Adjuvant modulation of the cytokine balance in Mycobacterium tuberculosis subunit vaccines; immunity, pathology and protection

Abstract

It is known that protection against tuberculosis is mediated primarily by T helper type 1 (Th1) cells but the influence of the Th1/Th2 balance of a vaccination response on the subsequent protection and pathology during infection has not been studied in detail. We designed a panel of Ag85B-ESAT-6 subunit vaccines based on adjuvants with different Th1/Th2-promoting activities and studied cellular responses, bacterial replication and pathology in the lungs of mice infected with Mycobacterium tuberculosis. All vaccines induced cell-mediated and humoral responses but with markedly different interferon-gamma : interleukin-5 (IFN-gamma : IL-5) and immunoglobulin G1 (IgG1) : IgG2 ratios. The vaccines promoted different levels of control of bacterial replication with the most efficient protection being exerted by cationic liposomes containing monophosphoryl lipid A and low to completely absent immunity with conventional aluminium. The level of protection correlated with the amount of IFN-gamma produced in response to the vaccine whereas there was no inverse correlation with the level of IL-5. Characterizing a protective response was an accelerated recruitment of IL-17 and IFN-gamma-producing lymphocytes resulting in the early formation of granulomas containing clustered inducible nitric oxide synthase-activated macrophages. In comparison, non-protected mice exhibited a different inflammatory infiltrate rich in neutrophil granulocytes. This study indicates that the adjuvant component of a tuberculosis vaccine may be crucial in determining the kinetics by which effective granulomas, pivotal in controlling bacterial growth, are formed.

Figure 1

Polarized vaccine-induced immune responses generated by T helper type 1 (Th1)/Th2 panel of adjuvants. Release of interleukin-5 (IL-5) and interferon-γ (IFN-γ) from blood lymphocytes isolated from C57BL/6 (a) or BALB/c (b) mice immunized with 2 μg Ag85B-ESAT-6 in Al(OH)₃, Al(OH)₃/DDA, DDA and DDA/MPL. Blood lymphocytes were isolated 5 weeks after the first immunization and restimulated in vitro with 5 μg Ag85B-ESAT-6. Antigen-specific antibody midpoint titres in serum of C57BL/6 mice (c) measured as IgG1 and IgG2b or BALB/c mice (d) measured as IgG1 and IgG2a. Antibodies were measured in the serum 7 weeks after the first immunization.

Figure 2

Accelerated emergence of cell subsets and vaccine-specific cytokine responses during infection with Mycobacterium tuberculosis in mice vaccinated with Ag85B-ESAT-6 in DDA/MPL. Lung tissue of unvaccinated C57BL/6 mice or mice immunized with Ag85B-ESAT-6 in Al(OH)₃ or DDA/MPL (n = 3) were disrupted and analysed for the presence of CD4 T cells, CD8 T cells, and B cells (CD19⁺) by flow cytometry before infection and at different time-points after infection with Mycobacterium tuberculosis Erdman. Interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), interleukin-17 (IL-17) and IL-5 responses were measured by ELISA after restimulation in vitro with 5 μg Ag85B-ESAT-6. *P < 0·05, **P < 0·01 and ***P < 0·001 compared to the corresponding values for naïve mice.

Figure 3

Lung interferon-γ (IFN-γ) responses predominant in the CD4 T-cell subsets. (a) Lung tissue was obtained from C57BL/6 mice immunized with Ag85B-ESAT in DDA/MPL 2 weeks after infection and the IFN-γ responses analysed in CD8 and CD4 T-cell subsets using flowcytometry. Percentage of IFN-γ-positive cells after restimulation in vitro with 5 μg Ag85B-ESAT-6 within the two subsets has been indicated with values for unstimulated control wells shown in parenthesis. (b) Lung tissue obtained from mice immunized with Ag85B-ESAT-6 in DDA/MPL 4 weeks after infection was cultured in the presence or absence of anti-CD4 or anti-CD8 antibodies and the IFN-γ response was measured by ELISA.

Figure 4

Computer-aided lesion morphometry indicated more numerous granulomas (a) covering a larger total pulmonary surface area (b) in C57BL/6 mice (n = 6) vaccinated with Ag85B-ESAT-6 in DDA/MPL adjuvant relative to those vaccinated using Al(OH)₃ adjuvant or unvaccinated at different time-points after challenge with 25 CFUs Mycobacterium tuberculosis Erdman by the aerosol route. *P < 0·05 and ***P < 0·001 compared to the corresponding values for naive mice.

Figure 5

Photomicrographs with insets of lung tissue 14 days after challenge with 25 CFUs Mycobacterium tuberculosis Erdman by the aerosol route illustrating reduced numbers of acid-fast bacteria in large tight clusters of inducible nitric oxide synthase (iNOS)-positive macrophages and reduced numbers of neutrophils in C57BL/6 mice vaccinated with Ag85B-ESAT-6 in DDA/MPL adjuvant (b), relative to those vaccinated using Al(OH)₃ adjuvant (a) or unvaccinated control animals (c). Images in the left-hand panels are stained using haematoxylin & eosin and in right-hand panels are double-stained using immunohistochemistry for iNOS followed by the modified Ziehl–Neelsen method for acid-fast bacteria. The images shown are from one representative mouse of six in each group. Magnification × 400.

Figure 6

Photomicrographs with insets of lung tissue 28 days post-challenge with 25 CFUs of Mycobacterium tuberculosis Erdman by the aerosol route illustrating reduced numbers of acid-fast bacteria within inducible nitric oxide (iNOS)-positive macrophages and increased numbers of lymphocytes in C57BL/6 mice vaccinated with Ag85B-ESAT-6 in DDA/MPL adjuvant (b), relative to those vaccinated using Al(OH)₃ adjuvant (a) or unvaccinated control animals (c). Images in left-hand panels are stained using haematoxylin & eosin (magnification × 200) and in right panels double stained using immunohistochemistry for iNOS followed by the modified Ziehl–Neelsen method for acid-fast bacteria (magnification × 400). The images shown are from one representative mouse of six in each group.