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Cited by 800SARS-CoV-2 and COVID-19 ResearchImmune responses and vaccinationsvaccines and immunoinformatics approaches

The DOI matches its Crossref record; 64 of the 64 references it cites were found in a registry or an index; 8 of the 35 sources its claims cite were checked for support.

Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection

Open access
Wang, Zijun, Muecksch, Frauke, Schaefer-Babajew, Dennis, Finkin, Shlomo, Viant, Charlotte, Gaebler, Christian, Hoffmann, Hans- Heinrich, Barnes, Christopher O., Cipolla, Melissa, Ramos, Victor, Oliveira, Thiago Y., Cho, Alice, Schmidt, Fabian, Da Silva, Justin, Bednarski, Eva, Aguado, Lauren, Yee, Jim, Daga, Mridushi, Turroja, Martina, Millard, Katrina G., Jankovic, Mila, Gazumyan, Anna, Zhao, Zhen, Rice, Charles M., Bieniasz, Paul D., Caskey, Marina, Hatziioannou, Theodora, Nussenzweig, Michel C.·2021·Nature
DOI 10.1038/s41586-021-03696-9PMC PMC8277577

Published version of a preprint

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Abstract

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies 1,2 . Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines 3,4 . In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals 2,5–8 . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern 4,9 . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Overview

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Written from the paper’s full text.

TL;DR

In a longitudinal cohort of 63 SARS-CoV-2 convalescent individuals, antibody reactivity, neutralizing activity, and RBD-specific memory B cells remained relatively stable between 6 and 12 months after infection in the absence of vaccination, while mRNA vaccination boosted humoral responses by roughly 30- to 50-fold. Over time, memory B cells continued to evolve through somatic mutation and clonal turnover, producing antibodies with increased potency and breadth against SARS-CoV-2 variants.

Key findings

  • Among unvaccinated convalescent individuals, anti-RBD IgM, IgG, and IgA titres were maintained at 103%, 82%, and 72% between 6 and 12 months, whereas anti-N titres decreased significantly; vaccination increased anti-RBD IgG titres nearly 30-fold.
  • At 12 months, unvaccinated individuals had a geometric mean neutralizing titre (NT50) of 75, not significantly different from 6.2 months; vaccinated individuals had a geometric mean NT50 of 3,684, nearly 50-fold higher. After vaccination, geometric mean NT50 values against variants were 11,493 for B.1.351, 48,341 for B.1.1.7, 22,109 for B.1.526, and 26,553 for P.1.
  • RBD-specific memory B cells were 1.35-fold lower at 12 versus 6.2 months in unvaccinated individuals (P = 0.027), but vaccinated individuals showed an average 8.6-fold increase. Among 89 clones detected at 12 months, 61% were not previously detected and 39% were present at an earlier time point.
  • Of 174 monoclonal antibodies tested at 12 months, 173 bound RBD; geometric mean ELISA EC50 was 2.6 ng ml−1, lower than at 6 months. Neutralizing IC50 improved from 171 ng ml−1 at 1.3 months to 116 ng ml−1 at 6 months to 79 ng ml−1 at 12 months. For matched clonal antibody pairs, 10 of 15 antibodies at 12 months neutralized all tested mutants versus 1 of 15 at 1.3 months, with IC50 values as low as 1 ng ml−1 against K417N/E484K/N501Y.

Methods

  • Longitudinal cohort of 63 adults aged 26–73 years (median 47; 43% female; 10% hospitalized) assessed at 1.3, 6.2, and 12 months after SARS-CoV-2 infection; 26 participants (41%) had received at least one dose of Moderna mRNA-1273 or Pfizer-BioNTech BNT162b2, on average 40 days before the visit (range 2–82 days) and 311 days after illness onset (range 272–373 days). All tested negative for active infection by saliva PCR at 12 months.
  • Plasma ELISAs measured RBD and N antibody binding; neutralizing activity was measured with HIV-1 pseudotyped with SARS-CoV-2 spike and with authentic SARS-CoV-2 WA1/2020 and B.1.351. Flow cytometry counted RBD-specific memory B cells; single-cell sorting, antibody sequencing, cloning, and expression generated monoclonal antibodies. Biolayer interferometry assessed epitope competition and affinity; computational analyses assessed clonal relationships, somatic hypermutation, and phylogenetics.
  • Statistical comparisons used Kruskal–Wallis with Dunn’s multiple comparisons, Friedman, Mann–Whitney, Wilcoxon, and Spearman tests as indicated. The paper states that no statistical methods were used to predetermine sample size, experiments were not randomized, and investigators were not blinded.

Limitations

  • Stated in the paper: no statistical methods were used to predetermine sample size; experiments were not randomized; investigators were not blinded to allocation during experiments and outcome assessment.
  • Evident from the text: the cohort was small (n = 63), mostly had mild initial infections (only 10% hospitalized), and 41% had received mRNA vaccination, with vaccination occurring at variable times before the 12-month visit (range 2–82 days).
  • Evident from the text: follow-up ends at 12 months, so conclusions about very long-lasting immunity and protection against future variants are inferred from this interval and from in vitro antibody measurements rather than direct clinical protection data.

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Registry

Crossref · 10.1038/s41586-021-03696-9

Cites

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Statistics

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Figures

Fig. 1 Plasma ELISAs and neutralizing activity. a–d, Plasma IgG antibody binding to SARS-CoV-2 RBD (a) and N protein (b) shown as area under the curve (AUC; numbers in red are mean geometric AUC), and plasma neutralizing activity (NT50) in unvaccinated (c) and vaccinated (vac) (d) individuals 12 months after SARS-CoV-2 infection (n = 63). n = 63 individuals, 37 convalescent unvaccinated (black) and 26 convalescent vaccinated (blue) individuals. a, b, Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons. c, d, Lines connect longitudinal samples from the same individual. Two-sided Friedman test with subsequent Dunn’s multiple comparisons. Two individuals who received their first dose of vaccine 24–48 h before sample collection are represented in purple. e, Plasma neutralizing activity against indicated SARS-CoV-2 variants of concern (n = 30, 15 convalescent and 15 convalescent vaccinated individuals). The B.1.526 variant used here contains the E484K substitution. Substitutions, deletions and insertions in S variants used here are described in Methods. Two-tailed Mann–Whitney test. Red numbers in c–e indicate the geometric mean NT50 at the indicated time point. All experiments were performed at least in duplicate.
Fig. 1 Plasma ELISAs and neutralizing activity. a–d, Plasma IgG antibody binding to SARS-CoV-2 RBD (a) and N protein (b) shown as area under the curve (AUC; numbers in red are mean geometric AUC), and plasma neutralizing activity (NT50) in unvaccinated (c) and vaccinated (vac) (d) individuals 12 months after SARS-CoV-2 infection (n = 63). n = 63 individuals, 37 convalescent unvaccinated (black) and 26 convalescent vaccinated (blue) individuals. a, b, Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons. c, d, Lines connect longitudinal samples from the same individual. Two-sided Friedman test with subsequent Dunn’s multiple comparisons. Two individuals who received their first dose of vaccine 24–48 h before sample collection are represented in purple. e, Plasma neutralizing activity against indicated SARS-CoV-2 variants of concern (n = 30, 15 convalescent and 15 convalescent vaccinated individuals). The B.1.526 variant used here contains the E484K substitution. Substitutions, deletions and insertions in S variants used here are described in Methods. Two-tailed Mann–Whitney test. Red numbers in c–e indicate the geometric mean NT50 at the indicated time point. All experiments were performed at least in duplicate.CC BY · PubMed Central
Fig. 2 SARS-CoV-2 RBD-specific B cell memory. a, Number of antigen-binding memory B cells per 2 × 106 B cells (Extended Data Fig. 5b, c) obtained at 1.3, 6.2 and 12 months after infection from 40 randomly selected individuals (vaccinated, n = 20; non-vaccinated, n = 20). Each dot represents one individual. Red horizontal bars indicate geometric mean values. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons. WT, wild type. b, The distribution of antibody sequences from 6 individuals 1.3 (top) or 6.2 (middle) or 12 (bottom) months after infection3,4. The number in the inner circle indicates the number of sequences analysed for the individual whose identifier is denoted above the circle. Pie slice size is proportional to the number of clonally related sequences. The outer black arc indicates the frequency of clonally expanded sequences detected in each participant. Coloured slices indicate persisting clones (same IGV and IGJ genes, with highly similar CDR3 sequences) found at both time points in the same participant. Grey slices indicate clones unique to the time point. White indicates sequences isolated once, and white slices indicate singlets found at both time points. c, Number of somatic nucleotide mutations (SHM) in the IGVH and IGVL genes (Supplementary Table 3) obtained after 1.3 or 6.2 or 12 months (1.3 month, n = 889; 6.2 month, n = 975; 12 month, n = 1,105 (unvaccinated, n = 417; vaccinated, n = 688)). Red horizontal bars indicate mean values. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons.
Fig. 2 SARS-CoV-2 RBD-specific B cell memory. a, Number of antigen-binding memory B cells per 2 × 106 B cells (Extended Data Fig. 5b, c) obtained at 1.3, 6.2 and 12 months after infection from 40 randomly selected individuals (vaccinated, n = 20; non-vaccinated, n = 20). Each dot represents one individual. Red horizontal bars indicate geometric mean values. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons. WT, wild type. b, The distribution of antibody sequences from 6 individuals 1.3 (top) or 6.2 (middle) or 12 (bottom) months after infection3,4. The number in the inner circle indicates the number of sequences analysed for the individual whose identifier is denoted above the circle. Pie slice size is proportional to the number of clonally related sequences. The outer black arc indicates the frequency of clonally expanded sequences detected in each participant. Coloured slices indicate persisting clones (same IGV and IGJ genes, with highly similar CDR3 sequences) found at both time points in the same participant. Grey slices indicate clones unique to the time point. White indicates sequences isolated once, and white slices indicate singlets found at both time points. c, Number of somatic nucleotide mutations (SHM) in the IGVH and IGVL genes (Supplementary Table 3) obtained after 1.3 or 6.2 or 12 months (1.3 month, n = 889; 6.2 month, n = 975; 12 month, n = 1,105 (unvaccinated, n = 417; vaccinated, n = 688)). Red horizontal bars indicate mean values. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons.CC BY · PubMed Central
Fig. 3 Anti-SARS-CoV-2 RBD monoclonal antibodies. a, EC50 for SARS-CoV-2 RBD of antibodies isolated at 1.3 (n = 152) 6.2 (n = 153) and 12 (n = 174) months after infection3,4, determined by ELISA. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons (1.3 months versus 6.2 months, P = 0.27; 1.3 months versus 12 months, P = 0.0075; 6.2 versus 12 months, P < 0.0001). b, SARS-CoV-2-neutralizing activity of monoclonal antibodies measured using a SARS-CoV-2 pseudovirus neutralization assay3,13. IC50 values for antibodies isolated at 1.3, 6.2 and 12 months after infection against wild-type SARS-CoV-2 (Wuhan-Hu-1 strain41) are shown. Each dot represents one antibody. Pie charts illustrate the fraction of non-neutralizing (IC50 > 1,000 ng ml−1) antibodies (grey slices), inner circle shows the number of antibodies tested per group. Horizontal bars and red numbers indicate geometric mean values. Statistical significance was determined through the two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons.
Fig. 3 Anti-SARS-CoV-2 RBD monoclonal antibodies. a, EC50 for SARS-CoV-2 RBD of antibodies isolated at 1.3 (n = 152) 6.2 (n = 153) and 12 (n = 174) months after infection3,4, determined by ELISA. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons (1.3 months versus 6.2 months, P = 0.27; 1.3 months versus 12 months, P = 0.0075; 6.2 versus 12 months, P < 0.0001). b, SARS-CoV-2-neutralizing activity of monoclonal antibodies measured using a SARS-CoV-2 pseudovirus neutralization assay3,13. IC50 values for antibodies isolated at 1.3, 6.2 and 12 months after infection against wild-type SARS-CoV-2 (Wuhan-Hu-1 strain41) are shown. Each dot represents one antibody. Pie charts illustrate the fraction of non-neutralizing (IC50 > 1,000 ng ml−1) antibodies (grey slices), inner circle shows the number of antibodies tested per group. Horizontal bars and red numbers indicate geometric mean values. Statistical significance was determined through the two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons.CC BY · PubMed Central
Fig. 4 Epitope targeting and evolution of anti-SARS-CoV-2 RBD antibodies. a, Schematic of the BLI experiment (left) and IC50 values for randomly selected neutralizing and non-neutralizing antibodies (Ab1 and Ab2) isolated at 1.3 and 12 months after infection (n = 15 antibodies per group, n = 60 antibodies in total). Red horizontal bars indicate geometric mean. Two-sided Mann–Whitney test. b, Dissociation constants (KD) of the n = 30 neutralizing (green) and n = 30 non-neutralizing (red) antibodies shown in a. Horizontal bars indicate geometric mean values. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons. BLI traces are shown in Extended Data Fig. 8. c, Heat map of relative inhibition of binding of a monoclonal antibody (Ab2) to preformed complexes of RBD with another monoclonal antibody (Ab1) (grey, no binding; orange, intermediate binding; red, high binding). Data are normalized by subtraction of the autologous antibody control. BLI traces are shown in Extended Data Fig. 9. d, Neutralization of the indicated mutant RBD proteins with antibodies shown in a–c. Pie charts illustrate the fraction of antibodies that are poorly or non-neutralizing (IC50 of 100–1,000 ng ml−1, red), intermediate neutralizing (IC50 of 10–100 ng ml−1, pink) and potently neutralizing (IC50 of 0–10 ng ml−1, white) for each mutant. The number in the inner circle shows the number of antibodies tested. e, Graphs show affinities (y-axis) plotted against neutralization activity (x-axis) for 18 clonal antibody pairs isolated 1.3 (top) and 12 months (bottom) after infection (n = 36 antibodies). Spearman correlation test. f, BLI affinity measurements for same n = 36 paired antibodies as in e. Two-tailed Wilcoxon test. g, IC50 values for n = 30 paired neutralizing antibodies isolated at indicated time points versus indicated mutant SARS-CoV-2 pseudoviruses. Antibodies are divided into groups I, II and III (left), on the basis of neutralizing activity: I, potent clonal pairs that do not improve over time; II, clonal pairs that show increased activity over time; and III, clonal pairs showing decreased neutralization activity after 12 months. Antibody class assignment based on initial (1.3 month after infection) sensitivity to mutation is indicated on the right. Red stars indicate antibodies that neutralize all tested RBD mutants. Colour gradient indicates IC50 values ranging from 0 (white) to 1,000 ng ml−1 (red).
Fig. 4 Epitope targeting and evolution of anti-SARS-CoV-2 RBD antibodies. a, Schematic of the BLI experiment (left) and IC50 values for randomly selected neutralizing and non-neutralizing antibodies (Ab1 and Ab2) isolated at 1.3 and 12 months after infection (n = 15 antibodies per group, n = 60 antibodies in total). Red horizontal bars indicate geometric mean. Two-sided Mann–Whitney test. b, Dissociation constants (KD) of the n = 30 neutralizing (green) and n = 30 non-neutralizing (red) antibodies shown in a. Horizontal bars indicate geometric mean values. Two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons. BLI traces are shown in Extended Data Fig. 8. c, Heat map of relative inhibition of binding of a monoclonal antibody (Ab2) to preformed complexes of RBD with another monoclonal antibody (Ab1) (grey, no binding; orange, intermediate binding; red, high binding). Data are normalized by subtraction of the autologous antibody control. BLI traces are shown in Extended Data Fig. 9. d, Neutralization of the indicated mutant RBD proteins with antibodies shown in a–c. Pie charts illustrate the fraction of antibodies that are poorly or non-neutralizing (IC50 of 100–1,000 ng ml−1, red), intermediate neutralizing (IC50 of 10–100 ng ml−1, pink) and potently neutralizing (IC50 of 0–10 ng ml−1, white) for each mutant. The number in the inner circle shows the number of antibodies tested. e, Graphs show affinities (y-axis) plotted against neutralization activity (x-axis) for 18 clonal antibody pairs isolated 1.3 (top) and 12 months (bottom) after infection (n = 36 antibodies). Spearman correlation test. f, BLI affinity measurements for same n = 36 paired antibodies as in e. Two-tailed Wilcoxon test. g, IC50 values for n = 30 paired neutralizing antibodies isolated at indicated time points versus indicated mutant SARS-CoV-2 pseudoviruses. Antibodies are divided into groups I, II and III (left), on the basis of neutralizing activity: I, potent clonal pairs that do not improve over time; II, clonal pairs that show increased activity over time; and III, clonal pairs showing decreased neutralization activity after 12 months. Antibody class assignment based on initial (1.3 month after infection) sensitivity to mutation is indicated on the right. Red stars indicate antibodies that neutralize all tested RBD mutants. Colour gradient indicates IC50 values ranging from 0 (white) to 1,000 ng ml−1 (red).CC BY · PubMed Central
Extended Data Fig. 1 Clinical correlations. a–d, Association of persistence of symptoms (Sx) 12 months after infection with various clinical and serological parameters in our cohort of individuals who recovered from COVID-19 (n = 63). a, b, Acute disease severity as assessed with the WHO Ordinal Scale of Clinical Improvement (a, P = 0.99) and duration of acute phase symptoms (b, P = 0.63) in individuals reporting persistent symptoms (+) compared to individuals who are symptom-free (−) 12 months post-infection. c, Proportion of individuals reporting persistent symptoms (black area) compared to individuals who are symptom-free (grey area) 12 months after infection grouped by vaccination status (P = 0.72). d, Anti-RBD IgG (P = 0.75), anti-N IgG (P = 0.15), the RBD/N IgG ratio (P = 0.73), and NT50 titers (P = 0.38) at 12 months after infection in individuals reporting persistent symptoms (+) compared to individuals who are symptom-free (−) 12 months post-infection. Statistical significance was determined using the two-tailed Mann–Whitney test in a, b, d, and using the two-sided Fisher’s exact test in c.
Extended Data Fig. 1 Clinical correlations. a–d, Association of persistence of symptoms (Sx) 12 months after infection with various clinical and serological parameters in our cohort of individuals who recovered from COVID-19 (n = 63). a, b, Acute disease severity as assessed with the WHO Ordinal Scale of Clinical Improvement (a, P = 0.99) and duration of acute phase symptoms (b, P = 0.63) in individuals reporting persistent symptoms (+) compared to individuals who are symptom-free (−) 12 months post-infection. c, Proportion of individuals reporting persistent symptoms (black area) compared to individuals who are symptom-free (grey area) 12 months after infection grouped by vaccination status (P = 0.72). d, Anti-RBD IgG (P = 0.75), anti-N IgG (P = 0.15), the RBD/N IgG ratio (P = 0.73), and NT50 titers (P = 0.38) at 12 months after infection in individuals reporting persistent symptoms (+) compared to individuals who are symptom-free (−) 12 months post-infection. Statistical significance was determined using the two-tailed Mann–Whitney test in a, b, d, and using the two-sided Fisher’s exact test in c.CC BY · PubMed Central
Extended Data Fig. 2 Plasma activity. a–h, ELISA results for plasma against SARS-CoV-2 RBD 12 months after infection (n = 63). Non-vaccinated individuals are depicted with black circles and lines, and vaccinated individuals are depicted in blue throughout. Two outlier individuals who received their first dose of vaccine 24–48 h before sample collection is depicted as purple circles. a–n, IgM (a–d) IgG (e–g) and IgA (h–k) antibody binding to SARS-CoV-2 RBD and IgG binding to N (l–n) 12 months after infection. a, e, h, i, ELISA curves from non-vaccinated (black lines) individuals, as well as individuals who received one or two doses (blue lines) of a COVID-19 mRNA vaccine (left panels). Area under the curve (AUC) over time in non-vaccinated (b, f, i, m) and vaccinated individuals (c, g, j, n). Lines connect longitudinal samples. d, k, Boxplots showing AUC values of all 63 individuals, as indicated. o, ranked average NT50 at 1.3 months (light grey) and 6.2 months (dark grey), as well as at 12 months for non-vaccinated (orange) individuals, and individuals who received one or two doses (blue circles) of a COVID-19 mRNA vaccine, respectively. Two individuals who received their first dose of vaccine 24–48 h before sample collection is depicted in purple. p–r, Correlation of serological parameters in non-vaccinated (black circles and black statistics) and vaccinated (blue circles and blue statistics) individuals. Two individuals who received their first dose of vaccine 24-48 h before sample collection is depicted as purple circles. Correlation of 12-month titers of anti- RBD IgG and NT50 (p), anti-RBD IgG and N IgG (q), and anti-N IgG and NT50 (r). s, Plasma neutralizing activity against authentic virus isolates WA1/2020 and B.1.351, as indicated (n = 6). Statistical significance was determined using two-sided Friedman test with subsequent Dunn’s multiple comparisons (b, c, f, g, i, j, m, n), or two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons (d, k) or using the Spearman correlation test for the non-vaccinated and vaccinated subgroups independently (p–r) or using two-tailed Mann–Whitney test (s). Red numbers indicate the geometric mean NT50 at the indicated time point. All experiments were performed at least in duplicate.
Extended Data Fig. 2 Plasma activity. a–h, ELISA results for plasma against SARS-CoV-2 RBD 12 months after infection (n = 63). Non-vaccinated individuals are depicted with black circles and lines, and vaccinated individuals are depicted in blue throughout. Two outlier individuals who received their first dose of vaccine 24–48 h before sample collection is depicted as purple circles. a–n, IgM (a–d) IgG (e–g) and IgA (h–k) antibody binding to SARS-CoV-2 RBD and IgG binding to N (l–n) 12 months after infection. a, e, h, i, ELISA curves from non-vaccinated (black lines) individuals, as well as individuals who received one or two doses (blue lines) of a COVID-19 mRNA vaccine (left panels). Area under the curve (AUC) over time in non-vaccinated (b, f, i, m) and vaccinated individuals (c, g, j, n). Lines connect longitudinal samples. d, k, Boxplots showing AUC values of all 63 individuals, as indicated. o, ranked average NT50 at 1.3 months (light grey) and 6.2 months (dark grey), as well as at 12 months for non-vaccinated (orange) individuals, and individuals who received one or two doses (blue circles) of a COVID-19 mRNA vaccine, respectively. Two individuals who received their first dose of vaccine 24–48 h before sample collection is depicted in purple. p–r, Correlation of serological parameters in non-vaccinated (black circles and black statistics) and vaccinated (blue circles and blue statistics) individuals. Two individuals who received their first dose of vaccine 24-48 h before sample collection is depicted as purple circles. Correlation of 12-month titers of anti- RBD IgG and NT50 (p), anti-RBD IgG and N IgG (q), and anti-N IgG and NT50 (r). s, Plasma neutralizing activity against authentic virus isolates WA1/2020 and B.1.351, as indicated (n = 6). Statistical significance was determined using two-sided Friedman test with subsequent Dunn’s multiple comparisons (b, c, f, g, i, j, m, n), or two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons (d, k) or using the Spearman correlation test for the non-vaccinated and vaccinated subgroups independently (p–r) or using two-tailed Mann–Whitney test (s). Red numbers indicate the geometric mean NT50 at the indicated time point. All experiments were performed at least in duplicate.CC BY · PubMed Central
Extended Data Fig. 3 Flow cytometry. a, Gating strategy. Gating was on singlets that were CD20+ and CD3-CD8-CD16-Ova-. Anti-IgG, IgM, and IgA antibodies were used for B cell phenotype analysis. Sorted cells were RBD-PE+ and RBD/KEN-AF647+. b, c, Flow cytometry showing the percentage of RBD-double positive (b) and 647-K417N/E484K/N501Y mutant RBD cross-reactive (c) memory B cells from 1.3 or 6- and 12-months post-infection in 10 selected participants. d, As in Fig. 2b, Pie charts show the distribution of antibody sequences from 4 individuals after 1.33 (upper panel) or 6.24 months (middle panel) or 12 months (lower panel). e, Circos plot depicts the relationship between antibodies that share V and J gene segment sequences at both IGH and IGL. Purple, green, and grey lines connect related clones, clones and singles, and singles to each other, respectively. f, Graph summarizes cell number (indicated in b, c) (per 2 million B cells) of immunoglobulin class of antigens binding memory B cells in samples obtained at 1.3, 6.2 and 12 months. Each dot is one individual. (Vaccinees, n = 20, and non-vaccinees, n = 20). Red horizontal bars indicate mean values. Statistical significance was determined using two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons.
Extended Data Fig. 3 Flow cytometry. a, Gating strategy. Gating was on singlets that were CD20+ and CD3-CD8-CD16-Ova-. Anti-IgG, IgM, and IgA antibodies were used for B cell phenotype analysis. Sorted cells were RBD-PE+ and RBD/KEN-AF647+. b, c, Flow cytometry showing the percentage of RBD-double positive (b) and 647-K417N/E484K/N501Y mutant RBD cross-reactive (c) memory B cells from 1.3 or 6- and 12-months post-infection in 10 selected participants. d, As in Fig. 2b, Pie charts show the distribution of antibody sequences from 4 individuals after 1.33 (upper panel) or 6.24 months (middle panel) or 12 months (lower panel). e, Circos plot depicts the relationship between antibodies that share V and J gene segment sequences at both IGH and IGL. Purple, green, and grey lines connect related clones, clones and singles, and singles to each other, respectively. f, Graph summarizes cell number (indicated in b, c) (per 2 million B cells) of immunoglobulin class of antigens binding memory B cells in samples obtained at 1.3, 6.2 and 12 months. Each dot is one individual. (Vaccinees, n = 20, and non-vaccinees, n = 20). Red horizontal bars indicate mean values. Statistical significance was determined using two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons.CC BY · PubMed Central
Extended Data Fig. 4 Frequency distribution of human V genes. Graph shows comparison of the frequency distributions of human V genes of anti-SARS-CoV-2 antibodies from donors at 1.33, 6.24, 12 months after infection. a, Graph shows relative abundance of human IGVH genes Sequence Read Archive accession SRP010970 (green), convalescent vaccinees (blue), and convalescent non-vaccinees (orange). Statistical significance was determined by two-sided binomial test. b, c, Same as in a, but showing comparison between antibodies from donors at 1.3 months3 (b), 6.2 month4 (c) and 12 months after infection. Two-sided binomial tests with unequal variance were used to compare the frequency distributions., significant differences are denoted with stars (* P < 0.05, ** P < 0.01, *** P < 0.001, **** = P < 0.0001).
Extended Data Fig. 4 Frequency distribution of human V genes. Graph shows comparison of the frequency distributions of human V genes of anti-SARS-CoV-2 antibodies from donors at 1.33, 6.24, 12 months after infection. a, Graph shows relative abundance of human IGVH genes Sequence Read Archive accession SRP010970 (green), convalescent vaccinees (blue), and convalescent non-vaccinees (orange). Statistical significance was determined by two-sided binomial test. b, c, Same as in a, but showing comparison between antibodies from donors at 1.3 months3 (b), 6.2 month4 (c) and 12 months after infection. Two-sided binomial tests with unequal variance were used to compare the frequency distributions., significant differences are denoted with stars (* P < 0.05, ** P < 0.01, *** P < 0.001, **** = P < 0.0001).CC BY · PubMed Central
Extended Data Fig. 5 Analysis of anti-RBD antibodies. a, Number of clonally expanded B cells (per 10 million B cells) at indicated time points in 10 individuals. Colours indicate shared clones appearing at different time points. Statistical significance was determined using two-tailed Wilcoxon matched-pairs signed rank test. Vaccinees are marked in red. Statistical significance was determined using Wilcoxon matched-pairs signed rank tests. Vaccinees are marked in red. b, Number of somatic nucleotide mutations in the IGVH (top) and IGVL (bottom) in antibodies obtained after 1.3 or 6.2 or 12 months from the indicated individual. c, Same as b, but graphs show comparison between new clones and conserved clones in 6 vaccinated convalescent individuals at 12 months after infection. d, The amino acid length of the CDR3 s at the IGVH and IGVL for each individual. Right panel shows all antibodies combined. (1.3m: n = 889; 6.2m: n = 975; 12m: n = 1105, (non-vax: n = 417; vax: n = 688)). The horizontal bars indicate the mean. Statistical significance was determined using two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons (a, b, d), or two-tailed Mann–Whitney U-tests (c).
Extended Data Fig. 5 Analysis of anti-RBD antibodies. a, Number of clonally expanded B cells (per 10 million B cells) at indicated time points in 10 individuals. Colours indicate shared clones appearing at different time points. Statistical significance was determined using two-tailed Wilcoxon matched-pairs signed rank test. Vaccinees are marked in red. Statistical significance was determined using Wilcoxon matched-pairs signed rank tests. Vaccinees are marked in red. b, Number of somatic nucleotide mutations in the IGVH (top) and IGVL (bottom) in antibodies obtained after 1.3 or 6.2 or 12 months from the indicated individual. c, Same as b, but graphs show comparison between new clones and conserved clones in 6 vaccinated convalescent individuals at 12 months after infection. d, The amino acid length of the CDR3 s at the IGVH and IGVL for each individual. Right panel shows all antibodies combined. (1.3m: n = 889; 6.2m: n = 975; 12m: n = 1105, (non-vax: n = 417; vax: n = 688)). The horizontal bars indicate the mean. Statistical significance was determined using two-sided Kruskal–Wallis test with subsequent Dunn’s multiple comparisons (a, b, d), or two-tailed Mann–Whitney U-tests (c).CC BY · PubMed Central
Extended Data Fig. 6 Evolution of anti-SARS-CoV-2 RBD antibody clone. Clonal evolution of RBD-binding memory B cells from ten convalescent individuals, a, Phylogenetic tree graph shows clones from convalescent non-vaccinees, b, Same as a, but from convalescent vaccinees. Numbers refer to mutations compared to the preceding vertical node. Colours indicate time point; grey, orange and red represent 1.3, 6 and 12 months respectively, black dots indicate inferred nodes, and size is proportional to sequence copy number; GL = germline sequence.
Extended Data Fig. 6 Evolution of anti-SARS-CoV-2 RBD antibody clone. Clonal evolution of RBD-binding memory B cells from ten convalescent individuals, a, Phylogenetic tree graph shows clones from convalescent non-vaccinees, b, Same as a, but from convalescent vaccinees. Numbers refer to mutations compared to the preceding vertical node. Colours indicate time point; grey, orange and red represent 1.3, 6 and 12 months respectively, black dots indicate inferred nodes, and size is proportional to sequence copy number; GL = germline sequence.CC BY · PubMed Central
Extended Data Fig. 7 WT RBD binding and pseudovirus neutralization. a, b, Binding curves (a) and EC50 dot plot (b) of mAbs isolated from non-vaccinated (black curves and dots) and from vaccinated (blue curves and dots) convalescents individuals 12 months after infection (P = 0.74). c, Avidity (dissociation rate) measuring plasma reactivity to RBD at the 1.3- and 12 month follow-up visit (n = 33). d–f, IC50 values of mAbs isolated 12 months after infection from non-vaccinated and vaccinated individuals; all 12 month antibodies irrespective of clonality (d), singlets only (e), and only antibodies belonging to a clone or shared over time (f). Statistical significance in b, d–f was determined using the two-tailed Mann–Whitney test; two-tailed Wilcoxon test (c). The geometric mean EC50 and IC50 are indicated in red. g, Heat map shows the neutralizing activity of clonally related antibodies against wt-SARS-CoV-2 over time. White tiles indicate no clonal relative at the respective time point. Clones are ranked from left to right by the potency of the 12 month progeny antibodies which are denoted below the tiles. h, IC50 values of shared clones of mAbs cloned from B cells from the initial 1.3- and 6.2-, as well as 12 month follow-up visit, divided by participant, as indicated. Lines connect clonal antibodies shared between time points. Antibodies with IC50 > 1,000 ng/ml are plotted at 1,000 ng/ml in d–h. i, IC50 values of 5 neutralizing antibody pairs against indicated authentic SARS-CoV-2 WA1/2020 and B.1.351 viruses (n = 10). Average EC50 and IC50 values of two independent experiments are shown.
Extended Data Fig. 7 WT RBD binding and pseudovirus neutralization. a, b, Binding curves (a) and EC50 dot plot (b) of mAbs isolated from non-vaccinated (black curves and dots) and from vaccinated (blue curves and dots) convalescents individuals 12 months after infection (P = 0.74). c, Avidity (dissociation rate) measuring plasma reactivity to RBD at the 1.3- and 12 month follow-up visit (n = 33). d–f, IC50 values of mAbs isolated 12 months after infection from non-vaccinated and vaccinated individuals; all 12 month antibodies irrespective of clonality (d), singlets only (e), and only antibodies belonging to a clone or shared over time (f). Statistical significance in b, d–f was determined using the two-tailed Mann–Whitney test; two-tailed Wilcoxon test (c). The geometric mean EC50 and IC50 are indicated in red. g, Heat map shows the neutralizing activity of clonally related antibodies against wt-SARS-CoV-2 over time. White tiles indicate no clonal relative at the respective time point. Clones are ranked from left to right by the potency of the 12 month progeny antibodies which are denoted below the tiles. h, IC50 values of shared clones of mAbs cloned from B cells from the initial 1.3- and 6.2-, as well as 12 month follow-up visit, divided by participant, as indicated. Lines connect clonal antibodies shared between time points. Antibodies with IC50 > 1,000 ng/ml are plotted at 1,000 ng/ml in d–h. i, IC50 values of 5 neutralizing antibody pairs against indicated authentic SARS-CoV-2 WA1/2020 and B.1.351 viruses (n = 10). Average EC50 and IC50 values of two independent experiments are shown.CC BY · PubMed Central
Extended Data Fig. 8 Biolayer interferometry affinity measurements. a, b, Graphs depict affinity measurements of neutralizing (green) and non-neutralizing (red) antibodies isolated 1.3 months (a) or 12 months (b) after infection.
Extended Data Fig. 8 Biolayer interferometry affinity measurements. a, b, Graphs depict affinity measurements of neutralizing (green) and non-neutralizing (red) antibodies isolated 1.3 months (a) or 12 months (b) after infection.CC BY · PubMed Central
Extended Data Fig. 9 Biolayer interferometry antibody competition experiment. a, b, Anti-SARS-CoV-2 RBD antibodies isolated 1.3 (a) or 12 months (b) after infection were assayed for competition with structurally characterized anti-RBD antibodies by biolayer interferometry experiments as in Fig. 4a. Graphs represent the binding of the second antibody (2nd Ab) to preformed first antibody (1st Ab)–RBD complexes. Dotted line denotes when 1st Ab and 2nd Ab are the same. For each antibody group identified in Fig. 4c the left graphs represent the binding of the class-representative C144, C121, C135 or C1053,23 (2nd Ab) to the candidate antibody (1st Ab)-RBD complex. The right graphs represent the binding of the candidate antibody (2nd Ab) to the complex of C144-RBD, C121-RBD, C135-RBD or C105-RBD (1st Ab). Antibodies belonging to the same groups are indicated to the left of the respective curves.
Extended Data Fig. 9 Biolayer interferometry antibody competition experiment. a, b, Anti-SARS-CoV-2 RBD antibodies isolated 1.3 (a) or 12 months (b) after infection were assayed for competition with structurally characterized anti-RBD antibodies by biolayer interferometry experiments as in Fig. 4a. Graphs represent the binding of the second antibody (2nd Ab) to preformed first antibody (1st Ab)–RBD complexes. Dotted line denotes when 1st Ab and 2nd Ab are the same. For each antibody group identified in Fig. 4c the left graphs represent the binding of the class-representative C144, C121, C135 or C1053,23 (2nd Ab) to the candidate antibody (1st Ab)-RBD complex. The right graphs represent the binding of the candidate antibody (2nd Ab) to the complex of C144-RBD, C121-RBD, C135-RBD or C105-RBD (1st Ab). Antibodies belonging to the same groups are indicated to the left of the respective curves.CC BY · PubMed Central

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  • The paper relies on this without support.

    The 63 convalescent participants are representative of the broader population of individuals who have recovered from COVID-19.

    “Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines3,4.”

  • The paper relies on this without support.

    Neutralizing antibody titres are a correlate of protection against SARS-CoV-2 variants in vivo.

    “The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.”

  • The paper relies on this without support.

    In vitro neutralization breadth measured with pseudotyped viruses predicts protection against authentic circulating SARS-CoV-2 variants.

    “Plasma neutralizing activity in 63 participants was measured using a human immunodeficiency virus 1 (HIV-1) pseudotyped with the SARS-CoV-2 spike (S) protein3,4,13”

  • The paper relies on this without support.

    The mRNA vaccines used (Moderna mRNA-1273 and Pfizer-BioNTech BNT162b2) are representative of available mRNA vaccines.

    “26 (41%) had received at least one dose of either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines”

  • The paper relies on this without support.

    Antibody sequences obtained from 10 individuals are representative of the memory B cell repertoire.

    “we obtained 1,105 paired antibody heavy- and light-chain sequences from 10 individuals who were also assessed at the earlier time points, 6 of whom were vaccinated”
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Metadata

Authors
Wang, Z., Muecksch, F., Schaefer-Babajew, D., Finkin, S., Viant, C., Gaebler, C., Hoffmann, H. H., Barnes, C. O., Cipolla, M., Ramos, V., Oliveira, T. Y., Cho, A., Schmidt, F., Da Silva, J., Bednarski, E., Aguado, L., Yee, J., Daga, M., Turroja, M., Millard, K. G., Jankovic, M., Gazumyan, A., Zhao, Z., Rice, C. M., Bieniasz, P. D., Caskey, M., Hatziioannou, T., & Nussenzweig, M. C.
Affiliations
  • Rockefeller University (US)
  • California Institute of Technology (US)
  • Weill Cornell Medicine (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
  • Bieniasz, Paul D.
  • Caskey, Marina
  • Nussenzweig, Michel C.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2021-06-14
Volume
595
Issue
7867
Pages
426-431
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-021-03696-9
PMC
PMC8277577
OpenAlex
W3167555428
Semantic Scholar
0f7808971438f11ba057dd2209a3aa8f4c8d28c8
Cited by
721
References
63
Citations by year
2026: 23 · 2025: 41 · 2024: 71 · 2023: 150 · 2022: 281 · 2021: 154
FWCI
51.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • CaltechAUTHORS (California Institute of Technology) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • Howard Hughes Medical Institute (UL1TR001866)
  • George Mason University (3 R01-AI091707-10S1)
  • California Institute of Technology
  • Wellcome Trust
  • Fast Grants
  • National Institutes of Health (P01-AI138398-S1, COVID-19, TR001866, UL1TR001866)
  • Beckman Institute, California Institute of Technology
  • National Institute of Allergy and Infectious Diseases (R01AI091707, R01AI078788, U19AI111825, R37AI064003)
  • National Center for Advancing Translational Sciences (TR001866)
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Immunology and Microbiology › Immunology › Immune responses and vaccinations
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
Keywords
SARS-CoV-2, COVID-19 pandemic, natural immunity, convalescent patients, neutralizing antibodies, receptor binding domain, memory B cells, mRNA vaccines, variants of concern, monoclonal antibodies

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