A New Adjuvant MTOM Mediates Mycobacterium tuberculosis Subunit Vaccine to Enhance Th1-Type T Cell Immune Responses and IL-2+ T Cells

Abstract

The only licensed vaccine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) cannot prevent the prevalence of tuberculosis (TB), which remains a major public health problem worldwide. A more effective TB vaccine than BCG is urgently needed. Subunit vaccine is a promising strategy, and suitable adjuvants will benefit the development of effective TB subunit vaccines. MTO, consisting of monophosphoryl lipid A (MPLA), trehalose-6,6'-dibehenate (TDB), and MF59, was developed as an adjuvant of TB vaccine because of its ability to evoke the Th1-type T cell responses, while it is insufficient to induce single and multifunctional IL-2⁺ T cells and has a limited ability to confer protection against Mycobacterium tuberculosis infection. Heat-killed Mycobacterium vaccae (Mv), which can evoke cytotoxic CD8⁺ and CD4⁺ T cell responses and has adjuvanticity, was, in this study, combined with MTO to produce a new adjuvant, called MTOM. The TB fusion protein Rv3407-PhoY2-Ag85A-Rv2626c-RpfB (WH121) was mixed with MTO, Mv, and MTOM to produce three subunit vaccines, and the protective efficacy and immune responses were compared in C57BL/6 mice. WH121/MTOM provided better protection against TB than the other two vaccines, matching the performance of BCG vaccine. MTOM showed stronger ability to increase single and multifunctional IL-2⁺ T cells and induce Th1-type responses than MTO or Mv. Therefore, MTOM might be a promising adjuvant that could contribute to the development of TB subunit vaccines.

Keywords: IL-2+ T cells; MTOM; Mycobacterium tuberculosis; Th1-type response; adjuvant.

Figure 1

Comparison of protective efficacy of different vaccines. (A) Six weeks after final immunizations, C57BL/6 mice (n = 6) were intranasally challenged with about 100 CFU virulent M. tb H37Rv strain. Four weeks after challenge, the bacterial load within lungs and spleens of mice in different groups was assessed and represented as mean (±SEM) log₁₀ CFU/organ (n = 6). (B) Lung tissue was sectioned and stained with hematoxylin and eosin (HE) (scale bar, 400 µm) and acid-fast (AF) staining (scale bar, 50 µm). Arrows indicate AF-positive bacteria.

Figure 2

Effect of MTOM-adjuvanted WH121 protein on serum antibody levels in immunized mice. Six weeks after final immunization, the levels of serum IgG, IgG1, and IgG2c (replaced with IgG2a) antibodies from immunized mice were detected using ELISA. (A) WH121/MTOM induced IgG, IgG1, and IgG2a antibodies specific to WH121. (B) Comparison of antibody levels induced by WH121/MTO, WH121/Mv, and WH121/MTOM. Results are shown as mean (±SEM) log₁₀ end point titers and the ratio of IgG2a:IgG1 in the differently vaccinated groups (n = 3).

Figure 3

The levels of IFN-γ, TNF-α, and IL-2 secreted by splenocytes from different groups. Six weeks after the last immunization, splenocytes were collected from each mouse (n = 3). A total of 5 × 10⁶ cells were added to each well of 24-well microtiter plates and incubated with WH121 protein (10 µg), PPD (10 µg), or complete RPMI1640 medium for 24 h (for IL-2 detection) or 72 h (for IFN-γ and TNF-α detection) at 37°C in 5% CO₂. The cytokine concentrations in the suspension were determined by ELISA. (A) PPD- or WH121-specific cytokines induced by Bacillus Calmette–Guérin (BCG), PBS, WHI121/MTOM, and MTOM. (B) PPD-specific cytokines induced by MTO, Mycobacterium vaccae (Mv) and MTOM, and WH121-specific cytokines induced by WH121/MTO, WH121/Mv, and WH121/MTOM. The results are shown as mean ± SD (pg/mL).

Figure 4

PPD- and WH121-specific multifunctional CD4⁺ and CD8⁺ T cell responses. Splenocytes from each mouse in each group were obtained and counted. A total of 5 × 10⁶ cells per well were added to 24-well plates and stimulated with WH121 protein (10 µg) or PPD (10 µg). Intracellular cytokine profiles for IFN-γ, TNF-α, and IL-2 in individual cells were detected using multicolor flow cytometry by gating for CD4⁺ or CD8⁺ T cells. (A) The numbers of multifunctional CD4⁺ and CD8⁺ T cells induced by WH121/MTOM. (B) PPD-specific CD4⁺ and CD8⁺ T cells elicited by MTO, Mycobacterium vaccae (Mv), and MTOM and WH121-specific CD4⁺ and CD8⁺ T cells induced by WH121/MTO, WH121/Mv, and WH121/MTOM. The number of cells in the PBS group, considered a background value, was subtracted. Each possible combination of cytokines is shown on the x-axis of the bar chart, and the absolute number of antigen-specific T cells expressing any combination of cytokines is shown as mean ± SEM in the different groups (n = 3). ^- represents P < 0.05.