The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide antibody has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide(s) (PPS) vaccine, Pneumovax®, such as children<2 years, the asplenic and a subset of Common Variable Immunodeficiency (CVID) patients are profoundly deficient or lack IgM memory cells (CD27+IgM+), they are also deficient in the switched memory (CD27+ IgM−) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study we labeled PPS14 and PPS23F with fluorescent markers. Fluorescent labeled PPSs were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and post-immunization with Pneumovax®. The labeled PPS were capable of inhibiting binding of antibody to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and post-immunization demonstrated a predominance of naïve CD27−IgM+ cells accounting for 61.4% of B cells. Likewise, the PPS-specific B cells obtained pre-immunization consisted primarily of naïve, CD27− B cells, 55.4–63.8%. In contrast, the PPS-specific B cells obtained post-immunization were predominantly IgM memory cells displaying the CD27+IgM+, 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27+IgM−) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.

Pneumococcal polysaccharide vaccines have been used to elicit a protective anti-pneumococcal polysaccharide antibody response against S. pneumoniae in healthy individuals. Identifying human B cells which respond to T-cell independent type-2 antigens, such as pneumococcal polysaccharides, has been challenging. We employed pneumococcal polysaccharides directly conjugated to fluorophores in conjunction with flow cytometry to identify the phenotype of B cells that respond to pneumococcal polysaccharide vaccination. We have previously identified that the majority of pneumococcal polysaccharide-selected cells responding to vaccination are CD27+IgM+ (IgM+ memory) cells. In this study, we further characterized pneumococcal polysaccharide-selected cells in the peripheral blood to better identify how the various B cell phenotypes responded 7 and 30 days post-immunization. We show that 7 days post-immunization the majority of pneumococcal polysaccharide-selected IgM+ memory cells (PPS14+ 56.5%, PPS23F+ 63.8%) were CD19+CD20+CD27+IgM+CD43+CD5+/−CD70−, which was significantly increased compared to pre-immunization levels. This phenotype is in alignment with recent publications describing human B-1 cells. PPS-responsive B cells receded to pre-immunization levels by day-30. These findings suggest that this B-1 like cell population plays an important role in early responses to S. pneumoniae infection and possibly other T-cell independent type-2 antigens in humans.

Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique K_D values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar K_D values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype