibodies). Pie charts illustrate the fraction of neutralization mediated by type-specific and cross-reactive antibodies. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. c, g, k, Kruskal-Wallis ANOVA with Dunn’s post-test.

Figure 2.. Low levels of Omicron-specific antibodies in humans boosted with a fourth dose of BA.1- or BA.5-matched vaccines.

a, g, Schemes of immunizations and blood draws. Clinical trial participants received a primary two-dose series of 100 μg of mRNA-1273, a third dose of 50 μg of mRNA-1273, and then were boosted at least six months later with 50 μg of Wuhan-1/BA.1 bivalent vaccine (mRNA-1273.214) (a, n = 10; data in b-f) or Wuhan-1/BA.5 bivalent vaccine (mRNA-1273.222) (g, n = 15; data in h-l). Sera were collected immediately before and one month after the fourth dose. Subjects with no prior SARS-CoV-2 infection history (anti-N negative) were selected for analysis. b, h, Paired analysis of pre- and post-boost serum antibody reactivity against Wuhan-1 and BA.1 (b) or BA.5 (h) spike proteins. c, i, Antibody binding to Wuhan-1, BA.1 (c), or BA.5 (i) spike proteins of pre- and post-boost sera pre-cleared with empty beads (no Dp), BA.1 spike-loaded beads (BA.1 Dp), BA.5 spike-loaded beads (BA.5 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) (connecting lines represent sera from the same individual; dotted lines show the LOD; values at the LOD are plotted slightly below). d, j, Percentages of Wuhan-1 (top), BA.1 (d, bottom), and BA.5 (j, bottom) spike-specific antibodies before and after the fourth immunization (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive antibodies. e, k, Paired analysis of pre- and post-boost serum neutralizing activity against Wuhan-1, BA.1 (e), and BA.5 (k) pseudoviruses. f, l, Neutralizing activity of pre-cleared sera against Wuhan-1 (top), BA.1 (f, bottom), and BA.5 (l, bottom) pseudoviruses (numbers above data are GMTs; fractions indicate the numbers of individuals with detectable neutralization by type-specific antibodies). Pie charts illustrate the fraction of neutralization by type-specific and cross-reactive antibodies. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. Statistical analysis was performed using a two-sided Wilcoxon signed-rank test.

Figure 3.. Humans administered a BA.5/XBB.1.5 bivalent vaccine after a previous BA.5-matched booster develop cross-neutralizing antibodies.

a, Scheme of immunizations and blood draws. Clinical trial participants received a primary two-dose mRNA-1273 series, a third dose of mRNA-1273, a fourth dose of mRNA-1273.222 (Wuhan-1/BA.5 bivalent vaccine), and a fifth dose of mRNA-1273.231 (BA.5/XBB.1.5 bivalent vaccine) (n = 10). Sera were collected immediately before and one month after the fifth dose. Subjects with no prior SARS-CoV-2 infection history were selected for analysis. b, Paired analysis of pre- and post-boost serum antibody reactivity against Wuhan-1 (top), BA.5 (middle), and XBB.1.5 (bottom) spike proteins. c, Antibody binding to Wuhan-1, BA.5, and XBB.1.5 spike proteins of pre-cleared pre- and post-boost sera. No Dp, depletion with empty beads; BA.5 Dp, depletion with BA.5 spike-loaded beads; BA.5 & XBB.1.5 Dp, depletion with a 1:1 mixture of BA.5-loaded and XBB.1.5-loaded beads; Wuhan-1 Dp, depletion with Wuhan-1 spike-loaded beads (connecting lines represent sera from the same individual; dotted lines show the LOD; values at the LOD are plotted slightly below). d, Percentages of Wuhan-1-specific (BA.5- and XBB.1.5-non-reactive), BA.5-specific (Wuhan-1-non-reactive), and XBB.1.5-specific (Wuhan-1-non-reactive) antibodies before and after the fifth immunization (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive antibodies. e, Paired analysis of pre- and post-boost serum neutralizing activity against Wuhan-1, BA.5, and XBB.1.5 pseudoviruses. f, Neutralizing activity of pre-cleared sera against Wuhan-1, BA.5, and XBB.1.5 pseudoviruses (numbers above data are GMTs; fractions indicate the numbers of individuals with detectable neutralization by type-specific antibodies). Pie charts illustrate the fraction of neutralization by type-specific and cross-reactive antibodies. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. Statistical analysis was performed using a two-sided Wilcoxon signed-rank test.

Figure 4.. Humans administered an XBB.1.5 monovalent vaccine after a previous BA.5-matched booster develop cross-neutralizing antibodies.

a, Scheme of immunizations and blood draws. Clinical trial participants received a primary two-dose mRNA-1273 series, a third dose of mRNA-1273, a fourth dose of mRNA-1273.222 (Wuhan-1/BA.5 bivalent vaccine), and a fifth dose of mRNA-1273.815 (XBB.1.5 monovalent vaccine) (n = 14). Sera were collected immediately before and one month after the fifth dose. Subjects with no prior SARS-CoV-2 infection history (anti-N negative) were selected for analysis. b, Paired analysis of pre- and post-boost serum antibody reactivity against Wuhan-1 and XBB.1.5 spike proteins. c, Antibody binding to Wuhan-1 and XBB.1.5 spike proteins of pre- and post-boost sera pre-cleared with empty beads (no Dp), XBB.1.5 spike-loaded beads (XBB.1.5 Dp), or Wuhan-1 spike-loaded beads (Wuhan-1 Dp) (connecting lines represent sera from the same individual; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD). d, Percentages of Wuhan-1 and XBB.1.5 spike-specific antibodies before and after the fifth immunization (bars and numbers at the top denote median values). Pie charts illustrate the fraction of type-specific and cross-reactive antibodies. e, Paired analysis of pre- and post-boost serum neutralizing activity against Wuhan-1 and XBB.1.5 pseudoviruses. f, Neutralizing activity of pre-cleared sera against Wuhan-1 and XBB.1.5 pseudoviruses (numbers above data are GMTs; fractions indicate the numbers of individuals with detectable neutralization by type-specific antibodies). Pie charts illustrate the fraction of neutralization by type-specific and cross-reactive antibodies. Serum samples with an NT₅₀ below the LOD after pre-clearing with empty beads (no DP) were not used in pie chart analyses. Statistical analysis was performed using a two-sided Wilcoxon signed-rank test.

Figure 5.. Neutralizing activity of vaccine-derived serum against distantly related sarbecoviruses.

1273/1273/1273 post-boost sera (first left panels, described in Extended Data Fig. 5), 1273/1273/1273/222 post-boost sera (second left panels, described in Fig. 2), or pre- and post-boost sera relative to immunization with mRNA-1273.815 (XBB.1.5 monovalent vaccine) of SARS-CoV-2 non-infected (third left panels, described in Fig. 4) and infected (right panels, described in Extended Data Fig. 7) individuals were pre-cleared with Wuhan-1 NTD, RBD, S2, RBD together with S2 (RBD & S2), or spike protein. Pre-cleared sera were tested for neutralizing activity against pseudoviruses expressing Wuhan-1 (a), XBB.1.5 (b), EG.5.1 (c), Pang/GD (d), or SARS-CoV-1 (e) spike proteins (bars and numbers at the top are GMTs; dotted lines show the LOD; values at the LOD are plotted slightly below the LOD; individuals that became infected (anti-N antibody negative before the fourth vaccine dose and positive before the fifth vaccine dose) are indicated with red borders in the right panels).