Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine

Abstract

Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic alpha-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the beta-lactamase signal sequence in the multicopy Asd(+) pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd(+) vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Delta crp-28 and Delta asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant Salmonella. After a single oral immunization in BALB/c mice with 10(9) CFU of the recombinant Salmonella vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.

FIG. 1.

Reduced expression of Asd protein by deletion of the asd gene promoter region. SDS-PAGE was performed with cell lysates of S. enterica serovar Typhimurium χ4550 with pYA3333 (entire asd gene with P_asd, pBR ori), pYA3334 (entire asd gene with P_asd, pUC ori), pYA3342 (promoterless SD-asd gene, pBR ori), and pYA3341 (promoterless SD-asd gene, pUC ori). Standards are indicated to the left, and Asd protein (39 kDa) is indicated by an arrow. Lanes; 1, pYA3333; 2, pYA3334; 3, pYA3342; 4, pYA3341.

FIG. 2.

Asd⁺ antigen expression vectors. (A) Asd⁺ vector pYA3342. The map of pYA3342 and the nucleotide sequences of the P_trc promoter region and multicloning sites are shown. (B) Periplasmic secretion Asd⁺ vector pYA3493. A DNA fragment encoding the β-lactamase signal sequence and 12 amino acid residues of the N terminus of mature β-lactamase of plasmid pBR322 was positioned under the control of the P_trc promoter of the Asd⁺ vector pYA3342 (pBR ori). The map of pYA3493 and the nucleotide sequences of the P_trc promoter region, β-lactamase signal sequence (bla SS), and multicloning sites are shown. The P_trc sequences for −35, −10, and SD are indicated, and the translation start codon is in boldface. An arrow within the sequence indicates the signal peptidase cleavage site. Unique restriction enzyme sites in the multicloning site are indicated. 5ST1T2 is a transcriptional terminator.

FIG. 3.

Recombinant plasmid pYA3494 for PspA expression. (A) The PspA region used in this study. Functional domains of native PspA from S. pneumoniae (PspA_Rx1) are diagramed. Dotted box, leader sequence (31 amino acids [aa]); open box, immunodominant α-helical region (aa 1 to 288); hatched box, proline-rich region (aa 289 to 370); 10 gray boxes, choline-binding repeats (aa 371 to 571); black box, C terminus (aa 572 to 588). Dotted lines represent the limit of the rPspA region used in this study. Bioinformatical analyses of the rPspA for antigenic index and surface probability are presented. Analyses were performed with the Protean module of the Lasergene sequence analysis software. (B) Map of recombinant plasmid pYA3494. A 0.7-kb EcoRI-HindIII fragment of PCR-amplified DNA of pspA_Rx1 was cloned into the EcoRI and HindIII sites of pYA3493 (Fig. 2B). The cloned fragment included the immunogenic α-helical region of PspA including amino acids 3 through 257 of mature PspA (255 amino acids).

FIG. 4.

Subcellular location of expressed rPspA in S. enterica serovar Typhimurium. Subcellular fractions were prepared from S. enterica serovar Typhimurium χ8599(pYA3494) cells grown in LB broth at 37°C by the procedures described in Materials and Methods. Fractions equivalent to 30-μl volumes of the culture at an OD₆₀₀ of 0.8, except for supernatant fluids, were analyzed by SDS-PAGE, and the rPspA was detected by immunoblotting with PspA-specific monoclonal antibody Xi126 (33). β-Galactosidase and OmpC were used as fractionation controls for cytoplasmic and outer membrane fractions, respectively. Standards are indicated to the left. Lanes: 1, total cell lysate; 2, cytoplasm; 3, periplasm; 4, outer membrane; 5, concentrated supernatant (750 μl); 6, supernatant (10 μl).

FIG. 5.

Expression of rPspA in the S. enterica serovar Typhimurium vaccine strain. χ8501 harboring pYA3494 (specifying rPspA) or pYA3493 (vector control) was cultured in LB broth at 37°C. Total cells (equivalent to 7.5 × 10⁸ cells) and concentrated culture supernatants (Sup.) (equivalent to 750 μl of supernatant of cultures at an OD₆₀₀ of 0.8) were subjected to SDS-PAGE analysis. Left panel, Coomassie brilliant blue-stained gel. Right panel, immunoblot of the duplicated gel with PspA-specific monoclonal antibody Xi126. Molecular markers are indicated to the left. PspA proteins are indicated by arrows. Lanes 1 and 2, protein profiles of χ8501(pYA3493) and χ8501(pYA3494), respectively.

FIG. 6.

Serum IgG responses to S. enterica serovar Typhimurium LPS and SOMPs and to rPspA. The data represent IgG antibody levels induced in mice orally immunized with χ8501(pYA3493) (vector control) and χ8501(pYA3494) (expressing rPspA) at the indicated weeks after immunization. ELISA and data analysis are described in Materials and Methods. Arrows indicate sublethal i.v. infection with S. pneumoniae WU2.

FIG. 7.

Secretory IgA responses to S. enterica serovar Typhimurium LPS and SOMPs and to rPspA. The data represent anti-LPS, -SOMP, and -rPspA IgA antibody levels in vaginal secretions of BALB/c mice orally immunized with χ8501(pYA3493) (vector control) and χ8501(pYA3494) (expressing rPspA) at weeks 4, 6, 8, and 10 after immunization.

FIG. 8.

Serum IgG2a and IgG1 responses to S. enterica serovar Typhimurium LPS and SOMPs and to rPspA. The data represent IgG2a and IgG1 subclass antibody levels to Salmonella LPS and SOMPs and to rPspA in sera of BALB/c mice orally immunized with χ8501(pYA3493) (vector control) and χ8501(pYA3494) (expressing rPspA) at the indicated weeks after immunization. Arrows indicate sublethal i.v. infection with S. pneumoniae WU2. Anti-rPspA IgG2a and IgG1 responses of χ8501(pYA3493) (negative control) are not shown. The overall IgG2a/IgG1 ratios (means ± standard deviations) for each antigen are shown above the bars. Statistical analyses were performed with a paired Student t test to compare the IgG2a/IgG1 ratios for antigens. P values of <0.05 were considered significant. The results were as follows: (i) LPS for χ8501(pY3493) versus χ8501(pYA3494), P > 0.05; (ii) SOMPs for χ8501(pY3493) versus χ8501(pYA3494), P > 0.05; (iii) LPS for χ8501(pYA3493) versus SOMPs for χ8501(pYA3493), P < 0.05; (iv) LPS for χ8501(pYA3494) versus SOMPs for χ8501(pYA3494), P < 0.05; (v) PspA before versus after sublethal challenge of S. pneumoniae, P < 0.05; (vi) LPS for χ8501(pYA3494) versus PspA for χ8501(pYA3494), P > 0.05 (before challenge) and P < 0.05 (after challenge); (vii) SOMPs for χ8501(pYA3494) versus PspA for χ8501(pYA3494), P < 0.05 (before and after challenge).