Pneumococcal conjugate and plain polysaccharide vaccines have divergent effects on antigen-specific B cells

Abstract

Background: A 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP), routinely administered at the age of 65, has limited effectiveness, and revaccination induces attenuated antibody responses. It is not known whether pneumococcal polysaccharide-protein conjugated vaccines (PCV), although highly effective in infants, offer any immunological advantages over 23vP in adults.

Methods: We immunized adults with schedules combining both PCV and 23vP and investigated B-cell responses to establish whether PCV7 (a 7-valent PCV) induced T-dependent responses in adults, to assess the role of memory B cells in 23vP-induced antibody hyporesponsiveness, and to identify the B-cell subtypes involved.

Results: A single dose of PCV7 induced significant increases in serotype-specific memory B-cell populations in peripheral blood indicating a T-dependent response. Conversely, immunization with 23vP resulted in a decrease in memory B-cell frequency. Furthermore, memory B-cell responses to subsequent immunization with PCV7, when given after 23vP, were attenuated. Notably, B1b cells, a subset important in protecting mice against pneumococci, were also depleted following immunization with 23vP in humans.

Conclusions: This study indicates that PCV7 may have an immunological advantage over 23vP in adults and that 23vP-induced depletion of memory and B1b-cell subsets may provide a basis for antibody hyporesponsiveness and the limited effectiveness of 23vP. Clinical Trials Registration. ISRCTN: 78768849.

Figure 1.

Downregulation of memory B cells by the 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP) and subsequent recovery by a 7-valent pneumococcal conjugate vaccine (PCV7). A, Participants were vaccinated with either 1 dose of 23vP (red) or 1 dose of PCV7 (blue). Calculation of the mean change in serotype specific memory B-cells (MBC) from before to after vaccination showed an increase for all serotypes after PCV7 but a decrease after 23vP. MBC responses significantly differed between the vaccines for serotypes 4 (P = .045), 9V (P = .042), 18C (P = .045), 19F (P = .035), and 23F (P = .0149) B, The decline in MBCs seen after 23vP continued for 6 mo after vaccination. C, When participants who had received 23vP were given a dose of PCV7 6 mo later, there was an increase in their MBCs, but the number of MBCs after 23vP-PCV7 (green) remained lower than after a single dose of PCV7 (blue) alone. Horizontal bar represents median value, and each spot represents 1 sample (serotype 4, P = .038; serotype 6B, P = .006; serotype 9V, P = .003; serotype 14, P = .0009; serotype 18C, P = .007; serotype 19F, P = .008; serotype 23F, P = .0009). D, After a second dose of PCV7, memory B-cell numbers recovered to baseline values. All comparisons were made using analysis of covariance and P values adjusted using a false recovery method. Abbreviation: PBMCs, peripheral blood mononuclear cells.

Figure 2.

Depletion of polysaccharide-specific B1b-cells in 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP)–primed individuals. A, Subjects were randomized to receive 7-valent pneumococcal conjugate vaccine (PCV7)-PCV7 (group 1; blue circles, n = 24) or 23vP-PCV7 (group 2; red circles, n = 19). CD19⁺ purified B-cells were obtained 28 d after the second vaccine dose. Following gating on CD20⁺ pneumococcal polysaccharide (PnPs⁺) B-cells, the proportions of naive (CD27⁻IgM^lo), B1 (CD27⁻IgM^hi), MZB (CD27^hiIgM^hi), FO (CD27⁺IgM^lo), and switched memory B-cells (CD27⁺IgM⁻) that bound PnPs–fluorescein isothiocyanate (FITC) were determined. The data in the graph show the percentage of PnPs-specific B-cells within each of the subsets for each vaccine group, and the bar represents the median. B, B1b-cell but not B1a-cell frequency was reduced in 23vP-primed individuals. Purified CD19⁺ B-cells were labeled with anti-CD5, and the frequency of B1a (CD19⁺PnPs⁺IgM^hiCD27⁻CD5^int) vs B1b (CD19⁺PnPs⁺IgM^hiCD27⁻CD5^neg) cells were determined for group 1 (blue, n = 9) and group 2 (n = 8). The data represent the percentage of CD19⁺PnPs⁺CD27⁻IgM^hi B cells that were CD5^int or CD5^neg, and the bar shows the median (CD5 bright cells were residual T cells remaining after the sort). C, Boosting with 23vP induced a large B1-cell efflux into peripheral blood on day 7: PCV7-primed individuals received a 23vP booster (group 1: n = 8, blue circles) whereas those primed with 23vP-PCV7 were boosted with PCV7 (group 2: n = 5, red circles). Purified CD19⁺ B cells were then obtained on day 7 postboost, and the frequency of CD19⁺ marginal zone B-cells (CD23⁻CD21^hi), follicular origin B-cells (CD23⁺CD21⁻), and B1 cells (CD23⁻CD21⁻) present in peripheral blood mononuclear cells (PBMCs) on day 7 postboost in each group were compared. The graph represents the percentage of CD19⁺ B-cells within each subset, and the line shows the median value.

Figure 3.

Boosting of 7-valent pneumococcal conjugate vaccine (PCV7)–primed individuals with the 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP) induced a large efflux of plasma cells 7 d postboost. Purified CD19⁺ B-cells were obtained 7 d after boosting with 23vP (group 1, blue) or PCV7 (group 2, red). A, Plasma cells were identified using the phenotype (CD19⁺CD20^lo/-CD38^hi) and intracellular immunoglobulin G (IgG), immunoglobulin A (IgA), or immunoglobulin M (IgM) expression was determined. Example density plots showing the plasma cell populations (IgG, IgA, and IgM) for each group are given, and the graphs show the frequencies (with median) as a percentage of CD19⁺ B-cells for group 1 (n = 12) and group 2 (n = 18). B, B-cells isolated from some of the same individuals were also seeded directly onto enzyme-linked immunosorbent spot assay (ELISpot) plates to detect the total number of IgG-, IgA-, and IgM-secreting cells for group 1 (n = 7) and group 2 (n = 5).

Figure 4.

Priming with 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP) induces depletion of peripheral blood B1b and marginal zone B (MZB) cells—a mechanism. The top section of each panel is a representation of the B-cell enzyme-linked immunosorbent spot assay (ELISpot) data obtained, for each serotype in 7-valent pneumococcal conjugate vaccine (PCV7), at each point during the time course (x-axis). The middle section of each panel describes the effects of each immunization on the B-cell compartment. The bottom sections of panels B and C show the interaction of multivalent, high-epitope-density T-independent antigens vs low-epitope-density T-dependent antigens with a responsive B cell. A, Prior to immunization, the baseline composition of the peripheral B-cell pool is a mix of preexisting memory B cells, B1 cells, and MZB cells that can be stimulated to form immunoglobulin G (IgG)-ASCs following in vitro stimulation with mitogens, and these ASCs are detected in B-cell ELISpots at day 0. B, The polysaccharides in 23vP consist of multivalent (repeating) epitopes that are able to cross-link many B-cell receptors (BCRs) on a single cell providing a strong activation signal. Thus, the high dose of multivalent polysaccharides from 23 serotypes in 23vP may induce exhaustion of the peripheral B-cell pool by preferentially driving extrafollicular proliferation of B1b, memory B, MZB, and naive B cells via cross-linking BCRs, inducing a large plasma cell response. The lack of T-cell recruitment and germinal center formation prevents replenishment of the memory B-cell pool. This is demonstrated by the reduced frequency of ASCs detected in the ELISpot 1 mo postimmunization. C, Reduced frequency of polysaccharide specific B cells is still evident 6 mo later, and a subsequent dose of PCV7 is unable to induce increased frequencies of polysaccharide-specific B cells. The conjugated polysaccharide has reduced epitope density compared with the plain polysaccharide and cross-links fewer BCRs. This results in a weaker signal, and thus T-cell help is required for the B cell to proliferate. This occurs in the germinal center and results in formation of new memory B cells. However, the hyporesponsiveness observed in the ELISpot assay shows that 1 dose of PCV7 is not enough to overcome the effects of 23vP. D, The booster dose of PCV7 induces an increase in memory B cells detected by ELISpot demonstrating that by month 13 the hyporesponsiveness induced by 23vP has been overcome for the 7 serotypes in PCV7 and that the memory B-cell pool has been replenished for these serotypes. Abbreviation: PMBC, peripheral blood mononuclear cell.