Aggregation of Streptococcus pneumoniae by a pneumococcal capsular polysaccharide-specific human monoclonal IgM correlates with antibody efficacy in vivo

Abstract

Acquired antibody immunity to Streptococcus pneumoniae (pneumococcus) has been linked to serotype (ST)-specific opsonic antibodies to the relevant pneumococcal capsular polysaccharide (PPS) that mediate protection by enhancing the bactericidal effect of host phagocytes. Despite the well-recognized role of opsonic IgG in host defense against pneumococcus, PPS-specific monoclonal antibodies (MAbs) that mediate protection against lethal challenge with ST3 pneumococcus in mice but do not promote phagocytic killing in vitro (nonopsonic antibodies) have been described. In this study, we sought to determine the biological activity of one such MAb, A7 (a human PPS3-specific IgM), and the mechanism by which it mediates protection. In vitro studies demonstrated that coincubation of A7 with ST3 in the absence of phagocytes or a complement source resulted in a reduction in CFU on blood agar plates that was largely reversible by sonication. A chromogenic cellular proliferation assay demonstrated that A7 did not affect replication of ST3 in liquid culture. The ability of A7 to induce aggregation of ST3 was confirmed by fluorescence microscopy and flow cytometry: A7 induced aggregation of ST3, and in the presence of a complement source, A7 promoted deposition of complement component 3 (C3) on aggregated bacteria in a dose-dependent fashion. Similarly, administration of preincubated mixtures of A7 and ST3 intraperitoneally to mice protected them from the lethality of ST3 in a dose-dependent fashion. These findings suggest that A7-mediated aggregation enhances resistance to ST3, most likely by enhancing C3 deposition on the ST3 capsule, thereby promoting host antipneumococcal activity in vivo.

FIG. 1.

A7 induces reversible reduction in CFU of ST3 on solid medium. The black bars represent CFU of ST3 (ATCC 6303) without sonication; gray bars represent CFU after 2 min of sonication. Each bar represents the mean for the designated group; the error bars show the standard errors of the means. *, P < 0.0001, comparing ST3 (no sonication) to 10 μg A7 (no sonication) (t test); **, P < 0.0022, comparing 10 μg A7 (no sonication) to 10 μg A7 (sonication) (t test); ***, P < 0.0001, comparing ST3 (sonication) to 10 μg A7 (sonication) (t test); #, P = 0.0223, comparing ST3 (no sonication) to 5 μg A7 (no sonication) (Mann-Whitney U test). The data shown are combined from four separate experiments performed in duplicate, except for data obtained with 5 μg A7, for which experiments were performed two times.

FIG. 2.

A7 induces aggregation of ST3. Oregon Green-labeled ST3 (ATCC 6303) was imaged by fluorescence microscopy. Total magnification, ×400 (40× objective lens × 10× ocular lens). Microscopic images represent 10 μg A7 (A), ST3 alone (B), 10 μg (human myeloma) IgM (C), 1 μg human IgM (D), and 1 μg A7 (E).

FIG. 3.

A7 has no effect on growth of ST3 in liquid culture. The figures depict the optical density at 450 nm (OD₄₅₀) (y axis) versus the time of assay development (x axis). The wells contained an initial inoculum of ∼10⁶ CFU of ST3 (ATCC 6303). Optical densities were determined at the indicated times after addition of XTT, with or without sonication and with or without 10 μg (A) or 1 μg (B) of A7. Closed symbols represent OD₄₅₀ values obtained without sonication; open symbols represent OD₄₅₀ values obtained after 2 min of sonication. Symbols: ▪, ST3 alone; □, ST3 alone; •, A7 + ST3; ○, A7 + ST3; ▴, 57E2 (human IgM to PPS8) + ST3; ▵, 57E2 + ST3; *, heat-killed ST3 alone; +, ST6B alone. Data represent OD₄₅₀ values from four separate experiments performed in duplicate.

FIG. 4.

A7 mediates aggregation of ST3. Flow cytometric analysis was performed on mixtures of MAbs and Oregon Green-labeled ST3 (ATCC 6303). Cell size and forward scatter were determined for ST3 alone (A), 10 μg 57E2 (human MAb to PPS8) (B), 1 μg 57E2 (C), 1 μg A7 (D), and 10 μg A7 (E). Insets depict scatter plot analyses of cellular size.

FIG. 5.

A7 mediates aggregation and complement deposition on ST3. Binding of IgM (red, middle) and C3 (green, right) and merged DIC bright-field images (overlay, left) are shown for the designated mixtures of ST3 (ATCC 6303), A7, and mouse serum (used as a complement source). Δ C, heat-inactivated complement source; G19, human IgM MAb to GXM. Magnification, ×100 (all images). The appearance of the A7-ST3 aggregate in panel D differs from that in panels A to C due to focusing on the aggregate, rather than the bacteria, in the bright-field image.

FIG. 6.

A7-ST3 mixtures enhance mouse survival after i.p. infection with ST3. The percent survival was determined for mice that received mixtures of ST3 (ATCC 6303) preincubated with PBS, control IgM MAbs, 10 μg A7, or 1 μg A7 (A) or with different amounts of A7 (B) prior to i.p. administration. (A) *, P = 0.003 for comparison of 10 μg A7 to controls, P = 0.14 for comparison of 1 μg A7 to PBS, and P = 0.051 for comparison of 10 to 1 μg A7. Symbols: □, PBS; ▴, 10 μg IgM (myeloma); ▾, 1 μg IgM (myeloma); •, 10 μg A7; ▪, 1 μg A7. Note that PBS, 10 μg IgM, and 1 μg IgM produced overlapping curves. (B) *, P < 0.0001 for comparison of 10, 5, and 1 μg A7 to PBS and 0.1 μg A7; #, P = 0.048 for comparison of 10 to 1 μg A7. Symbols: •, PBS; ⧫, 10 μg A7; ▾, 5 μg IgM; ▴, 1 μg A7; •, 10 μg A7; ▪, 0.1 μg A7. Survival was analyzed by the Kaplan-Meier log rank test (n = 5 [A] and 10 to 15 [B] mice per group).