Correlation of molecular characteristics, isotype, and in vitro functional activity of human antipneumococcal monoclonal antibodies

Abstract

Structure-function correlations of pneumococcal antibodies are important in predicting how changes in the pneumococcus (Pnc)-specific B-cell repertoire will influence humoral immunity against invasive Pnc disease. Using a unique panel of human hybridomas derived from memory B cells after pneumococcal conjugate vaccination, we analyzed the structure-function relationship of nine monoclonal antibodies (MAbs) reactive to Pnc polysaccharides. The avidities of the antibodies correlated with the avidity of donor immune serum (R, 0.7; P < 0.025), and this relationship was particularly strong for immunoglobulin A clones (R, 1; P < 0.0005), suggesting that the MAbs may represent important clones contributing to serological memory. Common heavy-light chain combinations and amino acid replacement mutations were seen for clones with the same serospecificity from different individuals. The two highest-avidity MAbs used Vh3-48, and two MAbs with the same serospecificity, using the same V gene pairings (Vh3-7 and Vk2A17), had similar avidities, suggesting that canonical V gene use makes an important contribution to avidity. Although all clones had mutation levels consistent with their being derived from memory B cells, low levels of replacement mutation were associated with high avidities. This relationship was strongest for Vh genes (R, 0.8; P < 0.01). Opsonophagocytosis was demonstrated for all clones, and there was a trend toward clones using canonical genes with low levels of mutation having high opsonophagocytic activities (R, 0.5). These data suggest that the use of canonical genes in the Pnc antibody response is associated with highly functional antibodies and that most somatic mutations seen in these genes are not antigen selected.

FIG. 1.

Amino acid translations for heavy and light chains of Db8C11, Db7D4, and CbB2 aligned to the V_h3-07 and V_kIIA17 germ line genes and for heavy chains of AbA5 and CbE2 aligned to the V_h3-48 germ line gene. Nucleotide mutations resulting in amino acid replacement are shown in bold.

FIG. 2.

AIs measured by a modified ELISA for nine Pnc-specific MAbs. Three MAbs were IgA, six were IgG, and all were isotype and serotype matched with pre- and postvaccination serum. SEM, standard error of the mean.

FIG. 3.

Opsonophagocytosis of pneumococci by human MAbs. Mean OPAs (reciprocals of the MAb concentrations [μg/liter] causing a 50% reduction in bacterial uptake) and standard errors of the means are given for a minimum of two experiments performed in duplicate with eight limiting dilutions, using fresh human PMNs from at least two donors.