A temperature sensitive Mycobacterium paragordonae induces enhanced protective immune responses against mycobacterial infections in the mouse model

Abstract

Recently, we introduced a temperature sensitive Mycobacterium spp., Mycobacterium paragordonae (Mpg). Here, we checked its potential as a candidate for live vaccination against Mycobacterium tuberculosis and Mycobacterium abscessus. Intravenous infections of mice with Mpg led to lower colony forming units (CFUs) compared to infection with BCG, suggesting its usefulness as a live vaccine. The analyses of immune responses indicated that the highly protective immunity elicited by Mpg was dependent on effective dendritic maturation, shift of cytokine patterns and antibody production toward a Th1 phenotype, and enhanced cytotoxic T cell response. Compared to BCG, Mpg showed a more effective protective immune response in the vaccinated mice against challenges with 2 different mycobacterial strains, M. tuberculosis H37Ra or M. abscessus Asan 50594. Our data suggest that a temperature sensitive Mpg may be a potentially powerful candidate vaccine strain to induce enhanced protective immune responses against M. tuberculosis and M. abscessus.

Figure 1

M. paragordonae (Mpg) led to the attenuated infection into murine macrophage and in an in vivo mice system. (a) Survival test of M. gordonae, M. marinum and Mpg strains (10 M.O.I. infection) at 30 °C (left) and 37 °C (right) in the murine macrophage J774.1. (b) Survival test of M. bovis BCG (BCG), Mpg and M. tuberculosis H37Ra (H37Ra) (10 M.O.I. infection) in the murine macrophage J774.1 at 37 °C in early time point (0, 2, and 24 hours). (c) Growth of BCG and Mpg in the organs (lungs, liver and spleen) after intravenous inoculation into BALB/c_nu (nude, up panels) and BALB/c (down panels) mice (n = 3–4 per group) (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (d) The viability of BMDCs was measured by MTS and 7-AAD staining assay after infection with 10 M.O.I. of BCG or Mpg overnight. Representative histograms of 7-AAD are presented (**P < 0.05, ***P < 0.001; Student’s t-test). All data are mean ± standard deviation of CFU or intensity values in each group.

Figure 2

M. paragordonae (Mpg) induced enhanced BMDC maturation. BMDCs were infected with BCG, Mpg (10 M.O.I.) or LPS (1 μg/ml) overnight, stained with antibodies against DC maturation surface markers (CD40, CD80, CD86 and MHC II), and then analyzed by flow cytometer. Representative histograms of CD11c-gated cells are presented, and the mean fluorescent intensity (MFI) of each BMDC maturation surface marker are shown in the graph (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test).

Figure 3

M. paragordonae (Mpg) induced BMDC or THP-1 -mediated immune responses. (a) Supernatants of the infected BMDCs were collected, and IFN-γ, TNF-α, IL-2, and IL-10 cytokine levels were analyzed by ELISA (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (b) qRT-PCR was used to measure the expression levels of IL-10, IL-12, CIITA and H2DMb mRNA in the BMDCs infected with BCG or Mpg. The expression levels represent relative fold changes based on β-actin. (c) qRT-PCR was used to measure the expression levels of IL-10 and IL-12 mRNA in the THP-1 cells infected with BCG or Mpg. The expression levels represent relative fold changes based on 18S rRNA. (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test).

Figure 4

M. paragordonae (Mpg) induced enhanced BMDC migration. Expression levels of CCR7 protein and mRNA were measured by (a) flow cytometry and (b) qRT-PCR, respectively. To show the rate of CCR7⁺ BMDCs, representative histograms of CD11c-gated cells are presented and the expression levels denote the relative fold changes based on β-actin (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (c) In vivo migration of the infected BMDC with BCG or Mpg was measured by comparisons of CFSE-labeled BMDCs in popliteal lymph nodes (pLN) 2 days after injection of the BCG- or Mpg-infected BMDCs into mice footpad. The CFSE-labeled BMDCs among the CD11c ⁺ cells in popliteal lymph node were measured by flow cytometry. The mean percentile of the CFSE-labeled BMDCs in the popliteal lymph nodes from separated experiments are presented (*P < 0.05; Student’s t-test).

Figure 5

Enhanced T cell proliferation induced by M. paragordonae (Mpg)-infected BMDCs. Proliferation of (a and c) CD4 and (b and e) CD8 T cells induced by BCG- or Mpg-infected BMDCs was examined by CFSE dilution method and detected by flow cytometry. The data represent the mean ± standard deviation of CFSE intensities in each group (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (d and f) IL-2 cytokine amounts in the supernatants of the CD4 and CD8 T cells used for the proliferation assay were measured by ELISA (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test).

Figure 6

M. paragordonae (Mpg) elicits an enhanced cell mediated immune response against M. abscessus or tuberculosis infections in vaccinated mice. (a) Splenocytes from vaccinated (with BCG or Mpg) and challenged (with Mab or Mtb) mice (n = 3–5 per group) were stimulated with whole cell lysates of Mab and Mtb. The IFN-γ secretion levels were detected by ELISPOT analysis (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (b) IFN-γ, TNF-α, IL-2, IL-10 and IL-12 cytokine levels were detected from the culture supernatants of the in vitro stimulated splenocytes with whole cell lysates of Mab and Mtb by ELISA (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). Each vaccinated group is annotated as follows: group I [PBS + PBS], group II [BCG + BCG], group III [BCG + Mpg], and group IV [Mpg + Mpg].

Figure 7

M. paragordonae (Mpg) elicits an enhanced mycobacterial-specific Th1-biased humoral response and cytotoxic T lymphocyte (CTL) response in immunized mice. (a) Mab- and Mtb-specific immunoglobulin subtypes (IgG2a and IgG1) were detected by ELISA from serum samples of vaccinated and challenged mice. Optical densities at 450 nm of IgG2a and IgG1 isotypes were detected and compared among all the vaccinated groups at each time point. The ratio of IgG2a/IgG1 was calculated by dividing the OD values for IgG2a by OD values for IgG1 (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (b) Splenocytes from BCG- or Mpg-immunized mice were stimulated with Ag85B protein for 6 days and used as effector cells. P815 cells (H-2^d) were transfected with pcDNA 3.3-Ag85B:ESAT-6 expression vector and used as target cells. CTL assay was conducted by co-culturing the target and effector cells at various ratio (E/T ratios of 10:1, 20:1, and 50:1). Supernatants of co-cultured cells were used for lactate dehydrogenase (LDH) cytotoxicity assay (*P < 0.05; Student’s t-test). Each vaccinated group is annotated as follows: group I [PBS + PBS], group II [BCG + BCG], group III [BCG + Mpg], and group IV [Mpg + Mpg].

Figure 8

M. paragordonae (Mpg) vaccine elicited an enhanced protective efficacy against M. abscessus or M. tuberculosis infections in a mouse model. (a) CFU enumeration from organs (liver, lung and spleen) of BALB/c mice (n = 4–6 per group) vaccinated subcutaneously with PBS, BCG (1 × 10⁶ CFU) or Mpg (1 × 10⁶ CFU) (vaccination groups are detailed in Supplementary Fig. 2) and challenged 4 weeks after final vaccination with Mab or Mtb (1 × 10⁶ CFU, intravenously) (*P <b 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). (b) Representative histopathologic images (H&E stained) are shown for the sectioned lung tissues of BALB/c mice vaccinated with PBS, BCG or Mpg (vaccination groups are detailed in Supplementary Fig. 2) and challenged with Mab (at Day 14) or Mtb (at Week 8). Scale bars correspond to 100 μm. Each vaccinated group is annotated as follows: group I [PBS + PBS], group II [BCG + BCG], group III [BCG + Mpg], and group IV [Mpg + Mpg].

Figure 9

Schematic representation showing enhanced vaccine efficacy of M. paragordonae (Mpg). Compared to BCG, Mpg plays a pivotal role as an attenuated live vaccine in protecting against infections with Mtb and Mab via enhanced innate and adaptive immune responses and is safer for mice intravenous infections as well as in infections of macrophages.