FRAMEWORK

• Context: Adaptation of the airway organoid technology to IPBS

• Duration: Dec 2018 – Present

• Status: Ongoing

• Role: Work Package Manager and adviser.

• Co-Principal Investigator: Dr. Céline Cougoule, CRCN/CNRS.

• Funding:

2022 — 2025 TB organoids, Role as Work Package Manager.

Bill & Mélinda Gates Foundation, TISSUSE

“Development of a novel TB multi-organoid chip-based model for screening of candidate vaccines and treatment modalities.”

• Personnel participating in this project:

  • Dr. Natacha Faivre, PhD graduate (2022-2025)

  • Dr. Geanncarlo Lugo-Villarino, DR2/CNRS.

  • Dr. Olivier Neyrolles, DRCE/CNRS.

• Collaboration: IPBS/CNRS, Toulouse

  • Partner Team: Dr. Etienne Meunier, DR2/CNRS

  • Key Personnel: 

    • Dr. Céline Cougoule, CRCN CNRS

    • Dr. Stephen Leon-Icaza, PhD graduate and post-doctoral fellow

    • Dr. Caio César Barbosa Bomfim, post-doctoral fellow

    • Dr. Thomas Benoist, post-doctoral fellow.

    • Dr. Étienne Meunier, DR2 CNRS

• Collaboration:  Maastricht University

  • Country: The Netherlands

  • Partner Unit: M4I Nanoscopy Division

  • Partner team: Laboratory of Dr. Peter J Peters

  • Key Personnel: 

    • Dr. Peter Peters, Principal Investigator.

    • Dr. Nino Iakobachvil, post-doctoral fellow (2016-2021).

• Collaboration:  Hubrecht Institute

  • Country: The Netherlands

  • Partner Unit: Utrecht University

  • Partner team: Laboratory of Dr. Hans Clevers

• Collaboration: TissUse GmbH 

  • Country: Germany

  • Partner Unit: TissUse GmbH

  • Key Personnel: 

    • Annika Winter.

    • Andy Kleemann.

    • Leopold Koening.

A. The Organoids4TB Projects: Advancing Human Lung 3D Systems to Evaluate Anti-TB Innovative Therapies and Vaccines

• Student: Stephen Leon-Icaza, PhD candidate (2018-2022).

• Postdoctoral fellow: Nino Iakobachvil (2016-2021).

• Postdoctoral fellow: Stephen Leon-Icaza, PhD graduate (2022-2024).

• Supervisors: Céline Cougoule and Geanncarlo Lugo-Villarino (France).

SUMMARY: To better investigate TB in human-relevant systems, we adapted bronchiolar airway organoids derived from primary human lung epithelial cells to model Mtb infection. By developing a microinjection strategy that delivers bacteria directly into the organoid lumen, we established a controlled infection model enabling visualization of bacterial dynamics and epithelial responses within a physiologically relevant 3D architecture. Using this system, we showed that airway epithelial cells can mount innate immune responses to mycobacterial infection, including the induction of antimicrobial and inflammatory pathways. Importantly, the organoid model also allowed the evaluation of anti-TB drugs, demonstrating that frontline antibiotics such as rifampicin and isoniazid effectively reduce the mycobacterial burden in this epithelial niche. These results highlighted the potential of lung organoids as an experimental platform to study both host–pathogen interactions and therapeutic responses in a human tissue context.

SIGNIFICANCE: This study provided the first demonstration that human bronchiolar organoids can support controlled mycobacterial infection and enable assessment of antibiotic efficacy. It established the technological and conceptual foundation of the Organoids4TB research axis, aimed at developing advanced human lung models to study tuberculosis pathogenesis and evaluate innovative therapeutic strategies. These results were published in Molecular Microbiology in 2021, with Nino and Stephen as co-first authors, and my role is acknowledged as a senior co-author (Iakobachvili et al., Molecular Microbiology, 2021).

B. Integration of Immune Cells into Human Lung Models to Evaluate Anti-TB Compounds

• Student: Natacha Faivre, PhD graduate (2022-2025)

• Postdoctoral fellow: Caio César Barbosa Bomfim (2023-Present)

• Supervisors: Céline Cougoule and Geanncarlo Lugo-Villarino (France)

SUMMARY: Building on the initial bronchiolar organoid platform, we extended our human lung modeling strategy by integrating key immune and epithelial components relevant to TB. In this work, we established complementary airway air–liquid interface (ALI) cultures and alveolar macrophage-like cells to reproduce two major pulmonary niches encountered by Mtb: the airway epithelial barrier and the intracellular alveolar macrophage compartment. These models enabled not only mechanistic analysis of host–pathogen interactions, but also benchmarking of anti-TB compounds. We showed that standard-of-care antibiotics exhibited compartment-specific activity, with isoniazid, rifampicin, and moxifloxacin active in both systems, whereas pyrazinamide was active mainly in the macrophage model. In parallel, host-directed therapies such as ibuprofen and doramapimod reduced inflammatory responses without consistently affecting bacterial burden, highlighting the ability of these human lung models to discriminate antimicrobial from immunomodulatory effects. Together, this work established a more physiologically relevant and versatile platform for evaluating both anti-infective and host-directed strategies against tuberculosis. 

SIGNIFICANCE: By combining epithelial and macrophage-based human lung models, this study moved beyond proof-of-concept organoid infection toward a translational platform capable of assessing drug efficacy across distinct pulmonary niches. It provides an important framework for preclinical testing of antibiotics and host-directed therapies in human-relevant TB models. These results are available as a preprint on BioRxiv 2025 and are under revision at Microbiology Spectrum, with Natacha and Caio as co-first authors, and Dr. Cougoule and me as the last authors (Bomfim et al., bioRxiv preprint, 2025).

C. Development of a Human Multi-Organ Chip to Evaluate TB Vaccines

• Postdoctoral fellow: Thomas Benoist (2023-Present)

• Supervisors: Céline Cougoule and Geanncarlo Lugo-Villarino (France)

SUMMARY: To overcome the limitations of existing experimental models for TB research, we launched a collaborative project with TissUse to develop a human multi-organ-on-chip system combining vascularized lung, liver, and lymph node compartments. This microphysiological platform is designed to reproduce both innate and adaptive immune responses to airborne infection in a controlled human setting. The lung compartment enables Mtb infection, while the lymph node module supports antigen presentation and T-cell activation, enabling the study of vaccine-induced immune responses. The liver compartment contributes to metabolic homeostasis and long-term stability of the co-culture system. On this platform, we are currently evaluating several candidate anti-TB vaccines provided by our partners and comparing their ability to induce protective immune responses against Mtb infection with that of the reference Bacille Calmette–Guérin (BCG) vaccine.

SIGNIFICANCE: This project aims to establish the first human multi-organ microphysiological model that can recapitulate key aspects of TB pathogenesis and vaccine-induced immunity. By enabling the comparative evaluation of novel TB vaccines alongside the classical BCG vaccine in a human-relevant system, this platform offers a promising alternative to animal models for preclinical TB vaccine development. This is an ongoing project that will result in an original research article to be published sometime in 2027.

Connection Between Studies A, B, and C. 

Together, the studies presented below illustrate the stepwise development of the Organoids4TB program, which aims to establish human-relevant platforms to study TB and evaluate innovative therapies and vaccines. In A, we first adapted bronchiolar airway organoid technology to model Mtb infection in a 3D epithelial context and demonstrated that this system can be used to analyze host responses and assess the efficacy of frontline anti-TB antibiotics. In B, we expanded this platform by integrating immune components and complementary lung models, including airway epithelial cultures and alveolar macrophage-like cells, to better reproduce the distinct pulmonary niches exploited by the pathogen and to evaluate both antimicrobial and host-directed therapies. Finally, in C, we are extending this strategy to a multi-organ microphysiological system that combines lung, lymph node, and liver compartments, enabling the study of infection dynamics and the comparative evaluation of candidate TB vaccines alongside the classical BCG vaccine.