Fine-tuning synthesis of Yersinia pestis LcrV from runaway-like replication balanced-lethal plasmid in a Salmonella enterica serovar typhimurium vaccine induces protection against a lethal Y. pestis challenge in mice

Abstract

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

FIG. 1.

(A) Amino acid sequence of the chimeric LcrV196 encoded by bla_SS-lcrV196-bla_CT. The nucleotide sequences above the underlined amino acid residues correspond to the endonuclease restriction sites generated to construct the chimeric bla_SS-lcrV196-bla_CT gene and to clone the NheI fragment containing it in pYA4534 to yield pYA4535. The β-lactamase signal peptide, the 12 amino acid residues of the mature β-lactamase N-terminal region, and the 21 amino acid residues of the β-lactamase carboxy-terminal region are indicated in boldface type. The vertical arrow indicates the β-lactamase signal peptide cleavages site. The asterisks above the italicized amino acid residues indicate the codons optimized for high-level expression in lcrV196. (B) Map of plasmid pYA4535.

FIG. 2.

(A) Shift from low-copy-number to runaway-like replication of pYA4534. The supercoiled plasmid DNA was obtained from χ9447(pYA4534) grown in the minimal medium NB or LB broth with several arabinose concentrations, separated by electrophoresis on a 1.2% agarose gel, and stained with ethidium bromide. (B) Immunoblotting of the cell fraction profile of Y. pestis LcrV196 synthesis in RASV χ9447(pYA4535). S. Typhimurium χ9447(pYA4535) was grown in LB broth with 0.02% arabinose or without arabinose until the culture reached an OD₆₀₀ of 0.8, and the total extracts (TE), supernatant (SN), or cell fractions from the membranes (M), cytoplasm (C), and periplasm (P) were obtained as described in Materials and Methods, separated by SDS-PAGE, and immunoblotted. Rabbit serum against LcrV was used to detect LcrV196. (C) Immunoblot of an analogous cell fraction profile of χ9447(pYA4535), with the mouse monoclonal antibody anti-σ⁷⁰ used as a loading marker. All the experiments were performed independently three times.

FIG. 3.

Plasmid stability of χ9447(pYA4534) and χ9447(pYA4535) grown in LB broth (0.02% arabinose and 0.2% mannose) under selective and nonselective conditions (presence of DAP). The proportions of cells retaining Asd⁺ plasmids were determined for each culture repeated for five consecutive days (approximately 50 generations), and the cultured cells from each day were diluted, spread onto LB agar plates (0.2% arabinose, 0.2% mannose, and 50 μg/ml X-gal) with 50 μg/ml DAP, and grown overnight. Afterwards, 100 colonies from each vaccine construct and from each day were picked and patched onto LB agar plates (0.2% mannose, 0.2% arabinose, and 50 μg/ml X-gal) with or without 50 μg/ml DAP. (A) Percentage of clones retaining plasmids from each culture. (B) Supercoiled plasmid DNA was obtained from χ9447(pYA4535) grown in LB broth with several arabinose concentrations or without arabinose until the culture reached an OD₆₀₀ of 0.8, separated by electrophoresis in a 1.2% agarose gel, and stained with ethidium bromide. (C) Immunoblot of Y. pestis LcrV196 protein synthesis from total extract of RASV χ9447(pYA4535). Ten microliters of 1 ml of culture, as described in Materials and Methods, was loaded, separated by SDS-PAGE, and immunoblotted. Rabbit serum against LcrV was used to detect LcrV196. (D) Immunoblot of an analogous total extract profile of χ9447(pYA4535), with mouse monoclonal antibody anti-σ⁷⁰ used as loading marker. Cultures were grown in the presence of DAP.

FIG. 4.

Growth curves, culture cell viability, and β-galactosidase activities of χ9447(pYA4534) (vector control) and χ9447(pYA4535) (expressing bla_SS-lcrV196-bla_CT) inoculated into NB medium. Strains χ9447(pYA4534) (triangles) and χ9447(pYA4535) (circles) were inoculated into medium containing 0.02% arabinose (ara) and without arabinose (inverted triangles and squares, respectively). (A) The OD₆₀₀ was measured to monitor the growth of the cultures each hour from 0 to 12 h and after each 24 h until the end of the experiment. (B) Dilutions were plated onto LB agar medium with 0.2% arabinose, 0.2% mannose, and 50 μg/ml X-gal to assess the cell viability of the cultures. (C) The β-galactosidase activities were determined each hour during the first 12 h of growth and after each 24 h to the end of the experiment. The values are means of results from three independent experiments. Error bars indicate standard deviations.

FIG. 5.

Anti-LcrV196 and anti-SOMP serum IgG and vaginal IgA responses in mice. BALB/c mice were orally immunized at days 0 and 9 with 2 × 10⁹ CFU of χ9447(pYA4534) (control) or χ9447(pYA4535) synthesizing LcrV196. The IgG titers were measured by ELISA in preimmune serum (PI) and immunized mice at 2 weeks (2W) and 3 weeks (3W) after the second immunization. Orally immunized mice were challenged with Y. pestis strain CO92 by the subcutaneous (s.c.) route with a low dose (LD) of 4.49 × 10² CFU (s.c. LD) or a high dose (HD) of 5.63 × 10³ CFU (s.c. HD) or by the intranasal (i.n.) route with a low dose of 4.1 × 10³ CFU (i.n. LD) or a high dose of 4.4 × 10⁴ CFU (i.n. HD) at 4 weeks after the second immunization, and the IgG titers were measured after 2 weeks for mice surviving postchallenge. Pooled sera were made up from seven and six surviving mice challenged by the s.c. and i.n. routes, respectively. (A) Serum IgG total response to LcrV196. ***, P < 0.001 for comparison with mice immunized with vector control strain χ9447(pYA4534) or preimmune serum; *, P < 0.05 for comparison with χ9447(pYA4535)-immunized mice and post-i.n. challenged mice. (B) Serum IgG total response to SOMP. ***, P < 0.001 for comparison with preimmune serum; *, P < 0.05 for comparison with surviving mice post-s.c. challenge. (C) Subclasses IgG1 and IgG2a in serum against LcrV196. ***, P < 0.001 for comparison with mice immunized with vector control strain χ9447(pYA4534) or preimmune serum and comparison between titers of IgG1 and IgG2a. (D) Mucosal IgA response to LcrV196 in vaginal secretions in preimmune and immunized mice. *, P < 0.05 for comparison with all other vaccine groups. The data represent end points of antibodies in pooled sera from eight mice immunized at the indicated times after immunization or from mice surviving postchallenge. Error bars represent variations between duplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey's posttest.

FIG. 6.

Antigen-specific stimulation of cytokine responses in spleen cells from Salmonella χ9447(pYA4535)-vaccinated mice. Antigen-specific IFN-γ, IL-4, and IL-10 cytokine-forming T cells were determined by ELISPOT (spot-forming units [SFU]), and LcrV196-specific IFN-γ, IL-4, and IL-10 cytokine secretions were quantified by ELISA. BALB/c mice were immunized with χ9447(pYA4534) (control), pYA4535(LcrV196), or BSG dosed at days 0 and 9. Three weeks after the second immunization, spleen cells from three BALB/c mice per group were harvested and pooled. (A to C) Cells were restimulated for 24 h (for IFN-γ) and 48 h (for IL-4 and IL-10) with 1 μg/well of recombinant LcrV or medium for ELISPOT analyses. (D and E) For ELISAs, pools of 5 × 10⁶ spleen cells/well from each group of controls and immunized mice were restimulated with 10 μg/well of recombinant LcrV or medium for 48 h, and the concentrations of IFN-γ, IL-4, and IL-10 in the supernatants were measured. The results are presented as ELISPOTs per million splenocytes minus background ELISPOTs from unpulsed mock controls. ***, P < 0.001 for comparison of the Salmonella χ9447(pYA4535) vaccine group with χ9447(pYA4534) vector control and the BSG group for antigen-specific IFN-γ by ELISPOT analysis. *, P < 0.05 for comparison of the Salmonella χ9447(pYA4535) vaccine group with χ9447(pYA4534) and BSG groups for antigen-specific IL-4 and IL-10 by ELISPOT; **, P < 0.01 for comparison of the Salmonella χ9447(pYA4535) vaccine group with the χ9447(pYA4534) and BSG groups for antigen-specific IFN-γ and IL-10 secretion levels by ELISA. Error bars represent variations between quadruplicate or triplicate wells by ELISA and ELISPOT assay, respectively, The statistical significance was calculated by one-way ANOVA and Tukey's posttest.

FIG. 7.

Oral immunization of BALB/c mice with χ9447(pYA4535) carrying lcrV196 confers protection against bubonic plague challenge. Mice were orally immunized on days 0 and 9 with χ9447(pYA4534) (vector control), χ9447(pYA4535) (synthesizing LcrV196), or BSG (control). Mice were challenged s.c. with 4.49 × 10² CFU (LD) or 5.63 × 10³ CFU (HD) of virulent Y. pestis CO92 28 days after the second immunization. Survival was monitored for 2 weeks after bubonic challenge. Survival fractions obtained from vaccinated mice were compared with BSG-dosed mice, and significance was determined. **, P = 0.0016; *, P = 0.012.

FIG. 8.

Oral immunization of BALB/c mice with S. Typhimurium χ9447(pYA4535) expressing lcrV196 confers protection against pneumonic plague challenge. Mice were orally immunized on days 0 and 9 with 2 × 10⁹ CFU χ9447(pYA4534) (vector control), χ9447(pYA4535) (synthesizing LcrV196), or BSG (control). Mice were i.n. challenged with 4.1 × 10³ CFU (LD) or with 4.4 × 10⁴ CFU (HD) of virulent Y. pestis CO92 28 days after the second immunization. Survival was monitored for 2 weeks after pneumonic challenge. Survival fractions obtained from vaccinated mice were compared with BSG-dosed mice, and significance was determined. **, P = 0.0016.