Characterization of gene use and efficacy of mouse monoclonal antibodies to Streptococcus pneumoniae serotype 8

Abstract

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines, the prevalence of antibiotic-resistant pneumococcal strains, serotype (ST) replacement in nonconjugate vaccine strains, and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study, we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs, including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC, were produced, and their nucleotide sequences, epitope and fine specificity, and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes, with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy.

FIG. 1.

PPS8 specificity of PPS8- and PC-binding MAbs. PPS binding of the PPS8-binding MAb 28H11 (A, B) and the PC-binding MAb 24A4 (C, D) as determined by inhibition ELISA. The soluble PPSs shown in the legend were used to inhibit the binding of the indicated MAbs to PPS8-coated plates. Similar binding patterns were obtained for the other PPS8- and PC-binding MAbs (not shown).

FIG. 2.

Specificity of PC-binding MAbs. The binding of PC-binding MAbs to the analogs of phosphorylcholine indicated in the legend was determined by inhibition ELISA for 24A4 (left) and 5G6 (right), which differs from 24A4 by 1 amino acid (Y to D) in the D region.

FIG. 3.

Quellung and immunofluorescence (IF) imaging of PPS- and PC-binding MAbs. Binding of the PC-binding MAb 24A4 to ST3 (WU2) and ST8 (6308) was determined by Quellung (bright-field) and IF microscopy. The PC-binding MAb binds both ST3 and ST8. PPS8-binding MAb 31B12 binds ST8 cells but not ST3 cells, and the PPS3-binding MAb 1E2 binds ST3 but not ST8.

FIG. 4.

Effect of MAb administration on lethal ST8 infection in mice. The survival of BALB/c mice treated with 10 μg IgM (A and B) and IgG1 (C and D) MAbs as indicated in each panel after ST8 infection is shown for i.p. (A and C) and i.n. (B and D) infection. The IgM MAbs used in this study were 28H11 (PPS8), 24A4 and 5G6 (PC), and 2D10 (IgM isotype control). The IgG MAbs used in this study were 31B12 (PPS8) and 1E2 (IgG1 isotype control). *, P < 0.01 (Kaplan Meier log rank survival test comparing PPS-binding MAb to isotype control; n = ∼8 to 10 mice per group).

FIG. 5.

Effect of MAb administration on lethal infection with ST3 in mice. The survival of BALB/c mice treated with 10 μg of the MAbs indicated in the figure after i.p. infection with 100 CFU of ST3 (WU2) is shown. The PPS3-binding MAb 1E2 was used as a positive control. The GXM-binding MAb 2D10 was used as a negative control. The survival curves were not statistically significant with comparison of 24A4 and 5G6 to 2D10 (Kaplan Meier log rank survival test; n = ∼8 to 10 mice per group).