A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

Abstract

SopB is a virulence factor of Salmonella encoded by SPI-5. Salmonella sopB deletion mutants are impaired in their ability to cause local inflammatory responses and fluid secretion into the intestinal lumen and also can enhance the immunogenicity of a vectored antigen. In this study, we evaluated the effects on immunogenicity and the efficacy of a sopB deletion mutation on two Salmonella enterica serovar Typhimurium vaccine strains with different attenuating mutations expressing a highly antigenic alpha-helical region of the Streptococcus pneumoniae surface protein PspA from an Asd(+)-balanced lethal plasmid. After oral administration to mice, the two pairs of strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens. The levels of antigen-specific serum immunoglobulin G (IgG) and mucosal IgA were higher in mice immunized with sopB mutants. Enzyme-linked immunospot assay results indicated that the spleen cells from mice immunized with a sopB mutant showed higher interleukin-4 and gamma interferon secretion levels than did the mice immunized with the isogenic sopB(+) strain. The sopB mutants also induced higher numbers of CD4(+) CD44(hi) CD62L(hi) and CD8(+) CD44(hi) CD62L(hi) central memory T cells. Eight weeks after primary oral immunization, mice were challenged with 100 50% lethal doses of virulent S. pneumoniae WU2. Immunization with either of the sopB mutant strains led to increased levels of protection compared to that with the isogenic sopB(+) parent. Together, these results demonstrate that the deletion of sopB leads to an overall enhancement of the immunogenicity and efficacy of recombinant attenuated Salmonella vaccine strains.

FIG. 1.

Western blot showing expression of PspA (Rx1) by different S. enterica serovar Typhimurium strains. Cell lysates of S. enterica serovar Typhimurium strains expressing PspA (Rx1) were subjected to SDS-PAGE, and the proteins transferred to a nitrocellulose membrane, which was subsequently probed with polyclonal antibody specific for PspA or GroEL. (A) Lane 1, χ9241(pYA3493); lane 2, χ9241(pYA3634); lane 3, χ9277(pYA3493); lane 4, χ9277(pYA3634); M, molecular-mass markers are labeled. (B) Lane 1, χ9373(pYA4088); lane 2, χ9373(pYA3493); lane 3, χ9402(pYA4088); lane 4, χ9402(pYA3493); M, molecular-mass markers are labeled.

FIG. 2.

Serum IgG responses to rPspA (A) and to SOMPs (B) as determined by ELISA. The data represent IgG antibody levels induced in mice orally immunized with χ9241(pYA3634), χ9241(pYA3493), χ9277(pYA3634), or χ9277(pYA3493) at the indicated weeks after immunization. (C) Serum IgG1 and IgG2a responses to rPspA. The data represent ELISA results determining IgG1 and IgG2a subclass antibody levels to rPspA in sera of BALB/c mice at the indicated weeks after immunization. (D) Vaginal-wash IgA responses to rPspA. The data represent ELISA results determining IgA antibody levels to rPspA in vaginal washes of BALB/c mice orally immunized with χ9241(pYA3634) (expressing rPspA) or χ9277(pYA3634) (expressing rPspA) at the indicated weeks after immunization. *, compared to χ9241(pYA3634)-immunized mice, the P value was <0.05; **, compared to χ9241(pYA3634)-immunized mice, the P value was <0.01.

FIG. 3.

Antigen-specific stimulation of IL-4 or IFN-γ production. Splenectomies were performed on BALB/c mice sacrificed at 8 weeks following vaccination; BSG controls were also included. Splenocytes were harvested from three mice per group, pooled, and plated onto a precoated antimouse IL-4 or IFN-γ MAb 96-well format at dilutions of 1 × 10⁶ (in triplicate) in RPMI medium containing IL-2 and PspA antigen as shown. Unstimulated (mock) splenocytes were also plated for each group. After 12 h at 37°C, ELISPOT analysis was performed (described in Materials and Methods). The results are presented as ELISPOTs per million splenocytes minus background ELISPOTs from unpulsed mock controls. The P value was <0.01 when comparing χ9241(pYA3634) with χ9277(pYA3634) for secretion levels of both IL-4 and IFN-γ. **, P < 0.01.

FIG. 4.

Flow cytometric analysis of polyclonal central memory T cells. Single-cell suspensions were prepared from pooled spleens derived from three mice per group and then stimulated for 12 h with rPspA antigen (5 μg/ml). Splenic T cells were gated on CD4 and CD8 markers separately and analyzed for the expression of the activation markers CD44 and CD62L. Data were derived from 100,000 events acquired from each sample. Representative results are shown from two experiments. Numbers are percentages of CD4⁺ CD44^hi CD62L^hi and CD8⁺ CD44^hi CD62L^hi cells.

FIG. 5.

(A) Serum IgG responses to rPspA. (B) Serum IgG responses to SOMPs. The data represent IgG antibody levels induced in mice orally immunized with χ9373(pYA4088) (expressing rPspA), χ9373(pYA3493), χ9402(pYA4088) (expressing rPspA), and χ9402(pYA3493) at the indicated weeks after immunization. (C) Serum IgG1 and IgG2a responses to rPspA. The data represent ELISA results determining IgG1 and IgG2a subclass antibody levels to rPspA in sera of BALB/c mice orally immunized with χ9373(pYA4088) (expressing rPspA) and χ9402(pYA4088) (expressing rPspA) at the indicated weeks after immunization. (D) Vaginal-wash IgA responses to rPspA. The data represent ELISA results determining IgA antibody levels to rPspA in vaginal washes of BALB/c mice orally immunized with χ9373(pYA4088) (expressing rPspA) and χ9402(pYA4088) (expressing rPspA) at the indicated weeks after immunization. *, compared to χ9373(pYA4088)-immunized mice, the P value was <0.05; **, compared to χ9373(pYA4088)-immunized mice, the P value was <0.01.