SARS-CoV-2-specific plasma cells are not durably established in the bone marrow long-lived compartment after mRNA vaccination

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines are effective at protecting from severe disease, but the protective antibodies wane rapidly even though SARS-CoV-2-specific plasma cells can be found in the bone marrow (BM). Here, to explore this paradox, we enrolled 19 healthy adults at 2.5-33 months after receipt of a SARS-CoV-2 mRNA vaccine and measured influenza-, tetanus- or SARS-CoV-2-specific antibody-secreting cells (ASCs) in long-lived plasma cell (LLPC) and non-LLPC subsets within the BM. Only influenza- and tetanus-specific ASCs were readily detected in the LLPCs, whereas SARS-CoV-2 specificities were mostly absent. The ratios of non-LLPC:LLPC for influenza, tetanus and SARS-CoV-2 were 0.61, 0.44 and 29.07, respectively. In five patients with known PCR-proven history of recent infection and vaccination, SARS-CoV-2-specific ASCs were mostly absent from the LLPCs. We show similar results with measurement for secreted antibodies from BM ASC culture supernatant. While serum IgG titers specific for influenza and tetanus correlated with IgG LLPCs, serum IgG levels for SARS-CoV-2, which waned within 3-6 months after vaccination, were associated with IgG non-LLPCs. In all, our studies suggest that rapid waning of SARS-CoV-2-specific serum antibodies could be accounted for by the absence of BM LLPCs after these mRNA vaccines.

Fig. 1. Demographics of the 19 BM subject cohort.

a, The general FACS gating strategy used for sorting BM ASC subsets. Age given in years. Cauc, Caucasian; Pre-inf, previous (COVID-19) infection. b, BMMCs were first gated for lymphocytes, singlets and viable cells (based on their forward scatter/side scatter (FSC/SSC) and live/death properties). CD3 and CD14 were then used as dump markers to capture CD19⁺ and CD19⁻ B cell populations. Subsequent subgating from CD19⁺ population on the IgD⁻ fraction (versus CD27) and using CD138 versus CD38 allowed the breakdown of BM ASC populations into three subsets of interest: PopA (CD19⁺CD38^hiCD138⁻), PopB (CD19⁺CD38^hiCD138⁺) and PopD (LLPC; CD19⁻CD38^hiCD138⁺). PE-Cy7, PE-Cyanine7 tandem fluorophore. c, Representative ELISpot scanned images (left: a vaccinated subject without previous COVID-19 infection; right: a vaccinated subject with previous COVID-19 infection). The numbers of input ASC that were incubated for total IgG: ~687 (left) or ~522 (right), and for antigen-specific ASC IgG: ~2,062 (left) or ~1,566 (right). Each circle represents an individual vaccine subject. The counts were provided by the sorters. Blood ASCs from subjects were collected at the peak time of response (which is 5–7 days post-vaccine). d, Frequencies (%) of antigen-specific IgG ASCs per total IgG ASCs. Data were generated from 3, 10, 13, 6, 15 and 8 different SARS-CoV-2-vaccinated subjects for SARS-CoV-2 antigens NTD, RBD, S1, S2, S2P and NP, respectively. Statistics were assessed using Student’s t-test (two-tailed unpaired t-test) in Excel (Microsoft), and differences were considered significant at P values less than 0.05. For additional antigen selection and validation, see Extended Data Fig. 2. In c and d, it can be seen that S2P was most sensitive to capture SARS-CoV-2-specific blood ASCs isolated after SARS-CoV-2 mRNA vaccines. Source data

Fig. 2. Absence of SARS-CoV-2 BM IgG LLPC after SARS-CoV-2 mRNA vaccines by detection of ASC and secreted IgG in the BM ASC culture supernatants.

a, Summary of the techniques and the experimental designs for detection of total, Flu, Tet and S2P ASCs and secreted IgG by ELISpots and MBBA, respectively. MNC, mononuclear cells. b, Representative ELISpot scanned images. The numbers of input ASC that were incubated were ~52 K, ~12.1 K and ~10.1 K for PopA, PopB and PopD, respectively. Each symbol represents an individual vaccine subject for total IgG and antigen-specific ASC from PopA, PopB and PopD. c, ELISpots measuring BM IgG ASC specific for Flu, Tet and S2P. Data were generated from 8, 15 and 17 different SARS-CoV-2-vaccinated subjects for PopA, PopB and PopD, respectively. For individual ratios and statistic comparisons between any two antigens for any subset or between any two subsets for any antigen, see Supplementary Tables 1 and 2, respectively. d, Fold difference (ratios) when comparing different vaccine specificities between non-LLPCs (combined PopA and PopB) versus LLPCs (PopD). e, MBBA measuring IgG specific for Flu, Tet and S2P (normalized to total IgG) from culture supernatant of PopA, PopB and PopD. Supernatant preps were collected from 18–24-h cultures of BM ASCs after revival from the FACS sorters and were quantified for total IgG and vaccine-specific IgG in neat (undiluted). Data were generated from eight different SARS-CoV-2-vaccinated subjects. For individual ratios and statistic comparisons between any two antigens for any subset or between any two subsets for any antigen, see Supplementary Tables 1 and 2, respectively. f, The fold difference (ratios) when comparing normalized vaccine-specific IgG in the supernatants from the culture of non-LLPCs (combined PopA and PopB) versus LLPCs (PopD). For ratio calculation, see Methods. For IgG standard versus MFI curve, see Extended Data Fig. 4. Counts were provided by the sorters. LLPC, boxes in b, c, and e. Sub, subject; Sups, BM ASC culture supernatant preps. For details of subjects and samples, see Table 1. Source data

Fig. 3. Vaccine-specific IgG levels in the serum: kinetics and magnitude, and correlation with BM IgG ASC responses.

a, Kinetics and magnitude of IgG titers from subjects (n = 8) with at least two sequential serum samples (collected before the additional SARS-CoV-2 vaccines). b–d, Serum IgG levels versus BM IgG LLPC, non-LLPC and total ASC responses for Flu (b), Tet (c) and S2P (d) specificities in all examined subjects (n = 19). Sera collected within 5 months of the time of BM aspiration. For b–d, data were generated from eight different SARS-CoV-2-vaccinated subjects and correlations were assessed using simple linear regression analysis performed with GraphPad Prism (GraphPad Software). The exact P values for vaccine-specific LLPCs, non-LLPCs and total ASCs are 0.0043, 0.0196 and 0.0075, respectively (b); <0.0001, 0.91 and <0.0001, respectively (c); and 0.6096, 0.0025 and 0.0008, respectively (d). All serum samples tested at dilutions of 1:1,000–1:100,000 (total IgG) or 1:200–1:16,000 (antigen-specific IgG). For serum total and vaccine-specific IgG standard curves, see Supplementary Fig. 1. For details of subjects and samples, see Table 1. Source data

Fig. 4. Kinetics and magnitude of BM IgG ASC responses and of total and vaccine-specific serum IgG levels in the subject who donated three longitudinal BM aspirates over 2 years (subject 14).

a, ELISpot scanned images. The numbers of input ASC that were incubated were ~21K, ~40K and ~4.9K (2.5 months); ~14K, ~12K and ~3.8K (14 months); and ~58K, ~22K and ~7.2K (23 months) for PopA, PopB and PopD, respectively. b,c, The kinetics and magnitude of antigen-specific BM IgG ASC responses (b) and of total and antigen-specific IgG levels in the serum (c). Sera were collected within 1–5 months of the time of BM aspiration. Part of Fig. 4c is reproduced from Fig. 3a for the purpose of kinetics comparison. Sub, subject; K, 1,000. Counts were provided by the sorters. LLPC, boxes in a and b. All serum samples were tested at dilutions of 1:1,000–1:100,000 (total IgG) or 1:200–1:16,000 (antigen-specific IgG). For serum total and vaccine-specific IgG standard curves, see Supplementary Fig. 1. For details of subjects and samples, see Table 1. Source data

Fig. 5. SARS-CoV-2 BM IgG LLPCs are not durably established after mRNA vaccination.

a, Antigen specificity strata of all individuals examined for each BM ASC subset. n, number of BM donors. b, Graphical summary. The majority of SARS-CoV-2 plasma cells are not established from the BM LLPC compartment 33 months after mRNA vaccination. Created with BioRender.com. Source data

Extended Data Fig. 1. General FACS gating strategy used for sorting blood ASC.

PBMC were first gated for lymphocytes, singlets, and viable cells (based on their FSC/SSC and Live/Death properties). CD3 and CD14 were then used as dump markers to capture CD19+ and CD19⁻ B cell populations. Subsequent sub-gating using CD38 versus CD27 on the IgD- fraction (of CD19+ population) allows for sorting for blood ASC (CD27hiCD38hi). See Methods for antibody panels.

Extended Data Fig. 2. Assessment of vaccine-specific ASC and validation of vaccine specificities with blood ASC.

(a) Summary of the techniques and the experimental designs. From the cultures of blood ASC, the cells were collected and ELISpot-quantitated for validating vaccine specificities. (b) Representative ELISpot scanned images shown. Blood ASC from subjects at the peak (5-7 days post-vaccine) assayed for Flu-, Tet-, and S2P-specific IgG secretion. The numbers of input ASC that were incubated were ~894, ~1,124, and ~796 (total), and ~4,471, ~4,496, and ~2,388 (Flu-specific) for S#1, S#2, and S#3, respectively (far left); ~1 K, ~1 K, and ~1 K (total), and ~3 K, ~4 K, and ~4 K (Tet-specific) for S#4, S#5, and S#6, respectively (left); and ~712, ~1,415, and ~1,386 (total), and ~2,139, ~4,245, and ~5,544 (S2P-specific) for S#7, S#8, and S#9, respectively (right). (c) Each circle represents an individual vaccinee. Data were generated from 3, 3, 3, 8, 6, and 5 different vaccinated subjects for Flu, Tet, S2P (v1), S2P (v2), S2P (v3), and S2P (v4), respectively. S: subject; ~: counts provided by the sorters; K: 1,000; vac: vaccinated; Flu: influenza; Tet: tetanus; v: (SARS-CoV-2 mRNA) vaccine dose. All ASC assayed at day 1 in culture. Source data

Extended Data Fig. 3. Exclusion of S2P BM IgA LLPC in SARS-CoV-2 mRNA vaccinees.

(a) Representative ELISpot scanned images. The numbers of input ASC that were incubated were ~9.6 K, ~3.7 K, and ~1.2 K (total) and ~58 K, ~22 K, and ~7.2 K (vaccine-specific) for PopA, PopB, and PopD, respectively. (b) Each symbol represents an individual vaccinee. Data were generated from 2, 3, and 3 different SARS-CoV-2 vaccinated subjects for PopA, PopB, and PopD, respectively. Statistics were assessed using Student’s t-test (two-tailed unpaired t-test) in Excel (Microsoft) and differences were considered significant at p values less than 0.05. ~: counts provided by the sorters; K: 1,000; LLPC: long-lived plasma cell (dotted boxes); Flu: influenza; Tet: tetanus. All ASC assayed at day 1 in culture. For individual ratios and statistic comparisons between any two antigens for any subset or between any two subsets for any antigen, see Supplementary Tables 3 and 4, respectively. Source data

Extended Data Fig. 4. The human IgG standardized concentrations versus MFI values.

The displayed equation was used to normalize MFI values for detection of antibodies in the culture supernatants of each BM ASC subset. Source data

Extended Data Fig. 5. No correlation between vaccine-specific IgG BM ASC responses and the time windows of the vaccine.

BM (a) Flu, (b) Tet, and (c) S2P IgG LLPC, Non-LLPC, and total ASC responses in all examined subjects following the vaccine exposure time (time since the most recent (a) Flu, (b) Tet, or (c) the first SARS-CoV-2 vaccine). Data were generated from 17, 15, and 19 different SARS-CoV-2 vaccinated subjects for BM LLPC, Non-LLPC, and total ASC, respectively. Correlations were assessed using simple linear regression analysis performed with GraphPad Prism (GraphPad Software). The exact p values for vaccine-specific LLPC, Non-LLPC, and total ASC are 0.9397, 0.8563, and 0.7455, respectively (a); 0.1806, 0.2898, and 0.0952, respectively (b); and 0.3202, 0.3635, and 0.2862, respectively (c). Subjects yielding sufficient ASC for LLPC and Non-LLPC subsets included. Source data

Extended Data Fig. 6. No correlation between S2P IgG BM ASC responses and the number of SARS-CoV-2 vaccine doses.

BM S2P IgG ASC responses in all examined subjects stratified by the number of SARS-CoV-2 vaccine doses (prior to BM aspiration). Data were generated from 17, 15, and 19 different SARS-CoV-2 vaccinated subjects for BM LLPC, Non-LLPC, and total ASC, respectively. The exact p values for S2P LLPC, Non-LLPC, and total ASC are 0.3929, 0.9110, and 0.9912, respectively. Subjects yielding sufficient ASC for LLPC and Non-LLPC subsets included. Source data

Extended Data Fig. 7. No correlation between S2P IgG BM ASC responses and age of the subjects.

BM S2P IgG ASC responses in all examined subjects stratified by age of the subjects at the time of BM collection. Data were generated from 17, 15, and 19 different SARS-CoV-2 vaccinated subjects for BM LLPC, Non-LLPC, and total ASC, respectively. The exact p values for S2P LLPC, Non-LLPC, and total ASC are 0.4950, 0.7976, and 0.7699, respectively. Subjects yielding sufficient ASC for LLPC and Non-LLPC subsets included. Source data

Extended Data Fig. 8. No correlation between S2P IgG BM ASC responses and the time windows of the SARS-CoV-2 vaccine in both vaccinees and infected vaccinees (Vaccine+Infection).

BM S2P IgG SC responses in vaccinated versus hybrid subjects stratified by time since (a) the first or (b) the most recent (prior to BM aspiration) SARS-CoV-2 vaccine. The exposure time for infection in the hybrid subjects not shown. For BM LLPC, data were generated from 12 and 5 different subjects of Vaccine and of Vaccine+Infection, respectively. For BM Non-LLPC, data were generated from 12 and 3 different subjects of Vaccine and of Vaccine+Infection, respectively. The exact p values for Vaccine/LLPC, Vaccine+Infection/LLPC, Vaccine/Non-LLPC, and Vaccine+Infection/Non-LLPC are 0.3192, 0.6529, 0.3446, and 0.9254, respectively (a); and 0.4933, 0.7301, 0.8004, and 0.7128, respectively (b). Subjects yielding sufficient ASC for LLPC and Non-LLPC subsets included. Source data

Extended Data Fig. 9. Kinetics of vaccine-specific IgG ASC responses in the subject who donated two sequential BM aspirates (at 28 and 33 months after the first SARS-CoV-2 vaccine).

(a) ELISpot scanned images. The numbers of input ASC incubated were ~3.04 K, ~1.27 K, and ~0.88 K (28 m, total), ~15.20 K, ~7.61 K, and ~2.65 K (28 m, vaccine-specific); and ~1.56 K, ~1.02 K, and ~0.75 K (33 m, total), and ~18.67 K, ~7.12 K, and ~2.98 K (33 m, vaccine-specific), for PopA, PopB, and PopD, respectively. (b) Vaccine-specific IgG ASC response kinetics. ~: counts provided by the sorters; K: 1,000; LLPC: long-lived plasma cell (box); Flu: influenza; Tet: tetanus; m: month. For details of the subject, see Table 1. Source data

Extended Data Fig. 10. Downregulation of CD45 in LLPC (PopD).

(a) Representative FACS gating strategy and CD45 staining for BM ASC subsets. For details on BM ASC gating, see Fig. 1b. For the antibody panels, see Methods. (b) CD45 staining is downregulated in PopD. Each circle represents an individual healthy BM donor. Data were generated from five different healthy BM donors. Statistic comparisons between any two CD45+ subsets were assessed using Student’s t-test (two-tailed unpaired t-test) in Excel (Microsoft) and differences were considered significant at p values less than 0.05. Shown are p values from comparisons with PopD; for p values from comparisons between other subsets, see Supplementary Table 5. Source data