A multistage mycobacterium tuberculosis subunit vaccine LT70 including latency antigen Rv2626c induces long-term protection against tuberculosis

Figures & data

Figure 1. Expression, purification and analysis of fusion protein LT70. (a) Expression of LT70 in E. coli. Coomassie Blue-stained 12% SDS-PAGE: E. coli BL21 lysate (lane 1), total E. coli BL21 expressing LT70 lysate (lane 2), supernatant of E. coli BL21 expressing LT70 lysate (lane 3) and sediment of E. coli lysate (lane 4). M, molecular weight. (b) Purification of LT70. The BL21 lysate containing LT70 (lane 1) was purified by 2 steps. First purification of LT70 by salting (lane 2), and followed with hydrophobic chromatography (lane 3). M, molecular weight. (c) Purified LT70 was verified by immunoblot: Mouse monoclonal anti-Rv2626c (lane 1), Negative control (lane 2), Mouse monoclonal anti-Ag85B (lane 3), Negative control (lane 4). M, molecular weight.

Figure 2. IFN-γ responses to protein EAMM, HspX, Rv2626c and LT70 in different groups of donors. In this study, active TB patients (TB, n = 20) and close contacts (n = 10) were recruited. Freshly isolated PBMCs from these subjects were plated in duplicate at 3×10⁵ cell per well in 96 spot and incubated with EAMM (5 μg/ml), HspX (5 μg/ml), Rv2626c (5 μg/ml) and LT70 (5 μg/ml) for 48 h at 37°C, 5% CO₂. IFN-γ was detected using Human IFN-γ ELISPOT kits. Data were shown as means± SD. * p < 0.01.

Figure 3. The immunogenicity of LT70 vaccine. Mice were immunized with 13 μg of LT70 formulated in DDA/poly(I:C) 3 times at 2-week intervals subcutaneously (s.c.) in week 0, 2 and 4 of the experiments. For EAMM+MH group, the mice were received EAMM (10 μg ) and MH (10 μg ) in DDA/poly(I:C). Mice immunized with 5×10⁵ BCG or PBS was used as controls. Six weeks after the final vaccination, spleen cells were stimulated with ESAT6 (10 μg /ml), Ag85B (5 μg /ml), Mtb8.4 (10 μg /ml), Rv2626c (10 μg /ml) and PPD (5 μg /ml) respectively in vitro. The IFN-γ was detected by ELISPOT kit and was showed as number of cells secreting IFN-γ/3×10⁵. Data were shown as means± SD. n = 4. *p < 0.05, relative to PBS and BCG groups.

Table 1. Serum antibodies induced by LT70 vaccine and BCG-prime, LT70-boost regimen.

	Anti-Ag85B			Anti-Rv2626c
Groups	IgG1	IgG2c	IgG2c/ IgG1	IgG1	IgG2c	IgG2c/ IgG1
PBS	—	—	—	—	—	—
BCG	2.47 ± 0.33	1.13 ± 0.05	0.46 ± 0.15	1.17 ± 0.05	—	—
EAMM+MH	4.40 ± 0.41	2.50 ± 0.16	0.56 ± 0.39	—	—	—
LT70	4.67 ± 0.32^*	3.55 ± 0.33^*	0.76 ± 1.03^*	4.52 ± 0.43^*	2.90 ± 0.27	0.64 ± 0.62
BCG prime, LT70 boost	4.67 ± 0.32^*	4.52 ± 0.43^*	0.97 ± 1.34^*	4.50 ± 0.36^*	4.45 ± 0.36	0.98 ± 1.00

Notes. Serum samples were collected 6 weeks after the last injection and the reciprocal end point titers of antigen specific IgG1 and IgG2c were analyzed by ELISA. Data were shown as means ± SD.

* p < 0.05, relative to PBS and BCG groups.

Table 2. The protective efficacy of vaccines and BCG-prime, vaccine boost regimen against H37Rv infection in mice.

Groups	Mean Log10 CFU ± SD(LUNG)	Mean Log10 CFU ± SD(SPLEEN)
PBS	6.53 ± 0.26	5.98 ± 0.27
BCG	6.01 ± 0.33^*(−0.52)	5.10 ± 0.45^*(−0.88)
EAMM+MH	5.78 ± 0.32^*(−0.75)	5.07 ± 0.47^* (−0.91)
LT69	5.68 ± 0.39^*(−0.85)	5.20 ± 0.50^* (−0.78)
LT70	5.41 ± 0.37^** (−1.12)	4.91 ± 0.17^* (−1.07)
BCG prime, LT70 boost	5.62 ± 0.64^* (−0.91)	4.70 ± 0.41^* (−1.28)

Notes. Ten weeks later, the protective efficacy was measured by detecting the bacteria load in lung tissuesand spleen tissues. Results are presented as means ± SD from groups of 7 mice.

* p < 0.05, relative to PBS group.

** p < 0.01, relative to PBS and BCG groups.

Figure 4. The immune responses in the BCG-prime and LT70-boost regimen. C57BL/6 mice were primed with BCG at 0 week, and boosted with 10 μg of LT70 vaccine by s.c. at 15 and 18 week separately. Six weeks after the last boosting, spleen cells were stimulated with ESAT6, Ag85B and Rv2626c in vitro, and IFN-γ production was assayed by ELISPOT. Results are presented as means ± SD. n=4. *p < 0.05, relative to PBS and BCG groups. **p < 0.05, relative to PBS, BCG and LT70 groups.

Figure 5. Pathologic reaction induced by vaccine immunization and M. tuberculosis challenge. Mice were immunized with BCG, EAMM+MH, LT69, LT70 and BCG prime, LT70 boost respectively. After the last vaccination, mice were aerosol-infected with M. tuberculosis H37Rv 50–100 CFU. The representative histological sections of the lungs from mice and the area ratio of granuloma in sections with HE of all groups were shown. n=5.