The Majority of SARS-CoV-2 Plasma Cells are Excluded from the Bone Marrow Long-Lived Compartment 33 Months after mRNA Vaccination

Abstract

The goal of any vaccine is to induce long-lived plasma cells (LLPC) to provide life-long protection. Natural infection by influenza, measles, or mumps viruses generates bone marrow (BM) LLPC similar to tetanus vaccination which affords safeguards for decades. Although the SARS-CoV-2 mRNA vaccines protect from severe disease, the serologic half-life is short-lived even though SARS-CoV-2-specific plasma cells can be found in the BM. To better understand this paradox, we enrolled 19 healthy adults at 1.5-33 months after SARS-CoV-2 mRNA vaccine and measured influenza-, tetanus-, or SARS-CoV-2-specific antibody secreting cells (ASC) in LLPC (CD19^-) and non-LLPC (CD19⁺) subsets within the BM. All individuals had IgG ASC specific for influenza, tetanus, and SARS-CoV-2 in at least one BM ASC compartment. However, only influenza- and tetanus-specific ASC were readily detected in the LLPC whereas SARS-CoV-2 specificities were mostly excluded. The ratios of non-LLPC:LLPC for influenza, tetanus, and SARS-CoV-2 were 0.61, 0.44, and 29.07, respectively. Even in five patients with known PCR-proven history of infection and vaccination, SARS-CoV-2-specific ASC were mostly excluded from the LLPC. These specificities were further validated by using multiplex bead binding assays of secreted antibodies in the supernatants of cultured ASC. Similarly, the IgG ratios of non-LLPC:LLPC for influenza, tetanus, and SARS-CoV-2 were 0.66, 0.44, and 23.26, respectively. In all, our studies demonstrate that rapid waning of serum antibodies is accounted for by the inability of mRNA vaccines to induce BM LLPC.

Figure 1. Demographics of the 19-BM-subject cohort. Age, years; Cauc, Caucasian; pre inf, previous (COVID-19) infection (a). General FACS gating strategy used for sorting BM ASC subsets.

BM MNC were first gated for lymphocytes, singlets, and viable cells (based on their FSC/SSC and Live/Death properties) (b). CD3 and CD14 were then used as dump markers to capture CD19⁺ and CD19⁻ B cell populations. Subsequent sub-gating from CD19⁺ population on the IgD⁻ fraction (versus CD27) and using CD138 versus CD38 allow for breaking down BM ASC populations into 3 subsets of interest: PopA (CD19⁺CD38^hiCD138⁻), PopB (CD19⁺CD38^hiCD138⁺), and PopD (LLPC; CD19⁻CD38^hiCD138⁻).

Figure 2. Exclusion of SARS-CoV-2-specific BM IgG LLPC after SARS2 mRNA vaccines by ELISpots.

The S2P antigen is most sensitive to capture SARS-CoV-2-specific blood ASC isolated after SARS2 mRNA vaccines. Blood ASC from subjects at the peak time which is 5–7 days post-vaccine (a,b). Representative ELISpot scanned images (left, a vaccinated subject without previous COVID-19 infection; right, a vaccinated subject with previous COVID-19 infection) (a). The numbers of input ASC that were incubated for total IgG ~687 (left) or ~522 (right), and antigen-specific ASC IgG ~2,062 (left) or ~1,566 (right). Frequencies (%) of Ag-specific IgG ASC per total IgG ASC. Each circle represents an individual vaccine subject (b). For additional Ag validation, see Suppl. Fig. S2. Summary of the techniques and the experimental design for total, Flu, Tet, and S2P-specific ASC by ELISpots (c). Total IgG, and antigen-specific ASC from PopA, PopB, and PopD for Flu-, Tet-, S2P-specific BM ASC. Representative ELISpot scanned images shown (d). The numbers of input ASC that were incubated were ~52K, ~12.1K, and ~10.1K 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 for Flu-,Tet-, S2P-specific BM ASC (e). The numbers indicate fold difference (i.e. ratios) when comparing different BM ASC subsets for the same Ag specificity or comparing different Ag specificities within the same BM ASC subset. ns, not significant; *p<0.05; **p<0.005; ***p<0.0005. Fold difference (i.e. ratios) when comparing different Ag specificities between non-LLPC (i.e. combined PopA and PopB) versus LLPC (PopD) (f). BM IgG ASC response kinetics in the SARS2 mRNA vaccinee who donated three longitudinal BM aspirates (g,h). Representative ELISpot scanned images. The numbers of input ASC that were incubated were ~21K, ~40K, and ~4.9K (10w); ~14K, ~12K, and ~3.8K (62w); and ~58K, ~22K, and ~7.2K (94w) for PopA, PopB, and PopD, respectively (g). Ag-specific BM IgG ASC response kinetics (h). Sub, subject; K: 1,000; LLPC: long-lived plasma cell (dotted boxes in d, e, g, and h); Flu: influenza; Tet: tetanus; Pre inf: previous (COVID-19) infection; d: day; w: week; m: month; y: year. For details of subjects and samples, see Table 1.

Figure 3. Exclusion of SARS-CoV-2-specific BM IgG LLPC in SARS2 mRNA vaccinees by IgG detection from the BM ASC culture supernatants.

Summary of the techniques and the experimental design (a). From the cultures of BM ASC, the supernatant preps were collected for quantitation of Ag-specific IgG. Sups, BM ASC culture supernatant preps; MBBA, multiplex bead binding assay. MBBA measuring IgG specific for Flu, Tet, and S2P (normalized to total IgG and ASC input numbers) from culture supernatant of PopA, PopB, and PopD of 8 individuals (b). All supernatants were collected for 18–24 hours in culture from BM ASC during revival from the FACS sorters and were tested undiluted (neat). Fold difference (i.e. ratios) when comparing normalized Ag-specific IgG in the supernatants from the culture of non-LLPC (combined PopA and PopB) versus LLPC (c). For ratio calculation, see Methods. For IgG standard vs MFI curve, see Suppl. Fig. S4. LLPC: long-lived plasma cell (dotted boxes in b); Flu: influenza; Tet: tetanus. For details of subjects and samples, see Table 1.

Figure 4. Antigen specificity strata of all individuals examined for each BM ASC subset.

n, number of BM donors (a). Graphical summary. The majority of SARS2 plasma cells are excluded from the BM LLPC compartment 33 months after mRNA vaccination (b). Created with BioRender.com.