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, 80 (2), 815-31

Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis to Confer Protection Against Mycobacterium Tuberculosis

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Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis to Confer Protection Against Mycobacterium Tuberculosis

María Dolores Juárez-Rodríguez et al. Infect Immun.

Abstract

Live recombinant attenuated Salmonella vaccine (RASV) strains have great potential to induce protective immunity against Mycobacterium tuberculosis by delivering M. tuberculosis antigens. Recently, we reported that, in orally immunized mice, RASV strains delivering the M. tuberculosis early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10) antigens via the Salmonella type III secretion system (SopE amino-terminal region residues 1 to 80 with two copies of ESAT-6 and one copy of CFP-10 [SopE(Nt80)-E2C]) afforded protection against aerosol challenge with M. tuberculosis. Here, we constructed and evaluated an improved Salmonella vaccine against M. tuberculosis. We constructed translational fusions for the synthesis of two copies of ESAT-6 plus CFP-10 fused to the OmpC signal sequence (OmpC(SS)-E2C) and amino acids 44 to 338 of antigen 85A (Ag85A(294)) flanked by the signal sequence (SS) and C-terminal peptide (CT) of β-lactamase (Bla(SS)-Ag85A(294)-Bla(CT)) to enable delivery via the Salmonella type II secretion system. The genes expressing these proteins were cloned as an operon transcribed from P(trc) into isogenic Asd(+)/MurA(+) pYA3681 lysis vector derivatives with different replication origins (pBR, p15A, pSC101), resulting in pYA4890, pYA4891, and pYA4892 for SopE(Nt80)-E2C/Ag85A(294) synthesis and pYA4893 and pYA4894 for OmpC(SS)-E2C/Ag85A(294) synthesis. Mice orally immunized with the RASV χ11021 strain engineered to display regulated delayed lysis and regulated delayed antigen synthesis in vivo and harboring pYA4891, pYA4893, or pYA4894 elicited significantly greater humoral and cellular immune responses, and the RASV χ11021 strain afforded a greater degree of protection against M. tuberculosis aerosol challenge in mice than RASVs harboring any other Asd(+)/MurA(+) lysis plasmid and immunization with M. bovis BCG, demonstrating that RASV strains displaying regulated delayed lysis with delayed antigen synthesis resulted in highly immunogenic delivery vectors for oral vaccination against M. tuberculosis infection.

Figures

Fig 1
Fig 1
Regulated delayed lysis and regulated delayed synthesis of heterologous antigens. (A) Arabinose-regulated araC PBAD activator promoter. In the presence of arabinose, the AraC protein changes its conformation and forms a dimer that binds the I1 and I2 sites, and then Crp and RNA polymerase bind the complex and activate the transcription of the araBAD genes. (B) In the absence of arabinose, two distal AraC molecules interact and one of them binds the O2 site and the other the I1 site, which generates a DNA loop that represses the transcription from the araC PBAD promoter. (C) In the RASV strains with regulated delayed lysis and synthesis of heterologous antigens, in the presence of arabinose, the transcription of the genes under the araC PBAD promoter is induced on the chromosome and on the plasmid, for the biosynthesis of the cell wall and the synthesis of the repressor proteins C2 and LacI. LacI negatively represses the expression from the Ptrc promoter. (D) In the animal tissues, arabinose is not available, and the synthesized proteins are diluted during each cell division, resulting in the synthesis of the heterologous antigens and bacterial cell lysis.
Fig 2
Fig 2
Synthesis of Ag85A294 in RASV strain χ9879. Immunoblot of whole-cell lysates (total extract) and supernatant fraction (20× concentrated) of χ9879(pYA3941) synthesizing M. tuberculosis Ag85A294 protein. RASV strains were grown as described in Materials and Methods. The total extract and supernatant fractions were separated by SDS-PAGE and immunoblotted with rabbit polyclonal anti-Ag85A antibody to detect Ag85A294. The numbers at the left of the blot are molecular masses. The unprocessed form of Ag85A (BlaSS-Ag85A294-BlaCT) is indicated by the arrow, and the mature form (Ag85A294) is shown by the asterisk; these forms have the expected molecular masses of 38 kDa and 36 kDa, respectively. Lanes: 1, molecular mass markers (MM); 2, χ9879(pYA3941).
Fig 3
Fig 3
Synthesis of Ag85A294 in RASV strain χ11021. Immunoblot of whole-cell lysates and supernatant fractions (20× concentrated) of χ11021 strains containing plasmids producing Ag85A294. (A) Strains were grown in LB broth with 0.2% arabinose, to repress chimeric protein synthesis. (B and C) Strains were grown in LB broth with 1 mM IPTG, to induce a maximal chimeric protein synthesis. Total extracts and supernatant fractions were separated by SDS-PAGE and immunoblotted with rabbit polyclonal anti-Ag85A antibody. The numbers at the left of the blots are molecular masses. The unprocessed protein is indicated by the arrows, and the mature form is shown by the asterisks; these forms have expected molecular masses of 38 kDa and 36 kDa, respectively. Lanes: 1, molecular mass markers (MM); 2, RASV χ11021(pYA3681) vector control; 3, χ11021(pYA4890 [SopENt80-E2C/BlaSS-Ag85A294-BlaCT, pBR ori]); 4, χ11021(pYA4891 [SopENt80-E2C/BlaSS-Ag85A294-BlaCT, p15A ori]); 5, χ11021(pYA4892 [SopENt80-E2C/BlaSS-Ag85A294-BlaCT, pSC101 ori]); 6, χ11021(pYA4893 [OmpCSS-E2C/BlaSS-Ag85A294-BlaCT, pBR ori]); 7, χ11021(pYA4894 [OmpCSS-E2C/BlaSS-Ag85A294-BlaCT, p15A ori]).
Fig 4
Fig 4
Synthesis of SopENt80-E2C or OmpCSS-E2C in RASV strain χ11021. Immunoblot of whole-cell lysates and supernatant fractions of χ11021 strains harboring Asd+ lysis plasmids and producing SopE-E2C or OmpC-E2C. Strains were grown as described in the legend to Fig. 2A to C. Total extracts and supernatant fractions were separated by SDS-PAGE and immunoblotted using rabbit polyclonal anti-ESAT-6 antibody to detect SopENt80-E2C or OmpCSS-E2C. pBR ori is high copy number, p15A ori is low copy number, and pSC101 ori is very low copy number. The numbers at the left of the blot are molecular masses. Squares, SopE80-E2C chimeric protein (expected molecular mass, 34 kDa); circles, SopE80-E2C chimeric protein breakdown products (expected molecular masses, 35 kDa and 32 kDa, respectively); arrows, unprocessed OmpCSS-E2C (expected molecular mass, 34 kDa); asterisks, the mature form of OmpCSS-E2C (expected molecular mass 32 kDa). Lanes: 1, molecular mass markers (MM); 2, RASV χ11021(pYA3681) vector control; 3, χ11021(pYA4890 [SopENt80-E2C/BlaSS-Ag85A294-BlaCT, pBR ori]); 4, χ11021(pYA4891 [SopENt80-E2C/BlaSS-Ag85A294-BlaCT, p15A ori]); 5, χ11021(pYA4892 [SopENt80-E2C/BlaSS-Ag85A294-BlaCT, pSC101 ori]); 6, χ11021(pYA4893 [OmpCSS-E2C/BlaSS-Ag85A294-BlaCT, pBR ori]); 7, χ11021(pYA4894 [OmpCSS-E2C/BlaSS-Ag85A294-BlaCT, p15A ori]).
Fig 5
Fig 5
Anti-Ag85A and anti-ESAT-6 serum IgG in mice. C57BL/6 mice were orally immunized at days 0, 7, and 49 with 1 × 109 CFU of RASV strain χ9879 harboring either pYA3620 (vector control) or its derivative, pYA3941 (synthesizing the BlaSS-Ag85A294-BlaCT protein), or RASV strain χ11021 independently harboring Asd+/MurA+ plasmids: pYA3681 (vector control) or the isogenic pBR, p15A, and pSC101 ori plasmids pYA4890, pYA4891, and pYA4892, respectively (synthesizing the SopENt80-E2C/BlaSS-Ag85A294-BlaCT protein), or harboring the isogenic pBR or p15A ori plasmids pYA4893 and pYA4894, respectively (synthesizing OmpCSS-E2C/BlaSS-Ag85A294-BlaCT), or were immunized simultaneously with both χ11021(pYA4892) and χ11021(pYA4894). The anti-Ag85A IgG titers in preimmune serum (PI) and in serum from immunized mice at 21 and 65 days (d) or at 77 days after the first immunization were measured by ELISA. (A) Serum IgG total response to Ag85A. ***, P < 0.001 for comparison with mice immunized with vector control strain χ11021(pYA3620), BSG-dosed mice, or preimmune serum. (B) Serum IgG total response to Ag85A. ***, P < 0.001 for comparison with mice immunized with vector control strain χ11021(pYA3681), BSG-dosed mice, or preimmune serum; P < 0.001 for comparison of mice immunized with χ11021(pYA4894) versus mice immunized with χ11021(pYA4890), χ11021(pYA491), χ11021(pYA4892), and χ11021(pYA4893); and P < 0.001 for comparison of mice immunized with χ11021(pYA4891) versus mice immunized with χ11021(pYA4890). (C) Serum IgG total response to ESAT-6. ***, P < 0.001, and **, P < 0.01, for comparison of mice immunized with the vector control strain, BSG-dosed mice, or preimmune serum; **, P < 0.01 for comparison of mice immunized with χ11021(pYA4892) versus χ11021(pYA4891) and P < 0.01 for comparison of mice immunized with χ11021(pYA4891) versus χ11021(pYA4890); ***, P < 0.001 for comparison of mice immunized with χ11021(pYA4894) versus mice immunized with χ11021(pYA4890), χ11021(pYA4891), χ11021(pYA4892), and χ11021(pYA4893). D Serum IgG total response to CFP-10. ***, P < 0.001; **, P < 0.01; *, P < 0.05 for comparison of mice immunized with the vector control strains. *, P < 0.05 for comparison of mice immunized with χ11021(pYA4892) versus χ11021(pYA4890) or χ11021(pYA4891); *, P < 0.05 for comparison of mice immunized with χ11021(pYA4894) versus χ11021(pYA4892 + pYA4894). The data represent endpoints of antibodies in pooled sera from 6 mice immunized at the indicated time after immunization. Error bars represent variations between duplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey's posttest.
Fig 6
Fig 6
Anti-SOMP total serum IgG and anti-Ag85A IgG2b and IgG1 in serum in mice. C57BL/6 mice were orally immunized at days 0, 7, and 49 with 1 × 109 CFU of χ9879 harboring either pYA3620 (control) or pYA3941 (specifying the BlaSS-Ag85A294-BlaCT protein) or with 1 × 109 CFU of χ11021 harboring either pYA3681 (lysis vector control) or the isogenic lysis plasmids pYA4890 (pBR ori), pYA4891 (p15A ori), and pYA4892 (pSC101 ori), each specifying SopENt80-E2C/BlaSS-Ag85A294-BlaCT, as well as pYA4893 (pBR ori) and pYA4894 (p15A ori), each synthesizing OmpCSS-E2C/BlaSS-Ag85A294-BlaCT, or were immunized simultaneously with both χ11021(pYA4892) and χ11021(pYA4894). The anti-Ag85A IgG2b and IgG1 titers and anti-SOMP titers in preimmune serum (PI) and in immunized mice at 21, 65, and 77 days after the first immunization were measured by ELISA. (A and B) Total serum IgG responses to SOMPs. ***, P < 0.001, **, P < 0.01, and *, P < 0.05, for comparison with mice immunized with the vector control strain, BSG-dosed mice, or preimmune serum. (C and D) Subclasses IgG2 and IgG1 in serum against Ag85A. The data represent endpoints of antibodies in pooled sera from 6 mice immunized at the indicated time after immunization. Error bars represent variations between duplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey's posttest.
Fig 7
Fig 7
Antigen-specific stimulation of cytokine responses in lymphocytes from mice vaccinated with Salmonella χ9879(pYA3620) or χ9879pYA3941. Antigen-specific IFN-γ (A), TNF-α (B), IL-2 (C), and IL-4 (D) cytokine-forming lymphocytes were determined by ELISPOT assay. C57BL/6 mice were orally immunized with χ9879(pYA3941) specifying BlaSS-Ag85A294-BlaCT or the χ9879(pYA3620) vector control or were BSG dosed at days 0, 21, and 49. Three weeks after the last immunization, spleen cells from three mice per group were harvested and pooled. The pool of spleen cells from the same group of mice was analyzed in triplicate. Cells were restimulated for 24 h (for IFN-γ- and TNF-α-secreting cells) or 48 h (for IL-2- and IL-14-secreting cells) with 1 μg/well of recombinant Ag85A or medium for ELISPOT assays. The results are presented as number of ELISPOTs per million lymphocytes minus background number of ELISPOTs from unpulsed mock controls. ***, P < 0.001 for comparison of mice immunized with χ9879(pYA3941) or with χ9879(pYA3620) and BSG group for Ag85A-specific IFN-γ-, TNF-α-, and IL-2-secreting cells; *, P < 0.05 for comparison of the mice immunized with χ9879(pYA3941) and mice immunized with χ9879(pYA3620) for Ag85A-specific TNF-α-secreting cells and P < 0.05 for comparison of mice immunized with χ9879(pYA3941) and BSG group for Ag85A-specific IL-4-secreting cells. Error bars represent variations between triplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey's posttest.
Fig 8
Fig 8
Antigen-specific stimulation of cytokine responses in spleen cells from mice vaccinated with RASV χ11021 harboring either Asd+/MurA+ lysis plasmid pYA3681 (vector control) or plasmids pYA4890, pYA4891, and pYA4892 (specifying synthesis of SopENt80-E2C/BlaSS-Ag85A294-BlaCT) or harboring plasmids pYA4893 and pYA4894 (specifying synthesis of OmpCSS-E2C/BlaSS-Ag85A294-BlaCT), or were immunized simultaneously with both χ11021(pYA4892) and χ11021(pYA4894). Ag85A-specific IFN-γ (A), TNF-α (B), IL-2 (C), and IL-4 (D) cytokine-forming lymphocytes were detected by ELISPOT assay. C57BL/6 mice were orally immunized with the Salmonella vaccine strains or BSG dosed at days 0, 21, and 49. Three weeks after the last immunization, spleen cells from three mice per group were harvested and pooled. Cells were restimulated for 40 h (for IFN-γ- and TNF-α-secreting cells) or 66 h (for IL-2- and IL-14-secreting cells) with 1 μg/well of recombinant Ag85A or medium for ELISPOT assays. The results are presented as the number of ELISPOTs per million lymphocytes minus background number of ELISPOTs from unpulsed mock controls. *, P < 0.05, and ***, P < 0.001, for comparison of BSG-dosed mice. (A) For Ag85A-specific IFN-γ secreting cells, *, P < 0.05 for comparison of mice immunized with either χ11021(pYA4890) or χ11021(pYA4891) and mice immunized with χ11021(pYA3681); **, P < 0.01 for comparison of mice immunized with both χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA4894); ***, P < 0.001 for comparison of mice immunized with both strains χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA3681). (B) For Ag85A-specific TNF-α-secreting cells; *, P < 0.05 for comparison of mice immunized with χ11021(pYA4890) and mice immunized with χ11021(pYA4892) or χ11021(pYA3681); **, P < 0.01 for comparison of mice immunized with both χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA4894) or χ11021(pYA3681). (C) For Ag85A-specific IL-2-secreting cells, **, P < 0.01 for comparison of mice immunized with both χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA4894). (D) For Ag85A-specific IL-4-secreting cells; *, P < 0.05 for comparison of mice immunized with χ11021(pYA3681) and mice immunized with χ11021(pYA4890) and P < 0.05 for comparison of mice immunized with χ11021(pYA4890) and mice immunized with χ11021(pYA4891); ***, P < 0.001 for comparison of mice immunized with both strains χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ1102(pYA4894). Error bars represent variations between triplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey's posttest.
Fig 9
Fig 9
RASV χ9879(pYA3941) confers significant protection against mycobacterial infection. C57BL/6 mice were orally immunized with χ9879(pYA3941), specifying synthesis of BlaSS-Ag85A294-BlaCT, the χ9879(pYA3620) vector control, or BSG at days 0, 21, and 49. One group of mice was immunized subcutaneously with M. bovis BCG at day 0. All mice were challenged with M. tuberculosis by aerosol 4 weeks after the last immunization and euthanized 6 weeks later, to determine the bacterial loads in the lungs (A) and spleens (B) in each group of mice. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, for significance of vaccinated groups compared with mice that received BSG as a control.
Fig 10
Fig 10
RASV χ11021 harboring the Asd+/MurA+ lysis plasmids confers significant protection against mycobacterial infection. C57BL/6 mice were orally immunized with the χ11021(pYA3681) vector control or with χ11021 harboring each of isogenic plasmids pYA4891, pYA4892, and pYA4893 (synthesizing SopENt80-E2C/BlaSS-Ag85A294-BlaCT) as well as either pYA4890 or pYA4894 (synthesizing OmpCSS-E2C/BlaSS-Ag85A294-BlaCT); were immunized simultaneously with both χ11021(pYA4892) and χ11021(pYA4894); or were BSG dosed at days 0, 7, and 49. One group of mice was immunized subcutaneously with M. bovis BCG at day 0. All mice were challenged with M. tuberculosis by aerosol 4 weeks after the last immunization and euthanized 6 weeks later, to determine M. tuberculosis loads in the lungs (A) and spleens (B) of each group of mice. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, for significance in numbers of CFU between vaccinated and BSG-dosed mice.

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