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Background Listeriosis is an orphan disease, which is nevertheless fatal in immunocompromised people. CRS0540 is a novel PolC DNA polymerase inhibitor that has demonstrated good in vitro and in vivo activity against Listeria monocytogenes. Methods Rodent-to-human allometry projection-based human population pharmacokinetics of CRS0540 were used for all studies. CRS0540 pharmacokinetics/pharmacodynamics studies in an intracellular hollow-fibre system model of disseminated listeriosis (HFS-Lister) examined the effect of eight treatment doses, administered daily over 7 days, in duplicate units. Total bacterial burden versus AUC/MIC exposures on each day were modelled using the inhibitory sigmoid Emax model, while CRS0540-resistant bacterial burden was modelled using a quadratic function. Ten thousand-subject Monte Carlo simulations were used to predict an optimal clinical dose for treatment. Results The mean CRS0540 intracellular/extracellular AUC0–24 ratio was 34.07 (standard error: 15.70) as measured in the HFS-Lister. CRS0540 demonstrated exposure-dependent bactericidal activity in the HFS-Lister, with the highest exposure killing approximately 5.0 log10 cfu/mL. The free drug AUC0–24/MIC associated with 80% of maximal kill (EC80) was 36.4. Resistance emergence versus AUC/MIC was described by a quadratic function, with resistance amplification at an AUC/MIC of 54.8 and resistance suppression at an AUC/MIC of 119. Monte Carlo simulations demonstrated that for the EC80 target, IV CRS0540 doses of 100 mg/kg achieved PTAs of >90% at MICs up to 1.0 mg/L. Conclusions CRS0540 is a promising orphan drug candidate for listeriosis. Future PK/PD studies comparing it with penicillin, the standard of care, could lead to this drug as a new treatment in immunocompromised patients.

Objectives The standard of care (SOC) for the treatment of pulmonary Mycobacterium avium complex (MAC) disease (clarithromycin, rifabutin, and ethambutol) achieves sustained sputum conversion rates of only 54%. Thus, new treatments should be prioritized. Methods We identified the omadacycline MIC against one laboratory MAC strain and calculated drug half life in solution, which we compared with measured MAC doubling times. Next, we performed an omadacycline hollow fibre system model of intracellular MAC (HFS-MAC) exposure–effect study, as well as the three-drug SOC, using pharmacokinetics achieved in patient lung lesions. Data was analysed using bacterial kill slopes (γ-slopes) and inhibitory sigmoid Emax bacterial burden versus exposure analyses. Monte Carlo experiments (MCE) were used to identify the optimal omadacycline clinical dose. Results Omadacycline concentration declined in solution with a half-life of 27.7 h versus a MAC doubling time of 16.3 h, leading to artefactually high MICs. Exposures mediating 80% of maximal effect changed up to 8-fold depending on sampling day with bacterial burden versus exposure analyses, while γ-slope-based analyses gave a single robust estimate. The highest omadacycline monotherapy γ-slope was −0.114 (95% CI: −0.141 to −0.087) (r2 = 0.98) versus −0.114 (95% CI: −0.133 to −0.094) (r2 = 0.99) with the SOC. MCEs demonstrated that 450 mg of omadacycline given orally on the first 2 days followed by 300 mg daily would achieve the AUC0-24 target of 39.67 mg·h/L. Conclusions Omadacycline may be a potential treatment option for pulmonary MAC, possibly as a back-bone treatment for a new MAC regimen and warrants future study in treatment of this disease.

Background Based on meta-analyses the current standard-of-care regimen (SOC) for Mycobacterium avium complex (MAC) lung disease achieves a sustained sputum conversion rate of ∼54% at best. Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. To identify EBO exposure-effect parameters we used the intracellular hollow fiber system model of intracellular pulmonary MAC (HFS-MAC). Methods EBO was administered once daily at 8 different doses to HFS-MAC replicates for 28 days to achieve a half-life (t½) of 10.4h and the 0-24h area under the concentration-time curves (AUC0-24) that cover the observed AUC values in humans. The SOC combination of clarithromycin (CLR), ethambutol (EMB), and rifabutin (RFB) at human intrapulmonary pharmacokinetic (PK) concentrations was used. The central compartment of each HFS-MAC unit was sampled throughout the 28 days to assess PK parameters as was the peripheral compartment for total bacterial burden and the EBO-resistant bacterial burden. EBO AUC versus total MAC burden was modeled using the inhibitory sigmoid maximal effect (Emax) model. EBO AUC versus EBO-resistant MAC burden was modeled using a quadratic function. Results Measured EBO concentrations demonstrated a t1/2 of 10h. For SOC, the AUC for CLR, EMB, and RFB was 60 mg*h/L (t1/2=6h), 39 mg*h/L (t1/2=8h), and 1.5 mg*h/L (t1/2=45h), respectively, similar to human lung concentrations. Changes in MAC burden over 28 days (Fig. 1) show that highest EBO exposures matched SOC until day 14. The exposure versus effect on each sampling day is shown in Fig. 2. The EBO AUC mediating 50% of Emax (EC50) was an AUC of 22 mg*h/L (95% confidence interval [CI]: 16-70), and the EC80 was an AUC of 47.5 mg*h/L (CI: 34.6-151.2). The relationship between AUC and EBO-resistant subpopulation (Fig. 3) shows that an AUC0-24 of 47.5 mg*h/L, the same as the EC80 for microbial kill, was associated with resistance suppression, as was the addition of SOC to EBO. Figure 1.Changes in bacterial burden over the 28-day dosing period. Bacterial burden on serial samples from all HFS-MAC units and replicates over 28days are shown. The highest epetraborole exposures demonstrated the same effectiveness as the three drug SOC until day 14, after which it failed as resistance emerged. On the other hand adding epetraborole at an AUC of 43.6 mg*h/L to the SOC led to much faster kill (i.e, higher kill slopes) than SOC or the epetraborole monotherapy at the same AUC. Figure 2.Epetraborole exposure versus effect on bacterial burden. Shown are inhibitory sigmoid Emax curves for epetraborole AUC versus log10 CFU/mL on each sampling day, as well as various extents of microbial kill such as stasis (holding burden similar to day 0), 1 log CFU/mL kill and 2 log CFU/mL kill. Beyond day 3, epetraborole killed > 1log CFU/mL, which means it was rapidly bactericidal. Figure 3.Epetraborole exposure versus epetraborole-resistant subpopulation. Drug-resistant burden is shown as log10 CFU/mL. The addition of SOC to EBO totally suppressed emergence of any resistance on all days. Conclusion EBO monotherapy with an AUC0-24 > 16.9 mg*h/L killed > 1.0 log10 CFU/mL of MAC compared to day 0. At the EC80, EBO killed at least 2.0 log10 CFU/mL, thus was highly bactericidal. EBO plus SOC demonstrated resistance suppression. Disclosures Moti Chapagain, MD, PhD, Praedicare Inc.: Stocks/Bonds Shruti Athale, PhD, Praedicare Inc.: Stocks/Bonds Jotam Pasipanodya, MD, PhD, Praedicare Inc.: Employee MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Tawanda Gumbo, MD, Hydronium Biopharma: Ownership Interest|Hydronium Biopharma: Stocks/Bonds|Praedicare Africa Pvt Ltd: Board Member|Praedicare Africa Pvt Ltd: Ownership Interest|Praedicare Africa Pvt Ltd: Stocks/Bonds|Praedicare Inc.: Patents|Praedicare Inc.: Ownership Interest|Praedicare Inc.: Stocks/Bonds.

Objective To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. Methods Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. Results In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. Conclusion These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Drug-resistant tuberculosis represents a global emergency, requiring new drugs. We found that minocycline was highly potent in laboratory strains of Mycobacterium tuberculosis and that 30 drug-susceptible and multidrug/extensively drug-resistant clinical strains were susceptible to clinically achievable concentrations. In the hollow fiber system model, lung concentration-time profiles of 7 mg/kg/day human-equivalent minocycline dose achieved bacterial kill rates equivalent to those of first-line antituberculosis agents. Minocycline killed extracellular bacilli directly. Minocycline also killed intracellular bacilli indirectly, via concentration-dependent granzyme A-driven apoptosis. Moreover, minocycline demonstrated dose-dependent antiinflammatory activity and downregulation of extracellular matrix-based remodeling pathways and, thus, could protect patients from tuberculosis immunopathology. In RNA sequencing of repetitive samples from the hollow fiber system and in independent protein abundance experiments, minocycline demonstrated dose-dependent inhibition of sonic hedgehog-patched-gli signaling. These findings have implications for improved lung remodeling and for dual immunomodulation and direct microbial kill-based treatment shortening regimens for drug-susceptible and drug-resistant latent and active M. tuberculosis infection.

Understanding the mechanisms underlying autoantibody development will accelerate therapeutic target identification in autoimmune diseases such as systemic lupus erythematosus (SLE)1. Follicular helper T cells (TFH cells) have long been implicated in SLE pathogenesis. Yet a fraction of autoantibodies in individuals with SLE are unmutated, supporting that autoreactive B cells also differentiate outside germinal centers2. Here, we describe a CXCR5-CXCR3+ programmed death 1 (PD1)hiCD4+ helper T cell population distinct from TFH cells and expanded in both SLE blood and the tubulointerstitial areas of individuals with proliferative lupus nephritis. These cells produce interleukin-10 (IL-10) and accumulate mitochondrial reactive oxygen species as the result of reverse electron transport fueled by succinate. Furthermore, they provide B cell help, independently of IL-21, through IL-10 and succinate. Similar cells are generated in vitro upon priming naive CD4+ T cells with plasmacytoid dendritic cells activated with oxidized mitochondrial DNA, a distinct class of interferogenic toll-like receptor 9 ligand3. Targeting this pathway might blunt the initiation and/or perpetuation of extrafollicular humoral responses in SLE.

Human dendritic cells (DCs) play a fundamental role in the initiation of long-term adaptive immunity during vaccination against influenza. Understanding the early response of human DCs to vaccine exposure is thus essential to determine the nature and magnitude of maturation signals that have been shown to strongly correlate with vaccine effectiveness. In 2009, the H1N1 influenza epidemics fostered the commercialization of the nonadjuvanted monovalent H1N1 California vaccine (MIV-09) to complement the existing nonadjuvanted trivalent Fluzone 2009-2010 vaccine (TIV-09). In retrospective studies, MIV-09 displayed lower effectiveness than TIV-09. We show that TIV-09 induces monocyte-derived DCs (moDCs), blood conventional DCs (cDCs), and plasmacytoid DCs (pDCs) to express CD80, CD83, and CD86 and secrete cytokines. TIV-09 stimulated the secretion of type I interferons (IFNs) IFN-α and IFN-β and type III IFN interleukin-29 (IL-29) by moDC and cDC subsets. The vaccine also induced the production of IL-6, tumor necrosis factor, and the chemokines IFN-γ-inducible protein 10 (IP-10) and macrophage inflammatory protein-1β (MIP-1β). Conversely, MIV-09 did not induce the production of type I IFNs in moDCs and blood cDCs. Furthermore, it inhibited the TIV-09-induced secretion of type I IFNs by these DCs. However, both vaccines induced pDCs to secrete type I IFNs, indicating that different influenza vaccines activate distinct molecular signaling pathways in DC subsets. These results suggest that subtypes of nonadjuvanted influenza vaccines trigger immunity through different mechanisms and that the ability of a vaccine to induce an IFN response in DCs may offset the absence of adjuvant and increase vaccine efficacy.

Smallpox was eradicated by vaccination with a related poxvirus, vaccinia virus, which was applied to superficially injured skin in a process called scarification. Recombinant poxvirus–based vaccines are attractive candidates for protecting against a number of different infections, but they are nowadays usually administered intramuscularly. Thomas Kupper and his colleagues now show that the traditional route of administration, scarification, enables poxvirus-based vaccines to mediate more potent immunity compared with the intramuscular route. In particular, scarification with the poxvirus vaccines induced the sort of immune responses that are required for protection of distant tissues, including the lung mucosa.

Autoantibodies against nucleic acids and excessive type I interferon (IFN) are hallmarks of human systemic lupus erythematosus (SLE). We previously reported that SLE neutrophils exposed to TLR7 agonist autoantibodies release interferogenic DNA, which we now demonstrate to be of mitochondrial origin. We further show that healthy human neutrophils do not complete mitophagy upon induction of mitochondrial damage. Rather, they extrude mitochondrial components, including DNA (mtDNA), devoid of oxidized (Ox) residues. When mtDNA undergoes oxidation, it is directly routed to lysosomes for degradation. This rerouting requires dissociation from the transcription factor A mitochondria (TFAM), a dual high-mobility group (HMG) protein involved in maintenance and compaction of the mitochondrial genome into nucleoids. Exposure of SLE neutrophils, or healthy IFN-primed neutrophils, to antiribonucleotide protein autoantibodies blocks TFAM phosphorylation, a necessary step for nucleoid dissociation. Consequently, Ox nucleoids accumulate within mitochondria and are eventually extruded as potent interferogenic complexes. In support of the in vivo relevance of this phenomenon, mitochondrial retention of Ox nucleoids is a feature of SLE blood neutrophils, and autoantibodies against Ox mtDNA are present in a fraction of patients. This pathway represents a novel therapeutic target in human SLE.

The mechanisms by which microbial vaccines interact with human APCs remain elusive. Herein, we describe the transcriptional programs induced in human DCs by pathogens, innate receptor ligands and vaccines. Exposure of DCs to influenza, Salmonella enterica and Staphylococcus aureus allows us to build a modular framework containing 204 transcript clusters. We use this framework to characterize the responses of human monocytes, monocyte-derived DCs and blood DC subsets to 13 vaccines. Different vaccines induce distinct transcriptional programs based on pathogen type, adjuvant formulation and APC targeted. Fluzone, Pneumovax and Gardasil, respectively, activate monocyte-derived DCs, monocytes and CD1c+ blood DCs, highlighting APC specialization in response to vaccines. Finally, the blood signatures from individuals vaccinated with Fluzone or infected with influenza reveal a signature of adaptive immunity activation following vaccination and symptomatic infections, but not asymptomatic infections. These data, offered with a web interface, may guide the development of improved vaccines., The effects of vaccines on human dendritic cells (DCs) are incompletely understood. Here the authors build a transcriptional modular network based on in vitro infection of human DCs and apply it for the characterization of the DC response to 13 different vaccines.