Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Abstract

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA3–286) from strain R36A. Abs to PspA3–286 were encoded by diverse VH and Vκ families/genes. The H chain CDR3 and L chain CDR3 lengths were 3–13 (7.8 ± 0.5) and 8–9 (8.7 ± 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack DH gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of DH gene-derived amino acids did not prevent anti-PspA3–286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA3–286 polyclonal Ab response in PspA3–286- and R36A-immunized mice. Compared with PspA3–286-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. DH genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of DH-less Abs.