CpG oligodeoxynucleotides act as adjuvants for pneumococcal polysaccharide-protein conjugate vaccines and enhance antipolysaccharide immunoglobulin G2a (IgG2a) and IgG3 antibodies

Abstract

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses against Streptococcus pneumoniae polysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM(197). BALB/c ByJ mice were injected with 19F-CRM(197) or 6B-CRM(197) with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM(197) alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM(197) alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM(197) IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.

FIG. 1

CpG ODN enhance antipolysaccharide antibody responses after immunization with 19F-CRM₁₉₇ conjugate vaccine. Groups of six mice were immunized on days 0 and 14 with 19F-CRM₁₉₇ (approximately 5 μg of polysaccharide equivalent) with or without 100 μg of CpG ODN 1826 or 100 μg of non-CpG ODN 1982 in saline. Serial dilutions of mouse sera obtained on day 28 were assayed by ELISA for anti-19F antibodies, the OD values for the dilutions were used to construct a curve for each mouse, and the anti-19F antibody titer was calculated for each mouse at an OD of 0.5, using two-point curve fit analysis (see Materials and Methods). The geometric mean titer is shown for each group of six mice (error bars indicate SEM); ∗ indicates P < 0.05 compared to immunization with 19F-CRM₁₉₇ alone. (A) Total anti-19F; (B) anti-19F IgG1; (C) anti-19F IgG2a; (D) anti-19F IgG3; (E) anti-19F IgM. In sera from unimmunized mice, the titers of total anti-19F and all isotypes of anti-19F were <1 (maximum OD < 0.5). These data are representative of three independent experiments of similar design plus three modified related experiments, all of which showed enhancement of anti-19F antibodies by CpG ODN.

FIG. 2

CpG ODN enhance antipolysaccharide antibody responses after immunization with 6B-CRM₁₉₇ conjugate vaccine. Groups of five mice were immunized on days 0 and 14 with 6B-CRM₁₉₇ (approximately 5 μg of polysaccharide equivalent) with or without 100 μg of CpG ODN 1826 or 100 μg of non-CpG ODN 1982 in saline. Serial dilutions of mouse sera obtained on day 28 were tested by ELISA for anti-6B antibodies, and titers of anti-6B antibodies were determined as in Fig. 1. (A) Total anti-6B antibody; (B) anti-6B IgG1; (C) anti-6B IgG2a; (D) anti-6B IgG3; (E) anti-6B IgM. The geometric mean titer is shown for each group of five mice (error bars indicate SEM); ∗ indicates P < 0.05 compared to immunization with 6B-CRM₁₉₇ alone. In sera from unimmunized mice, the titer of total anti-6B and the titers of all isotypes of anti-6B were <1 (maximum OD < 0.5 for ELISA). These data are representative of two independent experiments of similar design using 6B-CRM₁₉₇.

FIG. 3

Kinetics of the anti-19F antibody response after immunization with 19F-CRM₁₉₇ with or without ODN. Mice were bled on day 0 and then immunized as in Fig. 1. Sera were collected weekly, stored, and then assayed simultaneously at a single dilution. (A) Total anti-19F antibodies (sera assayed at 1:200); (B) anti-19F IgG1 (sera assayed at 1:300); (C) anti-19F IgG2a (sera assayed at 1:200); (D) anti-19F IgG3 (sera assayed at 1:300); (E) anti-19F IgM (sera assayed at 1:50). Each point represents the mean of six mice. Error bars indicate SEM. These data are representative of three independent experiments of similar design.

FIG. 4

Effect of CpG ODN on anti-CRM₁₉₇ antibodies after immunization with 19F-CRM₁₉₇. Groups of five mice were immunized as in Fig. 1. Sera collected on day 28 were assayed for anti-CRM₁₉₇ antibodies, and antibody titers were calculated as in Fig. 1. The geometric mean titer is shown for each group of five mice (error bars indicate SEM); ∗ indicates P < 0.05 compared to immunization with 19F-CRM₁₉₇ alone. (A) Total anti-CRM₁₉₇ antibody; (B) anti-CRM₁₉₇ IgG1; (C) anti-CRM₁₉₇ IgG2a; (D) anti-CRM₁₉₇ IgG3. In sera from unimmunized mice, the titer of total anti-CRM₁₉₇ and the titers of all isotypes of anti-CRM₁₉₇ were all <1 (maximum OD < 0.5). These data are representative of three independent experiments of similar design.