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. 2017 Aug 18;85(9):e00245-17.
doi: 10.1128/IAI.00245-17. Print 2017 Sep.

Listeria-Vectored Vaccine Expressing the Mycobacterium Tuberculosis 30-Kilodalton Major Secretory Protein via the Constitutively Active prfA* Regulon Boosts Mycobacterium Bovis BCG Efficacy Against Tuberculosis

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Listeria-Vectored Vaccine Expressing the Mycobacterium Tuberculosis 30-Kilodalton Major Secretory Protein via the Constitutively Active prfA* Regulon Boosts Mycobacterium Bovis BCG Efficacy Against Tuberculosis

Qingmei Jia et al. Infect Immun. .
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Abstract

A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective Mycobacterium bovis BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein (r30/antigen 85B [Ag85B]) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated L. monocytogenes vectors, L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII), we constructed five rLm30 vaccine candidates expressing r30 linked in frame to the L. monocytogenes listeriolysin O signal sequence and driven by the hly promoter (h30) or linked in frame to the ActA N-terminal 100 amino acids and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm30 expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the largest amount of r30 in broth culture, all five rLm30 vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting of BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T cells expressing the three cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) (P < 0.001) and splenic and lung CD8+ T cells expressing IFN-γ (P < 0.0001). In mice and guinea pigs, the rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 with an adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting of mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized M. tuberculosis (P < 0.01).

Keywords: 30-kDa major secretory protein; Listeria vector; Mycobacterium tuberculosis; PrfA; antigen 85B; heterologous prime-boost vaccination; prfA; r30; vaccine.

Figures

FIG 1
FIG 1
Construction and characterization of rLm30 vaccine candidates expressing M. tuberculosis r30. (a) Diagram showing the construction of attenuated recombinant L. monocytogenes expressing M. tuberculosis r30. The sequence for r30 was codon optimized for expression in L. monocytogenes, synthesized, and cloned into a conjugation vector downstream of the actA promoter (PactA) and ligated in frame with the coding sequence for the amino-terminal 100 amino acids of ActA (ActAN) (actA-30 fusion [a30]) or cloned into a conjugation vector downstream of the L. monocytogenes hly promoter (Phly) and ligated in frame with the coding sequence for the amino-terminal 28 amino acids of listeriolysin O for its signal sequence (LLOss) (hly-30 fusion [h30]). The resulting integration plasmid was subsequently integrated at the 3′ end of a tRNAArg gene in L. monocytogenes vectors through conjugation. CAT, chloramphenicol acetyltransferase; Erm, erythromycin; Pp60, L. monocytogenes p60 promoter; PSA int., PSA integrase. (b) Diagram of rLm30 vaccine candidates. Three L. monocytogenes vectors, the L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII) strains, were used to construct candidate vaccines expressing h30 or a30, as indicated. (c) Expression of M. tuberculosis proteins in the supernatant fluid of rLm30 broth cultures. (Left) Equivalent amounts of culture filtrates of each rLm30 strain were analyzed by Western blotting using rabbit polyclonal antibody to r30 (top) or L. monocytogenes P60 (bottom), followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The blot was processed by using the Bio-Rad imaging system (ChemiDoc XRS) and Quantity One software, which allows the overlap of a white-light image, for visualization of the protein standards (lanes 1 and 6), and a chemiluminescent image, for visualization of the antibody-labeled protein bands (lanes 2 to 5 and 7 to 9). (Right) The densities of the 31-kDa (h30) and 39-kDa (a30) protein bands in rLm30 vaccines were estimated by using Quantity One software (Bio-Rad), adjusted to those of the LmIII vector, and normalized to the density of the ∼60-kDa (P60) protein band. (d) Protein expression in mouse macrophage-like J774 cells. J774 cells were uninfected (UI) or infected at an MOI of 10 with each of the five rLm30 vaccines listed in panel b or the LmI or LmIII vector that had been grown to stationary phase. At 5.5 h postinfection, the infected cells were lysed and subjected to Western blotting using polyclonal antibody to r30 (top left) or to ActAN (top right), followed by a monoclonal antibody to P60 and/or β-actin (bottom). In the bottom left panel, a small quantity of the lysate in lane 2 that spilled over from lane 3 was detected by a monoclonal antibody to P60. On the right border of each panel are the proteins of interest (arrows). On the left border of each panel are the sizes of the molecular mass standards. M, molecular marker.
FIG 2
FIG 2
Immunogenicity of BCG-rLmI/h30 prime-boost vaccination in guinea pigs. (a) Animals (6/group) were primed i.d. with 103 CFU of BCG at week 0; not boosted (prime-alone control) or boosted i.d. at week 4 with 100 μg r30 in SAF (r30/SAF), 1010 virions of rAdv30 (rAdv30), 106 CFU of the LmI (L. monocytogenes ΔactA) vector, or 106 CFU rLmI/h30; and evaluated for cell-mediated and humoral immunity at week 8. LPA, lymphocyte proliferation assay; cDTH, cutaneous delayed-type hypersensitivity. (b) cDTH in response to r30 was assessed by measurement of the diameter of erythema and induration. SH, sham. (c and d) Subsequently, the animals were euthanized, their splenocytes were assayed for r30- or PPD-specific lymphocyte proliferation (c), and their sera were assayed for r30-specific antibody (d). Data are means ± standard errors. Only values that are significantly different from those of animals immunized with BCG only or animals primed-boosted with BCG-rLmI/h30 (indicated by the open ends of horizontal lines) are marked with asterisks over the comparison group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (by two-way [b and c] or one-way [d] ANOVA with Tukey's multiple-comparison test).
FIG 3
FIG 3
Efficacy of BCG-rLmI/h30 prime-boost vaccination against M. tuberculosis challenge in guinea pigs. Animals (15/group) were primed i.d. with 1 × 103 CFU BCG; not boosted (prime-alone control); or boosted i.d. once at week 4 only (1×) or twice at weeks 4 and 8 (2×) with 100 μg r30 in SAF, 1 × 1010 virions of rAdv30, 1 × 106 CFU of the L. monocytogenes vector, or 1 × 106 CFU rLmI/h30 (study I) (a to c) or 3 × 106 CFU rLmI/h30 (study II) (d to f). Animals were then challenged at week 20 with a low dose (study I) or a high dose (study II) of the M. tuberculosis (Mtb) Erdman strain, weighed weekly (b and e), and euthanized at week 30, and their lungs and spleens were assayed for M. tuberculosis CFU (c and f). Lung and spleen CFU were analyzed by one-way ANOVA with Tukey's multiple-comparison test, comparing the mean CFU of each group with the mean CFU of any other group. Values are the means ± standard errors. Only P values equating to statistically significant differences (P < 0.05) are shown in the graphs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 4
FIG 4
Comparative immunogenicity of rLm30 vaccine candidates administered as a booster vaccine in a prime-boost vaccination strategy. C57BL/6 mice (n = 4/group) were primed with BCG i.d. at week 0, boosted twice with the indicated vaccine i.d. at weeks 3 and 6, and euthanized at week 10 for immunogenicity studies. (a, c, and d) Their splenocytes were in vitro stimulated with the r30 protein or r30 peptides and assayed for r30-specific IFN-γ secretion (a); the r30- or r30 peptide-specific iMFI of each cytokine (c); and the r30- or r30 peptide-specific total frequency of polyfunctional CD4+ T cells expressing IFN-γ, IL-2, and/or TNF-α (d). (b) Sera were assayed for r30-specific antibody. Data are the means ± standard errors. Statistically significant differences between the group immunized with only BCG and any other group are indicated by a bracket between the groups marked with one or more asterisks indicating the P value. Statistically significant differences among other groups are not marked. With respect to differences between other groups, in panel d, mice primed with BCG and boosted with rLmI/h30 or rLmIII/a30 also had significantly higher percentages of polyfunctional T cells expressing IFN-γ, TNF-α, and IL-2 than did sham-immunized mice (P < 0.0001); mice immunized with only BCG (P < 0.0001); or mice primed with BCG and boosted with the L. monocytogenes ΔactA vector (P < 0.0001), rLmII/a30 (P < 0.0001), rLmII/h30 (P < 0.0001), or rLmIII/h30 (P < 0.0001). The polyfunctional T-cell response was also greater than that in mice primed with BCG and boosted with r30/SAF, but these differences were not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by one-way [a and b] or two-way [c and d] ANOVA with Tukey's multiple-comparison test).
FIG 5
FIG 5
Comparative efficacies of rLm30 vaccine candidates administered as a booster vaccine in a prime-boost vaccination strategy. (a) Schedule. C57BL/6 mice (n = 8/group) were primed with BCG i.d. at week 0, boosted i.d. with various vaccines twice at weeks 3 and 6, challenged with the aerosolized M. tuberculosis Erdman strain (average of 112 CFU delivered to the lung of each animal) at week 12, and euthanized at week 18, 22, or 27 (6, 10, or 15 weeks postchallenge), and their lungs and spleens were assayed for M. tuberculosis. (b) Organ CFU. Bacterial burdens of M. tuberculosis in the lung (top) and spleen (bottom) were assayed as described in Materials and Methods. Values are the means ± standard errors. Values that are significantly different between two groups are marked with one or more asterisks over an open horizontal line crossing above the two groups. For comparisons of values from three different time points, residual errors were confirmed to have a normal distribution by examining normal quantile plots (not shown). The assumption of variance homogeneity was confirmed by computing the Brown-Forsythe statistic. The P values for pairwise mean comparisons were adjusted for multiple comparisons under this model by using the Fisher-protected LSD criterion (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 6
FIG 6
Secretion of IFN-γ and IL-17A into the culture supernatant of lung and spleen cells after a single booster vaccination with rLmIII/a30. C57BL/6 mice (n = 4/group) were primed with BCG i.d.; boosted once i.d. with rLmIII/a30 or the LmIII (L. monocytogenes ΔactA ΔinlB prfA*) vector at week 12 or 15, as indicated; and euthanized 6 days later. Single-cell suspensions of lung (a and b) and spleen (c and d) cells were stimulated with r30 or PPD for 3 days, and the supernatant fluids were collected and assayed for IFN-γ (a and c) and IL-17A (b and d) by an enzyme-linked immunosorbent assay. Values are the means ± standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by two-way ANOVA with Tukey's multiple-comparison test). Data are representative of results from three independent experiments, although the rLm30 booster vaccines were given on different schedules and at different doses.
FIG 7
FIG 7
Frequency of cytokine-expressing CD4+ T cells in the lungs and spleens of mice primed with BCG and boosted 12 versus 15 weeks later with rLmIII/a30. C57BL/6 mice (n = 4/group) were primed with BCG i.d. and boosted with the LmIII (L. monocytogenes ΔactA ΔinlB prfA*) vector or rLmIII/a30 i.d. at week 12 or 15, as indicated on the left side of each row of panels. Six days later, mice were euthanized; their lungs (top two rows) and spleens (bottom two rows) were removed; single-cell suspensions were prepared and stimulated with r30, r30 peptides, or PPD in the presence of anti-CD28 monoclonal antibody or PMA (positive control) for 6 h, as indicated above the panels; and the cells were assayed by intracellular cytokine staining for IFN-γ, TNF-α, IL-2, and IL-17A, as indicated below each panel. Values are the means ± standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by two-way ANOVA with Tukey's post-multiple-comparison test). Data are representative of results from three independent experiments, although the rLm30 booster vaccines were given on different schedules and at different doses.
FIG 8
FIG 8
Frequencies of polyfunctional CD4+ T cells in the lungs and spleens of mice primed with BCG and boosted 12 or 15 weeks later with rLmIII/a30. C57BL/6 mice (n = 4/group) were primed with BCG i.d. at week 0 and boosted with rLmIII/a30 i.d. at week 12 or 15, as indicated at the left side of each row of panels. Six days later, mice were euthanized, and single-cell suspensions of lung and spleen cells were cultured with medium alone or with medium supplemented with r30, r30 peptides, or PPD, as indicated above panels, in the presence of anti-CD28 monoclonal antibody and assayed for intracellular cytokine staining. The frequencies of live CD4+ T cells producing any of the 15 possible combinations of four cytokines (IFN-γ, TNF-α, IL-2, and IL-17A) were uniquely distinguished by using logic combinations of the gates for IFN-γ, TNF-α, IL-2, and IL-17A and FACSDiva software (BD), as indicated below the panels. Background frequencies of cells producing cytokines without antigen stimulation were subtracted. Values are the means ± standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by ANOVA with Tukey's multiple-comparison test). Data are representative of results from three independent experiments, although the rLm30 booster vaccines were given on different schedules and at different doses.
FIG 9
FIG 9
Frequencies of IFN-γ-producing CD8+ T cells in the lungs and spleens of mice primed with BCG and boosted 12 weeks later with rLmIII/a30. C57BL/6 mice (n = 4/group) were primed i.d. with BCG at week 0, boosted i.d. with rLmIII/a30 at week 12, and euthanized 6 days later. Single-cell suspensions of lung (top) and spleen (bottom) cells were cultured with medium alone or with medium supplemented with r30, r30 peptides, or PPD, as indicated at the top of each panel, for 24 h in the presence of anti-CD28 monoclonal antibody and assayed by intracellular cytokine staining. Background frequencies of cells producing cytokines without antigen stimulation were subtracted. Frequencies of CD8+ T cells expressing IFN-γ were analyzed by two-way ANOVA. Values are means ± standard errors. *, P < 0.05; ****, P < 0.0001 (by two-away ANOVA with Tukey's multiple-comparison test). Data from a similar experiment are shown in Fig. S8 in the supplemental material.
FIG 10
FIG 10
Efficacy against aerosol challenge with M. tuberculosis of priming mice with BCG and boosting them once 15 weeks later with rLmIII/a30. (a) BALB/c mice (n = 8/group) were immunized i.d. with PBS (sham) or BCG at week 0. BCG-primed mice were either not boosted or boosted i.d. once at week 15 with rLmIII/a30. The mice were then challenged with the aerosolized M. tuberculosis Erdman strain (average of 32 CFU delivered to the lungs of each animal) at week 18 and euthanized at week 28. (b) Afterwards, lungs (left) and spleens (right) were removed and assayed for organ bacterial burdens. Values are the means ± standard errors. **, P < 0.01; ****, P < 0.0001 (by one-way ANOVA with Tukey's multiple-comparison test).

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