Durable SARS-CoV-2 B cell immunity after mild or severe disease

Abstract

Multiple studies have shown loss of SARS-CoV-2 specific antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from COVID-19. However, memory B cells (MBC) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multi-dimensional flow cytometric analysis of S protein receptor binding domain (S-RBD)-specific MBC in cohorts of ambulatory COVID-19 patients with mild disease, and hospitalized patients with moderate to severe disease, at a median of 54 (39-104) days after onset of symptoms. We detected S-RBD-specific class-switched MBC in 13 out of 14 participants, including 4 of the 5 participants with lowest plasma levels of anti-S-RBD IgG and neutralizing antibodies. Resting MBC (rMBC) made up the largest proportion of S-RBD-specific class-switched MBC in both cohorts. FCRL5, a marker of functional memory when expressed on rMBC, was dramatically upregulated on S-RBD-specific rMBC. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD-specific, class-switched MBC that phenotypically resemble germinal center-derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell-mediated immunity against SARS-CoV-2 after recovery from mild or severe COVID-19 disease.

Figure 1.. Quantifying S-RBD specific B cells.

(A) % of lymphocytes that are class switched MBC, class switched ASC, or non-class switched B cells in healthy, mild, and severe participants (N=7 for each group). (B) Gating strategy for S-RBD specific non-class switched B cells (CD3−, CD19+, IgD/IgM+, S-RBD+), S-RBD specific class switched MBC (CD3−, CD19+, IgM−, IgD−, CD38+/− (excluding ++), CD138−, S-RBD+), and S-RBD specific class switched ASC (CD3−, CD19+/−, IgM−, IgD−, CD38++, CD27+, S-RBD+) in “healthy” (COVID-19 negative), “mild” (COVID-19+, ambulatory), and “severe” (COVID-19+, hospitalized) participants. (C) % of class switched MBC, class switched ASC, and non-class switched B cells that are S-RBD specific in healthy, mild, and severe participants (N=7 for each group). Dotted line represents the true positive threshold, defined as the mean plus two standard deviations of the healthy group. For A and C box plots, horizontal lines indicate means, boxes are inter-quartile range, and whiskers are minimum to maximum. Three group comparisons in A were performed using one-way ANOVA, with p values adjusted for multiple comparisons using the Benjamini, Krieger and Yekutieli method. Two group comparisons in C were performed with t tests if data were normally distributed based on Shapiro Wilk normality test or Mann Whitney test if data were not normally distributed. Statistically significant comparisons are indicated (** P ≤ 0.01)

Figure 2.. Comparisons of serum anti S-RBD IgG and neutralizing antibody titers in mild and severe participants.

(A) Anti S-RBD IgG area under the curve (AUC) in mild and severe participants. (B) Neutralizing antibody AUC in mild and severe participants. (C) % of class switched MBC that are RBD specific, from participants with plasma anti-S-RBD AUC values in the lowest, middle, and highest tertiles. (D) % of class switched MBC that are RBD specific, from participants with plasma neutralizing antibody AUC values in the lowest, middle, and highest tertiles. Dotted line represents the true positive threshold, defined as the mean plus two standard deviations of the healthy group. For box plots, horizontal lines indicate means, boxes are inter-quartile range, and whiskers are minimum to maximum. P values for A-B were calculated using t tests and P values for C-D were calculated using one-way ANOVA test for linear trend. No statistically significant comparison was found.

Figure 3:. UMAP and histogram expression profiles of surface receptors on class switched MBC.

(A) Concatenated class switched MBC from healthy, mild, and severe subjects projected as UMAP with gated S-RBD+ class switched B cells. (B) Multigraph color mapping of the UMAP projection of S-RBD, CD21, CD27, CD38, FcRL5, CD22, CXCR5, and BTLA. (C) Overlay histograms comparing the expression of FCRL5, CD22, CXCR5, BTLA, CD21, CD27, and CD38 in S-RBD− or S-RBD+ healthy, mild and severe groups.

Figure 4:. Frequency of MBC subsets in S-RBD nonspecific (S-RBD−) or S-RBD specific (S-RBD+) class switched MBC from healthy, mild, or severe participants.

(A) intMBC (CD21+, CD27−). (B) rMBC (CD21+, CD27+). (C) actMBC (CD21−, CD27+). (D) atyMBC (CD21−, CD27−). For box plots, horizontal lines indicate means, boxes are inter-quartile range, and whiskers are minimum to maximum. Groups were compared by one-way ANOVA with p values adjusted for multiple comparisons using the Benjamini, Krieger and Yekutieli method. Statistically significant comparisons are indicated (* P ≤ 0.05).

Figure 5:. Surface expression of FcRL5, CXCR5, CD22, and CD38 on S-RBD nonspecific (S-RBD−) or S-RBD specific (S-RBD+) class switched MBC from healthy, mild, or severe participants. Expression is shown as either percent of cells positive or the mean fluorescent intensity (MFI).

(A) FcRL5 (B) CXCR5 (C) CD22 (D) CD38. For box plots, horizontal lines indicate means, boxes are inter-quartile range, and whiskers are minimum to maximum. Groups were compared by one-way ANOVA with p values adjusted for multiple comparisons using the Benjamini, Krieger and Yekutieli method. Statistically significant comparisons are indicated (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001).

Figure 6:. Surface expression of FcRL5, CXCR5, CD22, and CD38 on S-RBD nonspecific (S-RBD−) or S-RBD specific (S-RBD+) class switched intMBC, rMBC, actMBC, or atyMBC from healthy, mild, or severe participants. Expression is shown as either percent of cells positive or the mean fluorescent intensity (MFI).

(A) FcRL5 (B) CXCR5 (C) CD22 (D) CD38. For box plots, horizontal lines indicate means, boxes are inter-quartile range, and whiskers are minimum to maximum. Groups were compared by one-way ANOVA with p values adjusted for multiple comparisons using the Benjamini, Krieger and Yekutieli method. Statistically significant comparisons are indicated (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001).