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. 2015 Oct;22(10):1096-108.
doi: 10.1128/CVI.00301-15. Epub 2015 Aug 12.

Correlates of Vaccine-Induced Protection against Mycobacterium tuberculosis Revealed in Comparative Analyses of Lymphocyte Populations

Sherry L Kurtz  1 , Karen L Elkins  2
Affiliations

Correlates of Vaccine-Induced Protection against Mycobacterium tuberculosis Revealed in Comparative Analyses of Lymphocyte Populations

Sherry L Kurtz et al. Clin Vaccine Immunol. 2015 Oct.

Abstract

A critical hindrance to the development of a novel vaccine against Mycobacterium tuberculosis is a lack of understanding of protective correlates of immunity and of host factors involved in a successful adaptive immune response. Studies from our group and others have used a mouse-based in vitro model system to assess correlates of protection. Here, using this coculture system and a panel of whole-cell vaccines with varied efficacy, we developed a comprehensive approach to understand correlates of protection. We compared the gene and protein expression profiles of vaccine-generated immune peripheral blood lymphocytes (PBLs) to the profiles found in immune splenocytes. PBLs not only represent a clinically relevant cell population, but comparing the expression in these populations gave insight into compartmentally specific mechanisms of protection. Additionally, we performed a direct comparison of host responses induced when immune cells were cocultured with either the vaccine strain Mycobacterium bovis BCG or virulent M. tuberculosis. These comparisons revealed host-specific and bacterium-specific factors involved in protection against virulent M. tuberculosis. Most significantly, we identified a set of 13 core molecules induced in the most protective vaccines under all of the conditions tested. Further validation of this panel of mediators as a predictor of vaccine efficacy will facilitate vaccine development, and determining how each promotes adaptive immunity will advance our understanding of antimycobacterial immune responses.

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Figures

FIG 1
FIG 1
A panel of vaccines generates a hierarchy of protection against M. tuberculosis aerosol challenge. Groups of five male C57BL/6 mice were vaccinated with 2 × 102 CFU of either M. bovis BCG Pasteur (PAS), M. bovis BCG SSI (SSI), or heat-killed M. bovis BCG SSI (HK BCG). Eight weeks after vaccination, vaccinated and unvaccinated naive mice were aerogenically challenged with ∼200 CFU of M. tuberculosis Erdman. Four weeks after challenge, each mouse was euthanized, and the organs were aseptically removed, homogenized, and plated to enumerate bacterial burdens as CFU. Shown are the M. tuberculosis CFU in the lungs (A) and spleen (B). The bars represent the mean and standard deviation of CFU from five mice per group. This is one representative experiment of 14 of similar designs and outcomes. (C) The log10 reduction in CFU for the vaccine group was calculated compared to a naive group within the same experiment. The table represents the mean of the log10 reduction for each group for all 14 experiments. (A and B) *, P ≤ 0.05 by Student's t test compared to naive mice; #, P ≤ 0.05 by Student's t test compared to HK BCG within this representative experiment.
FIG 2
FIG 2
Peripheral blood lymphocytes from vaccinated mice control intracellular growth of M. bovis BCG Pasteur. C57BL/6 mice were vaccinated with M. bovis BCG Pasteur, M. bovis BCG SSI, or heat-killed M. bovis BCG SSI. Seventeen mice per group were sacrificed after 8 weeks, and splenocytes (SPL) and peripheral blood lymphocytes (PBLs) were isolated; splenocytes and PBLs were also obtained from naive mice. C57BL/6 BMDMϕ were infected with M. bovis BCG Pasteur at an MOI of 1:100, and then 5 × 106 splenocytes or 106 PBLs were added to triplicate wells of infected macrophages. Control wells (no cells) were infected, but no immune cells were added. The bars represent the means and standard deviations of CFU from triplicate wells. (A) Intracellular bacterial burdens (CFU) were assessed immediately after infection at day 0 (indicated) and at 7 days (all other data points) after infection of the cocultures. (B) Splenocytes and PBLs were recovered from cultures at 3 days after infection, and RNA was prepared for reverse transcription-quantitative PCR (qRT-PCR). For PAS (black bars), SSI (white bars), and HK BCG (gray bars), the expression of either IFN-γ or iNOS is shown as the fold change in immune cells compared to naive cells (N) using ABI TaqMan array cards. (C) Supernatants were harvested from cultures at 7 days after infection and the level of IFN-γ measured by sandwich ELISA. (D) Reactive nitrogen intermediates (RNI) in supernatants were also measured using Griess reagent. The results are shown for one representative experiment of 14 of similar designs and outcomes. Error bars represent standard deviations. *, P ≤ 0.05 by Student's t test compared to naive mice; #, P ≤ 0.05 by Student's t test compared to HK BCG.
FIG 3
FIG 3
Peripheral blood lymphocytes from vaccinated mice control intracellular growth of M. tuberculosis (MTB). C57BL/6 mice were vaccinated with M. bovis BCG Pasteur, M. bovis BCG SSI, or heat-killed M. bovis BCG SSI. Seventeen mice per group were sacrificed after 8 weeks, and splenocytes and peripheral blood lymphocytes (PBLs) were isolated; splenocytes and PBLs were also obtained from naive mice. C57BL/6 BMDMϕ were infected with M. tuberculosis at an MOI of 1:100, and then 5 × 106 splenocytes or 106 PBLs were added to triplicate wells of infected macrophages. The bars represent the means and standard deviations of CFU from triplicate wells. (A) Intracellular bacterial burdens (CFU) were assessed immediately after infection at day 0 (indicated) and at 7 days (all other data points) after infection of the cocultures. (B) Splenocytes and PBLs were recovered from cultures at 3 days after infection, and RNA was prepared for qRT-PCR. For PAS (black bars), SSI (white bars), and HK BCG (gray bars), the expression of either IFN-γ or iNOS is shown as the fold change in immune cells compared to naive cells (N) using ABI TaqMan array cards. (C) Supernatants were harvested from cultures at 7 days after infection and the level of IFN-γ measured by sandwich ELISA. (D) RNI in supernatants was also measured using Griess reagent. The results are shown for one representative experiment of 14 of similar designs and outcomes. Error bars represent standard deviations. *, P ≤ 0.05 by Student's t test compared to naive mice; #, P ≤ 0.05 by Student's t test compared to HK BCG.
FIG 4
FIG 4
Immune splenocytes (Imm. SPL) and PBLs recovered from cocultures with M. bovis BCG or M. tuberculosis differentially express selected genes. Immune splenocytes and PBLs obtained from the indicated vaccinated mice were incubated with either BCG-infected or M. tuberculosis-infected macrophages for 3 days. Cells were recovered from cocultures, and RNA was prepared for qRT-PCR amplification on ABI TaqMan array cards. For PAS (black bars), SSI (white bars), and HK BCG (gray bars), the relative expression in immune cells of each gene compared to naive cells is shown. Shown are the top five most highly upregulated genes from splenocytes cocultured with BCG-infected macrophages (A), PBLs cocultured with BCG-infected macrophages (B), splenocytes cocultured with M. tuberculosis-infected macrophages (C), and PBLs cocultured with BCG-infected macrophages. The data represent the median values obtained from eight experiments for BCG and four for M. tuberculosis.
FIG 5
FIG 5
Distribution of the most differentially expressed genes. As in Fig. 2 to 4, splenocytes and PBLs were incubated with either BCG- or M. tuberculosis-infected macrophages for 3 days. Cells were harvested from the culture supernatants, and RNA was prepared for qRT-PCR amplification on ABI TaqMan array cards. Gene expression was determined as the fold increase of the test sample (PAS, SSI, or HK) over the naive sample within that group. The experiments were repeated eight times for BCG and four times for M. tuberculosis. The median values for the fold change (immune/naive) for each gene were determined. The top 30 genes from each category, which were immune splenocytes cocultured with BCG (BCG/SPL), and immune PBLs cocultured with BCG (BCG/PBL), immune splenocytes cocultured with M. tuberculosis (M. tuberculosis/SPL), and immune PBLs cocultured with M. tuberculosis (M. tuberculosis/PBL) using all available experiments, were plotted into the four-dimensional Venn diagram. G-CSF, granulocyte colony-stimulating factor; VEGF, vascular endothelial growth factor; CTLA4, cytotoxic T-lymphocyte-associated protein 4; IKK, IκB kinase; M-CSF, macrophage colony-stimulating factor; LRP2, low-density lipoprotein receptor-related protein 2.
FIG 6
FIG 6
Supernatants recovered from cocultures of immune lymphocytes with M. bovis BCG or M. tuberculosis-infected macrophages differentially produce selected proteins. As in Fig. 2 to 4, splenocytes and PBLs were incubated with either BCG or M. tuberculosis-infected macrophages for 3 days. The supernatants were harvested and protein concentrations for 120 unique proteins determined using protein array chips. For PAS (black bars), SSI (white bars), and HK BCG (gray bars), the expression of each protein is shown as a fold change (immune/naive). Shown are the top five most highly upregulated proteins from splenocytes cocultured with BCG-infected macrophages (A), PBLs cocultured with BCG-infected macrophages (B), splenocytes cocultured with M. tuberculosis-infected macrophages (C), and PBLs cocultured with BCG-infected macrophages (D). The data represent the median values obtained from eight experiments for BCG and four for M. tuberculosis. TCA3, T-cell activation 3; P-sel, P-selectin.
FIG 7
FIG 7
Distribution of the most differentially expressed proteins. As in Fig. 2 and 3, splenocytes and PBLs were incubated with either BCG- or M. tuberculosis-infected macrophages for 3 days. The supernatants were harvested and protein concentrations determined using protein array chips. For three experimental groups, 120 unique proteins were measured in supernatants from immune splenocytes cocultured with BCG (BCG/SPL), immune PBLs cocultured with BCG (BCG/PBL), and immune splenocytes cocultured with M. tuberculosis (MTB/SPL). For the immune PBLs cocultured with M. tuberculosis (MTB/PBL), a subset of 40 unique proteins were measured. The experiments were repeated eight times for BCG and four times for M. tuberculosis. The median value for the fold increase for each protein was determined. All differentially regulated proteins from each category (BCG/SPL, BCG/PBL, MTB/SPL, and MTB/PBL) were plotted on the four-dimensional Venn diagram. TPO, thyroid peroxidase; PF4, platelet factor 4; TNFRI, tumor necrosis factor receptor I; MCP, monocyte chemoattractant protein.
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