Immunogenicity of a live recombinant Salmonella enterica serovar typhimurium vaccine expressing pspA in neonates and infant mice born from naive and immunized mothers

Abstract

We are developing a Salmonella vectored vaccine to prevent infant pneumonia and other diseases caused by Streptococcus pneumoniae. One prerequisite for achieving this goal is to construct and evaluate new recombinant attenuated Salmonella vaccine (RASV) strains suitable for use in neonates and infants. Salmonella enterica serovar Typhimurium strain chi9558(pYA4088) specifies delivery of the pneumococcal protective antigen PspA and can protect adult mice from challenge with S. pneumoniae. This strain is completely safe for oral delivery to day-old and infant mice. Here we assess the colonizing ability, immunogenicity, and protective efficacy of chi9558(pYA4088) in neonatal mice. Colonization was assessed in mice 0, 2, 4, or 7 days of age after oral inoculation. In the presence of maternal antibodies, the colonization of lymphoid tissues was delayed, but the immune responses were enhanced in mice born to immunized mothers. Both oral and intranasal routes were used to assess immunogenicity. All orally or intranasally immunized neonatal and infant mice born to either immunized or naïve mothers developed PspA-specific mucosal and systemic immune responses. Mice born to immunized mothers produced higher titers of PspA-specific antibodies in the blood and mucosa and greater numbers of PspA-specific interleukin-4 (IL-4)-secreting cells than mice born to naïve mothers. More importantly, mice born to immune mothers showed a significant increase in protection against S. pneumoniae challenge. These results suggest that strain chi9558(pYA4088) can circumvent some of the limitations of the immature immune system in neonatal and infant mice, generating enhanced protective immune responses in the presence of maternal antibodies.

FIG. 1.

Distribution of S. Typhimurium strain χ9558(pYA4088) in tissues of newborn mice born to naïve or immunized mothers. Groups of pups were orally inoculated with χ9558(pYA4088) on the day (d) after birth indicated in the key. For mice born to naïve mothers, the doses were 1.4 × 10⁸ CFU for 0-day-old mice, 1.6 × 10⁸ CFU for 2-day-old mice, 3.0 × 10⁸ CFU for 4-day-old mice, and 3.5 × 10⁸ CFU for 7-day-old mice. For mice born to immunized mothers, the doses were 1.5 × 10⁸ CFU for 0-day-old mice, 1.5 × 10⁸ CFU for 2-day-old mice, 2.0 × 10⁸ CFU for 4-day-old mice, and 1.0 × 10⁸ CFU for 7-day-old mice. Significant differences between results obtained from mice born to naïve or immunized mothers are indicated (*, P < 0.01; **, P < 0.05). Tissue samples were taken from 3 mice/group on days 3 and 7 (3d and 7d) after inoculation. The results from three experiments are summarized.

FIG. 2.

ELISA measurements of serum IgG and mucosal IgA responses in immunized mice. (A and B) Serum IgG responses against rPspA (A) and S. Typhimunium LPS (B) were measured using pooled sera from neonates and infants born to either naïve (N) or immunized (I) mothers. (C) Mucosal IgA responses against rPspA were measured in pooled vaginal washes. Mice were immunized orally with either χ9558(pYA4088) (pspA) or χ9558(pYA3493) (control) or were mock immunized with BSG on either day 7 (7d) or day 21 (21d) after birth. Only mice from naïve mothers were inoculated with χ9558(pYA3493). Mice were boosted 3 and 6 weeks after the primary immunization. Error bars represent differences between triplicate wells. Significant differences between groups are indicated (*, P < 0.05; **, P < 0.01). No immune responses to PspA were detected in mice immunized with χ9558(pYA3493). No antibody to PspA or LPS was detected in mice inoculated with buffer only or in preimmune sera from vaccinated mice (reciprocal titer, <1:50).

FIG. 3.

Serum IgG2a and IgG1 responses to rPspA measured by ELISA. The data represent IgG2a and IgG1 subclass antibody levels to rPspA in pooled sera from neonates and infants orally immunized with χ9558(pYA4088) at various times after immunization. Error bars represent differences between triplicate wells. Differences between IgG1 and IgG2a responses are indicated (*, P < 0.05; **, P < 0.01).

FIG. 4.

PspA-specific cytokine stimulation in mice immunized with χ9558(pYA4088) and in nonimmunized mice. Numbers of IFN-γ (A)- and IL-4 (B)-producing cells were determined by ELISPOT assays. Splenectomies were performed on euthanized mice 7 days after the final immunization. Mice mock immunized with BSG were included as controls. Splenocytes were harvested from 6 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 expressed as ELISPOTs per million splenocytes minus any background ELISPOTs from unpulsed cells from mock-immunized controls. Significant differences between groups are indicated (**, P < 0.01).

FIG. 5.

Oral immunization with χ9558(pYA4088) protects BALB/c mice against i.p. challenge with S. pneumoniae WU2. The survival of orally immunized or nonimmunized mice after intraperitoneal challenge with 2 × 10³ CFU of S. pneumoniae WU2 4 weeks after the final immunization is shown. Each experiment was performed twice with similar results, and the data have been pooled for presentation and analysis. All vaccine groups were significantly different from the vector control [χ9558(pYA3493)] and the PBS control (P < 0.01). **, P < 0.01 for survival of infants born to naïve mothers compared to that of infants born to immunized mothers; *, P < 0.05 for survival of neonates born to naïve mothers compared to that of neonates born to immunized mothers.