The proline-rich region of pneumococcal surface proteins A and C contains surface-accessible epitopes common to all pneumococci and elicits antibody-mediated protection against sepsis

Abstract

Pneumococcal surface protein A (PspA) and PspC of Streptococcus pneumoniae are surface virulence proteins that interfere with complement deposition and elicit protective immune responses. The C-terminal halves of PspA and PspC have some structural similarity and contain highly cross-reactive proline-rich (PR) regions. In many PR regions of PspA and PspC, there exists an almost invariant nonproline block (NPB) of about 33 amino acids. Neither the PR regions nor their NPB exhibit the alpha-helical structure characteristic of much of the protection-eliciting N-terminal portions of PspA and PspC. Prior studies of PspA and PspC as immunogens focused primarily on the alpha-helical regions of these molecules that lack the PR and NPB regions. This report shows that immunization with recombinant PR (rPR) molecules and passive immunization with monoclonal antibodies reactive with either NPB or PR epitopes are protective against infection in mice. PR regions of both PspA and PspC were antibody accessible on the pneumococcal surface. Our results indicate that while PspA could serve as a target of these protective antibodies in invasive infections, PspC might not. When antibody responses to rPR immunogens were evaluated by using flow cytometry to measure antibody binding to live pneumococci, it was observed that the mice that survived subsequent challenge produced significantly higher levels of antibodies reactive with exposed PR epitopes than the mice that became moribund. Due to their conservation and cross-reactivity, the PR regions and NPB regions represent potential vaccine targets capable of eliciting cross-protection immunity against pneumococcal infection.

FIG. 1.

Regions of whole PspA (top) and portions of the full protein used in the constructs (bottom; see labels on the left) used in this paper. Amino acid sequences to the right of the construct diagrams are distinguished by region as follows: unbolded amino acids represent PspA sequence outside the proline-rich region, bolded amino acids represent proline-rich regions, underlined amino acids represent the NPB within a PR region, and boxed amino acids represent a hypothesized epitope for MAb PR-5C4.7. Diagrams in this figure are not drawn to scale.

FIG. 2.

Reactivity of MAb to PR regions. Western blot of PR recombinant proteins reacted with MAb to PR regions (upper panels). Flow cytometry analysis of binding of MAb to pneumococcal strain D39 and its ΔpspA, ΔpspC, and ΔpspA ΔpspC mutants (lower panels). The ΔpspA ΔpspC mutant bound significantly less antibody than the wild type for all three MAb that bound the wild type (P < 0.0001 by ANOVA). Results for the Dunnett's multiple-comparison test against the wild-type control are as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Comparisons to the ΔpspA ΔpspC double mutant are as follows: ⁺, P < 0.05; ⁺⁺, P < 0.01; and ⁺⁺⁺, P < 0.001.

FIG. 3.

Active immunization with PR recombinant proteins protects against i.v. infection with pneumococcal type 3 strain 3JYP2670. CBA/N mice immunized with Imject alum control, vector tag protein, PR+NPB, PR-NPB, or NPB were infected i.v. with type 3 strain 3JYP2670 and monitored for the number of days to moribundity. Mice plotted as “Alive” were still not moribund at 21 days postinfection. None of the “Alive” mice showed ruffled fur, a hunched back, or any other signs of illness. All three proteins were found to protect significantly against infection compared to a pool of the alum and two vector controls (for Kruskal Wallis, P < 0.0001; P values were <0.001 [***] and <0.05 [*] compared to the pooled control using Dunn's multiple-comparison post hoc test). None of the control groups protected significantly more than any of the others in a separate post hoc analysis. The sequence of the PR region of infection strain 3JYP2670 PspA is distinguished by regions as in Fig. 1.

FIG. 4.

Surface binding of MAb (bars) and passive protection against infection (symbols) for pneumococcal challenge strain WU2. Monoclonal antibodies for each panel are listed on the x axis, and the sequence of the single PR region in the PspA of strain WU2 is given below. For each antibody, the log binding to the infection strain determined by flow cytometry is indicated as a bar graph along the left y axis, and the number of days to reach moribundity is indicated as a dot plot along the right y axis. During challenge, mice were injected with antibody and infected i.v. 1 h later. Two MAb, PR-1A4.7 and PR-6A5.12, protected significantly compared to the control group that received no antibody (Kruskal Wallis P value, 0.0002; **, P < 0.01 using Dunn's multiple-comparison test to the control). MAb 2A4, which interacts with the alpha-helical region of family 1 PspA, was used as a positive control. The protective MAb (PR-1A4.7, PR-6A5.12, and 2A4) bound significantly more strongly to the infection strain (WU2) than did the nonprotective MAb (PR-5C4.7) or the saline (no-antibody) control (ANOVA P value, <0.001. ***, P < 0.01 compared to nonprotective strains using Tukey's post hoc test).

FIG. 5.

Surface binding of MAb (bars) and passive protection against infection (symbols) for pneumococcal challenge strain BG12730. Monoclonal antibodies for each panel are listed on the x axis, and the sequence of the PR regions in the PspA and the PspC of strain BG12730 is given below the x axis. Mice indicated with solid symbols were scored as moribund and euthanized due to neurologic symptoms. Mice indicated with open symbols were scored as moribund and euthanized due to a surface temperature of <24°C. For each antibody, the binding to the infection strain determined by flow cytometry is indicated as a bar graph along the left y axis, and the number of days to reach moribundity is indicated as a dot plot along the right y axis. During challenge, mice were injected with antibody and infected i.v. 1 h later. In this challenge, MAb PR-5C4.7 protected significantly well compared to the control group that received no antibody (Kruskal Wallis P value, 0.01; **, P < 0.01 using Dunn's multiple-comparison test to the control). Monoclonal antibody 2A4, which does not bind the PspA of this strain, was included as a negative control.