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

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.

Carbohydrate Immune Adjuvants in Subunit Vaccines

“…A similarly designed vaccine against C. trachomatis co-administered with CAF01 also induced strong cellular and humoral immune responses and protected mice in C. trachomatis SvF challenge [ 179 ] when delivered via both subcutaneous and intranasal routes. This was consistent with another study, where parenteral (intramuscular) priming and airway intrapulmonary boost immunization were used for a vaccine formulation including H56 antigens and CAF01 [ 180 ]. The immunization strategy of intramuscular vaccination followed by an intrapulmonary boost induced higher lung and systemic H56-specific memory CD4 + T-cell activation, IgA and IgG antibody responses, and dose-dependent IFN-γ cytokine responses than the same vaccine delivered via a single administration route.…”

Intrapulmonary (i.pulmon.) Pull Immunization With the Tuberculosis Subunit Vaccine Candidate H56/CAF01 After Intramuscular (i.m.) Priming Elicits a Distinct Innate Myeloid Response and Activation of Antigen-Presenting Cells Than i.m. or i.pulmon. Prime Immunization Alone

“…Liposomes were prepared by using the thin film method and characterized for average intensity-weighted hydrodynamic diameter (z-average), polydispersity index (PDI) and zetapotential as previously described (24). Briefly, weighed amounts of DDA and TDB (5:1, w/w) were dissolved in chloroform/methanol (9:1, v/v) in a round bottom flask.…”

“…For the i.pulmon. immunizations, 10 µg Alexa Fluor R 647-labeled unadjuvanted H56 or 10 µg Alexa Fluor R 647labeled H56 adjuvanted with DiR-CAF01 (125/25 µg DDA/TDB) was used, and they were performed as described previously (24). All vaccines were formulated and administered in a dose-volume of 50 µL in isotonic Tris buffer.…”

“…priming followed by i.pulmon. mucosal pull immunization with the H56/CAF01 vaccine, which resulted in the induction of lung-localized, H56-specific T cells and systemic as well as lung mucosal IgA responses (24). However, the role of (i) H56/CAF01 deposition within lung tissue, (ii) CAF01 internalization by phagocytic cells, and (iii) antigen presentation in the lungs and the lung-draining LNs on the induction of immune responses after pulmonary administration are unknown.…”

Molecular Imaging of Pulmonary Inflammation and Infection

“…According to such evidence, 67 Ga-citrate has been suggested as an indicator of activated macrophages and bacterial load, guiding the prompt isolation and treatment of patients with active pulmonary disease. Recently, the association of 67 Ga-citrate with 111 In, using a novel dual-isotope radiolabeling approach conjugated with vaccines was successfully used in a preclinical animal model to assess the efficacy of a mucosal vaccines for tuberculosis [41].…”