Michelle Nelson

Background/Objectives: Q fever, caused by Coxiella burnetii, is a zoonotic disease usually treated with doxycycline, but alternative options are needed when doxycycline is unsuitable. This study evaluated inhaled liposomal ciprofloxacin (Apulmiq^®) as a post-exposure prophylaxis (PEP) compared with oral doxycycline in a common marmoset model. Methods: Marmosets were exposed to aerosolised C. burnetii and treated 24 h later with Apulmiq (0.8 mg/kg inhaled), doxycycline (6 mg/kg oral), or placebo for 7 days. Outcomes included fever, weight loss, bacterial burden, histopathology, liver enzymes, and immune responses. Results: Both treatments delayed fever onset and reduced disease severity versus placebo. Doxycycline prevented fever during treatment and significantly reduced bacterial loads in the lungs and spleen by Day 28. Apulmiq slightly delayed the onset of fever and reduced early bacteraemia but was less effective at bacterial clearance. Immune analyses showed macrophage activation and elevation of IFN-γ during acute infection, and adaptive immune responses by Day 28. Conclusions: Inhaled Apulmiq provided partial prophylactic protection but did not achieve bacterial clearance. Doxycycline reduced bacterial burden but did not completely eliminate disease. Inhaled antibiotics may have potential for use as an alternative approach for PEP treatment, although further optimisation and investigation are needed.

Introduction: Francisella tularensis is the causative agent of tularemia, a severe infection that can be fatal if untreated. There is a need for improved therapeutic options to enhance the efficacy of existing antibiotic regimens for severe tularemia and to reduce the risk of disease relapse. Novel formulations of ciprofloxacin have demonstrated protection against inhalational tularemia in murine and marmoset models; however, a detailed immunological characterisation of treatment response is required. Methods: Blood samples were collected from marmosets infected with a lethal dose of inhalational tularemia and treated with either oral ciprofloxacin or inhaled ciprofloxacin (Apulmiq®). Immunological responses were assessed longitudinally and compared with immunological changes previously observed in recovering human cases of tularemia. Results: Infected marmosets exhibited increased neutrophil levels, which, together with reduced HLA-DR expression, indicated severe infection. Rising circulating interferon-gamma (IFN γ) levels were followed by classical activation of macrophages as an initial attempt to control bacterial dissemination. Progressive disease was characterised by a marked depletion of neutrophils, excessive IFN γ production, and elevated levels of overactivated macrophages. In animals that responded successfully to treatment, these immunological parameters returned to baseline levels. Recovery was subsequently associated with increased T cell populations, particularly gamma delta T (γδ T) cells, and limited antibody production, suggesting the development of long-term immunity comparable to that observed in humans. Discussion: The observed immunological changes in marmosets closely mirror those reported in human tularemia, supporting the relevance of this model for studying disease progression and recovery. The capacity to monitor these immune parameters demonstrates the utility of the marmoset model in assessing the efficacy of new and novel antibiotic treatments for tularemia.

Background/Objectives: Q fever, caused by Coxiella burnetii, is typically treated with doxycycline, but its efficacy is limited in chronic cases and may be poorly tolerated. Systemic ciprofloxacin shows limited activity for acute Q fever. However, inhaled liposomal formulations may provide therapeutic benefit. Methods: Two inhaled ciprofloxacin formulations (Lipoquin^® and Apulmiq^®) were evaluated in an A/J mouse model of Q fever and compared with intraperitoneal ciprofloxacin and oral doxycycline. Initially, pharmacokinetic studies were performed to determine an appropriate dosing regimen for the inhaled ciprofloxacin formulations. A separate cohort of mice were then infected with C. burnetii and treated once daily via nebulisation with Lipoquin or Apulmiq, initiated at 24, 48, or 72 h post-challenge. Clinical signs, weight change, splenomegaly, bacterial burden, and lung histopathology were evaluated. Results: Pharmacokinetic analysis confirmed sustained lung concentrations of inhaled ciprofloxacin, supporting once-daily dosing. Inhaled Lipoquin and Apulmiq significantly reduced clinical signs, weight loss, splenomegaly, and pulmonary bacterial burden compared to untreated controls and doxycycline-treated mice. Histopathology revealed decreased lung inflammation and lesion severity following inhalational dosing. Systemic ciprofloxacin slightly reduced splenic bacterial burden but was less effective in controlling pulmonary infection. Conclusions: Inhaled liposomal ciprofloxacin demonstrated superior protection and reduced respiratory manifestations of Q fever compared to doxycycline and systemic ciprofloxacin. These findings suggest inhaled formulations may represent a viable alternative for the treatment of Q fever pneumonia. Further studies are needed to evaluate clinical applicability and long-term outcomes.

Francisella tularensis is a gram-negative, intracellular bacterium that causes the disease tularemia. Tularemia is prevalent in North America, Europe, and Asia and is typically treated with injected and orally administered antibiotics, including streptomycin, gentamicin, doxycycline, and ciprofloxacin, administered for 10 to 21 days. New therapeutic options are required to reduce the potential of a relapse of disease. Inhaled liposomal-encapsulated ciprofloxacin has demonstrated protection in a murine model of tularemia. The efficacy was further assessed in a nonhuman primate model of tularemia. Mixed-sex common marmosets were challenged with F. tularensis by the inhalational route, and the efficacy of ciprofloxacin delivered by either the inhalational (Apulmiq liposomal formulation) or oral route was compared. Antibiotics were initiated either at 24 h post-challenge (post-exposure prophylaxis) or at the onset of fever (treatment) and continued for 7 days. All control (untreated) animals succumbed to infection by 8 days post-challenge. All animals that received antibiotics, by either route, survived the duration of the study, with bacterial clearance in all but one animal that received inhalational ciprofloxacin. Antibiotic treatment also reduced the physiological and immunological responses observed when compared to animals that received no antibiotics. Histological changes in the lungs were less frequent, although mild, resolving lesions were present in animals treated with ciprofloxacin delivered at the onset of fever by either route.

Background: Inhalational antibiotics have been used effectively to treat chronic diseases such as Pseudomonas aeruginosa infections associated with cystic fibrosis. This approach may enhance treatment options for difficult-to-treat, acute pneumonic diseases. Liposomal encapsulated ciprofloxacin (Lipoquin and/or Apulmiq) has provided protection in murine models of plague, anthrax, Q fever and tularemia. Development of the ability to deliver these drugs to nonhuman primates (NHPs) would enable further extrapolation of the data observed in small animal models of infection to humans. Methods: In this study, the methodology was established to deliver Apulmiq to common marmosets (Callithrix jacchus). Marmosets were anaesthetised with a novel, reversible anaesthetic comprising fentanyl, medetomidine and midazolam (FMM). They were placed into plethysmography tubes with their heads in an exposure chamber. The LC Sprint jet nebuliser or Pari eFlow Rapid nebuliser were used to aerosolise Apulmiq into the exposure chamber. Animals were euthanised after dosing and the concentration of ciprofloxacin was assessed in the plasma and lungs of the animals. Results: Non-compartmental pharmacokinetic analysis determined that a 30 min exposure of drug was required to reach a human-equivalent target dose of 0.8 mg/kg body weight in the lungs. Conclusions: This approach can now be used to assess the efficacy of inhalational liposomal ciprofloxacin in NHP infection models.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

Burkholderia pseudomallei, the causative agent of the disease melioidosis, has been isolated from the environment in 45 countries. The treatment of melioidosis is complex, requiring lengthy antibiotic regimens, which can result in the relapse of the disease following treatment cessation. It is important that novel therapies to treat infections with B. pseudomallei be assessed in appropriate animal models, and discussions regarding the different protocols used between laboratories are critical. A ‘deep dive’ was held in October 2020 focusing on the use of the BALB/c mouse model and the inhalational route of infection to evaluate new antibiotic therapies.

Burkholderia pseudomallei is the causative agent of melioidosis and presents with diverse clinical manifestations. Naturally occurring infection occurs following contamination of cuts or skin abrasions, or ingestion of contaminated water, and occasionally through inhalational of infected soil or water particles.

Burkholderia pseudomallei is the causative agent of melioidosis, a multifaceted disease. A proportion of the mortality and morbidity reported as a result of infection with this organism may be due to the premature cessation of antibiotic therapy typically lasting for several months. The progression of re-emergent disease was characterised in Balb/c mice following cessation of a 14 day treatment course of co-trimoxazole or finafloxacin, delivered at a human equivalent dose. Mice were culled weekly and the infection characterised in terms of bacterial load in tissues, weight loss, clinical signs of infection, cytokine levels and immunological cell counts. Following cessation of treatment, the infection re-established in some animals. Finafloxacin prevented the re-establishment of the infection for longer than co-trimoxazole, and it is apparent based on the protection offered, the development of clinical signs of disease, bodyweight loss and bacterial load, that finafloxacin was more effective at controlling infection when compared to co-trimoxazole.

There is an enduring requirement to develop animal models of COVID-19 to assess the efficacy of vaccines and therapeutics that can be used to treat the disease in humans. In this study, six marmosets were exposed to a small particle aerosol (1–3 µm) of SARS-CoV-2 VIC01 that delivered the virus directly to the lower respiratory tract. Following the challenge, marmosets did not develop clinical signs, although a disruption to the normal diurnal temperature rhythm was observed in three out of six animals. Early weight loss and changes to respiratory pattern and activity were also observed, yet there was limited evidence of viral replication or lung pathology associated with infection. There was a robust innate immunological response to infection, which included an early increase in circulating neutrophils and monocytes and a reduction in the proportion of circulating T-cells. Expression of the ACE2 receptor in respiratory tissues was almost absent, but there was ubiquitous expression of TMPRSS2. The results of this study indicate that exposure of marmosets to high concentrations of aerosolised SARS-CoV-2 did not result in the development of clear, reproducible signs of COVID-19.