Live recombinant Salmonella Typhi vaccines constructed to investigate the role of rpoS in eliciting immunity to a heterologous antigen

Abstract

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (chi9639 and chi9640) were derived from the rpoS mutant strain Ty2 and one (chi9633) from the RpoS(+) strain ISP1820. In chi9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS(+) vaccines induced a balanced Th1/Th2 immune response while the RpoS(-) strain chi9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS(+) strain chi9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, chi9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.

Figure 1. Genealogy of the RASTyV strains construction.

This figure illustrates the steps used to construct S. Typhi vaccine vector strains χ9633, χ9639 and χ9640. Beginning at the top of the figure with parent strains ISP1820 and Ty2, individual mutations or other genetic modifications were introduced using suicide plasmids introduced either by conjugation or transduction. In one case, the mutation was introduced using the λ red method.

Figure 2. Protein expression and secretion in ISP1820 derivative χ9633 carrying either pYA4088 or pYA3493.

(A) Synthesis of proteins encoded by arabinose-regulated genes. Cells were grown in NB with arabinose (Lane 0) and then diluted 1∶10 into fresh NB without arabinose every 3.3 generations. This process was repeated 6 times (∼20 generations) (Lane 1–6). Synthesis of Crp, Fur, LacI and PspA was detected by western blot. GroEL was used as a control. (B) Location of PspA in χ9633(pYA4088) cell fractions from cells grown in the absence of arabinose and detected by western blot. σ⁷⁰ was used as a control to detect leakage of cytoplasmic contents into other fractions.

Figure 3. Sensitivity of RASTyV strains to transient deoxycholate exposure.

S. Typhi strains (either RASTyV or wild-type) were diluted to a concentration of 1×10⁹ CFU/ml and transiently challenged with varying concentrations of sodium deoxycholate for 2 hours at 37°C. Survival of the S. Typhi strains following challenge was assessed by plating on LB agar + 0.2% arabinose. The Ty2-derived RASTyV strains were significantly more sensitive than wild-type to all concentrations of deoxycholate *, all parental strains different from their vaccine derivatives, P<0.05; ** 2 of the 3 parent strains different from their vaccine derivatives.

Figure 4. Survival of RASTyV strains in chlorinated tap water and raw sewage and untreated surface water.

(A) Survival of RASTyV strains in chlorinated water. 1 ml of S. Typhi containing 1×10⁹ CFU was added to 19 ml of chlorinated water and viability was assessed by spread plating various dilutions at 0, 10, 30 and 60 min. (B) Survival of RASTyV strains in raw sewage. S. Typhi strains were inoculated into triplicate 20 ml sewage samples to a concentration of 1×10⁸ CFU/ml. Viability of the Salmonella strains was assessed on days 0, 1, 3, 7 and 10 after inoculation. (C) Survival of RASTyV strains in untreated surface water. S. Typhi strains were inoculated into triplicate 20 ml water samples to a concentration of 1×10⁸ CFU/ml. Viability of the Salmonella strains was assessed on days 0, 1, 3, 7 and 10 after inoculation. For B and C, *, all parental strains different from their vaccine derivatives, P<0.05; ** 2 of the 3 parent strains different from their vaccine derivatives.

Figure 5. Survival of RASTyV strains in human blood, peripheral human mononuclear cells, and guinea pig complement.

The bactericidal effects of (A) active whole blood and (B) heat-treated blood were compared by incubating 1×10⁶ CFU of each strain in 1.5 ml normal or heat-inactivated human whole blood. Bacterial survival was measured by spread plating at the indicated times after inoculation. **, P<0.01, for each RASTyV compared to its wild-type parent at 18 h. (C) S. Typhi survival in peripheral human mononuclear cells at 1 h, 4 h and 24 h after inoculation with 5×10⁶ CFU. **, P<0.01, for each RASTyV compared to its wild-type parent at 24 h, and significant differences between Ty21a and RASTyV are indicated. The assay was performed in duplicate and was repeated at least 3 times using blood from different individuals. The limit of detection was less than 10 CFU/ml. (D) S. Typhi survival in guinea pig complement three hours after inoculation with 1×10⁶ CFU. **, P<0.01, for the RASTyV strain compared to its wild-type parent at 3 h. Data shown are the arithmetic means of triplicate samples.

Figure 6. Distribution of RASTyV strains in tissues of newborn mice.

The numbers of Salmonella bacteria in the intestines (A), liver (B) and spleen (C) at 3 and 7 days after oral inoculation of newborn mice with 1±0.2×10⁹ CFU of the indicated strains are plotted. Bars represent the arithmetic mean ± standard deviations from two separate experiments with 5 mice per group. *, P<0.05; **, P<0.01 for CFU counts in the indicated tissues for vaccine strains compared to their wild-type parent strains. The assays were performed twice.

Figure 7. Immune responses against PspA, S. Typhi LPS and S. Typhi OMP in immunized mice.

Serum IgG responses against rPspA (A), S. Typhi LPS (B), and S. Typhi OMP (C), and mucosal IgA responses to rPspA (D) were measured by ELISA using pooled sera and vaginal washes from BALB/c mice intranasally immunized with the indicated strains carrying either plasmid pYA3493 (control) or pYA4088 (PspA). Mice were boosted at week 6. ELISAs were performed twice with identical results. Significant differences were indicated *, P<0.05; **, P<0.01. No immune responses were detected to any antigen tested in mice immunized with only BSG or in preimmune sera from vaccinated mice (reciprocal titer <1∶50).

Figure 8. Serum IgG1 and IgG2a responses to rPspA measured by ELISA.

Anti-rPspA IgG1 and IgG2a titers in pooled sera from BALB/c mice intranasally immunized with the indicated RASTyV strains at various times. Mice were boosted at week 6. The ratios of IgG1∶IgG2a at 8 weeks were 1∶1 for χ9633(pYA4088) (A) and χ9640(pYA4088) (C) immunized mice respectively; and 8∶1 for χ9639(pYA4088) (B) immunized mice. All ELISAs were performed twice with identical results.

Figure 9. Antigen-specific stimulation of IFN-γ (A) or IL-4 (B).

Splenectomies were performed on euthanized BALB/c mice 7 weeks after the primary immunization and one week after the boost. Buffer controls were also included. Splenocytes were harvested from 3 mice per group, and cells from each spleen were assayed in triplicate. Each symbol represents the results from a single well. The results from each well are presented as ELISPOTS per million splenocytes minus any background (≤4) ELISPOTS, from unpulsed mock controls. There were no significant differences between vaccine strains and the control group for secretion of IFN-γ. For secretion levels of IL-4, **, P<0.01 for χ9633(pYA4088) and χ9639(pYA4088) versus controls BSG group or empty vector group, *, P<0.05 for χ9640(pYA4088) versus BSG group or χ9640(pYA3493), and for χ9639(pYA4088) versus χ9640(pYA4088) as indicated.

Figure 10. Evaluation of protective efficacy.

Groups of eight 7-weekold mice were intranasally immunized twice at 6-week intervals with the indicated strains and challenged intraperitoneally with 1×10⁴ CFU of S. pneumoniae WU2 4 weeks later. The experiment was performed twice. Both experiments gave similar results, and the data have been pooled. The protection from each RASTyV strains was significantly different compared with control groups. *, P<0.05 for survival of mice immunized with χ9640(pYA4088) (Ty2 RpoS⁺) compared with survival of mice immunized with χ9633(pYA4088) (ISP1820).