Increased immunogenicity and induction of class switching by conjugation of complement C3d to pneumococcal serotype 14 capsular polysaccharide

Abstract

Previous studies have demonstrated an adjuvant effect for the C3d fragment of complement C3 when coupled to T-dependent protein antigens. In this study, we examined the antibody response to covalent conjugates of C3d and a T-independent antigen, the capsular polysaccharide of serotype 14 Streptococcus pneumoniae (PPS14). We prepared a conjugate of mouse C3d and PPS14 and compared its immunogenicity with that of a conjugate of PPS14 and ovalbumin (OVA). When BALB/c mice were immunized with PPS14-C3d, there was a significant increase in serum anti-PPS14 concentrations compared with either native PPS14 or control PPS14-glycine conjugates. This was accompanied by a switch in anti-PPS14 from predominantly immunoglobulin M (IgM) to IgG1 by day 25 following primary immunization. Following secondary immunization with PPS14-C3d, there was a marked booster response and a further increase in the ratio of IgG1 to IgM anti-PPS14. Although the primary antibody response to the PPS14-OVA conjugate exceeded that induced by immunization with PPS14-C3d, serum anti-PPS14 concentrations after a second injection of PPS14-C3d were nearly identical to those induced by secondary immunization with PPS14-OVA. Experiments with athymic nude mice suggested that T cells were not required for the adjuvant effect of C3d on the primary immune response to PPS14 but were necessary for enhancement of the memory response after a second injection of PPS14-C3d. These studies show that the adjuvant effects of C3d extend to T-independent antigens as well as T-dependent antigens. As a means of harnessing the adjuvant potential of the innate immune system, C3d conjugates may prove useful as a component of vaccines against encapsulated bacteria.

FIG. 1

Enhancement of the PPS14 antibody response by conjugation of C3d to PPS14. BALB/c mice (15 animals per group) were immunized subcutaneously on day 0 with either 0.1 μg (left) or 1.0 μg (right) of PPS14 either as the PPS14-glycine control (open circles) or as PPS14-C3d conjugate (closed circles). A second immunization with the same PPS14 preparation given in the primary immunization was performed 47 days following the first injection (arrows). Serum samples were obtained 10 and 25 days after the primary immunization and 10 and 23 days after the secondary immunization. Serum anti-PPS14 immunoglobulin concentrations were determined by RABA. Data points represent the GMC for each group of mice. The error bars represent the 95% CI of the geometric mean for each group of sera. Data were analyzed by Student's t test on log-transformed data; the P values for the PPS14-C3d conjugate versus the PPS14-glycine conjugate are shown above the error bar at each time point.

FIG. 2

Comparison of the PPS14 antibody response to PPS14-C3d with that induced by a conjugate of PPS14 and OVA, a T-dependent protein carrier. BALB/c mice (5 animals per group) were immunized subcutaneously on day 0 with 1 μg of PPS14 either as unmodified PPS14 (left graph) or as PPS14 conjugated to C3d (middle graph) or OVA (right graph). For mice receiving PPS14 conjugates (right two graphs), one group of mice received PPS14 conjugate on both day 0 and day 70 (closed circles) and another group of mice received PPS14 conjugate on day 0 followed by unmodified PPS14 on day 70 (open triangles). The mice were bled 10, 25, 45, and 63 days after the primary immunization and 10, 27, and 42 days after the secondary immunization. Serum anti-PPS14 immunoglobulin concentrations were determined by RABA. Data points represent the GMC for each group of mice. The error bars represent the 95% CI of the geometric mean for each group of sera. The arrowheads indicate the day of secondary immunization. Serum anti-PPS14 concentrations were significantly greater for mice receiving two injections of either conjugate preparation than for those receiving two injections of PPS14 (P ≤ 0.02 at all time points). They were also significantly increased for mice reimmunized with unmodified PPS14 after primary immunization with PPS14-C3d or PPS14-OVA (P ≤ 0.05 at all time points). Reimmunization with unmodified PPS14 after primary immunization with either conjugate did not cause a significant increase in serum anti-PPS14 compared with values 7 days prior to secondary immunization (P ≥ 0.1 at all time points after secondary immunization).

FIG. 3

ELISA heavy chain isotype analysis of anti-PPS14 antibodies in mice immunized with either PPS14, PPS14-C3d, or PPS14-OVA. BALB/c mice were immunized as described in the Fig. 2 legend, and serum samples obtained 10 days after primary and secondary immunization were analyzed for immunoglobulin isotype. For each assay, individual serum samples were diluted so that each had an equivalent concentration of total anti-PPS14 Ig. Isotype concentrations were determined by ELISA. Results are expressed as the absorbance at 405 nm for each sample. Within each horizontal pair of graphs, each symbol represents data for the same mouse; each pair of graphs displays data for a single group of five mice immunized as shown.

FIG. 4

Kinetics of anti-PPS14 heavy chain switching in mice immunized with PPS14, PPS14-C3d, or PPS14-OVA. BALB/c mice were immunized as described in the Fig. 2 legend; the arrow indicates the time of secondary immunization at day 70 post-primary immunization. Serum samples from all time points were analyzed for immunoglobulin isotype as described in the Fig. 3 legend. The predominant isotypes at all time points were IgM and/or IgG1. The ratio of the A₄₀₅ for IgG1 to the A₄₀₅ for IgM was calculated for each mouse. Each point represents the mean ratio for each group of five mice. Error bars are omitted for the sake of clarity.

FIG. 5

Comparison of the PPS14 antibody response to PPS14-C3d and PPS14-OVA in athymic nude mice. BALB/c nu/nu mice (seven animals per group) were immunized subcutaneously on day 0 with 1 μg of PPS14 either as PPS14 conjugated to OVA (open circles) or to C3d (closed circles). A second immunization with the same PPS14 preparation given in the primary immunization was performed 45 days following the first injection (arrow). The mice were bled 14, 28, and 44 days after the primary immunization and 15 and 29 days after the secondary immunization. Serum anti-PPS14 immunoglobulin concentrations were determined by RABA. Data points represent the GMC for each group of mice. Error bars represent the 95% CI of the geometric mean for each group of sera. Serum anti-PPS14 concentrations were significantly greater for mice receiving two injections of PPS14-C3d than for those receiving two injections of PPS14-OVA at day 14 (P = 0.01), day 28 (P = 0.02), and day 44 (P = 0.03) after primary immunization, but not at day 15 (P = 0.09) and day 29 (P = 0.15) after secondary immunization.