intradermal-inoculation-of-mycobacterium-avium-in-the-mouse-ear

Intradermal Inoculation of <em>Mycobacterium avium</em> in the Mouse Ear

In contrast to natural nontuberculous mycobacterial (NTM) exposures or infections whose pathology is founded on a slow-replicating organism, the aerosol ...

Seattle Children's Research Institute

The increase of global cases of drug resistant (DR) Mycobacterium tuberculosis (M.tb) is a serious problem for the tuberculosis research community and the goals to END TB by 2030. Due to the need for advancing and screening next generation therapeutics and vaccines, we aimed to design preclinical DR models of Beijing lineage M.tb HN878 strain in different mouse backgrounds. We found escalating sensitivities of morbidity due to low dose aerosol challenge (50-100 bacilli) in CB6F1, C57BL/6 and SWR mice, respectively. We also observed that pulmonary bacterial burden at morbidity endpoints correlated inversely with survival over time between mouse strains. Interestingly, with in vitro passaging and in the process of selecting individual DR mutant colonies, we observed a significant decrease in in vivo HN878 strain virulence, which correlated with the acquisition of a large genetic duplication. We confirmed that low passage infection stocks with no or low prevalence of the duplication, including stocks directly acquired from the BEI resources biorepository, retained virulence, measured by morbidity over time. These data help confirm previous reports and emphasize the importance of monitoring virulence and stock fidelity.

Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event.

For much of the last decade, tuberculosis (TB) was the leading cause of mortality due to an infectious pathogen (Mycobacterium tuberculosis, M.tb). Approximately 1.3 million deaths in 2023 worldwide were attributed to TB disease. Focused intervention strategies to block transmission would significantly reduce the global health burden of TB. Mycobacteriophages (phages) are a sorely underutilized biologic therapy for the pathogen M.tb, and here we aimed to address outstanding questions about their utility for clinical applications. We aimed to determine the impact of repeated mucosal or intravenous (IV) delivery of representative anti-M.tb phage FionnbharthΔ45Δ47 (Fionnbharth) in a preclinical mouse model. In addition, we specifically sought to understand which route induced anti-phage antibodies, which may reduce the long-term impact of phage therapy. C57BL/6 mice were dosed weekly for 6 weeks by either route and serum and bronchoalveolar lavage fluid (BALf) were evaluated for anti-phage humoral responses by ELISA. We found that aerosol delivery disperses phage across all lung lobes where M.tb is also found after experimental infection by the same route. Repeated aerosol delivery was well tolerated and did not induce robust neutralizing humoral immunity. In contrast, Mice receiving IV phage developed increasing magnitude and neutralizing total IgG and IgA responses over time. To determine whether pre-treatment environmental exposure to Fionnbharth-like phages could induce antibody responses that are potentially neutralizing, ~ 500 human plasma samples from normal donors were evaluated by ELISA. We observed that 5% of samples had antibodies to Fionnbharth (with end point titers > 10 dilution), although none were neutralizing. Furthermore, we found that highly-purified phage preparations did not activate mouse or human derived toll like receptor (TLR) 4 or TLR9 in HEKblue reporter assays. These data together support using Fionnbharth in anti-M.tb therapy phage cocktail strategies and that aerosol delivery should be prioritized for further efficacy testing.

Evaluation of host immune responses to Mycobacteriophage Fionnbharth by route of delivery.

Tuberculosis (TB) is again the deadliest infectious disease globally, and more efficacious vaccines are needed to reduce this mortality. Successful subunit TB vaccines need antigens and adjuvants that are immunogenic, inexpensive, and accessible. Here we evaluated the potential of synthetically produced Monophosphoryl lipid A (SyMLP), a TLR4-agonist, formulated in an oil-in-water emulsion (EmT4™) in combination with selected fusion proteins, to drive an effective vaccine-mediated immunogenic response in C57BL/6 mice against Mycobacterium tuberculosis (M.tb) HN878 and H37Rv challenge. We first observed that EmT4™ enhances activation of C57BL/6 bone-marrow derived macrophages and dendritic cells measured by CD40, CD86, and MHCII expression by flow cytometry. EmT4™ did not induce safety signals in a scaled tolerability study. In immunogenicity studies, mice immunized 3 times 3 weeks apart with ID93 antigen + EmT4™ produced a significantly higher magnitude of circulating proinflammatory cytokines and ID93-specific immunoglobulin G (IgG) antibodies pre- and post-challenge with M.tb than saline control animals. Ex vivo ID93 restimulated splenocytes and lung cells elicited significant polyfunctional CD4+ T-helper 1 responses. Importantly, ID93 + EmT4™ immunizations significantly reduced bacterial burden in C57BL/6 mice 4 weeks post-challenge. Interestingly, EmT4™ paired with a next generation protein fusion ID91 also afforded prophylactic protection against M.tb HN878 challenge in both young (6 to 8 wk) and aged (20 mo) immunocompromised Beige mice. These protection and immunogenicity findings suggest that synthetically derived EmT4™ adjuvant is not only suitable to help backfill the preclinical TB vaccine candidate pipeline but is also suitable for the needs of the global community.

Article	Year
Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 36375278	2022
Evaluation of host immune responses to Mycobacteriophage Fionnbharth by route of delivery. Virology journal\| PubMed ID: 39833928	2025
Characterizing TLR4 agonist EmT4™ as an anti-Mycobacterium tuberculosis vaccine adjuvant. ImmunoHorizons\| PubMed ID: 40285479	2025

Suhavi Kaur

Biography

JoVE Journal Publications

Other Publications