Evaluation of the humoral immune response in mice orally vaccinated with live recombinant attenuated Salmonella enterica delivering a secreted form of Yersinia pestis PsaA

Abstract

Yersinia pestis PsaA is an adhesin that is synthesized inside macrophages. Here, we evaluated the immune profile of codon-optimized Y. pestis PsaA synthesized in a live recombinant attenuated Salmonella vaccine (RASV) strain chi9558. Oral immunization of BALB/c mice with chi9558(pYA3705) delivering a secreted form of PsaA, elicited a systemic PsaA-specific immunoglobulin G (IgG) response but offered limited protection against lethal challenge with the intranasally introduced Y. pestis CO92 strain. Our results suggest that appropriate fine-tuning of Y. pestis PsaA delivery by RASV could improve its protective role in curtailing plague colonization and infection.

Figure 1

A. Map of the pYA3705 plasmid. B. Immunoblot of total extract and supernatant of the S. enterica serovar Typhimurium χ9558 harboring pYA3705 with a PsaA-specific polyclonal rabbit antibody. The χ9558 strain was grown in LB with 0.2% mannose (+) and 0.2% arabinose (+) or without arabinose at 37°C, with shaking to an OD_600nm of 0.8. C. Immunoblot of an analogous total extract and supernatant of χ9558 harboring pYA3705 blotted with anti-σ⁷⁰ antibody. The σ⁷⁰ was used as a cytoplasmic marker and as a loading control.

Figure 2

Humoral immune responses to PsaA and SOMPs in mice orally immunized with Salmonella strain χ9558(pYA3705) (PsaA) and χ9558(pYA3342) (control) measured by ELISA. A. Serum IgG total response to PsaA. B. S. Typhimurium anti-SOMP IgG. C. Subclasses (gG1 and IgG2a) of anti-PsaA serum. D. Mucosal IgA response to PsaA in vaginal secretions. The data represent the endpoint of antibodies in pooled pre-immune serum (PI) and in immunized mouse serum at 22 and 32 days after the first immunization. Error bars represent variations between triplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey’s post-test (*P < 0.05; **P < 0.01; *** < 0.001).

Figure 3

Survival percentage of immunized BALB/c mice after Y. pestis challenge. Mice were orally immunized on days 0 and 9 with χ9558(pYA3342) (vector control), χ9558(pYA3705) (synthesizing PsaA) or BSG (control). A. Mice were subcutaneously (s.c.) challenged with 4.49 × 10² CFU (LD) or 5.63 × 10³ CFU (HD) of virulent Y. pestis CO92 at 28 days after the second immunization. Survival was monitored for 2 weeks after bubonic challenge. B. Mice were intranasally (i.n.) challenged with 4.1 × 10³ CFU (LD) or with 4.4 × 10⁴ CFU (HD) of virulent Y. pestis CO92 at 28 days after the second immunization. Survival was monitored for 2 weeks after pneumonic challenge.

Figure 4

Humoral immune responses to PsaA and SOMPs in immunized mice surviving the post-intranasal challenge with Y. pestis. The humoral immune responses were measured by ELISA. Mice were orally immunized with Salmonella strain χ9558(pYA3705) (PsaA) and χ9558(pYA3342) (control) on days 0 and 9 and intranasally (i.n.) challenged with LD or HD of Y.pestis at 28 days after the second immunization. A. Serum IgG total response to PsaA. B. Subclasses (IgG1 and IgG2a) of anti-PsaA serum. C. S. Typhimurium anti-SOMP IgG. The data represent the endpoint of antibodies in pooled pre-immune serum (PI) and in immunized mice that survived i.n. challenge with virulent Y. pestis. Error bars represent variations between triplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey’s post-test (*P < 0.05; **P < 0.01; *** < 0.001).