A Promising Listeria-Vectored Vaccine Induces Th1-Type Immune Responses and Confers Protection Against Tuberculosis

Abstract

Deaths associated with tuberculosis (TB) is rising and accounted for 1.4 million deaths in 2015 many of which were due to drug-resistant bacteria. Vaccines represent an important medical intervention, but the current Bacilli Calmette-Guerin (BCG) vaccine is not ideal for the protection of teenagers and adults. Therefore, a safe and effective vaccine is urgently needed. In this study, we designed a novel vaccine using an attenuated Listeria monocytogenes strain carrying fusion antigen FbpB-ESAT-6 (rLM) and characterized its safety and protective efficacy against Mycobacterium tuberculosis (M.tb) infection in mice. Compared to the wild type strain yzuLM4 and parental strain LMΔactA/plcB (LM1-2), the virulence of rLM was significantly reduced as judged by its infectious kinetics and LD₅₀ dose. Further characterization of intravenous immunization showed that prime-boost vaccination significantly increased the levels of Th1 cytokines (IFN-γ, IL-17, and IL-6), and enhanced cytotoxic T lymphocyte (CTL) CTLs activity, suggesting that rLM could elicit potent Th1/Th17 responses. More importantly, rLM significantly conferred the protection against M.tb H37Rv challenge. Collectively, our findings indicated that rLM is a novel and useful tool to prevent M.tb infection, and can be potentially be used to boost BCG-primed immunity.

Keywords: FbpB-ESAT-6; Listeria monocytogenes; Mycobacterium tuberculosis; Th1/Th17; attenuated; protective efficacy.

Figure 1

Identification of recombinant L. monocytogenes strain. (A) Western blot analysis of expressed FbpB-ESAT-6 protein. Secreted proteins were TCA-precipitated and probed by a rabbit anti-ESAT-6 monoclonal antibody by western blot. M represents protein marker; lane 1 represents proteins from LM1-2, lane 2 represents the proteins from rLM. (B) Hemolytic activity of listeria strains. Culture supernatants were incubated with sheep erythrocytes at 37°C in two-fold serial dilutions (wells 1–7). The recombinant strain rLM had hemolysis titers of 2³, while the parental strains were 2⁴. (C,D) Kinetics of different LM strains during infection in spleens (C) and livers (D) of mice. C57BL/6 mice (20 mice per group) were injected intravenously (i.v.) with rLM and LM1-2 at a dose of 0.1 LD₅₀, respectively. Bacteria in spleen and liver were enumerated on day 1, 3, 5, and 7 post-inoculation by tissue homogenization and serial plating.

Figure 2

The level of inflammatory cytokines in the splenocytes of mice immunized with indicated LM strains or controls. Five groups of C57BL/6 mice (n = 5) were intravenously immunized with rLM, LM1-2, PBS, BCG, and BCG/rLM, respectively. IFN-γ (A), IL-17A (B), TNF-α (C), IL-6 (D), and IL-4 (E) were determined on the 8th day after immunization. Splenocytes were prepared and stimulated with peptide FbpB_240−259, ESAT-6₁₋₂₀, PPD and ConA. Cytokines in the supernatants from each well were determined by ELISA. The results were presented as the mean ± SD of three wells in triplicate. ^***indicated P < 0.001, ^**indicated P < 0.01, and ^*indicated P < 0.05 of the comparisons. (A–E) Cytokines in the splenocytes.

Figure 3

PPD-specific CTL responses in vivo. PPD-specific CTL activity in vivo in mice (n = 5 per group) immunized with rLM, LM1-2, PBS, BCG, or BCG/rLM, respectively. A mixture of CFSE-labeled splenocytes pulsed with PPD (CFSE^high) or without PPD(CFSE^low) was intravenously injected into each group of mice on day 7 after the second immunization. The histogram shows CFSE^low untreated and CFSE^high PPD pulsed target cells in the spleens of mice after 15 h of transfer, respectively. Results were representative of three experiments, each with five mice per group. ^*P < 0.05 rLM group vs. LM1-2 group; ^**P < 0.01 BCG/rLM group vs. BCG group.

Figure 4

Protective efficacy of prime-boost vaccination with rLM. After 42 days of immunization, mice were challenged with 5 × 10⁵ CFU virulent M.tb H37Rv via a lateral tail vein. Six weeks post-challenge, all mice were sacrificed. The spleens and livers of five mice per group were removed, homogenized and cultured for CFU of M.tb. The left lung of each mouse was excised and fixed in 10% phosphate-buffered formalin, sectioned, stained with hematoxylin and eosin, and examined for histological lesions with microscope. Alternatively, the tissues were subjected to acid-fast staining to visualize the bacilli. The challenge experiment was conducted twice. (A) Survival of the immunized mice challenged by M.tb H37Rv. (B) The area ratio of granulomas per slice in each group of mice; (C) Histopathology (HandE staining) of the spleen in mice in each group; (D,E) Bacteria load in lung (D) and spleen (E) of mice. (F) Lung tissue with anti-acid staining.