Prior cycles of anti-CD20 antibodies affect antibody responses after repeated SARS-CoV-2 mRNA vaccination

Abstract

BACKGROUNDWhile B cell depletion is associated with attenuated antibody responses to SARS-CoV-2 mRNA vaccination, responses vary among individuals. Thus, elucidating the factors that affect immune responses after repeated vaccination is an important clinical need.METHODSWe evaluated the quality and magnitude of the T cell, B cell, antibody, and cytokine responses to a third dose of BNT162b2 or mRNA-1273 mRNA vaccine in patients with B cell depletion.RESULTSIn contrast with control individuals (n = 10), most patients on anti-CD20 therapy (n = 48) did not demonstrate an increase in spike-specific B cells or antibodies after a third dose of vaccine. A third vaccine elicited significantly increased frequencies of spike-specific non-naive T cells. A small subset of B cell-depleted individuals effectively produced spike-specific antibodies, and logistic regression models identified time since last anti-CD20 treatment and lower cumulative exposure to anti-CD20 mAbs as predictors of those having a serologic response. B cell-depleted patients who mounted an antibody response to 3 vaccine doses had persistent humoral immunity 6 months later.CONCLUSIONThese results demonstrate that serial vaccination strategies can be effective for a subset of B cell-depleted patients.FUNDINGThe NIH (R25 NS079193, P01 AI073748, U24 AI11867, R01 AI22220, UM 1HG009390, P01 AI039671, P50 CA121974, R01 CA227473, U01CA260507, 75N93019C00065, K24 AG042489), NIH HIPC Consortium (U19 AI089992), the National Multiple Sclerosis Society (CA 1061-A-18, RG-1802-30153), the Nancy Taylor Foundation for Chronic Diseases, Erase MS, and the Claude D. Pepper Older Americans Independence Center at Yale (P30 AG21342).

Keywords: Autoimmune diseases; Autoimmunity; COVID-19; Multiple sclerosis.

Figure 1. Humoral vaccine responses to third mRNA vaccines after anti-CD20 mAb treatments.

(A and B) Dot plots of anti–SARS-CoV-2 spike antibody titers (A) and neutralizing antibody titers (B) were evaluated from before the first vaccine (baseline) to 6 months after the third vaccine (V3 + 24 weeks). The median is marked by a horizontal line. The proportion of seropositive participants at each time point is shown above the dot plots and the dotted line indicates the threshold for antibody detection (0.01 μg/mL). Circles represent participants without documented SARS-CoV-2 infections and triangles show those with known prior infection at each time point. Data were evaluated by independent-sample 2-tailed t test. (C) Box-and-whisker plots of the neutralization capacities between controls (pre-V3, n = 18; post-V3, n = 18; 24 weeks post-V3, n = 10) and B cell depletion (pre-V3, n = 67; post-V3, n = 63; 24 weeks post-V3, n = 29) is shown. The median is marked by a horizontal line, with whiskers extending to the farthest point within a maximum of 1.5 × IQR. Independent-sample 2-tailed t tests were performed. (D) Dot plots of anti-spike or anti-RBD antibody concentrations for each variant (Alpha, Beta, Gamma, and Delta) between controls (pre-V3, n = 16; post-V3, n = 15) and B cell–depleted participants (pre-V3, n = 66; post-V3, n = 59). The median is marked by a horizontal line. The proportion of seropositive participants at each time point is shown above the dot plots and the dotted line indicates the threshold for antibody detection (0.01 μg/mL). Independent-sample 2-tailed t tests with Bonferroni’s correction were performed. Ctrl, control participants; BCDT, patients with B cell depletion therapy.

Figure 2. Spike-specific B cell response to third mRNA vaccines after B cell depletion.

(A) Representative flow cytometry of CD19⁺ B cells and their proportions between controls (n = 10) and B cell–depleted participants (n = 48). Data are represented as mean ± SEM and independent-sample t tests with Bonferroni’s correction were performed. (B) Representative flow cytometry of spike⁺ B cells and their proportions between controls (n = 10) and B cell depleted (n = 48). Wilcoxon’s signed-rank test (middle) and independent-sample 2-tailed t tests with Bonferroni’s correction were performed. (C and D) Representative flow cytometry of each subset in spike⁺ B cells and their proportions between pre-V3 and post-V3 in controls (n = 10). UCSM, IgD⁺CD27⁺ non–class-switched memory B cells; CSM, IgD^–CD27⁺ class-switched memory B cells; DN, IgD^–CD27^– double-negative B cells. (E and F) Correlation between the proportion of CD19⁺ B cells before V3 and the proportion of spike⁺ B cells (E) or anti–SARS-CoV-2 spike antibody titers (F) after V3 in B cell–depleted participants (n = 47–48). The vertical dotted line represents the value of 0.25% (E). Linear regression is shown with 95% confidence intervals (gray area) and correlation statistics by 2-tailed Spearman’s rank correlation test were performed (F).

Figure 3. Spike-specific T cell response to third mRNA vaccine after anti-CD20 mAb therapy.

(A–C) Representative flow cytometry of CD137⁺OX40⁺ spike-specific CD4⁺ T cells (A) and their proportions between controls (n = 10) and B cell–depleted participants (n = 48) in relation to a third vaccination (B and C). Baseline, prevaccination samples for controls (n = 17), and B cell–depleted participants (n = 23) were also evaluated. Wilcoxon’s signed-rank test (B) and 2-tailed independent-sample t test (C) were performed. (D–F) Representative flow cytometry of CD137⁺CD69⁺ spike-specific CD8⁺ T cells (D) and their proportions between controls (n = 10) and B cell depletion (n = 48) (E and F). Wilcoxon’s signed-rank test (E) and 2-tailed independent-sample t test (F) were performed. (G) Body mass index (BMI, kg/m²) between B cell–depleted participants with increased spike-specific CD8⁺ T cells (n = 36) and without an increase (n = 12). Two-tailed independent-sample t test was performed. (H) The proportion of increased spike-specific CD8⁺ T cells before V3 based on BMI (kg/m²). WHO BMI classification was applied for the subgroups with B cell depletion: BMI < 18.5 (n = 0), 18.5–24.9 (n = 20), 25.0–29.9 (n = 10), 30.0–34.9 (n = 10), > 35.0 (n = 8).

Figure 4. The prediction of humoral immune responses after third vaccine in B cell–depleted participants.

(A) Sequential anti–SARS-CoV-2 spike antibody titers before and after V3 in B cell–depleted participants (n = 44). (B and C) Prior cycles of anti-CD20 antibodies between B cell–depleted participants with increased anti-spike antibodies (n = 14) and without an increase (n = 30). Two-tailed independent-sample t test was performed (C). (D) Box-and-whisker plots of sequential anti–SARS-CoV-2 spike antibody titers from before V3 to 24 weeks after the third vaccine (V3 + 24 weeks). The median is marked by a horizontal line, with whiskers extending to the farthest point within a maximum of 1.5 × IQR. Controls (blue, n = 7), B cell–depleted participants who increased anti-spike antibodies after V3 (green, n = 7), and B cell–depleted participants who did not increase anti-spike antibodies after V3 (purple, n = 14) are shown. Independent-sample 2-tailed t tests with Bonferroni’s correction were performed.