Front Immunol. 2018 Nov 30;9:2825. doi: 10.3389/fimmu.2018.02825. eCollection 2018.

Dual-Isotope SPECT/CT Imaging of the Tuberculosis Subunit Vaccine H56/CAF01: Induction of Strong Systemic and Mucosal IgA and T-Cell Responses in Mice Upon Subcutaneous Prime and Intrapulmonary Boost Immunization.

Thakur A¹, Rodríguez-Rodríguez C^2,³, Saatchi K², Rose F¹, Esposito T², Nosrati Z², Andersen P⁴, Christensen D⁴, Häfeli UO², Foged C¹.

Author information

1: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
2: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC, Canada.
3: Department of Physics and Astronomy, The University of British Columbia, Vancouver, BC, Canada.
4: Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark.

Abstract

Pulmonary tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a global pandemic, despite the widespread use of the parenteral live attenuated Bacillus Calmette-Guérin (BCG) vaccine during the past decades. Mucosal administration of next generation TB vaccines has great potential, but developing a safe and efficacious mucosal vaccine is challenging. Hence, understanding the in vivo biodistribution and pharmacokinetics of mucosal vaccines is essential for shaping the desired immune response and for optimal spatiotemporal targeting of the appropriate effector cells in the lungs. A subunit vaccine consisting of the fusion antigen H56 (Ag85B-ESAT-6-Rv2660) and the liposome-based cationic adjuvant formulation (CAF01) confers efficient protection in preclinical animal models. In this study, we devise a novel immunization strategy for the H56/CAF01 vaccine, which comply with the intrapulmonary (i.pulmon.) route of immunization. We also describe a novel dual-isotope (¹¹¹In/⁶⁷Ga) radiolabeling approach, which enables simultaneous non-invasive and longitudinal SPECT/CT imaging and quantification of H56 and CAF01 upon parenteral prime and/or i.pulmon. boost immunization. Our results demonstrate that the vaccine is distributed evenly in the lungs, and there are pronounced differences in the pharmacokinetics of H56 and CAF01. We provide convincing evidence that the H56/CAF01 vaccine is not only well-tolerated when administered to the respiratory tract, but it also induces strong lung mucosal and systemic IgA and polyfunctional Th1 and Th17 responses after parenteral prime and i.pulmon. boost immunization. The study furthermore evaluate the application of SPECT/CT imaging for the investigation of vaccine biodistribution after parenteral and i.pulmon. immunization of mice.

KEYWORDS:

H56/CAF01 vaccine; SPECT/CT imaging; T cells; drug delivery; dual-isotope 111In/67Ga; mucosal immunity; nanomedicine; pulmonary immunization

PMID:: 30555488
PMCID:: PMC6284049
DOI:: 10.3389/fimmu.2018.02825

Free PMC Article

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Figure 5

Robust T-cell responses in the lungs and the lung-draining tracheobronchial and mediastinal lymph nodes (TLN + MLN) by parenteral (i.m.) priming and airway (intrapulmonary, i.pulmon.) mucosal H56/CAF01 immunization. Mice were primed i.m. and boosted twice i.m. (5/250/50 μg H56/DDA/TDB) or primed i.m. (5/250/50 μg H56/DDA/TDB) and boosted twice i.pulmon. (10/125/25 or 10/250/50 or 10/500/100 μg H56/DDA/TDB) at 2 weeks interval with H56 adjuvanted with different concentrations of CAF01 (DDA/TDB). Lung cells and the lung draining LNs (TLN + MLN) were examined for CD4⁺CD44⁺ T cells and IFN-γ, TNF-α, and IL-17 cytokines by intracellular flow cytometry analysis after stimulation with H56, 2 weeks after the last booster immunization. (A) Gating strategy for quantification and localization of lung-associated CD44⁺ T cells in immunized mice (exemplified by lung cells from an immunized mouse from the vaccine group 125/25 i.m./2*i.pulmon.). Lung cells were examined for labeling with i.v. injected FITC-conjugated anti-CD45.2 mAb 2 weeks after the last booster immunization by intracellular flow cytometry analysis after stimulation with H56. Dot plot shows IFN-γ, TNF-α, or IL-17 expression in i.v.CD45⁺ (intravascular) and i.v.CD45⁻ (parenchymal) cells. (B) Number of cyt⁺CD4⁺CD44⁺ T cells (any cytokine IFN-γ, TNF-α, and IL-17), which are located in lung parenchyma (CD45⁻). Statistical analysis: one-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. ****p < 0.0001. (C) After Boolean gating analysis, the frequencies of the seven possible CD4⁺CD44⁺ T cell subpopulations expressing any combination of the IFN-γ, TNF-α, and IL-17 cytokines are shown for all immunization groups. The background from the control group was subtracted. Pie charts represent the fraction of CD4⁺CD44⁺ T cells expressing different cytokine combinations. Pie chart color-coding and the subpopulation association for each color is shown below the bar graph (E). Statistical analysis: two-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. *p < 0.05, **p < 0.01, and ****p < 0.0001. (D) Number of cyt⁺CD4⁺CD44⁺ T cells (any cytokine IFN-γ, TNF-α, and IL-17) in the lung draining LNs (TLN+MLN). Statistical analysis: one-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. **p < 0.01. (E) Boolean gating analysis and pie charts of CD4⁺CD44⁺ T cells expressing different cytokine combinations in the lung-draining LNs (TLN+MLN), 2 weeks after the last booster immunization. Statistical analysis: two-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. ****p < 0.0001.

Dual-Isotope SPECT/CT Imaging of the Tuberculosis Subunit Vaccine H56/CAF01: Induction of Strong Systemic and Mucosal IgA and T-Cell Responses in Mice Upon Subcutaneous Prime and Intrapulmonary Boost Immunization

Front Immunol. 2018;9:2825.

Figure 6

Parenteral (i.m.) prime and airway (i.pulmon.) mucosal boost immunization with H56/CAF01 induce equivalent T-cell responses in the spleen as parenteral (i.m.) prime-boost immunization. Mice were primed i.m. and boosted twice i.m. (5/250/50 μg H56/DDA/TDB) or primed i.m. (5/250/50 μg H56/DDA/TDB) and boosted twice i.pulmon. (10/125/25 or 10/250/50 or 10/500/100 μg H56/DDA/TDB) at 2 weeks interval with H56 adjuvanted with different concentrations of CAF01 (DDA/TDB). Spleen cells and the inguinal and popliteal lymph nodes (ILN+PLN) draining the site of i.m. injection were harvested 2 weeks after the last booster immunization, surface-stained for the CD4 and CD44 receptors and intracellular localized IFN-γ, TNF-α, and IL-17 cytokines by intracellular flow cytometry analysis after stimulation with H56. (A) Gating strategy for the evaluation of antigen-specific, cytokine-producing CD44⁺ T cells in spleen and lymph nodes of immunized mice (exemplified by TLN + MLN cells from an immunized mouse from the vaccine group 125/25 i.m./2*i.pulmon.). Dot plot shows IFN-γ, TNF-α, or IL-17 expression in CD44⁺ T cells. (B) Number of cyt⁺CD4⁺CD44⁺ T cells (any cytokine IFN-γ, TNF-α, and IL-17) in the spleen. Statistical analysis: one-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. *p < 0.05. (C) After a Boolean gating analysis, the frequencies of the seven possible CD4⁺CD44⁺ T cell subpopulations expressing any combination of the IFN-γ, TNF-α, and IL-17 cytokines are shown for all immunization groups. Background from the control group was subtracted. Pie charts represent the fraction of CD4⁺CD44⁺ T cells expressing different cytokine combinations. Pie chart color-coding and the subpopulation association for each color is shown below the bar graph (E). Statistical analysis: two-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. *p < 0.05 and ****p < 0.0001. (D) Number of cyt⁺CD4⁺CD44⁺ T cells (any cytokine IFN-γ, TNF-α and IL-17) in LNs draining the site of injection (ILN + PLN). Bars represent mean values ± s.e.m., n = 6. (E) Boolean gating analysis and pie charts of CD4⁺CD44⁺ T cells expressing different cytokine combinations in the LNs draining the site of injection (ILN+PLN) 2 weeks after the last booster immunization. Statistical analysis: two-way ANOVA and Tukey's post-test. Bars represent mean values ± s.e.m., n = 6. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Front Immunol. 2018;9:2825.