Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice

Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen.National Major Science and Technology Projects (China) (2012ZX10003-008-006)National Natural Science Foundation (China) (31470895)National Natural Science Foundation (China) (81072499)China. Ministry of Education (Doctoral Fund 20120211110038

Guinea pig infected with Mycobacterium tuberculosis via oral consumption

Guinea pig represents a highly suitable animal model for study of Mycobacterium tuberculosis(Mtb)infection, as it demonstrates similarities to Mtb pulmonary infection in humans. It is known that guinea pigs can be efficiently infected by the respiratory or subcutaneous exposure to Mtb. However, information on Mtb infection through oral route is almost absent in the literature. Here, we examined whether guinea pigs can be infected by drinking Mtb containing water. Our findings confirmed that the guinea pigs could be infected with Mtb via drinking virulent water. The infected guinea pigs developed uniform oval-craterform ulcers at the 30th day after infection. Bacterial cultures showed Mtb growth in the lungs and spleens from the guinea pigs infected with high dose of Mtb. In addition, the infected animals had histopathological granulomatous lesions in lungs, spleens and mesenteric lymph nodes. We provide the compelling evidence that the guinea pigs could be infected by drinking Mtb containing water. The clinical and pathological observations in the infected animals were similar to those found in guinea pigs infected via the respiratory or subcutaneous routes

Subunit vaccine consisting of multi-stage antigens has high protective efficacy against Mycobacterium tuberculosis infection in mice.

To search for more effective tuberculosis (TB) subunit vaccines, antigens expressed in different growth stages of Mycobacterium tuberculosis (M. tuberculosis), such as RpfE (Rv2450c) produced in the stage of resuscitation, Mtb10.4 (Rv0288), Mtb8.4 (Rv1174c), ESAT6 (Rv3875), Ag85B (Rv1886c) mainly secreted by replicating bacilli, and HspX (Rv2031c) highly expressed in dormant bacilli, were selected to construct six fusion proteins: ESAT6-Ag85B-MPT64190-198-Mtb8.4 (EAMM), Mtb10.4-HspX (MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6-Ag85B, and ESAT6-RpfE. The six fusion proteins were separately emulsified in an adjuvant composed of N,N'-dimethyl-N, N'-dioctadecylammonium bromide (DDA), polyribocytidylic acid (poly I:C) and gelatin to construct subunit vaccines, and their protective effects against M. tuberculosis infection were evaluated in C57BL/6 mice. Furthermore, the boosting effects of EAMM and MH in the adjuvant of DDA plus trehalose 6,6'-dimycolate (TDM) on BCG-induced immunity were also evaluated. It was found that the six proteins were stably produced in E. coli and successfully purified by chromatography. Among them, EAMM presented the most effective protection against M. tuberculosis. Interestingly, the mice that received EAMM+MH had significantly lower bacterial counts in the lungs and spleens than the single protein vaccinated groups, and had the same effect as those that received BCG. In addition, EAMM and MH could improve BCG-primed protective efficacy against M. tuberculosis infection in mice. In conclusion, the combination of EAMM and MH containing antigens from both replicating and dormant stages of the bacilli could induce robust immunity against M. tuberculosis infection in mice and may serve as promising subunit vaccine candidate

The protective efficacy of LT69 against <i>M</i>. <i>tuberculosis</i> H37Rv infection in mice.

<p>Mice were immunized subcutaneously with two different doses (10 μg and 2 μg) of LT69 formulated in DDA-Poly I:C three times at 2-wks interval or with a single dose of BCG (5×106 CFU). At the 34th week, 30 weeks after the last vaccination, mice were aerosol-infected with <i>M</i>. <i>tuberculosis</i> H37Rv 50–100 CFU. Ten weeks later, the protective efficacy was measured and was expressed as log10 of number of CFU in lungs (A) and spleens (B). Results are presented as means ± SD from groups of seven mice. * <i>p</i> < 0.05, relative to PBS; ** <i>p</i> < 0.05, relative to PBS，BCG and EAMM+MH groups.</p

The T cell immune responses in mice immunized with different dosage of LT69.

<p>Mice were immunized subcutaneously with three different doses（50 μg, 10 μg or 2 μg) of LT69 formulated in DDA-Poly I:C three times at 2-wks interval or with a single dose of BCG (5×106 CFU), and the number of vaccine-induced IFN-γ cells was determined at 6 and 24 weeks after the last immunization. Freshly isolated spleen lymphocytes were plated in duplicate at 5×10⁵ cell per well in 96 spot and incubated with ESAT6 (10 μg/ml), Ag85B (5 μg/ml) and HspX (10 μg/ml) for 20h. IFN-γ production was determined using mouse IFN-γ ELISPOT kits. Results are presented as means ± SD, n = 4</p

Expression and purification of LT69.

<p>(A) Expression of LT69 in <i>E</i>. <i>coli</i>. The LT69 protein was induced by IPTG in <i>E</i>. <i>coli</i> BL21 at 25℃ for 6h. Coomassie Blue-stained 12% SDS-PAGE of total <i>E</i>. <i>coli</i> lysate (lane 1), supernatant of <i>E</i>. <i>coli</i> lysate (lane 2), and sediment of <i>E</i>. <i>coli</i> lysate (lane 3). (B) Purification of LT69. LT69 was purified by two steps, and finally its purity is beyond 90%. The figure shows a Coomassie Blue-stained 12% SDS-PAGE of LT69 purified by gradient salt fractionation (<i>lane</i> 1) along with LT69 purified by gradient salt fractionation and gel filtration chromatography (<i>lane</i> 2, 3). M, molecular weight. (C) Purified fusion proteins MH (<i>lane</i> 2) and EAMM (<i>lane</i> 3).</p

The immunogenicity of LT69 vaccine in mice.

<p>C57BL/6 mice were immunized with 50μg or 10μg or 2μg of LT69 formulated in DDA/Poly(I:C) separately via subcutaneous injection three times (2 weeks apart) or with a single dose of BCG (5×106 CFU). For EAMM+MH group, the mice received EAMM (10 μg) plus MH (10 μg) in DDA/Poly(I:C). Six weeks after the final immunization, spleen cells were stimulated with ESAT6 (10 xg /ml), Ag85B (5 μg/ml), Mtb10.4 (10 μg/ml), HspX (10 μg/ml) and PPD (5 μg/ml) separately <i>in vitro</i>. (A) The IFN-γ secretion in splenocytes. (B) The IL-2 secretion in splenocytes. Results are presented as means ± SD, n = 4. * <i>p</i> < 0.05, relative to PBS，BCG; ** <i>p</i> < 0.05, relative to PBS，BCG and EAMM+MH groups</p

IFN-γ response profiles to recombinant proteins MH, HspX, EAMM and ESAT-6 in different groups of donors.

<p>Healthy BCG-vaccinated donors (HD, <i>n</i> = 5-11), close contacts (CC, <i>n</i> = 4-10) and active TB patients (TB, <i>n</i> = 21-28) were recruited. IFN-γ secreting cells were analyzed using human IFN-γ ELISPOT kits. Freshly isolated PBMCs from these subjects were plated in duplicate at 3 ×10⁵ cells per well in 96 spot and incubated with the recombinant proteins (10 µg/ml or 20 µg/ml) for 48 h at 37°C. Data are shown as mean ± SD. * <i>p</i> < 0.01.</p

IFN-γ and IL-17 secretion in splenocytes of mice primed with BCG and boosted with subunit vaccine candidates.

<div><p>C57BL/6 mice were primed with BCG, and boosted twice with EAMM or AMM emulsified in the adjuvant of DDA/TDM by s.c. at 12 and 14 week after prime. Six weeks after the last boosting, spleen cells were stimulated with purified protein derivative of tuberculin (PPD), ESAT6 or Ag85B <i>in vitro</i> respectively, and IFN-γ and IL-17 were assayed by ELISPOT. (A) IFN-γ secretion in splenocytes. (B) IL-17 secretion in splenocytes. Results are presented as mean ± SD, <i>n</i> = 4. * <i>p</i> < 0.05 vs. PBS and BCG groups;.</p> <p>** <i>p</i> < 0.05 vs. PBS, BCG and BCG/AMM groups.</p></div