Shared B cell memory to coronaviruses and other pathogens varies in human age groups and tissues



Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated convergent antigen-specific antibody genes of similar sequences shared between individuals in pediatric and adult blood, and deceased organ donor tissues. B cell memory varied for different pathogens. Polysaccharide antigen-specific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class-switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, pre-pandemic children had class-switched convergent clones to SARS-CoV-2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. The results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.

The clonal identity of a B cell can be traced by the sequence of its B cell receptor (BCR), which determines its antigen specificity (1). Immunoglobulin (Ig) sequences are formed via irreversible variable, diversity, and joining (VDJ) gene segment rearrangement and can be diversified through somatic hypermutation (SHM) and class-switch recombination (CSR) (2). Convergent BCRs with high sequence similarity in individuals exposed to the same antigen reflect antigen-driven clonal selection and form shared immunological memory between individuals (35). It is still unclear, however, how B cell memory to different antigens distributes in human tissues and changes during an individual’s lifespan.

Humoral immune responses can differ between children and adults, as seen in children utilizing more B cell clones to achieve neutralizing antibody breadth to human immunodeficiency virus 1 (HIV-1) (6). Children usually have milder disease following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection than adults (710), potentially due to differences of viral receptor expression and immune responses (11, 12). SARS-CoV-2-infected children, in contrast to adults, show lower antibody titers and more IgG specific for the spike (S) protein over the nucleocapsid (N) protein. The faster viral clearance and lower viral antigen loads in children have been attributed to these differences (13). Whether B cell clones specific for coronaviruses and other pathogens differ between children and adults is unclear. Blood-based studies survey only a fraction of an individual’s BCR repertoire. The lymph nodes, spleen, and gastrointestinal tract harbor greater numbers of B cells and are major sites for SHM and CSR (14, 15). Specialized responses in particular tissues have been reported, such as for polysaccharide antigen-specific B cells in functional splenic tissue (16, 17).

To study changes in antigen-specific B cell memory over the human lifespan and across tissues, we characterized convergent Ig heavy chain (IGH) repertoires specific to six common pathogens, and two viruses not encountered by the participants, Ebola virus (EBOV) and SARS-CoV-2, in pre-COVID-19 pandemic individuals. We analyzed 12 cord blood (CB) samples, 93 blood samples from 51 children aged 1 to 3 years (18), 114 blood samples from healthy human adults aged 17 to 87 years (18), and blood, lymph node, and spleen samples from eight deceased organ donors (table S1). Children were vaccinated for Haemophilus influenzae type b (Hib), Pneumococcus pneumoniae (PP), and tetanus toxoid (TT) at 2, 4, 6, and 12-15 months, had influenza virus (flu) vaccination and were very likely exposed to respiratory syncytial virus (RSV) but were not vaccinated for Neisseria meningitidis (NM) (19). Adult vaccination histories were unknown. Convergent IGH were identified by clustering with pathogen-specific reference IGH (table S2) sharing IGHV and IGHJ gene segment usage, CDR-H3 length, and minimum 85% CDR-H3 amino acid sequence identity.

B cell clones fell into three groups: (i) naïve clones containing only unmutated IgM or IgD (unmutM/D); (ii) antigen-experienced IgM/D with median SHM over 1% and without class-switched members (mutM/D); and (iii) antigen-experienced clones with class-switched members (CS). As we hypothesized, CB samples showed the lowest convergent IGH frequencies, consistent with limited fetal pathogen or vaccine exposures (Fig. 1A). Convergent clones in children and adults were largely mutM/D or CS (Fig. 1A and fig. S1). In adult blood, convergent clones for Hib, NM, and RSV were predominantly mutM/D clones, whereas PP, TT, and flu clones were predominantly CS (fig. S2A). Adults over 45 years of age had elevated mutM/D B cell clone frequencies to NM, potentially from exposures preceding widespread NM vaccination (20). Surprisingly, children had higher frequencies than adults of CS convergent clones for Hib, PP, TT, and RSV (fig. S2B), with mutated IgM or IgD also found in these clones (Fig. 1B). Convergent clone frequency in children’s blood was not significantly associated with vaccination timing (figs. S3 to S5 and table S3), indicating persistently elevated frequencies. Flu-specific convergent clone frequencies were comparable in children and adults (Fig. 1A), with age-related increases in IgG SHM potentially due to frequent exposures via vaccination or infection (Fig. 1C) (18).

Fig. 1 Frequency, class-switching, and SHM of pathogen-specific convergent clones in children and adults.

(A) Convergent clone frequencies for each pathogen, plotted on a square root scale. Ages in years; CB, cord blood. (B) Fractions of convergent clones expressing unmutated IgM/IgD, mutated IgM/IgD, class-switched, or combinations of these. Children have significantly higher fractions of class-switched convergent clones with mutated IgM/IgD clone members (colored in purple) than adults, (P = 5.08 × 10−32, 6.66 × 10−29, 2.39 × 10−29, 3.45 × 10−34, and 1.71 × 10−41 for Hib, NM, PP, TT, and RSV, respectively, by Wilcoxon–Mann–Whitney (WMW) test). (C) Median IGHV-gene SHM frequencies of each convergent clone in participants of different ages indicated in years. SHM frequencies of convergent clones expressing IgG or IgA were lower in children than in adults (P = 6.50 × 10−13 and 1.96 × 10−8, respectively, WMW test).

To test whether low frequencies of CS convergent clones in adult blood reflect preferential localization of clones in lymphoid tissues, we analyzed the blood, spleen, mediastinal lymph nodes (MDLN), and mesenteric lymph nodes (MSLN) of eight adult deceased organ donors. Lymph nodes and spleen showed greater clonal sharing with each other compared to blood (fig. S6A), suggesting larger clone sizes in lymphoid tissues and limited recirculation. Each tissue was dominated by different clones (fig. S6B) and SHM correlated with the number of tissues a clone occupied (fig. S7), consistent with greater prior antigen exposure leading to wider tissue distribution (21). Convergent clones frequencies for Hib, NM, PP, TT, RSV, and flu were higher in adult lymph nodes and spleen than blood (Fig. 2A). Adult lymph nodes and child blood shared more convergent clones than adult and child blood, showing differing distributions of these clones in children and adults (Fig. 2B and fig. S8, P = 0.0001181, Fisher’s exact test). B cells specific for bacterial capsular polysaccharides are reported to be enriched in the spleen, and splenectomized patients are vulnerable to these bacteria (16, 17). However, frequencies of convergent clones for Hib, NM, and PP are similar or higher in lymph nodes than in spleen. Moreover, estimated B cell numbers are greater in human lymph nodes than spleen (22, 23), indicating that spleen is not the sole reservoir of these clones. Convergent IGH for polysaccharides were usually IgM or IgD, with some CS clones for PP in lymph nodes and spleen (Fig. 2C). Thus, memory to these antigens spans both a diversity of lymphoid tissues and of isotype expression.

Fig. 2 Convergent B cell clone distribution in tissues.

(A) Convergent clone frequencies in adult blood (PBMC), MDLN, MSLN, and spleen. Frequencies are on a square root scale. Frequencies in tissues were higher than in blood (P = 0.00049, 0.0037, 0.016, 6.71 × 10−7, 0.012, and 0.00017 for Hib, NM, PP, TT, RSV, and flu respectively, WMW test). (B) Convergent antigen-specific IGH in CB and blood of children, healthy adults (Adult* PBMC), deceased organ donors (Donor PBMC), and donor spleen, MSLN and MDLN. Vertical bars: reference antigen-specific IGH sequences per specimen combination. Left bars: total convergent IGH unique sequences per tissue. (C) Fraction of convergent clones containing the indicated isotypes in tissues. Some clones contain multiple isotypes. Compared to protein antigen-specific clones, polysaccharide-specific clones more frequently express IgM/D and less often express IgG, (P = 0.035 and 0.0058, respectively, WMW test).

Recent reports describe SARS-CoV-2 binding antibodies in pre-pandemic children’s blood (12, 24). Such antibodies and other physiological distinctions are under investigation in adults and children (25) and could contribute to the generally milder COVID-19 disease in children. SARS-CoV-2 S-binding B cells in unexposed individuals have been analyzed in a former SARS-CoV patient (26), naïve B cells of healthy individuals (27), and memory B cells in pre-pandemic donors (26, 28). We detected rare convergent clones for EBOV, as unmutM/D in blood or tissues (Fig. 3A and fig. S9A). By contrast, convergent clones for SARS-CoV-2 (table S4) were more common in children’s blood. In 37 of 51 children, these clones displayed SHM with or without CS, indicating prior antigen experience (Fig. 3, A and B). Adult frequencies of SARS-CoV-2 convergent clones were lower in blood and lymphoid tissues compared to children’s blood, with few CS examples (Fig. 3A and fig. S9). Convergent clones specific for SARS-CoV-2 receptor-binding domain (RBD) and other S domains showed similar distributions (fig. S10). Reference antibodies for SARS-CoV-2, EBOV, and the pathogens in Fig. 1 used a wide diversity of IGHV genes (fig. S11).

Fig. 3 Convergent clones for SARS-CoV-2 and EBOV.

(A) Convergent clone frequencies on a square-root scale. CS and mutM/D convergent clone frequencies for SARS-CoV-2 are higher in children than adults (P = 1.22 × 10−13 and 0.0089, respectively, WMW test). (B) SHM frequencies of convergent clones for each isotype in participants of different ages (x axis). (C) CDR-H3 amino acid sequences of convergent IGH cross-reactive to SARS-CoV-2 and other HCoVs. Top row: CDR-H3 sequence logos for reported antigen-specific clones; Second row: sequence logos for convergent clones from children (blue indicates a match, cyan indicates sequence differences).

Three convergent clones from five children but no adults in this study had IGH sequences highly similar to SARS-CoV-2 S-binding clones isolated from a pre-pandemic donor that were reported to weakly bind other HCoV spikes (26) (Fig. 3C). Three other clones from six children had IGHs identical to known SARS-CoV-2 binders (fig. S12). We expressed 19 monoclonal antibody (mAb) clones for SARS-CoV-2 (table S5) with IGH from participants in this study and reference light chains, and identified 17 binders for SARS-CoV-2 S and S domains (Table 1). Four RBD-binders showed over 90% blocking of ACE2 binding to SARS-CoV-2 S (table S6). mAb FY11H1 showed evidence of S2 binding and did not block ACE2 binding. We characterized the breadth of mAb binding using a panel of HCoV spikes and SARS-CoV-2 viral variant RBDs and spikes. Three child-derived mAbs (FY7H1, FY7H2, and FY1H2) and one adult mAb (FY4H1) showed the strongest binding to B.1.1.7, B.1.351, and P.1 S and RBD variants (table S7). Cross-reactive binding to endemic HCoV spikes was very weak to negative for all mAbs, as previously noted for reference mAb-154 (similar to mAb FY13H1) isolated from a sorted cross-reactive B cell (26). The child-derived mAbs FY13H1 and FY9H2 had higher although still weak signal for binding HKU1. Thus, children’s convergent coronavirus-binding B cells may have greater cross-reactivity compared to those of adults, in addition to having higher frequencies.

Table 1 Convergent mAb binding data for SARS-CoV-2 spike, RBD, and nucleocapsid (N), and endemic HCoV spikes.

Testing by electrochemiluminescence immunoassay in duplicate wells, with the average arbitrary unit per milliliter (AU/ml) values displayed in the table. Antibodies with binding signal at least five standard deviations above the average of negative control antibodies (Neg1 to Neg5) are listed.

Childhood immune responses are particularly important in an individual’s life, as they form the initial memory B cell pool that shapes future responses (29). We find that in comparison to adults, children have higher frequencies of convergent B cell clones in their blood for pathogens they have encountered. Notably, pre-pandemic children also had class-switched convergent clones to SARS-CoV-2 and its viral variants, but not EBOV, at higher frequencies than adults. We hypothesize that previous HCoV exposures may stimulate cross-reactive memory, and that such clonal responses may have their highest frequencies in childhood. Caveats of our analysis are that convergent clones may not fully represent the properties of all pathogen-specific clones in an individual, and binding affinities for cross-reactivity that would be relevant in vivo are not known. Further study of the role of cross-reactive memory B cell populations in primary immune responses to related but divergent viruses, as well as better understanding of the determinants of long-lived B cell memory and plasma cell formation will be important for ongoing improvement of vaccines to SARS-CoV-2, its viral variants, and other pathogens.

References and Notes

  1. W. McKinney, “Data structures for statistical computing in Python,” Proceedings of the 9th Python in Science Conference, Austin, Texas, 28 June to 3 July 2010.

Acknowledgments: We thank all staff members of the California Transplant Donor Network (now Donor Network West), especially S. Swain, RN, MSN. We thank Dr. A. Z. Fire, Dr. E. A. Hope, and Dr. J. D. Merker for helpful discussions and contributions to the research. The authors thank Meso Scale Diagnostics for helpful collaboration and material support in this study. Funding: This work was supported by NIH/NIAID R01AI127877, R01AI130398, U19AI057229, U19AI090019 and NIH/NCI U54CA260517 (S.D.B.), NIH R01 HD063142 (J.P.), and an endowment from the Crown Family Foundation (S.D.B.). Author contributions: F.Y. and S.D.B. conceived of the project. F.Y., S.C.A.N., K.J.L.J., Y.L., and K.M.R performed data analyses. F.Y., S.C.A.N., and S.D.B. verified the analyses. F.Y., S.C.A.N., R.A.H., K.R., E.H., O.F.W., G.H.J., R.S.O., E.M.O., J.-Y.L., K.N., and T.D.P. contributed to sample preparation and carried out the experiments. T.D.P., K.C.N., C.U.N., J.P., and S.D.B. provided samples and supported the project. F.Y., S.C.A.N., and S.D.B. wrote the initial manuscript. All authors provided critical feedback and contributed to the final manuscript. Competing interests: S.D.B.: Consulting for Regeneron; stock ownership in AbCellera Biologics; collaboration with Meso Scale Diagnostics. K.N.: Director of World Allergy Organization (WAO) Center of Excellence at Stanford, Advisor at Cour Pharma; Co-founder of Before Brands, Alladapt, Latitude, and IgGenix; National Scientific Committee member at Immune Tolerance Network (ITN) and National Institutes of Health (NIH) clinical research centers; DSMB member for NHLBI. Data and materials availability: All data are available in the main text or the supplementary materials. Previously generated IGH repertoire data are available with BioProject number PRJNA503602 (child dataset) and PRJNA491287 [114 healthy human adult dataset was previously reported (18)]. The IGH sequences for mAbs tested in this study were deposited in GenBank (MW821491 to MW821509). The IGH repertoire data for the deceased organ donors and the cord blood infant samples is in BioProject: PRJNA674610. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.


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