Carrier Proteins Facilitate the Generation of Antipolysaccharide Immunity via Multiple Mechanisms

Abstract

Capsular polysaccharides (CPSs) are important antigenic targets against bacterial infections. As T-independent antigens, however, CPSs elicit short-lived immune responses in adults and are poorly immunogenic in young children. Coupling CPS with protein carriers enhances anti-CPS responses and generates long-lasting immune memory. However, the mechanisms whereby carrier proteins accomplish this are not fully understood. Here, we dissect different mechanisms whereby carrier proteins enhance anti-CPS immunity. We show how coupling CPS with protein carriers modifies the interaction of CPS with antigen-presenting cells, enables a dual-activation mechanism for CPS-specific B cells via interaction with CPS- or carrier-specific T helper cells, and potentiates the recall of anti-CPS responses by engaging memory T helper cells during subsequent vaccination or bacterial exposure. Our findings provide new insights into the immunological basis of carrier-mediated anti-CPS immunity and may help in the design of more effective CPS-based vaccines. IMPORTANCE Polysaccharide capsules, the outermost shells of many bacterial pathogens, play a role in pathogenesis and protect bacteria against the immune system. Generating antipolysaccharide antibodies by vaccination has provided effective protection against infectious diseases caused by encapsulated bacteria. However, most pure polysaccharide preparations are poorly immunogenic, particularly in young children. To circumvent this problem, vaccines have been developed using polysaccharides associated with protein carriers. The precise mechanism whereby protein carriers enhance the immunogenicity of the polysaccharide remains unclear. The significance of our research is in elucidating the different roles played by carriers in facilitating polysaccharide processing and presentation, priming polysaccharide-specific B cells, and potentiating recall antipolysaccharide responses. Overall, our work provides new insights into the immunological basis of carrier-mediated antipolysaccharide immunity and may help in the design of more effective polysaccharide-based vaccines.

Keywords: capsular polysaccharide; immune mechanisms; immunization.

FIG 1

Immunization with MAPS vaccines induces TD anti-CPS responses and immune memory. (A and B) C57BL/6 mice (n = 10 per group) received three subcutaneous immunizations with adjuvant alone (alum), the adjuvanted CPS14 or CPS14 MAPS vaccine, or the CPS14 conjugate vaccine (CV) (1 μg of CPS content per dose). (A) Anti-CPS IgM and IgG antibodies (Ab) in each group after one (P1), two (P2), or three (P3) immunizations. ns, not significant. (B) Avidity of anti-CPS IgG antibodies in CPS14 MAPS-immunized mice after two (P2) or three (P3) immunizations. AI, avidity index. (C) Anti-CPS IgM and IgG antibodies in wild-type (WT) or MHCII^−/− C57BL/6 mice (n = 5 per group) after one (P1) or two (P2) immunizations with the CPS14 MAPS vaccine. (D) Rag1^−/− mice (n = 8 to 10 per group) received an adoptive transfer of splenocytes isolated from naive mice (SpN) or from CPS14 MAPS-immunized mice (SpM). Eight days after adoptive transfer, Rag1^−/− mice received one immunization with uncoupled CPS14 (CPS14) or CPS14 MAPS (MAPS) (1 μg of PS content per dose). Anti-CPS IgG antibodies were measured 1 day before (Pre) and 14 days after (Post) immunization. For all panels, antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum sample for CPS14 antigen (see Materials and Methods). Bars represent geometric means and 95% confidence intervals (CIs). Statistical analyses were performed using the Mann-Whitney U test, in comparison to the alum group (A) or as indicated (B to D).

FIG 2

Coupling with carrier proteins enhances the uptake, processing, and MHCII-dependent and -independent surface presentation of CPS antigens in macrophages. (A and B) Peritoneal macrophages isolated from C57BL/6 mice were incubated in culture medium containing no CPS (control [Con]), 2.5 μg/mL of CPS14, or CPS14 MAPS (at 2.5 μg/mL of CPS content) at 4°C for 2 h (A) or at 37°C for the indicated periods (B). Intracellular and surface-associated CPS contents in different samples were measured by an inhibition ELISA and then normalized to the total cellular protein content (micrograms of CPS per milligram of protein). Bars represent means and standard errors of the means (SEM) (n = 12 in 4 independent experiments). (C) Internalization and presentation of purified CPS14, CPS14 in bacterial cells, or the MAPS complex in wild-type (WT) or MHCII^−/− macrophages. Peritoneal macrophages isolated from either WT or MHCII^−/− C57BL/6 mice were incubated in culture medium containing CPS14, heat-killed type 14 pneumococci (Pn14), or CPS14 MAPS (all at 2.5 μg/mL of CPS content) at 37°C for 18 h. Intracellular and surface-associated CPS contents in different samples were measured by an inhibition ELISA and then normalized to the total cellular protein content. Bars represent means and SEM (n = 9 in 3 independent experiments). Statistical analyses were performed using the Mann-Whitney U test between the indicated groups. (D) Peritoneal macrophages were incubated with CPS14 MAPS (2.5 μg/mL of CPS and 7.5 μg/mL of avidin), CPS14 (2.5 μg/mL), or avidin (Avi) (7.5 μg/mL) at 37°C for 18 h. After incubation, cells were washed with PBS twice and then lysed with lysis buffer. All cell lysates were then normalized by the total protein content measured by a BCA assay. For coimmunoprecipitation (Co-IP), each cell lysate was mixed with rabbit anti-CPS14 serum-pretreated protein A resins and incubated overnight at 4°C. After extensive washing with lysis buffer, the resins were boiled in SDS sample buffer, and the supernatants were then applied onto an SDS-PAGE gel. Western blotting was done using primary antibodies against β-actin (internal control) and MHCII.

FIG 3

T_carrier-mediated activation of naive B_CPS during MAPS vaccination. Rag1^−/− mice (n = 5 for group BN and n = 10 for the other groups) received an adoptive transfer of B cells isolated from naive mice (BN), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), carrier 1-primed mice (TC), or 5V-MAPS1-primed mice (TM). Eight days later, Rag1^−/− mice received one immunization with 5V-MAPS1 (1 μg per CPS). Anti-CPS IgG antibodies were measured 1 day before (preimmunization) and 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum sample for each CPS antigen. Dashed lines indicate geometric means of anti-CPS IgG titers of all groups preimmunization. Bars represent geometric means and 95% CIs of anti-CPS IgG titers of each group postimmunization. Statistical analyses were performed using the Mann-Whitney U test between the indicated groups.

FIG 4

T_CPS-mediated activation of naive B_CPS during MAPS vaccination. Rag1^−/− mice (n = 5 for group BN and n = 10 for the other groups) received an adoptive transfer of B cells isolated from naive mice (BN), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), carrier 1-primed mice (TC), or 5V-MAPS1-primed mice (TM). Eight days after adoptive transfer, Rag1^−/− mice received one immunization with 5V-MAPS2 (2.5 μg per CPS). Anti-CPS IgG antibodies were measured 1 day before (preimmunization) and 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum sample for each CPS antigen. Dashed lines indicate geometric means of anti-CPS IgG titers of all groups preimmunization. Bars represent geometric means and 95% CIs of anti-CPS IgG titers of each group postimmunization. Statistical analyses were performed using the Mann-Whitney U test between the indicated groups.

FIG 5

Anti-CPS recall responses in the absence or presence of naive, carrier-primed, or MAPS-primed CD4⁺ T cells. Rag1^−/− mice (n = 7 to 10 per group) received an adoptive transfer of B and CD4⁺ T cells isolated from naive mice (BN+TN) or B cells isolated from 5V-MAPS1-primed mice (BM), alone or in combination with CD4⁺ T cells isolated from naive mice (TN), carrier 1-primed mice (TC), or 5V-MAPS1-primed mice (TM). Eight days after adoptive transfer, Rag1^−/− mice received one immunization with 5V-MAPS1 (1 μg per CPS content). Anti-CPS IgG antibodies were measured 1 day before (preimmunization) and 14 days after immunization. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum sample for each CPS antigen. Dashed lines indicate geometric means of anti-CPS IgG titers of all groups preimmunization. Bars represent geometric means and 95% CIs of anti-CPS IgG titers of each group postimmunization. Statistical analyses were performed using the Mann-Whitney U test between the indicated groups.

FIG 6

The use of a pathogen-homologous carrier protein enhances anti-CPS IgG production in vaccinated mice during pneumococcal exposure. C57BL/6 mice (n = 10 for groups 1 and 2, and n = 15 for groups 3 and 4) received one immunization with adjuvant (alum) alone (groups 1 and 2) or adjuvanted CPS4 MAPS vaccine (groups 3 and 4) (2 μg of CPS content per mouse). Serum samples were collected 14 days after immunization (Pre-SP exposure). Two weeks after the bleed, mice were exposed to either the heat-killed wild-type (WT) (groups 1 and 3) or pneumolysin knockout (ΔPly) (groups 2 and 4) Tigr4 strain, each at 1 μg of CPS4 content (2.5 or 2.1 μg of protein content for the WT or ΔPly, respectively) per mouse via intraperitoneal injection. Serum samples were collected 14 days after exposure (Post-SP exposure). Anti-Ply (A) and anti-CPS IgG antibodies (B) were measured using an ELISA. Antibody titers are expressed in arbitrary units (a.u.) relative to a reference serum sample of Ply or CPS4 antigen. Bars represent geometric means and 95% CIs. Statistical analyses were performed using the Mann-Whitney U test between the indicated groups.