Apramycin efficacy against carbapenem- and aminoglycoside-resistant Escherichia coli and Klebsiella pneumoniae in murine blood stream infection models.

BACKGROUND: The aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, its potential in the treatment of drug-resistant bloodstream infections (BSIs) has yet to be assessed. METHODS: The resistance gene annotations of 40 888 blood culture isolates were analyzed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for nine Escherichia coli and Klebsiella pneumoniae isolates. RESULTS: Genotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, in comparison to 46.4% of colistin and 2.1% of apramycin resistance. Apramycin activity against methylated ribosomes was > 100-fold higher than other aminoglycosides. Frequencies of resistance were < 10-9 at 8  ×  MIC. Tentative epidemiological cutoffs (ECOFFs) were determined as 8 µg/mL for E. coli and 4 µg/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log CFU reduction in the blood and peritoneum. Two doses of 80 mg/kg, resulting in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans, resulted in complete eradication of carbapenem- and aminoglycoside-resistant bacteremias. CONCLUSION: Encouraging coverage and potent in vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants further consideration of clinical studies to validate apramycin as a drug candidate for the treatment and prophylaxis of BSI.

Pharmacokinetic and pharmacodynamic evaluation of the atypical tetracyclines chelocardin and amidochelocardin in murine infection models.

IMPORTANCE: There is a strong need to find novel treatment options against urinary tract infections associated with antimicrobial resistance. This study evaluates two atypical tetracyclines, namely chelocardin (CHD) and amidochelocardin (CDCHD), with respect to their pharmacokinetics and pharmacodynamics. We show CHD and CDCHD are cleared at high concentrations in mouse urine. Especially, CDCHD is highly effective in an ascending urinary tract infection model, suggesting further preclinical evaluation.

Expert workshop summary: Advancing toward a standardized murine model to evaluate treatments for antimicrobial resistance lung infections.

The rise in antimicrobial resistance (AMR), and increase in treatment-refractory AMR infections, generates an urgent need to accelerate the discovery and development of novel anti-infectives. Preclinical animal models play a crucial role in assessing the efficacy of novel drugs, informing human dosing regimens and progressing drug candidates into the clinic. The Innovative Medicines Initiative-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium is establishing a validated and globally harmonized preclinical model to increase reproducibility and more reliably translate results from animals to humans. Toward this goal, in April 2021, COMBINE organized the expert workshop "Advancing toward a standardized murine model to evaluate treatments for AMR lung infections". This workshop explored the conduct and interpretation of mouse infection models, with presentations on PK/PD and efficacy studies of small molecule antibiotics, combination treatments (ß-lactam/ß-lactamase inhibitor), bacteriophage therapy, monoclonal antibodies and iron sequestering molecules, with a focus on the major Gram-negative AMR respiratory pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Here we summarize the factors of variability that we identified in murine lung infection models used for antimicrobial efficacy testing, as well as the workshop presentations, panel discussions and the survey results for the harmonization of key experimental parameters. The resulting recommendations for standard design parameters are presented in this document and will provide the basis for the development of a harmonized and bench-marked efficacy studies in preclinical murine pneumonia model.

Variability of murine bacterial pneumonia models used to evaluate antimicrobial agents.

Antimicrobial resistance has become one of the greatest threats to human health, and new antibacterial treatments are urgently needed. As a tool to develop novel therapies, animal models are essential to bridge the gap between preclinical and clinical research. However, despite common usage of in vivo models that mimic clinical infection, translational challenges remain high. Standardization of in vivo models is deemed necessary to improve the robustness and reproducibility of preclinical studies and thus translational research. The European Innovative Medicines Initiative (IMI)-funded "Collaboration for prevention and treatment of MDR bacterial infections" (COMBINE) consortium, aims to develop a standardized, quality-controlled murine pneumonia model for preclinical efficacy testing of novel anti-infective candidates and to improve tools for the translation of preclinical data to the clinic. In this review of murine pneumonia model data published in the last 10 years, we present our findings of considerable variability in the protocols employed for testing the efficacy of antimicrobial compounds using this in vivo model. Based on specific inclusion criteria, fifty-three studies focusing on antimicrobial assessment against Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were reviewed in detail. The data revealed marked differences in the experimental design of the murine pneumonia models employed in the literature. Notably, several differences were observed in variables that are expected to impact the obtained results, such as the immune status of the animals, the age, infection route and sample processing, highlighting the necessity of a standardized model.

Topical niclosamide (ATx201) reduces Staphylococcus aureus colonization and increases Shannon diversity of the skin microbiome in atopic dermatitis patients in a randomized, double-blind, placebo-controlled Phase 2 trial.

BACKGROUND: In patients with atopic dermatitis (AD), Staphylococcus aureus frequently colonizes lesions and is hypothesized to be linked to disease severity and progression. Treatments that reduce S. aureus colonization without significantly affecting the skin commensal microbiota are needed. METHODS AND FINDINGS: In this study, we tested ATx201 (niclosamide), a small molecule, on its efficacy to reduce S. aureus and propensity to evolve resistance in vitro. Various cutaneous formulations were then tested in a superficial skin infection model. Finally, a Phase 2 randomized, double-blind and placebo-controlled trial was performed to investigate the impact of ATx201 OINTMENT 2% on S. aureus colonization and skin microbiome composition in patients with mild-to-severe AD (EudraCT:2016-003501-33). ATx201 has a narrow minimal inhibitory concentration distribution (.125-.5 µg/ml) consistent with its mode of action - targeting the proton motive force effectively stopping cell growth. In murine models, ATx201 can effectively treat superficial skin infections of methicillin-resistant S. aureus. In a Phase 2 trial in patients with mild-to-severe AD (N = 36), twice-daily treatment with ATx201 OINTMENT 2% effectively reduces S. aureus colonization in quantitative colony forming unit (CFU) analysis (primary endpoint: 94.4% active vs. 38.9% vehicle success rate, p = .0016) and increases the Shannon diversity of the skin microbiome at day 7 significantly compared to vehicle. CONCLUSION: These results suggest that ATx201 could become a new treatment modality as a decolonizing agent.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria.

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.

How preclinical infection models help define antibiotic doses in the clinic.

Appropriate dosing of antibiotics is key in the treatment of bacterial infections to ensure clinical efficacy while avoiding toxic drug concentrations and minimizing emergence of resistance. As collection of sufficient clinical evidence is difficult for specific patient populations, infection types and pathogens, market authorization, dosing strategies and recommendations often rely on data obtained from in vitro and animal experiments. The aim of this review is to provide an overview of commonly used preclinical infection models, including their strengths and limitations. In vitro, static and dynamic time-kill experiments are the most frequently used methods for assessing pharmacokinetic/pharmacodynamic (PK/PD) associations. Limitations of in vitro models include the inability to account for the effects of the immune system, and uncertainties in clinically relevant bacterial concentrations, growth conditions and the implications of emerging resistant bacterial populations during experiments. Animal experiments, most commonly murine lung and thigh infections models, are considered a necessary link between in vitro data and the clinical situation. However, there are differences in pathophysiology, immunology, and PK between species. Mathematical modeling in which preclinical data are integrated with human population PK can facilitate translation of preclinical data to the patient's clinical situation. Moreover, PK/PD modeling and simulations can help in the design of clinical trials aiming to establish optimal dosing regimens to improve patient outcomes.

ESAT-6 (EsxA) and TB10.4 (EsxH) based vaccines for pre- and post-exposure tuberculosis vaccination.

The ESX systems from Mycobacterium tuberculosis are responsible for the secretion of highly immunogenic proteins of key importance for bacterial survival and growth. The two prototypic proteins, ESAT-6 (EsxA from ESX-1) and TB10.4 (EsxH from ESX-3) share a lot of characteristics regarding genome organization, size, antigenic properties, and vaccine potential but the two molecules clearly have very different roles in bacterial physiology. To further investigate the role of ESAT-6 and TB10.4 as preventive and post-exposure tuberculosis vaccines, we evaluated four different fusion-protein vaccines; H1, H4, H56 and H28, that differ only in these two components. We found that all of these vaccines give rise to protection in a conventional prophylactic vaccination model. In contrast, only the ESAT-6-containing vaccines resulted in significant protection against reactivation, when administered post-exposure. This difference in post-exposure activity did not correlate with a difference in gene expression during infection or a differential magnitude or quality of the vaccine-specific CD4 T cells induced by ESAT-6 versus TB10.4-containing vaccines. The post-exposure effect of the ESAT-6 based vaccines was found to be influenced by the infectious load at the time-point of vaccination and was abolished in chronically infected animals with high bacterial loads at the onset of vaccination. Our data demonstrate that there are specific requirements for the immune system to target an already established tuberculosis infection and that ESAT-6 has a unique potential in post-exposure vaccination strategies.

CAF01 liposomes as a mucosal vaccine adjuvant: In vitro and in vivo investigations.

Mucosal administration of vaccines has many advantages compared to parenteral vaccination. Needle-free mucosal vaccination would be easily applicable, target the vaccine to the entry point of many pathogens, and reduce the risk of infection with other pathogens during vaccination as compared to invasive methods. CAF01 is a novel liposome-based vaccine adjuvant with remarkable immunostimulatory activity. The potential of CAF01 liposomes as adjuvant for mucosal vaccines was investigated using the Calu-3 epithelial cell culture in vitro model. Thus, the mucosal permeability of the antigen as well as the epithelial integrity and the metabolic activity of the well-differentiated cells were investigated after exposure to CAF01. Finally, the adjuvant was tested for nasal administration in mice, combined with an influenza vaccine. The results suggest that CAF01 enhanced transport of antigen through the mucus layer on Calu-3 cells, increasing the concentration of antigen in the cell layer, as well as the transport across the epithelial cells. Furthermore CAF01 was well tolerated by the Calu-3 cells and the in vivo studies demonstrated increased cell-mediated immunity (CMI) as well as humoral immune responses in mice after nasal application of the influenza vaccine when combined with CAF01. CAF01 is thus a promising adjuvant for mucosal delivery.

TB vaccine strategies--what is needed to solve a complex problem?

An estimated 2 billion people are latently infected with Mycobacterium tuberculosis, the majority of which are already BCG vaccinated and repeatedly sensitized to mycobacterial strains from the environment. To be successful in the high endemic regions, any future TB vaccine strategy will have to be tailored in accordance with the resulting complexity of the TB infection and anti-mycobacterial immune response. In this review we will discuss some of the most advanced attempts to address this challenge.