Human monoclonal antibodies isolated from a primary pneumococcal conjugate Vaccinee demonstrates the expansion of an antigen-driven Hypermutated memory B cell response

Abstract

Background: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination.

Methods: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity.

Results: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge.

Conclusions: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.

Keywords: Human; Monoclonal antibodies; Plasmablast B cell; Pneumococcal conjugate vaccine.

Fig. 1

mAbs from PCV13-induced plasmablast B cells reactive to individual polysaccharide antigens. A two-day polysaccharide-specific sandwich ELISA was used to identify polysaccharide-binding mAbs . mAb 1A2 is reactive to polysaccharide type 7F. mAb 1A4, 1C6, 1D4 are reactive to polysaccharide type 1. mAb 1A6, 1A10, 1B4, 1C3 are reactive to polysaccharide type 4. mAb 1B2 is reactive to polysaccharide type 18C, and mAb 1C4, 1D7 are reactive to polysaccharide types 6A and 6B

Fig. 2

S. pneumoniae serotype 4-specific mAb protects mice from S. pneumoniae serotype 4 challenge. a Survival rate of each treatment group at 96 h post-infection. Log-rank (Mantel-Cox) test was performed. Each group was compared to No Antibody Treatment Group. * p < 0.05, statistically significant difference. b Mean weight of survivors in each group at 0, 24, 48, 72 h post-infection. c Bacteremia of each animal at 48 h post-infection. Individual raw data plotted on Log₁₀ scale with geometric mean and 95% CI. Transformed data analyzed by One-way ANOVA with Dunnett post test to evaluate for significant reduction in blood CFU counts (compared to No Antibody Treatment Group), *** p < 0.001, highly statistically significant difference