Functional CVIDs phenotype clusters identified by the integration of immune parameters after BNT162b2 boosters

Abstract

Introduction: Assessing the response to vaccinations is one of the diagnostic criteria for Common Variable Immune Deficiencies (CVIDs). Vaccination against SARS-CoV-2 offered the unique opportunity to analyze the immune response to a novel antigen. We identify four CVIDs phenotype clusters by the integration of immune parameters after BTN162b2 boosters.

Methods: We performed a longitudinal study on 47 CVIDs patients who received the 3rd and 4th vaccine dose of the BNT162b2 vaccine measuring the generation of immunological memory. We analyzed specific and neutralizing antibodies, spike-specific memory B cells, and functional T cells.

Results: We found that, depending on the readout of vaccine efficacy, the frequency of responders changes. Although 63.8% of the patients have specific antibodies in the serum, only 30% have high-affinity specific memory B cells and generate recall responses.

Discussion: Thanks to the integration of our data, we identified four functional groups of CVIDs patients with different B cell phenotypes, T cell functions, and clinical diseases. The presence of antibodies alone is not sufficient to demonstrate the establishment of immune memory and the measurement of the in-vivo response to vaccination distinguishes patients with different immunological defects and clinical diseases.

Keywords: BNT162b2; COVID-19; CVIDs; SARS-CoV-2; antibodies; booster dose; mRNA vaccine; memory B cells.

Figure 1

Flow chart. Observational study on 47 adults with CVIDs naïve to SARS-CoV-2 infection immunized with the booster mRNA BNT162b2 vaccine.

Figure 2

Proportion of responders after third dose of BNT162b2 accordingly to Anti-S1 IgG antibodies and spike-specific memory B cells. (A) Individual anti-S1 IgG values in HCWs (green) and in patients with CVIDs. The values measured in the patients with anti-S1 IgG above the cut-off value (dotted line) are shown in black and those below in gray. Bars indicate median values. (B, C) Frequency of low (S+) and high-affinity (S++) MBCs in HCWs (green) and in Responders and Non-Responders CVIDs patients. (D) The pie charts depict the percentage of Responders or Non-Responders by considering only the presence of antibodies above the cut-off (left), by combining the presence of antibodies and S+ MBCs (centre), or by compounding antibodies S+ and S++ MBCs (right). Non-parametric Mann–Whitney t-test was used to evaluate statistical significance. Two-tailed P value significances are shown as **p< 0.01, ****p< 0.0001.

Figure 3

Specific immune response to SARS-CoV-2. (A) Spike-specific MBCs with low (S+) and high (S++) binding capacity are shown in patients representative of the four groups. (B) S+ MBCs, detectable only in patients of groups 1 and 2, are mostly of IgM+ isotype. (C) S++MBCs, found only in group 1, are IgM- MBCs. (D) RBD-specific MBCs were only detectable in group 1. (E) Dot plots represent the frequency of S+, S++, and RBD+ MBCs in the four groups of CVIDs patients and HCWs. (F) The plots show S1-specific IgG and neutralizing antibody titers in serum samples of HCWs (in green), and CVIDs patients. Dot line indicates the cut-off value. Bars indicate the median. Non-parametric Mann–Whitney t-test was used to evaluate statistical significance. Two-tailed P value significances are shown as **p< 0.01, ***p< 0.001, ****p< 0.0001.

Figure 4

Persistence of the specific response against SARS-CoV-2. (A–D) Graphs show the frequency of spike-specific S+ MBCs (A) S++ MBCs (B), RBD+ MBCs (C), the concentration of anti-S1 IgG (D) and the neutralizing antibody titers for the wild type and the BA.5 variant (E), after the 3^rd dose, before and 10 days after the 4^th dose in the four groups of CVIDs patients. Medians are shown as bars and the dotted line indicates the cut-off. Non-parametric Wilcoxon matched pair signed-rank test and Mann–Whitney t-test were used to evaluate statistical significance. Two-tailed P value significances are shown as *p<0.05, **p< 0.01, ***p< 0.001.

Figure 5

Percentage of IgM+ and IgM- specific MBCs. Pie charts depict the percentage of IgM+ and IgM- S+ (A) and S++ MBCs (B) in HCWs, and groups 1 and 2 CVIDs patients at the time points of analysis. MBCs were always undetectable in group 3 and group 4. Categorical variables were compared by Fisher exact test. Level of significance: **p< 0.01, ***p<0.001, ****p< 0.0001.

Figure 6

B-cell population in the 4 groups. (A) X-Shift B-cell cluster sets originated from the four concatenated groups and overlaid onto the Opt-SNE map, each cluster is indicated by a color. Based on the relative expression level of surface markers calculated by X-shift, we identified naïve B cells (clusters 11, 12, 13 and 15), switched MBCs (clusters 1, 2 and 3), IgM MBCs (cluster 6), transitional B cells (clusters 13 and 14), plasmablasts (cluster 5), and atypical MBCs IgM- (clusters 4 and 11) and IgM+ (cluster 7). (B) Expression of CD24, CD27, CD38 and IgM for population characterization and identification. For sake of simplicity, we represent the same population as one cluster with one color. (C) Merged Opt-SNE plots for each group with relative X-Shift cluster sets overlaid onto the Opt-SNE map. (D) Heat map depicts the cluster sets abundancy (%) in the four groups. Bar plots depict the frequency (E) and the absolute numbers (F) of MBCs (CD19+CD24+CD27+), IgM (IgM+), switched (IgM-) MBCs, in the four groups of CVIDs patients. The frequency of the B cell populations was evaluated in the lymphocyte gate. (G) Serum levels of IgM and IgA in the four groups. Non-parametric Mann–Whitney t-test was used to evaluate statistical significance. Two-tailed P value significances are shown as *p<0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.

Figure 7

CD4+ T-cell population in the 4 groups. (A) X-Shift B-cell cluster sets originated from the four concatenated groups and overlaid onto the Opt-SNE map, each cluster is indicated by a color. Naïve T cells were identified in two clusters 2 and 7 and were merged together. (B) Expression of CD45RO, CD40L, IFNγ, and TNFα for population characterization and identification. (C) Merged Opt-SNE plots for each group with relative X-Shift cluster sets overlaid onto the Opt-SNE map. (D) Heat map depicts the cluster sets abundancy (%) in the four groups.

Figure 8

(A) Clinical phenotypes (immune dysregulation, autoimmunity, and CLD) of CVIDs patients grouped according to post-immunization S+ and S++ MBCs response and anti-S1 IgG. (B) Paired dot plots depict the S+, S++ and RBD+ MBCs measured before and after an infection in CVID patients who had COVID-19 after 3^rd dose. Categorical variables were compared by Fisher exact test. Level of significance: *p<0.05.

Figure 9

Summary of the four CVIDs groups. The memory B-cell compartment includes GC- and T-independent IgM MBCs, as well as post-GC and T-dependent MBCs of both IgM and switched isotypes. Short-lived plasmablasts (PB) are generated by recent immune responses. Long-lived plasma cells (LLPCs) maintain serum antibody levels in time. Group 1 patients are able to produce all MBCs types. Hypogammaglobulinemia may be explained by the inability to produce LLPCs in this group of CVIDs patients. For patients of groups 2 and 3, the defective CD4+ T-cell function may impair GC development. Patients of group 2 retain the ability to generate T-independent IgM MBCs, whereas all MBCs are missing in group 3. In patients of group 4, the significant reduction of all B-cell populations past the transitional B-cell stage suggests a severe defect in all B-cell functions. The severity of the clinical disease increases in groups 3 and 4.