Engineered Mycobacterium tuberculosis triple-kill-switch strain provides controlled tuberculosis infection in animal...

Area of Science:

• Microbiology
• Vaccinology
• Genetic Engineering

Background:

• Human challenge experiments are crucial for accelerating tuberculosis vaccine development.
• A safe Mycobacterium tuberculosis (Mtb) strain that replicates in hosts and is reliably cleared is needed for these models.

Purpose of the Study:

• To engineer a genetically modified Mtb strain with multiple kill switches for enhanced safety in human challenge models.
• To evaluate the efficacy and safety of this engineered strain in preclinical models.

Main Methods:

• Genetic engineering of Mtb to incorporate two tetracycline-regulated mycobacteriophage lysin operons and a trimethoprim-regulated degron-NadE fusion (triple-kill-switch or TKS).
• In vitro assessment of TKS strain's growth kinetics and susceptibility to antibiotics.
• In vivo studies in mice to evaluate infection establishment, clearance upon antibiotic withdrawal, and relapse potential.

Main Results:

• The TKS strain exhibited growth and antibiotic susceptibility similar to wild-type Mtb under permissive conditions.
• The TKS strain was rapidly killed in vitro without specific antibiotics (trimethoprim and doxycycline).
• In mice, the TKS strain established infection during antibiotic treatment but was cleared upon cessation, with no observed relapse in immunodeficient mouse models. The strain demonstrated a very low mutation rate.

Conclusions:

• The engineered triple-kill-switch (TKS) Mtb strain shows promise as a safe and effective candidate for tuberculosis human challenge models.
• This TKS strain facilitates controlled infection and clearance, potentially accelerating the development of new tuberculosis vaccines.