Comparison of immunogenicity and vaccine efficacy between heat-shock proteins, HSP70 and GrpE, in the DnaK operon of Mycobacterium tuberculosis

Abstract

Antigens (Ags) in Mycobacterium tuberculosis (Mtb) that are constitutively expressed, overexpressed during growth, essential for survival, and highly conserved may be good vaccine targets if they induce the appropriate anti-Mtb Th1 immune response. In this context, stress response-related antigens of Mtb might serve as attractive targets for vaccine development as they are rapidly expressed and are up-regulated during Mtb infection in vivo. Our group recently demonstrated that GrpE, encoded by rv0351 as a cofactor of heat-shock protein 70 (HSP70) in the DnaK operon, is a novel immune activator that interacts with DCs to generate Th1-biased memory T cells in an antigen-specific manner. In this study, GrpE was evaluated as a subunit vaccine in comparison with the well-known HSP70 against the hyper-virulent Mtb Beijing K-strain. Both HSP70- and GrpE-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 in the lung and spleen of Mtb-infected mice, but GrpE only produced a similar level of IFN-γ to that produced by ESAT-6 stimulation during the late phase and the early phase of Mtb K infection, indicating that GrpE is highly-well recognised by the host immune system as a T cell antigen. Mice immunised with the GrpE subunit vaccine displayed enhanced antigen-specific IFN-γ and serum IgG2c responses along with antigen-specific effector/memory T cell expansion in the lungs. In addition, GrpE-immunisation markedly induced multifunctional Th1-type CD4⁺ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs of Mtb K-infected mice, whereas HSP70-immunisation induced mixed Th1/Th2 immune responses. GrpE-immunisation conferred a more significant protective effect than that of HSP70-immunisation in terms of bacterial reduction and improved inflammation, accompanied by the remarkable persistence of GrpE-specific multifunctional CD4⁺ T cells. These results suggest that GrpE is an excellent vaccine antigen component for the development of a multi-antigenic Mtb subunit vaccine by generating Th1-biased memory T cells with multifunctional capacity, and confers durable protection against the highly virulent Mtb K.

Figure 1

IFN-γ production and memory T cell phenotypes induced by recombinant antigen stimulation in the lung and spleen of Mtb K-infected mice. (a) HSP70 and GrpE antigens were successfully induced in E. coli BL21 by supplementation with 1 mM isopropyl β-d-thiogalactoside (IPTG) for 12 h. (a, 1 and 4) The expression of N-terminal His-tagged antigens was identified by SDS-PAGE (M, molecular weight markers; Lane 1, uninduced; Lane 2, induced). (a, 2 and 5) SDS–PAGE analysis of purified antigens by Ni-nitrilotriacetic acid (NTA). (a, 3 and 6) Western blot analysis of purified antigens using mouse anti-His antibodies. (b) HSP70 (10 μg/mL)- and GrpE (10 μg/mL)-specific IFN-γ production were measured in the spleen and lung cells of individual mice 4 and 8 weeks after challenge with aerosolised Mtb K strain (n = 4 mice/group). NI: Non-infected mice, K: Mtb K-infected mice. ESAT-6 (1 μg/mL) was used as a positive control. Data from one of two independent experiments are shown. (c) At the same time point, spleen and lung cells from Mtb K-infected mice (n = 4 mice) were stimulated in vitro with antigens, and numbers of CD4⁺ effector/memory and effector T cells were analysed by flow cytometry. Data from one of two independent experiments are shown. All results are expressed as means ± SD and statistical significance (*p < 0.05, **p < 0.01, or ***p < 0.001) is shown for treatments compared to the non-treated each T cells (non; effector or effector/memory T cells, respectively). The value of n.s. was defined as no significant effect.

Figure 2

Immunogenicity in the lung and spleen of HSP70- and GrpE-immunised mice. (a) Schematic diagram of the subunit vaccine experimental design. (b,c) Mice (n = 5 mice/group) were immunised with BCG (Red bars), HSP70 (Black bars), GrpE (Blue bars), or adjuvant alone (Green bars), and their spleen and lung cells were stimulated in vitro with no antigen, PPD, HSP70, or GrpE 4 weeks after the last vaccination. IFN-γ secretion by spleen and lung cells in response to PPD (b,c), HSP70 (b) and GrpE (c) stimulation was analysed by ELISA. (d) An example of the gating strategy for the analysis of antigen-specific IFN-γ producing T cells among lung T cells of one representative mouse from the GrpE-immunised group is shown. (e) The percentages of PPD-, HSP70- or GrpE-specific IFN-γ-producing CD3⁺CD4⁺CD44^high and CD3⁺CD8⁺CD44^high T cells were analysed in cells isolated from the spleen and lungs of BCG- and antigen-immunised mice (n = 5 mice/group). (f) Serum levels of antigen-specific IgG1 and IgG2c at 4 weeks after the final immunisation (n = 5 mice/group). Data from one of two independent experiments are shown. *p < 0.05, **p < 0.01, or ***p < 0.001, compared to the adjuvant control group (Green bars).

Figure 3

Protective efficacy of antigen immunisation against Mtb K infection. (a) H&E-stained sections (n = 5 mice/group) of the lung for each immunised mouse (Adjuvant alone, BCG, HSP70, or GrpE) at 4 and 8 weeks after challenge with the aerosolised Mtb K strain. One BCG vaccinated group was included as a positive control. (b) Lung inflammation values are presented as the mean percentages of the area of inflammation from lung sections of infected mouse groups. (c) The differences in bacterial numbers between vaccinated groups and adjuvant control groups at 4 and 8 weeks after challenge are shown. Data from one of two independent experiments are shown. *p < 0.05, **p < 0.01, or ***p < 0.001, when compared to adjuvant control group; green bars or plots (a one-way ANOVA followed by Dunnett’s test). ^#p < 0.05 (BCG vs. GrpE; unpaired t-test).

Figure 4

Antigen-specific multifunctional CD4⁺ T cell responses in antigen-immunised mice after challenge with Mtb K. BCG-, HSP70-, GrpE-, or adjuvant alone-immunised mice were infected by aerosol exposure to Mtb K. At different time points after infection, as indicated, mice (n = 5 mice/group at 4 and 8 weeks post-infection) were euthanised and their lung cells (a and b; top panel) and splenocytes (b; bottom panel) were stimulated in vitro with PPD (10 μg/mL), HSP70 (10 μg/mL) or GrpE (10 μg/mL). (a) The percentage of antigen-specific CD4⁺CD44^high T cells producing IFN-γ, TNF-α, or IL-2 was measured by use of the gating strategy shown in Supplementary Fig. S1, in cells isolated from lung cells and splenocytes at 4 and 8 weeks after challenge with Mtb K strain. (b) The pie charts present the mean frequencies of cells co-expressing IFN-γ, TNF-α, and/or IL-2. The cytokine profiles for individual cells were analysed by multi-colour flow cytometry by gating for lymphocytes, CD3⁺CD4⁺CD44^high. Data from one of two independent experiments are shown. *p < 0.05, **p < 0.01, or ***p < 0.001 relative to the control groups, respectively.

Figure 5

Antigen-specific multifunctional CD8⁺ T cell responses in antigen-immunised mice after Mtb K challenge. (a) The percentage of antigen-specific CD8⁺CD44^high T cells producing IFN-γ, TNF-α, or IL-2 measured in cells isolated from the lung (a and b; top panel) and splenocytes (a and b; bottom panel) of vaccinated mice (n = 5 mice/group at 4 and 8 weeks post-infection) at 4 and 8 weeks after challenge with Mtb K. The pie charts present the mean frequencies of cells co-expressing IFN-γ, TNF-α, and/or IL-2. The cytokine profiles for individual cells were analysed by multi-colour flow cytometry by gating for lymphocytes, CD3⁺CD8⁺CD44^high. Data from one of two independent experiments are shown. *p < 0.05, **p < 0.01, or ***p < 0.001 relative to the control groups, respectively.

Figure 6

T cell immunity induced by antigen immunization. (a,b) Spleen and lung cells from BCG-, adjuvant alone-, HSP70-, and GrpE-immunised mice (n = 5 mice/group at 4 and 8 weeks post-infection in each group) were stimulated with PPD, HSP70, or GrpE, and the supernatant was collected and assayed for cytokines by ELISA. Antigen-specific cytokine responses are shown for spleen and lung cells collected post-infection (4 and 8 weeks). Data from one of two independent experiments are shown. *p < 0.05, **p < 0.01, or ***p < 0.001 by unpaired t-tests.