PspA family fusion proteins delivered by attenuated Salmonella enterica serovar Typhimurium extend and enhance protection against Streptococcus pneumoniae

Abstract

Pneumococcal surface protein A (PspA) is highly immunogenic and can induce a protective immune response against pneumococcal infection. PspA is divided into two major families based on serological variability: family 1 and family 2. To provide broad protection, PspA proteins from pneumococcal strains Rx1 (family 1) and EF5668 (family 2) were combined to form two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. Each protein was fused to a type II secretion signal and delivered by a recombinant attenuated Salmonella vaccine (RASV). Both PspA/Rx1-EF5668 and PspA/EF5668-Rx1 were synthesized in the RASV and secreted into the periplasm and supernatant. The fusion proteins reacted strongly with both anti-PspA/Rx1 and anti-PspA/EF5668 antisera. Oral immunization of BALB/c mice with RASV synthesizing either PspA fusion protein elicited serum immunoglobulin G (IgG) and mucosal IgA responses against both families of PspA. Analysis of IgG isotypes (IgG2a and IgG1) indicated a strong Th1 bias to the immune responses to both proteins. Sera from mice immunized with RASV synthesizing PspA/Rx1-EF5668 bound to the surface and directed C3 complement deposition on representative strains from all five PspA clades. Immunization with RASV synthesizing either protein protected mice against intraperitoneal challenge with Streptococcus pneumoniae WU2 strain (family 1), intravenous challenge with S. pneumoniae 3JYP2670 strain (family 2), and intranasal challenge with S. pneumoniae A66.1 (family 1). The PspA/Rx1-EF5668 protein elicited significantly greater protection than PspA/EF5668-Rx1, PspA/Rx1, or PspA/EF5668. These results indicate an RASV synthesizing a PspA fusion protein representing both PspA families constitutes an effective antipneumococcal vaccine, extending and enhancing protection against multiple strains of S. pneumoniae.

FIG. 1.

Schematic diagram of PspA and PspA fusion proteins. At the top is the entire PspA molecule containing the N-terminal α-helical domain (region A), the proline-rich region (region B), the choline-binding domain (region C), and the C-terminal tail (region D). Each recombinant fusion protein is shown with its distinct domains.

FIG. 2.

Plasmid maps of pYA4432 and pYA4550, encoding pspA fusions Rx1-EF5668 and EF5668-Rx1, respectively.

FIG. 3.

PspA fusion proteins synthesized in S. Typhimurium χ9241. Cells were grown in LB broth at 37°C to an OD₆₀₀ of 0.8, and Western blots were performed on whole cells. GroEL was used as a marker to indicate loading of the same amount of protein sample in each lane. The expected molecular mass of PspA/Rx1 is 37 kDa, and that of PspA/EF5668 is 70 kDa, and both fusion proteins together have a mass of 107 kDa.

FIG. 4.

Kinetic analysis of anti-PspA serum IgG responses in mice. IgG responses to PspA (Rx1) and PspA (EF5668) were determined by ELISA at 2, 4, 6, and 8 weeks after the primary immunization (day 1) in sera from mice orally immunized with 1 × 10⁹ CFU of S. Typhimurium χ9241(pYA4088) (PspA/Rx1), χ9241(pYA4432) (PspA/Rx1-EF5668), χ9241(pYA4550) (PspA/EF5668-Rx1), or χ9241(pYA4326) (PspA/EF5668). Statistical significance was determined at week 8. All vaccine groups were significantly different from the vector and PBS controls (P < 0.05). Titers are the log₂ geometric mean titers of eight mice ± standard deviation.

FIG. 5.

Serum IgG2a and IgG1 responses to PspA/Rx1 (A) and PspA/EF5668 (B) and mucosal IgA responses to PspA/Rx1 and PspA/EF5668 (C). The IgG2a and IgG1 titers were determined in sera from BALB/c mice orally immunized with 1 × 10⁹ CFU of S. Typhimurium χ9241 harboring different pspA fusions at 6 weeks after primary immunization (day 1). IgA endpoint titers were determined in vaginal secretions taken on the same day as the sera. Titers are the log₂ geometric mean titer of eight mice ± standard deviation.

FIG. 6.

Binding of anti-PspA antibodies (A) and complement deposition (B) to the pneumococcal cell surface in the presence of sera from immunized and control mice. S. pneumoniae strains synthesizing PspAs of clades 1 (L81905), 2 (D39), 3 (EF3269), 4 (3JYP2670), and 5 (ATCC 6303) were incubated in the presence of sera from mice immunized with S. Typhimurium χ9241 synthesizing PspA/Rx1, PspA/EF5668, PspA/Rx1-EF5668, or PspA/EF5668-Rx1 and normal mouse serum. Cells were analyzed by flow cytometry. The percentage of fluorescent bacteria (greater than 1 fluorescence intensity unit) is shown for each sample. Serum from mice immunized with χ9241(pYA3493) was used as a control.

FIG. 7.

Protective efficacy of attenuated S. Typhimurium expressing pspA against S. pneumoniae challenge. BALB/c mice were orally immunized with 1 × 10⁹ CFU of S. Typhimurium strain χ9241 synthesizing the indicated PspA fusion proteins. Mice were challenged with 200 LD₅₀s virulent S. pneumoniae WU2 (2 × 10⁴ CFU) i.p. (A), with 100 LD₅₀s virulent S. pneumoniae 3JYP2670 (4 × 10⁵ CFU) i.v. (B), or with 20 LD₅₀s of virulent S. pneumoniae strain A66.1 (1 × 10⁸ CFU) i.n. (C) at week 8 after immunization. Mortality was monitored for 3 weeks. No change occurred after 9 days postpneumococcal challenge. The numbers in brackets represent surviving mice/total mice. All vaccine groups were significantly different from the χ9241(pYA3493) vector control and PBS controls (P < 0.01). For panels A and B, groups with different letters are significantly different. Where a is different from b or c, P is <0.01; where b is different from c, P is <0.05.