Characterisation of a live Salmonella vaccine stably expressing the Mycobacterium tuberculosis Ag85B-ESAT6 fusion protein

Abstract

A recombinant Salmonella enterica serovar Typhimurium (S. Typhimurium) vaccine strain was constructed that stably expressed the Mycobacterium tuberculosis fusion antigen Ag85B-ESAT6 from the chromosome. Live oral vaccination of mice with the Salmonella/Ag85B-ESAT6 strain generated a potent anti-Ag85B-ESAT6 T(H)1 response with high antibody titres with a IgG2a-bias and significant IFN-gamma production lasting over a 120-day period. When mice primed with the Salmonella/Ag85B-ESAT6 vaccine were mucosally boosted with the Ag85B-ESAT6 antigen and adjuvant the IFN-gamma responses increased markedly. To determine the protective efficacy of this vaccine strain, guinea pigs were immunised and followed for a 30-week period after aerosol challenge with M. tuberculosis. The heterologous prime-boost strategy of live Salmonella vaccine followed by a systemic boost of antigen and adjuvant reduced the levels of M. tuberculosis bacteria in the lungs and spleen to the same extent as BCG. Additionally, this vaccination regimen was observed to be statistically equivalent in terms of protection to immunisation with BCG. Thus, live oral priming with the recombinant Salmonella/Ag85B-ESAT6 and boosting with Ag85B-ESAT6 plus the adjuvant LTK63 represents an effective mucosal vaccination regimen.

Fig. 1

Schematic diagram for the construction of targeting construct and chromosomal integration of expression cassette. (A) Expression cassette consists of a constitutive promoter (lacZ), a gene fragment encoding the model vaccine fusion antigen Ag85B–ESAT6 (grey symbol). Expression cassette for expression of Ag85B–ESAT6 was inserted into the multiple cloning site of p2795 within SalI (black line) and BamH1 (grey line) sites. p2795 contains the kanamycin resistance gene (diagonal lines) and binding sites for primers (black symbol). (B) The targeting construct consisting of expression cassette and resistance gene was amplified by knock-in primers containing sequences complementary to the chromosomal target gene, phoN (dots). (C) S. Typhimurium strain SL3261 harbouring pKD46 for expression of Red recombinase was transformed with linear DNA of targeting construct. (D) Recombinant clones with replacements of a chromosomal target gene, phoN (white symbol) by the targeting construct were selected. Modified from .

Fig. 2

Ag85B–ESAT6 expression by recombinant S. Typhimurium vaccine strain. Bacterial lysates, were separated by SDS-PAGE electrophoresis (4–20% gradient gel; 2 × 10⁷ bacteria per lane), blotted onto nitrocellulose, and probed with a monoclonal antibody against native Ag85B. Lanes 1 and 2 shows log phase static cultures of SL3261mycolacZ and the SL3261 parental strain, respectively. The protein was identified by comparison to 1 μg of purified recombinant Ag85B–ESAT6 standard (Lane 3). The protein band corresponding to Ag85B–ESAT6 is indicated by the arrow. The * indicates dimers of Ag85B–ESAT6 in the purified protein.

Fig. 3

Serum Ig anti-Ag85B–ESAT6 antibody response in C57BL/6 mice following oral Salmonella prime and i.n. protein boost. Mice were orally vaccinated with approximately 5 × 10⁹ CFU of SL3261mycolacZ on day 0 and then boosted with 25 μg Ag85B–ESAT6 + 20 μg LTK63 or appropriate antigen controls on day 50. Negative control mice were immunised with wild-type SL3261 or PBS, and then also boosted on day 50 with suitable antigens. Positive controls received 1 μg LT plus 10 μg Ag85B–ESAT6 i.n. at day 0. Mice were left for 21, 50, 57 and 120 days and then sample bled to determine anti-Ag85B–ESAT6-specific Ig antibodies, which were determined by ELISA. (A) Graphs express total antibody titres using a cut off of OD 0.3 on days 50 (prior to boost) and 57 (plus boost) and on (B) day 120. The black bar shows the geometric mean from the group with the significant values of *p < 0.05; **p < 0.01 and ***p < 0.001 as determined using the Kruskal–Wallis test followed by Dunn's Multiple Comparison test compared to negative controls.

Fig. 4

Ag85B–ESAT6-specific IgG1:IgG2a antibody titre ratios. C57BL/6 mice were immunised as described in Fig. 3 legend. Negative control mice, i.e. PBS and SL3261 immunised had no detectable IgG subtype titres (data not shown). Titres shown were determined in individual serum samples obtained on days 50, 57 and 120 after immunisation. Bars represent geometric means. Statistical significance was determined by using the Kruskal–Wallis test followed by Dunn's Multiple Comparison test (*p < 0.05 and **p < 0.01).

Fig. 5

Systemic and mucosal IgA induced by immunisation with Salmonella live vector vaccine. Antibody titres were measured following oral prime immunisation with SL3261mycolacZ vaccine candidate and i.n. boosting with antigen plus adjuvant. (A) Graph shows titres obtained from individual serum samples collected on days 50 (prior to boost) and then on days 57 (plus boost). (B) Lung and nasal wash IgA titres are also shown. Serum and washes isolated from mice immunised with SL3261 parental strain or PBS served as negative controls. For group and statistics details see legend of Fig. 3.

Fig. 6

Ag85B–ESAT6-specific cytokine responses in C57BL/6 mice primed with Salmonella live vector vaccine and boosted with LTK63 and Ag85B–ESAT6. Ag85B–ESAT6-specific cytokine secretion of splenocytes from mice primed by oral immunisation with SL3261mycolacZ, SL3261, or PBS (day 0) and boosted by i.n. administration of Ag85B–ESAT6 plus LTK63 or appropriate controls (day 50) are shown. Spleens were harvested on days 50 and 57, and 120. (A) Figure shows cytokine levels from stimulated splenocytes on days 50 and 57. Cytokine responses were measured upon in vitro stimulation with Ag85B–ESAT6 for 36–42 h including IFN-γ, IL-4, IL-2, IL-5 and, IL-6. (B) IFN-γ levels from splenocytes isolated on day 120 are also shown. Cells were also stimulated with ConA (positive control) and RPMI⁺ media (negative control). Columns represent the mean (±SD) stimulation indices of splenocytes from 5 to 10 animals per group. The increase in cytokine levels between SL3261mycolacZ-primed, i.n.-boosted mice, compared to negative control mice, is indicated (*p < 0.05; **p < 0.01; ***p < 0.001) as determined using the Kruskal–Wallis test followed by Dunn's Multiple Comparison test. None of the cytokines tested were detected in splenocyte populations stimulated with medium alone. All cytokines were detected in splenocytes from immunised mice upon stimulation with ConA (data not shown).

Fig. 7

Protective responses in vaccinated animals. (A) Survival curve of guinea pigs that were aerosol-infected with M. tuberculosis H37Rv throughout an observation period of 210 days (n = 8 guinea pigs). Results are represented as Kaplan–Meier survival curves, and differences in survival were calculated by the log-rank test ***p ≤ 0.001. Influence of vaccination on bacterial load of M. tuberculosis in the (B) lungs and (C) spleens of guinea pigs infected by the aerosol route. Columns represent log₁₀ CFU (±SD). The reduction in bacterial numbers compared to PBS controls is indicated (*p < 0.05; **p < 0.01; ***p < 0.001) as determined using the Kruskal–Wallis test followed by Dunn's Multiple Comparison test.