Immunization of mice with single PspA fragments induces antibodies capable of mediating complement deposition on different pneumococcal strains and cross-protection

Abstract

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal alpha-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

FIG. 1.

Schematic diagram of the PspA fragments. The whole PspA molecule, containing the N-terminal α-helical domain (A and B), the proline-rich region (C), and the choline-binding domain, is shown on the top. The recombinant fragments PspA1, PspA2, PspA3, PspA4, PspA4AB, PspA4Pro, PspA5, and Psp3-NonPro are represented with the distinct domains. P indicates the proline blocks, and NP indicates the NonPro block. CDR, clade-defining region.

FIG. 2.

Cross-reactivity of sera against PspA. Sera from four BALB/c mice immunized with PspA1, PspA2, PspA3, PspA4, or PspA5 were collected after the second immunization and analyzed individually by ELISA against each PspA fragment. The results are shown as the log₁₀ of the titer. An asterisk indicates a statistically significant difference from animals immunized with alum (Student's t test, P ≤ 0.05). Results are representative of two experiments using sera from independent immunizations.

FIG. 3.

Binding of antibodies and complement deposition in the presence of the different anti-PspA sera. Sera from mice immunized with PspA from Fam1 (PspA1 or PspA2) (A and C) or from Fam2 (PspA3, PspA4, or PspA5) (B and D) were tested for the ability to bind (1% serum, A and B) and to mediate deposition of C3 (10% serum, C and D) on S. pneumoniae strains bearing PspA proteins from clades 1 to 5. Serum from mice immunized with alum was used as a control for each strain and is represented by the gray area in each graph. Results are shown as fluorescence intensity histograms, and the median fluorescence intensity is indicated for each sample. Results are representative of two experiments using sera from independent immunizations.

FIG. 4.

Binding of antibodies against the PspA4 and PspA3 constructs. Sera from mice immunized with PspA4AB, PspA4Pro, and PspA4 (A) or PspA3 and PspA3-NonPro (B) were tested for the ability to bind (1% serum) to S. pneumoniae strains bearing PspA proteins from clade 3 (M10, lacks NonPro) and clade 5 (ATCC 6303, has NonPro). Serum from mice immunized with alum was used as a control for each strain and is represented by the gray area in each graph. Results are shown as fluorescence intensity histograms, and the median fluorescence intensity is indicated for each sample. Results are representative of two experiments using sera from independent immunizations. FITC, fluorescein isothiocyanate.